SECTION 13 Endocrine disorders

13.1 Principles of hormone action 2245 Rob Fowkes,

13.1 Principles of hormone action 2245 Rob Fowkes, V. Krishna Chatterjee, and Mark Gurnell

ESSENTIALS Hormones, produced by glands or cells, are messengers which act locally or at a distance to coordinate the function of cells and organs. Types of hormone include (1) peptides (e.g. hypothalamic releasing factors) and proteins (e.g. insulin, growth hormone)—​these generally interact with membrane receptors located on the cell surface, causing activation of downstream signalling pathways leading to alteration in gene transcription or modulation of biochemical pathways to effect a physiological response; (2) steroids (e.g. cortisol, progesterone, tes- tosterone, oestradiol) and other lipophilic substances (e.g. vitamin D, retinoic acid, thyroid hormone)—​these act by crossing the plasma membrane to interact with intracellular receptors, with hormone ac- tion via nuclear receptors altering cellular gene expression directly. Hormone synthesis, processing, and secretion—​production of hor- mones can be regulated at many levels, including (1) gene transcrip- tion; (2) mRNA processing; (3) post-​translational modification. Some hormones are not significantly concentrated within cells and are re- leased via Golgi-​derived transport vesicles that fuse with the plasma membrane (a ‘constitutive’ pathway of secretion). By contrast, many endocrine cells contain an additional ‘regulated’ secretory pathway, which allows the export of high concentrations of hormone stored in cytoplasmic vesicles. Many hormones are released in a rhythmic or pulsatile manner. Control of hormone production—​the classical mechanism by which hormone-​producing glands are controlled is by negative feedback, e.g. triiodothyronine (T3) inhibits production of thyrotropin-​releasing hormone and thyroid-​stimulating hormone. Physiological roles of hormones—​these are enormously varied and include (1) control of growth and differentiation; (2) maintenance of homeostasis—​energy balance, metabolic pathways; fluid, electrolyte, and calcium balance; control of blood pressure; and (3) regulation of reproduction. Clinical features of endocrine disorders—​these comprise conditions of either hormone excess or hormone deficiency or hormone resist- ance, caused by acquired endocrine cellular dysfunction or germline or somatic defects in genes mediating hormone synthesis or action causing inherited syndromes. Definition Endocrinology is the study of hormones secreted by glands or cells which, acting locally or at a distance, facilitate communica- tion between cells and different organs, thus coordinating their activities. Classically, the production of hormones has been associated with specialized glands or tissues including the hypothalamus, pituitary, thyroid, parathyroids, gonads, pancreatic islet cells, ad- renal glands, and placenta. It is now recognized that hormones are also produced by a range of other organs and tissues which are not considered to be classical endocrine glands. The heart is the primary source of atrial natriuretic peptide factor which has effects blood pressure and intravascular volume; endothelin and nitric oxide are derived from vascular endothelium and regulate vascular tone. Endocrine cells are distributed throughout the gastrointestinal tract and are a rich source of hormones such as cholecystokinin, gastrin, secretin, and vasoactive intestinal pep- tide; many of these gastrointestinal hormones are also produced in the brain and central nervous system, where their role is less well understood. Erythropoietin, a circulating factor that stimu- lates erythropoiesis, is derived from the kidney. Adipose tissue produces leptin, a circulating hormone which acts centrally to control appetite. However, as understanding of intercellular communication has advanced, the lines of division that separate different physio- logical systems have become blurred. For example, neuroendo- crinology represents intimate connections between the nervous and endocrine systems: peptide hormones produced in the brain exert effects via the hypothalamus to control hormone secre- tion from the pituitary gland; in the periphery, the sympathetic nervous system modulates hormone production by the adrenal medulla and pancreatic islets. Similarly, there are complex interrelationships between the immune and endocrine systems (e.g. glucocorticoid hormones exert powerful immunosuppres- sive effects); conversely, cytokines (e.g. tumour necrosis factor α and interleukin (IL)-​6), produced by cells of the immune system, markedly influence hormone secretion by glands such as the pi- tuitary and adrenal. 13.1 Principles of hormone action Rob Fowkes, V. Krishna Chatterjee, and Mark Gurnell

SECTION 13  Endocrine disorders 2246 Nature of hormones In general, hormones can be classified into those that are based on proteins or peptides and those that are chemically derived. Small peptides include hypothalamic releasing factors produced by neuro- endocrine cells, which act locally on the pituitary; larger polypep- tides such as insulin or growth hormone (GH) are characteristically circulating hormones which act on more distant targets. Biogenic amines including catecholamines, dopamine, and serotonin are de- rived from amino acids. Most protein and peptide hormones interact with membrane receptors located on the cell surface. Binding to membrane receptors activates downstream signalling pathways leading to changes in cellular function which mediate responses to hormones. A second class of hormones includes steroids and other lipophilic substances which act by crossing the plasma membrane to interact with intracellular receptors. Steroid hormones are derived from cholesterol and include cortisol, progesterone, testosterone, and oestradiol. Vitamin D and retinoic acid, which are synthesized from dietary sources, and thyroid hormone produced by modification of tyrosines in thyroglobulin, are structurally dissimilar to steroids but also act via nuclear receptors. Development of endocrine glands The hypothalamus develops from forebrain tissue adjacent to the third ventricle. Neurons secreting releasing factors send cel- lular processes which terminate in portal capillaries that perfuse the pituitary gland. The latter develops from ectoderm to form the adenohypophysis or anterior pituitary; the posterior pituitary or neurohypophysis is formed directly from axonal terminals of hypothalamic neurons which grow downward. The thyroid gland develops from endoderm in the floor of the oropharynx with mi- gration of cells caudally to its final position in the neck. During its descent, parafollicular C cells derived from neural crest tissue within the ultimobranchial body and parathyroid glands from the third and fourth pharyngeal pouches, become incorporated into the thyroid gland. The adrenal glands comprise a steroid-​secreting cortex developing from mesoderm, together with a catecholamine-​ producing medulla composed of chromaffin cells derived from neural crest. Germ cells within indifferent gonadal prim- ordia differentiate to form the ovary or, in the presence of the Y chromosome-​encoded sex-​determining gene (SRY), develop into testes. Endocrine cells of the pancreas are derived from endoderm and differentiate to form the islets of Langerhans. Various tran- scription factors which control the development of cells within endocrine glands and their differentiation to hormone biosynthesis are listed in Table 13.1.1. Hormone synthesis, processing, and secretion The organization of endocrine genes is homologous to those encoding many other proteins, although there are some character- istic features. Gene transcription is usually regulated by the pro- moter, which is located in the upstream 5′ flanking region of the gene (Fig. 13.1.1). Typically, the promoter may contain three types of regulatory DNA sequence which are recognized by specific tran- scription factors; a hormone response element is recognized by nuclear receptors; a tissue-​specific element binds cell-​specific Table 13.1.1  Some transcription factors involved in endocrine gland development Gland Transcription factor(s) Pituitary HESX-​1, POU1F1, PROP-​1, TBX19 Thyroid TTF-​1, TTF-​2, PAX-​8 Adrenal cortex SF-​1, DAX-​1 Pancreatic islet cells IPF-​1 Testis SRY, SF-​1 Ovary SF-​1, DAX-​1 DAX-​1, dosage-​sensitive sex reversal adrenal hypoplasia critical region on the
X-​chromosome 1; HESX-​1, homeobox gene expressed in embryonic stem cells 1; IPF-​1, insulin promoter factor 1; PAX-​8, paired box gene 8; POU1F1, POU homeodomain containing pituitary transcription factor 1 (previously known as Pit-​1); PROP-​1, prophet of Pit-​1; SF-​1, steroidogenic factor 1; SRY, sex-​determining region of the Y chromosome; TBX-​19 (also known as TPIT), a T-​box containing transcription factor; TTF-​1, thyroid transcription factor 1; TTF-​2, thyroid transcription factor 2. Signal sequence Prepro- hormone Prohormone cleavage Mature hormone Post-translational modification Secretory granule Ca2+− dependent exocytosis Rough endoplasmic reticulum Golgi network Intracellular Extracellular Nucleus Cytoplasm mRNA Mature polypeptide Basal transcription factors

Transcription initiation site HRE TSE CRE AP-1 Cell specific transcription factor CREB Fos Nuclear receptor Jun Fig. 13.1.1  Pathway of hormone synthesis, processing, and secretion. See text for explanation.

13.1  Principles of hormone action 2247 transcription factors (see Table 13.1.1), which enhance the tran- scription of the hormone gene in a tissue-​specific manner; a third class of response element mediates transcriptional activation in re- sponse to second-​messenger signalling pathways. A rise in intra- cellular cAMP leads to the activation of protein kinase A  and subsequent phosphorylation of cAMP response element binding proteins (CREBs) which interact with CREs; cell signalling path- ways which activate protein kinase C induce phosphorylation of the Fos-​Jun (AP-​1) transcription factor complex which binds its cog- nate DNA regulatory sequence. Binding of transcription factors to regulatory DNA response elements, activates and stabilizes basal transcription factors, promoting gene transcription and mRNA synthesis (Fig. 13.1.1). Transcription of the gene generates mRNA which undergoes translation in ribosomes leading to polypeptide synthesis. In some endocrine genes, alternative exon splicing allows substitution or re- moval of particular exons, such that peptides of differing sequence can be produced. For example, alternative splicing of the calcitonin gene in a tissue-​specific manner directs the production of calcitonin in the C cells of the thyroid, whereas calcitonin gene-​related peptide is produced preferentially in the brain. Secreted polypeptide hormones incorporate a signal sequence at the amino terminus of the protein which directs its translocation across the endoplasmic reticulum where this sequence is cleaved (Fig. 13.1.1). Many hormones are synthesized as larger polypep- tides (prohormones) which undergo proteolytic cleavage to generate smaller functional peptides. Such proteolytic processing is mediated by specific proteases, such as prohormone convertase 1 and 2 (PC1, PC2), which are highly expressed in cells of neuroendocrine lineage. Examples of hormone processing include the cleavage of proinsulin with removal of an internal C peptide to yield insulin, the active hormone. Processing of the polypeptide precursor can also yield multiple functioning products. For example, pro-​opiomelanocortin (POMC) is cleaved by endopeptidases to yield adrenocorticotropic hormone (ACTH), melanocyte-​stimulating hormone (MSHα, β, γ), β-​endorphin, and lipocortin. Hormones may also undergo post-​translational modification such as amidation of neuropeptides, acylation, or glycosylation. Modification of amino acids by addition of carbohydrate side chains is a particular characteristic of the glycoprotein hormones—​ luteinizing hormone (LH), follicle-​stimulating hormone (FSH), thyroid-​stimulating hormone (TSH), and human chorionic gonadotropin (hCG)—​and such glycosylation affects both their biological activity as well as their half-​life in the circulation (see Fig. 13.1.1). Hormones such as growth factors and cytokines are not concen- trated within cells significantly but released via small, clear, Golgi-​ derived transport vesicles which fuse with the plasma membrane, representing a ‘constitutive’ pathway of secretion. In contrast, many endocrine cells contain an additional ‘regulated’ secretory pathway, which allows the export of high concentrations of hormone stored in cytoplasmic dense-​core vesicles. Chromogranin B, an acidic protein, and polypeptide proteases are additional constituents of secretory vesicles. Adrenal cells secreting catecholamine hor- mones contain chromaffin granules which include enzymes (e.g. dopamine β hydroxylase) that catalyse catecholamine biosynthesis. Dense-​core vesicle exocytosis is mediated by a rise in intracellular calcium which activates cytoskeletal machinery, promoting vesicle translocation and docking with the plasma membrane (see Fig. 13.1.1). Cells secreting steroid hormones contain abundant mito- chondrial and smooth endoplasmic reticulum which contain en- zymes that mediate steroid biosynthesis. Mitochondrial side-​chain cleavage enzyme converts cholesterol to pregnenolone and the latter is converted to glucocorticoid, mineralocorticoid, or sex steroids de- pendent on the cell-​specific expression of steroidogenic enzymes. Steroid hormones are not stored to any extent and are secreted constitutively. Control of hormone production The classic mechanism by which hormone-​producing glands com- municate is by endocrine pathways, whereby the products from one gland are secreted into the circulation (and exert effects on a dif- ferent, distant target gland). Such endocrine pathways integrate the hypothalamus, pituitary, and various end organs to control the pro- duction of major hormones (Fig. 13.1.2). Thus, peptide-​releasing factors (e.g. GnRH, TRH, GHRH, CRH) from the hypothalamus, stimulate production of tropic hormones from specific pituitary cell types; exceptions to this are somatostatin, which inhibits pituitary GH release, and dopamine, which is secreted continuously to in- hibit prolactin secretion. The pituitary hormones act on end organs to generate products which, in turn, exert a negative feedback ef- fect at both hypothalamic and pituitary levels to regulate their own synthesis. Triiodothyronine (T3) inhibits TRH and TSH produc- tion; gonadal steroids and inhibin negatively regulate hypothalamic GnRH and pituitary gonadotropins; cortisol suppresses CRH and ACTH generation; circulating insulin-​like growth factor 1 (IGF-​1) inhibits GHRH and GH secretion (Fig. 13.1.2). Osmoreceptors in the hypothalamus sense changes in serum osmolality to control the release of vasopressin from the posterior pituitary. In addition to these endocrine control mechanisms, other types of local regulatory pathways are recognized. Paracrine regulation re- fers to factors that are released by one cell and act upon a nearby cell in the same tissue. For example, somatostatin produced by δ cells in pancreatic islets inhibits the local production of insulin from β cells; in the testis, testosterone produced from Leydig cells exerts an effect on nearby Sertoli cells to enhance spermatogenesis. Autocrine control refers to a factor which acts upon the same cell in which it is produced. Examples include gonadotroph secretion of activin which stimulates production of FSH from the same cell; similarly, T cells produce IL-​2 which acts to promote their own proliferation. In addition to discrete hormonal responses, endocrine systems can respond to environmental stimuli by the integrated production of multiple hormones. For example, stress activates an array of path- ways, with sympathetic activation mediating catecholamine release from the adrenals, and stimulation of the hypothalamus inducing multiple axes, resulting in the production of cortisol, GH, prolactin, and vasopressin. The hormonal responses to starvation are also inte- grated by the hypothalamus. Here, diminished production of leptin from adipose tissue inhibits hypothalamic GnRH and TRH secre- tion with a consequent reduction in the production of both gonadal steroids and thyroid hormone to limit reproduction and energy expenditure. In addition to the feedback regulatory mechanisms just outlined, many hormones are released in a rhythmic or pulsatile manner.

SECTION 13  Endocrine disorders 2248 Insulin is secreted in rapid (c. every 10 min) pulses in response to changes in glucose concentration in the pancreatic β cell. GnRH is secreted from the hypothalamus at a lower pulse frequency of every 1.5 to 3 h, stimulating similar pulses of pituitary LH and FSH release; differential release of LH and FSH is controlled by varying GnRH pulse frequency, with low frequency pulses favouring FSH secre- tion and high frequency pulses stimulating LH secretion. Another hypothalamic peptide (kisspeptin) can augment GnRH secretion in a paracrine manner. This hormonal rhythm controls ovarian folliculogenesis and steroid production to establish the female re- productive and menstrual cycle. Pituitary GH secretion is regulated by pulses of stimulatory GHRH and inhibitory somatostatin from the hypothalamus, which are out of phase with each other, corres- ponding to peaks and troughs of circulating GH. Many hormonal pathways are influenced by the light–​dark cycle, with circadian variation in their circulating levels. For example, the hypothalamic–​pituitary–​adrenal axis exhibits most activity in the early morning with peak cortisol production, followed by a nadir in glucocorticoid levels in the evening. Sleep is another environmental regulator: GH secretion is enhanced nocturnally and the release of vasopressin during sleep inhibits diuresis; puberty is associated with nocturnal surges of LH. Hormone-​binding proteins Thyroid hormones and many steroids are transported in the circula- tion with serum binding proteins. Thus, thyroxine (T4) and triiodo- thyronine (T3) are bound to thyroxine-​binding globulin, albumin, and thyroxine binding prealbumin. Cortisol and progesterone are bound to cortisol binding globulin, while oestrogens and andro- gens are bound to sex hormone-​binding globulin. The role of serum binding proteins is to provide a reservoir of circulating hormone. The interaction of hormones with binding proteins is relatively weak compared to their affinity for receptors, enabling them to dis- sociate easily. Only free hormone interacts with receptor to elicit a biological response. Hormone-​binding proteins are produced by the liver and their synthesis can be increased (e.g. by oestrogens or in pregnancy) or decreased (e.g. in liver disease), affecting the circulating concentration of total hormones. Accordingly, wher- ever possible, the concentration of free hormones in the circulation (e.g. T4, T3) or urine (cortisol) is measured. Some protein hor- mones also circulate associated with binding proteins, which may modulate their action. A range of insulin-​like growth factor binding proteins bind to IGF-​1, with some inhibiting and others facilitating the action of this peptide on target tissue receptors. GH circulates bound to the extracellular domain of its receptor derived by cleavage from the membrane, with the complex prolonging the circulating half-​life of the hormone. Functions of hormones The physiological roles of the major hormones can be broadly clas- sified into three areas: control of growth and differentiation; main- tenance of homeostasis; and regulation of reproduction. Some hormones have multiple functions and play a role in more than one area. In addition, some biological effects are mediated by the com- bined action of several different hormonal pathways. The principal actions of major hormones are outlined in Table 13.1.2. Linear growth is dependent on a complex interplay of many hor- mones and growth factors. GH plays a key role and exerts many of its effects by stimulating the hepatic production of IGF-​1. Thyroid hormone also stimulates the epiphyseal growth plate in childhood Hormone Effect(s) Effect(s) Target organ Pituitary gland Hypothalamus GnRH −+ Vasopressin Oxytocin TRH CRH Dopamine LH/FSH Gonads TSH Thyroid gland GH Liver ACTH Adrenal cortex PRL Breast Distal nephron Uterus Breast Somatostatin Anterior pituitary Oestrogen Progesterone Testosterone Inhibin ↑Plasma osmolality − − +

13.1  Principles of hormone action 2249 whereas, at puberty, production of sex steroids leads to epiphyseal closure. Other important actions of thyroid hormone include en- hancement of myocardial contractility and differentiation of the central nervous system. The maintenance of homeostasis includes the control of en- ergy balance, metabolic pathways, fluid, electrolyte and calcium balance, and regulation of blood pressure. Energy homeostasis in- volves regulation of food intake and energy expenditure. Leptin, an adipose tissue-​derived hormone, acts via hypothalamic path- ways (e.g. melanocortin 4)  to reduce food intake; conversely, rising gastrointestinal production of ghrelin preprandially stimu- lates food intake. Thyroid hormone is an important determinant of resting energy expenditure or basal metabolic rate. Metabolic effects are mediated by several hormones:  insulin lowers blood glucose by enhancing its cellular uptake and promotes glycogen synthesis; conversely, GH, cortisol, glucagon, and adrenaline act as counterregulatory hormones to raise blood glucose. Glucagon and adrenaline stimulate glycogenolysis and, together with cortisol, promote gluconeogenesis. Other metabolic pathways are also in- fluenced by these hormones: GH and cortisol are lipolytic whereas insulin mediates lipogenesis; insulin and GH are also anabolic by promoting protein biosynthesis, whereas cortisol increases protein breakdown. Adiponectin, another adipose tissue-​derived hormone, enhances tissue insulin sensitivity. Circulating concentrations of ions and water balance are also under hormonal control. Vasopressin promotes water reabsorption via membrane channels (aquaporins) in the distal collecting ducts of the kidney; aldosterone acts at the renal distal convoluted tubule to stimulate sodium reabsorption and potassium excretion, effects which are antagonized by atrial natriuretic peptide (ANP). Both parathyroid hormone and vitamin D increase serum calcium levels; PTH mediates Ca2+ resorption from bone and kidney, whereas vitamin D acts on the gastrointestinal tract as well as these sites. Catecholamines and angiotensin II are potent vasoconstrictors and, together with cortisol, control blood pressure. Hormones involved in reproduction exert effects from early in de- velopment. During embryogenesis, Müllerian inhibiting substance (MIS) from the testis causes regression of female structures (uterus, fallopian tube) and testosterone promotes the development of male structures (vas deferens, epididymis, seminal vesicles) which are Table 13.1.2  Major actions of hormones Hormone Action Homeostasis Energy balance Leptin Reduces food intake Ghrelin Increases hunger Fluid and electrolyte balance Aldosterone Renal Na+/​K+ exchange Vasopressin ↓Renal free water clearance Metabolism Insulin ↑Cell glucose uptake; ↑glycogen synthesis; lipogenic; ↑protein synthesis Glucagon Glycogenolysis; gluconeogenic Cortisol Gluconeogenic; lipolysis; ↑protein breakdown Growth hormone Lipolysis; ↑protein synthesis Testosterone ↑Protein synthesis Calcium Parathyroid hormone ↑Ca2+ resorption from bone and kidney; ↑renal 1α hydroxylation of vitamin D Vitamin D ↑Ca2+ absorption from gastrointestinal tract; ↑Ca2+ resorption from bone and kidney Growth and development Growth hormone Growth Thyroid hormone Growth, regulation of basal metabolic rate, central nervous system development Retinoic acid Embryonic development; morphogenesis C-​type natriuretic peptide Bone growth, meiosis inhibition, axonal development Reproduction Testosterone Sexual differentiation, virilization, spermatogenesis Dihydrotestosterone Male external genitalia Oestradiol Female external genitalia; mammary gland development Progesterone Uterotrophic Prolactin Lactation Oxytocin Uterine contraction; milk reflex

SECTION 13  Endocrine disorders 2250 derived from the Wolffian duct. Dihydrotestosterone promotes de- velopment of the male external genitalia. In both sexes, the gonadal axes are quiescent in childhood and become reactivated at puberty. Testosterone mediates virilization, secondary sexual characteristics, and spermatogenesis in the male; in females, ovarian production of oestrogen and progesterone induces secondary sexual features and controls the menstrual cycle. In both sexes, gonadal steroids are re- quired for the attainment of peak bone density at the end of puberty and its subsequent maintenance. During pregnancy, prolactin acts in concert with oestrogen to promote lactation; oxytocin stimulates uterine contraction at parturition and smooth muscle contraction in the mammary gland during suckling. Hormone action Hormones induce biological responses by interacting with receptors located either on the membrane or intracellularly in the cytoplasm or nucleus. Hormones bind to receptors with high affinity, such that low concentrations of free hormone associate and dissociate from receptors rapidly in a dynamic equilibrium. The interaction of hor- mones with receptors is usually highly specific, with individual receptors being highly selective for a single hormone even within a class of structurally related molecules (e.g. steroid hormones). However, there are exceptions to this: parathyroid hormone (PTH) and parathyroid hormone-​related peptide (PTHrP) or LH and hCG share a common receptor, generating similar biological responses; insulin and IGF-​1 exhibit some degree of cross-​reactivity with their respective receptors; the mineralocorticoid receptor binds cortisol with equal or higher affinity than aldosterone. Hormones that bind to membrane receptors act via effector pro- teins to activate second-​messenger signalling pathways. In turn, the second messengers stimulate a cascade of kinases, which then act upon target substrates in the cell membrane, the cytoplasm or nucleus, to alter gene transcription or modulate a biochemical pathway, leading to a physiological response. Hormones that act through nuclear receptors are transported passively, or pumped ac- tively, across the plasma membrane to interact with their targets. The hormone–​receptor complex interacts with DNA sequences in target genes to either stimulate or repress their expression. The cellular ac- tions of nuclear receptors are mediated by changes in target gene transcription, altering mRNA synthesis and, in turn, the levels of protein product. Signalling by membrane receptors Membrane receptors can be divided into several groups (Table 13.1.3) depending on the signalling pathways that they utilize. The largest group consists of receptors with multiple transmembrane domains which are coupled to G proteins; a second class of receptor contains an intracellular domain with tyrosine kinase activity; several hormones signal via membrane proteins that are homologous to cytokine recep- tors; a fourth class of hormone receptor contains an intracellular do- main with serine or threonine kinase activity. G protein-​coupled receptors (GPCRs) are characterized by seven separate hydrophobic domains that traverse the membrane phospholipid bilayer (Fig. 13.1.3a). They possess an extracellular domain of variable size, enabling further subclassification of these receptors: glycoprotein hormones or small molecule ligands (e.g. calcium, GABA) interact with large N-​terminal extracellular domains; biogenic amines (e.g. catecholamines, serotonin) bind to residues that lie within the transmembrane domain; other polypep- tide hormones interact with residues in both the extracellular and transmembrane domains. The intracellular domains of the receptor enable interaction with G proteins. G proteins typically form a heterotrimeric complex of α, β, and γ subunits which bind the guanine nucleotides GTP and GDP. The complex transduces signals from the receptor to downstream ef- fectors such as adenylate cyclase, phospholipase C, or membrane voltage-​dependent calcium channels. A family of different G pro- teins (Gs, Gi, Gq, and others) exists with the ability to couple to dif- ferent receptors and effectors, allowing a large array of potential receptor–​G protein–​effector complexes, leading to diversity of cel- lular signalling. Several hormones signal via the cAMP pathway (Table 13.1.4) and this mechanism is considered in further detail (Fig. 13.1.4). In the resting state, the G protein complex is inactive and bound to GDP (Fig. 13.1.4a). Following hormone binding to the receptor (Fig. 13.1.4b), the Gα subunit binds GTP, becomes activated and dissociates from the βγ complex, to interact with adenylate cyclase (Fig. 13.1.4c). The latter converts ATP to the second messenger, cAMP. This rise in intracellular cAMP activates protein kinase A (PKA), which can phosphorylate certain cellular targets: phos- phorylation of a transcription factor, CREB, stimulates transcription of genes containing CREs; other targets for PKA include enzymes in biochemical pathways or membrane ion channels. Several mechanisms serve to terminate signalling via a hormone–​ receptor complex: first, hydrolysis of GTP to GDP by the Gα subunit promotes its reassociation with βγ subunits to reform an inactive complex; second, the hormone–​receptor complex is internalized via Table 13.1.3  Membrane receptor families G protein-​coupled Glycoprotein hormones FSH, TSH, LH/​CG Biogenic amines Adrenaline, noradrenaline, serotonin, histamine, dopamine Peptides Calcitonin, PTH/​PTHrP Ghrelin, GHRH, CRH, GnRH, kisspeptin, SRIF, TRH Vasopressin, oxytocin Angiotensin Glucagon, secretin, VIP, gastrin Small molecules Calcium, GABA Tyrosine kinase Insulin, IGF-​1 Guanylyl cyclase Atrial natriuretic peptide, CNP, guanylin Cytokine GH, PRL, EPO, leptin Serine/​threonine kinase Activin, inhibin, MIS CG, chorionic gonadotrophin; CRH, corticotropin releasing hormone; EPO, erythropoietin; FSH, follicle-​stimulating hormone; GABA, γ-​aminobutyric acid; GH, growth hormone; GHRH, growth hormone releasing hormone; GnRH, gonadotropin releasing hormone; IGF-​1, insulin-​like growth factor 1; LH, luteinizing hormone; MIS, Müllerian inhibiting substance; PRL, prolactin; PTH, parathyroid hormone; PTHrP, parathyroid hormone-​related peptide; SRIF, somatostatin; TRH, thyrotropin-​releasing hormone; TSH, thyroid-​stimulating hormone; VIP, vasoactive intestinal polypeptide.

13.1  Principles of hormone action 2251 cell surface vesicles and targeted for lysosomal degradation; third, following hormone binding, the GPCRs undergo phosphorylation of their intracellular domains by either PKA or other specific kinases (GRKs). Such phosphorylation prevents further coupling to G pro- teins and promotes receptor internalization desensitizing the cell to hormone action, until further surface receptor is expressed. Activation of their receptors by hormones such as somatostatin or dopamine, is known to decrease intracellular cAMP. Here, the hormone–​receptor complex associates with a G protein (Gi), whose α subunit inhibits adenylate cyclase. Although many GPCRs signal via cAMP, some receptors (e.g. TRH, GnRH, Table 13.1.4) are linked to different pathways. These receptors are coupled to Gq, whose α subunit activates membrane phospholipase C (PLC) (Fig. 13.1.5). This enzyme catalyses the hydrolysis of phosphatidylinositol 4,5-​ bisphosphate (PIP2) to generate the second messengers, inositol 1,4,5-​triphosphate (IP3) and 1,2-​diacylglycerol (DAG). IP3 inter- acts with a specific receptor located on smooth endoplasmic re- ticulum, inducing opening of intracellular channels leading to a rise in cytoplasmic calcium (Fig. 13.1.5). Interaction of calcium with calmodulin, a cytoplasmic calcium-​binding protein, activates a specific kinase (CAM kinase), which regulates several processes including hormone secretion, gene transcription, and metabolic enzymes. The rise in cellular calcium also facilitates DAG activa- tion of protein kinase C (PKC), leading to phosphorylation of the NH2 COOH Transmembrane domain Intracellular domain Extracellular domain (a) (b)

SECTION 13  Endocrine disorders 2252 Fos-​Jun transcription factor complex, inducing target gene expres- sion (Fig. 13.1.5). Hormones do not signal exclusively via a single pathway, with glycoprotein hormones and some peptides for ex- ample (Table 13.1.4) activating both cAMP and phosphoinositide signalling. The tyrosine kinase class of receptors is a diverse family that transduces signalling by insulin and IGF-​1 but also epidermal, nerve, fibroblast, and platelet-​derived growth factors. Growth factor signalling differs from insulin and the latter pathway will be con- sidered (Fig. 13.1.6). Hormone interaction with receptor promotes autophosphorylation of tyrosine residues in their cytoplasmic do- mains. In turn, this promotes phosphorylation of substrates, for example, Shc and insulin receptor substrate 1 (IRS-​1), followed by recruitment of adaptor proteins (Grb2/​SOS). The Grb2/​SOS com- plex recruits Ras, a GTP-​binding protein. Ras activation induces signalling via a series of kinases (Raf, Mek, MAP kinase), culmin- ating in the phosphorylation and activation of transcription factors which regulate target genes involved in mitogenesis or cellular dif- ferentiation. On the other hand, IRS-​1 recruits phosphatidylinositol-​ 3′-​OH-​kinase (PI3-​kinase), which in turn activates the AKT cascade. The latter mediates several of the metabolic effects of in- sulin, enhancing translocation of a glucose transporter to the mem- brane to promote cellular glucose uptake, and activating pathways involved in glycogen, lipid, or protein synthesis. Hormones such as prolactin and GH interact uniquely with their receptors; a single polypeptide interacts simultaneously with two receptors promoting their dimerization (Fig. 13.1.7). The hormone–​ receptor complex recruits Janus kinases (JAKs) which phosphorylate STATs (signal transducers and activators of transcription). STATs translocate to the nucleus, interact with regulatory DNA elements, and promote target gene transcription. Activin and inhibin belong to the transforming growth factor class of peptides which signal via a heterodimeric transmembrane receptor complex with intrinsic protein serine/​threonine kinase activity (Fig. 13.1.8). Here, hormone binding promotes the asso- ciation of two surface receptors (type I and type II) with differing properties. Subsequent transphosphorylation of the type I receptor by the intracellular kinase domain of the type II receptor leads to phosphorylation and dimerization of cytoplasmic Smad proteins. The Smad complex translocates to the nucleus to activate target gene expression (Fig. 13.1.8). The membrane guanylyl cyclases act as receptors for natriuretic peptides. ANP and CNP bind their selective receptors (guanylyl cyclase A  and B, respectively), which exist as phosphorylated homodimers, leading to intrinsic guanylyl cyclase activity. This leads to elevated cGMP generation, and subsequent activation of cGMP-​binding proteins such as phosphodiesterases (PDEs), cyclic nucleotide-​gated ion channels, and protein kinase G (Fig. 13.1.9). As just described, GPCR signalling is usually coupled to responses (e.g. hormone secretion) by Gα subunit activation of cAMP or phosphoinositide pathways. However, following receptor activation in some cellular contexts, the dissociated Gβ/​γ dimer subunit com- plex is also capable of stimulating effectors (e.g. Ras, PI3-​kinase), to enhance MAP kinase activity and elicit a mitogenic response. Nuclear receptor signalling The nuclear receptors are a family of transcription factors which mediate the action of steroid and other lipophilic hormones. The human genome encodes approximately 60 to 70 different recep- tors and it is clear that only a minority of these are targets for the action of major hormones (Table 13.1.5). The remainder com- prise a large group classified as ‘orphan receptors’, reflecting the fact that either their ligands and/​or physiological roles remain to be elucidated. Based on homologies in their primary amino acid sequence, nu- clear receptors can be divided into distinct domains which mediate specific functions (Fig. 13.1.3b). A central DNA binding domain contains cysteine-​rich peptide motifs which chelate zinc to form two ‘zinc fingers’. The latter mediate receptor binding to specific DNA sequences or hormone response elements, usually located in target gene promoters. The C-​terminal region of receptors encompasses their hormone-​binding function as well as their ability to dimerize. Nuclear receptors can be divided into two major subclasses, the steroid receptors and heterodimeric receptors, which differ in their mode of action. Steroid receptors (e.g. GR, MR, ER, PR, AR) bind to hormone response elements as homodimers (Fig.  13.1.10b). Some recep- tors (e.g. GR, PR, AR) are bound to cytosolic heat shock proteins. Hormone binding to receptors promotes their dissociation from these, enabling translocation to the nucleus, dimerization, and inter- action with DNA. In contrast, the thyroid, retinoid, and vitamin D receptors are constitutively nuclear and form heterodimers with a Table 13.1.4  Signalling pathways of membrane receptors Signalling pathway Hormone/​receptor Gsα/​cAMP↑ β-​Adrenergic receptor CRH GHRH ACTH Giα/​cAMP↓ Somatostatin Dopamine α-​Adrenergic receptor Gqα/​IP3 and DAG TRH GnRH Gsα/​cAMP↑ and Gqα/​IP3 and DAG LH FSH TSH PTH Calcitonin JAK-​STAT GH PRL EPO Leptin cGMP↑ ANP CNP Tyrosine kinase/​MAP kinase Insulin IGF-​1 Ser/​Thr kinase/​SMAD Activin, inhibin, MIS Abbreviations as for Table 13.1.3.

13.1  Principles of hormone action 2253 common partner (retinoid X receptor, RXR), to interact with DNA even in the absence of hormone or ligand (Fig. 13.1.10a). In some target gene contexts, RXR can also form homodimers to mediate retinoid signalling. In contrast to other transcription factors whose activity is con- trolled by post-​translational modification (e.g. phosphorylation), the hallmark of nuclear receptors is their ability to modulate gene expression in a hormone-​dependent manner. Thus, in the absence of ligand, the thyroid and retinoic acid receptors actively silence target gene transcription by recruiting a corepressor complex of cofactors (Fig. 13.1.10a). For all nuclear receptors, hormone binding induces a conformational change with dissociation of corepressors and re- cruitment of coactivator proteins (Fig. 13.1.10b). This latter com- plex acts to relax the interaction between histone proteins and DNA in chromatin, thereby facilitating the access of basal transcription factors and RNA polymerase, which induce gene transcription. A further mechanism which controls signalling via nuclear recep- tors is regulation of the supply of their ligands to cells and tissues. A specific membrane transporter (MCT8) mediates cellular entry of thyroid hormone in the central nervous system. T3, the ligand Hormone Extracellular Plasma membrane Intracellular Adenylate cyclase Adenylate cyclase Adenylate cyclase Hormone GTP GTP (a) (b) (c) ATP cAMP PKA CRE CREB CREB Nucleus Cytoplasm P Gsα Gsα β γ +

GDP Hormone Gsα GDP β β P γ γ Fig. 13.1.4  G protein-​coupled receptor signalling via the cAMP pathway. See text for explanation.

SECTION 13  Endocrine disorders 2254 Hormone Extracellular Plasma membrane Intracellular P Jun Fos Jun Fos + ++ PKC PLC DAG + Cytoplasm Nucleus Calmodulin Smooth endoplasmic reticulum CAM kinase IP3R IP3 PIP2 Ca2+ P β γ Gqα Fig. 13.1.5  G protein-​coupled receptor signalling via the phosphoinositide pathway. See text for explanation. α α β β Insulin P P P P PI3 Kinase Plasma membrane Extracellular Intracellular P P P P P P P P IRS-1 IRS-2 IRS-3 IRS-4 AKT ‘cascade’ Glycogen synthesis Cbl CAP P Glucose GLUT4 vesicle Shc Grb2 P SOS Ras Raf MAPK MEK Mitogenesis Differentiation GTP GDP GSK3 Protein synthesis mTOR Lipid synthesis Fig. 13.1.6  Insulin action via its tyrosine kinase receptor and signalling cascade. See text for explanation.

13.1  Principles of hormone action 2255 for TR, is generated from circulating thyroxine by the action of type 1 or type 2 deiodinase enzymes expressed in the liver and central nervous system respectively; the enzyme 5α reductase converts tes- tosterone to dihydrotestosterone in tissues of the male external geni- talia. In contrast, the enzyme 11β-​hydroxysteroid dehydrogenase type 2 catabolizes cortisol in the renal cells, thereby enabling the mineralocorticoid receptor to respond selectively to aldosterone ra- ther than to glucocorticoid, which it is also capable of binding with high affinity. Finally, in contrast to classical effects of steroid hormones to modulate gene expression, recent evidence indicates that they can also modulate cellular functions such as hormone secretion or neur- onal excitability within seconds or minutes. These rapid effects of steroid hormones occur independent of the genome and can occur either by hormone interaction with a cell surface receptor or by direct interaction of the nuclear receptor with cytoplasmic signalling molecules. Genetic defects and endocrine disease Most endocrine diseases can be divided into conditions of hor- mone excess, hormone deficiency, and hormone resistance. Defects in genes involved in hormone synthesis and action give rise to a spectrum of disorders (Tables 13.1.6 and 13.1.7). Both germline gene defects causing inherited syndromes and somatic mutations leading to acquired endocrine cellular dysfunction have been described. Defects in developmental transcription factors are usually associ- ated with endocrine gland hypoplasia: mutations in HESX-​1 cause optic and pituitary hypoplasia with agenesis of the corpus callosum; Plasma membrane Extracellular Intracellular P P P P JAK JAK P P P P S T A T S T A T ++ + Nucleus Cytoplasm Hormone S T T A S T T A Fig. 13.1.7  Hormone signalling via the JAK-​STAT pathway. See text for explanation. Plasma membrane Extracellular Intracellular P P Hormone RII RI GS Smad2 P Smad4

Nucleus Cytoplasm Smad2 P Smad4 P Smad2 Smad4 Fig. 13.1.8  Hormone signalling by the transforming growth factor peptide family. See text for explanation. Plasma Membrane Extracellular Intracellular PDE Hormone PKG GTP cGMP Ion channel Guanylyl cyclase domain Kinase homology domain Fig. 13.1.9  Hormone signalling via membrane guanylyl cyclases, which act as receptors for peptides. See text for explanation.

SECTION 13  Endocrine disorders 2256 both Pit-​1 (POU1F1) and PROP-​1 mutations disrupt development of multiple pituitary cell types resulting in a combination of hor- mone deficiencies; defects in TTF-​1, TTF-​2, and PAX-​8 result in thyroid dysgenesis manifesting as neonatal hypothyroidism; muta- tions in the SRY gene lead to failure of testis development and sex reversal in XY males. Mutations in DAX-​1 or SF-​1, orphan members of the nuclear receptor family, disrupt both adrenal and gonadal development. Defects in other nuclear receptors (e.g. VDR, TR, GR) are char- acterized by tissue resistance to their respective hormone ligands. Vitamin D resistance leads to rickets together with abnormalities of skin differentiation, hair growth, and lymphocyte function, em- phasizing its important extraskeletal actions. Point mutations in the androgen receptor are associated with a spectrum of pheno- types ranging from complete feminization of XY individuals to mildly impaired virilization in men. In addition, expansion of a polyglutamine repeat sequence in the N-​terminal domain of AR is associated with adult-​onset neuronal degeneration leading to spinal and bulbar muscular atrophy. A homozygous defect in the oestrogen receptor in a male led to failure of epiphyseal closure re- sulting in tall stature together with severe osteoporosis. These mani- festations suggest that testosterone effects on the male skeleton are, in part, mediated by its enzymatic conversion to oestrogens. A growing number of disorders associated with defects in trans- membrane receptors or their signalling intermediates have been described (Table 13.1.7). However, in addition to mutations which disrupt protein function, gain-​of-​function mutations causing con- stitutive activation of the receptor or signalling protein also occur. With GPCRs, diverse loss-​of-​function mutations, occurring most frequently in the extracellular domain, block hormone binding or signalling, leading to insensitivity to hormone action. Such hor- mone resistance can lead to both hypofunction (e.g. ACTH, TSH receptors) or hypoplasia (e.g. LH, FSH receptors) of target glands expressing the receptor. Conversely, gain-​of-​function mutations in GPCRs typically occur in the third intracellular loop, causing constitutive activation of the receptor in the absence of hormonal ligand. Again, the functional consequence is either autonomous hyperfunction (e.g. calcium, LH, FSH receptors) or excessive neoplastic proliferation (e.g. TSH receptor, RET tyrosine kinase receptor) of the target tissues in which the receptor is expressed Table 13.1.5  Hormone signalling via nuclear receptors Nuclear receptor Hormone Homodimeric GR Cortisol MR Aldosterone ERα/​β Oestradiol PR Progesterone AR Testosterone, dihydrotestosterone Heterodimeric TRα/​β Triiodothyronine RARα/​β/​γ all-​trans-​Retinoic acid RXRα/​β/​γ 9-​cis-​Retinoic acid VDR 1,25-​Dihydroxyvitamin D3 PPARα/​β/​γ Unsaturated fatty acids, eicosanoids AR, androgen receptor; ER, oestrogen receptor α or β subtypes; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PPAR, peroxisome proliferator-​activated receptor α, β, or γ subtypes; PR, progesterone receptor; RAR, retinoic acid receptor α, β or γ subtypes; RXR, retinoid X receptor α, β, or γ subtypes; TR, thyroid hormone receptor α or β subtypes; VDR, vitamin D receptor. Nucleus Cytoplasm RXR NR Coactivator ‘complex’ HRE Histone Acetylation Activation RXR NR HRE BTFs Corepressor ‘complex’ Histone deacetylation Repression NR NR or Nucleus Cytoplasm (a) (b) L L BTFs Fig. 13.1.10  Transcriptional regulation by nuclear receptors. (a) In the absence of hormone, a subset of heterodimeric nuclear receptors (thyroid, retinoic acid) recruit corepressors to inhibit gene transcription. (b) Hormone occupancy of homodimeric or heterodimeric receptors promotes their association with coactivators, leading to transcriptional activation. Table 13.1.6  Genetic defects in transcription factors or nuclear receptors and endocrine disorders Gene Disorder or phenotype Transcription factors HESX-​1 Septo-​optic dysplasia POU1F1/​PROP-​1 GH, PRL, TSH deficiencies TBX19 ACTH deficiency TTF-​1/​TTF-​2/​PAX-​8 Thyroid dysgenesis SRY XY female Nuclear receptors DAX-​1/​SF-​1 Adrenal insufficiency, hypogonadism, and disorders of sex development (DSD) VDR Hereditary vitamin D-​resistant rickets AR Androgen insensitivity syndrome or spinal and bulbar muscular atrophy ERα Tall stature and osteoporosis GR Glucocorticoid resistance TRβ Resistance to thyroid hormone PPARγ Lipodystrophic insulin resistance

13.1  Principles of hormone action 2257 (Table 13.1.7). Constitutive activation of signal transduction may also result from G protein mutations. Here, specific amino acid sub- stitutions in Gsα inhibit its intrinsic GTPase activity, and the GTP-​ bound protein constitutively activates adenylate cyclase leading to cAMP accumulation. Somatic Gsα mutations occur in a proportion of pituitary GH secreting and thyroid adenomas; more widespread expression of a somatic Gsα mutation occurring early in develop- ment, leads to polyostotic fibrous dysplasia, café au lait skin pig- mentation, and hyperfunction of multiple endocrine glands, constituting the McCune–​Albright syndrome. Similarly, germline loss-​of-​function mutations which reduce cellular Gsα activity, are associated with resistance to multiple hormones together with char- acteristic bone anomalies (Albright’s hereditary osteodystrophy). FURTHER READING Braverman LE, Cooper D (eds) (2012). Werner & Ingbar’s the thyroid; a fundamental and clinical text, 10th edition. Lippincott Williams & Wilkins, Philadelphia. Jameson JL, DeGroot LJ (eds) (2015). Endocrinology, 7th edition. Elsevier, Philadelphia. Lodish H, et  al. (2016). Molecular cell biology, 8th edition. W.H. Freeman, San Francisco, CA. Melmed S, et al. (eds) (2016). Williams’ textbook of endocrinology, 13th edition. Elsevier, Philadelphia. Strauss JF, Barbieri RL (eds) (2019). Yen & Jaffe’s reproductive endocri- nology, 8th edition. Elsevier Saunders, Philadelphia. Table 13.1.7  Genetic defects in membrane receptors or signalling and endocrine disorders Gene Loss-​of-​function mutation Gain-​of-​function mutation G protein-​coupled receptor TRH Central hypothyroidism GHRH GH deficiency with short stature GnRH Central hypogonadotropic hypogonadism KiSS 1 Central hypogonadotropic hypogonadism Precocious puberty NK3R (TACR3) Central hypogonadotropic hypogonadism Arginine vasopressin 2 (V2) Nephrogenic diabetes insipidus Nephrogenic syndrome of inappropriate antidiuresis Melanocortin 2 (ACTH) Family (isolated) glucocortisol deficiency Ca Familial hypocalciuric hypercalcaemia (FHH) Familial hypercalciuric hypocalcaemia TSH TSH resistance Familial non​autoimmune hyperthyroidism, familial pregnancy-​limited hyperthyroidism, autonomous thyroid adenomas LH Leydig cell hypoplasia (males); primary amenorrhoea (females) Male-​limited precocious puberty FSH Hypofertility (males); ovarian dysgenesis (females) FSH-​independent spermatogenesis (males); spontaneous ovarian hyperstimulation syndrome (females) PTH/​PTHrP Blomstrand chondrodysplasia Jansen’s metaphyseal chondrodysplasia Melanocortin 4 Extreme obesity Tyrosine kinase receptor RET Hirschprung’s disease MEN2: medullary thyroid carcinoma, phaeochromocytoma parathyroid neoplasia Insulin Insulin resistance Cytokine receptors GH Laron dwarfism Leptin Obesity Guanyly cyclase receptors CNP Acromesomelic dysplasia, type Maroteaux Skeletal overgrowth and macrodactyly Signalling pathway Gsα PTH, TSH, LH resistance Albright’s hereditary osteodystrophy Somatotroph adenomas, thyroid adenomas, McCune–​Albright syndrome Giα Ovary, adrenal, thyroid tumours AKT2 Insulin resistance

13.10 Hormonal manifestations of nonendocrine dise

13.10 Hormonal manifestations of nonendocrine disease 2541

ESSENTIALS Tumours (usually but not invariably malignant), other ‘non​endocrine conditions’ and drugs can be associated with a wide variety of endo- crine syndromes. ‘Ectopic’ hormone secretion, defined as the release of a hormone from a site different from the gland that normally produces it, has classically been recognized in the context of neo- plasia, but it is now apparent that many hormones are synthesized by ‘non​endocrine’ tissue. Although a particular endocrinopathy may be associated with a specific type of tumour in a particular organ, the relationship is not invariable, and many neoplasms elaborate more than one hormonal substance at the same or at different times and thus produce a mixed endocrine picture. Syndromes of ectopic hormone secretion Most syndromes of ectopic hormone secretion are due to peptide hormones. Clinically evident syndromes are much less common than laboratory abnormalities, which are frequently found if exten- sive biochemical and hormonal assays are applied to patients with cancer. Well-​described syndromes include the following: Ectopic calciotropic hormones—​hypercalcaemia in the absence of detectable bony metastases occurs in about 15% of patients with squamous cell carcinoma (usually bronchial), carcinoma of the kidney, ovary, or breast. Parathyroid hormone-​related protein (PTHrP) is responsible for most cases, but sometimes increased pro- duction of 1,25-​dihydroxyvitamin D3 (lymphoproliferative tumours) or transforming growth factor α (TGFα) may be involved. Syndrome of inappropriate antidiuresis (SIAD)—​is reported in 40% of cases of small cell lung cancer; usually associated with high levels of circulating AVP, but other unidentified antidiuretic substances are sometimes involved. Presentation is with hyponatraemia, with diagnosis requiring exclusion of the very many other causes of this condition. Ectopic ACTH secretion—​pro-​opiomelanocortin (POMC), the pre- cursor for ACTH and other polypeptides, can be secreted by a var- iety of non​pituitary tumours (e.g. small cell lung cancer, carcinoids), which are responsible for about 10–​20% of patients with Cushing’s syndrome. Presentation is variable, but with rapid onset and pro- gression the physical manifestations of Cushing’s syndrome may
not have time to develop, and selected features may predominate (e.g. weight loss, proximal muscular weakness, oedema, type 2 dia- betes mellitus (T2DM), and hypokalaemic alkalosis). Ectopic secretion of insulin-​like growth factors (IGFs)—​IGF-​2 is most typically (although rarely) secreted by large mesenchymal tumours; presentation is with symptoms of neuroglycopenia. Endocrine manifestations of non​malignant
non​endocrine diseases Systemic disease of non​endocrine glands may influence endo- crine function due to (1) a specific effect of the disease itself (e.g. hypercalcaemia in sarcoidosis driven by 1,25 dihydroxyvitamin D3 produced by alveolar macrophages); opportunistic infections, lymphoma, or Kaposi’s sarcoma involving the adrenal glands in
HIV/​AIDS; and (2) as a general response to either acute or chronic illness, for example, non​thyroidal illness (‘sick euthyroid syndrome’), where reduced peripheral conversion of thyroxine (T4) to tri-​ iodothyronine (T3) is associated with a normal or reduced thyroid stimulating hormone in association with low T3 (± T4). Drug-​induced endocrine manifestations Drugs may (1)  induce manifestations of endocrine disease (e.g. amiodarone may cause hyperthyroidism because of its high iodine content or due to a destructive thyroiditis) and (2) influence the re- sults of hormonal assays and lead to mistaken diagnosis (e.g. oes- trogen increases thyroid-​binding globulin, hence women on the combined oral contraceptive pill have high total T4 concentrations but are euthyroid). Oral oestrogen also increases cortisol-​binding globulin, which can lead to spurious interpretation of results from short Synacthen testing during assessment for hypocortisolaemia. Introduction Several endocrine syndromes may develop in association with dis- eases that are not primarily disorders of an endocrine gland. In most the cause is a tumour, usually but not invariably malignant, that develops in tissue which is not normally the origin of the particular hormone synthesized. Other non​endocrine condi- tions may also be associated with either hormonal excess or defi- ciency (e.g. sarcoidosis and AIDS). Certain drugs may also modify 13.10 Hormonal manifestations of non​endocrine disease Thomas M. Barber and John A.H. Wass

section 13  Endocrine disorders 2542 hormonal biochemistry and cause hormonal imbalance syndromes. Albright suggested that the hypercalcaemia sometimes associated with malignant disease without osteolytic metastases might be due to the secretion by the tumour of a parathyroid hormone (PTH)-​ like peptide; we now know that this is true (parathyroid hormone-​ related protein, PTHrP). Later it was shown that hypersecretion of ACTH, not from the pituitary but from an ectopic site, was the cause of ACTH-​dependent Cushing’s syndrome in up to one-​fifth of pa- tients with this condition. Syndromes of ectopic hormone secretion—​general considerations Although ectopic hormone secretion has classically been recog- nized in the context of neoplasia, and defined as the release of a hor- mone from a gland different from that which normally produces the hormone, it is increasingly recognized that many hormones are synthesized by non​endocrine tissue. Thus, the syndromes of neo- plastic ectopic hormone secretion are actually due to the patho- logical oversecretion and/​or inappropriate production of hormones within non​endocrine tissue. Increasing recognition of the import- ance of paracrine secretion of hormones such as insulin-​like growth factors (IGF-​1), their modulation by growth factors and binding proteins (e.g. IGFBP1, 2, and 3), and their role in progression of neo- plasia adds greatly to these complexities. Many different hormones are ectopically secreted by neoplasms arising in diverse organs, notably the bronchus, breast, pancreas, kidney, and ovary as well as in mesenchymal tissue. Although a particular endocrinopathy may be associated with a specific type of tumour in a particular organ, the relationship is not invariable. An example is the lung, where squamous cell carcinomas may be associated with hypercalcaemia due to production of PTHrP, while small cell lung cancer and bronchial carcinoid tumours are both as- sociated with ectopic ACTH secretion, but with very different clin- ical manifestations. Many neoplasms produce ectopically more than one hormonal substance concurrently or at different times and thus may produce a mixed endocrine picture (e.g. pancreatic endocrine tumours producing ACTH and insulin). The amount of ectopic hormone(s) produced may fluctuate from time to time (e.g. cyclical Cushing’s syndrome in ectopic ACTH secretion). The clinical and biochemical changes induced by the ectopic hormone may mimic very closely, and be clinically indistinguishable, from those found in the true endocrinopathy. In other patients, clinical features are less characteristic and dominated more by abnormalities of bio- chemistry or hormone levels. Thus, in many cases of ectopic ACTH production by small cell lung cancer, the deteriorating nature of the underlying illness may be too rapid for the classical features of florid Cushing’s syndrome to develop, and hypokalaemic alkalosis with T2DM predominate. Definition The diagnosis of ectopic hormone production depends on many criteria, although it is seldom practicable or possible to confirm them all: 1. There is an association of the tumour with an endocrine syndrome. 2. Even though the endocrine syndrome may not be clinically florid, there is an elevated or inappropriately raised plasma level of the putative hormone. 3. Removal or suppression of the tumour induces a regression of the endocrinopathy and a fall in the hormone level. 4. The clinical picture and hormone levels are uninfluenced by re- moval of the gland that normally secretes the hormone. 5. The putative hormone level is higher in venous blood draining the tumour than in the arterial blood supplying it. 6. Extraction or immunohistochemical staining shows a higher concentration of the hormone in the tumour than in adjacent, non​involved tissue. 7. Demonstration can be made of tumour cell synthesis of identifi- able hormones in vitro or of mRNA coding for the hormone. Chemical structure Most syndromes of ectopic hormone secretion are due to peptide hormones. It is rare for tumours to secrete steroid hormones because of the complexity of the enzyme cascade required for steroid bio- synthesis. Tumours may, however, be associated with altered steroid metabolism (e.g. increased aromatase activity in hepatocellular car- cinoma leads to feminization and gynaecomastia due to androgen conversion to oestrogens). The precise amino acid sequences of hormones of ectopic origin are being increasingly defined. In general, they appear to resemble closely those of their normally occurring counterparts (except PTH and PTHrP). There is a tendency for a greater proportion of higher molecular weight precursors, prohormones, subunits, and fragments to be associated with an ectopic origin than with true endocrinopathies, but it is not always clear whether this is due to differences in biosynthesis or in intracellular or extracellular pro- cessing. Minor differences in molecular structure are sometimes re- flected in disparities between bioassay and immunoassay. Prevalence Clinically evident syndromes are less common than biochemical or hormonal abnormalities. The prevalence of ectopic production of ACTH, corticotrophin-​releasing hormone (CRH), PTHrP, calci- tonin, human chorionic gonadotrophin (hCG), prolactin, or growth hormone (GH), without clinical manifestations, is high when exten- sive biochemical and hormonal assays are applied to patients with cancer. These assays bring closer the prospect of identifying a diag- nostic marker for tumours in general and, in particular, as is already the case with the monitoring of hCG or its subunits, to determine the response of tumours to treatment. Hypercalcaemia in the absence of detectable bony metastases is the most common abnormality resulting from ectopic hormone se- cretion. It occurs in about 15% of patients with squamous cell car- cinoma (usually of the bronchus), carcinoma of the kidney, ovary, or breast. Next most common in neoplastic diseases is the syndrome of inappropriate antidiuresis, usually associated with a small cell lung cancer and reported in 40% of such cases. Cushing’s syndrome due to ectopic ACTH or CRH secretion occurs in about 5% of patients with small cell lung cancer, and in association with other neoplasms. Biochemical accompaniments of Cushing’s syndrome in the absence of the clinical features are much more common, occurring in up to 50% of patients with small cell lung cancer.

13.10  Hormonal manifestations of nonendocrine disease 2543 Pathogenesis As techniques for molecular analyses have evolved, it has become clear that every somatic cell is capable of synthesizing every polypep- tide hormone. However, only under pathological circumstances is that capability ever likely to be expressed. A variety of hypotheses for ectopic hormone synthesis and secretion have been proposed. None explains all of the observed facts. Fundamentally, all cells inherit an identical complement of DNA. They are therefore totipotential and have all the coded information required for the synthesis of all proteins and peptides, including protein hormones. The normal in- ability of non​endocrine tissue to synthesize hormones is ascribed to repressors that mask specific segments of the DNA molecule. It seems possible that when a cell becomes malignant this normal repression becomes ineffective, allowing the unmasked DNA to synthesize pro- teins or peptides foreign to the cell concerned. Such a derepression hypothesis does not explain why certain tumours are more prone to secrete specific ectopic hormones. Neuroendocrine cells, character- ized by the presence of peptide hormone granules, are likely to be the origin of some tumours associated with hormone secretion, such as small cell lung cancer and bronchial carcinoids. Another hypothesis suggests that there are a small number of special proliferative cells in normal mature tissues that have fetal characteristics with the ability to produce peptide hormones—​a process of dysdifferentiation ra- ther than de-​repression. There is currently no unifying mechanism with supportive experimental evidence to explain ectopic hormone production. Further information on the control of gene expression and hormone production, the role of oncogenes, and paracrine growth factors may provide further insight. Treatment Treatment of the clinical or biochemical abnormalities associated with endocrinopathies of non​endocrine origin is best directed at the primary disorder. In neoplastic disease, this may involve sur- gical excision, radiotherapy, or chemotherapy. Sometimes, the tumour secreting the ectopic hormone is extremely difficult to lo- cate even with the use of sophisticated imaging techniques such as MRI, radiolabelled isotope scanning (e.g. indium-​111 pentetreotide imaging), or using selective venous catheterization. More specific therapy may be necessary to manage the associated metabolic abnormality until such time as the underlying disorder can be controlled. For example, immediate measures may be re- quired to reduce hypercalcaemia with fluids and bisphosphonates, or steps taken (administration of metyrapone) to diminish cortico- steroid secretion from adrenal glands stimulated by ectopic ACTH secretion. Particular syndromes of ectopic hormone  secretion Ectopic secretion of calciotropic hormones Malignancy is the most common cause of hypercalcaemia in hos- pital inpatients and may be due to direct tumour spread to the bones or related to ectopically secreted calcium-​releasing factors. Often several different mechanisms are involved in the same patient. After its discovery in 1987, it was shown that PTHrP is responsible for hypercalcaemia in up to 70% of patients with tumour-​associated hypercalcaemia. Many of these patients also have bone metastases. PTHrP shares amino acid homology with PTH between positions 2 and 13 of the 84 residues of PTH and acts via the PTH receptor, resulting in an elevation of extracellular calcium concentration. The PTHRP gene is located on the short arm of chromosome 12; that of PTH is on chromosome 11. The PTHRP gene may be activated by transactivation, hypomethylation (renal carcinomas), or the effect of growth factors and cytokines, including IGF-​1 and epidermal growth factor, while glucocorticoids and vitamin D3 suppress PTHrP levels. Unlike PTH-​mediated hypercalcaemia, dihydroxycholecalciferol is suppressed in PTHrP-​mediated hypercalcaemia. PTHrP is pro- duced by squamous carcinomas as well as renal, bladder, ovary, skin, pancreas, and breast carcinomas, and lymphomas. Other factors can be involved in hypercalcaemia unassoci- ated with osseous metastases. It is not uncommon for 1,25-​ dihydroxyvitamin D3 to be produced by lymphoproliferative tumours, which are either high grade or widely disseminated. Transforming growth factor-​α (TGFα) which stimulates osteo- clastic bone resorption, is also made by squamous carcinoma, and renal and breast carcinomas. Some tumours cosecrete both TGFα and PTHrP. Interleukin-​1 (IL-​1), which is a very powerful stimu- lator of osteoclastic bone resorption, is also made by squamous carcinomas as well as some haematological malignancies. Tumour necrosis factor (TNF) and lymphotoxin also stimulate osteoclastic bone resorption. These related cytokines cause hypercalcaemia in vivo; lymphotoxin is also produced by cultured myeloma cells in vitro and accounts for the hypercalcaemia seen in this condi- tion. Prostaglandins of the E series may also cause hypercalcaemia. Finally, vascular endothelial growth factor (VEGF) and IL-​8 and IL-​11 may be implicated in the development of hypercalcaemia of malignancy. It is important to remember that primary hyperparathyroidism itself is common, particularly in older people; two diseases may co- exist. For this reason, primary hyperparathyroidism should always be considered when hypercalcaemia occurs, even if it is in a patient within the setting of malignant disease. It is now possible to differ- entiate between these two conditions by using the PTH two-​site radioimmunoassay. Paraneoplastic hypercalcaemia may be either asymptomatic or dominate the clinical picture and be life-​threatening as a consequence of dehydration and renal failure. The features of hypercalcaemia and its general management are discussed elsewhere (see Chapter 13.4). Oncogenic osteomalacia, an acquired phenotype, is a rare syn- drome characterized clinically by reduced mineralization of newly formed bone and the features of osteomalacia (including fractures, bone pain, and muscle weakness). It is usually associated with benign mesenchymal or mixed connective tissue tumours (par- ticularly haemangiopericytomas) that have a propensity to arise in the head and neck. The use of imaging (including octreotide scintigraphy or PET) is important in the localization of such tu- mours. Biochemical features of oncogenic osteomalacia include an excessive renal loss of phosphate that results in phosphaturia and hypophosphataemia. The serum calcium level is usually normal and serum alkaline phosphatase is usually elevated. Circulating levels of 1,25-​dihydroxyvitamin D3 are usually suppressed (des- pite ambient hypophosphataemia). Circulating levels of fibroblast growth factor 23 (FGF-​23), a secretory product of tumours associ- ated with oncogenic osteomalacia, are usually elevated. It is possible

section 13  Endocrine disorders 2544 that FGF-​23 plays an important role in renal phosphate wasting or impairs regulation of vitamin D metabolism, although there may be other unknown phosphaturic factors which also inhibit the 1α-​hydroxylase enzyme. Removal of the causative tumour is the treatment of choice. Syndrome of inappropriate antidiuresis (SIAD) Syndrome of inappropriate antidiuresis is a disorder of sodium and water balance characterized by impaired water excretion, with re- sultant hyponatraemia, reduced plasma osmolality, and inappropri- ately high urine osmolality. A diagnosis of this syndrome requires the absence of hypovolaemia, hypotension, deficiency of cardiac, renal, thyroid, or adrenal function, or any known stimulus for the secretion of AVP, an antidiuretic hormone. The syndrome is usually, but not invariably, associated with high levels of circulating AVP, al- though other, as yet unidentified antidiuretic substances are some- times involved. Inappropriate secretion of AVP can either be from an ectopic or eutopic (posterior pituitary) source. The most common ec- topic source of AVP associated with a syndrome of inappropriate antidiuresis is bronchogenic carcinoma. Inappropriate eutopic se- cretion of AVP can be induced by a wide variety of diseases and drugs. Thus, although a syndrome of inappropriate antidiuresis in association with malignancy may be due to inappropriate ectopic secretion of AVP from the tumour itself, it may also result from inappropriate eutopic AVP secretion. The latter may be caused by treatment of the tumour (e.g. chemotherapy such as cyclophospha- mide), an intercurrent illness such as pneumonia, a complication such as hydrocephalus or cerebrovascular accident, or even by the tumour itself (see Table 13.10.1). The treatment of this syndrome is often restriction of fluid intake (e.g. 500 ml/​24 h). Occasionally, it may also be necessary to administer hypertonic saline. Use of aquaretic agents such as tolvaptan, which increase excretion of free water through antagonism of the V2 receptor, thereby blocking the action of AVP within the distal nephron, is an important develop- ment in the management of SIAD and a useful addition to our treat- ment options for this condition. In a small number of individuals, the syndrome of inappropriate antidiuresis occurs in the absence of ADH secretion and is attribut- able to activating mutations in the arginine vasopressin receptor 2 (AVPR2). Ectopic ACTH secretion Pro-​opiomelanocortin (POMC) is a 31-​kDa precursor for both ACTH and β-​lipotropin as well as for other polypeptides derived from it, including γ-​lipotropin and β-​endorphin. A variety of non-​ pituitary tumours are capable of secreting POMC-​derived pep- tides. Approximately 50% of ectopic ACTH-​producing tumours are in the lung and the rest are present in a variety of other tissues (Table 13.10.2). Some tumours, particularly pancreatic islet cell tumours which are seldom (<5%) associated with Cushing’s syn- drome, can, in addition to ACTH, also secrete many other hor- mones that include insulin, gastrin, and glucagon. This accounts for the usefulness, when screening for ectopic ACTH, of measuring other hormones (e.g. calcitonin, hCG) which may be cosecreted, the presence of which raises the suspicion of an ectopic hormone-​ secreting tumour. Very rarely, CRH is secreted ectopically in asso- ciation with ACTH. Neuroendocrine tumours are the most common source of ectopically-​derived ACTH. These include bronchial carcinoid tu- mours most frequently, but also include carcinoids at other sites including the foregut, pancreas, and thymus. Other endocrine and non​endocrine tumours that can secrete ectopic ACTH include small cell lung carcinoma, phaeochromocytoma, medullary carcinoma of the thyroid, mesothelioma, and small cell colorectal carcinoma (see Table 13.10.2). The exact mechanism of synthesis of ectopic POMC-​derived peptides is still debated. POMC mRNA can be found in most tu- mours, but ACTH secretion is much less common, probably due to the lack of the signal sequence required for translocation. Changes Table 13.10.1  Conditions associated with the syndrome of inappropriate antidiuresis (SIAD) Malignancies Small cell lung Pancreas—​islet cell Duodenum Colon Bladder Prostate Thymus Cervix Lymphoma Lung diseases Pneumonia •​ Viral •​ Bacterial •​ Fungal Tuberculosis Lung abscess Asthma Pneumothorax Chest wall injury Mechanical ventilation Central nervous system diseases Cerebral trauma Cerebrovascular accident Meningitis Encephalitis Cerebral abscess Brain tumours—​primary or secondary Hydrocephalus Guillain-​Barré syndrome Delirium tremens Acute intermittent porphyria General surgery Drugs Vasopressin Desmopressin (DDAVP) Oxytocin Thiazides Vincristine, vinblastine Cyclophosphamide Phenothiazines Tricyclic antidepressants Carbamazepine Chlorpropamide Clofibrate Serotonin-​reuptake inhibitors Metabolic causes Porphyria

13.10  Hormonal manifestations of nonendocrine disease 2545 in promoter usage and also in POMC processing may lead to ec- topic secretion of ACTH. In addition, many tumours associated with ectopic ACTH secretion are of neuroendocrine morphology and may arise from progenitor cells associated with ACTH secre- tion. Differential gene expression profiles have been demonstrated in surgical tissue specimens taken from carcinoid tumours causing ectopic ACTH syndrome and pituitary tumours causing Cushing’s disease. These include more abundant mRNA expression in ectopic ACTH syndrome compared with Cushing’s disease from genes such as Ikaros family zinc finger protein 1 (IKZF1, a DNA-​binding pro- tein), proprotein convertase 2 (PC2) and somatostatin receptors 2 and 5 (SSTR-​2 and -​5). Enhanced SSTR-​2 and -​5 expression in tu- mours associated with ectopic ACTH syndrome has therapeutic im- plications for use of more selective somatostatin agonists in these patients. Presentation The clinical picture is variable and independent of the mass of the ectopically ACTH-​secreting tumour. In patients with small cell lung cancer who have a rapidly progressive tumour, the physical features of Cushing’s syndrome may not have time to develop. The major fea- tures are weight loss, proximal muscular weakness, polyuria, thirst, oedema, carbohydrate intolerance with glycosuria, and sometimes pigmentation due to melanocyte-​stimulatory effects of ectopically produced POMC-​related peptides. Hypokalaemic alkalosis is a characteristic finding; the plasma potassium is typically less than 3.2 mmol/​litre and plasma bicarbonate more than 30 mmol/​litre, urinary potassium loss being an important contributor to these bio- chemical features. This hypokalaemia is in part due to the very high cortisol levels, which have a mineralocorticoid action, and cortico- sterone and 11-​deoxycorticosterone, which may also be produced in excess. The 11β-​hydroxysteroid dehydrogenase enzyme may also function abnormally, causing decreased inactivation of cortisol and corticosterone. The serum cortisol level is usually greatly ele- vated (>1000 nmol/​litre) and the plasma ACTH level is also raised (>200 µg/​litre). These high levels do not usually occur in pituitary-​ dependent Cushing’s disease. However, there is some overlap in plasma ACTH levels between patients with ectopic ACTH secretion and patients with Cushing’s disease, with very high ACTH levels having been reported in a cohort of patients with Cushing’s disease originating from pituitary macroadenomas. When ectopic sources of ACTH originate from tissues other than small cell lung cancer, the clinical manifestations may be quite in- distinguishable from Cushing’s disease, and cushingoid features (including proximal myopathy, thinning of the skin, bruising, and psychiatric disorders) may antedate by months or years any evidence of a tumour causing ectopic ACTH secretion. The de- gree of elevation of ACTH is less marked than with small cell lung cancer and is proportional to tumour size. Some carcinoid tu- mours may be small and difficult to locate. The real problem is to differentiate ectopic ACTH secretion from pituitary-​dependent disease (Table 13.10.3). The presence of a hypokalaemic alkalosis (K<3.2 mmol/​litre) is a very useful test in the differential diagnosis. Lack of suppression on high-​dose dexamethasone testing is found in 90% of patients with ectopic ACTH production, but also in up to 20% with Cushing’s disease. However, the CRH test is very useful in differentiation as patients with ectopic ACTH secretion show an absent rise in cortisol whereas pituitary-​dependent Cushing’s dis- ease is associated with an exaggerated response in 95% of patients. Because most tumours that ectopically secrete POMC are located either within the chest or abdomen, MRI or CT scans will often re- veal the source of ectopic hormone secretion. In patients in whom the lesion is not readily visible by imaging techniques, selective venous catheterization and sampling may help to determine the ec- topic source of ACTH secretion by comparing levels at various sites within the venous system. Such sampling should include inferior pe- trosal sinuses in case of pituitary-​dependent disease. Radionuclide imaging (including octreotide scintigraphy) may occasionally be helpful in localizing the source of ectopic ACTH secretion. In a sig- nificant minority of patients with presumed ectopic ACTH secre- tion, the source of ACTH cannot be identified. Treatment Rapid control of hypercortisolaemia is the initial aim of manage- ment following diagnosis. Removal or debulking of the primary tu- mour or its control with radiotherapy, chemotherapy or, in the case Table 13.10.2  Types of neoplasm causing ectopic pro-​ opiomelanocortin (ACTH) secretion Small cell carcinoma of the bronchus Bronchial carcinoid Thymic carcinoid Islet cell pancreatic tumour Phaeochromocytoma Medullary carcinoma of the thyroid Breast carcinoma Tracheal carcinoma Oesophageal carcinoma Gastric carcinoma Ileal carcinoma Appendicular carcinoma Colonic carcinoma Ovarian carcinoma Prostatic carcinoma Squamous carcinoma of the cervix Adrenal medullary paraganglioma Melanoma Mesothelioma Table 13.10.3  Response to tests used to differentiate ectopic ACTH secretion from Cushing’s disease (from Howlett et al., 1986) Ectopic ACTH
(% of cases) Cushing’s disease
(% of cases) Hypokalaemia <3.2 mmol/​litre 100 10 Diabetes mellitus 78 38 Dexamethasone 8 mg/​day
(no suppression) 89 22 CRH test excessive response 0

90 CRH, corticotrophin-​releasing hormone.

section 13  Endocrine disorders 2546 of neuroendocrine tumours, 131I-​m-​iodobenzylguanidine therapy, will relieve the endocrine manifestations. A  relapse may occur if metastases develop because these, too, usually secrete ACTH. When it proves impossible to control a primary tumour, or when the source of ectopic ACTH cannot be identified, adrenocortical hypersecretion may be reduced by medical adrenalectomy. This can usually be achieved through the administration of steroidogenesis inhibitors, including metyrapone (500–​4000 mg/​day), an 11β-​ hydroxylase inhibitor of the conversion of 11-​deoxycortisol to cor- tisol. Aminoglutethimide (1000–​1500 mg/​day) may also be used but frequently causes a skin rash. Ketoconazole (400–​800 mg/​ day), which can cause fatal liver damage, and the adrenolytic drug mitotane are also useful. Mifepristone (RU-​486), a glucocorticoid antagonist at the receptor level, has been used as palliative therapy for some patients (10–​30 mg/​kg per day). Lastly, the long-​acting somatostatin analogue, octreotide (0.3 mg/​day, subcutaneously), has also been used in the treatment of ectopic ACTH syndrome. Bilateral adrenalectomy is an alternative approach, but frequently it is not practical for patients with rapidly progressive metastatic disease. It may be possible to embolize the arterial supply of the adrenal gland if patients are not suitable surgical candidates for adrenalectomy. Medical treatment needs to be monitored carefully so that adrenal insufficiency is avoided. The prognosis of patients with ectopic ACTH secretion is poor in patients with small cell lung carcinoma but can be excellent in patients with neuroendocrine tumours, depending on tumour hist- ology and the presence of lymph node metastases. Ectopic secretion of insulin-​like growth factors The insulin-​like growth factors, IGF-​1 and IGF-​2, share some sequence homology and actions of insulin. IGF-​2 is important in fetal growth, whereas IGF-​1, synthesized in the liver, mediates most of the actions of GH. IGFs circulate bound to one of six binding pro- teins (IGFBPs). Of these, the most important is IGFBP3, which itself is GH-​dependent and binds 75% of IGF-​1 and IGF-​2. IGF-​2 secretion from tumours may be associated with hypo- glycaemia. Usually the tumour is large and of mesenchymal origin, arising in the abdomen or thorax. Symptoms are those of neuroglycopenia—​sweating, tachycardia, disorientation, drowsi- ness, fits, and coma. Histology shows a mesothelioma, a fibrosar- coma, or other sarcoma such as a leiomyosarcoma. Other neoplasms associated with hypoglycaemia are haemangiopericytoma, hepa- toma, adrenal carcinoma, lung carcinoma, Wilms’ tumour, and co- lonic carcinoma. IGF-​2 secretion leads to suppression of GH and insulin, and re- duced production of IGFBP3, IGF-​1, and acid-​labile subunit (ALS), leading to reduced formation of the IGF–​IGFBP3–​ALS complex which protects the IGFs from degradation. IGF-​2 circulates as a smaller complex which has enhanced tissue and receptor bioavail- ability, allowing access to the insulin receptor. There is also an in- crease in the large molecular weight molecules and increased levels of big IGF-​2 not detected on radioimmunoassay. GH deficiency, decreased gluconeogenesis, and increased glucose metabolism by the tumour, which is usually large, may also contribute to hypogly- caemia. Treatment of these tumours is difficult. The hypoglycaemia is often not responsive to diazoxide, glucagon, octreotide, or cor- ticosteroids. However, administration of GH may be effective—​ increasing IGFBP3 and IGF-​1 and antagonizing the effect of excess IGF-​2. The underlying tumour may be resistant to radiotherapy; surgery, although effective when possible, is not always feasible. IGF-​1 and IGF-​2 may also play an important role in tumour pro- gression. Studies of breast cancer cells have suggested that IGF-​1 may have local mitogenic effects, and a role for IGF-​2 has recently been proposed in hepatocellular, colorectal, and adrenocortical tumours. Ectopic hCG secretion hCG is a glycoprotein consisting of an α and a β subunit. The α sub- unit is species specific and is the same for all glycoprotein hormones—​ luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH). The β-​subunit determines re- ceptor interaction and specific hormone activity. The β subunit of hCG is very similar to that of LH and this can cause problems with cross-​reaction in assays: the LH value may be spuriously elevated in the presence of increased hCG levels. Clinically silent, ectopic secre- tion of hCG, with or without its free α and β subunits, occurs in many patients (Table 13.10.4). Patients with ectopic secretion of gonadotrophins usually pre- sent with abnormalities in the reproductive system. In the first decade of life, ectopic hCG production may cause isosexual preco- cious puberty in boys with hepatoblastoma or a germ cell tumour. hCG, through its LH-​like action, causes Leydig cell stimulation in the testes. In turn, testosterone levels reach those of a normal adult, and secondary sexual characteristics develop together with prema- ture skeletal maturity. The testes remain small because there is no seminiferous tubule growth as this is dependent on FSH. Precocious puberty is rare in girls. Intracranial teratoma, choriocarcinoma, and pinealoma are as- sociated with ectopic hCG secretion. In some patients, cosecretion of ectopic hCG with oestrogen may be associated with gynaeco- mastia in men, and with dysfunctional uterine bleeding in women. Hirsutism and amenorrhoea are also presenting features of women with ectopic hCG secretion. Other tumours associated with ectopic Table 13.10.4  Human chorionic gonadotrophin (hCG) in sera of patients with malignant tumours (from Vaitukaitis, 1991) Tissue Percentage of cases with ectopic secretion of hCG Breast 21 Lung 10 Gastrointestinal tract 18 Pancreas (more commonly hCG -​α) 33 Stomach 22 Liver 21 Small intestine 13 Large intestine 12 Biliary tract 11 Ovary (adenocarcinoma) 40 Testis 62 Seminoma 38 Embryonal cell carcinoma 58 Choriocarcinoma 100 Mixed 73

13.10  Hormonal manifestations of nonendocrine disease 2547 hCG secretion include dysgerminomas, testicular tumours, ovarian adenocarcinoma, and stomach, pancreatic, bronchogenic, hep- atic, and renal cell carcinomas. Rarely, ectopic LH secretion has been described in adrenal tumours and pancreatic neuroendocrine tumours. hCG is a useful tumour marker in gestational trophoblastic dis- ease (choriocarcinoma) and in some men with testicular tumours, and provides an early warning of recurrent disease. However, it is important to measure other tumour markers (e.g. α-​fetoprotein), which may also be secreted by non​seminomatous germ cell tu- mours. Discordance of marker levels and tumour progress may be seen. In central nervous system disease, cerebrospinal fluid/​plasma ratios may help in the correct localization of tumours, as hCG does not cross the blood–​brain barrier and levels in cerebrospinal fluid remain undetectable in pregnancy. Thus, cerebrospinal fluid con- centrations higher than plasma suggest primary central nervous system disease. In some patients, most commonly with choriocarcinoma and massive elevation of hCG, the latter, through its weak TSH ac- tivity, due to its biochemical similarity to TSH, may cause goitre and hyperthyroidism. This most frequently occurs in women, is not associated with eye signs, and is usually associated with modest biochemical abnormalities. Treatment of the tumour results in a re- sumption of a euthyroid state but, if this is not possible, carbimazole or propylthiouracil may be required. Ectopic human placental lactogen Human placental lactogen (hPL), also called human chorionic somatomammotropin (hCS), is a trophoblastic hormone which may be secreted ectopically in association with lung tumours, testicular tumours, and trophoblastic disease. It is usually associated with gy- naecomastia in men, and these tumours may also be associated with increased levels of oestradiol and hCG. Ectopic GHRH and GH secretion Most patients with acromegaly (98%) have benign GH-​producing pituitary adenomas. Less than 2% of patients with acromegaly have ectopic growth hormone-​releasing hormone (GHRH) production which causes hyperstimulation of the somatotroph cells within the anterior pituitary, and consequently increased GH secretion. Indeed, the presence of anterior pituitary somatotroph hyperplasia differentiates histologically the minority of acromegalic patients with ectopic GHRH syndrome from those with GH-​producing pitu- itary adenomas. A patient with a carcinoid tumour of the pancreas producing GHRH enabled the final elucidation of the structure of this important hypothalamic peptide. Ectopic GHRH syndrome is caused most frequently by carcinoid tumours, especially of the lung and gastrointestinal tract. Although many carcinoid tumours ex- press immunoreactive GHRH, the development of acromegaly is uncommon (although many of these patients may display abnormal GH secretory dynamics). Other tumours reported to secrete GHRH ectopically include small cell lung cancer, adrenal adenomas, endo- metrial tumours, and phaeochromocytoma. GHRH can also be se- creted by hypothalamic hamartomas which also result in anterior pituitary somatotroph hyperplasia. Determination of the cause of acromegaly (pituitary GH excess versus ectopic GHRH syn- drome) is extremely important in the management of acromegaly. Ectopically GHRH-​secreting tumours are usually clinically apparent and GHRH levels in the circulation are elevated. Surgical resection of such tumours is the logical approach to management. Long-​ acting somatostatin analogues can also be used in those patients with ectopic GHRH syndrome caused by disseminated or recurrent carcinoid tumours. Ectopic GH secretion has been reported in patients with bron- chial, pancreatic, and gastrointestinal carcinoma, and cells cultured from an undifferentiated lung cancer have been shown to synthesize GH in vitro. Breast carcinoma and ovarian tumours may also occa- sionally secrete GH but no clinical syndrome has been clearly iden- tified as caused by ectopic GH. Ectopic prolactin secretion Prolactin may be secreted by bronchial carcinoma and renal cell carcinoma; the usual endocrine manifestation is galactorrhoea and there may be marked hyperprolactinaemia. These abnormalities are reversed if the tumour is controlled or removed. Difficulties in differential diagnosis may arise unless the underlying abnormality is clinically obvious or suspected, because in most instances the hyperprolactinaemia will be attributed to a prolactin-​secreting ad- enoma. Suspicion of an ectopic source may only arise when the pro- lactin level is not lowered by treatment with dopamine agonists. An autocrine role for prolactin in breast and prostate cancer has recently been postulated. Ectopic calcitonin secretion Increased serum calcitonin levels are encountered in a variety of cancers apart from medullary carcinoma of the thyroid. The most common of these are small cell lung cancer, leukaemia, and neo- plasms of the breast and pancreas. It is often produced as part of a multihormonal profile in conjunction with gastrin, ACTH, and somatostatin, among others. Ectopic calcitonin may differ from the normal hormone in having more components of high molecular weight; it does not cause any apparent symptoms and does not pro- duce hypocalcaemia. Ectopic renin secretion Although hypertension associated with hyperreninism and in- creased aldosterone production is usually due to a renal lesion, ec- topic secretion of renin has also been described in association with cancer of the lung, pancreas, ovary, and, rarely, testicle. The clinical picture is usually dominated by the underlying neoplasm, but the patient has hypertension and the cause of this may be suspected from the associated hypokalaemia and its accompanying muscle weakness. Effective treatment of the primary lesion will reduce the increased renin and aldosterone levels and hence the raised blood pressure. When the underlying cause cannot be eradicated, the use of an angiotensin-​converting enzyme inhibitor will control the hypertension. Ectopic aldosterone secretion Hypertension and hypokalaemia related to ectopic secretion of al- dosterone from a non​adrenal neoplasm have been described in pa- tients with ovarian tumours. Its pathogenesis is different from the others described in this section. The aberrant production of a steroid, aldosterone, rather than a peptide, is presumably due to biochemical change within the ovarian steroidogenic cells. Attention is likely to be focused on a suspected lesion of the adrenal zona glomerulosa

section 13  Endocrine disorders 2548 because the hyperaldosteronism is associated with low plasma renin activity. The ovarian lesion may initially be clinically silent and only revealed by pelvic imaging. Endocrine manifestations of non​malignant, non​endocrine diseases Systemic disease of non​endocrine glands may influence endocrine function due to a specific effect of the disease itself, due to a general response to either acute or chronic illness, or due to drug therapy used to treat the illness itself (Table 13.10.5). Often, hormonal per- turbations may be a complex mixture of all of these mechanisms, as may be seen in AIDS or critically ill patients on intensive therapy units. This section includes examples of systemic disease-​causing endocrine disorders. A commonly observed hormonal disturbance encountered in many hospital inpatients is the sick euthyroid syndrome. Peripheral conversion of thyroxine (T4) to tri-​iodothyronine (T3) is reduced, and typical thyroid function tests in this syndrome are a normal or reduced TSH in association with reduced T3 and T4 (and in- creased reverse T3 if measured). Severe illness may also interfere with hypothalamopituitary function and lead to hypogonadotropic hypogonadism. Possible mechanisms include increased cortisol levels, stress, cytokines, or opioids given as analgesia. Disorders influencing hypothalamopituitary function Anorexia nervosa is associated with complex changes in hypothalamopituitary function, with reduction in GnRH and gonadotrophin secretion leading to hypogonadotropic hypo- gonadism, and increased GH secretion which is associated with in- creased peripheral resistance to GH. Iron overload due to haematological conditions such as β-​thalassaemia major and to haemochromatosis may cause iron deposition in the anterior pituitary gland, and in particular in the gonadotrophs. This leads to hypogonadotropic hypogonadism, which may be ameliorated to a degree by venesection and iron che- lation therapy. Haemochromatosis may also lead to other hormonal changes due to pancreatic involvement causing diabetes mellitus, and cirrhosis associated with secondary hyperaldosteronism and hypogonadism. Thyroid Morning sickness in the first trimester of pregnancy may be asso- ciated with clinical and biochemical features of thyrotoxicosis, as the molecules hCG and TSH share very similar β subunits, allowing cross-​reactivity when high levels of hCG occur. Opportunistic infections of the thyroid gland may occur, in conditions associated with immunosuppression such as AIDS. Infections with cytomegalovirus, cryptococcus, and pneumocystis have been described. In addition, some patients with HIV infec- tion have increased T4 and T3 due to increased thyroid-​binding globulin. As the disease progresses, T4 and T3 levels fall as patients develop biochemical features of sick euthyroidism. Adrenal Opportunistic infections (cytomegalovirus, atypical mycobac- teria, cryptococci, toxoplasma, and pneumocystis), lymphoma, and Kaposi’s sarcoma may involve the adrenal glands in HIV and AIDS. The adrenal gland is the most commonly involved endo- crine gland at autopsy. However, frank adrenal insufficiency is rare because this requires destruction of over 90% of the adrenal cortex. Gonads Chemotherapy and irradiation may be associated with gonadal failure due to hypothalamopituitary gonadotrophin deficiency (e.g. following cranial irradiation or due to testicular/​ovarian damage following cytotoxic drug therapy such as cyclophospha- mide, cisplatin, and busulfan). Table 13.10.5  Hormonal abnormalities associated with non​endocrine disorders Disease Endocrine abnormality Severe illness Sick euthyroid syndrome (↓TSH ↓T4 ↓T3 ↑rT3) Hypogonadism (↓LH ↓testosterone/​oestradiol) Anorexia nervosa Hypogonadotropic hypogonadism (↓GnRH ↓LH/​FSH ↑GH) Iron overload Hypogonadotropic hypogonadism (↓LH/​FSH ↓T or E2) Hyperemesis gravidarum Thyrotoxicosis (↓TSH ↑T4, ↑hCG) HIV infection and AIDS ↑T4 ↑T3 ↑TBG(HIV) Opportunistic infections may cause goitre, hypo-​ or hyperthyroidism Adrenal infiltration (infection, lymphoma, and Kaposi’s sarcoma), however Addison’s rare Impaired aldosterone and adrenal androgen secretion, with preferential glucocorticoid production Cytotoxic chemotherapy and radiotherapy Hypogonadotropic hypogonadism (↓ LH/​FSH ↓T/​E2) Premature ovarian and testicular failure due to direct cytotoxic effect Coeliac disease Reversible androgen resistance (↑FSH/​LH ↓testosterone) Alcoholic liver disease Androgen deficiency (↓testosterone ↑SHBG ↑E2) Sarcoidosis and other granulomatous disorders ↑1,25 DHCC ↑calcium HTLV-​1 infection ↑PTHrP ↑calcium

13.10  Hormonal manifestations of nonendocrine disease 2549 Coeliac disease is associated with reversible male infertility due to androgen resistance, and improves on a gluten-​free diet. Alteration of gonadal steroid metabolism may occur in chronic liver dis- ease, particularly if alcohol related. Elevated sex hormone-​binding globulin and oestradiol levels are associated with a reduction in bio- available testosterone leading to testicular atrophy, gynaecomastia, and erectile impotence. Gynaecomastia Palpable breast glandular tissue is prevalent in population studies of men and boys. Subareolar glandular tissue of more than 2 cm in diameter is found in 35 to 60% of men. Gynaecomastia may occur as a result of various underlying conditions (Table 13.10.6) as well as drug therapy, and results from an alteration in the ratio of oes- trogen to androgen. Gynaecomastia can occur in association with testicular and adrenal neoplasms, Klinefelter’s syndrome, thyrotoxi- cosis, cirrhosis, primary hypogonadism, malnutrition, and ageing (see Table 13.10.6). An increase in levels of free oestrogen, reduced levels of free endogenous androgens and androgen receptor defects may underlie these changes. Increased aromatization of oestrogen precursors occurs in patients with obesity, liver disease, and hyper- thyroidism, and as a result of ageing. Calcium Hypercalcaemia in sarcoidosis is due to increased circulating 1,25-​dihydroxyvitamin D3, which undergoes dysregulated over- production in alveolar macrophages in a dose-​dependent fashion, stimulated by γ-​interferon, which is one factor responsible for the maintenance of the inflammatory process in sarcoidosis. Other granulomatous disorders (tuberculosis, histoplasmosis, coccidiomycosis, ruptured silicone breast implants) may rarely be associated with hypercalcaemia due to the same mechanism. Treatment with glucocorticoids or hydroxychloroquine are effective in lowering 1,25-​dihydroxycholecalciferol and calcium. HTLV1 infection may be associated with hypercalcaemia, due to transactivation of the PTHRP gene on chromosome 12. Endocrine manifestations of obesity Our understanding of adipose tissue has been transformed from a role of fat storage to one that incorporates a complex interplay of hormones with both endocrine and paracrine effects, and plays important roles in the regulation of appetite, energy expenditure, and body fat mass. A detailed description of the complexities of the endocrine manifestations of obesity is beyond the scope of this chapter (see Chapter 11.6), but the major endocrine manifestations are outlined. Leptin is an adipokine, levels of which increase with weight gain. Leptin has effects within the hypothalamus to sup- press food intake and promote energy expenditure pathways. Leptin may also have direct effects on ovarian function in women, which may play a role in the link between menstrual cyclicity and fer- tility with optimum weight. Other adipokines such as visfatin and resistin play important roles in reducing insulin sensitivity and pro- moting a proinflammatory state in obesity. Levels of adiponectin (which has anti-​inflammatory and immunoregulatory effects and improves insulin sensitivity) are reduced in obesity. Increased aromatase expression within the adipocyte in obesity may explain in part the association of male obesity with feminizing effects and, through suppression of the hypothalamo-​pituitary regulation of the gonadal axis through oestradiol, the association of male obesity with hypogonadotropic hypogonadism. Finally, there has been much interest in the effects of obesity on steroid hormone regu- lation within the adipocyte, including the effects on the enzyme 11β-​hydroxysteroid dehydrogenase type 1 (11β-​HSD1) that inter- converts inactive cortisone and active cortisol. Inhibition of 11β-​ HSD1 remains an exciting therapeutic strategy for patients with obesity, to improve the associated dysmetabolic sequelae. Drug-​induced endocrine manifestations Several pharmaceutical drugs may induce manifestations of endo- crine disease. More commonly they may influence the results of hor- monal assays and lead to mistaken diagnosis. It may not be a major problem when it is known that the patient is taking a particular com- pound and, from its molecular structure, it is appreciated that such a substance could influence the endocrine system or the results of hormonal assays. The problem is greater, however, when the drug in question has no clear relationship to a hormone and the mechanism Table 13.10.6  Non​endocrine conditions associated with gynaecomastia Neoplasms Ectopic production of human chorionic gonadotrophin or human placental lactogen Liver disease (18%) Starvation during recovery phase (refeeding) Renal disease and dialysis (1%) Drugs (10–​20%)   Antiandrogens/​inhibitors of androgen synthesis     Cyproterone     Flutamide     Spironolactone   Antibiotics     Ketoconazole   Antiulcer medication     Cimetidine     Omeprazole     Ranitidine   Cancer chemotherapeutic agents     Alkylating agents   Cardiovascular drugs     Captopril     Digoxin     Methyldopa     Nifedipine   Psychoactive drugs     Haloperidol     Phenothiazines   Drugs of abuse     Cannabis

section 13  Endocrine disorders 2550 by which it induces an endocrine manifestation, or interferes with an assay procedure, is not readily apparent. Thyroid Abnormalities of thyroid function test measurements Drugs can interfere with thyroid function tests. Some act by inhibiting the conversion of T4 to T3, others by increasing thyroid-​ binding globulin. β-​Blockers with membrane stabilizing proper- ties, such as propranolol, inhibit peripheral conversion of T4 to T3. Oral cholecystographic agents and amiodarone, a heavily iodinated antiarrhythmic agent, are also potent inhibitors of T4 to T3 conver- sion and produce decreased serum T3 concentrations and an in- crease in reverse T3. Oestrogen increases thyroid-​binding globulin, due to an increase in the sialic acid content of thyroxine-​binding globulin, which prolongs its half-​life in the circulation. Thus, women on oestrogens (e.g. the contraceptive pill) have high total T4 concen- trations but are euthyroid. Such results may also be seen in patients on tamoxifen. Heroin and methadone addicts also have raised levels of thyroxine-​binding globulin, as do patients on the lipid-​lowering agent, clofibrate. A decreased serum T4 does not necessarily indicate the presence of hypothyroidism. Many pharmacological agents lower the total T4 concentration by interfering with the binding of T4 to one or more of the thyroid-​binding proteins. Therapeutic levels of pheny- toin lower the level of serum T4 and high concentrations are capable of inhibiting the binding of T4 and T3 to thyroid-​binding globulin. High doses of salicylates have the same effect. Diclofenac, a non-​ steroidal anti-​inflammatory drug structurally similar to thyroxine, also interferes with thyroid hormone binding. Phenylbutazone, anabolic steroids, and glucocorticoids may also be associated with a low total T4 and normal thyroid function. Measurement of free thyroxine (FT4) will obviate the problems of misleading results from the measurement of total T4. Drug-​induced hyperthyroidism Amiodarone may cause hyperthyroidism due to its high iodine con- tent, or due to a destructive thyroiditis. Biochemically, there may be a marked elevation of total thyroxine, a relatively normal level of T3, and a suppressed TSH. Often, thyrotoxicosis is masked by the β-​blocking effect of the drug. Because of the large iodine load, it may be very difficult to treat with antithyroid drugs, and steroids may also be necessary to suppress thyroid hormone levels into the normal range. Even if amiodarone is stopped, its effects continue for many weeks because it is predominantly stored in adipose tissue. Contrast media and iodine-​containing cough medicines may simi- larly induce hyperthyroidism (Jod–​Basedow phenomenon). Drug-​induced hypothyroidism Increased iodide intake may also lead to decreased iodide trapping and a decrease in synthesis of thyroid hormones, hypothyroidism, and goitre. Iodine is contained in several tonics and cough medi- cines. Amiodarone, besides producing thyrotoxicosis, may cause iodine-​induced hypothyroidism in patients replete with iodine. Lithium blocks iodine uptake and the release of thyroid hormones. It also interferes with cAMP formation and thus inhibits the effects of TSH stimulation and may lead to goitre, although only 2% of patients on lithium actually develop clinical features of hypothyroidism. Adrenal cortex Abnormalities of adrenal hormone measurements Drugs may interfere with tests of adrenal function. Thus, the drug phenytoin accelerates metabolism of dexamethasone, and patients on phenytoin may not suppress cortisol normally during dexa- methasone suppression tests. Furthermore, during the assessment of adrenal reserve, chronic topical application of steroids, as well as inhalation of steroids for asthma, may suppress adrenal function. Oestrogens, by enhancing hepatic production of cortisol-​binding globulin, which binds between 90 and 97% of circulating cortisol, increases cortisol-​binding globulin two-​ to threefold. Thus, assess- ment of glucocorticoid replacement in patients on oestrogens is in- fluenced by this effect and oestrogens should be stopped 6 weeks prior to the test. Drug-​induced Cushing’s syndrome Chronic, excessive intake of alcohol causes alcoholic pseudo-​ Cushing’s syndrome. These patients behave biochemically as if they have Cushing’s syndrome with absent dexamethasone suppres- sion. This occurs through a centrally mediated mechanism with hypersecretion of pituitary ACTH and secondary secretion of cor- tisol by the adrenals. Drug-​induced primary aldosteronism Primary aldosteronism can be mimicked by the mineralocorticoid effect of glycyrrhizic acid contained in both carbenoxolone and li- quorice. Cortisol is normally inactivated by conversion to the in- active metabolite, cortisone, by the enzyme 11β-​hydroxysteroid dehydrogenase but these compounds inhibit the enzyme, which is important in the kidney because it protects renal mineralocorticoid receptors from cortisol. This can result in the syndrome of apparent mineralocorticoid excess in which renal mineralocorticoid recep- tors are stimulated locally by cortisol. Drug-​induced adrenal insufficiency The antifungal agent, ketoconazole, and the short-​acting anaes- thetic, etomidate, are imidazole derivatives with significant inhibi- tory effects on 11β-​hydroxylase. While they do not usually produce clinical insufficiency, they may do so in subjects with limited pi- tuitary or adrenal reserve. Rifampicin and phenytoin, which both accelerate the metabolism of cortisol by inducing hepatic mixed-​ function oxygenase enzymes, can also provoke adrenal insufficiency in similar patients with limited pituitary or adrenal reserve. In such patients, increased doses of replacement therapy are necessary. Gonads Several drugs can affect testicular function, leading to hypogonadism and infertility. Mechanisms include the direct inhibition of testos- terone synthesis or competitive inhibition of androgen action at receptor level. Spironolactone acts as a partial androgen receptor antagonist. Alcohol reduces testosterone levels acutely and chronic- ally, by both a central and a gonadal effect on testosterone synthesis, secretion, and metabolism. Cimetidine has antiandrogen effects due to direct interaction with the androgen receptor and it may also exert antiandrogen effects at the pituitary and hypothalamus leading to gynaecomastia and impotence in males. Anticonvulsants (e.g. phenytoin) increase sex hormone-​binding globulin and therefore

13.10  Hormonal manifestations of nonendocrine disease 2551 decrease free testosterone levels. They also enhance conversion of testosterone to oestradiol. Sulfasalazine causes reversible male infer- tility associated with oligospermia. Infertility may occur as a result of cytotoxic therapy, caused in particular by alkylating agents such as cyclophosphamide. These produce depletion of the germinal epithelium and lead to a raised FSH level, and oligo-​ or azoospermia, but normal LH and testos- terone levels in males, and may lead to premature ovarian failure in women. In women, hirsutism can be caused by several drugs, including danazol, phenytoin, diazoxide, and minoxidil. Pharmacological doses of glucocorticoids may lead to hypo- gonadism because of inhibited gonadotrophin release. Drugs such as tricyclics, benzodiazepines, antihypertensives, and antipsychotics may also lead to hypogonadotropic hypogonadism in both sexes. Prolactin Prolactin is controlled predominantly by a hypothalamic inhibitory mechanism through dopamine secretion. Some drugs can cause hyperprolactinaemia and galactorrhoea, usually acting through a dopaminergic mechanism. They may elevate prolactin to a suf- ficient extent to cause a clinical suspicion of prolactinoma, and in such patients a careful drug history is particularly important. Metoclopramide, pimozide, and sulpiride all act as dopamine ant- agonists and may considerably elevate prolactin, with all the at- tendant effects thereof. Fluoxetine may also lead to elevated serum prolactin, although tricyclic antidepressants are not usually associ- ated with hyperprolactinaemia. Phenothiazines, chlorpromazine, perphenazine, and trifluo- perazine also act as dopamine antagonists, as do haloperidol and butyrophenone. Reserpine and methyldopa both decrease dopa- mine stores and may cause hyperprolactinaemia. Oestrogens, in high doses, may slightly elevate prolactin but normal contraceptive pills do not. Verapamil, by decreasing dopaminergic tone, may also increase prolactin levels. Gynaecomastia Gynaecomastia may occur due to treatment with various drugs (see Table 13.10.6). Drugs such as spironolactone and ketoconazole, which can displace steroids from sex hormone-​binding globulin, displace oestrogens more easily than androgens. Activation of the oestrogen receptors in breast tissue may take place with drugs that have structural homology with oestrogen, such as digoxin; griseofulvin and cannabis may have the same effect. A decrease in androgen levels occurs in older men and with drugs such as spir- onolactone and ketoconazole that inhibit the biosynthesis of tes- tosterone. The mechanism for the induction of gynaecomastia by captopril and calcium channel blockers (nifedipine) is unclear. With cimetidine and omeprazole, this effect may be due to a direct antiandrogen effect or the inhibition of liver cytochrome P450. Posterior pituitary The syndrome of inappropriate antidiuresis is characterized by normovolaemic hyponatraemia with persistent secretion of AVP, despite a reduced plasma osmolality. Several drugs can cause this syndrome through the inappropriate stimulation of eutopic AVP secretion. These include thiazide diuretics, vincristine, vin- blastine, cyclophosphamide, chlorpropamide, phenothiazines, carbamazepine, clofibrate, tricyclic antidepressants, and serotonin-​ reuptake inhibitors (see Table 13.10.1). The syndrome of inappro- priate antidiuresis can also be caused by the administration of desmopressin (a V2 selective analogue of AVP) or oxytocin. Nephrogenic diabetes insipidus can be induced by lithium in the therapeutic range, and up to 20% of patients receiving long-​term therapy may develop this complication. Demethylchlortetracycline produces dose-​dependent nephrogenic diabetes insipidus, and both the concentrating defect and the unresponsiveness to vasopressin are reversible on cessation of the drug. Parathyroid Lithium therapy can cause an increase in parathyroid gland size, either with hyperplasia or adenoma. This hyperparathyroidism leads to mild hypercalcaemia and sometimes osteoporosis. Thiazide diuretics, by causing haemoconcentration and hypocalciuria, may also result in mild hypercalcaemia but this is usually transient (4–​6 weeks); after this time, other causes of hypercalcaemia should be sought. Vinblastine and colchicine inhibit parathyroid hormone secretion which may result in hypocalcaemia. FURTHER READING Alexandraki KI, Grossman AB (2010). The ectopic ACTH syndrome. Rev Endocr Metab Disord, 11, 117–​26. Bell NH (1991). Endocrine complications of sarcoidosis. Endocrinol Metab Clin North Am, 20, 645–​54. Braunstein GD (1993). Current concepts: gynecomastia. N Engl J Med, 328, 490–​5. Carpenter TO (2003). Oncogenic osteomalacia—​a complex dance of factors. N Engl J Med, 348, 1705–​8. Chattopadhyay N (2006). Effects of calcium-​sensing receptor on the secretion of parathyroid hormone-​related peptide and its impact on humoral hypercalcemia of malignancy. Am J Physiol Endocrinol Metab, 290, E761–​70. Chopra IJ (1997). Clinical review 86: euthyroid sick syndrome: is it a misnomer? J Clin Endocrinol Metab, 82, 329–​34. Daughaday WH, Deuel TF (1991). Tumour secretion of growth fac- tors. Endocrinol Metab Clin North Am, 20, 539–​63. Docter R, et al. (1993). The sick euthyroid syndrome: changes in thy- roid hormone serum parameters and hormone metabolism. Clin Endocrinol (Oxf), 39, 499–​510. Gola M, et  al. (2006). Neuroendocrine tumors secreting growth hormone-​releasing hormone:  pathophysiological and clinical aspects. Pituitary, 9, 221–​9. Grinspoon SK, Bilezikian JP (1992). HIV disease and the endocrine system. N Engl J Med, 327, 1360–​5. Guise TA, Mundy GR (1998). Cancer and bone. Endocr Rev, 19, 18–​54. Hayes AR, Grossman AB (2018). The ectopic adrenocorticotropic hor- mone syndrome: rarely easy, always challenging. Endocrinol Metab Clin North Am, 47, 409–25. Hirshberg B, et al. (2003). Ectopic luteinizing hormone secretion and anovulation. N Engl J Med, 348, 312–​17. Howlett TA, et  al. (1986). Diagnosis and management of ACTH-​ dependent Cushing’s syndrome:  comparison of the features in
ectopic and pituitary ACTH production. Clin Endocrinol (Oxf), 24, 699–​713. Hung W, et al. (1963). Precocious puberty in a boy with hepatoma and circulating gonadotropin. J Pediatr, 63, 895–​903.

section 13  Endocrine disorders 2552 Isidori AM, et  al. (2006). The ectopic adrenocorticotropin syn- drome:  clinical features, diagnosis, management, and long-​term follow-​up. J Clin Endocrinol Metab, 91, 371–​7. Kovacs L, Robertson GL (1992). Syndrome of inappropriate anti­ diuresis. Endocrinol Metab Clin North Am, 21, 859–​76. Melmed S (1991). Extrapituitary acromegaly. Endocrinol Metab Clin North Am, 20, 507–​18. Penny E, et al. (1984). Circulating growth hormone releasing factor concentrations in normal subjects and patients with acromegaly. Br Med J, 289, 453–​55. Robertson GL (2006). Regulation of arginine vasopressin in the syndrome of inappropriate antidiuresis. Am J Med, 119, Suppl 1, S36–​S42. Schrier RW, et al. (2006). Tolvaptan, a selective oral vasopressin V2-​receptor antagonist, for hyponatremia. N Engl J Med, 355, 2099–​112. Singla P (2010). Metabolic effects of obesity: a review. World J Diabetes, 1, 76–​88. Tani Y, et al. (2011). Differential gene expression profiles of POMC-​ related enzymes, transcription factors and receptors between non-​ pituitary and pituitary ACTH-​secreting tumours. Endocr J, 58, 297–​303. Tomlinson JW, et al. (2004). 11 beta-​hydroxysteroid dehydrogenase type 1: a tissue-​specific regulator of glucocorticoid response. Endocr J, 25, 831–​66. Turner HE, Wass JAH (1997). Gonadal function in men with chronic illness. Clin Endocrinol (Oxf), 47, 379–​403. Vanderpump MPJ, Tunbridge WMG (1993). The effects of drugs on endocrine function. Clin Endocrinol (Oxf), 39, 389–​97. Verbalis JG, et al. (2011). Efficacy and safety of oral tolvaptan therapy in patients with the syndrome of inappropriate antidiuretic hor- mone secretion. Eur J Endocrinol, 164, 725–​32. Wass JAH, et al. (1982). HCGB producing pineal choriocarcinoma. Clin Endocrinol (Oxf), 17, 423–​31. White A, Clark AJL (1993). The cellular and molecular basis of the ec- topic ACTH syndrome. Clin Endocrinol (Oxf), 39, 131–​41.

13.11 The pineal gland and melatonin 2553

13.11 The pineal gland and melatonin 2553

ESSENTIALS The pineal gland transduces light–​dark cycles for the timing of body rhythms by secretion of melatonin, an endogenous indoleamine de- rived from tryptophan, the concentrations of which in plasma and cerebrospinal fluid are up to 100 times higher at night than in the daytime. This exerts its effects through transmembrane, G-​protein coupled receptors (MT1 and MT2), and nuclear receptors primarily in the suprachiasmatic nucleus, pars tuberalis of the pituitary gland and hypothalamus. The natural period of the human circadian system is on average 24.1 to 24.3 h, and the principal resetting agent is light. Exogenous melatonin can shift the timing of the internal clock to earlier and later times, and synchronize a free-​running clock that is not properly entrained to the 24-​h day, hence it may have a therapeutic role for disorders of sleep rhythm including jet lag, in shift workers, and in blind people. It has proved useful for sleep disorders in neurologic- ally disabled children. Exogenous melatonin also has rapid transitory ‘soporific’ or sleep-​inducing effects and may be used to hasten sleep onset in suitable circumstances. A combination of sleep induction and circadian realignment is most effective. Numerous reports of antioxidant/​free-​radical scavenging/​neuroprotective/​anticancer ef- fects (in large doses) have yet to find a confirmed clinical application. Introduction The mammalian pineal gland is a secretory organ. Whereas in fish and amphibians it is directly photoreceptive, in reptiles and birds it has a mixed photoreceptor and secretory function. Although an endocrine function was considered for many years, this was only given credibility in 1958 by the pioneering work of Lerner, who iso- lated a small molecule from bovine pineal glands that he named melatonin because it caused blanching of melanophores in am- phibian skin. The primary function of the pineal gland in all species studied to date is to transduce information concerning light–​dark cycles to body physiology, particularly for the organization of body rhythms, via the secretion of its major hormone melatonin. In some birds and lower vertebrates, it serves as a rhythm generating system, or biological clock. In mammals, it is concerned with the coordin- ation of rhythm physiology without having the capacity to act as a rhythm generator. In humans, the gland has been known since an- tiquity. Many questions remain unanswered about the function of the human pineal gland, but its secretion of the chronobiotic mol- ecule melatonin has prompted enormous interest in the fields of travel medicine, neurophysiology, and endocrine research. The pineal gland Structure The pineal gland is less than 1 cm in its longest diameter and weighs less than 0.2 g; it lies above the posterior aspect of the third cerebral ven- tricle. The major cellular component of the normal mammalian pineal gland, the pinealocyte, is believed to have evolved from truly photo- receptive cells in lower vertebrates and structural remnants of the outer segments of photoreceptor cells are reported in higher vertebrates. It also contains neuroglial components, principally of astrocytic type, which occasionally become malignant. Human pineal tissue calcifies with age, but this does not necessarily diminish its secretory activity. The pineal gland is considered to reside outside the func- tional blood–​brain barrier. Pathology Tumours of the pineal region in children are frequently associated with abnormal pubertal development. Much evidence suggests that preco- cious puberty in such cases is due to the production of human chori- onic gonadotrophin (β-​hCG) by germ cell tumours of the pineal gland. Delayed puberty has also been associated with pineal tumours. Pineal tumours are heterogeneous and may arise from germ cells (teratomas, germinomas, choriocarcinomas, endodermal sinus tumours, mixed germ cell tumours), pineal parenchymal cells (pineoblastoma and pineocytoma), and the supporting stroma (gliomas). Classification of pineal parenchymal tumours is complicated by the presence of mixed pineocytoma-​pineoblastoma types, some with intermediate differ- entiation. A new classification has been proposed recently, based on histological features, which is closely related to patient survival. Treatment of pineal tumours by surgical excision or radiation ap- pears to suppress melatonin secretion leading to sleeping difficulties; 13.11 The pineal gland and melatonin J. Arendt and Timothy M. Cox

section 13  Endocrine disorders 2554 melatonin replacement therapy has been reported to benefit such pa- tients with defective melatonin release. However not all reports are con- sistent and two recent studies showed no effect of pinealectomy on sleep assessed pre- and post-operatively in humans by polysomnography, and in rats. Melatonin is a darkness hormone not a sleep hormone. Melatonin Biosynthesis Melatonin, like serotonin, is an endogenous indoleamine derived from tryptophan. The first step in indoleamine synthesis is the 5-​ hydroxylation of tryptophan by tryptophan hydroxylase—​an en- zyme with requirements for dioxygen, iron, and tetrahydrobiopterin. 5-​Hydroxytryptophan (5HTP) is then decarboxylated to serotonin or 5-​hydroxytryptamine (5HT). The enzyme arylalkylamine N-​ acetyltransferase (AANAT), regulated by the sympathetic trans- mitter noradrenaline, then acetylates 5HT to N-​acetylserotonin (NAS) and this appears to be the rate-​limiting step in melatonin syn- thesis. The enzyme is localized principally in the pineal gland but also in the retina, the skin, within specific cells in the upper gastrointes- tinal tract, and with minor expression in bone marrow, lymphocytes, and certain epithelia. The final step in the synthesis is carried out by hydroxyindole-​O-​methyl transferase (HIOMT) (Figs. 13.11.1a and 13.11.1b). This enzyme is now known as N-acetylserotonin O-methyltransferase (ASMT). Melatonin receptors Melatonin binds to specific receptors including the seven transmem- brane G-​protein-​coupled MT1 and MT2 receptors, as well as nuclear receptors RZR/​ROR orphan receptor family and downstream tran- scription factors that are associated with melatonin signalling. A third site, named MT3, is the enzyme quinone reductase type 2, which mela- tonin and its congeners bind and inhibit with high affinity. This action is shared with the natural product resveratrol, and indicates a potential application for melatonin as an antioxidant in pleiotropic detoxifica- tion processes, including the treatment and prevention of cancer. Membrane G-​protein receptors for melatonin are principally expressed in the nervous system but they have been found in nu- merous other locations. The nuclear transcription factor appears to be expressed in the periphery. There is emerging evidence that a nuclear signalling pathway with ligand-​induced control of target gene transcription, mediates some functions of melatonin. The MT2 melatonin receptors have been implicated in mediating learning and memory in experimental mice, and there is also evidence that their activation alters electrophysiological phenomena associated with memory, such as long-​term potentiation in neurones; loss of these receptors also decreases hippocampal synaptic plasticity. Melatonin has been reported to control expression of the 5-​lipoxygenase gene; Third ventricle PVN Pineal SCN SCG Spinal cord (a) Pituitary Eye NH2 CH2CH COOH NH2 CH2CH CH2CH2NH2 CH2CH2NHCOCH3 CH2CH2NHCOCH3 CH3O HO HO HO N H N H N H N H N H COOH TRYPTOPHAN TRYPTOPHAN-5-HYDROXYLASE 5 HTP- DECARBOXYLASE SEROTONIN-N-ACETYLTRANSFERASE NAT or AANAT HYDROXYINDOLE-0-METHYL TRANSFERASE (HIOMT) MELATONIN 5-HYDROXYTRYPTOPHAN (5HTP) 5-HYDROXYTRYPTAMINE (5HT, SEROTONIN) N-ACETYSEROTONIN (NAS) (N-ACETYL-5-METHOXYTRYPTAMlNE) Fig. 13.11.1  (a) Melatonin synthesis and the principal neural pathways innervating the pineal gland. SCN, suprachiasmatic PVN, paraventricular nucleus; SCG, superior cervical ganglion. (b) Control of melatonin synthesis in the mammalian pineal gland. (a) Source data from Tamarkin K, Baird CJ, Almeida OF (1985). Melatonin: A coordinating signal for mammalian reproduction. Science, 227, 714–​20. (b) Reprinted by permission from Springer Nature: Springer Cell and Tissue Research. Ganguly S, Coon SL, Klein DC (2002). Control of melatonin synthesis in the mammalian pineal gland: the critical role of serotonin acetylation. Cell Tissue Res, 309, 127–​37, Copyright © 2002.

13.11  The pineal gland and melatonin 2555 the cognate enzyme is not implicated in circadian rhythms but is expressed in myeloid cells and participates in allergic and inflam- matory reactions. Melatonin (formal chemical name, N-​acetyl-​5-​methoxytryptamine) has been found within membrane-​bound bodies in pinealocytes. In experimental animals these show light-​dependent morphological changes associated with melatonin secretion under altered envir- onmental light conditions. The pineal gland is the principal source of circulating melatonin in mammals, indeed pinealectomy leads to undetectable melatonin concentrations in blood. Synthesis else- where (e.g. in the retina), seems to change local concentrations only. Melatonin appears to exert its main effects through MT1 receptors in the infundibular part of the pituitary gland, through MT1 and MT2 re- ceptors in the central biological rhythm generating system of the brain (the suprachiasmatic nuclei of the hypothalamus) and other regions of the hypothalamus that modulate the secretion of pituitary hormones, and that influence core body temperature and other functions. The role of melatonin and the pineal gland in photoperiodism In all species studied to date, melatonin is normally synthesized and secreted at night. This rhythm is circadian in nature (i.e. it is en- dogenously driven by the activity of the suprachiasmatic nuclei). Exposure to light influences the secretion of melatonin, and mela- tonin release is suppressed particularly by blue light (wavelength 460–​480 nm) in a manner that increases with length of exposure and intensity of luminance. The length of the day (photoperiod) strongly influences melatonin secretion: the longer the night, the longer the duration of secretion. The long-​standing tendency of humans to alter their light environment since the discovery of fire renders this relationship hard to show, except under conditions in which the dur- ation of total darkness is altered. However, the seasonal physiology of many animals is regulated by the photoperiod and changing dur- ation of melatonin secretion is critical for inducing several specific seasonal responses (e.g. reproduction, coat growth). Melatonin influences production of gonadotrophins and gonadal hormones via actions within the hypothalamus. There is recent evi- dence for a molecular mechanism within the pars tuberalis of the pi- tuitary, which links the short duration, long day melatonin signal to a hypothalamic increase of triiodothyronine (T3) through a thyroid-​ stimulating hormone/​deiodinase2 paracrine mechanism. The local synthesis of type 2 deiodinase (Dio2) promotes triiodothyronine (T3) production and summer biology, whereas type 3 deiodinase (Dio3) promotes T3 degradation and winter biology. However, melatonin controls seasonal variations in prolactin by a direct ac- tion on the pars tuberalis of the pituitary. This structure is the major site of melatonin receptors (MT1) in most species. Photoperiod-​ dependent gene expression in the pars tuberalis is directly modified by melatonin (Fig. 13.11.2). Exogenous melatonin can be used as the photoperiodic signal and has been commercialized to permit regulation of the breeding season in useful domesticated species such as sheep, goats, and mink. In animals and humans there is now evidence for an alter- native photoreceptive system, which is independent of retinal rods Fig. 13.11.1  Continued

section 13  Endocrine disorders 2556 SUMMER Long days Short melatonin profile WINTER Short days Long melatonin profile (a) Cry Per CCG BMal Clk MT1 & MT2 PVN (b) SCG NA WINTER TSH DIO2 T4 T3 TSH DIO2 Prolactin LH GnRH T4 T3 WINTER SUMMER SUMMER RETINA HYPO- THALAMUS RHT-GLUTAMATE SCN MELATONIN PINEAL GLAND PARS TUBERALIS Per NAT MT1 MT2 Per Cry Fig. 13.11.2  Photoperiodic and circadian mechanisms. (a) The duration of melatonin secretion changes with daylength, providing an internal time cue for the organization of daylength-​dependent seasonal functions in photoperiodic species. This changing duration can be seen in humans if they are maintained in different durations of total darkness. However, domestic intensity light at night is sufficient to greatly diminish or eliminate this photoperiodic response. (b) Diagrammatic representation of the control of production and the functions of melatonin with regard to seasonal and circadian timing mechanisms. RHT, retino-​hypothalamic tract; NA, noradrenalin; SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; SCG, superior cervical ganglion; TSH, thyroid-​stimulating hormone, DIO2, type II thyroid hormone deiodinase. MT1 and MT2, melatonin receptor subtypes. The melatonin rhythm is generated by a closed-​loop negative feedback of clock gene expression in the SCN. Clk and BMal, positive stimulatory elements; Per and Cry, negative elements; CCG, clock-​controlled genes. Per and NAT mRNA oscillate in the pineal, although posttranscription control is evident in some species. Melatonin influences SCN activity via two or more receptors. MT2 appears to be primarily the phase-​shifting receptor in rodents, whereas MT1 is associated with suppression of SCN electrical activity. The MT2 receptor was first characterized in the retina and influences dopamine release. Melatonin conveys photoperiodic information influencing the patterns of clock gene expression in the pars tuberalis for the control of seasonal prolactin variations via an MT1 receptor. (b) Adapted (with permission) from an original diagram by Elisabeth Maywood, MRC Laboratory of Molecular Biology, Neurobiology Division, Cambridge, United Kingdom. Modified from Encyclopedia of Endocrine diseases.

13.11  The pineal gland and melatonin 2557 or cones, utilizes light-sensitive retinal ganglion cells and a distinct photopigment (melanopsin). It serves to mediate the physiological (but non​visual) effects of light (usually in concert with rods and cones). This photoreceptive apparatus responds preferentially to short-​wavelength light, 460–480 nm. Melatonin and circadian rhythms Rhythmic melatonin secretion leads to concentrations in the plasma or cerebrospinal fluid that are up to 100 times greater at night than in the daytime, with very large interindividual but con- sistent intraindividual variation. These fluctuations are used to assess the timing of the human biological clock: the secretion pro- file of melatonin provides, in the periphery, the most accurate and sensitive index of the activity of the suprachiasmatic nuclei. In the diagnosis of circadian rhythm disorders, blood, and salivary deter- minations of melatonin and/​or measures of the principal metabolite, 6-​sulphatoxymelatonin (aMT6s) in urine are useful. Maximum con- centrations are observed in childhood and melatonin concentra- tions decline thereafter with age. The role of endogenous melatonin in humans is unclear. Peak night-​time concentrations in the plasma are closely associated with the nadirs of core temperature, alertness, performance, and metabolism. The profile of secretion is strongly associated with increasing sleep propensity, and sleep is longer and of better quality when taken in phase with peak melatonin secretion (and with the nadir in core temperature; see Fig. 13.11.3). Melatonin is able to reinforce night-​time physiology (e.g. in con- tributing to the propensity to sleep and the decreased nocturnal core temperature). Melatonin appears to play a supporting role in the influence of the light–​dark cycle for synchronizing the circadian rhythms to the 24-​h day. In the absence of time cues (free-​running) the natural period of the human circadian system is on average 24.1 to 24.3 h and the principal resetting agent is light (Fig. 13.11.4). Exogenous melatonin clearly shifts the timing of the internal clock to earlier and later times and synchronizes a free-​running clock that is incompletely entrained to the 24-​h day (Fig. 13.11.5). Several syn- dromes associated with long-​term insomnia in humans appear to result from slower, faster, or free-​running sleep–​wake cycles. These include the non-​24-​h sleep–​wake cycle of blind people (with no light perception at all), delayed sleep-​phase syndrome, advanced sleep-​phase syndrome, and irregular sleep–​wake cycles. In addition, abrupt shifts of time cues such as are found in shift work and jet lag lead to circadian asynchrony with resultant difficulties affecting sleep, fatigue, and alertness, and with possible long-​term health con- sequences. In these circumstances, melatonin has actual and poten- tial therapeutic benefit as a result of its chronobiotic activity. Timed exposure to light at high luminance may improve disorders of the circadian rhythm that affect sleep. However, in many circumstances, the correct timing and intensity of light exposure (and avoidance) is hard to achieve. Notably, blind people cannot have access to light treatment and for the non-​24-​h sleep–​wake disorder of the blind, melatonin, correctly timed, is the treatment of choice. Pharmaceutical use of melatonin In addition to its use in blind circadian rhythm disorder, melatonin has proved successful in normalizing delayed sleep timing in de- layed sleep-​phase syndrome, stabilizing irregular sleep–​wake cycles in neurologically disabled children, and in treating the symptoms of jet lag. Melatonin treatment has also been suggested as a means to improve sleep in night-​shift workers, in older individuals with in- somnia (for which a registered preparation is available on prescrip- tion), and in patients with pineal tumours. Melatonin pg/ml Core body temperature°C Triacylglycerol mmol/litre Alertness 0 = not alert, 100 = very alert Reaction time sec Clock time h Sleep propensity Road accidents 60 40 20 37.1 37.0 36.9 36.8 36.7 36.6 1.8 1.6 1.4 1.2 60 40 20 1.8 1.7 1.6 1.5 1.4 12 16 20 24 04 08 12h Fig. 13.11.3  Relationship of plasma melatonin to other major circadian rhythms. Note the close correspondence between the core temperature nadir and the melatonin peak. Reprinted from The Lancet, Vol. 358, Rajaratnam SM, Arendt J, Health in a 24-​h society, Pages 999–​1005, Copyright 2001, with permission from Elsevier. Melatonin Free-run Synchronised Biological night (time of daily endogenous melatonin secretion) Time E.g. blindsubjects, Lockley et al., J Endocrinol, 2000 Fig. 13.11.4  Melatonin can synchronize free-​running rhythms in both blind and sighted subjects. Before treatment, the subject shows circadian rhythms of melatonin, and sleep which are longer than 24 h.

section 13  Endocrine disorders 2558 Many studies have been carried out to investigate the efficacy of melatonin as a chronobiotic agent for the alleviation of symptoms of jet lag. The results of one meta-​analysis to assess the effective- ness of oral melatonin, taken in different dosing regimens for al- leviating jet lag after travel across several time zones, showed that the agent is effective in preventing or reducing jet lag and that its short-​term use appears to be safe on an occasional basis. Side-​effect reporting has been low, except in patients with epilepsy or those who are taking warfarin in whom convulsant effects or increased bleeding, respectively, have been reported. Melatonin may theoret- ically influence reproductive development in children and reduce sexual activity, if overused, in adults. No evidence of these effects has yet been reported and the prolonged-​release agent has been found to be effective and safe in mitigating the disordered sleep of children with neurodevelopmental diseases. Recent studies of jet lag tend to recommend the use of preflight timed melatonin (0.5 mg) to initiate an advance or delay as required of the circadian system, and to use postflight higher doses (3–​5 mg) again timed correctly, to reinforce the shift in timing and to acutely induce sleepiness. Another meta-​analysis was less positive. Melatonin is nevertheless recommended as a treatment for jet lag, delayed sleep-​phase syn- drome, and irregular sleep–​wake cycles by the American Academy of Sleep Medicine. Its use in shift work has proved inconsistent, not all studies have been successful regarding its use in insomnia in older people, and there is insufficient data to evaluate properly its effect in pineal tumours. The timing of treatment with respect to in- ternal circadian timing is very important and judging such timing is often not simple, especially in shift work and jet lag. As a reinforcer of circadian phase melatonin may be useful in other conditions, for example in reduction of blood pressure in pa- tients with essential hypertension. There are increasing associations of melatonin with glucose metabolism and animal data and human genetic studies suggest that either low melatonin secretion or re- duced melatonin signalling can impair insulin sensitivity and lead to type 2 diabetes. However there are considerable controversies in this field. It is likely that any aspect of physiology which depends on per- ception of daylength change, and/​or which is driven by the master circadian clock in the suprachiasmatic nucleus (SCN), is susceptible to the effects of melatonin. Melatonin is freely available in the United States of America, and a melatonin formulation has been registered for use in insomnia in older people in Europe. It is available as a prolonged-​release prescrip- tion drug (Circadin) and is approved by the European Medicines Agency as a single treatment in a dose of 2 mg for patients aged at least 55 years, for the short-​term treatment (up to 13 weeks) of pri- mary insomnia characterized by poor sleep quality. Moreover, the findings of a randomized controlled clinical trial showed that mela- tonin had beneficial effects on delirium in geriatric patients. There is little evidence concerning the advantages or disadvantages of slow as compared to fast release preparations. The European Food Safety Agency (EFSA) Panel on Dietetic Products, Nutrition and Allergies (NDA) considers that reduction of sleep onset latency might be a beneficial physiological effect of mela- tonin (assumed to be a food constituent). The target population is as- sumed to be the general population. The Panel considers that in order to obtain the claimed effect, 1 mg of melatonin should be consumed close to bedtime. It should be noted however that 1 mg melatonin taken or- ally leads to pharmacological, not physiological plasma concentrations in most people, and that the marked circadian dependency of the ef- fects of melatonin is not taken into account by these recommendations. The melatonin MT1/​MT2 receptor agonist ramelteon/​Rozerem has been approved by the United States Food and Drug Administration (FDA), again for insomnia. The manufacturer Takeda has however withdrawn its application for marketing in Europe. The product re- mains available in the United States and Japan. Most recently the MT1/​MT2 receptor agonist tasimelteon (Hetlioz) has been approved for non-​24 h sleep–​wake disorder in the blind, including paedi- atric use, both by the FDA and in Europe. The manufacturer Vanda 100 75 50 25 12 16 20 0 4 8 12 12 Clock time (h) after mel after plac after mel 100 75 50 25 during mel Sleep efficiency (%) DIRECT EFFECT *

CIRCADIAN EFFECT 16 20 0 4 8 12 Fig. 13.11.5  Exogenous melatonin has both direct and circadian effects on sleep. Healthy young men (n = 8) received 1.5 mg surge sustained release melatonin (mel) or placebo (plac) in a double-​blind cross-​over design at 16:00 h daily for 8 days, recumbent, less than 5 lux, 16:00–​08:00 h, sleep was evaluated by polysomnography on the night after the last dose of melatonin or placebo and on the subsequent night after melatonin washout. These data confirm the utility of melatonin for the treatment of delayed sleep. In a treatment situation the two effects can be maximized by suitable timing of the dose. Hormones (melatonin, cortisol, TSH) and heart rate variability showed similar substantial phase advances as seen for sleep. From Rajaratnam SMW, Middleton B, Stone BM, Arendt J, Dijk D-​J (2004). Melatonin advances the circadian timing of EEG sleep and directly facilitates sleep without altering its duration in extended sleep opportunities in humans. J Physiol, 561, 339–​51, by permission. Copyright © 2004, John Wiley and Sons.

13.11  The pineal gland and melatonin 2559 Pharmaceuticals has recognized the important circadian effects of melatonin and its development has targeted circadian disorders and correctly timed treatment. Other formulations and deriva- tives of melatonin are under development and a distinct agonist (agomelatine/​Valdoxan) is registered for use in major depression; the potential therapeutic effect is postulated to be mediated by an antag- onist effect on the serotonin receptor, 5HT2C. Its use is constrained by possible hepatic toxicity in patients with liver dysfunction, especially in older people. It is interesting to note that the doses used for all the agonists mentioned are much higher than the recommended doses for melatonin itself. In summary, there is evidence indicating that oral ingestion of melatonin may be beneficial to correct sleep timing, and when used occasionally after transmeridian flights that would induce daytime fatigue and sleep disturbance associated with gastrointestinal complaints, weakness, malaise, loss of mental efficiency, and other symptoms that characterize jet lag. Clearly, since the drug is not as yet licensed in all countries, routine pharmaceutical quality control must be established and the use and safety of melatonin in pregnancy has not yet been completely established. Given that prion-​related diseases result from the ingestion or injection of material derived from brain or other animal tissues, only pure biosynthetic melatonin should be considered for human use. Melatonin derived from bovine pineal or other biological sources should be avoided. Recently, partial deficiency of melatonin, induced by excess noc- turnal exposure to light, has been suggested to explain an increased risk of cancer in shift workers. It seems more likely that a general dis- turbance of the circadian system rather than selective suppression of melatonin provides the mechanistic explanation for the increased frequency of cancers in this group. Melatonin also has antioxidant properties and is widely taken in certain communities, particularly in the United States of America, where it is claimed to provide un- specified protection against ageing, degenerative diseases, cancer, and impaired immune function, as well as reproductive and psychi- atric illness. Nonetheless, it should be acknowledged that, as in other vertebrates, melatonin has diverse physiological actions in humans, many of which are not fully understood. At present, the principal authenticated indication for exogenous melatonin is for the control of sleep disorders in adults, in children with neurodevelopmental abnormalities, and the treatment of symptoms associated with jet lag, rather than the many conditions for which our scientific under- standing of its proposed benefits is as yet incomplete. FURTHER READING Al-​Aama T, et  al. (2011). Melatonin decreases delirium in elderly patients:  a randomized, placebo-​controlled trial. Int J Geriatr Psychiatry, 26, 687–​94. Arendt J (1995). Melatonin and the mammalian pineal gland. Chapman & Hall, London. Arendt J (2000). Melatonin, circadian rhythms, and sleep. N Engl J Med, 343, 1114–​6. Arendt J (2011). Chapter 15: The pineal gland and pineal tumours. http://​ www.endotext.org/​neuroendo/​neuroendo15/​neuroendoframe15.htm. Auger RR, et al. (2015). Clinical practice guideline for the treat- ment of intrinsic circadian rhythm sleep-​wake disorders: advanced sleep-​wake phase disorder (ASWPD), delayed sleep-​ wake phase disorder (DSWPD), non-​24-​hour sleep-​wake rhythm disorder (N24SWD), and irregular sleep-​wake rhythm disorder (ISWRD). An update for 2015. J Clin Sleep Med, 11(10), 1199–​236. Buscemi N, et al. (2006). Efficacy and safety of exogenous melatonin for secondary sleep disorders and sleep disorders accompanying sleep restriction: meta-​analysis. BMJ, 332, 385–​93. Carlberg C, Wiesenberg I (2007). The orphan receptor family RZR/​ ROR, melatonin and 5-​lipoxygenase: an unexpected relationship. Pineal Research, 18, 171–​8. De Leersnyder H, et  al. (2011). Prolonged-​release melatonin for
children with neurodevelopmental disorders. Pediatr Neurol, 45, 23–​6. Hardeland R (2009). Melatonin: signaling mechanisms of a pleiotropic agent. Biofactors, 35, 183–​92. Herxheimer A, Petrie KJ (2002). Melatonin for the prevention and treatment of jet lag. Cochrane Database Syst Rev, 2, CD001520. Hut RA (2011). Photoperiodism: shall EYA compare thee to a summer’s day? Curr Biol, 21, R22–​5. Johnsa JD, Neville MW (2014). Tasimelteon:  a melatonin receptor agonist for non-​24-​hour sleep-​wake disorder. Ann Pharmacother, 48, 1636–​41. Morgenthaler TI, et al. (2007). Practice parameters for the clinical evaluation and treatment of circadian rhythm sleep disorders. An American Academy of Sleep Medicine report. Sleep, 30, 1445–​59. Poeggeler B, et al. (1994). Melatonin—​a highly potent endogenous radical scavenger and electron donor: new aspects of the oxidation chemistry of this indole accessed in vitro. Ann N Y Acad Sci, 738, 419–​20. Posadzki PP, et al. (2018). Melatonin and health: an umbrella review of health outcomes and biological mechanisms of action. BMC Med, 16(1), 18. Riemann D, et al. (2017). European guideline for the diagnosis and treatment of insomnia. J Sleep Res, 26, 675–700. Wade AG, et al. (2011). Prolonged release melatonin in the treatment of primary insomnia: evaluation of the age cut-​off for short-​ and long-​term response. Curr Med Res Opin, 27, 87–​98. Williams WP, McLin DE, Dressman MA, Neubauer DN (2016). Comparative Review of Approved Melatonin Agonists for the Treatment of Circadian Rhythm Sleep-Wake Disorders. Pharmacotherapy, 36(9), 1028–41.

SECTION 14 Medical disorders in pregnancy Section editor: Catherine Nelson-Piercy 14.1 Physiological changes of normal pregnancy  2563 David J. Williams 14.2 Nutrition in pregnancy  2568 David J. Williams 14.3 Medical management of normal pregnancy  2575 David J. Williams 14.4 Hypertension in pregnancy  2583 Fergus McCarthy 14.5 Renal disease in pregnancy  2589 Kate Wiles 14.6 Heart disease in pregnancy  2597 Catherine E.G. Head 14.7 Thrombosis in pregnancy  2606 Peter K. MacCallum and Louise Bowles 14.8 Chest diseases in pregnancy  2613 Meredith Pugh and Tina Hartert 14.9 Liver and gastrointestinal diseases
of pregnancy  2619 Michael Heneghan and Catherine Williamson 14.10 Diabetes in pregnancy  2627 Bryony Jones and Anne Dornhorst 14.11 Endocrine disease in pregnancy  2638 David Carty 14.12 Neurological conditions in pregnancy  2642 Pooja Dassan 14.13 The skin in pregnancy  2648 Gudula Kirtschig and Fenella Wojnarowska 14.14 Autoimmune rheumatic disorders and vasculitis in pregnancy  2655 May Ching Soh and Catherine Nelson-Piercy 14.15 Maternal infection in pregnancy  2671 Rosie Burton 14.16 Fetal effects of maternal infection  2678 Lawrence Impey 14.17 Blood disorders in pregnancy  2687 David J. Perry and Katharine Lowndes 14.18 Malignant disease in pregnancy  2696 Robin A.F. Crawford 14.19 Maternal critical care  2701 Rupert Gauntlett 14.20 Prescribing in pregnancy  2706 Lucy MacKillop and Charlotte Frise 14.21 Contraception for women with
medical diseases  2711 Aarthi R. Mohan

13.2 Pituitary disorders 2258

13.2 Pituitary disorders 2258

13.2.1 Disorders of the anterior pituitary gland 2

13.2.1 Disorders of the anterior pituitary gland 2258 Niki Karavitaki and John A.H. Wass

CONTENTS 13.2.1 Disorders of the anterior pituitary gland  2258 Niki Karavitaki and John A.H. Wass 13.2.2 Disorders of the posterior pituitary gland  2277 Niki Karavitaki, Shahzada K. Ahmed, and John A.H. Wass 13.2.1  Disorders of the anterior pituitary gland Niki Karavitaki and John A.H. Wass ESSENTIALS The anterior pituitary gland produces growth hormone (GH), lutein- izing hormone (LH), follicle-​stimulating hormone (FSH), adrenocor- ticotropic hormone (ACTH), thyroid-​stimulating hormone (TSH), and prolactin. Their secretion is regulated by hypothalamic-​releasing and inhibitory factors delivered via portal capillaries, and by negative feedback inhibition of the cognate hormones produced by target endocrine glands such as the thyroid and adrenal cortex. Clinical features—​presentation of pituitary disease, mostly associ- ated with a space-​occupying lesion, may result from (1) local mass effects—​often causing headache, visual field defects (most typic- ally bitemporal hemianopia or upper temporal quadrantanopia) and ocular nerve palsies; (2) pituitary hormone deficits—​producing wide-​ranging effects as a result of single or multiple deficiencies, with GH and gonadotropins (LH and FSH) usually affected first, fol- lowed much later by ACTH and TSH; and/​or (3) pituitary hormone hypersecretion, usually arising as a consequence of neoplastic prolif- eration of particular cell types within the gland, producing complex and disabling syndromes such as Cushing’s disease or acromegaly. Investigation—​this includes testing for (1)  hormonal hyper-​ or hyposecretion—​measurements of basal levels of pituitary hormones with target gland hormone secretion are usually sufficient for as- sessment of TSH (thyroxine), FSH/​LH (testosterone or oestra- diol) and prolactin; dynamic testing is required for the ACTH/​ cortisol axis and determination of GH deficiency or excess; (2) radio- logical assessment—​MRI is the modality of choice; and (3) neuro-​ ophthalmological evaluation, including assessment of visual acuity, visual fields, and fundoscopy. Management—​the availability of sensitive hormonal assays, re- placement hormones, and hypothalamic peptides, together with refined neuroimaging methods and neurosurgical techniques, has increased our ability to identify precisely and successfully treat most patients with diseases of the anterior pituitary gland. Growth hormone GH deficiency—​in children this causes growth failure, and in adults features including decreased energy and quality of life, and increase in fat mass/​decrease in muscle mass. The insulin tolerance test is con- sidered the ‘gold standard’ for diagnosis. Goals of GH treatment in adults are to achieve an appropriate clinical response while avoiding side effects, and an IGF-​1 level in the middle of the reference range. GH excess—​this causes acromegaly, which develops insidiously with multiple clinical features, most notably including local tumour effects, and increase in size of hands, feet, jaw, and skull. Biochemical diagnosis is made by confirming absence of suppression of GH in the oral glucose tolerance test, and by increased serum IGF-​1 levels. Management options include:  (1) surgery—​with trans-​sphenoidal surgery the treatment of choice for most patients; (2)  drugs—​ including dopamine receptor agonists, somatostatin receptor lig- ands (e.g. octreotide, lanreotide) and GH receptor antagonists (e.g. pegvisomant); and (3) radiotherapy—​generally offered for tumours that have recurred or persisted after surgery in patients with resist- ance to or intolerance of medical treatment. FSH and LH Gonadotropin deficiency—​this presents in women with oligo/​amenor- rhoea, loss of libido, dyspareunia, hot flushes, and infertility; and in men with loss of libido; impaired sexual function; mood impairment; loss of facial, scrotal, and trunk hair; decreased muscle bulk and en- ergy. Diagnosis in women is based on clinical features in association with FSH and LH levels that are ‘inappropriately normal’ or low in 13.2 Pituitary disorders

13.2.1  Disorders of the anterior pituitary gland 2259 premenopausal women and low in postmenopausal women; in men there is low morning serum testosterone with low or ‘inappropriately normal’ gonadotropins. Treatment comprises appropriate replace- ment therapy. Prolactin Prolactinomas are the most common pituitary adenomas and typ- ically present with galactorrhoea and hypogonadism, manifesting in men as impotence, infertility, and decreased libido, and in women as oligo/​amenorrhoea and infertility. Secondary causes of hyperprolactinaemia must be excluded in any patient with an ele- vated serum prolactin and serum prolactin levels usually parallel tu- mour size in those with prolactinomas. Dopaminergic agonists (e.g. cabergoline, bromocriptine, pergolide, quinagolide) are the primary therapy. ACTH Chronic ACTH deficiency is associated with fatigue, pallor, anorexia, weight loss, hypotension, hyponatraemia, hypoglycaemia, and eo- sinophilia. The insulin tolerance test is considered the ‘gold standard’ for diagnosis. Glucocorticoid deficiency can be life-​threatening and hence replacement with hydrocortisone (or other steroid) in a dose and timing to mimic the normal pattern of cortisol secretion should begin as soon as the diagnosis is confirmed. Cushing’s disease is caused by chronic exposure to endogenous glucocorticoids (Cushing’s syndrome) produced by the adrenal cortex in response to excess ACTH production by a pituitary corticotroph adenoma. TSH Central hypothyroidism is diagnosed when the concentration of thy- roxine is decreased and the level of TSH levels is usually normal or low. Clinical presentation is as for primary hypothyroidism. Treatment is with thyroxine. Other conditions Hypopituitarism—​can be caused by a range of conditions including pituitary and non​pituitary tumours, hypophysitis, pituitary apoplexy, Sheehan’s syndrome (postpartum), brain injury (traumatic, surgical, irradiation, postinfective), and granulomatous diseases. Clinical manifestations depend mainly on the underlying disease, as well as the type and the degree of the hormonal deficits. Diagnosis and treatment of each pituitary hormone deficit is as just described. Pituitary adenomas—​the most common cause of pituitary disease; may be functioning (resulting in syndromes of hormonal excess) or non​functioning (presenting with mass effects). Treatment in- volves surgery, radiotherapy, or medical therapy as described earlier. Pituitary carcinomas are very rare. Pituitary apoplexy—​occurs primarily in patients with pre-​existing pituitary adenomas; results from acute haemorrhage or infarction of the pituitary gland and is characterized by sudden onset of head- ache, vomiting, visual disturbance, ophthalmoplegia, and altered consciousness. Initial management requires close monitoring of fluid and electrolyte balance and immediate replacement of deficient hormones, especially corticosteroids. Some authorities recommend urgent surgical decompression in some cases. Craniopharyngiomas—​these epithelial tumours can present with pressure effects and/​or compromised hypothalamo-​pituitary function. First-​line treatment usually comprises surgery with or without adjuvant external beam irradiation. Hypophysitis—​may be primary (granulomatous, xanthomatous or lymphocytic) or caused by a known agent or systemic disease. Differential diagnosis is from pituitary adenoma. Optimal treatment for the inflammatory process has not been established and replace- ment of defective endocrine function is required. Introduction The pituitary gland or hypophysis cerebri was first described by Galen of Pergamon in the 2nd century ad, and is considered to be the ‘master gland’ integrating hormonal signals that control nu- merous endocrine and metabolic functions. Since the demonstra- tion of the hypothalamic control of pituitary function by Harris in Oxford in the 1950s, our understanding of the physiology and pathophysiology of the pituitary gland has broadened. The devel- opment of radioimmunoassays in the 1960s, the extraction of hypo- thalamic factors principally by Schally and Guillemin in the 1970s, the advances in immunocytochemistry, electron microscopy, and in situ hybridization methods, as well as the expansion of molecular biology have increased this understanding. Finally, the advances in modern imaging techniques and in pituitary surgery combined with the development of medical treatments for pituitary tumours have greatly expanded the therapeutic possibilities, providing successful and safe outcomes in most patients. Anatomy and embryology The pituitary gland consists of the anterior lobe (adenohypophysis), the posterior lobe (neurohypophysis), and an intermediate zone. The adenohypophysis derives from the stomodeal ectoderm which in- vaginates by the third week of gestation forming Rathke’s pouch. In the sixth week of gestation it comes in contact with the infun- dibulum. A remnant of the pharyngeal hypophysis may be found in adults, forming the pharyngeal pituitary located in the midline of the nasopharynx. The posterior lobe originates from the neural primordium as an outpouching from the floor of the third ventricle at the fourth week of gestation. The intermediate lobe arises from the posterior portion of Rathke’s pouch. This area normally contains microcytic remnants of Rathke’s pouch, which rarely become clin- ically significant. The portal system starts developing at the seventh week and is completed at around the 20th week of gestation. The body of the sphenoid bone and the sella turcica arise from the fusion of hypophyseal cartilage plates on either side of the developing pi- tuitary. The sella is well formed by the seventh week and matures by endochondral ossification. The pituitary measures around 13  mm transversely, 9  mm anteroposteriorly, and 6  mm vertically. It weighs approximately 100 mg. It increases during pregnancy to almost twice its normal size, and decreases in older people. The gland is centrally located at the base of the brain in the sella turcica within the sphenoid bone. It is attached to the hypothalamus by the pituitary stalk and a fine vascular network. The gland lacks leptomeninges. The sella turcica is lined by periosteal dura mater; the dura properly covers

SECTION 13  Endocrine disorders 2260 the lateral aspects of the cavernous sinuses and constitutes the sellar diaphragm. The cavernous sinuses are on either side of the sella, lat- eral and superior to the sphenoid sinuses, and contain important neurovascular structures including the cavernous segments of the internal carotid arteries and the cranial nerves III, IV, V, and VI. The optic chiasm is located superiorly and is separated from the pituitary by the suprasellar cistern and the sellar diaphragm (Figs. 13.2.1.1 and 13.2.1.2). The anterior lobe comprises nearly 80% of the gland and includes the pars distalis, pars intermedia, and pars tuberalis. Staining char- acteristics divide the pars distalis into a central ‘mucoid wedge’ and two ‘lateral wings’. On light microscopy the cells of the anterior lobe show variation in size, shape, and histochemical staining features. They are organized in nests and cords, and are separated by a com- plex capillary network. The pars intermedia is poorly developed in humans and lies between the pars distalis and the posterior pituitary. Large numbers of cells in the central zone are basophilic and pro- duce adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), follicle-​stimulating hormone (FSH), and thyrotropic hor- mone (TSH). Most of the cells in the lateral wings are acidophilic and produce growth hormone (GH) and, less frequently, prolactin (PRL) (Table 13.2.1.1). The pars tuberalis is an extension of the anterior lobe along the pituitary stalk. It is formed by normal acini of pituitary cells dis- tributed around surface portal vessels. The anterior lobe also in- cludes follicular cells, derived from secretory cells and constituting follicles within the gland, and folliculostellate cells (less than 5% of the adenohypophyseal cells), which have a physiological role that is not clear. The anterior pituitary receives most of its blood supply from the hypothalamo-​hypophyseal portal system (primary plexus, long portal venous system, and secondary plexus), which origin- ates from the capillary plexus of the median eminence and su- perior stalk derived from the terminal ramifications of the superior and inferior hypophyseal arteries. This system carries blood and hypophysiotropic hormones down to the stalk. The remainder of (a) Lamina terminalis Optic chiasm Sphenoid sinus Pituitary stalk with upper capillary loop of portal vessels Anterior pituitary Posterior pituitary Marrow in sphenoid bone Third ventricle Pineal gland Mamillary body Median eminence of hypothalamus Fourth ventricle Pons Medulla (b) Fig. 13.2.1.1  Sagittal MRI scan of normal pituitary gland and an anatomical line drawing. TRH Hypothalamus Somatostatin Pituitary − + T4 T3 TSH T3T4 Thyroid − − + Fig. 13.2.1.2  Diagram of the hypothalamo-​pituitary–​thyroid axis showing negative feedback loops. TRH, thyrotropin-​releasing hormone; TSH, thyroid-​stimulating hormone. Table 13.2.1.1  Hormone-​producing cells in anterior pituitary gland Type of cell Approximate percentage of adenohypophyseal cells Distribution Somatotrophs or
GH cells 50% Greatest density in lateral wings Lactotrophs or PRL cells 20% Mainly in posterior portions of lateral wings Corticotrophs or
ACTH cells 15–​20% Mainly middle and posterior portions of mucoid edge Gonadotrophs or FSH and LH cells (produce FSH and LH in isolation or by the same cell) 10% Evenly distributed throughout anterior lobe Thyrotropes or TSH cells 5% Mainly in anterior part of mucoid edge ACTH, adrenocorticotropic hormone; GH, growth hormone; FSH, follicle-​stimulating hormone; LH, luteinizing hormone; PRL, prolactin; TSH, thyrotropic hormone.

13.2.1  Disorders of the anterior pituitary gland 2261 the blood supply is through the pituitary capsular vessels originating from the superior hypophyseal arteries. The venous drainage from the anterior pituitary is through the cavernous sinuses into the pe- trosal sinuses and the internal jugular veins. The anterior lobe has no direct innervation, apart from a few sym- pathetic nerve fibres spreading to the anterior lobe along blood ves- sels. The hypothalamic regulation is exerted via the neurohormonal link with the hypothalamic regulatory peptides reaching the pitu- itary via the portal vessels. General physiology The secretion of the anterior pituitary hormones is under elegant regulation exerted by hypothalamic peptides and, with the exception of prolactin, by the negative feedback (at both the hypothalamic and pituitary level) of hormones from the target glands (Fig. 13.2.1.3). The hypothalamic peptides are secreted in the median eminence and are transferred to the anterior pituitary gland via the hypothalamic–​ pituitary portal system. They integrate environmental and neural in- formation and bind to specific high affinity cell membrane receptors of the particular pituitary cell type. Failure of the target gland results in decreased negative feedback and increased hypothalamic and pi- tuitary secretion. Primary overactivity of the target gland results in increased negative feedback and decreased hypothalamic and pitu- itary secretion. Additional ‘short-​loop’ feedback, in which pituitary hormones affect the secretory activity of the hypothalamus, is also implicated in the network contributing to the meaningful function of the pituitary gland. Finally, the anterior pituitary synthesizes sev- eral peptides, growth factors, and cytokines that play an important part in autocrine and/​or paracrine control of pituitary secretion and/​or cell proliferation. Clinical features of pituitary disease The clinical features of pituitary disease, mostly associated with a space-​occupying lesion, may result from local mass effects and/​or pituitary hormone deficits or hypersecretion. The local mass effects depend on the size of the tumour and its anatomical position. Headache is usually the consequence of dural stretching. It can be variable (occipital, retro-​orbital, bitemporal) and is often non​specific. The neuro-​ophthalmological effects in- clude visual field defects (usually bitemporal hemianopia or upper temporal quadrantanopia or any unilateral or bilateral visual field defect) from compression of the optic chiasm (Fig. 13.2.1.4) and squint, ptosis, or papillary dilatation from ocular nerve palsies caused by lateral tumour extension. Compression of the first or second branch of the trigeminal nerve may rarely result in facial pain. Very large pituitary tumours obstructing the fourth ventricle or the foramen of Monro cause hydrocephalus and expansion of the lateral ventricles. Inferior invasion and erosion of the sellar floor may result in recurrent sinusitis, cerebrovascular fluid rhinorrhoea, and recur- rent meningitis. Extension into the temporal lobe may rarely be as- sociated with temporal lobe epilepsy and to the cerebral peduncles with motor and/​or sensory disturbances. Superior expansion to the hypothalamus may be associated with hypothalamic dysfunction Central input Posterior pituitary Target glands Peripheral hormones ADH Oxytocin LH/FSH Anterior pituitary Hypothalamus Negative feedback PRL TSH ACTH GH Releasing hormones and inhibiting factors Fig. 13.2.1.3  Regulation of the hypothalamic–​pituitary–​peripheral function. The anterior pituitary produces GH, LH/​FSH, ACTH, TSH, and PRL. The secretion of these hormones is regulated by hypothalamic-​ releasing and hypothalamic-​inhibiting factors and by negative feedback inhibition of their peripheral hormones. Reprinted from Schneider HJ, et al. (2007). Hypopituitarism. Lancet, 369, 1461–​70, © (2007), with permission from Elsevier. Temporal visual field Nasal visual field Temporal retinal fibres Nasal retinal fibres compressed by tumour Optic nerve Optic chiasm X X Fig. 13.2.1.4  Neuro-​ophthalmological pathways and the classical bitemporal hemianopia that results from compression of the central optic chiasm by a pituitary tumour. However, any degree of unilateral or bilateral visual deficit can occur depending on the anatomical site of the lesion.

SECTION 13  Endocrine disorders 2262 including disorders of appetite, thirst, temperature regulation, and consciousness. The pituitary hormone deficits attributed to a pituitary tumour tend to occur in a specific order, with GH and gonadotropins af- fected first, followed later by ACTH and TSH. Diabetes insipidus is almost never a presenting feature of pituitary adenomas. The clinical manifestations of hypopituitarism are presented separately for each anterior pituitary hormone. In case of a functioning pituitary adenoma, the clinical manifest- ations depend on the type or types of anterior pituitary hormone(s) hypersecreted and are also presented separately for each anterior pi- tuitary hormone. Pituitary assessment strategy The investigation of suspected anterior pituitary disease includes testing for hormonal hyperfunction or hypofunction, radiological assessment, and neuro-​ophthalmological evaluation. Testing of pituitary function The optimum methods for testing anterior pituitary function and the interpretation of the results are the subject of continuing de- bate. The diagnostic evaluation of pituitary hypofunction has many complementary limbs. First, it is necessary to demonstrate target organ hormonal insufficiency. Paired testing of both hormones in the pituitary–​target organ feedback loop (e.g. serum testosterone and gonadotropins), sometimes in combination with provocative testing, will prove that target organ failure is consequent upon lack of stimulation by the relevant pituitary tropic hormone. Additional tests may be necessary to clarify whether the pituitary itself is at fault or whether the pituitary failure is secondary to understimulation by the hypothalamus. Basal pituitary function tests Measurements of basal levels of pituitary hormones with target gland hormone secretion are usually sufficient for cases of pituitary dysfunction involving TSH (thyroxine), FSH/​LH (testosterone or oestradiol), and PRL. When interpreting basal measurements of pituitary hormones, several factors need to be taken into account: • With the exception of PRL, the interpretation of the anterior pitu- itary hormonal levels should be done in relation to the level of the target hormone. • The pulsatile secretion of the anterior pituitary hormones may make a single, random blood sample not representative of the total secretion (e.g. GH). • Specific factors including time of day, stress, fed, or fasting, asleep or awake, and stage of pubertal development may influence the results. Currently, the modern chemiluminescent assays and mass spec- trometry methods are used for measurement of hormone concentra- tions; these methods have the advantages of increased automation and improved sensitivity and specificity. GH The marked pulsatile secretion of GH results in values in a normal subject ranging from undetectable to more than 80 mU/​litre. Furthermore, the GH secretion is influenced by several factors including nutritional status and stress. As a result of these variables, random levels are of limited value in clinical practice and dynamic endocrine tests are usually needed. FSH/​LH Serum FSH and LH are secreted in a pulsatile manner. However, it is rare for tests other than basal measurements of gonadotropin hormones and sex-​steroid levels to be required for the evaluation of the pituitary–​gonadal axis. The gonadotropins need to be in- terpreted taking into account the simultaneous levels of the target gland hormone, as well as the clinical picture of the patient. Thus, in men, a low serum testosterone in conjunction with low or ‘inappro- priately normal’ gonadotropins suggests hypogonadotropic hypo- gonadism. In women, the interpretation is more complex because of the significant changes in the levels of the gonadotropins during the various phases of the menstrual cycle. A  normal menstrual cycle with normal luteal phase serum progesterone (days 18–​25 of the cycle) makes gonadotropin deficiency very unlikely. In cases of amenorrhoea, the measurements of gonadotropins, PRL, oestra- diol, and human chorionic gonadotropin (possible pregnancy) are needed. ACTH The ACTH molecule undergoes rapid proteolytic degradation and, therefore, blood samples must be collected in a cold syringe, placed in an ethylenediaminetetraacetic acid (EDTA) tube at 4°C, and immediately frozen. ACTH secretion is pulsatile with a circa- dian rhythm and it increases during stress. As a result, the inter- pretation of the measurements should take into account the time of sample collection, whether the sample was taken from an indwelling cannula in place for at least 30 min, and whether the patient was stressed. Simultaneous measurement of plasma cortisol is important to check the appropriateness of the ACTH levels. PRL PRL is secreted in a pulsatile fashion and also shows an increase in the early morning hours. Stress may cause mild elevations of the hormone and, therefore, the stress of venepuncture should be taken into account when assessing the results. TSH The new ultrasensitive assays for TSH have made dynamic testing unnecessary and random sampling therefore provides meaningful information. Dynamic endocrine tests In general, the more dynamic the physiological system in health, the more likely will be the need for a dynamic test to investigate its possible malfunction in disease. The provocative tests include those that stimulate hormone release indirectly (e.g. insulin tolerance test) and those that stimulate hormone release directly by injecting pharmacological doses of synthetically manufactured peptides (e.g. short Synacthen test). Currently, the combined anterior pituitary function test with the administration of LH-​releasing hormone (LHRH) and thyrotropin-​releasing hormone (TRH) is not used in clinical practice, as basal hormone measurements provide the ne- cessary diagnostic information. Thus, only disorders of ACTH and

13.2.1  Disorders of the anterior pituitary gland 2263 GH secretion need dynamic endocrine testing with stimulation or suppression tests, according the presenting picture. The most commonly used tests in clinical practice are described next. Insulin tolerance test This test is considered the gold standard for assessing the integrity of the ACTH–​cortisol axis, as well as the GH reserve. The hypogly- caemia induced by the intravenous injection of insulin is a powerful stimulus, which in the intact pituitary and hypothalamus induces ACTH and GH release, as well as a rise, therefore, in the serum cor- tisol levels. It has been proposed that the peak cortisol levels of pa- tients undergoing major surgery are comparable to those achieved during the insulin-​induced hypoglycaemia. The test should be undertaken only under close supervision at skilled centres. The pa- tient should be fasted from midnight and the test started at 9.00 a.m. At 0 min, 0.1 to 0.15 IU/​kg (or 0.3 IU/​kg for those with acromegaly, Cushing’s syndrome, or other conditions with insulin resistance) of soluble human insulin is injected intravenously and blood is drawn at times 0, 30, 45, 60, 90, and 120 min. During the procedure, pulse rate, blood pressure, and manifestations of hypoglycaemia should be recorded. Blood glucose must fall to less than 2.2 mmol/​litre (to provide an adequate stimulus) in order to interpret the cortisol and GH levels. A normal cortisol response is a rise to 500–​550 nmol/​ litre or above (depending on the hormone assay). Severe GH defi- ciency in adults is diagnosed if the peak GH is less than 3 μg/​litre. Contraindications include ischaemic heart disease, epilepsy, or unexplained blackouts, severe long-​standing hypoadrenalism, un- treated hypothyroidism, and glycogen storage disease. Many phys- icians are uncomfortable with the use of this test in older people. Short Synacthen test This test has been advocated as an alternative to the insulin tolerance test for assessing the ACTH reserve. The rationale for its use is that chronic underexposure of the adrenal glands to ACTH (following prolonged corticosteroid therapy or due to hypothalamic–​pituitary disease) will result in a blunted cortisol response to exogenously administered ACTH. It involves the injection of a pharmacological dose (250 µg) of ACTH with measurement of the cortisol response 30 min later. The correlation between cortisol levels 30 min after the injection of Synacthen and the peak cortisol achieved during the in- sulin tolerance test is excellent; stimulated cortisol concentrations of 500–​550 nmol/​litre or less (depending on assay) suggest ACTH de- ficiency. This test does not differentiate primary from secondary ad- renal insufficiency. It requires no specialist staff and the only reported side effect is allergy in patients with atopy. It cannot be used in cases of acute hypopituitarism as it takes at least 2 weeks for the adrenal zona fasciculata to involute following withdrawal of ACTH stimu- lation. In recent years, much interest has arisen in the use of a lower dose of ACTH (1 µg), as the injected bolus of 250 µg is considered supraphysiological; this has not gained widespread acceptance. Glucagon stimulation test The glucagon stimulation test is used as an alternative to the insulin tolerance test for assessment of the ACTH/​cortisol and GH reserve. The subcutaneous injection of glucagon causes a transient rise in plasma glucose and, during the subsequent fall in glucose, ACTH and GH are released. The test involves the administration of 1 mg (or 1.5 mg if body weight >90 kg) glucagon subcutaneously with blood sampling for cortisol, GH, and glucose at times 0, 90, 120, 150, 180, and 210 min. The contraindications for this test include phaeo- chromocytoma or insulinoma, glycogen storage disease, and severe hypocortisolaemia. The interpretation of results relies on criteria established for the insulin tolerance test. The injection may cause nausea, abdominal pain, and vomiting. Glucagon is a less powerful stimulus to ACTH release and false-​negative results may be seen in up to 20% of patients. In some false-​negative results, no rise in the blood glucose is achieved after the glucagon injection. Growth hormone-​releasing hormone (GHRH) plus arginine test The GHRH plus arginine test is used as an alternative to the insulin tolerance or glucagon test for assessment of the GH reserve. The protocol involves intravenous infusion of GHRH 1 µg/​kg (maximum dose 100 µg) followed by arginine infusion 0.5 g/​kg (maximum 35 g) over 30 minutes. With blood sampling for GH at times 0, 30, 45, 60, 75, 90, 105, and 120 min. The cut-​offs for GH response are BMI-​ related and false normal GH response may be seen if GH deficiency is attributed to hypothalamic damage (e.g. following irradiation). Oral glucose tolerance test The evaluation of GH hypersecretion requires a suppression test. Increased blood glucose levels inhibit GH secretion and the admin- istration of oral glucose is used in suspected acromegaly. The test is performed at 9.00 a.m. with the patient fasted from midnight. Blood samples are drawn for measurement of glucose and GH at times 0, 30, 60, 90, and 120 min. Immediately after the first blood sample, 75 g of glucose are dissolved in water and given to the patient. In normal individuals serum GH should reach undetectable levels. Failure of suppression or a paradoxical rise in GH suggest acro- megaly. False-​positive results may be seen in uncontrolled diabetes mellitus, obesity, liver disease, renal insufficiency, malnutrition, or anorexia. During late adolescence, when GH secretion is maximal, GH may also fail to be suppressed. Radiological assessment Currently, the imaging modality of choice for patients with suspected pituitary pathology is MRI. CT is an acceptable alternative if MRI is contraindicated. The advantages of MRI are direct multiplanar scanning, lack of ionizing radiation, and good anatomical tissue dis- crimination without the need for pharmaceutical contrast agents. The evaluation of the pituitary and the hypothalamus is optimal in sagittal and coronal planes. The only disadvantage of MRI (apart from the cost) is its relative insensitivity to pathological calcifica- tion and lack of signal from corticated bone. CT or even plain film radiography may be required to demonstrate pathological calcifi- cation. The structures of the sellar region are best visualized using T1-​weighted sequences, which produce images with dark cerebro- spinal fluid, grey brain and pituitary, and white fat. Corticated bone returns low signal and appears dark, but bone marrow fat returns high signal and appears white. The nuclei of the hypothalamus cannot be distinguished, but if phospholipid vesicles are present in the neuro- hypophysis they are apparent as high signal areas. The need for routine intravenous administration of paramagnetic agents is controversial (however, it increases the pick-​up rate of pituitary microadenomas). These agents do not cross the blood–​brain barrier and, therefore, the pituitary gland and stalk enhance and appear whiter on T1-​weighted

SECTION 13  Endocrine disorders 2264 images. The hypothalamus and the optic chiasm do not enhance if the blood–​brain barrier is intact. Blood vessels, meninges, and mucosa of the paranasal sinuses will enhance. Dynamic MRI has been used to study the timing of intravenously administered gadolinium uptake by the hypophysis (see Figs. 13.2.1.1, 13.2.1.2). Apart from its complementary role to MRI in detecting patho- logical calcification, CT scanning is the imaging modality of choice for patients who are unable to undergo MRI (extreme claustrophobia, presence of cardiac pacemakers, or other implants such as intracra- nial aneurysm clips or traumatic metallic fragments). Multislice spiral CT scanners produce images of sufficient quality to demon- strate sellar anatomy on unenhanced images. Intravenous injection of iodinated contrast media is used to improve tissue contrast and it is taken up by the hypophysis in the same way as gadolinium. Thus, macroadenomas or craniopharyngiomas enhance and are better de- lineated, but demonstration of microadenomas within a morpho- logically normal pituitary depends on differential uptake rates. Neuro-​ophthalmological evaluation The neuro-​ophthalmological evaluation in suspected pituitary pathology includes assessment of the visual acuity (with the use of Snellen charts), assessment of the visual fields (by confrontation using a red pin and formally by the Goldmann perimetry test or by visual evoked responses), and fundoscopy (to check for optic at- rophy, retinal vein engorgement, or papilloedema). Vision is usually lost gradually, except in cases of pituitary apoplexy when it may be sudden. Successful decompression of the optic nerves and chiasm achieved surgically or by medical therapy results in marked im- provement of visual function; this becomes apparent within hours or days of surgery continuing thereafter for 6 months or more. The chance of complete reversal of any visual field defects is higher if the duration of compression of the optic chiasm is short (<1 year). Pituitary surgery Currently, the main aims of pituitary surgery are to cure any endo- crine excess and to reverse the pressure effects (particularly the visual compromise and the pituitary dysfunction) without causing mor- bidity or mortality. For all pituitary tumours, except prolactinomas, surgery is the treatment of choice. It is also indicated when other therapies have not been successful or in case of tumour recurrence. The trans-​sphenoidal approach (via the translabial or transethmoidal route) with the microscopic or endoscopic technique) is most com- monly used and, compared with the transfrontal route, it is less time consuming, less traumatic, and associated with less morbidity. The trans-​sphenoidal approach is less successful for large tumours with significant invasion to neighbouring structures. In pituitary aden- omas the tumour is usually soft and white and can be easily removed by curettes and suction. Other tumours may also be recognized during surgery, including meningiomas or craniopharyngiomas. Complications include cerebrospinal fluid leakage, impaired anterior pituitary function, diabetes insipidus (most commonly temporary), the syndrome of inappropriate secretion of antidiuretic hormone (usually transient), visual deterioration, meningitis, headache (at- tributed to haematoma in the air sinuses, meningitis, hyponatraemia, or abscess), vascular damage, epilepsy, frontal lobe damage, hypo- thalamic damage, and intracranial oedema/​haemorrhage. The success and complication rates are mainly associated with the size and extensions of the tumour and any previous therapy, as well as the experience and expertise of the neurosurgeon. Pituitary radiotherapy After the improvement of surgical techniques and the availability of medical therapy for prolactinomas, pituitary irradiation is no longer prescribed routinely for the management of pituitary tumours. It is mainly reserved for patients who are not fit to undergo surgery, for those who have had an unsuccessful operation, or for those showing tumour recurrence. Conventional irradiation uses a linear accelerator and is admin- istered in a fractionated manner to a total dose of 4500 cGy in daily doses not exceeding 180 cGy over a 5-​ to 6-​week period. Hormonal hypersecretion shows a rapid fall within the first 2 years with the de- cline continuing for up to 20 years. Radiotherapy is also considered an effective modality for decreasing the recurrence rates of pituitary tumours. With modern technology and careful planning, the use of multiple fixed fields from linear accelerators, and careful fractionation, the risk of radiation-​induced late complications is small. These include hypopituitarism and visual impairment; oncogenesis and cognitive impairment occur infrequently. With increasing time after irradi- ation, anterior pituitary function assessment will show compromised reserve in gonadotropins and GH, followed later by ACTH and TSH. It has been reported that by 10 years after radiotherapy, 47% of pa- tients were hypogonadal, 30% were hypoadrenal, and 16% were hypo- thyroid. Therefore, any patient who has received pituitary irradiation needs lifelong follow-​up aiming for the early diagnosis of hormonal deficits. Notably, the total dose and the dose per daily fraction influ- ence the risk of hypopituitarism. Optic nerve/​chiasmal damage can be avoided by keeping the daily fractionated dose to less than 200 cGy. From the available data, the incidence of late carcinogenesis cannot be estimated with certainty, but it is unlikely to be more than 1 to 2%. Recently introduced techniques include intensity modulated ra- diation therapy, proton beam radiation therapy, stereotactic radio- therapy, and stereotactic radiosurgery (producing a highly localized deposition of radiation on the target, at the perimeter of which there is a fast ‘fall off’ of the isodoses, thereby sparing the surrounding normal tissue from high doses of irradiation). Following stereo- tactic radiosurgery, there is a faster early reduction of the excessive secretory hormone product; hypopituitarism can also occur. The selection of the optimal radiation treatment modality should be based on the size and extent of the adenoma, the postoperative endocrine situation for secretory tumours, and the pituitary hor- mone reserve. For small, discrete tumours located in the fossa, radiosurgery seems to be a good option. This technique is also useful for patients with recurrence who have already received conventional radiotherapy. Anterior pituitary hormones GH Human GH is a single chain protein of 191 amino acids containing two disulphide bonds. It is produced by the somatotroph cells and it

13.2.1  Disorders of the anterior pituitary gland 2265 has several similarities to prolactin and the placental lactogen mol- ecule. Nearly 75% of the hormone circulates as a 22-​kDa protein, 5 to 10% as a smaller 20-​kDa isoform, and the remainder consists of glycosylated and sulphated isoforms. GH is secreted in an episodic manner (pulses occurring every 3–​4 h) that is modified by age and sex. The most profound discharge occurs during deep sleep (phases III and IV). Its secretion is under complex neuroregulatory control. The hypothalamic participation is exerted through GH-​releasing hormone (GHRH) and somatostatin, which reach the pituitary gland via the hypothalamo-​pituitary portal vessels. GHRH stimulates both synthesis and secretion of GH, whereas somatostatin inhibits the re- lease but not the synthesis of the hormone. Ghrelin, the endogenous ligand of the GH secretagogue receptor localized mainly in the stomach, may also be implicated in the control of GH secretion. The somatotroph cell is also regulated by negative feedback at the pitu- itary level by the circulating insulin-​like growth factor 1 (IGF-​1) and by ‘short-​loop’ feedback on the hypothalamus by GH. The secretion of GH is greater in women and it shows a decrease with age in both sexes. Amino acids (e.g. arginine, leucine), sleep, exercise, stress, a fall in blood glucose, poorly controlled diabetes mellitus type 1, hep- atic cirrhosis, anorexia nervosa, central α-​adrenergic agonists, and acetylcholine agonists enhance GH secretion. Oestrogens increase the pulse amplitude of GH. β-​Antagonists augment the stimulatory effect of other stimuli. Agents lowering acetylcholine tone suppress GH release. Hyperglycaemia in normal subjects acutely suppresses GH secretion. Obesity is associated with decreased GH release and emotional deprivation may inhibit GH secretion in children. GH levels are decreased in pregnancy due to the negative feedback by the GH variant secreted by the placenta. GH exerts its actions through a specific 638-​amino acid receptor belonging to the class I haematopoietin or cytokine/​GH/​PRL re- ceptor superfamily. It shows a wide distribution including muscle, adipose and immune tissues, liver, mammary gland, bones, kidneys, brain, and embryonic stem cells. It is a single membrane-​spanning type I glycoprotein with an extracellular ligand-​binding domain, a single 24-​amino acid hydrophobic transmembrane region, and an intracellular domain. The signal transduction requires dimerization of the receptor, which is facilitated by the GH binding. The down- stream signalling pathways resulting in GH actions include, but are probably not limited to, the signal transducer and activator of tran- scription, mitogen-​activated-​kinase (MAP), and phosphoinositide 3-​kinase (PI3) pathways. Abnormalities in the GH receptor occur in Laron’s dwarfism, which is characterized by failure of growth, high levels of GH, and low IGF-​1. Up to 60% of the circulating serum GH is bound to the GH-​ binding protein, which corresponds to part of the extracellular do- main of the GH receptor. The binding reduces the clearance rate of the hormone and thus prolongs its half-​life. The effects of GH are mediated either directly or mainly indir- ectly via the production of IGF-​1 by the liver, bones, and other types of tissues. IGF-​1 is a polypeptide of 70 amino acids and acts in an endocrine, paracrine, or autocrine fashion. It circulates bound to a group of binding proteins; IGF binding protein-​3 is the main carrier of IGF-​1 and plays an important role in regulating its bio- activity. IGF-​1 induces cell proliferation and inhibits apoptosis. Its levels are determined by sex and genetic factors, are highest during late adolescence, and decline throughout adulthood. The produc- tion of IGF-​1 is suppressed in malnourished patients, as well as in those with liver disease, hypothyroidism, or poorly controlled dia- betes. Although IGF-​1 levels usually reflect the integrated secretory activity of GH, subtly elevated GH levels may not uniformly induce high IGF-​1. The main actions of GH are the promotion of skeletal growth, mainly of long bones, and the regulation of several metabolic ac- tions. In the muscles GH promotes the incorporation of amino acids and protein synthesis, and in the adipose tissue it promotes free fatty acid release. Disorders of GH secretion GH deficiency The manifestations of GH deficiency are shown in Table 13.2.1.2. Given its pulsatile secretion, the measurements of random GH levels do not distinguish between normal and compromised GH secretion; multiple sampling for GH would be ideal but in clinical practice this is not a practical procedure. Therefore, the diagnosis of GH deficiency requires stimulation testing, unless all other pituitary hormones are deficient and the IGF-​1 is low (in these patients the likelihood of GH deficiency is 99%). The biochemical criteria for the diagnosis of adult GH deficiency are complicated by the lack of normative data that are age-​adjusted and sex-​adjusted, by the assay variability, and by the stimulus used. Among the available tests the insulin tolerance test is considered the gold standard. Severe GH de- ficiency in adults is diagnosed if the peak GH is less than 3 μg/​litre. In children the secretory capacity of GH is higher and a cut-​off of 10 μg/​litre is used. The GHRH plus arginine test has been shown to re- liably detect severe GH deficiency in a lean adult population when a cut-​off of 9 μg/​litre is used. The response to this test declines greatly with increasing body mass index and the above cut-​off in obese pa- tients is associated with a high proportion of false-​positive results. Furthermore, as GHRH directly stimulates the pituitary, it can give a falsely normal GH response in patients with GH deficiency of hypo- thalamic origin. Other alternative tests, but less commonly used in clinical practice, include the GHRH plus GH-​releasing peptide-​ 6 test, the glucagon test, the arginine test, the l-​dopa test, and the clonidine test. Normal serum levels of IGF-​1 do not exclude a diag- nosis of GH deficiency. The main targets of GH therapy in children with GH deficiency are to normalize height during childhood and to reach normal adult height. The benefits of treatment with GH among adult patients have been reported in several domains: body composition (decrease in total body fat content, increase in muscle mass), exercise capacity, bone health (increase in bone mineral density with greater effects Table 13.2.1.2  Manifestations of GH deficiency Children Adults Growth failure Relative increase in fat mass Relative decrease in muscle mass Increased serum low-​density lipoprotein cholesterol Decreased bone mineral density Increased risk of cardiovascular disease Increased inflammatory cardiovascular risk markers Decreased energy and quality of life

SECTION 13  Endocrine disorders 2266 at vertebral sites), cardiovascular risk factors (decrease in blood pressure, reduction of C-​reactive protein, increase in high-​density lipoprotein, and decrease in low-​density lipoprotein and total chol- esterol), and quality of life. In adults, the dosing plans have evolved from weight-​based dosing to individualized dose-​titration strategies with the goals being an appropriate clinical response, an avoidance of side effects, and an IGF-​1 level in the middle of the age-​adjusted reference range. In general, women require higher doses of GH to achieve the same IGF-​1 response (much higher GH doses were also needed to achieve the same IGF-​1 levels in women receiving oral oestrogen replacement, but this does not apply when oestro- gens are offered as patches). Furthermore, as GH secretion normally decreases with age, older patients require lower doses of GH. The duration of GH replacement therapy in adults is unclear; if bene- fits are apparent, there is no particular reason to stop treatment. On the other hand, if there are no benefits following around 1 year of treatment, discontinuing GH therapy may be appropriate. Most adverse effects are dose related and are attributed to fluid reten- tion (paraesthesias, joint stiffness, peripheral oedema, arthralgia, and myalgia). In children, there is a risk of slipped capital femoral epiphysis. With the current dosing regimens, there may be a slight excess risk of diabetes mellitus. Other complications of GH therapy include retinopathy, benign intracranial hypertension, and gynae- comastia. GH replacement may cause a decrease of serum free thy- roxine levels (perhaps due to increased deiodination of thyroxine) and of serum cortisol levels (revealing central hypoadrenalism that had been masked, probably due to enhanced conversion of cortisone to cortisol during the GH-​deficient state). GH treatment is contra- indicated in the presence of an active malignancy. Acromegaly Acromegaly is the syndrome resulting from GH hypersecretion. Its incidence is estimated to be approximately three cases per 1  million persons per year, and its prevalence is about 60 per million. More than 90% of patients with acromegaly have a be- nign monoclonal GH-​secreting pituitary adenoma surrounded by non​hyperplastic pituitary tissue. Cosecretion of PRL has been described in about 25% of GH-​secreting adenomas. More than 70% of somatotroph tumours are macroadenomas at diagnosis. Younger patients usually present with more rapidly growing tu- mours. Rarely, acromegaly is associated with familial occurrence [including multiple endocrine neoplasia type 1 or 4, familial isolated pituitary adenoma (related in several cases with muta- tions in the aryl hydrocarbon receptor-​interacting protein (AIP) gene), and Carney’s syndrome]. X-​linked acrogigantism (X-​LAG) is a new syndrome of pituitary early-​onset gigantism, caused by microduplications on chromosome Xq26.3, encompassing the gene GPR101. Acromegaly may also be seen in the McCune-​ Albright syndrome. Excess production of GH-​releasing hormone (as in central hypothalamic tumours, usually gangliocytomas, and peripheral neuroendocrine tumours, e.g. of the lung) can result in somatotroph hyperplasia and acromegaly. Clinical features  The clinical features of acromegaly are shown in Box 13.2.1.1. Disease features develop insidiously, and the diagnosis may often take 10 years from presentation. GH-​secreting adenomas arising before the closure of epiphyseal bone are associated with ac- celerated growth and gigantism. Box 13.2.1.1  Clinical features of acromegaly Local tumour effects • Pituitary enlargement • Visual field defects • Cranial nerve palsy • Headache Somatic systems • Acral enlargement including thickness of soft tissue of hands and feet Musculoskeletal system • Gigantism • Prognathism • Jaw malocclusion • Arthralgias and arthritis • Carpal tunnel syndrome • Acroparesthaesia • Proximal myopathy • Hypertrophy of frontal bones Skin and gastrointestinal system • Hyperhidrosis • Oily texture • Skin tags • Colon polyps Cardiovascular system • Left ventricular hypertrophy • Asymmetric septal hypertrophy • Cardiomyopathy • Hypertension • Congestive heart failure Pulmonary system • Sleep disturbances • Sleep apnoea (central and obstructive) • Narcolepsy Visceromegaly • Tongue • Thyroid gland • Salivary glands • Liver • Spleen • Kidney • Prostate Endocrine and metabolic systems Reproduction • Menstrual abnormalities • Galactorrhoea • Decreased libido, impotence, low levels of sex hormone-​binding globulin Multiple endocrine neoplasia type 1 • Hyperparathyroidism • Pancreatic islet cell tumours Carbohydrate • Impaired glucose tolerance • Insulin resistance and hyperinsulinaemia • Diabetes mellitus Lipid • Hypertriglyceridaemia

13.2.1  Disorders of the anterior pituitary gland 2267 Whether acromegaly is associated with an increased relative risk of cancer is controversial and has been extensively reviewed. Some studies have suggested increased risk of colon cancer in patients with acromegaly necessitating screening colonoscopy at diagnosis with follow-​up according to standard guidelines. Acromegaly is associated with increased mortality and factors contributing to this include the higher prevalence of hypertension, hyperglycaemia, or overt diabetes, cardiomyopathy, and sleep ap- noea in this population. In some studies, increased IGF-​1 levels are associated with higher mortality. However, GH levels seem to be more consistently independent predictors of mortality than are IGF-​1 levels. Diagnosis  The biochemical diagnosis is made by confirming ab- sence of suppression of GH in the oral glucose tolerance test and by increased serum IGF-​1 levels. With the use of most commercial assays, nadir GH levels of less than 1 µg/​litre during the oral glucose tolerance test rule out the diagnosis. With very sensitive GH assays this cut-​off is less than 0.4 µg/​litre. Treatment  The goals of treatment in patients with acromegaly include the improvement/​reversal of the manifestations and comorbidities related with the GH hypersecretion, the reduction of the mortality risk and restoration of the abnormal biochemistry. Further aims include the decrease or stabilization of the tumour size, improvement or preservation of the pituitary function, and prevention of recurrence. It should be noted that, with the currently available therapeutic options, normal GH secretion dynamics are only rarely achieved and it is, therefore, more appropriate to de- fine disease control. A biochemical target goal of an age normalized serum IGF-​I value suggests control of the acromegaly. Furthermore, a random GH less than 1 mg/​litre is another therapeutic goal and correlates with control of the disease. It has also been proposed that a normal IGF value and undetectable GH value are sufficient for indicating remission after surgery. Given the variability between Gh and IGF-​I assays, it is important to use the same assay in the same patient throughout his management. Management options include surgery, drugs, and radiotherapy. Trans-​sphenoidal surgery remains the treatment of choice for most patients. The success rate depends on the size and extensions of the tumour, the presurgical GH levels, as well as the experience and expertise of the neurosurgeon. Biochemical control has been reported for up to 80% of microadenomas and for up to 40% of macroadenomas. Tumours that have invaded the cavernous sinus cannot be completely removed surgically and the hypersecretion of GH almost invariably persists postoperatively in such patients. Although up to 10% of tumours recur, many recurrences prob- ably represent persistent growth of residual non​resectable tumour tissue. Medical treatment includes dopamine receptor agonists, som- atostatin receptor ligands, and GH receptor antagonists. Dopamine receptor agonists are less costly than other agents, but are only oc- casionally effective in selected patients. The doses required are usually much higher than in prolactinomas and biochemical con- trol has been reported in around 15% of the patients. Side effects include gastrointestinal upset, nasal congestion, fatigue, postural hypotension, and headache. Cardiac valve abnormalities occur with high doses of cabergoline used for patients with Parkinson’s dis- ease but have not been observed in most studies of patients with prolactinomas treated with conventional doses. Somatostatin re- ceptor ligands, such as octreotide and lanreotide, bind to somato- statin receptors resulting in suppression of GH secretion. They also act on the liver to block the synthesis of IGF-​1. Octreotide and lanreotide are selective for somatostatin receptors type 2 and 5, which are expressed in more than 90% of the GH-​secreting ad- enomas. Depot preparations—​long-​acting-​release octreotide and a long-​acting aqueous-​gel preparation of lanreotide—​allow for injec- tions every 14 to 28 days maintaining highly effective drug levels. IGF-​I normalization has been reported in 17–​35% of both drug-​ naive and postoperative patients. In 59% of patients, there is reduc- tion in tumour volume of more than 50% and tumour shrinkage usually correlates with hormonal control. Surgical debulking of macroadenomas not amenable to total resection enhances the effi- cacy of subsequent somatostatin analogue treatment. Somatostatin analogues are indicated following unsuccessful surgery and after ra- diation therapy, during the period when GH levels remain elevated. They can also be offered as primary treatment to patients with large extrasellar tumours who have no evidence of central compressive effects, those who are too frail to undergo surgery, and those who decline an operation. Transient diarrhoea, nausea, and abdominal discomfort may occur, but typically resolve within 8 to 10 weeks. Blood glucose levels may rise in some patients. Gallbladder sludge or asymptomatic gallstones develop within 18 months in up to 20% of patients and these conditions should be managed according to standard guidelines. Selective activation of other somatostatin re- ceptors by specific somatostatin receptor ligands results in additive suppression of GH; the ligand pasireotide (SOM230) suppresses levels of GH in patients with resistance to octreotide but is associated with hyperglycaemia in 57% of the patients. Lastly, pegvisomant, a pegylated GH receptor analogue showing enhanced activity for the GH receptor, also prevents the functional GH-​receptor signalling. As a result of this, it blocks the GH-​mediated IGF-​1 generation in nearly 90% of patients, although in a surveillance study IGF-​I was controlled in 63% of the patients (this may be attributed to com- pliance and inadequate dose titration). It is indicated for patients whose GH levels are inadequately controlled with other modalities or in those experiencing significant drug side effects. During treat- ment with pegvisomant, GH levels increase and IGF-​I is the bio- marker for monitoring the efficacy of treatment. Elevated hepatic aminotransferase levels have been reported requiring monitoring of the liver function tests monthly for the first 6 months of treatment and 6-​monthly thereafter with consideration of discontinuation of pegvisomant if the transaminases are greater than 3-​fold elevated. Mineral • Hypercalciuria, increased levels of 25-​hydroxyvitamin D3 • Urinary hydroxyproline Electrolyte • Low renin levels • Increased aldosterone levels Thyroid • Low thyroxine binding globulin levels • Goitre Source data from Melmed S (2006). Acromegaly. N Engl J Med, 355, 2558–​73, with permission. © 2006 Massachusetts Medical Society. All rights reserved.

SECTION 13  Endocrine disorders 2268 Tumour growth may occur in 3–​5% of patients, but it is not clear whether this is due to the tumour natural history or to reduced nega- tive feedback by the lower IGF-​1 levels. Probable tumour size should be monitored at 6-​month intervals to detect possible continued enlargement for the first year of treatment. If the tumour remains stable, yearly imaging is recommended. Combined treatment with somatostatin analogues and pegvisomant restores IGF-​1 concentrations to normal in over 90% of patients and in about 20% decreases tumour size, whereas pegvisomant (PEG-​V) monotherapy does not induce a clinically relevant decrease in the size of pituitary tumours. Transient elevations in serum transaminase ac- tivities are the main adverse effects of this combination treatment re- ported, in up to 27% of patients. Radiotherapy is generally offered for tumours that have recurred or persisted after surgery in patients with resistance to or intolerance of medical treatment. Twenty-​two per cent of patients achieved a level less than 2.5 ng/​ml (5 mU/​litre) by 2 years, 60% by 10 years, and 77% by 20 years. The time taken to achieve this depends on the pre-​ irradiation concentration of GH. IGF-​I concentrations normalize in 63% of patients by 10 years. In patients offered stereotactic radio- therapy and followed up to 15 years, remission rates of 10–​60% have been reported. Although the overall efficacy of stereotactic radio- therapy may be similar to conventional radiotherapy, time to remis- sion may be shorter with stereotactic radiotherapy. FSH/​LH The gonadotropins, TSH, and human chorionic gonadotropin β belong to the family of glycoprotein anterior pituitary hormones. They share a common α subunit and each has a unique β subunit conferring biological and immunological specificity. The LH β and FSH β subunits both have 115 amino acids and two carbohydrate side chains. A terminal sialic acid may be present on the carbohy- drate side chain of the FSH β subunit, thus decreasing its metabolic clearance. The regulation of FSH and LH secretion is exerted by the inte- gration of the gonadotropin-​releasing hormone (a hypothalamic decapeptide) signal and the (stimulatory and inhibitory) feedback effects of gonadal steroids and peptides. Gonadotropin-​releasing hormone interacts with the membrane receptor and regulates the synthesis and release of gonadotropins. The major feature of the pituitary–​gonadal axis is that its constituents exhibit a pulsatile pattern of hormonal release. The frequency and the amplitude of gonadotropin-​releasing hormone pulses are important in differen- tially regulating LH and FSH secretion. Gonadotropin concentra- tions are very low in children and the nocturnal augmentation of gonadotropin release marks the onset of sexual development. In women, LH levels rise slightly during the follicular phase, peaking at the time of the midcycle surge and then decline during the luteal phase. The FSH levels start rising during the late luteal phase, increase during the early follicular phase of the next cycle, and decline just before the midcycle FSH surge. During the luteal phase, the FSH levels show a decline and increase again before the next menses. In the follicular phase, most of the LH pulses are fol- lowed by a release of oestrogens from the ovary, whereas in the mid and late luteal phase the LH pulses induce progesterone se- cretion. Both oestradiol and progesterone inhibit the release of LH acting at the hypothalamic and pituitary level. However, in the fol- licular phase there is enhanced release of oestradiol, which acts in a stimulatory way and induces the large discharge of LH responsible for ovulation. During this surge, LH levels remain increased for 36 to 48 h, during which time ovulation occurs, oestradiol levels de- cline, and luteinization of the follicle results in increasing produc- tion of progesterone. The ovary exerts a negative feedback on FSH secretion mainly through the secretion of inhibin, a glycoprotein hormone synthesized in the granulosa cells of the ovarian follicle and counterbalanced by activin. In the late follicular phase, inhibin levels increase and, in combination with oestradiol, inhibit the syn- thesis and release of FSH, an inhibition that it is overcome at the preovulatory gonadotropin discharge. The hypothalamic control of FSH and LH secretion is very sensitive to environmental conditions, such as stress or changes to nutrition or energy homeostasis. Stress activates the corticotropin-​releasing hormone pathways, which may inhibit the gonadotropin-​releasing hormone neurons through opiate pathways. Reduction in the daily food intake leads to a reduc- tion in the gonadotropin-​releasing hormone secretion translated into a reduced and non​pulsatile secretion of FSH and LH into the circulation. The regulation of the gonadal axis is equally complex but more static in men. It is assumed that gonadotropin pulses in men follow the scarce pulses of gonadotropin-​releasing hormone and, in fact, are highly variable and of small amplitude. Testosterone exerts a negative feedback on LH secretion, and Sertoli cells secrete activin and inhibin in order to regulate FSH secretion. The gonadotropins are responsible for the gonadal sex-​steroid production by the Leydig cells of the testis and the ovarian follicles, secondary sexual development, maintenance of secondary sexual characteristics, and fertility. Disorders of FSH/​LH secretion FSH/​LH deficiency Gonadotropin deficiency presents with oligomenorrhoea/​amen- orrhoea, loss of libido, dyspareunia, hot flushes, and infertility in women, and with loss of libido, impaired sexual function, mood im- pairment, loss of facial, scrotal, and trunk hair, and decreased muscle bulk and energy in men. Hypogonadism in both sexes is associated with decreased bone mineral density. In children, gonadotropin de- ficiency causes delayed or arrested puberty. The diagnosis in women is based on oligomenorrhoea/​amenor- rhoea combined with low or ‘inappropriately normal’ FSH and LH levels. In postmenopausal women, the FSH and LH levels are in- appropriately low. In men, there is low serum testosterone (9.00 a.m. sample, as levels show considerable diurnal variation) with low or ‘inappropriately normal’ gonadotropins. The treatment consists of appropriate replacement therapy (un- opposed oestrogens for women who have undergone hysterectomy or combined oestrogen-​progestogen preparations for those with in- tact uterus to prevent endometrial hyperplasia). Oestrogens are avail- able in many forms with different potency (oral, transdermal, topical gels and lotions, intravaginal creams and tablets, vaginal rings). The choice of the oestrogen (and progestin) preparation needs to rely on the risk of adverse effects, cost, patient convenience, and pref- erence. The follow-​up of gonadal hormone replacement includes evaluating the effectiveness by symptom control and monitoring for oestrogen/​progestogen side effects. The patients require annual re- view to evaluate use and assess risk and benefit profile. Hormonal replacement therapy should be used until the mean age of natural

13.2.1  Disorders of the anterior pituitary gland 2269 menopause; further management decisions should rely on current recommendations for the general female population. For induc- tion of fertility, gonadotropins, or pulsatile gonadotropin-​releasing hormone (the latter only in hypothalamic dysfunction) are used. Serum androgen levels in women with hypopituitarism are low; the pros and cons of replacing androgens in such cases are under inves- tigation. In men, testosterone replacement using one of the avail- able preparations (gel, intramuscular injections) is suggested. The dose is adjusted to normal testosterone concentrations. Serum LH cannot be used to monitor the adequacy of therapy. Monitoring for prostate-​specific antigen, prostate size, and haematocrit (erythro- poietin is stimulated by testosterone) are recommended regularly. For the induction of fertility, gonadotropin therapy, or pulsatile gonadotropin-​releasing hormone (the latter only in hypothalamic dysfunction) are available options. Gonadotroph adenomas Gonadotropinomas are pituitary adenomas secreting intact LH and/​ or FSH. They are rare, as most tumours expressing gonadotropins secrete a subunit without causing biological effects (non​functioning pituitary adenomas). Both types of tumours are most commonly diagnosed in middle-​aged men and present with symptoms related to a pituitary mass. FSH-​secreting tumours cause testicular enlarge- ment in men and ovarian hyperstimulation syndrome in premeno- pausal women. Treatment includes surgical excision combined or not with radiotherapy. PRL PRL is released by the lactotroph cells of the adenohypophysis. It is composed of 199 amino acids and has three disulphide intramo- lecular bonds. Its molecular structure is similar to GH and to pla- cental lactogen with which it shares a common phylogenetic origin. PRL physiology differs from that of other anterior pituitary hor- mones in that its secretion is mainly under tonic inhibition (affecting both synthesis and release) by dopamine released from the hypo- thalamus. Hypothalamic stressors, such as the insulin tolerance test, are able to release PRL and exogenous administration of TRH re- leases PRL in addition to TSH, operating through specific lactotroph receptors. Oestrogens induce hyperplasia of the lactotroph cells and enhance PRL secretion. The increase in pituitary volume in preg- nant women may be in part due to the large oestrogenic production by the fetoplacental unit. In the third trimester, PRL concentrations may rise as high as 8000 mU/​litre. During pregnancy, the develop- ment of the synthetic and secretory potential of the breast appar- atus is under the influence of several hormones (oestrogen, insulin, cortisol, placental mammotrophic hormones). Before delivery, lac- tation is inhibited by high levels of oestrogen and progesterone. The rapid fall of these hormones after delivery allows PRL to initiate lac- tation. After delivery, maternal levels of PRL fall if there is no breast- feeding, but remain increased in response to suckling. Conditions associated with hyperprolactinaemia are shown in Box 13.2.1.2. PRL receptors have a wide distribution in the body. They are mostly found in the mammary gland and their activation is respon- sible for the initiation and maintenance of physiological lactation. In mammary tissue primed with oestrogens and progesterone, PRL induces the synthesis of milk proteins. The actions of PRL at other sites have not been clarified; it has been suggested that PRL is also involved in the immune functions. Disorders of PRL secretion PRL deficiency A clinical syndrome associated with PRL deficiency is not recog- nized and the only known manifestation of PRL deficiency is the inability to lactate following delivery. Prolactinomas Prolactinomas are pituitary adenomas expressing and secreting PRL. They are the most common pituitary adenomas with an estimated prevalence in the adult population of 44 per 100 000 people. Their frequency varies with age and sex; between the ages of 20 and 50 years they are most commonly diagnosed in women. In the paediatric/​ado- lescent age group, prolactinomas are rare but represent about half of all pituitary adenomas. Most of the prolactinomas are small, intrasellar tumours. Occasionally, they can be aggressive or locally invasive and cause compression of vital structures. Malignant prolactinomas that are resistant to treatment and disseminate inside and outside the central nervous system are very rare. Mixed GH-​secreting and PRL-​ secreting tumours are well recognized and are usually associated with acromegaly and hyperprolactinaemia. Occasionally, prolactinomas may be a component of multiple endocrine neoplasia1 (MEN1); they are the most common pituitary adenomas in this syndrome). Box 13.2.1.2  Causes of hyperprolactinaemia Physiological • Stress • Pregnancy • Lactation • Nipple stimulation/​suckling • Sexual intercourse • Sleep Drugs (commonly used in clinical practice) • Antipsychotics–​neuroleptics (e.g. phenothiazines, butyrophenones) • Antidepressants (e.g. tricyclic and tetracyclic antidepressants, mono- amine oxidase inhibitors, selective serotonin reuptake inhibitors) • Opiates • Cocaine • Antihypertensive medications (e.g. verapamil, methyldopa, reserpine) • Gastrointestinal medications (e.g. metoclopramide, domperidone) • Protease inhibitors? • Oestrogens Pathological • Primary hypothyroidism • Hypothalamic–​pituitary disease — Hypothalamic tumours — Granulomatous disease (sarcoidosis, tuberculosis, Langerhans cell histiocytosis) — Cranial irradiation — Pituitary stalk section (e.g. following surgery) — Prolactinoma — Mixed GH/​PRL-​secreting adenoma — Tumours causing stalk compression • Renal or hepatic failure • Polycystic ovarian syndrome • Chest wall stimulation (e.g. repeated breast self-​examination, following herpes zoster infection) • Ectopic secretion (e.g. bronchogenic carcinoma, hypernephroma)

SECTION 13  Endocrine disorders 2270 Presentation  The clinical features of a prolactinoma predominantly result from hyperprolactinaemia. These include galactorrhoea and primary (in children) or secondary hypogonadism (in men, impo- tence, infertility, and decreased libido; in women, oligomenorrhoea/​ amenorrhoea and infertility). Hyperprolactinaemia interrupts the pulsatile secretion of gonadotropin-​releasing hormone, in- hibits the release of LH and FSH, and directly impairs gonadal steroidogenesis. In the case of large tumours, symptoms related to pressure effects may also be present. Most prolactinomas in women are microadenomas. Men usually present with larger tumours and neurological manifestations probably due to the delayed recognition of symptoms associated with the high PRL levels. Postmenopausal women with hyperprolactinaemia are often recognized only when a large adenoma produces mass effects. Investigations  When evaluating a patient with persistently ele- vated serum PRL, secondary causes of hyperprolactinaemia should first be ruled out by a careful clinical history, physical examination, pregnancy test, routine biochemical analysis (to assess kidney and liver function), and TSH measurement. If the patient is on a medi- cation known to increase serum PRL, it is important to determine if the drug is indeed the cause by withdrawing it (if this can be done safely). When the drug cannot be stopped, the evaluation should include MRI of the sella to exclude a mass lesion. In the case of prolactinomas, serum prolactin levels usually parallel tumour size. PRL values between the upper limits of normal and 100 mg/​litre (2000 mU/​litre) may be due to psychoactive drugs, oestrogen, func- tional (idiopathic) causes, or microprolactinoma. Macroadenomas are typically associated with levels of more than 250 mg/​litre (5000 mU/​litre). It should be stressed, though, that such values are not absolute; prolactinomas may present with variable elevations in PRL and there may be dissociation between tumour mass and hor- monal secretion. Furthermore, the interpretation of a moderate elevation of PRL in a patient with a pituitary macroadenoma needs to be done cautiously, as the hyperprolactinaemia may be attrib- uted to compression of the pituitary stalk by a tumour other than a prolactinoma. Recent data suggest that serum PRL of more than 2000 mU/​litre is almost never encountered in non​functioning pi- tuitary macroadenomas. Values above this limit in the presence of a macroadenoma are probably associated with a prolactinoma (after acromegaly or Cushing’s disease have been excluded). Alternatively, the empirical confirmation of the diagnosis can be obtained by treat- ment for several months with dopamine agonists with serial assess- ment of serum PRL levels and adenoma size. Normalization of PRL combined with a substantial reduction of the initial adenoma size confirms the diagnosis of a prolactinoma. Normalization of PRL with no change or only a small reduction in tumour volume may suggest a pituitary adenoma other than a prolactinoma. No change in serum PRL and no reduction in tumour volume indicate a drug-​ resistant prolactinoma (5% to 10% of cases). Two potential pitfalls in the diagnosis of a prolactinoma should always be taken into account: (1) the presence of macroprolactin and (2) the ‘hook effect’. Macroprolactin is a complex of PRL and, gen- erally, an immunoglobulin G antibody. Serum PRL concentrations are increased due to the reduced rate of clearance of this complex. Macroprolactin has reduced bioactivity and is present in signifi- cant amounts in up to 20% of hyperprolactinaemic sera, resulting in pseudohyperprolactinaemia and the potential for misdiagnosis (thus, macroprolactinaemia is suggested in the presence of high PRL levels with no menstrual irregularities). For confirmation of macroprolactinaemia, polyethylene glycol precipitation is the most practical method. The ‘hook effect’ may be observed in cases of very high serum PRL concentrations, such as those observed in giant prolactinomas. The high levels of circulating PRL causes antibody saturation in the immunoradiometric assay, leading to artefactually low results. To overcome this effect, an immunoradiometric PRL assay should be performed at a serum dilution of 1:100 or, alter- natively, should include a washout between the binding to the first antigen and the second step in order to eliminate excess unbound PRL. Currently, dynamic tests of PRL secretion are not used in clin- ical practice. Treatment  The primary goal of therapy in patients with microprolactinomas is to restore gonadal and sexual function by normalizing PRL levels. In patients with macroadenomas, reduc- tion of tumour size is also important. Dopaminergic agonists such as cabergoline, bromocriptine, pergolide, and quinagolide are the primary therapy. Bromocriptine, pergolide, and cabergoline are all ergot derivatives. The only non-​ergot derivative that is used in clinical practice is quinagolide. These drugs normalize PRL levels and significantly reduce the volume of the tumour in most pa- tients. Dopamine inhibition of PRL secretion is mediated by the D2-​ dopamine receptors expressed by normal and tumorous lactotrophs. For microprolactinomas, bromocriptine is successful in 80% to 90% of patients in normalizing serum PRL levels, restoring gonadal func- tion, and shrinking tumour mass. For macroprolactinomas, normal- ization of serum PRL levels and tumour shrinkage occur in about 70% of patients. Tumour shrinkage can often be observed within a week or two after commencing treatment, but in some cases may not be observed for several months. Continued tumour shrinkage may occur for many months or even years. Visual field defects im- prove in most patients. The therapeutic doses are in the range of 2.5 to 15 mg/​day and most patients respond while on 7.5 mg/​day or less. Cabergoline is effective in most patients, including those who did not previously respond to bromocriptine. Normalization of serum PRL has been reported in 86% of cases (92% with idio- pathic hyperprolactinaemia or microprolactinoma, and 77% with macroprolactinoma). Following 12 to 24 months of treatment with cabergoline, a greater than 20% decrease of baseline tumour size has been reported in more than 80% of cases, with complete disappear- ance of tumour mass in 26% to 36% of them. The initial dose of cabergoline is 0.5 mg once weekly and doses up to 1 mg twice weekly are usually effective. Large comparative studies of cabergoline and bromocriptine have suggested the superiority of cabergoline in terms of patient tolerability and convenience, reduction in serum PRL, res- toration of gonadal function, and decrease in tumour size. Although there is less experience with pergolide and quinagolide in the pri- mary treatment of patients with prolactinomas, these drugs appear to have similar efficacy and adverse event profiles when compared with bromocriptine. The adverse effects of dopamine agonists may be grouped into gastrointestinal (most commonly nausea, vomiting, constipation, dry mouth, dyspepsia), cardiovascular (most com- monly postural hypotension, digital vasospasm causing blanching of the extremities in response to cold), and neurological (headache and drowsiness). Other less common adverse effects are psychiatric manifestations (including psychosis and hypomania), cerebrospinal

13.2.1  Disorders of the anterior pituitary gland 2271 fluid rhinorrhoea (due to tumour shrinkage and an eroded pituitary sellar floor), leg cramps, flushing, and nasal congestion. Symptoms tend to occur after the initial dose and with dosage increases, but can be minimized by introducing the drug at a low dosage at bedtime, by taking it with food, and by very gradual dose escalation. Rarely, in patients with Parkinson’s disease treated with very high doses of bromocriptine, pulmonary infiltrates, fibrosis, pleural effusions, pleural thickening, and retroperitoneal fibrosis have been reported; however, these effects are dose-​dependent and are unlikely to occur at the low doses used for the treatment of prolactinomas. Recently, the occurrence of valvular insufficiency in patients who have been treated with high doses of cabergoline or pergolide for Parkinson’s disease has been described. Nonetheless, the relevant literature on patients with prolactinoma (managed with lower doses that those used in Parkinson’s patients) is generally reassuring. When a patient does not respond adequately to a dopamine agonist, the prolactinoma is considered resistant. For these patients possible treatment approaches include a trial of an alternative dopamine agonist, escalation of the dopamine agonist beyond conventional doses, surgical tumour resection, or radiotherapy. Trans-​sphenoidal surgery does not reliably lead to a long-​term cure, and recurrence of hyperprolactinaemia is frequent. The surgical outcomes are highly dependent on the experience of the neurosurgeon, the size of the tumour, and the serum PRL levels. The success rate of surgery in microadenomas is about 75% and for macroprolactinomas approxi- mately 34%. The indications for surgery mainly include resistance or intolerance to medical therapy and unstable pituitary apoplexy. The very high rate of efficacy of dopamine agonists combined with the high complication rates of radiotherapy render treatment with this modality rarely necessary; it is reserved for patients who do not re- spond to dopamine agonists, those who are not cured by surgery, or for malignant prolactinomas. When beginning dopaminergic treatment, women must be warned that restoration of fertility may be immediate (even before their first normal menstruation). In women with a macroprolactinoma wishing to become pregnant, it is necessary to plan conception to occur after the normalization of the serum PRL and the significant reduction of the adenoma size. The considerable experience with patients taking bromocriptine during pregnancy suggests that the incidence of abortions, ectopic pregnancies, or congenital malfor- mations is no higher than that in the general population. The ex- perience with cabergoline is less, but there is no evidence to suggest that such treatment may be unsafe. The relevant information on pergolide and quinagolide is much more limited and, therefore, they should not be used in pregnancy or when pregnancy is desired. Given that for women with microprolactinomas the risk of clinic- ally relevant tumour expansion is less than 2% during pregnancy, dopamine agonists can be safely stopped as soon as pregnancy has been confirmed. The patients need to be advised to report severe headaches or visual deterioration. In women with macroadenomas, symptomatic tumour expansion occurs in 20 to 30% of them; man- agement options include stopping the dopamine agonist when preg- nancy is confirmed with close surveillance thereafter, or continuing the dopamine agonist throughout the pregnancy. If visual field de- fects or progressive headaches develop, an MRI without gadolinium (not a CT) should be performed, and if the tumour has significantly increased in size, a dopamine agonist should be restarted. If the en- larged tumour does not respond to medical treatment, alternatives include delivery if the pregnancy is far enough advanced or trans-​ sphenoidal surgery. There are no data to suggest that breastfeeding leads to prolactinoma enlargement. Treatment withdrawal  When the serum PRL has been normal for at least 2 years and the size of the tumour decreased by more than 50%, the dose of the dopamine agonist can be gradually decreased, as at this stage low doses are likely to maintain stable PRL levels and tumour size. After pregnancy, normoprolactinaemia may occur. Women with hyperprolactinaemia who pass through the meno- pause require reassessment of the need for continuing treatment. If a patient has normal PRL levels after treatment with dopamine agonists for at least 5 years and the tumour volume is markedly de- creased, a trial of tapering and discontinuation of these drugs may be initiated. In such cases, careful monitoring for detection of recur- rence of hyperprolactinaemia and tumour enlargement in advised. ACTH ACTH is a single chain 39-​amino acid peptide released by the corticotroph cells. The initial synthesis is of a larger 231-​amino acid peptide (pro-​opiomelanocortin, POMC) that following proteo- lytic cleavage produces several peptides and hormones, including ACTH, melanocyte-​stimulating hormone, and β-​endorphin. ACTH is secreted in a pulsatile manner. The secretion is under positive hypothalamic control by the corticotropin-​releasing hormone, which exerts tropic and releasing actions on the corticotroph cells. Arginine vasopressin also stimulates ACTH release acting synergis- tically with corticotropin-​releasing hormone. ACTH exerts a nega- tive feedback effect at the hypothalamic level. ACTH and cortisol secretion follow a diurnal rhythm, with highest amounts in the early morning and lowest concentrations around midnight. The secretory bursts start at around 3.00 a.m. and are maximal in the last few hours before waking up. This pattern is mainly regulated by the light–​dark and the sleep–​wake cycles and may be altered by a major time shift. Any stressful event may induce a large discharge of ACTH into the circulation followed by a similar increase in the release of cortisol. Serum cortisol exerts feedback regulatory action on the pituitary but also at the hypothalamic level reducing ACTH release. This nega- tive feedback may be imitated by synthetic glucocorticoids such as dexamethasone. The first 24 amino acids of the ACTH are identical across species and are associated with its biological activity. ACTH acts through G protein-​coupled receptors predominantly found in the fascicular and reticular zones in the adrenal cortex where it stimulates the se- cretion of glucocorticoids and androgens. It also contributes to the release of mineralocorticoids. ACTH is also responsible for the maintenance of the adrenal growth and size. Disorders of ACTH secretion ACTH deficiency Chronic ACTH deficiency is associated with fatigue, pallor, an- orexia, weight loss, hypotension, hyponatraemia, hypoglycaemia, and eosinophilia. Children may present with delayed puberty and failure to thrive. In its most severe form, when left untreated, it may be fatal due to vascular collapse especially during superim- posed illness. In contrast to primary adrenal insufficiency, in which the ACTH levels are increased, there is no hyperpigmentation or hyperkalaemia.

SECTION 13  Endocrine disorders 2272 Given the diurnal rhythm of ACTH and cortisol secretion, random serum cortisol measurements are not always helpful in the diagnosis of ACTH deficiency. It has been proposed that sec- ondary adrenal insufficiency is present when morning cortisol con- centrations are less than 100 nmol/​litre (in the absence of previous steroid treatment); values greater than 500–​550 nmol/​litre exclude this diagnosis. Levels between these values need a stimulation test. Hypoglycaemia (blood glucose <2.2 mmol/​litre) induced by the in- sulin tolerance test is considered the gold standard for the assessment of the entire hypothalamic–​pituitary–​adrenal axis. A maximum cor- tisol response to a peak concentration greater than 500–​550 nmol/​ litre generally excludes adrenal insufficiency. ACTH deficiency causes adrenal atrophy and ACTH-​receptor downregulation. Based on these, the standard 250 μg (1–​24) ACTH (Synacthen) test may be useful for the diagnosis of secondary adrenal insufficiency, if done at least 4 weeks after the onset of ACTH deficiency. Stimulated cor- tisol concentrations at 30 min of 500–​550 nmol/​litre or less suggest ACTH deficiency. Other tests checking the ACTH reserve, which are less commonly used, include the glucagon and the metyrapone test. Glucocorticoid deficiency can be life-​threatening and, therefore, substitution should begin as soon as the diagnosis is confirmed. The replacement involves the administration of hydrocortisone or other steroids (prednisolone or dexamethasone) in a dose and timing to mimic the normal pattern of cortisol secretion. The most commonly used regime involves 10 to 25 mg hydrocortisone/​day; it is divided into two or three doses/​day (e.g. 10 mg, 5 mg, and 5 mg). There is no reliable biochemical test to assess the adequacy of replacement and the least dose necessary to relieve clinical symptoms is recom- mended. All patients should be supplied with an emergency card or bracelet giving information about their diagnosis, and instructions on stress-​related dose adjustments should be clearly offered. In case of vomiting or during the perioperative period, parenteral steroid administration is needed. GH replacement may unmask ACTH de- ficiency and glucocorticoid replacement may unmask underlying diabetes insipidus. Cushing’s disease Cushing’s disease refers to the chronic exposure to endogenous glucocorticoids (Cushing’s syndrome) produced by the adrenal cortex caused by excess ACTH production by a pituitary corticotroph adenoma (see ‘Pituitary adenomas’ and ‘Pituitary carcinomas’, next). Nelson’s syndrome  Nelson’s syndrome is defined by the association of an expanding pituitary tumour and high levels of ACTH secretion after bilateral adrenalectomy for Cushing’s disease. Its prevalence ranges from 8% to 29% with a time interval between adrenalectomy and the diagnosis of the syndrome of 0.5 to 24 years (most com- monly thought to be within 2 years). Apart from high basal ACTH levels after adrenalectomy and the presence of a pituitary adenoma remnant after adrenalectomy, there is no general agreement on the predictive factors for the development of Nelson’s syndrome. Notably, pituitary irradiation prior to adrenalectomy has been found to be protective in some studies, but not all. The clinical manifest- ations include those from the mass effect (headaches, deterioration of vision, ophthalmoplegia) and hyperpigmentation as a result of the increased ACTH levels. Early diagnosis is important and moni- toring with measurement of ACTH levels and pituitary imaging at 6  months and yearly after the adrenalectomy are recommended. Cases of pituitary tumour with distant metastases have also been described. Treatment should be aggressive and includes surgical ex- cision and pituitary irradiation. TSH TSH is a glycoprotein hormone consisting of two non​covalently bound subunits: the α subunit, which is common to all anterior pi- tuitary glycoprotein hormones and the β subunit, which is unique for TSH and confers its biological specificity. The α subunit has a molecular weight of 20 to 22 kDa, is composed of 92 amino acid res- idues, and contains two N-​linked carbohydrate groups. The β sub- unit has a molecular weight of 18 kDa, is composed of 110 amino acid residues, and contains one N-​linked complex carbohydrate. The glycosylation is important for the normal bioactivity of TSH. TSH is secreted in a pulsatile manner (with low amplitude peaks), as well as in a circadian fashion (with elevation in the late hours of the evening). The hypothalamic tripeptide TRH stimulates TSH release, whereas thyroid hormones exert a negative feedback at the hypo- thalamic and pituitary levels. Somatostatin and dopamine inhibit TSH secretion. TSH binds to specific thyroid cell plasma membrane receptors and regulates both synthesis and secretion of thyroid hormones. Disorders of TSH secretion TSH deficiency Central hypothyroidism is diagnosed when the concentrations of thyroxine are decreased and the TSH levels are usually normal or low. The clinical manifestations include tiredness, cold intolerance, constipation, hair loss, dry skin, hoarseness, cognitive slowing, leth- argy, weight gain, bradycardia, facial puffiness, delayed relaxation phase of the deep tendon reflexes, and hypotension. In children, de- velopmental delay and growth retardation are also present. TSH de- ficiency is treated with l-​thyroxine with the aim of achieving normal serum free thyroxine levels (serum TSH cannot be used as a guide of adequate replacement). An increase in the dose of l-​thyroxine may be necessary during pregnancy or new oestrogen or GH re- placement. As thyroid hormone replacement increases the rate of metabolism of glucocorticoids and may therefore lead to an adrenal crisis in cases of coexisting hypoadrenalism, thyroxine should be ad- ministered after hydrocortisone substitution has been initiated. Thyrotropinomas TSH-​secreting pituitary adenomas account for less than 1% of all pi- tuitary adenomas with an overall prevalence of about 1 in one million of the population. They show no gender difference and the majority are diagnosed between the third and sixth decade of life. The signs of thyrotoxicosis may vary from severe to absent. More than 90% of the patients present with goitre. Compression features including head- aches and visual field defects due to tumour invasion or suprasellar extension may be also present. Coexistent oversecretion of other pi- tuitary hormones may also occur resulting in additional symptoms. The biochemical profile of TSH-​secreting pituitary adenomas in- cludes elevated circulating thyroxine with normal or increased TSH levels (as opposed to undetectable levels of TSH in Graves’ disease, which causes primary hyperthyroidism). Other markers of thyroid hormone action may be increased as well, such as sex hormone-​ binding globulin, cholesterol, angiotensin-​converting enzyme, or

13.2.1  Disorders of the anterior pituitary gland 2273 C-​terminal cross-​linked telopeptide of type 1 collagen. Most tu- mours are macroadenomas. Diagnosis  It is essential to differentiate a TSH-​secreting pituitary adenoma from the syndrome of pituitary resistance to thyroid hormone, in which mutations in the gene coding for the thyroid hormone receptor β prevent the detection of peripheral thyroid hormones by the pituitary resulting in increased levels of TSH and hyperthyroidism. While it is mainly the pituitary gland being in- sensitive to thyroid hormones, other tissues do not show resistance to thyroid hormones in this form of the syndrome. Detection of a mutation in the gene coding for thyroid hormone receptor β con- firms the diagnosis. Nevertheless, in about 10% of patients, no muta- tions can be found. The combination of the TRH stimulation test, α subunit levels, and the α subunit/​TSH ratio are helpful in the differ- ential diagnosis. Thus, in cases of TSH-​secreting pituitary adenoma, the α subunit level is increased, the molar ratio of α subunit to TSH is more than 1.0, and TRH administration is associated with a less than twofold increase of TSH. Treatment  The first-​line therapy for patients with TSH-​secreting pituitary adenoma is trans-​sphenoidal resection of the tumour, after which about one-​third of all patients will be cured. If surgery is contraindicated or declined, the administration of somatostatin analogues should be considered. About 85% of the patients re- spond to somatostatin analogues with decrease of thyroxine levels. Occasionally, external pituitary irradiation is indicated. Criteria of cure go beyond the establishment of euthyroidism and include the normalization of α subunit levels, α subunit/​TSH ratio, peripheral markers of thyroid hormone action, and dynamic tests, as well as pi- tuitary imaging. Long-​term follow-​up including clinical, biochem- ical, and radiological monitoring is mandatory. Hypopituitarism Hypopituitarism, first described by Simmonds in 1914, results from the decreased secretion of pituitary hormones. It is caused by an inability of the gland to produce hormones and/​or an insufficient supply of hypothalamic-​releasing hormones. It is associated with an increased mortality (with causes of premature mortality being cardiovascular and cerebrovascular disease) and its main causes are shown in Box 13.2.1.3. The clinical manifestations of hypopituitarism depend mainly on the underlying disease, as well as the type and degree of the hor- monal deficits. Tumoural masses in the sellar region with suprasellar extension may be associated with visual impairment, headaches, oculomotor nerve palsy, or damage to other cranial nerves within the cavernous sinus. Hypopituitarism may be subclinical, diagnosed only following hormonal investigations, or of acute and severe clin- ical onset requiring hospital admission. ACTH, TSH, and antidiur- etic hormone deficiency are potentially life-​threatening, whereas FSH/​LH and GH deficiencies are associated with chronic morbidity. The clinical manifestations and the diagnosis and treatment of each pituitary hormone deficit are described in previous paragraphs. It should be noted that as thyroid hormone replacement increases the rate of metabolism of the glucocorticoids and may lead to an adrenal crisis, glucocorticoid substitution should begin before thyroid hor- mone treatment is offered. Pituitary adenomas Pituitary adenomas are the most common cause of pituitary dis- ease. They are benign lesions arising from adenohypophyseal cells and account for up to 25% of intracranial tumours. Based on their secretory activity, they are classified as functioning (resulting in the syndromes of hormonal excess previously described) and non-​ functioning (presenting with mass effects). Tumours measuring less than 10 mm in diameter are considered microadenomas and those larger than 10 mm macroadenomas. Immunohistochemically they are grouped according to their hormone content; generally, the clinical classification overlaps the histopathological one. On CT, macroadenomas are isodense or hypodense relative to brain tissue with variable patterns of enhancement after contrast administration. Calcification may be seen occasionally. On MRI, they have homoge- neous low intensity on T1-​weighted images and, after contrast ad- ministration, homogeneous enhancement that is less intense than the enhancement in the adjacent pituitary. Cysts or areas of necrosis cause foci of moderate hypointensity on T1-​weighted sequences, foci of hyperintensity on T2-​weighted sequences, and heteroge- neous enhancement with gadolinium. Haemorrhage in the subacute or chronic phase shows high signal on T1-​weighted images. On CT, microadenomas show little inherent contrast to the normal pituitary Box 13.2.1.3  Causes of hypopituitarism • Pituitary and non​pituitary tumours — Pituitary adenomas — Craniopharyngiomas — Meningiomas — Gliomas — Chordomas — Ependymomas — Primary or metastatic (especially lung and breast) cancer — Hamartomas — Germinomas — Optic gliomas • Lymphocytic hypophysitis • Pituitary apoplexy • Sheehan’s syndrome (postpartum hypopituitarism) • Cranial irradiation • Pituitary surgery • Traumatic brain injury • Subarachnoid haemorrhage • Haemochromatosis • Granulomatous diseases — Sarcoidosis — Tuberculosis — Langerhans’ histiocytosis • Empty sella syndrome • Genetic causes Mutations of genes encoding transcription factors including HESX1 (homeobox gene expressed in embryonic stem cells 1), LHX3 (LIM-​domain homeobox gene 3), LHX4 (LIM-​domain homeobox gene 4), PROP1 (prophet of Pit1), POU1F1 (POU domain, class 1, transcription factor 1) • Infections • Abscess • Meningitis • Encephalitis

SECTION 13  Endocrine disorders 2274 tissue and intravenous contrast demonstrates non​enhancement against a background of normal gland enhancement. On MRI, they are hypointense on T1-​weighted sequences and this contrast may or may not be amplified after gadolinium. The treatment of pituitary adenomas involves surgery, radio- therapy, or medical therapy and has been described in previous sections. Pituitary carcinomas Pituitary carcinomas are defined as pituitary tumours with sub- arachnoid, brain, or systemic metastasis. They account for less than 0.5% of symptomatic pituitary tumours. They mainly arise from the transformation of initially large, but benign, adenomas. Their pathogenetic mechanism remains unclear; it has been pro- posed that under the influence of unknown growth-​enhancing stimuli, an early proliferative stage of polyclonality is followed by monoclonal or multiclonal mutations, leading to selective growth advantage and a state of invasiveness. Alterations in the function of oncogenes and/​or tumour suppressor genes may also be impli- cated. Their malignant nature is not usually obvious in their micro- scopic appearance and the reliable distinction between carcinoma and adenoma is impossible on the basis of standard histological cri- teria. Their clinical manifestations are similar to invasive and non-​ invasive pituitary adenomas (pressure effects to the surrounding tissues and/​or consequences of hormonal hypersecretion). The great majority of pituitary carcinomas are hormonally active, most commonly ACTH-​secreting or PRL-​secreting. No differ- ences in the hormone levels differentiating pituitary carcinomas from other invasive and/​or non​invasive macroadenomas have been identified. Although on imaging pituitary carcinomas ap- pear as invasive macroadenomas, there are no reliable features distinguishing tumours that could behave in a malignant manner from other types of invasive adenomas. Metastases can occur in every part of the central nervous system (usually cortex, cere- bellum, and cerebellopontine angle) or in distant sites (usually liver, lymph nodes, bone, and lung). The treatment of pituitary carcinomas is similar to that of large and aggressive pituitary tumours and includes surgery, external beam radiotherapy, and adjuvant medical treatment (chemotherapy). In the last years, the alkylating agent temozolomide has been used for aggressive pituitary adenomas and carcinomas. The therapy of pitu- itary carcinoma is mainly palliative and may not prolong survival to any major extent (mean survival after the development of metastatic disease is reported to be less than 4 years). Pituitary apoplexy Pituitary apoplexy is a clinical syndrome resulting from acute haemorrhage or infarction of the pituitary gland. It is potentially life-​threatening and is characterized by the sudden onset of head- ache, vomiting, visual disturbance, ophthalmoplegia, and altered consciousness. This constellation of findings occurs primarily in patients with pre-​existing pituitary adenomas and can be due to ex- tensive tumour infarction or haemorrhage. The term has also been used to describe spontaneous infarction and haemorrhage within a non​tumorous pituitary gland with similar clinical effects. The age range of occurrence is broad, from the first to the ninth decade. The incidence of pituitary apoplexy presenting with classic symptoms is reported to be in the order of 0.6 to 9.1% of surgically treated pi- tuitary adenomas. However, clinically silent pathological evidence of pituitary haemorrhage (‘subclinical pituitary apoplexy’) has been reported in up to 25% of surgically removed pituitary aden- omas. The clinical syndrome of pituitary apoplexy usually evolves fully within hours to 2 days and its pathophysiology remains un- certain. Predisposing factors for pituitary apoplexy include major surgery, warfarin, aspirin, arterial hypertension, oral contraceptive pill, gonadotropin-​releasing hormone analogue, dynamic pituitary function tests, and head trauma. A  variety of pituitary tumours, both endocrinologically active and inactive, have been documented in association with pituitary apoplexy, but opinions differ as to whether there is a predominance of a particular type of pituitary tu- mour. Following apoplexy, hypofunctioning (partial or complete) of normal pituitary tissue appears to be the rule. Hyponatraemia, noted in 44% of patients, may be caused either by inappropriate antidiur- etic hormone secretion, hypocortisolism, or hypothyroidism or by a combination of these. On pituitary MRI performed in the first 3 to 5 days, the haemorrhage within the sella is isointense or hypointense on T1-​weighted images and hypointense on T2-​weighted sequences. When pituitary apoplexy is suspected, the initial management con- sists of close monitoring of fluid and electrolyte balance coupled with immediate replacement of deficient hormones, in particular corticosteroids. Although not widely accepted, it has been suggested that in patients with visual field or visual acuity defects, surgical de- compression should be performed as soon as possible, preferably within the first week, as this appears to optimize visual outcome and to improve pituitary function. Following apoplexy, the risk of tu- mour recurrence is small, but careful follow-​up initially with annual imaging is indicated. Craniopharyngiomas Craniopharyngiomas are epithelial tumours (grade I, World Health Organization classification) arising along the path of the craniopharyngeal duct (the canal connecting the stomodeal ectoderm with the evaginated Rathke’s pouch). Their overall in- cidence is 0.13 per 100 000 person-​years and they account for 2 to 5% of all the primary intracranial neoplasms (5.6–​15% of the intracranial tumours in children). They may be diagnosed at any age (peak incidence rates between 5 and 14  years and 50 and 74 years). Histologically, two primary subtypes have been recog- nized, the adamantinomatous and the papillary, but transitional or mixed forms have also been described. The adamantinomatous craniopharyngioma is the most frequently reported and may be diagnosed at all ages. Macroscopically, it shows cystic and/​or solid components, necrotic debris, fibrous tissue, and calcification. The liquid within the cysts ranges from ‘machinery oil’ to shimmering cholesterol-​laden fluid and it is mostly composed of desquamated squamous epithelial cells, rich in membrane lipids and cytoskel- eton keratin. In this subtype, the flat squamous epithelial cells may be desquamated in distinctive stacked clusters forming the path- ognomonic nodules of ‘wet’ keratin. The papillary subtype has been almost exclusively found in adult patients. Macroscopically

13.2.1  Disorders of the anterior pituitary gland 2275 it is usually solid or mixed, calcification is rarely seen, and the cyst content is mostly viscous and yellow. Mutations in CTNNB1, encoding β-​catenin and in the BRAF gene have been identified in craniopharyngiomas and their significance in the pathogenesis of these tumours remains to be established. Presentation Most of the craniopharyngiomas are detected in the sellar/​parasellar region (a suprasellar component is present in 94–​95% of cases). They may exert pressure effects to various brain structures resulting in multiple clinical features (neurological, visual, hypothalamo-​ pituitary); headaches, nausea/​vomiting, visual disturbances, growth failure (in children), and hypogonadism (in adults) are the most frequently described symptoms. A substantial number of patients present with compromised hypothalamo-​pituitary function; re- ported rates for pituitary hormone deficits include 35 to 95% for GH, 38 to 82% for FSH/​LH, 21 to 62% for ACTH, 21 to 42% for TSH, and 6 to 38% for antidiuretic hormone. Useful imaging tools for the diagnosis of craniopharyngiomas include plain skull radio- graphs, CT, MRI, and, occasionally, cerebral angiography. The con- sistency of the tumours is purely or predominantly cystic in 46% to 64%, purely or predominantly solid in 18% to 39%, and mixed in 8% to 36%. Calcification is present in 45% to 57% and is probably more common in children (78–​100%). Hydrocephalus has been re- ported in 20% to 38% and is probably more often seen in childhood populations (41–​54%). Plain skull radiographs may show calcifica- tion and an abnormal sella. CT is helpful for the evaluation of the bony anatomy, the identification of calcifications, and the discrim- ination of the solid and the cystic components (the cystic fluid is hypodense and the solid portions, as well as the cyst capsule show enhancement following contrast administration). The MRI is useful for the topographic and structural analysis of the tumour. A solid lesion appears isointense or hypointense relative to the brain on precontrast T1-​weighted images, shows enhancement following gadolinium administration, and is usually of mixed hypointensity or hyperintensity on T2-​weighted sequences. Large amounts of cal- cification present as areas of low signal on both T1-​weighted and T2-​weighted images. A cystic element is usually hypointense on T1-​ weighted sequences and hyperintense on T2-​weighted sequences. On T1-​weighted images a thin peripheral contrast-​enhancing rim of the cyst is demonstrated. Protein, cholesterol, and methaemo- globin may cause high signal on T1-​weighted images. The differen- tial diagnosis includes several sellar or parasellar lesions, including Rathke’s cleft cyst, dermoid cyst, epidermoid cyst, pituitary ad- enoma, germinoma, hamartoma, suprasellar aneurysm, arachnoid cyst, suprasellar abscess, glioma, meningioma, sarcoidosis, tubercu- losis, and Langerhans cell histiocytosis. Treatment Surgery combined or not with adjuvant external beam irradiation is currently the most widely used first therapeutic approach for these tumours. Craniopharyngiomas remain challenging tumours, even in the era of modern neurosurgery. This is mainly attributed to their sharp, irregular borders and to their tendency to adhere to vital neurovascular structures making surgical manipulations potentially hazardous to vital brain areas. Consequently, the at- tempted degree of excision has been a subject of long-​standing debate. The advances in neuroimaging, microsurgical techniques, perioperative care, and hormone replacement therapy have signifi- cantly improved the perioperative mortality, which according to recent reports is between 1.7 and 5.4% for the primary operations. The mean interval for the diagnosis of recurrence following various primary treatment modalities ranges between 1 and 4.3 years. The recurrence rates following gross total removal range between 0 and 62% at 10 years follow-​up and are significantly lower than those after partial or subtotal resection (25–​100% at 10 years follow-​up). In cases of limited surgery, adjuvant radiotherapy significantly im- proves the local control rates (recurrence rates 10–​63% at 10 years follow-​up). Finally, radiotherapy alone provides 10-​year recur- rence rates ranging between 0 and 23%. For predominantly cystic tumours, fluid aspiration provides relief of the obstructive mani- festations and facilitates the consecutive removal of the solid tu- mour portion; the latter should not be delayed for more than a few weeks due to the significant risk of the cyst refilling. The man- agement of recurrent tumours remains difficult, as scarring and/​ or adhesions from previous operations or irradiation decrease the chances of successful excision. In such cases, total removal is achieved at a substantially lower rate when compared with pri- mary surgery (0–​25%) and is associated with increased periopera- tive morbidity and mortality (10.5–​24%). The beneficial effect of radiotherapy (preceded or not by second surgery) in recurrent lesions is well established. Other treatment modalities include brachytherapy (stereotactically guided instillation of β-​emitting isotopes into cystic craniopharyngiomas), intracystic installation of the antineoplastic agent bleomycin, stereotactic radiosurgery or radiotherapy, and systemic chemotherapy. Morbidity and mortality Craniopharyngiomas are associated with significant long-​term mor- bidity (mainly endocrine, visual, hypothalamic, neurobehavioural, and cognitive sequelae), which compromise normal psychosocial integration and quality of life. These complications are attributed to the damage to critical structures by the primary or recurrent tu- mour and/​or to the adverse effects of the therapeutic interventions. In studies with variable follow-​up periods and after different treat- ment modalities, the rates of individual hormone deficits range from 88 to 100% for GH, 80 to 95% for FSH/​LH, 55 to 88% for ACTH, 39 to 95% for TSH, and 25 to 86% for ADH. Compromised vision has been reported in up to 62.5% of the patients treated by surgery combined or not with radiotherapy during observation periods of 10 years. Hypothalamic damage may result in hyperphagia and un- controllable obesity, disorders of thirst and water/​electrolyte balance, behavioural and cognitive impairment, loss of temperature control, and disorders in the sleep pattern. Among these, obesity is the most frequent (affecting 26–​61% of the patients treated by surgery com- bined or not with radiotherapy) and is a consequence of the dis- ruption of the mechanisms controlling satiety, hunger, and energy balance. Other rare long-​term irradiation-​attributed morbidities include vasculopathy and second brain tumours. The overall mor- tality rates of patients with craniopharyngioma have been reported to be 3 to 6 times higher than that of the general population. The 10-​ year survival rates range between 83 and 92.7% and are significantly lower in cases of recurrent disease. Apart from the deaths directly attributed to the tumour (pressure effects to critical structures) and to the surgical interventions, the risk of cardiovascular/​cerebrovas- cular and respiratory mortality is increased.

SECTION 13  Endocrine disorders 2276 Hypophysitis Inflammatory processes of the hypophysis are classified as pri- mary, when the inflammation is confined to the pituitary gland with no identifiable aetiological association, and secondary, when the inflammatory pituitary reaction is triggered by a def- inite aetiological agent or a known systemic disease (local lesions such as germinomas, Rathke’s cleft cysts, craniopharyngiomas, or pituitary adenomas, or systemic diseases such as sarcoidosis, Langerhans cell histiocytosis, or tuberculosis). In the latter cases, the infiltrate is mainly lymphocytic or xanthogranulomatous and focuses around the lesion rather than diffusing to the entire gland. Drug-​related autoimmune hypophysitis (e.g. after immune modulation using anticytotoxic T-​lymphocyte-​associated antigen 4 biological therapy) is a new form of hypophysitis being increas- ingly recognized under a spectrum of immune-​related adverse events. Recently, IgG4-​related hypophysitis has emerged as a part of IgG4-​related disease with multiple coexisting organ involve- ment. Anti-PIT-1 hypophysitis is a newly described condition associated with a thymoma or other neoplasm that ectopically ex- presses PIT-1 protein. Primary hypophysitis is histologically classified into three types: granulomatous, xanthomatous, and lymphocytic. Granulomatous hypophysitis has an unclear pathogenesis, affects men and women in equal proportions, and presents with nausea, vomiting, dia- betes insipidus, and hyperprolactinaemia. It is characterized by diffuse collections of multinucleated giant cells and histiocytes with surrounding lymphocytes and plasma cells. Xanthomatous hypophysitis is an infiltrating process of the pituitary of unclear cause; it consists of cystic-​like areas of liquefaction infiltrated by lipid-​rich foamy histiocytes and lymphocytes. Lymphocytic hypophysitis is a rare condition, but insufficient population-​based data exist to estimate its real incidence. Based on the published cases, women are affected more frequently than men in a ratio of about 5 to 1 or 8 to 1. It shows a striking temporal association with pregnancy, with most of these patients presenting in the last month of pregnancy (without causing complications to the fetus or the outcome of pregnancy) or in the first 2 months after delivery. Its clinical presentation is variable and comprises four categories of symptoms: sellar compression (headaches, visual disturbances, diplopia), hypopituitarism (mainly ACTH followed by TSH, gonadotropins, and PRL—​it should be noted that the usual order of loss of anterior pituitary hormones does not occur in patients with hypophysitis), diabetes insipidus, and hyperprolactinaemia. The defining pathological feature is the infiltration of the pituitary gland with lymphocytes. The immune infiltrate also contains other cells including plasma cells, eosinophils, macrophages, histiocytes, and neutrophils. The role of antipituitary antibodies remains to be established but their detection has amplified the diagnostic criteria, also suggesting a possible pathogenetic role. The mechanisms by which the infiltrate causes loss of function/​destruction of the endo- crine cells or impairment of vasopressin release are not clear. It has been suggested that the disease progresses through various stages. Initially, the pituitary gland is inflamed, infiltrated by lymphocytes, and oedematous, causing mass effect symptoms; subclinical hypo- pituitarism may be present. If the inflammation resolves and the pituitary parenchyma is not destroyed, remission occurs. If the inflammation continues, the pituitary is replaced by fibrotic tissue, becomes atrophic, and loses its function. Even when using modern MRI studies, nearly 40% of the cases are misdiagnosed as pituitary adenomas. The typical precontrast MRI findings include a symmetrical enlargement of the pituitary gland, a thickened but rarely displaced stalk, and a usually intact sellar floor. The pattern of signal enhancement after gadolinium may be helpful in differentiating hypophysitis from macroadenoma. A strong and homogenous enhancement of the anterior pituitary gland is more suggestive of an inflammatory infiltrative process. A strip of enhanced tissue along the dura mater (‘dural tail’) has also been described. Macroadenomas enhance less or more slowly than the normal pi- tuitary on dynamic MRI. If the infundibuloneurohypophysitis is in- volved, there is the loss of T1 hyperintensity in the neurohypophysis, swelling of the posterior pituitary, and thickening of the pituitary stalk of more than 3 mm at the level of the median eminence of the hypothalamus. The treatment of this condition is controversial and includes re- placing the defective endocrine function and/​or reducing the size of the pituitary mass. The role of surgery remains controversial; it should be performed only in the presence of serious and progressive deficits of visual fields, visual acuity, or ocular movements. It is also performed when a pituitary adenoma is suspected and the diagnosis is subsequently made by histology. Cases of spontaneous resolution without any treatment have been reported. Rathke’s cleft cysts Rathke’s cleft cysts are common benign sellar and/​or suprasellar le- sions, found in 13 to 33% of routine autopsies. Symptomatic cases are rare and they probably arise from remnants of Rathke’s pouch, a structure apparent during the third week of gestation and formed by the infolding of the simple ciliated columnar epithelium lining the roof of the stomodeum. Rathke’s cleft cysts are smoothly marginated cysts with size usually ranging from a few millimetres to 1 to 2 cm. Their contents vary from a clear cerebrospinal fluid-​like liquid to a thick mucoid material made up of cholesterol and protein. They are lined by single or pseudostratified cuboidal or columnar epithelium with or without cilia and with goblet cells. The presenting mani- festations are the result of compression to adjacent structures. The most frequent ones include headaches, hypopituitarism of varying degrees, hyperprolactinaemia, visual disturbance, and diabetes insipidus. Their imaging features are highly variable. Forty per cent are completely intrasellar, whereas 60% have some suprasellar ex- tension. On CT, the cyst density ranges from hypodense to isodense or is mixed. On MRI, they have a variable T1 signal (hyperintense, hypointense, or isointense) depending on their biochemical content. Cysts with high protein concentration show high T1 signal intensity and usually have a low intracystic water content leading to T2 signal decrease. Small intracystic nodules corresponding to proteinaceous concentrations may be demonstrated presenting with lower T2 and higher T1 signal intensity than the rest of the cyst. The nodules do not enhance and are virtually pathognomonic for Rathke’s cleft cysts. Symptomatic cases are managed by surgery. The risk of recur- rence following evacuation and biopsy ranges between 8 and 33%. Although not widely accepted, the extent of removal (gross total vs. partial) may predict relapse.

13.2.2 Disorders of the posterior pituitary gland

13.2.2 Disorders of the posterior pituitary gland 2277

13.2.2  Disorders of the posterior pituitary gland 2277 FURTHER READING Ajithkumar T, et al. (2011). Pituitary radiotherapy. In:  Wass JAH, Stewart P (eds) Oxford textbook of endocrinology and diabetes (2nd edn), pp. 176–87. Oxford University Press, Oxford. Assie G, et  al. (2007). Corticotroph tumor progression after adrenalectomy in Cushing’s disease: a reappraisal of Nelson’s syn- drome. J Clin Endocrinol Metab, 92, 172–​9. Baldeweg SE, et al. (2016). Society for Endocrinology Endocrine Emergency Guidance: Emergency management of pituitary apo- plexy in adult patients. https://ec.bioscientifica.com/view/journals/ ec/5/5/G12.xml Bernabeu I, et al. (2011). General concepts of hypothalamus-pituitary anatomy. In: Wass JAH, Stewart P (eds) Oxford textbook of endocri- nology and diabetes (2nd edn), pp. 71–82. Oxford University Press, Oxford. Byrne JV (2011). Imaging of the pituitary. In: Wass JAH, Stewart P (eds) Oxford textbook of endocrinology and diabetes (2nd edn), pp. 136–​45. Oxford University Press, Oxford. Capatina C, et al. (2015). Management of endocrine disease: pituitary tumour apoplexy. Eur J Endocrinol, 172, 179–​90. Caturegli P, et al. (2005). Autoimmune hypophysitis. Endocr Rev, 26, 599–​614. Colao A, et al. (2019). Acromegaly. Nat Rev Dis Primers, 5(1), 20. doi: 10.1038/s41572-019-0071-6. Drake WM, et al. (2011). Pituitary assessment strategy. In: Wass JAH, Stewart P (eds) Oxford textbook of endocrinology and diabetes (2nd edn), pp. 127–​36. Oxford University Press, Oxford. Endocrine Society (2016). Hormone Replacement in Hypopituitarism Guideline Resources https://​www.endocrine.org/​guidelines-​and- ​clinical-​practice/​clinical-​practice-​guidelines/​hormone-​replacement- s​in-​hypopituitarism Freda PU, et al. (2011). Pituitary incidentaloma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab, 96, 894–​904. Gillam MP, et al. (2006). Advances in the treatment of prolactinomas. Endocr Rev, 27, 485–​534. Herring N, et  al. (2009). Valvular heart disease and the use of cabergoline for the treatment of prolactinoma. Clin Endocrinol, 70 104–​8. Kaltsas GA, et  al. (2005). Diagnosis and management of pituitary carcinomas. J Clin Endocrinol Metab, 90, 3089–​99. Karavitaki N (2007). Benign cysts: Rathke’s cysts, mucoceles, arach- noid cysts, and dermoid and epidermoid cysts. In: Wass JAH, Hay I (eds) Clinical endocrine oncology. Blackwell Publishing, Oxford. Karavitaki N, et  al. (2006). Craniopharyngiomas. Endocr Rev, 27, 371–​97. Katznelson L, et al. (2014). Acromegaly: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 99, 3933–​51. Kienitz T, et al. (2007). Long-​term management in five cases of TSH-​ secreting pituitary adenomas: a single center study and review of the literature. Eur J Endocrinol, 157, 39–​46. Melmed S, et al. (2011). Diagnosis and treatment of hyperprolactinemia: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 96, 273–​88. Molitch ME, et al. (2006). Evaluation and treatment of adult growth hormone deficiency:  an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 91, 1621–​34. Ntali G, Karavitaki N (2015). Efficacy and complications of pituitary irradiation. Endocrinol Metab Clin North Am, 44, 117–​26. Schneider HJ, et al. (2007). Hypopituitarism. Lancet, 369, 1461–​70. Trifanescu R, et al. (2012). Rathke’s cleft cysts. Clin Endocrinol, 76, 33–​7. Vroonen L, Daly AF, Beckers A (2019). Epidemiology and manage- ment challenges in prolactinomas. Neuroendocrinology, 109, 20–7. Wass JA, Karavitaki N (2009). Nonfunctioning pituitary adenomas: the Oxford experience. Nat Rev Endocrinol, 5, 519–​22. Yamamoto M, et al. (2019). Autoimmune pituitary disease: new concepts with clinical implications. Endocr Rev, pii: bnz003. doi: 10.1210/endrev/bnz003. 13.2.2  Disorders of the posterior pituitary gland Niki Karavitaki, Shahzada K. Ahmed,
and John A.H. Wass ESSENTIALS The posterior pituitary produces arginine vasopressin, which has a key role in fluid homeostasis, and oxytocin, which stimulates uterine contraction during birth and ejection of milk during lactation. Cranial diabetes insipidus is the passage of large volumes (>3 litres/​24 h) of dilute urine (osmolality <300 mOsm/​kg) due to vasopressin deficient synthesis and/​or release. The most common cause is lesions of the neurohypophysis or the hypothalamic me- dian eminence damaging the magnocellular neurons. It is diag- nosed by a water deprivation test revealing urine osmolality less than 300 mOsml/​kg with concurrent plasma osmolality more than 290 mOsml/​kg after dehydration and with urine osmolality rising to more than 750 mOsml/​kg after desmopressin. MRI of the neuro- hypophysis is required to delineate the cause. Mild polyuria can be managed simply by ensuring adequate fluid intake; treatment with the long-​acting vasopressin analogue, desmopressin (desamino, D-​8 arginine vasopressin; DDAVP), is used for more severe cases. The syndrome of inappropriate antidiuresis is diagnosed when there is hyponatraemia with hypotonic plasma (osmolality <270 mOsm/​kg), inappropriate urine osmolality (>100 mOsm/​kg) and urinary sodium more than 20 mmol/​litre, together with (1) no evidence of volume overload or hypovolaemia, and (2) normal renal, adrenal, and thyroid function. Few patients satisfy these strict criteria, but many conditions (e.g. malignant diseases, chest diseases, central nervous system dis- orders, and drugs) have been implicated. Aside from treatment (when possible) of the underlying cause, management requires fluid restric- tion and (rarely) infusion of hypertonic saline. Blockade of renal vaso- pressin receptors (V2) would be another option. Anatomy of the posterior pituitary gland The posterior pituitary gland, lying dorsally and caudally to the anterior pituitary, is connected by the hollow pituitary stalk to the hypothalamus in the floor of the third ventricle and is sometimes referred to as the neurohypophysis as it acts as an extension of the nervous system.

SECTION 13  Endocrine disorders 2278 In contrast to the anterior pituitary which develops from ecto- derm, is highly cellular, and is connected to the hypothalamus via the circulatory system, the posterior pituitary is derived from fore- brain and consists of nerve fibres which extend directly from the axonal terminals of hypothalamic neurons. The posterior pituitary hormones are synthesized in the hypothal- amic supraoptic nucleus (located just lateral to and above the optic chiasm) and the paraventricular nuclei (located on each side of the third ventricle). They then migrate along the axons of these neurons as neurosecretory granules in the supraoptic–​hypophyseal tract to the posterior pituitary before release into the circulation via branches of the inferior hypophyseal artery. The sensory signals that affect release of vasopressin and oxytocin are accumulated from the afferent fibres of osmoreceptors close to the hypothalamic nuclei, the brainstem, and also from the vagus and glossopharyngeal nerves receiving input from the pharynx and baroreceptors of the heart and great vessels (Fig. 13.2.2.1). The hypothalamic nuclei receive their blood supply from deriva- tives of the circle of Willis: the suprahypophyseal, anterior commu- nicating, anterior cerebral, posterior communicating, and posterior cerebral arteries. The inferior and superior hypophyseal arteries, formed from branches of the internal carotid artery, supply the pos- terior pituitary. The venous supply of the system drains to the dural, cavernous, and inferior petrosal sinuses. Vasopressin and oxytocin: Structure and synthesis Vasopressin and oxytocin have molecular weights of 1087 Da and 1007 Da, respectively, and are both non​apeptides with a disulphide bridge between the cysteine residues at positions 1 and 6 (Fig. 13.2.2.2). Oxytocin differs from vasopressin by only two amino acids with isoleucine for phenylalanine at position 3 and leucine for arginine at position 8. The genes for both these hormones lie 8 kb apart on chromosome 20q13. They encode 145-​amino acid pre- cursors comprising a signal peptide, the specific vasopressin, or oxy- tocin sequence, a hormone-​specific peptide called a neurophysin, and a C-​terminal peptide. The vasopressin precursor also has a glycoprotein at the C-​terminus. The hormones are initially packaged in granules as a precursor complex of neurophysin and oxytocin or vasopressin. During transport of these neurosecretory granules to the posterior pituitary, endopeptidases cleave off the active hormone from the neurophysin and the final products are stored in the nerve termini in the gland. The synthesis of vasopressin and oxytocin occurs in separate neurons within the paraventricular and supraoptic nucleus which allows the individual release of hormones. On stimulus of the ap- propriate magnocellular cell body, an action potential propagates along the axon, causing an influx of calcium at the axon terminal and releasing the hormone’s neurosecretory granules into the peri- vascular space. Neurophysin is also released but has no further role after acting as a carrier protein in the neurons. The hormones are unbound in the circulation and their half-​life is short, that of vasopressin being about 10 min. They are degraded by endothelial and circulating endopeptidases and aminopeptidases; vasopressin is mainly cleared in the liver and kidneys, while oxytocin is also cleared in the uterus. Oxytocin: Physiology and functions Oxytocin binds to a G protein-​coupled cell surface receptor on target cells to mediate a variety of physiological effects, but princi- pally regulation of lactation, parturition, reproductive, and maternal behaviour. It is named after the Greek phrase meaning ‘rapid birth’. It has no known role in men but has been postulated to aid con- traction of the seminal vesicles. In women oxytocin receptors are ex- pressed predominantly in uterine and breast myometrial cells. Their numbers are increased by oestrogen and during pregnancy. The hor- mone causes uterine contraction when cervical dilatation triggers oxytocin release during parturition. It is also released in response Anterior pituitary Posterior pituitary Median eminence Third ventricle Floor of ventricle Optic chiasm Paraventricular nucleus Supraoptic nucleus Brainstem Fig. 13.2.2.1  Schematic representation of the neuronal pathways from the paraventricular and supraoptic nuclei. The nerves project to the posterior pituitary, the median eminence, the floor of the third ventricle, and the brainstem. Afferent fibres from the osmoreceptors and thirst centre are shown. This article was published in Clinical Endocrinology, Besser GM, Thorner MO (eds) pp. 5.1–​5.14, © Mosby-​Wolfe (2002). s s s s s s NH2 3. Phe 4. Gln 2. Tyr 5. Cys 7. Pro 8. Arg 9. Gly Desmopressin (1-desamino-8-D-arginine casopressin DDAVP) Oxytocin Argininie vascopressin NH2 5. Cys 7. Pro 8. Leu 9. Gly 4. Glu 3. Ile 5. Cys 7. Pro 8. D-Arg 9. Gly

  1. Asn
  2. Asn
  3. Phe
  4. Cys
  5. Tyr
  6. Glu
  7. Tyr
  8. Asn
  9. Cys
  10. Cys Fig. 13.2.2.2  The structure of vasopressin, oxytocin, and desmopressin. Amino acid differences are highlighted in bold.

13.2.2  Disorders of the posterior pituitary gland 2279 to suckling when breast duct smooth muscle contraction leads to ejection of breast milk during breastfeeding. The importance of oxytocin in maintaining milk secretion is demonstrated in trans- genic mice with a knockout of oxytocin synthesis. These animals deliver their young normally, showing the involvement of several other hormones (prostaglandins, endothelins, adrenergic agonists, corticotropin-​releasing hormone, glucocorticoids, and cytokines) in the initiation and completion of labour. They also produce their milk normally, demonstrating the role of prolactin. However, the mice are unable to release milk during suckling and the young die of dehydration. Administration of oxytocin to the knockout mothers restores milk secretion and the young survive. Vasopressin: Physiology and functions Vasopressin is also known as antidiuretic hormone (ADH) and these two names relate to the two physiological entities regulated by this hormone: pressure/​volume and osmosis. However, there are sep- arate sensory inputs to these two systems and distinct receptors at the end-​organs of response. There are three known vasopressin receptors: V1 receptors occur in vascular smooth muscle, V2 receptors are expressed in the col- lecting tubules of the kidney, and V3 receptors on anterior pituitary corticotrophs. The receptors are all seven-​transmembrane-​domain, G protein-​coupled receptors; V1 and V3 signal by inositol phosphate pathways, while the V2 receptor activates adenylate cyclase with an increase in intracellular cAMP. The V2 receptor mediates the prin- cipal physiological effect of vasopressin, that of regulation of water reabsorption in the distal nephron. The presence of selective water channel proteins (aquaporins) in the wall of the distal nephron al- lows reabsorption of water from the lumen along an osmotic gra- dient with excretion of concentrated urine. Eight aquaporins have been cloned so far. The V2 receptor activation and subsequent re- lease of intracellular cAMP results in the insertion of aquaporin-​ 2 into the apical membrane of the collecting duct. The subsequent movement of water into the cell and renal interstitium accounts for the antidiuretic action of vasopressin. The function of the remaining vasopressin receptors is summarized in Table 13.2.2.1. Activation of the V1 receptor in vascular smooth muscle results in vasocon- striction and a rise in blood pressure with higher concentrations of vasopressin. The V3 receptor acts as one of the central regulators of secretion of ACTH in synergy with corticotropin-​releasing hor- mone. In addition, V2 receptors stimulate the production of clotting factor VIII. Vasopressin release occurs in response to three key stimuli: (1) a rise in plasma osmolality, (2) a drop in blood pressure, and (3) a stressful event. These changes are sensed by osmoreceptors in the hypothalamus and baroreceptors in the heart, aorta, and the great vessels. The principal physiological stimulus is a rise in plasma osmolality detected in the osmoreceptor cells. Figure 13.2.2.3 illus- trates the tight linear positive correlation between the plasma osmo- lality and release of vasopressin and thus, the exquisite sensitivity of this system which maintains plasma osmolality within the narrow range of 285 to 295 mosmol/​kg. This correlation also exists between osmolality and thirst. A  loss of extracellular water will stimulate vasopressin secretion to conserve water, accompanied by thirst, and a drive to drink. The regulation of thirst by osmolality is more im- portant physiologically than that induced by hypovolaemia. Most humans consume the bulk of their ingested water as a result of rela- tively unregulated fluid intake such as consumption of drinks with food or in tea, coffee, and soft drinks. This explains why in the syn- drome of inappropriate antidiuretic hormone described next, water intake must be consciously restricted to avoid overconsumption. Hypotension stimulates vasopressin release through the activation of baroreceptors in the carotid sinus and aortic arch and low-​pressure receptors in the atria and pulmonary venous system. A significant drop in circulating volume (i.e. falls of 5–​10% of arterial blood pres- sure), are required to increase vasopressin concentrations. However, in contrast to osmoregulated vasopressin secretion, a progressive decrease in blood pressure produces an exponential increase in plasma vasopressin. The subsequent water retention helps to restore blood volume. Vasopressin is also released under non​specific stress. Although the precise role of vasopressin in the stress response is unknown, it is classified as a stress hormone as it is released, in re- sponse to, for example, neuroglycopenia, nausea, and emesis among other stimuli. There are also normal physiological states where the system is modified. In pregnancy, there is a resetting of the osmostat such that both increases and decreases in plasma vasopressin occur at an osmolality approximately 10 mosmol/​kg less than the normal vaso- pressin concentration/​plasma osmolality relationship. Furthermore, Table 13.2.2.1  Vasopressin receptor functions V1 V2 V3 Location Vascular smooth muscle Basolateral membrane of distal nephron Pituitary corticotroph Liver Platelets Central nervous system Function Smooth muscle contraction Increased production of aquaporin-​2 and antidiuresis Enhanced ACTH release Stimulation of glycogenolysis Platelet adhesion Neurotransmitter Adapted from Ball SG, Baylis PH (2002). The neurohypophysis. In: Wass JAH, Shalet SM (eds) Oxford textbook of endocrinology and diabetes. Oxford University Press, Oxford.

SECTION 13  Endocrine disorders 2280 during pregnancy, there is degradation of the vasopressin by the pla- cental enzyme cysteine aminopeptidase; despite this, the hormone levels are often normal. Vasopressin concentrations increase with age, as does the response of vasopressin to osmotic stimulation. However, thirst recognition and thus fluid intake is reduced. These changes, along with a lessened ability to excrete a water load, pre- dispose older people to both hypernatraemia and hyponatraemia. Disorders of vasopressin secretion Diabetes insipidus Clinical manifestations and causes This is the passage of large volumes (>3 litres/​24 h) of dilute urine (osmolality <300 mosmol/​kg) and may be caused by: (1) deficient synthesis and/​or release of vasopressin (cranial diabetes insipidus); this is the most common form of polyuric and polydipsic dis- order which occurs mainly due to lesions of the neurohypophysis or the hypothalamic median eminence (usually 80–​90% of the magnocellular neurons of the hypothalamus need to be damaged before the manifestations of diabetes insipidus arise), (2)  renal resistance to the actions of vasopressin (nephrogenic diabetes insipidus), and (3) excessive fluid intake (primary polydipsia) when the hypotonic polyuria is an appropriate physiological response. A further type of diabetes insipidus is the gestational one and relates to the increased metabolism of vasopressin by the placental cysteine aminopeptidase. It usually develops in the second or third trimester of pregnancy and remits spontaneously 4 to 6 weeks after delivery. The clinical features of diabetes insipidus are polyuria, polydipsia, nocturia, and, in children, nocturnal enuresis and failure to thrive. The causes of cranial and nephrogenic diabetes insipidus are listed in Box 13.2.2.1. The most common causes of cranial diabetes insipidus are trauma (head injury, neurosurgery) and tumours. Removal of or damage to the posterior pituitary usually results in temporary diabetes insipidus lasting 6 weeks to 6 months as the proximal nerve endings grow out to capillaries in scar tissue formed and resume secretion. As hormone synthesis actually occurs higher up in the hypothal- amus, destruction here or at the level of the upper pituitary stalk or median eminence results in permanent diabetes insipidus. The most common solid tumour to produce diabetes insipidus is a craniopharyngioma. Suprasellar germinomas or pinealomas also commonly cause diabetes insipidus. Metastases to the pituitary 20 Plasma vasopressin (pmol/litre) 15 10 5 0 Lowest detectable level Normal detectable level Plasma osmolality (mosmol/kg) Theoretical threshold of vasopressin release 10 Thirst (cm) 8 4 2 0 Plasma osmolality (mosmol/kg) 6 280 300 320 280 300 320 Fig. 13.2.2.3  The relationship between plasma osmolality and plasma arginine vasopressin concentration, and between plasma osmolality and thirst. AVP concentrations and thirst sensation rise in a linear fashion in relation to plasma osmolality. This article was published in Clinical Endocrinology, Besser GM, Thorner MO (eds) pp. 5.1–​5.14, © Mosby-​Wolfe (2002). Box 13.2.2.1  Causes of cranial and nephrogenic diabetes insipidus Cranial diabetes insipidus Familial • Vasopressin-​neurophysin gene mutations (autosomal dominant) • DIDMOAD syndrome (diabetes insipidus, diabetes mellitus, optic at- rophy, deafness) (autosomal recessive) • X-​linked recessive Acquired • Trauma (head injury, neurosurgery, cerebral hypoxia) • Tumours (e.g. craniopharyngioma, germinoma, pinealoma, metastases) • Inflammatory conditions (e.g. sarcoidosis, Langerhans cell histiocytosis, tuberculosis, lymphocytic hypophysitis, Guillain–​Barré syndrome) • Infections (e.g. meningitis, encephalitis) • Autoimmune (antivasopressin neuron antibodies) • Vascular (aneurysm, arteriovenous malformations, infarction, Sheehan’s syndrome, sickle cell disease) • Drug/​toxin-​induced (e.g. ethanol, snake venom) • Idiopathic Nephrogenic diabetes insipidus Familial • X-​linked recessive (V2-​receptor defect) • Autosomal recessive (aquaporin-​2 defect) • Autosomal dominant (aquaporin-​2 defect) Acquired • Drugs (e.g. lithium, demeclocycline) • Metabolic (hypercalcaemia, hypokalaemia) • Chronic renal disease (polycystic kidneys, obstructive uropathy) • Osmotic diuresis (diabetes mellitus) • Infiltrative (e.g. amyloidosis, multiple myeloma, Sjogren’s disease)

13.2.2  Disorders of the posterior pituitary gland 2281 hypothalamic area are more likely to cause diabetes insipidus than a deficiency of anterior pituitary hormones because they lodge in the portal system of the hypothalamus. Lymphoma and infiltration with leukaemia are rare causes of diabetes insipidus. If the thirst centre is destroyed as part of the hypothalamic lesion (whether a tumour or any other cause), dangerous dehydration may ensue. Familial dia- betes insipidus is rare, accounting for approximately 5% of cranial diabetes insipidus, and may be caused by an autosomal dominant mutation in the vasopressin gene in the sequence encoding the precursor molecule or the signal peptide but not the peptide hor- mone itself. It is postulated that this causes abnormal folding of the precursor protein which then accumulates within the neurons and leads to cell death. As the pathology develops over time, diabetes insipidus becomes manifest when approximately 80% of the neurons have been destroyed. This is probably why symptoms are not present at birth but gradually develop between 1 and 6 years of age. Until recently there were two known causes of congenital nephro­ genic diabetes insipidus, an X-​linked recessive mutation of the V2 receptor, which accounts for 90% of cases, and an autosomal reces- sive mutation of the aquaporin-​2 water channels. The two types can be discriminated by an infusion of desmopressin which leads to an increase in blood pressure, and in circulating von Willebrand factor and factor 8 in the autosomal recessive condition. These effects are expressed via V2 receptor signalling and, therefore, will not be seen in the X-​linked form. Furthermore, an autosomal dominant muta- tion of the C-​terminal intracellular tail of aquaporin-​2 has been de- scribed. In all cases, in contrast to familial cranial diabetes insipidus, nephrogenic diabetes insipidus usually presents from birth with poly- uria and hypernatraemia. Without recognition the hypernatraemia, polyuria, vomiting, constipation, fever, irritability, and a failure to thrive may result in long-​term cognitive impairment. More com- monly nephrogenic diabetes insipidus is due to acquired metabolic or pharmacological causes. The most common drugs leading to nephro- toxicity and to diabetes insipidus are lithium and demeclocycline. In primary polydipsia, there is inappropriate ingestion of ex- cess fluid. This leads to a slight decrease in plasma osmolality and suppressed vasopressin secretion which results in polyuria. As the kidney can excrete up to 18 litres of dilute urine per day, serum osmolality is usually maintained in the normal range. The volumes of urine passing through the collecting duct reduce inner medulla osmolality and the sustained reduction in vasopressin release leads to less aquaporins in the collecting duct cells. These abnormalities lead to an inability to concentrate urine maximally. This dysfunction returns to normal within days to weeks of decreased fluid ingestion. The syndrome may occur as a behavioural abnormality in patients with psychiatric disease. Diagnosis Diabetes insipidus can be diagnosed by simultaneously measuring serum and urine osmolarity in patients with polyuria (> 3 litres per 24 hours). In the presence of high serum osmolarity (>295 mosmol/​litre), normally urine osmolarity should reach approximately 600 mosmol/​ litre (urine osmolality/​plasma osmolality should be ≥2). After the con- firmation of hypotonic polyuria, history and clinical presentation can provide useful diagnostic information. It should be noted however, that clinical data may be of limited value as patients with a preserved thirst mechanism may not differ biochemically to a significant de- gree. In complete absence of vasopressin, urine can reach a maximum dilution of 50 mosmol/​kg and the deficiency will lead to passage of anywhere between 3 and 20 litres of urine in 24 h. With unrestricted access to water, normal circulating volume and sodium concentration are maintained by an intact thirst centre. Cranial diabetes insipidus can be masked by cortisol deficiency as glucocorticoids are necessary for renal excretion of a water load. Therefore, diabetes insipidus may only become manifest with the introduction of corticosteroids. Before the vasopressin axis is investigated, other aetiologies of polyuria such as diabetes mellitus, renal failure, hypokalaemia, and hypercalcaemia should be excluded. Following this, the most common investigation to discriminate normality from the various causes of diabetes insipidus is the water deprivation test (Box 13.2.2.2). This dynamic test assesses the ability to concentrate urine during con- trolled water deprivation and is then followed by an assessment of re- sponse to exogenous vasopressin to confirm renal sensitivity. Cranial diabetes insipidus can be diagnosed with paired urine osmolality Box 13.2.2.2  Water deprivation test Preparation The patient is allowed fluids overnight (if primary polydipsia is suspected, consider fluid deprivation overnight to avoid morning overhydration). A light breakfast is taken at 6.30 a.m.; no tea, coffee, or smoking. Deprivation of fluids for 8 hours (starting from 8:00 a.m.—​check weight at the beginning of the test). Measure weight of patient and urine volume hourly during the test. Measure plasma and urine osmolalities every 2–​3 hours. At 4:00 p.m. administer desmopressin 2 mcg IM and allow the patient to drink freely. If plasma osmolality >305 mOsm/​kg or if 3% loss of body weight with plasma osmolality >305 mOsm/​kg, proceed to desmopressin adminis- tration earlier. If the urine output has not decreased and/​or urine osmolality/​plasma osmolality <2 but the plasma osmolality has not become concentrated to >295 mOsm/​kg, continue water deprivation for a further hour and measure plasma and urine osmolalities –​offer desmopressin after this. Continue measuring urine osmolality for the next 4 hours hourly (after the desmopressin administration) and measure hourly urine volumes. Stop test if >3% weight loss occurs. Plasma osmolality >295 mOsm/​litre with inappropriately hypotonic urine (urine osmolality/​plasma osmolality <2) during the fluid deprivation confirms diabetes insipidus (test is stopped). After administration of desmopressin, urine concentrates >800 mOsm/​kg in cases of central diabetes insipidus and <300 mOsm/​kg in nephrogenic diabetes insipidus. In partial diabetes insipidus or primary polydipsia, urine concentrates par- tially during the water deprivation (300–​800 mOsm/​kg) and further in- vestigations are needed (including prolonged water deprivation test or therapeutic trial of desmopressin).

SECTION 13  Endocrine disorders 2282 less than 300 mosmol/​kg and plasma osmolality more than 290 mosmol/​kg after dehydration; urine osmolality should rise more than 750 mosmol/​kg after desmopressin. However, if the urine osmo- lality does not rise more than 300 mosmol/​kg after dehydration and desmopressin, nephrogenic diabetes insipidus is confirmed. Patients with primary polydipsia should concentrate urine appropriately after dehydration, without a significant rise in plasma osmolality. There is no contraindication to the test providing the patient is fully hydrated. Interpretation also relies on normal thyroid and adrenal function; if function is impaired, the patient must be adequately treated before undergoing the test. In reality, further investigation is sometimes required, particularly when patients have partial forms of diabetes insipidus. Plasma vaso- pressin is measured directly in response to an infusion of 0.05 ml/​kg per min of 5% hypertonic saline for 2 h. In cranial diabetes insipidus, there is no increased vasopressin, whereas in nephrogenic diabetes insipidus the vasopressin level is high with no increased urine osmo- lality. Alternatively, the diagnosis of diabetes insipidus can be made with a therapeutic trial of desmopressin with monitoring of plasma and urine osmolalities and plasma sodium. Patients with primary polydipsia develop progressive dilutional hyponatraemia, whereas those with nephrogenic diabetes insipidus remain unaffected. In cranial diabetes insipidus, there is an improvement in polyuria and polydipsia. Imaging of the neurohypophysis with MRI should also be undertaken to identify any possible cause of cranial diabetes insipidus. Treatment In cranial diabetes insipidus where polyuria is mild (<4 litres/​24 h), patients with an intact thirst mechanism can be managed by advising an adequate fluid intake. With more severe symptoms, the treatment is the long-​acting vasopressin analogue desmopressin (1-​desamino-​8-​d arginine vasopressin), which acts predominantly on the V2 receptors in the kidney with almost no action at the V1 receptors in vascular smooth muscle. Desmopressin is given orally (100–​1000 µg daily), intranasally (10–​40 µg daily), or parenterally (0.1–​2 µg daily). There is wide individual variation in bioavail- ability and, therefore, dose required for symptom control. Women with pre-​existing cranial diabetes insipidus who become pregnant can be treated successfully with oral desmopressin which unlike the native hormone, is resistant to degradation by the cysteine aminopeptidase; the dose may be slightly higher than the one re- quired in the non​gravid state. The main adverse effect desmopressin is dilutional hyponatraemia, making monitoring of serum sodium and osmolality essential. In nephrogenic diabetes insipidus, causative drugs should be withdrawn although their effects are not always reversible. High-​ dose desmopressin (up to 5 µg intramuscularly) can be effective in partial nephrogenic diabetes insipidus. Thiazide diuretics which re- duce urine output by increasing sodium excretion can be helpful. In addition, prostaglandin synthase inhibitors, such as indomethacin may be effective as prostaglandins locally inhibit the renal actions of vasopressin. Diabetes insipidus with loss of thirst sensation (adipsic dia- betes insipidus) as a result of hypothalamic damage by tumours, infiltrative diseases, or neurosurgical interventions is a challenging condition managed by desmopressin and a prescribed regular fluid intake. These patients need to be monitored with daily weighing and fluid balance. In primary polydipsia, management is difficult, with reduced fluid intake being the only effective treatment; treatment of any underlying psychiatric condition is crucial. Syndrome of inappropriate antidiuretic hormone
secretion (SIADH) SIADH is a common cause of hyponatraemia and constitutes normovolaemic hyponatraemia as the increased water is dispersed though all compartments. The increased inappropriate vasopressin secretion leads to inappropriately concentrated urine, dilute plasma, and hyponatraemia with ongoing renal sodium excretion. The diag- nosis is considered when renal, adrenal, and thyroid function are normal; there is no evidence of volume overload or hypovolaemia and the biochemistry is consistent, that is, hyponatraemia and hypotonic plasma (osmolality <270 mosmol/​kg), inappropriate urine osmolality (>100 mosmol/​kg), and high urinary sodium (>20 mmol/​litre). There are many causes (Box 13.2.2.3) and thus the Box 13.2.2.3  Causes of SIADH Malignant disease • Carcinoma (lung, duodenum, stomach, pancreas, bladder, ureter, prostate) • Thymoma • Lymphoma, leukaemia • Mesothelioma • Sarcoma • Carcinoid Chest disease • Infection (pneumonia, tuberculosis, empyema) • Pneumothorax • Asthma • Positive pressure ventilation • Cystic fibrosis Central nervous system disorders • Head injury • Infections (meningitis, encephalitis, abscess) • Tumour • Vascular disorders (haemorrhage, thrombosis) • Guillain–​Barré syndrome • Acute intermittent porphyria • Psychosis • Hydrocephalus Drugs • Psychiatric drugs (phenothiazines, monoamine oxidase inhibitors, se- lective serotonin reuptake inhibitors) • Chemotherapy (vincristine, vinblastine, cisplatin) • Thiazides • Anticonvulsants (carbamazepine) • Clofibrate • Chlorpropamide • 3,4-​Methylenedioxymethamphetamine (MDMA, ‘ecstasy’) • Lansoprazole Other • Hypothyroidism • Glucocorticoid deficiency • Idiopathic • Abdominal surgery

13.2.2  Disorders of the posterior pituitary gland 2283 diagnosis itself should prompt a hunt for underlying pathology. For further discussion see Chapter 21.2.1. Initially the condition is most often asymptomatic, as devel- opment of hyponatraemia is gradual. However, as sodium falls to 120 mmol/​litre or less, it is associated with confusion, drowsiness, and seizures; rapid reduction in sodium or severe hyponatraemia can cause coma and death. Management of the condition is the same whatever the cause or type of SIADH. The underlying cause should be treated appropri- ately, and fluid restriction instituted to between 500 and 750 ml/​24 h. This generally restores sodium levels and osmolality within a few days. Very rarely, hypertonic saline infusion may be required if the hyponatraemia is acute and symptomatic. This approach requires great caution because an overly rapid correction of hyponatraemia may cause brain damage, ultimately leading to the osmotic demye- lination syndrome. See Chapter 21.2.1 for further discussion. If the symptoms are not temporary and long-​term fluid restric- tion is difficult for the patient, drugs such as demeclocycline that induce nephrogenic diabetes insipidus were used historically and can be effective, but this is no longer an appropriate approach. The most specific treatment for SIADH is to block the V2 receptor in the kidney. Vasopressin receptor antagonists, known as vaptans (e.g. Tolvaptan, a V2-​receptor antagonist, and conivaptan, a combined V1/​V2-​receptor antagonist), can effectively correct hyponatraemia, but caution is required and patients need to be monitored closely in order to avoid rapid correction or overcorrection. Further studies comparing vaptans with other available treatments are needed to de- termine the place of these agents in the management algorithm, and also to assure regarding their safety. A rare but important differential diagnosis of SIADH is cerebral salt wasting. This is a rare complication of pituitary surgery or more commonly occurs after subarachnoid haemorrhage. It tends to occur 5 to 10 days following a neurological event and is associated with hypovolaemia and hyponatraemia. It must be differentiated from SIADH as the treatments are quite different; fluid replacement in cere- bral salt wasting and fluid restriction in SIADH. The diagnosis of cere- bral salt wasting usually needs central venous pressure measurement as this demonstrates hypovolaemia compared with normovolaemia in SIADH. In cerebral salt wasting, the urinary sodium is often ex- tremely high and the plasma urate and haematocrit may be raised. FURTHER READING Bockenhauer D, Bichet DG (2015). Pathophysiology, diagnosis and management of nephrogenic diabetes insipidus. Nat Rev Nephrol, 11, 576–​88. Capatina C, et al. (2015). Diabetes insipidus after traumatic brain in- jury. J Clin Med, 13, 1448–​62. Christ-Crain M, et al. (2019). Diabetes insipidus. Nat Rev Dis Primers, 5(1), 54. doi: 10.1038/s41572-019-0103-2. Ellison DH, Berl T (2007). The syndrome of inappropriate antidiuresis. N Engl J Med, 356, 2064–​72. Endocrine Society (2016). Guidelines on management of hypopituit- arism. https://​www.endocrine.org/​news-​room/​press-​release-​archives/​ 2016/​endocrine-​society-​experts-​issue-​clinical-​practice-​guideline-​on-​ hypopituitarism Fenske W, Allolio B (2012). Clinical review: current state and future perspectives in the diagnosis of diabetes insipidus: a clinical review. J Clin Endocrinol Metab, 97, 3426–​37. Marlin BJ, et  al. (2015). Oxytocin enables maternal behavior by balancing cortical inhibition. Nature, 520, 499–​504. Peri A, Combe C (2012). Considerations regarding the management of hyponatraemia secondary to SIADH. Best Pract Res Clin Endocrinol Metab, 26, S16–​S26. Peri A (2013). The use of vaptans in clinical endocrinology. J Clin Endocrinol Metab, 98, 1321–​32.

13.3 Thyroid disorders 2284

13.3 Thyroid disorders 2284

13.3.1 The thyroid gland and disorders of thyroid

13.3.1 The thyroid gland and disorders of thyroid function 2284

CONTENTS 13.3.1 The thyroid gland and disorders of thyroid
function  2284 Anthony P. Weetman and Kristien Boelaert 13.3.2 Thyroid cancer  2302 Kristien Boelaert and Anthony P. Weetman 13.3.1  The thyroid gland and disorders of thyroid function Anthony P. Weetman and Kristien Boelaert ESSENTIALS The iodine-​containing thyroid hormones triiodothyronine (T3) and thyroxine (T4) have diverse effects on metabolism and are essen- tial for normal development, particularly of the fetal brain. The active principle, T3, binds to nuclear receptor isoforms and serves as a transcriptional regulatory factor, thus explaining the protean actions. Thyroid hormone release is regulated by thyrotropin (TSH) from the anterior pituitary, which is itself modulated by the hypothalamic tripeptide, thyrotropin-​releasing hormone. Thyroid hormones exert negative feedback control on the pituitary gland and on the synthesis of thyrotropin-​releasing hormone. A normal TSH level rules out primary thyroid dysfunction, but when TSH levels are abnormal, or when pituitary or hypothal- amic abnormalities are possible, it is essential to confirm thyroid status by measuring circulating thyroid hormone levels, which is best achieved by immunoassay of free T3 and free T4. Thyroid-​ antibody measurement and imaging by scintiscanning are useful in determining the aetiology of thyroid disease when this is not obvious clinically. Goitre Endemic goitre—​which is particularly common in the Himalayas, the Andes, and parts of Africa—​is mainly due to iodine deficiency, can cause massive thyroid enlargement, but rarely leads to compressive symptoms. Its main impact on health is the association with en- demic cretinism, which can be prevented by iodine supplementa- tion, achievable by iodization of salt or bread, intramuscular or oral iodized oil as a single annual dose, or iodination of drinking water. Sporadic goitre—​cause unknown; presentation is with neck swelling or sensation of pressure or discomfort; most patients are euthyroid and do not require treatment. Hypothyroidism Aetiology—​iodine deficiency and neonatal hypothyroidism remain major challenges for public health in many countries, but the most frequent cause of thyroid dysfunction in iodine-​sufficient areas is autoimmunity, where the follicular gland structure is destroyed by autoreactive T cells. Clinical features—​manifests in the adult with the gradual onset of a constellation of symptoms and signs including tiredness, feeling cold, weight gain, hoarseness of the voice, and slow-​relaxing tendon reflexes. A goitre may (Hashimoto’s thyroiditis) or may not (atrophic thyroiditis/​primary myxoedema) be present. Biochemical diagnosis of primary hypothyroidism is confirmed by a high serum TSH and a low free T4, with autoimmune hypothyroidism associated with the presence of thyroid peroxidase autoantibodies. Treatment is with levothyroxine (typically 100–​150 µg/​day for total hypothyroidism, but beginning with a lower dose in older people or those with heart disease). Myxoedema coma—​this is the most dramatic presentation of hypothyroidism and a medical emergency with high mortality: man- agement requires (1)  supportive treatment; (2)  identification and treatment of any precipitating condition, often infective; (3) paren- teral thyroid hormone replacement. Thyrotoxicosis Aetiology—​Graves’ disease, which is caused by TSH receptor stimu- lating autoantibodies, is responsible for 60 to 80% of cases, and nodular thyroid disease (toxic multinodular goitre and toxic ad- enoma) accounts for most of the rest. 13.3 Thyroid disorders

13.3.1  The thyroid gland and disorders of thyroid function 2285 Clinical features—​presents with a wide range of symptoms and signs including hyperactivity, palpitation, fatigue, weight loss (despite increased appetite), sinus tachycardia (or atrial fibrillation), tremor, and eye signs (including lid retraction and lid lag). Biochemical diag- nosis of primary hyperthyroidism is confirmed by a low serum TSH and a high free T4 and/​or T3, with autoimmune hyperthyroidism associated with the presence of thyroid peroxidase autoantibodies in many and anti-​TSH receptor antibodies in most patients with Graves’ disease. β-​blockers can rapidly relieve symptoms, but de- finitive treatment requires antithyroid drugs (usually carbimazole or propylthiouracil), radio-​iodine (131I), or surgery. Thyroid-​associated ophthalmopathy—​this often causes anxiety and social embarrassment, but severe cases are a threat to vision and may require treatment with corticosteroids, radiotherapy, other im- munosuppressive agents, or orbital decompression. Thyrotoxic crisis or storm—​this is the most dramatic presentation of hyperthyroidism and a medical emergency with high mortality. Manifestations include fever (>38.5ºC), delirium or coma, seizures, vomiting, diarrhoea, and jaundice. Management requires (1) sup- portive treatment; (2) identification and treatment of any precipitating condition, including infection; (3) antithyroid treatment (e.g. loading dose of carbimazole or propylthiouracil), followed 1 h later by stable iodine (e.g. Lugol’s iodine). Other conditions Acute thyroiditis—​usually caused by bacterial infection; presents with severe thyroid pain, fever, and malaise; thyroid function is rarely disturbed. Subacute (or de Quervain’s) thyroiditis—​due to viral infection and commonly presents with thyroid pain; there may be transient thyro- toxicosis, followed by hypothyroidism, before restoration of normal thyroid function; diagnosis depends on demonstration of raised inflammatory markers and low/​absent radio-​iodine uptake by the thyroid. Amiodarone—​inhibits T4 deiodination and hence leads to free T4 levels that are in the upper half of the reference range or mildly elevated; may cause hypothyroidism or hyperthyroidism, the latter being difficult to treat. Structure of the thyroid gland Development The human thyroid develops as a diverticulum in the pharyngeal floor at around 3 weeks of gestation. This median anlage moves caudally and remains connected to the pharynx via the thyroglossal duct, which is subsequently obliterated when the thyroid begins to expand as two distinct lobes at around 2 months of gestation. The foramen caecum marks the point in the tongue where the thyroid develops and there is sometimes an upward extension of thyroid tissue from the isthmus, the pyramidal lobe, arising from the lower part of the thyroglossal duct. At the same time, the lateral anlage ultimobranchial bodies, derived from the fifth branchial pouches, fuse with the developing thyroid to which they contribute the parafollicular calcitonin-​secreting clear cells. Synthesis of thyroid hormone begins at week 12, at the same time as TSH production by the pituitary. There is significant maternal-​to-​fetal T4 transfer so that babies with no endogenous thyroid hormone production are none- theless protected from the adverse effects of fetal hypothyroidism on development of the brain, lung, and skeleton. Preterm infants may have transient hypothyroxinaemia in the first weeks of life but trials of thyroid hormone supplementation have been inconclusive. Anatomy and histology The adult thyroid weighs 15 to 20 g; each lobe is around 4 cm long and 2 cm wide, although the right lobe is often larger than the left. The isthmus connecting the two lobes lies just below the cricoid car- tilage. The blood supply on each side is derived from the external ca- rotid artery via the superior thyroid artery and from the subclavian artery via the inferior thyroid artery. There is adrenergic and cholin- ergic innervation which regulates blood flow. The thyroid is attached to the trachea by connective tissue, and the recurrent laryngeal nerves lie between the trachea and the posterior aspect of the lobes. The gland is made up of lobules each comprising 20 to 40 spher- ical follicles. The follicles vary considerably in size, but average 200 µm in diameter, and are made up of a single layer of thyroid fol- licular epithelial cells (Fig. 13.3.1.1). The cells are cuboidal when quiescent and columnar when active, and have a microvillous apical membrane. The follicular lumen contains colloid, the principal con- stituent of which is the glycoprotein thyroglobulin secreted by the thyroid cells. Each follicle is surrounded by a rich capillary network. Clear cells lie scattered between follicular epithelial cells or in the interstitium, and account for around 1% of the epithelial mass. Thyroid hormone synthesis and metabolism Synthesis and secretion Thyroid hormone synthesis requires iodide uptake and oxidation, iodination of certain tyrosine molecules on thyroglobulin, and coupling of the iodotyrosines to form the thyroid hormones T3 and T4 (Fig. 13.3.1.2). Iodide is actively transported into the thyroid cell by the Na+/​I− symporter, which is also expressed in breast tissue and the salivary glands. Perchlorate, thiocyanate, and pertechnetate are also transported by the same symporter and these anions can Fig. 13.3.1.1  Histology of a normal thyroid. Thyroid epithelial cells are arranged in follicles containing colloid. Original magnification ×200. Photomicrograph by courtesy of Dr K. Suvarna.

SECTION 13  Endocrine disorders 2286 competitively inhibit iodide uptake. The World Health Organization (WHO) recommended daily intake of iodine is 150 µg for adults (250 µg during pregnancy and lactation) but there is wide variation in actual intake with many countries having borderline or frankly deficient intakes of less than 50 to 100 µg. In some parts of western Europe and North America intake has been excessive (up to 750 µg/​ day), although there is recent evidence that an increasing number of individuals in these countries remain at risk of iodine deficiency, especially during pregnancy. Iodide is oxidized by thyroid peroxidase, a haem-​containing enzyme located at the apical border of the thyroid cell, using hydrogen peroxide generated by dual oxidase (DUOX) and DUOX maturation factor 2, and is then rapidly incorporated into tyro- sine residues to form monoiodotyrosine and di-​iodotyrosine. Thyroid peroxidase is also responsible for the coupling of these iodotyrosines, with different sites in the thyroglobulin molecule being responsible for the formation of T3 or T4. Normally, each thyroglobulin molecule contains three or four T4 molecules, but only 20% of thyroglobulin molecules contain a T3 molecule. Thyroglobulin acts as slow turnover reservoir for thyroid hor- mone, thus ensuring maximum use is made of often scarce dietary iodine. Around a 7-​week supply of T4 is contained in the normal thyroid. Thyroid hormone is released from the gland after endo- cytosis of colloid and lysosomal hydrolysis of the thyroglobulin to yield T4 and T3, which are secreted from the basal membrane into the capillaries in a molar ratio of 14:1. Released iodotyrosines are deiodinated for iodide recycling. Thyroid hormone transport Up to 90% of the total T3 in the circulation is derived from per- ipheral conversion of T4 to T3 by deiodinase enzymes (see next) rather than thyroid secretion. Only 0.03% of T4 and 0.3% of T3 in the circulation exist as free hormone that is able to diffuse into tissues; the remainder is protein bound. T4 binds predominantly to T4-​binding globulin, and to a lesser extent to transthyretin (or prealbumin); a little is bound to albumin. T3 binds to T4-​binding globulin and albumin, with little bound to transthyretin. Alteration in the concentration or binding capacity of thyroid hormone-​ binding proteins can produce major changes in total but not free thyroid hormone levels (Table 13.3.1.1). Several transporters mediate thyroid hormone uptake by cells; monocarboxylate transporter 8 is particularly important in the uptake of T3 by brain, and mutations in this gene cause severe psychomotor 1 Iodide trapping 2 Oxidation 3 TG iodination 4 Coupling 5 Colloid resorption 6 TG hydrolysis 7 Deiodination of MIT+ DIT 8 Deiodination of T4 9 T3 and T4 secretion Follicular lumen Apical membrane containing TPO, catalysing steps 2–4 Thyroid epithelial cell TG synthesis MIT DIT I− T4 T3 9 8 6 1 I− 2 3 4 TG TG TG I− T3 T4 5 7 T4 T3 DIT MIT Fig. 13.3.1.2  Steps in the synthesis of thyroid hormones. DIT, di-​iodotyrosine; MIT, monoiodotyrosine; TG, thyroglobulin; TPO, thyroid peroxidase. Table 13.3.1.1  Conditions in which there is altered binding of thyroid hormones to binding proteins TBG Increased binding Genetic variation in TBG Oestrogens (pregnancy, oral contraception, hormone replacement therapy, tamoxifen) Other drugs (perphenazine, opiates,
5-​fluorouracil, clofibrate, mitotane) Hepatitis, cirrhosis Acute intermittent porphyria Decreased binding Genetic variation in TBG Steroids (testosterone, anabolic steroids, glucocorticoids) Acromegaly Nephrotic syndrome Protein malnutrition Acute severe illness L-​asparaginase Albumin Decreased binding Any cause of hypoalbuminaemia Increased binding Genetic variation Transthyretin Increased binding Genetic variation Competition for binding sites Drugs Phenytoin Carbamazepine Salicylates and non​steroidal anti-​inflammatory drugs Non​esterified fatty acids TBG, thyroid-​binding globulin.

13.3.1  The thyroid gland and disorders of thyroid function 2287 retardation and hypotonia due to brain-​specific hypothyroidism during development. Metabolism of thyroid hormone The half-​life of T4 in the circulation is 7 days, contrasting with the much shorter half-​life of T3 of 24 h. The most important metabolic pathway for T4 is outer ring (5′) deiodination to T3
(Fig. 13.3.1.3). This is catalysed by type 1 and type 2 deiodinase (EC 1.97.1.10), while type 3 deiodinase (EC 1.97.1.11) catalyses inner ring (5) deiodination leading to hormone inactivation. Type 1 deiodinase can also catalyse inner ring deiodination of T3 and T4. All three enzymes have a selenocysteine moiety as the active catalytic site. Type 1 deiodinase is expressed predominantly in the liver, kidney, thyroid, and brain, type 2 in the pituitary, brain, placenta, skeletal muscle, and heart (tissues critically dependent on thyroid hormone for development or function), and type 3 in the brain, placenta, and skin. The type 1 deiodinase is largely re- sponsible for the generation of circulating T3 from T4, whereas T3 generated by the type 2 enzyme mainly provides intracellular T3 at specific sites. Around 40% of T4 is metabolized to T3 and 40% is converted to reverse T3 by the type 3 deiodinase. This same enzyme is responsible for the main metabolic pathway for T3 which is converted to 3,3′-​ di-​iodothyronine. Starvation, trauma, and drugs (propylthiouracil, amiodarone, glucocorticoids, propranolol) impair T4 to T3 conver- sion and must be borne in mind when interpreting tests of thyroid function (see next). In addition to deiodination, a small proportion of thyroid hormone is metabolized by conjugation of the phenolic hydroxyl group with sulphate or glucuronic acid, which increases water solubility and allows urinary and biliary excretion. Biliary iodothyronine glucuronides can be reabsorbed, constituting an enterohepatic cycle. Thyroid hormone action Thyroid hormone acts primarily as a transcription regulatory factor, mediated by T3 binding to nuclear receptor isoforms that belong to the same superfamily as steroid and retinoic acid receptors. All such receptors possess a conserved DNA-​binding domain containing two zinc fingers, which interact with specific DNA response elem- ents, and a hormone-​binding domain. Alternative splicing results in two pairs of thyroid hormone receptors (Fig. 13.3.1.4) whose tissue expression varies during development. Thyroid hormone receptors bind to DNA as homodimers or heterodimers (with the retinoid X receptor). Without ligand, basal gene transcription is in- hibited by a corepressor. When T3 binds, homodimers dissociate releasing corepressor and allowing gene transcription; the stable heterodimer binds coactivators in the presence of T3 with the same outcome. The α2 thyroid hormone receptor does not bind T3 and may act as a natural inhibitor of receptor activity. A cell surface re- ceptor for T3, involving integrin αvβ3 and leading to protein kinase signal transduction, has been delineated and there are further path- ways for thyroid hormone action involving cytoplasmic and mito- chondrial receptors. DI1,DI2 T3 HO I I O CH COOH I DI3,(DI1) reverse T3 HO I I O CH COOH NH2 NH2 I HO I I O CH COOH T4 HO I(5′) I(3′) I(3) O I(5) CH COOH DI3,(DI1) di-iodothyronine (T2) DI1,DI2 NH2 CH2 CH2 CH2 NH2 CH2 Fig. 13.3.1.3  Main deiodination pathway for thyroid hormones. DI, deiodinase enzyme; parentheses denote a minor contribution. Deiodination of T3 also yields 3,5-​T2 and deiodination of reverse T3 also yields 3′,5′-​T2. T2 is further deiodinated to monoiodothyronine and thyronine. Tissue distribution Brain, muscle fat Brain, testis Liver, kidney Pituitary, hypothalamus 1 2 1 2 410 492 461 514 T3 binding domain DNA binding domain Fig. 13.3.1.4  Structure of the thyroid hormone receptors. The numbers indicate the amino acid content. Homologous areas are shaded; the lack of homology in the T3-​binding domain of the α2 receptor (hatched area) prevents T3 binding and the function of this receptor is unknown.

SECTION 13  Endocrine disorders 2288 Regulation of thyroid function The main regulator of thyroid function is TSH (thyrotropin), se- creted by thyrotrophs in the anterior pituitary gland in response to the tripeptide thyrotropin-​releasing hormone derived from the hypothalamic supraoptic and paraventricular nuclei. Thyroid hor- mones exert a classic negative feedback effect on thyrotrophs; the acute effect is mediated by T3 in the pituitary which is derived from T4 by type 2 deiodination. Thyroid hormones also inhibit hypo- thalamic thyrotropin-​releasing hormone synthesis. TSH secretion stimulated by thyrotropin-​releasing hormone is inhibited by dopa- mine and somatostatin, while α-​adrenergic activation stimulates TSH release. Cytokines, particularly interleukin-​1, interleukin-​6, and tumour necrosis factor, inhibit TSH synthesis and may be re- sponsible for the suppression of TSH seen in severe illness. Within the thyroid, TSH binds to the G protein-​coupled TSH re- ceptor, leading to intracellular signalling predominantly via cAMP. TSH increases iodide transport and organification, endocytosis of colloid, and thyroid hormone secretion, as well as thyroid follicular epithelial cell division. Autoregulatory mechanisms can modulate thyroid function when TSH levels are constant. The most important is iodine intake. Increased iodide transport transiently decreases organification and reduces thyroid hormone synthesis (the Wolff–​ Chaikoff effect); after several weeks under normal conditions, the thyroid escapes and resumes hormone production. Sudden in- creases in iodine intake can also acutely block thyroid hormone re- lease. In iodine deficiency, thyroid hormone production is switched to preferential T3 synthesis, but this effect is largely TSH-​mediated rather than autoregulatory. Laboratory investigation of thyroid function Determining thyroid status The introduction of sensitive immunoradiometric assays for circu- lating TSH, with a detection level of 0.1 mU/​litre or less, has trans- formed the evaluation of thyroid status. A normal TSH level rules out primary thyroid dysfunction. Low levels of thyroid hormones elevate TSH as a result of negative feedback, while excessive thyroid hormone suppresses TSH. The thyrotropin-​releasing hormone test for detecting low TSH levels is now redundant. As well as primary thyroid disorders, other conditions may alter TSH levels and must be borne in mind when using TSH as a screening test for thyroid dysfunction (Table 13.3.1.2), as must the possibility of secondary (pituitary or hypothalamic) disturbances of thyroid function. It is, therefore, essential to confirm thyroid status when TSH levels are abnormal, or when pituitary or hypothalamic abnor- malities are possible, by measuring circulating thyroid hormone levels. Methods which measure total T3 or T4 are prone to artefacts caused by abnormal thyroid hormone binding (Table 13.3.1.1), al- though in the absence of such abnormalities these tests are reliable. When altered binding is suspected or found, compensation can be made by calculation of the free T3 or free T4 index. These indices are derived from the total hormone levels and measurement of the differential distribution of radiolabelled T3 between unoccupied protein binding sites in the sample and an absorbent resin (hence the term ‘resin uptake test’). T4-​binding globulin levels can also be measured directly. However, the ready availability of immunoassays for free T3 and free T4 has generally supplanted these methods. The immunoassays rely on the ability of a radiolabelled thyroid hormone analogue to bind to thyroid hormone antibody but not to plasma binding pro- teins. The analogue then competes for antibody binding with the free thyroid hormone in the sample. Despite initial concerns about the theoretical basis and performance of such assays, recent im- provements allow generally reliable estimation of free thyroid hor- mones. In cases of doubt, free hormone levels can be measured by physical separation from bound hormone using ultracentrifugation or equilibrium dialysis. Several indirect methods can be used to determine thyroid status. The thyroidal uptake of radio-​iodine (123I, 131I) or 99mTc pertechnetate is increased in hyperthyroidism and decreased in hypothyroidism, but can be affected by excessive dietary iodine and destructive processes in the thyroid so that uptake is low when the patient is thyrotoxic (see ‘Destructive thyroiditis’). Serum thyro- globulin is raised in hyperthyroidism of all types but is also raised in destructive thyroiditis and thyroid cancer. Its main role in inves- tigation is follow-​up of treated thyroid cancer (see Chapter 13.3.2). Several non-​specific tests have also been used to determine end-​ organ responses to thyroid hormones, including basal metabolic rate, tendon relaxation time, and serum levels of cholesterol, ferritin, sex hormone-​binding globulin, and liver enzymes although these should not be used routinely to determine thyroid status. Thyroid function in non​thyroidal illness and pregnancy Assessing thyroid function in severely ill patients often reveals abnor- malities termed the ‘sick euthyroid syndrome’. Many of the changes are due to cytokine release, but therapeutic agents such as dopamine Table 13.3.1.2  Causes of abnormal serum TSH concentrations TSH level Cause Free thyroid hormone levels Raised Overt hypothyroidism ↓ Subclinical hypothyroidism N Sick euthyroid syndrome ↓ or N Dopamine antagonists (acute effect) N TSH-​secreting pituitary adenoma ↑ Thyroid hormone resistance syndrome ↑ Adrenal insufficiency ↓ or N Lowered Overt thyrotoxicosis ↑ Subclinical thyrotoxicosis N Recently treated hyperthyroidism N Thyroid-​associated ophthalmopathy without Graves’ disease N Excessive thyroxine treatment N or ↑ Sick euthyroid syndrome ↓ or N First trimester of pregnancy N or ↑ Pituitary or hypothalamic disease N or ↓ Anorexia nervosa N or ↓ Dopamine, somatostatin (acute effect) N Glucocorticoids N N, normal; TSH, thyroid-​stimulating hormone; ↑, increased; ↓, decreased.

13.3.1  The thyroid gland and disorders of thyroid function 2289 and glucocorticoids also contribute, as do unknown factors. Any major, acute illness or starvation can result in a decrease in circu- lating T3 (total and free) with normal levels of T4 and TSH. Reverse T3 levels rise. The severity of the illness correlates with the magni- tude of the fall in T3, and in very sick patients total T4 levels also fall. Analogue-​based free T4 assays generally produce normal results but sometimes high or low values occur. In 10 to 15% of sick individuals, TSH levels are abnormal (raised or lowered). Psychiatric illness can be associated with raised total and free T4 levels with normal T3. There is no proven benefit from thyroid hormone administration in the sick euthyroid syndrome and the hormone changes may be protective by limiting catabolism (although this view is regularly challenged). The importance of these alterations lies in their potential to cause diagnostic confusion. Thyroid function tests should only be requested in ill patients when thyroid disease is genuinely suspected. Abnormal thyroid function tests due to the sick euthyroid syndrome return to normal after recovery and, therefore, repetition of testing is the simplest way to confirm the reason for unusual results. Pregnancy also affects thyroid function testing. The most obvious change is the rise in T4-​binding globulin secondary to high oes- trogen concentrations, which elevates total but not free T3 and T4 levels. In addition, the reference ranges for free T3 and T4 are higher than normal in the first half of pregnancy because placental human chorionic gonadotropin, at high levels, acts as a weak stimulator of the TSH receptor. There is a reciprocal fall in TSH levels during the first trimester, but TSH returns to normal in the second trimester as human chorionic gonadotropin levels decline. Current guidelines advise the use of population and trimester-​specific reference ranges during pregnancy. Occasionally, the changes in circulating hormone concentrations are sufficiently pronounced to cause transient ‘ges- tational’ hyperthyroidism associated with high circulating HCG (human chorionic gonadotrophin) concentrations (e.g. in those with hyperemesis) during pregnancy. Antithyroid drugs are usually un- necessary in this condition, and attention should be directed to con- trolling the vomiting and giving parenteral fluids. Renal clearance of iodine is increased in pregnancy, leading to maternal and neonatal goitre and mild hypothyroidism in areas where iodine intake is mar- ginal (50 µg/​day). These complications can be prevented by ensuring an adequate iodine intake of 250 µg/​day during pregnancy. Determining the cause of thyroid dysfunction The most frequent cause of thyroid dysfunction in iodine-​sufficient areas is autoimmunity, and the simplest test for this is measurement of thyroid autoantibodies, particularly those directed against thyroid peroxidase. Antibodies against thyroglobulin are also easily meas- ured but are usually accompanied by thyroid peroxidase antibodies, so testing for the latter alone is usually adequate. Different methods, including haemagglutination, immunofluorescence, radioimmuno- assay, and enzyme-​linked immunosorbent assay, give different prevalence rates for thyroid autoantibodies. Almost all patients with autoimmune hypothyroidism and around 75% with Graves’ disease have thyroid peroxidase antibodies. Generally lower levels are found in 5 to 15% of healthy women and 2% of men, and in slightly higher proportions of patients with nodular goitre and thyroid cancer, and results therefore need to be interpreted carefully. Individuals with positive thyroid autoantibodies but normal thyroid function are at increased risk of developing autoimmune hypothyroidism (c.2% per year). Measurement of antibodies to the TSH receptor may be useful to confirm an underlying diagnosis of Graves’ disease and reliable assays to perform this test are now widely available. Thyroid imaging by scintiscanning is useful in determining the aetiology of thyroid disease when this is not obvious clinically, par- ticularly in hyperthyroidism and ectopic thyroid tissue. Its role in the evaluation of a solitary thyroid nodule is considered in Chapter 13.3.2. 99Tcm pertechnetate is usually used as it has a short half-​life (6 h) which allows safe administration of high activity and rapid scanning. 123I is not as readily available but is preferable to 131I, especially in children, as it too has a short half-​life and does not emit β-​radiation. Thyroid ultrasound is being increasingly used as an alternative to scintiscanning. The technique allows accurate determination of thy- roid size, which may be useful in follow-​up of goitre, and can help to determine the nature of an atypical neck mass. Its role in evaluating nodular thyroid disease is considered in Chapter 13.3.2. CT scanning is particularly valuable in determining the extent of a retrosternal goitre and assessing tracheal compression (Fig. 13.3.1.5). In contrast, a standard chest radiograph can be misleading in evaluating tracheal compression, particularly in the anterior–​posterior plane. Goitre The distribution of thyroid size in any population forms a continuous, positively skewed curve, whose shape depends on the age, sex, and country of residence of the individuals assessed. Hence a precise definition of goitre is impossible. Ultrasound is the most accurate method to assess thyroid size, and estimates of goitre prevalence based on inspection and palpation underestimate the true frequency. However, simple schemes, such as that shown in Box 13.3.1.1, are useful in field studies of goitre prevalence. Fig. 13.3.1.5  CT scan of the chest of a patient with a large retrosternal goitre causing tracheal compression. Box 13.3.1.1  WHO/​UNICEF grading of goitre Grade 0 = no visible or palpable thyroid Grade 1 = thyroid enlargement that is palpable but not visible when the neck is in the neutral position Grade 2 = thyroid enlargement that is both visible and palpable when the neck is in the neutral position Grade 3 = goitre visible at a considerable distance

SECTION 13  Endocrine disorders 2290 Of the many causes of goitre (Box 13.3.1.2), those associated with disturbances of thyroid function are considered later. The remainder can be classified broadly as endemic and sporadic non​toxic goitres. Endemic goitre Prevalence Goitre is said to be endemic when the prevalence exceeds 10% in children aged 6 to 12 years, although this figure is arbitrary and it has recently been suggested that a prevalence of more than 5% should be used. Over 200 million people are affected worldwide, especially in the Himalayas, Andes, and parts of Africa, although Eastern and Southern Europe are also involved. Aetiology The main cause is iodine deficiency, with goitre prevalence ex- ceeding 30% in areas with very low iodine intakes (<30 µg/​day). However, endemic goitre is not exclusively related to iodine defi- ciency. Naturally occurring goitrogens, such as those in vegetables of the cabbage family and in cassava and millet, exaggerate the effects of iodine deficiency by the action of thiocyanates and fla- vonoids on thyroid hormone synthesis. Where selenium and iodine deficiency coincide, thyroid cell destruction and gland fibrosis min- imize goitre formation. In Japan, endemic goitre actually results from iodine excess, as well as goitrogens in seaweed, and in Kentucky, chemically polluted water from the coal industry is goitrogenic. Clinical presentation Diffuse goitre is more frequent in girls, and gradually becomes nodular with age and increasing iodine deficiency. Endemic goitres can be massive but give few compressive symptoms. In areas of mar- ginal iodine deficiency, such as Belgium, a modest goitre only ap- pears when demands on thyroidal iodide metabolism are increased during puberty or in pregnancy. The major impact of endemic goitre and iodine deficiency on health is the association with endemic cretinism. Two forms of cret- inism can be delineated in separate geographical areas, but there is considerable overlap. First, when maternal iodine intake is severely reduced causing hypothyroidism there is reduced placental transfer of T4 to the fetus, resulting in a profound neurological deficit in the infant, with mental deficiency, deafness, speech defects, and spastic gait. Second, hypothyroidism in the infant after birth produces the typical features of cretinism, in particular stunted growth. The thy- roid in such patients may be enlarged or atrophic and it is clear from field studies that iodine deficiency alone cannot account for the multiple forms of endemic cretinism. Management Iodine supplementation is perhaps the simplest and cheapest of all remedies and it prevents a condition that has devastating con- sequences; it is sobering that iodine deficiency still persists. There are few complications from iodine supplementation, although thyrotoxicosis may result in a variable proportion of individuals (the Jod–​Basedow phenomenon) some of whom have avoided this previously through lack of sufficient iodine. Political, social, and economic inertia are at the heart of continuing iodine defi- ciency. Effective programmes are best targeted at children and women intending pregnancy. Iodization of salt or bread is widely used in developed countries, but intramuscular or oral iodized oil, as a single annual dose, or iodination of drinking water is preferable in areas where distribution of iodized foodstuffs is a problem. Sporadic goitre Prevalence Goitre occurs in around 5% of the iodine-​sufficient population and is four times more common in women. However, the preva- lence varies with area and generally declines with age; over 60% of goitres found in adolescents regress over the next 20  years. The character also changes over time, from a diffuse (sometimes called simple) goitre to a multinodular goitre. The presentation of single thyroid nodules is dealt with in Chapter 13.3.2, but it is worth mentioning here that solitary thyroid nodules increase in frequency with age. Aetiology The aetiology of sporadic goitre is largely unknown. Unidentified goitrogens may be responsible in a few patients, and in others mild iodine deficiency in infancy may initiate goitrogenesis which persists despite a subsequently normal iodine intake. A  large proportion are probably the result of mild defects in hormone synthesis; compensatory growth ensures normal thy- roid function and current tests cannot identify the nature of the defect. Familial clustering of sporadic goitre supports this idea. Although TSH is the most obvious thyroid growth factor, TSH levels by definition are normal in sporadic goitre, which may therefore be the result of other autocrine and paracrine growth Box 13.3.1.2  Causes of goitre Endemic goitre Iodine deficiency Goitrogens, including drugs with an antithyroid action Sporadic goitre Simple, non​toxic goitre: diffuse or multinodular (colloid goitre) Toxic multinodular goitre Hashimoto’s thyroiditis Graves’ disease Destructive thyroiditis Postpartum thyroiditis Silent thyroiditis Subacute thyroiditis Amiodarone Genetic disorders Dyshormonogenesis Thyroid hormone resistance syndrome McCune–​Albright syndrome TSH receptor mutation Infiltration Riedel’s thyroiditis Amyloidosis Sarcoidosis Secondary TSH-​secreting pituitary tumour Excessive stimulation from human chorionic gonadotropin in pregnancy or choriocarcinoma

13.3.1  The thyroid gland and disorders of thyroid function 2291 factors (e.g. insulin-​like growth factor-​1, epidermal growth factor, fibroblast growth factor). Progression to a multinodular goitre occurs when unencapsulated nodules form in a long-​standing diffuse goitre. These nodules con- tain colloid-​rich polyclonal follicles and are usually distinct from ad- enomas, which are encapsulated and derived from a single thyroid follicular cell with a somatic mutation conferring growth advan- tage. However, some goitres contain both nodules and adenomas, suggesting a spectrum of pathological changes. Because thyroid follicular cells are heterogeneous, nodules generally develop with varying degrees of function, giving rise to ‘hot’ and ‘cold’ areas on scintiscanning with radio-​iodine. Some nodules develop autonomy and may eventually cause hyperthyroidism, completing the evolu- tion from non​toxic to toxic multinodular goitre (see next). Other nodules undergo degeneration with haemorrhage, fibrosis, and cyst formation. Clinical presentation Patients usually seek attention because of the appearance of the neck or a sensation of pressure or discomfort. Equally, they may be unaware of a long-​standing small goitre which is noticed on exam- ination. Careful palpation is sufficient to distinguish true goitre, which moves on swallowing, from a prominent pad of fat over the front of the neck. Very large goitres can cause dysphagia or even stridor when the trachea is compressed, but these symptoms are uncommon. Venous compression at the thoracic inlet is even rarer; this sign is exacerbated by asking the patient to raise his/​her arms (Pemberton’s sign). Pain in the thyroid, which radiates to the jaw, is uncommon and suggests either destructive thyroiditis (see next) or haemorrhage into a cyst in a multinodular goitre. In the latter, the pain is usually unilateral, acute, and associated with a rapid change in thyroid size; symptoms resolve spontaneously in a few days. Investigations Thyroid function should be assessed by checking TSH levels, and then free T3 and T4 levels if the TSH is abnormal to rule out goitre associated with thyroid dysfunction. The presence of thyroid per- oxidase antibodies is also useful as a marker of an underlying auto- immune thyroiditis, which occurs in 10 to 20% of multinodular goitres. The use of imaging varies between centres. Ultrasound is useful in determining thyroid size and nodularity accurately and may reassure an anxious patient that the thyroid is not enlarging. Thyroidal uptake of radioisotopes (especially 99Tcm pertechnetate) is indicated if destructive thyroiditis is suspected as a cause of goitre. Otherwise, the major role for imaging is to ensure there is no tra- cheal compression or intrathoracic/​retrosternal component in a pa- tient with suggestive symptoms, and a CT scan is then the preferred investigation (Fig. 13.3.1.5). Treatment Most patients with euthyroid sporadic goitre do not require treat- ment. Neck discomfort or cosmetic concerns may prompt inter- vention but it is necessary to take a careful history to ensure that discomfort or difficulty swallowing is indeed caused by the goitre. T4, given at doses to maintain slightly suppressed TSH levels (0.1–​0.3 mU/​litre), leads to a reduction in goitre size in up to 60% of patients but is unlikely to have any effect on a very nodular goitre or when the TSH level is already low (so-​called subclinical hyperthyroidism, discussed next). This treatment is now much less used than previously as there are concerns about the long-​term ef- fects of suppressive doses of T4 on the heart and skeleton, and treat- ment must be continued long-​term to maintain any improvement. Radio-​iodine, by contrast, is increasingly being given to reduce goitre size. Doses of 131I range from 600 to 3400 MBq (hospitalization is required for doses >800 MBq). Recombinant TSH administration may allow lower 131I doses to be given, increasing the potential for outpatient treatment. Goitre size is usually reduced by more than 50% at 2 years, and most of the improvement occurs within 2 to 3 months. Long-​term follow-​up data are not yet available, although hypothyroidism certainly occurs in 20 to 40% by 5 years. Tracheal compression by a goitre can be treated with 131I despite theoretical concerns over acute worsening due to a radiation thyroiditis. Surgery is used in other centres and is particularly indicated for stridor, severe tracheal compression, or retrosternal goitres and if there is any suspicion of malignancy. Thyroidectomy is the most ef- fective treatment available for goitre, but there may be a recurrence in around 20% of patients within 10 years and is not avoidable by giving T4 replacement. Complications, including recurrent laryn- geal nerve damage, hypoparathyroidism, and hypothyroidism, are more likely with the biggest goitres, near-​total thyroidectomy, and reoperation. Hypothyroidism Impaired production of thyroid hormones is usually due to a pri- mary abnormality of thyroid gland or iodine deficiency; occasion- ally it is secondary to pituitary or hypothalamic disorders, dealt with in Chapters 13.2.1 and 13.2.2. The onset of primary hypothyroidism is gradual and may be detected when the TSH is elevated (to com- pensate for impaired thyroid output) but the free thyroid hormone levels are normal. This state is subclinical hypothyroidism (diag- nosis depends on ensuring that the TSH elevation is not due to a sick euthyroid syndrome by repeat measurement after 3 months). As thyroid damage continues, TSH levels rise further but free T4 levels fall. When serum TSH concentrations rise above 10 mU/​litre, symp- toms usually become apparent, and the patient is said to have overt or clinical hypothyroidism. Aetiology The causes of hypothyroidism are listed in Box 13.3.1.3. The com- monest cause worldwide is iodine deficiency, discussed earlier. In iodine-​sufficient areas, autoimmune hypothyroidism and thyroid damage after radio-​iodine or surgical treatment for hyperthyroidism are the major causes. Epidemiology The prevalence of overt hypothyroidism in white populations is around 2% in women and 0.2% in men, with a mean age of 60 at diagnosis: rates are lower among black and Asian/​Pacific Islander populations. Subclinical hypothyroidism is even more common (6–​8% of women and 3% of men). Around 4% of these individuals progress to overt hypothyroidism annually if thyroid peroxidase antibodies accompany the elevated TSH. Half this number progress in the absence of thyroid peroxidase antibodies. Focal lympho- cytic infiltration of thyroid associated with thyroid autoantibody

SECTION 13  Endocrine disorders 2292 positivity occurs in up to 15% of healthy women and 2% of men without an elevated TSH level, representing the earliest manifest- ation of thyroid autoimmunity; 2% of these people progress to overt hypothyroidism annually. Congenital hypothyroidism occurs in about 1 in 3000 births and this high frequency has led to the widespread introduction of neonatal screening; recent increases in prevalence are likely to be due to the inclusion of milder cases. Pathogenesis Autoimmune hypothyroidism is primarily the result of auto­ reactive T-​cell-​mediated cytotoxicity directed against thyroid follicular cells. Cytokines derived from the locally infiltrating T cells, macrophages, and dendritic cells impair thyroid cell func- tion and enhance T-​cell-​mediated cytotoxicity. The role of thyroid autoantibodies in thyroid cell destruction is unclear, but thyroid peroxidase antibodies fix complement and may cause secondary damage. In 10 to 20% of patients, antibodies which block the TSH receptor are partially or wholly responsible for hypothyroidism, and transplacental passage of these antibodies (but not thyroid peroxidase antibodies) occasionally causes transient neonatal hypothyroidism. Genetic and environmental factors are in- volved in the aetiology but, as with most autoimmune disorders, the complex interaction of these factors has so far prevented a full understanding. Polymorphisms in HLA-​DR, CTLA4 and other immunoregulatory genes are associated with autoimmune hypothyroidism, and a high iodine intake may be an important environmental factor in some cases. Congenital hypothyroidism is caused by thyroid aplasia or hypoplasia in 60% of cases and in 30% there is an ectopic gland. Mutations in thyroid-​specific transcription factors have been found in some of these cases. In the remaining 10%, hypothyroidism is due to dyshormonogenesis (see Box 13.3.1.3). Clinical features The cardinal features in adults with hypothyroidism are shown in Box 13.3.1.4. However, the ready availability of reliable screening tests for hypothyroidism, especially TSH assays, has led to the recog- nition of many patients in whom there are only vague or non​specific symptoms, such as tiredness, weight gain, and poor concentration. The differential diagnosis is accordingly vast, but the high frequency of hypothyroidism should prompt its exclusion when any suggestive features are present, particular in middle-​aged women with chronic fatigue or depression. Autoimmune hypothyroidism may present with a goitre (Hashimoto’s thyroiditis) or without (atrophic thyroiditis or primary myxoedema); these entities represent the ends of a spectrum of pro- gressive thyroid destruction. When present, the goitre is of variable size but is often hard and irregular, sometimes giving rise to a sus- picion of malignancy, which then requires exclusion by fine needle aspiration biopsy. Primary lymphoma of the thyroid is a rare but important association (Chapter 13.3.2). Thyroid pain due to auto- immune thyroiditis is also a rare complication. Patients may notice a Hashimoto goitre before any thyroid dysfunction has developed and annual follow-​up is then needed. The most dramatic presentation of hypothyroidism is myxoe- dema coma, which is fortunately rare. In addition to the usual fea- tures, there is hypothermia (as low as 23 °C) and coma, sometimes with seizures. Mortality is 30–​50% even with intensive treatment. Patients are typically older and either previously undiagnosed or poorly compliant with medication. There is generally an additional precipitant, such as respiratory depression due to drugs, chest infec- tion, heart failure, stroke, blood loss, or exposure to cold. Box 13.3.1.3  Causes of hypothyroidism Primary Iodine deficiency Autoimmune hypothyroidism Hashimoto’s thyroiditis Primary myxoedema Iatrogenic 131I treatment Subtotal or total thyroidectomy External irradiation for lymphoma or cancer involving the neck Drugs Iodine-​containing contrast media Amiodarone Lithium Antithyroid drugs p-​Aminosalicylic acid Interferon-​α and other cytokines Aminoglutethimide Tyrosine kinase inhibitors (e.g. sunitinib) Congenital hypothyroidism Absent or ectopic thyroid gland Dyshormonogenesisa TSH receptor mutation Destructive thyroiditis Postpartum thyroiditis Silent thyroiditis Subacute thyroiditis Infiltrative disorders Amyloidosis Sarcoidosis Haemochromatosis Scleroderma Cystinosis Riedel’s thyroiditis Consumptive hypothyroidism due to increased type 3 deiodinase ex- pression (e.g. infantile haemangiomas) Secondary Hypopituitarism Pituitary tumours Trauma (head injury) Pituitary surgery or irradiation Infiltrative disorders Infarction Isolated TSH deficiency or inactivity Hypothalamic disease Idiopathic Drugs Bexarotene a The following types of dyshormonogenesis are due to mutations in the genes encoding the proteins given in parentheses: iodide transport defect (Na+/​I− symporter), defective iodide organification (thyroid peroxidase, dual oxidase 2, pendrin), loss of iodide reutilization (dehalogenase), deficient thyroid hormone synthesis (thyroglobulin). Defects in monoiodotyrosine coupling also occur but are, so far, poorly characterized.

13.3.1  The thyroid gland and disorders of thyroid function 2293 Autoimmune hypothyroidism is frequently associated with other autoimmune conditions. In the type 2 autoimmune polyglandular syndrome, autoimmune thyroid disease (hypothyroidism or Graves’ disease) is associated with type 1 diabetes and/​or Addison’s disease. This syndrome is autosomal dominant with variable penetrance. In the rare, autosomal recessive type 1 autoimmune polyglandular syn- drome (chronic mucocutaneous candidiasis, Addison’s disease, and hypoparathyroidism), autoimmune hypothyroidism is found in 5 to 10% of patients. Other commoner associations include pernicious anaemia, vitiligo, and alopecia areata, and there is a significant ex- cess of autoimmune hypothyroidism in coeliac disease, dermatitis herpetiformis, chronic active hepatitis, premature ovarian failure, rheumatoid arthritis, systemic lupus erythematosus, and Sjögren’s syndrome. Breast cancer patients and individuals with Down’s and Turner’s syndromes have a higher than expected frequency of thy- roid autoimmunity. Around 5% of patients with thyroid-​associated ophthalmopathy, discussed later in this chapter, have autoimmune hypothyroidism and 15% of patients with Graves’ disease success- fully treated with antithyroid drugs develop hypothyroidism 10 to 20 years later. This relationship with Graves’ disease is further em- phasized by rare patients who oscillate between hyperthyroidism and hypothyroidism over a period of months. The likely explanation is fluctuation in the relative levels of TSH receptor stimulating and blocking antibodies, but the cause of these changes is unknown. Juvenile hypothyroidism is uncommon. The features of adult hypothyroidism (Box 13.3.1.4) may be present, but the diagnosis is usually suggested by retarded growth and dentition, and an infantile face. Myopathy with muscle enlargement is common. Puberty is usually delayed, although sometimes it is precocious. Congenital hypothyroidism is typically unrecognizable at birth but, if not iden- tified by screening, gives rise to prolonged jaundice, failure to thrive, impaired growth, feeding difficulties, constipation, and hypotonia. Left untreated, even for a few weeks after birth, there is permanent neurological damage resulting in intellectual impairment. Pathology In Hashimoto’s thyroiditis there is a prominent diffuse and focal lymphocytic infiltrate with germinal centre formation. The thyroid follicles show varying degrees of destruction and little or no colloid. The remaining thyroid follicular cells have an increased number of mitochondria, giving rise to oxyphil metaplasia (Askanazy or Hürthle cells). There is a variable degree of fibrosis. In atrophic thyroiditis, fibrosis is the most prominent feature, with a less ob- vious lymphocytic infiltrate than in Hashimoto’s thyroiditis. Thyroid follicles are usually sparse, reflecting the later stage at which this form of autoimmune hypothyroidism is diagnosed. Whether there is a natural progression from Hashimoto’s to atrophic thyroiditis is unclear, although the goitre usually decreases with T4 replacement. Laboratory diagnosis Measuring serum TSH is the first step in diagnosing hypothyroidism, with the important caveat that this approach will miss most cases of secondary hypothyroidism in which the serum TSH measured by immunoassays may be low, normal, or even slightly raised, due to the secretion of bioinactive forms of the hormone. If secondary hypothyroidism is suspected, for instance in the follow-​up of a pa- tient with treated pituitary disease, it is essential to check the free T4 level. The TSH is elevated in other settings besides primary overt hypothyroidism (Table 13.3.1.2). It is therefore important to confirm the diagnosis by measuring the free T4 in all samples in which the TSH is elevated. Measurement of free T3 adds nothing to the diagnosis, es- pecially as values may be within the reference range in a quarter of hypothyroid patients due to extrathyroidal conversion of T4. If myxoedema coma is expected, it is essential that treatment is initiated immediately without awaiting confirmation of the diagnosis. These patients often have dilutional hyponatraemia, hypoglycaemia, and electrocardiography changes (low voltage, prolonged QT interval, flat or inverted T waves, and heart block). Other non​specific features which may be found in any patient with hypothyroidism are elevation in serum liver and muscle enzymes (raised creatine phosphokinase concentrations particularly may cause unnecessary concern), raised cholesterol, and anaemia. The anaemia is usually normocytic or macrocytic, but microcytosis oc- curs when hypothyroidism is accompanied by menorrhagia. The aetiology is usually easily established. In the absence of a his- tory of treated hyperthyroidism or iodine exposure, most juvenile or adult onset primary hypothyroidism in iodine-​sufficient countries is due to autoimmune hypothyroidism. Transient hypothyroidism due to destructive thyroiditis is considered later. The diagnosis of auto- immune hypothyroidism is confirmed by the presence of thyroid peroxidase antibodies, usually at high levels, although occasionally these antibodies are absent. Cytological diagnosis of Hashimoto’s thyroiditis is possible using fine needle aspiration biopsy, but is only necessary if there is uncertainty over the cause of a nodular goitre. Once congenital hypothyroidism is diagnosed by routine testing after birth, it is usual to initiate T4 immediately. The aetiology can be Box 13.3.1.4  Clinical features of hypothyroidism Symptoms Tiredness, weakness Dry skin Altered facial appearance Feeling cold Hair dry, unmanageable, and thinning Poor memory and concentration Constipation Weight gain with poor appetite Dyspnoea Hoarse voice Menorrhagia (later, oligomenorrhoea or amenorrhoea), decreased libido Paraesthesias Deafness Signs Dry coarse skin Cool peripheries Puffy face, hands, and feet Yellow skin due to carotene accumulation Diffuse alopecia Bradycardia Peripheral oedema Slow-​relaxing tendon reflexes Carpal tunnel syndrome Serous cavity effusions Galactorrhoea (raised prolactin) Enlarged salivary glands Rarely: ataxia, dementia, psychosis, coma

SECTION 13  Endocrine disorders 2294 established by scintiscanning and/​or ultrasound: if the exact cause is not established at birth, treatment can be stopped without neuro- logical consequences at age 3 to 4 years to repeat imaging and establish whether life-​long T4 replacement is necessary. Dyshormonogenesis, suspected when there is detectable thyroid tissue and a family his- tory, requires specialized investigation to establish the diagnosis and increasingly this is possible by direct analysis of gene mutations. The commonest of these defects is Pendred’s syndrome in which there are mutations in the pendrin gene (SLC26A4) encoding a chloride/​ iodide transporter present in the thyroid and cochlea, leading to goitre, mild hypothyroidism, and deafness. The thyroid abnormal- ities usually appear in the second or third decade, rather than at birth. The diagnosis can be made easily by the perchlorate discharge test, which shows an excessive decline of radioactivity in the thyroid when potassium perchlorate is given 2 to 3 h after allowing the thy- roid to take up a tracer dose of radio-​iodine. Treatment In adult patients without heart disease and below the age of 65, treat- ment can begin with the estimated replacement dose of T4. If there is no remaining thyroid tissue (indicated by a very high TSH level and very low or undetectable free T4), the daily replacement dose is 1.6 µg T4/​kg body weight, which is around 100 to 150 µg/​day. In practice, the typical starting dose is 50 to 100 µg T4 daily, the lower dose being re- served for patients with mild to moderate biochemical abnormalities and those with significant cardiac disease. Dosage changes should be based on TSH levels measured 2 to 3 months after starting treatment, the main goal of treatment being to normalize the TSH. A similar period is required to assess the effect of any change to the dosage, made as 25 or 50 µg increments or decrements depending on the degree of abnormality of the TSH. Treatment is usually straightfor- ward, although if there is only partial thyroid failure when treatment is begun, the dose of T4 may require adjustment over many months. Once on a full replacement dose, TSH levels should be checked an- nually. Fluctuating or elevated TSH levels in a previously stable patient, or T4 requirements in excess of 200 µg/​day, usually indicate adher- ence problems. It is important to rule out malabsorption, including coeliac disease, Helicobacter pylori infection, excessive soya intake, or interactions with drugs: cholestyramine, ferrous sulphate, lovastatin, aluminium hydroxide, rifampicin, amiodarone, carbamazepine, and phenytoin all alter the absorption or clearance of T4. A common cause for poor adherence is worsening angina. Optimization of antianginal treatment is then required, although some patients may simply prove intolerant of full T4 replacement if their coronary artery disease is ex- tensive and irremediable. It is important to remind poorly adherent patients that, because of the long half-​life of T4, missed tablets should always be taken and that this is safe. It should be emphasized that, in the absence of coronary artery disease, T4 has no adverse effects when given at doses that return TSH levels to normal. In older patients or in individuals with heart disease, the usual starting dose is 25 µg T4 daily (or on alternate days when there is severe angina), although in the elderly person without any car- diac comorbidity such caution is probably unnecessary. Dosage should be increased slowly with increments of 12.5 to 25 µg T4. Proportionately higher doses of T4 are needed during the first year of life than in adults, and the starting daily dose of T4 for con- genital hypothyroidism is 8–​15 µg/​kg body weight. There is a con- tinuing debate on the benefit of T4 in subclinical hypothyroidism. It is reasonable to commence T4 when (i) the TSH levels are above 10 mU/​litre or (ii) above 5 mU/​litre prior to and during pregnancy. When TSH levels are below 10 mU/​litre, routine treatment is not indicated, but this could be considered if patients have symptoms suggestive of hypothyroidism or positive TPO antibodies. Modest improvements in mental function and lipid levels occur when T4 is given to some patients with subclinical hypothyroidism, but con- clusive long-​term studies on the benefits of treatment have not been conducted. All patients with subclinical hypothyroidism or positive thyroid peroxidase antibodies should be offered annual testing for the development of overt hypothyroidism if T4 is not given. Another problem is posed by the occasional patient with overt hypothyroidism who continues to feel unwell or who fails to lose weight after the TSH is normalized with T4 replacement. It can take around 3 months from achieving full replacement for all symptoms to disappear, and weight gained during hypothyroidism will gener- ally only be lost by following an appropriate diet. It is sensible to en- sure that the TSH level is in the lower half of the reference range and sometimes a small increment of T4 can achieve this, improving symp- toms but not suppressing the TSH. Higher doses of T4 that suppress the TSH should be avoided, as there is an increased risk of atrial fibril- lation due to subclinical thyrotoxicosis. The other recognized adverse effect of excessive T4 is a decrease in bone mineral density, particu- larly in postmenopausal women who have previously had hyperthy- roidism and therefore already have a low skeletal mass. Changes in bone mineral density are modest but an increase in fracture rate has been reported as a result of T4 given at supraphysiological doses in those aged over 65. There has some recent interest in the concept that thyroid hormone replacement should consist of both T4 and T3, based on the observation that deiodinase activity varies between tis- sues, suggesting that in some organs the level of the active thyroid hormone, T3, is insufficient when only T4 is given. The short half-​life of current T3 preparations makes T3 alone unsuitable for replacement and there is no evidence of any consistent benefit from trials of com- bined T4 and T3 treatment. Treatment of myxoedema coma is a medical emergency (see Box 13.3.1.5). Prognosis T4 treatment is usually life-​long and, properly taken, restores normal health and lifespan. Occasional patients may discontinue T4 and re- main euthyroid. Errors in initial diagnosis account for some of these; in others, a spontaneous decline in TSH receptor blocking antibody levels may be responsible. There is no easy means of ascertaining whether a patient continues to need T4, short of stopping it and measuring the TSH 6 weeks later. Because remission is uncommon and of uncertain duration, few endocrinologists attempt T4 with- drawal once started. Special problems in pregnant women Untreated hypothyroidism impairs fertility and increases the risk of miscarriage. Children born to such mothers have varying degrees of intellectual impairment. It is therefore essential that T4 replacement is monitored closely in women with hypothyroidism who intend to become or who are pregnant. Ideally TSH and free T4 should be checked prior to conception, and then every 4 weeks once preg- nancy is confirmed up to mid-​pregnancy. The requirement for T4 can increase by up to 50% during pregnancy but reverts to normal

13.3.1  The thyroid gland and disorders of thyroid function 2295 after delivery. Women can be advised to anticipate this by taking two extra doses of T4 each week as soon as pregnancy is confirmed. The TSH should be maintained below 2.5 mU/​litre during the first tri- mester. There are no implications for breastfeeding. Areas of uncertainty or needing further research Although present combination regimens of T3 and T4 have shown no additional benefit compared to T4 alone, development of a sus- tained release preparation of T3 would be worth assessment. Because hypothyroidism is frequent, routine screening of certain groups or even the entire population has been advocated (Box 13.3.1.6), but the cost–​benefit of setting up new screening programmes is unclear. If widely adopted, screening will turn up many individuals with subclinical hypothyroidism for whom the benefits of early treat- ment with T4 have not yet been fully established. Recent data show that there is an increased risk of miscarriage in thyroid peroxidase antibody-​positive women who are euthyroid and this may be re- duced by T4 treatment, but more work is needed to confirm whether this is due to a subtle defect in thyroid hormone levels or a conse- quence of autoimmunity. Thyrotoxicosis Thyrotoxicosis is defined as the state produced by excessive thyroid hormone. Hyperthyroidism exists when thyrotoxicosis is caused by thyroid overactivity but there are several types of thyrotoxicosis that are not due to hyperthyroidism, the most obvious being ad- ministration of excessive T4. Aetiology The causes of thyrotoxicosis are shown in Box 13.3.1.7. Graves’ disease is responsible for 60 to 80% of cases and nodular thyroid disease (toxic multinodular goitre and toxic adenoma) accounts for most of the rest. Destructive thyrotoxicosis is dealt with in the next section. Epidemiology The prevalence of thyrotoxicosis in white people is 2 to 3% in women and 0.2 to 0.3% in men and higher still in black and Asian/​Pacific Islander people. The peak age of onset for Graves’ disease is between 20 and 50 years of age, whereas toxic multinodular goitre occurs more often in later life. Box 13.3.1.5  Treatment of myxoedema coma • Thyroid hormone replacement — A single intravenous bolus of 200–​400 µg T4; thereafter 50–​100 µg T4 daily — Some centres add a supplementary bolus of T3 5–​20 µg, followed by 2.5–​10 µg T3 every 8 h, with lower doses in older people or those with heart disease • Supportive treatment — Ventilation usually required — Space blankets for hypothermia — Intravenous infusion of hypertonic saline or glucose as required — Parenteral hydrocortisone 50 mg every 6 h • Identify and treat underlying precipitant (including any cause of re- spiratory depression, infection, cardiac and renal failure, and myocar- dial infarction) • Broad-​spectrum antibiotics if infection suspected Box 13.3.1.6  Indications for screening for hypothyroidism Established Congenital hypothyroidism Previous treatment for hyperthyroidism Previous neck irradiation (e.g. for lymphoma) Pituitary tumours, including follow-​up after surgery or irradiation Treatment with lithium or amiodarone Subclinical hypothyroidism Worthwhile Antepartuma in type 1 diabetes Three months postpartum after a prior episode of postpartum thyroiditis Unexplained infertility Non​specific symptoms in women over 40 years of age Refractory depression or bipolar affective disorder with rapid cycling Turner’s syndrome Down’s syndrome Autoimmune Addison’s disease Uncertain Patients with a family history of thyroid autoimmunity Dementia or obesity without other evidence of thyroid disease Antepartum to detect unsuspected hypothyroidismb Breast cancer a Also measure thyroid peroxidase antibodies; screen euthyroid antibody-​ positive women 3 months postpartum for postpartum thyroiditis. b It is also uncertain whether all pregnant women should be checked for thy- roid peroxidase antibodies as predictors of postpartum thyroiditis. Box 13.3.1.7  Causes of thyrotoxicosis Primary hyperthyroidism Graves’ disease Toxic multinodular goitre Toxic adenoma Drugs: iodine excess (Jod–​Basedow phenomenon), lithium, amiodarone (type 1 amiodarone-​induced thyrotoxicosis) Thyroid carcinoma or functioning metastases Activating mutation of the TSH receptor Activating mutation of the Gsα protein (McCune–​Albright syndrome) Struma ovarii (ectopic thyroid tissue) Thyrotoxicosis without hyperthyroidism Ingestion of excess thyroid hormone (factitious thyrotoxicosis) Subacute thyroiditis Silent thyroiditis Other causes of thyroid destruction: amiodarone (type 2 amiodarone-​ induced thyrotoxicosis), 131I or external irradiation (acute effect), in- farction of an adenoma Secondary hyperthyroidism TSH-​secreting pituitary tumour Chorionic gonadotropin-​secreting tumours Gestational thyrotoxicosis Thyroid hormone resistance (usually euthyroid)

SECTION 13  Endocrine disorders 2296 Pathogenesis Graves’ disease is caused by TSH receptor stimulating antibodies, clearly demonstrated by the occurrence of transient, neonatal thyro- toxicosis in babies born to mothers with Graves’ disease whose antibody levels are high enough for transplacental transfer to affect the fetus. As with autoimmune hypothyroidism, genetic factors, including HLA-​DR, CTLA4, and TSHR gene polymorphisms, are associated with the disease; the concordance rate in monozygotic twins is about 20% and much less in dizygotic twins. A high iodine intake, smoking, and stress have all been identified as environmental factors, but in many patients the genetic and environmental trig- gers remain elusive. Immune reconstitution after alemtuzumab and highly active antiretroviral therapy are associated with Graves’ dis- ease. Smoking is a major risk factor for the development of thyroid-​ associated ophthalmopathy. These eye signs are due primarily to swelling of the extraocular muscles, the result of fibroblast activa- tion by cytokines released by infiltrating T cells and macrophages, leading to glycosaminoglycan accumulation, oedema, and fibrosis. The close correlation between ophthalmopathy and thyroid disease is best explained by a shared orbital and thyroid autoantigen (prob- ably the TSH receptor). Toxic multinodular goitre evolves from a non​toxic sporadic goitre (see earlier) and is particularly likely when iodine intake increases, ei- ther gradually as a result of changes in the diet, or acutely when iodine-​ containing agents (amiodarone, some contrast media) are given. More than 50% of toxic adenomas are due to a somatic activating mutation in the genes encoding the TSH receptor or the associated Gsα protein, and a similar but unknown mechanism leading to constitutive activa- tion of a clone of thyroid cells must underlie the remainder. Clinical features The typical features of thyrotoxicosis from any cause are shown in Box 13.3.1.8, but their presence and severity depend on the duration of disease and the age of the patient. Occasionally there are para- doxical manifestations, such as the weight gain that can occur in up to 10% of patients when the increase in appetite exceeds the effects of increased metabolism, and apathetic or masked thyrotoxicosis in older patients which mimics depression. The most dramatic but rare presentation is thyrotoxic crisis or storm, with a mortality rate of 10 to 30% even with treatment. Patients typically are previously undiagnosed or partially treated and have an acute exacerbation of thyrotoxicosis precipitated by acute illness (infection, stroke, dia- betic ketoacidosis) or trauma, especially directly to the thyroid (sur- gery or radio-​iodine). Exact diagnostic criteria for thyrotoxic crisis are not agreed and its frequency is sometimes exaggerated. There is marked fever (>38.5 °C), delirium or coma, seizures, vomiting, diar- rhoea, and jaundice, with death being caused by arrhythmias, heart failure, or hyperthermia. The differential diagnosis of thyrotoxicosis includes any cause of weight loss, anxiety, and phaeochromocytoma, but simple biochem- ical testing can readily distinguish thyrotoxicosis from these con- ditions. Once the diagnosis of thyrotoxicosis is made, it is essential to determine the cause (see Box 13.3.1.7), as this determines treat- ment. Graves’ disease is usually clinically distinctive; there is a small to moderate, diffuse, firm goitre and around one-​half of these pa- tients have signs of thyroid-​associated ophthalmopathy (Fig. 13.3.1.6
and Table 13.3.1.3). There may be evidence of another autoimmune disorder, in the patient or his/​her family, with the same associ- ations as autoimmune hypothyroidism just described. Less than 1% of patients have pretibial myxoedema, which is better called thy- roid dermopathy as it can occur anywhere, especially after trauma Box 13.3.1.8  Clinical features of thyrotoxicosis of any cause Symptoms Hyperactivity, irritability, altered mood Heat intolerance, sweating Palpitations Fatigue, weakness Weight loss with increased appetite Diarrhoea, steatorrhoea Polyuria Oligomenorrhoea, amenorrhoea, loss of libido Signs Sinus tachycardia, atrial fibrillation in older patients Fine tremor Warm, moist skin Goitre Palmar erythema, onycholysis, pruritus, urticaria, diffuse pigmentation Diffuse alopecia Muscle weakness and wasting, proximal myopathy, hyperreflexia Eyelid retraction or lag Gynaecomastia Rarely: chorea, periodic paralysis (usually in Asian men), psychosis, im- paired consciousness (a) (b) Fig. 13.3.1.6  Thyroid-​associated ophthalmopathy. (a) Upper lid retraction, periorbital oedema, and scleral injection. (b) Chemosis (conjunctival oedema) and proptosis.

13.3.1  The thyroid gland and disorders of thyroid function 2297 (Fig. 13.3.1.7). These patients almost always have moderate-​to-​severe ophthalmopathy and 10 to 20% have clubbing (thyroid acropachy). Thyroid dermopathy most commonly occurs as non​pitting plaques with a pink or purple colour but no inflammatory signs. Nodular and generalized forms, the latter mimicking elephantiasis, also occur. Hyperplasia of lymphoid tissue, including splenomegaly and thymic enlargement, is rarely found in Graves’ disease. The absence of these features of Graves’ disease and the pres- ence of a multinodular goitre strongly suggest toxic multinodular goitre, although nodular thyroid disease is so common that occa- sional patients with Graves’ disease may cause confusion when their thyrotoxicosis arises in a pre-​existing multinodular gland. In toxic adenoma, the solitary thyroid nodule is usually readily palpable. Other, rare causes of thyrotoxicosis can usually be easily identified from the history and biochemical investigations. Pathology In Graves’ disease, there is thyroid hypertrophy and hyperplasia. The follicles show considerable folding, contain little colloid, and are composed of tall columnar cells. Gland vascularity increases. There is a focal and diffuse lymphocytic infiltrate and lymphoid hyper- plasia may occur in the lymph nodes, spleen, and thymus. These changes are all reversed by antithyroid drugs. Toxic multinodular goitre comprises a mixture of areas of follicular hyperplasia and nodules filled with colloid. There is a variable degree of fibrosis, haemorrhage, and calcification. Toxic adenomas are encapsulated and cellular, sometimes with little evidence of follicle formation, and occasionally containing unusual cell forms suggesting malignant change. However, capsular invasion is absent and this is the cardinal feature which distinguishes a follicular adenoma from carcinoma. Laboratory diagnosis Measuring the serum TSH is the simplest way to exclude primary thyrotoxicosis. A normal or slightly raised TSH level can rarely be associated with secondary hyperthyroidism in the case of a TSH-​ secreting pituitary adenoma. A low TSH level is not always the re- sult of thyrotoxicosis (see Table 13.3.1.2), therefore the diagnosis of thyrotoxicosis must be confirmed by measuring thyroid hor- mone levels. Free hormone assays are preferable to those for total hormone, to eliminate binding protein effects (see Table 13.3.1.1). Measuring free T4 alone is adequate in most cases of thyrotoxicosis, which can be confirmed by the presence of a suppressed TSH and elevated free T4 level. However, in up to 5% of patients, only free T3 levels are elevated (T3 toxicosis), especially during the earliest phase of the disorder. Therefore, if both free T3 and T4 are not measured routinely by a laboratory, it is essential to request free T3 analysis in any sample showing a suppressed TSH but normal free T4 level. Rarely, the free T4 is elevated but the free T3 is normal. This usu- ally arises when Graves’ disease or nodular thyroid disease is pre- cipitated by the administration of excess iodine (the Jod–​Basedow phenomenon). TSH receptor antibodies can be measured by methods which test the ability of such antibodies to inhibit the binding of TSH to its re- ceptor, thus called TSH binding inhibiting immunoglobulin (TBII) assays. The presence of these antibodies in a thyrotoxic patient proves the existence of Graves’ disease; TBIIs are also detectable in patients with hypothyroidism caused by TSH receptor blocking anti- bodies. Thyroid peroxidase antibodies are present in around 75% of patients with Graves’ disease. In cases of diagnostic uncertainty or when TBII assays are not available, a thyroid scintiscan will dem- onstrate a diffuse goitre with high isotope intake in Graves’ disease and reveal nodular thyroid disease, as well as ectopic thyroid tissue in the extremely rare struma ovarii. In destructive and factitious Table 13.3.1.3  Clinical features of thyroid-​associated ophthalmopathy Signs and symptoms Assessment Approximate frequencya (%) Lid lag, lid retraction Measure lid fissure width 50–​60 Grittiness, discomfort, excessive tearing, retrobulbar pain, periorbital oedema Self-​assessment score by patient; activity score by clinician 40 Proptosis Exophthalmometry or CT/​MRI-​based measurement 20 Extraocular muscle dysfunction (typically causing diplopia looking
up and out) Hess chart or similar; CT/​MRI scan to detect muscle size 10 Corneal involvement, causing exposure keratitis Rose bengal or fluorescein staining <5 Loss of sight due to optic nerve compression Visual acuity and fields, colour vision; CT/​MRI scan <1 a In patients with Graves’ disease. Patients often have multiple signs and in 5–​10% of them signs are unilateral. Fig. 13.3.1.7  Thyroid dermopathy (pretibial myxoedema) affecting the lateral aspect of the shin and the dorsum of the foot; the patient also had thyroid acropachy.

SECTION 13  Endocrine disorders 2298 thyrotoxicosis, the thyroid scan shows virtually no isotope uptake and the diagnosis of factitious thyrotoxicosis can be confirmed by measuring serum thyroglobulin levels, which are suppressed in con- trast to the raised levels in all other causes of thyrotoxicosis. When a TSH-​secreting pituitary adenoma is suggested biochemically, the diagnosis is made by demonstrating both an elevated level of the α-​ subunit common to glycoprotein hormones including TSH and a pi- tuitary tumour on CT, or preferably MRI. Prolonged thyrotoxicosis can cause several non​specific biochemical abnormalities, especially abnormal liver function tests, hypercalciuria, and elevated serum levels of ferritin. Less commonly, serum calcium and phosphate may be raised, glucose intolerance or diabetes may occur, and rarely there may be a microcytic anaemia or thrombocytopenia. Treatment Definitive diagnosis is the most important determinant of treat- ment selection for thyrotoxicosis. In particular, antithyroid drugs only achieve a cure in Graves’ disease. When due to a subacute or silent thyroiditis, discussed next, spontaneous resolution of thyro- toxicosis is expected and symptomatic treatment with β-​blockers such as propranolol, 20 to 80 mg three times daily, is indicated. Although β-​blockers will rapidly alleviate symptoms in all types of hyperthyroidism, definitive treatment is also necessary, and when euthyroidism is restored β-​blockers can be gradually withdrawn. There are three types of treatment for Graves’ disease: antithyroid drugs, radio-​iodine (131I), and surgery. Local policy and patient age dictate the order of their use. For young or middle-​aged adults, antithyroid drugs are generally used initially in Europe and Japan, whereas radio-​iodine is preferred in North America. Surgery is par- ticularly useful in patients with a large goitre, but is less frequently used in North America than elsewhere. The local availability of an experienced surgeon is crucial. There is more international agree- ment over the preferential use of radio-​iodine for a recurrence after antithyroid drugs and as first-​line treatment in older people with Graves’ disease. The main antithyroid drugs are carbimazole, its active metabolite methimazole and propylthiouracil. All exert their principal action by inhibiting iodide oxidation and organification by thyroid peroxidase. Propylthiouracil additionally inhibits the activity of type 1 deiodinase, reducing T3 formation in many tissues, but this activity is only of clin- ical importance in very severe hyperthyroidism, and more frequent dosing is necessary with this drug. However, propylthiouracil may induce toxic effects on the liver, particularly in children, in whom it is contraindicated unless no other therapy is available. Two regimens are used to avoid antithyroid drug-​induced hypothyroidism and achieve the best chance of remission, which oc- curs in 40 to 50% of patients and is inversely proportional to dietary iodine intake. The first method is to titrate the dose of antithyroid drug, giving carbimazole (or methimazole) 20–​40 mg once daily, and then lowering the dose every 3 to 4 weeks or so, based on free T4 measure- ments, until a maintenance dose of 5 to 10 mg once daily is achieved. Equivalent starting and maintenance doses of propylthiouracil are 100 to 200 mg 2 to 3 times daily and 50 mg once or twice daily. Maximum remission rates occur after 18 to 24 months of treatment. The second regimen is to start with the same dose of antithyroid drug but then to add 100 µg T4 daily after 3 to 4 weeks when free T4 levels are usually becoming normal, rather than lowering the dose of drug. Thereafter the patient is maintained on 40 mg carbimazole or methimazole once daily (alternatively, 100 to 150 mg propylthiouracil three times daily) and T4, the latter being adjusted if necessary 4 weeks after starting to achieve normal free T4 levels. This block–​replace regimen achieves the same remission rate as the titra- tion regimen within 6 months; continuation beyond this time is not necessary but can be used if a patient wishes to ensure euthyroidism for a particular period of time. Patients with the biggest goitres al- most always relapse after antithyroid drug treatment, and reduced cure rates have been described in those with detectable anti-​TSH receptor antibodies at completion of a course of antithyroid drugs, in males and in younger patients. However not all studies are con- sistent and there are no reliable predictors of which other patients will relapse. It is therefore usual practice to follow patients closely (e.g. every 3  months) in the first year after stopping treatment. Thereafter, an annual check of thyroid function is warranted as re- currence occurs in 10 to 20% 1 to 5 years after treatment, and auto- immune hypothyroidism may supervene in around 15%. The side effects of antithyroid drugs are shown in Box 13.3.1.9; most occur in the first 3 months of treatment and there is a moderate dose dependency. Substituting propylthiouracil for carbimazole or methimazole usually reverses the common side effects but further antithyroid drugs should be avoided if bone marrow disturbance de- velops. Lower doses of antithyroid drugs can be used in areas of low iodine intake. Lithium and potassium perchlorate have antithyroid actions and are alternatives when antithyroid drugs are not toler- ated, but these drugs are difficult to use, their side effects are serious, and they are given as a last resort. Anticoagulation with warfarin or newer anticoagulants should be considered in all patients with atrial fibrillation; only 50% of patients revert to sinus rhythm when euthyroidism is restored. In the remainder, attempts at cardioversion should be made, ideally when hyperthyroidism has been defini- tively treated with radio-​iodine. Digoxin is useful to control atrial fibrillation acutely, but higher doses than normal are needed in the thyrotoxic state. Box 13.3.1.9  Side effects of antithyroid drugs Common Rash (typically maculopapular) Urticaria Arthralgia Fever, sometimes with malaise Rare Gastrointestinal symptoms Abnormal taste and smell Arthritis Agranulocytosisa Very rare Thrombocytopenia Aplastic anaemia Hepatitis and liver failure (propylthiouracil; avoid in children) Cholestasis (methimazole, carbimazole) Lupus-​like syndrome, vasculitis Hypoglycaemia due to the insulin autoimmune syndrome a All patients must be warned in writing, before treatment commences, to seek immediate medical advice and stop medication if features suggesting agranulocytosis (fever, mouth ulcers, sore throat) develop.

13.3.1  The thyroid gland and disorders of thyroid function 2299 Accurate dosimetry for radio-​iodine administration, based on uptake tests, has now largely fallen out of favour as the results have been little or no better than more empirical methods of dose calcula- tion. Typical 131I doses are 400 to 600 MBq in uncomplicated Graves’ disease, but local policies vary, not least because less 131I is needed when iodine intake is low. Around 5 to 10% of patients treated this way require a second dose of 131I, while hypothyroidism rates are 40–​60% after 1 year and 5 to 10% annually thereafter, depending on the dose of 131I administered. Close follow-​up is needed in the first year after treatment, and an annual test of thyroid function there- after is recommended. Transient cytoplasmic, rather than nuclear, damage may cause hypothyroidism in the first 2 to 3 months after 131I treatment, which then resolves. It is usual to delay a second dose of 131I for 6 months after the first, as hyperthyroidism is controlled only slowly by radiation-​induced nuclear damage. Antithyroid drugs or β-​blockers are useful in the interim. Radio-​iodine is contraindicated in pregnancy and breastfeeding. There are no teratogenic risks if men or women attempt concep- tion 6 months or more after treatment. Overall mortality rates from cancer are not increased by radio-​iodine. There is a theoretical risk of an increase in the frequency and aggressiveness of thyroid cancer in children, which makes some endocrinologists reluctant to use 131I, but this view is gradually changing with increasing usage of radio-​iodine in this group. Another concern is the precipitation of thyrotoxic crisis by 131I, but in practice this is extremely rare. To minimize the risk, antithyroid drugs can be given for up to 4 weeks or more prior to radio-​iodine, particularly in older people who are at special risk, but should be stopped 3–​5 days before radio-​iodine administration. Thyroid-​associated ophthalmopathy may appear or worsen after radio-​iodine, especially if the patient smokes. A 6-​week tapering course of prednisolone, starting with 0.2 mg/​kg bw daily at the time of 131I administration, will prevent such worsening but an extended course of antithyroid drugs, with scrupulous maintenance of euthyroidism, may be preferable to radio-​iodine in the case of any severe and active ophthalmopathy until this becomes inactive. Surgery for Graves’ disease consists of near-​total or total thyroidectomy, and in the best centres achieves cure in more than 99% of patients but with a hypothyroidism rate similar to radio-​ iodine. Lower rates of hypothyroidism are inevitably associated with a higher recurrence rate. Patient preference is the main determinant of when surgical treatment is used to treat relapses after antithyroid drugs. Euthyroidism must be achieved with a further course of these drugs prior to surgery to avoid thyrotoxic crisis. Stable iodine (e.g. Lugol’s iodine three drops three times daily) is often also given for 7 to 10 days prior to surgery to block hormone synthesis acutely. Specific complications of surgery include haemorrhage leading to laryngeal oedema, damage to the recurrent laryngeal nerves, and hypopara- thyroidism. These problems occur in less than 1% of patients in ex- perienced hands and the last two problems are often transient. The management of thyroid-​associated ophthalmopathy is sum- marized in Box 13.3.1.10. Symptoms and signs are usually mild to moderate, although still capable of creating considerable anxiety and disturbance of social function. Severe ophthalmopathy is fortu- nately rare (1–​5% of cases) and requires specialist ophthalmological management. Signs usually stabilize 12 to 18 months after onset, and may improve thereafter in 30 to 50% of patients, although improve- ment is less likely for marked proptosis or diplopia. Corrective sur- gery for diplopia or cosmetic problems should only be considered in this stable phase. Thyroid dermopathy is usually left untreated and often resolves spontaneously. Meticulous control of thyroid func- tion and compression stockings may help. Surgical removal typically worsens the situation and, when troublesome, the best treatment is topical, high-​potency corticosteroids. Toxic multinodular goitre is usually managed by radio-​iodine treatment. Antithyroid drugs will control the hyperthyroidism but relapse is inevitable when the drugs are stopped. Long-​term use of antithyroid drugs may be indicated in the very old or frail, or when incontinence poses an insuperable problem for the safe dis- posal of excreta after 131I. The therapeutic dose of 131I used for toxic multinodular goitre is generally higher than for Graves’ disease (500–​800 MBq) because there is uneven uptake of the isotope and usually a large goitre. Surgery is sometimes used as an alternative in patients with a retrosternal goitre or if there is any suspicion of a ma- lignancy. Toxic adenoma is also usually treated with 131I (500 MBq) and the rate of subsequent hypothyroidism is low because the func- tion of the normal thyroid tissue is suppressed at the time the patient is hyperthyroid and therefore receives little irradiation. When there is a large (>5 cm) nodule or in young patients, surgical excision is preferable and subsequent hypothyroidism is uncommon. Treatment of rare forms of primary hyperthyroidism is by surgical removal of the source of thyroid hormone or by radio-​iodine. TSH-​secreting pituitary adenomas causing secondary hyperthyroidism are usually treated by trans-​sphenoidal surgery, with radiotherapy for any re- sidual tumour. Octreotide can also be used to lower TSH secretion. Thyrotoxic crisis is a medical emergency (Box 13.3.1.11). Prognosis Although spontaneous remission occurs in Graves’ disease, its exact frequency is unknown and is unlikely to be more than 10%, with no guarantee of persistence. Remission does not occur in other types of hyperthyroidism. Mortality rates in untreated hyperthyroidism are also uncertain but are probably around 30%. Even after successful Box 13.3.1.10  Treatment of thyroid-​associated ophthalmopathy Mild to moderate disease Reassurance and explanation Avoid hypothyroidism and hyperthyroidism Stop smoking Protect eyes from dust and bright light Artificial tears; simple eye ointment at night Sleep with more pillows or the head of the bed elevated Diuretics Stick-​on prisms Severe disease (worsening diplopia, exposure keratitis, sight loss) Corticosteroids (e.g. pulse therapy with 500 mg methylprednisolone once weekly for 6 weeks, then 250 mg once weekly for 6 weeks) Radiotherapy (10 fractionated doses of 2 Gy) Immunosuppressive agents (azathioprine, ciclosporin A, rituximab) Intravenous immunoglobulin Orbital decompression (usually transantral) Stable, burnt-​out disease Prisms Surgery to extraocular muscle Cosmetic eyelid surgery

SECTION 13  Endocrine disorders 2300 treatment, there is a threefold increased risk of death from osteo- porotic fracture and a 1.3-​fold increased risk of death from cardio- vascular disease and stroke. It is important that the patient with Graves’ disease understands that the course of ophthalmopathy is sometimes independent of the thyroid disorder; eye signs appear one or more years before or after the onset of hyperthyroidism in one-​quarter of patients and progression of the orbital disease can occur despite restoration of euthyroidism. Special problems in pregnant women Graves’ disease during pregnancy is usually treated with propy­ lthiouracil, especially in the antenatal period and in the first trimester of pregnancy, as carbimazole and methimazole have been associated with fetal aplasia cutis and other fetal abnormalities. The block–​ replace regimen is contraindicated in pregnancy, as preferential pla- cental transfer of antithyroid drug will cause fetal hypothyroidism. Instead, the dose of antithyroid drug should be titrated to the lowest dose that results in maternal free T4 levels in the upper part of the reference range. TSH receptor stimulating antibodies decline during pregnancy and it is usually possible to stop treatment in the second or third trimester. Subtotal thyroidectomy can be performed in the second trimester for women intolerant of antithyroid drugs. Transplacental passage of TSH receptor antibodies causes fetal and neonatal thyrotoxicosis in 1 to 5% of mothers with Graves’ disease, and can be predicted by demonstrating a high level of these antibodies in the maternal circulation at 20 weeks of gesta- tion. Poor intrauterine growth and a high fetal heart rate also sug- gest this diagnosis. Fetal thyrotoxicosis is treated by giving the mother antithyroid drugs and the neonate requires treatment for 1 to 3 months after delivery. Failure to treat intrauterine and neo- natal thyrotoxicosis causes low birth weight, premature closure of the sutures, and intellectual impairment. Breastfeeding is safe with low doses of antithyroid drugs, but when high doses are needed (e.g. 20 mg or more carbimazole daily) thyroid function should be checked monthly in the baby. Patients with Graves’ disease who have entered remission prior to or during pregnancy have an increased risk of relapse around 3 to 6 months after delivery and should be offered thyroid function testing at this time. Areas of uncertainty or needing further research The pathogenesis of thyroid-​associated ophthalmopathy is poorly understood, and this remains an obstacle to developing better treat- ments. Outcome after antithyroid drug treatment in Graves’ dis- ease cannot yet be predicted, but improved assays for TSH receptor antibodies may permit better assessment in the future. Antithyroid drugs modulate the autoimmune response favourably in those pa- tients whose Graves’ disease remits, indicating the potential for more specific immunotherapy aimed at the cause of the disease, which would be preferential to present treatments which merely block or destroy the thyroid. The evolution of hyperthyroidism is gradual and patients with multinodular goitre in particular are now recognized at the stage of subclinical hyperthyroidism (i.e. with a low or suppressed TSH but normal free T3 and T4 levels). Their optimum management is uncer- tain. There is a twofold to threefold increased risk of atrial fibrilla- tion over 10 years in subclinical thyrotoxicosis, as well as deleterious effects on bone mineral density, but no clinical trials have been per- formed to show a clear benefit from early intervention. Many endo- crinologists simply follow such patients carefully if the TSH is still detectable (i.e. >0.1 mU/​litre) and if they are less than 65 and in otherwise good health, electing to treat when overt hyperthyroidism is shown by an abnormal free T3 level (T3 usually increases before T4). However, in older patients, those with known cardiac disease or those who are symptomatic with a persistent TSH value less than 0.1 mU/​litre, there is an increasing shift to the use radio-​iodine for subclinical hyperthyroidism. Destructive thyroiditis Acute thyroiditis is rare and is usually caused by bacterial infection of the thyroid via a pyriform sinus connecting the gland with the oropharynx. Most such patients are children or young adults. There is severe thyroid pain with fever and malaise, but thyroid function is rarely disturbed. Diagnosis is made by fine needle aspiration biopsy with culture of the specimen, and treatment consists of antibiotics, surgical drainage of any abscess, and excision of the sinus which is identified by barium swallow. Immunocompromised patients may also develop acute thyroiditis. Subacute (or de Quervain’s) thyroiditis is due to thyroid infec- tion by any of several viruses, especially mumps, Coxsackie, influ- enza, adenoviruses, and echoviruses. The most prominent symptom is pain in the thyroid, often radiating to the ears. A small, tender goitre can be palpated which is usually diffuse, but there can be asymmetrical involvement. Systemic upset with fever is variable but sometimes profound, and symptoms of a prodromal viral infec- tion several weeks earlier may be recalled. Serum C-​reactive protein levels and the erythrocyte sedimentation rate are elevated. There is a granulomatous thyroid inflammation with follicular destruc- tion and the release of thyroid hormones often results in a transient thyrotoxicosis lasting 1 to 4 weeks. Continuing thyroid destruction then leads to a phase of hypothyroidism once stored hormone is de- pleted. This lasts 4 to 12 weeks before euthyroidism is restored, but relapses occur in 10 to 20% of patients. Sometimes only one phase of thyroid disturbance is seen. Confirmation of the clinical diagnosis is made by finding an elevated erythrocyte sedimentation rate and low or absent radio-​iodine uptake by the thyroid. Thyroid function requires continuous monitoring as the disease evolves. Mild cases Box 13.3.1.11  Treatment of thyrotoxic crisis (‘thyroid storm’) • Antithyroid treatment — Propylthiouracil 500–​1000 mg as a loading dose; then 250 mg every 4 h, given orally, by nasogastric tube, or per rectum — Stable iodine given 1 h after starting propylthiouracil (e.g. Lugol’s iodine five drops every 6 h); ipodate 500 mg every 12 h is an al- ternative with additional deiodinase blocking activity but the avail- ability of this agent is limited — Propranolol 60 mg orally or 2 mg intravenously every 4 h to con- trol heart rate; careful monitoring necessary in heart failure — Severe cases may respond to plasmapheresis or dialysis • Supportive treatment • Oxygen • External cooling • Intravenous saline • Hydrocortisone 300 mg bolus intravenously, then 100 mg every 8 h • Diuretics and digoxin for heart failure • Identify and treat underlying precipitant (including trauma, infection, diabetic ketoacidosis, and myocardial infarction) — Broad-​spectrum antibiotics if infection suspected

13.3.1  The thyroid gland and disorders of thyroid function 2301 may resolve spontaneously with paracetamol or a non​steroidal anti-​ inflammatory drug as symptomatic treatment, but most patients benefit from prednisolone 15–40 mg daily as this rapidly alleviates the pain. The dose is tapered over 6 to 8 weeks depending largely on symptoms. Propranolol may be useful for thyrotoxic symptoms, and temporary T4 replacement is sometimes needed during the hypo- thyroid phase. Permanent late-​onset hypothyroidism may develop in around 15% of patients. Silent thyroiditis is an autoimmune disorder in which there is a transient but painless thyroid destruction, giving rise to the same kind of thyroid function disturbances as subacute thyroiditis. As well as the absence of thyroid pain, there is no sign of a systemic in- flammatory response (including normal C-​reactive protein levels and erythrocyte sedimentation rate) and the two conditions are therefore readily distinguished. The commonest setting for silent thyroiditis is in the postpartum period in women with pre-​existing thyroid peroxidase antibodies and a mild autoimmune thyroiditis, exacerbated for unknown reasons at this time. Such postpartum thyroiditis is common, being detectable in up to 5% of women 3 to 6 months after delivery when repeated biochemical testing is done, although in many of these women the changes in thyroid function are mild and asymptomatic. Postpartum thyroiditis is three times more common in type 1 diabetes. Thyroid uptake tests are useful in the postpartum period to distinguish thyrotoxicosis due to postpartum thyroiditis from Graves’ disease. 99mTc pertechnetate is used in pref- erence to 131I and only requires cessation of breastfeeding for a day. Treatment is with propranolol for thyrotoxic symptoms and T4 for hypothyroidism. As 90% of women recover normal thyroid function, T4 should be withdrawn 1 year after delivery and thyroid function tested 6 weeks later. However, annual follow-​up is needed as around 20% of these women have permanent hypothyroidism 5 years later. The condition usually recurs in subsequent pregnancies. Riedel’s thyroiditis is a rare disorder which, in some patients, may be with associated with IgG4-​related systemic disease causing multi- focal fibrosclerosis (retroperitoneum, mediastinum, biliary tree, orbit). The typical presentation is a hard, painless goitre suggestive of malignancy, often with symptoms due to compression of the oe- sophagus, trachea, neck veins, or recurrent laryngeal nerves. Some cases respond to corticosteroids or tamoxifen, and surgery can re- lieve compressive symptoms. Amiodarone inhibits T4 deiodination, and in all amiodarone-​ treated patients free T4 levels are in the upper half of the reference range or mildly elevated. Several months to years after starting amiodarone, however, effects on the thyroid may become manifest. In patients with mild thyroid dysfunction, especially autoimmune thyroiditis and positive thyroid peroxidase antibodies, the excessive iodine released from the drug causes hypothyroidism. This is treated as usual with levothyroxine. Paradoxically, the high level of iodine causes hyperthyroidism in other subjects who are predisposed to this because of an underlying multinodular goitre or incipient Graves’ disease (Jod–​Basedow phenomenon). This is called type 1 amiodarone-​induced thyrotoxicosis; type 2 amiodarone-​induced thyrotoxicosis is due to thyroid destruction via drug-​induced lyso- somal activation. Colour-​flow Doppler thyroid scanning shows an increase in vascularity in type 1 but not type 2 amiodarone-​induced thyrotoxicosis, but mixed forms sometimes make an exact diag- nosis impossible. Treatment of amiodarone-​induced thyrotoxicosis can be diffi- cult and biochemical changes are often out of proportion to the symptoms. Amiodarone should be stopped if possible, but often this cannot be done and in any case the drug has a very long half-​ life. Antithyroid drugs alone can be very slow to take effect in type 1 amiodarone-​induced thyrotoxicosis; High doses are often re- quired and potassium perchlorate may need to be added, 200 mg 4 or 5 times daily. There is a high frequency of agranulocytosis (up to 1%) with this drug. Prednisolone can also be used at doses of 40 to 60 mg daily and is particularly helpful in type 2 amiodarone-​ induced thyrotoxicosis. Thyroidectomy is another alternative in severe cases. Thyroid hormone resistance syndrome Mutations in one allele of the β thyroid hormone receptor gene (Fig. 13.3.1.4) cause thyroid hormone resistance. The mutations af- fect the hormone-​binding domain and the mutant receptor inhibits the activity of normally encoded receptors, so-​called dominant negative inhibition, resulting in an autosomal dominant pattern of inheritance. The condition is usually discovered during screening for a goitre, but children may sometimes present with short stature, hyperactivity, or mild learning difficulties. Thyrotoxic features in some patients were originally ascribed to selective pituitary resist- ance to thyroid hormone, leading to increased thyroid hormone secretion and therefore thyrotoxicosis in the peripheral tissues. However, the same receptor mutations occur in generalized and pituitary resistance syndromes, and although differential tissue ex- pression of receptor subtypes presumably underlies the occasional expression of thyrotoxic signs and symptoms, the exact molecular basis is unknown. The diagnosis is suggested by the presence of a normal or elevated TSH level with elevated free T3 and T4 levels. Biochemical changes of thyrotoxicosis such as elevated ferritin, sex hormone-​binding globulin, and liver enzymes are absent. The main differential diag- nosis is a TSH-​secreting adenoma. Thyroid hormone resistance can be confirmed by direct mutational analysis. Treatment is usu- ally not required as reducing thyroid hormone levels to normal causes hypothyroidism. If thyrotoxic symptoms do occur, treat- ment is with β-​blockers or thyroid hormone analogues (e.g. tri-​ iodothyroacetic acid) aimed at lowering TSH secretion. Recently, mutations in the α thyroid hormone receptor gene have been identified; patients have early-​onset severe hypothyroidism with a normal TSH level, low or normal T4 levels and normal or elevated T3 levels. FURTHER READING Akamizu T, et al. (2012). Diagnostic criteria, clinical features, and in- cidence of thyroid storm based on nationwide surveys. Thyroid, 22, 661–​74. Alexander EK, et al. (2017). 2017 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid, 27, 315–89. Bahn RS (2010). Graves’ ophthalmopathy. N Engl J Med, 366, 726–​38. Beck-​Peccoz P, et  al. (2013). 2013 European Thyroid Association guidelines for the diagnosis and treatment of thyrotropin-​secreting pituitary tumors. Eur Thyroid J, 2, 76–​82.

13.3.2 Thyroid cancer 2302

13.3.2 Thyroid cancer 2302

SECTION 13  Endocrine disorders 2302 Bernal J, Guadaño-​Ferraz A, Morte B (2015). Thyroid hormone transporters—​functions and clinical implications. Nat Rev Endocrinol, 11, 406–​17. Biondi B, Wartofsky L (2014). Treatment with thyroid hormone. Endocr Rev, 35, 433–​512. Bonnema SJ, Fast S, Hegedüs L (2014). The role of radioiodine therapy in benign nodular goitre. Best Pract Res Clin Endocrinol Metab, 28, 619–​31. Braverman LE, Cooper D (eds) (2012). Werner and Ingbar’s the thyroid, 10th edition. Lippincott Williams & Wilkins, Philadelphia. Chen AY, et al. (2014). American Thyroid Association statement on optimal surgical management of goiter. Thyroid, 24, 181–​9. DeGroot LJ, et  al. (2019). Thyroid Disease Manager. https://​www. thyroidmanager.org de Vries EM, Fliers E, Boelen A (2015). The molecular basis of the non-​thyroidal illness syndrome. J Endocrinol, 225, R67–​81. Fagman H, Nilsson M (2010). Morphogenesis of the thyroid gland. Mol Cell Endocrinol, 323, 35–​54. Franklyn JA, Boelaert K (2012). Thyrotoxicosis. Lancet, 379, 1155–​66. Jonklaas J. et  al. (2014). Guidelines for the treatment of hypothyroidism:  prepared by the American Thyroid Association task force on thyroid hormone replacement. Thyroid, 24, 1670–​751. Kahaly GJ, et al. (2018). 2018 European Thyroid Association guideline for the management of Graves’ hyperthyroidism. Eur Thyroid J, 7, 167–86. Koulouri O, et al. (2013). Pitfalls in the measurement and interpret- ation of thyroid function tests. Best Pract Res Clin Endocrinol Metab, 27, 745–​62. Lazarus J, et al. (2014). 2014 European Thyroid Association guidelines for the management of subclinical hypothyroidism in pregnancy and in children. Eur Thyroid J, 3, 76–​94. Léger J, et al. (2014). European Society for Paediatric Endocrinology consensus guidelines on screening, diagnosis, and management of congenital hypothyroidism. J Clin Endocrinol Metab, 99, 363–​84. Marcocci C, Marinò M (2012). Treatment of mild, moderate-​to-​severe and very severe Graves’ orbitopathy. Best Pract Res Clin Endocrinol Metab, 26, 325–​37. Mullur R, Liu YY, Brent GA (2014). Thyroid hormone regulation of metabolism. Physiol Rev, 94, 355–​82. Okosieme O, et al. (2016). Management of primary hypothyroidism: statement by the British Thyroid Association Executive Committee. Clin Endocrinol (Oxf), 84, 799–​808. Paes JE, et al. (2010). Acute bacterial suppurative thyroiditis: a clinical review and expert opinion. Thyroid, 20, 247–​55. Papini E, Pacella CM, Hegedüs L (2014). Thyroid ultrasound (US) and US-​assisted procedures: from the shadows into an array of applica- tions. Eur J Endocrinol, 170, R133–​46. Persani L, et al. (2018). 2018 European Thyroid Association (ETA)
guidelines on the diagnosis and management of central hypothyroidism. Eur Thyroid J, 7, 225–37. Ross DS, et al. (2016). 2016 American Thyroid Association Guidelines for Diagnosis and Management of Hyperthyroidism and Other Causes of Thyrotoxicosis. Thyroid, 26, 1343–421. Szinnai G (2014). Genetics of normal and abnormal thyroid develop- ment in humans. Best Pract Res Clin Endocrinol Metab, 28, 133–​50. Verloop H, et al. (2014). Genetics in endocrinology: genetic variation in deiodinases: a systematic review of potential clinical effects in hu- mans. Eur J Endocrinol, 171, R123–​35. Weetman AP (2011). Diseases associated with thyroid autoimmunity: explanations for the expanding spectrum. Clin Endocrinol (Oxf), 74, 411–​8. Zimmermann MB, Boelaert K (2015). Iodine deficiency and thyroid disorders. Lancet Diabetes Endocrinol, 3, 286–​95. 13.3.2  Thyroid cancer Kristien Boelaert and Anthony P. Weetman ESSENTIALS Thyroid cancers are the most common endocrine malignancies and their incidence is rising globally, largely due to significant increases in small, incidentally detected low-​risk tumours. Follicular epithelial cell cancer—​the commonest type; usually well differentiated tumours with an excellent prognosis but occasionally highly undifferentiated; may be induced by exposure to ionizing ra- diation; typically present with asymptomatic thyroid enlargement; usually diagnosed by ultrasonography and fine needle aspiration bi- opsy; treatment is typically by hemi-​or total thyroidectomy, followed by radio-​iodine ablation (administered to remove any remaining thyroid tissue) and short-​ to longer-​term thyroid-​stimulating hor- mone suppression depending on clinical risk stratification Medullary thyroid carcinoma—​arises from parafollicular C cells; comprises 3–​5% of all thyroid cancers; hereditary autosomal dom- inant forms associated with germline point mutations in the RET proto-​oncogene; occurs in 20% as part of multiple endocrine neoplasia (MEN) type 2A or 2B, or as isolated familial medullary carcinoma; typically presents with a solitary thyroid nodule, accom- panied in 50% of cases by cervical lymphadenopathy; can be asso- ciated with unusual hormonal effects, including secretory diarrhoea; diagnosis often made by fine needle aspiration biopsy and by raised serum calcitonin concentration; treatment is by total thyroidectomy (requiring long-​term thyroid hormone replacement) and neck dis- section (if required), followed by monitoring of serum calcitonin levels and tyrosine kinase inhibitors in advanced cases; Genetic testing for the presence of RET mutations allows family testing, with prophylactic thyroidectomy recommended for affected individuals. Rare thyroid tumours—​include (1) anaplastic carcinomas—​present as a rapidly enlarging and fixed thyroid masses, sometimes with local pain; rap- idly fatal; (2) sarcomas; and (3) primary lymphomas—​ usually present as a rapidly enlarging thyroid mass in a patient with Hashimoto’s thyroiditis. Primary thyroid follicular epithelial tumours Aetiology Differentiated thyroid cancers are the most common tumours arising from thyroid follicular epithelial cells and account for 95% of all thyroid carcinomas (Table 13.3.2.1). Papillary carcinomas Around 85% of all thyroid cancers are papillary carcinomas, and these carry the best prognosis. Most of these tumours are indo- lent and harbour mutually exclusive mutations of genes encoding effectors that signal through the mitogen-​activated protein kinase (MAPK) pathway. BRAF V600E accounts for 60% of these muta- tions, followed by RAS (15%) and chromosomal rearrangements (12%) including RET, NTRK, and ALK resulting in aberrant expres- sion of BRAF or of receptor tyrosine kinases. The remaining 13% have no known driver mutations. Different mutations are associated

13.3.2  Thyroid cancer 2303 with varying histological subtypes of papillary thyroid cancers and confer distinct patterns of gene expression, signalling, and clinical characteristics. BRAF-​mutated papillary thyroid cancers tend to be more aggressive with high frequency of lymph node metastases and recurrence rates. Exposure to ionizing radiation is a risk factor for the devel- opment of papillary thyroid cancer and sharp increases in the incidence of these tumours, especially in young children, were observed following the nuclear reactor accident in Chernobyl in 1986 and following the atomic bomb explosions in Hiroshima and Nagasaki in 1945. Radiation-​induced papillary cancers have a high prevalence of fusion oncogenes activating RET or NTRK and RET/​ PTC3 is particularly linked to radiation. Low-​dose external beam radiation (10–​1500 cGy) to the head and neck increases the risk of papillary thyroid cancer over 10 to 30 years. Higher thyroid radi- ation doses, including those arising from radio-​iodine given for treatment of hyperthyroidism, are not associated with an increased risk of malignancy because thyroid cells are destroyed rather than transformed. Mutations in the TERT gene have been identified in more aggressive subsets of papillary cancers. Follicular carcinomas Follicular thyroid cancers account for 2–​5% of thyroid cancers and usually harbour mutations of either RAS or the PAX8-​PPARG fu- sion oncogene. These tumours are also more prevalent in areas of iodine-​deficiency and excessive stimulation of thyroid cell growth by thyrotropin (TSH) plays a role in their pathogenesis. Rarely fol- licular carcinomas are associated with activating mutations of the genes encoding the TSH receptor or Gsα protein, similar to those found in toxic adenoma, and it is thought that some follicular carcinomas arise from adenomas. The prognosis of patients with follicular cancers depends on the size of the tumour, the patient’s age, and the degree of angioinvasion which predicts the risk of dis- tant metastases. Hürthle cell cancers These are classified as a variant of follicular carcinomas and are genetically distinct. They may be widely invasive and may behave aggressively, with lung and bone metastases and refractoriness to radio-​iodine. Anaplastic carcinomas These may arise from and can coexist with differentiated cancers but can also occur de novo. They have been linked to mutations in TP53 (p53 tumour suppressor gene), the TERT promoter, effectors of the phosphatidylinositol 3-​kinase (PI3K)-​AKT, mammalian target of rapamycin (mTOR), and genes involved in epigenetic regulation. Germline mutations Several germline variations in chromosomes 9q22.23 and 14q13.3 have been linked with high risk of differentiated thyroid cancers, due to their proximity to genes encoding proteins involved in regulating thyroid development and differentiation including FOXE1 and NKX2-​1. Familial forms of papillary and follicular carcinomas exist but are unusual (3–​9% of cases), including Cowden’s disease (mul- tiple hamartoma syndrome associated with PTEN mutations OMIM 158350), familial adenomatosis polyposis, including the Gardner syndrome variant (associated with loss of function mutations in the APC gene OMIM 175100), and Werner’s syndrome (adult pro- geria associated with mutation in the WRN gene). Other familial forms may be found in Peutz–​Jeghers syndrome (OMIM 175200), the Carney complex (OMIM 160980), and ataxia–​telangiectasia (OMIM 208900). Epidemiology Thyroid cancer incidence rates have increased dramatically in most Westernized countries and between 2014–2016 there were an average of 3527 new diagnoses annually in the United Kingdom, rep- resenting more than 155% rise since the early 1990s. In the United States, the incidence of thyroid cancer has more than tripled from 1975 through to 2015, and it is estimated that there will be 52 070 new thyroid cancer cases and 2170 deaths from the disease in 2019. Thyroid cancer is about three times more common in women than in men, and the peak incidence is between 30 and 50 years of age. Most of the recent increase in disease burden is due to the inci- dental detection of small, low-​risk tumours, although there is some evidence that other factors including ionizing radiation, obesity, and environmental pollutants may be contributing factors. Papillary microcarcinomas, defined as tumours less than 1 cm in diameter, occur in up to 36% of autopsy specimens and up to 24% of surgical thyroidectomies. Clearly most of these do not significantly impact on long-​term outcomes and life expectancy. Clinical features Most patients present with thyroid enlargement in the form of a thy- roid nodule, which is often asymptomatic. This may be noticed by the patient or their relatives, or sometimes the abnormality is de- tected during physical examination for another complaint. The diffi- culty for diagnosis arises because thyroid nodules are frequent, and only about 5% of palpable thyroid nodules are malignant. Diffuse or multinodular thyroid enlargement occurs in around 10% of the population and is four times more common in women than in men. Between 4 and 7% of adults have visible thyroid nodules and these are usually hyperplastic or colloid nodules. Overall, the prevalence of malignancy in thyroid nodules is estimated between 4 and 6.5%. There are usually no symptoms or signs to distinguish benign from malignant nodules because most cancers progress slowly and Table 13.3.2.1  Classification of thyroid malignancies Primary thyroid follicular epithelial tumours Differentiated (papillary, follicular, Hürthle cell) Poorly differentiated Undifferentiated (anaplastic) C-​cell epithelial tumours (medullary carcinoma) Primary non​epithelial tumours Lymphoid origin (lymphoma, plasmacytoma) Mesenchymal cell origin (sarcoma) Other (teratoma) Secondary non​thyroidal tumours Metastases Extension of tumour from adjacent structures

SECTION 13  Endocrine disorders 2304 present before disease is advanced. Clinical parameters associated with increased risk of malignancy include male sex and being at the extremes of age (less than 20 or over 60 years). Previous exposure to radiation and a family history of thyroid cancer should also arouse suspicion. A carcinoma is more likely if thyroid enlargement has grown recently or is hard, irregular, or fixed on palpation. Clinical assessment should include careful examination of the cer- vical, submental, and supraclavicular lymph nodes. Late-​presenting features include hoarseness, dysphagia, or dyspnoea which may in- dicate local invasion, but these symptoms can occasionally occur with an enlarging benign goitre. Rarely the diagnosis only becomes apparent when metastatic disease is detected in bone or lung. The relatively indolent presentation of papillary and follicular thyroid carcinoma contrasts with that of anaplastic carcinoma, in which a rapidly enlarging and fixed thyroid mass occurs, sometimes with local pain. Extension to the oesophagus, trachea, and/​or recur- rent laryngeal nerves is frequent, and the overlying skin may also be infiltrated. Pathology Papillary carcinomas There are several variants of papillary thyroid carcinoma united by their characteristic cytological features. The nuclei are large, clear (‘Orphan Annie’, after the eyes of the cartoon character), and have lon- gitudinal grooves and invaginations of cytoplasm causing a papillary growth pattern (Fig. 13.3.2.1a). One-​half of papillary carcinomas contain degenerate calcified papillae, termed psammoma bodies. The tumour is multicentric in up to 80% of cases if the resected thy- roid is examined carefully. Metastasis is via the lymphatics and local lymph nodes are infiltrated in 40 to 50% of cases (more in young patients). Distant metastases are found in less than 5% of patients at presentation, with the lung being the most common site. There are more than 10 different variants of papillary thyroid cancer and those with a less favourable outcome include tall cell, col- umnar cell, and hobnail variants. These tumours generally present at a later age and more advanced stage than classical-​type papillary thyroid cancer. Data regarding outcomes of solid variant and scler- osing (including diffuse sclerosing) variants remain controversial. The follicular variant of papillary thyroid carcinoma is a tumour composed of neoplastic follicles rather than papillae, but with fol- licular cells showing nuclear features characteristic of papillary thyroid cancer. There are two main subtypes:  infiltrative (non-​ encapsulated) and encapsulated. The latter form has increased in incidence by an estimated 2-​to 3-​fold over the past two to three decades and makes up 10–​20% of all thyroid cancers currently diagnosed in Europe and North America. These tumours have a very low risk of adverse outcomes and are now termed non​invasive follicular thyroid neoplasm with papillary-​like nuclear features (NIFTP). No additional treatment with surgery or radio-​iodine ab- lation is required for these tumours, and no further pathological staging is needed. Follicular carcinomas Follicular carcinoma is characterized by follicular differentiation with a solid growth pattern and without the nuclear features of pap- illary carcinoma. The tumour is encapsulated, but there is invasion of the capsule and vessels, thereby distinguishing it from a follicular adenoma (Fig. 13.3.2.1b). They may be minimally invasive when there is only microscopic capsular invasion, or angioinvasive when there is vascular invasion. The former are indolent tumours with 10-​year mortality rates of less than 5%, whereas the latter are asso- ciated with mortality rates of 5–​30% depending on the number of invaded blood vessels. (a) (b) (c) Fig. 13.3.2.1  Histopathological features of thyroid follicular epithelial carcinoma. (a) Papillary carcinoma, with psammoma bodies and typical nuclear appearance. (b) Metastatic follicular carcinoma, eroding vertebral bone. (c) Anaplastic carcinoma showing pleomorphic spindle cells. All sections, original magnification ×200. Photomicrographs by courtesy of Dr K. Suvarna.

13.3.2  Thyroid cancer 2305 Hürthle cell cancers Oncocytic (Hürthle cells) cells are characterized by oxyphilic staining due to mitochondrial accumulation, resulting in abundant eosinophilic granular cytoplasm and hyperchromatic nuclei. They are classified into Hürthle cell adenomas or Hürthle cell carcinomas, depending on the presence or absence of capsular and vascular invasion. Poorly differentiated and anaplastic carcinomas Poorly differentiated thyroid cancers are aggressive tumours with partial loss of features of thyroid differentiation occupying a pos- ition between well-​differentiated thyroid tumours and completely dedifferentiated anaplastic cancers. They are associated with poor outcomes and 10-​year survival rates are around 50%. In anaplastic carcinoma there is no capsule, the cells are atypical, including spindle, multinuclear, and squamoid forms, and mitoses are frequent (Fig. 13.3.2.1c). Investigation and diagnosis Blood tests Thyroid epithelial cancers generally fail to affect thyroid function, but this should be evaluated in all patients presenting with a thyroid nodule; a low circulating TSH concentration strongly suggests an autonomous benign nodule. Anaplastic carcinoma may occasionally cause hypothyroidism, but the most frequent cause of an elevated TSH concentration with a hard, nodular thyroid is Hashimoto’s thyroiditis. Some of the glands in these cases are so irregular that the presence of malignancy may be suspected. Differentiated thy- roid cancer is more common in patients with autoimmune thyroid disease—​both Graves’ disease and Hashimoto thyroiditis—​and thyroid lymphoma almost always occurs in association with auto- immune thyroiditis. Any dominant or atypical area in a Hashimoto’s goitre therefore requires careful evaluation. Thyroid peroxidase and/​or thyroglobulin antibodies are raised in about one-​quarter of patients with thyroid follicular epithelial carcinoma, coincident with the presence of a lymphocytic infil- trate which, in turn, is associated with a slightly more favourable prognosis. Although measurement of serum thyroglobulin concentrations is extremely useful in follow-​up, as discussed next, this investi- gation is unhelpful in diagnosing thyroid cancer; levels may not be elevated in some patients and, even when elevated, cannot be causally distinguished from those that occur in benign adenoma, multinodular goitre, Graves’ disease, destructive thyroiditis, or Hashimoto’s. Imaging Thyroid nodules may be detected in up to 60% of adults when using high resolution ultrasonography, and they are found incidentally in 16% of cross-​sectional CT or MRI scans, 10% of carotid Doppler investigations, and 2–​3% of positron emission tomography (PET) scans. FDG-​PET positive nodules carry a higher risk of malignancy and require further evaluation. Several ultrasonographic criteria are associated with a higher suspicion for malignancy: current guide- lines combine these into algorithms guiding practitioners regarding the selection of thyroid nodules that warrant further evaluation with fine needle aspiration biopsy. Ultrasonography High-​resolution ultrasonography is a very sensitive tool to diag- nose thyroid malignancy and may be specific for the identification of papillary thyroid cancer. Most current guidelines recommend ultrasonography, performed by a competent operator, as the first line imaging tool in patients presenting with thyroid enlargement (Figs. 13.3.2.2 and 13.3.2.3). Ultrasonographic features associated with a higher risk of malignancy are illustrated in Table 13.3.2.2. Ultrasonographic features should be used in summation to identify thyroid lesions that may be malignant and warrant fine needle aspiration biopsy (FNAB). Ultrasonography also increases the yield of FNAB. The presence or absence of abnormal cervical lymph nodes should be documented when undertaking thyroid ultrasonography. Fig. 13.3.2.2  Ultrasound appearances of a cystic thyroid nodule with tiny echogenic foci with subtle ‘ring-​down’ or ‘comet tail’ artefact (arrow) due to inspissated colloid. From Scoutt LM, Hamper UM, Angtuaco TL (eds) (2016). Ultrasound. By permission of Oxford University Press, USA. Fig. 13.3.2.3  Ultrasound appearances of papillary thyroid carcinoma. There is a large, solid nodule in the inferior pole of the right lobe of the thyroid with punctate echogenic, non​shadowing microcalcifications (arrows); T, normal thyroid parenchyma. From Scoutt LM, Hamper UM, Angtuaco TL (eds) (2016). Ultrasound. By permission of Oxford University Press, USA.

SECTION 13  Endocrine disorders 2306 In the United Kingdom, a scoring system of U1-​5 is recommended to classify thyroid nodules based on ultrasound findings: U1: normal thyroid; U2: benign thyroid lesion; U3: indeterminate thyroid lesion (likely follicular lesion); U4:  suspicious thyroid lesion; U5:  diag- nostic for thyroid cancer. Nodules graded as U3-​5 warrant further cytological evaluation. The US guidelines recommend use of similar system classifying thyroid nodules as benign, very low, low, inter- mediate, or high risk of malignancy, and also take nodule size into account when selecting nodules that warrant FNAB. Radionuclide scanning Radionuclide scanning with 99mTc pertechnetate or radio-​iodine (123I or 131I) may be indicated if patients have thyroid enlargement and a low serum TSH concentration. The presence of a ‘hot’ nodule taking up significant amounts of radionuclide with failure of the sur- rounding tissue to take up the tracer is consistent with the presence of an autonomous nodule which is almost invariably benign. Most thyroid cancers fail to take up radionuclide (‘cold’ nodules), but benign lesions may behave in a similar way. Overall radionuclide scanning is not routinely recommended to diagnose thyroid cancer. Fine needle aspiration biopsy Fine needle aspiration biopsy and subsequent cytological evaluation remains the gold standard for the diagnosis of thyroid cancer, with sensitivity and specificity rates of more than 90%. This is now usually performed under ultrasound guidance. In the United Kingdom, the use of the ‘THY classification’ is re- commended:  THY1:  non​diagnostic (insufficient cells sampled); THY2: non​neoplastic; THY3: neoplasm possible, which is further sub- divided into THY3a: atypical features present, and THY3f: follicular neoplasm; THY4: suspicious of malignancy and THY5: diagnostic of thyroid cancer. US guidelines recommend the use of the Bethesda clas- sification, which is very similar to the United Kingdom system. Surgery is usually required for lesions classified as THY3f, THY4, and THY5 and repeat biopsy should be undertaken for THY1 and THY3a nodules. In all cases the clinical, ultrasonographic, and cytological findings should be considered together and where ap- propriate should be discussed in a multidisciplinary team (MDT) setting, especially if there is non​concordance between findings. Papillary carcinoma is readily diagnosed by fine needle aspir- ation biopsy, and medullary carcinoma and lymphoma can also be detected by the use of immunohistochemical staining, although lymphoma frequently requires core or open biopsy for confirmation. Follicular carcinomas cannot be distinguished cytologically from follicular adenomas and are usually labelled as THY3F. Many pa- tients with these lesions will undergo diagnostic hemithyroidectomy. However, there is a rapidly growing body of literature describing the use of gene expression classifiers, mutation analysis panels, and protein expression chips to distinguish benign from malignant fol- licular lesions. Some centres, especially in the United States, use mo- lecular analysis of FNAB specimens routinely in decision-​making processes regarding the recommendation of surgery for these in- determinate lesions, although this is not common practice in the United Kingdom and most European countries. Thyroid cysts are usually benign and may be aspirated during bi- opsy, although reaccumulation of fluid is common and may indicate presence of malignancy; if this occurs, surgery is usually required for definitive diagnosis. Management of differentiated thyroid cancer Surgical excision The initial form of treatment is surgery, which should be performed by a surgeon with the required training and expertise who is a core member of the multidisciplinary team. Assessment of extrathyroidal extension and presence (or absence) of central compartment and lateral neck lymph node disease through ultrasonography and cross-​ sectional imaging with CT or MRI is recommended. Thyroid lobectomy is recommended for patients with unifocal papillary microcarcinoma (<1 cm) with no other risk factors. Total thyroidectomy and central compartment lymph node dis- section is recommended for patients with tumours greater than 4 cm in diameter or tumours of any size if associated with any of the following characteristics:  multifocal disease, bilateral disease, extrathyroidal spread (pT3 and pT4a), positive familial history, clinically or radiologically involved lymph nodes and/​or distant metastases. Tumours between 1 and 4  cm in maximum diameter may be treated with lobectomy or total thyroidectomy, depending on judge- ment by the multidisciplinary team taking additional risk factors into account. Lobectomy is deemed sufficient and central lymph node dissection unnecessary for tumours between 1 and 4 cm in patients aged less than 45 years, with unifocal classical type papillary cancer, without a positive family history, and without extrathyroidal spread or lymph node metastases. Neck dissection is indicated if there is clinical or radiological evidence of lateral compartment lymph node involvement. Postoperative risk stratification UK and US guidelines recommend postoperative staging using the TNM classification (Table 13.3.2.3)/​AJCC (American Joint Commission on Cancer) system (Table 13.3.2.4). This classification system predicts the risk of death from dis- ease and is a valuable indicator of overall prognosis, but it does not take individual responses to treatment into account, which may alter prognosis or the risk of recurrence. The British and American Thyroid Association guidelines therefore recommend the use of a three-​tier system to predict postoperative risk of re- currence (Table 13.3.2.5). Table 13.3.2.2  Ultrasonographic features of benign and malignant thyroid nodules Benign nodule Malignant nodule Follicular lesion Spongiform/​ honeycomb Solid and hypoechoic Hyperechoic/​ homogeneous halo benign Purely cystic Irregular margin Hypoechogenicity/​loss of halo suspicious Egg shell calcification Intranodular vascularity Iso/​hyperechoic (hypoechoic halo) Absence of halo Peripheral vascularity Taller than wide Microcalcifications

13.3.2  Thyroid cancer 2307 Radio-​iodine remnant ablation and therapy
for differentiated thyroid cancer After surgery, radio-​iodine is usually administered to remove any remaining thyroid tissue, which then allows thyroglobulin or 131I total body scanning to be used in follow-​up to detect metastases. This treatment may also destroy occult carcinoma and, by scan- ning after ablation, metastatic disease is revealed. Similar to the multidisciplinary approach recommended for decision-​making re- garding the extent for surgery, a risk-​based approach is now recom- mended when selecting patients requiring radio-​iodine remnant ablation. Patients in the definite indications category are those with tumours more than 4 cm or tumours of any size with gross thyroidal extension (pT4) or distant metastases. Remnant ablation is not in- dicated in patients with tumours not more than 1 cm, unifocal or multifocal, and in cases where histology reveals classical papillary or follicular variant or minimally invasive follicular tumours (i.e. without capsular or angioinvasion). Indications to treat tumours between 1 and 4 cm with 131-​I ab- lation remain uncertain and MDT discussion and decision-​making is required. Factors favouring intervention include tumours more than 2 cm, unfavourable histological subtype, widely invasive tu- mours, more than five resected metastatic lymph nodes, resected lymph nodes more than 6  mm in maximum diameter, ratio of positive/​negative nodes more than 0.7 and extracapsular nodal involvement. Results from two large randomized controlled trials have shown that an ablative 131-​I dose of 1.1 GBq is as effective as 3.7 GBq for low to intermediate risk tumours, with fewer adverse effects in the 1.1 GBq group. It is recommended that patients with T1-​2 and R0 re- section should receive the lower dosage. For patients with pT3 and/​ or N1 disease, the activity of 131-​I should be decided by the MDT on an individual case basis taking all prognostic factors into account. Iodine exposure, including iodine-​containing contrast media, may prevent accumulation of 131I during treatment. Guidelines regarding implementation of a low iodine diet prior to radio-​iodine ablation have recently been devised in the United Kingdom (see ‘Further reading’). High levels of stimulation by TSH (>30 mU/​litre) are required to produce maximum uptake of 131I. This may be achieved by levothyroxine withdrawal for 4 weeks, resulting in development of severe hypothyroid symptoms, or by the administration of recom- binant human TSH (rhTSH) for 48 hours prior to administration of radio-​iodine, thereby avoiding the need for cessation of thyroid hormone replacement. If thyroid hormone withdrawal is used, pa- tients are usually switched to the shorter-​acting liothyronine (20 µg three times daily) as replacement therapy 28 days before 131-​I abla- tion and this treatment is then discontinued 14 days before radio-​ iodine administration. Randomized controlled trials have shown Table 13.3.2.3  TNM classification of thyroid cancer (adapted from 2014 BTA Guidelines for the management of thyroid cancer) Primary tumour—​T Tx Primary tumour cannot be assessed T0 No evidence of primary tumour T1 Tumour ≤2 cm limited to the thyroid T2 Tumour >2 cm but ≤4 cm limited to the thyroid T3 Tumour >4 cm limited to the thyroid or any tumour with minimal extrathyroidal extension T4a Tumour of any size beyond the thyroid capsule and invading surrounding tissues T4b Tumour invades prevertebral fascia or encases carotid artery or mediastinal vessels Regional lymph nodes (cervical and upper mediastinal)—​N Nx Regional lymph nodes cannot be assessed N0 No regional lymph node metastases N1 Regional lymph node metastases N1a Metastases to Level VI (pretracheal, paratracheal, and prelaryngeal/​ Delphian lymph nodes) N1b Metastases to unilateral, bilateral, or contralateral cervical (levels I, II, III, IV, or V) or retropharyngeal or superior mediastinal lymph nodes (level VII) Distant metastases—​M M0 No distant metastases M1 Distant metastases Residual tumour—​R Rx Cannot assess presence of residual primary tumour R0 No residual primary tumour R1 Microscopic residual primary tumour R2 Macroscopic residual primary tumour Based on the TNM classification tumours are then staged as follows (AJCC/​UICC staging system, Table 13.3.2.4). Table 13.3.2.4  Staging of differentiated (papillary and follicular) thyroid carcinomas (adapted from 2014 BTA guidelines for the management of thyroid cancer) Patients aged <55 Patients aged ≥55 y Stage TNM description Stage TNM description I Any T, any N, M0 I T2, N0, M0 II Any T, any N, M1 II Any T, N1, M0 III T4a, any N, M0 IVa T4b, any N, M0 IVb Any T, any N, M1 Table 13.3.2.5  Postoperative risk stratification for risk of recurrence of differentiated thyroid cancer (adapted from 2014 BTA Guidelines for the management of thyroid cancer) Low risk Intermediate risk (any) High risk (any) No local/​distant metastases Microscopic tumour invasion (perithyroidal soft tissues T3) Extrathyroidal invasion All macroscopic
tumour resected
(R0/​R1) Cervical lymph node metastases N1a/​N1b Incomplete tumour resection (R2) No locoregional
tumour invasion No aggressive histological features Aggressive histology, e.g. Tall cell, insular or angioinvasion Distant metastases (M1)

SECTION 13  Endocrine disorders 2308 equal efficacy of 131-​I remnant ablation when using thyroid hor- mone withdrawal and rhTSH, with improved quality of life in the rhTSH group due to absence of troublesome hypothyroid symp- toms. The use of rhTSH is therefore recommended in patients with low to intermediate risk tumours (pT1 to pT3, pN0 or Nx, or N1 and M0 and R0). Radio-​iodine-​avid thyroid cancer metastases may be treated with further radio-​iodine therapy; doses between 3.7 and 5.5 GBq are usually administered. Dynamic risk stratification Patients who have undergone total thyroidectomy and remnant ablation with radio-​iodine should undergo dynamic risk stratifica- tion to define the response to initial therapy. This is usually done 9–​12 months following treatment, when patients are recommended to have a neck ultrasound and measurement of rhTSH stimulated thyroglobulin (Tg). Subsequent intensity and duration of follow-​up, as well as targets for TSH suppression, is then based on the assign- ment of patients to different response groups:  excellent, indeter- minate, or incomplete response (Table 13.3.2.6). Long-​term levothyroxine replacement therapy Following thyroidectomy, it is necessary to maintain euthyroidism through lifelong levothyroxine replacement in all patients who have undergone a total thyroidectomy and, in some patients, fol- lowing lobectomy. Suppression of serum TSH is required in inter- mediate and high-​risk patients since thyrotropin is a growth factor for thyroid cells. Following initial treatment with surgery and radio-​iodine rem- nant ablation, and before dynamic risk stratification, serum TSH should be suppressed below 0.1 mIU/​litre. It is unnecessary to suppress serum TSH in subjects who have undergone less than total thyroidectomy and in those who have not undergone rem- nant ablation. In patients who are stratified into the excellent response category following dynamic risk stratification, serum TSH can be maintained in the lower half of the normal reference range (0.3–​2.0 mIU/​litre). For historic patients who have not undergone dynamic risk stratification, it is appropriate to keep serum TSH suppressed below 0.1 mIU/​litre for 5–​10 years and then to allow serum TSH to rise to the lower half of the normal reference range if there is no evidence of recurrence either bio- chemically or structurally. The long-​term consequences of serum TSH concentrations suppressed below 0.1 mIU/​litre include car- diovascular morbidity and mortality as well as osteoporosis, and evaluation of probability of osteoporotic fragility fracture risk may be required. Systemic therapies for metastatic radio-​iodine-​refractory thyroid cancer Metastatic thyroid cancer occurs in less than 10% of patients, about two-​thirds of whom ultimately present with radio-​iodine refractory disease. If patients have radio-​iodine avid disease, repeated adminis- tration of radio-​iodine therapy every 4–​6 months may be beneficial, especially if lung metastases are present. There is little benefit above a cumulative dose of 18.5 GBq. Bone metastases may respond to 131I or external beam radiotherapy, and orthopaedic intervention may be required to stabilize pathological fractures. Palliative external beam radiotherapy has a limited role in controlling locoregional dis- ease when further surgery is ineffective or impractical. Radiotherapy can be used alone or in combination with low-​dose chemotherapy. Targeted therapies Improved understanding of the molecular pathogenesis of thyroid cancer has resulted in an explosion of targeted therapies in patients with advanced metastatic thyroid cancer. Two multikinase inhibi- tors, sorafenib and lenvatinib, have been approved for the treat- ment of radio-​iodine refractory tumours on the basis of prospective, double-​blind randomized placebo-​controlled trials that showed longer progression-​free survival. Lenvatinib appears to have the greatest efficacy. Significant adverse effects of both drugs often make the maintenance of full dose therapy a challenge. Common adverse effects include hypertension, hand–​foot skin reactions, diarrhoea, rash, fatigue, weight loss, and stomatitis. Longer-​term effects on quality of life and cumulative toxic effects of these agents remain to be determined. Phase 2 trials using several other multikinase inhibitors have commenced, including sunitinib, pazopanib, axitinib, cabozantinib, and motesanib, all of which have multifunctional actions including antiangiogenic properties. Targeted therapies including RAF-​kinase inhibitors used alone or in combination with MEK inhibitors are also being explored in several combination trials. Treatment with selumetinib, a MAPK kinase (MEK) 1 and MEK2 inhibitor, has been shown to produce a clinically meaningful increase in radio-​ iodine uptake in tumours previously refractory to radio-​iodine treatment through loss of the sodium iodide symporter, particu- larly those with a RAS mutation. This is a rapidly developing field and more trials will help address how best to stratify patients for treatment. Follow-​up Lifelong follow-​up is necessary for papillary and follicular cancer because they may recur many years after apparent cure. As well as monitoring the concentration of serum TSH and performing Table 13.3.2.6  Dynamic risk stratification determining response to treatment (adapted from 2014 BTA Guidelines for the management of thyroid cancer) Excellent response Indeterminate response Incomplete response All the following: Suppressed and stimulated Tg <1 mcg/​litre -​ Neck USS without evidence of disease -​ Cross-​sectional and/​or nuclear medicine imaging negative (if performed) Any of the following: Suppressed Tg <1 mcg/​ml and stimulated Tg ≥1 and <10 mcg/​litre -​ Neck USS with non​specific changes or stable lymph nodes (<1 cm) -​ Cross-​sectional and/​or nuclear medicine imaging with non​specific changes, not completely normal Any of the following: Suppressed Tg ≥1 mcg/​litre or stimulated Tg ≥10 mcg/​litre -​ Rising Tg values -​ Persistent or newly identified disease on cross-​ sectional and/​or nuclear medicine imaging USS, ultrasound scan.

13.3.2  Thyroid cancer 2309 careful neck palpation, serum thyroglobulin should be measured. Detectable levels of thyroglobulin after thyroid ablation indicate persistent or recurrent disease. Measuring thyroglobulin levels is especially valuable when the patient is not taking thyroxine replacement, or after recombinant TSH stimulation, as the rise in TSH will promote thyroglobulin production and exaggerate any increase. The use of rhTSH stimulated thyroglobulin is recom- mended as part of dynamic risk stratification, but for low-​risk patients (i.e. those who have not undergone radio-​iodine rem- nant ablation and those assigned to the excellent response dy- namic risk stratification category), it is appropriate to measure unstimulated Tg while the patient is on levothyroxine therapy. The presence of thyroglobulin antibodies may interfere with thyroglobulin measurements on most assays, hence these anti- bodies should be evaluated by a quantitative method whenever serum Tg is measured. If thyroglobulin is detectable, the patient should have a total body 131I scan and any recurrent disease can then be treated with a therapeutic dose of radioactive iodine (Fig. 13.3.2.4). If the total body scan is negative and serum Tg is consistently raised, cross-​sectional imaging and occasionally FDG-​PET scanning is indicated. Prognosis Differentiated thyroid cancer Most papillary thyroid cancers are indolent tumours and have an excellent prognosis, with 10-​year relative survival rates of more than 98% (Table 13.3.2.7). The risk of death increases with age, hence most prognostic scoring systems take age into account. The 10-​year cause-​specific survival rate is lower in those with follicular carcinoma, especially if angioinvasion is present, and is only 50% in those with poorly differentiated tumours. Patients who present with or develop metastatic disease (around 10–​15% of patients) have poor survival rates which are less than 10% at 10 years, especially in those whose disease becomes refractory to radio-​iodine. Anaplastic carcinoma Anaplastic carcinoma (Fig. 13.3.2.5) is usually rapidly fatal. Initial as- sessment should focus on the identification of the small proportion of patients with localized disease and good performance status that Fig. 13.3.2.4  131I scans of three patients. Patient A had undergone near-​total thyroidectomy three months previously: two foci of remnant thyroid tissue remain in the neck. Patients B and C had both had thyroidectomy followed by 131I therapy two years previously and been found to have elevated serum thyroglobulin on routine monitoring. In patient B there are numerous iodine-​avid lymph node metastases in the neck/​mediastinum and bilateral pulmonary metastases. No iodine-​avid disease is identified in patient C where there is physiological iodine accumulation in salivary glands, saliva, nasal secretions, and stomach, with excreted iodine in bowel and urinary tract. From Kim CK (ed) (2015). Nuclear medicine and PET/​CT cases. By permission of Oxford University Press, USA. Table 13.3.2.7  Ten-​year relative survival for differentiated thyroid cancer (adapted from 2014 BTA Guidelines for the management of thyroid cancer) Stage 10-​year relative survival (%) I 98.5 II 98.8 III 99.0 IVA 75.9 IVB 62.5 IVC 63

SECTION 13  Endocrine disorders 2310 may benefit from surgical resection and other adjuvant therapies. Surgery may help to relieve obstructive symptoms and external beam radiotherapy is useful in palliation, but the tumour does not take up radio-​iodine. The place of chemotherapy (usually doxorubicin com- bined with other drugs) remains unclear. The median survival time for anaplastic carcinoma is 4 to 12 months and those with distant me- tastases at presentation have a median survival time of only 3 months. Prevention In the event of a nuclear accident, prompt administration of stable iodine prevents the uptake of inhaled and ingested radioactive iodine isotopes. Emergency arrangements should be in place close to nuclear installations to provide for distribution of potassium iodate tablets. Advice is available from the World Health Organization (see ‘Further reading’). Special problems in pregnancy A solitary nodule in a pregnant woman should be evaluated by ultra- sonography and fine needle aspiration biopsy. If the biopsy suggests malignancy and the nodule is growing significantly, or if lymph node metastases develop, surgery can be undertaken in the second trimester, but otherwise this is best deferred until after delivery. Regular ultrasound surveillance is required in patients with newly diagnosed thyroid cancer who do not undergo surgery during preg- nancy. Breastfeeding should be discontinued at least 8 weeks before radio-​iodine remnant ablation, and pregnancy should be avoided for 6 to 12 months following radio-​iodine treatment. The preconception TSH goal in women with pre-​existing differ- entiated thyroid cancer, which is determined by risk stratification, should be maintained during pregnancy. It is likely that higher doses of levothyroxine will be required to achieve this. Medullary carcinoma of the thyroid Epidemiology and pathology Medullary carcinoma accounts for 3–​5% of all thyroid cancers. About 80% are sporadic with a peak incidence at 40 to 60 years of age. Hereditary autosomal dominant forms occur as part of multiple endocrine neoplasia type 2A (MEN2A) or type 2B (MEN2B) or as isolated familial medullary carcinoma. These forms are associated with germline point mutations in the RET proto-​oncogene (different from those in papillary carcinoma) and preneoplastic C-​cell hyper- plasia (Table 13.3.2.8). (a) (b) Fig. 13.3.2.5  MRI and CT appearances of anaplastic thyroid carcinoma. Panel (a): transverse fat-​saturated T2-​weighted MRI image demonstrating a large right-​sided mass involving the right thyroid lamina and extending around the posterior margin of the cartilage towards the larynx. Panel (b): CT (bone windows) on the same patient demonstrating the markedly abnormal involved right thyroid lamina with ill definition, permeative destruction, and reactive sclerosis. From Hoskin P, Goh V (eds) (2010). Radiotherapy in practice—​imaging. By permission of Oxford University Press. Table 13.3.2.8  Types of medullary carcinoma of the thyroid Type Frequency (%) Associated lesions RET gene mutation Sporadic 80 None RET (in approximately 50%), HRAS, NRAS, or KRAS (in 0–​43%) MEN2A 10 Phaeochromocytoma (20–​50%), hyperparathyroidism (12–​30%) Hirschsprung disease or cutaneous lichen amyloidosis in some 95% of RET mutations in exon 10 (codon 609, 611, 618 or 620) or exon 11 (codon 634) MEN2B 3 Phaeochromocytoma, mucosal neuromas, marfanoid habitus, typical facies, medullated corneal nerves, and aerodigestive tract ganglioneuromatosis RET M918T mutation in more than 95% and RET A833F in the remainder Familial medullary thyroid carcinoma 7 None Broad range of RET mutations MEN, multiple endocrine neoplasia.

13.3.2  Thyroid cancer 2311 The pathological findings are of an encapsulated tumour with round, spindle-​shaped, or polyhedral cells arranged in a variety of patterns that have no prognostic significance. There is vari- able fibrosis and three-​quarters of tumours show marked depos- ition of amyloid—​a feature associated with a good prognosis. Heterogeneous staining for calcitonin, a hormone of C cells, is as- sociated with a poorer outcome, reflecting dedifferentiation. Even the smallest medullary tumours may be associated with local lymph node metastases. Clinical features, diagnosis, and investigation The presentation of sporadic medullary carcinoma is typically with a solitary thyroid nodule, accompanied by cervical lymphadenop- athy in 50% of cases. Lung, liver, or bone metastases are present at diagnosis in 10% of cases. Symptoms due to local invasion or the paraneoplastic production of polypeptides and prostaglandins, such as flushing, diarrhoea, and Cushing’s syndrome, are less common presenting features. The diagnosis is typically made through ultrasonography and fine needle aspiration biopsy. Immunohistochemical staining for calci- tonin in aspirated cells or in washout fluid of the fine needle aspir- ation may be required. Basal serum calcitonin concentrations are almost invariably elevated and confirm the diagnosis. There is con- troversy over the utility of routine serum calcitonin measurement in the work-​up of all thyroid nodules; most centres perform aspir- ation biopsy initially. Newly diagnosed patients should be screened for other evidence of MEN and a careful family history is essential. In particular, phaeochromocytoma occurring as part of an inherited cancer syndrome must be excluded before surgery through meas- urement of serum or urine metanephrines or catecholamines. In all cases of confirmed MTC, RET mutation analysis to establish the possible genetic basis for the disease should be performed, even in the absence of a positive family history. Testing genomic DNA for RET mutations in the germline is now widely available and should ideally be carried out on leucocyte DNA from all new patients. The absence of the most common mutations, coupled with a negative family history and the absence of C-​cell hyperplasia or multicentric tumours in the resected thyroid, indicates that further family testing is not warranted. When a RET mutation is detected, there is a clear benefit from family testing as prophylactic thyroidectomy in affected individuals improves outcome. However, there are some kindreds in whom familial medullary carcinoma occurs without a recognizable RET mutation and family screening must then be undertaken an- nually, up to the age of 35 to 40 years, using serum calcitonin and carcinoembryonic antigen (CEA) measurements as a guide to the presence of the inherited abnormality. Management Surgery is the primary treatment for patients with sporadic or hereditary medullary thyroid carcinoma and ranges from thy- roid lobectomy in selected patients with sporadic disease to total thyroidectomy with central neck dissection and unilateral or bi- lateral lymph-​node compartment dissection. In patients who have inherited a mutated RET allele, the reliable indicator for timing thyroidectomy is the serum calcitonin concentration rather than the specific mutation. Levothyroxine replacement is needed at physiological doses rather than doses that suppress TSH. After surgery, the patient should be monitored by measurement of serum calcitonin concentration. The most accurate measurement of medullary cancer progression and ag- gressiveness is the calcitonin or CEA doubling time (the time it takes for the marker to double). Doubling times that are less than 6 months are associated with a very poor prognosis and those that are more than 2 years are associated with much better long-​term survival rates. Although many patients with metastatic or persistent medullary thyroid carcinoma can be monitored, patients with progressive or symptomatic disease should undergo further treatment. Local re- currence with identifiable lymph node involvement should be treated surgically. Standard chemotherapy and radiotherapy regi- mens are largely ineffective. Profuse (secretory) watery diarrhoea is frequently a troublesome feature of extensive disease. This may respond to treatment with loperamide, whereas somatostatin analogues have an inconsistent benefit. Systemic therapy with vandetanib and cabozantinib, RET tyrosine kinase inhibitors with additional inhibitory effects on the vascular endothelial growth factor receptor, increase progression-​ free survival in locally advanced or metastatic progressive medullary thyroid cancer and provide relief from symptoms. Prognosis Cure, defined as a persistently normal calcitonin level, occurs in only about one-​third of patients, but 80 to 90% of patients with an ele- vated calcitonin level and only nodal disease survive for 10 years. Age, stage and size of tumour, and completeness of surgical removal are important prognostic features. Familial medullary carcinoma has the best outcome; in contrast the tumour associated with MEN2B is very aggressive. The overall 10-​year survival is around 70%, but is over 90% in those detected early by family screening. Primary thyroid lymphoma Less than 5% of thyroid malignancies are non-​Hodgkin’s B-​cell lymphoma. The peak incidence is between 50 and 80 years of age, and women are affected three times more frequently than men. The typical presentation is a rapidly enlarging thyroid mass in a patient with Hashimoto’s thyroiditis. The clinical features may suggest anaplastic carcinoma. The diagnosis can be made by fine needle aspiration biopsy and confirmed by large-​needle or open bi- opsy. Accurate staging is then necessary to plan treatment, which is with external beam radiotherapy and anthracycline-​based chemo- therapy. Intensive treatment has produced 8-​year survival rates of over 90%. Rituximab, a monoclonal antibody directed against B cells, has shown some evidence of therapeutic benefit. FURTHER READING Agrawal N, et al. (2015). Integrated genomic characterization of pap- illary thyroid carcinoma. Cell, 159, 676–​90. British Thyroid Association (2016). Thyroid Cancer: Low Iodine Diet. http://​www.btf-​thyroid.org/​projects/​thyroid-​cancer-​campaign/​ low-​iodine-​diet Brose MS, et  al. (2014). Sorafenib in radioactive iodine-​refractory, locally advanced or metastatic differentiated thyroid cancer: a ran- domised, double-​blind, phase 3 trial. Lancet, 384, 319–​28.

SECTION 13  Endocrine disorders 2312 Cabanillas ME, McFadden DG, Durante C (2016). Thyroid cancer. Lancet, 388, 2783–​95. Fagin JA, Wells SA Jr (2016). Biologic and clinical perspectives on thy- roid cancer. N Engl J Med, 375, 2307. Filetti S, et al. (2019). Thyroid cancer: ESMO clinical practice guide- lines for diagnosis, treatment and follow-up. Ann Oncol, pii: mdz400. doi: 10.1093/annonc/mdz400. Harrington KJ, et al. (2001). Gene therapy for prostate cancer: current status and future prospects. J Urol, 166, 1220–​33. Haugen BR, et al. (2016). 2015 American Thyroid Association man- agement guidelines for adult patients with thyroid nodules and differentiated thyroid cancer:  the American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer. Thyroid, 26, 1–​133. Haugen BR, et al. (2017). American Thyroid Association guidelines on the management of thyroid nodules and differentiated thyroid cancer task force review and recommendation on the proposed renaming of encapsulated follicular variant papillary thyroid car- cinoma without invasion to noninvasive follicular thyroid neoplasm with papillary-​like nuclear features. Thyroid, 27, 481–​3. Ho AL, et al. (2013). Selumetinib-​enhanced radioiodine uptake in ad- vanced thyroid cancer. N Engl J Med, 368, 623–​32. Leboulleux S, et al. (2016). Papillary thyroid microcarcinoma: time to shift from surgery to active surveillance? Lancet Diabetes Endocrinol, 4, 933–​42. Morris LG, Tuttle RM, Davies L (2016). Changing trends in the in- cidence of thyroid cancer in the United States. JAMA Otolaryngol Head Neck Surg, 142, 709–​11. Perros P, et  al. (2014). Guidelines for the management of thyroid cancer. Clin Endocrinol (Oxf), 81 Suppl 1, 1–​122. Schlumberger M, et al. (2012). Strategies of radioiodine ablation in patients with low-​risk thyroid cancer. N Engl J Med, 366, 1663–​73. Schlumberger M, Tahara M, Wirth LJ (2015). Lenvatinib in radioiodine-​refractory thyroid cancer. N Engl J Med, 372, 1868. Spitzweg C, Morris JC, Bible KC (2016). New drugs for medullary thy- roid cancer: new promises? Endocr Relat Cancer, 23, R287–​97. Tufano RP, et al. (2015). Management of recurrent/​persistent nodal disease in patients with differentiated thyroid cancer: a critical re- view of the risks and benefits of surgical intervention versus active surveillance. Thyroid, 25, 15–​27. Viola D, et al. (2016). Treatment of advanced thyroid cancer with targeted therapies: ten years of experience. Endocr Relat Cancer, 23, R185–​205. Viola D, Elisei R (2019). Management of medullary thyroid cancer. Endocrinol Metab Clin North Am, 48, 285–301. WHO (2011). Ionizing Radiation: Use of Potassium Iodide for Thyroid Protection During Nuclear or Radiological Emergencies. http://​www. who.int/​ionizing_​radiation/​pub_​meet/​tech_​briefings/​potassium_​ iodide/​en/

13.4 Parathyroid disorders and diseases altering c

13.4 Parathyroid disorders and diseases altering calcium metabolism 2313

ESSENTIALS The control of body calcium involves a balance—​chiefly under the control of parathyroid hormone (PTH)—​between the amounts that are absorbed from the gut, deposited into bone and into cells, and excreted from the kidney. Under normal physiological circumstances PTH secretion from the parathyroid glands is increased by hypocal- caemia and diminished by hypercalcaemia. Hypercalcaemia Clinical presentation—​this is very variable, ranging from a mild asymptomatic biochemical abnormality to (in extreme cases) a life-​ threatening medical emergency. Clinical manifestations can be renal (nephrocalcinosis, kidney stones), musculoskeletal (bone pain, mus- cular weakness), gastrointestinal (anorexia, nausea, vomiting, con- stipation, peptic ulceration, pancreatitis), neurological (depression, confusion, coma), and cardiac (arrhythmia). Aetiology—​primary hyperparathyroidism and malignancy account for more than 90% of patients with hypercalcaemia. Other causes include (1)  excess vitamin D—​exogenous or endogenous (e.g. granulomatous disorders); (2) drugs (e.g. thiazide diuretics, lithium, milk-​alkali syndrome); (3) non​parathyroid endocrine disorders (e.g. thyrotoxicosis, immobilization); (4) inappropriate PTH levels due to altered set point (e.g. familial benign hypocalciuric hypercalcaemia). Management—​aside from appropriate treatment of the underlying condition, management of hypercalcaemia depends on its severity and the presence of symptoms. Asymptomatic patients with serum calcium less than 3.00 mmol/​litre do not usually need urgent treat- ment. Patients with serum calcium below 3.50 mmol/​litre, or above 3.00 mmol/​litre with symptoms, require (1) vigorous hydration with 0.9% saline (assuming adequate renal function), with diuresis en- couraged with a loop diuretic (e.g. furosemide) if necessary; (2) par- enteral bisphosphonate (e.g. pamidronate, zoledronic acid); with (3) glucocorticoids—​if the hypercalcaemia is mediated by the actions of 1,25-​dihydroxy vitamin D (e.g. granulomatosis disease, lymphoma, myeloma) and in exceptional circumstances; (4) haemodialysis. Specific diseases causing hypercalcaemia Primary hyperparathyroidism—​due to excessive secretion of PTH by parathyroid tumour(s); of unknown cause in most instances, but 10% of cases are associated with hereditary disorders, for example, multiple endocrine neoplasia type 1 (MEN1, with combined occurrence of parathyroid, pancreatic islet cell and anterior pituitary tumours) and type 2 (MEN2, with association of medullary thyroid carcinoma, phaeochromocytoma and parathyroid tumours); biochemical diag- nosis typically achieved by finding an elevated PTH concentration in the presence of hypercalcaemia; parathyroidectomy is the definitive cure, but cinacalcet—​an allosteric activator of the calcium-​sensing receptor—​can be effective. Tertiary hyperparathyroidism—​secondary hyperparathyroidism arises in the context of chronic kidney disease, but eventually the para- thyroid cells become autonomous, secreting excessive PTH despite hypercalcaemia, which is known as tertiary hyperparathyroidism. Malignancy—​hypercalcaemia is usually due to increased bone resorption, which may either be directly due to skeletal metastases (most commonly from breast, lymphoma, or multiple myeloma) or indirectly due to tumour production of a humoral factor (usu- ally parathyroid hormone-​related peptide, PTHrP, secreted from squamous carcinomas or other cancers) that stimulates osteoclastic bone resorption. Aside from measures just described, management involves reducing the tumour load by surgery, radiotherapy, and/​or chemotherapy. Granulomatous disorders—​hypercalcaemia is due to extrarenal synthesis of 1,25-​dihydroxy vitamin D; most common diagnosis is sarcoidosis, when hypercalcaemia should respond within 10 days to treatment with glucocorticoids. Familial benign hypocalciuric hypercalcaemia—​autosomal dom- inant due to heterozygous inactivating mutations of the calcium-​ sensing receptor, G-​protein subunit α11, or adaptor protein 2 sigma subunit; causes (usually) asymptomatic hypercalcaemia in asso- ciation with an inappropriately low urinary calcium excretion and normal serum PTH. Hypocalcaemia Clinical presentation—​this is variable, including (1)  a mild, asymp- tomatic, biochemical abnormality; (2) in chronic cases with ectopic calcification, subcapsular cataract, papilloedema, and abnormal dentition; and (3) in severe cases with neuromuscular irritability. Aetiology—​may be associated with (1)  low serum PTH—​ hypoparathyroidism, most often caused by autoimmune disease, surgical removal of the parathyroid glands, or hypomagnesaemia; 13.4 Parathyroid disorders and diseases altering calcium metabolism R.V. Thakker

SECTION 13  Endocrine disorders 2314 or (2)  high serum PTH—​secondary hyperparathyroidism, most com- monly due to vitamin D deficiency and/​or renal failure. Management—​aside from appropriate treatment of the underlying condition, management of acute hypocalcaemia depends on its se- verity, rapidity of onset, and the degree of neuromuscular irritability. Patients with seizures or tetany may require intravenous calcium gluconate, as do asymptomatic patients with serum calcium below 1.90 mmol/​litre as well as oral vitamin D. Specific diseases causing hypocalcaemia Pluriglandular autoimmune hypoparathyroidism—​characterized by hypoparathyroidism, Addison’s disease, and candidiasis in the pres- ence of other organ-​specific autoimmune diseases; autosomal re- cessive inheritance due to mutation of an autoimmune regulator gene, with antibodies directed against the adrenal, thyroid, and para- thyroid glands sometimes present. Hypomagnesaemia—​may be caused by malabsorption or renal tubular disorder; leads to functional hypoparathyroidism because magnesium is required for the release of PTH from the parathyroid gland and also for PTH action via adenyl cyclase. A variety of rare syndromes may cause hypoparathyroidism, and similar functional consequences can be caused by resistance to the effects of PTH (e.g. pseudohypoparathyroidism, of which there are five variants, some with somatic features such as shortening of one or more metacarpals). Introduction Calcium plays an important role in many physiological pathways that include muscle contraction, the secretion of neurotransmitters and hormones, and coagulation. The control of body calcium involves a balance between the amounts that are absorbed from the gut, depos- ited into bone and cells, and excreted from the kidney (Fig. 13.4.1). This fine balance, involving all these organs, is chiefly under the control of parathyroid hormone (PTH), which is synthesized and secreted by the parathyroid glands. Thus, hypocalcaemia will lead to an increased secretion of PTH, whereas hypercalcaemia will result in diminished PTH secretion. Abnormalities of the parathyroid glands themselves will cause derangements of calcium homeostasis and several clinical disorders. PTH oversecretion due to parathyroid tumours, which af- fect 3 in 1000 of the population, is a major cause of hypercalcaemia which may be associated with kidney stones, osteoporosis, and peptic ulcers. PTH deficiency, which results in hypocalcaemia and occurs in 1 in 4000 live births, may be associated with epilepsy, tetany, cataracts, skeletal malformations, and abnormal dentition. This chapter will re- view the physiological and biochemical mechanisms underlying extra- cellular calcium homeostasis, the clinical features of hypercalcaemia and hypocalcaemia, the clinical disorders associated with abnormal calcium homeostasis and their management, and the genetic basis for disorders of calcium metabolism. Historical perspective The discovery of the parathyroids in the latter part of the 19th century and their function in regulating calcium homeostasis has evolved over 150 years and has involved studies in humans and other mammals (Table 13.4.1). In the past few decades with the advent of the advances in molecular biology, several cellular and molecular mechanisms involving G-​protein-​coupled receptors, intracellular second messengers, and transcription factors have been shown to be involved in calcium homeostasis and in the aetiology of parathyroid disorders (Fig. 13.4.2). These advances have elucidated the roles of the parathyroids and PTH in regulating calcium. Moreover, these advances have helped in defining new treatments for patients. For example, cinacalcet, which is an allosteric modulator of the calcium-​ sensing receptor (CaSR), is now used in the treatment of secondary hyperparathyroidism in dialysis patients with end stage renal dis- ease and for the treatment of hypercalcaemia in parathyroid car- cinoma, and PTH, which has been shown to reduce the incidence of vertebral and non​vertebral fractures, has now been approved as the first anabolic agent for the treatment of osteoporosis. Calcium homeostasis Most of the total of 1 kg of calcium in the healthy adult is present within the crystal structure of bone mineral and less than 1% is in sol- uble form in the extracellular and intracellular fluid compartments. In the extracellular fluid compartment about one-​half of the total Dietary Ca2+, Vitamin D Vitamin D PARATHYROID ECF PTH INTESTINE UV LIGHT 7-Dehydro- cholesterol LIVER KIDNEY 1,25(OH)2Vitamin D 25(OH) Vitamin D 1α S K I N + + + B O N E Ca2+ + + Ca2+ Ca2+ Ca2+ Fig. 13.4.1  Regulation of extracellular fluid (ECF) calcium (Ca2+) by parathyroid hormone (PTH) action on kidney, bone, and intestine. A decrease in ECF Ca2+ is sensed by the calcium-​sensing receptor (CaSR) (Fig. 13.4.2), and this leads to an increase in PTH secretion which predominantly acts directly on kidney and bone that possess the PTH receptor (PTHR, Fig. 13.4.2). The skeletal effects of PTH are to increase (+) osteoclastic bone reabsorption, but as osteoclasts do not have PTHRs, this action is mediated via the osteoblasts, which do have PTHRs and in response release cytokines and factors that activate osteoclasts. In the kidney, PTH stimulates (+) the 1α-​hydroxylase (1α) to increase the conversion of 25-​hydroxyvitamin D (25(OH)D) to the active metabolite 1,25-​dihydroxyvitamin D (1,25(OH)2D). In addition, PTH increases (+) the reabsorption of Ca2+ from the renal distal tubule and inhibits the reabsorption of phosphate from the proximal tubule, thereby leading to hypercalcaemia and hypophosphataemia. PTH also inhibits Na+–​H+ antiporter activity and bicarbonate reabsorption, thereby causing a mild hyperchloraemic acidosis. The elevated 1,25(OH)2D acts on the intestine to increase (+) absorption of dietary calcium and phosphate, and it is important to note that PTH does not appear to have a direct action on the gut. Thus, in response to hypocalcaemia and the increase in PTH secretion, all of these direct and indirect actions of PTH on the kidney, bone, and intestine will help to increase ECF Ca2+, which in turn will act via the CaSR to decrease PTH secretion.

13.4  Parathyroid disorders and diseases altering calcium metabolism 2315 calcium is ionized and the rest is principally bound to albumin or complexed with counterions. Ionized calcium concentrations range from 1.17 to 1.33 mmol/​litre, and the total serum calcium concentra- tion ranges from 2.12 to 2.62 mmol/​litre. Measurements of free ion- ized calcium are not often undertaken because they are difficult; most laboratories report total serum calcium concentration for routine clinical use. However, the usual 2:1 ratio of total to ionized calcium may be disturbed by disorders such as metabolic acidosis, which re- duces calcium binding by proteins, or by changes in protein concen- tration caused by cirrhosis, dehydration, venous stasis, or multiple myeloma. In view of this, total serum concentrations are adjusted or ‘corrected’ to a reference albumin concentration; thus, the corrected serum calcium may be related to a reference albumin concentration of 41 g/​litre and for every 1 g/​litre of albumin above or below the ref- erence value the calcium is adjusted by 0.016 mmol/​litre up or down, respectively. For example, a total serum calcium of 2.70 mol/​litre with an albumin concentration of 47 g/​litre would be equivalent to a cor- rected serum calcium of 2.60 mmol/​litre, thereby correcting the ini- tial apparent hypercalcaemic value to a normal value. The extracellular concentration of calcium is closely regulated within the narrow physiological range that is optimal for those cel- lular functions that are affected by calcium (Fig. 13.4.1). Indeed, both hypercalcaemia and hypocalcaemia impair the function of many different organ systems. Regulation of extracellular cal- cium takes place through complex interactions at the target or- gans of the major calcium-​regulating hormone PTH (Fig. 13.4.2) and by vitamin D and its active metabolite 1,25-​dihydroxyvitamin D.  The parathyroid glands secrete PTH at a rate that is appro- priate to and depending on the prevailing extracellular calcium ion concentration. Aetiology and genetics Parathyroid gland disorders cause either hypercalcaemia or hypo- calcaemia, and these can be classified according to whether they arise from an excess of PTH, its deficiency, or an insensitivity to its effects (Table 13.4.2 and Fig. 13.4.2). Table 13.4.1  Some historical landmarks elucidating the role of the parathyroids in calcium homeostasis Date Discovery 1852 Sir Richard Owen, curator of the Natural History Museum (London) discovers the parathyroids when dissecting a rhinoceros that had died in London Zoo 1880 Sandstrom, in Uppsala, describes parathyroids in man 1881 Weiss in Billroth’s clinic (Vienna) reports tetany following thyroidectomy 1891 Gley shows that parathyroidectomy alone can cause tetany and death 1891 Von Recklinghausen reports first case of osteitis fibrosis cystica 1906 Erdheim describes parathyroid ‘overgrowth’ in calcium-​deficient state of osteomalacia 1909 McCallum and Voegtlin demonstrate that postparathyroidectomy tetany and hypocalcaemia can be corrected by administration of parathyroid extract or calcium 1925 Collip establishes the role of parathyroids as endocrine glands that secrete PTH 1925 Mandl operates on a patient with severe bone demineralization and fractures and removes an enlarged parathyroid, resulting in a dramatic improvement of the patient’s condition; this represents the first successful parathyroidectomy 1939 Drake et al. report six cases of idiopathic hypoparathyroidism 1942 Albright et al. report three cases of pseudohypoparathyroidism 1959 Aurbach, and Rasmussen and Craig independently isolate PTH 1978 Keutmann et al. report complete amino acid sequence of human PTH 1983 Vasicek et al. characterize nucleotide sequence of human PTH gene 1987 Mosely et al. Suva et al. and Strewler et al. independently identify PTHrP as the humoral factor causing the hypercalcaemia of malignancy 1991 Juppner et al. identify a G-​protein-​coupled receptor that mediates actions of PTH and PTHrP 1993 Brown et al. identify the CaSR, a G-​protein-​coupled receptor, and that CaSR mutations cause familial hypocalciuric hypercalcaemia type 1 (FHH1) 1996 Winer et al. report that administration of synthetic human PTH to patients with hypoparathyroidism, maintains normocalcaemia and reduces urinary calcium excretion 2001 Neer et al. report that administration of PTH reduces the occurrence of osteoporotic vertebral and non​vertebral fractures in postmenopausal women 2004 Block et al. show that cinacalcet, a calcimimetic agent that acts on the CaSR, lowers PTH levels, and improves calcium and phosphate homeostasis in patients on dialysis and with uncontrolled secondary hyperparathyroidism 2005 Peacock et al. report that cinacalcet reduces serum calcium and PTH in patients with primary hyperparathyroidism, thereby providing a potential medical therapy for this condition 2013 Nesbit et al. show that mutations of Gα11 cause FHH2 2013 Nesbit et al. report that mutations of AP2σ cause FHH3 2015 FDA approves use of human recombinant PTH (1-​84) for management of refractory hypoparathyroidism 2016 Howles et al. report that cinacalcet can reduce the symptoms of hypercalcaemia in patients with FHH3 CaSR, calcium-​sensing receptor; Gα11, G-​protein subunit alpha 11; AP2σ, adaptor protein 2 sigma subunit; PTH, parathyroid hormone; PTHrP, parathyroid hormone-​related peptide.

SECTION 13  Endocrine disorders 2316 Fig. 13.4.2  Schematic representation of some of the components involved in calcium homeostasis. Alterations in extracellular calcium are detected by the calcium-​sensing receptor (CaSR), which is a 1078 amino acid G-​protein-​coupled receptor. The PTH/​PTH-​related peptide (PTHrP) receptor, which mediates the actions of PTH and PTHrP, is also a G-​protein-​coupled receptor. Thus, Ca2+, PTH, and PTHrP involve G-​protein-​coupled signalling pathways, and interaction with their specific receptors can lead to activation of Gs, Gi, and Gq, respectively. Gs stimulates adenyl cyclase (AC) which catalyses the formation of cAMP from ATP. Gi inhibits AC activity. cAMP stimulates protein kinase A which phosphorylates cell-​specific substrates. Activation of Gα11/​Gq stimulates phospholipase C (PLC), which catalyses the hydrolysis of the phosphoinositide (PIP2) to inositol triphosphate (IP3), which increases intracellular calcium, and diacylglycerol (DAG), which activates protein kinase C (PKC). These proximal signals modulate downstream pathways, which result in specific physiological effects. Abnormalities in several genes, which lead to mutations in proteins in these pathways, have been identified in specific disorders of calcium homeostasis (Table 13.4.1). ADH1 and ADH2, autosomal dominant hypocalcaemia types 1 and 2; AIRE, autoimmune regulator protein; AP2σ, adaptor protein 2 sigma subunit; APECED, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome; CCND1, cyclin D1; CDC73, cell division cycle protein 73; FAMIIIA, family with sequence similarity 111A; FHH1–​3, familial hypocalciuric hypercalcaemia types 1–​3; GATA3, GATA binding protein 3; GCM2, glial cells missing homologue 2; HDR, hypoparathyroidism, deafness, and renal dysplasia syndrome; HPT-​JT, hyperparathyroidism–​jaw tumour syndrome; KSS, Kearns–​Sayre syndrome; MELAS, mitochondrial encephalopathy, lactic acidosis, and stroke-​like episodes; MEN1, multiple endocrine neoplasia type 1 syndrome; MTPDS, mitochondrial trifunctional protein deficiency syndrome; NSHP, neonatal severe primary hyperparathyroidism; Rb, retinoblastoma; TBCE, tubulin-​specific chaperone. Adapted from Thakker RV (2000). Parathyroid disorders. Molecular genetics and physiology. In: Morris PJ, Wood WC (eds) Oxford textbook of surgery, 2nd edition, pp. 1121–​9. Oxford University Press, Oxford.

13.4  Parathyroid disorders and diseases altering calcium metabolism 2317 Table 13.4.2  Parathyroid diseases and their chromosomal locations Metabolic abnormality Disease Inheritance Gene/​gene product Chromosomal location Hypercalcaemia MEN1 (OMIM 131100) Autosomal dominant Menin 11q13 MEN2 and MEN3 (OMIM 171400) Autosomal dominant RET 10q11.2 MEN4 (OMIM 610755) Autosomal dominant CDNK1B 12p13.1 HPT-​JT (OMIM 145001) Autosomal dominant CDC73/​Parafibromin 1q31.2 Sporadic hyperparathyroidism (OMIM 145000) Sporadic CCND1 11q13 Retinoblastoma 13q14 Unknown 1p32-​pter Parathyroid carcinoma (OMIM 608266) Sporadic Parafibromin 1q31.2 Retinoblastoma 13q14   FHH1 (OMIM 145980) Autosomal dominant CASR 3q21.1   FHH2 (OMIM 139313) Autosomal dominant Gα11 19p13   FHH3 (OMIM 600740) Autosomal dominant AP2S1/​AP2σ 19q13 NSHP (OMIM 239200) Autosomal recessive CASR 3q21.1 Autosomal dominant Jansen’s disease (OMIM 156400) Autosomal dominant PTHR/​PTHrPR 3p21.3 William’s syndrome (OMIM 194050) Autosomal dominant ELN, LIMK1 (and other genes) 7q11.23 Infantile hypercalcaemia (OMIM 143880) Autosomal recessive CYP24A 20q13.2-​q13.3 McCune–​Albright syndrome (OMIM 174800) Mutations during early embryonic development? Gsα 20q13.3 Hypocalcaemia Isolated hypoparathyroidism (OMIM 146200) Autosomal dominant PTH 11p15a Autosomal recessive PTH, GCM2 11p15a, 6p24.2 X-​linked recessive SOX3 Xq26–​27 ADH1 (OMIM 601198) Autosomal dominant CaSR 3q21.1 ADH2 (OMIM 615361) Autosomal dominant Gα11 19p13 Hypoparathyroidism associated with polyglandular autoimmune syndrome (APECED) (OMIM 240300) Autosomal recessive AIRE1 21q22.3 Hypoparathyroidism associated with Kearns–​Sayre (OMIM 530000) and MELAS (OMIM 540000) syndromes Maternal Mitochondrial genome Hypoparathyroidism associated with complex congenital syndromes   DiGeorge syndrome type 1 (OMIM 188400) Autosomal dominant TBX1 22q11.2/​10p   DiGeorge syndrome type 2 (OMIM 601362) Autosomal dominant NEBL 10p14-​p13   HDR syndrome (OMIM 146255) Autosomal dominant GATA3 10p15   Blomstrand lethal chondrodysplasia (OMIM 215045) Autosomal recessive PTHR/​PTHrPR 3p21.3   Kenney–​Caffey type 1 (OMIM 244460), and
  Sanjad–​Sakati syndromes Autosomal dominant TBCE 1q42.3   Kenney–​Caffey type 2 (OMIM 127000) Autosomal recessive FAM111A 11q12.1   Barakat syndrome Autosomal recessiveb Unknown ?   Lymphoedema Autosomal recessive Unknown ?   Nephropathy, nerve deafness Autosomal dominantb Unknown ?   Nerve deafness without renal dysplasia Autosomal dominant Unknown? ? PHP (type Ia) (OMIM 103580) Autosomal dominant parentally imprinted GNAS exons 1–​3 20q13.2 PHP (type Ib) (OMIM 603233) Autosomal dominant parentally imprinted GNAS, upstream deletion 20q13.3 CDC73, cell division cycle protein 73; CDNK1B, cyclin-​dependent kinase inhibitor 1B, GATA3, GATA binding protein 3; GCM2, glial cells missing homologue 2; HDR, hypoparathyroidism, deafness, and renal dysplasia; MELAS, mitochondrial encephalopathy, lactic acidosis, and stroke-​like episodes; NEBL, nebulette; PTHR, parathyroid hormone receptor; PTHrPR, parathyroid hormone-​related peptide receptor;  ? location not known. a Mutations of PTH gene identified only in some families. b Most likely inheritance.

SECTION 13  Endocrine disorders 2318 The PTH gene is located on chromosome 11p15 and consists of three exons (transcribed regions) which are separated by two in- trons. Exon 1 of the PTH gene is 85 bp in length and is untranslated whereas exons 2 and 3 code for the 115 amino acid pre-​proPTH peptide. Exon 2 is 90 bp in length and encodes the initiation (ATG) codon, the prehormone sequence, and part of the prohormone sequence. Exon 3 is 612 bp in length and encodes the remainder of the prohormone sequence, the mature PTH peptide, and the 3′ untranslated region. The 5′ regulatory sequence of the human PTH gene contains a vitamin D response element 125 bp upstream of the transcription start site, which down-​regulates PTH mRNA tran- scription in response to vitamin D receptor binding. PTH gene transcription (as well as PTH peptide secretion) is also dependent on the extracellular calcium concentration, although the presence of a specific upstream ‘calcium response element’ has not yet been demonstrated. The mature PTH peptide is secreted from the parathyroid chief cell as an 84 amino acid peptide; however, when the PTH mRNA is first translated it is as pre-​proPTH peptide. The ‘pre’ sequence con- sists of a 25 amino acid signal peptide (leader sequence) which is responsible for directing the nascent peptide into the endoplasmic reticulum to be packaged for secretion from the cell. The ‘pro’ sequence is six amino acids in length and, although its function is less well defined than that of the ‘pre’ sequence, it is also essential for correct PTH processing and secretion. After the 84 amino acid ma- ture PTH peptide is secreted from the parathyroid cell, it is cleared from the circulation, with a short half-​life of about 2 min, via non-​ saturable hepatic uptake and renal excretion. PTH shares a receptor with PTH-​related peptide (PTHrP); this PTH/​PTHrP receptor (Fig. 13.4.2) is a member of a subgroup of the G-​protein-​coupled receptor family. The PTH/​PTHrP receptor gene is located on chromosome 3p21.3 and is expressed in kidney and bone, where PTH is its predominant agonist. Expression of the PTH/​ PTHrP receptor also occurs in the brain, heart, skin, lung, liver, and testis where it mediates the actions of PTHrP. Mutations involving the genes that encode these proteins and receptors in this calcium regulating pathway (Fig. 13.4.2) are associated with hypercalcaemic and hypocalcaemic disorders (Table 13.4.2). Hypercalcaemia Clinical features and investigations The clinical presentation of hypercalcaemia varies from a mild, asymptomatic, biochemical abnormality detected during routine screening to a life-​threatening medical emergency. In general, the presence or absence of symptoms correlates with the severity and rapidity of onset of the hypercalcaemia. Thus, symptoms do not usu- ally develop when serum calcium is below 3.00 mmol/​litre and are in- variably present when the hypercalcaemia exceeds 3.50 mmol/​litre. However, there is a considerable variability and some patients may be symptomatic with mild hypercalcaemia (2.65–​2.90 mmol/​litre). Although there are many causes of hypercalcaemia (Box 13.4.1), the signs and symptoms of hypercalcaemia are similar, regardless of aetiology. Indeed, the clinical manifestations of hypercalcaemia in- volve several organ systems that include the renal, musculoskeletal, gastrointestinal, neurological, and cardiac systems (Box 13.4.2), and many of these have been referred to as ‘moans, groans, pains, and stones’. Investigations should be directed at confirming the presence of hypercalcaemia and establishing the cause (Box 13.4.1). The causes of hypercalcaemia can be classified according to whether serum PTH concentrations are elevated (i.e. primary hyperparathyr- oidism) or low (i.e. not due to a parathyroid tumour). Primary hyper- parathyroidism and malignancy are the most common causes and account for more than 90% of patients with hypercalcaemia. Detailed clinical history and examination will usually help to differentiate be- tween these two diagnoses. In primary hyperparathyroidism, the hypercalcaemia is often less than 3.00 mmol/​litre, asymptomatic, and may have been present for months or years. If symptoms such Box 13.4.1  Causes of hypercalcaemia High PTH levels • Primary hyperparathyroidisma (adenoma, hyperplasia, or carcinoma): non​familial or familial (e.g. MEN1, MEN2, HPT-​JT, FIHP) • Tertiary hyperparathyroidism (hyperplasia or adenoma in chronic renal failure) Low PTH levels Malignancya Primary • PTH-​related protein, PTHrP (carcinoma of lung, oesophagus, renal cell, ovary, and bladder) • Excess production of 1,25-​dihydroxyvitamin D (lymphoma) Secondary • Lytic bone metastasesa (multiple myelomaa and breast carcinomaa) • Other location, ectopic factors (e.g. cytokines) • Excess vitamin D • Exogenous vitamin D toxicity by parent D compound, 25-​hydroxyvitamin D3, or 1,25-​dihydroxyvitamin D3 in vitamin preparations, cod liver oil, herbal medicines • Endogenous production of 25-​hydroxyvitamin D3—​William’s syndrome • Endogenous production of 1,25-​dihydroxyvitamin D3, for example, granulomatous disorders (sarcoidosis, HIV, tuberculosis, histo- plasmosis, coccidioidomycosis, leprosy), lymphoma, and infantile hypercalcaemia Drugs • Thiazide diuretics • Lithium • Total parenteral nutrition • Oestrogens/​antioestrogens, testosterone • Milk-​alkali syndrome • Vitamin A toxicity • Foscarnet • Aluminium intoxication (in chronic renal failure) • Aminophylline Non​parathyroid endocrine disorders • Thyrotoxicosis • Phaeochromocytoma • Acute adrenal insufficiency • Vasoactive intestinal polypeptide hormone producing tumour (VIPoma) • Immobilization Inappropriate PTH levels due to altered set point • Familial benign hypocalciuric hypercalcaemia (FHH or FBH) types 1–​3 a Most common causes.

13.4  Parathyroid disorders and diseases altering calcium metabolism 2319 as nephrolithiasis are present, then they have usually been present for several months. However, in malignancy the patients are usually acutely ill, often with neurological symptoms, the hypercalcaemia is more than 3.00 mmol/​litre, and the cancer (e.g. lung, breast, or mye- loma) is often readily apparent. Hypercalcaemia from causes other than primary hyperparathyroidism or malignancy may also occur (Box 13.4.1) and a careful history (e.g. for vitamin D ingestion, drugs, renal disease) and examination (e.g. for thyrotoxicosis, adrenal dis- ease, granulomatosis diseases), together with appropriate investiga- tions (Box 13.4.3) are essential for establishing the diagnosis. Management of hypercalcaemia The management of hypercalcaemia depends on the severity of the hypercalcaemia and the presence of symptoms. Thus, asymptom- atic patients with mild hypercalcaemia (i.e. serum calcium below 3.00 mmol/​litre), do not usually need urgent treatment. However, a patient with severe hypercalcaemia (i.e. a serum calcium above 3.50 mmol/​litre), would require treatment regardless of symptoms, while a patient with moderate hypercalcaemia (i.e. a serum calcium in the range 3.00 to 3.50 mmol/​litre), would require urgent treatment if symptomatic. Before instituting treatment, it is always important to consider the underlying causes (Box 13.4.1) and to initiate in- vestigations (Box 13.4.3). In addition, drugs such as thiazides and vitamin D compounds that cause hypercalcaemia should be discon- tinued and, if appropriate, dietary calcium restricted. The acute management of hypercalcaemia involves general meas- ures to enhance hydration and diuresis, and specific measures using drugs to lower serum calcium. Dehydration due to hypercalcaemic symptoms (e.g. anorexia, nausea, vomiting, and polyuria) because of defective urinary concentration, is very common and patients may require 5 to 10 litres of 0.9% sodium chloride over a 24-​ to 48-​h period. This vigorous hydration with normal saline may lower serum calcium by 0.25 to 0.75 mmol/​litre; it enhances urinary calcium ex- cretion by increasing glomerular filtration and reducing proximal and distal renal tubular reabsorption of calcium and sodium. The saline diuresis may need adjuvant therapy with a loop diuretic (e.g. furosemide 10 to 20 mg), as necessary, to control complications due to volume overload, especially in elderly people and those with im- paired cardiovascular and renal function. It is important to note that excessive use of furosemide before intravascular volume has been restored may worsen the hypercalcaemia by exacerbating volume depletion. Saline diuresis may lead to hypokalaemia, hypomagnes- aemia, and electrolyte imbalance, which will need correction. If saline diuresis is not successful, and particularly if the hypercalcaemia is very severe, then more specific measures (e.g. dialysis and/​or drugs), will be required. The drugs of choice are pamidronate or zoledronic acid, which are potent bisphosphonates that are administered parenterally. Recommended regimens are to administer pamidronate (60–​90 mg) or zoledronic acid (4 mg) intra- venously as a single infusion. Other bisphosphonates (e.g. etidronate and clodronate), and other agents such as mithramycin, calcitonin, and gallium nitrate have also been used in the past. Glucocorticoid therapy (e.g. hydrocortisone 120 mg/​day in three divided doses) is particularly effective when the hypercalcaemia is mediated by the actions of 1,25-​dihydroxyvitamin D, for example, in granulomatosis disease or lymphoma (Box 13.4.1), or myeloma. Dialysis using a low or zero calcium dialysate should be considered if these treatments are not effective or if the patient has renal failure. Once the acute management of hypercalcaemia has been completed, then appro- priate treatment for the underlying cause (e.g. parathyroidectomy for primary hyperparathyroidism), needs to be undertaken. Box 13.4.2  Clinical features of hypercalcaemia Renal Stones (nephrolithiasis) and nephrocalcinosis Polyuria Polydipsia Musculoskeletal Bone pain Osteopenia Fractures Muscular weakness, especially proximal myopathy Gastrointestinal Nausea Vomiting Lack of appetite Constipation Peptic ulcers Pancreatitis Neurological Tiredness Lethargy Inability to concentrate Increased sleepiness Depression Confusion Coma Cardiac Bradycardia First-​degree AV block Arrhythmias Shortened QT interval Box 13.4.3  Preliminary investigations for hypercalcaemia Blood Two or three estimations of serum calcium, phosphate, albumin, urea and electrolytes, creatinine, alkaline phosphatase, and liver function tests PTH Haemoglobin, full blood count, ESR Electrophoretic protein strip 25-​hydroxyvitamin D3 (and, if indicated, 1,25-​dihydroxyvitamin D3) Thyroid function tests Magnesium PTHrP (if malignancy suspected) Urine Two or three estimations of 24-​h urinary calcium and creatinine, and clearance ratios Imaging Chest radiograph Radiograph of hands Ultrasound of kidneys

SECTION 13  Endocrine disorders 2320 Hypercalcaemic diseases Hypercalcaemia may arise through one or more of three mech- anisms:  increased bone resorption, increased gastrointestinal absorption of calcium, and decreased renal calcium excretion (Fig. 13.4.1). For example, lytic bone metastases cause increased bone resorption, thiazide diuretics lead to a decrease in calcium excretion, and excessive PTH will either directly or indirectly, by increasing 1,25-​dihydroxyvitamin D production, stimulate bone resorption and calcium absorption from the gut and renal tubules. The hypercalcaemic diseases may be classified according to whether serum PTH concentrations are elevated or reduced (Box 13.4.1). In addition, hypercalcaemia may be classified as being due to an excess of PTH (e.g. primary or tertiary hyperparathyroidism) from parathyroid tumours, an excessive production of PTHrP, a defect in the PTH receptor (i.e. the PTH/​PTHrP receptor), an excess pro- duction of downstream mediators (e.g. 1,25-​dihydroxyvitamin D), or an altered set point in the CaSR (Fig. 13.4.2). Hyperparathyroidism Hyperparathyroidism is characterized by high concentrations of serum immunoreactive PTH, and three types, referred to as pri- mary, secondary, and tertiary, are recognized. Primary and tertiary hyperparathyroidism are associated with hypercalcaemia (Box 13.4.1), whereas secondary hyperparathyroidism is associated with hypocalcaemia (see next). Primary hyperparathyroidism may arise as an isolated endocrinopathy or as part of a multiple endocrine neo- plasia (MEN) syndrome, and tertiary hyperparathyroidism usually arises in association with chronic renal failure. Primary hyperparathyroidism Primary hyperparathyroidism, which affects 3 in 1000 adults, is one of the two most common causes of hypercalcaemia and is due to an excessive secretion of PTH from one or more parathyroid tumours. Epidemiological studies have estimated that the global prevalence of parathyroid tumours is 4 million. In 80% of patients this tumour is a solitary parathyroid adenoma, and in 15 to 20% of patients hyperplasia involving all four parathyroids is present. Parathyroid carcinoma accounts for less than 0.5% of patients with primary hyperparathyroidism. Primary hyperparathyroidism usu- ally occurs between the ages of 40 to 65 years, and is three times more common in women than men. The underlying causes of pri- mary hyperparathyroidism are largely unknown, but abnormalities of several genes have been identified. Thus, abnormalities of the cyclin D1 (CCNDI), retinoblastoma, CaSR (CASR), parafibromin, MEN type 1 (MEN1), and MEN type 2 (MEN2) genes, together with other genes yet to be identified, for example, on chromosome 1p (Table 13.4.2), are associated with the development of some parathyroid tumours. Clinical features  Many patients with primary hyperparathyr- oidism will be asymptomatic and the hypercalcaemia, which is usually mild, will have been detected by chance at the time of bio- chemical screening for other reasons. However, it is important to note that nearly one-​half of the patients will have subtle neuromus- cular symptoms such as fatigue and weakness and this becomes ap- parent only in retrospect after a successful parathyroidectomy. Symptomatic hypercalcaemia (Box 13.4.2) predominantly af- fects the skeletal, renal, and gastrointestinal systems; peptic ulcers and pancreatitis may develop. The skeletal changes of osteitis fibrosa cystica due to subperiosteal resorption of the distal phalanges (Fig. 13.4.3), tapering of the distal clavicles, a ‘salt and pepper’ ap- pearance of the skull, bone cysts, and brown tumours of the long bones are now identified in less than 5% of patients. However, osteopenia, as assessed by bone mineral density, occurs in 25% of patients. Renal stone disease (nephrolithiasis and nephrocalcinosis) occurs in 20% of patients and hypercalciuria occurs in 30% of pa- tients; renal impairment may complicate this disease. (f) (e) (d) (c) (b) (a) Fig. 13.4.3  Renal osteodystrophy over a 9-​year period in a patient with chronic renal failure. Marked periosteal erosions were seen (a) despite treatment with 1α-​hydroxycholecalciferol, and a resolution was observed following dialysis (b). Note the vascular calcification. One year later a relapse was noted with periosteal erosions (c) and the use of calcitriol resolved these (d). Unfortunately, a relapse occurred 2 years later (e), and following renal transplantation a marked resolution was observed (f).

13.4  Parathyroid disorders and diseases altering calcium metabolism 2321 Investigations  In the presence of hypercalcaemia, the finding of elevated circulating PTH concentrations establishes the diag- nosis, as the PTH will be elevated in approximately 90% of pa- tients with primary hyperparathyroidism who will invariably have hypercalcaemia. However, it is important to make sure that the immunoradiometric and immunochemiluminometric as- says for PTH are used to measure the intact molecule, rather than the older radioimmunoassays which are not as reliable. The only other hypercalcaemic disorders in which PTH may occasion- ally be elevated are those related to familial benign hypocalciuric hypercalcaemia (FBH), immobilization, or lithium or thiazide use (Box 13.4.1), and a careful history and a cessation of drug use will help to exclude these possibilities. About one-​third of patients with primary hyperparathyroidism will have a low serum phosphate and in the others, it will be in the lower range of normal. In addition, some patients will have a small increase in serum chloride concen- tration and a concomitant decrease in bicarbonate concentration. Serum alkaline phosphatase activity may be elevated in some pa- tients, and urinary calcium excretion is increased in 30% of pa- tients. The circulating 1,25-​dihydroxyvitamin D concentration is elevated in some patients with primary hyperparathyroidism, al- though it is not of diagnostic value as it is also elevated in other hypercalcaemic disorders such as sarcoidosis and lymphomas. The serum 25-​hydroxyvitamin D concentration is within the normal range. Densitometric scanning is of use in detecting early skeletal changes. Patients with primary hyperparathyroidism develop re- duced bone mineral densities (osteopenia) primarily of the cortical bone (e.g. distal one-​third of forearm) rather than the cancellous bone (e.g. lumbar spine). The hip bones, which are an equal mix- ture of cortical and cancellous bone, show intermediate reductions in bone mineral density. Overall, the risk of bone fractures in pa- tients with mild primary hyperparathyroidism is similar to those in matched, normal controls. However, successful parathyroidectomy does lead to an increase in bone mineral density over a 6-​ to 12-​ month period and this continues for up to 10 years. Indeed, bone mineral density measurements are used in the evaluation of pa- tients with primary hyperparathyroidism and in deciding on con- servative as opposed to surgical management (Box 13.4.4). Preoperative localization to define the site(s) of the parathy- roid tumours may be undertaken. The noninvasive tests consist of ultrasonography, CT, MRI, and scintigraphy with technetium-​99m sestamibi. Sestamibi scintigraphy has now become established as the best and most convenient localization test; this can be performed with CT techniques (single photon emission CT, SPECT) to give a three-​dimensional image with greater anatomical resolution. It is important to note that there is an appreciable incidence of false-​ positive rates with all the noninvasive localization procedures and so a confirmation using two methods is preferable. Invasive local- ization tests consist of arteriography and selective venous sampling for PTH in the veins draining the thyroidal region. These tests are time-​consuming, expensive, difficult, and dependent on the skill of the radiologist. It is generally accepted that these preoperative local- ization tests are indicated in those patients who have had previous neck surgery. However, their role in patients who have not had prior surgery remains to be established and at present the preferences and expertise of the local medical, radiology, and surgery teams usually determine the use of venous sampling procedures. Management and treatment  Parathyroidectomy, which is the de- finitive cure, is a generally successful and safe procedure if under- taken by an experienced surgeon. There have also been major advances in surgery that have facilitated a surgical approach to be undertaken under local, as opposed to general, anaesthesia. An ex- ample of this is the use of minimally invasive parathyroidectomy in the patient with single gland disease that has been successfully lo- calized by the combined use of sestamibi scintigraphy and ultrason- ography. Surgery is recommended for symptomatic patients and for those who have skeletal and renal complications. However, the de- cision to recommend surgery, which does have a small risk, may be difficult in asymptomatic patients, who may constitute over 50% of patients with primary hyperparathyroidism. The natural history of primary hyperparathyroidism in most patients is to progress slowly or not at all. For example, among asymptomatic patients only 25% will have progressive disease, which is usually manifested as a de- crease in bone mineral density during a 10-​year period. This has led to a controversy regarding the indications for surgery, and guidelines have been provided by the Consensus Development Conference on the Management of Asymptomatic Primary Hyperparathyroidism (Box 13.4.4). However, these guidelines may not exclusively influ- ence the decision for or against surgery, and a careful evaluation and assessment of the risks and benefits is considered by most medical and surgical teams in conjunction with the patient. Clearly, some patients will not wish to continue living with a curable disease and will prefer surgery despite the guidelines (Box 13.4.4), while other patients will decline surgery, despite having guideline indications for it, because they may have coexisting medical conditions that make them feel that the risks of surgery are too great. Patients who do not undergo parathyroidectomy (e.g. those with asymptomatic primary hyperparathyroidism) should be evaluated clinically, and also monitored for serum calcium, creatinine, and es- timated glomerular filtration rate (eGFR), annually; bone mineral density at three sites, 1–​3 year intervals with X-​ray, or vertebral frac- ture assessment of spine if there has been height loss or back pain; and if nephrolithiasis is suspected then appropriate assessments of 24 hour urine collections and renal imaging. In addition, the fol- lowing medical guidelines are recommended. First, they should Box 13.4.4  Guidelines for the management of primary hyperparathyroidism, recommended by the Fourth International Workshop (2013) Surgery is recommended if the patient meets any one of the following criteria: • Serum calcium is more than 0.25 mmol/​litre above upper limit of normal • Any complication of primary hyperparathyroidism (e.g. nephrolithiasis, nephrocalcinosis, bone erosions of osteitis fibrosa cystica) • An episode of acute primary hyperparathyroidism with life-​threatening hypercalcaemia • Marked hypercalciuria (>10 mmol/​litre per 24 h or >400 mg/​24 h) and increased stone risk by biochemical stone risk analysis • Significant reduction in creatinine clearance (i.e. <60 ml/​min) • Reduction in bone mineral density (BMD) (i.e. T score <–​2.5 at spine, total hip, femoral neck, or distal third of radius; and/​or vertebral frac- ture shown by X-​ray, CT, MRI, or vertebral fracture assessment) • Age less than 50 years

SECTION 13  Endocrine disorders 2322 avoid dehydration and remain ambulant. Second, vitamin D defi- ciency should be corrected, and the serum 25-​hydroxy vitamin D should be maintained above 50 nmol/​litre. Third, dietary intake of calcium should be normal; limiting calcium intake is not recom- mended. Fourth, thiazide diuretics, herbal and tonic remedies that may contain vitamin D or vitamin A, should be avoided. These meas- ures may help. Drugs that have been used include oral phosphate, oestrogens, or selective oestrogen receptor modulators (SERMs) in postmenopausal women, and bisphosphonates. Phosphate is not used because of concerns related to soft tissue ectopic calcification. Oestrogens and SERMs such as raloxifene do increase bone density in postmenopausal women with primary hyperparathyroidism but they have only small effects on the serum calcium and PTH con- centrations. The bisphosphonates (e.g. alendronate) inhibit bone re- sorption, improve bone mineral density (BMD) at the lumbar spine without altering serum calcium and PTH concentrations. However, these effects are not sustained. The calcimimetic drug, cinacalcet, which increases the sensitivity of the parathyroid CaSR (see next) to extracellular calcium and thereby reduces PTH secretion, is effective in lowering serum calcium concentrations to normal values with modest reductions in PTH levels in patients with primary hyperpara- thyroidism. However, bone mineral density in the treated patients remained unchanged. The use of cinacalcet is approved for adult (i.e. over the age of 18 years) patients, who are on dialysis with uncon- trolled secondary hyperparathyroidism, have hypercalcaemia due to inoperable parathyroid carcinoma, or have severe hypercalcaemia due to primary hyperparathyroidism and are unable to undergo parathyroidectomy. The use of these drugs should be dictated by the aims of the treatment. For example, bisphosphonate therapy should be chosen if the aim is to increase BMD, and cinacalcet should be chosen if the aim is to reduce serum calcium concentrations. Combined use of cinacalcet and alendronate has been reported, by one study, to normalize hypercalcaemia and improve BMD in pa- tients with primary hyperparathyroidism. Uraemic hyperparathyroidism Serum PTH levels rise in response to hypocalcaemia and this sec- ondary hyperparathyroidism usually resolves with treatment of the underlying cause of hypocalcaemia (Box 13.4.5). However, in chronic renal failure the secondary hyperparathyroidism may persist for a longer time, and eventually the parathyroid cells gain an autono- mous function, secreting excessive PTH despite hypercalcaemia; this state is referred to as tertiary hyperparathyroidism (Box 13.4.1). The cause of progression from the early, presumably polyclonal, secondary hyperplasia of the parathyroids to the later, presumably monoclonal, tumours is not understood and appears to involve genes other than those involved in the aetiologies of the sporadic and familial forms of primary hyperparathyroidism (Table 13.4.2). Clinical features and treatment  In chronic renal failure, the ensuing phosphate retention and decreased production of 1,25-​ dihydroxyvitamin D result in hypocalcaemia and secondary hyper- parathyroidism. This combination of biochemical abnormalities results in a severe bone disease that shows combined features of hyper- parathyroidism and vitamin D deficiency (i.e. osteomalacia). Thus in renal osteodystrophy, bone erosions (Fig. 13.4.3) and osteomal- acia are simultaneously observed. Treatment is based on correcting the hypocalcaemia (e.g. with oral administration of calcium salts), which also ameliorates the hyperphosphataemia by chelating phos- phate in the intestines, and with calcitriol (1,25-​dihydroxyvitamin D). The use of the most appropriate phosphate binder is not well established, but it is clear that aluminium-​containing compounds are to be avoided. Aluminium in these preparations and as a con- taminant of dialysis solutions contributed in the recent past to the osteomalacic osseous disease and other aspects of metal toxicity in patients with renal failure (e.g. hypochromic anaemia and enceph- alopathy). Early treatment of the metabolic disturbance will prevent or delay the onset of severe secondary hyperparathyroidism and tertiary hyperparathyroidism, which requires parathyroidectomy. For patients who have end stage renal failure and are on dialysis, Box 13.4.5  Causes of hypocalcaemia Low PTH levels (hypoparathyroidism) Parathyroid agenesis • Isolated or part of complex developmental anomaly (e.g. DiGeorge’s syndrome) Parathyroid destruction • Surgerya • Radiation • Infiltration by metastases or systemic disease (e.g. haemochromatosis, amyloidosis, sarcoidosis, Wilson’s disease, thalassaemia) • Autoimmune • Isolated • Polyglandular (type 1)a Reduced parathyroid function (i.e. PTH secretion) • PTH gene defects • Hypomagnesaemiaa • Neonatal hypocalcaemia (may be associated with maternal hypercalcaemia) • Hungry bone disease (postparathyroidectomy) • CaSR or Gα11 mutations High PTH levels (secondary hyperparathyroidism) Vitamin D deficiencya • As a result of nutritional lacka, malabsorptiona, liver disease, or vitamin D receptor defects Vitamin D resistance (rickets) • As a result of renal tubular dysfunction (Fanconi’s syndrome) or vitamin D receptor defects PTH resistance • (e.g. pseudohypoparathyroidism, hypomagnesaemia) Drugs • Calcium chelators (e.g. citrated blood transfusions, phosphate; cow’s milk is rich in phosphate) • Inhibitors of bone resorption (e.g. bisphosphonates, calcitonin, plicamycin) • Altered vitamin D metabolism (e.g. phenytoin, ketoconazole) • Foscarnet Miscellaneous • Acute pancreatitis • Acute rhabdomyolysis • Massive tumour lysis • Osteoblastic metastases (e.g. from prostate or breast carcinoma) • Toxic shock syndrome • Hyperventilation a Most common causes.

13.4  Parathyroid disorders and diseases altering calcium metabolism 2323 cinacalcet—​the allosteric activator of the CaSR—​can be used to treat the severe secondary hyperparathyroidism. Cinacalcet will re- duce the PTH concentrations and may also have an antiproliferative effect. Familial primary hyperparathyroidism Primary hyperparathyroidism is most frequently encountered as a non​familial disorder. However, approximately 10% of patients with primary hyperparathyroidism will have a hereditary form which may either be part of the MEN type 1 (MEN1) and type 2 (MEN2) syndromes, or part of the hereditary hyperparathyroidism–​jaw tu- mour (HPT-​JT) syndrome. In addition, hereditary primary hyper- parathyroidism may develop as a solitary endocrinopathy and this has also been referred to as familial isolated hyperparathyroidism (FIHP). Investigations of these hereditary and sporadic forms of primary hyperparathyroidism have helped to identify some of the genes and chromosomal regions that are involved in the aetiology of parathyroid tumours (Table  13.4.2). FIHP has been reported in several kindreds, and some have been shown to harbour muta- tions of the MEN1 gene or the gene encoding parafibromin. These familial syndromes associated with parathyroid tumours will be briefly reviewed. MEN1  MEN1 is characterized by the combined occurrence of tu- mours of the parathyroids, pancreatic islet cells, and anterior pi- tuitary. Parathyroid tumours occur in 95% of MEN1 patients, and the resulting hypercalcaemia is the first manifestation of MEN1 in about 90% of patients. Pancreatic islet cell tumours occur in 40% of MEN1 patients, and gastrinomas, leading to the Zollinger–​Ellison syndrome, are the most common type and also the important cause of morbidity and mortality in MEN1 patients. Anterior pituitary tumours occur in 30% of MEN1 patients, with prolactinomas rep- resenting the most common type. Associated tumours that may also occur in MEN1 include adrenal cortical tumours, carcinoid tumours, lipomas, angiofibromas, and collagenomas. The gene causing MEN1, which is located on chromosome 11q13 and repre- sents a putative tumour suppressor gene, consists of 10 exons that encode a 610 amino acid protein, known as menin. Menin is pre- dominantly a nuclear protein in non​dividing cells, but in dividing cells it is found in the cytoplasm. Menin has been shown to interact with many proteins that are involved in transcriptional regulation, genome stability, and cell division. The majority (>80%) of the germ-​ line MEN1 mutations in families are inactivating. Mutational ana- lysis of the MEN1 gene is helpful in the diagnosis and management of patients and their families. MEN2 and MEN3  MEN2 describes the association of medullary thyroid carcinoma (MTC), phaeochromocytomas, and parathyroid tumours. Three clinical variants of MEN2 are recognized: MEN2a, MEN2b (also referred to as MEN3), and MTC-​only. MEN2a is the most common variant, where the development of MTC is associ- ated with phaeochromocytomas (50% of patients), which may be bilateral, and parathyroid tumours (20% of patients). MEN2b, which represents 5% of all MEN2 cases, is characterized by the oc- currence of MTC and phaeochromocytoma in association with a marfanoid habitus, mucosal neuromas, medullated corneal fibres, and intestinal autonomic ganglion dysfunction leading to multiple diverticulae and megacolon. Parathyroid tumours do not usually occur in MEN2b. MTC-​only is a variant in which MTC is the sole manifestation of the syndrome. The gene causing all three MEN2 variants was mapped to chromosome 10q11.2, a region containing the c-​RET proto-​oncogene which encodes a tyrosine kinase re- ceptor with cadherin-​like and cysteine-​rich extracellular domains and a tyrosine kinase intracellular domain. Specific mutations of c-​RET have been identified for each of the three MEN2 variants. Thus in 95% of patients, MEN2a is associated with mutations of the cysteine-​rich extracellular domain and mutations in codon 634 (Cys→Arg) account for 85% of MEN2a mutations. MTC-​only is also associated with missense mutations in the cysteine-​rich extracel- lular domain and most mutations are at codon 618. MEN2b is asso- ciated with mutations in codon 918 (Met→Thr) of the intracellular tyrosine kinase domain in 95% of patients. Mutational analysis of c-​RET to detect mutations in codons 609, 611, 618, 634, 768, and 804 in MEN2a and MTC-​only, and codon 918 in MEN2b, has been used in the diagnosis and management of patients and families with these disorders. MEN4  Approximately 5% to 10% of MEN1 patients do have muta- tions of the MEN1 gene, and about 3% of these may have mutations involving the gene encoding a cyclin-​dependent kinase inhibitor (CDNK1B) and are referred to as having MEN4. MEN4, an auto- somal dominant disorder, is characterized by occurrence of parathy- roid adenomas in association with pituitary adenomas, pancreatic neuroendocrine tumours, and tumours of the gonads, adrenals, kid- neys, and thyroid. HPT-​JT  The HPT-​JT syndrome is an autosomal dominant dis- order characterized by the occurrence of parathyroid tumours that may be carcinomas in approximately 15% of patients and ossifying fibromas that usually affect the maxilla and/​or mandible. In addition, some patients may also develop Wilms’ tumours, renal cysts, renal hamartomas, renal cortical adenomas, papillary renal cell carcinomas, uterine tumours that may be malignant, pancre- atic adenocarcinomas, testicular mixed germ cell tumours with a major seminoma component, and Hürthle cell thyroid adenomas. It is important to note that the parathyroid tumours may occur in isolation and without any evidence of jaw tumours, and this may cause confusion with other hereditary hypercalcaemic disorders such as MEN1, familial hypocalciuric hypercalcaemia (FHH), and FIHP. HPT-​JT can be distinguished from FHH, as in FHH serum calcium concentrations are elevated from the early neonatal or in- fantile period whereas in HPT-​JT such elevations are uncommon in the first decade. In addition, HPT-​JT patients, unlike those with FHH, will have associated hypercalciuria. The distinction between HPT-​JT patients and MEN1 patients, who have only developed the usual first manifestation of hypercalcaemia (>90% of patients), is more difficult and is likely to be influenced by the operative and histological findings and the occurrence of other characteristic le- sions in each disorder. It should be noted that HPT-​JT patients will usually have single adenomas or a carcinoma, while MEN1 patients will often have multiglandular parathyroid disease. The distinc- tion between FIHP and HPT-​JT in the absence of jaw tumours is difficult but important as HPT-​JT patients may be at a higher risk of developing parathyroid carcinomas. These distinctions may be helped by the identification of additional features, and a search for jaw tumours and renal, pancreatic, thyroid, and testicular abnor- malities may help to identify HPT-​JT patients. The jaw tumours in HPT-​JT are different from the brown tumours observed in some

SECTION 13  Endocrine disorders 2324 patients with primary hyperparathyroidism, and do not resolve after parathyroidectomy. Indeed, ossifying fibromas of the jaw are an important distinguishing feature of HPT-​JT from FIHP, and the occurrence of these may occasionally precede the development of hypercalcaemia in HPT-​JT patients by several decades. The gene causing HPT-​JT is CDC73, located on chromosome 1q31.2 and consisting of 17 exons that encode a ubiquitously ex- pressed 531 amino acid protein, parafibromin. This gene is also re- ferred to as HRPT2 (i.e. hyperparathyroidism type 2). Parafibromin has been shown to be associated with the human homologue of the Paf1 protein complex which interacts with RNA polymerase II, and, as part of this protein complex, parafibromin may regu- late post-​transcriptional events and histone modification. The ma- jority (>80%) of the germ-​line mutations in HRPT-​JT families are inactivating and are predicted to result in a functional loss of the parafibromin protein because of premature truncation. In addition, patients with non​familial parathyroid carcinomas may harbour germ-​line mutations, and mutational analysis of the gene encoding parafibromin is now undertaken in patients who have non​familial parathyroid carcinoma, FIHP, and HPT-​JT. Malignancy Hypercalcaemia may occur in c.25% of patients with a malignancy and this is usually due to increased bone resorption, which may be either directly due to skeletal metastases or indirectly due to tumour production of a humoral factor that stimulates osteoclastic bone re- sorption. The cancers that typically metastasize to produce lytic bone lesions are from the breast, lymphomas, or multiple myeloma (Box 13.4.1). The associated osteolysis, mediated by recruitment and ac- tivation of osteoclasts, involves cytokines. Denosumab, which a hu- manized neutralizing monoclonal antibody to RANKL may be used to prevent the recruitment and activation of osteoclasts. The cancers that are typically associated with the humoral hypercalcaemia of ma- lignancy (HHM) are squamous carcinomas of the lung, oesophagus, cervix, vulva, skin, head, or neck, but other types from the kidney, bladder, ovary, and breast may also occur. HHM accounts for up to 80% of patients with malignancy-​associated hypercalcaemia. The most common factor causing HHM is PTHrP, which can be meas- ured in the serum by immunoassay. However, these assays may be relatively insensitive and the failure to detect serum PTHrP does not exclude the diagnosis of HHM. Patients with HHM generally have hypercalcaemia associated with lower or undetectable serum PTH levels, marked hypercalcaemia, and a reduced plasma 1,25-​ dihydroxyvitamin D level. Therapy of HHM is aimed at: (1) reducing the tumour load by surgery, radiotherapy, and/​or chemotherapy; (2) reducing osteoclastic bone resorption by use of bisphosphonates or calcitonin; and (3) increasing renal calcium clearance by a saline diuresis. Granulomatous disorders Several granulomatous disorders are associated with hypercalcaemia (Box 13.4.1) and this is invariably associated with elevated circu- lating concentrations of 1,25-​dihydroxyvitamin D, which is due to extrarenal synthesis. Sarcoidosis is the most frequently encountered granulomatous disorder associated with hypercalcaemia, and 10% of patients with sarcoidosis will have hypercalcaemia and about one-​half will become hypercalciuric. The finding of raised serum angiotensin-​converting enzyme activity may help in confirming the diagnosis. Glucocorticoids (e.g. 40–​60 mg prednisolone) de- crease 1,25-​dihydroxyvitamin D production and restore the calcium concentration to normal. Failure to achieve normal serum calcium concentrations within 10 days of glucocorticoid therapy (e.g. hydro- cortisone 40 mg, three times per day), which is referred to as the steroid suppression test, should suggest the coexistence of another cause for the hypercalcaemia (e.g. primary hyperparathyroidism) or malignancy. Endocrine causes of hypercalcaemia other than hyperparathyroidism Several non​parathyroid disorders (Box 13.4.1) are associated with hypercalcaemia and these include thyrotoxicosis, phaeochromo- cytoma, Addison’s disease, VIPomas, FHH (also referred to as fa- milial benign hypercalcaemia (FBH)), Jansen’s disease, and William’s syndrome. Thyrotoxicosis Mild hypercalcaemia (<3.00 mmol/​litre) frequently accompanies thyrotoxicosis, which leads to increased bone turnover and re- sorption. The hypercalcaemia may respond to treatment with β-​ adrenergic blockers. FHH and NHPT FHH is an autosomal dominant disorder with a high degree of pene- trance. It is characterized by lifelong asymptomatic hypercalcaemia in association with an inappropriately low urinary calcium excretion (i.e. ratio of calcium clearance to creatinine clearance (CCR) <0.01), and normal circulating PTH concentrations in 80% of patients. Mild hypermagnesaemia is also typically present. Although most patients with FHH are asymptomatic, chondrocalcinosis and acute pancreatitis have occasionally been observed. Patients with FHH have been mis-​diagnosed as having primary hyperparathyroidism, as 20% of FHH patients may have elevated plasma PTH concentra- tions. In addition, 20% of FHH patients may have a CCR more than 0.01, and therefore be indistinguishable from patients with primary hyperparathyroidism. Moreover, low CCR are observed in patients with primary hyperparathyroidism who have vitamin D deficiency, or renal insufficiency, or are of an African-​American origin. It is important to distinguish FHH patients from those with primary hyperparathyroidism, as the hypercalcaemia in FHH is generally benign and does not result in sequelae (Table 13.4.3). Moreover, parathyroidectomy does not correct the hypercalcaemia in FHH. Mutational analysis may help in identifying FHH patients from those with primary hyperparathyroidism. FHH is genetically heterogenous, with three reported variants FHH1, FHH2, and FHH3, whose loci are on chromosomes 3q21.1, 19p, and 19q13, respectively (Table 13.4.1). FHH1 is due to heterozy- gous loss-​of-​function mutations of the CaSR, which is a G-​protein-​ coupled receptor (GPCR) that signals via Gαq and Gα11. The human CaSR, a 1078 amino acid cell surface protein encoded by the CaSR gene located on chromosome 3q21.1, is expressed in parathy- roids, thyroid cells, and kidney (Fig. 13.4.2), and is a member of the family of G-​protein-​coupled receptors. Approximately two-​thirds of FHH kindreds will have unique heterozygous loss-​of-​function mu- tations of the CaSR. FHH2 is due to loss-​of-​function mutations in the G-​protein subunit α11 (Gα11) (Fig. 13.4.2), and these may occur in less than 5% of FHH patients. FHH3 is due to loss-​of-​function

13.4  Parathyroid disorders and diseases altering calcium metabolism 2325 mutations of the adaptor protein 2 (AP-​2) sigma subunit (AP2σ) (Fig. 13.4.2). AP2 is a central component of clathrin-​coated vesicles (CCVs) and is pivotal in clathrin-​mediated endocytosis, which in- ternalizes plasma membrane constituents such as GPCRs. AP2 is a heterotetramer of α, β, μ and σ subunits and links clathrin to vesicle membranes and binds to tyrosine-​ and dileucine-​based motifs of membrane-​associated cargo proteins. The FHH3-​associated AP2σ mutations, which all involve an Arg15 residue that forms key con- tacts with the dileucine-​based motifs of CCV cargo proteins, reduce CaSR endocytosis, whose disruption likely decreases intracellular signalling. Such AP2σ loss-​of-​function mutations occur in more than 5% of FHH patients. FHH1, FHH2, and FHH3 have similar clinical features, and thus genetic analysis may help to identify the relevant mutations. However, the hypercalcaemia in FHH3 patients may be more severe and symptomatic than that in FHH1 patients, and cinacalcet can correct the hypercalcaemia of FHH3 patients with an improvement in the symptoms. Some patients, who have the clinical features of FHH1, but not CaSR mutations, may have autoimmune hypocalciuric hypercalcaemia (AHH). Such patients may have multiple clinical autoimmune manifestations, including antithyroid antibodies, antigliadin, or antiendomyseal antibodies. These patients were shown to have cir- culating antibodies to the extracellular domain of the CaSR, and these antibodies stimulated PTH release from dispersed human parathyroid cells in vitro, probably by inhibiting the activation of the CaSR by extracellular calcium. The effects of treatment with gluco- corticoids have been variable, with the hypercalcaemia responding in one patient but not in another. Thus, AHH is a condition of extra- cellular calcium-​sensing that should be considered in FHH1 pa- tients who do not have CaSR mutations. NSHP  Neonatal severe primary hyperparathyroidism (NSHP) is defined as symptomatic hypercalcaemia with skeletal manifestations of hyperparathyroidism in the first 6 months of life. NSHP children often present in the first few days or weeks of life with failure to thrive, dehydration, hypotonia, constipation, rib cage deformities, and multiple fractures due to bony undermineralization. Children with NSHP often have life-​threatening hypercalcaemia and require urgent parathyroidectomy, which corrects the PTH-​dependent hypercalcaemia and bone demineralization. FHH is due to heterozy- gous inactivating mutations of the calcium-​sensing receptor (CaSR) and NSHP is often associated with inactivating homozygous CaSR mutations when the children are from consanguineous parents with FHH1 (Fig. 13.4.2). However, NSHP has also been observed in chil- dren where only one parent had clinically apparent FHH, and many other NSHP patients appear to be sporadic, that is both parents have normal serum calcium concentrations. In such NSHP patients with heterozygous CaSR mutations, the mutant CaSR may exert a dom- inant negative action on the normal CaSR. Jansen’s disease Jansen’s disease is an autosomal dominant disease that is characterized by short-​limbed dwarfism, due to metaphyseal chondrodysplasia, and severe hypercalcaemia and hypophosphataemia, despite normal or undetectable serum levels of PTH. These abnormalities are asso- ciated with activating mutations of the PTH receptor (Fig. 13.4.2) and thus this represents a PTH-​independent activation of the PTH receptor. William’s syndrome William’s syndrome is an autosomal dominant disorder character- ized by supravalvular aortic stenosis, elfin-​like facies, psychomotor retardation, and infantile hypercalcaemia. The underlying abnor- mality of calcium metabolism remains unknown but abnormal 1,25-​dihydroxyvitamin D3 metabolism or decreased calcitonin pro- duction have been implicated, although no abnormality has been consistently demonstrated. Hemizygosity due to a microdeletion at the ELN locus on chromosome 7q11.23 in over 90% of patients with the classic William’s phenotype has been demonstrated. This microdeletion has been reported to involve another gene, desig- nated LIMK1, that is expressed in the central nervous system. The Table 13.4.3  Clinical, biochemical, and genetic features of hypoparathyroid and pseudohypoparathyroid disorders Hypoparathyroidism Pseudohypoparathyroidism PHPIa PPHP PHPIb PHPIc PHPII AHO manifestations No Yes Yes No/​Rarely Yes No Serum calcium ↓ ↓ N ↓ ↓ ↓ Serum PO4 ↑ ↑ N ↑ ↑ ↑ Serum PTH ↓ ↑ N ↑ ↑ ↑ Response to PTH:   Urinary cAMPa (Chase–​Aurbach test) ↑ ↓ ↑ ↓ ↓ ↑   Urinary PO4 (Ellsworth–​Howard test) ↑ ↓ ↑ ↓ ↓ ↓ Gsα activity N ↓ ↓ N N N Inheritance AD/​AR/​X AD AD AD/​Sporadic AD Sporadic Molecular defect PTH/​CaSR/​GATA3/​GCM2/​others GNAS1 GNAS1 GNAS1b GNAS1 ?cAMP targets Other hormonal resistance No Yes No No (Usually) Yes No AD, autosomal dominant; AHO, Albright’s hereditary osteodystrophy; AR, autosomal recessive; CaSR, calcium-​sensing receptor; GATA3, GATA binding protein 3; GCM2, glial cells missing homologue 2; N, normal; PHP, pseudohypoparathyroidism; PPHP, pseudopseudohypoparathyroidism; X, X-​linked; ↓, decreased; ↑, increased;  ?, presumed, but not proven. a Plasma cAMP responses are similar to those of urinary cAMP. b Involves deletions located upstream of GNAS1

SECTION 13  Endocrine disorders 2326 calcitonin receptor gene has been localized to chromosome 7q21 and close to the region deleted in William’s syndrome. However, the calcitonin receptor gene was not involved in the deletion found in four patients with William’s syndrome, indicating that it is un- likely to be implicated in the hypercalcaemia of such children. While the involvement of the ELN and LIMK1 genes in the deletions of William’s syndrome patients can explain the respective cardiovas- cular and neurological features of this disorder, it seems possible that another, as yet uncharacterized gene that is within this contigu- ously deleted region is likely to be involved to explain the abnormal- ities of calcium metabolism. Infantile hypercalcaemia Infantile hypercalcaemia is associated with failure to thrive and is characterized by:  severe hypercalcaemia; hypercalciuria; nephrocalcinosis; and elevated circulating 1,25(OH)2D concen- trations. Some infants with this disorder have homozygous or compound heterozygous mutations of the gene encoding the 24-​ hydroxylase (CYP24A1) enzyme which metabolizes the active 1,25(OH)2D to the inactive 1,24,25(OH)3D form (Fig. 13.4.1). Drugs Several drugs (Box 13.4.1) can cause hypercalcaemia by different mechanisms. Compounds containing vitamin D and vitamin A are common and frequently associated with hypercalcaemia. The use of thiazide diuretics is often associated with hypercalcaemia. The hypercalcaemia appears to be largely renal in origin, as thiazides enhance distal renal tubular calcium reabsorption. Hypercalcaemia reverses rapidly with discontinuation of the drug. The milk-​alkali syndrome was first described in the 1930s, gen- erally in the context of ulcer treatment with large quantities of milk together with sodium bicarbonate. Today, the responsible agent is usually calcium carbonate, although consumption of large quantities of dairy products (milk, cheese, and yoghurt) may still contribute. Classic features include moderate to severe hypercalcaemia with al- kalosis and renal impairment. The amount of calcium ingested by patients with this syndrome is usually 5 to 15 g/​day. Treatment con- sists of: (1) discontinuing the ingestion of the calcium-​containing compound(s) and antacids, (2) rehydration, and (3) saline diuresis. Hypocalcaemia Clinical features and investigations The clinical presentation of hypocalcaemia (serum calcium <2.12 mmol/​litre) ranges from an asymptomatic biochemical ab- normality to a severe, life-​threatening condition. In mild hypo- calcaemia (serum calcium 2.00–​2.12 mmol/​litre), patients may be asymptomatic. Those with more severe (serum calcium <1.9 mmol/​ litre) and long-​term hypocalcaemia may develop acute symptoms of neuromuscular irritability (Box 13.4.6), ectopic calcification (e.g. in the basal ganglia, which may be associated with extrapyramidal neurological symptoms), subcapsular cataract, papilloedema, and abnormal dentition. Investigations should be directed at confirming the presence of hypocalcaemia and establishing the cause. Hypocalcaemia (Box 13.4.5) can be classified by cause, according to whether serum PTH concentrations are low (i.e. hypoparathyroid disorders) or high (i.e. disorders associated with secondary hyperparathyr- oidism). Hypocalcaemia is most commonly caused by hypopara- thyroidism, a deficiency or abnormal metabolism of vitamin D, acute or chronic renal failure, or hypomagnesaemia. In hypopara- thyroidism, serum calcium is low, phosphate is high, and PTH is undetectable; renal function and concentrations of the 25-​hydroxy and 1,25-​dihydroxy metabolites of vitamin D are usually normal. The features of pseudohypoparathyroidism are similar to those of hypoparathyroidism except for PTH, which is markedly increased. In chronic renal failure, which is the most common cause of hypo- calcaemia, phosphate is high and alkaline phosphatase, creatinine, and PTH are elevated; 25-​hydroxyvitamin D3 is normal and 1,25-​ dihydroxyvitamin D3 is low. In vitamin D deficiency and osteomal- acia, serum calcium and phosphate are low, alkaline phosphatase and PTH are elevated, renal function is normal, and 25-​hydroxyvitamin D3 is low. The most frequent artefactual cause of hypocalcaemia is hypoalbuminaemia, such as occurs in liver disease or the nephrotic syndrome. Management of acute hypocalcaemia The management of acute hypocalcaemia depends on the severity of the hypocalcaemia, the rapidity with which it developed, and the degree of neuromuscular irritability (Box 13.4.6). Treatment should be given to symptomatic patients (e.g. with seizures or tetany) and asymptomatic patients with a serum calcium of less than 1.90 mmol/​litre who are at high risk of developing compli- cations. The preferred treatment for acute symptomatic hypocal- caemia is calcium gluconate, 10 ml 10% weight per volume (w/​v) (2.20 mmol calcium), diluted in 50 ml of 5% dextrose or 0.9% so- dium chloride and given by slow intravenous injection (>5 min); this can be repeated as required to control symptoms. Serum calcium concentrations should be assessed regularly. Persistent hypocalcaemia may be managed acutely by administration of a calcium gluconate infusion as follows. Dilute 10 ampoules of cal- cium gluconate, 10 ml 10% w/​v (22.0 mmol calcium), in 1 litre of 5% dextrose or 0.9% sodium chloride, start the infusion at 50 ml/​ h, and titrate to maintain serum calcium concentrations in the normal range. Generally, 0.3 to 0.4 mmol/​kg elemental calcium in- fused over 4 to 6 h increases serum calcium by 0.5 to 0.75 mmol/​ Box 13.4.6  Hypocalcaemic clinical features of neuromuscular irritability • Paraesthesia, usually of fingers, toes, and circumoral regions • Tetany, carpopedal spasm, muscle cramps • Chvostek’s signa • Trousseau’s signb • Seizures of all types (i.e. focal or petit mal, grand mal, or syncope) • Prolonged Q–​T interval on ECG • Laryngospasm • Bronchospasm a Chvostek’s sign is twitching of the circumoral muscles in response to gentle tapping of the facial nerve just anterior to the ear; it may be present in 10% of normal individuals. b Trousseau’s sign is carpal spasm elicited by inflation of a blood pressure cuff to 20 mm Hg above the patient’s systolic blood pressure for 3 min.

13.4  Parathyroid disorders and diseases altering calcium metabolism 2327 litre. If hypocalcaemia is likely to persist, oral vitamin D therapy (see next) should also be administered. In hypocalcaemic patients who are also hypomagnesaemic, the hypomagnesaemia must be corrected before the hypocalcaemia will resolve. This may occur in the postparathyroidectomy period or in patients with severe malabsorption (e.g. those with established coeliac disease). Management of persistent hypocalcaemia The two main agents available for the treatment of hypocalcaemia are supplemental calcium (c.10–​20 mmol calcium every 6–​12 h), and vitamin D preparations. Patients with hypoparathyroidism seldom require calcium supplements after the early stages of sta- bilization with vitamin D.  A  variety of vitamin D preparations have been used. These include vitamin D3 (cholecalciferol) or vitamin D2 (ergocalciferol), 25 000 to 100 000 IU (1.25–​5 mg/​ day); dihydrotachysterol (now seldom used), 0.25 to 1.25 mg/​ day; alfacalcidol (1α-​hydroxycholecalciferol), 0.25 to 1.0 µg/​day; and calcitriol (1,25-​dihydroxycholecalciferol), 0.25 to 2.0 µg/​day. In children, these preparations are prescribed in dosages based on body weight. Cholecalciferol and ergocalciferol are the least expensive preparations but have the longest durations of action and may result in prolonged toxicity. The other preparations, which do not require renal 1α-​hydroxylation, have the advantage of shorter half-​lives and thereby minimize the risk of prolonged toxicity. Calcitriol is probably the drug of choice because it is the active metabolite and, unlike alfacalcidol, does not require hepatic 25-​hydroxylation. Close monitoring (at about 1-​ to 2-​week inter- vals) of the patient’s serum and urine calcium concentrations are required initially, and at 3-​ to 6-​month intervals once stabilization is achieved. The aim is to avoid hypercalcaemia, hypercalciuria, nephrolithiasis, and renal failure. It should be noted that hypercalciuria may occur in the absence of hypercalcaemia. The use of PTH replacement, using recombinant human PTH 1-​84 (rhPTH) by subcutaneous injections, in patients with hypopara- thyroidism has been reported to:  decrease the requirements of supplemental calcium and calcitriol; and to restore serum cal- cium concentrations to within the normal range without produ- cing hypercalciuria. The use of rhPTH has been approved by the Food and Drug Administration (FDA) for treatment of refractory hypoparathyroidism. Hypocalcaemic diseases Hypocalcaemic diseases (Box 13.4.5) may arise because of destruc- tion of the parathyroid glands, failure of parathyroid gland develop- ment, or reduced PTH secretion or PTH-​mediated actions in target tissues. Thus, these diseases may be classified as being due to a de- ficiency of PTH, a defect in the PTH receptor (i.e. the PTH/​PTHrP receptor), or an insensitivity to PTH caused by defects downstream of the PTH/​PTHrP receptor (Fig. 13.4.2). The diseases may also be classified as being part of the hypoparathyroid disorders, of the CaSR abnormalities, or of the pseudohypoparathyroid disorders. Hypoparathyroidism Hypoparathyroidism is characterized by hypocalcaemia and hyperphosphataemia, which are the result of a deficiency in PTH secretion or action. Serum concentrations of immunoreactive PTH are low or undetectable and the concentrations of 1,25-​ dihydroxyvitamin D3 are usually in the low normal to low range, but alkaline phosphatase activity is unchanged. The daily urinary excretion of calcium is reduced, although the fractional excretion of calcium is increased. Nephrogenous cAMP excretion is low and renal tubular reabsorption of phosphate is elevated. Urinary cAMP, plasma cAMP, and urinary phosphate excretion increase markedly after administration of exogenous bioactive PTH (Chase–​Aurbach and Ellsworth–​Howard tests). Hypoparathyroidism may result from agenesis (e.g. DiGeorge’s syndrome) or destruction of the parathyroid glands (e.g. following neck surgery, in autoimmune diseases), from reduced secretion of PTH (e.g. neonatal hypocal- caemia or hypomagnesaemia), or resistance to PTH (which may occur as a primary disorder (e.g. pseudohypoparathyroidism) or secondary to hypomagnesaemia). In addition, hypoparathyroidism may occur as an inherited disorder (Table 13.4.2) that may either be part of a complex congenital defect (e.g. DiGeorge’s syndrome), or as part of a polyglandular autoimmune disorder, or as a solitary endocrinopathy, which has been referred to as isolated or idiopathic hypoparathyroidism. Hypoparathyroidism may also complicate iron storage disease, especially secondary haemochromatosis in children and adolescents. In thalassaemic children, destruction of the parathyroids is associated with ill health and frank tetany, which may elude diagnosis and effective treatment unless hypoparathyr- oidism is suspected. Isolated hypoparathyroidism Isolated hypoparathyroidism may either be inherited, or it may be acquired by damage to the parathyroids at surgery, by infiltrating metastases, or following systemic disease (Box 13.4.5). Inherited hypoparathyroidism  Patients with inherited forms of hypoparathyroidism may develop hypocalcaemic seizures in the neonatal or infantile periods and require lifelong treatment with oral vitamin D preparations (e.g. calcitriol). Autosomal dominant, autosomal recessive, and X-​linked recessive inheritances for hypo- parathyroidism have been observed (Table 13.4.2). Some of the autosomal forms are due to mutations of the PTH gene, the CaSR and Gα11 (see next), and the transcriptional factor GCM2 (glial cells missing homologue 2). CaSR and Gα11 mutations causing ADH1 and ADH2  ADH1 and ADH2 are due to gain-​of-​function mutations of the CaSR and Gα11, respectively (Table 13.4.1, Fig. 13.4.2). ADH1 is character- ized by lifelong mild or severe hypocalcaemia in association with normal serum PTH concentrations in c.40% of patients or low serum PTH concentrations in c.60% of patients. Serum phosphate and magnesium concentrations may be elevated or low, respectively. Approximately 50% of ADH1 patients have asymptomatic hypo- calcaemia, and the remaining 50% may experience paraesthesia, muscle cramps, carpo-​pedal spasms, and seizures, which may be associated with a febrile illness. In addition, about 10% of ADH1 patients may have absolute hypercalciuria which may be associated with nephrocalcinosis and kidney stones in 35% of patients. Vitamin D preparations and calcium supplementation to correct the hypo- calcaemia may worsen the hypercalciuria and lead to renal impair- ment. Basal ganglia or ectopic calcification may be found in more than 35% of patients. About 20% of ADH1 patients do not have a previously reported family history, as they have de novo mutations. ADH2 patients appear to have clinical features that are similar to those in ADH1 patients.

SECTION 13  Endocrine disorders 2328 Acquired forms of  hypoparathyroidism  Hypoparathyroidism may occur after neck surgery, irradiation, or because of infiltration by metastases or systemic disease, for example, haemochroma- tosis, amyloidosis, sarcoidosis, Wilson’s disease, or thalassaemia (Box 13.4.5). Surgical damage to the parathyroids occurs most com- monly after a radical neck dissection; for example, laryngeal or oe- sophageal carcinoma treatment, a total thyroid resection, or after repeated parathyroidectomies for multiglandular disease (e.g. in MEN1 or MEN2, see earlier). Hypocalcaemic symptoms begin 12 to 24 h postoperatively and may need treatment with oral or intra- venous calcium. Parathyroid function often returns, but persistent hypocalcaemia requires treatment with vitamin D preparations. Neonatal hypoparathyroidism resulting in hypocalcaemia may occur in the baby of a mother with hypercalcaemia caused by pri- mary hyperparathyroidism. Maternal hypercalcaemia results in in- creased calcium delivery to the fetus, and this fetal hypercalcaemia suppresses fetal PTH secretion. Postpartum, the infant’s suppressed parathyroids are unable to maintain normocalcaemia. The disorder is usually self-​limiting, but occasionally therapy may be required. In addition, the feeding to babies of cow’s milk, which has a high phos- phate content, may also result in hypocalcaemia in some infants. Functional hypoparathyroidism may result from severe hypomag- nesaemia (<0.40 mmol/​litre), which may be due to a severe intestinal malabsorption disorder (e.g. Crohn’s disease) or a renal tubular dis- order. It is associated with hypoparathyroidism because magnesium is required for the release of PTH from the parathyroid gland and also for PTH action via adenyl cyclase. Magnesium chloride, 35 to 50 mmol intravenously in 1 litre of 5% glucose or other isotonic so- lution given over 12 to 24 h may be repeatedly required to restore normomagnesaemia. Complex syndromes associated with hypoparathyroidism Hypoparathyroidism may occur as part of a complex syndrome which may either be associated with a congenital developmental anomaly or with an autoimmune syndrome. The congenital devel- opmental anomalies associated with hypoparathyroidism, which occurs in 1 in 4000 live births, include the DiGeorge, the HDR (hypoparathyroidism, deafness, and renal anomalies), the Kenney–​ Caffey, and the Barakat syndromes, and also syndromes associ- ated with either lymphoedema or dysmorphic features and growth failure (Table 13.4.2). DiGeorge’s syndrome types 1 and 2  Patients with DiGeorge’s syn- drome (DGS) have neonatal hypoparathyroidism, T-​cell immuno- deficiency, congenital heart defects, and deformities of the ear, nose, and mouth (e.g. cleft lip and/​or palate). Children with DGS often die from infections related to the immunodeficiency. The disorder arises from a congenital failure in the development of the deriva- tives of the third and fourth pharyngeal pouches with resulting ab- sence or hypoplasia of the parathyroids and thymus. Most cases of DGS are sporadic but an autosomal dominant inheritance of DGS has been observed, and an association between the syndrome and an unbalanced translocation and deletions involving chromosome 22q11.2 have also been reported. Such patients with the 22q11.2 deletions are referred to as DGS type 1 (DGS1), and studies of the DGS1 deleted region have revealed four genes (RNEX40, NEX2.2-​ NEX3, UDFIL, and TBX1) to be involved. However, point muta- tions in DGS1 patients have been detected only in the TBX1 gene, and TBX1 is now considered to be the gene causing DGS1. TBX1 encodes a DNA-​binding transcriptional factor of the T-​box family that is known to have an important role in vertebrate and inverte- brate organogenesis and pattern formation. The TBX1 gene is de- leted in approximately 96% of all DGS1 patients, and some of those without deletions have been shown to harbour mutations of TBX1. In some other patients, deletions of another locus on chromosome 10p13–​p14 have been observed in association with DGS and this is referred to as DGS type 2 (DGS2). The nebulette (NEBL) gene has been reported to be heterozygously deleted in cell lines from DGS2 patients, and may be the responsible gene. Hypoparathyroidism, deafness, and renal anomalies (HDR) syn- drome  HDR is an autosomal dominant disorder in which patients often have asymptomatic hypocalcaemia with undetectable or in- appropriately normal serum concentrations of PTH, and normal brisk increases in plasma cAMP in response to the infusion of PTH. Bilateral, symmetrical, sensorineural deafness involving all frequen- cies occurs, and the renal abnormalities consist mainly of bilateral cysts that compress the glomeruli and tubules and lead to renal impairment. Cytogenetic abnormalities involving chromosome 10p14–​10pter have been identified in HDR patients. HDR patients do not have immunodeficiency or heart defects, which are key fea- tures of DGS2, and indeed there are two non​overlapping regions; thus, the DGS2 region is located on 10p13–​14 and HDR on 10p14–​ 10pter. HDR patients have deletions or mutations of the zinc finger transcription factor GATA3. Mitochondrial disorders associated with  hypoparathyr- oidism  Hypoparathyroidism has been reported to occur in three disorders associated with mitochondrial dysfunction: the Kearns–​ Sayre syndrome (KSS), the mitochondrial encephalopathy, lactic acidosis, and stroke-​like episodes syndrome (MELAS), and a mitochondrial trifunctional protein deficiency syndrome. Kearns–​ Sayre syndrome is characterized by progressive external ophthal- moplegia and pigmentary retinopathy before the age of 20 years, and is often associated with heart block or cardiomyopathy. The MELAS syndrome consists of a childhood onset of mitochondrial encephalopathy, lactic acidosis, and stroke-​like episodes. In add- ition, varying degrees of proximal myopathy can be seen in both conditions. Both the Kearns–​Sayre and MELAS syndromes have been reported to occur with insulin-​dependent diabetes mellitus and hypoparathyroidism, and mitochondrial gene abnormalities have been identified in some patients. Mitochondrial trifunctional protein deficiency is a disorder of fatty acid oxidation that is associ- ated with peripheral neuropathy, pigmentary retinopathy, and acute fatty liver degeneration in pregnant women who carry an affected fetus. Hypoparathyroidism has been observed in one patient with trifunctional protein deficiency. Kenney–​Caffey, Sanjad–​Sakati, and Kirk-​Richardson syn- dromes  Hypoparathyroidism has been reported to occur in over 50% of patients with the Kenney–​Caffey syndrome, which is as- sociated with short stature, osteosclerosis, and cortical thickening of the long bones, delayed closure of the anterior fontanel, basal ganglia calcification, nanophthalmos, and hyperopia. Parathyroid tissue could not be found in a detailed post-​mortem examination of one patient and this suggests that hypoparathyroidism may be due to an embryological defect of parathyroid development. In the Kirk-​Richardson and Sanjad–​Sakati syndromes, which are similar, hypoparathyroidism is associated with severe growth failure and

13.4  Parathyroid disorders and diseases altering calcium metabolism 2329 dysmorphic features. This has been reported in patients of Middle Eastern origin, whose parents were consanguineous, thereby indicating that these are autosomal recessive disorders. Molecular genetic investigations have identified mutations of the tubulin-​ specific chaperone (TBCE) to be associated with the Kenney–​Caffey and Sanjad–​Sakati syndromes. TBCE encodes one of several chap- erone proteins required for the proper folding of α-​tubulin subunits and the formation of α-​β tubulin heterodimers. Kenney–​Caffey type 2 is due to mutations of the family with sequence similarity 111, member A (FAM111A) gene, located on chromosome 11q12.1. Additional familial syndromes  Single familial syndromes in which hypoparathyroidism is a component have been reported (Table 13.4.2). Thus, an association of hypoparathyroidism, renal insufficiency, and developmental delay has been reported in one Asian family in whom autosomal recessive inheritance of the dis- order was established. The occurrence of hypoparathyroidism, nerve deafness, and a steroid-​resistant nephrosis leading to renal failure, which has been referred to as the Barakat syndrome, has been re- ported in four brothers from one family, and an association of hypo- parathyroidism with congenital lymphoedema, nephropathy, mitral valve prolapse, and brachytelephalangy has been observed in two brothers from another family. Molecular genetic studies have not been reported from these two families. Blomstrand’s disease  Blomstrand’s chondrodysplasia is an auto- somal recessive disorder characterized by early lethality, dramat- ically advanced bone maturation, and accelerated chondrocyte differentiation. Affected infants, who usually have consanguin- eous unaffected parents, develop pronounced hyperdensity of the entire skeleton with markedly advanced ossification that results in extremely short and poorly modelled long bones. Mutations of the PTH/​PTHrP receptor that impair its function are associated with Blomstrand’s disease. Thus, it seems likely that affected infants will, in addition to the skeletal defects, have abnormalities in other or- gans, including secondary hyperplasia of the parathyroid glands, presumably due to hypocalcaemia. Polyglandular autoimmune hypoparathyroidism  This syn- drome (Fig. 13.4.4) comprises hypoparathyroidism, Addison’s disease, candidiasis, and two or three of the following:  insulin-​ dependent diabetes mellitus, primary hypogonadism, autoimmune thyroid disease, pernicious anaemia, chronic active hepatitis, steatorrhoea (malabsorption), alopecia (totalis or areata), and viti- ligo. The disorder has also been referred to as either the autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) syndrome or the polyglandular autoimmune type 1 syndrome. Antibodies directed against the adrenal, thyroid, and parathyroid glands are detected in the sera of some patients. The polyglandular autoimmune type 2 syndrome is characterized by adrenal insuffi- ciency, insulin-​dependent diabetes mellitus, and thyroid disease, and does not involve hypoparathyroidism. APECED syndrome, which has an autosomal recessive inheritance, has a high incidence in Finland and among Iranian Jews. The affected gene in APECED syndrome is the AIRE (autoimmune regulator) gene, which has been located to chromosome 21q22.3. It encodes a 545 amino acid protein that contains motifs suggestive of a transcriptional factor and includes a nuclear localization signal, two zinc finger motifs, a proline-​rich region, and three LXXLL motifs. Four AIRE mutations are commonly found in APECED families: Arg257Stop in Finnish, German, Swiss, British, and Northern Ireland families; Arg139Stop in Sardinian families; Tyr85Cys in Iranian Jewish families; and a 13-​ bp deletion in exon 8 in British, Dutch, German, and Finnish fam- ilies. These mutations likely abolish the E3 ubiquitin ligase activity of the AIRE1 protein, which has been shown to regulate the elimin- ation of organ-​specific T cells in the thymus. Thus, APECED is likely to be caused by a failure of this specialized mechanism for deleting forbidden T cells and establishing immunological tolerance. Autoimmune acquired hypoparathyroidism (AH) Twenty per cent (20%) of patients who had acquired hypopara- thyroidism in association with autoimmune hypothyroidism, were found to have autoantibodies to the extracellular domain of the CaSR (Table 13.4.1, Fig. 13.4.2). The CaSR autoantibodies did not persist for long; 72% of patients who had AH for less than 5 years had detectable CaSR autoantibodies; whereas only 14% of patients with AH for more than 5 years had such autoantibodies. The ma- jority of the patients who had CaSR autoantibodies were females, a finding that is similar to that found in other auto-​antibody medi- ated diseases. Indeed a few acquired hypoparathyroidism patients have also had features of autoimmune polyglandular syndrome type 1. The epitopes for the anti-​CaSR antibodies were localized to the N-​terminal of the extracellular domain of the receptor. These findings establish that the CaSR is an autoantigen in acquired hypoparathyroidism. Pseudohypoparathyroidism (PHP) Patients with PHP, which may be inherited as an autosomal dominant disorder, are characterized by hypocalcaemia and hyperphosphataemia due to PTH resistance rather than PTH deficiency. Five variants are recognized on the basis of bio- chemical and somatic features (Table 13.4.3) and three of these—​PHP type Ia (PHPIa), PHP type 1b (PHPIb), and pseudopseudohypoparathyroidism (PPHP)—​will be reviewed in further detail. Fig. 13.4.4  Candidiasis and hyperpigmentation of the hands, particularly over the knuckles, are seen in this 8-​year-​old patient with hypoparathyroidism and Addison’s disease. The patient also had vitiligo, and thus had some of the features of the polyglandular autoimmune syndrome type 1. Reproduced with permission from Thakker RV (1997). Hypocalcaemic disorders. In: Thakker RV, Wass JAH (eds) Endocrine disorders, medicine, vol. 25, pp. 68–​70. The Medicine Group (Journals), Abingdon.

SECTION 13  Endocrine disorders 2330 Patients with PHPIa exhibit PTH resistance (hypocalcaemia, hyperphosphataemia, elevated serum PTH, and an absence of an increase in serum and urinary cAMP and urinary phosphate following intravenous human PTH infusion), together with the features of Albright’s hereditary osteodystrophy (AHO), which in- cludes short stature, obesity, subcutaneous calcification, mental re- tardation, round facies, dental hypoplasia, and brachydactyly (i.e. shortening of the metacarpals (Fig. 13.4.5), particularly the third, fourth, and fifth). In addition to brachydactyly, other skeletal ab- normalities of the long bones and shortening of the metatarsals may also occur. Patients with PHPIb exhibit PTH resistance only and do not have the somatic features of AHO, while patients with PPHP ex- hibit the somatic features of AHO in the absence of PTH resist- ance. The absence of a normal rise in urinary excretion of cAMP excretion after an infusion of PTH in PHPIa indicates a defect at some site of the PTH receptor–​adenyl cyclase system (Fig. 13.4.2). This receptor system is regulated by at least two G proteins, one of which stimulates (Gsα) and another which inhibits (Giα) the ac- tivity of the membrane-​bound enzyme that catalyses the forma- tion of the intracellular second messenger cAMP. Interestingly, patients with PHPIa may also show resistance to other hormones (e.g. thyroid-​stimulating hormone, follicle-​stimulating hormone, and luteinizing hormone) that act via G-​protein-​coupled recep- tors. Inactivating mutations of the Gsα gene (referred to as GNAS1), which is located on chromosome 20q13.2, have been identified in PHPIa and PPHP patients. However, GNAS1 mutations do not fully explain the PHPIa or PPHP phenotypes, and studies of PHPIa and PPHP that occurred within the same kindred re- vealed that the hormonal resistance is parentally imprinted. Thus, PHPIa occurs in a child only when the mutation is inherited from a mother affected with either PHPIa or PPHP, and PPHP occurs in a child only when the mutation is inherited from a father affected with either PHPIa or PPHP. PHPIb is due to deletions that are located upstream of the GNAS1 gene. Moreover, in affected in- dividuals the deletion involved the maternal allele, whereas its occurrence on the paternal allele resulted in unaffected healthy carriers. This is consistent with parental imprinting of the GNAS1 abnormality causing PHPIb. FURTHER READING Bilezikian JP, et al. (2014). Guidelines for the management of asymp- tomatic primary hyperparathyroidism:  summary statement from the Fourth International Workshop. J Clin Endocrinol Metab, 99, 3561–​9. Bilezikian JP, et al. (2016). Management of hypoparathyroidism: pre- sent and future. J Clin Endocrinol Metab, 101, 2313–​24. Bollerslev J, et al. (2015). European Society of Endocrinology Clinical Guideline: Treatment of chronic hypoparathyroidism in adults. Eur J Endocrinol, 173, G1–20. Brandi ML, et al. (2016). Management of hypoparathyroidism: sum- mary statement and guidelines. J Clin Endocrinol Metab, 101, 2273–​83. Clarke BL, et al. (2016). Epidemiology and diagnosis of hypoparathyr- oidism. J Clin Endocrinol Metab, 101, 2284–​99. Eastell R, et al. (2014). Diagnosis of asymptomatic primary hyperpara- thyroidism: proceedings of the Fourth International Workshop. J Clin Endocrinol Metab, 99, 3570–​9. Fraser WD (2009). Hyperparathyroidism. Lancet, 374, 145–​58. Hannan FM, Thakker RV (2013). Investigating hypocalcaemia. BMJ, 9, 346–​f2213. Hannah FM, et al. (2018). The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases. Nat Rev Endocrinol, 15, 33–51. Howles SA, et al. (2016). Cinacalcet for symptomatic hypercalcemia caused by AP2S1 mutations. N Engl J Med, 374, 1396–​8. Erratum in: N Engl J Med. 2016, 374, e30. Marcocci C, Cetani F (2011). Primary hyperparathyroidism. N Engl J Med, 365, 2389–​97. Nesbit MA, et al. (2013). Mutations affecting G-​protein subunit alpha11 in hypercalcemia and hypocalcemia. N Engl J Med, 368, 2476–​86. Nesbit MA, et  al. (2013). Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3. Nat Genet, 45, 93–​7. Pallan S, et al. (2012). Diagnosis and management of primary hyper- parathyroidism. BMJ, 344, e1013. Peacock M, et al. (2009). Cinacalcet treatment of primary hyperpara- thyroidism: biochemical and bone densitometric outcomes in a five-​ year study. J Clin Endocrinol Metab, 94, 4860–​7. Rubin MR, et al. (2016). Therapy of hypoparathyroidism with PTH(1-​ 84): a prospective six year investigation of efficacy and safety. J Clin Endocrinol Metab, 101, 2742–​50. Shoback DM (2008). Clinical practice. hypoparathyroidism. N Engl J Med, 359, 391–​403. Shoback DM, et al. (2016). Presentation of hypoparathyroidism: eti- ologies and clinical features. J Clin Endocrinol Metab, 101, 2300–​12. Silverberg SJ, et al. (2014). Current issues in the presentation of asymp- tomatic primary hyperparathyroidism: proceedings of the Fourth International Workshop. J Clin Endocrinol Metab, 99, 3580–​94. Thakker RV (2016). Genetics of parathyroid tumours. J Intern Med, 280, 574–83. Thakker RV, et  al. (2012). Clinical practice guidelines for multiple endocrine neoplasia type 1 (MEN1). J Clin Endocrinol Metab, 97, 2990–​3011. Udelsman R, et al. (2014). The surgical management of asymptom- atic primary hyperparathyroidism:  proceedings of the Fourth International Workshop. J Clin Endocrinol Metab, 99, 3595–​606. Wysolmerski JJ (2012). Parathyroid hormone-​related protein: an up- date. J Clin Endocrinol Metab, 97, 2947–​56. Fig. 13.4.5  Radiograph of both hands of a patient with pseudohypoparathyroidism type 1a. The patient has a normal right hand, but there is shortening of the left fourth metacarpal (brachydactyly). Metatarsals may be similarly shortened. Reproduced with permission from Thakker RV (1997). Hypocalcaemic disorders. In: Thakker RV, Wass JAH (eds) Endocrine disorders, medicine, vol. 25, pp. 68–​70. The Medicine Group (Journals), Abingdon.

13.5 Adrenal disorders 2331

13.5 Adrenal disorders 2331

13.5.1 Disorders of the adrenal cortex 2331 Mark S

13.5.1 Disorders of the adrenal cortex 2331 Mark Sherlock and Mark Gurnell

CONTENTS 13.5.1 Disorders of the adrenal cortex  2331 Mark Sherlock and Mark Gurnell 13.5.2 Congenital adrenal hyperplasia  2360 Nils P. Krone and Ieuan A. Hughes 13.5.1  Disorders of the adrenal
cortex Mark Sherlock and Mark Gurnell ESSENTIALS Three classes of steroid hormone are produced by the adrenal cortex after uptake of precursor cholesterol from the plasma—​ mineralocorticoids, glucocorticoids, and sex steroids—​with classical endocrine feedback loops controlling their secretion. Adrenocortical diseases are relatively uncommon, but they have detrimental clinical consequences and can be treated effectively. Hormonal deficiency or excess is usually the result of abnormal secretion. Glucocorticoid excess Cushing’s syndrome may be ACTH dependent (usually due to a pituitary adenoma, Cushing’s disease) or ACTH independent (most often caused by an adrenal adenoma or exogenous glucocorticoid exposure). Clinical features—​presentation may be with ‘classical’ manifestations of centripetal obesity, moon face, hirsutism, and plethora, with signs (when present) that best distinguish from simple obesity being bruising and muscle weakness (typically proximal), but cases may be more subtle. Diagnosis—​the presence of Cushing’s syndrome can be confirmed by finding one or more of elevated Urine free cortisol, elevated late-​night salivary cortisol, or failure to suppress 09.00 h cortisol to less than 50 nmol/​litre following a dexamethasone suppression test (1 mg overnight, or 2 mg/​d for 48 h). ACTH-​dependent causes can be distinguished from ACTH-​independent causes by measurement of plasma ACTH (09.00 h sample). Determining whether elevated ACTH is coming from the pituitary (Cushing’s disease) or from an ectopic source can be difficult, but may be achieved by consider- ation of plasma potassium, high-​dose dexamethasone suppression test, a corticotropin-​releasing factor test, and bilateral inferior pe- trosal sinus sampling/​selective venous catheterization. Imaging—​pituitary MRI is the investigation of choice once bio- chemical testing suggests Cushing’s disease. In ectopic ACTH se- cretion, CT scanning of the neck, thorax, abdomen, and pelvis is required, often in conjunction with isotope (functional) imaging. Adrenal CT scanning is required if biochemical testing suggests ACTH-​independent Cushing’s syndrome. Management—​drugs that interfere with cortisol synthesis (e.g. metyrapone, ketoconazole) can lower cortisol levels, but definitive treatment depends on the cause: (1) adrenal adenomas—​unilateral adrenalectomy; (2) Cushing’s disease—​trans-​sphenoidal removal of the pituitary tumour; (3) ectopic ACTH—​surgical removal of the tu- mour can lead to cure, but this is not always possible. Glucocorticoid deficiency Glucocorticoid deficiency can be due to adrenal disease (primary, in which case mineralocorticoids and adrenal androgens are also de- ficient) or as a result of ACTH deficiency (secondary, in which case glucocorticoids and sex steroids are deficient). Aetiology—​primary hypoadrenalism (Addison’s disease) is most commonly caused by autoimmune disease (>70% cases in the Western world) or infection, for example, tuberculosis (the com- monest cause worldwide). The commonest cause of secondary hypoadrenalism is stopping of exogenous glucocorticoid therapy or its inadequacy in stressful situations. Clinical features—​primary adrenal failure may present (1) acutely—​ with hypotension and acute circulatory failure (Addisonian crisis); or (2)  chronically—​with vague features of ill health, sometimes including gastrointestinal symptoms, features suggestive of postural hypotension, and salt craving. Skin pigmentation is nearly always present in primary adrenal insufficiency (but not in secondary). 13.5 Adrenal disorders Acknowledgement: The chapter on disorders of the adrenal cortex in the fifth edition of this textbook was written by Professor P M Stewart. Some of his chapter is retained here.

SECTION 13  Endocrine disorders 2332 Biochemical diagnosis—​this depends on an ACTH stimulation test: plasma cortisol should rise to over 450 nmol/​litre (16.3µg/​dl) in response to injection of tetracosactide (Synacthen, 250 µg) and failure to do so indicates adrenal insufficiency. Management—​patients presenting acutely should be treated in a critical care setting with immediate intravenous hydrocortisone
(100 mg, followed by 200 mg per 24 h). Appropriate fluid replace­ ment and glucose/​ electrolyte monitoring and treatment are also central to effective management, along with treatment of any precipitating condition. Long-​term treatment requires glucocorticoid replacement in divided doses, with the largest dose on waking to mimic the circadian rhythm, and with the dose typically doubled in the event of intercurrent stress or illness. Patients should be supplied with, and trained in how to use, an emergency hydrocortisone injec- tion. Mineralocorticoid replacement is usually required in primary adrenal failure. Every patient should be advised to wear a medical alert bracelet or necklace and to carry a ‘steroid card’. Mineralocorticoid excess Primary aldosteronism (Conn’s syndrome) accounts for between 5 and 10% of all hypertension and 20–​25% of ‘resistant hyper- tension’. The diagnosis is often challenging, but in the absence of confounding influences is suggested by a high random plasma aldosterone/​renin ratio, especially if plasma aldosterone concen- tration is over 415 pmol/​litre (15 ng/​dl). The cause of primary aldos- teronism can also be difficult to establish: adrenal MRI/​CT scanning may demonstrate a unilateral adenoma but cannot exclude bilateral disease. Adrenal vein cannulation with sampling for estimation of aldosterone/​cortisol ratio may be required to ensure appropriate lateralization. Treatment of a unilateral adrenal adenoma is by sur- gical excision and of bilateral adrenal hyperplasia is medical, usually with spironolactone. Several single gene defects can cause mineralocorticoid excess, including 17α-​hydroxylase deficiency, 11β-​hydroxylase deficiency, glucocorticoid-​suppressible hyperaldosteronism, and apparent min- eralocorticoid excess (mutations in 11β-​hydroxysteroid dehydro- genase type 2 gene). Mineralocorticoid deficiency This is most commonly seen in the context of primary hypoadrenalism but is also caused (rarely) by conditions including primary defects in aldosterone biosynthesis, defects in aldosterone action, and hyporeninaemic hypoaldosteronism (most commonly in the context of diabetic nephropathy or chronic interstitial nephritis). Introduction An initial rate-​limiting step in adrenal steroidogenesis is the uptake of cholesterol from circulating cholesterol bound to low-​density lipoprotein, by mitochondria in the adrenal cortex. This process is dependent upon steroidogenic acute regulatory (StAR) protein. Thereafter, the functional zonation of the adrenal cortex is in part achieved through the discrete expression and regulation of the genes for the final steroidogenic enzymes:  aldosterone synthase (EC 1.14.15.5) in the zona glomerulosa, and 11β-​hydroxylase (EC 1.14.15.4) in the zona fasciculata (Fig. 13.5.1.1). Aldosterone acts physiologically to stimulate sodium transport across epithelial cells in the distal nephron, colon, and salivary gland. This involves the interaction of aldosterone with the min- eralocorticoid receptor, and the induction of the expression of the genes for the basolateral Na+, K+ –​ATPase pump and the apical sodium channel. This is mediated by the induction of SGK1, the gene for serum/​glucocorticoid-​regulated kinase 1.  The mineralo- corticoid receptor, however, is non​selective in vitro; paradoxically, cortisol and aldosterone have the same intrinsic affinity for the min- eralocorticoid receptor, raising the question of why aldosterone is the preferred mineralocorticoid in vivo. This selectivity is achieved at a prereceptor level through the production of an enzyme, 11β-​ hydroxysteroid dehydrogenase type 2 (11β-​HSD2; HSD11B2; EC 1.1.1.1.46), which efficiently inactivates cortisol to cortisone, al- lowing aldosterone to occupy the mineralocorticoid receptor. Inhibition of 11β-​HSD2 results in cortisol, conventionally regarded as a glucocorticoid, acting as a potent mineralocorticoid. Glucocorticoids have more diverse and extensive roles than mineralocorticoids, regulating sodium and water homeostasis, glucose and carbohydrate metabolism, inflammation, and stress. These effects are mediated by the interaction of cortisol with ubi- quitous glucocorticoid receptors, and the induction or repres- sion of target gene transcription (via glucocorticoid response elements, GREs). Adrenocortical diseases are most readily classified by whether they are characterized by hormone excess or deficiency (Table 13.5.1.1). Glucocorticoid excess: Cushing’s syndrome Harvey Cushing first described a case of polyglandular syndrome secondary to pituitary basophilia in 1912, and several years later linked this to bilateral adrenal hyperplasia. The first case of an adrenal adenoma was probably reported by H G Turney in 1913 (Fig. 13.5.1.2). StAR Pregnenolone Cholesterol SCC Aldosterone ACTH Progesterone CYP 21 DOC 3β-HSD CYP11β1 Corticosterone CYP11β2 Mineralocorticoid Zona Glomerulosa Zona Fasciculata Zona Reticularis CYP 17 17-OH-Pregnenolone 17-OH-Progesterone 11-Deoxycortisol Glucocorticoid DHEA A’dione Androgens CYP11β1 Cortisol CYP 17 Fig. 13.5.1.1  Pathways of adrenocortical steroid biosynthesis. Key: 3β-​ HSD, 3β-​hydroxysteroid dehydrogenase; ACTH, adrenocorticotrophic hormone; CYP, cytochrome enzymes; DOC, deoxycorticosterone; SCC, side-​chain cleavage enzyme; StAR, steroidogenic acute regulatory protein.

13.5.1  Disorders of the adrenal cortex 2333 Definition Cushing’s syndrome comprises the symptoms and signs associ- ated with prolonged exposure to inappropriately elevated levels of free plasma glucocorticoids. This definition thus takes into account the elevated corticosteroid levels that may be found in severely depressed patients, but which appear to be appropriate to the condition, and also the increased total (but normal free) glucocorticoid levels found when there is an increase in circu- lating cortisol-​binding globulin (e.g. in patients on oral oestrogen therapy). The use of the term glucocorticoid in the definition covers both endogenous (cortisol) and exogenous steroid excess (e.g. prednisolone, fluticasone, budesonide, beclomethasone, dexamethasone, etc). The condition is most readily classified into ACTH-​dependent and ACTH-​independent causes (Table 13.5.1.2). The term ‘Cushing’s syndrome’ is used to describe all causes, whereas ‘Cushing’s disease’ is reserved for cases of pituitary-​dependent Cushing’s syndrome. ACTH-​dependent causes Cushing’s disease When iatrogenic causes are excluded, the most frequent cause of Cushing’s syndrome is Cushing’s disease, which accounts for ap- proximately 70% of cases. The adrenal glands show bilateral adrenocortical hyperplasia, with widening of the zona fasciculata and zona reticularis. Nodules may form within the hyperplastic glands. Table 13.5.1.1  Adrenocortical diseases Adrenal abnormality Glucocorticoid excess -Cushing’s syndrome ACTH dependent Cushing’s disease (pituitary) Ectopic ACTH production ACTH independent Exogenous glucocorticoid therapy Adrenal tumours Glucocorticoid deficiency Primary Congenital adrenal hyperplasia (21-​hydroxylase, P450 oxidoreductase, 3β-​hydroxysteroid dehydrogenase, 17-​hydroxylase, 11 β-​hydroxylase, and StAR deficiencies) Addison’s disease Hereditary adrenocortical unresponsiveness to ACTH Secondary Postcorticosteroid therapy Hypothalamic/​pituitary disease Mineralocorticoids Excess Primary aldosteronism (aldosterone) Congenital adrenal hyperplasia (deoxycorticosterone)   11β-​Hydroxylase deficiency   17α-​Hydroxylase deficiency Glucocorticoid receptor resistance (deoxycorticosterone) Glucocorticoid receptor mutations Metyrapone, RU486 ingestion Deoxycorticosterone—​secreting adrenal tumour Liddle’s syndrome 11 β-​Hydroxysteroid dehydrogenase deficiency (apparent mineralocorticoid excess and liquorice and carbenoxolone ingestion) (cortisol) Ectopic ACTH syndrome (cortisol) Deficiency Addison’s diseases Congenital adrenal hyperplasia Congenital adrenal hypoplasia Disorders of terminal part of aldosterone biosynthetic pathway Pseudohypoaldosteronism Hyporeninaemia Adrenal androgens Excess Congenital adrenal hyperplasia (21-​hydroxylase, 11 β-​ hydroxylase deficiency) Polycystic ovary syndrome (PCOS) Tumours Deficiency Congenital adrenal hyperplasia (17-​hydroxylase, 3β-​hydroxysteroid dehydrogenase deficiency) Adrenal incidentalomas and carcinomas Fig. 13.5.1.2  H.G. Turney’s case of Cushing’s syndrome before and after developing the condition.

SECTION 13  Endocrine disorders 2334 Cushing himself raised the question of whether his disease was a primary pituitary condition or secondary to an abnormality of the hypothalamus. There is abundant evidence to indicate that the con- dition is related to the pituitary rather than the hypothalamus. In over 90% of cases it is caused by a pituitary adenoma of monoclonal origin; basophilic hyperplasia is very rare, and selective surgical re- moval of the microadenoma usually results in remission. A somatic mutational hotspot is found in the USP8 gene in 30–​ 60% of corticotrophinomas, resulting in impaired down regulation of the epidermal growth factor receptor (EGFR) which enables its constitutive signalling. Epidermal growth factor (EGF) is an im- portant regulator of corticotroph function and its receptor is highly expressed in Cushing’s pituitary tumours, where it leads to increased ACTH synthesis in vitro and in vivo. The mutational hotspot found in corticotrophinomas hyperactivates USP8, enabling it to rescue EGFR from lysosomal degradation and facilitate its stimulatory signalling. Ectopic ACTH syndrome Cushing’s syndrome may be caused by non​pituitary tumours pro- ducing ACTH, most commonly a malignant small-​cell carcinoma of the bronchus (Table 13.5.1.3). However, the most challenging diagnostic problems relate to ACTH secretion from more benign and indolent neuroendocrine tumours (which may be very small), which may present with Cushing’s syndrome many years before the occult tumour manifests, indeed in one large series 1 in 8 patients did not have identification of the source of ACTH secretion despite prolonged follow-​up. Ectopic production of corticotropin-​releasing factor (CRF) This is a very rare cause of pituitary-​dependent Cushing’s disease. However, cases have been described in which a tumour (e.g. medul- lary thyroid, prostate carcinoma) has been shown to produce CRF. ACTH-​independent causes Iatrogenic Cushing’s syndrome Estimates suggest that up to 3% of the population of the United Kingdom and United States are currently taking glucocorticoid therapy. Long-​term prescription rates for oral glucocorticoids have increased in recent decades, as has the use of inhaled, intranasal, and topical glucocorticoid therapy. In most cases, the doses of prescribed glucocorticoids are sufficient to cause hypothalamic–​ pituitary–​adrenal (HPA) axis suppression (total daily doses >5 mg prednisolone or equivalent, Table 13.5.1.4). Glucocorticoid pre- scriptions are often extended for a sustained period of time; as a result, iatrogenic Cushing’s syndrome and subsequent suppression of the HPA axis may be frequent and potentially overlooked. In a recent study assessing rates of hypoadrenalism in patients receiving exogenous steroids there was a high percentage who failed a short Synacthen test, indicating suppression of endogenous cortisol se- cretion as one would see in patients who have iatrogenic Cushing’s syndrome. In patients who received oral or intravenous steroids the rate of failure on Synacthen testing was 44.3% and in those receiving inhaled, intranasal, or topical steroids 24.6%. While overt iatrogenic Cushing’s syndrome is clinically easy to diagnose, more subtle cases may often evade diagnosis. If left on long-term glucocorticoids, patients with ‘mild’ iatrogenic Cushing’s syndrome may develop all the adverse effects seen in patients with endogenous Cushing’s syndrome. Indeed, studies have shown that patients who receive ex- ogenous glucocorticoids at doses 7.5 mg or more of prednisolone per day have increased cardiovascular mortality. Importantly other concomitant medications can increase the potency of exogenous glucocorticoids (e.g. drugs which inhibit glucocorticoid metabolism via cytochrome P450-​3A4). Concomitant adrenal insufficiency is a clinically important factor in patients who develop iatrogenic Cushing’s syndrome. If these pa- tients have their steroids stopped abruptly, or if they are unwell and do not follow directions for stress dose steroids, they may develop an adrenal crisis. As such, if these patients are discontinuing their exogenous glucocorticoid therapy, they may require replacement doses of glucocorticoids until their hypothalamic–​pituitary–​adrenal axis recovers. Table 13.5.1.2  Classification of causes of Cushing’s syndrome ACTH dependent ACTH independent Cushing’s disease (pituitary-​dependent) Ectopic ACTH syndrome Ectopic corticotrophin-​releasing factor syndrome Iatrogenic (treatment with ACTH1–​39 or Synacthen®, ACTH1–​24) Iatrogenic (such as pharmacological doses of prednisolone, fluticasone, budesonide, beclomethasone, or dexamethasone) Adrenal adenoma Adrenal carcinoma Carney’s complex McCune–​Albright syndrome Aberrant receptor expression Table 13.5.1.3  Tumours associated with the ectopic ACTH syndrome Tumour type Approximate incidence (%) Small-​cell lung carcinoma 50 Non-​small-​cell lung carcinoma 5 Pancreatic neuroendocrine tumours (NETs) 10 Thymic NETs 5 Lung NETs 10 Other NETs 2 Medullary carcinoma of thyroid 5 Phaeochromocytoma and related tumours 3 Rare carcinomas of prostate, breast, ovary, gallbladder, colon 10 Table 13.5.1.4  Equivalent anti-​inflammatory effects of commonly used glucocorticoids Anti-​inflammatory effects equivalent to 20 mg hydrocortisone 5 mg prednisolone 25 mg cortisone acetate 0.75 mg dexamethasone 0.75 mg beclomethasone 4 mg methylprednisolone 4 mg triamcinolone 6 mg deflazacort

13.5.1  Disorders of the adrenal cortex 2335 Adrenal adenoma and carcinoma With the exclusion of iatrogenic Cushing’s syndrome, a solitary cortisol-​secreting adrenal adenoma is the cause in about 10% of cases. Adrenal carcinomas are rare, have a poor prognosis, and may be associated with the secretion of other hormones in addition to cortisol (usually adrenal androgens) or may be ‘endocrine inactive’ (although steroid profiling often reveals evidence of excess adrenal corticosteroid production even in the absence of overt clinical features). Carney’s complex Carney complex (CNC) is a rare multiple neoplasia syndrome, inherited in an autosomal-​dominant manner or occurring sporadically as a result of a de novo genetic defect, and first de- scribed by J. Aidan Carney as ‘the complex of myxomas, spotting pigmentation and endocrine over-​reactivity’. It is characterized by pigmented lesions of the skin and mucosae, cardiac, cutaneous, and other myxomatous tumours, and multiple other endocrine (pituitary, adrenal, thyroid, gonads) and non​endocrine neoplasms (eye, breast, uterus, liver, bone). In patients with Carney com- plex who develop Cushing’s syndrome the adrenal glands contain multiple small, pigmented nodules (primary pigmented nodular adrenocortical disease (PPNAD), see Fig. 13.5.1.3). Carney com- plex is associated with mutations in the regulatory subunit R1A of protein kinase A, PRKAR1A gene. PRKAR1A, which is situated at the 24.2–​24.3 locus of the long arm of chromosome 17 and has 11 (a) (b) (d) (f) (h) (g) (e) (i) (c) Fig. 13.5.1.3  Characteristics of patients with Carneys Complex. Characteristic distribution of the lentigines on the eyelids (a), the vermillion border of the lips and the cheeks (b), and the ears, including the ear canal (c) in patients with CNC; such typical pigmentation on the face is only present in less than one-​third of the patients but it is rather diagnostic when present. (d) a pigmented macule (arrow) on the outer canthus of a patient with CNC who had minimal other pigmentation; inner or outer canthal pigmentation such as the one shown here is only seen in CNC and Peutz–​ Jeghers syndrome making it diagnostic for these two conditions. (e) Nipple myxoma in a female patient with CNC. (f) Ear myxoma complicated by chronic infection and tissue overgrowth in a toddler with CNC. (g and h) Large myxoma (circled) between the left atrium and ventricle detected by echocardiography in an adolescent with CNC (g) who had surgery immediately thereafter, and a much smaller myxoma (arrow) of the left ventricle originating from the cardiac diaphragm detected by cardiac MRI in an older patient with CNC (h); this myxoma was followed by serial echocardiogram and has yet to be operated, as it is not growing and poses no immediate risks. (i) 5× magnification haematoxylin and eosin staining of the adrenal gland of a patient with CNC: the characteristic nodules of PPNAD are shown by the arrows; the overall size of the gland is normal and the nodules may not be visible by imaging studies. From Correa R, Salpea P, Stratakis CA (2015). Carney complex: an update. Eur J Endocrinol, 173, M85–​97.

SECTION 13  Endocrine disorders 2336 exons, of which exons 2–​11 are protein-​coding. More than 70% of the patients diagnosed with Carney complex carry mutations on the PRKAR1A gene (CNC1 locus) and this percentage increases to 80% for those with Cushing’s syndrome due to PPNAD. A second genetic locus is associated with Carney complex and is referred to as the ‘CNC2’ locus (CNC1 being the PRKAR1A gene 17q locus). CNC2 is a 10 Mb region in the 2p16 locus. McCune–​Albright syndrome In this condition, fibrous dysplasia and cutaneous pigmentation may be associated with pituitary, thyroid, gonadal, and adrenal hyperfunction, with the latter manifesting as Cushing’s syndrome. McCune–​Albright syndrome (MAS) is caused by mosaicism for a mutation in the guanine nucleotide-​binding protein, α-stimulating activity polypeptide (GNAS) gene, which maps to chromosome 20q13, and encodes the ubiquitously expressed stimulatory sub- unit α of the G protein (Gsa). Gsa activates adenyl cyclase and leads to the generation of cAMP. Causative mutations in GNAS result in the G protein being constitutively activated, which, in the ad- renal gland, mimics constant ACTH stimulation leading to adrenal hypersecretion and nodule formation. Aberrant receptor expression Initially, patients were described with nodular hyperplasia, ACTH-​ independent Cushing’s syndrome, and enhanced adrenal respon- siveness to gastric inhibitory polypeptide (GIP). The biochemical clues were the presence of subnormal morning levels of plasma cortisol and a rise in cortisol after food. This food-​dependent form of Cushing’s syndrome results from the normal increase in GIP after eating. Not surprisingly, the clinical syndrome is related to food intake; fasting can produce adrenal insufficiency. More recently, several novel mechanisms which regulate cortisol secre- tion from adrenal nodules have been uncovered. This includes constitutive activation of the cAMP system and steroidogenesis, or its regulation, as a consequence of aberrant adrenal expression of several hormone receptors, particularly G-​protein coupled hormone receptors (GPCR) and their ligands. When surgical samples of patients with bilateral macronodular adrenal hyper- plasia and unilateral adrenal tumours (with Cushing’s syn- drome) are analysed there are frequent aberrant expression of G-​protein coupled receptors and frequent coexpression of sev- eral receptors. Aberrant hormone receptors can also exert their activity by regulating the paracrine secretion of ACTH or other ligands. The aberrant expression of hormone receptors is not limited to adrenal Cushing’s syndrome but can be implicated in other endocrine tumours including primary aldosteronism and Cushing’s disease. Alcohol-​associated pseudo-​Cushing’s syndrome In the original description of this syndrome, urinary and plasma cor- tisol levels were elevated, but were not suppressed with dexametha- sone. Plasma ACTH may be normal or suppressed. The frequency and pathogenesis of this condition remain unknown, but a two-​hit hypothesis has been put forward to explain its aetiology. Chronic liver disease, irrespective of the cause, is associated with impaired cortisol metabolism, but in those consuming excess alcohol this is associated with an increase in the cortisol secretion rate, rather than concomitant suppression in the face of impaired metabolism. With abstinence from alcohol the biochemical abnormalities rapidly re- vert to normal. Micronodular and macronodular adrenal hyperplasia There is a spectrum of recently recognized bilateral hyperplasias including micronodular adrenal disease (and its pigmented variant, primary pigmented nodular adrenocortical disease, see Carney complex) and macronodular bilateral adrenal hyperplasia (ACTH-​ independent macronodular hyperplasia or massive macronodular adrenocortical disease). Micronodular hyperplasia is commonly due to a genetic defect (including PRKAR1A, PDE11A, PDE8B) and is associated with Cushing’s syndrome in children and young adults. Macronodular adrenal hyperplasia is less frequently linked to a genetic cause (e.g. MENIN, APC (familial adenomatous polyposis coli), GNAS, FH (fumarate hydratase), ectopic G-​protein coupled receptors, WNT (encoding Wnt (wingless-​related integration site) and WISP-​2 (Wnt-​inducible signalling pathway protein 2)), and very rarely manifests in childhood, but rather presents with atypical Cushing’s syndrome in middle-​aged or elderly adults (for more de- tails, see Table 13.5.1.5). Clinical features The classical features of Cushing’s syndrome—​centripetal obesity, moon face, hirsutism, and plethora—​are well known following Cushing’s initial description in 1912 (Fig. 13.5.1.2 and Fig. 13.5.1.4), but this gross clinical picture is not always present. The signs and symptoms in patients with Cushing’s syndrome are listed in Table 13.5.1.6, together with the most discriminatory features distinguishing Cushing’s syndrome from simple obesity. Weight gain and obesity are the most common symptom and sign, but the distribution of fat is not invariably centripetal—​a ‘buffalo hump’ or cervicodorsal fat pad is present in about one-​half of patients. Gonadal dysfunction is very common, with menstrual irregu- larity in females and loss of libido in males, resulting from a sup- pressive effect of cortisol on gonadotropin secretion. Hirsutism is frequently found in female patients, as is acne, and typically reflects ACTH-​stimulated hyperandrogenism, but may also be seen in the context of an adrenal carcinoma cosecreting cortisol and androgens. Psychiatric abnormalities have been reported in all series of pa- tients with Cushing’s syndrome, regardless of cause. Depression and lethargy are among the most common problems, but poor con- centration, paranoia, and overt psychosis are also well recognized. Lowering of plasma cortisol by medical or surgical therapy usually results in a rapid improvement in the psychiatric state. Many patients with long-​standing Cushing’s syndrome have lost height because of osteoporotic vertebral collapse. Pathological frac- tures, either spontaneous or after minor trauma, are not uncommon. Rib fractures, by contrast with those of the vertebrae, are often pain- less. The radiographic appearance is typical, with exuberant callus formation at the site of the healing fracture. The plethoric appearance of the patient with Cushing’s syndrome results from thinning of the skin, not true polycythaemia. The typical red-​purple livid striae of the syndrome are found most frequently on the abdomen, but may also be present on the upper thighs and axilla. They are very common in younger patients, and less so in those over 50 years of age. Myopathy and bruising are two of the most discriminatory fea- tures of the syndrome. The myopathy involves the proximal muscles

13.5.1  Disorders of the adrenal cortex 2337 of the lower limbs and the shoulder girdle. Complaints of weakness, such as an inability to climb stairs or get up from a deep chair, are relatively uncommon, but observation of whether the patient can rise from a crouching position often reveals the problem. Bruising of the skin may be extensive and occurs with unknown or trivial trauma. Hypertension is another prominent feature. Even though epi- demiological data show a strong association between blood pressure and obesity, hypertension is much more common in patients with Cushing’s syndrome than in those with simple obesity due to several factors including action on angiotensin II, catecholamine sensitivity, and cortisol action at the mineralocorticoid receptor. Pigmentation is rare in Cushing’s disease, but common in ectopic ACTH syndrome. However, in some pituitary tumours there is ab- normal processing of the pro-​opiomelanocortin (POMC) precursor molecule, with resulting pigmentation. Infections are more common in patients with Cushing’s syn- drome than in unaffected individuals. In many instances these are asymptomatic, as the normal inflammatory response may be sup- pressed. Reactivation of tuberculosis has been reported. Fungal in- fection of the skin is frequently found. Glucose intolerance may be a predisposing factor, with overt diabetes being present in up to one-​ third of patients in some series. Ocular effects may include raised intraocular pressure, chemosis, and exophthalmos (present in up to one-​third of patients in Cushing’s original series). Cataracts, a well-​recognized complication of exogenous corticosteroid therapy, seem to be uncommon, except as a complication of diabetes. In patients with ectopic ACTH syndrome caused by small-​cell lung carcinoma, the clinical presentation more commonly resem- bles Addison’s disease than Cushing’s syndrome. The patients are commonly pigmented (associated with high ACTH concentrations) and have lost weight, but the association of these with hypokalaemic alkalosis and alterations in glucose homeostasis should alert the clinician to the diagnosis. Patients with more indolent causes, such as bronchial carcinoids, present with the more typical features of Cushing’s syndrome. Patients with Cushing’s syndrome have increased rates of venous thromboembolic disease. As with all cases of thrombo- embolic disease there are alterations in one or more of Virchow’s triad (endothelial dysfunction, hypercoagulability, and venous stasis). Patients with Cushing’s syndrome have abnormalities in Table 13.5.1.5  Adrenocortical causes of Cushing’s syndrome Type of lesion Frequency (percentage of cases of Cushing’s syndrome) Age group Condition (gene/​protein) Benign Common adenoma 10% All ages MEN 1 (menin) FAP (APC) MAS (GNAS) HLRCS (FH) CNC (PRKAR1A, 2p16) Others Primary macronodular adrenal hyperplasia (PMAH, nodules

10 mm diameter) <1% 40–​60 yr MEN 1 (menin) FAP (APC) MAS (GNAS) HLRCS (FH) (ARMC5) (MC2R) (PDE11A) Micronodular hyperplasias (nodules <10 mm diameter) <1% Isolated primary pigmented nodular adrenocortical disease (i-​PPNAD) Children and young adults (PRKAR1A) (PDE11A) (2p16) CNC-​associated PPNAD (c-​PPNAD) CNC (PRKAR1A, 2p16) Isolated micronodular adrenocortical disease (i-​MAD) (PDE11A) (PDE8B) Malignant Cancer (sporadic) 8% All ages (TP53) (Wnt/​β-​catenin) (INHA) Others Cancer (syndromic) Children and young adults Li-​Fraumeni (TP53, CHEK2) Beckwith–​Wiedemann syndrome (chromosome 11 abnormalities, IGF2, H19) Rubenstein–​Taybi syndrome (CREBBP, EP300) Brazil variant Children and young adults MEN 1, multiple endocrine neoplasia type 1; FAP, familial adenomatous polyposis (polyposis coli); MAS, McCune–​Albright syndrome; HLRCS, hereditary leiomyomatosis and renal cancer syndrome; FH, fumerate hydratase; AD, autosomal dominant; CNC, Carney complex; GPCR, G-​protein-​coupled receptors; LFS, Li-​Fraumeni syndrome; BWS, Beckwith–​ Wiedemann syndrome; RTS, Rubinstein-​Taybi syndrome; AD, autosomal dominant; PMAH is also known as ACTH-​independent macronodular adrenocortical hyperplasia (AIMAH); PPNAD, primary pigmented nodular adrenocortical disease.

SECTION 13  Endocrine disorders 2338 the coagulation cascade such that pro-​coagulant factors are in- creased (factors VIII, IX, and von Willebrand factor) and fibrino- lytic ability is decreased (elevation in plasminogen activator inhibitor 1). There may also be a rise in platelets, thromboxane B2, and fibrinogen. The result of the aforementioned abnormal- ities is a reduction of activated partial thromboplastin time and increased thrombin generation. Therefore, these patients are at high risk of thromboembolic disease and should be treated with prophylactic anticoagulation in keeping with guidance for high-​ risk patients. Special features Cyclical Cushing’s syndrome Of particular clinical interest has been a group of patients with cyc- lical Cushing’s syndrome, characterized by periods of excess cor- tisol production, followed by intervals of normal (or decreased) (a) (b) (c) (d) Fig. 13.5.1.4  Clinical features of a patient with Cushing syndrome before (a and b) and after (c and d) treatment. From Jolly E, Fry A, Chaudry A (eds) (2016). Training in medicine. By permission of Oxford University Press.

13.5.1  Disorders of the adrenal cortex 2339 cortisol production. The length of each cycle, and the intervening period between episodes, can vary markedly from days to months and even years. Some of these patients demonstrate a paradoxical rise in plasma ACTH and cortisol when treated with dexametha- sone. Most patients have been thought to have pituitary-​dependent disease, and in many instances, basophil adenomas have been re- moved, some with long-​term cure. However, cortisol secretion may show cyclicity in other causes of Cushing’s syndrome, notably ec- topic ACTH syndrome. Children All the aforementioned features occur in children, but growth arrest is almost invariable. The dissociation between height and weight on the growth chart is obvious. If the child is growing along the same centile lines, then the diagnosis of Cushing’s syndrome is highly unlikely. In addition to glucocorticoid-​induced growth arrest, an- drogen excess may result in precocious puberty. Pregnancy Pregnancy is rare in women with Cushing’s syndrome because of associated amenorrhoea resulting from androgen excess or hypercortisolism. However, several cases have been reported, 50% of which resulted from adrenal adenomas. A few cases of true pregnancy-​induced Cushing’s syndrome have been reported, with postpartum regression. In these cases, the aeti- ology is unknown. Establishing a diagnosis and cause can be dif- ficult; normal pregnancy is associated with a threefold increase in plasma cortisol caused by increased production rates and increases in cortisol-​binding globulin. Urinary free cortisol also rises, and dexamethasone does not suppress plasma cortisol to the same de- gree as in the non​pregnant state. However, salivary cortisol levels/​ profiles are potentially helpful (as they reflect unbound free cortisol levels). Untreated, the condition has high maternal and fetal mor- bidity and mortality. Adrenal and/​or pituitary adenomas should be excised (most frequently performed in the second trimester). Metyrapone has been effective in controlling the hypercortisolism in many cases, but a risk benefit discussion regarding possible fetal tox- icity is required (there is limited data available and hence it is not re- commended during pregnancy for the management of endogenous Cushing’s syndrome unless clearly necessary). Metyrapone is ex- creted in breast milk. Adrenocortical carcinomas In addition to features of glucocorticoid excess the patient may pre- sent with other problems relating to: (1), the tumour (e.g. abdom- inal pain from the primary tumour or secondary deposits), or (2), the secretion of other steroids such as androgens or mineralocor- ticoids. Thus, in addition to hirsutism, there may be other features of virilization in females, including clitoromegaly, breast atrophy, deepening of the voice, temporal recession, and severe acne. Men can present with gynaecomastia due to increased oestrogen syn- thesis. Adrenocortical carcinomas are discussed later in the chapter in more detail. Investigation There are two stages in the investigation of a patient with suspected Cushing’s syndrome: (1) Does the patient have Cushing’s syndrome? (2) If the answer to (1) is yes, what is the cause? Many investigators fail to make this distinction and ill-​advisedly use tests that are relevant to question (2) to try to answer question (1), this leads to difficulties when trying to appropriately interpret the results of investigations. Accurate diagnosis can be challenging as no single test confers 100% sensitivity and specificity. However, because of the poten- tial seriousness of untreated Cushing’s syndrome, highly sensitive tests are recommended to avoid missing the diagnosis. In all cases this brings with it a high prevalence of false positives, and the clin- ical pretest probability of the patient having Cushing’s syndrome, based on clinical symptoms and signs, remains of paramount importance. In particular, it is essential that appropriate radiological inves- tigations are not undertaken until Cushing’s syndrome has been confirmed biochemically (given the high rate of incidentalomas re- ported in pituitary and adrenal imaging). Diagnostic tests Four screening tests with high sensitivity can be used to confirm Cushing’s syndrome. Depending on the index of clinical suspicion these can be performed in isolation or combination. 1. Urine free cortisol (UFC; at least two/​three measurements) 2. Late-​night salivary cortisol (at least two measurements) Table 13.5.1.6  Prevalence of symptoms and signs in Cushing’s syndrome and discriminant index compared with prevalence of features in patients with simple obesity % Discriminant index Weight gain 91 Menstrual irregularity 84 1.6 Hirsutism 81 2.8 Psychiatric 62 Backache 43 Muscle weakness 29 8.0 Fractures 19 Loss of scalp hair 13 Obesity Truncal Generalized 97 46 55 1.6 0.8 Plethora 94 3.0 Moon face 88 Hypertension 74 4.4 Bruising 62 10.3 Red/​purple striae 56 2.5 Muscle weakness 56 Ankle oedema 50 Pigmentation 4 Other findings Hypertension Diabetes Overt Impaired GTT Osteoporosis Renal calculi 74 50 13 37 50 15 Data from Ross EJ, Linch DC (1982). Cushing’s syndrome—​killing disease: discriminatory value of signs and symptoms aiding early diagnosis. Lancet, 2, 646–​9.

SECTION 13  Endocrine disorders 2340 3. 1 mg overnight dexamethasone suppression test (DST) 4. 48-​hour low-​dose DST (LDDST) (2 mg/​day for 48 hours) Urinary free cortisol For many years, the diagnosis of Cushing’s syndrome was based on the measurement of urinary metabolites of cortisol (24-​h urinary 17-​ hydroxycorticosteroid or 17-​oxogenic steroid excretion, depending on the method used). However, the sensitivity and specificity of these methods is poor, and these assays have now been replaced with the much more sensitive measurement of urinary free cortisol excretion. Urinary free cortisol is an integrated measure of plasma-​ free cortisol. As cortisol secretion increases, the binding capacity of cortisol-​binding globulin is exceeded, resulting in a dispropor- tionate rise in urinary free cortisol. This is a useful screening test, but even so, it is accepted that urinary free cortisol may be normal in 7 to 10% of patients with Cushing’s syndrome. False positives may occur with high urine volumes, and certain medications may increase urinary free cortisol including carbamazepine, liquorice, carbenoxolone, fenofibrate (if measured by HPLC), and some syn- thetic glucocorticoids (if measured by immunoassays). False nega- tive results may occur in renal impairment when the creatinine clearance falls to less than 60 ml/​min, if the patient has cyclical dis- ease, or if the patient has mild Cushing’s syndrome. Because UFC levels in a patient with Cushing’s syndrome are variable at least two/​ three collections should be performed. Measurement of the cortisol:creatinine ratio in the first urine spe- cimen passed on waking obviates the need for a timed collection, and has been used by some as a sensitive screening test, particularly if multiple assessments are needed, for example if cyclical Cushing’s syndrome is suspected. However, this test has largely been replaced by midnight salivary cortisol for this indication. Late-​night plasma/​salivary cortisol In normal subjects, plasma cortisol concentrations are at their highest first thing in the morning and reach a nadir at around mid- night (with plasma cortisol <50 nmol/​litre at midnight effectively excluding Cushing’s syndrome). This circadian rhythm is lost in patients with Cushing’s syndrome, such that in most patients the 09.00 level of plasma cortisol is normal, but nocturnal levels are raised. Random morning levels of plasma cortisol are therefore of little value in making the diagnosis. In addition, various factors, such as the stress of venepuncture, intercurrent illness, and admis- sion to hospital, may result in normal subjects losing their circadian rhythm. It is therefore good practice not to measure plasma cor- tisol until the patient has been in hospital for 48 hours. For practical reasons midnight plasma cortisol is not routinely used as a first-​line screening test. By contrast, midnight salivary cortisol can be collected at home and offers greater accuracy. The most commonly accepted salivary cortisol cut-​off is less than 4 nmol/​litre at midnight. Using a range of cut-​offs, the midnight salivary cortisol test has 92–​100% sensi- tivity and 93–​100% specificity in diagnosing Cushing’s syndrome. Other screening and confirmatory tests may be required to evaluate false-​positive results. The performance and reliability of immuno- assays at these levels of cortisol may limit their interpretation and therefore liquid chromatography-tandem mass spectrometry (LC- MS/MS) is the preferred analytical method if available. Overnight dexamethasone suppression test In normal subjects, administration of a supraphysiological dose of a glucocorticoid results in suppression of ACTH and hence reduc- tion in cortisol secretion. In Cushing’s syndrome of whatever cause there is failure of this suppression when low doses of the synthetic glucocorticoid dexamethasone are given. The overnight test is often used as an outpatient screening test. Various doses of dexametha- sone have been used, usually given at 23:00 h, but most experience is with a dose of 1 mg. A plasma cortisol of less than 50 nmol/​litre (1.8µg/​dl) between 08.00 and 09.00 the following morning has a sensitivity of 95% and specificity of 80% in excluding Cushing’s syn- drome. Thus, the outpatient 1 mg dexamethasone suppression test has high sensitivity but low specificity, and further investigation is often required. Low-​dose dexamethasone suppression test (LDDST) The LDDST may require inpatient admission, although with clear instructions this can be performed reliably as an outpatient. Here, plasma cortisol is measured at 09.00 on day 0 and 48 h later, fol- lowing dexamethasone given at a dose of 0.5 mg every 6 h for 48 h; a result less than 50 nmol/​litre (1.8µg/​dl) is normal. This test is re- ported as having a sensitivity of 96% and a specificity of between 70 and 80%. There are some medications which may interfere with the dexa- methasone suppression test by either accelerating dexametha- sone metabolism or impairing dexamethasone metabolism via their interaction with CYP 3A4. Drugs which accelerate dexa- methasone metabolism include phenobarbital, phenytoin, carba- mazepine, primidone, rifampicin, rifapentine, ethosuximide, pioglitazone. Drugs which impair dexamethasone metabolism in- clude itraconazole, ritonavir, fluoxetine, diltiazem, cimetidine, and aprepitant/​fosaprepitant. Healthy women who receive oral oestrogen therapy will often fail the dexamethasone suppression test (50% false-​positive rate) due to elevated levels of cortisol-​binding globulin. Therefore, oral oes- trogens should be discontinued for six weeks prior to assessment with DST. Other tests Occasionally, other (second line) screening tests (e.g. the dexa- methasone suppressed corticotrophin-​releasing hormone (CRH) test or the desmopressin stimulation test) may be used to try and discriminate true Cushing’s syndrome from unaffected subjects or pseudo-​Cushing’s states—​however, these tests are less well validated than their more commonly employed counterparts and clinicians may be less familiar with interpretation, which can therefore add to diagnostic uncertainty. Differential diagnostic tests Once the biochemical diagnosis has been made, other investigations are required to determine the cause of the Cushing’s syndrome. Plasma ACTH at 09.00 h This will differentiate ACTH-​dependent from ACTH-​independent causes. ACTH is either within the normal reference range (50% of cases) or elevated in patients with Cushing’s disease. ACTH levels in ectopic ACTH syndrome are typically high, but overlap the values

13.5.1  Disorders of the adrenal cortex 2341 seen in Cushing’s disease in 30% of cases and cannot therefore be used to differentiate these two conditions (Fig. 13.5.1.5). The meas- urement of ACTH precursors (pro-​ACTH, POMC) is not routinely available, but may be more useful in detecting an ectopic source of ACTH. In patients with autonomous adrenal tumours, plasma ACTH is invariably undetectable. This can also occur with degradation of ACTH; consequently, non​haemolysed blood samples should be taken on ice and immediately separated. Diagnosis is a problem in those patients whose plasma ACTH levels are in the low normal range or intermittently detectable. This may occur in macronodular hyperplasia. The danger is that in some patients with ACTH-​dependent disease the asymmetry of the nodular hyperplasia may lead to a diagnosis of adrenal adenoma, the plasma ACTH is ignored, and an inappropriate adrenalectomy is performed. Conversely, in some patients with this syndrome an au- tonomous adrenal tumour develops and, despite detectable ACTH, unilateral adrenalectomy is required. Plasma potassium Hypokalaemic alkalosis is a marker of severity of hypercortisolaemia as it reflects the overwhelming of the protective effect of 11 β-​HSD2 at the level of the mineralocorticoid receptor, resulting in cortisol-​ induced mineralocorticoid hypertension (see ‘Apparent mineralo- corticoid excess syndrome’ later). Hypokalaemic alkalosis is present in many patients with ectopic ACTH syndrome, but in fewer than 10% of patients with Cushing’s disease. In addition, these patients have higher levels of the ACTH-​dependent mineralocorticoid deoxycorticosterone. High-​dose dexamethasone suppression test The rationale for this test is that in Cushing’s disease there is nega- tive feedback control of ACTH but set at a higher level than normal. Thus, in Cushing’s disease, cortisol levels are not suppressed with a low dose of dexamethasone, but are suppressed with a higher dose. The original test introduced by Liddle was based on giving dexa- methasone at a dose of 2 mg every 6 h for 48 h and measuring urinary 17-​oxogenic steroids. Suppression was defined as a greater than 50% fall in 24-​h urinary 17-​oxogenic steroids. In the modern test, plasma cortisol is measured at 0 and 48 h or, less commonly, plasma cor- tisol is measured at 08.00 (basal sample), 8 mg dexamethasone is given orally at 23.00 on the same day, and plasma cortisol is meas- ured again at 08.00 on the following morning. In both these tests, greater than 50% suppression of plasma cortisol in comparison with the basal sample has been used to define a positive response. In Cushing’s disease about 90% of patients have a positive 48-​h test, compared with 10% with ectopic ACTH syndrome. With overnight 8 mg DST testing, 89% sensitivity and 100% specificity has been re- ported for Cushing’s disease. Corticotrophin-​releasing factor (CRF) test CRF is a peptide of 41 amino acids, identified by Vale in 1981 from ovine hypothalami. The ovine sequence differs by seven amino acid residues from that of the human peptide, but despite this stimulates the release of ACTH in humans. The test involves the intravenous injection of either ovine or human CRF at a dose of 1 µg/​kg body weight (or a single dose of 100 µg). The test can be performed in the morning or afternoon, and—after basal sampling—blood samples for ACTH and cortisol are taken every 15 min for 1 to 2 hours after administering CRF. There are differences in the performance of the CRF test depending on whether ovine or recombinant human CRF are used and this needs to be taken into account when interpreting the test results. In normal subjects CRF elicits a rise in ACTH and cortisol, and this response is exaggerated in Cushing’s disease. It is typically absent in ectopic ACTH syndrome and patients with adrenal tumours. In distinguishing pituitary-​dependent Cushing’s disease from ectopic ACTH syndrome, the response of ACTH to CRF has a specificity of 90%, and with cortisol as the endpoint, 95%, using as endpoints an ACTH increase of 100% over basal or a cortisol rise of 50%. Inferior petrosal sinus sampling/​selective venous catheterization This is the most robust test for distinguishing Cushing’s disease from ectopic ACTH syndrome, but also the most costly and tech- nically demanding. As blood from each half of the pituitary drains Post- adrenal- ectomy Untreated Adrenal tumour Ectopic ACTH Cushing’s disease 110000 4000–12000 2000–4000 1000–2000 900 700 500 300 250 200 150 100 50 0 37 49 9 42 Normal range 08.00–10.00 Plasma immunoreactive ACTH (ng/litre) Fig. 13.5.1.5  Immunoreactive N-​terminal ACTH levels in plasma samples taken between 08.00 and 10.00 in normal subjects (hatched area), and patients with Cushing’s disease (either untreated or postadrenalectomy), adrenal tumours, or ectopic ACTH syndrome. Courtesy of Professor LH Rees.

SECTION 13  Endocrine disorders 2342 into the ipsilateral inferior petrosal sinus (in most cases), catheter- ization of both sinuses with simultaneous sampling of venous blood can distinguish a pituitary from an ectopic source, and aid in the lateralization of a pituitary microadenoma (Fig. 13.5.1.6). The re- sults for lateralization are not as robust as the results for determining whether the ACTH comes from a pituitary versus ectopic sources, as many patients have anatomical variations in drainage. However, because of the problem of intermittent ACTH secretion, it is useful to make measurements before and at intervals (e.g. 2, 5, and 10 min) after intravenous injection of 100 µg of CRF. If the pituitary-​to-​peripheral ratio of ACTH is greater than 2 the patient has Cushing’s disease, with a sensitivity and specificity of up to 100%. In Cushing’s disease an ipsilateral:contralateral ACTH more than 1.4 may be helpful in localizing the adenoma to an indi- vidual side, however this is far from 100% accurate and an experi- enced surgeon on review may find an adenoma on the opposite side. If the pituitary-​to-​peripheral ratio of ACTH is less than 1.5 the patient has ectopic Cushing’s syndrome. Rarely, selective catheter- ization of vascular beds may be required to identify the source of ectopic ACTH secretion (e.g. from a small pulmonary or thymic neuroendocrine tumour). Tumour markers Many tumours responsible for ectopic ACTH syndrome also produce peptide hormones other than ACTH or its precursors. Calcitonin, chromogranin A, and gut hormones such as gastrin and vasoactive intestinal polypeptide should be measured for assess- ment of secretion from neuroendocrine tumours. Imaging High-​resolution contrast-​enhanced imaging of thin sections of the pituitary by MRI and adrenals by either CT or MRI has revolution- ized the investigation of Cushing’s syndrome. However, if mistakes are to be avoided it is essential that full biochemical assessment takes place prior to imaging and the results of any imaging technique must always be interpreted in the light of the biochemical results. In Cushing’s disease imaging, the adrenals often reveal asym- metrical nodular hyperplasia which may lead to a false diagnosis of adrenal adenoma (Fig. 13.5.1.7) and inappropriate adrenal resec- tion. Similarly, in patients with Cushing’s syndrome secondary to an adrenal lesion there may be a pituitary incidentaloma on MRI. Thus, assessment of biochemistry and in particular ACTH is key to directing and interpreting imaging. Pituitary MRI is the investigation of choice if the biochemical tests suggest Cushing’s disease, and has a sensitivity of 70% and specificity of 87% (Fig. 13.5.1.8). Approximately 90% of ACTH-​secreting pitu- itary tumours are microadenomas (i.e. less than 10 mm in diameter). The classical features of a pituitary microadenoma are a hypodense lesion after contrast and a convex upper surface of the pituitary gland (Fig. 13.5.1.8). With such small tumours it is not surprising that the sensitivity of CT scanning is relatively low (20–​60%), with a similar specificity, and therefore CT scanning of the pituitary should not be used unless MRI is contraindicated. By contrast, CT scanning is the investigation of choice for adrenal imaging and affords good spatial resolution (Fig. 13.5.1.9), with MRI serving as an alternative. Once again, adrenal incidentalomas are present in up to 5% of normal subjects (this number increases with age), and thus adrenal imaging should not be performed unless biochemical investigation suggests a primary adrenal cause. Adrenal carcinomas are large and often associated with metastatic spread at presentation (Fig. 13.5.1.10). Cavernous sinus Inferior petrosal sinus Pituitary veins Jugular vein Fig. 13.5.1.6  Positions of bilateral catheters in inferior petrosal sinus sampling. Fig. 13.5.1.7  CT scan of patient with Cushing’s disease with asymmetrical nodular hyperplasia (right > left).

13.5.1  Disorders of the adrenal cortex 2343 In patients with occult ectopic ACTH syndrome, high-​definition MRI/​CT scanning of the neck, thorax, and abdomen/​pelvis, with im- ages every 0.5 cm, may be required to detect small ACTH-​secreting carcinoid (NET) tumours. Functional imaging (e.g. with 18F-​fluorodeoxyglucose (18F-​FDG) or 68Gallium DOTATATE (68Ga-​DOTATATE) positron emission tomography (PET)/​CT may aid localization of small neuroendo- crine tumours or confirm sites of metastatic disease. 11C-​methionine PET/​CT coregistered with MRI has also been proposed as a method for localizing small corticotroph adenomas not readily visualized on MRI. Management Studies carried out before the introduction of effective therapy suggested that 50% of patients with untreated Cushing’s syn- drome died within 5 years, causing some physicians to label this the ‘killing disease’. Even with modern management, an increased prevalence of cardiovascular risk factors persists for many years after an apparent remission. Close follow-​up of all patients is recommended. Adrenal causes Adrenal adenomas should be removed by unilateral adrenalectomy, which has a 100% cure rate. Laparoscopic adrenalectomy is now the surgical treatment of choice for unilateral adenomas as it reduces surgical morbidity and postoperative hospital stay compared with open approaches. After surgery it may take many months or even years for the contralateral suppressed adrenal to recover (due to chronic lack of stimulation as a result of low ACTH levels). Patients who undergo a unilateral adrenalectomy for Cushing’s syndrome secondary to an adrenal adenoma will require hydrocortisone re- placement peri-​ and postoperatively. To assess if the contralateral adrenal gland has recovered from suppression intermittent meas- urement of the 08.00 level of plasma cortisol after having omitted therapy the evening before and morning of test is advised. When the morning plasma cortisol is above 180 nmol/​litre a stimulation test such as the Synacthen test (250 mcg), may then demonstrate whether the contralateral adrenal gland has recovered from its suppression. Adrenocortical carcinomas have a very poor prognosis. It is usual practice to try to remove the primary tumour, even though metas- tases may be present, so as to enhance the response to the adrenolytic agent mitotane (see ‘Medical treatment of Cushing’s syndrome’ later). Radiotherapy to the tumour bed and to some metastases, such as those in the spine, may be of limited value (see the ‘Adrenocortical carcinomas’ section). Pituitary-​dependent Cushing’s disease In most cases, the treatment of Cushing’s disease involves transsphenoidal surgery. Before the selective removal of a pitu- itary microadenoma became routine, the treatment of choice was bilateral adrenalectomy. This had an appreciable mortality, even in the best centres (c.4%), as well as postoperative morbidity. The main risk was the subsequent development of Nelson’s syn- drome (postadrenalectomy hyperpigmentation with locally ag- gressive pituitary tumour) (Fig. 13.5.1.11). To avoid this, pituitary Fig. 13.5.1.8  MRI scan of pituitary demonstrating the typical appearance of a pituitary microadenoma. A hypodense lesion is seen in the centre of the gland. Following a biochemical diagnosis of Cushing’s disease, this patient was cured following transsphenoidal hypophysectomy. Fig. 13.5.1.9  Typical solitary left-​sided non​secretory adrenal adenoma with low HU on
non​contrast adrenal CT scanning.

SECTION 13  Endocrine disorders 2344 irradiation is often carried out in patients who have undergone bi- lateral adrenalectomy. These patients required lifelong replacement therapy with hydrocortisone and fludrocortisone. Today, bilateral adrenalectomy is reserved for patients with Cushing’s disease in whom no pituitary tumour can be found, or when pituitary surgery has failed to control hormone hypersecretion, or the condition has recurred or is life-​threatening. After selective removal of a microadenoma, the surrounding corticotrophs are normally suppressed (Fig. 13.5.1.12). In these cases, plasma cortisol concentrations are also suppressed postoperatively, and glucocorticoid replacement therapy is required, but gradual recovery of the HPA axis can be anticipated (Figs 13.5.1.12(c) and 13.5.1.13), particularly in patients with normal pituitary function as it relates to other endocrine axes. There is no consensus on the criteria for defining remission after resection of an ACTH producing tumour. However, remission is generally considered likely if morning serum/​plasma cortisol is less than 138 nmol/​litre (5µg/​dl) and/​or urinary free cortisol less than 28–​56 nmol/​24 hours (<10–​20 µg/​24 hours) within 7 days of surgery, but a number of alternative cut-​offs have been suggested. Patients who are eucortisolaemic following surgery may still have residual tumours, even though cortisol secretion may have fallen to normal or subnormal values. These patients are at high risk of recurrence. An important caveat to this relates to patients with mild or cyc- lical Cushing’s disease and those rendered eucortisolaemic prior to surgery (following treatment with medical therapy), who may not have suppressed corticotropes in the pituitary. In this situation their 24-​hour urinary free cortisol assessments and morning cortisol may be non​suppressed, but they may be in true remission from their Cushing’s disease. In such patients the return of a normal circadian rhythm (as demonstrated by the finding of low midnight salivary cortisol levels) is helpful in assessing disease control. In the past, pituitary irradiation was often used in the treat- ment of Cushing’s disease. However, improvements in pituitary surgery have resulted in far fewer patients receiving radiotherapy. Radiotherapy is not recommended as a primary treatment, but is reserved for patients not responding to pituitary surgery (e.g. when bilateral adrenalectomy has been performed), or in those with es- tablished Nelson’s syndrome. Conventional pituitary radiotherapy has been associated with increased risk of stroke in patients with non​functioning adenomas and acromegaly. Conventional radio- therapy has also been associated with development of hypopituit- arism and rarely secondary intracranial malignancies and damage to the optic apparatus. There is increasing data relating to radiosurgery techniques in the management of Cushing’s disease, which appear to show favourable results when compared with fractionated conven- tional radiotherapy. Radiosurgery may offer more rapid biochem- ical response and less risk of radiation damage to surrounding brain structures, but careful case selection is paramount, and more data is required in this area. Ectopic ACTH syndrome Treatment of ectopic ACTH syndrome depends on the cause. If the tumour can be found and has not spread, then its removal can lead to cure (e.g. bronchial carcinoid tumours, or thymomas). However, the prognosis for small-​cell lung cancer associated with ectopic ACTH syndrome is poor. The cortisol excess and associated hypo- kalaemic alkalosis and diabetes mellitus can be ameliorated by med- ical therapy (see later). Treatment of the small-​cell tumour itself will also, at least initially, produce improvement (see Chapter 18.19.1). Sometimes, if the ectopic source of ACTH cannot be found, it may be necessary to perform bilateral adrenalectomy and then follow the patient carefully clinically and with directed imaging (sometimes for several years) to find the primary tumour. Medical treatment of Cushing’s syndrome Several drugs have been used in the treatment of Cushing’s syndrome. Most commonly, metyrapone or ketoconazole has been given, often to lower cortisol concentrations before definitive therapy, or while awaiting benefit from pituitary irradiation. The daily dose has to be determined by measuring either plasma or urinary free cortisol to guide dose titration. Metyrapone Metyrapone inhibits 11-​β hydroxylase which catalyses the conver- sion of 11–​deoxycortisol to cortisol. Metyrapone is usually given in doses ranging from 250 mg twice daily to 1.5 g every 6 h (due to its short half-​life of 2 hours). Nausea is a common side effect and can be alleviated (if not caused by adrenal insufficiency) by giving the drug with milk. Adverse effects of metyrapone include gastrointestinal disturb- ances (need to ensure not related to unrecognized hypoadrenalism), hirsutism, acne (due to stimulation of androgenic precursors) and hypertension, hypokalaemia, and oedema (due to accumulation of mineralocorticoid precursors). Importantly when assessing re- sponse of cortisol hypersecretion to metyrapone it is best to use a Fig. 13.5.1.10  CT scan of a patient with rapidly progressing Cushing’s syndrome and virilization as a result of a left-​sided adrenal carcinoma. An irregular left adrenal mass is shown.

13.5.1  Disorders of the adrenal cortex 2345 LC-​MS/​MS assay rather than an immunoassay as significant levels of 11-​deoxycortisol can accumulate while on metyrapone, which can cross react with immunoassays for cortisol. In a recent study there was a significant difference in cortisol concentrations reported in the same sample between the two methods. The difference between LC-​ MS/​MS versus immunoassay in the metyrapone therapy group posi- tively correlated with metyrapone dose and serum 11-​deoxycortisol concentrations. The immunoassay read higher cortisol concentra- tions than LC-​MS/​MS due to interference from 11 deoxycortisol—​if not recognized, this may lead to an increase in dose of metyrapone with subsequent unrecognized adrenal insufficiency. A recent large study of metyrapone use in clinical practice showed higher doses were required to control patients with adrenocortical cancer, Cushing’s disease, and ectopic ACTH syndromes than be- nign adrenal nodules. Several approaches may be used to assess the (a) (b) (c) Fig. 13.5.1.11  A young woman with Cushing’s disease, photographed initially alongside her identical twin sister (a). In this case treatment with bilateral adrenalectomy was undertaken and several years later the patient re-​presented with Nelson’s syndrome and right third cranial nerve palsy (b) following cavernous sinus infiltration from a locally invasive corticotropinoma (c). (a) +++ Secretion above normal (c) (b)

−ve Secretion suppressed ND not detectable ACTH secreting tumour ACTH Cortisol ND ACTH−ve ACTH+ Cortisol+ ACTH−ve ACTH−ve ACTH+++ Cortisol+++ Fig. 13.5.1.12  Selective removal of a microadenoma and its effect on the hypothalamic–​pituitary–​adrenal axis. Because the surrounding normal pituitary corticotrophs are suppressed in a patient with an ACTH-​ secreting pituitary adenoma (a), successful removal of the tumour results in ACTH and hence adrenocortical deficiency with an undetectable (<50 nmol/​litre) level of plasma cortisol (b). With time the HPA axis can recover (c). Courtesy of Professor P Trainer. I.H.T. Trans-sphenoidal microadenomectomy 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 5 10 15 20 25 Weeks 0900h Plasma cortisol (μmol/litre) I.H.T. Insulin hypoglycaemia test Fig. 13.5.1.13  Gradual recovery of function of the hypothalamic–​ pituitary–​adrenal axis after removal of a pituitary ACTH-​secreting microadenoma. The insulin hypoglycaemia test eventually demonstrated the return of a normal stress response.

SECTION 13  Endocrine disorders 2346 degree of control in patients receiving metyrapone, and include cor- tisol day profiles and measurement of 24-​hour urinary free cortisol. A second 11β-​hydroxylase inhibitor (LCI699) is currently in development. Ketoconazole Ketoconazole (an imidazole previously used as an antifungal agent) blocks a variety of steroidogenic cytochrome P450-​dependent en- zymes (in both the adrenal gland and gonads), and thus lowers plasma cortisol levels. Its main effect is via inhibiting side-​chain cleavage, 17,20-​lyase, and 11-​β hydroxylase enzymes. For effective control of Cushing’s syndrome, 400 to 800 mg ketoconazole daily is typically required, but liver function tests must be monitored closely since hepatic failure is a potentially serious complication. Normalization of cortisol hypersecretion is seen in between 25 and 93% of cases depending on the study reviewed. Abnormal liver function tests occur in 10 to 15% of patients and a severe idiosyncratic hepatic reaction in 1 in 15 000 exposed individuals. The Food and Drug Administration (FDA) issued a black box warning and the European Medicines Agency has restricted access to the agent to physicians specialized in treating Cushing’s syndrome. Levoketoconazole (the pure 2 S,4R enantiomer of ketoconazole) is currently undergoing phase 3 studies, and may show a more favourable efficacy, safety, and tolerability profile. Mitotane Mitotane is an adrenolytic drug that is taken up by both normal and malignant adrenal tissue, causing adrenal atrophy and necrosis. Because of its toxicity, it has been used mainly in the management of adrenal carcinoma. Doses of up to 8 g/​day are required to control glucocorticoid excess. The drug will also produce mineralocorticoid deficiency, and both glucocorticoid and mineralocorticoid replacement therapy may be required. Importantly, mitotane increases the levels of cortisol-​ binding globulin within serum and also increases the metabolism of cortisol in the liver and therefore higher doses of glucocorticoid re- placement (e.g. hydrocortisone 20 mg thrice daily) are required to avoid hypoadrenalism. Mitotane can cause several biochemical and endo- crine abnormalities, including thyroid dysfunction (a mixed picture of primary and secondary thyroid dysfunction) that may require replace- ment. Abnormalities in liver function, in particular raised γ-glutamyl transferase and hyperlipidaemia, are frequently seen. Clinical side effects are common and include fatigue, skin rashes, and gastrointestinal disturbance. Levels of mitotane need to be closely monitored as the drug has a narrow therapeutic window in the treatment of adrenocortical carcinoma. Mitotane toxicity is associated predominantly with gastrointestinal and neurological symptoms, which are reversible on discontinuation of the drug. Mitotane doses at levels below the therapeutic window may be bene- ficial in controlling hypercortisolaemia, but will not have an impact on growth of adrenocortical cancers. Glucocorticoid receptor antagonist (mifepristone) Mifepristone works by blocking the glucocorticoid receptor, there- fore ACTH or cortisol estimation cannot be used to assess disease control, indeed ACTH levels increased twofold in 31/​44 patients in one study due to the loss of negative feedback. Assessment of disease control in patients on mifepristone include clinical measures such as improvement in hypertension, hyperglycaemia, weight, quality of life, clinical appearance, and symptoms. As it is difficult to as- sess efficacy, it is generally recommended to start at a low dose of 300 mg per day and titrate up as clinically indicated (max dose of 1200 mg/​day). Adverse effects include those associated with adrenal insufficiency (fatigue, nausea, vomiting, arthralgias and headache), antiprogestin (endometrial thickening) and mineralocorticoid ex- cess (hypertension, oedema, hypokalaemia). Given the blockade of the glucocorticoid receptor, treatment of adrenal crisis can be chal- lenging; mifepristone should be held, haemodynamic support given, and one study reported the use of high-​dose dexamethasone. Etomidate Etomidate (an anaesthetic agent) can be used in patients with severe Cushing’s syndrome who cannot take oral medications or are critically unwell due to their Cushing’s syndrome. It is an imidazole derivative (similar to ketoconazole) which can be given at subhypnotic doses to rapidly decrease cortisol levels by inhibiting side-​chain cleavage, 17,20-​ lyase and 11-​β hydroxylase enzymes. Etomidate should only be given under careful monitoring conditions such as is possible in an intensive care unit because of its potential hypnotic properties. A loading dose of 3–​5 mg can be followed by a continuous infusion of 0.03–​0.1 mg/​ kg/​hr (this may need to be adjusted in patients with renal impairment as etomidate is renally excreted). During treatment, cortisol should be measured 4–​6 hourly and the etomidate infusion titrated to achieve a serum cortisol level between 280 and 560 nmol/​litre (10–​20 µg/​dl). Pasireotide Pasireotide is a second-​generation somatostatin receptor ligand (SRL) with a different receptor subtype affinity to octreotide or lanreotide. Pasireotide has a much stronger affinity to SSTR1 and SSTR5 than SSTR2 (unlike octreotide and lanreotide). Corticotroph tumours have a high expression of SSTR5. A phase 3 trial of twice daily subcuta- neous pasireotide in patients with Cushing’s disease in whom surgery had failed (or who were not suitable for surgery) reported that 20% of treated patients had a normal 24-​hour urinary free cortisol and 44% had a reduction in tumour size. Adverse effects were similar to those seen in patients receiving first generation SRL’s, with the exception of hyperglycaemia which was significantly more frequent in patients treated with pasireotide than is seen in patients treated with octreotide or lanreotide, hence close monitoring of glycaemic control is required in patients receiving pasireotide. Studies investigating the use of a long acting pasireotide LAR in Cushing’s disease are underway. Cabergoline Cabergoline is a long acting agonist of the D2 dopamine receptor (which is frequently expressed on corticotroph adenomas). There is limited data regarding its efficacy in Cushing’s disease, with reports of between 30 and 40% of patients responding with lowering of cor- tisol levels; however, in some of these patients the initial response did not persist despite high doses of cabergoline therapy. Adverse effects of cabergoline (less frequent than in patients receiving older dopamine agonists such as bromocriptine) include nausea, dizzi- ness, constipation, and there has recently been a concern regarding valvular fibrosis in patients who receive a high cumulative dose of cabergoline for Parkinson’s disease (although the data in patients re- ceiving cabergoline at doses used for the treatment for prolactinoma are reassuring). Cabergoline is also under investigation in combin- ation with some of the other agents discussed.

13.5.1  Disorders of the adrenal cortex 2347 Aminoglutethimide Aminoglutethimide is a more toxic drug that in high doses blocks the initial steps in the biosynthetic pathway, and thus affects the secretion of steroids other than cortisol. In doses of 1.5 to 3 g daily (starting with 250 mg every 8 h) it commonly produces nausea, marked lethargy, and a skin rash. Trilostane Trilostane, a 3β-​hydroxysteroid dehydrogenase inhibitor, is in- effective in Cushing’s disease since the block in steroidogenesis is overcome by the rise in ACTH. However, it can be effective in patients with adrenal adenomas. Targeted therapies in ectopic ACTH production Neuroendocrine tumours which secrete ectopic ACTH may ex- press SSTR2 and D2 receptors and may therefore be responsive to SRL (octreotide and lanreotide) therapy or cabergoline. The use of these agents may decrease ACTH secretion, but typically has little effect on tumour growth rates. Glucocorticoid deficiency: Primary and secondary hypoadrenalism Primary hypoadrenalism refers to glucocorticoid deficiency occurring in the setting of adrenal disease, whereas secondary hypoadrenalism arises from a deficiency of ACTH (due to hypothalamic/​pituitary dysfunction), the major trophic hor- mone controlling cortisol secretion. The principal distinc- tion between these two conditions is that mineralocorticoid deficiency invariably accompanies primary hypoadrenalism, but this does not occur in secondary hypoadrenalism because only ACTH is deficient; the renin–​angiotensin–​aldosterone axis is intact. Primary hypoadrenalism Congenital adrenal hyperplasia Various inherited enzyme defects have been identified in the syn- thetic pathway of adrenocortical hormones, which cause a spectrum of glucocorticoid and/​or mineralocorticoid deficiency. Adrenal androgens may be increased or decreased, depending upon the underlying enzyme block. This group of conditions is addressed in Chapter 13.5.2. Addison’s disease Thomas Addison described this condition in his classic monograph published in 1855. Addison worked with Bateman, a dermatolo- gist who produced one of the first classifications of skin disease. It seems likely that this stimulated Addison’s interest in the skin pigmentation that is so characteristic of this disease. Addison’s disease is a rare condition, with an estimated incidence in the developed world of 0.8 cases per 100 000 popu- lation. The causes of primary adrenal insufficiency are listed in Table 13.5.1.7. Table 13.5.1.7  Aetiology of adrenocortical insufficiency Primary adrenal insufficiency Secondary adrenal insufficiency Addison’s disease Tuberculosis Autoimmune: Sporadic Polyglandular deficiency type I (Addison’s disease, chronic mucocutaneous candidiasis hypoparathyroidism, dental enamel hypoplasia, alopecia, primary gonadal failure) Polyglandular deficiency type II (Schmidt’s syndrome) (Addison’s disease, primary hypothyroidism, primary hypogonadism, insulin-​dependent diabetes, pernicious anaemia, vitiligo) Bilateral adrenalectomy Metastatic tumour Lymphoma Amyloid Haemochromatosis Intra-​adrenal haemorrhage (Waterhouse–​Friderichsen syndrome) following meningococcal septicaemia Adrenal infarction or infection other than tuberculosis (especially AIDS) also Ebola and other haemorrhagic fevers Adrenoleukodystrophies Congenital adrenal hypoplasia (DAX-​1 mutations) Hereditary adrenocortical unresponsiveness to ACTH Type 1: ACTH receptor, melanocortin 2 receptor gene MC2R Type 2: MRAP Familial glucocorticoid deficiency (MCM4, NNT, TXNRD2) Triple A (Allgrove’s) syndrome, achalasia, Addison’s disease, alacrima, AAAS gene mutation Drug-​induced adrenal enzyme inhibitors: mitotane, ketoconazole, metyrapone, etomidate, aminoglutethimide, drugs that may accelerate cortisol metabolism and induce adrenal insufficiency CTLA-​4 inhibitors may enhance autoimmunity and cause PAI Other metabolic disorders Mitochondrial disease (rare) Wolman’s disease Adrenal hypoplasia Congenita—​X-​linked NROB1, Xp21 deletion (with Duchenne’s muscular deficiency), SF-​1 mutations (XY sex reversal), IMAGe syndrome Exogenous glucocorticoid therapy Hypopituitarism: Selective removal of ACTH-​secreting pituitary adenoma Pituitary tumours and pituitary surgery, craniopharyngiomas Pituitary apoplexy Granulomatous disease (tuberculosis, sarcoid, eosinophilic granuloma) Secondary tumour deposits (breast, bronchus) Postpartum pituitary infarction (Sheehan’s syndrome) Pituitary irradiation (effect usually delayed for several years) Isolated ACTH deficiency

SECTION 13  Endocrine disorders 2348 Infectious causes Worldwide, infectious diseases are the most common cause of pri- mary adrenal insufficiency. Leading causes include tuberculosis, fungal infections (histoplasmosis, cryptococcosis), and cytomegalo- virus. Adrenal failure may occur in AIDS and has been reported in haemorrhagic fevers such as Ebola. In tuberculous Addison’s disease the adrenals are initially enlarged, with extensive epithelioid granu- lomas and caseation. Chronic atrophy can occur, and calcification eventually ensues in most cases (Fig. 13.5.1.14): both the cortex and the medulla are affected. Autoimmune causes In the Western world, autoimmune adrenalitis accounts for over 70% of all cases of Addison’s disease. Pathologically, the adrenal glands are atrophic, with loss of most of the cortical cells, but the adrenal medulla is usually intact. However, catecholamine syn- thesis may be impaired due the need for intra-​adrenal cortisol to regulate catecholamine synthesis (norepinephrine to epinephrine by phenylethanolamine N-​methyltransferase). Adrenal autoanti- bodies can be detected in up to 75% of newly diagnosed cases and have helped elucidate the cause of the disease. Fifty per cent (50%) of patients with Addison’s disease have an associated autoimmune disease, and these polyglandular autoimmune syndromes have been classified into two distinct variants: Type I is inherited as an autosomal recessive condition and com- prises Addison’s disease, chronic mucocutaneous candidiasis, and hypoparathyroidism. The condition is rare and usually presents in childhood with either candidiasis or hypoparathyroidism. Other autoimmune conditions, such as pernicious anaemia, thyroid dis- ease, chronic active hepatitis, and gonadal failure may occur, but are rare. Autoantibodies to the cholesterol side-​chain cleavage enzyme and 17α-​hydroxylase may be detected, but not to 21-​hydroxylase. The condition occurs because of mutations in the autoimmune regu- lator gene, AIRE. Type II polyglandular autoimmune syndrome is more common, comprising Addison’s disease, autoimmune thyroid disease, dia- betes mellitus, and hypogonadism. The condition has an inherited basis, with linkage to the HLA major histocompatibility complex, notably HLA DR3 and DR4. Autoantibodies to 21-​hydroxylase are usually present and are predictive for the development of adrenal destruction. Other causes With the exception of tuberculosis and autoimmune adrenal failure, other causes of Addison’s disease are rare (Table 13.5.1.7). Adrenal metastases (most commonly from primary lung and breast tu- mours) are often found at post-​mortem examinations, but adrenal insufficiency from these is uncommon (unless associated with ad- renal haemorrhage). Necrosis of the adrenals following intra-​adrenal haemorrhage should be considered in any severely ill patient, and may result from infection, trauma, or hypercoagulability. Intra-​adrenal bleeding may be found in severe septicaemia of any cause, particularly in children. When this is caused by meningococci, the association with adrenal insufficiency is known as Waterhouse–​Friderichsen syndrome. Adrenal infiltration/​ replacement leading to glandular failure may also occur with amyloidosis and haemochromatosis. Congenital adrenal hypoplasia is an X-​linked disorder com- prising congenital adrenal insufficiency and hypogonadotropic hypogonadism. The condition is caused by mutations in the DAX1 (NR0B1) gene, a member of the nuclear receptor family that is ex- pressed in the adrenal cortex, gonads, and hypothalamus. X-​linked adrenoleukodystrophy causes adrenal insufficiency in association with demyelination within the nervous system, and re- sults from a failure of β-​oxidation of fatty acids within peroxisomes. Increased accumulation of very long-​chain fatty acids (VLCFA) oc- curs in many tissues, and serum measurement can be used diagnos- tically. Male patients have the fully expressed condition, and female carriers are increasingly recognized to be affected by it (albeit with different timings of symptom development). X-​linked adrenoleukodystrophy (X-​ALD), which accounts for about 10% of cases of adrenocortical failure in boys and men, is clin- ically characterized by two main phenotypes: adrenomyeloneuropathy (AMN) and the cerebral demyelinating form of X-​ALD (cerebral ALD). AMN and cerebral ALD occur frequently within the same family and there is no correlation between X-​ALD phenotype and mutations in the ABCD1 gene. Cerebral ALD presents usually with a rapidly progressive inflammatory demyelination within the brain re- sulting in severe cognitive and neurologic disability, a vegetative state within two to five years of clinical symptom onset, and death there- after. This phenotype is most common during childhood and adoles- cence, but up to 20% of adult males initially presenting with AMN will develop cerebral ALD later in life. The pathology of AMN is fun- damentally different from that of cerebral ALD and is characterized predominantly by a non​inflammatory distal axonopathy involving mostly the long tracts of the spinal cord that results in a progressive spastic paraplegia. Adrenal insufficiency is usually present, but does not appear to correlate with the neurological deficit. Both the childhood and adult conditions result from muta- tions in the ABCD1 gene on chromosome Xq28, which encodes an ATP-​binding cassette peroxisomal membrane protein involved in the import of VLCFA into the peroxisome. Monounsaturated fatty acids that block the synthesis of saturated VLCFA have been used for treatment. A combination of erucic acid and oleic acid (Lorenzo’s oil) has led to normal levels of VLCFA, but this has not altered the rate of neurological deterioration. Bone marrow trans- plantation appears to be more effective if undertaken in the early stages of the disease. Familial glucocorticoid deficiency (FGD) is a rare autosomal re- cessive cause of hypoadrenalism that usually presents in childhood. Fig. 13.5.1.14  Plain radiograph of the abdomen showing adrenal calcification in a patient with tuberculous Addison’s disease.

13.5.1  Disorders of the adrenal cortex 2349 The renin–​angiotensin–​aldosterone axis is intact, and children usually present either with neonatal hypoglycaemia, or later with increasing pigmentation, often with enhanced growth velocity. Patients have glucocorticoid deficiency with very high plasma ACTH levels; this occurs because of mutations in the melanocortin 2 receptor (MC2R; ACTH receptor) or an accessory protein in- volved in the cellular trafficking of MC2R (MRAP). Mutations in mini chromosome maintenance-​deficient 4 homologue (MCM4) and nicotinamide nucleotide transhydrogenase (NNT), genes in- volved in DNA replication and antioxidant defence respectively, have been recognized in FGD cohorts. A variant syndrome is called the triple A or Allgrove’s syndrome, and refers to a triad of adrenal insufficiency, namely ACTH resist- ance, achalasia, and alacrima. Allgrove syndrome is characterized by mutation(s) in the AAAS gene, located on chromosome 12q13, that codes for ALADIN protein. Secondary hypoadrenalism (ACTH deficiency) This is a common clinical problem and most often results from a sudden cessation of exogenous glucocorticoid therapy, or a failure to give adequate glucocorticoid cover for intercurrent stress in a patient who has been on long-​term glucocorticoid therapy. Such therapy suppresses the hypothalamic–​pituitary–​adrenal axis, with consequent adrenal atrophy that may last for months to years after stopping glucocorticoid treatment. Adrenal atrophy and subsequent deficiency should be anticipated in any subject who has taken more than the equivalent of 30 mg of oral hydrocortisone per day (ap- proximately 7.5 mg/​day prednisolone or 0.75 mg/​day dexametha- sone) for longer than 1 month. In addition to the magnitude of the dose of glucocorticoid, the timing of administration may affect the degree of adrenal suppression. Thus, prednisolone in a dose of 5 mg at night and 2.5 mg in the morning will produce more marked sup- pression of the hypothalamic–​pituitary–​adrenal axis than 2.5 mg at night and 5 mg in the morning because the larger evening dose blocks the early morning surge of ACTH. Other causes of secondary adrenal insufficiency are rare (Table 13.5.1.7), and reflect inadequate ACTH production from the anterior pituitary gland. In many of these, other pituitary hor- mones are deficient in addition to ACTH, so that the patient pre- sents with partial or complete hypopituitarism. However, if there is isolated ACTH deficiency this diagnosis may be readily missed. Lymphocytic hypophysitis and mutations in a transcription factor gene, Tpit (TBX19), involved in dictating the corticotroph lineage within the anterior pituitary, are rare diseases that may cause iso- lated ACTH deficiency. Hypoadrenalism may also complicate critical illness, even in in- dividuals with a previously intact hypothalamic–​pituitary–​adrenal axis. This functional adrenal insufficiency is usually transient and not caused by a structural lesion. This is a controversial area and debate continues regarding its diagnosis, aetiology, and treatment. This is beyond the scope of this chapter (for further information, see suggested reading). Clinical features The most obvious feature differentiating primary from secondary hypoadrenalism is skin pigmentation (Fig. 13.5.1.15), which is nearly always present in primary adrenal insufficiency (unless of short duration) and absent in secondary. The pigmentation is seen in sun-​exposed areas, recent rather than old scars, axillae, nipples, palmar creases, pressure points, and in mucous membranes (buccal, vaginal, vulval, anal). The pigmentation reflects increased mel- anocyte activity induced by POMC-​related peptides acting via the melanocortin 1 receptor (MC1R). In autoimmune Addison’s disease there may be associated vitiligo (Fig. 13.5.1.16). Patients with primary adrenal failure usually have both gluco- corticoid and mineralocorticoid deficiency. By contrast, those with secondary adrenal insufficiency have an intact renin–​angiotensin–​ aldosterone system. This accounts for differences in salt and water balance in the two groups of patients, which in turn result in dif- ferent clinical presentations. (a) (b) Fig. 13.5.1.15  Pigmentation in a patient with Addison’s disease. This is increased in sun-​exposed areas (panel (a)) and in the palmar creases (panel (b), where the patient’s hand is on the right side of the image, compared to an unaffected control subject’s hand on the left side). With permission from Medical Masterclass, 3rd edition, RCP London.

SECTION 13  Endocrine disorders 2350 Primary adrenal failure may present with hypotension and acute circulatory failure (Addisonian crisis). Anorexia may be an early fea- ture that progresses to nausea, vomiting, diarrhoea, and sometimes, abdominal pain. These crises may be precipitated by intercurrent in- fection or by stress, such as surgery. Alternatively, the patient may pre- sent with vague features of chronic adrenal insufficiency—​weakness, tiredness, weight loss, nausea, intermittent vomiting, abdominal pain, diarrhoea or constipation, general malaise, muscle cramps, and symptoms suggestive of postural hypotension. Salt craving may be a feature, and there may be a low-​grade fever. The lying blood pressure is usually normal, but almost invariably there is a fall in blood pres- sure on standing. In adrenal insufficiency secondary to hypopituitarism, the presen- tation may relate to deficiency of hormones other than ACTH, not- ably luteinizing hormone/​follicle-​stimulating hormone (infertility, oligo-​/​amenorrhoea, poor libido), thyroid-​stimulating hormone (weight gain, cold intolerance), and growth hormone (hypogly- caemia). Patients with isolated ACTH deficiency present with mal- aise, weight loss, and other features of chronic adrenal insufficiency. By contrast with primary adrenal failure, patients are usually pale. Investigation Routine biochemical profile In established primary adrenal insufficiency, hyponatraemia is pre- sent in about 90% of cases and hyperkalaemia in 65%. The blood urea concentration is usually elevated. In secondary adrenal failure there may be dilutional hyponatraemia, with normal or low blood urea, be- cause glucocorticoids are required to maintain the glomerular filtra- tion rate and excrete a water load. Therefore, patients with secondary adrenal failure (glucocorticoid deficiency only) may be misdiagnosed as having the syndrome of inappropriate ADH secretion (SIADH). Hypoglycaemia has been found in up to 50% of patients with chronic adrenal insufficiency. Plasma cortisol/​ACTH Clinical suspicion of the diagnosis should be confirmed with de- finitive diagnostic tests. Basal plasma cortisol concentrations are often in the low normal range and cannot be used to exclude the diagnosis. In primary adrenal insufficiency the simultaneous meas- urement of plasma cortisol and plasma ACTH reveals an ACTH level that is disproportionately elevated in comparison with plasma cortisol (Fig. 13.5.1.17). A plasma ACTH concentration exceeding 66 pmol/​litre (300 ng/​litre) provides maximum stimulation of glucocorticoid synthesis, hence in the setting of a low cortisol a level of ACTH more than 66 pmol/​litre indicates the inability of the adrenal cortex to respond to ACTH stimulation. Mineralocorticoid status In primary hypoadrenalism there is usually mineralocorticoid de- ficiency, with elevated plasma renin activity or concentration and either low or low-​normal plasma aldosterone. This aspect of inves- tigation is all too frequently ignored in patients with Addison’s dis- ease. By contrast, in secondary adrenal failure, only ACTH drive to the adrenal cortex is lacking; the renin–​angiotensin–​aldosterone axis is intact. Stimulation tests In practice, all patients suspected of having adrenal insufficiency should have an ACTH stimulation test. This involves the intra- muscular or intravenous administration of 250 µg of tetracosactide (Synacthen, cosyntropin), a peptide comprising the first 24 amino acids of normally secreted 1–​39 ACTH. Plasma cortisol levels are measured at 0 and 30 min after tetracosactide administration, and a normal response is defined by a peak plasma cortisol of more than 450–​500 nmol/​litre (the exact threshold is assay dependent). Levels less than this in response to tetracosactide are found in both pri- mary and secondary adrenal insufficiency. False-​positive results have occasionally been reported, particularly in cases of sudden-​ onset secondary hypoadrenalism. For example, in patients fol- lowing pituitary surgery or apoplexy assessment of ACTH reserve indirectly by ACTH stimulation test should be delayed for six weeks postoperatively to allow adrenal atrophy to occur in patients with ACTH deficiency (glucocorticoid cover may of course be required in the intervening period). Fig. 13.5.1.16  Vitiligo: smooth depigmented patches. From Barge S, Matin R, Wallis D (eds). Oxford handbook of medical dermatology, 2nd edition. By permission of Oxford University Press. PRIMARY
SECONDARY Addison’s Congenital adrenal hyperplasia Organic latrogenic 15 000 1000 500 100 Plasma ACTH (ng/litre) Fig. 13.5.1.17  Morning immunoreactive ACTH values in patients with hypoadrenalism. The reference range is indicated by the shaded bar. Courtesy of Professor LH Rees.

13.5.1  Disorders of the adrenal cortex 2351 A low-​dose ACTH stimulation test giving only 1 µg ACTH has been proposed to screen for adequate function of the hypothalamic–​ pituitary–​adrenal axis, with the suggestion that it may be more sensitive than the conventional 250 µg test. At present there are insufficient data to support this. A prolonged ACTH stimulation test, involving the administration of depot tetracosactide in a dose of 1 mg by intramuscular injection, with measurement of plasma cortisol at 0, 4, and 24 h, will differentiate primary from secondary hypoadrenalism, but this test is now rarely required if plasma ACTH has been appropriately measured at baseline. The insulin-​induced hypoglycaemia or insulin tolerance test re- mains one of the most useful in assessing ACTH and growth hor- mone reserves. It should not be performed in patients with ischaemic heart disease (check ECG before test), epilepsy, or severe hypopitu- itarism (i.e. plasma cortisol at 09.00 <100 nmol/​litre). The test in- volves the intravenous administration of soluble insulin in a dose of 0.1 to 0.15 U/​kg body weight, with measurement of plasma cortisol at 0, 30, 45, 60, 90, and 120 min. Adequate hypoglycaemia (blood glucose <2.2 mmol/​litre, with signs of neuroglycopenia—​sweating and tachycardia) is essential. In normal subjects the peak plasma cortisol exceeds 500 nmol/​litre. However, the response to hypogly- caemia can be reasonably reliably predicted by the response to acute ACTH stimulation (see earlier); a safer, cheaper, and quicker test. If the ACTH test is normal, insulin-​induced hypoglycaemia testing is not necessary in the vast majority of cases, unless there is a need to document endogenous growth hormone reserve in a patient with pituitary disease. Other tests Radioimmunoassays to detect autoantibodies, such as those against the 21-​hydroxylase antigen, are available and should be undertaken in patients with primary adrenal failure. In autoimmune Addison’s disease it is also important to look for evidence of other organ-​ specific autoimmune disease (e.g. thyroid dysfunction, pernicious anaemia, coeliac disease, and premature ovarian failure). In long-​ standing tuberculous adrenal disease there may be adrenal at- rophy with calcification on plain radiographs or CT scanning. Early morning urine samples should be cultured for mycobacteria or QuantiFERON checked if tuberculosis is suspected. Management Acute adrenal insufficiency This is an emergency and treatment should not be delayed while waiting for definitive proof of diagnosis. However, in addition to the measurement of plasma electrolytes and blood glucose, appropriate samples for ACTH and cortisol determination should be taken be- fore giving corticosteroid therapy. If the patient is not critically ill, an acute ACTH stimulation test can be performed, but if necessary this can be delayed and carried out with the patient on corticosteroid therapy. Once the patient has been stabilized, the patient can have a Synacthen test (with omission of evening and morning dose of hydrocortisone prior to test; once the test is complete the patient can receive their usual morning dose of hydrocortisone). In the acute setting, patients should be treated in a critical care set- ting and intravenous hydrocortisone should be given immediately at a dose of 100 mg, followed by 200 mg of hydrocortisone per 24 hours (either as a continuous infusion or 50 mg by injection every 6 hours). If this is not possible then the intramuscular route should be used. In the patient with shock, 1 litre of 0.9% sodium chloride should be given intravenously over the first hour. Because of possible hypogly- caemia, 5% dextrose is often also required. Subsequent intravenous fluid replacement will depend on biochemical monitoring and the patient’s condition. Inotropic support may be required initially until the patient’s condition is improved. Clinical improvement, espe- cially in blood pressure, should be seen within 4 to 6 h if the diag- nosis is correct. It is important to recognize and treat any associated condition, such as an infection that may have precipitated the acute adrenal crisis. Table 13.5.1.8  Management of primary adrenal insufficiency Condition Suggested action Home management of illness with fever Hydrocortisone replacement doses doubled (>38°C) or tripled (>39°C) until recovery (usually 2 to 3 d); increased consumption of electrolyte-​containing fluids as tolerated Unable to tolerate oral medication due to gastroenteritis or trauma Adults, IM or SC hydrocortisone 100 mg; children, IM hydrocortisone 50 mg/​m2 or estimate; infants, 25 mg; school-​age children, 50 mg; adolescents, 100 mg Minor to moderate surgical stress Hydrocortisone, 25–​75 mg/​24 h (usually 1 to 2 d) Major surgery with general anaesthesia, trauma, delivery, or disease that requires intensive care Hydrocortisone, 100 mg per IV injection followed by continuous iv infusion of 200 mg hydrocortisone/​24 h (alternatively 50 mg every 6 h IV or IM) Children, hydrocortisone 50 mg/​m2 iv followed by hydrocortisone 50–​100 mg/​m2/​d divided q 6 h Weight-​appropriate continuous iv fluids with 5% dextrose and 0.2 or 0.45% NaCl Rapid tapering and switch to oral regimen depending on clinical state Acute adrenal crisis Rapid infusion of 1000 ml isotonic saline within the first hour or 5% glucose in isotonic saline, followed by continuous iv isotonic saline guided by individual patient needs Hydrocortisone 100 mg IV immediately followed by hydrocortisone 200 mg/​d as a continuous infusion for 24 h, reduced to hydrocortisone 100 mg/​d the following day Children, rapid bolus of normal saline (0.9%) 20 ml/​kg. Can repeat up to a total of 60 ml/​kg within 1 h for shock. Children, hydrocortisone 50–​100 mg/​m2 bolus followed by hydrocortisone 50–​100 mg/​m2/​d divided q 6 h For hypoglycaemia: dextrose 0.5–​1 g/​kg of dextrose or 2–​4 ml/​kg of D25W (maximum single dose 25 g) infused slowly at rate of 2–​3 ml/​min. Alternatively, 5–​10 ml/​kg of D10W for children <12 years old Cardiac monitoring: rapid tapering and switch to oral regimen depending on clinical state Abbreviation: D10W, 10 % dextrose solution; D25W, 25% dextrose solution. From Bornstein SR, et al. (2016). Diagnosis and treatment of primary adrenal insufficiency: an endocrine society clinical practice guideline. J Clin Endocrinol Metab, 101, 364–​89. By permission of Oxford University Press.

SECTION 13  Endocrine disorders 2352 After 24 hours of clinical improvement (and if there is no ongoing precipitant) the dose of hydrocortisone can be reduced, usually to 100 mg per 24 h (either by continuous infusion or 25 mg every 6 hours). If the patient continues to recover and the precipitant has been treated then, if the patient can take by mouth, hydrocortisone can be switched to the oral route, 40 mg in the morning and 20 mg at 18.00. This can then be rapidly reduced to the normal replacement dose. Some patients will require more than 30 mg/​day, but most pa- tients require less than this (usually 15–​25 mg/​day). Chronic adrenal insufficiency Glucocorticoid replacement Long-​term treatment requires glucocorticoid replacement; doses vary between 15 and 25 mg hydrocortisone (or 20–​35 mg cortisone acetate) in divided doses (either twice or thrice daily), with the lar- gest dose on waking to mimic the circadian rhythm. Hydrocortisone is to be doubled or trebled in the event of intercurrent stress or illness. Higher doses may be required in some patients but there needs to be caution in using higher doses for prolonged periods due to the potential of chronic glucocorticoid overexposure. Monitoring of glucocorticoid replacement should be performed by clinical assess- ment including body weight, postural blood pressure, energy levels, and signs of frank glucocorticoid excess. A newly marketed modi- fied release hydrocortisone preparation can be administered once/​ twice daily and other slow release preparations are in development. Patients receiving glucocorticoid replacement therapy should be advised to double/​treble the dose in the event of an intercurrent fe- brile illness, accident, or in some cases of psychological stress. If the patient is vomiting and cannot take by mouth, parenteral hydro- cortisone must be given urgently, as indicated earlier. For minor surgery, 50 to 100 mg of hydrocortisone is given with the premedica- tion. For major procedures this is then followed by the same regimen as for acute adrenal insufficiency. Every patient on glucocorticoid therapy should be advised to register for an alert bracelet or necklace and to carry a steroid card giving information on the treatment being given. Many patients also carry hydrocortisone emergency kits for self-​injection in case imme- diate access to medical care is not possible, and all patients should be trained in giving themselves hydrocortisone injections. The hydro- cortisone emergency kit should only be used as a stopgap in order to allow the patient to get to hospital for urgent medical attention. Mineralocorticoid replacement In primary adrenal failure, mineralocorticoid replacement is usu- ally also required in the form of fludrocortisone at a dose of 50 to 100 µg/​day. After the acute phase has passed, the adequacy of min- eralocorticoid replacement can be assessed by measuring electro- lytes, supine and erect blood pressure, and plasma renin activity; too little fludrocortisone may cause postural hypotension with elevated plasma renin activity, and too much causes the converse. If patients develop hypertension while receiving fludrocortisone an initial dose reduction may be helpful; equally a dose reduction of hydrocortisone replacement may be required. If hypertension persists, fludrocortisone should not be discontinued but rather an antihypertensive agent added. If the patient is euvolaemic the antihypertensive of choice is an angiotensin II receptor blocker. If these cannot be used or tolerated, then a dihydropyridine calcium channel blocker can be used, but diuretics should be avoided and eplerenone and spironolactone are contraindicated. Adrenal androgen replacement For patients with both primary and secondary adrenal failure, bene- ficial effects have been reported for adrenal androgen replacement therapy with 25 to 50 mg/​day dehydroepiandrosterone (DHEA). Benefit is principally confined to female patients and includes im- provement in sexual function and well-​being, hence those who have low libido, depressive symptoms, and low energy levels despite op- timized glucocorticoid and mineralocorticoid replacement may be helped by DHEA. If there are no improvements in symptoms after six months the treatment should be discontinued. Morning dehydroepiandrosterone sulphate (DHEAS) levels before the inges- tion of DHEA dose can be used for monitoring with a target DHEAS in the mid-​normal range. Mineralocorticoid excess Blood pressure is a quantitative trait that significantly affects car- diovascular and cerebrovascular risk and mortality. Based on this, arbitrary cut-​offs define a hypertensive population that, depending on age, constitutes 10 to 25% of the population. In most cases, no underlying cause for the patient’s raised blood pressure can be found, and they are given a diagnosis of essential hypertension. However, mineralocorticoid-​based hypertension may account for a significant proportion of secondary causes of hypertension, and classically re- fers to hypertension caused by increased sodium and water retention by the kidney, and expansion of the extracellular fluid compartment, resulting in suppression of endogenous plasma renin activity. The implicated mineralocorticoid is most commonly aldosterone. Mineralocorticoid production and action Several key steps govern the production and action of mineralocorticoids: • Angiotensinogen is converted to Angiotensin I under the control of renin (produced in the juxtaglomerular apparatus in response to reduced plasma volume and renal perfusion); Angiotensin I is, in turn, converted to angiotensin II by the action of Angiotensin Converting Enzyme (ACE). • Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex from corticosterone by aldosterone synthase (CYP11B2), which is regulated by serum potassium and angiotensin II (with ACTH having a lesser effect). • Aldosterone binds to its (mineralocorticoid) receptor in the distal renal tubules and results in increased activity of the α subunit of the epithelial sodium channel, Na/​K ATPase, and Aquaporin 2, which increase retention of salt and water and excretion of potas- sium and hydrogen ions. Aetiology Mineralocorticoid excess may be seen in several contexts: • Primary (hyper)aldosteronism • Secondary (hyper)aldosteronism (e.g. due to renovascular disease)

13.5.1  Disorders of the adrenal cortex 2353 • Non​aldosterone mediated (e.g. some types of congenital adrenal hyperplasia; syndrome of apparent mineralocorticoid excess; excess liquorice or carbenoxolone ingestion; Cushing’s syndrome; gluco- corticoid resistance; deoxycorticosterone-​producing adrenal tumour) A comprehensive list of the causes of mineralocorticoid hyperten- sion is given in Table 13.5.1.9. For a perspective from a specialist in hypertension, see Chapter 16.17.3 for further information. Primary aldosteronism Epidemiology and aetiopathogenesis First described by Jerome Conn in 1955, primary aldosteronism was for a long time considered to account for only a small proportion of cases of hypertension. However, it has become clear in recent years that this classical teaching no longer holds true, with recent studies demonstrating a much higher prevalence of primary aldosteronism (5–​10% of all patients), especially among those with resistant hyper- tension (up to 25%). Aldosterone-​producing adenomas (APA, also known as Conn’s adenomas) and bilateral idiopathic hyperaldosteronism (IHA, also known as bilateral adrenal hyperplasia) account for most cases. Rarer causes include unilateral hyperplasia, adrenal carcinoma, ec- topic aldosterone production, and familial/​inherited syndromes. Several inherited and acquired forms of primary aldosteronism are now recognized. Familial (hyper)aldosteronism (FH) Type I: fusion of the CYP11B1 (11-​β hydroxylase) and CYP11B2 (aldosterone synthase) genes creates a chimera in which the ACTH-​responsive 11-​β-​hydroxylase promoter drives expression of aldosterone synthase; autosomal dominant (AD) inheritance, with early-​onset severe hypertension; responds to glucocorticoid treatment [to suppress ACTH  =  glucocorticoid-​remediable (suppressible) aldosteronism (GRA)]. Type II: aetiology remains unknown, although linkage has been shown to chromosome 7p22; AD inheritance with variable phenotype. Type III: caused by germline mutations in the KCNJ5 gene (encoding a subunit for an inwardly rectifying potassium channel GIRK4), which reduces potassium channel selectivity, and thus facilitates enhanced aldosterone production/​secretion and possibly cell proliferation; AD. Type IV: caused by germline mutations in the CACNA1H gene, which encodes the α subunit of an L-​type voltage-​gated calcium channel (Cav3.2). Sporadic primary aldosteronism Somatic mutations in several genes have recently been identified in patients with sporadically-​occurring APAs. These include: KCNJ5: the same gene as implicated in FH type III; mutations re- sult in chronic depolarization and Ca2+ influx; estimated to be present in up to 40% of all APAs (but with a particular predom- inance in young females with APAs). ATP1A1: encodes the α subunit of the Na+/​K+-​ATPase 1; mutations result in chronic depolarization, with opening of voltage-​gated calcium channels leading to aldosterone production/​secretion; estimated to be present in c.5% of APAs. ATP2B3: encodes the plasma membrane Ca2+-​ATPase 3; estimated to be present in c.1–​2% of APAs. CACNA1D: encodes an L-​type voltage-​gated calcium channel (Cav1.3); mutations result in channel activation at less depolar- ized potentials and may in some instances impair channel inacti- vation; estimated to be present in c.10% of APAs. CTNNB1: activating mutations of CTNNB1 gene (β-​catenin) in the Wnt signalling pathway. Clinical features Symptoms are often absent or non​specific, but include tiredness, muscle weakness, thirst, polyuria, and nocturia resulting from hypokalaemia. Spontaneous hypokalaemia (<3.5 mmol/​litre) is rare in untreated hypertension; when it is found in a patient on diuretics these should be withdrawn, and potassium stores replenished and Table 13.5.1.9  Differential diagnosis of mineralocorticoid excess Cause Offending mineralocorticoid Primary aldosteronism Aldosterone Congenital adrenal hyperplasia Deoxycorticosterone 11β-​Hydroxylase deficiency 17α-​Hydroxylase deficiency Glucocorticoid receptor resistance Deoxycorticosterone Glucocorticoid receptor mutations Metyrapone, RU486 ingestion Deoxycorticosterone-​secreting adrenal tumour Deoxycorticosterone Liddle’s syndrome None 11 β-​Hydroxysteroid dehydrogenase deficiency Cortisol Apparent mineralocorticoid excess Liquorice and carbenoxolone ingestion Ectopic ACTH syndrome Liddle’s syndrome is caused by a gain-​of-​function mutation in the epithelial sodium channel (ENaC) and is therefore strictly speaking independent of mineralocorticoid.

SECTION 13  Endocrine disorders 2354 remeasured 2 weeks later. Despite this, it is now accepted that most patients with confirmed primary aldosteronism have normal serum potassium concentrations. Screening for primary aldosteronism Screening for primary aldosteronism should be considered in the following circumstances: •​ Hypertension and hypokalaemia (especially unprovoked) •​ Young age of onset of hypertension and/​or severe hypertension •​ Treatment-​resistant hypertension (≥3 antihypertensive agents and poor control) •​ Hypertension in the setting of an incidental adrenal mass •​ Whenever secondary causes of hypertension are being considered. Case detection can be performed by measuring ambulatory, paired random plasma renin activity (PRA) (or plasma renin mass/​ concentration—​PRC) and plasma aldosterone concentration (PAC), to yield an aldosterone-​renin-​ratio. It has been suggested that screening should preferably be performed using an early morning sample, but testing at other times of day is acceptable and often more practical. Interpretation of PRA and PAC measurements is laboratory and assay dependent and, wherever possible, locally derived PRA/​PAC cut-​offs should be defined (similarly, centres using plasma renin mass/​concentration assays will need to determine thresholds for triggering further investigation). With a few notable exceptions including mineralocorticoid receptor antagonists, amiloride, renin inhibitors (which should be discontinued for at least 4–​6 weeks before testing), and possibly β-​adrenergic blockers (which require discontinuation for at least two weeks—​although see comments to follow), PRA and PAC can be measured while the patient is taking antihypertensive therapy. However, careful interpretation under- pinned by a clear understanding of the effects of different agents on the renin–​angiotensin system (RAA) system is required if ‘false positives’ and ‘false negatives’ are to be avoided. ACE-​inhibitors (ACE-​I), angiotensin receptor antagonists/​ blockers, and non​potassium-​sparing diuretics can all elevate PRA/​ renin mass, thereby masking a diagnosis of primary aldosteronism (false negative screen). By contrast, the finding of a low/​suppressed PRA/​renin mass in this treatment context is very strongly suggestive of PA. β-​adrenergic blockers (and centrally ​acting α-2-​agonists) sup- press renin and aldosterone secretion in subjects without primary aldosteronism, but the fall in aldosterone is typically less marked, resulting in an increase in the PAC/​PRA ratio, although absolute al- dosterone levels do not usually exceed 415 pmol/​litre. It is important to note, however, that uncorrected hypokalaemia and/​or calcium an- tagonist therapy (see later) may confound interpretation of absolute aldosterone levels. Dihydropyridine calcium antagonists/​blockers can suppress aldosterone secretion, resulting in a false negative test. Some calcium channel antagonists (e.g. verapamil, diltiazem), α 1-​adrenergic blockers (e.g. doxazosin) and the direct-​acting smooth muscle relaxant hydralazine appear to have little effect on PRA and PAC and hence remain the ‘agents of choice’ when screening for primary aldosteronism in patients requiring antihypertensive therapy, but not all patients can be satisfactorily controlled on these drugs alone. It is also important to correct hypokalaemia before measuring PAC as a low serum potassium level can impair aldosterone secretion and, if left uncorrected, can result in false negative screening in milder cases of primary aldosteronism. Other (non-​antihypertensive) agents may also interfere with measurement of PRA and PAC, most notably non​steroidal anti-​ inflammatory drugs (NSAIDs), which lower renin and aldosterone levels. Patients should also be advised to avoid ingestion of liquorice-​ containing products given the potential to cause confusion by inducing a state of apparent mineralocorticoid excess (see next). Confirmatory testing for primary aldosteronism Several different tests have been proposed to confirm autonomous aldosterone secretion. Oral sodium loading test Sodium (>200  mmol per day, equivalent to c.12 g of sodium chloride) is administered for three days, with potassium supple- mentation as required to prevent exacerbation of hypokalaemia. At completion of the test a 24 h urine specimen is collected (from the morning of day 3 to the morning of day 4) for estimation of sodium, creatinine, and aldosterone to confirm (i) adequate so- dium loading, (ii) ongoing inappropriate/​autonomous aldosterone secretion. This is more time-​consuming than the intravenous sa- line infusion test and relies on ability of laboratory to accurately measure urinary aldosterone. It should be avoided in patients with severe uncontrolled hypertension, cardiac failure, arrhythmias, or renal impairment. Intravenous saline infusion test Two litres of 0.9% saline is infused over 4 h with measurement of PAC before and after the infusion to demonstrate failure of suppres- sion of aldosterone secretion. Hypokalaemia should be corrected, and blood pressure and heart rate must be monitored during the test, which is normally performed in the morning. It should be avoided in patients with severe uncontrolled hypertension, cardiac failure, arrhythmias, or renal impairment. False negative results (i.e. apparent normal suppression) have been described in some patients with primary aldosteronism. Fludrocortisone suppression test 0.1 mg of oral fludrocortisone is given every 6 h for 4 days, with oral sodium supplementation as required and potassium replacement sufficient to avoid hypokalaemia. Caution must be exercised in pa- tients with severe hypertension and in those with a history of cardiac or renal impairment. Captopril test This is possibly less reliable than the other tests described, with false negative or equivocal results reported. Distinguishing unilateral and bilateral (sporadic) primary aldosteronism In patients with unilateral primary aldosteronism (40–​50% of cases) due to an APA or unilateral hyperplasia, adrenalectomy offers the potential for biochemical cure, and resolution/​improvement of hypertension; by contrast, IHA is most appropriately treated with medical therapy, hence it is extremely important to achieve a correct diagnosis.

13.5.1  Disorders of the adrenal cortex 2355 Investigations used to establish laterality in primary aldoster- onism include: Cross-​sectional imaging Thin slice adrenal CT or MRI are the standard techniques (Fig. 13.5.1.18). Although the finding in a young patient (<35 y) of a classical solitary Conn’s adenoma (1–​2 cm diameter lesion) with an entirely normal contralateral gland may be sufficient to proceed directly to surgery, CT or MRI alone are generally con- sidered insufficient to lateralize primary aldosteronism in most patients. Adrenal vein sampling This remains the gold-​standard for distinguishing unilateral and bilateral primary aldosteronism, but is technically challenging and in many centres the right adrenal vein (which drains directly into the inferior vena cava) is successfully cannulated in only 50–​80% of cases. Matters are further complicated by the fact that there are several different sets of criteria for defining successful ad- renal vein cannulation and lateralization based on measurement of plasma cortisol and aldosterone levels in both adrenal veins and in the inferior vena cava (IVC). In recognition that both cortisol and aldosterone secretion can be pulsatile, many centres advocate performing adrenal vein sampling with tetracosactide (Synachen, cosyntropin) stimulation (either bolus or continuous infusion) and/​ or simultaneous (as opposed to sequential) adrenal vein sampling. These modifications minimize the chance of creating artificial gra- dients, and cosyntropin infusion can increase confidence that both adrenal veins have been successfully cannulated by enhancing the plasma cortisol gradients between adrenal veins and IVC. However, simultaneous sampling (at least theoretically) increases the risk of adrenal vein thrombosis due to longer catheter occupancy of the vein on the side catheterized first. A recently published expert consensus statement offers a de- tailed summary of the literature and makes helpful recommenda- tions regarding how to perform and interpret adrenal vein sampling, although a recent study (SPARTACUS trial) has questioned the as- sumption that adrenal vein sampling is superior to cross-​sectional imaging with respect to guiding management and yielding favour- able outcomes, but these findings remain strongly debated. 11C-​metomidate PET-​CT Emerging data suggests that functional adrenal imaging may offer a non​inferior alternative to adrenal vein sampling for distinguishing unilateral and bilateral primary aldosteronism. An immediate at- traction of this technique is its non​invasive nature, but further studies are awaited to confirm early findings. Clinical prediction score The inherent difficulties associated with performing adrenal vein sampling stimulated Kupers and colleagues to assess the potential utility of a clinical scoring system in predicting unilateral disease. In 87 patients with primary aldosteronism and successful adrenal vein sampling, lateralization was demonstrated in 49 patients. All 26 pa- tients with a typical Conn’s adenoma and serum potassium level less than 3.5 mmol/​litre or estimated glomerular filtration rate (eGFR) at least 100 ml/​min/​1.73 m2 (or both) had unilateral primary aldoster- onism; this rule had 100% specificity and 53% sensitivity. However, in two follow-​up studies by independent workers, neither group was able to reproduce the 100% specificity (88.5% and 80%, respectively). Management Unilateral laparoscopic adrenalectomy remains the preferred treat- ment option for patients with an APA or unilateral hyperplasia. It offers the potential to ameliorate/​correct hypertension, abolish hypo- kalaemia, and correct hyperaldosteronism. However, patients must be carefully counselled that successful surgery (as judged by the correc- tion of biochemical hyperaldosteronism) does not translate into nor- malization of blood pressure in some cases, although the number of antihypertensive agents required is usually reduced postoperatively. Two potential complications must be looked out for following unilateral adrenalectomy:  hypocortisolism secondary to cortisol cosecretion by an aldosterone-​producing adenoma with suppres- sion of the hypothalamic-​pituitary-​contralateral adrenal gland, and hypoaldosteronism manifest as hypotension (especially postural) and hyperkalaemia due to persistent suppression of the RAA system. Recovery of normal endogenous function is recognized in both con- texts and should be checked for periodically. Hyperaldosteronism per se is associated with excess cardiovas- cular morbidity and mortality independent of its effects on blood (a) (b) Fig. 13.5.1.18  (a) Adrenal CT scan demonstrating a solitary adrenal adenoma in a patient with Conn’s syndrome and (b) the characteristic yellow appearance of the cut surface of the excised tumour reflecting the high cholesterol content of these tumours.

SECTION 13  Endocrine disorders 2356 pressure (e.g. through promoting myocardial fibrosis). Accordingly, mineralocorticoid receptor antagonist therapy is the treatment of choice when preparing patients for adrenalectomy and as long-​term primary medical therapy in those unfit/​unwilling to consider sur- gery or in whom there is evidence of bilateral disease. Spironolactone remains the mineralocorticoid receptor antagonist of choice in many centres, but side effects (gynaecomastia in males; menstrual irregularity in females) may limit its use. Eplerenone, a competitive and selective mineralocorticoid receptor antagonist, is a useful al- ternative, although its use in this context remains off-​licence in most countries and its potency appears to be less than that of spironolac- tone. Combination with amiloride (to block the epithelial sodium channel) increases the efficacy of mineralocorticoid antagonism. Calcium antagonists are a useful adjunct for controlling hyperten- sion in primary aldosteronism. Single gene defects resulting in mineralocorticoid excess Hypertension is a phenotype of some well-​documented gene mu- tations; 17α-​hydroxylase deficiency and 11β-​hydroxylase deficiency cause forms of congenital adrenal hyperplasia in which mineralocor- ticoid excess occurs because of ACTH-​driven deoxycorticosterone excess. A similar process is thought to explain the hypertension seen in patients with glucocorticoid resistance resulting from mutations in the glucocorticoid receptor gene. A significant advance in our understanding of the molecular basis of cardiovascular disease has been the elucidation of other single gene defects causing mineralo- corticoid hypertension (Fig. 13.5.1.19). Glucocorticoid-​suppressible hyperaldosteronism (See Chapter 16.17.4.) Liddle’s syndrome (See Chapter 16.17.4.) Apparent mineralocorticoid excess and abnormalities of 11β-​ hydroxysteroid dehydrogenase type 2 (See Chapter 16.17.4 for discussion of apparent mineralocorticoid excess.) Liquorice has been associated with a mineralocorticoid excess state since the late 1940s, when Reevers, a Dutch physician, used a liquorice preparation, succus liquoritiae, to treat patients with dys- pepsia. This was the origin of the antiulcer drug, carbenoxolone, which also results in mineralocorticoid side effects in up to 50% of patients. The active ‘mineralocorticoids’ in both cases are glycyrrhizic acid and its hydrolytic product, glycyrrhetinic acid, which them- selves have little inherent mineralocorticoid activity, but cause hyper- tension and hypokalaemia by inhibiting 11β-​HSD2 (Fig. 13.5.1.20). Such patients will also have an increase in the urinary ratio of cortisol to cortisone metabolites (THF+allo-​THF/​THE), although not to the same extent as patients with apparent mineralocorticoid excess. Cortisol is also the offending mineralocorticoid in patients with some forms of Cushing’s syndrome. In ectopic ACTH syndrome, for example, the high cortisol secretion rate overwhelms renal 11β-​HSD2, resulting in spillover to the mineralocorticoid receptor. A high THF+allo-​THF/​THE ratio is also observed in some patients with pituitary-​dependent Cushing’s syndrome, and this may explain the hypertension in these cases. Activating mutations in the mineralocorticoid receptor One kindred has been reported with a homozygous point mutation in the mineralocorticoid receptor that results in a serine to leucine change at amino acid 810, with severe hypertension at a young age. An interesting facet of this mutation is that the mutated receptor is induced by progesterone and some of its hydroxylated derivatives, thereby explaining pregnancy-​induced hypertension in affected fe- male members of the kindred. Glucocorticoid resistance A few patients have been described who have increased cortisol secretion, but none of the stigmas of Cushing’s syndrome. These patients are resistant to the suppression of cortisol with low-​dose dexamethasone but respond to high doses. ACTH levels are elevated and lead to increased adrenal production of androgens and deoxy- corticosterone, hence patients may present with the features of an- drogen and/​or mineralocorticoid excess. Treatment with a dose of dexamethasone adequate to suppress ACTH (usually 3 mg/​day) re- sults in a fall in adrenal androgens and often the return of plasma po- tassium and blood pressure to normal levels. Many of these patients have been found to have point mutations in the steroid-​binding Na+ Apparent mineralocorticoid excess Liddle’s syndrome Cortisone Apical Na channel Mineralocorticoid receptor Type 2 11β-HSD Cortisol Basolateral Na/K ATPase Target gene transcription Cortisol Aldosterone Aldosterone Aldosterone excess Glucocorticoid- suppressible hyperaldosteronism Na+ α β γ Fig. 13.5.1.19  A schematic diagram representing an epithelial cell in the distal colon or distal nephron. In normal physiology, aldosterone interacts with the mineralocorticoid receptor (MR) to stimulate sodium reabsorption via induction of the apical sodium channel and serosal Na+,K+-​ATPase pump. GSH (glucocorticoid-​suppressible hyperaldosteronism) is a cause of aldosterone excess that results from the production of a chimaeric gene, 11β-​hydroxylase/​aldosterone synthase, within the adrenal cortex. Apparent mineralocorticoid excess results because cortisol cannot be inactivated to cortisone by the type 2 isoform of 11β-​hydroxysteroid dehydrogenase (11β-​HSD2); cortisol can then act as a potent mineralocorticoid. Liddle’s syndrome occurs because of constitutively active mutations in the β-​ or γ-​subunits of the apical sodium channel. Activating mutations in the MR can also lead to inappropriate sodium retention.

13.5.1  Disorders of the adrenal cortex 2357 domain of the glucocorticoid receptor, with consequent reduction of glucocorticoid-​binding affinity. Mineralocorticoid deficiency These syndromes are listed in Table 13.5.1.10. They can be divided into those that are congenital and others that are acquired. Adrenal insufficiency Mineralocorticoid deficiency may occur in some forms of con- genital adrenal hyperplasia and these are discussed elsewhere (see Chapter 13.5.2). Similarly, other causes of adrenal insufficiency (e.g. Addison’s disease and congenital adrenal hypoplasia) are discussed earlier. Primary defects in aldosterone biosynthesis Before the characterization of the CYP11B2 gene, the disease was termed corticosterone methyl oxidase type I (CMO I) deficiency and corticosterone methyl oxidase type II (CMO II) deficiency. Subsequently, both variants were shown to be secondary to muta- tions in aldosterone synthase and are now termed type I and type II aldosterone synthase deficiency. Both variants are rare and in- herited as autosomal recessive traits. The type II deficiency is found most frequently among Jews of Iranian origin. Presentation is usu- ally in neonatal life as a salt-​wasting crisis with severe dehydration, vomiting, and failure to grow and thrive. Hyperkalaemia, meta- bolic acidosis, dehydration, and hyponatraemia are found. Plasma renin activity is elevated, and plasma aldosterone levels are low. Plasma 18-​hydroxycorticosterone levels and the ratio of plasma 18-​ hydroxycorticosterone to aldosterone and their urinary metabolites are used to differentiate the type I and II variants. In most infants the disorders become less severe as the child ages; in older children, adolescents, and adults, the abnormal steroid pattern described may be present and may persist throughout life without clinical mani- festations. Mineralocorticoids (fludrocortisone) are given during infancy and early childhood, but this therapy can be discontinued in most adults. Spontaneous normalization of growth can occur in untreated patients. Rarely, presentation can be in adulthood. Defects in aldosterone action: Pseudohypoaldosteronism (See Chapter 16.17.4.) Hyporeninaemic hypoaldosteronism Angiotensin II is a key stimulus for aldosterone secretion, and damage or blockade of the renin–​angiotensin system may result in mineralocorticoid deficiency. Various renal diseases have been as- sociated with damage to the juxtaglomerular apparatus and hence renin deficiency. These include systemic lupus erythematosus, mye- loma, amyloidosis, AIDS, and the use of NSAIDs, but the most common (>75% of cases) is diabetic nephropathy. The usual picture is of an older patient with hyperkalaemia, acid- osis, and mild to moderate impairment of renal function. Plasma renin activity and aldosterone are low and fail to respond to sodium depletion, erect posture, or furosemide administration. By contrast with adrenal insufficiency, patients have normal or elevated blood pressure and no postural hypotension. Muscle weakness and cardiac arrhythmias may also occur. Other factors may contribute to the hyperkalaemia, including the use of potassium-​sparing diuretics, potassium supplementation, insulin deficiency, and β-​adrenoceptor blockers and prostaglandin synthase inhibitors that inhibit renin release. The treatment of primary renin deficiency is with fludrocortisone in the first instance, together with dietary potassium restriction. However, these patients are not salt depleted and may become MR F (a) (b) Kidney, colon, salivary gland MR AME 11β-HSD2 F MR aldo MR aldo MR aldo MR aldo MR aldo MR aldo MR aldo MR aldo F E F Kidney, colon, salivary gland MR F MR aldo MR F MR F MR F MR F MR F MR F E Fig. 13.5.1.20  (a) The role of 11β-​hydroxysteroid dehydrogenase (11β-​HSD2) in protecting the non​specific mineralocorticoid receptor (MR) from cortisol, and (b) with congenital or acquired deficiency of the enzyme, F (cortisol) cannot be inactivated to E (cortisone) and acts as a potent mineralocorticoid. Table 13.5.1.10  Causes of mineralocorticoid deficiency Addison’s disease Adrenal hypoplasia Congenital adrenal hyperplasia:   17-​hydroxylase   3β-​hydroxysteroid dehydrogenase deficiencies Pseudohypoaldosteronism types I and II Hyporeninaemic hypoaldosteronism Aldosterone biosynthetic defects Drug induced

SECTION 13  Endocrine disorders 2358 hypertensive with fludrocortisone. In such a scenario the addition of a loop-​acting diuretic such as furosemide is appropriate. This will increase acid excretion and improve the metabolic acidosis. Adrenal incidentalomas An adrenal incidentaloma is an adrenal mass detected on imaging not performed for suspected adrenal disease. With the more wide- spread use of high-​resolution cross-​sectional imaging procedures (CT and MRI), incidentally discovered adrenal masses have be- come common, being uncovered in 4 to 7% of patients over the age of 40 years who are imaged for non​adrenal pathology. Over 80% of cases are non​functioning, with phaeochromocytomas and cor- tisol or aldosterone secreting adenomas making up most of the re- mainder. A few incidentalomas will be adrenocortical carcinomas or rarer causes such as lymphangioma or primary adrenal lymphoma. Importantly, in patients with prior history of malignancy, an adrenal incidentaloma may reflect a metastatic deposit (e.g. up to 20% of patients with lung cancer have adrenal metastases on CT scanning). The data regarding relative frequency of different underlying tu- mour type is variable depending whether the series is surgical or one which takes all patients with an adrenal mass. Investigation In general, when an adrenal lesion is discovered, two questions need to be addressed: is this lesion malignant? And is this lesion secretory? These questions are answered by a combination of endo- crinological and radiological tests. Endocrinological tests Some incidentalomas may cause abnormal hormone secretion without obvious clinical manifestations of a hormone excess state, the best example of which relates to autonomous cortisol secretion (often without any clinical evidence of Cushing’s syndrome) that may occur in up to 10% of all cases. As a result, all patients with incidentally discovered adrenal masses should undergo appro- priate endocrine screening tests:  plasma or urine metanephrines and normetanephrines to exclude a phaeochromocytoma; over- night dexamethasone suppression test/​late-​night salivary cortisol/​ 9am ACTH/​DHEAS (low levels are suggestive of a lack of ACTH) to exclude autonomous hypercortisolism; plasma aldosterone:renin ratio to screen for primary aldosteronism; and adrenal androgens if hyperandrogenism is suspected. Adrenal imaging There are three main radiological techniques that can help deter- mine whether an adrenal lesion is benign or malignant: CT, MRI, and FDG-​PET/​CT. CT scanning CT scanning has high spatial and quantitative contrast reso- lution, which allows assessment of tissue density as measured by Hounsfield units (HU—​the HU value of water is 0 and tissues are compared to this). An adrenal lesion on a non​contrast CT with a density less than 10 HU is strongly suggestive of a lipid rich benign adenoma (Fig. 13.5.1.9), but 30% of benign adenomas will have a HU more than 10 which overlaps with more malignant lesions and phaeochromocytomas. In an attempt to further characterize these lesions (with HU >10) a contrast-​enhanced washout CT can be per- formed. Benign adenomas take up intravenous contrast rapidly, but also exhibit a rapid loss (washout) of contrast; malignant lesions are described as having slower contrast washout. During a contrast-​ enhanced washout scan, a measure of HU is taken at baseline before contrast injection (HUnative), 60 seconds following contrast injec- tion (HUmax), and then 10 or 15 minutes postcontrast injection (HU 10/​15). This allows calculation of the relative contrast enhance- ment washout (=100 × (HUmax –​HU10/​15 min)/​HUmax) and ab- solute contrast enhancement washout (=100 × (HUmax –​ HU10/​ 15 min)/​ (HUmax –​ HUnative)). A relative washout more than 40% and an absolute washout more than 60% is suggestive that an adrenal lesion is benign. MRI imaging This has the advantage of not exposing the patient to ionizing radiation and not requiring iodinated contrast. To differentiate between benign and malignant adrenal lesions the technique of chemical shift is utilized. This allows separate images to be gen- erated, with fat and water oscillating in phase or out of phase with each other (Fig. 13.5.1.21). Adrenal adenomas, which Fig. 13.5.1.21  Patient with primary hyperaldosteronism secondary to lipid rich right Conn’s adenoma. In (right) and out (left) of phase images showing signal dropout on the out of phase sequence (compare the grey and black appearances of the adrenal nodule during the in and out of phase images, respectively).

13.5.1  Disorders of the adrenal cortex 2359 have high intracellular lipid content usually, lose signal inten- sity on out of phase images compared with in phase images. Phaeochromocytoma or malignant lesions which are lipid poor do not have this characteristic. FDG-​PET/​CT This nuclear medicine modality provides quantitative tomography images after intravenous injection of 18F-​fluorodeoxyglucose (18F-​ FDG-​PET/​CT). Positivity is not specific for cancer but rather a marker of cells which have increased requirement for glucose/​ glucose metabolism. Quantitative measures of 18F concentrations within tissues can be determined by using the standardized uptake value, which compares the intensity of uptake of 18F in the adrenal lesion to the average of the whole body. Lesions which can be 18F-​ FDG-​PET/​CT avid include phaeochromocytomas/​paragangliomas, adrenocortical carcinomas and metastases from non​adrenal pri- maries (Fig. 13.5.1.22). Other investigations Despite the aforementioned imaging modalities there may still be some lesions which are indeterminate. In this setting there are three options: 1. Consider further imaging using a different modality 2. Interval imaging in 6–​12  months (using non​contrast CT or MRI) 3. Surgical resection without further delay The choice of the best approach for each individual patient should be made in a multidisciplinary team environment. Biopsy of adrenal lesions In general, biopsy of an adrenal lesion should be avoided. This is important in patients who have an adrenocortical carcinoma as a biopsy breaches the tumour capsule, which can lead to seeding of the tumour and a poorer outcome. Similarly, a biopsy should not be undertaken until a thorough endocrine work up has been per- formed: most notably phaeochromocytoma should be excluded given the risk of inducing a phaeochromocytoma crisis. Adrenal biopsy should only be performed if the result of the biopsy will significantly alter the management of the patient, for example in assessment of someone with other malignant disease (e.g. lung cancer), in order to allow accurate staging. An adrenal resection rather than biopsy is often a more appropriate method of assessment. Adrenocortical carcinoma Adrenocortical carcinoma is a rare and highly aggressive malig- nancy with an annual incidence of 0.7–​2.0 cases per million popula- tion. Alterations have been found in several genes in this condition, including TP53, CTNNB1, SF1, 11p15 locus, mismatch repair genes, microRNAs, and Jag1. Autonomous hormone secretion is reported in more than 80% of cases. Detailed analysis of urinary corticosteroid metabolites by Gas Chromatography/​ Mass Spectrometry reveals characteristic abnor- malities of metabolites which may serve as a ‘fingerprint’ for malig- nancy and may in future be used as a potential biomarker. Imaging of adrenocortical carcinomas often show large heterogenous tumours with central necrosis and in many cases metastatic disease at time of diagnosis (Fig. 13.5.1.10). All adrenocortical carcinomas show ele- vated Hounsfield units more than 10 on non​contrast imaging. On surgical resection specimens the diagnosis is made based on the Weiss score, comprised of certain histopathological features. The higher the score the more likely a diagnosis of adrenocortical carcinomas, a score of more than 3 particularly predicting increased risk of malignant behaviour. Treatment of adrenocortical carcinomas involves radical resec- tion were possible, with or without irradiation to the tumour bed as adjuvant therapy. Other adjuvant therapies include mitotane and cytotoxic chemotherapy. In recurrent, metastatic, or advanced disease the treatment options include repeat surgery (limited to isolated disease) and mitotane with the addition of cytotoxic chemo- therapy (FIRM-​ACT protocol etoposide, doxorubicin, cisplatin, and mitotane). This treatment regimen has been shown to have a greater survival in a randomized control trial compared to streptozotocin and mitotane. FURTHER READING Cushing’s syndrome Correa R, Salpea P, Stratakis CA (2015). Carney complex: an update. Eur J Endocrinol, 173, M85–​M97. Daniel E, et al. (2015). Effectiveness of metyrapone in treating Cushing’s syndrome: a retrospective multicenter study in 195 patients. J Clin Endocrinol Metab, 100, 4146–​54. El Ghorayeb N, Bourdeau I, Lacroix A (2015). Multiple aberrant hormone receptors in Cushing’s syndrome. Eur J Endocrinol, 173, M45–​60. Fig. 13.5.1.22  18F-​FDG-​PET/​CT scan of large right sided adrenocortical carcinoma.

13.5.2 Congenital adrenal hyperplasia 2360 Nils P.

13.5.2 Congenital adrenal hyperplasia 2360 Nils P. Krone and Ieuan A. Hughes

SECTION 13  Endocrine disorders 2360 Isidori AM, et  al. (2006). The ectopic adrenocorticotropin syn- drome:  clinical features, diagnosis, management, and long-​term follow-​up. J Clin Endocrinol Metab, 91, 371–​7. Monaghan PJ, et al. (2011). Comparison of serum cortisol measure- ment by immunoassay and liquid chromatography-​tandem mass spectrometry in patients receiving the 11β-​hydroxylase inhibitor metyrapone. Ann Clin Biochem, 48(Pt 5), 441–​6. Nieman LK, et al. (2008). The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 93, 1526–​40. Nieman LK, et al.; Endocrine Society (2015). Treatment of Cushing’s syndrome: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 100, 2807–​31. Sbiera S, et al. (2015). The new molecular landscape of Cushing’s dis- ease. Trends Endocrinol Metab, 26, 573–​83. Stratakis CA (2008). Cushing syndrome caused by adrenocortical tumors and hyperplasias (corticotropin-​independent Cushing syndrome). In: Flück CE, Miller WL (eds) Disorders of the human adrenal cortex. Endocr Dev. Basel, Karger, vol 13, pp. 117–​32. Theodoropoulou M, et al. (2015). Decoding the genetic basis of Cushing’s disease: USP8 in the spotlight. Eur J Endocrinol, 173, M73–​83. Tritos NA, Biller BMK (2019). Current management of Cushing’s dis- ease. J Intern Med, doi: 10.1111/joim.12975. Vaidya A, et al. (2019). The evaluation of incidentally discovered adrenal masses. Endocr Pract, 25, 178–92. Adrenal insufficiency Agha A, et al. (2006). The long-​term predictive accuracy of the short synacthen (corticotropin) stimulation test for assessment of the hypothalamic-​pituitary-​adrenal axis. J Clin Endocrinol Metab, 91, 43–​7. Arlt W, Allolio B (2003). Adrenal insufficiency. Lancet, 361, 1881–​93. Arlt W, et al. (1999). Dehydroepiandrosterone replacement in women with adrenal insufficiency. N Engl J Med, 341, 1013–​20. Boonen E, Bornstein SR, Van den Berghe G (2015). New insights into the controversy of adrenal function during critical illness. Lancet Diabetes Endocrinol, 3, 805–​15. Boonen E, Van den Berghe G (2016). Mechanisms in endocrin- ology: new concepts to further unravel adrenal insufficiency during critical illness. Eur J Endocrinol, 175, R1–​9. Bornstein SR, et al. (2016). Diagnosis and treatment of primary ad- renal insufficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 101, 364–​89. Erturk E, Jaffe CA, Barkan AL (1998). Evaluation of the integrity of the hypothalamo-​pituitary adrenal axis by insulin hypoglycaemia test.
J Clin Endocrinol Metab, 83, 2350–​4. Johannsson G, et al. (2012). Improved cortisol exposure-​time profile and outcome in patients with adrenal insufficiency: a prospective randomized trial of a novel hydrocortisone dual-​release formula- tion. J Clin Endocrinol Metab, 97, 473–​81. Marik PE, et  al. (2008). Recommendations for the diagnosis and management of corticosteroid insufficiency in critically ill adult patients: consensus statements from an international task force by the American College of Critical Care Medicine. Crit Care Med, 36, 1937–​49. Oelkers W (1996). Adrenal insufficiency. N Engl J Med, 335, 1206–​12. Rushworth RL, Torpy DJ, Falhammar H (2019). Adrenal crisis. N Engl J Med, 381, 852–61. Stewart PM, et  al. (1988). A rational approach for assessing the hypothalamo-​pituitary adrenal axis. Lancet, 1, 1208–​10. Teblick A, et al. (2019). Adrenal function and dysfunction in critically ill patients. Nat Rev Endocrinol, 15, 417–27. Woods CP, et al. (2015). Adrenal suppression in patients taking in- haled glucocorticoids is highly prevalent and management can be guided by morning cortisol. Eur J Endocrinol, 173, 633–​42. Mineralocorticoids Burton TJ, et al. (2012). Evaluation of the sensitivity and specificity of (11)C‐metomidate positron emission tomography (PET)-​CT for lateralizing aldosterone secretion by Conn’s adenomas. J Clin Endocrinol Metab, 97, 100–​9. Funder JW, et  al. (2016). The management of primary aldoster- onism:  case detection, diagnosis, and treatment:  an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 101, 1889–​916. Hundemer GL, et al. (2018). Cardiometabolic outcomes and mortality in medically treated primary aldosteronism: a retrospective cohort study. Lancet Diabetes Endocrinol, 6, 51–​9. Monticone S, et  al. (2018). Cardiovascular events and target organ damage in primary aldosteronism compared with essential hyper- tension:  a systematic review and meta-​analysis. Lancet Diabetes Endocrinol, 6, 41–​50. Monticone S, et al. (2018). Genetics in endocrinology: the expanding genetic horizon of primary aldosteronism. Eur J Endocrinol, 178, R101–​11. Rossi GP (2011). A comprehensive review of the clinical aspects of pri- mary aldosteronism. Nat Rev Endocrinol, 7, 485–​95. Rossi GP, et al. (2014). An expert consensus statement on use of ad- renal vein sampling for the subtyping of primary aldosteronism. Hypertension, 63, 151–​60. Adrenal tumours Chortis V, et  al. (2013). Mitotane therapy in adrenocortical cancer induces CYP3A4 and inhibits 5α-​reductase, explaining the need for personalized glucocorticoid and androgen replacement. J Clin Endocrinol Metab, 98, 161–​71. Fassnacht M, et al. (2016). Management of adrenal incidentalomas: European Society of Endocrinology clinical practice guideline in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol, 175, G1–​G34. Fassnacht M, Kroiss M, Allolio B (2013). Update in adrenocortical carcinoma. J Clin Endocrinol Metab, 98, 4551–​64. 13.5.2  Congenital adrenal hyperplasia Nils P. Krone and Ieuan A. Hughes ESSENTIALS Congenital adrenal hyperplasia results from enzymatic defects in the pathways of adrenal steroidogenesis, with over 90% of cases being due to 21-​hydroxylase deficiency caused by autosomal recessive mutations in the CYP21A2 gene.

13.5.2  Congenital adrenal hyperplasia 2361 Classical presentation—​this is in the neonatal period with virilized external genitalia of a female infant, with the phenotype tradition- ally subdivided according to the presence (75%) or absence of salt wasting, which in affected males is the sole manifestation, and can, if unrecognized, be life-​threatening. Delayed presentations can occur, manifesting in women as hirsutism, oligomenorrhoea, and infertility, and in men as infertility or testicular adrenal rest tumours. Biochemical diagnosis—​in the newborn this is made on the basis of an elevated plasma concentration of 17-​OH-​progesterone; the diagnosis of non​classic congenital adrenal hyperplasia often re- quires a Synacthen (ACTH1-​24) stimulation test, with confirmation by sequencing of the CYP21A2 gene for specific mutations. Management—​this requires glucocorticoid and mineralocorticoid replacement sufficient to replenish salt balance and control ACTH hyperstimulation without incurring steroid side effects. In the ado- lescent and young adult attention is focused on continuing optimal steroid replacement, with clinical endpoints being potential repro- ductive function rather than linear growth. Fertility in women is compromised by scarring effects of surgery following genitoplasty in childhood, inadequate adrenal suppression that leads to anovulation, and an overall reduced maternal desire in women with congenital adrenal hyperplasia. Men with congenital adrenal hyperplasia should be screened for testicular adrenal rest tumours after puberty, and semen preservation should be considered in young adulthood. Genetic testing of the index case, their partner, and fetus allows pre- vention of major congenital malformation in an affected female in- fant by maternal treatment with dexamethasone during pregnancy. Introduction Congenital adrenal hyperplasia (CAH) comprises a family of in- herited autosomal recessive disorders of adrenal steroidogenesis (Fig. 13.5.2.1), characterized by deficiency of cortisol and an accu- mulation of substrate precursors. A pathophysiological consequence of inadequate cortisol production is ACTH hypersecretion associ- ated with hyperplastic adrenal glands. Genital anomalies are not a universal feature of all forms of CAH, and the original adrenogenital syndrome nomenclature is now seldom used. The rate-​limiting step is the delivery of cholesterol from the outer to the inner mitochondrial membrane to act as substrate for P450 side-​chain cleavage enzyme (CYP11A1), a mixed-​function oxidase side-​chain cleavage enzyme. The intracellular transport of cholesterol is controlled by several proteins, including steroidogenic acute regu- latory protein (StAR). The synthesis of cortisol is predominantly con- trolled by ACTH, acting via a G-​protein-​coupled receptor activation of cAMP. Table 13.5.2.1 is a summary of the types of enzymes involved in adrenal steroidogenesis and the location of the genes that encode each enzyme. Deficiency of 21-​hydroxylase activity is the cause of CAH in more than 90% of cases; it occupies the bulk of this chapter. CAH resulting from 21-​hydroxylase deficiency Clinical presentation Deficiency of 21-​hydroxylase presents with a wide range of clinical manifestation representing a disease continuum that can manifest from birth to adult life (Table 13.5.2.2). The classical form pre- sents in infancy, with ambiguous genitalia of the female newborn. An affected female fetus becomes virilized in utero as a result of the effect of excess adrenal androgens, converted peripherally to testos- terone, masculinizing the external genital anlagen. CAH is the com- monest cause of ambiguous genitalia of the newborn, now classified as 46,XX DSD (disorder of sex development; see Chapter 13.7.3). Milder forms of virilization manifest either as isolated clitoromegaly or as isolated labial fusion. Clinically relevant impairment of aldos- terone biosynthesis can be found in at least 75% of cases; in affected males, salt loss is initially the sole manifestation, as the onset of vir- ilization in males is delayed beyond infancy. Left unrecognized, this can lead to a life-​threatening salt-​losing crisis. The male without clinically apparent salt loss may not manifest until the second year of life or beyond, with signs of precocious sexual development, rapid growth, and tall stature with acceleration of the bone age. The testes remain prepubertal in size (<4 ml in volume), which is a useful distinguishing feature from central precocious puberty associated with increased gonadotropin secretion due to activation of the hypothalamic-​pituitary-​gonadal axis. Biochemistry In the absence of 21-​hydroxylase, accumulation of 17OH-​ progesterone (17OHP) occurs due to reduced cortisol pro- duction, initiating a compensatory ACTH-​induced increased steroidogenesis. 17OHP is generally an inert steroid, apart from displaying some competitive inhibition of aldosterone binding to the mineralocorticoid receptor when produced in excessive amounts. However, 17OHP serves as the substrate for increased androgen production that leads to the profound virilization characteristics of the newborn female with CAH. The pathways to excess androgen production are threefold. First, some 17OHP is converted to andro- stenedione and thence to testosterone in the liver and most likely the adrenal cortex. Similarly, increased dehydroepiandrosterone (DHEA) production leads via androstenedione conversion to tes- tosterone synthesis by action of enzymes with 17-​β hydroxysteroid dehydrogenase activity. Finally, another pathway has been pro- posed as a source of the increased androgen production in CAH that involves 5α and 3α reduction of 17OH-​progesterone to 17OH-​ allopregnanolone, which—​via androsterone and androstanediol intermediary steps—​leads to production of DHT (dihydrotes- tosterone), the most potent androgen. This has been dubbed the ‘backdoor pathway’ of adrenal steroidogenesis and is referred to later in the section on P450 oxidoreductase (POR) deficiency. It has first been suggested that this alternative pathway to androgens in humans is only present during fetal life and up to 1 year after birth; however, recent evidence suggests that this pathway might serve as source of androgens in untreated patients with CAH beyond the first year of life. Non​classical forms of 21-​hydroxylase deficiency are also recog- nized, and have an incidence as high as 1 in 500 to 1 in 1000 among white populations. The non​classical form in females may present with early onset of pubic hair growth, or after puberty with signs of hirsutism and symptoms of menstrual dysfunction. It is important to exclude an adrenal tumour as the cause of late-​onset signs of viril- ization. In adult females the symptoms and signs are similar to those associated with polycystic ovary syndrome. Male infertility has also been ascribed to 21-​hydroxylase deficiency. Tumours arising from

SECTION 13  Endocrine disorders 2362 the testicular adrenal rests may also be a presenting feature (see ‘Reproductive function’, next). The characteristic biochemical hallmark is an elevated plasma con- centration of 17OHP, generally greater than 300 nmol/​litre (normal <10 nmol/​litre). In most cases, 17OHP concentrations in patients with SW-​CAH are higher than in non-​salt-​losing patients. A more specific marker for 21-​hydroxylase deficiency is the metabolite 21-​ deoxycortisol, which is generated by 11-​hydroxylation of 17OHP (Fig. 13.5.2.1). The analysis of a spot urine steroid profile is diag- nostic with increased metabolites of 17OHP and 21-​deoxycortisol. A  24-​h-​urine collection is generally not required to achieve the diagnosis of inborn errors of steroidogenesis. Plasma testosterone can reach adult male concentrations. The patient with salt loss has hyponatraemia, hyperkalaemia, and elevated plasma renin levels. The newborn with salt-​losing CAH may also have hypoglycaemia. The diagnosis of non​classic CAH often requires a Synacthen (ACTH1-​24) stimulation test. This might be necessary to distinguish it from premature adrenarche, which is characteristically accom- panied by elevated dehydroepiandrosterone sulphate (DHEAS) and androstenedione concentrations. It is also useful to test if pa- tients with non​classic CAH are glucocorticoid replete and re- quire glucocorticoid stress cover. Studies of women with signs of hyperandrogenism show only about 5% of hormone profiles consistent with non​classic CAH. Polycystic ovary syndrome is a Cholesterol Cholesterol outer mitochondrial membrane inner ADR/Adx POR POR POR POR POR H6PDH H6PDH POR PAPSS2 b5 b5 ADR/Adx ADR/Adx ADR/Adx ADR/Adx ADR/Adx ADR/Adx ADR/Adx StAR CYP11A1 CYP17A1 CYP17A1 CYP11B1 CYP11B1 CORTISOL TESTOSTERONE ALDOSTERONE CYP21A2 HSD3B2 17OH-Pregnenolone 17OH-Progesterone 11-deoxycortisol 21-deoxycortisol CYP17A1 Dehydroepi androsterone Androste- nedione 11OH-Andro- stenedione 11keto-Andro stenedione CYP11B1 CYP11B1 CYP17A1 DHEA-S SULT2A1 HSD3B2 HSD11B1 HSD11B1 HSD11B2 HSD11B2 11keto-Testosterone 11OH-Testosterone Androstenediol HSD17B HSD17B HSD17B HSD3B2 HSD17B1 HSD3B2 CYP21A2 CYP11B2 CYP11B2 CYP11B2 18OH-corticosterone Corticosterone 11-deoxycorticosterone Pregnenolone Pregnenediol Pregnenediol THDOC THA, THB 5α THA, 5α THB 18OH-THA THALDO THF, 5α THF THS Pregnanetriolone 11O-Etiocholanolone 11O-Androsterone 11OH-Etiocholanolone Androsterone Etiocholanolone Androsterone Etiocholanolone DHEA, 16αOH-DHEA 11OH-Androsterone Pregnanetriol 17OH-pregnanolone Pregnanetriol Progesterone Fig. 13.5.2.1  Pathways of adrenal steroid biosynthesis. Enzymes are marked with boxes. Mitochondrial P450 cytochrome (CYP) type I enzymes requiring electron transfer via adrenodoxin reductase (ADR) and adrenodoxin (Adx) CYP11A1, CYP11B1, CYP11B2, are marked with a labelled box ADR/​Adx. Microsomal CYP II enzymes receive electrons from P450 oxidoreductase, CYP17A1, CYP21A2, are marked by circled POR. The 17,20-​lyase reaction catalysed by CYP17A1 requires in addition to POR also cytochrome b5 indicated by a circled b5. Hexose-​6-​ phosphate dehydrogenase (H6PDH) is the cofactor to HSD11B1 and given as ellipse. Recent evidence suggests that androstenedione and testosterone can be further metabolized by the adrenal. Urinary steroid hormone metabolites are given in italics below the plasma hormones. The asterisk (*) indicates the pathognomonic 11-​hydroxylation of 17OHP to 21-​deoxycortisol in 21-​hydroxylase deficiency. The conversion of androstenedione to testosterone is catalysed by HSD17B3 in the gonad and aldo-​keto reductase (AKR) 1C3 (HSD17B5) in the adrenal. StAR, steroidogenic acute regulatory protein; CYP11A1, P450 side-​chain cleavage enzyme; HSD3B2, 3β-​hydroxysteroid dehydrogenase type 2; CYP17A1, 17α-​hydroxylase; CYP21A2, 21-​hydroxylase; CYP11B1, 11β-​hydroxylase; CYP11B2, aldosterone synthase; HSD17B, 17β-​ hydroxysteroid dehydrogenase facilitated by AKR1C3 and/​ or HSD17B3; SULT2A1, sulfotransferase 2A1; PAPPS2, 3’-​phosphoadenosine 5’-​ phosphosulfate synthase 2; PAPPS2, 3’-​phosphoadenosine 5’-​phosphosulfate synthase 2.

13.5.2  Congenital adrenal hyperplasia 2363 well-​recognized and more frequent cause of hirsutism and infer- tility, although ultrasonographic evidence of polycystic ovaries is common in CAH. The definitive diagnosis of idiopathic hirsutism as being the result of 21-​hydroxylase deficiency can be confirmed by sequencing the CYP21A2 gene for one of the mutations associated with the non​classic form of CAH. Management in infancy and childhood Medical For the infant in salt-​losing crisis, treatment with intravenous saline and hydrocortisone is required. Blood glucose levels need moni- toring for hypoglycaemia. Otherwise, the well infant with CAH re- quires glucocorticoid replacement with oral hydrocortisone. Since most affected infants are losing salt, mineralocorticoid replacement is recommended in all patients with classic CAH at least during infancy. The average daily hydrocortisone replacement dose is aligned to the daily physiological cortisol production, which is approximately 6–​8 mg/​m2 per day and enterohepatic cortisol circulation, together yielding an average hydrocortisone replacement dose of 8–​10 mg/​m2 per day. In CAH, where suppression of the HPA-​axis is required to control androgen excess, a higher dose of up to 15 mg/​m2 per day is recommended. Importantly, doses of more than 17 mg/​m2 per day during puberty have a significant deleterious effect on growth vel- ocity and final height. In infancy there is evidence to suggest that an- drogen excess is not associated with increased height velocity and that lower hydrocortisone replacement doses can therefore be used (8–​10 mg/​m2 per day). Due to the lack of circadian rhythm of cortisol secretion in the first months of life, the dose can be split into ideally four even doses. However, after about 6 months of age half to two-​ thirds of the daily hydrocortisone dose should be replaced as early as possible in the morning. The total daily dose is divided into three or four daily doses in view of the short half-​life of hydrocortisone. In all forms of CAH a degree and spectrum of aldosterone de- ficiency and salt loss is present. During infancy there is relative aldosterone resistance with the immature kidney tubular system being unable to adequately respond to aldosterone action to regu- late water and sodium homeostasis. Thus, in the neonatal period and in early infancy a higher dose of fludrocortisone is required. Fludrocortisone doses during the first year of life are commonly 150 µg/​m2 per day. Sodium supplementation is also required to replace the sodium losses. If hyponatraemia persists on a standard dose of Table 13.5.2.1  Genes and proteins involved in adrenal steroidogenesis Activity Protein and synonyms Site of protein Gene/​chromosome Cholesterol transport Steroidogenic acute regulatory protein StAR Mitochondrial surface STAR/​8p11.2 Cholesterol side-​chain cleavage Cytochrome P450, subfamily XIA, polypeptide 1 P450scc Mitochondrion CYP11A/​15q23-​q24 3β-​hydroxysteroid dehydrogenase/​ isomerase 3βHSD Endoplasmic reticulum HSD3B1, HSD3B2/​1p11–​p13 17α-​hydroxylase and 17,20-​lyase Cytochrome P450, family 17, subfamily A, polypeptide 1 P450c17 Endoplasmic reticulum CYP17A1/​10q24–​q25 Oxidoreductase Cytochrome P450, oxidoreductase P450 Endoplasmic reticulum (membrane bound) POR/​7q11.2 21-​hydroxylase Cytochrome P450, subfamily XXIA, polypeptide 2 P450c21 Endoplasmic reticulum CYP21A2, CYP21P/​6p21.3 11β-​hydroxylase Cytochrome P450, subfamily XIB, polypeptide 1 P450c11 Mitochondrion CYP11B1/​8q21–​q22 Aldosterone synthase Cytochrome P450, subfamily XIB, polypeptide 2 Mitochondrion CYP11B2/​8q21–​q22 Table 13.5.2.2  Clinical manifestations of 21-​hydroxylase deficiency from birth to adulthood Type Female Male Age Clinical signs Age Clinical signs Classic Neonatal Ambiguous genitalia Occasional male phenotype Salt loss in 75% Late neonatal Occasional pigmented scrotum Salt loss in 75% Unexpected death Early childhood Penile growth Pubic hair Rapid linear growth Increased musculature Adult Testicular adrenal rest tumour Oligospermia Non​classic Late infancy Clitoromegaly Late infancy Occasional delayed salt loss Childhood Pubic hair Rapid growth Childhood Pubic hair Tall stature Adolescence Abnormal menses Hirsutism Acne Adolescence Not known Adult Hirsutism Oligomenorrhoea Adult Infertility

SECTION 13  Endocrine disorders 2364 fludrocortisone (150 µg/​m2 per day), the dose of fludrocortisone should only be increased further after 10–​12 mmol/​kg/​day of so- dium supplements have failed to normalize serum sodium. If higher fludrocortisone doses are necessary, close monitoring of blood pressure and renin is indicated to avoid iatrogenic hypertension. Commonly, sodium supplementation can safely be discontinued from usually 6 to 8 months of age when salt intake is sufficient via food. This should be approached on an individualized basis taking growth, development, and compliance into account. Commonly, the fludrocortisone requirement decreases with age. The principle of longer-​term medical treatment for CAH in child- hood is to provide sufficient glucocorticoid and, if necessary, min- eralocorticoid replacement for adequate homeostasis, but not at the expense of steroid side effects such as growth suppression, obesity, arterial hypertension, and other metabolic effects. There is a ten- dency to overtreat during infancy in particular when hydrocorti- sone doses above 10 mg/​m2 per day are given. This is compounded by the need to increase the hydrocortisone dose during episodes of intercurrent infection with fever, which often occur at this time. This may result in the suppression of the infantile growth spurt as well as the impairment of later growth and the development of obesity, a problem more common in adolescent girls with CAH. Serial measurements of growth are thus the clinical mainstay of monitoring treatment for CAH in childhood. This is supplemented by calculating the bone age at intervals of about 1 to 2 years. The assessment is particularly valuable as an index of undertreatment, where the resulting increase in adrenal androgens leads to an ad- vanced bone age. If left unchecked, this will eventually lead to a sig- nificant reduction in adult height. The child with non​classic CAH presenting in mid childhood often already has a marked increase in bone age (often >3–​4 years in advance of chronological age). In those cases, final height will be considerably reduced. Furthermore, the advanced bone age is asso- ciated with an earlier puberty, thus further shortening the period for statural growth. Linear growth ceases when bony epiphyses fuse at the ends of the long bones. This is mediated by oestrogens in both sexes, based on studies of a rare male with a disrupted oestrogen receptor, and several reported males with aromatase deficiency. These individuals were excessively tall because of continued growth in young adulthood resulting from lack of closure of the growth plate. Oestrogen treatment was effective in fusing the epiphyses in aromatase-​deficient patients, but not in the man with an oestrogen-​ receptor defect. There is a wide age range for the onset of puberty in normal chil- dren, but in girls with CAH the onset of menarche at a normal age (12–​13 years) and subsequent regular menses is a reasonable index of adequate control. Hydrocortisone is the predominant glucocorticoid used in infancy and childhood and remains the glucocorticoid of first choice during later life. However, when growth is mostly complete, a longer-​acting glucocorticoid such as prednisolone is occasionally substituted when therapy adherence is an issue. Dexamethasone is in current practice rarely used. It has a potency 80-​ to 100-​fold greater than hydrocortisone in suppressing ACTH-​induced steroidogenesis and its use is reserved for specific treatments. For example, it can usefully restore regular menses in adolescent girls with poorly controlled CAH or shrink testicular adrenal rest tumours in males. However, the dose must be carefully titrated to avoid side effects such as weight gain, striae, metabolic effects, and hypertension. Effective doses can be as small as 0.1 to 0.3 mg daily; its long half-​life enables a single daily dose to be employed. CAH requires biochemical monitoring to complement clinical indices of control, not dissimilar to the use of serial blood glu- cose and glycosylated haemoglobin measurements in diabetes. Figure 13.5.2.1 indicates that the equivalent analytes in CAH are 17OHP, androstenedione, and testosterone. Several monitoring strategies have been proposed; however, no data on validating a su- perior marker regarding long-​term outcomes exists. 17OHP has a marked diurnal rhythm and shows stress-​induced increases in con- centrations. Consequently, random single measurements can be misleading. Some have suggested a daily profile made up of samples collected in the early morning, at midday, in the late afternoon, and at bedtime might be more appropriate and informative. Capillary blood spot and saliva assays of 17OHP enable families to undertake home sampling. However, 17OHP concentrations can be used to monitor overtreatment with glucocorticoids indicated by normal- ization to suppression of 17OHP concentrations. Plasma andro- stenedione and testosterone are both useful longer-​term markers of control, and an age-​ and sex-​related increase usually reflects prolonged undertreatment, which in due course leads to excessive linear growth and an advancing bone age. Testosterone measure- ments are not reflective of CAH control in males from puberty on- wards because of the predominance of testicular testosterone at this age. Recent reports suggest that the use of additional plasma ster- oids such as 11-​keto-​testosterone, specific for adrenal testosterone production, might be a specific biomarker of control in adult males. Urinary steroid analysis by specific chromatographic techniques is primarily for diagnosis, but is also used in some centres to monitor treatment. The adequacy of mineralocorticoid replacement is best assessed by renin measurement. Renin values are normally higher in infants and young children than in adults and should be kept in the upper normal range to slightly elevated concentrations to avoid overexposure to mineralocorticoids. Surgical The degree of virilization of the external genitalia in female infants born with CAH can vary from mild clitoromegaly and some labial fusion, to marked clitoromegaly, complete labial fusion resembling a scrotal sac without palpable gonads, and a urethral opening on the tip of the phallus. In this circumstance the infant may initially be wrongly designated male. However, the absence of palpable gonads in the ‘scrotal sac’ on routine newborn examination should alert to the need for further investigation. A Prader scoring system, as shown in Fig. 13.5.2.2, is used to denote the degree of clitoromegaly and the site of insertion of the vagina into the common urogenital sinus. The surgery that might be required is a reduction clitoroplasty and a vaginoplasty to enable separate urethral and vaginal open- ings to be exposed on the perineum. There has been a change in policy regarding the threshold for deciding that the clitoris is too large and needs reducing in size. The current practice is not to op- erate on a clitoris of Prader stage less than III. Some centres have adopted the practice delaying surgery until the patient can provide full informed consent. Decisions about early surgery have been in- fluenced by the results of studies in women with CAH who report dissatisfaction with sexual function, purported to be the result of clitoral surgery undertaken when they were infants. In the presence

13.5.2  Congenital adrenal hyperplasia 2365 of marked clitoromegaly (Prader stages III–​V), parents generally want surgery performed early to make the appearance consonant with the female sex of rearing, even though they understand this may have consequences for their daughter in adulthood. The number of clitoroplasties can be significantly reduced by assessing the situation after 12–​18 months of age under adequate hormone re- placement therapy, when the clitoris is often partly or fully covered by the labia majora. Technical details of clitoroplasty can be found in surgical textbooks, but it is vitally important to preserve as much of the highly innervated neurovascular bundle surrounding the clitoris as possible. It is questionable whether vaginoplasty is needed before puberty, but many surgeons also undertake this procedure early to take advantage of favourable tissue healing at a young age. A fur- ther examination under anaesthetic is usually required at puberty to assess the vaginal anatomy and the need for any revision surgery or the use of vaginal dilators. For those infants where a decision has been taken not to perform a clitoroplasty, medical treatment must be adequate to avoid further clitoral enlargement generated by elevated testosterone levels. Genetics of 21-​hydroxylase deficiency CAH due to 21-​hydroxylase deficiency is an autosomal recessive condition. The CYP21A2 gene (also known as CYP21) is closely linked to the highly polymorphic major HLA histocompatibility complex on chromosome 6p21.3. It is 98% homologous with a pseudogene, CYP21AP1 (also known as CYP21P), which has ac- cumulated several mutations that render it functionally inactive. The genes are in tandem repeat with neighbouring genes such as tenascin TNXA/​B, complement C4A/​B, and the serine/​threonine nuclear protein kinase RP. The CYP21A2 gene comprises 10 exons. Misalignment and unequal crossing over between sister chroma- tids during meiosis leads to large gene deletions and chimeric genes (previously called large gene conversions). These are, when homo- zygous, always associated with the severe, salt-​losing form of CAH. The allele frequency of large gene deletions and chimeric genes as a cause of 21-​hydroxylase deficiency is about 25–​30% and is highest in northern European populations. The majority of CYP21A2-​inactivating mutations have occurred by gene-​conversion events between CYP21A2 and its pseudo- gene (CYP21AP1) that are small-​scale in nature. These include the eight most common point mutations and an 8bp deletion in exon 3. In most populations, pseudogene derived mutations can be de- tected in similar frequencies. Novel or rare mutations account for about 3–​5% of detected mutations in large cohorts. To date, over 90 additional rare pseudogene-​independent mutations have been described (http://​www.hgmd.cf.ac.uk; http://​www.cypalleles.ki.se/​ cyp21.htm). Linked microsatellites are useful for prenatal diagnosis when the family genotype has previously been ascertained. About 65–​75% of patients with 21-​hydroxylase deficiency are compound heterozygous (e.g. they are affected, but carry different mutations on each chromosome). The clinical phenotype of CAH correlates well with the less severely mutated allele, and conse- quently with the allele encoding for the higher residual activity of 21-​hydroxylase. The mutations that cause more than 90% of cases of 21-​hydroxylase deficiency are shown in Fig. 13.5.2.3 in relation to the expected phenotype. A common mutation in classic 21-​hydroxylase deficiency affects mRNA splicing and results from a nucleotide base change in the second intron. A stretch of nucleotides that is normally spliced out is retained, so that the translational reading frame is altered and an inactive protein synthesized. Most patients with this muta- tion have the salt-​losing form of CAH, but some patients who are homozygous for this mutation are salt replete. Presumably, enough normally spliced mRNA is generated to produce some enzyme activity (sometimes referred to as leaky transcription). Other ex- amples leading to salt loss are shown in Fig. 13.5.2.3. In vitro func- tional assays of wild-​type and mutant CYP21A2 enzymes using progesterone and 17-​hydroxyprogesterone as substrates show total absence of enzyme activity for mutations leading to salt loss. A specific mutation not associated with salt wasting occurs in exon 4, changing isoleucine to asparagine (Ile172Asn). This mutation results in an enzyme with about 1 to 2% of normal activity, suffi- cient for adequate aldosterone production. The non​classic form of 21-​hydroxylase deficiency is associated with a mutant enzyme that displays 20 to 50% of normal activity in functional in vitro assays. An example is Val281Leu in exon 7; this single mutation accounts for most non​classical cases of CAH. Other common examples of non​classic alleles include Pro30Leu and Pro453Ser. The definitive diagnosis of non​classic CAH as a cause of premature adrenarche in a child or idiopathic hirsutism in a woman may not even be Normal I II III IV V Normal Fig. 13.5.2.2  Prader genital scores (reproduced from Helv Paediatrc Acta). The upper panel denotes the stages of virilization, resulting in a penile urethra and high insertion of the vagina to a common urogenital sinus by stage V. The lower panel depicts the degree of clitoral hypertrophy with stage V resembling a penis.

SECTION 13  Endocrine disorders 2366 confirmed by an ACTH1-​24 stimulation test, but only secured by CYP21A2 analysis. Prenatal diagnosis and treatment Prenatal treatment is effective to prevent severe genital virilization in 46,XX individuals with 21-​hydroxylase deficiency. However, it remains controversial as its safety, including metabolic and psycho-​ intellectual consequences, remains to be fully defined. Counselling regarding prenatal dexamethasone therapy should be carried out well before conception, involving an endocrinologist, fetal medicine specialist, and clinical geneticist. Chorionic villus sampling and molecular analysis of the CYP21A2 gene has enabled an earlier and more reliable diagnosis to be made. A recent report suggests that the fetal CAH status can be correctly determined by targeted massively parallel sequencing of DNA in maternal plasma, as early as 5 weeks 6 days of gestation. Dexamethasone is the chosen glucocorticoid as it crosses the pla- centa unmetabolized by the placental 11β-​hydroxysteroid enzyme and is not protein-​bound. Maternal dexamethasone treatment needs to start once pregnancy is confirmed and ideally before the sixth and no later than the eighth week of gestation to achieve significant benefit in preventing 46,XX DSD, as fetal adrenal steroidogenesis is established by 7 to 8 weeks of gestation. CYP21A2 genotyping of the index case, parents, and unaffected siblings should have been performed previously. DNA analysis is then more reliable and can be coupled with using additional linked microsatellite markers. The conventional starting dose is 20–​25 μg/​kg per day based on prepregnancy body weight, administered in three divided doses (total maximum dose 1.5 mg/​day). In the traditional protocols, treatment was only continued to term in the case of an affected female fetus. Thus, seven out of eight fetuses will be exposed unnecessarily to dexamethasone for about 6 weeks during early gestation. However, analysis of free fetal DNA in the maternal circulation enables Y chromosome material to be detected by specific probes (e.g. for SRY, the male sex-​determining gene) as early as 7 weeks of gestation if the fetus is male. Thus, dexametha- sone exposure would be avoided in male fetuses. Both approaches have raised significant ethical concerns due to the high number of unaffected fetuses unnecessarily treated. With the most recent de- velopment determining the CAH status from maternal plasma, it appears possible that only affected fetuses will be treated overcoming previous ethical problems. gene del/conversion ∆8bp E6 cluster p.Leu307PhefsX6 p.Gin318X p.Arg356Trp intron splice p.lle172Asn p.Pro30Leu p.Val281Leu p.Pro453Ser Nonclassic Simple virilizing Salt wasting Mutation group Phenotypic severity 21-hydroxylase deficiency CAH form Speiser et al., 1992 96% Positive predictive values Prader genital stages 100% 97% 91% 100% 85% 90% 96% 92% 84% 73% 74% 53% 85% 87% 63% 65% 100% 98% 100% Krone et al., 2000 Stikkelbroeck et al., 2003 Finkielstain et al., 2011 Marino et al., 2011 Speiser et al., 1992 II–IV (IV) III–V (IV) II–IV III–V (IV) III–IV II–V II–V (IV) II–V (III) II–V I–IV I–IV (III) 0–IV (0) 0–IV (0) 0 0–V Wedell et al., 1994 Jääskeläinen et al., 1997 Krone et al., 2000 Null A B C 0–1% 1–5% 20–30% 30–50% 0% Fig. 13.5.2.3  Genotype–​phenotype correlation for most common mutations causing 21-​hydroxylase deficiency. The genotype–​phenotype correlation in CAH due to 21-​hydroxylase deficiency is based on in vitro CYP21A2 activity. Mutation groups Null and A are associated with the salt wasting (SW) form of 21-​hydroxylase deficiency (21OHD), group B with the simple virilizing (SV) form, and group C with the non​classic (NC) form. Positive predictive values are calculated from the cited publications. The variability in the degree of virilization of the female external genitalia in the different mutation groups (grading according to Prader genital stages) is shown in the lower panel. Modal values are provided in brackets where possible. Intron 2 splice, refers to the c.293–​13A/​C>G mutation (other names: I2G, IVS2-​13A/​C>G), Δ8bp refers to the p.Gly110ValfsX21 mutation, E6 cluster to the p.Ile236Asn, p.Val237Glu, and p.Met239Leu mutation cluster at exon 6; and the p.Leu307PhefsX6 mutation is also described as insT exon 7.

13.5.2  Congenital adrenal hyperplasia 2367 Fetal adrenal suppression is monitored by serial measurement of maternal plasma or urinary oestriol concentrations. This steroid metabolite is formed as a result of placental aromatization of weak androgen substrates uniquely produced by the fetal adrenal gland. This monitoring also enables the dexamethasone dose to be lowered in later pregnancy. More direct evidence of adrenal suppression can be obtained by collecting amniotic fluid for measurement of 17OHP and testosterone. The outcome of prenatal treatment is satisfactory in most cases when treatment is started early and continues uninterrupted to term. Thus the external genitalia in affected females are completely normal, or so mildly affected that surgery is not required. There have been isolated reports of other abnormalities in dexamethasone-​exposed infants, but no cluster of anomalies that appear to be teratogenically specific to glucocorticoids. In animal studies, exposure to steroids has resulted in growth restriction, cleft palate, thymic hypoplasia, and features of metabolic syndrome, such as hypertension and im- paired glucose tolerance. The hippocampus was also smaller in some species. However, the sensitivity of rodents to prenatal dexametha- sone is significantly higher than in human and studies in primates used significantly higher prenatal dexamethasone doses than being employed in human prenatal dexamethasone treatment. Thus, a direct translation of results from animal experiments into the human context has to be done with some caution. Studies of cognitive function and verbal and visuospatial working memory, in a controlled study of children aged 7 to 17 years who had been prenatally exposed to dexamethasone, generally gave normal results, with perhaps poorer verbal working memory in the treated group. There is some evidence that gender role behaviour may be affected in males who were exposed to prenatal dexamethasone. A more recent report analysing behavioural and emotional problems as well as social anxiety did not reveal differences between prenatally dexamethasone-​exposed unaffected individuals and controls. Maternal side effects occur in 10% of treated pregnancies, com- prising excess weight gain, striae, and hypertension in some. It is clear that virilization of the external genitalia in a female fetus with CAH can largely be prevented if appropriate doses of dexamethasone are started soon enough in pregnancy. However, a number of fetuses will be exposed unnecessarily to such treatment, and the uncertain- ties about effects on behavioural development during childhood and metabolic effects in adulthood mandates that prenatal treatment of CAH should be considered experimental and conducted in the con- text of clinical trials. Neonatal screening for CAH It is possible to screen newborn infants for CAH by measurement of 17OHP in dried blood spots collected on the Guthrie card cur- rently used for other conditions such as phenylketonuria and con- genital hypothyroidism. Most centres use standard immunoassays, but improved positive predictive values can be achieved with the use of techniques such as tandem mass spectrometry. False-​positive results may occur from sampling on the day of birth in low birth weight and sick preterm infants, and because of assay interference by cross-​reacting steroids. It is essential that laboratories establish cut-​off values of 17OHP that are specific for birth weight and gesta- tional age. The classic form of CAH has an incidence of 1 in 10 000 to 1 in 15 000 live births, based on newborn screening. Non​classical CAH is much more common (at least 1 in 1000), but is often not detected by newborn 17OHP measurement. A false-​negative result can occasionally occur with the simple virilizing form of CAH, or if the mother has been treated with glucocorticoids during pregnancy. CAH forms other than 21-​hydroxylase deficiency can be detected if tandem mass spectrometry is used as a second-​tier method. The main benefit of newborn screening for CAH is the detection of affected males early enough to prevent a life-​threatening salt-​ losing adrenal crisis and to avoid the risk of delayed diagnosis pre- senting with precocious pseudopuberty. Retrospective case studies have shown a preponderance of females over males with CAH, suggesting an increased male mortality when screening is not em- ployed. Other benefits include the avoidance of incorrect sex assign- ment (the Prader V virilized female thought to be a boy at birth) and earlier treatment, which may improve later growth and pubertal de- velopment. Not all countries, including the United Kingdom, have yet incorporated CAH in the panoply of conditions included in the newborn blood-​spot screening programme. Longer-​term outcome in CAH CAH is a condition that extends across the lifespan, with manage- ment issues that vary according to development and maturation in adulthood (Fig. 13.5.2.4). It is during adolescence and young adult- hood that the longer-​term outcomes of treatment instigated during infancy, early childhood, and even before birth become manifest. Adult stature and medical management Management of CAH in childhood has primarily focused on growth, but increasing awareness exists regarding the prevention of long-​term comorbidities. Growth velocity is a dynamic biomarker of control, and is sensitive to any deviation in age-​appropriate gluco- corticoid replacement doses. Closure of the growth plate at a bone-​ age of around 16 to 17 years of age signals the end of linear growth, and final height adjustment. Most adults with CAH are shorter than predicted from mean parental height, but are generally within the normal population range for adult height. Two meta-​analyses from 2001 and 2010 showed similar impairment of final height in patients with 21-​hydroxylase deficiencies with mean final height standard deviation scores (SDS) of –​1.37 and –​1.38, which, when corrected for target height in a subgroup, resulted in a mean final height SDS of –​1.21 and –​1.03, respectively. Interestingly, the more recent meta-​analysis did not find a significant association with age at diagnosis, gender, type and dose of steroid, and age of onset of pu- berty; however, patients treated with mineralocorticoids had better height outcomes compared to non​users. Final height outcome was better in a single large clinic population based in Munich, Germany, where final height SDS corrected for target height was –​0.6 for fe- males and –​0.9 for males with the salt-​losing form of CAH. The infantile growth spurt during the first two years of life deserves spe- cial attention as this growth phase is characterized by the highest postnatal growth velocity. An impaired growth velocity during this time significantly impacts on final height outcome. Thus, the lowest optimal dose for glucocorticoid replacement and normalization of sex hormone needs to be found as early in life as possible. An add- itional decrement in final height is attributed to a reduction in total pubertal growth and the use of longer-​acting glucocorticoids such as prednisolone. There is also a relationship between total cumula- tive glucocorticoid dose and a reduction in bone mineral density, the impact being more pronounced during puberty. Paradoxically,

SECTION 13  Endocrine disorders 2368 some studies show that the milder form of CAH that presents in later childhood might have a worse outcome for final height because of the combination of advanced bone age and earlier onset of puberty. In such a situation, the addition of a long-​acting gonadotropin-​ releasing hormone analogue to delay puberty, and supplementation with growth hormone, can have a beneficial effect on final height. Glucocorticoid replacement is preferably provided as hydrocor- tisone in adulthood, but longer-​acting glucocorticoid preparations, such as prednisolone and dexamethasone, are more often used. There is no fixed dose; the amount has to be calculated according to general well-​being, the absence of steroid side effects, and biochem- ical monitoring using indices such as serum 17OHP, androstene- dione, and testosterone. No current oral glucocorticoid preparation can replicate the normal diurnal cortisol rhythm, characterized by a rise in early morning levels. Modified-​release formulations of hydro- cortisone that better mimic circadian cortisol profiles are becoming available and are likely to optimize future glucocorticoid replace- ment in CAH. The requirement for mineralocorticoid replacement is lower in adulthood, so lower relative doses of fludrocortisone are used. Plasma renin activity or renin (upper normal range) and blood pressure measurements are needed to adjust the dose to avoid hypertension. There is a tendency for a higher body mass index in CAH, which is related to glucocorticoid dose. This can be associated with frank obesity, particularly occurring in the female during adolescence. At this time, control of CAH (as indicated by regularity of menses and measurements of 17OHP and testosterone) may be inad- equate, yet increasing the glucocorticoid dose merely compounds the weight problem. This is also associated with insulin resistance, which perpetuates the irregular menses and anovulation. A problem of compliance may be the explanation, but medical manipulation with longer-​acting steroids, the combined oral contraceptive pill, gonadotropin-​releasing hormone analogues, or antiandrogens can all be to no avail in restoring control towards regular menses, ovu- lation, and reduced hyperandrogenism. Bilateral adrenalectomy is sometimes undertaken in these circumstances to good effect redu- cing the adrenal hyperandrogenism; however, this procedure leaves the patient completely adrenal insufficient increasing the risk of ad- renal crisis. In the last decade, several studies explored the health status in adults with CAH. Prevalent features were obesity, hypercholester- olaemia, insulin resistance, osteopenia, and reduced quality of life and more recently the finding of increased cardiovascular and meta- bolic morbidity. Some studies identified a significant shortfall in the expected numbers of patients who should be attending specialized adult services for CAH, in stark contrast to facilities provided for children with this condition. Furthermore, individuals with 21-​ hydroxylase deficiency might be at increased risk to suffer from psychiatric disorders emphasizing the requirement for a multidis- ciplinary approach for all age groups. Prenatal

16 years to adulthood Fig. 13.5.2.4  CAH is a disorder that extends across the lifespan, with management issues that vary according to the development and maturation in adulthood. It is during adolescence and young adulthood that the longer-​term outcomes of treatment instigated during infancy, early childhood, and even before birth become manifest. It is now widely accepted that psychological support plays an important role, but is often not provided due to lack of resources. Due to a current lack of evidence, variable strategies to screen for comorbidities and monitor biochemical parameters exist. Prenatal dexamethasone treatment, surgery, and the use of additional drugs is controversially discussed.

13.5.2  Congenital adrenal hyperplasia 2369 Reproductive function Female The overall fertility rate is reduced in women with CAH. However, pregnancy rates for women with classical CAH trying to conceive appear to be normal. A number of factors appear to contribute to impaired fertility; these include vaginal stenosis and unsatisfactory sexual intercourse, ovulatory dysfunction from inadequate adrenal suppression, elevated progesterone levels acting like a contracep- tive mini-​pill, and a lower desire of women with CAH to become mothers. Studies of clitoral sensation and measures of sexual func- tion in adult women with CAH show impaired genital sensitivity and difficulties in sexual function (vaginal penetration and inter- course frequency) in those who had feminizing genitoplasty, com- pared with the minority of CAH women who did not have surgery and with non-​CAH controls. Elevated progesterone levels during the follicular phase of the cycle are associated either with anovula- tory cycles or a thin endometrium that is not receptive to blastocyst implantation. It is important to maintain androgen concentrations within the age-​related range for females throughout childhood and adolescence, as permanent effects such as voice lowering can be a feature in adulthood. Studies of psychosexual issues in women with CAH indicate overall satisfaction with their gender assignment, irrespective of the degree of prenatal masculinization. However, the rates of bisexual and homosexual orientation are increased, even in the milder forms of CAH. Women with CAH are less likely to have partners, are de- layed in their sexual debut, and have decreased frequency of sexual intercourse. These features are more evident with higher Prader vir- ilization scores. Even so, pregnancy rates in CAH have improved with better hor- monal control, and rates in non-​salt-​losers are similar to those in women without CAH. Indeed, the pregnancy rate does not differ between the types of CAH or even the normal population when the rate is expressed as the proportion of women who are actively wanting to become pregnant. Glucocorticoid replacement for the mother should not be dexamethasone, as this steroid will readily cross the placenta unmetabolized. Prepregnancy doses of hydrocortisone or prednis- olone can be used without the need to oversuppress slightly elevated levels of 17OHP and testosterone. The latter is efficiently converted to oestrogens by placental aromatase, thus protecting a female fetus from being virilized. Fludrocortisone requirements may sometimes increase due to the antimineralocorticoid properties of progesterone. The mineralocorticoid dose is adjusted according to postural blood pressure response, and serum sodium and potassium concentrations. Renin is physiologically increased during pregnancy and thus cannot serve as a monitoring tool. Parenteral hydrocortisone is provided during labour, although most women are likely to need a caesarean section because of previous genital surgery. Women may have an in- creased risk of gestational diabetes, but the pregnancies are other- wise normal. The offspring have normal birthweight, no increased frequency of malformations, and normal development. Intriguingly, there is a 2:1 ratio of girls to boys born to mothers with CAH. Male The adult male with CAH is not devoid of problems of a repro- ductive nature. Non​adherence with treatment is prevalent, which can result in elevated adrenal androgen secretion and suppression of pituitary luteinizing hormone secretion following aromatization to oestrogens. Some men have not gone through true puberty due to suppression of the hypothalamic-​pituitary-​gonadal axis if CAH control has already slipped during adolescence. Hypogonadotropic hypogonadism with small soft testes may ensue, with consequent oligospermia. This can be potentially rectified by reinstituting gluco- corticoid replacement. Adult males with CAH may develop benign testicular adrenal rest tumours that are the result of hyperstimulation of adrenal rest cells by ACTH. The origin of such cells is the common site of adrenal and gonadal development at the urogenital ridge. The prevalence of testicular adrenal rest tumours is reported to be up to 90% in males with CAH. They are evident on routine testicular ultrasound examination while often not palpable on clinical examination. Histological examination shows sheets of polygonal cells separated by dense fibrous tissue with focal lymphocytic infiltrates. There is a decrease in diameter of the tubules and reduced spermatogen- esis. Infertility is probably the result of long-​standing obstruction of the seminiferous tubules. Testis-​sparing surgery has not been suc- cessful in restoring normal pituitary–​gonadal function. Ultrasound examination shows that nearly one-​quarter of prepubertal boys with CAH have testicular adrenal rest tumours. It is now recommended that routine ultrasound screening should start at puberty. Early op- timization of treatment to reduce these tumours appears to be key to improve long-​term fertility outcomes. Semen preservation in young adulthood is an option to consider. Adrenal myelolipomas are also frequently found in adults with CAH. Other forms of CAH Steroid acute regulatory protein (StAR)
deficiency—​lipoid adrenal hyperplasia The first key initial step to enable cholesterol to be utilized as a sub- strate for steroidogenesis is the intracellular cholesterol transport from the outer to the inner mitochondrial membrane, mediated by the StAR (Fig. 13.5.2.1). StAR-​independent cholesterol transport only occurs at a low rate. Therefore, a defect in StAR leads to almost no substrate provision for P450 side-​chain cleavage enzyme (CYP11A1) and the production of all steroid hormones from adrenal and gonad is severely reduced. StAR is not necessary for placental progesterone production and does therefore not affect placental progesterone syn- thesis. In contrast to other CAH forms, the adrenals show an ac- cumulation of lipids, predominantly cholesterol esters resulting in large adrenals of yellow appearance. The most severe form presents with 46,XY DSD and combined adrenal insufficiency. Salt wasting typically develops in the neonatal period or after few weeks of life, but later onset also occurs. Females can show spontaneous pubertal development. The pathophysiology of StAR deficiency is explained by a ‘two-​hit’ hypothesis, whereby there is reduced transport of chol- esterol into the mitochondria which, under the continued stimula- tion of ACTH, leads to engorgement of steroid cells from cholesterol accumulation that ultimately severely disrupts cell function. Girls with StAR deficiency may start puberty spontaneously, but might later develop hypergonadotropic hypogonadism. Since fetal infant ovarian follicles are quiescent until puberty these are undamaged

SECTION 13  Endocrine disorders 2370 by the cholesterol accumulation and the length of the ovulatory cycle is too short to impair follicular hormone production. It is only after puberty, with elevated gonadotropins, that the ovarian cells become engorged and any cycles that occur are likely to be an- ovulatory. Polycystic changes may also ensue. A milder non​classical form of StAR deficiency is now recognized. Non​classic Lipoid CAH manifests later in life with adrenal insufficiency resembling non-​ autoimmune Addison’s disease and mild 46,XY DSD or normal male genital development. Adult phenotypes suggest that fertility may also be compromised in the non​classic form. In some patients, hypocortisolism is the only clinical manifestation. In such cases, non​classic CAH might be misdiagnosed as familial glucocorticoid deficiency. Treatment consists of glucocorticoid and mineralocor- ticoid replacement, and substitution of sex hormones in later life tailored to the individual requirements. P450 Side-​chain cleavage enzyme (CYP11A1) deficiency Cholesterol is converted to pregnenolone via three enzymatic re- actions (20α-​hydroxylation, 22-​hydroxylation, and cleavage of the cholesterol side chain) via the mitochondrial enzyme, CYP11A1, also known as P450scc. These initial steps in steroidogenesis are rate limiting and ACTH dependent. In contrast to StAR, CYP11A1 is generally required to maintain sufficient progesterone synthesis in pregnancy. However, a few cases of P450scc deficiency as a re- sult of mutations in the CYP11A1 gene have now been reported. The clinical presentation varies from prematurity and early onset of salt-​losing adrenal failure, to adrenal failure presenting in later childhood. Generally affected XY male patients have female external genitalia, but sometimes with clitoromegaly. However, some milder mutations seem to allow for normal masculinization of the external genitalia in 46,XY individuals clinically presenting with ‘isolated’ adrenal insufficiency. By contrast with StAR deficiency, the adrenals are small rather than grossly enlarged. 3β-​Hydroxysteroid dehydrogenase deficiency 3β-​Hydroxysteroid dehydrogenase/​isomerase (3βHSD) is a non-​ P450 membrane-​bound enzyme which converts Δ5 to Δ4 steroids in the adrenals and gonads. Hence it is needed for the synthesis of glucocorticoids, mineralocorticoids, progesterone, androgens, and oestrogens. HSD3B2 on chromosome 1p13.1 expresses the type II enzyme in the adrenals and gonads. The type I enzyme is expressed predominantly in the placenta and peripheral tissues, and is there- fore essential for maintaining high levels of progesterone in preg- nancy. Consequently, deficiency of 3βHSD activity is only associated with mutations in HSD3B2. Genital abnormalities occur, mainly in males, from inadequate masculinization resulting from the produc- tion of weak androgens by the testis and peripheral tissues. Affected females may be mildly virilized. Salt loss generally occurs. Diagnosis is confirmed by an elevated ratio of Δ5 (17-​hydroxypregnenolone, DHEA) to Δ4 (17OHP, androstenedione) steroids and analysis of urinary steroid metabolites. Molecular studies show that most patients have missense mu- tations in the HSD3B2 gene, and many are compound heterozy- gotes. There is close concordance between genotype and phenotype with respect to salt-​wasting forms. Significant conversion of Δ5 to Δ4 steroids occurs in peripheral tissues through the action of type I  3βHSD. Consequently, some patients have elevated concentra- tions of Δ4 steroids (17OHP, androstenedione), which has led to a mistaken diagnosis of 21-​hydroxylase deficiency. Spontaneous onset of puberty and menarche may occur in females, and gynaecomastia in males, presumably the result of conversion of Δ5 to Δ4 steroids by the type I enzyme. Based on HSD3B2 genotyping, the hormonal criteria have been refined for the diagnosis of HSD3B2 deficiency. 17OH-​pregnenolone concentrations and 17OH-​pregnenolone to cortisol ratios at baseline and after ACTH stimulations are of the highest discriminatory value in differentiating between patients af- fected by HSD3B2 deficiency and patients with milder biochemical abnormalities, who are negative for HSD3B2 mutations 17α-​Hydroxylase deficiency A single P450c17 (CYP17A1) microsomal enzyme catalyses 17α-​hydroxylase and 17,20-​lyase reactions. Both are required for the synthesis of sex hormones (C19 steroids), whereas only 17α-​hydroxylase activity is required for the synthesis of cortisol (C21, 17-​hydroxysteroids). The conversion of pregnenolone to 17-​ hydroxypregnenolone, and progesterone to 17-​hydroxyprogesterone, is 17α-​hydroxylase dependent, whereas the 17,20-​lyase reaction converts 17-​hydroxypregnenolone to DHEA and with by far lesser efficiency 17OHP to androstenedione. Mineralocorticoid biosyn- thesis is not dependent on the presence of the CYP17A1 enzyme, thus ACTH-​stimulated, low-​renin hypertension is a typical feature of the combined 17α-​hydroxylase/​17,20-​lyase deficiency from ex- cess production of C21,17-​deoxysteroids such as deoxycortico- sterone (DOC). There is an accompanying hypokalaemic metabolic alkalosis. Inadequate androgens in affected males cause a phenotype ranging from female genitalia to an ambiguous appearance or fea- tures of a hypospadic male. Female patients lack breast development and have primary amenorrhea. Patients with 46,XY karyotype may only present at adolescence because of pubertal failure. The external genitalia are female in appearance, with a blind-​ending vagina, testes that may be abdominal or inguinal in location, and absent pubic and axillary hair. The presentation is not unlike complete androgen in- sensitivity syndrome, apart from the lack of breast development. The excess production of corticosterone, a weak glucocorticoid, gener- ally prevents adrenal crisis in patients with CYP17A1 deficiency. Increased corticosterone, deoxycorticosterone, and progesterone and decreased concentrations of testosterone, oestradiol, and renin characterize this enzyme defect. Measurements of steroid metabol- ites delineate patterns indicative of 17α-​hydroxylase or 17,20-​lyase deficiency alone or combined. A rare variant of CYP17A1 deficiency has been described, as isolated 17,20 lyase deficiency, with largely preserved 17α-​hydroxylase activity. This manifests clinically with impaired sex steroid biosynthesis only, without apparent clinical evidence of mineralocorticoid excess or glucocorticoid deficiency. Affected boys have genital anomalies, whereas in girls the presenta- tion is one of delayed puberty. The 6.5 kb human CYP17A1 gene on chromosome 10q24.3 has eight exons. Over 100 different mutations have been described, without evidence of a hot spot apart from mutations in specific population. A frequent mutation is a 4 bp duplication in exon 8, which, as a result of altering the reading frame, leads to a short- ened C-​terminal sequence. This mutation is shared by Mennonites and other individuals in the Friesland region of the Netherlands, suggesting a founder effect. There are other geographic clusters in Southeast Asia (in-​frame deletion of residues 487–​489) and Brazilians of Portuguese and Spanish ancestry (Arg362Cys and

13.5.2  Congenital adrenal hyperplasia 2371 Trp406Arg, respectively). CYP17A1 mutations underlying the iso- lated 17,20 lyase deficiency variant are located within the area of the CYP17A1 molecule that is thought to interact with the cofactor cytochrome b5, thereby disrupting the electron transfer from P450 oxidoreductase to CYP17A1 specifically disrupting the conversion of 17OH-​pregnenolone to DHEA. Further biochemical work-​up has revealed that subclinical impairment of 17-​hydroxylation is present in these patients. Interestingly mutations in cytochrome b5 (CYB5A), a key redox cofactor for the 17,20-​lyase enzyme have been shown to be the cause for ‘true’ isolated 17,20-​lyase deficiency. P450 oxidoreductase (POR) deficiency Apparent combined deficiencies of the 17α-​hydroxylase (CYP17A1) and 21-​hydroxylase (CYP21A2) enzymes were reported in patients with ambiguous genitalia, but in whom analysis of the CYP17A1 and CYP21A2 genes revealed no mutations. The phenotypes com- prised mild degrees of virilization of affected female infants (and in some cases their mothers during pregnancy), which was self-​ limiting after birth, and some undermasculinization in affected males. This diagnostic conundrum was resolved when mutations were found in P450 oxidoreductase (POR), which is a membrane-​ bound flavoprotein that functions to transfer electrons from NADPH to all microsomal cytochrome P450 enzymes, including CYP17A1 and CYP21A2. Most patients with POR deficiency have skeletal malformations similar to malformations observed in Antley-​Bixler syndrome (OMIM 207410), adrenal and gonadal insufficiency, and disorder of sex development in both sexes (46,XX, DSD, and 46,XY DSD). However, normal development of the external genitalia has also been observed in either sex. Classic Antley–​Bixler syndrome is caused by autosomal dominant mutations in the fibroblast growth factor receptor 2 (FGFR2) gene, and manifests neither with abnor- malities of steroid metabolism nor ambiguous genitalia. Impairment of sterol biosynthesis, specifically of POR-​dependent 14α-​lanosterol demethylase (CYP51A1), may be causative. This is supported by the finding that children born to mothers treated during pregnancy with the CYP51A1 inhibitor fluconazole show evidence of Antley–​Bixler-​ like skeletal malformations. Skeletal abnormalities associated with POR deficiency include craniosynostosis, radiohumeral synostosis, choanal atresia, femoral bowing, and joint contractures. The dual deleterious effects of virilizing an affected girl and causing undermasculinization in an affected boy have two explan- ations. First, placental aromatase is also POR-​dependent and thus requires this cofactor for adequate aromatization of fetal adrenal androgen. Mutations in POR would thus explain virilization of an affected female at birth, as well as the mother, and the self-​limiting nature of the disorder. A second, more speculative, suggestion for the virilizing effect is the presence of a fetus-​specific ‘back door’ pathway of dihydrotestosterone production from 17OHP, which does not utilize androstenedione and testosterone as intermedi- aries. Such a scheme has been documented in the tammar wallaby (Macropus eugenii), and there is evidence that a similar pathway may be functional in the human fetus. The affected XY male is probably undermasculinized because of partial disturbance of 17,20-​lyase activity. POR deficiency can also manifest in the form of delayed and disordered puberty (ovarian cysts in girls). Milder de- fects in POR can manifest as polycystic ovary syndrome in women or as gonadal dysfunction in men. The POR gene is located on chromosome 7q11.2. More than 80 different mutations have been described, which are distrib- uted throughout all four functional domains of the POR protein. Most missense mutations are located within the central electron-​ transfer domain; Arg287Pro is prevalent in patients of European ancestry, whereas Arg457His is common in Japan. Baseline gluco- corticoid secretion is often sufficient, but the cortisol response to stress is often impaired, whereas mineralocorticoid deficiency is not common. Some patients have increased excretion of mineralo- corticoid metabolites and mild hypertension. Since most clinically used drugs are metabolized by POR-​dependent hepatic P450 en- zymes, POR mutations and polymorphisms can impact on variant drug metabolism. 11β-​Hydroxylase deficiency Deficient 11β-​hydroxylase activity accounts for 5 to 8% of cases of CAH, with an incidence of about 1 in 100 000 in non​consanguineous populations. The enzyme is required for the terminal conversion of 11-​deoxycortisol to cortisol, and DOC to corticosterone, but is lacking noteworthy 18-​hydroxylase and 18-​oxidase activity. The impaired cortisol production leads to increased ACTH stimulation and as a consequence to salt and water retention, low-​renin hyper- tension, and virilization, which appear to be more profound than in 21-​hydroxylase deficiency. Hypertension is commonly identi- fied in late childhood or adolescence, the severity not necessarily correlating with plasma concentrations of DOC. Complications of long-​standing hypertension include cardiomyopathy, retinal vein occlusion, blindness, and stroke. There is typically hypernatraemia, hypokalaemia, and sup- pressed renin concentrations. The diagnosis is confirmed by ele- vated concentrations of 11-​deoxycortisol and DOC in plasma, and their tetrahydro metabolites in urine. Plasma concentrations of androstenedione and testosterone are increased. Moderately elevated levels of 17OHP may lead to an erroneous diagnosis of 21-​hydroxylase deficiency. Of note 17OHP concentrations are commonly magnitudes lower than in 21-​hydroxylase deficiency and often paired with excessively increased androstenedione con- centrations. Treatment requires only glucocorticoid replacement, although transient salt wasting may follow an initial fall in levels of the mineralocorticoid, DOC. Treatment can be monitored by measuring 11-​deoxycortisol, androstenedione, and testosterone as well as normalization of renin and aldosterone concentrations. Antihypertensive treatment may be necessary if hypertension has been long-​standing and should be initiated with a mineralo- corticoid receptor blocker. Milder or non​classic deficiency also occurs, and manifests similar features to the non​classic form of 21-​hydroxylase deficiency. Steroid 11β-​hydroxylase activity is a function of the CYP11B1 gene located on chromosome 8q21–​22 in tandem with CYP11B2, which encodes aldosterone synthase, the enzyme involved in mineralocorticoid synthesis. CYP11B1 is expressed in the zona fasciculata, whereas CYP11B2 is exclusively expressed in the zona glomerulosa, where it not only catalyses the 11β-​hydroxylation of DOC to corticosterone, but also catalyses the terminal steps of al- dosterone synthesis. More than 90 mutations causing 11β-​hydroxylase deficiency are distributed throughout the CYP11B1 gene. CYP11B1-​inactivating mutations have been shown to be distributed over the entire

SECTION 13  Endocrine disorders 2372 coding region consisting of 9 exons. Although a cluster is reported in exons 2, 6, 7, and 8, real hot spots such as in 21OHD do not exist. A  higher incidence of this form of CAH occurs in some populations such as in Moroccan Jews, and is associated with an Arg448His mutation. Prenatal treatment with dexamethasone has been used successfully in this form of CAH to prevent virilization of an affected female fetus. A non​classic form of 11β-​hydroxylase deficiency is described that can manifest as premature adrenarche, or hirsutism and infertility in late childhood, adolescence, or adulthood. Aldosterone synthase, encoded by CYP11B2, catalyses the ter- minal steps of aldosterone synthesis: the 11β-​hydroxylation of DOC, 18-​hydroxylation to 18-​hydroxycorticosterone, and 18-​oxidation to aldosterone. Patients with CYP11B2 deficiency generally respond well to fludrocortisone (start dose 150 µg/​m2 per day in neonates and infancy) and can also benefit from salt supplementation. Patients who presented with failure to thrive generally show a good catch-​up growth after initiation of treatment. The need for mineralocorticoid treatment lessens in later life. A chimaeric form of the two CYP11B genes, under the control of the CYP11B1 promoter, leads to an autosomal dominant form of hyper- tension, termed glucocorticoid remediable hyperaldosteronism. This condition is suppressible with dexamethasone because of its ACTH dependence. FURTHER READING Arlt W, et al. (2010). Health status of adults with congenital adrenal hyperplasia:  a cohort study of 2013 patients. J Clin Endocrinol Metab, 95, 5110–​21. Bidet M, et  al. (2010). Fertility in women with nonclassical con- genital adrenal hyperplasia due to 21-​hydroxylase deficiency. J Clin Endocrinol Metab, 95, 1182–​90. Carroll AE, Downs SM (2006). Comprehensive cost-​utility analysis of newborn screening strategies. Pediatrics, 117, 5287–​95. Casteras A, et al. (2009). Reassessing fecundity in women with classical congenital adrenal hyperplasia (CAH): normal pregnancy rate but reduced fertility rate. Clin Endocrinol, 70, 833–​7. Crouch NS, et al. (2008). Sexual function and genital sensitivity fol- lowing feminizing genitoplasty for congenital adrenal hyperplasia. J Urol, 179, 634–​8. Dhir V, et  al. (2009). Steroid 17alpha-​hydroxylase deficiency: functional characterization of four mutations (A174E, V178D, R440C, L465P) in the CYP17A1 gene. J Clin Endocrinol Metab, 94, 3058–​64. Falhammer H, et al. (2014). Increased mortality in patients with con- genital adrenal hyperplasia due to 21-​hydroxylase deficiency. J Clin Endocrinol Metab, 99, E2715–​21. Falhammar H, et al. (2015). Increased cardiovascular and metabolic morbidity in patients with 21-​hydroxylase deficiency:  a Swedish population-​based national cohort study. J Clin Endocrinol Metab, 100, 3520–​8. Finkielstain GP, et al. (2012). Clinical characteristics of a cohort of 244 patients with congenital adrenal hyperplasia. J Clin Endocrinol Metab, 97, 4429–​38. Hagenfeldt K, et al. (2008). Fertility and pregnancy outcome in women with congenital adrenal hyperplasia due to 21-​hydroxylase defi- ciency. Hum Reprod, 23, 1607–​13. Hirvikoski T, et al. (2007). Cognitive functions in children at risk for congenital adrenal hyperplasia treated prenatally with dexametha- sone. J Clin Endocrinol Metab, 92, 542–​8. Hirvikoski T, et  al. (2011). Gender role behaviour in prenatally dexamethasone-​treated children at risk for congenital adrenal hyperplasia—​a pilot study. Acta Paediatr, 100, e112–​9. Hughes IA (2006). Prenatal treatment of congenital adrenal hyper- plasia: do we have enough evidence? Treat Endocrinol, 5, 1–​6. Hughes IA (2007). Congenital adrenal hyperplasia: a lifelong disorder. Horm Res, 68, Suppl 5, 84–​9. Kamrath C, et  al. (2012). Increased activation of the alternative ‘backdoor’ pathway in patients with 21-​hydroxylase deficiency: evi- dence from urinary steroid hormone analysis. J Clin Endocrinol Metab, 97, E367–​75. Khattab A, Marshall I (2019). Management of congenital adrenal hyperplasia: beyond conventional glucocorticoid therapy. Curr Opin Pediatr, 31, 550–4. Kim CJ, et al. (2008). Severe combined adrenal and gonadal deficiency caused by novel mutations in the cholesterol side chain cleavage en- zyme, P450ssc. J Clin Endocrinol Metab, 93, 696–​702. Krone N, Arlt W (2009). Genetics of congenital adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab, 23, 181–​92. Lajic S, et al. (2011). Long-​term outcome of prenatal dexametha- sone treatment of 21-​hydroxylase deficiency. Endocr Dev, 20, 96–​105. Mallappa A, et al. (2015). A phase 2 study of chronocort, a modified-​ release formulation of hydrocortisone, in the treatment of adults with classic congenital adrenal hyperplasia. J Clin Endocrinol Metab, 100, 1137–​45. Meyer-​Bahlburg HFL, et  al. (2008). Sexual orientation in women with classical or non-​classical congenital adrenal hyperplasia as a function of degree of prenatal androgen excess. Arch Sex Behav, 37, 85–​99. Miller WL, Auchus RJ (2011). The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev, 32, 81–​151. Nermoen I, et al. (2011). High frequency of adrenal myelolipomas and testicular adrenal rest tumours in adult Norwegian patients with classical congenital adrenal hyperplasia due to 21-​hydroxylase de- ficiency. Clin Endocrinol, 75, 753–​9. New MI, et al. (2014). Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-​free fetal DNA in maternal plasma. J Clin Endocrinol Metab, 99, E1022–​30. Nimkaru S, New MI (2008). Steroid 11β-​hydroxylase deficiency con- genital adrenal hyperplasia. Trends Endocrinol Metab, 19, 96–​9. Nordenskjöld A, et al. (2007). Type of mutation and surgical procedure effect long-​term quality of life for women with congenital adrenal hyperplasia. J Clin Endocrinol Metab, 93, 380–​6. Nordenstrom A (2011). Adult women with 21-​hydroxylase deficient congenital adrenal hyperplasia, surgical and psychological aspects. Curr Opin Pediatr, 23, 436–​42. Ogilvie CM, et al. (2006). Congenital adrenal hyperplasia in adults: a review of medical, surgical and psychological issues. Clin Endocrinol, 64, 2–​11. Parajes S, et al. (2009). Functional consequences of seven novel mu- tations in the CYP11B1 gene:  four mutations associated with nonclassic and three mutations causing classic II beta-​hydroxylase deficiency. J Clin Endocrinol Metab, 95, 779–​88. Prader A, Gurtner HP (1955). The syndrome of male pseudoherm- aphroditism in congenital adrenocortical hyperplasia without

13.5.2  Congenital adrenal hyperplasia 2373 overproduction of androgens (adrenal male pseudohermaphrodism). Helv Paediatr Acta, 10, 397–​412. Reisch N (2019). Pregnancy in congenital adrenal hyperplasia. Endocrinol Metab Clin North Am, 48, 619–41. Reisch N (2019). Review of health problems in adult patients with classic congenital adrenal hyperplasia due to 21-hydroxylase defi- ciency. Exp Clin Endocrinol Diabetes, 127, 171–7. Scott RR, Willer WL (2008). Genetic and clinical features of P450 oxidoreductase deficiency. Horm Res, 69, 266–​75. Speiser PW (2009). Nonclassic adrenal hyperplasia. Rev Endocr Metab Disord, 10, 77–​82. Speiser PW, et al. (2010). Congenital adrenal hyperplasia due to steroid 21-​hydroxylase deficiency: an Endocrine Society Clinical Practice guideline. J Clin Endocrinol Metab, 95, 4133–​60. Turcu AF, et  al. (2016). Adrenal-​derived 11-​oxygenated 19-​carbon steroids are the dominant androgens in classic 21-​hydroxylase defi- ciency. Eur J Endocrinol, 174, 601–​9. Wallensteen L, el al. (2016). Evaluation of behavioral problems after prenatal dexamethasone treatment in Swedish adolescents at risk of CAH. Horm Behav, 85, 5–​11. White PC (2009). Neonatal screening for congenital adrenal hyper- plasia. Nat Rev Endocrinol, 5, 490–​8.

13.6 Reproductive disorders 2374

13.6 Reproductive disorders 2374

13.6.1 Ovarian disorders 2374 Stephen Franks, Kate

13.6.1 Ovarian disorders 2374 Stephen Franks, Kate Hardy, and Lisa J. Webber

CONTENTS 13.6.1 Ovarian disorders  2374 Stephen Franks, Kate Hardy, and Lisa J. Webber 13.6.2 Disorders of male reproduction and
male hypogonadism  2386 P.-​M.G. Bouloux 13.6.3 Benign breast disease  2406 Gael M. MacLean 13.6.4 Sexual dysfunction  2408 Ian Eardley 13.6.1  Ovarian disorders Stephen Franks, Kate Hardy, and Lisa J. Webber ESSENTIALS The ovary produces (1) oocytes—​germ cells in the ovary have under- gone the first meiotic division to become oocytes in primordial fol- licles by the time of birth, with about 400 of these ovulating during reproductive life; and (2) hormones—​oestradiol, progesterone, an- drogens, and two non​steroidal glycopeptides, inhibin A and B. Ovulation and hormonal secretion are regulated by the pituitary gonadotropins, follicle-​stimulating hormone (FSH) and luteinizing hormone (LH), production of which is controlled by pulsatile release of the decapeptide gonadotropin-​releasing hormone from the hypo- thalamus. LH and FSH act on maturing ovarian follicles: LH inducing androgen secretion from the thecal layer, and FSH stimulating the inner granulosa cell layer to aromatize androgens to generate oestro- gens. After ovulation, the corpus luteum produces oestradiol as well as progesterone: these two hormones, together with inhibins, exert feedback inhibition on gonadotropin release. In the normal menstrual cycle, differential sensitivity to FSH leads to enhanced growth of a dominant follicle which becomes respon- sive to LH, with enhanced steroidogenesis and greatly increased oes- tradiol concentrations. This triggers a surge in LH, a positive feedback phenomenon that induces resumption of meiosis in the oocyte and ovulation by rupture of the follicle, which is then induced to secrete abundant progesterone. Progesterone suppresses gonadotrophin release and—​if trophoblastic gonadotrophin secretion fails to occur (in the absence of fertilization and pregnancy)—​the corpus luteum breaks down, inducing the onset of a new cycle. Involuntary infertility affects about 15% of couples, with ovulatory disorders accounting for 25 to 30%. Disorders of ovulation Disorders of ovulation usually result in perturbation of normal cyc- lical menses, leading to amenorrhoea, oligomenorrhoea (more than 6 weeks between periods) or very irregular menses. Aetiology—​the condition may be (1)  primary—​menarche de- layed beyond 16  years, no previous periods; may be caused by developmental disorders; or (2)  secondary—​at least one previous spontaneous period; causes include premature (primary) ovarian in- sufficiency, hypothalamic/​pituitary dysfunction, and polycystic ovary syndrome, which is the commonest cause of oligomenorrhoea. Premature (primary) ovarian insufficiency  —​defined as ovarian failure at less than 40 years; cause unknown in most cases but may be associated with organ-​specific autoimmune diseases, chromosomal abnormalities (e.g. Turner syndrome, 45X) or be iatrogenic (young adult cancer survivors); high FSH, low oestrogen; often treated with hormone replacement therapy, but oocyte donation is the only op- tion for conception. Hypothalamic/​pituitary disorder—​characterized by low FSH, low oestrogen; most commonly related to (a)  weight loss—​often as- sociated with an underlying eating disorder; fertility treatment is unwise until normal body mass index has been achieved; or (b) hyperprolactinaemia. Polycystic ovary syndrome—​typically presents with amenorrhoea in association with clinical signs of hyperandrogenism (hirsutism, persistent acne, male-​pattern alopecia); wider definition requires two of (1) oligo-​ and/​or anovulation, (2) clinical and/​or biochem- ical signs of hyperandrogenism, and (3)  polycystic ovaries. Is as- sociated with a metabolic disorder including insulin resistance/​ hyperinsulinaemia/​impaired glucose tolerance and dyslipidaemia. Management is mainly targeted at relief of symptoms/​complications with diet, antiandrogens (e.g. cyproterone acetate, spironolactone). Anovulatory women who wish to conceive usually respond to ovula- tion induction therapy (e.g. clomiphene). 13.6 Reproductive disorders

13.6.1  Ovarian disorders 2375 Hirsutism Mild to moderate long-​standing hirsutism in women with regular menses is very likely to be associated with polycystic ovary syn- drome, which can be confirmed by finding normal/​slightly elevated serum testosterone concentration and pelvic ultrasonog- raphy to determine ovarian morphology. Patients with a short history of hirsutism (particularly if severe), symptoms suggesting other endocrine disorders (e.g. Cushing’s syndrome), and/​or serum testosterone above 5 nmol/​litre
(normal range 0.5–​3.0) require further investigation including ovarian and/​or adrenal imaging (for androgen-​secreting tumour) and biochemical tests for Cushing’s syndrome and congenital ad- renal hyperplasia. Introduction Ovarian disorders are very common. Involuntary infertility affects an estimated 15% of couples and disorders of ovulation account for 25–​30% of the causes of infertility. In most cases, disorders of ovulation are attributable to a treatable, endocrine abnor- mality. Indeed, polycystic ovary syndrome, a major contributor to endocrine-​related infertility, is the commonest endocrine disorder in women, with a prevalence in excess of 5% in the female popu- lation of reproductive age. Disorders of ovarian function usually manifest themselves as irregular, infrequent, or absent menses. Hirsutism, or excess, male-​pattern body hair is another common manifestation of ovarian (and, less often adrenal) dysfunction. In the following chapter, ovarian development, the physiology of the hypothalamic–​pituitary–​gonadal axis, and the hormonal changes of the normal menstrual cycle will be described as a preface to the description of disorders of ovulation, their investigation, and man- agement. The causes, investigation, and management of hirsutism will also be elucidated. Ovarian development Ovarian development is essentially complete by about six months of fetal life and at this time the ovaries contains some 6–​7 million germ cells. By the time of birth, the number of germ cells has fallen to 1–​2 million and the remaining germ cells have entered the first meiotic division to form oocytes. Each oocyte is surrounded by a single layer of flattened, somatic (pregranulosa) cells, to form the primordial follicle. It is the primordial follicles that constitute the ‘resting’ pool that must provide sufficient oocytes to last a normal reproductive lifespan. Ovarian organogenesis and follicle formation The fetal ovary is formed from three embryonic cell lineages: the coelomic epithelium, the mesenchyme of the mesonephros (primi- tive kidney) and primordial germ cells, which are first observed within the extraembryonic tissue of the yolk sac. At about four weeks the coelomic epithelium thickens over the mesial aspect of the mesonephros forming the gonadal ridge. The underlying mes- enchymal cells of the mesonephros also proliferate and the gonadal ridge protrudes into the coelomic cavity as the gonadal anlagen or primordium. Concurrently, primordial germ cells begin to migrate from the yolk sac with an amoeboid-​like action. Before leaving the yolk sac, as well as en route, they divide by mitosis and enter the primordium. Once populated by primordial germ cells, the primor- dium becomes the indifferent gonad. This initial development of the ovary is identical to that of the testis until morphological changes occur at around the sixth week of embryonic life that makes the male and female gonad distinguishable. Evidence from the mouse indi- cates that primordial germ cells differentiate once they have reached the female gonad, lose their migratory ability, and are then known as oogonia. The distinct morphology of the ovary is recognizable by the end of the sixth week, a few days after that of the testis. Oogonia dramat- ically increase in number by mitosis, germ cell number reaching a maximum of about seven million at mid-​gestation. Although mi- tosis can continue until birth, by the third trimester germ cell loss, occurring by apoptosis or (programmed cell death) exceeds the rate of mitosis and germ cell number falls. Proliferation of the coelomic epithelium forms protrusions into the mesenchyme which gives rise to the sex cords, surrounding nests of primordial germ cells/​oogonia. Once enclosed by these cords of somatic cells, the germ cells cease mitosis and enter mei- osis several weeks after sex-​specific gonadal differentiation. This division is arrested, one to two weeks later, at the diplotene stage of the first meiotic division, resulting in the formation of oocytes. The oocyte remains arrested in the first meiotic division and meiosis is completed only in the mature follicle that is destined to ovulate. Resumption of meiosis is triggered by the surge of LH preceding ovulation, which may be many decades later. Newly formed oocytes become enclosed in a single flattened layer of somatic, pregranulosa cells surrounded by a basement membrane to form the primordial follicle (Fig. 13.6.1.1). Follicle formation begins close to the cortico-​medullary boundary and primordial follicles appear to separate from the sex cords. Oogonia and oocytes that have not formed follicles by association 20 mm 0.2 mm Preantral (gonadotrophin independent) several months Antral (gonadotrophin dependent) 6 weeks Primordial Primary Secondary Early antral Graafian (preovulatory) Fig. 13.6.1.1  Follicle development in the human ovary. The various stages of preantral and antral (gonadotropin-​dependent) development of the follicle are depicted ranging from the primordial (quiescent) stage in which the oocyte is enclosed in a single layer of pregranulosa cells, to the preovulatory stage. This process takes several months. Adapted from Hardy K, et al. (2000). In vitro maturation of oocytes. Br Med Bull, 56, 588–​602. Copyright (2000), with permission from Oxford University Press.

section 13  Endocrine disorders 2376 with somatic cells generally remain within the sex cords, where they undergo apoptosis. The origin of granulosa cells is still not completely certain, and may vary from one species to another but it is likely that they are derived from the ovarian surface epithelium. Follicle development in the normal ovary Primordial follicles provide the ‘stock’ of oocytes which must last for up to 50 years. Initiation of follicle growth (i.e. progression of the follicle from the primordial to the early growing phases) must be tightly regulated to ensure a steady supply of oocytes for ovula- tion during a normal reproductive life-​time. However, the factors responsible for controlling initiation of growth are yet to be deter- mined. The first indication of growth of the follicle is proliferation and a resultant change in shape of the granulosa cells, which be- come more cuboidal in appearance (Fig. 13.6.1.1). Follicles pass through a transitional, or intermediary, stage in which a propor- tion of the granulosa cells are cuboidal, and the rest remain flat- tened. This is followed by the primary stage in which the oocyte is enclosed in a single layer of completely cuboidal cells. By this stage, the oocyte has increased significantly in volume. Follicle develop- ment progresses by formation of a second layer of granulosa cells, and at this stage the first theca cells, derived from surrounding stroma, begin to organize around the granulosa layer. This is fol- lowed by formation of further layers of granulosa and theca cells (with enlargement of the oocyte) to form a multilayered preantral follicle. The outer layers of the theca comprise cells which are similar to those in surrounding stroma and constitute the theca externa. The cells of the inner layers become polyhedral and form the theca interna, the site of androgen production in large preantral and antral follicles. Eventually, the theca interna of each follicle receives its own blood supply. Development of the follicle to the multilayered preantral stage can progress without the need for gonadotropins. It is unclear how long it takes for a follicle to pro- gress from the primordial to large preantral follicle, but estimates suggest that this may be at least three months. Granulosa cells continue to proliferate, and a fluid-​filled space (the antrum) eventually forms between them and continues to en- large: it is now an antral follicle, and this is the stage at which the gonadotropins take over as the major regulators of follicle develop- ment (Fig. 13.6.1.1). It is from this stage that the greatest expansion of the follicle occurs, in terms of granulosa and theca cell numbers, antrum size as well as oocyte growth and overall follicle diameter. Follicles that reach this stage are considered to be part of a ‘select- able’ follicle pool from which the so-​called ‘dominant’ follicle will arise (i.e. that which is the most likely to complete maturation and ovulate). This pool may number 10–​15 follicles between the two ovaries in young women and declines with age, averaging 10 at 30 years and 5 at 40 years. It will be evident that only a small frac- tion of the total pool of follicles is destined to ovulate. The rest will undergo atresia (death by apoptosis) along the way. Although it is likely that follicle loss by atresia occurs at all stages of follicle devel- opment, the highest proportion of atretic follicles is seen during the gonadotropin-​dependent, antral stages. As described later, selection of a single follicle for ovulation in the human menstrual cycle, inev- itably involves regression and demise of subsidiary follicles within the same cohort. The hypothalamic–​pituitary–​ovarian axis Like the testis, the ovary has two major functions: (1) the produc- tion of gametes and (2) the secretion of hormones (particularly sex steroids) that affect development and function of the reproductive tract, as well as having important peripheral effects on muscle, bone, and skin. Like all classic endocrine organs, the function of the ovary is dependent upon regulation by pituitary hormones, which, in turn are regulated by hypothalamic signals (Fig. 13.6.1.2). Gonadotropin-​releasing hormone (GnRH) is a decapeptide, secreted by the hypothalamus in a pulsatile manner, the frequency of pulses (between 60 and 180 minutes, according to the stage of the menstrual cycle), having a profound influence on the response of the pituitary gonadotropins (which are glycoprotein hormones) to GnRH. The episodic secretion of GnRH is reflected in the pattern of circulating gonadotropins, the ‘pulses’ of luteinizing hormone (LH) being more discrete than those of follicle-​stimulating hormone (FSH) because of the shorter half-​life of LH in the circulation. LH and FSH act in con- cert on the maturing large ovarian follicles. LH stimulates the theca layer of the follicle to produce androgens (androstenedione and tes- tosterone) while FSH acts specifically on the inner, granulosa cell layer of the mature follicle—​which lacks the capacity to synthesize androgens—​to convert androgens to oestrogens (the so-​called two-​ cell, two gonadotropins hypothesis). Following ovulation, oestradiol continues to be produced by the corpus luteum but the principal circulating steroid at this stage of the cycle is, of course, proges- terone. Oestradiol, during the mid-​follicular phase of the cycle (see Hypothalamus LH FSH Oestradiol Progesterone Ovary Blood Pituitary (GnRH) Gonadotrophin releasing hormone − + Fig. 13.6.1.2  The hypothalamic–​pituitary–​ovarian axis. Courtesy of Prof K Hardy.

13.6.1  Ovarian disorders 2377 next section) and progesterone (luteal phase) exert a negative feed- back effect on both pituitary and hypothalamus to inhibit the secre- tion of gonadotropins. The ovary also produces two closely related, non​steroidal (glycopeptide) hormones that selectively inhibit FSH and contribute to negative-​feedback inhibition: inhibin B (produced by developing follicles in the follicular phase) and inhibin A (pro- duced mainly by the corpus luteum). The extraordinary feature of the hypothalamic–​pituitary–​ovarian axis is the phenomenon of ‘positive feedback’ stimulation of gonadotropins by oestradiol in mid-​cycle, which results in the LH ‘surge’ and ovulation, as described next. The menstrual cycle The endocrine events of the menstrual cycle are summarized in Fig. 13.6.1.3. At the beginning of each normal cycle (convention- ally taken as the first day of menses), there is a cohort of follicles, ranging between 2 and 5 mm in diameter, which are dependent on, and responsive to FSH. Between the late luteal phase of the previous cycle and the early follicular phase, the negative feedback signal pro- vided primarily by progesterone is removed and the concentrations of FSH rise. This intercycle rise of FSH exceeds a notional threshold level that encourages follicle maturation. Of the cohort of follicles that arrive at this FSH ‘window’, only one (or occasionally two) is destined to complete the journey to ovulation. This is the follicle that is most responsive to FSH. It is often the largest of the cohort but not necessarily so. As the follicles grow in response to FSH, oestradiol (and inhibin B) levels rise in the circulation and exert a negative feedback effect on FSH. As a result of the fall in FSH in the mid-​follicular phase, most of the follicles in the cohort will re- gress and die by atresia, leaving only the most FSH sensitive, ‘dom- inant’ follicle to continue to grow and to secrete oestradiol. By this time, the granulosa cells of the dominant follicle have acquired LH receptors (the only time in the life of the follicle when this occurs) and are also responsive to LH. The dual effect of FSH and LH en- hances granulosa cell differentiation and steroidogenesis so that in the preovulatory phase of the cycle serum oestradiol levels in the cir- culation have increased more than 10-​fold compared with the early follicular phase, 95% of circulating oestradiol being attributable to that single preovulatory follicle. The steeply rising levels of oestra- diol (probably assisted by a small increase in circulating proges- terone, indicating granulosa cell, and perhaps oocyte ‘maturation’) then trigger the LH surge—​the only example of a positive feedback effect of a target hormone on the hypothalamic-​pituitary unit. The LH surge has three main functions: (1) it triggers resumption of mei- osis in the oocyte ready for fertilization, (2) it leads to follicle rupture and ovulation and (3) it stimulates formation of the corpus luteum, converting the follicle from a mainly oestrogen-​producing unit to a highly vascularized, progesterone-​producing ‘factory’. Progesterone suppresses gonadotropins during the luteal phase and finally, if con- ception does not occur, luteolysis ensues after an apparently prepro- grammed interval of 12–​14 days, triggering the onset of a new cycle. 5 8 6 4 2 0 0 50 0 10 15 Follicle diameter (mm) 20 Menses Oestradiol 0 26 1 5 10 15 Days of cycle 20 25 28 100 200 300 400 Oestradiol (pg/ml) Progesterone (ng/ml) FSH LH (U/litre) LH FSH Progesterone Menses CL Ovulation 25 Fig. 13.6.1.3  The human menstrual cycle. Adapted from Baird DT (1983). Prediction of ovulation: biophysical, physiological and biochemical coordinates. In: Jeffcoate SL (ed). Ovulation: methods for its prediction and detection, pp. 1–​17, with permission from John Wiley and Sons Inc.

section 13  Endocrine disorders 2378 Disorders of ovulation Clinical presentation and causes of anovulation Disorders of ovulation usually result in perturbation of normal cyclical menses. It is uncommon to have regular but anovula- tory cycles, the exception being during adolescence when cyclical ovarian activity without ovulation (a feature of ‘immaturity’ of the hypothalamic–​pituitary–​ovarian axis) is characteristic of the early months post menarche. Thus, anovulation is generally characterized by amenorrhoea, oligomenorrhoea (more than 6 weeks between periods) or very irregular menses. Amenorrhoea may be primary (i.e. no previous periods) or secondary (at least one previous spon- taneous period). Primary amenorrhoea is less common and al- though its causes overlap with those of secondary amenorrhoea, it is not surprising that disorders of development of the ovaries and/​ or reproductive tract are overrepresented in this category. In some cases, primary amenorrhoea (defined as menarche delayed be- yond 16 years of age) is accompanied by delayed pubertal develop- ment. Menstrual disturbance may be accompanied by symptoms of oestrogen deficiency, including vaginal dryness and hot flushes. Interestingly, vasomotor symptoms are common in women with premature ovarian insufficiency (POI) but not in younger women with POI or those with oestrogen deficiency due to hypothalamic-​ pituitary dysfunction. Patients with hyperprolactinaemia may complain of inappropriate lactation (galactorrhoea) but it is im- portant to recognize that this affects only 30–​50% of women with hypersecretion of prolactin. Menstrual abnormalities accompanied by symptoms of androgen excess—​hirsutism, acne, or alopecia—​are typical of polycystic ovary syndrome. The causes of amenorrhoea are summarized in Table 13.6.1.1. The most prevalent cause of secondary amenorrhoea is that caused by hypothalamic and/​or pituitary dysfunction. Hyperprolactinaemia, weight loss-​related, and idiopathic amenorrhoea are all associated with a functional, rather than structural, hypothalamic disorder of gonadotropin regulation (see later). Polycystic ovary syn- drome accounted for a further 32% of cases and primary ovarian failure for 11%. Among women presenting with oligomenorrhoea (Table 13.6.1.2), the underlying cause was polycystic ovary syn- drome in the great majority of cases, making this syndrome the most common overall cause of anovulation, as discussed in more detail next. Differential diagnosis of amenorrhoea and oligomenorrhoea Examination should include routine measurement of height and weight and calculation of body mass index. With the aid of a small number of endocrine investigations, it is usually possible to dif- ferentiate between the various causes of ovarian disorders and to guide management (Table 13.6.1.3). Measurement of serum FSH will distinguish premature ovarian insufficiency (wherein FSH is elevated) from other causes of amenorrhoea and oligomenorrhoea (in which FSH is normal or low). Assessment of oestrogen status is an important step in investigation of women with amenorrhoea. This can be achieved by direct measurement of serum oestradiol, by ultrasound measurement of endometrial thickness or by a proges- togen challenge test. Serum oestradiol measurements are valuable if results are unequivocally low (i.e. lower than in the early follicular phase) or normal (equivalent to mid-​follicular phase levels) but concentrations in the early follicular phase range may not exclude chronic oestrogen deficiency. The advantage of ultrasound scan- ning or the progestogen challenge test is that these provide what amounts to an in vivo bioassay of endogenous oestrogen action (on the endometrium). A combination of low oestrogen and low (or normal) FSH is indi- cative of hypothalamic/​pituitary dysfunction. In such cases, serum prolactin should be measured and if elevated, pituitary imaging per- formed (see later and in Chapter 13.2.1). Overview of management of disorders of ovulation The simple schema for differential diagnosis of ovarian disorders provides a basis for selection of appropriate treatment, as outlined in Table 13.6.1.3. Details of management of the individual disorders are given in the appropriate following sections. The first principle must al- ways be to treat any underlying cause, if possible; for example, helping women with weight loss-​related amenorrhoea to gain weight. For women with POI (high FSH, low oestrogen), gamete donation is the only option for fertility treatment but oestrogen replacement (with a progestogen for endometrial protection when the uterus is present) is required for treatment of symptoms of oestrogen deficiency (see later). In women with a hypothalamic or pituitary cause of anovulation (low or normal FSH with low oestrogen), ovulation can be induced by gonadotropins or GnRH (or, in the case of hyperprolactinaemia by dopamine agonists), but patients not requiring fertility treatment will, like those with POI, need sex hormone replacement. Ovulation can be induced in most women with polycystic ovary syndrome (PCOS; normal FSH, normal oestrogen) by antioestrogens, but some may Table 13.6.1.1  Causes of amenorrhoea Primary ovarian failure (11%) Hypothalamic/​pituitary dysfunction (55%) Hyperprolactinaemia (11%) Weight loss-​related (35%) Idiopathic (9%) Polycystic ovary syndrome (32%) Genital tract disorder (2%) Table 13.6.1.2  Causes of oligomenorrhoea Polycystic ovary syndrome (87%) Peri-​menopausal (3%) Recovered weight loss (9%) Uncertain cause (1%) Table 13.6.1.3  Differential diagnosis and guide to management of women with amenorrhoea Results of investigations Diagnosis Management High FSH, low oestrogen Primary ovarian failure Hormone replacement therapy (HRT) Normal/​low FSH, low oestrogen —​if prolactin high Hypothalamic/​pituitary disorder Hyperprolactinaemia GnRH or FSH (or HRT) Dopamine agonists Normal FSH, normal oestrogen (± high LH) Polycystic ovary syndrome Clomiphene, FSH Cyclical progestogens or oral contraceptive

13.6.1  Ovarian disorders 2379 require gonadotropin therapy. In those not wishing to conceive, man- agement of erratic periods and treatment of attendant symptoms of androgen excess are important considerations. Premature (primary) ovarian insufficiency (POI) Amenorrhoea due to loss of ovarian function under the age of 40 years affects approximately 1% of women and is known as POI. Other terms that have been used for this condition include premature (or primary) ovarian failure, premature menopause, hypergonadotropic amenor- rhoea, and gonadal dysgenesis. It results from an irreversible prema- ture depletion of ovarian follicles, although it is not unusual to have episodes of spontaneous ovarian function. In a very small proportion of women with POI (up to 5%) this may even result in pregnancy. This is in contrast to the normal menopause, or last ever menstrual period, usually occurring after the end of the fifth decade (average age 51 years). Menopause is a normal physiological event, occurring as a result of permanent ovarian failure once the number of ovarian fol- licles falls below a critical number, believed to be about 1000. No data exist on follicle number at the onset of POI and in some rare causes of POI, ovarian follicles remain within the ovary but are unresponsive to gonadotropin stimulation (e.g. FSH receptor mutations). Normal menopause and POI share the characteristic endocrine features of oestrogen deficiency associated with elevated serum con- centrations of FSH (and LH). It is important to investigate the cause of POI as it may have im- portant other clinical implications for the individual and sometimes her family. Idiopathic Most cases of POI are idiopathic, and it is not clear if the underlying cause is related to an initial reduced population of primordial fol- licles within the fetal ovary, an increased rate of atresia throughout reproductive life or a combination of both. Smokers may experience menopause at a younger age than non​smokers, although there is no evidence for causation in POI. Case reports have described various infections preceding POI (e.g. HIV, cytomegalovirus, tuberculosis, shigella), but only mumps oophoritis is considered causative, ex- plaining only a low percentage of cases of POI. Chromosomal and genetic Around 10% of POI is due to an abnormal karyotype, Turner syn- drome (45X) and its mosaic forms being the commonest. Turner syndrome (TS) usually results in delayed puberty and primary amen- orrhoea, but Turner mosaics may undergo normal puberty and can present with secondary amenorrhoea, sometimes even over the age of 35 years. An oocyte requires two normal X chromosomes to protect it from early atresia: this is thought to be the mechanism leading to the depleted number of ovarian follicles in TS and TS mosaic females. TS females have a structurally normal vulva, vagina, uterus, Fallopian tubes, and ovaries, although the latter may be small. In contrast, in XY-​ gonadal dysgenesis (Swyer’s syndrome) the 46XY karyotype invariable results in delayed puberty and primary amenorrhoea. A deletion on the Y chromosome results in a non​functioning streak gonad, which is un- able to produce anti-​Müllerian hormone (AMH: normally a product of Sertoli cells in the fetal testis) and therefore the Müllerian structures do not regress. The result is an anatomically normal vulva, vagina, uterus, and fallopian tubes. The gonads are thought to be at increased risk of malignant change and surgical removal is usually recommended. The gonads often lie high on the pelvic side-​wall or may even be found above the pelvis and can be removed laparoscopically. Autosomal dominant, autosomal recessive, and X-​linked patterns of inheritance have also been described in primary ovarian failure. It is now recognized that the mutation responsible for Fragile X syndrome, a cause of mental retardation, can also cause premature ovarian failure in carriers, and Fragile X (FRAXA) ‘premutations’ are probably the most common genetic cause of ovarian failure. Women with POI should be offered FRAXA testing after appro- priate counselling, although it is not currently thought necessary to test routinely for autosomal gene mutations unless clinical features should suggest a particular underlying condition. It is important to investigate potential chromosomal and gen- etic causes of POI, as these may be significant in counselling family members or any children of affected women. In addition, there may be important clinical implications for the woman with POI attached to the underlying diagnosis. TS females are prone to congenital heart disease, aortic dissection, and their pregnancies (both natural and egg donation) are high risk. A minority of TS women (around 10%) may have a small amount of Y chromosome material present, which may put them at increased risk of gonadal malignancy. Iatrogenic Iatrogenic POI is increasingly seen in young adult cancer sur- vivors, especially of childhood haematological malignancies. Other common groups are young women with breast cancer having ad- juvant chemotherapy and those with cervical cancer treated with chemo/​radiotherapy. Stem cell transplants (which require chemo- therapy) are being increasingly used for benign conditions such as β thalassaemia and multiple sclerosis. The cause is loss of germ cells and follicles as a consequence of chemotherapy and/​or irradiation. Not all chemotherapy regimens are equally toxic to the ovary and the effects are variable. Alkylating agents, especially cyclophospha- mide and procarbazine, are particularly associated with subsequent reduced ovarian reserve and POI, although platinum-​based agents, anthracyclines, and taxoids are less so and the antimetabolites (e.g. methotrexate, 5-​fluorouracil) probably have the lowest risk. Ovarian surgery and oophorectomy for benign and malignant disease is another important cause of iatrogenic POI and whenever possible, fertility-​sparing surgery should be performed. It is possible that endometriosis destroys ovarian follicles but surgical treatment to ovarian endometriosis may also have an effect, especially if per- formed repeatedly. Autoimmune POI may be associated with a range of autoimmune conditions, including thyroid disease, but the most clinically important one is with autoimmune Addison’s disease. However, a direct autoimmune cause for POI, where there is histological evidence of inflammatory infiltration of the ovarian cortex, has only ever been described when steroid cell autoantibodies are identifiable in the peripheral blood. Treatment Treatment of POI falls into five categories: • induction of puberty (using low-​dose oestradiol) in girls with de- layed puberty; • control of symptoms of oestrogen deficiency;

section 13  Endocrine disorders 2380 • preservation (or improvement) of bone mineral density; • fertility; • psychological. Hypo-​oestrogenic symptoms include hot flushes and night sweats, dyspareunia, urinary frequency, and loss of libido. Bone mineral density is likely to be low in untreated POI and can be preserved and increased with exogenous oestradiol. Oestrogen replacement should be in the form of a sequential or continuous combined regimen if the uterus is intact: progesterone being required to protect the endomet- rium from the effects of unopposed oestrogen. The psychological impact of diagnosis can be devastating, with a sense of loss, low self-​esteem, socioeconomic disadvantage, and overall reduction in quality of life. Life expectancy appears to be re- duced in women with POI, mainly due to cardiovascular disease, and although the evidence is sparse, oestrogen replacement may at- tenuate the effect. Oestrogen replacement should ideally be in the form of oestra- diol, the active component of which is the same as the main ovarian oestrogen, 17-​β oestradiol. Conjugated equine oestrogens are prob- ably best avoided for women with POI. Some young women find the combined oral contraceptive pill (COCP) more acceptable than standard oestrogen replacement, which is marketed for older, nor- mally menopausal women. Compliance can be an issue, especially in the under 25s, and the pill can be a useful alternative. However, most COCPs contains the synthetic oestrogen, ethinyloestradiol, which is more potent and may carry slightly higher risks. In addition, symp- toms of low oestrogen may occur during the pill-free week. It may also be less beneficial for bone health and should not be used for in- duction of puberty. There are some new COCPs containing oestra- diol, but it is not known how the risk of venous thromboembolism compares to those containing ethinyloestradiol. In addition, if taken for 21 out of every 28 days, the overall dose of oestrogen provided is lower than that required. Another advantage, though, of relying on the pill for oestrogen replacement is that it is contraceptive, whereas conventional hor- mone replacement therapy is not. Although pregnancy in POI is uncommon, it may occur. Unplanned pregnancy for any women can be traumatic, but when it occurs in the context of POI, the re- sults can be particularly upsetting. An important part of the man- agement of POI is therefore contraceptive advice, if pregnancy is undesirable. In contrast, a woman who would welcome a preg- nancy can be reassured that hormone replacement treatment will not decrease her chances of a pregnancy occurring and may pos- sibly be facilitating by maintaining uterine health (an oestrogen-​ dependent organ). Fertility Sadly, no fertility treatment will assist conception for women with POI and oocyte donation is the only option. Pelvic irradiation is associated with a poor outcome of pregnancy, both natural and oocyte donation, because of attendant uterine damage. The endo- metrium may be unable to support implantation and even if preg- nancy occurs, obstetric risks are increased, including miscarriage, premature delivery, and intrauterine growth restriction. The risks of pregnancy must also be considered seriously for women with Turner syndrome as maternal deaths from aortic arch dissection have occurred and there appears to be an increased incidence of placental abruption. It is vital that women with TS in whom preg- nancy may occur are suitably assessed and cared for by a specialist team with appropriate expertise. Hypothalamic/​pituitary dysfunction Most cases of amenorrhoea due to hypothalamic/​pituitary dysfunc- tion are of hypothalamic, rather than pituitary origin, and most are a function of an underlying disorder. Weight loss-​related amen- orrhoea is very common. Nutritional status is an important de- terminant of reproductive function and being underweight (BMI <19 kg/​m2) is very likely to result in abnormalities in the pulsatile secretion of GnRH leading, in turn, to reduced frequency and amp- litude of LH and FSH pulses, and oestrogen deficient amenorrhoea. Cyclical ovarian function can usually be restored by weight gain, but this is often difficult to effect. In some cases, even once weight has been restored, there may still be a time delay of months or even years before periods resume. Most women with weight loss-​related amenorrhoea have an underlying eating disorder and it is often ne- cessary to seek the help of specialist psychological or psychiatric services. Fertility treatment should generally be delayed in under- weight women until a normal body mass index (or close to normal) has been achieved and maintained. This is because a low maternal BMI is associated with intrauterine growth restriction. Correction of oestrogen deficiency is usually appropriate while treatment to aid weight gain is underway. Hyperprolactinaemia is another common cause of oestrogen-​ deficient amenorrhoea (discussed in greater detail in Chapter 13.2.1). Amenorrhoea is the typical presenting symptom of hyper­ prolactinaemia. It is important to exclude primary hypo­thyroidism or concurrent medication as possible causes before embarking upon pituitary radiology. In particular, dopamine antagonists (e.g. pheno- thiazines and metoclopramide) are well-​recognized causes of ele- vated serum prolactin. Magnetic resonance imaging is the preferred method of detecting pituitary abnormalities. A  microadenoma of the pituitary may be found in up to 50% of women with hyperprolactinaemic amenorrhoea. Larger tumours (≥10 mm) are much less common. Management of hyperprolactinaemic amenor- rhoea, even in women with an obvious prolactinoma, is primarily by the use of long-​acting dopamine agonists such as bromocrip- tine or cabergoline. These drugs lower prolactin, restore ovulatory function and, typically, reduce the size of prolactin-​secreting tu- mours. Pituitary surgery is rarely needed, even in women with large prolactinomas. In about 10% of cases, the underlying cause of hypothalamic amenorrhoea is uncertain. Recent studies of women with idiopathic (or ‘functional’) hypothalamic amenorrhoea have suggested that this category of patients represent what is essentially a stress-​related hypothalamic disorder. Such patients respond very well to cogni- tive behavioural therapy (CBT), which results in resumption of ovulatory cycles without the need for endocrine treatment. If CBT is unsuccessful, ovulation can be induced, by pulsed gonadotropin-​ releasing hormone (GnRH) or gonadotropins, in women seeking fertility treatment (Fig. 13.6.1.4). However, at the time of writing, there are no GnRH delivery systems available commercially. Otherwise oestrogen/​progestogen replacement is desirable to treat symptoms of oestrogen deficiency and/​or to maintain bone density.

13.6.1  Ovarian disorders 2381 Finally, it is important to recognize that other hypothalamic-​ pituitary disorders, while themselves being rare causes of amenor- rhoea, may first present with menstrual dysfunction. Congenital deficiency of GnRH, best illustrated by Kallmann syndrome (in which gonadotropin deficiency is associated with anosmia), often presents as delayed puberty but may manifest as primary amenor- rhoea in girls who have completed pubertal development. In the last decade, there has been an exponential increase in our know- ledge of the network of neuropeptides and their receptors that im- pact on the hypothalamic control of gonadotropin secretion. These include the network of kisspeptin, neurokinin B, dynorphin and their receptors (so-​called KnDY neurones). Important informa- tion has emerged regarding mutations and polymorphism in genes coding for these neuropeptides and receptors that are responsible for most causes of hypothalamic congenital gonadotropin defi- ciency. Amenorrhoea is a common presenting symptom in women with acromegaly or Cushing syndrome. Hypothalamic tumours or granulomas may cause deficiency of not only GnRH but also other hypothalamic hormones. It is not necessary routinely to screen for these rarer causes of amenorrhoea, but it is important to be alert to features in history and examination that may suggest a more un- usual diagnosis. Polycystic ovary syndrome Polycystic ovary syndrome (PCOS) is the commonest of all the ovarian disorders and the commonest endocrine disorder in women of reproductive age, with a prevalence of greater than 5% in the gen- eral population. The typical clinical presentation is the association of features of anovulation or oligo-​ovulation (amenorrhoea or men- strual irregularity) with clinical and/​or biochemical evidence of an- drogen excess (hirsutism, persistent acne, or male-​pattern alopecia) in women with polycystic ovaries (Fig. 13.6.1.5). However, the recog- nition that there may be a broader spectrum of clinical and biochem- ical presentation has led to a revision of the diagnostic criteria for PCOS (see next). The aetiology of PCOS remains uncertain. There is strong evidence for an ovarian origin of androgen excess although the hypersecretion of adrenal androgens can also be found, albeit in the minority of patients with PCOS. Genetic factors clearly play a part in the aetiology of the syndrome. There is clustering of cases of PCOS within families and a twin study showed that the concord- ance of features of PCOS is significantly greater in identical than in non​identical twins. The mode of inheritance is unclear, but it is not a simple Mendelian trait. Rather, like type 2 diabetes, it is a complex endocrine disorder in which several genes are likely to play a part. Case-​control studies of over 100 candidate genes have failed to re- veal a clear candidate locus (or loci) but recent genome-​wide asso- ciation studies (GWAS) have proved more fruitful, albeit with some unexpected findings, which may lead to a better understanding of the aetiology of PCOS. A recently meta-analysis of the available GWAS studies has helped to clarify the genetic basis of PCOS. There was a similar pattern in the genetic architecture between the various diagnostic criteria, indicating a common genetic basis amongst the different diagnostic subtypes and suggesting a common aeti- ology despite heterogeneity of clinical and biochemical features. In addition, as in type 2 diabetes, the phenotype is modified by en- vironmental factors and obesity clearly exacerbates endocrine and metabolic dysfunction and is associated with more severe symptoms. A new International Guideline has been published which attempts to collate the various aspects of diagnosis and management of PCOS (https://www.monash.edu/medicine/sphpm/mchri/pcos/guideline). In so doing, it points to the relative paucity of large, randomized con- trolled trials in assessing therapeutic options. Hypothalamus LH FSH FSH GnRH Oestradiol Ovary Blood Pituitary (GnRH) Gonadotrophin releasing hormone − + Fig. 13.6.1.4  Induction of ovulation in patients with hypothalamic amenorrhoea. Exogenous GnRH may be administered by pulsatile infusion pump, leading to restoration of gonadotropin secretion and allowing normal negative feedback regulation of LH and FSH by ovarian steroids, thus limiting the risk of multiple follicle development. An alternative strategy is to give gonadotropins by daily injection but the risk of hyperstimulation is greater than with GnRH. Normal Ovary Polycystic Ovary Growing follicle Preovulatory follicle Fig. 13.6.1.5  Ultrasound images of a normal and polycystic
ovary, and of a growing and a preovulatory follicle.

section 13  Endocrine disorders 2382 Definition and diagnostic criteria The ‘classic’ definition of PCOS, that is, hyperandrogenism associated with chronic anovulation (in the absence of any confounding pituitary or adrenal disorders) is notable for its lack of reference to the ovarian morphology and yet almost all women who meet these criteria will have polycystic ovaries. In addition, polycystic ovaries can be found in women with symptoms of hyperandrogenism but who have regular menstrual cycles, as well as in those with anovulation but no evi- dence of androgen excess. A consensus meeting held in 2003 revised the diagnostic criteria allowing a more inclusive definition (Table 13.6.1.4). This revision has inevitably led to some controversy about definition of PCOS but there is ample evidence that both women with polycystic ovaries who present with hyperandrogenism but have regular cycles, and those with oestrogen-​replete amenorrhoea or oligomenorrhoea but who have no features of androgen excess, simply have varying forms of the same underlying condition. Indeed, prelim- inary data from the ongoing GWAS studies, across various popula- tions of women with PCOS, support this notion. Endocrine features The heterogeneity of the clinical features of PCOS extends to the endocrine abnormalities associated with it. As a result, specific endocrine indices are not a requirement for diagnosis, although measurement can be helpful to support it and, importantly, to ex- clude other conditions. Until the advent of widely available high-​ resolution ultrasound, the diagnosis of PCOS was usually based on a combination of biochemical and clinical features. Raised serum testosterone concentration is the most commonly found biochemical abnormality in PCOS, occurring in about 70% of cases. Free androgen index (FAI), calculated from total testosterone and sex hormone binding globulin (SHBG), has been found a useful marker by some clinicians. However, since SHBG is closely associ- ated with BMI and, more particularly, abdominal circumference, the increased FAI found in PCOS is, at least in part, a reflection of an in- creased abdominal adiposity which is characteristic of the syndrome. Serum concentrations of the weak androgen, androstenedione, are also elevated in PCOS. In practical terms, measuring serum testos- terone is usually preferable to androstenedione as the process is auto- mated in most clinical laboratories and therefore more cost-​efficient. This is however some concern about the specificity and precision of many commonly available androgen assays, particularly in women, with an emphasis being placed on the use of gas chromatography with mass spectrometry or, at least, well validated immunoassays. In 10–​20% of patients with PCOS, serum levels of the weak adrenal androgen, dehydroepiandrosterone sulphate (DHEAS) are also mod- estly elevated, suggesting that there may be, at least in some patients with PCOS, an adrenal contribution to increased circulating androgens. Among women with PCOS, clinical signs of hyperandrogenaemia (e.g. hirsutism) are associated with higher testosterone levels than those without. The presence or absence of features of hyperandrogenism, however, does not accurately predict serum androgen levels as clinical expression depends on the peripheral conversion of testosterone to its active metabolite, 5α-​dihydrotestosterone (DHT) by 5α-​reductase, as well as end-​organ sensitivity (androgen receptor activity) (see section on hyperandrogenism). Obesity in PCOS is associated with higher free testosterone levels in comparison to lean counterparts and, in part, this reflects the lower SHBG levels found in the former group. In addition, obesity may have an independent effect on peripheral an- drogen metabolism since androsterone glucuronide levels, a marker for peripheral 5α-​reductase activity, are raised in this group. Genetic factors may affect end-​organ sensitivity: for example, PCOS occurs in the Chinese and Japanese, but hirsutism is relatively uncommon in these races. In contrast, hirsutism features commonly in women with PCOS from the Indian subcontinent. Women with PCOS tend to have higher LH levels compared to those with normal ovaries. The highest prevalence of elevated LH levels is in those with anovulatory menses or amenorrhoea but even in this group more than 40% will have normal LH. In contrast, FSH levels are normal but tend to be lower than in the normal early fol- licular phase. Many have cited a raised LH:FSH ratio (either 2.5:1 or 3:1) as a diagnostic feature of PCOS, but it is neither sensitive nor specific enough to be used as a reliable diagnostic criterion. Oestrogen levels in women with all variants of PCOS are normal. As discussed previously, this can be used to distinguish between oligo/​amenorrhoeic women with PCOS and those with other causes of anovulation such as hypothalamic or pituitary disorders or ovarian failure. Plasma oestradiol levels in PCOS lie within the range normally seen in the early to mid-​follicular phase of the menstrual cycle, but oestrone levels are typically higher than normal. This is probably due to the increased peripheral conversion of high circu- lating androstenedione to oestrone in adipose tissue. Hyperprolactinaemia has been described in association with PCOS, but this usually reflects spurious fluctuations in serum pro- lactin, probably occurs no more commonly than in the normal population and is rarely a persistent problem. Metabolic abnormalities Polycystic ovary syndrome is not just a reproductive disorder it is also associated with a characteristic metabolic abnormality, cen- tral to which is peripheral insulin resistance and compensatory hyperinsulinaemia. Insulin resistance is independent of body weight but the difference between women with PCOS and controls is amp- lified with increasing body weight. Reduced insulin sensitivity is related to an abnormality in energy balance, specifically reduced postprandial thermogenesis, which may contribute to develop- ment of obesity. Interestingly, insulin resistance in PCOS appears to be confined to (or at least is most apparent in) the major sub- group of women who have both anovulation and hyperandrogenism (Table 13.6.1.5). Typically, women with PCOS have increased abdom- inal adiposity (and visceral fat accumulation) and this is correlated with insulin resistance. There is also an associated dyslipidaemia, characterized by lower than normal serum concentrations of Table 13.6.1.4  Diagnostic criteria for polycystic ovary syndrome (PCOS) according to National Institutes of Health (NIH) conference on PCOS 1990 and the joint ESHRE/​ASRM consensus conference, Rotterdam 2003 NIH 1990a Rotterdam 2003b Chronic anovulation Oligo-​ and/​or anovulation Clinical and/​or biochemical signs of hyperandrogenism Clinical and/​or biochemical signs of hyperandrogenism Polycystic ovaries a Both criteria needed; b 2 of 3 criteria required. Diagnosis, using either set of criteria, assumes that other aetiologies that may mimic PCOS (e.g. non​classical 21-​hydroxylase deficiency) have been excluded.

13.6.1  Ovarian disorders 2383 high-​density lipoprotein cholesterol (HDL-​C) and elevated levels of low-​density lipoprotein cholesterol (LDL-​C). Although glucose toler- ance is often normal in these women, impaired glucose tolerance has been noted in 10–​40% of young obese women with PCOS with frank diabetes in 5–​10%. The defect in insulin action associated with PCOS appears to be secondary to a defect in postreceptor signal transduc- tion and shows subtle differences from that found in other insulin re- sistant states. The major defect associated with PCOS, independent of obesity, is within insulin signalling in classic insulin target tissues such as muscle. Suppression of hepatic gluconeogenesis is reduced, but only in obese PCOS women. In contrast, obesity alone has a smaller effect on the sensitivity of insulin-​mediated glucose utilization but a greater effect on the rate of glucose utilization. There is some debate as to whether this insulin resistance in PCOS represents a primary defect in insulin action or whether it is secondary to hyperandrogenism and/​or the result of increased truncal-​abdominal fat. The interaction of insulin and androgens is complex. Experimental data suggest that androgens affect flux of free fatty acids from visceral fat deposits, which may, in turn, affect insulin sensitivity. However therapeutic reduction of serum androgen levels does not appear to im- prove insulin sensitivity. On the other hand, hyperinsulinaemia clearly affects androgen production. Insulin has gonadotropic activity and can influence ovarian steroidogenesis by both theca and granulosa cells by an interaction with LH. In addition, hyperinsulinaemia reduces hepatic production of SHBG and thereby raises levels of non-​protein-​ bound (i.e. biologically available) testosterone. Reproductive consequences Polycystic ovary syndrome is by far the most common cause of anovulatory infertility, accounting for more than 75% of cases. Anovulation is the undoubtedly the principal reason for subfertility in women with polycystic ovaries but there has been some specu- lation that polycystic ovaries, in the absence of the syndrome, may contribute to problems with fertility. Polycystic ovaries are found more commonly than in the general population in infer- tile, ovulatory women with tubal disease (50%), in women whose partners have sperm dysfunction (53%) and in couples with unex- plained infertility (44%). Women with polycystic ovaries are also overrepresented among women with a history of recurrent miscar- riage (three or more consecutive miscarriages). However, the live birth rate, of ovulatory women with polycystic ovaries, after spon- taneous conception, is the same as that in a well-​matched population of women with normal ovaries. Long-​term consequences The significance of PCOS for women’s health at a population level is increasingly being recognized. While the management of symptoms of PCOS such as infertility and hirsutism is important, consider- ation must also be given to management, and, if possible, prevention of long-​term effects of the disorder. These include increased risk of developing endometrial cancer, and the consequences of metabolic abnormalities, namely diabetes and cardiovascular disease. Endometrial carcinoma. PCOS has been recognized as a risk factor for endometrial carcinoma since the 1950s and there are reports of the disease occurring in young (premenopausal) women with PCOS. In women with PCOS who have amenorrhoea or infrequent menses, the endometrium is exposed to prolonged stimulation with oestrogen in the absence of cyclical progesterone (so-​called ‘unopposed’ oestrogen). This may lead to endometrial hyperplasia and, if untreated, to endometrial car- cinoma. Obesity adds to the risk of developing endometrial cancer by several interrelated intermediary factors including increased oes- trogen production, hyperinsulinaemia, and reduced serum SHBG. Gestational diabetes. The link between PCOS, insulin resistance and impaired glucose tolerance suggests that women with PCOS are at increased risk of developing both type 2 diabetes and gestational diabetes (GDM). The physiological insulin resistance of pregnancy adds to that in- trinsic to PCOS and may unmask impaired pancreatic β cell func- tion. The evidence for increased risk of GDM among women with PCOS is suggestive but not yet compelling. Most of the studies to date have been small and retrospective, and involve ethically mixed populations. Meta-​analysis of these smallish studies suggested a 3-​fold increase in risk of GDM in women with PCOS but a more recent prospective, multicentre study in the Netherlands found a prevalence of GDM in their PCOS population of 22% (against an expected population prevalence of around 5%). Type 2 diabetes mellitus. As indicated earlier, impaired glucose tolerance (IGT) and even frank diabetes are common in obese young women with PCOS. Longitudinal studies have been limited both in number and in duration of follow-​up but those that are available indicate that the prevalence of both IGT and diabetes increase, as might be predicted, with age (and inevitably BMI). Likewise, population studies have been few, but the results sup- port the view that PCOS is a significant risk factor for development of type 2 diabetes. The relative risk is around twofold after adjustment for obesity but rises to 3–​7-​fold in obese women with PCOS. Cardiovascular risk. Polycystic ovary syndrome is associated with well-​recognized risk factors for cardiovascular disease: namely obesity, insulin resistance, dyslipidaemia, diabetes, and (in some but not all studies) hyperten- sion. In addition, surrogate markers of cardiovascular disease have also been found to be abnormal. Endothelial function is impaired in young women with PCOS. Carotid artery intima–​media wall thickness (associated with an adverse cardiovascular risk profile in middle-​aged and elderly general populations) is increased in women with PCOS over the age of 45 years and carotid plaques are more common. Coronary artery calcification is a marker for coronary ath- erosclerosis and is also more common in women with PCOS than BMI-​matched controls. Left ventricular mass index was found to be increased and diastolic dysfunction present in obese and non​obese young women with PCOS, suggesting a detrimental effect on the cardiovascular system, although this is yet to be confirmed. Table 13.6.1.5  Typical metabolic features of PCOS. Metabolic abnormalities are much more prevalent in women who have both anovulation and androgen excess and are exacerbated by obesity Metabolic features of PCOS Insulin resistance and hyperinsulinaemia Abnormal energy expenditure (reduced postprandial thermogenesis) Dyslipidaemia Impaired glucose tolerance

section 13  Endocrine disorders 2384 It might be expected from the presence of multiple risk factors for cardiovascular disease that women with PCOS, especially if obese, would have an increased morbidity and mortality from the condition. There are few epidemiological studies and no substantial longitudinal studies, but the data so far suggest that there are fewer cardiovascular events than would have been predicted from the cluster of risk fac- tors. The two largest studies give a similar odds ratio (1.5) for the risk of cardiovascular events. In both studies, the populations were under 60 years of age, so it remains possible that the relative risk of heart attacks (and stroke) will increase with age. An alternative explan- ation is that there are factors in women with PCOS that are protective against cardiovascular disease, for example, as a result of unopposed oestrogen or even raised androgen levels. Perhaps most importantly of all, it must be appreciated that the presence of risk factors does not prove the presence of the disease and that surrogate markers are not necessarily reliable predictors of outcome. Management of PCOS The management of PCOS is mainly targeted at relief of symptoms. Symptoms of androgen excess, including hirsutism, acne, or alo- pecia can be attenuated by use of antiandrogens, such as cyproterone acetate (CPA) and spironolactone (which, in the absence of CPA, is widely used in the United States). Flutamide is a pure antiandrogen (i.e. unlike, CPA, it has no progestogenic activity) but its place is less secure in management of symptoms of androgen excess because there are fewer studies to support its routine use and there have been reports of hepatic toxicity. Low-​dose CPA or drospirenone (a derivative of spironolactone) may be conveniently combined with ethinyl oestradiol (co-​cyprindiol or Dianette® and Yasmin®, respect- ively) and these preparations are particularly useful in those women with accompanying menstrual disturbance. They are also effective contraceptives. For those in whom oestrogen is contra-​indicated, CPA or spironolactone can be given alone but non​hormonal contraception should be advised in those at risk of pregnancy because of the theoretical risk of feminization of a male fetus. Eflornithine inhibits the enzyme ornithine decarboxylase (ODC) which is involved in the production of the hair shaft. When applied topically, it can slow the growth of hair, although the effect can take 2 to 4 months to become apparent. Women often seek medical help with hirsutism when ‘beauty treatments’ such as waxing, plucking, electrolysis, and laser become inconvenient or too expensive. It is important to ensure a realistic expectation of treatment, which is that antiandrogens should be used as adjuncts, not replacements, to beauty treatments. In addition, hormone treatment may take 9 to 12 months to become fully effective: an improvement in hirsutism is often best judged by a reduced frequency of hair-​removal treat- ment. Antiandrogen therapy is also effective for acne but, unfortu- nately, alopecia rarely improves with antiandrogen treatment and the objective here is to limit further hair loss. It is therefore im- portant to treat early signs of androgen-​dependent hair loss. Anovulatory women with PCOS who wish to conceive usually re- spond to ovulation induction therapy (Fig. 13.6.1.6). The principle is to raise serum FSH levels to encourage development of a single, healthy dominant follicle (and therefore limit the risk of multiple pregnancy). The first-​line treatment is the antioestrogen clomi- phene. Around 75–​80% of women will ovulate in response to clomi- phene. In those who do not respond, or do not conceive after six or more ovulatory cycles, treatment with exogenous gonadotropin is appropriate. The modern approach is to start with a low dose of FSH and, if necessary, make small increments in dose to find the ‘threshold’ for development of a single dominant follicle. Even low-​ dose FSH treatment, requires close monitoring to reduce the risks of multiple pregnancy. An alternative to gonadotropin treatment is laparoscopic ovarian diathermy, a single, if invasive, procedure. However, surgery alone will result in ovulatory cycles in less than 50% of subjects and adjuvant clomiphene or FSH treatment is often required. In anovulatory women with PCOS who do not wish to conceive, regulation of menses can be ensured by treatment with a combined oral contraceptive or cyclical progestogen treatment. Because of the risk of endometrial hyperplasia or cancer, it is important to offer such treatment even in women with amenorrhoea or oligomenorrhoea who are not concerned about lack of periods, In obese women with PCOS calorie restriction is not only de- sirable but also surprisingly effective in improving symptoms of PCOS, particularly menstrual pattern and fertility. Dietary restric- tion leading to merely a 5 to 10% reduction weight is associated with much improved ovarian function. Overweight and obese women re- spond poorly to induction of ovulation and from an obstetric view- point, the risks of GDM and pregnancy-​related hypertension are increased. Weight reduction before fertility treatment, though never easy to achieve, is therefore an important aspect of management. Evidence from the Diabetes Prevention Program, a prospective study of men and women with IGT, suggests that calorie restric- tion, coupled with lifestyle changes (including increased exercise) will reduce the risk of conversion to diabetes. While there are, as yet, no such studies in women with PCOS, it is logical that such an approach will also reduce the chance of developing diabetes in this ‘at risk’ group. Hypothalamus LH FSH FSH antioestrogens (e.g. clomiphene) Oestradiol Ovary Blood Pituitary (GnRH) Gonadotrophin releasing hormone − + Fig. 13.6.1.6  Induction of ovulation in polycystic ovary syndrome. The treatment of first choice is the antioestrogen clomiphene which leads to elevation of serum FSH. If this is ineffective, exogenous FSH can be given by daily, low-​dose injection.

13.6.1  Ovarian disorders 2385 The Diabetes Prevention Program also showed that the biguanide, metformin—​which has long been used for treatment of type 2 diabetes—​was effective in reducing conversion from IGT to dia- betes (although significantly less so than diet and lifestyle changes). In recent years, metformin has been enthusiastically advocated for management of PCOS, even in the absence of IGT. A large number of publications have supported its use in fertility treatment (par- ticularly in combination with clomiphene), menstrual regulation, and management of hirsutism. However, there have been few large randomized controlled trials of metformin in management of PCOS, and those few adequately powered studies that have been performed to date have failed to support those claims. It remains to be seen whether metformin has a role in diabetes prevention in women with PCOS. Other causes of hyperandrogenism in women Hyperandrogenism, which, in this context, is defined as clinical evi- dence of androgen excess in women, is a common and distressing problem. Hyperandrogenism manifests itself as hirsutism (or ex- cess, unwanted male-​pattern hair), persistent acne or androgenic alopecia (male-​pattern balding). Although PCOS is the commonest cause of androgen excess, it is important to consider other possible diagnoses. Physiology of androgen-​dependent hair growth
and androgen production in women During puberty, circulating androgen concentrations rise and the familiar pattern of androgen-​dependent body (terminal) hair growth is seen. In normal, premenopausal women, the adrenal is the predominant source of androgens. Testosterone is the most important circulating androgen and is secreted by both ovaries and adrenals. But about 50% of circulating testosterone is derived by conversion from androstenedione (a weak androgen) in per- ipheral tissues such as skin and adipose. More than 90% of circu- lating testosterone is bound either to SHBG or albumin. Only the unbound (and possibly albumin-​bound) testosterone is available to target tissues. Testosterone is further metabolized within the hair follicle to the more potent androgen, dihydrotestosterone (DHT) by the enzyme 5-​α-​reductase. Both testosterone and (with a higher affinity) DHT bind to specific androgen receptors within the hair follicle to affect growth of terminal hair. The biological effect of an- drogens may also be regulated at the level of the androgen receptor itself. Recent evidence suggests that heterogeneity of the androgen receptor is conferred by epigenetic modification of the androgen receptor gene and that these modifications are related to clinical in- dices of androgenicity. Causes of hirsutism The causes of hirsutism are summarized in Table 13.6.1.6. Hirsutism is most commonly caused by PCOS. It accounts for about 90% of cases, including those who might previously have been labelled as having idiopathic hirsutism. However, hirsutism may be a mani- festation of other, much rarer but more serious, endocrine disorders such as Cushing syndrome and adrenal or ovarian tumours. Careful clinical evaluation is the key to differential diagnosis. Long-​standing mild to moderate hirsutism, with or without menstrual disturbance is suggestive of PCOS (or idiopathic hirsutism) while a short history of increasing hirsutism in a previously non​hirsute subject should alert the physician to the possibility of alternative diagnoses. Hirsutism and menstrual disturbances are common presenting fea- tures in women with Cushing syndrome or androgen-​secreting tu- mours. In the case of Cushing syndrome, the presence of additional features such as hypertension, easy bruising, and striae, help to make the diagnosis more likely. Hyperthecosis refers to the histological finding of islands of theca cells within dense ovarian stroma and is almost certainly a variant of PCOS. Its clinical presentation is indistinguishable from that of PCOS but it tends to be associated with severe hirsutism and there is often also cutaneous evidence of significant insulin resistance (acanthosis nigricans). Another well-​recognized cause of hirsutism that may be diffi- cult to distinguish clinically from PCOS non​classical (late onset) congenital adrenal hyperplasia (CAH) due to 21-​hydroxylase de- ficiency. Such cases tend to present first during adolescence with symptoms of anovulation and androgen excess, but rarely can pre- sent in adulthood. Androgen-​secreting ovarian and adrenal tumours are rare. Causes, diagnosis, and management of adrenal tumours are de- scribed elsewhere (Chapter  13.5.1). Ovarian tumours may be benign or (less commonly) malignant and are classified as ei- ther sex cord stromal tumours (Sertoli-​Leydig cell tumours), or adrenal-​like tumours (e.g. virilizing lipoid cell tumours, adrenal rest tumours). Investigation and diagnosis of hirsutism A guide to investigation of hirsutism is given in Table 13.6.1.7. Mild-​moderate long-​standing hirsutism in women with regular menses is very likely to be either idiopathic or, much more com- monly, associated with polycystic ovaries. Serum testosterone con- centrations are usually modestly elevated or within the normal range. It could be argued that no investigations are strictly necessary in this category of patients but measuring serum testosterone and determining ovarian morphology by pelvic ultrasound scan, means a specific diagnosis can be offered to the patient. The principal reason for measuring testosterone in women with hirsutism is to screen for the more serious causes of androgen excess that will require further investigation. It is not measured to diagnose hyperandrogenism since this is already manifested clinically as hirsutism. It is not ne- cessary to measure routinely androstenedione, SHBG, or free tes- tosterone. Some laboratories offer androstenedione as a reasonable alternative to testosterone assays. Table 13.6.1.6  Causes of hirsutism Ovarian Polycystic ovary syndrome (>80%) Hyperthecosis (5–​10%) Ovarian tumours (<1%) Adrenal Congenital adrenal hyperplasia (classical, 1%; non​classical (late onset, 3%) Cushing syndrome (<1%) Adrenal tumours (<1%) Idiopathic With raised androgens (5%) Without raised androgens (7%)

13.6.2 Disorders of male reproduction and male hyp

13.6.2 Disorders of male reproduction and male hypogonadism 2386

section 13  Endocrine disorders 2386 In women with hirsutism and menstrual disturbance, the most likely diagnosis is again PCOS. In such cases, however, it is legit- imate to extend biochemical tests to include measurements of gonadotropins and, in amenorrhoeic women, prolactin and oestra- diol. Further investigations are necessary in those patients with a short history of hirsutism (particularly if this is severe), with symp- toms suggesting other endocrine disorders (e.g. Cushing syndrome) and/​or those with a high serum testosterone (loosely defined as a level more than twice the upper limit of the normal range for the laboratory). In female patients with a serum testosterone concen- tration in the normal male range (>10 nmol/​litre), the presence of an androgen-​secreting tumour must be excluded. Imaging of the ovaries and adrenals by MRI is important. In experienced hands, ultrasonography of the adrenals and, particularly the ovaries may also be helpful. Selective catheterization of adrenal or ovarian veins to localize suspected a suspected tumour is difficult to execute and rarely informative. Measurement of dehydroepiandrosterone sul- phate (DHEAS) is a useful specific index of adrenal function in patients with a suspected androgen-​secreting tumour but it is less helpful as a routine test in hirsute patients. The prevalence of non​classical CAH is very low (<1% of cases of hirsutism) so it is not necessary to routinely measure basal and ACTH-​stimulated concentrations of 17-​hydroxyprogesterone to screen for this disorder. However, it is appropriate to do so in women with severe hirsutism and a raised serum testosterone. Management of hirsutism The approach to the management of women with hirsutism is described earlier in the section about PCOS. The principles of symptomatic management are similar in women with idiopathic hirsutism. In those with a specific underlying diagnosis, treatment is directed towards that primary disease or disorder. For example, removal of a pituitary corticotroph adenoma or an ovarian tumour is a very effective way of treating hirsutism. FURTHER READING Baird DT (1983). Prediction of ovulation: biophysical, physiological and biochemical coordinates. In:  Jeffcoate SL (ed) Ovulation: methods for its prediction and detection, pp. 1–​17. John Wiley, Chichester. Berga SL, Loucks TL (2005). The diagnosis and treatment of stress-​ induced anovulation. Minerva Ginecol, 57, 45–​54. Boehm U, et  al. (2015). Expert consensus document:  European Consensus Statement on congenital hypogonadotropic hypogonadism—​pathogenesis, diagnosis and treatment. Nat Rev Endocrinol, 11, 547–​64. Dunaif A (2016). Perspectives in polycystic ovary syndrome: from hair to eternity. J Clin Endocrinol Metab, 101, 759–​68. Gillam MP, et al. (2006). Advances in the treatment of prolactinomas. Endocr Rev, 27, 485–​534. Gougeon A (1996). Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev, 17, 121–​54. Hardy K, et al. (2000). In vitro maturation of oocytes. Br Med Bull, 56, 588–​602. Jayasena CN, Franks S (2014). The management of patients with poly- cystic ovary syndrome. Nat Rev Endocrinol, 10, 624–​36. Koulouri O, Conway GS (2008). A systematic review of commonly used medical treatments for hirsutism in women. Clin Endocrinol (Oxf), 68, 800–​5. Norman RJ, et  al. (2007). Polycystic ovary syndrome. Lancet, 370, 685–​97. Teede HJ, et al. (2018). Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Human Reproduction, 33, 1602–18. Webber L, et al. (2016). ESHRE guideline: management of women with premature ovarian insufficiency. Human Reproduction, 31, 926–​37. 13.6.2  Disorders of male reproduction and male hypogonadism P.-​M.G. Bouloux ESSENTIALS The adult testis performs two principle functions: the synthesis and secretion of androgens, and the production of male germ cells, the spermatozoa. Gonadotrophin releasing hormone, released from the hypothalamus, stimulates pituitary release of luteinizing and follicle-​ stimulating hormones (LH and FSH). LH acts on Leydig cells of the testes promoting synthesis and release of the principle male androgen testosterone. Testosterone is essential for male sexual differentiation, growth, and function of the male genital tract, secondary sexual char- acteristics, sexual potency, and production of spermatozoa. Hypogonadism Hypogonadism may be due to disorders of the pituitary/​hypothal- amus (secondary or hypogonadotropic hypogonadism) or testes (primary or hypergonadotropic hypogonadism). Clinical features—​symptoms and signs depend on the age of onset of androgen deficiency. Prepubertal presentation is with sexual in- fantilism, delayed puberty, and eunuchoidal body proportions. Postpubertal presentation is with diminished sex drive and erection, loss of ejaculation, muscle atrophy, poor stamina, decreased sec- ondary sexual hair, decreased shaving frequency, and regression of spermatogenesis (reduced testicular volume). Diagnosis—​hypogonadism is confirmed by low serum testos- terone, best measured between 08.00 and 09.00 h. Measurement of Table 13.6.1.7  Guide to investigation of hirsutism Presenting features Investigations Mild, chronic hirsutism, regular cycles Testosterone, ultrasound of ovaries Moderate hirsutism and/​or cycle disturbance Testosterone, (LH, FSH), ultrasound of ovaries Severe hirsutism and/​or short history and/​or testosterone

5 nmol/​litre DHEAS, 17-​hydroxyprogesterone, dexamethasone suppression test, 24 h urine free cortisol ovarian &/​or adrenal imaging fasting glucose/​insulin

13.6.2  Disorders of male reproduction and male hypogonadism 2387 LH and FSH differentiates between primary (high gonadotrophins) and secondary (low gonadotrophins) hypogonadism. Specific causes of primary gonadal failure include Klinefelter’s syndrome (eunuchoid proportions, typical karyotype 47XXY) and dystrophia myotonica, and of secondary gonadal failure in- clude Kallmann’s syndrome (anosmia, red–​green colour blindness, synkinesis, nerve deafness, cleft lip or palate, and renal malforma- tions). Cryptorchidism, the absence of one or both testes from the scrotum and the commonest birth defect of the male genitals, results from the failure of the testis to descend during fetal development from an abdominal position into the scrotum and is associated with increased risk of testicular cancer. Management—​the aims of treatment are to:  (1) relieve the symptoms of androgen deficiency; (2)  prevent the long-​term consequences of androgen deficiency such as osteopenia;
(3) reproduce physiological circulating and tissue levels of testos- terone, dihydrotestosterone, and oestradiol; (4)  induce fertility, if required, in hypogonadotropic patients; (5)  treat any specific underlying diseases. The mainstay of treatment is androgen re- placement therapy. Infertility Male infertility may affect 5% of men of reproductive age and is caused by a heterogeneous group of disorders. The commonest cause (60% of cases) is ‘idiopathic’ azoo/​oligozoospermia, although many cases are now recognized as due to discrete gene defects asso- ciated with impaired spermatogenesis. Other causes include crypt- orchidism, testicular tumours, genital tract infection, obstructive azoospermia, and sperm autoimmunity. Laboratory investigation—​conventional parameters of the semen analysis provide a semiquantitative index of fertility potential. Measurement of plasma testosterone, LH, FSH, and chromosome karyotyping is indicated in some cases. Management—​no medical treatment has been shown to im- prove fertility in subfertile men. Assisted conception techniques are increasingly applied to overcome idiopathic male infertility, including intrauterine insemination, In vitro fertilization, and micro- injection of a single live spermatozoon directly into harvested oo- cytes, which is the treatment of choice for severe oligozoospermia. Cryopreservation of semen should be offered to all men of repro- ductive age before anticancer chemotherapy, orchidectomy, or tes- ticular irradiation. Physiology of the hypothalamo-​pituitary-​
testicular axis The testes The adult testis performs two principle functions: the synthesis and secretion of androgens, and the production of male germ cells, the spermatozoa. The testicular parenchyma is surrounded by a solid capsule (tunica albuginea) and consists of seminiferous tu- bules in which gametes are produced. Septa of connective tissue divide the testis into 200–​300 lobules which coalesce to form the rete testes. Each lobule contains two to three seminiferous tubules and each testis contains 600–​900 seminiferous tubules. Testicular function is under the regulation of the hypothalamo-​pituitary axis (Fig. 13.6.2.1). Central control of testicular function The septo-​preoptic region of the hypothalamus contains about 2000 dispersed gonadotrophin releasing hormone (GnRH) neurons, whose nerve endings converge on the capillary plexus of the me- dian eminence, where episodic neurosecretion of the 10 amino acid decapeptide GnRH occurs every 90–​120 minutes in the adult. The episodic secretion of GnRH represents an intrinsic property of GnRH neurones, a basic rhythm essential for correct functioning of the gonadotroph that is modulated by numerous neurotransmit- ters, which modulate pulse amplitude and frequency. Prolactin is a potent negative modulator of GnRH pulse frequency. GnRH in turn stimulates the gonadotrophs cells of the anterior pituitary to synthesize and secrete the glycoproteins luteinizing and follicle-​ stimulating hormones (LH and FSH). These hormones are secreted in an episodic manner, entrained by the pulsatile GnRH secretion. LH acts on Leydig cells of the testes promoting synthesis and release of the principle male androgen testosterone. Negative feedback con- trol of LH secretion by testosterone is in part mediated by aroma- tization into oestradiol by the hypothalamus. By contrast, FSH binds to FSH receptors of the Sertoli cells of the testes, leading to the elab- oration of inhibin B, which negatively feeds back on pituitary FSH secretion. Locally produced activin in the pituitary, stimulates FSH secretion. There are no FSH receptors on the germ cells themselves (Fig. 13.6.2.2). In the adenohypophysis, GnRH binds to a specific G-​protein coupled receptor on gonadotrophs, initiating gene expression of the α and β subunits of FSH and LH and their secretion by induction of inositol 1,4,5-​triphosphate, mobilization of intra- cellular calcium, and stimulation of calcium influx. Androgens reduce the expression of GnRH receptors. Experimentally, con- tinuous exposure of gonadotrophs to exogenous GnRH leads to GnRH receptor desensitization and subsequent profound suppression of gonadotrophin secretion and therefore gonadal steroid production. This desensitizing property of continuous GnRH exposure on GnRH receptors is exploited clinically in the use of GnRH super-​active analogues to produce effective med- ical castration in conditions such as prostatic carcinoma and endometriosis. Mode of action of gonadotrophins The glycoproteins LH and FSH comprise non​covalently bound α and β chains, which form a heterodimer. The α chain (encoded on chromosome 6) is common to all glycoprotein hormones, whereas the β chain is specific (LH β chain chromosome 19; FSH β chain chromosome 11); the biological activity of these glycoprotein hormones is mediated by the β chain, and isolated subunits and homodimers have no biological activity. The carbohydrate content of glycoprotein hormones differs significantly and influence the ter- tiary structure of these molecules, exerting a powerful influence on their biological half-​life, binding to specific receptors, and also intra- cellular signal transduction after receptor binding in target cells. A high concentration of sialic acid residues prevents their metab- olism in the liver; this prolongs the half-​life and biological activity

section 13  Endocrine disorders 2388 of FSH. LH has a half-​life of 20 minutes whereas FSH has a half-​life of 3 hours. LH binds to its specific receptor on Leydig cells, and via the action of adenyl cyclase, induces cAMP formation, effecting an increase in formation of intracellular cholesterol and gene expres- sion of enzymes involved in steroidogenesis, in particular the key enzyme 20,22-​desmolase, which initiates the formation of testos- terone by cleavage of the cholesterol side chain. The feedback control of LH production in man is mediated via testosterone and its metabolite oestradiol. Testosterone has an in- hibitory effect on GnRH neurons with only a minor effect on the gonadotroph, whereas oestradiol exerts negative feedback at both hypothalamic and gonadotroph levels. FSH binds to its receptor on the Sertoli cells, and induces activity of the aromatase enzyme which converts testosterone into oestradiol. It also induces formation of inhibin B and activin. Inhibin B is a heterodimer composed of α and β subunits; there are two variants (β α, and β β). Hetero-​ and homodimers of the β chain are called activin A and B, respectively. Inhibin is an important component in the negative feedback regu- lation of FSH secretion, and in isolated functional disturbances of the Sertoli cells (Sertoli cell only, following radiotherapy or chemo- therapy), low inhibin B levels are associated with elevation of FSH, while LH levels remain unchanged. Activins can stimulate FSH secretion at the pituitary level. Testosterone and E2 exert negative feedback on FSH via an effect on GnRH neurons. Spermatogenesis Spermatogenesis is a complex process involving both mitotic di- visions and meiosis processes, the latter the process whereby dip- loid spermatogonia become haploid spermatids (Fig. 13.6.2.3). Spermatids are transformed into flagellated spermatozoa, a pro- cess known as spermiogenesis. After spermiogenesis is complete, spermatozoa are released from the germinal epithelium into the epididymis with the fluid from the tubules. The entire process takes 60–​72 days in the human. Sertoli cells are essential to the early devel- opmental stages of spermatogenesis, up to the stage of spermiation. Sertoli cells extend from the basement membrane of the semin- iferous tubules deep into the lumen. They express the androgen receptor and, under the influence of testosterone and FSH, secrete electrolytes and fluid into the lumen. In the postpubertal stage, spermatogenesis can be maintained without FSH by sufficiently high local testosterone concentrations alone. Sertoli cells form tight junctions with each other at their base, sealing the intercellular gaps, thereby forming the anatomical basis of the ‘blood-​testes’ barrier. Hypothalamus Anterior pituitary Inhibin Testes Second messenger Cell products Sertoli cell Sertoli cell Androgen-binding protein (ABP) (ABP) To body for secondary effects Testosterone (T) Leydig cells Spermatogonium – – – Tissue response Integrating centre KEY Efferent pathway Effector Spermatocyte GnRH LH T FSH Fig. 13.6.2.1  Organization of hypothalamo-​pituitary-​testicular axis. GnRH is secreted in a pulsatile manner into the portal circulation and this in turn leads to pulsatile release of LH and FSH. FSH is required for spermatogenesis and inhibit B secretion from Sertoli cells, and LH, acting on Leydig cells promotes synthesis and release of testosterone. Inhibin B and testosterone (via oestradiol) participate in negative feedback regulation of gonadotrophin secretion

13.6.2  Disorders of male reproduction and male hypogonadism 2389 Spermatogonia are embedded between Sertoli cells; during sperm- atogenesis, developing cells migrate from the basement membrane to the lumen, where the spermatozoa are released. In the fetus, Sertoli cells produce the anti-​Müllerian hor- mone (AMH), which prevents the development of the uterus and Fallopian tubes from the Müllerian duct during sexual differenti- ation in the male. AMH can be detected in serum until puberty and then levels fall. Spermatogenesis is a complex, repetitive series of cytodifferentia­ tion processes in the seminiferous epithelium, whereby cohorts of undifferentiated diploid germ cells (spermatogonia) proliferate and transform into the greatly expanded populations of haploid spermatozoa. The human testes produce around 200 million sperm- atozoa per day. Mitotic divisions of spermatogonial stem cells form subpopulations of spermatogonia which, at regular intervals of 16 days, differentiate into primary preleptotene spermatocytes to ini- tiate meiosis. Meiotic reduction divisions of spermatocytes generate round spermatids which are then transformed (spermiogenesis) into compact, virtually cytoplasm free, elongated spermatids. Condensed nuclear DNA forms the sperm head within an overlying HYP PRL KiSS-1 GnRH PIT Testosterone oestradiol LH FSH Inhibin B Testis Norepinephrine, Galanin-like peptide (GALP), glutaminergic, Neuropeptide Y (NPY) β-endorphin, corticotrophin releasing hormone (CRH), γ-amino butyric acid (GABA) Fig. 13.6.2.2  GnRH neurons are located in the hypothalamus in the septo-​preoptic region and release GnRH at the median eminence capillary plexus. GnRH neutrons receive both stimulatory (norepinephrine, kisspeptin, galanin-​like peptide (GALP), glutaminergic, neuropeptide Y (NPY)) as well as inhibitory inputs (β endorphin, corticotrophin-​releasing hormone (CRH), γ-​amino butyric acid (GABA)). Kisspeptin neurons express oestrogen, progesterone, and androgen receptors, and leptin receptors which are also expressed on NPY neurons. FSH, follicle-​stimulating hormone; GABA, γ-​amino butyric acid; GnRH, gonadotropin-​releasing hormone; HYP, hypothalamus; KiSS-​1, Kisspeptin; LH, luteinizing hormone; PIT, pituitary gland; PRL, prolactin; T, inhibitory signal; ↑, stimulatory signal. Spermatogonium 2n 2n 2n 1n 1n 1n 1n 1n 1n (a) Spermatogenesis Mitosis Primary spermatocyte Secondary spermatocyte Meiosis I Meiosis II Spermatid Spermiogenesis Spermatozoa (sperm) Peritubular cells (b) Sertoli cells Round spermatids Elongated spermatids Spermatogonia Leydig cells Spermatocytes Fig. 13.6.2.3  (a) Spermatogenesis. Spermatogonia are located on the basement membrane of the seminiferous tubules, wedged between Sertoli cells. They undergo mitosis into primary spermatocytes, which in turn undergo a meiotic division into secondary spermatocytes, and a after a second meiotic division, spermatids are formed. These undergo spermatogenesis into mature spermatozoa. (b) Cross section of a seminiferous tubule. (b) Reproduced from Wass JAH, Stewart PM, Amiel SA, Davies MJ (2011). Oxford textbook of endocrinology and diabetes, 2nd edn. By permission of Oxford University Press.

section 13  Endocrine disorders 2390 Golgi–​derived acrosome and a tail (containing nine pairs of micro- tubules arranged around a central pair) capable of propelling, fla- gellar movements. Mature spermatozoa are released from Sertoli cell cytoplasm into the tubular lumen some 60 to 74 days after the initial development from spermatogonia. The control systems regulating germ-​cell divisions and development are poorly understood. Testosterone and the androgen receptor Testosterone biosynthesis Testosterone is the most important steroid synthesized in the testes, 5–​7 mg being produced by the Leydig cells of an adult man each day. Leydig cells have a large endoplasmic reticulum and copious mitochondria. The parent substance of testosterone biosynthesis is cholesterol, mainly synthesized by Leydig cells, only a small amount being taken up from the circulation. Cholesterol is stored in the form of esters in fat vacuoles in these cells, until further processing through a total of five enzymatic steps converting cholesterol (C27) through hydrolytic steps into testosterone (C19). The rate limiting step in testosterone biosynthesis is the conversion of cholesterol to pregnenolone, a process which occurs on the inner mitochondrial membrane where the cytochrome P450sc (sc, side-​chain cleavage), 20, 22 desmolase, encoded on chromosome 15) enzyme catalyses three consecutive processes: hydroxylation on atom C20, followed by hydroxylation on atom C22, and thereafter cleavage between C20 and C22, thereby generating pregnenolone and isocaproic acid. This process depends on LH binding to the LH receptor, activation of adenylyl cyclase, generation of cAMP, and activation of protein kinase A. Pregnenolone is the parent steroid of all biologically ac- tive steroid hormones, and exits the mitochondrion by simple diffu- sion to undergo further modification on the endoplasmic reticulum. The ∆5 pathway leads to the initial C17 hydroxylation (via 17 α-​ hydroxylase) forming 17 α-​hydroxypregnenolone. The weak an- drogens DHEA (dehydroepiandrosterone) and androstenediol are produced by the enzymes 17,20 desmolase, and 17-​β hydroxysteroid dehydrogenase, respectively (Fig. 13.6.2.4). An additional step is the conversion of the less biologically active ∆5 steroids 17 α-​ hydroxypregnenolone, DHEA and androstenediol to the corres- pondingly more potent ∆4 steroids 17 α-​hydroxyprogesterone, androstenedione, and testosterone, respectively, steps catalysed by the enzyme 3 β-​hydroxysteroid dehydrogenase. This is effected by the initial oxidation of the 3β-​hydroxy group to a ketone, followed by the subsequent transfer of the C5–​C6 gene group from the B ring to the C4–​C5 site on the A ring. Newly synthesized testosterone cannot be stored in the testes and is immediately released into the circulation via the spermatic vein and lymphatics. Testosterone transport in the blood The lipophilic molecule testosterone leaves the Leydig cell by diffu- sion, and in the blood is largely bound to transport proteins (98%), leaving approximately 2% free and biologically active. About 60% is transported via high affinity binding to the 92.5 KDa β globulin glycoprotein molecule SHBG (sex hormone binding globulin), en- coded on chromosome 17, whereas approximately 38% is loosely bound to albumin. SHBG circulates as a homodimer with two binding sites for steroids. It is predominantly synthesized in the liver, with a little manufactured by the mammary gland and prostate. SHBG may also bind to specific membrane receptors, and account for the rapid non​genomic effects of testosterone and oestradiol (E2). SHBG binds testosterone (T) with greater affinity than oestradiol (E2), and conditions where SHBG are elevated will lead to a shift in the free E2 to T ratio. Several factors regulate SHBG concentration as shown in Table 13.6.2.1. Some conditions, such as cirrhosis due to hepatitis C, can cause significant rises in SHBG concentrations, thereby reducing free T concentrations; when inadequately com- pensated by a rise in LH, a state of hypogonadism will ensue. HO Cholesterol 1 4 HO HO HO HO HO OH OH O OH Pregnenolone 17-OH-Pregnenolone 17-OH-Progesterone Progesterone Dehydroepi- androsterone 5-androstene- 3β, 17β-diol Oestradiol 5α-dihydrotestosterone Testosterone Androstenedione C O C O C O OH OH OH O O O O CH3 CH3 CH3 C O O CH3 4 4 4 3 3 5 6 2 2 2 2 Fig. 13.6.2.4  Steroid biosynthesis in Leydig cells in response to LH binding onto its receptor. Bold arrows depict the Δ5 pathway preferred in the human testis. The circled numbers indicate the enzymes used by the metabolic steps: (1) cholesterol side-​chain cleavage enzyme = 20,22 desmolase; (2) 17α-​hydroxylase/​17,20 desmolase; (3) 17β-​hydroxysteroid dehydrogenase; (4) 3β-​hydroxysteroid dehydrogenase; (5) aromatase; and (6) 5α-​reductase. In addition, many of the steroid intermediates are sulphate-​conjugated within the testis (not shown). Reproduced from Wass JAH, Stewart PM, Amiel SA, Davies MJ (2011). Oxford textbook of endocrinology and diabetes, 2nd edn. By permission of Oxford University Press.

13.6.2  Disorders of male reproduction and male hypogonadism 2391 Metabolism of testosterone Free testosterone that has diffused into tissues (e.g. prostate, scalp) may be metabolized into dihydrotestosterone (DHT) or oestradiol (E2), depending on the availability of the enzymes 5 α-​reductase (Type 1: liver, skin: type 2 prostate, adrenal, seminal vesicles, genital skin, hair follicles and cerebral cortex) and aromatase respect- ively (Fig. 13.6.2.4). About 80% circulating DHT originates from tissue metabolism of T and about 20% from release from the testes. DHT and T bind to the same androgen receptor (AR), DHT having a greater than ×10 greater affinity for the AR compared to T, and therefore being the more potent. About 30 µg E2 is produced by extratesticular aromatization of testosterone and androstenedione each day, particularly in adipose tissue, bone cells and prostate, in contrast to the 10 µg secreted by the testes. Thus, T is a pro-​hormone as well as a classical hormone, and its endocrine effects are both directly and indirectly mediated. Both T and DHT are catabolized in the liver by oxidation, reduction, or hydroxylation reactions, followed by conjugation with glucuronic acid or sulphation on C3 or C17. The half-​life of T in the circulation is only about 10 min. The androgen receptor The androgen receptor is encoded by eight exons by a gene lo- cated near the centromere of the long arm of the X chromosome (Fig. 13.6.2.5). It is a polypeptide of 910 amino acids, with a mo- lecular weight of 98.5 kDa. It is a DNA-​binding protein comprising three domains. The N terminus, encoded by Exon 1,  contains a (CAGn) repeat sequence encoding glutamine repeated between 8 to 35 times. The number of repeats affects the transcriptional effi- ciency of the receptor. The greater the number of CAGn repeats, the weaker the transcriptional efficiency. It is known that the shorter the number of repeats, the greater the binding to coactivators, and therefore the greater the magnitude of the androgen effect. It is of interest that CAG repeats totalling less than 22 are associated with a greater risk of prostate cancer. A large number of repeats (>38) is associated with the androgen resistance seen in Kennedy’s syn- drome, a degenerative bulbospinal motor neuropathy. The cen- trally located hydrophilic DNA-​binding domains (Exons 2 and 3) carry two zinc fingers that bind to specific DNA sequences in or next to androgen sensitive genes, and influence transcription. The carboxyterminus (exons 4–8) domains carries the hydrophobic an- drogen binding responsible for binding testosterone and DHT. Prior to androgen binding, the AR is associated with several chaperone molecules, which stabilize it. Androgen binding to AR induces a conformational change which leads to AR dissociation from these molecular chaperones (Hsp90, Hsp70, Hsp56, and immunophilins). These proteins help in maintaining the correct conformation of the receptor necessary for efficient ligand binding. Activated AR com- plexes form dimers which bind to the androgen responsive elements Table 13.6.2.1  Regulation of SHBG production Stimulation of SHBG production Inhibition of SHBG production Oestrogens (e.g. oral contraceptive
pill, pregnancy) Androgen therapy Growth hormone deficiency Obesity Androgen deficiency Acromegaly Hyperthyroidism Hypothyroidism Hepatitis (especially HCV) Nephrotic syndrome Phenytoin Glucocorticoids Ageing Hyperinsulinism Antiretroviral therapies Progestogens (e.g. danazol) X CHROMOSOME Introns: Intron size (kb): Exon size (bp): Exons:

26 15 26 5.6 4.8 0.8 0.7 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 4 3 2 1 p q q11–12 5 6 7 3' 5' 8 1613 152 117 288 145 131 158 155 -COOH Zn++ Zn++ Domains Transcription-regulation DNA-binding Hinge Steroid-binding NH2- GENE cDNA RECEPTOR PROTEIN Fig. 13.6.2.5  Structure of androgen receptor. The gene is made up of 8 exons, the mRNA encoding 910 amino acids. The zinc finger configuration is characteristic of all steroid hormones.

section 13  Endocrine disorders 2392 that are found in the promoter regions of androgen sensitive genes. The transcriptional activity of the androgen receptor is modulated by numerous coactivators, including SRC 1/​NCoA-​1, SRC2/​GRIP1-​ TIF2 and SRC3/​ACTR/​AIB1, and negatively regulated by AP-​1, NFκB, TR4, HBO1, and AES. A defective androgen receptor may lead to variable phenotypes of androgen insensitivity in humans. Physiological effects of testosterone The functions of T are age-​related (Table 13.6.2.2). In the embryo, androgens are responsible for sexual differentiation (i.e. virilizing the external and internal genitalia). Thus, during the sexual dif- ferentiation phase, development, and growth of the Wolffian duct, epididymis, vas deferens, and seminal vesicles are pro- moted by testosterone. The enzyme 5 α-​reductase, which converts T to DHT, is expressed in scrotal skin, the penile shaft, and the prostate. These tissues depend on DHT for their development (Fig. 13.6.2.6). Failure of 5 α-​reductase activity leads to micropenis and incomplete/​deficient labioscrotal fusion giving rise to am- biguous genitalia. At puberty, androgens, acting with growth hormone, are re- sponsible for the adolescent growth spurt, in particular for ver- tebral (i.e. upper segment) growth. It also induces promotion of secondary sexual characteristics. In adulthood, it maintains the male phenotype, sexual function as well as mediating ana- bolic effects. The endocrine (androgen synthesis) and gameto- genic (spermatogenesis) functions of the testis are interlinked. Although testosterone is important as the principal circulating androgen, its local paracrine action within the testis is crucial, together with FSH, for the initiation and maintenance of normal spermatogenesis and hence fertility. Since germ cells do not pos- sess AR, these hormones signals are transduced through the Sertoli cells and peritubular cells. Sertoli cells create an insular microenvironment in the seminiferous tubules by providing the physical framework and elaborating a chemical myriad of growth Table 13.6.2.2  Consequences of androgen deficiency before and after the onset of puberty. Hypogonadism occurring at the time of expected puberty results in a different phenotype to that acquired postpubertally Physiological action of androgen Onset of androgen deficiency before puberty Onset of androgen deficiency after puberty Increase bone mass and density Osteoporosis Osteoporosis, female fat distribution Fusion of long bone epiphyses Tall, eunuchoid habits Decrease subcutaneous/​visceral fat Female fat distribution Female fat distribution Laryngeal enlargement Unbroken, high pitched voice Secondary sexual hair development Lack of pubic, axillary, and facial hair, no temporal recession Decrease facial and pubic hair, no temporal recession Increase pilosebaceous activity Lack of sebum, pale smooth skin Atrophy, fine wrinkles, pallor Stimulation of erythropoiesis Moderate anaemia Moderate anaemia Increase in muscle mass Underdeveloped, poor physical stamina Decrease strength and physical stamina Penile growth Infantile Prostate and seminal vesicle growth Underdeveloped, no ejaculate Atrophy, low volume, or absence of ejaculate Stimulation of spermatogenesis Not initiated, very small testes Regression, small testes Stimulation of sexual interest Not developed Decrease Stimulation of erectile function Low/​absent spontaneous erection Decrease erection Effect on mood and behaviour Placid Low moods, unassertiveness, tiredness 5-α reductase Aromatase Sexual differentiation Secondary sexual hair growth Production of sebum Prostatic growth Sexual differentiation Muscle growth Increase in bone mass Erythropoiesis Erythropoietin synthesis Sexual potency and libido Psychotropic effects Dihydrotestosterone Oestradiol Bone mass Epiphyseal fusion Psychotropic effects Negative feedback modulation of gonadotrophin secretion Prostate growth (complex effects) TESTOSTERONE Fig. 13.6.2.6  Testosterone is metabolized to dihydrotestosterone by the enzyme 5-​α reductase and to oestradiol via the aromatase enzyme. These hormones exert different effects to testosterone.

13.6.2  Disorders of male reproduction and male hypogonadism 2393 factors and cytokines for the developing germ cells. Sertoli cells also secrete inhibin B, a glycoprotein hormone which inhibits FSH secretion by the pituitary. Male hypogonadism Male hypogonadism is a descriptive term for the clinical complex associated with androgen deficiency due to failure of Leydig cell function. Concomitant impairment of spermatogenesis is likely since the seminiferous tubules will also be androgen deficient or directly involved by the same pathological process. However, infertility is usually an isolated abnormality of spermatogenesis where patients seldom show any clinical of androgen deficiency. In the past several years, an increasing number of specific genetic defects have been identified by genomic DNA mapping to be asso- ciated with abnormal gonadal function and development. New light has been shed on the pathogenesis of these conditions. Aetiologies There are a large number of pathological conditions that can lead to de- struction or malfunction of the hypothalamo-​pituitary-​testicular axis (Tables 13.6.2.3 and 13.6.2.4). It is important to identify the underlying cause of hypogonadism and distinguish between pituitary/​ hypothalamic Table 13.6.2.3  Classification and aetiologies of male reproductive disorders Site of lesion Clinical picture Androgen deficiency Infertility Hypothalamus and pituitary (hypogonadotropic hypogonadism) Isolated GnRH deficiency Congenital GnRH deficiency + + Kallmann syndrome Anosmia and GnRH deficiency + + GnRH insensitivity GnRH receptor gene mutation + + Fertile eunuch (Pasqualini syndrome) Partial GnRH deficiency, low LH +

Hypogonadotropic hypogonadism/​adrenal hypoplasia DAX-​1 gene mutation + + Constitutional delayed puberty Self-​limiting + + Male anorexia nervosa Weight-​related, reversible GnRH deficiency + + Hyperprolactinaemia Pituitary tumour, drug induced + + Congenital hypopituitarism PROP 1 gene mutation, hypogonadotropic hypogonadism prolactin, GH, ACTH deficiencies + + Acquired hypopituitarism Pituitary tumour, craniopharyngioma, haemachromatosis irradiation, hypophysitis, transfusion haemosiderosis, sarcoidosis, tuberculosis, histiocytosis X + + Biologically inactive LH LH β gene mutation + + Isolated FSH deficiency FSH β gene mutation ? + Testicular (hypergonadotropic hypogonadism) Klinefelter’s syndrome 47XXY, 48XXXY,47XXY/​46XY mosaic, and so on + + 46XX male SRY gene translocation to X chromosome + + Sex chromosome/​autosomal abnormalities Translocation, deletion + –​ Mixed gonadal dysgenesis XY/​XO, true hermaphroditism + + Testicular agenesis Absence of testes postnasally + + Testicular torsion Destruction of testicular tissue + + Surgical orchidectomy, testicular trauma, tumour orchitis Destruction of testicular tissue + + Sickle cell disease Microinfarcts of the testes + + Noonan–​Leopard syndrome 12q22 gene defect, autosomal-​dominant cryptorchidism, Turner’s stigmata, with short stature, webbed neck, pectus excavatum hypertelorism, ptosis, right sided congenital heart disease Persistent Müllerian duct syndrome AMH gene or AMR receptor type II gene mutation, Fallopian tube, and uterus present with cryptorchidism +/​-​ + Congenital steroidogenic enzyme deficiencies 10q.24.3 CYP17 17,20-​desmolase, 9q22 HDD17b3, 17OH-​steroid DH gene mutation + + (continued)

section 13  Endocrine disorders 2394 Site of lesion Clinical picture Androgen deficiency Infertility LH insensitivity LH receptor mutation, pseudohermaphroditism + + Idiopathic infertility Defective spermatogenesis of uncertain aetiology –​ + Varicocele Reflux in spermatic vein –​ + Microdeletions Yq Deletion of azoospermic factor –​ + Cryptorchidism Congenital deficiency of testosterone or AMH action, dysgenetic gonads +/​–​ + Immotile cilia syndrome Absent dynein arms of sperm tail microtubules –​ + Globozoospermia Absence of acrosome cap on sperm head –​ + FSH insensitivity 2q21 FSH receptor gene mutation ? + Post-​testicular Immunological Sperm antibodies –​ + Immotile cilia Dynein arms absent in sperm tail –​ + Young’s syndrome Mercury poisoning? –​ + Congenital bilateral absence of vas deferens CFTR gene mutation and intronic variant –​ + Genital tract infection Postinfection, postvasectomy, herniorrhaphy –​ + Accessory gland/​prostate infection Bacterial, chlamydia, abnormal seminal fluid –​ + Retrograde ejaculation Autonomic neuropathy, postprostatectomy –​ + Coital insufficiency Defective vaginal insemination –​ + Target tissues Androgen insensitivity syndromes Xq11-​12 androgen receptor gene mutation + + Androgen receptor defects Xq11-​12 androgen receptor gene CAG repeat expansion + + 5-​α reductase deficiency 2p23 5 α reductase 2 gene mutation + + Oestrogen insensitivity ER α gene mutation –​ ? Aromatase CYP19 gene mutation –​ ? Systemic diseases Acute critical illness Cytokine or cortisol-​induced multilevel dysfunction + –​ Chronic illness: congestive cardiac failure, neoplasia, uncontrolled diabetes mellitus Cytokine or caloric deprivation induced multilevel dysfunction in HPT axis + + Liver cirrhosis Primary testicular failure, followed by gonadotrophin deficiency + + Chronic renal failure Hypogonadotropic hypogonadism + + Thyrotoxicosis Increased SHBG, gonadotrophins, oestradiol + + Cushing’s syndrome Multilevel dysfunction in HPT axis + + Haemochromatosis Hypogonadotropic + + HIV infection Hypogonadotropic + + Morbid obesity Hypogonadotropic, low SHBG, total, free T + + Obstructive sleep apnoea Hypogonadotropic + + Rheumatoid arthritis Suppression of testosterone during flare up + ? Acute febrile illness Temporary suppression of spermatogenesis –​ + Untreated congenital adrenal hyperplasia Suppression of gonadotrophin –​ + Neurological diseases Dystrophia Myotonin protein kinase (MT-​PK) gene CTG repeat expansion + + Prader–​Willi syndrome Deletion/​mutation of imprinting centre in paternal 15q11-​13, hypogonadotropic, mental retardation hypotonia, hyperphagia, obesity, short stature + + Laurence–​Moon syndrome Hypogonadotropic, retinitis pigmentosa, mental retardation, obesity, polydactyly + + Table 13.6.2.3  Continued

13.6.2  Disorders of male reproduction and male hypogonadism 2395 (secondary or hypogonadotropic hypogonadism), and testicular (pri- mary or hypergonadotropic hypogonadism) disorders. The causal lesion may require specific treatment (e.g. as in the case of a prolactin se- creting pituitary tumour and haemochromatosis). Hypogonadotropic conditions are amenable to treatment aimed at inducing or restoring spermatogenesis, while in primary testicular failure—which is usually irreversible—only testosterone replacement therapy is possible. Clinical features General clinical features of hypogonadism The age of onset of androgen deficiency critically influences the manifestations of hypogonadism (Table 13.6.2.2). Prepubertal onset of testosterone deficiency gives rise to sexual infantilism and patients present with delayed puberty. Eunuchoid body proportions (arm span greater than height and heel to pubis exceeding crown to pubis lengths by at least 5 cm) develop due to the continued growth of long bones (growth hormone mediated, allowed by the delayed closure of epiphyses due to lack of testosterone/​oestradiol-​induced spinal growth in late puberty). Postpubertal onset of testosterone deficiency leads to regression of spermatogenesis, low libido, erectile malfunction, loss of ejacula- tion, sarcopenia, poor stamina, and decreased secondary sexual hair and shaving frequency. However, no change is observed in body and penile proportions nor voice. Symptoms and signs of hypogonadism usually develop and progress insidiously. It is therefore common for patients to present many years following the onset of hypogonadism. Furthermore, younger patients who have never been adequately an- drogenized may not be aware, or even deny, that sexual function is subnormal. By contrast, after surgical or traumatic/​inflammatory cas- tration, adults may experience hot flushes from acute withdrawal of androgens. Fetal onset of defective androgen action due to androgen receptor abnormalities or defects of steroidogenic enzymes, will cause failure of masculinization of the genitalia resulting in intersexual states. Clinical findings associated with hypogonadism Hypothalamo-​pituitary tumours should be considered in the pres- ence of headache, defects of visual acuity or visual field loss, polyuria and polydipsia suggesting diabetes insipidus, or clinical /​biochemical Site of lesion Clinical picture Androgen deficiency Infertility Bardet–​Biedl syndrome Defects in BBS loci 16q11, 15q23.3, or 3p12 hypogonadotropic, retinitis pigmentosa, mental retardation, polydactyly + + Familial spinocerebellar degeneration 9p frataxin gene GAA repeat expansion, hypogonadotropic, progressive ataxia + + Kennedy syndrome X911-​12 androgen receptor gene CAG repeats expansion, late-​onset androgen resistance, progressive spinobulbar muscular atrophy + + Temporal lobe epilepsy Unknown + –​ Spinal cord injury Abnormal thermoregulation or neuroregulation of testes –​ + Fragile X syndrome FMR 1 gene CCG repeats expansion, mental retardation, macro-​orchidism –​ –​ Drugs/​chemical or physical agents Digitalis, spironolactone, cyproterone acetate, flutamide, bicalutamide, cimetidine Antiandrogenic + + Corticosteroids Multilevel dysfunction in HPT axis + + Ketoconazole Inhibits steroidogenesis + + Aminoglutethimide Antipsychotics, sedatives Hyperprolactinaemia, gonadotrophin suppression + + Anticonvulsants Increase SHBG, decreased free testosterone + + Ethanol Direct suppression of testicular function, hepatotoxic + + Opiate, cocaine, cannabis abuse Suppression of gonadotrophin + + Cytotoxic drug Agent specific, dose-​related germ cell loss –​ + Ionizing radiation Dose-​dependent loss of spermatogenesis, spermatocytes –​ + Sulfasalazine Abnormal sperm morphology and motility –​ + Nitrofurantoin Direct suppression or antiandrogenic –​ + Anabolic steroids, oestrogens, progestins Gonadotrophin suppression or antiandrogenic (+) + Lead, mercury, cadmium Adverse effects on spermatogenesis –​ + Pesticides, fungicides, amoebicides Direct toxic effects on spermatogonia –​ + HPT, hypothalamic–​pituitary–​thyroid axis. Table 13.6.2.3  Continued

section 13  Endocrine disorders 2396 evidence of pituitary hormone excess such as that found in Cushing’s disease, acromegaly, and hyperprolactinaemia. Hyperprolactinaemia causes loss of libido even in the presence of apparently normal tes- tosterone concentrations. Primary testicular failure is suggested by a history of orchitis, torsion, testicular trauma, surgery, chemotherapy, irradiation. An increasing number of chronic systemic diseases (Table 13.6.2.3) are associated with compromised hypothalamo-​ pituitary-​testicular function. With improved survival resulting from specific treatments, the role of gonadal dysfunction in the quality of life of these patients is becoming increasingly recognized. A history of excess alcohol consumption, use of recreational drugs and consumption of medications that interfere with pituitary-​ testicular function or androgen action should be specifically eluci- dated. Ethanol causes a lowering of plasma testosterone through a direct toxic effect on Leydig cell steroidogenesis. Testicular atrophy and gynaecomastia, found in 50% of men with liver cirrhosis, are due to disordered androgen steroid metabolism, an increase in sex hormone binding globulin, coupled with an increased oestrogen production. These changes are usually irreversible. Neurological diseases can be associated with hypogonadism. Postpubertal atrophy of the seminiferous tubules occurs in 80% of patients with dystrophia myotonica, an autosomal-​dominant disorder characterized by myotonia, distal muscle atrophy, lens opacification, and premature frontal balding. Variable degrees of androgen deficiency also coexist. Hypogonadotropic hypogonadism is associated with familial cerebellar ataxia, Gordon Holmes ataxia, Lawrence Moon, Bardet–​Biedl, and Prader–​Willi syndromes. Defective spermatogenesis is common in paraplegia or quadriplegia following spinal cord injury, perhaps due to the inability to maintain a low scrotal temperature. Specific conditions Primary gonadal failure Klinefelter’s syndrome Klinefelter’s syndrome is the commonest cause of male hypo- gonadism with an incidence of 2 per 1000 live births. It is a devel- opmental disorder of the testes resulting from the presence of an Table 13.6.2.4  Phenotypic characteristics associated with genes that cause hypogonadotropic hypogonadism (HH) Gene Gene product Inheritance Clinical phenotype GnRH GnRH Autosomal recessive Normosmic HH GnRHR Gonadotrophin releasing hormone receptor Autosomal recessive Normosmic HH LH β Luteinizing hormone β chain Autosomal recessive Normosmic HH SEMA3A Semaphorin-​3A Autosomal dominant IHH and anosmia SOX10 SOX10 Autosomal dominant IHH and anosmia, Waardenburg syndrome NR0B1 DAX-​1 X-​linked X-​linked adrenal hypoplasia and HH KAL-​1 Anosmin-​1 X-​linked IHH and anosmia, synkinesis, solitary kidney FGFR1 FGF receptor 1 Autosomal dominant Normosmic HH, craniofacial defects, digital anomalies of toes, dental agenesis FGF8 Fibroblast factor 8 Autosomal dominant Normosmic HH FGF17 Fibroblast growth factor 17 Single allelic defect insufficient Normosmic HH HS6ST1 Heparan-​sulphate 6-​0-​sulphotransferase Non​mendelian HH +/​-​ anosmia IL17RD Interleukin 17 receptor D Single allelic defect insufficiency IHH and anosmia, hearing loss DUSP6 Dual-​specific phosphatase 6 Single allelic defect insufficient HH SPRY4 Sprouty homologue 4 Single allelic defect insufficient HH FLTR3 Fibronectin leucine-​rich transmembrane protein 3 Single allelic defect insufficient HH NELF Nasal embryonic LHRH factor Single allelic defect insufficient HH+/​-​ anosmia CHD7 Chromodomain helicase DNA-​binding protein-​7 Autosomal dominant HH+/​-​anosmia, CHARGE syndrome PROK2 Ligand for G protein coupled prokinecitin receptor-​2 Heterozygous mutations, oligogenic inheritance HH +/​-​ anosmia PROKR2 G-​protein-​coupled prokineticin receptor-​2 Heterozygous mutations, oligogenic inheritance HH +/​-​ anosmia WRD11 WD repeat containing protein-​2 Heterozygous missense mutations HH+/​-​ anosmia GPR54 KISS1-​derived peptide receptor Recessive mutations HH KISS1 Metastin or kisspeptin Recessive mutations HH TACR3 Neurokinin B receptor Recessive mutations HH TAC3 Neurokinin B Recessive mutations HH LEP Leptin Recessive mutations HH LEPR Leptin receptor Recessive mutations HH IHH, ‘isolated’ or ‘idiopathic’ HH; LHRH, luteinizing hormone-​releasing hormone.

13.6.2  Disorders of male reproduction and male hypogonadism 2397 additional X chromosome derived from non​disjunction of parental (maternal origin in two-​thirds of cases) germ cells during meiosis. In fewer than 5% of patients, the non​disjunction occurs during mi- tosis of the zygote. Such postfertilization mitotic non​disjunction will result in mosaicism. The most common karyotype is 47XXY (80 to 90%), but rarer variants include 46XY/​47XXY mosaic, mul- tiple X+Y, and the so-​called XX male syndrome. Unlike many other numeral aberrations of chromosomes, KS is not associated with an increased rate of miscarriage. The risk of having a child with KS increases with both increasing maternal and paternal age. The signs of Klinefelter’s syndrome are almost unnoticeable in childhood. Accelerated atrophy of germ cells before puberty and hyalinization of the seminiferous tubules give rise to very few intact gametes, and the usual outcome is sterility and small firm (around 4 ml) testes. Individual tubules with intact spermatogenesis are seen in patients with the mosaic form 46,XY/​47,XXY and very occasion- ally motile sperms are found in the semen. Leydig cells appear rela- tively hyperplastic, although Leydig cell mass is in fact normal. The degree and impact of the Leydig cell steroidogenic defect (of uncer- tain aetiology) is very variable, ranging from the virilized adult male presenting with infertility, to the eunuchoid youth who fails to com- plete sexual maturation. In KS, the extra X chromosome carrying the androgen receptor with a short CAG repeat length in Exon 1 (i.e. greater AR activity) undergoes activation preferentially. Thus, the skewed inactivation of the X chromosome resulting in the preferen- tial activity of the long CAG repeat may contribute to the phenotypic severity and variability of KS. In adults, libido and potency are ini- tially normal, but decrease between the ages of 25 and 35, reflecting an increasing insufficiency of Leydig cells. In mid-​adulthood, 80% of patients have reduced testosterone with elevated LH/​FSH and oes- tradiol levels. Other clinical features include gynaecomastia, reduced body hair, long legs, tall stature (eunuchoid proportions), and learning (verbal and cognitive) difficulties, poor school performance, behavioural disturbances, and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren’s syndrome, as well as type 2 diabetes mellitus. Infants with KS may manifest with micropenis, hypospadias, cryptorchidism, or developmental delay. In addition to relatively tall stature, some patients have clinodactyly, hypertelorism, elbow dysplasia, high arched palate, hypotonia, lan- guage delay or reading and learning disabilities requiring therapy occurs in up to 70%. Character and personality disorders and be- havioural problems occur commonly, possibly in part because of the psychosocial consequences of androgen deficiency. IQ scores may be reduced by 10–​15%, though not into the intellectual disability range. There is also an increased incidence of osteopenia, mitral valve prolapse, breast tumours (3–​5%: the risk increases if there is a family history of breast cancer among female relatives), testicular and extratesticular germ-​cell tumours (especially mediastinal and retroperitoneal), varicose veins, and leg ulcers. Taurodontism, char- acterized by enlarged molar teeth resulting from enlargement and ex- tension of the pulp chamber, is present in 40% of men with KS. Mental retardation is associated with higher order X chromosome polysomy. Diagnosis and laboratory findings  Testicular volumes of less than 4 ml should always lead to a suspicion of KS. Serum testos- terone (T) is often reduced or lies in the lower reference range (40% patients), and free T is more frequently reduced than total T as the SHBG level tends to be increased. As LH stimulation is increased, Leydig cells produce relatively more oestradiol, with increase in free E2 to free T ratios. The Barr chromatin body test is a rapid investi- gation used to determine a chromosomal abnormality in a swab of cheek mucosa, but the definitive diagnosis rests on karyotyping of lymphocytes. Therapy  Androgen deficiency, as evidenced by a rise in LH, should prompt replacement therapy with testosterone. Androgen treatment should start early in the adolescent as this significantly promotes psychosocial development, and may prevent the development of gy- naecomastia and reduce the risk of breast cancer. It is important also for adequate development of secondary sexual characteristics, at- tainment of peak bone mass and bone mineral density and strength, energy, motivation, mood, and behaviour. If gynaecomastia is cos- metically unacceptable, mastectomy may be indicated. Infants with micropenis may benefit from topical testosterone, and early inter- vention with speech and language therapy is important if speech delay and dyslexia are present. Dystrophia myotonica This is an autosomal-​dominant inherited condition, characterized by delayed muscle relaxation after contraction, dystrophy of the distal musculature and pharyngeal muscles, cataracts, hyperacusis, and frontotemporal hair loss. 80% of affected males develop a pro- gressive untreatable primary hypogonadism with testosterone defi- ciency and damage to the germinal epithelium, with accompanying reduced testicular volumes, and hyalinization of the seminiferous tubules and vacuolation of Sertoli cells. 60% of affected males have testosterone deficiency, with loss of libido and impotence. Gonadotrophins are increased, particularly FSH. Tuberculosis Tuberculous orchitis is rare, but should be included in the differ- ential diagnosis of testicular tumours. The scrotal mass is usu- ally painless, and haematospermia, sterile pyuria, hydrocele, and oligoasthenoteratozoospermia may be present. Leprosy This can cause a granulomatous infiltration of the testes and up to 60% of affected males are hypogonadal, particularly with lep- romatous leprosy, though rarely with the tuberculoid form. Gynaecomastia may be present, and testosterone levels are low with increased gonadotrophin. Transverse section of the spinal cord Severe trauma to the spinal cord often leads to exocrine testicular insufficiency. Testosterone secretion often returns to normal spon- taneously, but spermatogenesis is usually permanently impaired in paraplegics and tetraplegics. This is thought to occur as a conse- quence of disorders of thermoregulation and circulation. Secondary gonadal failure This can result from absent or defective GnRH secretion, or failure of the anterior pituitary to respond to GnRH released from the me- dian eminence, leading to hypogonadotropic hypogonadism (HH). Kallmann syndrome and other forms of inherited HH This has an incidence of 1 in 7500 males, and is a sporadic or familial (X-​linked or autosomal) form of congenital hypogonadotropic

section 13  Endocrine disorders 2398 hypogonadism associated with several somatic congenital abnor- malities including anosmia or hyposmia (defective smell sense), hereditary bimanual synkinesis (mirror hand movements), nerve deafness, cleft lip or palate, renal malformations, and dental abnormalities. There are now over 20 genes whose mutations have been in- criminated in the pathogenesis of congenital hypogonadotropic hypogonadism. One of the most severe phenotypes is seen in X-​linked Kallmann syndrome, caused by mutations or deletions within the KAL-​1 gene, located in the Xp22.3 region, encoding the cell adhesion protein anosmin-​1. This is associated with faulty embryonic migration of GnRH secreting neurons from their site of origin in the medial olfactory placode into the hypo- thalamus, thereby preventing normal neurosecretion of GnRH (gonadotrophin releasing hormone) into the median eminence capillary circulation such that it does not reach the gonadotrophs of the anterior pituitary, resulting in hypogonadotropic hypo- gonadism. Associated maldevelopment of the olfactory bulb is responsible for anosmia. Patients present with delayed puberty, but the diagnosis may be suspected when neonatal males have undescended testes. Several additional genes are mutated in Kallmann syndrome that affect the fate and migration of GnRH neurons. Some of these will be associated with ‘isolated’ or ‘idiopathic’ hypogonadotropic hypogonadism (IHH) and anosmia, while others are associated with IHH alone (normosmic forms of IHH) (Table 13.6.2.4). Genes encoding fibroblast growth factor 8 (FGF8) signalling pathway proteins, chromodomain helicase DNA-​binding protein 7 (CHD7) and sex determining region Y-​Box 10 (SOX10) affect the neurogenic niche in the nasal area and craniofacial development. Prokineticin-​2 and prokineticin receptor 2 (encoded by PROK2 and PROKR2, respectively), WD repeat domain 11 (encoded by WDR11), semaphorin 3A (encoded by SEMA3A) and FEZ family zinc finger 1 (encoded by FEZF1) influence the migration of GnRH neurons. Postmigratory GnRH neurons are embedded in a complex neur- onal network of afferents that send information about permis- sive reproductive cues such as steroid and metabolic hormones to these cells (Fig. 13.6.2.2). Individual components of the underlying neural circuits are increasingly recognized, and some key molecules have been discovered through the study of the genetics of isolated hypogonadotropic hypogonadism. Inactivating mutations in genes encoding kisspeptin-​1 (KISS1) and its receptor (KISS1R) arrest pubertal development in humans. Extensive experimental studies in various species have demonstrated that kisspeptin-​producing neurons are major afferents to GnRH neurons and essential for dif- ferent aspects of GnRH function, ranging from the tonic feedback control of GnRH and/​or gonadotropin secretion to generation of the preovulatory surge responsible for ovulation. Although kisspeptins are not essential for GnRH neuron migration, experimental data has documented that populations of kisspeptin neurons undergo a dynamic process of prenatal and postnatal maturation enabling them to establish connections with GnRH neurons early in devel- opment (under the control of steroid hormones). Similarly, identi- fication of mutations in TAC3 (encoding tachykinin-​3, cleaved to form neurokinin B) and TACR3 (encoding tachykinin receptor 3; also known as neuromedin-​K receptor or NKR) in patients with IHH have underlined the importance of the tachykinin family in the control of GnRH neurons. These findings led to the identification of a subpopulation of afferent neurons in the arcuate and/​or infundibular hypothalamic region, coexpressing kisspeptins and neurokinin B (NKB), and it appears that the actual number of kisspeptin and/​or NKB neurons change during development. Mutations in proteins that regulate ubiquitination such as OTU domain-​containing pro- tein 4 (encoded by OTUD4) and E3 ubiquitin-​protein ligase RNF216 (also known as ring finger protein 216; encoded by RNF216), as well as in proteins involved in lipid metabolism such as neuropathy target esterase (also known as patatin-​like phospholipase domain-​ containing protein 6; encoded by PNPLA6), have been identified in patients with Gordon Holmes syndrome (with associated IHH and ataxia). Thus, mutations in these three genes give rise to a broad and progressive neurodegenerative syndrome that includes IHH. Haploinsufficiency of DMXL2, which encodes synaptic pro- tein DmX-​like protein 2, has been shown to cause a complex new syndrome associating IHH with polyendocrine deficiencies and polyneuropathies. Peripheral signals that convey information about metabolic status indirectly modulate GnRH neurosecretion as evidenced by the reproductive phenotype of absent pubertal development and hypogonadotropic hypogonadism in patients with inactivating mu- tations in the genes encoding leptin (LEP) or its receptor (LEPR). Experimental data suggest that kisspeptin neurons are sensitive to changes in leptin concentrations and metabolic conditions by an in- direct mechanism. Mutations of the GnRH locus and in the GnRH receptor cause IHH with normal olfactory function. This also occurs in patients with LH β-​gene mutations (Table 13.6.2.4). Oligogenicity and reversibility in IHH  Recent evidence suggests that in a few cases the coexistence of mutations in several genes incriminated in IHH may be necessary for full phenotypic expres- sion. This may explain why phenotypic penetrance can be variable with an identical genotype at one locus shared by several members of a kindred. It has also been shown that the HH phenotype is po- tentially reversible in up to 10% of patients with IHH. Late-​onset hypogonadism Total and free testosterone decline gradually and in varying degrees in men from the age of 40 onwards. This is amplified by the age-​ related increase in SHBG levels exacerbated by concomitant sys- temic diseases and the use of some medications. Differentiation of non​specific symptoms of ageing such as frailty, decreased muscle strength, lack of stamina and vitality, decline in libido, from those of mild classical hypogonadism can be difficult. A significant percentage of men over 60 years of age have serum testosterone levels below the lower limits of normal for young male adults (20 to 30 years), and some longitudinal studies have suggested that as many as 20% of men in their 60s and approximately 50% of men in their 80s have serum total testosterone (TT) levels signifi- cantly below those of normal young men. However, the European Male Ageing Study (EMAS) estimated a much lower prevalence (2.1%) of symptomatic late-​onset hypogonadism in the popula- tion. It is evident that the clinical symptoms/​manifestations in this age group may be more difficult to recognize because of masking by comorbid illnesses. There is controversy as to the significance of falling testosterone levels with age, some believing that it is a

13.6.2  Disorders of male reproduction and male hypogonadism 2399 medically significant condition resulting in significant detriment to the quality of life and adversely affecting the function of multiple organ systems, while others suggest that it is a chemical marker of generalized illness. Although significant advances have been made in improving the understanding of the pathophysiology of the hypogonadism, the diagnostic methods used to diagnose low testosterone levels, and testosterone replacement therapy, a great deal of confusion and mis- understanding still exists among clinicians and patients about the diagnosis of hypogonadism in ageing men, and benefits and risks associated with testosterone therapy. The important questions as yet unanswered questions are: (1) How to diagnose late-onset hypo- gonadism in ageing males? (2) What are the best treatment options for late-onset hypogonadism? (3)  Will older hypogonadal men benefit from testosterone treatment? (4) What are the risks associ- ated with such interventions? Investigation Confirmation of hypogonadism The clinical suspicion or diagnosis of hypogonadism must be con- firmed by demonstration of low circulating testosterone before re- placement therapy can be envisaged. It is recommended that blood samples be obtained between 8 to 9 AM, avoiding the circadian fall in levels of testosterone seen later in the day. The interpretation of total testosterone requires measurements of SHBG, which can alter in ageing, obesity, with the use of anticonvulsive medications, dia- betes, iron overload, and liver disease. The free testosterone can be calculated from the total testosterone, SHBG and albumin concen- trations using the Vermeulen formula (see http://​www.issam.ch/​ freetesto.htm for calculator). Assessment of the hypothalamo-​pituitary-​testicular axis and the target tissue androgen resistance Measurement of LH, FSH, and testosterone are required to dis- tinguish between primary and secondary hypogonadism. In primary gonadal failure, LH and FSH levels are elevated and testosterone levels low, whereas in secondary gonadal failure low testosterone levels are associated with inappropriately low gonadotrophins. Causes of hypo and hypergonadotropic hypo- gonadism are listed in Table 13.6.2.3, as are other conditions that can impair fertility. Pathologies in the hypothalamus and pituitary give rise to low or low normal gonadotrophins and low testosterone (hypogonadotropic hypogonadism or secondary testicular failure), where the potential for stimulating testicular function by exogenous gonadotrophin or GnRH replacement is maintained. Conditions affecting the testes will interrupt normal testicular negative feedback. This results in elevated gonadotrophin levels with a low testosterone, characteristics of a hypergonadotropic hypogonadal state (primary testicular failure). Failure of spermatogenesis with reduced testicular size is commonly associated with a rise in FSH alone. The value of es- timation of circulating inhibin B and Müllerian inhibiting hor- mone (MIH) for diagnostic purposes is currently being assessed. Patients with androgen insensitivity syndromes have elevated testosterone with high LH, but normal to low FSH. Increased LH or FSH is associated with a very rare LH and FSH resistance syndromes. Human chorionic gonadotropin (HCG) stimulates Leydig cell steroidogenesis and increases plasma testosterone level over 4 to 7 days. Administration of HCG is useful for detecting the presence of functional testicular tissue in patients with impalpable testes, and to assess functional reserve of the testes prior to treatment with exogenous gonadotrophin or GnRH, and in differentiating hypergonadotropic hypogonadism from rare causes who produce immunologically detectable but biologically inactive LH excess. Stimulation tests of gonadotrophin secretory reserve using clomi- phene and GnRH seldom give additional information and have be- come largely obsolete, especially with the improved sensitivity and range of modern gonadotrophin assays. Assessment of the pituitary Patients with hypogonadotropic hypogonadism without the stigmata of Kallmann syndrome should undergo full anterior pitu- itary functional evaluation and an anatomical basis sought for their gonadotrophin deficiency (e.g. a mass lesion in the hypothalamo-​ pituitary region). They require pharmacological tests of growth hormone and ACTH reserve, thyroid function tests, visual field charting, and MR or CT scanning of the hypothalamus-​pituitary region. Other investigations Ultrasound and MR scanning are useful in locating ectopic or intra-​ abdominal testes. DNA analysis can help confirm the diagnosis of androgen resistant syndromes and an increasing number of rare causes of hypogonadism such as haemochromatosis. Treatment objectives The treatment objectives are to: 1. Relieve the symptoms of androgen deficiency 2. Prevent the long-​term consequences of androgen deficiency such as osteopenia 3. Reproduce physiological circulating and tissue levels of plasma testosterone, dihydrotestosterone, and oestradiol 4. Induce fertility if required in hypogonadotropic patients 5. Treat any specific underlying disorder The mainstay of treatment of the hypogonadal male is androgen re- placement therapy. Although hypogonadotropic patients have the potential for fertility, gonadotrophin and pulsatile GnRH therapy should only be employed where there is a requirement for fertility because of the expense and complexity of these regimens. Previous testosterone exposure does not jeopardize response to gonado- trophins, hence younger hypogonadal subjects should be treated by testosterone in the same manner as hypergonadotropic patients to initiate and maintain virilization and sexual function. Modalities of androgen replacement therapy The circulating half-​life of free testosterone is around 10 minutes due to rapid metabolism by the liver. To achieve sustained physio- logical circulating concentrations, testosterone must be adminis- tered in a modified form or by a parenteral route so that its rate of metabolism or absorption is retarded. Injectable testosterone esters are the commonest first-​line an- drogen preparations. A  mixture of four different testosterone

section 13  Endocrine disorders 2400 esters (propionate, phenylpropionate, isocaproate, and decanoate) (Sustanon, 250 mg 2 to 3 weekly) and testosterone enanthate (Primoteston Depot) are the most popular. While undoubt- edly effective, these preparations inevitably give rise to high supraphysiological peak testosterone levels within the first week, which then fall sharply to the lower limit of normal before the next dose. Some patients are disturbed by fluctuations in libido, mood and stamina associated with the repeated rise and fall of testosterone levels, as well as by the painful deep intramuscular injections. Crystalline testosterone compressed into cylindrical palates, surgically implanted subcutaneously under local anaesthesia, pro- vide a depot source of testosterone which last 6 to 8 months. Peak testosterone levels are achieved after 2 to 4 weeks followed by a gradual decline over the subsequent months. A total dose of 800 mg can maintain physiological concentrations of testosterone for between 6 to 8 months, which some patients find more convenient than more frequent injections. The implantation procedure can be conducted as an outpatient, although rarely can be complicated by haemorrhage or infection, and in inexperienced hands 10% of im- planted pellets may be extruded, often quite late. Implants should only be used as maintenance therapy in patients who have already shown satisfactory tolerance to the androgen effects of shorter acting preparations. Testosterone undecanoate is administered orally, but low bio- availability (<0.5%), variable absorption, the requirement for mul- tiple daily dosing, and higher costs, have restricted its use despite the obvious appeal of oral administration. To maintain testosterone consistently within the physiological range, two to three times daily administration of 80 mg of testosterone undecanoate is required. Moreover, intestinal 5 α-​reductase activity gives rise to a dispropor- tionate and unphysiological increase in dihydrotestosterone relative to testosterone. Oral testosterone undecanoate is useful in the in- duction of puberty in adolescence, where lower doses are preferable, and as second line treatment in adults who are intolerant of injec- tions or implants. 17 α-​alkylated androgens are relatively weak androgens, but some may have more potent anabolic effects. 17 α-​alkylated compounds cause cholestatic jaundice in a reversal and dose-​related manner, while long-​term treatment has been associated with peliosis hepatis (haemorrhagic cysts) in the liver, and rarely with liver adenomas or tumours. Consequently, 17 α-​methyl testosterone, oxymetholone, fluoxymesterone have now been withdrawn from the market in several countries. As a group, 17 α-​alkylated androgens are not re- commended for clinical use, but they remain commonly abused as anabolic steroids. Mesterolone, which is not hepatotoxic, is a weak androgen with low clinical efficacy and remains commercially available. Transdermal testosterone preparations offer stable physiological levels of testosterone without peaks and troughs, painless self ad- ministration, and minimal risks of overdose and low potential for abuse. Testogel (50 mg), Testoderm (50 mg), and Tostran gel (50 mg) are three transcutaneous preparations available in the United Kingdom. These gel preparations have a bioavailability of around 10% and deliver 5 mg or so into the body (equivalent to the daily testosterone production rate of the testes). They are applied to the skin in the shoulder or central abdominal areas, and increase serum testosterone levels into the normal range within one hour of application. Steady state levels are achieved 48–​72 hours after initiation of therapy. The gels have a low incidence of skin irrita- tion, ease of application, invisibility of the dried gel, and have the ability to deliver testosterone dose-​dependently to the low, mid, or upper normal range. Passive transfer of applied testosterone to women and children can be avoided by covering the skin by clothing or showering 6 hours after application. The gels should not be applied to the genitalia and breast area. The choice of preparation depends on age of the patient, the patient’s own preference, facilities for injections, and available ex- pertise for surgical implants. Many boys with constitutional delayed puberty will spontaneously enter or progress into puberty after a short course of testosterone, for example intramuscular testos- terone enanthate 50 mg monthly or oral testosterone undecanoate 40 mg daily for 3 to 6 months. The low doses of testosterone will stimulate linear growth and promote virilization without prema- ture epiphyseal fusion. In patients with no evidence of spontan- eous progression, gradually increasing doses of testosterone over 3 to 4 years will ensure full virilization, except for testicular growth. They can be maintained on adult replacement doses subsequently if hypogonadotropic hypogonadism appears to be permanent. Treatment can be safely started after the age of 14. Indeed, de- layed treatment can be associated with permanently impaired peak bone mass. The invasive nature of the implantation procedure and the long duration of action makes implants less than ideal for the induc- tion of puberty in adolescence and the initiation of treatment in androgen—​naive young adults, where a more gradual and flexible increase in dose is desirable. For these reasons, implants are usually reserved for maintenance treatments in young adults, replacement therapy having been initiated with intramuscular or oral transcu- taneous preparations. Almost all adult patients respond well to testosterone enanthate 200 mg (2-​weekly), 300 mg (weekly), or Sustanon 250 mg (2–​3 weekly). In the absence of a satisfactory biological marker for androgen action, monitoring of treatment is best gauged by clinical response and documenting that plasma testosterone is in the low–​normal range immediately before the next dose, so that appropriate adjustments of dosing intervals can be made. Hypogonadal patients over the age of 50 starting testosterone treatment for the first time should be checked for pre-​existing occult prostatic cancer with the digital rectal examination and prostate-​ specific antigen (PSA) estimation, and these should be repeated in the first 3 to 6 months after initiating treatment to ensure there is no significant change. Subsequent monitoring for prostatic disease should not differ from eugonadal men of comparable age since there is no increased relative risk in hypogonadal patients on long-​term testosterone. Testosterone replacement therapy is safe and side effects are rare, although these may include acne, transient priapism, gynae- comastia, fluid retention, increasing haematocrit, obstructive sleep apnoea, and exacerbation of existing behavioural disturbances. Testosterone is contraindicated in patients with known prostatic or breast cancer. In older patients with benign prostatic hyperplasia, sleep apnoea, polycythaemia, dyslipidaemia, cardiac failure, liver disease, or renal failure, a cautious approach with reduced doses of testosterone, careful dose titration, and close supervision or specific management of the coexisting problems, usually allow patients to benefit from androgen replacement.

13.6.2  Disorders of male reproduction and male hypogonadism 2401 Infertility Infertility is defined as the inability of a couple to initiate a preg- nancy after 12 months unprotected intercourse. It is estimated that 8 to 15% of couples experience involuntary infertility. Of these, male factors alone are estimated to be responsible in up to 30%, and contributory in a further 20% of subfertile couples. Thus, male in- fertility may affect 5% of men of reproductive age. A secular trend of declining semen quality (sperm density) in men over the last 50 years has been reported in some but not other regions of Europe. This, together with a concurrent increase in incidence of testicular cancer, hypospadias, and cryptorchidism, has raised the question of possible environmental endocrine disruptors with oestrogenic or antiandrogenic actions influencing prenatal or neonatal testicular and genital tract development. The concern prompted the recent development of sensitive techniques for monitoring potential dele- terious reproductive effects of environmental chemicals. However, there is currently no evidence that the incidence of male infertility is increasing. Aetiologies Male infertility, comprising a heterogenous group of disorders, rep- resents the male partner’s contribution to the couple’s failure to con- ceive. This implied failure to fertilize normal ova is usually associated with defective spermatogenesis giving rise to absent (azoospermia) or low sperm output (oligospermia <20 million per ml) and/​or ab- normal spermiogenesis giving rise to spermatozoa with poor motility (asthenozoospermia: <50% of spermatozoa showing progressive mo- tility) and abnormal morphology (teratozoospermia: <4% normal forms). The pathogenic basis of defective spermatogenesis or spermiogenesis remains poorly understood. Testicular histology may show quanti- tative reduction in all germ-​cell types (hypospermatogenesis), Sertoli cell only syndrome, or maturation arrest at the primary spermatocyte (premeiotic) or spermatid (postmeiotic) stage. Idiopathic azoospermia/​oligospermia By far the commonest form of male infertility (60%) is idiopathic azoo/​oligospermia (absence of or too few sperm), usually asso- ciated with asthenozoospermia (reduced sperm motility) and teratozoospermia (sperm with abnormal morphology). This prob- ably represents the end result of a multitude of ill-​defined patholo- gies which disrupt normal seminiferous tubular functions. However, recent molecular analyses have revealed that in many cases hitherto classified as idiopathic, there are discrete gene defects associated with impaired spermatogenesis. Asthenozoospermia Reduced velocity or vigour of sperm motility may be due to meta- bolic/​functional defects or ultrastructural abnormalities in the axonemal complex of the sperm tail, usually associated with oligozoospermia or a high percentage of dead and abnormally-​ shaped sperm. The latter finding may indicate a recently recognized condition, epididymal necro/​asthenozoospermia. Testicular sperm- atozoa are normal, the defects occurring during epididymal transit. Rarely, complete asthenozoospermia (with normal sperm density), may result from absence of dynein arms (sites of Na/​K ATPase ac- tivity) linking individual microtubules. This is associated with similar defects in respiratory cilia and a history of chronic respiratory infection, bronchiectasis, and sinusitis (immotile cilia syndrome). In addition, some of these patients have situs inversus (Kartagener’s syndrome). Absence of the central pair of microtubules in the sperm tail is an even rarer cause of complete asthenozoospermia—​the 9+0 syndrome. Teratozoospermia An extreme example of abnormal sperm morphology is the failure of acrosomal cap development the sperm head, leading to forma- tion of rounded spermatozoa (globozoospermia) which are unable to bind to the zona pellucida, a prerequisite for fertilization. Chromosome disorders Chromosome abnormalities identified by cytogenetic studies of blood lymphocytes are found 15% of azoospermic patients, 90% of whom are found to have Klinefelter’s syndrome. Other chromosomal abnormalities encountered include reciprocal X or Y autosomal translocations, XXY, and XX males, reciprocal and robertsonian autosomal translocations, supernumerary autosomes, and inversion of autosomes. Klinefelter’s patients (XXY) are azoospermic. Spontaneous preg- nancies have been reported in the partners of such patients, usually in the context of 46 XY /​47XXY mosaicism. The mechanism whereby an extra X chromosome gives rise to spermatogenic failure is un- clear. Inactivation of the X chromosome in primary spermatocytes is necessary for spermatogenesis to proceed normally through mei- osis. Hyalinized seminiferous tubules devoid of germ cells are perva- sive in the atrophic testes. Occasionally isolated foci of tubules with preserved spermatogenesis can be identified in the testicular biopsy of 47 XXY patients. These can be used for micro testicular sperm ex- traction (TESE) procedures and subsequent intracytoplasmic sperm injection to enable fertility in Klinefelter’s syndrome patients. Y-​chromosome micro deletions A major breakthrough in the understanding of the molecular genetics of male infertility was the characterization of three non-​ overlapping regions (designated azoospermic factors AZFa, AZFb, and AZFc) on the long on the Y-​chromosome (Yq11), which con- tain multiple genes involved in spermatogenesis. Micro deletions in these AZFa loci, identifiable only by polymerase chain reaction (PCR) amplification of DNA but not routine karyotyping, have been found in 3 to 37.5% of patients previously considered to have idio- pathic azoospermia and severe oligozoospermia, but not in fertile control populations. Larger deletions (involving more than one AZF locus) are associated with more severe testicular phenotypes, and the incidence of microdeletions is highest among azoospermic pa- tients with Sertoli cell only histology. AZFc is by far the most fre- quently encountered deletion. Y-​chromosome micro deletions are emerging as the second most common specific aetiology of male in- fertility (after varicoceles). Several cloned genes have been mapped to each of the AZF inter- vals. At least one strong candidate gene is associated with each dele- tion cluster (DFFRY in AZFa, RBMY in AZFb, and DAZ in AZFc). These are multicopy gene families scattered in both arms of the Y-​ chromosome, with the latter two expressed only in the testes. The specific products of these candidate genes and their functional sig- nificance remain unclear. Male infertility associated with micro

section 13  Endocrine disorders 2402 deletions of Y chromatin is probably attributable to reduced copy number of more than one of these gene families. Other as yet un- identified genes important in spermatogenesis within or outside the AZF loci of the Y-​chromosome are highly likely. Some patients with microdeletions of the Y-​chromosome have oligozoospermia and not azoospermia. Transmission of specific Y-​chromosome micro dele- tions to male offspring by assisted conception techniques has been clearly documented. Defects in target tissue Mutations in the ligand binding or DNA-​binding domains of the androgen receptor cause defects in androgen action and varying degrees of failure of masculinization during primary sexual devel- opment (androgen insensitivity syndromes), despite raised levels of testosterone being produced by inguinal or intra-​abdominal testes. These defects are, in descending order of severity: 1. Complete testicular feminization (female phenotype and female external genitalia with absent uterus and Fallopian tubes pre- senting with primary amenorrhoea) 2. Incomplete testicular feminization (female phenotype and fe- male external genitalia with minimal virilization such as clitoral hypertrophy and partial fusion) 3. Reifenstein’s syndrome (ambiguous genitalia with perineoscrotal hypospadias, poor penile development, bifid scrotum, and gy- naecomastia at puberty.). In contrast to the aforementioned points, extension of CAG polyglutamate repeats to greater than 40 in the N terminal domain of the receptor causes X-​linked bulbar muscular atrophy (Kennedy’s disease) associated with gynaecomastia, poor virilization, and azoo- spermia due to ‘late-​onset’ androgen resistance. Expansion of CAG glutamine repeats to between 25 to 40 is associated with a fourfold increased risk of oligospermia or azoospermia without clinical evi- dence of neuromuscular degeneration. This may represent an exclu- sively testicular form of androgen insensitivity. Deficient 5 α-​reductase type II action in the genital tract causes external genitalia that are often predominantly female at birth in combination with a male internal urogenital tract. There is typic- ally clitoral hypertrophy with perineoscrotal hypospadias, inguinal testes and epididymes, with ejaculatory ducts emptying into a blind ending vagina. Usually raised girls, these patients dramatically virilize at puberty without gynaecomastia. Males with oestrogen resistance and aromatase deficiency are normally virilized at birth and have normal pubertal development except for non​fusion of epiphyses resulting in extreme tall stature, and osteoporosis in adulthood. The effects on spermatogenesis and fertility are currently unclear. Cryptorchidism Cryptorchidism has a prevalence of 2.5–​5% at birth, declining to 1% by one year. Spontaneous descent rarely occurs after this age. Undescended testes can be a feature of many hypogonadotropic conditions, intersexual and dysgenetic states such as androgen in- sensitivity syndromes, and Noonan’s syndrome. The persistent Müllerian duct syndrome is caused by defects in anti-​Müllerian hormone production or action during fetal development. The pres- ence of Fallopian tubes and uterus obstructs testicular descent. The lower temperature in the scrotum is a prerequisite for normal spermatogenesis. Undescended testes are therefore exposed to the harmful effects of the higher temperature in the abdomen and in- guinal region. A testis which is not permanently in a low scrotal pos- ition by the age of two years will sustain permanent damage to the seminiferous epithelium, hence orchidopexy after two years of age for undescended testes does not improve fertility. For these reasons, treatment should ideally be undertaken between one and two years of age. HCG or intranasal GnRH are being increasingly used for early initial treatment of cryptorchidism, with orchidopexy carried out by the age of two if this is unsuccessful. The risk of testicular tumour in a patient with a history of undescended testes, whether successfully treated by orchidopexy or not, is 4-​ to 5-​fold higher than in the general population. Testicular tumours It is important to remember that infertility can be a presenting symptom of testicular tumours, the commonest malignancy in young adult men. With increasing use of a testicular ultrasound it has become clear that there is a significantly higher risk of testicular tumours in infertile men (in absence of cryptorchidism) compared to the general population. Carcinoma in situ, an obligatory precan- cerous state, is occasionally encountered incidentally in diagnostic testicular biopsies. Without treatment, 50% of carcinoma in situ progress to malignant seminoma or non​seminomatous germ-​cell tumours. Varicocele Varicocele is a dilatation of the scrotal portion of the pampiniform plexus due to reflux of blood in the internal spermatic veins, usually involving the left side from the renal vein. It usually gives rise to a reduction in ipsilateral testicular volume, but varying degrees of hypospermatogenesis are often seen in both testes. Although a vari- cocele is clinically detectable in up to 40% of male partners of infer- tile couples, its significance in male infertility remains controversial. Increased scrotal temperature, hypoxia, and exposure of the testes to adrenal metabolites have been postulated as possible mechanisms by which spermatic vein reflux can induce seminiferous tubular damage. Since varicoceles can be detected clinically in 15% of fertile young men, it should not be assumed that this condition is invari- ably or solely responsible for infertility without actively excluding other possible aetiologies, including those in the female partner. Sperm autoimmunity Immunological infertility is a specific disorder caused by sperm membrane-​bound IgA antibodies found around 5% of men pre- senting with infertility. Conditions predisposing to sperm auto- immunity include vasectomy, testicular injury/​inflammation, genital tract infections/​obstruction, and family history of auto- immune disease. Male patients with significant antisperm anti- body titres usually have severely suppressed fertility potential due to sperm agglutination, poor sperm transit through cervical mucus, and blocked sperm-​oocyte fusion. Genital tract infection Infection in the lower genital tract is a major cause of male infer- tility in a global context. Chlamydia, gonococcus, Gram-​negative enterococci, and tuberculosis are the usual pathogens. If not treated by appropriate antibiotics promptly, inflammation of the accessory

13.6.2  Disorders of male reproduction and male hypogonadism 2403 gland’s excurrent ducts may give rise to disturbed function, forma- tion of anti-sperm antibodies, and permanent structural damage with obstruction of the outflow tract. Asymptomatic prostatitis due to occult and usually focal infection is best diagnosed by transrectal ultrasound examination and culture of an ejaculate. Excurrent duct obstruction Vasectomy and previous genitourinary infections, usually sexually transmitted or tuberculous, are the most common causes of ob- structed azoospermia. Congenital bilateral agenesis of the Wolffian duct-​derived structures—the corpus/​cauda epididymis, vas def- erens and seminal vesicles (CBAVD)—is characterized by impalp- able scrotal vasa, distended caput epididymis, acidly non​coagulating semen of reduced volume (<2 ml) devoid of fructose and sperm, is present in 95% of males with cystic fibrosis. More commonly (6% of azoospermic men and 1–​2% of infertile males), patients present with CBAVD without frank respiratory tract disease or pancreatic insufficiency. These have milder heterozygous mutations of the cystic fibrosis transmembrane regulator (CFTR) gene and /​or the 5T variant in intron 8, giving rise to a predominantly genital phenotype of cystic fibrosis. Renal and urinary tract abnormalities are common in these patients. In Young’s syndrome progressive epididymal obstruction is due to progressive inspissation of amorphous secretion in the lumen. In these patients, the high incidence of chronic sinopulmonary infection from childhood and bronchiectasis is presumably the consequence of the same abnormality in the respiratory tract. Epidemiological data has raised the possibility of mercury poi- soning as the cause of this condition. Coital disorders Inadequate coital frequency, technique (including the use of vaginal lubricants with spermicidal properties) and faulty timing of inter- course may contribute to continue infertility but are rarely the only etiological factor in the infertile couple. Diagnosis History Particular attention should be paid to the following aspects. Previous surgery such as herniorrhaphy in childhood, trauma, or torsion, suggest possible damage to the vas or testis. History of cryptorchidism and genitourinary infections are important etio- logical factors. Delayed onset of puberty may suggest the possi- bility of gonadotrophin deficiency. A  history of recurrent chest infections, sinusitis, or bronchiectasis may be obtained in patients with epididymal obstruction (Young’s syndrome), immotile cilia syndrome, and CBAVD associated with cystic fibrosis. Chronic disorders such as renal failure, liver disease, malignancy, diabetes mellitus, and multiple sclerosis are associated with a variety of tes- ticular and sexual dysfunctions. Patients should be asked about episodes of pyrexia within the past 12 weeks because of transient suppression of spermatogen- esis. Careful enquiry should also be made about occupational or environmental exposure to testicular toxins, current medications, previous and/​or current use of recreational drugs. Painful ejacu- lation, haematospermia, and pain in the perineum are symptoms suggestive of chronic infection in the prostate and seminal vesicles. It is important to establish that vaginal intercourse takes place with appropriate frequency, timing and without the use of the vaginal lubricants. Examination Assessment of height, weight, body habitus, and secondary sexual de- velopment should be carried out in all patients. Measurement of tes- ticular volumes by use of a Prader orchidometer provide a convenient clinical index of seminiferous tubular mass. Normal adult testicular volume ranges between 15 and 25 ml, and testicular volume is a key finding in differentiating between azoospermia due to seminiferous tubular failure (reduced volume) and that arising from excurrent duct obstruction (normal volume). Testicular size is also a useful indicator of the degree of testicular development in hypogonadotropic patients. If not in the scrotum, the lowest position of the testes should be de- fined with the patient upright. Irregular contour, induration, or ab- normal consistency of the testis suggest previous orchitis, surgery, or malignancy. Special attention should also be paid to the palpation of the epididymis and scrotal vas. An enlarged caput epididymis may be palpable in cases of obstructive azoospermia. Irregularity and indur- ation of the epididymis and vas suggest previous infection. In con- genital agenesis of the Wolffian duct-​derived structures, the scrotal vasa are either impalpable or extremely thin. The patient should be examined standing so that varicoceles can become visible (grade 3) or palpable (grade 2), or detected as a venous impulse in the spermatic cord during Valsalva manoeuvre (Grade 1). Rectal examination may detect irregular contour or abnormal consistency and tenderness in the prostate in the presence of chronic prostatitis, and enlarged sem- inal vesicles due to ejaculatory duct obstruction. Investigations Conventional parameters of the semen analysis such as sperm density, percentage of motile sperm, quality of sperm movements, and sperm morphology provide a semiquantitative index of fertility potential. Although a variety of tests of sperm function, such as com- puter aided sperm movement analysis, cervical mucus penetration, acrosome reaction, sperm-​zona pellucida binding, and hamster oo- cyte penetration, have been devised, none is sufficiently reliable and accurate to be used routinely in clinical practice. Infertile men with oligozoospermia produce spermatozoa harbouring abnormal DNA with strand breaks and redundant cytoplasm, which may produce excess reactive oxygen species. Chromatin structure and cytoplasmic enzyme (LDH-​X or CK-​M) assays are being applied to assess functional integrity of sperm- atozoa, and these may provide more reliable quantitative biochem- ical measures of male fertility to guide management in the future. Measurement of plasma FSH is useful in distinguishing pri- mary and secondary testicular failure and in identifying patients with obstructive azoospermia. In the presence of azoospermia or oligozoospermia, an elevated FSH, particularly with reduced tes- ticular volume, is presumptive evidence of severe and usually ir- reversible seminiferous tubular damage. Low or undetectable FSH (usually associated with low LH and testosterone with clinical evi- dence of androgen deficiency) is suggestive of hypogonadotropic hypogonadism. Conversely, azoospermia with normal FSH and a normal testicular volume usually indicates the presence of bilateral genital tract obstruction. The potential role of inhibin B measurement as a circulating marker of Sertoli cell function in routine diagnostic workup of male

section 13  Endocrine disorders 2404 infertility is currently being evaluated. Testosterone and LH meas- urements are only indicated in the assessment of the infertile male when there is clinical suspicion of androgen deficiency, Klinefelter’s syndrome, or sex steroid abuse. A high LH and testosterone should raise the possibility of abnormalities in androgen receptors, while low LH and testosterone suggest gonadotrophin deficiency. Hyperprolactinaemia is not a recognized cause of male infertility but prolactin measurement should be undertaken if there is evidence of sexual dysfunction (particularly diminished libido) or pituitary dis- ease leading to secondary testicular failure. Oestradiol measurement is rarely indicated, except in the presence of gynaecomastia. Chromosomal analysis by karyotyping or fluorescent in situ hy- bridization should be carried out in patients with azoospermia, tes- ticular atrophy and elevated FSH, primarily to confirm the diagnosis of Klinefelter’s syndrome. Screening for Y-​chromosome micro dele- tions should be considered in all patients with sperm density of less than 5 million/​ml by an appropriate number of PCR based DNA markers and confirmed by Southern blotting. The need for testicular biopsy has largely been superseded by the use of plasma FSH in recent years to differentiate between primary testicular failure and obstructive lesions. Undetectable or very low levels of seminal fructose is used to con- firm the clinical diagnosis of congenital bilateral absence of the vas deferens (CBAVD) or blocked ejaculatory ducts in the presence of obstructive azoospermia. An increasing number (more than 1 mil- lion/​ml) of peroxidase-​positive or monoclonal antibody-​detected leucocytes in the semen may indicate genital tract infection. Semen culture for pathogens is difficult because of the bactericidal prop- erties of seminal plasma and the presence of urethral and skin commensals. Antisperm antibodies are detected by mixed agglutination reac- tion where either sheep red blood cells or polyacrylamide beads are coated with rabbit antibodies to specific classes of human Igs. These will attach to motile spermatozoa carrying specific IgA on the sur- face of the sperm head or tail. Ultrasound examination of the testes has become a routine inves- tigation for infertile males with non​obstructive azoospermia or se- vere oligospermia, or to detect occult testicular tumours. In patients with persistent or treated cryptorchidism, testicular ultrasound should be carried out annually. Ultrasound of the urinary tract is indicated in patience with CBAVD. Transrectal ultrasound can aid the diagnosis of asymptomatic chronic prostatitis. Management Pregnancies can occur in subfertile couples without treatment, al- beit with a much-​reduced probability depending on the duration of infertility, age and coexisting subtle abnormalities in the female partner, in addition to any defects in sperm quality. Since most pa- tients with male infertility present no recognizable or reversible aeti- ologies, management remains largely empirical. Subfertility due to idiopathic hypospermatogenesis Although a wide variety of empirical medical treatments, including gonadotrophins, androgens, and antioestrogens have been tried in attempts to improve fertility in subfertile men, none has been shown to be effective when assessed in randomized control thera- peutic trials and are therefore none are recommended. Instead, as- sisted conception techniques are increasingly applied to overcome idiopathic male infertility. This is based on the premise that placing a large number of prepared motile spermatozoa in close proximity to ovulated or retrieved oocytes in vivo or in vitro can enhance the probability of fertilization. Intrauterine insemination (IUI) of more than 1 million washed and motile spermatozoa (freed of seminal plasma, leucocytes, and abnormal/​dead spermatozoa) is a rela- tively simple and inexpensive techniques with few complications. Pregnancy rates of 5–​10% per cycle can be expected. This can be combined with controlled ovarian stimulation of the female using gonadotrophin, but the risk of multiple pregnancies increases. In vitro fertilization (IVF) involves more intensive gonadotrophin simulation of the female, suppression of spontaneous ovulation using a GnRH antagonist, and collection of multiple oocytes by laparoscopy or transvaginal ultrasound guided ovarian puncture, which are then coincubated with prepared spermatozoa in culture medium. In patients with moderate oligozoospermia, average fer- tilization rates of 30% and live birth rates of 5 to 12% per treatment cycle can be anticipated. In those with severe and multiple defects in semen parameters, standard IVF is less effective. For these cases, microinjection of single live spermatozoa directly into harvested oocytes (intracytoplasmic sperm injection, ICSI) has become the treatment of choice. This by- passes the sperm-​oocyte interactions normally required for fertil- ization in natural conception or IVF and can achieve a remarkably high fertilization and live birth rates (55 and 26% per cycle respect- ively), even with the most severely abnormal samples. Since only a few spermatozoa are required, ICSI has revolutionized management of extreme oligozoospermia and azoospermia irrespective of aeti- ology. Non​obstructive azoospermia is frequently intermittent and careful examination of centrifuged deposits of semen to detect and harvest occasionally ejaculated spermatozoa for ICSI should be at- tempted repeatedly before resorting to alternatives. Even in patients with persistent nonobstructive azoospermia, isolated foci of sperm- atogenesis may be preserved so that testicular sperm extraction by multiple biopsies can often yield viable testicular spermatozoa (including several patients Klinefelter syndrome) for ICSI, and in persistent obstructive azoospermia epididymal spermatozoa can be aspirated by an open procedure or percutaneous needle punching of the proximal epididymitis. In these circumstances, cryostorage of harvested spermatozoa for subsequent ICSI is required. This does not appear to compromise efficacy. In children born after successful ICSI treatment, the incidence of major congenital abnormalities are not increased compared with natural pregnancies, but there is a small increase in sex chromosome aneuploidy in some series. Concern re- garding the developmental potential of children born after ICSI has been raised and long-term follow-​up of ICSI births is indicated. Specific treatable conditions Removal or withdrawal from antispermatogenic agents or drug ex- posure may lead to improvement in fertility. This is most frequently seen in patients with inflammatory bowel diseases, when changing treatment from sulphasalazine to 5-​aminosalicylic acid removes the offending moiety, sulphapyridine. Withdrawal from anabolic ster- oids abuse invariably leads to recovery of spermatogenesis, although this may take several months because of the long half-​lives of some agents. Cryopreservation of semen should be offered to all male pa- tients of reproductive age before commencing anticancer chemo- therapy or testicular irradiation.

13.6.2  Disorders of male reproduction and male hypogonadism 2405 When patients with hypogonadotropic hypogonadism desire fer- tility, they can discontinue exogenous androgen replacement and start on human chorionic gonadotropin (HCG 1500–​2000 IU sub- cutaneously twice weekly) for 6 to 12 months. This should maintain normal testosterone concentrations. Patients with postpubertally acquired gonadotrophin deficiency (e.g. from pituitary tumour), where spermatogenesis has been previously established, usually re- spond to HCG treatment alone to reinitiate germ-​cell development. If there are no spermatozoa in the ejaculate at the end of 12 months, human menopausal gonadotrophin (HMG), which contain both FSH/​LH or recombinant FSH (Puregon, Gonal F), should be added at 75 to 150 international units SC thrice weekly. Combined treat- ment may be required for a further 12 months. Most patients with congenital forms of hypogonadotropic hypogonadism will require FSH to stimulate Sertoli cell division and initiate spermatogenesis. In general, around 70% should show active spermatogenesis and 50% could be expected to achieve spontaneous pregnancies in their partners, even if sperm density remains in the oligospermic range. Patients with hypothalamic GnRH deficiency can be treated by pul- satile GnRH delivered two-​hourly by a battery driven portable in- fusion minipump, although many find this form of chronic therapy impractical and too demanding. The outcome of treatment is similar to that obtained with exogenous gonadotrophin therapy. Active infection in the genital tract should be treated with ap- propriate antibiotics (erythromycin, doxycycline, norfloxacin) for four weeks for the patient and his partner. Obstructive azoo- spermia due to epididymal obstruction can be treated by micro- surgical epididymovasostomy, but high pregnancy rates are only be achieved by experienced microsurgeons. A more feasible alterna- tive is to obtain spermatozoa from the caput epididymis or efferent ducts proximal to the site of obstruction by direct needle aspiration (microepididymal sperm aspiration or percutaneous epididymal sperm aspiration) for use in assisted fertilization procedures (usu- ally ICSI). In patients with CBAVD, CFTR mutation screening of the partner and genetic counselling should be undertaken beforehand because of the risks of cystic fibrosis in offspring. Sperm antibody can be treated by suppression with high-​dose prednisolone (0.75 mg per kilogram per day, or prednisone 20 mg twice daily on days 1 to 10 and 5 mg on days 11 and 12 of the partner’s cycle for 3 to 6 cycles). Side effects are common, including irritability, sleeplessness, arthralgia, muscle weakness, peptic ulceration, glucose intolerance, and aseptic necrosis of fem- oral head. Result of controlled trials have been conflicting. IVF and ICSI are increasingly being applied to manage immunological male infertility. Varicocele can be treated either by open surgical ligation or transfemoral embolization of the internal spermatic veins. Results of treatment of varicocele in eight prospective controlled therapeutic trials are confusing. Coexisting female factors contributing to infer- tility, insufficient sample size, high dropout rates, and lack of ran- domization/​blinding or sham procedures, are some of the more important confounding variables which typify difficulties of treat- ment trials in male infertility. Nevertheless, the Royal College of Obstetricians and Gynaecologists concluded that treatment of vari- cocele in oligozoospermic but not normospermic subfertile men can significantly improve semen quality and pregnancy rates. The cost of varicocele treatment per live birth is less with surgical ligation (and embolization) than for assisted conception techniques. Retrograde ejaculation can be treated medically with oral sym- pathomimetics and anticholinergics, but evidence of their efficacy is limited. If unsuccessful, spermatozoa can be recovered by bladder catheterization and after irrigation with culture medium used for artificial insemination or IVF. Semen can be obtained by mastur- bation, vibrators, or electroejaculation from patients with various coital dysfunctions. Untreatable sterility Patients with persistent non​obstructing azoospermia without re- trievable postmeiotic germ cells, unable to undergo or failed to be helped by ICSI, should be counselled regarding the options of con- tinuing childlessness, adoption, and donor insemination. Genetic screening and counselling This has become important with the realization that genetic dis- orders account for an increasing proportion of infertility previ- ously believed to be idiopathic, and that there is a high probability of transmitting infertility to male offspring if assisted reproductive treatment is successful. Furthermore, the long-​term health of ICSI offspring remains an unsettled question. All couples considering micro assisted fertilization techniques should therefore be coun- selled. It is also recommended that chromosomal karyotyping and Y-​chromosome screening be performed in patients with azoo- spermia and severe oligozoospermia (<5 million per ml), regardless on the coexistence of other clinical abnormalities such as varicocele or cryptorchidism. This not only allows a firm diagnosis to be made, but also encourages the clinician to forego empirical treatment and couples who have conceived by assisted reproduction techniques can inform their son at suitable age that he is likely to have fertility problems. As stated previously, patients with obstructive azoo- spermia due to CBAVD and their partners should undergo CFTR gene screening followed by a genetic counselling if positive. Erectile dysfunction Erectile failure maybe caused by neurological disorders such as autonomic neuropathy (usually complicating diabetes mellitus), multiple sclerosis, and spinal injuries, as well as vascular disease involving pelvic vessels, retroperitoneal and bladder neck surgery, medications (commonly α- and β-​adrenergic antagonists, psycho- tropic agents), alcohol abuse, severe systemic disease, psychological dysfunction (including depression), relationship problems, an- drogen deficiency, and hyperprolactinaemia. Loss of libido charac- terizes androgen deficiency and hyperprolactinaemia, while normal spontaneous morning erection is suggestive of psychogenic impo- tence. Testosterone deficiency is uncommon (less than 5%) in pa- tients who presents with erectile dysfunction without loss of libido. Management should aim to correct reversal of any underlying cause (e.g. prolactinoma treated by a dopamine agonist) or substi- tute offending medications. Androgen replacement is only indicated in patients with total or free plasma testosterone in the hypogonadal range. The use of a phosphodiesterase inhibitor (PGE5 inhibitors) such as sildenafil, tadalafil, vardenafil, and avanafil to enhance the neurovascular cGMP-​mediated nitric oxide smooth muscle relax- ation in penile vasculature is a remarkably successful way of treating a wide variety of erectile dysfunction. In refractory cases vacuum devices and intracavernous injection of vasodilator agents such as PGE1 can be considered.

13.6.3 Benign breast disease 2406

13.6.3 Benign breast disease 2406

section 13  Endocrine disorders 2406 FURTHER READING Fernandez-​Balsells MM, et al. (2010). Clinical review 1: adverse effects of testosterone therapy in adult men: a systematic review and meta-​analysis. J Clin Endocrinol Metab, 95, 2560–​75. Jungwirth A, et al. (2012). European Association of Urology
guidelines on male infertility: the 2012 update. Eur Urol, 62, 324–32. Kanakis GA, Nieschlag E (2018). Klinefelter syndrome: more than hypogonadism. Metabolism, 86, 135–44. Krausz C, Riera-Escamilla A (2018). Genetics of male infertility. Nat Rev Urol, 15, 369–84. Kwong JCC, Krakowsky Y, Grober E (2019). Testosterone deficiency: a review and comparison of current guidelines. J Sex Med, 16, 812–20. Mulhall JP, et al. (2018). Evaluation and management of testosterone deficiency: AUA guideline. J Urol, 200, 423–32. Nieschlag E, Behre HM (eds) (2012). Testosterone: action, deficiency, substitution. Cambridge University Press, Cambridge. Petak SM, et al. (2002). AACE Hypogonadism Task Force. Medical guidelines for clinical practice for the evaluation and treatment of hypogonadism in adult male patients—​2002 updated. Endocr Pract, 8, 440–​56. 13.6.3  Benign breast disease Gael M. MacLean ESSENTIALS Benign conditions of the breast are very common, but they cause great anxiety, often leading the patient to be concerned that she has breast cancer. Symptoms may include: (1) a mass in the breast, commonly due to fibroadenoma, benign cystic change, or macrocysts; (2)  discharge from the nipple, which may be
caused by hyperprolactinaemia, intraduct papilloma, or duct ectasia; and (3) mastalgia. Management involves exclusion of ma- lignancy, often by triple assessment of any palpable abnormality (clinical examination, radiological and pathological assessment), followed by reassurance, with appropriate specific treatment if required. Introduction Benign conditions of the breast are very common, and can lead to great anxiety that the symptoms may be caused by a breast cancer. For example, a mass in the breast is commonly due to a fibroadenoma or cysts (either micro or macro). Management of symptomatic patients involves the exclusion of malignancy, usually by triple assessment of the palpable area (for medical management of breast cancer, see Chapter  5.7) followed by reassurance and appropriate specific treatment if required. Congenital abnormalities Complete failure of breast development (amastia) is rare. Failure of breast development presenting with the absence of the pectoralis major muscle and often ipsilateral upper limb deformity is a condition known as Poland’s syndrome (named after Alfred Poland the 19th-​century sur- geon who first described the condition). Varying degrees of hypoplasia are common, ranging from asymmetry to tubular breasts. Tubular breasts are characterized by lack of breast tissue, enlarged areolae, and narrow base width, resulting in widely spaced narrow breasts which are pendulous. Accessory nipples are a common finding, occurring most frequently on the milk line, often at the inframammary fold. Accessory breast tissue is found most commonly in the axilla. Aberrations of normal breast development and involution Fibroadenoma This is a common aberration of normal lobular development and occurs after puberty in young women. In women under the age of 20 years they account for 60% of palpable lumps. Fibroadenomas are discrete lumps with a smooth or bosselated surface; they are char- acteristically painless and not fixed to deep structures or skin. Their characteristic mobility has led to these lesions being known as ‘breast mice’ (see Fig. 13.6.3.1). They also have a characteristic appearance on ultrasound scanning. A core biopsy (or fine needle aspirate) can confirm the diagnosis. The term ‘giant fibroadenoma’ is variably ap- plied to lesions greater than 5 cm. Most are asymptomatic and in- volute with age, and calcified fibroadenomas can be an incidental finding on a mammogram in older women. Surgical excision is only indicated if there is diagnostic uncertainty or at the patient’s request. Benign cystic change (benign breast change) The monthly hormonal cycle of breast stimulation and involution fre- quently results in areas of nodularity which can be discrete or diffuse. These may be tender and the patient may notice they are more prom- inent premenstrually and resolve, in part or completely, with menstru- ation. Areas of persistent asymmetrical nodularity should be assessed with imaging and biopsy. The pathology may show a spectrum of Fig. 13.6.3.1  Excised fibroadenoma, showing whorled surface.

13.6.3  Benign breast disease 2407 benign appearances, from non-​proliferative changes through to atypical hyperplasias. These latter lesions are associated with an increased risk of developing breast cancer and excision is normally recommended. Macrocysts Palpable cysts are a common cause of a lump in the involuting, perimenopausal breast. The diagnosis is made by ultrasound or by aspiration of the fluid. The aspirate is classically serous and green, yellow, brown, or inky blue, which can be discarded. A bloody as- pirate should be sent for analysis and prompt further investigation, as should a lump which persists after aspiration. Cysts occurring in the postmenopausal, involuted breast are unusual, and an intracystic neoplasm should be excluded. Other benign breast lumps Fat necrosis As the breast has a predominantly fatty stroma, trauma to the breast, even quite minimal, can result in an entity known as fat necrosis. The resultant lump can be hard and irregular and needs to be differenti- ated from cancer, often requiring tissue analysis. Phyllodes tumour Pathologically these fibroepithelial tumours range from benign (80%) through borderline to malignant. Phyllodes tumours are dis- tinct from fibroadenomas but can be difficult to differentiate clinic- ally as they present similarly as discrete solid masses, although they tend to occur in older patients than fibroadenomas and typically grow rapidly. Treatment, even for the benign lesions, is by excision with a margin as there is a high rate of local recurrence. Larger ma- lignant lesions may require a mastectomy. Fibromatosis This is an unusual condition which presents with a diffuse mass in the breast that can be locally invasive, infiltrating surrounding tis- sues. As the features can mimic malignancy, both clinically and radio- logically, a core biopsy is needed to provide the diagnosis. A range of treatments are described, ranging from radical excision (including chest wall) to more conservative approaches with non​steroidal anti-​inflammatory drugs. Diabetic mastopathy (lymphocytic lobulitis) This is a diffuse rubbery thickening of the breast in insulin de- pendent diabetics. Once malignancy is excluded, there is no specific treatment. Hamartoma Hamartomas of the breast are clinically very similar to fibroaden- omas, although often slightly softer on palpation. Benign problems of the nipple areolar complex Nipple discharge Non​physiological lactation (Fig. 13.6.3.2) can be caused by hyperprolactinaemia as a result of a pituitary prolactinoma, which can be treated by surgery or bromocriptine. Single duct serous or serosanguinous discharge is most com- monly due to an intraductal papilloma. Surgical excision with micro­ ductectomy (the duct affected is cannulated and removed) is advised to exclude papillary malignancy. In the peri-​ and postmenopausal age group, investigations should include mammography and duct exci- sion as ductal carcinoma in situ may present in this way. Intraduct papillomas and carcinomas may both present with blood stained nipple discharge, hence referral to a breast unit is advised. Coloured discharge (green, yellow, brown, or inky blue) from multiple ducts is usually caused by duct ectasia. Total duct excision (Hadfield’s operation) is only necessary if the discharge is profuse or distressing to the patient. Periductal fibrosis can result in nipple in- version (classically resulting in a linear, slit-​like inversion). Miscellaneous Benign conditions of the nipple/​areolar complex include retention cysts of Montgomery’s glands, benign pedunculated polyps, and nipple adenomas. The nipple is a common site for eczema, which must be differentiated (with a punch biopsy) from Paget’s disease, as the latter is usually associated with an underlying intraductal carcinoma. Breast pain (mastalgia) Mastalgia can be cyclical or non​cyclical dependent on its relation- ship to the menstrual cycle. Classic cyclical pain may need no treat- ment but reassurance and advice. Some women find relief from oral evening primrose oil or starflower oil, although the active in- gredient (GLA: γ-linolenic acid) does not help all women and the treatment needs to be taken daily for at least 3 months to determine whether or not it is effective. If severe, danazol, bromocriptine, and tamoxifen have been used, although they all have significant side effects. Costochondritis or chest wall pain is sometimes erroneously described as ‘breast pain’; it can be treated successfully with non-​ steroidal anti-​inflammatory drugs. Breast inflammation Obstruction to milk drainage can result in lactational mastitis or an abscess. This should be dealt with promptly with antibiotics and Fig. 13.6.3.2  Nipple showing discharge from two ducts.

13.6.4 Sexual dysfunction 2408

13.6.4 Sexual dysfunction 2408

section 13  Endocrine disorders 2408 percutaneous drainage under ultrasound guidance. In smokers, in- flammation around the ducts, known as periductal mastitis, leads to an abscess formation if secondary infection occurs. This can pro- gress to form a mammary fistula. Granulomatous mastitis is uncommon, the diagnosis being made after excluding infection, especially tuberculosis, and sarcoidosis. Mondor’s disease is the name given to superficial thrombophle- bitis of a chest wall vein. In the breast this results in a tender cord-​ like thickening running vertically and superficially. Benign conditions of the male breast Gynaecomastia, which is often unilateral, is hypertrophy of the ductal and stromal tissue of the male breast and is a common finding at puberty and in older men with failing testicular function. Pseudogynaecomastia, which is increase in the fat underlying the male breast, is more common than true gynaecomastia. This may be idiopathic, but a careful history of prescribed and recreational drugs may be fruitful. Common causes are digoxin, anabolic steroids, heroin, spironolactone, omeprazole, and methyldopa. Testicular tumours can cause gynaecomastia, which is also seen in Klinefelter’s syndrome. Other benign conditions of the male breast are very rare. Male breast cancers account for less than 1% of all breast can- cers in the United Kingdom, but suspicious features such as a hard, rugged lump in the breast should prompt triple assessment to ex- clude malignancy. FURTHER READING Chinyama CN (2014). Benign breast diseases, 2nd edition. Springer, London. Dixon MJ (ed) (2012). ABC of breast diseases, 4th edition. Wiley-​ Blackwell, Oxford. Hughes LE, Mansel RE, Webster DJ (1987). Aberrations of normal development and involution (ANDI): a new perspective on patho- genesis and nomenclature of benign breast disorders. Lancet, 2, 1316–​19. 13.6.4  Sexual dysfunction Ian Eardley ESSENTIALS Male sexual dysfunction, particularly erectile dysfunction, is common and becomes commoner with increasing age. It is often associated with cardiovascular disease and its risk factors. First line management should seek to identify and treat causative risk factors along with oral pharmacotherapy with a phosphodiesterase type 5 inhibitor. Premature ejaculation is a common but poorly under- stood condition that can be treated effectively by psychosexual therapy or by the use of selective serotonin reuptake inhibitors. Penile deformity is relatively uncommon and is usually due to Peyronie’s disease. It is possible to treat established disease by in- jection of collagenase, but for most patients who actually require treatment, surgical correction is the appropriate therapy. Prolonged erection or priapism is a rare medical emergency that requires ur- gent therapy or else erectile function may be lost. In women, sexual desire disorders are commoner in older postmenopausal women and a new treatment, flibanserin, has recently been licensed for this indication. Sexual arousal dis- orders in women also become more common postmenopausally and have a multifactorial aetiology. Treatment should be directed at the aetiological factor in the first instance; trials of oral pharma- cotherapy have been disappointing. While true vaginismus is un- common, dyspareunia is relatively common. Again, there may be a multifactorial aetiology, with vulvar vestibulitis being perhaps the most common aetiological condition. Treatment is often unsatisfactory. Male sexual dysfunction Male sexual function is a complex neurovascular event which can be affected by several disease processes that result in problems of penile erection, ejaculatory difficulties, and disorders of desire. Of these, the commonest dysfunctions encountered in clinical practice are erectile dysfunction, penile deformity, prolonged erections, and rapid (or premature) ejaculation. Erectile dysfunction Erectile dysfunction (ED) is defined as ‘The inability to attain or maintain an erection satisfactory for sexual intercourse’. It is a very common symptom which affects around 50% of men over the age of 40 years. The prevalence increases with age, and there are asso- ciations with a range of conditions including diabetes, hyperten- sion, dyslipidaemia, and depression. The first modern (and perhaps most important) epidemiological study was the Massachusetts Male Aging study which reported the age-​related prevalence in North American men. A plethora of subsequent epidemiological studies have explored the issues. The physiology of penile erection is complex and involves co- ordinated interaction of the neurological, vascular, and endocrine systems. As such, there are many diseases of these systems that can lead to the development of ED, some of which are listed in Table 13.6.4.1. It is now recognized that in most men with ED, both psychogenic and organic risk factors coexist, albeit to varying degrees. Certainly secondary ‘performance related anxiety’ often complicates the pathophysiology of men who have a primary or- ganic aetiology. The commonest cause of ED is that which arises secondary to vascular disease, with ED being a common coexisting symptom in men with hypertension, diabetes, hyperlipidaemia, and cardiovascular disease. Indeed, there is emerging evidence that ED may be one of the very earliest features of systemic vas- cular disease. Since ED is a self-​declared symptom, the fundamental basis of diagnosis is the clinical history. Mild cases are characterized by a history of difficulty in maintaining an erection, while in more

13.6.4  Sexual dysfunction 2409 severe cases, there may be difficulty in initiating an erection or even be loss of erections altogether. Assessment should seek to identify treatable causes (Tables 13.6.4.1, 13.6.4.2) and remediable risk factors. A predominantly psychogenic aetiology is suggested by the continued presence of nocturnal or early morning erections. Current guidelines suggest a focussed physical examination of the genitalia, assessment of secondary sexual characteristics, and as- sessment of blood pressure. More extensive genitourinary and vascular examination is only performed when clinically indicated. Baseline investigations should include a fasting blood sugar, a lipid screen, and an assessment of a serum testosterone. Further assess- ment is usually unnecessary, although in selected cases vascular investigations (including colour Doppler assessment of penile vas- culature) and more complex endocrine investigations might be indicated. In a few patients, specific remedies can cure ED. For instance, in men with a predominantly psychogenic aetiology, psychosexual counselling is often valuable. In men with an endocrine cause (such as hypogonadism or hyperprolactinaemia), appropriate endocrine therapy is also helpful. Finally, in men who have developed ED fol- lowing pelvic or perineal trauma, reconstructive vascular surgery may be effective. In most men, however, treatment is symptomatic, rather than aiming to cure the underlying problem. Given the prevalence of sec- ondary psychogenic problems, patient education and counselling are always valuable. Management of risk factors is an important part of patient man- agement. Those with hypertension should have their blood pressure optimally controlled, with emerging evidence that the angiotensin receptor antagonists actually improve erectile function, while many other drugs used for hypertension have an adverse effect upon erec- tions. Similarly, there is some evidence that improved diabetic con- trol improves erections, and there are some studies suggesting that lifestyle interventions can also improve erectile function. However, the improvements seen are usually modest and some form of pharmacological treatment is usually required. The mainstay of therapy in most men who present with ED will be oral therapy using a phosphodiesterase type 5 inhibitor (PDE5i). While sildenafil was the first such licensed preparation, three other drugs—​tadalafil, vardenafil, and avanafil—​have been marketed in recent years in Europe and North America. There appears to be little difference in efficacy and tolerability between these drugs, with the major differences relating to pharmacokinetics. The drugs are usu- ally used in an ‘on demand’ fashion, with ingestion 1–​2 hours prior to sexual activity. Tadalafil can be used in a daily dosing regimen given its extended half-​life. They are ineffective in the absence of sexual stimulation and a heavy meal may delay absorption. Around Table 13.6.4.1  Common causes of erectile dysfunction System Disease Neurological disease Multiple sclerosis Spinal cord injury Multiple system atrophy Lumbar disc prolapse Cauda equine syndrome Peripheral autonomic neuropathy (e.g. diabetes) Vascular disease Atherosclerosis Hypertension Hyperlipidaemia Diabetes Endocrine disease Diabetes mellitus Hypogonadism Hyperprolactinaemia Thyrotoxicosis Psychiatric disease Depression Anxiety states Iatrogenic Pelvic surgery (with nerve damage) Pelvic radiotherapy Drugs (see Table 13.6.4.2) Multifactorial aetiologies Renal failure Table 13.6.4.2  Drugs that can cause sexual dysfunction in men Drug type Drug or class of drug Effect Antihypertensive drugs Diuretics β-​blockers Centrally acting antihypertensive agents, e.g. clonidine, methyl DOPA ED ED ED Centrally acting agents Phenothiazines Butyrophenones Serotonin reuptake inhibitors Tricyclic antidepressants Phenytoin ED, reduced libido, ejaculatory dysfunction ED ED, ejaculatory dysfunction ED, reduced libido ED, reduced libido Endocrine drugs LHRH analogues Antiandrogens Oestrogens ED, reduced libido ED, reduced libido ED, reduced libido Recreational drugs Alcohol Marijuana Cocaine Opiates Amphetamines Anabolic steroids ED, reduced libido, ejaculatory dysfunction ED ED ED, reduced libido Reduced libido, ejaculatory dysfunction ED, reduced libido Other drugs Cimetidine Metoclopramide Digoxin ED, reduced libido ED, reduced libido ED

section 13  Endocrine disorders 2410 70–​80% of men will respond to such therapy, although the response rate is lower in certain patient groups, such as diabetics and in men who have undergone radical pelvic surgery. Side effects include headache, flushing, indigestion, and nasal congestion, although these are usually mild and well tolerated. Prolonged erections are ex- tremely rare with these drugs. Extensive research has not identified any significant cardiac risk with this class of drugs, although they are contraindicated in men using nitrate medication, and sexual activity is inadvisable in men with unstable cardiac disease. A detailed sum- mary of the management of ED in men with cardiac disease can be found in the ‘Princeton Consensus Statements’. In men who fail to respond to oral PDE5i therapy, several ap- proaches can be tried, although there is little evidence that use of a different PDE5i is beneficial. Regular (daily) dosing appears to help some patients, the rationale reflecting experimental evidence that a PDE5i might improve endothelial function when taken regularly. Alternatively, optimization of coexistent medical conditions is occa- sionally helpful in this respect. There is developing evidence that in some men with a low serum testosterone, the response to a PDE5i can be improved by normalizing the testosterone levels. In men who fail to respond to these manoeuvres, alternative therapies such as vacuum erection devices or intracavernosal self-​ injection of alprostadil are often effective. In a few patients, notably those with severe diabetes, even these treatments are ineffective or poorly tolerated, and under these circumstances insertion of a penile prosthesis may be indicated. Rapid (premature) ejaculation Premature ejaculation (PE) occurs when a man ejaculates before he or his partner want climax to happen. For some men, the problem starts with their first sexual experience (primary PE). For others, it happens after a period of normal sexual functioning (secondary PE). In recent years there have been consensus panels that have agreed a unified definition of both acquired and lifelong PE as a male sexual dysfunction characterized by ejaculation which always or nearly al- ways occurs prior to or within about one minute of vaginal pene- tration from the first sexual experiences (lifelong PE), or a clinically significant and bothersome reduction in latency time, often to about 3 minutes or less (acquired PE), associated with the inability to delay ejaculation on all or nearly all vaginal penetrations, and negative personal consequences such as distress, bother, frustration, and/​or the avoidance of sexual intimacy. The aetiology of primary PE is poorly understood with many po- tential pathophysiological mechanisms being proposed and none proven. Acquired PE is commonly associated with erectile dysfunc- tion and there are known associations with thyroid disease, alcohol abuse, opiate withdrawal, and possibly with prostatitis. Many men with PE do not seek treatment, although there is evidence that there can be deleterious effects upon self-​esteem, interpersonal relation- ships, and upon quality of life. When patients do seek medical at- tention, it is important to look for coexistent sexual dysfunctions (erectile dysfunction is commonly present) and to identify potential causes, while it is also important to assess the degree to which rela- tionship issues are important. Therapeutic options include sex therapy and psychotherapy, se- lective serotonin reuptake inhibitors (SSRIs), and topical anaes- thetics. While psychological approaches are commonly successful in the short term, they typically do not lead to long term cure. There is longstanding data that ‘off-​label’ use of SSRIs is an effective treatment for PE, with both on-​demand and daily dosing regimens in use. The latter provides a greater prolongation of the intravaginal ejaculatory latency time (IELT), but the potential risks of regular dosing with these drugs are well documented. Recently, a short acting SSRI has been licensed in Europe for this indication and appears to prolong the IELT two-​ to threefold, while improving control and distress. Local anaesthetic gels or sprays are also potential treatments, but none are currently licensed for clinical use. Penile deformity and Peyronie’s disease While young men or adolescents can occasionally present with a penile deformity that develops during adolescence (congenital curvature of the penis), the commonest cause of penile deformity is Peyronie’s disease, which is a localized connective tissue dis- ease of the penis leading to fibrotic plaque formation in the tu- nica albuginea of the corpus cavernosum. It was first described by Fallopius in 1561, although the condition is named after Francois Gigot De La Peyronie, surgeon to King Louis XV of France, who described it in 1743. While in pathological studies there is subclinical disease in over 20% of men, the clinical prevalence is around 1%. In some cases the condition can be familial, and there are associations with Dupuytren’s contracture, plantar fasciitis, and tympanosclerosis. The peak incidence occurs in the sixth decade, but cases have been reported throughout adult life. The pathophysiology is poorly understood, but the most commonly held view suggests that there is damage of the tunica albuginea associated with microvascular trauma. This leads to extravasation and perivascular inflammation with a round cell infiltrate, which in turn leads to fibrin deposition and subsequently fibrosis. Patients present with a palpable plaque within the penis which is most commonly felt on the dorsum, and there is a curvature of the penis, most commonly in the dorsal direction. The clinical pic- ture usually has two distinct phases, the ‘acute phase’, characterized by painful erections with progressive change in plaque size and deformity, which typically last 9–​18 months, and the subsequent ‘chronic’ phase where the deformity stabilizes (and occasionally im- proves) and pain with erection disappears. Numerous medical treatments have been tried in men with Peyronie’s disease. The oral agents that are most commonly used in clinical practice are vitamin E, tamoxifen, colchicine, and Potaba (potassium para-aminobenzoate, a form of vitamin B), but con- trolled studies have failed to demonstrate a consistent benefit for any agent. The most commonly used intralesional (injectable) agents are steroids, verapamil, and collagenase—​and following formal ran- domized controlled trials, the latter has been licensed for this indi- cation. In patients in the chronic phase of the disease, collagenase is injected into the plaque on several occasions with evidence that the deformity is improved in many, even if it does not disappear com- pletely. The cost of the treatment and the variability of the response currently limit the clinical utility of collagenase. Surgical treatment is reserved for those patients whose penile de- formity prevents sexual activity or those in whom sexual activity is painful either for the patient or his partner because of the deformity. The disease needs to be stable prior to surgical intervention. This point is particularly important, since disease progression after corrective surgery should be avoided. The most common surgical

13.6.4  Sexual dysfunction 2411 procedure is a Nesbit’s Procedure which involves surgical excision of an ellipse of a tunica albuginea on the opposite side of the penis to the plaque. The edges of the ellipse are apposed. Occasionally a grafting procedure can be performed whereby the plaque is incised and grafted typically with a patch of saphenous vein. In severe cases associated with coexistent erectile dysfunction, a penile prosthesis is indicated. Priapism A priapism is a penile erection which is unduly prolonged and which persists in the absence of a sexual stimulus. The classification of priapism divides cases into those where the aetiology is arterial (so-​called high flow priapism), and those cases where the aetiology reflects reduced venous drainage of the corpus cavernosum (so-​ called low flow priapism). Low flow priapism is by far the most common form of priapism and reflects stasis of blood within the penis, with sludging within the cavernosal sinusoids and subsequent thrombosis. Ischaemia, hypoxia, and acidosis develop as a consequence, and this prevents contraction of the penile smooth muscle which in turn exacerbates the condition. Low flow priapism is a medical emergency and re- quires urgent treatment to avoid irreversible damage to the vascular endothelium and smooth muscle of the penis and its erectile cap- acity. While comprehensive evidence is lacking, there are data to suggest that if treatment is successful within 24 hours, 56% of men will recover erectile function, while if treatment is delayed beyond that only 11% will recover potency. Causes of low flow priapism are shown in Table 13.6.4.3. The commonest are intracavernosal injec- tions, sickle cell disease, other hyperviscosity syndromes, and the use of some psychotropic drugs. Men with low flow priapism typically present with a painful erec- tion, in contrast to high flow priapism which is usually painless, but a careful assessment should seek to differentiate the low flow from the high flow condition (Table 13.6.4.4) and to identify the underlying cause. Aspiration of penile blood reveals thick dark venous blood, which on blood gas analysis shows hypoxia, hypercapnia, and acid- osis. Doppler scanning confirms lack of blood flow within the penis. Treatment for low flow priapism initially involves aspiration of enough blood via a wide bore cannula to produce detumescence and, if necessary, irrigation with warm heparinized normal saline (Fig. 13.6.4.1). If the erection reappears then intracavernosal in- jection of a smooth muscle α-agonist such as phenylephrine is appropriate, with concurrent monitoring of the systemic blood pres- sure. If this fails, then a surgical shunting procedure is appropriate. If treatment is delayed too long (see earlier) then even shunting procedures fail and the only hope for long term potency is a penile prosthesis. When there is a coexistent condition, such as sickle cell disease, specific treatment may be appropriate alongside treatment of the priapism. However, the traditional regime of oxygen, intravenous hydration, and analgesia is often unsuccessful, and only in selected cases is exchange transfusion indicated. High flow or non​ischaemic priapism develops following perineal trauma, when the penile artery is lacerated, resulting in a fistula be- tween the artery and the sinusoidal spaces of the penis. The priapism may appear at the time of injury, but more commonly it develops some time later, following spasm of the artery. Because the blood flowing through the penis is arterial (i.e. there is no ischaemia), there Table 13.6.4.3  Causes of low flow priapism Type Example Hypercoagulability disorders Sickle cell disease Myeloma Leukaemia Thalassaemia Total parenteral nutrition Drugs Intracavernosal agents (e.g. alprostadil) Psychotropic agents (e.g. phenothiazines, butyrophenones, trazadone) Anticoagulants (e.g. warfarin, heparin) Antihypertensives (e.g. α-blockers, calcium channel blockers) Miscellaneous Lumbar disc disease Solid tumours affecting the base of the penis Genitourinary sepsis Amyloidosis Table 13.6.4.4  Features that help to differentiate low flow from high flow priapism Findings Low flow priapism High flow priapism History of perineal trauma Rare Common History of coexistent blood disorder Sometimes Rare History of recent intracavernosal injection Sometimes Rare Corpora cavernosa rigid Usually Rare Penile pain Usually Rare Cavernosal blood gases Hypoxia, hypercapnia, acidosis Similar to arterial blood Colour Doppler penis Sluggish or absent flow Normal flow with evidence of arterial turbulence Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Urology (Gonzalez-​Cadavid NF, Rajfer J, 2005, Mechanisms of Disease: new insights into the cellular and molecular pathology of Peyronie’s disease, Nat Clin Pract Urol, 2, 291–​7), copyright © 2005. Initial measures Aspiration Irrigation Injection Surgery

section 13  Endocrine disorders 2412 can be some circumspection in relation to treatment. The diagnosis is usually made by Doppler scanning and subsequent selective pu- dendal arteriography, which typically shows an arterial blush in the penile artery. The treatment of choice is embolization of the fistula with autologous blood clot. There is a rare variant of ischaemic priapism called ‘stuttering pri- apism’ where men present with recurrent episodes of painful erec- tions, usually at night. These episodes often resolve spontaneously. The commonest cause is sickle cell disease, but in some cases there is no obvious explanation. Treatment is difficult, with a variety of ap- proaches including androgen suppression being used. Female sexual dysfunction The sexual problems of women have, until recently, received much less attention from the medical and scientific community than male sexual problems. This is reflected in a less well-​developed under- standing of physiological and pathophysiological processes, in a less well-​defined classification of dysfunctions, and in a smaller range of therapeutic interventions. One classification of female sexual dysfunction is shown in Table 13.6.4.5. Epidemiological studies, al- though relatively immature, suggest that female sexual disorders are common and that they relate in part to age and to hormonal status. Comorbidities are common and should be identified, when present. The different sexual dysfunctions commonly coexist. One contentious area involves the recent DSM-​5 classification, which introduces the diagnosis of female sexual interest/​arousal dis- order, and eliminates the previous separate diagnoses of hypoactive sexual desire disorder and female sexual arousal disorder. This re- classification has been controversial and for ease of presentation the conditions are described here in a traditional format, since that is how much of the clinical data has been developed. Sexual desire disorders Hypoactive sexual desire disorder (HSDD) can be defined as ‘absent or diminished feelings of sexual interest or desire, absent thoughts or fantasies, and a lack of responsive desire’. This lack of interest will be greater than that which is normally seen with ageing and with the length of a relationship. Epidemiological studies suggest that it affects up to 32% of women under the age of 50 years. It becomes commoner with increasing age and with the onset of the menopause, whether natural or sur- gically induced. Around half the women who have this condition are ‘distressed’ by it, with the degree of distress diminishing with increasing age. Women with a surgically induced menopause are more likely to be distressed than those who have undergone a nat- ural menopause. The physiology and pathophysiology of desire is poorly under- stood, but probably resides biologically in the limbic system of the brain, where hormonal influences, including oestrogens and an- drogens, are important. Other hormonal abnormalities including hyperprolactinaemia and thyroid dysfunction can also result in sexual desire disorders. The dominant neurotransmitters include dopamine, which is important in the seeking-​appetite-​lust system, and oxytocin. Aetiology is often difficult to ascertain. There may be biological factors including natural or premature menopause, urinary in- continence, alcohol excess, and recreational drug abuse, while there are certain drug classes that can be associated with HSDD, including psychotropic agents, antihypertensive agents, drugs with an endocrine mechanism, and anticholinergics (Table 13.6.4.6). Psychological factors including a history of sexual abuse, anxiety and (especially) depression are also important, and their interaction with the biological issues makes this condition notoriously difficult to treat. Finally, the close relationship to other female sexual dis- orders and to male sexual dysfunction means that these aspects should be explored. Clinical assessment includes a sexual history and a full medical history. A careful history will seek to identify any psychological or physical issues, and it is important to find out whether the problem is universal or situational, and whether it is acquired or lifelong. A careful gynaecological assessment is important and a full endo- crine evaluation may be appropriate, which will include measure- ment of serum testosterone, dehydroepiandrosterone sulphate, prolactin, 17-​β oestradiol, and SHBG. Table 13.6.4.5  A classification of female sexual dysfunction Category Subcategory Disorders of desire Interest desire disorders Sexual aversion disorders Disorders of arousal Subjective arousal disorders Genital arousal disorders Combined subjective and genital arousal disorders Persistent arousal disorder Orgasmic disorders Pain disorders Dyspareunia Vaginismus Table 13.6.4.6  Drugs that cause sexual dysfunction in women Medication Desire disorder Arousal disorder Orgasm disorder Psychotropics Antipsychotics Benzodiazepines Lithium SSRIs Venlafaxine + + + + + + + + + + + CV drugs Beta blockers Clonidine Digoxin Spironolactone + + + + + + Hormonal preparations Contraceptive pill Antiandrogens Tamoxifen GnRH analogues + + + + + + + + Other H2 receptor antagonists Ondomethicin Ketoconazole Phenytoin Anticholinergics Antihistamines + + + + + +

13.6.4  Sexual dysfunction 2413 Treatment has been difficult and often unsuccessful, partly be- cause the aetiology is commonly multifactorial; partly because of the complex interaction with relationship issues; but also because the motivation to be treated is often low. Where physical causes can be identified, they should be treated, and indeed outcomes are best in the group of patients who have definite endocrine abnormalities and who are highly motivated. Recently a new agent, flibanserin, has been licensed in the United States for the treatment of this condition. Flibanserin is a non-​ hormonal 5-​HT1A receptor agonist and 5-​HT2A receptor antag- onist. In two 24-​week clinical trials, treatment was associated with significant improvements in sexual desire, increased sexually sig- nificant experiences, and a decrease in sexual distress. The most commonly reported adverse events were dizziness, nausea, feeling tired, sleepiness and trouble sleeping, and drinking alcohol while on flibanserin resulted in low blood pressure in some patients. Although there is some data regarding the use of testosterone for this indication, the evidence is mixed and its use is not recommended. Non​pharmacological treatments include sex therapy and cognitive behavioural therapy, but the results are often disappointing. Sexual aversion disorders Defined as ‘severe anxiety or disgust at the thought of sexual ac- tivity’, sexual aversion disorder may arise as a result of incest, rape, molestation, and psychological abuse. It may coexist with other anxiety disorders, and psychosexual therapy is the mainstay of treatment. Sexual arousal disorders Female sexual arousal includes the physiological responses of in- creased blood flow to the clitoris, the labia, and the vagina, leading to vasocongestion and engorgement. Vaginal lubrication appears to be a purely hydrostatic event with transudation from the vaginal capil- laries into the extracellular space. The physiology of these responses is poorly understood, but involves parasympathetic activation, with release of several neurotransmitters of which VIP and nitric oxide are almost certainly the most important. There is concurrent relax- ation of the vaginal smooth muscle with lengthening and dilation of the vagina. Systemically, there is an increase in the heart rate, flushing, and erection of the nipples. There are three categories of arousal disorder. Firstly, there is the so-​called subjective arousal disorder, associated with a reduction in the feelings of sexual arousal (including sexual excitement and sexual pleasure), but with normal vaginal lubrication still occurring. Alternatively, there may be genital sexual arousal disorder, when there is a definite and reduced degree of genital arousal in response to a sexual stimulus. Finally, the two may be found in combination. Sexual arousal disorders affect up to 24% of women. They are com- moner in older women, especially after the menopause, and the aeti- ology may be multifactorial. Psychological factors include reduced desire, sexual inhibition, anxiety, lack of intimacy, and male erec- tion problems. Physical factors that may be important include endo- crine abnormalities (e.g. reduced oestrogens, hyperprolactinaemia), vascular disease (e.g. diabetes), neuropathy (e.g. diabetes, mul- tiple sclerosis), drugs (Table 13.6.4.6), iatrogenic problems (e.g. posthysterectomy, postpelvic radiotherapy) and pain disorders (e.g. genital pain such as vaginismus, bladder pain due to recurrent urinary tract infection (UTI) or interstitial cystitis). Assessment should involve a careful history and examination, seeking to identify the range of risk factors that are present in an individual. Investigations might include an endocrine screen and, when indicated, Doppler assessment or plethysmography can evaluate vaginal and clitoral blood flow. Treatment should be directed towards the predominant aetio- logical issues. Psychosexual methods are important for cases where ‘subjective’ problems predominate. Physical treatments that may be of value include local lubricants, topical oestrogens (when vaginal atrophy is present and oestrogen deficiency is present), and local physical devices analogous to the vacuum erection devices used in male erection difficulties. Systemic hormone replacement therapy may be appropriate in some, but there are no licensed non​ hormonal therapies for this condition. Given the physiological finding that nitric oxide was important in the control of vaginal blood flow, it was thought that the phosphodiesterase inhibitors such as sildenafil might have a role, but the results of clinical trials were disappointing. Persistent sexual arousal disorder This is a poorly documented but uncommon condition character- ized by persistent genital arousal in the absence of a sexual stimulus. The pathophysiology is unknown, and there is no recognized therapy. Orgasmic disorders in women Anorgasmia is reported to occur in up to 37% of women, while in some there may be marked delay or diminution in the intensity of the orgasm. The female orgasm is characterized by a transient sen- sation of intense pleasure, associated with rhythmic contractions of the pelvic floor musculature, often associated with uterine and anal muscular contractions. Although the nature of the orgasm changes with age (becoming shorter and less intense), there is no evidence that the prevalence of orgasmic disorders becomes commoner with increasing age. The physiology of the female orgasm is poorly understood, but in most cases involves clitoral stimulation in association with an intact sacral reflex arc. While it would seem logical that intact ascending neural pathways are necessary for a woman to ex- perience an orgasm, patients with complete spinal cord transac- tion can, on occasions, experience them. It has been suggested therefore that ascending fibres within the vagus nerve may be important. There are multiple psychological and cultural influences on the ability of a woman to experience an orgasm. Similarly, there are number of physical and psychological problems that influence the ability of a woman to experience orgasm, including neuropathy, endocrine changes, thyroid disease, drugs (Table 13.6.4.6) and the presence of a weak pelvic floor. Psychological and social issues can be important: for instance, there is an inverse relationship between the degree to which a woman holds serious religious views and her ability to experience an orgasm. From a physical perspective, arousal disorders can result in delayed or absent orgasm. Therapy is primarily psychosexual, particularly when the problem is lifelong. When the problem is more recent in onset and associ- ated with a physically induced sexual arousal disorder, then therapy should be directed at the arousal disorder. No pharmacological agents have shown any value in the treatment of this condition.

section 13  Endocrine disorders 2414 Sexual pain disorders in women Dyspareunia is pain associated with attempted or complete vaginal entry, while vaginismus indicates difficulties in the woman allowing entry of a penis (or any other object) into the vagina, despite her desire for this to happen. It is traditional to separate these two con- ditions, although in reality they may overlap, both in causality and in clinical presentation. Dyspareunia is reported by up to 15% of sexually active women, and it becomes much commoner in the postmenopausal population. Vaginismus is much less common, af- fecting less than 1% of sexually active women. Dyspareunia can be caused by several different conditions (see Table 13.6.4.7), and while these conditions can also cause vagin- ismus as well, there are cases where no clear organic aetiology ap- pears to exist. Clinically, it is important to identify such causes when they are present, and to identify and treat any sexual comorbidities. Vulvar vestibulitis is one of the most important causes of vagin- ismus and is characterized by pain with penetration or attempted penetration, tenderness of the vestibular area to even light touch, and erythema of the vestibular area. Its aetiology is unknown, but there is evidence of chronic inflammation, although the mechanism by which this arises is unclear. Treatment is often unsatisfactory, and involves analgesia (perhaps including tricyclic antidepressants and gabapentin), preventative hygienic measures, and sexual ab- stinence. There is preliminary evidence that vaginal electromyog- raphy (EMG) biofeedback, pelvic floor physiotherapy, and possibly vestibulectomy may have a role in treatment, although controlled studies are required for confirmation. FURTHER READING Basson R (2006). Clinical practice: sexual desire and arousal disorders in women. N Engl J Med, 354, 1497–​506. Basson R, et al. (2004). Revised definitions of women’s sexual dysfunc- tion. J Sex Med, 1, 40–​8. BAUS Section of Andrology Genitourethral Surgery (2018). BAUS consensus document for the management of male genital emergen- cies: priapism. BJU Int, 121, 835–9. Broderick GA, et  al. (2010). Priapism:  pathogenesis, epidemiology, and management. J Sex Med, 7, 476–​500. Brotto LA, et al. (2010). Women’s sexual desire and arousal disorders. J Sex Med, 7, 586–​614. Buvat J, et al. (2011). Hypogonadal men nonresponders to the PDE5 inhibitor tadalafil benefit from normalization of testosterone levels with a 1% hydroalcoholic testosterone gel in the treatment of erectile dysfunction (TADTEST study). J Sex Med, 8, 284–​93. Clayton AH, Vallardares Juarez EM (2017). Female sexual dysfunc- tion. Psychiatr Clin North Am, 40, 267–84. Ciocanel O, Power K, Eriksen A (2019). Interventions to treat erectile dysfunction and premature ejaculation: an overview of systematic reviews. Sex Med, 7, 251–69. Eardley I (2013). The incidence, prevalence, and natural history of erectile dysfunction. Sex Med Rev, 1, 3–​16. Eardley I, et al. (2010). Pharmacotherapy for erectile dysfunction. J Sex Med, 7, 524–​40. Feldman HA, et al. (1994). Impotence and its medical and psycho- social correlates:  results of the Massachusetts Male Aging Study.
J Urol, 151, 54–​61. Fugl-​Meyer KS, et al. (2013). Standard operating procedures for female genital sexual pain. J Sex Med, 10, 83–​93. Gandaglia G, et al. (2014). A systematic review of the association be- tween erectile dysfunction and cardiovascular disease. Eur Urol, 65, 968–​78. Gelbard MK, Chagan L, Tursi JP (2015). Collagenase clostridium histolyticum for the treatment of Peyronie’s disease:  the devel- opment of this novel pharmacologic approach. J Sex Med, 12, 1481–​9. Gonzalez-​Cadavid NF, Rajfer J (2005). Mechanisms of disease: new insights into the cellular and molecular pathology of Peyronie’s dis- ease. Nat Clin Pract Urol, 2, 291–​7. Gupta BP, et al. (2011). The effect of lifestyle modification and cardio- vascular risk factor reduction on erectile dysfunction: a systematic review and meta-​analysis. Arch Intern Med, 171, 1797–​803. Hackett G (2009). The burden and extent of comorbid condi- tions in patients with erectile dysfunction. Int J Clin Pract, 63, 1205–​13. Hackett G (2011). Cardiovascular drugs and sexual dysfunction. Primary Care Cardiovascular Journal, 4, 124–​6. Hatzichristou D, et al. (2010). Recommendations for the clinical evalu- ation of men and women with sexual dysfunction. J Sex Med, 7, 337–​48. Table 13.6.4.7  Causes of dyspareunia Organic Superficial and introital Infections (vulvitis, vulvar vestibulitis, cystitis, vaginitis) Hormonal (vaginal atrophy) Anatomic (fibrous hymen, vaginal agenesis) Muscular (hyperactivity of levator ani) Iatrogenic (postsurgical, postradiation) Neuropathic Deep Endometriosis Pelvic inflammatory disease Chronic pelvic pain syndrome Iatrogenic (postsurgical, postradiation) Psychological Comorbidity with other disorders of female sexual function Previous sexual abuse or rape Depression and anxiety Couple related Inadequate foreplay Couple conflicts Sexual dissatisfaction Anatomic compatibility issues

13.6.4  Sexual dysfunction 2415 Hatzimouratidis K, et al. (2010). Guidelines on male sexual dysfunction: erectile dysfunction and premature ejaculation. Eur Urol, 57, 804–​14. Hatzimouratidis K, et al. (2012). EAU guidelines on penile curvature. Eur Urol, 62, 543–​52. Ishak WW, et al. (2010). Disorders of orgasm in women: a literature review of etiology and current treatments. J Sex Med, 7, 3254–​68. Jaspers L, et al. (2016). Efficacy and safety of flibanserin for the treat- ment of hypoactive sexual desire disorder in women: a systematic review and meta-​analysis. JAMA Intern Med, 176, 453–​62. Kheirandish P, Chinegwundoh F, Kulkarni S (2011). Treating stut- tering priapism. BJU Int, 108, 1068–​72. Leiblum S, et al. (2005). Persistent sexual arousal syndrome: a descrip- tive study. J Sex Med, 2, 331–​7. Lewis RW, et  al. (2010). Definitions/​epidemiology/​risk factors for sexual dysfunction. J Sex Med, 7, 1598–​607. Nehra A, et al. (2012). The Princeton III Consensus recommenda- tions for the management of erectile dysfunction and cardiovas- cular disease. Mayo Clin Proc, 87, 766–​78. Pryor JL, et  al. (2006). Efficacy and tolerability of dapoxetine in treatment of premature ejaculation: an integrated analysis of two double-​blind, randomised controlled trials. Lancet, 368, 929–​37. Romeo JH, et al. (2000). Sexual function in men with diabetes type 2: association with glycemic control. J Urol, 163, 788–​91. Salonia A, et al. (2014). European association of urology guidelines on priapism. Eur Urol, 65, 480–​9. Seftel AD, Sun P, Swindle R (2004). The prevalence of hypertension, hyperlipidemia, diabetes mellitus and depression in men with erectile dysfunction. J Urol, 171, 2341–​5. Serefoglu EC, et  al. (2014). An evidence-​based unified definition
of lifelong and acquired premature ejaculation:  report of the second International Society for Sexual Medicine Ad Hoc Committee for the Definition of Premature Ejaculation. J Sex Med, 11, 1423–​41. Sharma KL, Alom M, Trost L (2019). The etiology of Peyronie’s disease: pathogenesis and genetic contributions. Sex Med Rev, pii: S2050- 0521(19)30069-1. doi: 10.1016/j.sxmr.2019.06.004. Sungur MZ, Gunduz A (2014). A comparison of DSM-​IV-​TR and DSM-​5 definitions for sexual dysfunctions: critiques and challenges. J Sex Med, 11, 364–​73. Tommola P, Unkila-​Kallio L, Paavonen J (2010). Surgical treatment of vulvar vestibulitis:  a review. Acta Obstet Gynecol Scand, 89, 1385–​95.

13.7 Disorders of growth and development 2416

13.7 Disorders of growth and development 2416

13.7.1 Normal growth and its disorders 2416

13.7.1 Normal growth and its disorders 2416

CONTENTS 13.7.1 Normal growth and its disorders  2416 Gary Butler 13.7.2 Normal puberty and its disorders  2428 Fiona Ryan and Sejal Patel 13.7.3 Normal and abnormal sexual differentiation  2435 S. Faisal Ahmed and Angela K. Lucas-​Herald 13.7.1  Normal growth and its disorders Gary Butler ESSENTIALS Normal growth has three phases: rapid in infancy and adolescence, steady during mid childhood. Height should always be interpreted within the context of the family: short or tall stature is often familial; idiopathic short stature occurs when the height of a normal child is below their target range. Failure of growth Aetiology and investigation—​constitutional growth delay is a common normal variant, but poor growth and/​or weight gain may be associated with recognized and unrecognized chronic disease, and also with psy- chosocial deprivation. Investigation must exclude conditions including hypothyroidism, coeliac disease, inflammatory bowel disease, and chronic kidney disease. Turner syndrome (karyotype 45,X) should be suspected in all girls presenting with growth failure, and skeletal dys- plasia when a child is either short for their family or has one parent of significant short stature. Growth hormone deficiency—​confirmed by a poor response to stimulation tests and low IGF-​1 levels—​may occur in isolation or in association with one or more additional pituitary hor- mone deficiencies, and may be genetic or acquired (usually from intra- cranial tumours or following traumatic brain injury). Management—​growth hormone (given by daily subcutaneous injection) may restore growth potential completely in children with growth hormone deficiency, and (usually in larger doses) can improve growth and may be appropriate for some children with conditions, including chronic kidney disease, Turner syndrome, Prader–​Willi syndrome, SHOX deficiency, and those who were born small for gestational age. Excessive growth Aetiology and investigation—​constitutional tall stature, often asso- ciated with obesity, is a common normal variant, but conditions that can present with tall stature include: (1) genetically identifiable syndromes—​for example, Marfan’s syndrome, Klinefelter’s syndrome (karyotype 47,XXY), 47,XYY boys; (2) any condition leading to pre- cocious sexual maturation; (3) pituitary gigantism—​a rare condition caused by a pituitary somatotroph macroadenoma secreting large quantities of growth hormone. Management—​attempts at growth limitation with high-​dose sex steroids are not often effective and may have short-​ and long-​term complications, but induction of early puberty with conventional hor- mone doses may offer some help. Absolute cessation of limb growth can only be obtained by epiphysiodesis. Introduction: What is normal growth? The growth of a person from a fertilized egg to a mature individual is a remarkable process involving many hundreds of thousands of synchronized steps, with size having increased a million-​fold. Yet it is noteworthy that not only do the heights of adult men and women each fall within quite a narrow range, but so does the growth of children at each age. For growth, we usually mean height and weight. Therefore, it is relatively easy to define what is normal and this normal range for age is represented on standard growth charts (see Figs. 13.7.1.1 and 13.7.1.2). Even though some variability exists between people of different ethnic backgrounds, the pattern of growth is remarkably constant, which is why the World Health Organization has been able to produce truly international growth reference standards. Of course, not all parts of the body grow at the same rate at the same time and spurts of growth can be seen at many different ages. 13.7 Disorders of growth and development

13.7.1  Normal growth and its disorders 2417 Fig. 13.7.1.1  UK-​WHO growth standards 0–​4 years in boys. © 2009 Department of Health.

section 13  Endocrine disorders 2418 Fig. 13.7.1.2  UK-​WHO growth standards 0–​4 years in girls. © 2009 Department of Health.

13.7.1  Normal growth and its disorders 2419 Before considering the problems of growth (summarized in Table 13.7.1.1), it is helpful to understand the three phases of postnatal growth separately: infancy, childhood, and adolescence. Infancy Growth is most rapid in the first year of life. Despite a normal weight loss of up to 10% in the first two weeks of life, weight will triple from a mean of 3.3 kg at birth to 10 kg and length increases by 50% from 50 cm to 75 cm, with a height velocity of 25 cm per year. Head cir- cumference, which reflects brain growth, increases by one-​third over the year, two-​thirds of this occurring within the first 6 months. Much tracking of growth measurements (movement upwards or downwards across centile bands) takes place over this first 6 months. Weight, length, and head circumference may each shift as much as one centile band which, if in a downwards move, may pose difficul- ties in the differential diagnosis of failure to thrive, but in normal growth variants this will cease once the genetic or preprogrammed centile has been attained. There are several reasons for this. First, size at birth principally tends to reflect maternal height and also pla- cental function. Second, in the absence of disease, growth will follow a genetically determined trajectory. Adequate nutrition is a highly important factor in determining normal growth and has a funda- mental influence during infancy. A good example of shifts in growth centiles is catch-​up growth exhibited by infants who have been subject to intrauterine growth retardation. Unless there are other constraints, more than 95% will show full catch-​up of length and weight by the end of the first year and 98% by 2 years of age. This should not be confused with in- fants of preterm birth whose relative size may be attributed to their prematurity. Here, by convention, measurements are adjusted for gestational age up until their first birthday for those born after 32 weeks’ gestation and their second birthday for those below 32 weeks’ gestation. Childhood The childhood phase of growth lasts from the second year of life until the clinical onset of puberty. The rate of height and weight gain is less rapid than in infancy and is similar between boys and girls until the onset of puberty. This phase of growth is largely under the control of hormonal factors most notably growth hormone (GH) and insulin-​like growth factor 1 (IGF-​1). There are also environ- mental influences such as the seasons and health and endogenous rhythms which produce spurts of growth, the largest of which at around about 6 years of age is known as the mid-​childhood growth spurt. This is one of a series of prepubertal growth spurts occurring approximately every 2 years. Although there is a temporal associ- ation with adrenarche, which is the maturation of the adrenal cortex to produce adrenal androgens, there is no direct link between this phase of adrenal physiology and growth as the prepubertal growth spurts are multiple and the mid-​childhood spurt, although the most consistent, is not necessarily the largest prepubertal growth spurt. Adolescence The most marked difference in growth between the sexes starts during puberty. The total height gain in the male during this period is 25 to 30 cm, greater than in the female which is 20 to 25 cm. However, most of the 14 cm average difference between men and women occurs before the onset of the adolescent growth spurt. Approximately 11 to 12 cm is accounted for by boys continuing to grow at the prepubertal growth rate for a further 2 years until the onset of the faster pubertal growth spurt, which contributes 2 to 3 cm more height than in girls. Although the rapid acceleration in growth is coincident with the clinical and hormonal onset of puberty in both sexes, it is much more intense and hence more immediately noticeable in girls and they will attain the maximum growth rate (peak height velocity) 1 year after starting puberty, whereas in boys the acceleration is far more gradual and peak growth occurs 2 years after the onset of puberty. Disorders of growth Simple errors Quite often children who are thought to have a growth disorder turn out not to; the apparent problem is due to an error in measurement or in plotting on a growth chart. Simply repeating these procedures if an odd pattern of growth is seen will usually reveal the problem. Genuine growth disorders demonstrate a continued trend, whether this is acceleration or deceleration of growth at inappropriate times or weight loss or gain when unexpected or at an unusual age. Table 13.7.1.1  Principal causes of abnormalities in growth and stature Short stature Tall stature Familial short stature Familial tall stature Constitutional delay of growth and/​or puberty Constitutionally advanced growth and/​or puberty (constitutional tall stature) Idiopathic short stature Obesity Small for gestational age/​intrauterine growth retardation Precocious sexual maturation Psychosocial short stature Supernumerary sex chromosomes, e.g. Klinefelter’s, XYY, XXX Chronic disease: recognized and unrecognized Genetic/​dysmorphic tall stature syndromes, e.g. Marfan’s, Sotos, Beckwith–​Wiedemann Endocrine gland disorders including GH deficiency Genetic abnormalities of the GH axis Chromosomal variations including SHOX deletions Genetic/​dysmorphic short stature syndromes, e.g. Turner’s, Russell–​Silver, Noonan’s Skeletal dysplasias

section 13  Endocrine disorders 2420 Growth standards Current UK-​WHO charts aged 0–​4 years are based on the WHO growth standards which are derived from prospectively collected data from over 8000 infants in six countries around the world until 5 years of age (see Figs. 13.7.1.1 and 13.7.1.2). These infants were breast fed and reared in optimal socioeconomic circumstances. They represent the ideal pattern of growth in health and are suitable for all ethnic groups. Charts are available in centile format (commonly used in the United Kingdom and United States) and in standard de- viation format preferred in many European countries. Beyond age 4 years, the UK 1990 growth data (see Figs. 13.7.1.3 and 13.7.1.4) continues to be used. Familial short stature The term ‘genetic short stature’ should be avoided in this situation as it is imprecise, and the meaning can be confused with small size from a chromosomal or single gene abnormality. From short parents generally come short children. The target centile range can generally be estimated from the information pro- vided on national growth standards. In the United Kingdom a rela- tively simple formula is applied. The target or mid-​parental height is a simple mean of the parents’ height adjusted for the 14 cm mean male–​female difference. Thus, target height for a boy is the parental mean plus 7 cm, and for a girl the target height is the parental mean minus 7 cm. The centile range within which most children of these biological parents will fall is 10 cm either side of the target height for boys and 8.5 cm for girls. A simpler method is the graphical Parent Height Comparator (see Figs. 13.7.1.3 and 13.7.1.4; top right). To estimate the mid-​parental centile, plot the mother’s and father’s heights on their respective scales and join the two points with a line. The mid-​parental centile is where this line crosses the centile line in the middle. Then compare the mid-​parental centile to the child’s current height centile, plotted on the adult height predictor scale (Figs. 13.7.1.3 and 13.7.1.4; bottom right). Nine out of ten children’s height centiles are within plus or minus two centile spaces of the mid-​parental centile. Children with short stature of familial origin grow normally with no deviation from their centile position. Assessment of height velocity over 1 year can confirm this. Clinical examination is un- remarkable, children having normal body proportions, and patho- logical estimations are also normal. However, in real life situations, several causes of short stature may coexist or overlap (e.g. familial short stature and constitutional growth delay, causing a greater than expected slowing of growth). Experienced clinical judgement is often necessary in situations like this and investigations may need to be performed for reassurance purposes. Once a familial cause of short stature is confirmed, the prin- cipal approach to management is reassurance of the child and their family. Some parents may wish to seek advantage for their child by requesting growth-​promoting treatments such as GH but evidence suggests that there is no significant height gain in either the short or the long term in these children. Constitutional delay of growth and puberty Although a delayed process of growth and maturation may present with short stature and delayed puberty, most often in boys, the pro- cess of slowing of growth starts much earlier and generally has a predictable pattern. Infants are of normal size at birth, but weight and length may begin decelerating in the first year, and can cause significant cause for concern as non​organic failure to thrive may be considered. The rate of growth is low normal, rarely subnormal, but height and weight may show only barely acceptable gains, with the children remaining below the normal centile range until catch-​up growth occurs during puberty. These children are phenotypically normal and in very good health, and the only usual finding on in- vestigation is a moderately delayed bone age (1 to 3 years). When the height for bone age is plotted on the centile chart this often falls within the target centile range. Once identified, reassurance is all that is required, but a boost to growth and sexual development may be given with low-​dose sex steroids if there is anxiety related to slow sexual development and growth in the mid-​teenage years. Idiopathic short stature This term, more precisely defined as non​familial, non-​GH defi- cient short stature, is inclusive of many pathologies. Children whose height lies 2.5 standard deviations below the mean and whose height falls below their target centile range are considered to have idiopathic short stature. Some children exhibit an appropriate height velocity and maintain their growth centile position, whereas others may grow slowly on the borderline of abnormal (c.4 cm/​year). General investigations are unremarkable, and bone age may only be slightly delayed (<2 years). The GH response to stimulation tests is normal. The disparity between this normal response to GH provocation and an abnormal disorganized pattern of physiological secretion has been described as neurosecretory dysfunction, but in reality there may be many causes. One possibility is polymorphisms in the GH receptor with various alleles showing a different GH responsiveness. If GH treatment is given to children whose abnormal growth pattern falls tightly within this definition, a good growth response to GH can be seen almost equivalent to that of true GH deficiency itself. Given diagnostic uncertainties in any case, there may be a case for GH treatment using the same dosage regimen, and this is an approved indication in the United States of America, but not in Europe. However, there are inevitable accusations of advantage ma- nipulation and social engineering which could be true if the precise definitions are not adhered to. Small for gestational age Infants born with weight and/​or length two standard deviations below the mean (2nd centile) are stated to be small for gestational age. This usually arises by a process of intrauterine growth retard- ation, but the terms are not interchangeable and indeed definitions differ. Obstetric practice will usually refer to intrauterine growth re- tardation as fetal measurements falling below the 10th centile for gestational age and maternal size. The causes of intrauterine growth retardation may be extrinsic to the fetus such as placental dysfunc- tion or twin–​twin placental steal syndrome, or intrinsic such as in- sulin resistance or genetic reasons such as deletion of a paternally imprinted growth gene or the equivalent maternal uniparental di- somy. This latter cause may account for approximately 10% of those children with clinical Russell–​Silver syndrome (see next). The diagnosis is reached from the obstetric history and subsequent postnatal growth pattern. In addition to intrauterine growth retard- ation there is often a sustained period of feeding difficulties and poor growth, often provoking much anxiety as general pathology

13.7.1  Normal growth and its disorders 2421 Fig. 13.7.1.3  United Kingdom 2013 childhood and puberty close monitoring chart for height, weight in boys 2–​20 years. © Royal College of Paediatrics and Child Health 2013.

section 13  Endocrine disorders 2422 Fig. 13.7.1.3  Continued

13.7.1  Normal growth and its disorders 2423 Fig. 13.7.1.4  United Kingdom 2013 childhood and puberty close monitoring chart for height, weight in girls 2–​20 years. © Royal College of Paediatrics and Child Health 2013.

section 13  Endocrine disorders 2424 Fig. 13.7.1.4  Continued

13.7.1  Normal growth and its disorders 2425 investigations are normal and fears of child protection issues are often raised. The infants are usually thin with little adipose tissue and may have a disproportionately large head, head circumference being within the normal centile range, length, and weight two standard deviations below the mean. In some cases, Russell–​Silver syndrome may be defined clinically. These children show a typical triangular facies with frontal bossing, a pointed chin, clinodactyly of the fifth fingers and toes, and in about 50% there is some hemihypertrophy which may be quite subtle. In about 7 to 10% of suspected cases, uniparental disomy of the maternal chromosome 7 can be identified, suggesting that it is an imprinted paternal gene which is required for normal growth. Recent studies have also suggested that imprinting defects within the 11p15 region play a role. Bone age is often isochronological in children who do not show any catch-​up growth, and consequently height prognosis is below the target centile range and often more than two standard deviations below the mean. For children over 4 years of age, GH treatment at 35 µg/​kg per day (1.0 mg/​m2 per day) has shown an initial catch-​up and gradual acceleration to adult heights within the normal range. Overall growth gain may be predicted from the first-​year response. Although some slight increase in insulin sensitivity and baseline glucose may be found, this is non​progressive and reversible on stopping GH and long-​term studies show continued benefits of having had GH treat- ment with lower blood pressure and an improved metabolic profile. Pathological causes of growth failure Recognized and unrecognized chronic disease Part of the assessment process of any child presenting with an ab- normality of growth is exclusion of other chronic conditions which may manifest initially as a variant of growth. Excess growth can result from overactivity of the thyroid gland as well as other non-​ endocrine causes of early puberty such as obesity. Slow growth is the more common, but usually less marked than it used to be in child- hood diseases such as chronic renal insufficiency, cystic fibrosis, type 1 diabetes, and inflammatory bowel disease as a result of much improved nutrition and clinical management of these conditions. Growth failure maybe the only presenting feature of conditions such as hypothyroidism, coeliac disease, inflammatory bowel dis- ease, or chronic renal insufficiency, so an initial screen should always include a full biochemical profile including vitamin D levels, a full blood count, iron status, inflammatory markers, thyroid function, and coeliac antibodies. Juvenile arthritis may, in its own right, and as a result of steroid treatment cause significant retardation of growth which may not be amenable to full catch-​up if this is longstanding. However, GH treatment at standard doses may help restore growth in some chil- dren. Chronic eczema and other atopic manifestations may subtly slow the tempo of growth, resulting in short stature and delayed pu- berty, but height usually catches up completely although this may not occur until the late teens or early twenties. Excess endogenous glucocorticoid secretion in Cushing’s syn- drome (adrenal pathology) or Cushing’s disease (ACTH-​secreting pituitary tumour) may present with slow or absent growth as well as other clinical signs such as central obesity, malar flushing, and striae. This can almost always be differentiated from children with exogenous obesity who usually grow faster than the norm and are taller than expected for their age. GH treatment is indicated in children with chronic renal insuf- ficiency in a high-​dose regimen of 50 µg/​kg per day (1.4 mg/​m2 per day) as soon as growth failure occurs, as long as nutrition is opti- mized. The main benefit is abolishing or slowing the decline in height velocity resulting from the uraemia. Juvenile hypothyroidism Bone age is often markedly delayed in juvenile acquired hypo­ thyroidism. This is usually due to autoimmune thyroiditis and may often present insidiously with growth failure alone. Early recogni- tion and treatment with levothyroxine at an initial dose of 100 µg/​m2 per day, titrated consequently to normalize thyroid function, will usually result in complete catch-​up of growth. Psychosocial short stature Psychosocial short stature occurs in children and adolescents in association with psychological harassment and/​or emotional de- privation and may be associated with transient abnormalities of the GH–​IGF-​1 axis. The clinical features can be very similar to those of GH deficiency, but children suffering from this syndrome usually have a disturbed family environment with a history or current evi- dence of child abuse, most commonly occult sexual abuse. A child with psychosocial growth failure tends to be isolated and may not participate in family activities. Behavioural disturbance and bizarre eating habits are common, with a tendency to hyperphagia rather than undernourishment. Two main subtypes of psychosocial short stature have been iden- tified. Early-​onset growth failure (type I or infantile psychosocial short stature) occurs during the first 2 years of life, is very common, and the cause is thought to be undernutrition. This manifests clinic- ally as failure to thrive. If sufficient nutrition is given, these children usually begin to grow normally again. Type II or childhood psychosocial short stature occurs in chil- dren older than 3 years. There is often a greater psychological com- ponent compared with type I psychosocial short stature. Failure to thrive is not such a marked feature and there may be a component of this in some patients diagnosed with constitutional growth delay. Abnormalities of GH secretion can be demonstrated, and these often resolve when the child is removed from the home environment. The clinical presentation is often a short child with growth failure and a normal body mass index, but paradoxical hyperphagia. In some situations, growth may not recover. In such children, full investiga- tion into the abnormality is needed and if inadequate GH secretion remains, replacement will be needed. Turner’s syndrome Turner’s syndrome should be suspected in all girls presenting with short stature and/​or poor growth. The incidence of all karyotypes together with complete or partial absence of one X chromosome, or a structurally abnormal X is approximately 1 in 2500. Girls with karyotype 45,X are more likely to show more features of the syn- drome, including peripheral oedema in infancy, webbed neck, low ears, low posterior hairline, cubitus valgus (increased carrying angle at the elbow), widely spaced nipples, a high arched palate, multiple small pigmented naevi, small convex nails, recurrent ot- itis media and deafness, delayed puberty, amenorrhea, and infer- tility due to ovarian dysgenesis, a higher prevalence of autoimmune hypothyroidism, and congenital heart disease of which coarctation of the aorta is the most common form.

section 13  Endocrine disorders 2426 Growth is decreased in utero resulting in a slightly reduced size at birth, and remains slow during infancy. The height velocity is subnormal during childhood and there is no pubertal acceleration of growth due to the absence of spontaneous oestrogen secretion. In untreated individuals with Turner’s syndrome, mean final height is approximately 143 cm. The short stature may be partly associated with haploinsufficiency of a homeobox-​containing gene SHOX lo- cated on the pseudoautosomal region of the X and Y chromosomes. Treatment with GH is now the norm, starting as soon as possible after diagnosis, even in infancy. Most benefit is gained with an early initiation of treatment, and predicted when there is a good first year response. The target GH dose is 45 to 50 µg/​kg per day (1.4 mg/​m2 per day). Significant height gain may thus occur, with some girls at- taining adult heights within the predicted normal range. The link with family height is retained. Puberty may be induced with low-​dose oestrogen (ethinyloestradiol), starting at 1 to 2 µg daily, increasing over approximately 2 years to 20 µg daily, at which time a cyclical pro- gestogen should be added often using the combined oral contracep- tive pill for convenience, but the timing should be as close to normal as possible as delaying this does not bring additional height gains. Cotreatment of the short stature with oxandrolone (a mild ana- bolic steroid) in Turner’s syndrome at a dose of 0.05 mg/​kg per day (maximum 2.5 mg) from 9 years of age has been shown in random- ized controlled trials (RCTs) to improve final height by 4.6–​7.1 cm. This is a similar result to that achieved by delaying the exogenous induction of puberty with low-​dose oestrogen until 14 years of age. Another RCT has confirmed a very similar growth-​promoting effect of early introduction of ultra-​low-​dose oestrogen treatment. SHOX deficiency Deletions or mutations of a homeobox-​containing gene SHOX lo- cated on the pseudoautosomal region of the X and Y chromosomes are a rare cause of short stature which may respond to GH treatment. This gene also has a role in the aetiology of abnormal bone morph- ology (such as the radio-​ulnar synostosis in Leri-​Weill syndrome) and sensorineural hearing loss. Prader–​Willi syndrome This condition is caused by either a deletion of the paternally im- printed genes on 15q or maternal uniparental disomy of chromo- some 15. Although often presenting at birth with extreme hypotonia and feeding difficulties, it is more often known for its association with obesity and hyperphagia and other hypothalamic dysfunc- tion such as delayed or absent puberty and slow growth due to a reduction in the numbers of gonadotropin releasing hormone and GH-​releasing hormone secretory neurons. Although adult stature is only modestly reduced (155 cm in men, 147 cm in women), GH replacement at 35 µg/​kg per day (1.0 mg/​m2 per day) may reverse the slowdown in growth, but additionally improves body composition with increased lean body mass and may enhance motor develop- ment. Caution should be exercised in treating children with extreme obesity as sudden death within the first 6 months of GH therapy due to obstructive sleep apnoea has been reported. Skeletal dysplasia Skeletal dysplasia may be suspected when a child is either short for their family or has one parent of significant short stature also. Body disproportion may exist, such as discrepancy between upper and lower segment measurements, short arms (span less than height) or a disproportionately large head circumference. Most of the skel- etal dysplasias are monogenic. The most common of the non​lethal genetic defects is achondroplasia, which is caused by mutations in the FGFR3 gene in the region 4p16.3. Several skeletal dysplasias are caused by mutations in genes that encode the family of fibroblast growth factor receptors which are tyrosine kinases and mutations within these proteins are thought to slow the rate of endochondral bone growth. Approximately 90% of cases of this disorder arise from de novo mutations. GH treatment may slow deceleration in some conditions, and may produce height gains in milder phenotypes, but is less effective than surgical limb-​lengthening procedures. Individual evaluation should be performed. GH deficiency (secondary IGF-​1 deficiency) The prevalence of GH deficiency has been variously reported as from 1 in 4000 to 1 in 9000 live births. Childhood-​onset GH defi- ciency may be congenital or acquired, and may occur in isolation (isolated GH deficiency) or in association with one or more add- itional pituitary hormone deficiencies (multiple pituitary hormone deficiency), male–​female ratio 2.2:1. Congenital GH deficiency may be caused by an inherited mutation or by a developmental abnor- mality. Acquired GH deficiency most frequently results from an intracranial tumour (craniopharyngioma, pituitary adenomas, or destructive lesions arising close to the hypothalamo-​pituitary axis such as optic glioma associated with neurofibromatosis or sec- ondary to treatment with surgery or irradiation (doses >2400 cGy) which can damage the hypothalamus, or following traumatic brain injury. Neurofibromatosis type 1 may directly affect pituitary func- tion, leading to growth failure and/​or precocious puberty. Genetic causes of GH deficiency Four distinct familial types of isolated GH deficiency are caused by mutations in the GH1 gene: type IA is inherited in an autosomal recessive manner and results in a complete absence of endogenous GH; type IB is also inherited in an autosomal recessive manner; type II is inherited in an autosomal dominant manner; and type III is X-​linked. Endogenous GH levels are diminished compared with normal in types IB, II, and III. The development of the pituitary gland is controlled by a large number of genes and transcription factors. Several mutations in the gene encoding POU1F1, a pituitary-​specific transcription factor responsible for pituitary development and hormone expression, have been shown to result in combined pituitary hormone deficiencies often of delayed onset. Other mutations in genes encoding pituitary transcription fac- tors, such as PROP1, LHX3, LHX4, HESX1, OTX2, SOX2, SOX3, GLI2, GLI3, FGFR1, FGF8, and PROKR have also been described. Clinical features The severity and duration of GH deficiency is usually reflected by the height deficit and the clinical appearance. Although GH has effects on fetal growth, manifested by a slightly reduced birth length and size, children with congenital GH deficiency may not present with growth failure until after the second year of life. Children with GH deficiency have skeletal proportions that are normal for their age and some are overweight for their height. Central adiposity if it occurs has a classical marbled appearance. Head circumference is within the normal range for age, but growth of the facial bones may be delayed, with a tendency for crowding of the facial features in the centre of the face, giving a doll-​like facies.

13.7.1  Normal growth and its disorders 2427 Dentition and skeletal maturity are also delayed. The voice may be high pitched due to the small size of the larynx. Significant hypoglycaemia only occurs in infants with severe iso- lated GH deficiency, and this tendency to hypoglycaemia usually wanes beyond the age of 5 years. However, if there is concurrent defi- ciency of ACTH (i.e. multiple pituitary hormone deficiency), hypo- glycaemia will be exaggerated. In these cases, treatment with both GH and glucocorticoids is required. The clinical features are less marked in children with milder or partial forms of GH deficiency. If a child’s height velocity over 1 year is low and other causes of growth failure have been excluded, the GH status should be assessed, along with other aspects of hypothalamo-​ pituitary function. Biochemical diagnosis Measurements of random serum GH concentrations are not helpful in diagnosing GH deficiency, as GH is secreted in a pulsatile manner. Subnormal IGF-​1 and IGFBP3 levels are only confirmatory of the diag- nosis, as low levels are often found in short slowly growing children. The diagnosis is made on the basis of a provocation test, which assesses GH secretory reserve, in combination with an IGF-​1 level which, if low, confirms an IGF-​1 deficiency secondary to the GH deficiency. Various stimuli may be used, including insulin, glucagon, ar- ginine, clonidine, and levodopa. The tests should be done using a standardized protocol after a prescribed fast dependent on the child’s age and only where there are experienced medical and nursing staff supervising the child constantly, and where full resuscitation facil- ities are immediately available. A peak GH concentration of less than 6.7 μg/​litre has tradition- ally been used to support the diagnosis of GH deficiency. This value depends on the assay used and the recombinant GH refer- ence preparation, and when there are different conversion factors between international reference units and mass-​based units. Lower spontaneous levels are seen prior to puberty and in obesity, both of which can give false positive results if not correctly interpreted. Peripubertal patients should have sex steroid treatment to ‘prime’ the somatotrophs to release GH prior to the stimulation test. It is clear, therefore, that diagnosing GH deficiency can be impre- cise due to an overlap with the spectrum of normality. The growth re- sponse to a trial of GH therapy is ultimately the best diagnostic test. Treatment with GH GH treatment in the standard regimen of 25 to 35 µg/​kg per day given by subcutaneous injection starting as soon as possible after diagnosis may allow complete catch-​up of growth, with final heights in both idiopathic GH deficiency and multiple pituitary hormone deficiency falling between –​1.0 and –​1.6 standard deviations (–​0.1 to –​0.3 standard deviations below mid-​parental height). Although physiological GH secretion increases during puberty, there are no current recommendations for altering the GH dose during adoles- cence. Recommendations for the diagnosis and treatment of growth hormone deficiency and other licensed indications reflect modifica- tions of the UK National Institute of Health and Clinical Excellence (NICE) clinical guideline. Primary IGF-​1 deficiency (GH insensitivity,
Laron’s syndrome) This is a very rare disorder which phenotypically may be similar to the much more common severe GH insufficiency, but the growth retardation is much more severe. In primary IGF-​1 de- ficiency there is excessive secretion of GH by the pituitary gland; the fault lies in the GH receptor, which is either absent or non​ functioning, leading to undetectable levels of IGF-​1 and its acid-​ labile subunit before and after a brief course of GH (the IGF-​1 generation test). The abnormality is due to one of several mu- tations in the receptor gene, which is inherited according to an autosomal recessive pattern. Recombinant IGF-​1 is licensed in Europe and in the United States of America, following successful clinical trials. Tall stature Given our ‘heightist’ society, which values tallness over shortness, and the tendency for a secular trend in increasing stature from one generation to the next (by c.1 cm per decade), referrals for concern about tall stature are far fewer than for short stature. By definition, the extremes of stature in men and women (i.e. the 99.6th centile) of 196 cm and 180 cm respectively, seem shorter than those actually tolerated and accommodated in society. As with short stature, diag- nosis of a disorder depends on whether there is rapid growth for chronological age or not. Causes of tall stature with rapid growth Genetically identifiable syndromes will cause rapid growth from infancy, but the clue to diagnosis is in the phenotype (e.g. large head circumference, classic elongated facies, and mild developmental delay in Sotos syndrome; hemihypertrophy, hyperinsulinism, and Wilms’ tumour (nephroblastoma) in Beckwith–​Wiedemann syndrome). Marfan’s syndrome is an autosomal dominant condition affecting connective tissue where tall stature may be associated with scoliosis, chest deformities, high arched palate and arachnodactyly, disloca- tion of the lenses, and dissection of the aorta. Tall stature is noticed from infancy with general accelerated growth. Cardiac assessment and follow-​up are essential in all suspected cases. Klinefelter’s syndrome (47,XXY) males are of normal size in in- fancy, but height acceleration throughout childhood is more rapid than usual. Extreme adult tall stature can be predicted early on as all the excess growth occurs in the prepubertal years, the adoles- cent growth spurt being of normal size and duration, which is in contrast with 47,XYY males who may exhibit tall stature during infancy and childhood, but in whom the adolescent growth spurt is exaggerated. Precocious sexual maturation Rapid growth may occur as a result of premature sexual matur- ation resulting from any cause, and it is essential that this is ex- cluded in a child with tall stature and increased height velocity (see Chapter 13.7.2). Familial tall stature Children coming from tall parents are expected to be tall, and are often long at birth; the same approach to diagnosis and predicting adult height can be followed as for familial short stature (see earlier). Height velocities are not excessive for their age, but in general tall children grow more quickly than short children and secrete larger amounts of GH and IGF-​1.

13.7.2 Normal puberty and its disorders 2428

13.7.2 Normal puberty and its disorders 2428

section 13  Endocrine disorders 2428 Constitutionally advanced growth and puberty Just as growth delay is a variant of growth at one end of the normal range, so is constitutional tall stature. Assessments such as bone age and dental age are advanced toward the upper end of the predicted range and children usually enter puberty within the early normal range. Consequently, the adolescent growth spurt is accelerated and growth will cease according to the normal pattern of events. This can cause distress for someone who is used to being a tall child but who may end up at an average or below average height as an adult. As better nutrition is leading to obesity, this accelerated growth pattern is becoming more common. Pituitary gigantism Pituitary somatotroph macroadenomas secreting large quantities of GH are extremely rare, but may present insidiously at any age. The classic phenotype of the pituitary giant with acromegaloid features is a late finding, but this diagnosis should be suspected in children of any age who are taller than predicted for their family and who do not show the clinical and radiological features of constitutional advance. As random GH and IGF-​1 levels have low specificity, a GH suppression test with glucose loading and a cranial MRI scan may be required. Treatment is with a combination of surgery and somatostatin analogues. Treatment of tall stature Attempts at growth limitation with high-​dose sex steroids have not in general been successful and may have short-​ and long-​term complications. An early and rapid induction of puberty with con- ventional hormone doses may offer some help. Absolute cessation of limb growth can only be obtained by epiphysiodesis. High-​dose ethinyl oestradiol treatment to limit height in girls may impair long-​ term fertility, but no long-​term adverse effects are seen with high-​ dose testosterone treatment in boys. FURTHER READING Allen DB, Cuttler L (2013). Short stature in childhood—​challenges and choices. N Engl J Med, 368, 1220–​8. Butler G, Kirk J (2011). Oxford specialist handbook of paediatric endo- crinology and diabetes. Oxford University Press, Oxford. Corredor B, et al. (2019). Tall stature: a challenge for clinicians. Curr Pediatr Rev, 15, 10–21. Dattani MT, Brook CGD (eds) (2020). Brook’s clinical pediatric endocri- nology, 7th edition. Wiley-Blackwell, Hoboken, NJ, USA. Jee YH, Baron J, Nilsson O (2018). New developments in the genetic diagnosis of short stature. Curr Opin Pediatr, 30, 541–7. Leung AKC, Leung AAC, Hon KL (2019). Tall stature in children. Adv Pediatr, 66, 161–76. Murray PG, Butler GE (2013). How to assess tall stature. Paediatrics and Child Health, 23, 409–​13. NICE (2010). Human Growth Hormone (Somatropin) for the Treatment of Growth Failure in Children (review). https://​www.nice.org.uk/​ guidance/​ta188 Royal College of Paediatrics and Child Health, Department of Health. UK-​WHO Growth Charts. http://​www.growthcharts. rcpch.ac.uk Vasques GA, Andrade NLM, Jorge AAL (2019). Genetic causes of isolated short stature. Arch Endocrinol Metab, 63, 70–8. World Health Organization. WHO Child Growth Standards. http://​ www.who.int/​childgrowth/​en/​index.html 13.7.2  Normal puberty and its disorders Fiona Ryan and Sejal Patel ESSENTIALS Puberty is the physiological sequence of events when secondary sexual characteristics develop, reproductive capacity is achieved, and final adult stature reached. The outward signs usually develop over 3 to 5 years, with significant variation both in the age that pu- berty starts and the pace at which development proceeds. The events that lead to the triggering of puberty remain uncer- tain, but clinical presentations may arise because the process is ab- normally early (precocious puberty) or abnormally late (delayed or absent puberty). Several variants of the normal processes may also present for clinical assessment, for example, premature isolated thelarche (breast development) or adrenarche (pubic and axillary hair development), which do not require treatment. Precocious puberty Aetiology—​this is classified into the more common central preco- cious puberty which is gonadotropin dependent, resulting from early activation of the hypothalamic-​pituitary-​gonadal axis, and the much rarer gonadotropin independent precocious puberty, sometimes known as peripheral or pseudo precocious puberty. The latter is related to sex steroid secretion which may be gonadal or extragonadal or due to the administration of exogenous sex steroids, but is not under gonadotropin control and the sequence of pubertal development is usually non​consonant. Investigation—​this requires measurement of sex steroids, thyroid function, a gonadotropin-​releasing hormone provocation test with additional pituitary function testing, usually combined with radio- logical imaging of the pituitary gland. A non​dominant wrist radio- graph for determination of bone age (advanced in precocious puberty) helps to estimate the extent of precocity and the possible impact on growth prognosis. In girls, a pelvic ultrasound scan is re- quired to determine ovarian and uterine dimensions and hence esti- mate the degree of pubertal maturation. Management—​the goals are to stop pubertal progression, improve final height prognosis where possible, reduce pubertal mood swings and behavioural changes, and diminish psychological distress. When possible, any underlying cause should be treated. The treatment of choice for central precocious puberty is with a gonadotropin-​ releasing hormone partial agonist. Delayed or absent puberty Aetiology—​this is classified into hypogonadotropic hypogonadism, with constitutional delay much the commonest cause of delayed/​ absent puberty, and hypergonadotropic hypogonadism due to go- nadal failure, including chromosomal abnormalities (e.g. Turner’s syndrome, Klinefelter’s syndrome). Investigation and management—​investigation is largely as for precocious puberty. Treatment goals are to induce puberty, accel- erate height gain, and improve self-​confidence. When possible, any underlying cause should be treated. Hormone replacement therapy is effective when used judiciously.

13.7.2  Normal puberty and its disorders 2429 Introduction Puberty is the physiological sequence of events when secondary sexual characteristics develop, reproductive capacity is achieved, and final adult stature reached. The start of puberty is characterized by the appearance of a palpable breast bud (stage B2) in girls and tes- ticular volumes of more than 3.5 ml in boys. Normal pubertal pro- gression occurs in a specific sequence. Consonance is important as in normal, centrally mediated puberty, there is a temporal relation- ship between the development of the physical features of puberty, the timing of menarche, and the pubertal growth spurt. It is im- portant to understand the normal course of pubertal developmental in order to identify what is a normal variant and what may indicate underlying pathology. Physiology of puberty Puberty begins with the reactivation of the hypothalamic-​pituitary-​ gonadal (HPG) axis. It is still unclear as to the precise trigger; however, neurotransmitters such as GABA, NMDA (N methyl D as- partate) and KISS1 are involved. In the fetus, the pituitary-​gonadal axis is active, and levels of gonadotrophins and sex steroids are high in the first few months of life, before dropping to very low levels during childhood. The gonadotrophin-​releasing hormones (GnRH) neurons are then inhibited by poorly defined pathways. Recent evi- dence suggests reactivation of these pathways is not only centrally controlled but gonadal contribution also plays a part. It is still un- clear why the GnRH neurons are usually inhibited after infancy until the start of puberty. Fat mass (via leptin signalling) and kisspeptin-​ secreting neurons stimulating the GnRH system certainly play a role. Physical signs of puberty are preceded by an increase in secre- tion of luteinizing hormone (LH) and follicle-​stimulating hormone (FSH). Gonadotrophin-​releasing hormone (GnRH) is released in a pulsatile fashion from the hypothalamus and stimulates the pulsatile release of LH and FSH from the anterior pituitary. LH stimulates the Leydig cells in the testes to produce testosterone which induces secondary sexual development in boys. FSH stimulates germ cell maturation leading to spermatogenesis. LH and FSH work together to stimulate follicular development in the ovaries resulting in oes- trogen production and the development of secondary sexual char- acteristic in girls. Ovulation is triggered by the interaction of LH, FSH, and oestrogen. Timing of puberty This is variable and influenced by a multitude of intrinsic and ex- trinsic factors including nutrition, genetic factors, ethnicity, and environmental exposure. Anorexia nervosa can cause both inhib- ition of pubertal initiation and pubertal arrest. Certain foods, cos- metics, and hair products are potential sources of exogenous sex steroids. Exogenous oestrogens and ‘endocrine disrupters’ have been postulated to be involved in the development of earlier sexual maturation, but this is unproven. A change in environment can also have a significant effect on pubertal timing as seen in children adopted into the United Kingdom from lower-​income countries. This is likely multifactorial although nutrition probably plays a key role. There has been a reported increase in earlier breast development in girls over recent decades, although the mean age of menarche has remained fairly stable during the last two decades, implying that the duration of puberty may be increasing in girls. In the United Kingdom, the ranges for normal onset of puberty are shown on the growth charts. Due to the significant variability of normal pubertal development, guidance to consider evaluation of abnormalities in puberty is important. This is usually defined at 2–​2.5 SD outside the population’s mean (Fig. 13.7.2.1). Puberty before the age of 8 in girls and 9 in boys is defined as precocious while puberty after the age of 13 in girls and 14 in boys in defined as delayed in the United Kingdom. The definition of the lower age at which puberty is acceptable in girls has been chal- lenged recently. Recent evidence from the United States suggests Breast budding (breast stage 2) Precocious Puberty Precocious puberty Age (years) Delayed puberty Delayed Puberty 16 yrs 14 12 10 8 6 4 2 0 Testicular enlargement (4 ml) BOYS GIRLS Fig. 13.7.2.1  The mean ages +/​-​ 2 standard deviations of the onset of puberty. The definition of delayed and precocious puberty in both sexes currently accepted in the United Kingdom has also been shown. Data from UK-​WHO growth charts.

section 13  Endocrine disorders 2430 earlier sexual maturation is occurring in girls especially in the Afro-​ Caribbean population. The most widely used standard of assessment of pubertal staging was developed by Marshal and Tanner (1969) based on their study of 192 English girls (Fig. 13.7.2.2). A more re- cent study from the United States by Herman-​Giddens et al. (1997) analysed data collected from 17 077 US girls between the age of 3 and 12 by the Paediatric Research in Office Settings (PROS) net- work. In 1999, the Lawson Wilkins Paediatric Endocrine Society re- commended lowering the normal age of the onset of puberty from 8 to 7 years in white girls and to 6 years in African-​American girls based largely on this study. However, this has not been formally adopted in the United Kingdom or any other European country. Fig. 13.7.2.2  Diagrammatic representations of Tanner stages 1 to 5, for males and females. Stage 1
is prepubertal; stage 5 adult. Source data from Butler, G., and Kirk, J. (eds) (2011). Paediatric Endocrinology and Diabetes (Oxford Specialist Handbooks in Paediatrics).

13.7.2  Normal puberty and its disorders 2431 Lowering of the age of menarche has been documented in African-​ American girls between the 1960s and 1990s but longitudinal data has not substantiated this for European girls. Data for a secular trend in the age of onset of puberty in boys is insufficient to suggest a sig- nificant change. Lowering age of pubertal onset has been linked to the emergence of the epidemic of obesity in childhood. It has been postulated that this could be due to greater aromatization of androgens to oes- trogens in subcutaneous fat tissue. Although there is certainly a clear association between childhood obesity and early sexual mat- uration this is not necessarily causal and these may be occurring independently. There is some evidence that the earlier sexual de- velopment is more likely to be innocent thelarche rather than true puberty. The balance of evidence currently suggests there has been no sig- nificant change in the timing of true puberty, but robust longitu- dinal studies are needed to determine this for sure. Precocious puberty This is classified into the more common central precocious puberty (CPP) which is gonadotropin dependent, resulting from early ac- tivation of the hypothalamic-​pituitary-​gonadal axis and the much rarer gonadotropin-​independent precocious puberty (GIPP) (sometimes known as peripheral or pseudoprecocious puberty). The latter is related to sex steroid secretion which may be gonadal or extragonadal or due to the administration of exogenous sex steroids but is not under gonadotropin control and the sequence of pubertal development is usually non-​consonant. Central pre- cocious puberty is more common in Afro-​Caribbean or mixed-​ race children especially if they are overweight. The appearance of signs of puberty before the age of 8 in girls and 9 in boys requires investigation and is defined as precocious. It is important to distinguish normal variants of puberty (adrenarche and thelarche) from central or peripheral precocious puberty. Central precocious puberty has an incidence of about 1 in 5000 children and is 10 times more common in girls. This commences with breast development in girls and is mostly idiopathic. However, the cause is more likely to be pathological in boys. The second most common cause in both boys and girls are tumours around the pi- tuitary stalk and hypothalamus. Clinical evaluation of symptoms related to an intracranial lesion, signs of an associated syndrome and accurate pubertal Tanner staging are of important. This will aid focus on appropriate investigations later on. A list of possible causes is given in Table 13.7.2.1. Normal variants of puberty Premature adrenarche At 5–​7 years, the zona reticularis develops in the adrenal cortex and the production of adrenal androgens are increased. This re- sults in increased height velocity (the mid childhood growth spurt). This is a benign condition and typically presents with the following clinical features: 1. Pubic/​axillary hair 2. Adult body odour 3. Greasy skin and/​or acne There are no other signs of pubertal development. It must be dis- tinguished from virilizing tumours and non​classical congenital ad- renal hyperplasia, both of which would be expected to give signs of virilization with penile enlargement or clitoromegaly. It requires no treatment but there is an association with later polycystic ovary syn- drome (PCOS) and the family should be advised to avoid excessive weight gain. Final height is not affected. Premature thelarche This is a self-​limiting condition characterized by unilateral or bilat- eral breast development in girls. The breast development often fluc- tuates in size. Most often, it occurs before the age of 2 with no other pubertal advancement. This represents partial activation of the HPG axis, resulting from pulsatile FSH secretion while LH secretion is prepubertal. Investigation is usually unnecessary, but some patients show an intermediate clinical picture between thelarche and central precocious puberty. In this ‘thelarche variant’ breast development occurs but is accompanied by an increased height velocity and often cyclical vaginal bleeding. This can be associated with some bone ad- vancement but low oestradiol secretion. Initial conservative man- agement is reasonable but follow-​up is essential. Investigations Several biochemical and radiological investigations are essential for children with precocious puberty. A GnRH (LHRH) test will aid differentiation between CPP and GIPP. An LHRH (luteinizing hormone-​releasing hormone) test is mandatory to confirm pubertal response. This test is performed by stimulating the HPG axis with 2.5 µg/​kg of LHRH with LH and FSH levels measured at 0, 30, and 60 minutes after stimulation and a baseline oestradiol or testosterone measurement. Table 13.7.2.1  Causes of precocious puberty Idiopathic Includes familial Central: Gonadotropin dependent Central nervous system (CNS) abnormalities • Septo-​optic dysplasia • Arachnoid and pineal cysts • Hypothalamic hamartoma • Hydrocephalus • Hypothalamic-​pituitary tumours –​ Optic nerve glioma (+/​-​ NF type 1) • CNS irradiation • Hydrocephalus Prolonged exposure to sex steroids Late onset CAH (21-​hydroxylase deficiency) Ectopic source of HCG Hepatoblastoma Choriocarcinoma Mediastinal germ cell tumours Prolonged hypothyroidism Peripheral: Gonadotropin independent Adrenal adenomas/​carcinomas Congenital adrenal hyperplasia Ovarian overactivity McCune–​Albright syndrome (GS ɑ-​activation) Ovarian cysts and tumours Testicular overactivity Testotoxicosis GS ɑ-​activation in McCune–​Albright syndrome Leydig cell and Sertoli cell tumours Exogenous sex steroids Drugs or diet sources

section 13  Endocrine disorders 2432 A pubertal LHRH test shows LH peak more than 5 units/​litre with an LH response usually greater than the FSH response. This confirms CPP. In GIPP the LHRH test shows a prepubertal response with a peak LH less than 5 units/​litre, LH response being less marked than the FSH response. Gonadotrophin levels may be completely sup- pressed and sex steroids may be very high. Serum levels of oestradiol in girls are not as helpful as levels of testosterone in boys, as low levels of oestradiol do not exclude precocious puberty in girls. Bone age and thyroid function tests are checked as routine. A radiograph of the non​dominant wrist is used to assess for bone age advancement which is associated with precocious puberty. Bone age is advanced usually by more than two standard devi- ations compared to chronological age. Hyperthyroidism advances bone age and may precipitate central precocious puberty. A high thyroid-​stimulating hormone (TSH) level in hypothyroidism can stimulate the FSH receptor and induce ovarian and testicular enlargement. A pelvic ultrasound is recommended in girls to confirm any oes- trogen effect on the uterus and any gonadotrophin effect on the ovaries stimulating follicular development. A  radiologist experi- enced in taking measurements of ovaries and the uterus is recom- mended and these measurements should be compared to standard centile charts for prepubertal and pubertal uterus and ovaries. MRI brain (if not available then CT) is essential in both boys and girls with a diagnosis of central precocious puberty (Figs. 13.7.2.3 and 13.7.2.4). Tumour markers such as ɑ-​fetoprotein and β-​HCG should be measured. β-​HCG is a tumour marker that is produced by tes- ticular, pineal, and hepatic tumours. Due to molecular similarities, it mainly acts on LH receptors leading to markedly higher androgen levels than expected for the testicular volumes in boys. ɑ-​fetoprotein may also help diagnose extragonadal causes of gonadotropin-​ independent precocious puberty. Treatment The requirement for treatment needs careful discussion with the family as the psychosocial impact of precocious puberty on the child and family is considerable. The aims of treatment are to: 1. Arrest pubertal progression 2. Attenuate skeletal maturation with the aim of improving final height (only effective in children <6 years) 3. Improve mood swings and behavioural changes to reduce psy- chosocial impact Whether or not treatment is appropriate depends on a multitude of factors, mainly age of onset, pubertal status, and parental pref- erence. For progressive puberty in boys and girls, the mainstay of treatment is GnRH analogues. These are partial agonists of GnRH receptors causing continuous release of LH and FSH briefly. This is followed by a paradoxical downregulation and hence inhibition of gonadotropin secretion. Subcutaneous depot injections have dur- ation of action between 4 to 12 weeks. Clinical response to treat- ment includes stopping further breast development or testicular enlargement, reduced mood swings, and improved behaviour. Close monitoring of response is needed as adjustment of dose or frequency of injections may be necessary if breakthrough occurs. Treatment usually continues until an appropriate time for pu- berty to commence. GnRH analogue treatment is safe and effective. Children with precocious puberty are taller than expected at pres- entation but with the advanced bone age may not achieve an adult height in the predicted range for the family. GnRH analogues slowly reduce epiphyseal fusion rate so will protect future height potential but only in children presenting under 6 years. Growth potential is therefore maximal for children with precocious puberty presenting at a younger age. Sex steroid antagonists like cyproterone acetate can be useful ad- junct therapies especially for the stimulatory effect of GnRH ana- logue treatment. Management of GIPP requires identification and treatment of the cause. Delayed puberty Delayed puberty is generally regarded as the absence of signs of sec- ondary sexual development in a girl by the age of 13 years and a boy by 14 years. Of note, failure of progression through puberty or pri- mary failure of menstruation by 16 years also warrants investigation. Pubertal delay is relatively common, occurring in approxi- mately 3% of the population and is much more frequent in boys (male:female ratio 7:1). It is relatively rare in girls and is more likely to be secondary to underlying pathology. Delayed puberty is most Pituitary stalk Microadenoma within pituitary gland Fig. 13.7.2.3  MRI brain, T1-​weighted sagittal image of an 18-​month-​old with precocious puberty found to have a pituitary microadenoma. Fig. 13.7.2.4  MRI abdomen. Coronal section showing a large right adrenal tumour in a 2-​year-​old girl presenting with peripheral precocious puberty.

13.7.2  Normal puberty and its disorders 2433 often due to constitutional delay in growth and puberty. Children present with short stature and no secondary sexual characteristics. However, some features arising from adrenal androgens may be pre- sent (adrenarche). It is important to assess body proportion as well as height as this may indicate a genetic cause for the delayed puberty (e.g. Turners syndrome or Klinefelter syndrome). An associated micropenis may suggest hypopituitarism while an absent/​dimin- ished sense of smell warrants investigation for Kallmann syndrome. Causes of pubertal delay The causes of puberty delay can be divided into three main categories (see Table 13.7.2.2): 1. Those with an intact hypothalamic-​pituitary-​gonadal axis, but a functional problem 2. Hypogonadotropic hypogonadism 3. Hypergonadotropic hypogonadism Functional hypogonadotropic hypogonadism In this instance the hypothalamic-​pituitary-​gonadal axis is intact but remains inactive past the usual time for the initiation of puberty. The most frequent cause is constitutional delay of growth and pu- berty, representing 60% of delayed puberty in boys and 30% in girls. However, this is a diagnosis of exclusion and needs monitoring over time. There is often a history of short stature noted in later child- hood, associated with a bone age delay of around 2 years. A positive family history of pubertal delay, which is not sex specific, is found in 50–​75%. FSH, LH, and sex steroid levels are low. Progression through puberty is slowed. Not all patients reach a final height within parental range, but the majority do. Any underlying chronic disease can lead to delay in activation of the hypothalamic-​pituitary-​gonadal axis and so lead to delayed puberty as can psychosocial deprivation and intensive exercise. Anorexia and malnutrition also commonly lead to pubertal delay and this is thought to be a secondary adaptation to prevent repro- duction in a less than ideal circumstance. However, simple delay is sometimes difficult to distinguish from idiopathic hypogonadotropic hypogonadism (also known as iso- lated hypogonadotropic hypogonadism). Follow-​up and possible testing may determine if puberty is going to spontaneously occur. Hypogonadotropic hypogonadism In this instance there is an inability to produce gonadotrophins from the pituitary (LH & FSH). Congenital, or isolated, hypogonadotropic hypogonadism can be difficult to differentiate from constitutional delay initially. Both have low levels of sex steroids and gonadotrophins and often a positive family history of pubertal delay. There is no reliable single test to dif- ferentiate between the two conditions; hence follow-​up is of utmost importance. The gonads are normal, but as they are not stimulated they remain prepubertal in size. It is 3 to 5 times less common in girls. In boys, there may be a history of micropenis or undescended testes at birth, due to prenatal gonadotrophin and androgen defi- ciency. Associated with lack of smell (anosmia) in approximately 60% of patients, it is defined as Kallmann syndrome. In this condi- tion, during embryonic development, there is a failure of migration of GnRH neurones from the olfactory placode to the brain and the olfactory bulbs, hence the association with anosmia. It is also as- sociated with cleft lip/​palate, sensorineural deafness, and cerebellar ataxia. The KAL1 gene is implicated in the X-​linked form, but in 60–​70% of cases, the gene is unknown. It is also inherited in auto- somal dominant and recessive forms, giving significant variation in the features present. Prevalence is 1:10, 000 births with a male: fe- male ratio of 5:1. Other causes of congenital hypothalamic–​pituitary axis dysfunc- tion include Prader–​Willi and Bardet–​Biedl syndromes, both of which can present as delayed or absent puberty. Hypogonadotropic hypogonadism may be acquired as part of multiple pituitary hor- mone deficiencies for pituitary adenoma, for example. The causes of hypogonadotropic hypogonadism are listed in Table 13.7.2.2. There may be either a relevant past medical history or the presence of other pituitary deficiencies. Often these children are already being moni- tored for their growth and pubertal delay is diagnosed promptly. Hypergonadotropic hypogonadism In this case the hypothalamic-​pituitary part of the axis is intact but levels of LH and FSH are high indicating a lack of negative feedback to the hypothalamus. In hypergonadotropic hypogonadism testos- terone or oestrogen levels are low, indicating testicular or ovarian failure. Klinefelter syndrome (47XXY/​mosaicism) is the commonest sex chromosome disorder, but diagnosis is delayed in around 60% of cases, as there is significant phenotypic variation with many individ- uals having only subtle features. Prepuberty, boys have small testes, increased incidence of developmental delay and behavioural prob- lems. The onset of puberty is often not delayed, as there is enough testosterone to initiate puberty; however, the testes remain small and firm. They often present with slow pubertal progression rather than delayed puberty. Testosterone levels rise, but cannot be main- tained, and subsequently oestrogen levels increase. Typically, boys are tall with gynaecomastia and echinoid features. As adults, they Table 13.7.2.2  Causes of delayed or absent puberty Hypogonadotropic hypogonadism Functional Constitutional delay of growth and puberty Chronic disease Anorexia nervosa Hypothyroidism Hyperprolactinaemia Intensive exercise Noonan’s/​Down’s syndromes Permanent Kallmann’s syndrome and variants Isolated gonadotropin deficiency Congenital multiple pituitary hormone deficiency: Unknown aetiology Pituitary transcription factor disorders, e.g. PROP1, LHX1, HESX1 CNS tumours: Craniopharyngioma Hypothalamic tumours Pituitary adenoma CNS: Irradiation (high dose) Infiltration Histiocytosis Traumatic brain injury Post CNS infection Syndromes: Prader–​Willi Bardet–​Biedl CHARGE CHARGE - Coloboma, Heart disease, choAnal atresia, Retarded Growth and Ear abnormalities (due to mutations in CHD7 gene).

section 13  Endocrine disorders 2434 have reduced facial and body hair and are infertile. In comparison to the general population, the risks of other medical complications are increased including diabetes mellitus, osteoporosis, breast cancer, and thromboembolic events. Turner syndrome (45 XO, mosaicism occurs) is the commonest cause of gonadal dysgenesis in girls. Diagnosis can occur at any age, including with pubertal delay or primary or secondary amen- orrhoea. Turner syndrome therefore must be considered in any girl with delayed puberty even if they do not show any phenotyp- ical features of the condition. Up to 20% show spontaneous onset of puberty and spontaneous menstruation may occur, typically in mosaicism. Short stature is present due to insufficiency of the SHOX gene. Streak ovaries may be seen on ultrasound scan. Other causes of hypergonadotropic hypogonadism are listed in Table 13.7.2.3. Many will have a relevant history (e.g. testicular/​ab- dominal radiotherapy or total body irradiation). Arrested puberty If puberty has commenced but then regresses or stops, a dysfunc- tion of the pituitary-​gonadal axis is suspected. If a cause is not ob- vious (severe malnutrition, steroid treatment) urgent investigation is needed. Low levels of FSH and LH relative to pubertal status suggests failure in the hypothalamic-​pituitary axis and intracranial pathology needs to be excluded. Prolactinomas may primarily pre- sent with pubertal arrest. Autoimmune hypothyroidism should al- ways be excluded. PCOS is increasingly common with associated obesity and meta- bolic syndrome. This mainly presents with amenorrhoea or irregular periods. Due to high insulin levels related to insulin resistance, sex hormone binding globulin levels are low and leads to raised free testosterone. This in turn causes hirsutism and amenorrhoea or oligomenorrhoea. Investigation In cases of delayed puberty, constitutional delay in males is the most common diagnosis. However, as it is a diagnosis of exclusion other causes need to be considered. A comprehensive history and exam- ination are paramount. Most patients may not need a significant number of investigations, and the emphasis must be placed on serial monitoring of growth and pubertal status at 6 monthly intervals. An initial bone age assessment is performed. Baseline bloods are dictated by the clinical assessment. Concerning features would include evi- dence of midline abnormalities, dysmorphic features, learning diffi- culties, tall stature, neonatal history of bilateral crypto-​orchidism, or small penis, gynaecomastia if prepubertal, and anosmia. Tests may include a GnRH test, sex steroid levels, other pituitary function tests, and system-​related investigations. If appropriate, an MRI brain would exclude a space occupying lesion such as a craniopharyngioma or pituitary adenoma (Fig. 13.7.2.5). All girls with delayed puberty should have karyotype testing and a pelvic ultrasound. It is difficult to differentiate between constitutional delay of pu- berty and idiopathic hypogonadotropic hypogonadism even if a GnRH test is conducted. Prepubertal levels of LH and FSH are seen in both cases, although sometimes much lower levels in idiopathic hypogonadotropic hypogonadism. The psychosocial impact of pu- bertal delay, especially for males can be significant so either way, a trial of sex steroids may be beneficial and could aid diagnosis. Low dose testosterone (50 mg, IM every 4 weeks) in males and oestradiol (2–​5 µg, daily) in females can be given for 4–​6 months. In constitu- tional delay of growth and puberty, this course is usually sufficient Table 13.7.2.3  Hypergonadotropic hypogonadism Genetic abnormalities Turner’s syndrome and variants Klinefelter’s syndrome Androgen insensitivity syndrome Gonadal dysgenesis Gonadal failure Autoimmune Metabolic (e.g. galactosaemia) Pelvic/​spinal irradiation Chemotherapy Anorchia/​vanishing testes Cryptorchidism Torsion Post infection (e.g. mumps) Idiopathic Disorders of steroid synthesis
and action Inactivating gonadotropin mutations Adrenal steroid enzyme defects Fig. 13.7.2.5  MRI brain; left: axial view and right: sagittal view. This is a 15-​year-​old with pubertal delay, found to have a large craniopharyngioma with mass effect.

13.7.3 Normal and abnormal sexual differentiation

13.7.3 Normal and abnormal sexual differentiation 2435

13.7.3  Normal and abnormal sexual differentiation 2435 to trigger puberty and ensure its continuation after treatment is stopped. Permanent hypogonadism however needs continuation of treatment to ensure pubertal progression. In hypergonadotropic hypogonadism, baseline and GnRH tests show high levels of LH and FSH (FSH >LH) with low sex steroid levels due to gonadal failure. Here, chromosomal disorders need to be considered and a karyotype would be important. Treatment In constitutional delay, treatment is not necessary and often reassur- ance, understanding, and regular review is sufficient. However, due to the distress for boys in particular, treatment may be initiated to induce puberty although if stated at too young an age may affect final height achieved Treatment for constitutional delay is in the form of low dose sex steroids as detailed earlier. The aim of this is to stimulate puberty by activating the pituitary to release gonadotropins and spontaneously continue pubertal progression. Oxandrolone (1.25–​2.5 mg daily) is a mild anabolic steroid and another option. It may augment height but has no effect on puberty. However, as Oxandrolone is not aroma- tized, unlike testosterone it does not produce gynaecomastia. For those with permanent hypogonadotropic hypogonadism, treatment needs to continue into adult life. The main aim is start at a low dose with slow increments over a 2–​3-​year period. In males, this entails starting testosterone esters, IM at 50 mg every 4 weeks. This is slowly increased over 2–​3 years to an adult dose of 250 mg every 3–​4 weeks. Other preparations of testosterone such as transdermal gels or longer acting IM testosterone are available and can be substituted as adult replacement. Treatment in females follows a similar approach. For prolonged treatment, transdermal patches are recommended to induce secondary sexual changes. Initially 25 μg 17 β-oestradiol ma- trix patch ¼ patch for 3 days per week building up over 2–3 years depending on response and age at initiation to adult replacement. Low dose oral ethinyloestradiol (2 μg/day) is a potential alternative. Progestogens should be introduced only after a suitable duration of unopposed oestrogen (usually 2–3 years) or if more than one episode of significant breakthrough bleeding occurs. For Turner syndrome and other congenital causes of short stature, there was a tendency to delay pubertal induction in an attempt to allow optimal growth, but recent evidence has demonstrated that growth hormone plus Oxandrolone results in maximal final adult height and therefore puberty can be induced at an appropriate age. Gonadal stimulants may be appropriate for young males with hypogonadotropic hypogonadism to induce testicular growth. This is in the form of HCG injections twice a week but has variable re- sults. Normal testicular function is required to commence HCG treatment. FURTHER READING Alikasifoglu A, et al. (2015). Changing etiological trends in male pre- cocious puberty: evaluation of 100 cases with central precocious puberty over the last decade. Horm Res Paediatr, 83, 340–​4. Heger S, et al. (2006). Kisspeptin-​GPR54 signaling in the neuroendo- crine reproductive axis. Mol Cell Endocrinol, 25, 91–​6. Howard SR (2018). Genes underlying delayed puberty. Mol Cell Endocrinol, 476, 119–28. Hughes IA, Kumanan M (2006). Long-​term GnRH agonist treatment for female central precocious puberty does not impair reproductive function. Mol Cell Endocrinol, 25, 217–​20. Nathan BM, Palmert MR (2005). Do all girls with apparent idio- pathic precocious puberty require gonadotropin-​releasing hormone agonist treatment? J Pediatr, 137, 819–​25. Wales J (2012). Disordered pubertal development. Arch Dis Child Educ Prac Ed, 97, 9–​16. Wei C, et al. (2015). Recent advances in the understanding and man- agement of delayed puberty. Arch Dis Child, 101, 481–​8. Wei C, et al. (2017). The investigation of children and adolescents with abnormalities of pubertal timing. Ann Clin Biochem, 54, 20–32. Wolfenden H, Ryan F (2014). Delayed puberty. Paediatric and Child Health, 24, 124–​30. 13.7.3  Normal and abnormal sexual differentiation S. Faisal Ahmed and Angela K. Lucas-​Herald ESSENTIALS Human sex development follows an orderly sequence of embryo- logical events coordinated by a cascade of gene expression and hor- mone production in a time-​ and concentration-​dependent manner. Underpinning the entire process of fetal sex development is the simple mantra: sex chromosomes (XX or XY) dictate the gonadotype (ovary or testis), which then dictates the somatotype (female or male phenotype). The constitutive sex in fetal development is female. The develop- ment of the gonad into a testis or an ovary and the development of the internal reproductive organs and external genitalia is due to (1) critical genes involved in gonadal development, such as SRY (sex chromosome related gene on the Y chromosome), which is first ex- pressed in the XY gonad at 6 to 7 weeks of gestation and leads to the development of the testis; and (2) production by the testis of hor- mones such as anti-​Müllerian hormone—​to repress Müllerian ducts forming the uterus and fallopian tubes, and androgens (testosterone and dihydrotestosterone) that stabilize the Wölffian ducts and pro- mote the masculinization of the external genitalia. An understanding of these basic principles is essential to formulate a logical approach to the diagnosis of disorders of sex development. Disorders of sex development (DSD) can be classified into three broad categories based on the knowledge of the karyotype: (1) sex chromosome abnormality (e.g. X/​XY, mixed gonadal dysgenesis); (2) XX DSD (e.g. congenital adrenal hyperplasia); (3) XY DSD (e.g. partial androgen insensitivity syndrome). Clinical features—​the commonest presentations of DSD are (1) atypical genitalia of the newborn; and (2) abnormalities of pu- bertal development in the adolescent. Investigation and management—​an extensive repertoire is avail- able, but the choice of genetic, endocrine, and imaging tests should

section 13  Endocrine disorders 2436 be based on the DSD classification and aimed at reaching a con- sensus about the choice of sex assignment. Any surgical procedure required to alter the external phenotype consonant with sex assign- ment need not be undertaken urgently. It is essential that families of children with DSD have the benefit of support and counselling by a multidisciplinary team that comprises at a minimum an endocrin- ologist, urologist/​gynaecologist, geneticist, and psychologist. Normal fetal sex development The following key facts underpin the mechanism of dimorphic sex development: • The constitutive sex is female. • Typical male development requires the presence of a Y chromo- some, a testis, and the production and action of testosterone during a critical time in early gestation. • Fetal experiments in mammals indicate that early castration leads to a female phenotype, despite the presence of the Y chromosome (Jost’s hypothesis). • Absence of an ovary does not affect the female phenotype at birth (e.g. Turner syndrome). • Oestrogen is not required for fetal female development, but an- drogen is essential for fetal male development. The link between sex chromosomes, gonad determination and the expression of the phenotype (somatotype) is illustrated in simple configuration in Fig. 13.7.3.1. The events that occur during fetal male development are depicted in Fig. 13.7.3.2. The indifferent gonad develops in the urogenital ridge, where the kidney and ad- renals also have their origins. This is germane to the frequent as- sociation of urinary tract anomalies with atypical genitalia and the occasional occurrence of nests of adrenal remnants found in the testis of males with congenital adrenal hyperplasia (see Chapter 13.5.2). Germ cells migrate from the yolk sac to take their position within the developing gonad. The testis is histologically defined initially by its seminiferous tubules and predates com- parable maturation of the ovary by a few weeks. Three products of the somatic component of this testis are key to development of the male phenotype (i.e. sex differentiation). Anti-​Müllerian hor- mone (AMH), a product of Sertoli cells, acts on its type II receptor to repress Müllerian ducts forming the uterus and fallopian tubes. This process is permitted to occur in the female because of the ab- sence of AMH at this stage in gestation. Testosterone produced by Leydig cells under the control initially of placental human chori- onic gonadotrophin (hCG) acts locally in high concentration to sta- bilize the Wölffian ducts. These form the vas deferens, epididymis, and seminal vesicles. A further product of the Leydig cells, insulin-​ like factor 3 (INSL3), is required for the transabdominal phase of migration of the testis from the urogenital ridge to its site at birth within the scrotum. The inguinoscrotal phase of testis descent in late gestation is under the control of androgens. All these events take place in a specific chronological order and are controlled by genes and hormones expressed at critical dosage thresholds and timing. The genetic and hormonal control of the events in male sex development is shown in Fig. 13.7.3.3. Not all the genes charac- terized for mammalian development are shown, but those identi- fied in the human and relevant to disorders of sex development are emphasized. The formation of the urogenital ridge is dependent on factors such as WT1 and SF1, their roles vividly illustrated by mouse gene knockout studies (absence of gonads and kidneys or adrenals, respectively) and syndromes of urogenital anomalies (WAGR, Denys–​Drash, Frasier syndromes) and combined gonadal dysgenesis/​adrenal insufficiency in humans with inactivating mu- tations of WT1 and SF1 genes, respectively. The master regulator of testis development (sex determination) is SRY (sex chromosome related gene on the Y chromosome) which is first expressed in the XY gonad at 6 to 7 weeks of gestation, just before the indifferent gonad differentiates as a testis. SRY is a 204 amino acid protein functioning as a high mobility group (HMG) box transcription factor. The HMG box of 79 amino acids is related to similar proteins such as SOX (SRY-​like HMG box) which is also a key protein in the regulation of testis development. That SRY is essential for testis development is supported by the following ob- servations: (1) translocation of SRY to the X chromosome during paternal meiosis is present in 90% of XX males; (2) mutations in SRY in 15 to 20% of XY females with complete gonadal dysgenesis leads to complete sex reversal; (3) induction of a male phenotype occurs by transgenic insertion of Sry in XX mice. However, the observation that 10% of XX males lack SRY and the vast majority of XY gonadal dysgenic females have a normal SRY indicates that other genes must also be involved in testis determination. Candidates such as SOX9 and SF1 play some role, but their inactivation in humans leads to various syndromes of which gonadal dysgenesis is only one compo- nent. The role of clinicians recording detailed phenotypes in cases of disordered sex development is essential to continue the search for the multitude of genes that must be involved in testis determination. What is known in the human is summarized in Fig. 13.7.3.3. The ovary is devoid of known factors, although genes such as WNT4, RSPO1, and DAX1 may act in female development by suppressing testis-​determining genes. The differentiation of the internal genital ducts and external genitalia into male structures is entirely androgen dependent. Chromosomal sex (genotype) Phenotypic sex (somatotype) XY XX Gonadal sex Fig. 13.7.3.1  The basic components of fetal sex development.

13.7.3  Normal and abnormal sexual differentiation 2437 For this to occur, an intact pathway of gonadotrophin-​induced steroidogenesis is required to produce the potent androgens, testos- terone and dihydrotestosterone (DHT), which in turn promote an- drogen signalling by ligand activating the nuclear androgen receptor (AR) in target cells. The pathways of testicular steroidogenesis and the ligand-​activated AR signalling are shown in Figs. 13.7.3.4 and 13.7.3.5, respectively. The enzymatic steps in androgen produc- tion are encoded by genes, each of which, when mutated, results in syndromes of undermasculinization. Some of the more proximal enzyme defects also involve adrenal steroidogenesis and lead to syn- dromes that include adrenal insufficiency (see Chapter 13.5.1). Androgen signalling is mediated by a single AR encoded by an X-​linked gene at Xq11–​12. The AR is a member of a large family of nuclear receptors that comprise four general functional do- mains:  an N-​terminal transactivation domain, a central DNA-​ binding domain, a hinge region, and a C-​terminal domain to which the ligand binds. Subdomains are involved in dimerization, nuclear localization, and transcriptional regulation. Circulating andro- gens are bound to carrier proteins such as sex hormone binding globulin (SHBG) and albumin but diffuse freely into target cells where the ligand binds to cytoplasmic AR complexed to heat shock proteins. The hormone–​receptor complex translocates to the nu- cleus where it binds to DNA response elements as a homodimer. An added refinement in androgen signalling is provided by inter- action with several coactivator proteins to enhance upregulation of androgen-​responsive genes via the general transcriptional Mesoderm Sertoli cells Leydig cells Germ cell migration Testosterone Testis descent External genital growth Male external genital differentiation Mullerian duct regression Wolffian duct stabilization Gestation (weeks) 4 5 6 7 8 9 10 11 12 13 14 15 40 10 nmol/litre Fig. 13.7.3.2  The embryology of fetal sex development in the male. Mesoderm represents the tissue source for the somatic components of testis development. The solid line denotes the rise in fetal serum testosterone levels (nmol/​litre). Gonadal development Genital development Intermediate mesoderm Testis WT1, NR5A1 Bipotential gonad Ovary Leydig cells Sertoli cells Testosterone AMH Male internal genitalia Regression of Müllerian ducts Male external genitalia NR5A1, SRY FGF9, SOX9, DAX1 DHT WT1, NR5A1 AMH-R LHR Steroid synthesis: SRD5A2 HSD17B3 AR WNT4, RSPO1, SOX3 FOXL2 Ovary (mature) MAMLD1 Fig. 13.7.3.3  Genetic and hormonal control of fetal sex development. Emphasis is placed on genes relevant to human development.

section 13  Endocrine disorders 2438 machinery. Androgens have pleiotropic effects beyond fetal male development. These include the development of secondary sexual characteristics at puberty, muscle and skeletal growth, stimulation of sebaceous glands, and elongation and thickening of the vocal cords that give rise to the ‘voice breaking’ characteristic of the later stages of male puberty. Prostate development and growth is also androgen dependent, a feature that is countered by antiandrogenic forms of treatment for prostate cancer. It is also clear that androgens have effects on brain development, with prenatal influences being especially relevant to the sex dimorphism in gender role behaviour. Much of the evidence for the role of androgens in psychosocial functioning has come from studies of females exposed to excess androgens (e.g. congenital adrenal hyperplasia) and syndromes associated with defects in androgen signalling (e.g. complete an- drogen insensitivity syndrome). Definitions, terminology, and nomenclature Clarity of thought is required when faced with a newborn infant whose external genitalia are so unusual in appearance that sex as- signment is not instantaneously possible. The problem does not need compounding by the use of unclear, ambiguous terms such as ‘true hermaphroditism’ and ‘pseudohermaphroditism’. The following LH/hCG Dihydrotestosterone Oestradiol 17–20 lyase 17βHSD Androstenedione 17α-hydroxylase CYP17 Pregnenolone 17-OH-Pregnenolone Dehydroepiandrosterone Androstenediol Testosterone HSD17B3 17-OH-Progesterone Progesterone CYP17 CYP17 CYP17 17α-hydroxylase 17–20 lyase 17βHSD HSD17B3 SRD5A2 5αRD CYP19 P450arom LHR P450scc Cholesterol StAR CYP11A1 HSD3B2/ 3βHSD Fig. 13.7.3.4  Pathway of androgen biosynthesis, including aromatase conversion to oestrogen. 3βHSD, 3β-​hydroxysteroid dehydrogenase; 5αRD, 5α-​reductase; 17βHSD, 17β-​hydroxysteroid dehydrogenase; P450arom, P450 aromatase. The cognate genes are depicted in italics.

13.7.3  Normal and abnormal sexual differentiation 2439 definitions are relevant to the understanding of normal sex develop- ment, both somatic and psychosexual: • development of the gonads—​transformation of the indifferent gonad into a testis or an ovary, also referred to as sex determination • development of the genitalia—​development of the phenotype as an expression of hormones produced by the gonad • sex assignment—​allocation of male or female at birth, usually instantaneous • gender identity—​the sense of self as being male or female • gender role—​sex-​typical behaviours and preferences in which males and females differ (e.g. toy preferences, aggression) • sexual orientation—​refers to the target of sexual arousal • gender attribution—​assigning as male or female on first encounter with an individual ‘Gender dysphoria’ is a condition characterized by a mismatch be- tween the body habitus (phenotype as male or female) and gender identity as perceived by the affected individual (‘I feel like a woman trapped in a man’s body’). The desire to ‘convert’ from male to fe- male, or vice versa, is a state of transsexualism. In most cases, there is no clear evidence of a genetic or endocrine explanation for gender dysphoria. However, rarely, gender dysphoria may be encountered in older individuals with a disorder of sex development such as an androgen biosynthetic defect. The term ‘intersex’ has traditionally been applied to the clinical scenario of an infant born with ambiguous genitalia and in whom the sex is indeterminate. That allocation has often strayed beyond this defined scenario to include conditions such as severe hypo- spadias, milder forms of congenital adrenal hyperplasia, and the complete androgen insensitivity syndrome, where intersex is an in- appropriate term. In response, a consensus statement produced by a faculty of experts in genetics, endocrinology, surgery, and psych- ology redefined the terminology in 2006. While the new phrase ‘disorders of sex development’ is nowadays used commonly, many members of the affected community feel that the term ‘disorders’ may lead to unnecessary pathologizing of their condition. Disorders of sex development (DSD) classification The DSD nomenclature is simple, reflecting procedures followed during initial investigation and is flexible enough to be adapted as new conditions are recognized and defined. Subtending the entire process is knowledge of the karyotype, a starting point which is now routine for the investigation of DSD. Since fertilization of the ovum with an X-​ or Y-​bearing spermatozoon and sex chromosome aneu- ploidy or mosaicism are so fundamental to normal fetal sex devel- opment, it is logical to consider the causes of DSD in three broad categories. Table 13.7.3.1 contains a fairly comprehensive list of the causes of DSD; Table 13.7.3.2 focuses on the causes of ambiguous genitalia from a functional standpoint. Sex chromosome DSD It can be argued that Klinefelter’s and Turner’s syndromes are not ex- amples of DSD in the context of abnormalities of the external geni- talia. Klinefelter syndrome (47,XXY) has a genital component that typically comprises small, soft testes with oligo-​ or azoospermia, and infertility. The syndrome affects 1 in 500 to 1 in 1000 live births. The problem of infertility is not absolute now that advances in artificial reproductive technologies have enabled some men with Klinefelter’s syndrome to father children through testicular sperm extraction combined with intracytoplasmic sperm injection. Small testes are al- ready evident in childhood and the penis may be small. Hypospadias may also occur and genitalia can be sufficiently undermasculinized to lead to ambiguity and even a sex-​reversed phenotype. Klinefelter variants such as 48,XXXY, and 49,XXXXY may have associated genital anomalies. Turner’s syndrome without evidence of Y chromosomal material is seldom associated with external genital abnormalities. The DSD classification has included this syndrome since it is a congenital disorder characterized by gonadal (ovarian) dysgenesis and a sex chromosome aneuploidy. Turner’s syndrome has an incidence of 1 in 2500. The classic form is associated with a 45,X karyotype, which accounts for more than 50% of cases. Genital anomalies are more evidenced in mosaic forms of Turner’s syndrome characterized by a 45,X/​46,XY karyotype. The external genitalia can range in ap- pearance from normal female or mild clitoromegaly, through overt ambiguous genitalia to a male spectrum of simple hypospadias or normal male genitalia. Indeed, most individuals with this karyotype appear to be normal males based on amniocentesis analyses that have revealed fetuses with a 45,X/​46,XY karyotype. The minority with ambiguous genitalia are detected at birth and can pose difficul- ties for assignment of gender. A multitude of factors need to be con- sidered, including the genital appearance and urogenital anatomy, Fig. 13.7.3.5  Schematic of androgen action in a target cell. ARA70, an androgen receptor specific coactivator; GTA, general transcriptional apparatus; HSP, heat shock protein; P, phosphorylation; SHBG, sex hormone binding globulin.

section 13  Endocrine disorders 2440 risk of gonadal tumour, fertility and reproductive options, gender identity, and psychosexual function. Those infants who are severely undermasculinized and have a uterine remnant are likely to be as- signed female and any dysgenetic gonad should be removed. Infants assigned male will require several hypospadia procedures and re- moval of any dysgenetic gonads. Long-​term outcome studies are not available. Furthermore, finding a 45,X/​46,XY karyotype during in- vestigations for male infertility or in men presenting with a tumour of the testis is rare. Ovotesticular DSD is a rare cause of abnormal genital develop- ment and is characterized by the presence of testicular and follicle-​ containing ovarian tissue. The external genitalia can be variable, comprising ambiguous genitalia or just severe hypospadias. The gonadal distribution may be a testis on one side and an ovary on the other, bilateral ovotestes, or, most frequently, an ovotestis on one side and a testis or ovary on the other. An ovary will be sited in its normal pelvic position whereas a testis or an ovotestis can be located anywhere along the migratory path to the scrotum, but usually in the inguinal region. The pattern of internal genital ducts can also be variable, but generally follows that of the ipsilateral gonad. A ru- dimentary uterus is often found adjacent to an ovary or ovotestis. The karyotype is 46,XX in most cases, but only in about 30% is the SRY gene X-​translocated. Familial cases are reported, with evidence for both autosomal recessive and sex-​limited autosomal dominant transmission. The syndrome of XX male is similar to Klinefelter’s syndrome where the external genitalia usually differentiate normally as male but the testes are small. Hypospadias may occur infrequently. The in- cidence is 1 in 20 000 male births and, unlike Klinefelter’s syndrome, height is below average for normal males. Infertility is invariable; around 90% of XX males are SRY positive. However, the coexistence of ovotesticular DSD and XX male within families and the reported occurrence of 46,XX SRY-​negative monozygotic twins with genital anomalies suggests that these two forms of DSD are manifestations of the same underlying disorder in gonad determination. Mutations in RSPO1, an ovarian-​specific determining gene, results in female-​ to-​male XX sex reversal and hence may explain the phenotype in some SRY-​negative XX males. The phenotype is also replicated in an Rspo1 (–​/​–​) XX mouse model. SOX3 is another SRY-​related HMG box-​containing gene which is postulated to have evolved from SRY. Table 13.7.3.2  Causes of atypical genitalia: a functional classification Type/​cause Illustrative examples Masculinized female (46,XX DSD) Fetal androgens CAH, placental aromatase deficiency Maternal androgens Ovarian and adrenal tumours Undermasculinized male (46,XYDSD) Abnormal testis determination Partial (XY) and mixed (XO/​XY) gonadal dysgenesis Androgen biosynthetic defects LH receptor inactivating mutations 17β-​OH-​steroid dehydrogenase deficiency 5α-​reductase deficiency Resistance to androgens Androgen insensitivity syndrome variants Ovotesticular DSD Presence of testicular and ovarian tissue Karyotypes XX,XY,XX/​XY Syndromal Denys–​Drash, Frasier’s Smith–​Lemli–​Opitz Table 13.7.3.1  Classification of disorders of sex development (DSD) Sex chromosome DSD 46,XY DSD 46,XX DSD A: 47,XXY (Klinefelter’s syndrome and variants) B: 45,X (Turner’s syndrome and variants) C: 45,X/​46,XY (mixed gonadal dysgenesis) D: 46,XX/​46,XY (chimerism) A: Disorders of gonadal (testicular) development

  1. Complete or partial gonadal dysgenesis (e.g. SRY, SOX9, SF1, WT1,
    DHH, and so on)
  2. Ovotesticular DSD
  3. Testis regression A: Disorders of gonadal (ovary) development
  4. Gonadal dysgenesis
  5. Ovotesticular DSD
  6. Testicular DSD (e.g. SRY+, dup SOX9, RSP01) B: Disorders in androgen synthesis or action
  7. Disorders of androgen synthesis LH receptor mutations Smith–​Lemli–​Opitz syndrome Steroidogenic acute regulatory protein mutations Cholesterol side chain cleavage (CYP11A1) 3β-​hydroxysteroid dehydrogenase 2 (HSD3B2) 17α-​hydroxylase/​17,20-​lyase (CYP17) P450 oxidoreductase (POR) 17β-​OH steroid dehydrogenase (HSD17B3) 5α-​reductase 2 (SRD5A2)
  8. Disorders of androgen action Androgen insensitivity syndrome Drugs and environmental modulators B: Androgen excess 1. Fetal 3β-​hydroxysteroid dehydrogenase 2 (HSD3B2) 21-​hydroxylase (CYP21A2) P450 oxidoreductase (POR) 11β-​hydroxylase (CYP11B1) Glucocorticoid receptor mutations
  9. Fetoplacental Aromatase (CYP19) deficiency Oxidoreductase (POR) deficiency
  10. Maternal Maternal virilizing tumours (e.g. luteomas) Androgenic drugs C: Other
  11. Syndromic associations of male genital development (e.g. cloacal anomalies, Robinow, Aarskog, hand–​foot–​genital, popliteal pterygium)
  12. Persistent Müllerian duct syndrome
  13. Vanishing testis syndrome
  14. Isolated hypospadias (CXorf6, MAMDL1)
  15. Congenital hypogonadotropic hypogonadism
  16. Cryptorchidism (INSL3, GREAT)
  17. Environmental influences C: Other
  18. Syndromic associations (e.g. cloacal anomalies)
  19. Müllerian agenesis/​hypoplasia (e.g. MURCS)
  20. Uterine abnormalities (e.g. MODY5)
  21. Vaginal atresias (e.g. McKusick–​Kaufman)
  22. Labial adhesions

13.7.3  Normal and abnormal sexual differentiation 2441 When overexpressed in XX mice, it induces testis development and male sex reversal. Furthermore, genomic rearrangements of the SOX3 regulatory region were identified in three XX males who were SRY negative. It is likely that the mechanism of testis determination in the absence of SRY is via ectopic gonadal expression of SOX3 and subsequent activation of the male pathway of development. SOX3 rearrangements are quite frequent in XX males and the observation goes some way to explain the paradox of XX male development in the absence of an SRY gene. 46,XX DSD In most instances, this broad category of DSD is characterized by a list of conditions where the effect of an abnormal excess of endogenous or exogenous androgens is superimposed on the constitutive female sex development. Thus, internal genital devel- opment is normal (ovaries, uterus, and fallopian tubes; absence of Wölffian ducts) whereas the external genitalia are virilized to a vari- able degree. The prime example of XX, DSD is congenital adrenal hyperplasia. Congenital adrenal hyperplasia (CAH) This autosomal recessive disorder of adrenal steroidogenesis is covered in detail in Chapter 13.5.2. CAH is the commonest cause of ambiguous genitalia of the newborn, and mutations affecting the CYP21A2 gene account for more than 90% of cases. The degree of external masculinization can be so extreme as to resemble a new- born male. However, routine newborn examination should reveal the absence of palpable testes, a sign that must prompt urgent inves- tigation. The diagnosis is straightforward and must be undertaken promptly in view of the potential life-​threatening consequences for the infant from glucocorticoid and mineralocorticoid deficiency. The much rarer enzyme deficiencies affecting early steps in adrenal steroidogenesis are also potentially life-​threatening and are de- scribed in Chapter 13.5.2. Other causes of endogenous fetal androgen excess The fetal adrenals are unique in containing a large fetal zone which involutes after birth. Its peculiar role in producing large amounts of androgens does not become established again until late childhood when the zona reticularis differentiates functionally and is mani- fest as adrenarche. Large quantities of dehydroepiandrosterone (DHA) and its sulphated moiety (DHAS) are produced greatly in excess of the principle adrenal glucocorticoid, cortisol, yet the pre- cise function of this steroid in both fetal and postnatal life remains a mystery. DHEAS is 16-​hydroxylated in the adrenals and liver before transfer to the placenta where the sulphate is cleaved by placental sulphatase. The substrates DHEA and 16-​OH-​DHEA are converted to more potent androgens such as androstenedione and testosterone and all three androgens are converted to oestrogens (oestrone, oes- tradiol, oestriol). This reaction is mediated by a single gene, CYP19, which encodes for the placental aromatase enzyme via a tissue-​ specific promoter. Measurement of maternal serum or urinary oestriol was previously used as a non​specific marker of placental dysfunction. However, the levels are specifically low in CAH, in pla- cental sulphatase deficiency (as the sulphate moiety is uncleaved to allow substrate for aromatization), and as a marker of suppression of fetal adrenal steroidogenesis during prenatal treatment for CAH. The effects of placental aromatase deficiency are profound on both the fetus and the mother. Exposing a female fetus to large amounts of adrenal-​derived androgens leads to virilization of the external genitalia as severe as in CAH. Salt wasting is not an asso- ciated feature. The mother is also virilized during pregnancy, with evidence of hirsutism, acne, and sometimes clitoromegaly. These signs resolve postnatally but can recur in subsequent pregnancies. Affected girls in later life can present with delayed puberty due to primary gonadal failure where there are cystic ovarian changes and hypergonadotropism. A male fetus exposed to excess adrenal androgens, as with CAH, is not affected at birth. However, males with aromatase deficiency are very tall in young adulthood be- cause of failure in oestrogen-​induced closure of the growth plate. The aromatase enzyme has a massive capacity to convert androgens to oestrogens as the mother escapes virilization if only 1 to 2% ac- tivity of mutant enzyme remains. The mechanism of virilization in a 46,XX infant with P450 oxidoreductase (POR) deficiency may also be partly related to a disturbance in aromatase deficiency as POR is also an electron donor for P450 aromatase. It has also been sug- gested that an alternative fetal pathway to DHT synthesis is adopted secondary to POR deficiency (see Chapter 13.5.2). Maternal androgen excess The fetus is protected against excess androgens because of a highly efficient placental aromatase enzyme that converts androgens to oestrogens. Women with CAH who become pregnant have relatively high serum androgen levels during pregnancy, yet virilization of a female fetus has not been observed. However, the enzyme can be- come overwhelmed by excess androgens originating from maternal adrenal or ovarian androgen-​secreting tumours. Ovarian luteomas can be recurrent in pregnancies and are most common in multip- arous women of Afro-​Caribbean descent. Other virilizing ovarian tumours include hyperreactio luteinalis, arrhenoblastoma, hilar cell tumour, and a Krukenberg tumour. Occasionally, polycystic ovarian syndrome may result in fetal virilization. Danazol, a synthetic de- rivative of 17β-​ethinyltestosterone with androgenic, antioestrogenic, and antiprogestogenic properties, readily crosses the placenta. It is used for several conditions as diverse as endometriosis, benign fibrocystic breast disease, for unexplained female infertility, and in hereditary angioedema. A female fetus can become virilized and the use of danazol is contraindicated in pregnancy. 46,XY DSD A lengthy list of causes is included in this category of DSD and the frequency is quite high if more common conditions such as hypo- spadias and cryptorchidism are included. Although the problem of XY DSD is more complex than XX DSD in terms of diagnosis and management, knowledge of the normal process of male fetal sex de- velopment allows the causes to be subdivided into (1) disorders of gonadal development or gonadal dysgenesis, (2) defects in androgen biosynthesis, and (3) resistance to androgens. Gonadal dysgenesis The pivotal role of SRY in human testis development is vividly illus- trated by the phenotype of XY sex reversal as a manifestation of an inactivating mutation of the SRY gene. There is complete gonadal dysgenesis with the histological appearance of streak gonads and no discernible testis development in the form of seminiferous tubules

section 13  Endocrine disorders 2442 or Leydig cells. Lack of Sertoli cells and hence no AMH production leads to a uterine remnant and the external genitalia are female as a result of no androgen production. Presentation does not usually occur until adolescence on account of delayed puberty and primary amenorrhoea. The condition when presenting in adulthood has often been referred to as Swyer’s associated with a gonadal tumour, typic- ally as a dysgerminoma. Other tumours include gonadoblastoma, teratoma, and embryonal carcinoma. The risk of gonadal tumours is in the range of 15 to 35%. Müllerian structures are preserved and the uterus increases in size when oestrogen replacement is started. Bone mineral density is reduced in the majority. Adult height in a United Kingdom series was 174 cm as compared with 164 cm in the normal female population. Successful pregnancies have been achieved fol- lowing egg donation. Mutations in the SRY gene are found in only 15 to 20% of cases of complete XY gonadal dysgenesis. The majority are located within the HMG box, a DNA-​binding domain which appears to function by modulating local chromatin structure in order to transcribe adjacent target genes. Most mutations are de novo, yet there is a curious subset of cases where the mutation is familial and found in the phenotypically normal and fertile father. Explanations for this paradox include expression of the gene to a sufficient threshold against a particular genetic background that is not present in the 46,XY daughter. Alternatively, paternal gonadal mosaicism may be an explanation for familial cases. Partial gonadal dysgenesis refers to evidence of some virilization in the form of clitoromegaly and partial labial fusion or a more male pattern of external genital devel- opment with severe hypospadias, bifid scrotum, and undescended gonads. This phenotype is common to many other causes of 46,XY DSD. Mutations in SRY are not identified in this form of gonadal dysgenesis but mutations in NR5A1 are increasingly being found in partial gonadal dysgenesis without adrenal insufficiency as well as in phenotypes as diverse as hypospadias, anorchia, and male infertility. Several syndromes are described in association with XY gonadal dysgenesis where the genital anomalies can constitute complete sex reversal or varying degrees of external genital ambiguity. Denys–​ Drash syndrome (OMIM 194080)  is characterized by gonadal dysgenesis, an early-​onset nephropathy due to diffuse mesangial sclerosis and a high risk of Wilms’ tumour. A related condition is Frasier’s syndrome (OMIM 136680)  which is also characterized by gonadal dysgenesis but of a greater severity (streak gonads), a later-​onset nephropathy due to focal segmental glomerulosclerosis, and a high risk of gonadal tumours. The two syndromes may rep- resent a continuum of phenotypes. Both have in common muta- tions in the Wilms’ tumour-​related gene, WT1, as the underlying cause. The gene encodes for a four-​zinc-​finger transcription factor expressed in the developing urogenital ridge, kidney, and gonads. A  macrodeletion affecting the region on chromosome 11p13 where WT1 resides causes the WAGR syndrome (Wilms’ tumour, aniridia, genitourinary abnormalities, and mental retardation; OMIM 194072). Denys–​Drash and Frasier’s syndromes are caused by heterozygous point mutations in WT1 that have a dominant-​ negative effect on the wild-​type protein. In the former syndrome, these affect the DNA-​binding zinc-​finger region of WT1. Most mu- tations causing Frasier’s syndrome involve the donor splice site of exon 9. The use of an alternative splice donor site for exon 9 re- sults in the addition of three amino acids—​lysine (K), threonine (T), and serine (S)—​between the third and fourth zinc fingers. The +KTS and –​KTS isoforms are thought to have differential effects on gonad and renal development and an imbalance in the ratio of these isoforms may be the explanation for the phenotype of Frasier’s syndrome. It has been proposed that all cases of XY DSD with ex- ternal genital anomalies should have routine urinalysis for protein- uria and renal ultrasound to exclude a Wilms’ tumour. Rarely, a WT1 mutation has been identified in a case of isolated hypospadias but this does not justify routine WT1 screening for this common genital anomaly. Another syndrome associated with gonadal dysgenesis and genital anomalies is caused by mutations in SOX9 which encodes for an SRY-​related HMG box protein of 509 amino acids. The protein is expressed in the developing testis shortly after SRY ex- pression. The protein is also expressed in cartilage; heterozygous mutations in SOX9 (chromosome 17q24–​25) cause campomelic dysplasia (long bone bowing, hypoplastic scapula and rib cage, de- formed pelvis, cleft palate, macrocephaly, cardiac and renal defects) as well as the associated genital anomalies. SOX9 mutations do not occur without the skeletal abnormalities. There are rare examples of gonadal dysgenesis occurring in association with mutations in desert hedgehog (DHH) and testis-​specific protein-​like-​1 (TSPYL1) genes and with chromosomal deletions at 9p24-​pter, 10q26-​pter, and Xq13.3. Chromosomal duplications at Xp21.2 (DAX1 candi- date gene implicated) and 1p35 (WNT4 and RSP01 candidate genes implicated). Another gene now found to be associated with XY sex reversal is CBX2, the human homologue of M33, which, when ablated in mice, leads to XY sex reversal. A loss-​of-​function CBX mutation in a girl whose prenatal karyotype was 46,XY was shown to be the cause of her sex reversal based on detailed functional studies. This gene appears to actively repress ovarian development in XY gonads. Remarkably, a uterus and histologically normal ovaries were present in this girl. A linkage analysis study successfully identified mutations in MAP3K1 in a familial form of XY gonadal dysgenesis as well as in some sporadic cases. The mutations cause alterations downstream in the MAP kinase signalling pathway, possibly by altering SOX9 or β-​catenin activities. Genes implicated in XY gonadal dysgenesis can cause other comorbidities, in particular in the cardiovascular system. GATA-​ binding protein 4 (GATA4) mutations result in a variable pheno- type ranging from 46,XY partial gonadal dysgenesis to a more severe phenotype with ambiguous genitalia and azoospermia. Systolic car- diac murmurs have been reported in the milder cases of atypical genitalia. XX carriers of these mutations may have congenital heart disease. Similarly, zinc finger protein multitype 2 (FOG2/​ZFPM2) mutations have been reported in cases with XY partial gonadal dysgenesis and atrial septal defects. Neurodevelopmental disorders are also associated with XY go- nadal dysgenesis. Aristaless-​related homeobox (ARX) mutations result in a varied phenotype of X-​linked developmental disorders associated with anomalies of the male genitalia. Arx-​/​-​ knockout mice have also been shown to lack fetal Leydig cell differentiation. Finally, 80% of individuals with α-​thalassaemia/​mental retardation syndrome X-​linked (ATRX) demonstrate gonadal abnormalities ranging from gonadal dysgenesis to hypospadias. Analysis of rare mutations in candidate gonad-​determining genes is chipping away at the current low frequency of determination of the molecular mechanism of XY gonadal dysgenesis.

13.7.3  Normal and abnormal sexual differentiation 2443 Defects in androgen biosynthesis The steps in androgen production from LH-​induced steroidogenesis via cholesterol through to testosterone and DHT are illustrated in Fig. 13.7.3.4. Many of the early steps in androgen biosynthesis are also essential for adrenal steroid biosynthesis and are described in Chapter  13.5.1. The same G-​protein-​coupled gonadotrophin re- ceptor (LHR) on Leydig cells binds both placental hCG and later, fetal pituitary luteinizing hormone (LH) for the initiation of an- drogen biosynthesis. Inactivating mutations in the LHR gene in 46,XY DSD lead to a variety of phenotypes that include complete sex reversal, ambiguous genitalia, severe hypospadias, or even just isolated micropenis. Why the phenotype should be so heteroge- neous is not entirely clear, although partial loss-​of-​function muta- tions that result in a milder phenotype such as micropenis tend to localize within the seventh transmembrane domain of the receptor. The biochemical profile comprises elevated LHRH-​stimulated LH and follicular stimulating hormone (FSH) levels and low androgen concentrations which do not respond to prolonged hCG stimula- tion. Leydig cells are absent or decreased in number on histological examination. Sertoli cells and seminiferous tubules are present but spermatogenic arrest attests to the importance of intracellular androgen concentrations in mediating the final stages of sperm- atogenesis. A  range of homozygous or compound heterozygous mutations of the LHR gene are reported in this syndrome of Leydig cell hypoplasia. Their pathogenicity can be confirmed in vitro by demonstrating impaired hCG stimulation of intracellular cAMP due to disturbances in hCG binding, receptor stability, or receptor trafficking. The extracellular N-​terminal ligand-​binding domain of LHR consists of nine leucine-​rich repeats flanked by cysteine-​rich regions. The C-​terminal cysteine-​rich region is referred to as a hinge region of the receptor within which amino acid residues Asp330 and Tyr331 are key components of LH/​hCG signalling. An XX female has been reported with an LHR-​inactivating mutation. The pheno- type comprised normal onset of puberty but associated primary amenorrhoea and elevated LH levels. Studies on a rare gene muta- tion in this context provides information to indicate that while the LHR is not required for oestrogen synthesis, it is necessary for the induction of ovulation and fertility, although some affected females may still have regular cycles. Two penultimate steps in androgen biosynthesis essential for normal male sex differentiation are shown in Fig. 13.7.3.4. Both conversion steps are characterized by the respective substrates being the subject of catalysis by different isoenzymes and not involving steroid biosynthesis in the adrenals. The forms of XY DSD resulting from deficiencies in the two enzymatic steps have in common a se- vere degree of undermasculinization at birth but profound viriliza- tion at puberty. Thus, if unrecognized at birth and the affected infant is assigned female, the clinical presentation occurs at puberty with distressing signs of clitoromegaly, hirsutism, and deepening of the voice in a pubertal girl. There are 14 known 17β-​hydroxysteroid dehydrogenase (17HSD) isoenzymes, of which 12 are present in humans. They belong to a family of oxidoreductases involved in the metabolism of steroids, prostaglandins, and retinols. Of most relevance to XY DSD is 17HSD type 3, which is predominantly expressed in the testis and converts androstenedione to testosterone. The reaction is reversible and util- izes nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. The cognate gene, HSD17B3, is located on chromosome 9q22. A spectrum of mutations in this gene generally results in com- plete XY sex reversal at birth and can be mistaken for complete an- drogen insensitivity syndrome. Presentation in infancy may be in the form of an inguinal hernia or labial swelling where investigation reveals the presence of a testis. If sex assigned female, gonadectomy must be undertaken before puberty to avoid a pubertal girl be- coming virilized. The mechanism of such profound androgenic ef- fects is postulated to be the result of extraglandular conversion to androgens utilizing other isoenzymes such as types 1, 2, and 5. Some 17HSDB3 mutations are associated with retention of 15 to 20% of normal 17βHSD3 activity that leads to sufficient virilization of the external genitalia at puberty for sex reassignment. The biochemical profile shows elevated androstenedione and decreased testosterone levels so that the ratio of testosterone to androstenedione is typic- ally 0.8 or less in this disorder. However, there are cases that are de- scribed with a higher ratio than this. Wölffian ducts are stabilized to form the vas deferens, epididymis, and seminal vesicle which is pre- sumably the result of high local concentrations of androstenedione. About 20 mutations in the 17HSDB3 gene are now reported, most being homozygous or compound heterozygous missense mutations. Females with 17βHSD type 3 deficiency are asymptomatic. Testosterone is converted irreversibly to dihydrotestosterone (DHT) by the 5α-​reductase type 2 enzyme which is expressed in the primordium of the prostate and external genitalia, but not in the Wölffian ducts until after their differentiation to male internal genital ducts. As with 17βHSD deficiency, the male internal genital ducts develop normally in 5α-​reductase deficiency. The phenotype is as- sociated with some external virilization so that presentation is more frequent at birth because of ambiguous genitalia or severe hypospa- dias. This cause of XY DSD became well characterized through de- tailed descriptions of a genetic isolate in the Dominican Republic where males were born with severely undermasculinized external genitalia but then virilized to varying degrees at puberty. The testes enlarge appropriately at this stage, but the prostate gland remains hypoplastic, indicative of the DHT-​dependent growth of this organ. Histology of the testes shows Leydig cell hyperplasia and decreased spermatogenesis due to maldescent of the testes. However, there are reports of male fertility either following artificial reproductive tech- niques or even spontaneously after hypospadias repairs had been completed. Gender role changes occur frequently in this condition. The biochemical profile is classically an elevated ratio of serum tes- tosterone to DHT of more than 25:1 after puberty (or following hCG stimulation in a prepubertal child) and a reduced ratio of urinary 5α-​ to 5β-​reduced C19 steroids. However, cases of 5α-​reductase de- ficiency without a marked abnormality in these ratios have been de- scribed. The 5α-​reductase enzyme is also utilized in the metabolism of glucocorticoids, so C21 5α/​5β steroids can usefully be analysed even when gonadectomy has already taken place. There are two iso- enzymes of 5α-​reductase, the type 2 enzyme being affected in this condition. SRD5A2 is located on chromosome 2p23 and encodes for a 254 amino acid protein. The type 1 enzyme is expressed in skin and may contribute to the virilization which takes place at puberty. More than 40 mutations have been detected in the SRD5A2 gene. The ma- jority are missense mutations, including the Gly183Ser substitution observed in the Dominican Republic population. A complete gene deletion is found in an affected New Guinea population.

section 13  Endocrine disorders 2444 Defects in androgen action Androgen resistance is defined as a failure in complete male sex differentiation despite the presence of a normal 46,XY karyotype in association with testes that produce age-​appropriate circulating concentrations of androgens. The androgen insensitivity syndromes (AIS) are subdivided into complete (CAIS) and partial (PAIS) forms as defined by complete XY sex reversal (female phenotype) and par- tial virilization of the external genitalia. The degree of virilization in the latter category can vary from mild, isolated clitoromegaly to normal male development with oligospermia. Total resistance to androgens leading to CAIS is the sine qua non of a hormone resistance syndrome and this condition typically pre- sents in adolescence with primary amenorrhoea. There is normal breast development as male-​typical androgen levels are aromatized to oestrogens, but there is absent or scanty pubic and axillary hair growth. The external genitalia are female, and a shortened vagina is blind-​ending. The upper part of the vagina, together with the uterus and fallopian tubes, are structures derived from the Müllerian duct, hence these are absent in CAIS as a result of normal AMH action by the testes. CAIS may also present in infancy because of the ap- pearance of inguinal herniae which, at surgical repair, are found to contain testes. Thus, a karyotype check in all female infants with an inguinal hernia is highly recommended. The increasing trend towards prenatal tests that reveal the karyotype is also a mode of presentation when the phenotype at birth is realized to be a mis- match with the prenatal genotype. A defect in any one of the steps in androgen signalling may underlie the pathophysiology of CAIS. The problem is generally located with the AR where numerous mu- tations have been identified that result in CAIS or PAIS. These are recorded on an international database (http://​androgendb.mcgill. ca/​). Mutations are distributed throughout the coding region of the AR gene and include deletions, insertions, premature stop codons, and splice site, as well as missense mutations. The majority are lo- cated within the ligand-​binding domain and codons such as Arg840 and Arg855 appear to be relative ‘hotspots’ for mutagenesis. AIS is an X-​linked disorder and approximately 30% of AR gene muta- tions are spontaneous. The same mutation may manifest as different phenotypes, between and within affected families and to the extent of different sex assignments. The reasons for phenotypic variability are unclear but may include somatic mosaicism and differences in the lengths of the two AR trinucleotide repeats in the N-​terminal domain, glutamine, and glycine. Hyperexpansion of the triplet re- peat (>50) underlies the pathogenesis of spinal and bulbar muscular atrophy (Kennedy’s disease). Males affected with this neurological disorder display signs of mild androgen insensitivity. Certain asso- ciations are also described for the glycine (GGN) repeat, either alone or in combination with variations in the CAG repeat. Gender assignment and sex of rearing in CAIS is female, as is later gender identity. There is a reported 5% risk of gonadal tumours. A precursor lesion to tumour development is intratubular germ-​cell neoplasia unclassified (ITGNU), also referred to as carcinoma in situ. This may subsequently lead to a gonadoblastoma, an occurrence which is rare before puberty. The timing of gonadectomy is vari- able but there is merit in delaying until young adulthood to enable spontaneous puberty to occur. There is no evidence that the slightly reduced bone mineral density in CAIS is ameliorated by this man- agement approach, suggesting that androgens have a direct role in normal bone architecture. Oestrogen replacement needs to be started at about 11 years of age when gonadectomy is performed early. Final height rests between the average height of adult males and females. Mutations are also distributed throughout the AR gene in PAIS, but are predominantly missense in nature. The partial androgenic effect can be verified by functional assays in vitro which demonstrate reporter gene responses close to the normal AR, but usually only after induction with very high concentrations of androgens. Such in- formation can be valuable for predicting outcome at puberty in PAIS patients assigned male. Establishing a precise diagnosis in PAIS can be difficult as so many other disorders can be associated with the typical phenotype of severe hypospadias, micropenis, bifid scrotum, and undescended testes. These include androgen biosynthetic de- fects, partial gonadal dysgenesis, and mixed gonadal dysgenesis. In many instances, no single genetic cause can be found for a PAIS-​like phenotype:  external genitalia as described and normal androgen production. There is a strong association with low birth weight for gestational age in PAIS cases that have no AR gene mutation. This suggests placental dysfunction being a common link between early fetal growth restriction and inadequate placental hCG-​induced early Leydig cell steroidogenesis. The infant with PAIS assigned male may require several surgical procedures to correct hypospa- dias, orchidopexy for undescended testes, and high supplemental androgen treatment to induce puberty. The risk of gonadal tu- mours is probably higher than in CAIS, but once in the scrotum, the testes can be monitored by self-​examination and periodic tes- ticular ultrasonography. Outcome data in adult males with PAIS are sparse but sexual function is reported to be impaired; fertility is rare. Those assigned female require genitoplasty procedures in infancy, gonadectomy before puberty, and oestrogen treatment to induce fe- male secondary characteristics. Other conditions within the XY DSD category Some disorders are associated with incomplete male development but do not raise any doubt that sex assignment at birth should be male. These include hypospadias, undescended testes, and the per- sistent Müllerian duct syndrome (PMDS). Isolated hypospadias has a birth prevalence of 3 to 4 per 1000 live births. Although the cause in most cases is unknown, it is possible that more comprehensive endocrine and genetic analysis of a wider panel of genes involved in testis development, androgen synthesis, and action will reveal more abnormalities. Familial cases occur with a 7% incidence of one or more additional family members being af- fected with hypospadias. There is an association with increased ma- ternal age, paternal subfertility, maternal vegetarian diet, maternal smoking, assisted reproductive techniques, exposure to pesticides, and twinning. The aforementioned low birth weight is also a further association, which is strong. Hypospadias is generally classified as mild to severe based on the site of the urethral meatus being distal, mid-​shaft, or proximal (severe). There is often an associated chordee in the severe form. Numerous surgical techniques are described to resite the urethral opening on to the glans penis and may require several procedures. The initial procedure is usually undertaken in infancy. Complications include fistulas, meatal stenosis, and ur- ethral strictures and may occur in about 25 of the cases and espe- cially in the more severe forms of hypospadias. Undescended testes or cryptorchidism is the commonest birth defect in boys, affecting 2 to 9% of male live births. Again, there is a strong association with low birth weight as well as disorders

13.7.3  Normal and abnormal sexual differentiation 2445 that affect pituitary–​gonadal function and androgen action. These observations emphasize the importance of androgens in mediating complete descent of the testes into the scrotum by their action during the inguinoscrotal phase of descent. Other associations include maternal smoking or use of nicotine substitutes, alcohol use, and gestational diabetes. There is an association with intra- uterine insemination, but not with other forms of artificial repro- ductive technology. Genetic factors also play a part, particularly for first-​degree relatives among brothers and maternal half-​brothers. Cryptorchidism can be unilateral or bilateral with the testis sited in the abdomen (non​palpable), inguinal canal, suprascrotal, or high scrotal (where it is not possible to manipulate the testis to the bottom of the scrotum). Undescended testis must be distinguished from a retractile testis which ascends in response to a pronounced cremasteric reflex but can be manipulated completely into the scrotum. A testis may be descended at birth but found to be un- descended at a later age. This has been termed the ‘ascending’ testis or an acquired form of cryptorchidism. Studies indicate that the phenomenon is more likely with a history of retractile testis, the processus vaginalis may be patent, and the testis is usually located in the inguinal region. Ascending testis accounts for nearly one-​ half of the cases of undescended testis and mostly explains why late orchidopexies occur around 7 years of age. It is recommended that orchidopexy for congenital cryptorchidism is undertaken between 6 to 12 months of age. Early surgery is associated with improved growth of the testis, less evidence of abnormal germ-​cell devel- opment and a lower risk of developing a seminoma in adulthood. Hormonal treatment has low efficacy. A  short 3-​week period of hCG stimulation that is performed as a means of assessing testes function is associated with a 25% chance of testes decent but pri- marily in those with inguinal testes. Despite evidence for the role of INSL3 and its receptor in testis descent, mutations in the genes that encode these proteins are found only in a minority of boys with cryptorchidism. The components of a quartet of male reproductive tract disorders—​ hypospadias, cryptorchidism, abnormal spermatogenesis, testis cancer—​are each interlinked, for which there is some epidemio- logical evidence to suggest an increase in frequency. Environmental factors have been proposed to explain the observation through the development of a testicular dysgenesis syndrome which has its origin in fetal life. Humans are exposed to more than 80 000 chemicals in the environment with any adverse effects assumed to be more pro- found on the developing fetus. Evidence that chemicals such as pes- ticides and phthalates can disrupt the androgen/​oestrogen balance critical for normal fetal sex development is present in wildlife and in animal experiments. It is more difficult to prove similar effects in humans. However, such chemicals labelled as endocrine disruptors are reported to be present in higher concentrations in cord blood, placentas, and breast milk samples of mothers having male offspring with hypospadias or cryptorchidism, compared with normal control offspring. Furthermore, the anogenital distance, which is a sensitive index of androgen action used in rodent reproductive studies, is re- duced in male infants of mothers who had higher prenatal exposure to phthalates. Bilateral anorchia, also referred to as the vanishing testis syn- drome, in an otherwise normal male infant indicates that testes were present and functioning normally in early gestation in order to programme normal male sex differentiation. It is hypothesized that interruption of the vascular supply to the testes must have oc- curred in later gestation (akin to bilateral torsion). This is supported by surgical findings which show a preserved vas deferens entering the internal inguinal ring at the end of which is only a nubbin of fi- brous tissue containing haemosiderin-​laden macrophages and dys- trophic calcification. The diagnosis is confirmed by demonstrating elevated LH and FSH concentrations, no testosterone response to hCG stimulation, and an undetectable serum AMH. Even with this endocrine scenario, surgeons generally still perform a laparoscopy to ensure that any gonadal remnant is removed to avoid the risk of malignancy. PMDS is a very rare phenomenon but given that it will be as- sociated with a phenotypic spectrum, its exact prevalence will be difficult to determine. It is associated with testis maldescent but in this instance normal testes are prevented from descending to the scrotum because of being attached to a fallopian tube. The uterus and tubes in this syndrome are retained from early fetal develop- ment because of the lack of AMH action. This can either be the result of a mutation in the AMH gene with low or undetectable serum AMH, or serum AMH concentrations may be normal but the protein is unable to bind to its receptor because of a mutation in the gene coding for the AMH type II receptor. A mutation is found in the majority of cases with equal distribution between the two causative mutant genes. The phenotypes are identical. The external genitalia are otherwise normal; both testes may be descended to one hemiscrotum. Such transverse testicular ectopia is diagnostic of PMDS. The diagnosis is usually made at orchidopexy or for an inguinal hernia repair where the sac is found to contain a uterus or a fallopian tube. Care must be taken to resite the testis to its normal position as such mobilization may damage the vas deferens. The uterus is often left in place. Aphallia is a condition with clear urological and psychological consequences which is reported to be as rare as 1 in 30 million births. It is believed to be as a result of a failure of development of the genital tubercle but is otherwise associated with normal testes function and virilization. Over 50% of cases have associated genitourinary malformations and no genetic abnormality has yet been identified. Mortality is reported to be higher in those cases with an associ- ated malformation and those where the urethral opening is in the rectum and proximal to the anal sphincter as opposed to distal to the sphincter. Communication The initial contact with the parents of a child with a DSD is im- portant as first impressions from these encounters often persist. A key point to emphasize is that the child with a DSD has the po- tential to become a well-​adjusted, functional member of society. The use of the phrase ‘differences or variations in sex development’ may be useful in introducing the concepts of the extent of variation in sex development. An analogy between a common condition such as variations in stature and associated functional disability may be easy to explain and understand, both for the parent as well as the health professional. Most differences in stature do not have any con- sequence but marked tall or short stature can affect function. In addition, in many cases, although the abnormality in stature itself may not be profound and may not have a functional consequence, it

section 13  Endocrine disorders 2446 may be a pointer towards coexisting health issues and, thus, requires thorough clinical evaluation. While it is likely DSD may be a more complex and challenging group of conditions, discussions that use the aforementioned approach as the first step may reduce the stigma that is often experienced by families. In those cases where there are no doubts about sex assignment, it should not be assumed that the parents’ need for information and psychological help are any less as the parents’ perception of their own child’s condition may be quite different from the clinician’s perception of the severity of illness. In those cases where there is true genital ambiguity, it should be ex- plained to the parents that the best course of action may not initially be clear, but the healthcare team will work with the family to reach the best possible set of decisions in the circumstances. The healthcare team should discuss with the parents the information to be shared in the early stages with family members and friends. In the case of an affected adolescent, the initial assessment should not only start the process of diagnosis but should also be used to develop a rapport with the patient. In adolescents with an existing DSD, conversation should always start with a review of the patient’s own understanding of their condition. The process of transferring to adult services is also an opportunity to review the diagnosis and consider more novel investigations that may not have been available earlier. Assessment of a DSD Atypical genitalia of the newborn and concerns about secondary sexual characteristics at puberty are the two key stages in life when a problem of DSD requires careful assessment based on clinical examination followed by a focused and logical investigation plan. It must also be recognized that while a definitive diagnosis may not be possible in some cases, this must not delay a decision on sex assign- ment unduly and lessen the importance for a management plan. Examination It is important that the affected person undergoes a full systematic examination. Approximately 25% of cases of DSD may have an asso- ciated malformation. Assessment of blood glucose and urinalysis for proteinuria should be routine. For the infant with atypical genitalia, the following details need to be recorded: the size of the phallus, presence of chordee, and whether the appearance is indicative of clitoromegaly or a micropenis; site of urethral opening; single or dual openings on the perineum; development of labioscrotal folds or a bifid scrotum; whether gonads are palpable and their site. Allied to the examination are salient points in the clinical history such as family history and exposure to potential reproductive tract terato- gens. Problems arising only at the time of puberty include signs of virilization occurring in a girl, delayed pubertal development, and primary amenorrhoea. Although scoring systems such as the Prader scoring system for XX DSD and modifications of this system for XY DSD may provide an integrated summary description of the geni- talia, these scoring systems are not sufficiently discriminate to por- tray the full spectrum of the variation encountered in the external genitalia. The external masculinization score (EMS), which indi- vidually scores external genitalia for scrotal fusion, microphallus, location of urethral meatus, and location of each gonad, may be a more discriminate and objective method of describing the external appearance. Investigations Infants with suspected DSD who require further clinical evalu- ation and need to be considered for investigation by a specialist should include those with isolated proximal hypospadias, isolated micropenis, isolated clitoromegaly, any form of familial hypospadias and those who have a combination of genital anomalies with an EMS of less than 11 (Box 13.7.3.1). This will avoid unnecessary detailed investigations of boys with isolated glandular or mid-​shaft hypo- spadias and boys with unilateral inguinal testis. In approximately 25% of affected cases, DSD is part of a complex condition and the coexistence of a systemic metabolic disorder, other malformations, or dysmorphic features, would lower the threshold for investiga- tion as would a family history of consanguinity, stillbirths, multiple miscarriages, fertility problems, genital abnormalities, hernias, de- layed puberty, genital surgery, unexplained deaths, and the need for steroid replacement. In addition, maternal health, and pregnancy history itself may hold key information. In those with ambiguous genitalia and/​or bilateral impalpable gonads, a first tier of investiga- tions should be undertaken to define the sex chromosomes and de- lineate, by pelvic ultrasound, the internal genitalia and exclude CAH (Box 13.7.3.2). This first tier should, therefore, also include plasma Box 13.7.3.1  Newborn problems that merit DSD investigation • Genitalia that are so unusual that sex assignment cannot be performed (i.e. ambiguous genitalia) • Apparent female genitalia with: — Enlarged clitoris — Posterior labial fusion — Inguinal/​labial mass • Apparent male genitalia with: — Non​palpable testes — Isolated perineoscrotal hypospadias — Severe hypospadias, undescended testes, micropenis • Genital anomalies associated with syndromes • Family history of DSD, such as CAIS • Discordance between genital appearance and prenatal karyotype Box 13.7.3.2  Investigating an affected infant • Genetics — FISH (X centromeric and SRY probes) — Karyotype (high resolution; abundant mitoses) — Save DNA with consent • Endocrine — 17-​OH progesterone, 11-​deoxycortisol (plus routine biochemistry; save serum), renin — ACTH, 24-​h urinary steroids (also check proteinuria) — Testosterone, androstenedione, DHT — LH, FSH, AMH, inhibin B — hCG stimulation test (define dose, timing) • Imaging — Pelvic, adrenal, renal ultrasound — MRI — Cystourethroscopy and sinogram • Surgical — Laparoscopy — Gonadal biopsies — Genital skin biopsy (extract DNA and RNA)

13.7.3  Normal and abnormal sexual differentiation 2447 glucose, serum 17OH-​progesterone (17OHP) and serum electro- lytes. Serum 17OHP is usually unreliable before the age of 36 hours, and in the salt-​losing form of CAH, serum electrolytes usually do not become abnormal before day 4 of life. The results of polymerase chain reaction or FISH analysis using Y-​ and X-​specific markers should be available within one to two working days, and labs should attempt to report 17OHP results in such a circumstance within two working days. In situations where the level of suspicion of CAH is very high and the infant needs immediate steroid replacement therapy, further serum samples should be collected and stored be- fore starting therapy. These should be of a sufficient volume to assess 17OHP, testosterone, androstenedione and, possibly, renin activity or concentration, in that order of priority. At least one spot or 24-​h urine sample (at least 5 ml) for a urine steroid profile should be col- lected before starting therapy. The results of these initial investiga- tions shall often dictate the second tier of investigations. In an infant with impalpable gonads, a karyotype of 46,XX, a significantly elevated serum 17OHP and the presence of a uterus makes congenital adrenal hyperplasia (CAH) due to 21-​hydroxylase deficiency very likely. A urine steroid profile can confirm this diag- nosis and can also identify other rare forms of CAH, which may also be associated with a raised 17OHP in the newborn such as 11β-​ hydroxylase deficiency and 3β-​hydroxysteroid dehydrogenase defi- ciency. For the XY or X/​XY infant with DSD, AMH and testosterone measurement will provide information about the presence of func- tioning testes. Depending on the age of the child, an hCG stimula- tion test may be required. Confirmation of a specific diagnosis will often require further biochemical identification of a defect in the an- drogen biosynthesis pathway and detailed genetic analysis. Imaging studies (ultrasonography and MRI) may locate the site of gonads but often laparoscopy is the only reliable method to identify gonads. This also provides the opportunity to obtain biopsies for histology, the only sure way to establish a diagnosis of ovotesticular DSD. Management Only the principles of DSD management can be described since each cause of DSD has specific requirements, some of which have been covered for CAH in Chapter 13.5.2. The greatest challenges for the clinician are to manage the newborn with ambiguous geni- talia and the pubertal child who develops physical signs incongruent with the sex of rearing. It is axiomatic that management should only be undertaken by a multidisciplinary team that comprises, at a min- imum, a paediatric endocrinologist, urologist, gynaecologist, a gen- eticist, and a clinical psychologist. There is consensus that all infants with DSD should have a gender assignment, but this may have to be delayed until the results of relevant investigations are available. Surgery required to make the genitalia concordant with gender as- signed may be deferred, even to an age where the child is of suf- ficient cognitive development to be involved with the discussions. Psychological support is required for the family from the outset, as misinformation given early can impact adversely in the longer term. As the child grows older, explanation of the diagnosis must be care- fully planned with the parents. In a girl with XY DSD (CAIS, for example), this will entail explanation concerning the nature of the gonads, the presence of a Y chromosome, absence of a uterus, lack of menses, and future infertility. Such explanation requires skilled counselling delivered as appropriate to the child’s development. Transitional care from adolescence to young adulthood is a further level of complexity that requires the recruitment of adult specialists relevant to whether sex assignment has remained male or female. Longer-​term studies in women with CAH are now being conducted and provide valuable information on surgical, endocrine, and psy- chosexual outcomes. In terms of XY DSD, outcome data are reason- ably robust for miscellaneous conditions such as cryptorchidism, hypospadias, mixed gonadal dysgenesis (XO/​XY), and CAIS. In contrast, data remain sparse in PAIS and some androgen biosyn- thetic defects, conditions where sex reassignment may arise in later childhood and adolescence. FURTHER READING Achermann JC, et al. (2015). Disorders of sex development: effects of molecular diagnostics. Nat Rev Endo crinol, 11, 478–​88. Ahmed SF, et al. (2013). Understanding the genetic aetiology in pa- tients with XY DSD. Br Med Bull, 106, 67–​89. Ahmed SF, Bryce J, Hiort O (2014). International networks for sup- porting research and clinical care in the field of disorders of sex de- velopment. In: Hiort O, Ahmed SF (eds) Understanding differences and disorders of sex development, 27, pp. 284–​92. Karger, Basel. Ahmed SF, et al. (2004). Prevalence of hypospadias and other genital anomalies among singleton births, 1988–​1997, in Scotland. Arch Dis Child, 89, F149–​51. Ahmed SF, Gardner M, Sandberg DE (2013). Management of children with disorders of sex development: new care standards explained. Psychology & Sexuality, 5, 5–​14. Ahmed SF, Iqbal A, Hughes IA (2000). The testosterone: andro- stenedione ratio in male undermasculinisation. Clin Endocrinol, 53, 697–​702. Ahmed SF, Khwaja O, Hughes IA (2000). The role of a clinical score in the assessment of ambiguous genitalia. BJU Int, 85, 120–​4. Ahmed SF, O’Toole S (2012). Management of boys and men with dis- orders of sex development. Curr Opin Endocrinol Diabetes Obes, 19, 190–​6. Ahmed SF, Rodie M (2010). Investigation and initial management of ambiguous genitalia. Best Pract Res Clin Endocrinol Metab, 24, 197–​218. Ahmed SF, et  al. (2000). Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab, 85, 658–​65. Ahmed SF, et al. (2011). UK guidance on the initial evaluation of an in- fant or an adolescent with a suspected disorder of sex development. Clin Endocrinol, 75, 12–​26. Ahmed SF, et al. (2015). Society for Endocrinology UK guidance on the initial evaluation of an infant or an adolescent with a suspected disorder of sex development (Revised 2015). Clin Endocrinol (Oxf), 84, 771–​88. Barseghyan H, Délot E, Vilain E (2015). New genomic technologies: an aid for diagnosis of disorders of sex development. Horm Metab Res, 47, 312–​20. Bashamboo A, McElreavey K (2015). Human sex-​determination and disorders of sex-​development (DSD). Semin Cell Dev Biol, 45, 77–​83. Baskin LS, Ebbers MB (2006). Hypospadias: anatomy, etiology, and technique. J Pediatr Surg, 41, 463–​72. Blackless M, et al. (2000). How sexually dimorphic are we? Review and synthesis. Am J Hum Biol, 12, 151–​66. Brain CE, et al. (2010). Holistic management of DSD. Best Pract Res Clin Endocrinol Metab, 24, 335–​54.

section 13  Endocrine disorders 2448 Cools M, et al. (2018). Caring for individuals with a difference of sex development (DSD): a consensus statement. Nat Rev Endocrinol, 14, 415–29. Cox K, et al. (2014). Novel associations in disorders of sex develop- ment: findings from the I-​DSD Registry. J Clin Endocrinol Metab, 99, e348–​55. di Clemente N, Belville C (2006). Anti-​Müllerian hormone receptor defect. Best Pract Res Clin Endocrinol Metab, 20, 599–​610. Duguid A, et al. (2007). The psychological impact of genital anomalies on the parents of affected children. Acta Paediatr, 96, 348–​52. Hughes IA (2010). The quiet revolution. Best Pract Res Clin Endocrinol Metab, 24, 159–​62. Hughes IA, Acerini C (2008). Factors controlling testis descent. Eur J Endocrinol, 159 Suppl 1, S75–​82. Hughes IA, et  al. (2006). Consensus statement on management of intersex disorders. Arch Dis Child, 91, 554–​63. Josso N, Rey RA, Picard J-​Y (2013). Anti-​müllerian hormone: a valu- able addition to the toolbox of the pediatric endocrinologist. Int J Endocrinol, 2013, Article ID 674105. Knarston I, Ayers K, Sinclair A (2016). Molecular mechanisms associ- ated with 46,XX disorders of sex development. Clin Sci, 130, 421–​32. Kolesinska Z, et al. (2014). Changes over time in sex assignment for disorders of sex development. Pediatrics, 134, e710–​15. Lee PA, et al. (2015). Global disorders of sex development update since 2005: perceptions, approach and care. Horm Res Paediatr, 85, 158–​80. Leon NY, Reyes AP, Harley VR (2019). A clinical algorithm to diagnose differences of sex development. Lancet Diabetes Endocrinol, 7, 560–74. Looijenga LH, et al. (2011). Dissecting the molecular pathways of (tes- ticular) germ cell tumour pathogenesis; from initiation to treatment-​ resistance. Int J Androl, 34(4 Pt 2), e234–​51. Main KM, et al. (2010). Genital anomalies in boys and the environ- ment. Best Pract Res Clin Endocrinol Metab, 24, 279–​89. Meyer-​Bahlburg HFL, et al. (2016). Gender assignment, reassignment and outcome in disorders of sex development: update of the 2005 consensus conference. Horm Res Paediatr, 85, 112–​18. Paterski V, et al. (2010). Consequences of the Chicago consensus on disorders of sex development (DSD): current practices in Europe. Arch Dis Child, 95, 618–​23. Rodie M, et al. (2011). Factors that influence the decision to perform a karyotype in suspected disorders of sex development: lessons from the Scottish Genital Anomaly Network Register. Sex Dev, 5, 103–​8. Schneuer FJ, et al. (2015). Prevalence, repairs and complications of hypospadias: an Australian population-​based study. Arch Dis Child, 100, 1038–​43. Sim H, et al. (2008). Boys, girls and shuttling of SRY and SOX9. Trends Endocrinol Metab, 19, 213–​22. Thankamony A, et  al. (2009). Anogenital distance from birth to 2 years: a population study. Environ Health Perspect, 117, 1786–​90. Verkauskas G, et al. (2007). The long-​term follow up of 33 cases of true hermaphroditism: a 40 year experience with conservative gonadal surgery. J Urol, 177, 726–​31. Vidal I, et al. (2010). Surgical options in disorders of sex development (DSD) with ambiguous genitalia. Best Pract Res Clin Endocrinol Metab, 24, 311–​24. Vorona E, et  al. (2007). Clinical, endocrinological, and epigenetic features of the 46,XX male syndrome, compared with 47,XXY Klinefelter patients. J Clin Endocrinol Metab, 92, 3458–​65. Wang MH, Baskin LS (2008). Endocrine disruptors, genital develop- ment and hypospadias. J Androl, 29, 499–​505.

13.8 Pancreatic endocrine disorders and multiple e

13.8 Pancreatic endocrine disorders and multiple endocrine neoplasia 2449

ESSENTIALS Pancreatic neuroendocrine tumours (islet-​cell tumours) are rare and usually sporadic, but they may be associated with complex familial endocrine cancer syndromes. Recognized types of pan- creatic neuroendocrine tumours are those that are non​functioning (often advanced at diagnosis and presenting with mass ef- fects due to the absence of symptoms attributable to hormone
hypersecretion), insulinoma (the most frequent type), and others including: Gastrinoma—​90% located in the pancreatic region; present with severe, multiple peptic ulcers that are often associated with compli- cations such as haemorrhage, perforation, and stricture formation (Zollinger–​Ellison syndrome); diagnosis requires demonstration of a raised fasting plasma gastrin concentration associated with increased basal gastric acid secretion; symptomatic treatment is with high-​dose proton pump inhibitors. VIPoma—​90% occur in the pancreas; present with large-​volume diarrhoea without steatorrhoea (Verner–​Morrison syndrome, pancreatic cholera); hypokalaemia may be profound; diagnosis can be confirmed by finding of an elevated plasma level of pep- tide histidine–​methionine (PHM, produced from the prepro-​VIP molecule); diarrhoea responds well to somatostatin analogues (octreotide, lanreotide) and parenteral hydrocortisone treatment. Glucagonoma—​rare α-​cell tumours of the pancreas; presenting features include weight loss, diarrhoea, anorexia, abdominal dis- comfort due to hepatomegaly from metastases, and diabetes, also necrolytic migratory erythema; diagnosis is made on the basis of an elevated fasting plasma glucagon in association with characteristic clinical features; skin rash and other symptoms may respond to som- atostatin analogues; topical zinc sulphate pastes and oral zinc sul- phate supplementation may be of benefit. Management—​the following should be considered in addition to the symptomatic treatments for pancreatic neuroendocrine tu- mours described: (1) surgical resection—​the only curative treatment, but not possible in many cases; (2) tyrosine kinase inhibitors which inhibit specific kinases involved in tumour cell proliferation, growth, and angiogenesis; (3)  mammalian Target of Rapamycin (mTOR) inhibitors; (4) peptide-​receptor radionuclide therapy (radiolabelled somatostatin analogues). Multiple endocrine neoplasia (MEN) There are two main MEN syndromes, which are rare hereditary con- ditions characterized by a predisposition to cancer development within two or more endocrine organs. MEN-​1—​typical features are parathyroid adenomas, pancreatic neuroendocrine tumours (gastrinomas > insulinomas > others) and pituitary adenomas; caused by mutation of the MEN1 gene, which encodes a nuclear protein (menin) that is presumed to be a tumour suppressor gene, with diagnosis confirmed by genetic analysis; fol- lowing identification of an index case, genetic analysis in first-​degree relatives allows identification of affected family members; minimal surveillance programme for individuals with MEN1 syndrome or a family-​specific mutation of the MEN1 gene should include annual measurement of serum prolactin (from age 5 years), fasting serum calcium and PTH (from age 8 years), and fasting serum gastrin con- centration (from age 20 years), and consideration of serial imaging of the pituitary and pancreas using MRI scanning at 1–​3 yearly intervals. MEN-​2—​there are three variants:  (1) MEN-​2A (Sipple’s syn- drome)—​medullary thyroid carcinoma, phaeochromocytoma, and parathyroid hyperplasia/​adenomas; (2) MEN-​2B—​medullary thyroid carcinoma and phaeochromocytoma, with other features including marfanoid habitus and mucosal neuromas; (3) familial medullary thy- roid carcinoma. These are caused by mutations in the RET oncogene, with diagnosis confirmed by genetic analysis and strong genotype–​ phenotype correlation. The codon location and type of mutation allows for risk-​assessment and informs the management of index cases and affected family members (e.g. prophylactic thyroidectomy is recommended for those possessing mutations conferring the highest risk of aggressive medullary thyroid carcinoma; annual measurement of urinary metanephrines for those with high risk of phaeochromocytoma). Other syndromes of MEN include (1)  Carney complex, (2)  McCune–​Albright syndrome, (3)  neurofibromatosis type 1, (4) von Hippel–​Lindau syndrome, (5) familial paraganglioma syn- dromes, including Carney–​Stratakis syndrome. 13.8 Pancreatic endocrine disorders
and multiple endocrine neoplasia B. Khoo, T.M. Tan, and S.R. Bloom

section 13  Endocrine disorders 2450 Pancreatic neuroendocrine tumours Pancreatic neuroendocrine tumours (islet-​cell tumours) are rare tu- mours representing 1 to 2% of all pancreatic neoplasms and have an incidence of approximately 1.8 to 2.6 per million per year. Although pancreatic neuroendocrine tumours may be associated with com- plex familial endocrine cancer syndromes such as multiple endo- crine neoplasia (MEN), the majority are non​familial (sporadic) cases. Pancreatic neuroendocrine tumours have a wide range of clinical manifestations. Between 15 and 30% are clinically silent (non​functioning) and usually present with mass effect or meta- static disease. However, even so-​called non​functioning pancreatic neuroendocrine tumours may secrete detectable amounts of tumour markers such as pancreatic polypeptide, chromogranin A, neuron-​ specific enolase, and human chorionic gonadotrophin subunits. Those pancreatic neuroendocrine tumours associated with a spe- cific endocrine hyperfunction syndrome are termed ‘functional’, with insulinomas and gastrinomas being the most common. Other types of neuroendocrine or ‘carcinoid’ tumours are described in Chapter 15.9.2. This section will consider the biochemical confirm- ation and localization of pancreatic neuroendocrine tumours, spe- cific clinical presentations of functional tumour types, management options, and discussion of the clinical features of MEN types 1 and 2 (MEN-​1 and MEN-​2). Introduction and definition The gastrointestinal tract is the largest endocrine organ in the body, which includes endocrine cells of the gut and pancreas. Current evi- dence suggests that these cells are derived from endodermal, om- nipotent stem cells. Since these enteroendocrine cells share many of the properties exhibited by neural cells, this has led to their de- scription as neuroendocrine cells. Criteria for defining neuroendo- crine cells include production of bioactive substances that provide transmitter functions, release of hormones via exocytosis from dense-​core secretory vesicles following an external stimulus, and an absence of axons or synapses. The histopathological hallmark of tumours arising from these cells, so-​called neuroendocrine tu- mours, is the expression of neuroendocrine markers, for example, chromogranins A, B, and C, synaptophysin, and neuron-​specific enolase. Pancreatic neuroendocrine tumours, the subject of this chapter, are now recognized to be a subtype of neuroendocrine tu- mour, which also include gastrointestinal neuroendocrine tumours (so-​called ‘carcinoid’ tumours), bronchial neuroendocrine tumours, and medullary thyroid carcinoma. Aetiology and genetics Pancreatic neuroendocrine tumours are associated with com- plex familial endocrine neoplasia syndromes, including MEN-​1 and MEN-​2, von Hippel–​Lindau (VHL) syndrome as well as the phacomatoses neurofibromatosis type 1 (NF-​1) and tuberous scler- osis. Nevertheless, most pancreatic neuroendocrine tumours are actually sporadic (i.e. non​inherited). Pancreatic neuroendocrine tumours are a principal feature of multiple endocrine neoplasia type 1.  Most patients with MEN-​1 display germline-​inactivating mutations in the MEN1 gene, a tumour suppressor gene located on chromosome 11q13 which encodes the menin protein. Of all these familial endocrine neoplasia syndromes, it is MEN-​1 that has the strongest association with pancreatic neuroendocrine tumours and these tumours occur in up to 80% of MEN-​1 patients. With regards to sporadic pancreatic neuroendocrine tumours, three pathways have been shown to be associated with the patho- genesis of these tumours. 44% of these tumours possess somatic mu- tations in MEN1, again supporting the significant role of menin in pathogenesis. However, these same studies also show that pathways controlled by DAXX (death-​domain associated protein) and ATRX (α-thalassaemia/​mental retardation syndrome X-​linked) are also aetiologically linked. Mutations in DAXX and ATRX activate the ‘al- ternative lengthening of telomeres’ (ALT) pathway, which immor- talizes cells. 61% of sporadic pancreatic neuroendocrine tumours show signs of ALT activity, supporting the role of this pathway in pathogenesis. Lastly, 15% of tumours possess mutations in the mam- malian Target of Rapamycin (mTOR) pathway, notably the tumour suppressor gene PTEN, the tuberous sclerosis complex gene TSC2, and the PI3 kinase subunit PIK3CA. The mTOR pathway controls translation, ribosomal assembly, cell growth, and cell proliferation. The clinical implication is that inhibition of this pathway with in- hibitors such as everolimus, and these have found application as tar- geted treatments for pancreatic neuroendocrine tumours (see next). Approximately 14% of patients with VHL have pancreatic neuroendocrine tumours, which are usually non​functioning and often multiple. VHL mutations lead to reduced clearance of the hypoxia-​induced transcription factor HIF-​1α via ubiquitination and proteasomal degradation, with inappropriate activation under normoxic conditions of the hypoxic response. In turn this induces angiogenesis via vascular endothelial growth factor (VEGF), cel- lular growth via platelet-​derived growth factor (PDGF) and trans- forming growth factor (TGF) α, metabolic changes promoting the transport of glucose, and enhanced cell survival. This ‘pseudo-​ hypoxic’ response is classically implicated in the pathogenesis of renal cell carcinomas, phaeochromocytomas and paragangliomas, and haemangioblastomas (i.e. the components of VHL syndrome). Although the exact link between activation of this pathway and the development of pancreatic neuroendocrine tumours is also obscure, it is of interest to note that sunitinib and other angiogenesis inhibi- tors are known to be effective against pancreatic NETs (see next). Interestingly, the ‘pseudo-​hypoxic’ pathway does not appear to be involved in the pathogenesis of sporadic pancreatic neuroendocrine tumours, as somatic mutations in the components of this pathway are not seen in these tumours. Again, the clinical implication is that ‘pseudo-​hypoxic’ pathway inhibitors such as sunitinib have been shown to be useful for treatment of metastatic pancreatic neuro- endocrine tumours. Pancreatic neuroendocrine tumour markers Serum markers The chromogranins are acidic glycoproteins stored in the dense-​core secretory granules of neuroendocrine cells. Chromogranin A is re- leased into the circulation by neuroendocrine cells and, to date, is considered to be the most useful non​specific neuroendocrine tumour marker. Chromogranin A concentrations may correlate with tumour burden and can be also useful in monitoring response to treatment or recurrence of disease. Several commercial immunoassays for the measurement of chromogranin A are available. The standardization of chromogranin A measurement is confounded by the multiplicity

13.8  Pancreatic endocrine disorders and multiple endocrine neoplasia 2451 of methods and by the fact that chromogranin A is processed to multiple post-​translational variants, leading to variable perform- ance between assays. Nevertheless, chromogranin A  is useful for the following purposes: (1) prognostication, higher levels connoting a worse prognosis; (2) for the longitudinal assessment of tumour burden during treatment, provided the same assay is used for each measurement; (3) for prediction of the response to somatostatin ana- logue treatment. Other conditions that may lead to false-​positive ele- vations of chromogranin A include chronic atrophic gastritis leading to achlorhydria, proton pump inhibitor therapy, chronic kidney dis- ease, pregnancy, untreated hypertension, and glucocorticoid treat- ment. The most important practical point concerns proton pump inhibitor therapy, which should be withdrawn for 3–​5 days before measurement of chromogranin A. Chromogranin B coexists with chromogranin A, but differs in its amino acid sequence. It has been reported to be less influenced by renal failure and proton pump inhibitors. Immunoassays directed against the 74 amino acid C-​terminal fragment of chromogranin B, known as GAWK, possess a better diagnostic accuracy for pancre- atic neuroendocrine tumours than chromogranin A. Pancreatic polypeptide is produced by the pancreatic poly- peptide (PP) or F cells of the normal pancreas. Plasma levels of pancreatic polypeptide are frequently increased in patients with non-​ functioning pancreatic neuroendocrine tumours. Some of these are explained by PP cell hyperplasia in the normal islets surrounding a pancreatic NET, and some due to incorporation of PP cells within the tumour itself. More rarely, some patients have a primary pan- creatic polypeptide-​secreting PNET, a so-​called PPoma. Pancreatic polypeptide alone is a less sensitive neuroendocrine tumour marker than chromogranin A, yet its diagnostic sensitivity may be signifi- cantly increased when combined with chromogranin A. Cocaine-​amphetamine-​regulated transcript (CART) peptide was originally characterized as a transcript up-​regulated in neurones in response to cocaine and amphetamines. CART peptide is made in various tissues within the body including endocrine and nervous tis- sues. Plasma CART peptide is elevated in patients with various types of neuroendocrine tumour, and relevantly in pancreatic neuroendo- crine tumours, although its diagnostic performance appears to be similar to that of chromogranin B, and better than chromogranin A. Pancreastatin, a cleavage product of chromogranin A from res- idues 250–​301, can also be measured by commercially available immunoassay, although it is unclear whether this has any concrete advantages over chromogranin A assays. 5-​hydroxyindole acetic acid (5-​HIAA), the principal metabolite of 5-​hydroxytryptamine, is not at all useful for the diagnosis and monitoring pancreatic neuroendocrine tumours, and should not be measured. In addition to general pancreatic neuroendocrine tumour markers, specific gut hormones produced by these tumours can be measured by RIA using a single fasting plasma sample, and for certain syndromes a small number of confirmatory tests. As with chromogranins, several non​neoplastic conditions are asso- ciated with increased levels of specific circulating gut hormones (Table 13.8.1). Gut hormone RIAs are not well standardized, and there is considerable variation between laboratories. However, con- centrations are usually of the same order of magnitude in all assays and show a similar percentage increase above normal. Immunohistochemical markers Assessment of pancreatic neuroendocrine tumours using immunohistochemistry involves general neuroendocrine tumour markers derived either from the cytosol such as neuron-​specific enolase and the protein gene product 9.5 (PGP9.5), or granular markers such as chromogranin A and synaptophysin. The tumour should be stained for Ki-​67 protein, to generate a proliferative index, which is useful for grading tumours (Table 13.8.2) and for predic- tion of tumour response to treatment. Table 13.8.1  Causes of elevated gut hormones other than pancreatic endocrine tumours All hormones Non​fasting sample Chronic kidney disease Gastrin Hypercalcaemia Achlorhydria (most commonly proton pump inhibitor
or other antacid therapy, also chronic atrophic gastritis) Antral gastrin cell hyperfunction Small bowel resection Gastric outlet obstruction VIP Hepatic cirrhosis Bowel ischaemia Glucagon Hepatic failure Oral contraceptives and danazol Stress Prolonged fast Familial hyperglucagonaemia PP Elderly Pernicious anaemia Hypercalcaemia Neurotensin Fibrolamellar hepatoma PP, pancreatic polypeptide; VIP, vasoactive intestinal polypeptide. Table 13.8.2  WHO 2010 and ENETS grading systems for NETs WHO 2010 classification WHO grading criteria ENETS classification ENETS grading criteria NE neoplasm grade 1 <2 mitoses/​10 hpf and no necrosis NET grade 1 (G1) <2 mitoses/​10 hpf and <3% Ki-​67 index NE neoplasm grade 2 <2 mitoses/​10 hpf or foci of necrosis NET grade 2 (G2) 2–​20 mitoses/​10 hpf or <3–​20% Ki-​67 index Small cell NE carcinoma Large cell NE carcinoma

10 mitoses/​10 hpf NE carcinoma grade 3 (G3)—​small
cell or large cell 20 mitoses/​10 hpf or >20% Ki-​67 index hpf, high power fields

section 13  Endocrine disorders 2452 Imaging in pancreatic neuroendocrine tumours Investigations used in the radiographic localization of pancreatic neuroendocrine tumours include ultrasonography, CT, and MRI. Transabdominal ultrasonography may have limited use in the de- tection of small pancreatic neuroendocrine tumours, but can be used to take biopsies from liver metastases for histopathological analysis. In experienced hands, endoscopic ultrasonography may be more sensitive than conventional imaging, with a resolution of 2 mm and a detection rate of over 75% for tumours in the pancre- atic head (Fig. 13.8.1). However, visualization using ultrasound is poorer for lesions in the pancreatic tail. Intraoperative ultrasonog- raphy may be particularly effective in identifying pancreatic neuro- endocrine tumours, especially when it is combined with palpation by the surgeon. Neuroendocrine tumour cells express somatostatin receptors, and hence somatostatin receptor imaging with radiolabelled somatostatin analogues is the mainstay in most neuroendo- crine tumours, particularly those arising from the pancreas. Somatostatin receptor imaging is the most sensitive imaging mo- dality for pancreatic neuroendocrine tumours (80–​90%). There are five somatostatin receptor subtypes (SSTRs 1–​5) which all avidly bind endogenous somatostatin. The clinically used som- atostatin analogues, octreotide and lanreotide, used for diagnosis and treatment, bind to SSTR-​2 and SSTR-​5. DOTATATE binds to SSTR-​2 primarily, but also SSTR-​4 and -​5. In cases of non-​ functioning pancreatic neuroendocrine tumours presenting as otherwise undifferentiated pancreatic masses, somatostatin re- ceptor imaging is helpful in differentiating these from pancreatic adenocarcinomas. Somatostatin receptor imaging is also useful in detecting metastatic disease (Fig. 13.8.2), and is more sensi- tive than conventional 99Tc bone scintigraphy in identifying bony metastases. Traditionally, somatostatin receptor imaging has utilized indium-​111 octreotide (111In-​octreotide), with more than 80% sensitivity for neuroendocrine tumours. This radiotracer is less suitable for detecting small tumours because of the limited spa- tial resolution of single-​photon emission computed tomog- raphy (SPECT). This is significant, since 40% of gastrinomas and insulinomas are microadenomas (<1 cm). The recent combination of higher-​resolution positron emission tomography (PET)/​CT with somatostatin receptor scintigraphy using newer radio-​tracers such as gallium-​68 (68Ga) DOTATATE, 68Ga DOTANOC and 68Ga DOTATOC has improved tumour detection with improved sen- sitivities of more than 90% and has the advantage of identifying patients suitable for radionuclide therapy with yttrium-​90 (90Y) or lutetium-​177 (177Lu) labelled somatostatin analogues. 18F fluorodeoxyglucose (FDG) PET imaging is not sensitive for well differentiated neuroendocrine tumours, as they are metabol- ically relatively inactive, but is better for detecting high-​grade tu- mours, and may well suggest that systemic chemotherapy is an option. 11C 5-​hydroxytryptophan imaging shows some promise as a more sensitive marker for pancreatic neuroendocrine tumours but requires further clinical validation. Fig. 13.8.1  Endoscopic ultrasound scan showing a 0.7 cm insulinoma in the head of the pancreas (arrowed). (a) (b) Fig. 13.8.2  (a) Rugal hypertrophy (arrowed) in a patient with Zollinger–​ Ellison syndrome; (b) gastrinoma with metastatic lymph-​node and periduodenal uptake (arrowed) of 68Ga-​DOTATATE on PET scanning.

13.8  Pancreatic endocrine disorders and multiple endocrine neoplasia 2453 Even with 68Ga DOTATATE PET scanning, insulinomas remain the most elusive of pancreatic neuroendocrine tumours. Only 50% of non​metastatic insulinomas express SSTR-​2, although insulinomas with metastatic spread are more likely to be positive for these recep- tors. Therefore, somatostatin receptor imaging’s role in detecting non​metastatic insulinomas is limited. Capitalizing on the fact that these tumours express receptors for glucagon-​like peptide-​1 (GLP-​1), exendin-​4 based tracers are emerging as a much more specific and sensitive marker for insulinomas at 95% sensitivity vs. 47% for cross-​ sectional imaging (Fig. 13.8.3). Small pancreatic neuroendocrine tumours may be detected by arterial stimulation venous sampling (ASVS). This operates on the principle of instilling a secretagogue into the main pan- creatic arteries (gastroduodenal, superior mesenteric, inferior pancreaticoduodenal, and splenic) and measuring the secretion of hormonal markers such as insulin and gastrin in the effluent from the hepatic vein. In addition to anatomical localization with the tumour ‘blush’ (Fig. 13.8.4), this enables biochemical localiza- tion. Calcium (chloride or gluconate) is the secretagogue of choice, having replaced secretin for this purpose. Injection of calcium into the artery supplying the tumour causes a marked rise in hor- mone levels in the hepatic vein, and hence allows equivocal lesions to be verified, particularly in the presence of multiple pancreatic neuroendocrine tumours as occurs in MEN-​1. Visualization of a tumour blush on angiography prior to calcium stimulation further increases the sensitivity of this investigation. Furthermore, since the hepatic artery is cannulated at the end of the procedure, the presence of microscopic hepatic metastases may also be detected by measuring rises in hormone levels after secretagogue injection into the hepatic artery. Natural history The spontaneous course of disease in pancreatic neuroendocrine tumours is difficult to ascertain because of their low incidence, heterogeneous behaviour, and a relative absence of controlled pro- spective clinical trials to assess the efficacy of different therapeutic strategies. Those tumours that secrete functionally active peptides present early with smaller tumours, morbidity and mortality re- sulting from the effects of peptide hypersecretion rather than tu- mour bulk. Non​functioning pancreatic neuroendocrine tumours are often more advanced at diagnosis because of the absence of symptoms attributable to hormone hypersecretion. They frequently present with mass effects (e.g. biliary tract obstruction, liver me- tastases, and local lymphadenopathy). Poorly differentiated, large (>3  cm) tumours associated with metastases are indicators of a poor prognosis. Metastatic spread to liver (in 42% of patients at presentation) and bone (in 8% of patients at presentation) is the major cause of death in patients with pancreatic neuroendocrine tumours. The overall 5-​ year survival for pancreatic neuroendocrine tumours is 50 to 80%, with insulinomas and gastrinomas having up to 94% 5-​year survival, primarily because their obvious clinical presentations prompt early surgical intervention. Pancreatic neuroendocrine tumours associ- ated with familial disease such as MEN-​1 may have a less favourable outcome than sporadic tumours, since these are frequently multiple and diffuse, which may limit surgical cure. Specific tumour syndromes Insulinomas Insulinomas are the most frequent functional pancreatic neuro- endocrine tumours and are discussed in Chapter 13.9.2. Gastrinoma The gastrinoma syndrome was first described in 1955 by Zollinger and Ellison, who reported the triad of fulminating ulcer diathesis, recurrent ulceration with a poor response to therapy, and pancreatic non-​β-​cell islet tumours. The syndrome, also known as Zollinger–​ Ellison syndrome, is the result of excess gastrin-​stimulated gastric acid secretion. This causes severe, multiple peptic ulcers, which are usually duodenal, but may occur in the oesophagus and jejunum, and are often associated with complications such as gastric rugal hypertrophy, haemorrhage, perforation, and stricture formation (Fig. 13.8.2). The excess gastric acid secretion inactivates pancreatic enzymes and damages the intestinal mucosa, resulting in diarrhoea and steatorrhoea, which may be prominent features that precede symptoms of peptic ulcer disease. Overall, gastrinomas are the second most frequent functionally active pancreatic neuroendocrine tumour. At the time of diagnosis, 50 to 60% are malignant. Over 90% of gastrinomas are in the pan- creatic region of the ‘gastrinoma triangle’, an area bounded by the Fig. 13.8.3  68Ga DOTA-​exendin-​4 PET scan showing the presence of an insulinoma within the neck/​body of pancreas. Fig. 13.8.4  Venous phase of coeliac axis angiogram demonstrating gastrinoma blush in duodenal wall (arrowed).

section 13  Endocrine disorders 2454 junction between the cystic duct and the common bile duct, the junction between the second and third parts of the duodenum, and the junction between the head and neck of pancreas. Those arising in the duodenum (50–​88%) are frequently multiple, relatively small, and therefore difficult to localize. Those gastrinomas arising in the pancreas are most frequently situated in the pancreatic head. In MEN-​1 patients, gastrinomas are the most common functional pan- creatic neuroendocrine tumour. Approximately 25% of gastrinomas are associated with MEN-​1, and a higher proportion (70–​100%) are situated in the duodenum rather than the pancreas. Gastrinomas are also less commonly found outside the ‘gastrinoma triangle’ in the stomach, liver, bile duct, ovary, heart, and lung. The diagnosis of the gastrinoma syndrome requires the dem- onstration of a raised fasting plasma gastrin concentration (>40 pmol/​litre), associated with increased basal gastric acid secre- tion. Hypercalcaemia may increase plasma gastrin concentrations, which may be of consequence in patients with MEN-​1 and coex- istent primary hyperparathyroidism. Since plasma gastrin levels in gastrinomas overlap with those seen with the use of antacids such as proton pump inhibitors, ideally patients should not take H2-​blockers for 3 days or proton pump inhibitors for 2 weeks before gastrin meas- urement. However, patients with true gastrinomas may be at risk of peptic ulcer perforation if antacids are stopped for plasma gastrin measurements. It is therefore often necessary to wean patients off antacids, for example by switching proton pump inhibitors to H2- blockers for 2 weeks before switching the H2-blockers to high-dose calcium/magnesium-based antacids for 3 days, prior to gastrin sam- pling. Hypergastrinaemia and raised acid output may also arise from other causes (Table 13.8.1). The intravenous secretin test (2 U/​kg body weight) distinguishes these conditions from gastrinoma and can aid diagnosis when other investigations are equivocal. Under normal physiological conditions, secretin inhibits serum gastrin. In contrast, secretin provokes a paradoxical rise in serum gastrin of at least 120 pg/​ml in patients with gastrinoma. Alternatively, an intra- venous calcium infusion can be used diagnostically, with a rise in plasma gastrin observed in gastrinoma patients. Since ingestion of food is a stimulus for gastrin secretion from the antral and duodenal mucosa, it has been proposed that a standard test meal may differ- entiate hypergastrinaemia of antral and tumoural origin. However, more recently the usefulness of this test has been questioned. If gas- tric acid output studies are not possible, a basal gastric pH above 2 virtually excludes the diagnosis of Zollinger–​Ellison syndrome and instead suggests the alternative diagnoses of chronic atrophic gastritis or antacid treatment. Endoscopy may be valuable in demonstrating oesophageal and duodenal ulceration and hypertrophy of the gastric mucosa. Localization of microgastrinomas may be aided preopera- tively by endoscopic ultrasound, somatostatin receptor imaging, or selective visceral angiography and venous sampling. Survival depends on the presence of hepatic metastases at pres- entation, which is more commonly seen with pancreatic rather than duodenal gastrinomas. Overall, the 5-​year survival rate is about 65%. VIPoma Tumours secreting vasoactive intestinal polypeptide (VIP) are termed VIPomas. Ninety per cent of VIPomas occur in the pancreas, most frequently arising from the pancreatic tail. Extrapancreatic VIPomas may be of neural origin, such as gangliomas or ganglioneuroblastomas which arise from the sympathetic chain or adrenal medulla, and these tumours are especially common in chil- dren. Most extrapancreatic tumours are benign, but more than 50% of pancreatic VIPomas have metastasized at the time of diagnosis, usually to local lymph nodes and the liver. Approximately 9% of VIPomas are associated with MEN-​1. The features of the VIPoma (Verner–​Morrison, or ‘pancreatic cholera’) syndrome reflect the known biological actions of VIP. Large-​ volume diarrhoea without steatorrhoea is the cardinal symptom, with most patients excreting more than 3 litres per day. It is often intermit- tent at first, but in severe crises the volume loss coupled with the vaso- dilatory effects of VIP and the associated hypokalaemia may precipitate cardiovascular collapse. Hypokalaemia in the VIPoma syndrome may be profound, resulting from gastrointestinal losses and activation of the renin–​angiotensin system. The loss of bicarbonate in the stool leads to a paradoxical acidosis, which may mask the true potassium deficit. Achlorhydria or hypochlorhydria occurs in more than 50% of patients and distinguishes this diarrhoeal syndrome from that associated with gastrinoma. Nevertheless, the absence of this feature in a significant proportion of VIPoma patients makes the acronym WDHA (watery diarrhoea, hypokalaemia, and achlorhydria) syndrome inappropriate. In up to 50% of cases there is glucose intolerance as a result of the glucagon-​like actions of VIP. Other biochemical abnormalities include hypercalcaemia, probably due to secretion of parathyroid hormone-​ related peptide (PTHrP) and exacerbated by the dehydration and hypomagnesaemia, due to loss in stools. The vasodilatory action of VIP may cause flushing of the head and neck and, particularly on tumour palpation, may be associated with a marked fall in systemic blood pres- sure. In advanced cases, extreme weight loss may occur. VIPomas are usually associated with markedly raised plasma VIP concentrations (>30 pmol/​litre). Since the half-​life of circulating VIP is only 2 min it may be difficult to always confirm an elevation. Peptide histidine–​methionine (PHM) produced from the prepro-​ VIP molecule, is more stable in plasma than VIP, and is cosecreted by VIPomas. Therefore, in patients with features consistent with VIPoma syndrome, the finding of an elevated PHM may confirm the diagnosis. Pancreatic polypeptide concentrations levels are ele- vated in 75% of cases and neurotensin in 10%. Primary pancreatic VIPomas are usually large (>2 cm) and so localization is rarely a problem. Occasionally, selective visceral angiography and venous sampling may be necessary to detect small pancreatic lesions. In those with non​metastatic VIPomas, the 5-​year survival rate is more than 90%; when metastases are present it is about 60%. Glucagonoma Glucagonomas are rare α-​cell tumours of the pancreas which se- crete various forms of glucagon and other peptides derived from the preproglucagon molecule. Primary glucagonomas most commonly arise in the pancreatic tail and extrapancreatic glucagonomas are rare. Glucagonomas are usually more than 2 cm in diameter at pres- entation. Smaller glucagonomas tend to be benign and increased tumour size correlates with risk of malignancy. In most cases of sporadic glucagonomas, metastases have occurred at presentation. Up to 17% of glucagonomas are associated with MEN-​1 and these patients tend to present at a younger age. Common presenting features of glucagonoma syndrome are weight loss, diarrhoea, anorexia, and abdominal discomfort, with the latter often reflecting tumour bulk from hepatomegaly. Necrolytic migratory erythema is a frequent presenting feature of glucagonoma

13.8  Pancreatic endocrine disorders and multiple endocrine neoplasia 2455 syndrome; it is cyclical in nature, consisting of macules, central bulla formation, and crusted plaques occurring mainly at friction sites such as perineum, buttocks, groin, lower abdomen, and lower extremities. Mucous membranes are also affected, leading to angular chelitis, sto- matitis, glossitis, and blepharitis. The exact pathogenesis of this un- usual skin eruption remains unclear and is likely to be multifactorial. Hypoaminoacidaemia, zinc deficiency, hypovitaminosis B, and hep- atic dysfunction have all been implicated. Diabetes mellitus is present in approximately two-​thirds of those with the glucagonoma syndrome and this may predate necrolytic migratory erythema. Neurological and psychiatric symptoms may also be a presenting feature, including ataxia, dementia, optic at- rophy, and proximal muscle weakness. Thromboembolism has been described in up to 30% of all cases of glucagonoma syndrome, which is not a feature of other pancreatic neuroendocrine tumours and is a significant cause of death in glucagonoma syndrome. A normocytic anaemia may also occur. The diagnosis of glucagonoma is made on the basis of an elevated fasting plasma glucagon (>50 pmol/​litre), in association with charac- teristic clinical features and a demonstrable neuroendocrine tumour and/​or metastatic deposits. Glucagonomas are usually of significant size at presentation to be identified by contrast-​enhanced CT or MRI. Endoscopic ultrasonography may be of limited use in glucagonomas, as these are usually located in the pancreatic tail. Glucagonomas and their metastases are commonly hypervascular, making selective vis- ceral angiography and venous sampling particularly useful in local- izing the tumour and identifying small hepatic metastases. Although most patients with glucagonoma syndrome present with evidence of metastases, the slow-​growing nature of these tumours can result in a relatively good prognosis. The 5-​year survival ranges from 66 to 85%. Somatostatinoma Somatostatinomas are extremely rare, with an estimated annual incidence of about 1 in 40 million per year. Fifty per cent of these tumours are pancreatic, the remainder arising in the duodenum. Unlike other functional pancreatic neuroendocrine tumours, somatostatinomas are rarely associated with MEN-​1. Pancreatic somatostatinomas are usually large, more than 2 cm at diagnosis, and thus present with local symptoms, biliary obstruction, or fea- tures relating to excess somatostatin secretion. Somatostatin has pan-​inhibitory effects on gut motility, transit and absorption, gallbladder contraction and secretion, and endocrine and exo- crine pancreatic functions. The so-​called somatostatin syndrome resulting from somatostatin hypersecretion therefore consists of steatorrhoea (due to inhibition of pancreatic exocrine function), cholelithiasis (due to reduction of cholecystokinin secretion and inhibition of gallbladder contraction), hyperglycaemia (due to sup- pression of insulin secretion), and hypochlorhydria (due to suppres- sion of gastrin secretion). Hypoglycaemia has occasionally been described, possibly due to larger molecular forms of somatostatin having a greater inhibitory effect on counterregulatory hormones such as glucagon than on insulin. In comparison to pancreatic somatostatinomas, duodenal somatostatinomas are smaller, fre- quently associated with neurofibromatosis type 1, seldom associated with a recognizable ‘somatostatin syndrome’ and often containing psammoma bodies (a round concretion of calcium said to originate from the calcification of abnormal collagen produced by the neo- plastic cells). Duodenal somatostatinomas usually present with obstructive jaundice, pancreatitis, intestinal obstruction, or gastro- intestinal haemorrhage. Diagnosis of a somatostatinoma is secured by demonstrating elevated plasma somatostatin levels (>150 pmol/​ litre) in the context of a relevant clinical history and the presence of a pancreatic mass. Multiple molecular weight forms of somato- statin may be demonstrated by column chromatography of plasma or tumour extracts, and these may explain unusual clinical features. Localization is rarely a problem due to the large size at presentation. Pancreatic and duodenal somatostatinomas appear to have similar rates of metastases and malignancy. The overall 5-​year survival rate is 75%, or 60% if metastases are present. GLP-​1omas Neuroendocrine tumours that cosecrete GLP-​1 together with other bioactive peptides have been described. In one case, an ovarian neuroendocrine cosecreting GLP-​1 and somatostatin was associ- ated with diabetes mellitus during oral and IV glucose tolerance tests followed by a profound reactive hypoglycaemia, due to the subsequent glucose-​dependent potentiation of insulin secretion by GLP-​1. In a second case, a patient with a pancreatic neuroendo- crine tumour cosecreting glucagon and GLP-​1 manifested diabetes mellitus (due to the hyperglucagonaemia) together with a fasting hyperinsulinaemic hypoglycaemia, attributed to chronic hyper- trophy of β cells and autonomous secretion of insulin. In two other cases, cosecretion of GLP-​1 and the related peptide glucagon-​like peptide-​2 (GLP-​2) caused hyperplasia of the small intestinal mucosa (due to the excess GLP-​2), prolonged intestinal transit time, intract- able constipation, and recurrent vomiting. Other rare pancreatic neuroendocrine tumour syndromes Other functional peptides can present with characteristic syn- dromes such as: • Ectopic ACTH production by pancreatic neuroendocrine tu- mours, resulting in Cushing’s syndrome, is well documented in the literature and virtually all cases are highly malignant with a poor prognosis. These patients can develop a pseudo-​Nelson’s syndrome which is due to the evolution of α-​MSH from the tu- mour, stimulating skin pigmentation. • Secretion of parathyroid hormone-​related peptide (PTHrP) by pancreatic neuroendocrine tumours can manifest with intractable hypercalcaemia. • Neurotensinomas are rare and truly difficult to separate from the symptom complex produced by VIP excess. • Patients can present with a secondary acromegaly and gigantism as a result of ectopic GHRH secretion by pancreatic neuroendo- crine tumours. • Arginine vasopressin (AVP) leading to hyponatraemia due to the syndrome of inappropriate antidiuresis. • Calcitonin, which can be released by neuroendocrine tumours other than medullary thyroid carcinoma, and which leads to a syndrome of flushing and diarrhoea. • Insulin-​like growth factor-​II (IGF-​2) causing hypoglycaemia. Other peptides produced by islet-​cell tumours include neuropep- tide Y, neuromedin B, calcitonin gene-​related peptide, bombesin, and motilin, but these are not associated with recognized clinical syndromes.

section 13  Endocrine disorders 2456 Pancreatic polypeptide-​secreting tumours (PPomas) PP is secreted by up to 75% of pancreatic neuroendocrine tumours, and as noted above PP appears to be secreted in many cases as an epiphenomenon of an otherwise non​functioning neuroendocrine tumour. Where the tumour is secreting PP as its primary secretion, PPomas may present with either watery diarrhoea or weight loss, due to the known appetite-​suppressive effects of PP. Non​functioning pancreatic neuroendocrine tumours Non​functional tumours represent 15 to 30% of pancreatic neuro- endocrine tumours. These most frequently arise in the pancreatic head and are most often diagnosed in the fifth to sixth decades of life. Twenty to thirty per cent (20–​30%) of these tumours are associated with MEN-​1. They usually present late with symptoms attributable to either tumour bulk, such as anorexia and weight loss, or to ef- fects on local structures, such as obstructive jaundice or intestinal obstruction. The clinical silence of these non​functioning pancreatic neuroendocrine tumours may also reflect secretion of neuropeptides at low circulating concentrations, biologically inactive molecular forms, downregulation of peripheral receptors, or simultaneous pro- duction of an inhibitor such as somatostatin. Non​functioning pan- creatic neuroendocrine tumours are often mistakenly diagnosed as adenocarcinomas, but the presence of elevated circulating markers such as chromogranins A and B, CART, pancreatic polypeptide, or neurotensin, uptake on somatostatin receptor imaging and the pres- ence of neuroendocrine markers on biopsy samples can establish the correct diagnosis. The overall 5-​year survival is about 50%. Management of pancreatic neuroendocrine tumours As can be seen from the following sections, the treatment of pan- creatic neuroendocrine tumour runs the entire gamut of modalities including gastroenterology, endocrinology, surgery, nuclear medi- cine, interventional radiology, and oncology. Treatment decisions must essentially be taken in a suitably constituted multidisciplinary meeting so that all suitable modalities are considered. Surgical treatment Surgery is the only curative treatment for pancreatic neuroendo- crine tumours and should be considered for tumours of G1 or G2 grade. Generally, tumours less than 2 cm have a low propensity to metastasize with only 6% being malignant when discovered. These can be surveyed with cross-​sectional imaging without the neces- sity for surgery. Localized pancreatic NETs less than 2 cm, or those causing significant hormonal syndromes may be considered for curative resection, for example, enucleation for lesions close to the surface, pancreaticoduodenectomy for head of pancreas lesions, a distal pancreatectomy ± splenectomy for lesions in the body and head of pancreas. The question as to whether the primary tumour should be resected in the presence of unresectable liver metastases is not yet answered. Although there is some evidence that suggests some improvement in median overall survival with resection of the primary tumour under these circumstances, this should be taken with caution as the quality of evidence in this area is relatively poor. The surgical management of pancreatoduodenal neuroendo- crine tumours in MEN-​1 remains controversial because of the multifocal nature of the associated pancreatic disease. In cases where patients present with functional syndromes such as insulinomas or Zollinger–​Ellison syndrome, detailed investigation (e.g. with exendin-​4 imaging or ASVS) is sometimes necessary to distinguish those tumours which are secretory from those that are non​functional, so that the right procedure may be planned. In pa- tients with MEN-​1, surgical cure rates are high for insulinomas, but are significantly lower for gastrinomas, as gastrinomas are frequently multifocal and situated in the duodenum. Even so, re- section of gastrinomas can reduce the rate of subsequent liver metastases, thus improving their overall prognosis, and this op- tion should be seriously considered where there is no evidence of extrapancreaticoduodenal spread. Some centres advocate an aggressive surgical approach to MEN-​1-​associated pancreatoduodenal neuroendocrine tumours, including the choices of total pancreaticoduodenectomy or distal subtotal pancreatectomy combined with preservation of the pancre- atic head, enucleation of any neuroendocrine tumours remaining in the pancreatic head and in the duodenal wall. This approach may significantly reduce the morbidity and mortality associated with pancreatic neuroendocrine tumours in MEN-​1 patients, but comes at the cost of perioperative morbidity and mortality, as well as the morbidity associated with endocrine and exocrine pancreatic insuf- ficiency, particularly brittle diabetes mellitus. Somatostatin analogues Somatostatin analogues such as octreotide and lanreotide are the standard of care in the medical treatment of neuroendocrine tu- mours. Octreotide and lanreotide bind most avidly to SSTR-​2 and -​5 with a lower affinity for SSTR-​3. A newer SSTA, pasireotide, pos- sesses a different affinity for SSTR-​1, -​3, and -​5 and is still being studied for its effects in neuroendocrine tumour therapy. SSTR-​2 is believed to mediate the biochemical responses to somatostatin ana- logues, whereas both SSTR-​2 and -​5 subtypes are believed to me- diate their antiproliferative effects. Somatostatin analogues have two principal effects:  (1) antisecretory, leading to relief of functional syndromes. This is par- ticularly useful for glucagonomas and VIPomas, although analogues are markedly less reliable for suppressing insulinomas (50% effect- iveness) and gastrinomas (see next); (2) antiproliferative, supported by the results of the CLARINET study in patients with pancreatic, jejeuno-​ileal, colonic, and rectal NETs of grades G1 and G2, which demonstrated that lanreotide Autogel therapy was capable of signifi- cantly extending progression-​free survival. Octreotide has a half-​life of several hours in the circulation and requires frequent subcutaneous injected administration three times a day. Depot injection preparations of somatostatin analogues such as octreotide LAR or lanreotide Autogel are more commonly used since these allow sustained release, typically necessitating an intra- muscular or deep subcutaneous injection every 4 weeks. Patients are often initially stabilized on short acting subcutaneous octreotide to ensure that treatment is tolerated well and that there are no ad- verse reactions, before converting to longer acting depot prepar- ations. Tachyphylaxis is said to occur with time, but this is not a frequent issue. Peptide-​receptor radionuclide therapy (PRRT) Following the binding of labelled somatostatin analogues to SSTRs, neuroendocrine tumour cells internalize and retain the analogues, leading to the retention of tracer. By binding high-​energy ß-​particle

13.8  Pancreatic endocrine disorders and multiple endocrine neoplasia 2457 emitting radionuclides such as 177Lu and 90Y to somatostatin analogues such as lanreotide and DOTATATE, tumour cells can be selectively ex- posed to high-​energy ß-​radiation, leading to cell apoptosis. Such PRRT acts systemically and is a particularly useful palliative option for pa- tients with inoperable or multisite disease. A key prerequisite prior to treatment is to demonstrate that there is high tumour uptake of the SSTA relative to non​target tissues on quantitative somatostatin receptor imaging, preferably with the same peptide (e.g. DOTATATE) that will be used for therapy. Patients must also have stable haematological and renal function. In uncontrolled case series, PRRT is associated with fa- vourable partial responses or tumour stabilization, with relatively mild adverse reactions—​acute nausea, bone marrow suppression, and renal toxicity being the chief problems. The results from a randomized con- trolled trial in mid-​gut neuroendocrine tumours (NETTER-​1) show that 177Lu DOTATATE therapy is capable of increasing progression-​ free survival and overall survival compared to treatment with high- dose somatostatin analogue therapy. Definitive randomized controlled trials in patients with pancreatic neuroendocrine tumours are cur- rently lacking, but it should be noted that 177Lu DOTATATE therapy for pancreatic NETs is now approved by cost-effectiveness bodies such as the UK National Institute for Health and Care Excellence based on appraised data from single-arm studies. Systemic chemotherapy Traditional antiproliferative chemotherapy has a relatively limited role in the treatment of neuroendocrine tumours. It is more effective against high-​grade G3 tumours, those exhibiting high-​grade FDG up- take or biologically aggressive behaviour. Typical chemotherapeutic regimens include combinations of etoposide and cisplatin, fluorouracil with streptozocin, fluorouracil with doxorubicin, or fluorouracil with streptozocin and cisplatin. Temozolomide ± capecitabine has also been advocated for the treatment of pancreatic NETs. Temozolomide is a cytotoxic alkylating agent, with comparable antitumour activity to streptozotocin, and is particularly effective in neuroendocrine tumours expressing low levels of the DNA repair enzyme O6-​ methylguanine DNA methyltransferase (MGMT). Capecitabine with streptozocin has also recently been shown to be reasonably effective in the Phase II NET-​01 trial, producing a radiological response or stabil- ization in 74%, a median progression-​free survival of 9.7 months and median overall survival of 27.5 months. The addition of cisplatin to the regimen did not improve outcomes. Targeted molecular therapy Targeted molecular therapies, for example the tyrosine kinase in- hibitor sunitinib (inhibiting VEGFR, PDGFR, c-​KIT, and FLT3) and everolimus (inhibiting mTOR), have been shown to be active against pancreatic neuroendocrine tumours in recent Phase III trials. In a placebo-​controlled randomized controlled trial in 171 patients with advanced and non​resectable pancreatic neuroendocrine tumours, sunitinib therapy was shown to double median progression-​free survival compared to placebo. Overall survival was improved. The main adverse effects were diarrhoea, nausea, and vomiting, tired- ness, hypertension, and neutropenia. The RADIANT-​3 Phase III randomized controlled trial showed that everolimus was also capable of doubling median progression-​free survival in patients with progressive and metastatic pancreatic neuro- endocrine tumours. Unlike sunitinib, no benefit on overall survival was noted. The most common adverse effects noted were stomatitis/​ aphthous ulceration, rash, diarrhoea, fatigue, neutropenia, and an in- creased rate of infections. The latter side effect is due to its activity as an immunosuppressant. Everolimus also induces some metabolic adverse effects:  hypertriglyceridaemia and diabetes mellitus. The metabolic effects are controllable with insulin and hypolipidaemic treatment. Indeed, its hyperglycaemic effect is sometimes advantageous in the case of metastatic insulinomas. The main dose-​limiting adverse effect of everolimus is pneumonitis, interstitial lung disease, and sometimes lung fibrosis. This can be potentially very serious and may require steroid treatment and withdrawal or dose reduction of everolimus. Other angiogenesis inhibitors such as sorafenib (a tyrosine kinase in- hibitor of VEGFR2, PDGFRB, FGFR1 and FLT3), pazopanib (targeting VEGFR-1, -2 and -3, PDGFRα and β, FGFGR-1, -2 and -3, c-Kit, Itk, Lck, c-Fms and B-Raf) and bevacizumab (a monoclonal antibody against VEGF-​A) have shown some promising activity in smaller trials but are not currently in routine use. Pazopanib, brivanib, cabozantinib, sulfatinib, famitinib and lenvatinib are newer tyrosine kinase inhibitors also targeting VEGFR which are currently under evaluation. External beam radiotherapy This may be effective in relieving pain from bone metastases and, in a small number of cases, has been curative in patients with locally unresectable pancreatic neuroendocrine tumours. Orthotopic liver transplantation Because of the generally less aggressive biological behaviour of neuroendocrine tumours, orthotopic liver transplantation (OLT) is an accepted, if uncommon, treatment of disease limited to liver me- tastases. The 5-​year survival after OLT has been shown in various series to vary between 36% and 90%. Contraindications include high-​grade tumours (G3), extrahepatic disease, or disease draining to the systemic circulation. As the experience of OLT for this indi- cation is limited, there remains considerable uncertainty about the true benefit of OLT and this treatment is usually recommended only on a case-​by-​case basis. Interferon alpha Interferon alpha (IFN-α) exhibits an antiproliferative activity and has been used for the treatment of gastrointestinal and pancreatic neuroendocrine tumours, with an overall response rate of 20% and a biochemical response rate of 63% (26). However, its adverse effects—​flu-​like symptoms, myelotoxicity, weight loss, and fatigue, depression, and on occasion, suicidal ideation—​limit the dose and duration of treatment, making this usually a third-​line therapy. Local ablation Ablation of liver metastases may be considered if the number of lesions is small (<5) and the lesion size limited (<5 cm). The types of ablative techniques include radiofrequency (most commonly used), micro- wave, laser, and cryotherapy. The experience with radiofrequency ablation suggests that this is a well-​tolerated procedure, although local recurrence is fairly common in approximately 1 in 5, and the majority of patients go on to develop either new liver metastases or extrahepatic disease at a median time of 30 months or so. Transarterial embolization therapies Hepatic metastases obtain their blood supply from the hepatic ar- tery, and the liver parenchyma is supplied with blood both from the

section 13  Endocrine disorders 2458 hepatic artery and the portal vein. Exploiting this, interventional intra-​arterial therapies which serve to block off the hepatic arterial supply to liver metastases (transarterial embolization, TAE), deliver cytotoxic chemotherapeutic agents such as doxorubicin (transarterial chemoembolization, TACE) or radionuclide therapy (selective in- ternal radiotherapy, SIRT) have been employed with some success in the treatment of patients presenting with liver metastases. The most clinical experience has been accumulated with TAE/​ TACE, which is successful in reducing syndromic symptoms in the majority of patients, and in reducing the size of metastases in 35–​74%. Median progression-​free survival is 18 months and 5-​year survival of patients undergoing this type of therapy is 40–​83%. Many patients experience a postembolization syndrome (fever, abdom- inal pain, elevated liver transaminases); there is severe morbidity in 10% (acute liver and renal failure, carcinoid crisis, cholecystitis, gastrointestinal bleeding) and an associated mortality of up to 5.6%. Contraindications to embolization therapy include complete portal vein thrombosis, previous pancreaticoduodenectomy, and liver in- sufficiency. The overall usefulness of embolization therapy lies in the fact that it can be applied repeatedly to control liver metastases and for symptomatic relief. SIRT, in which 90Y is delivered directly to liver metastases ei- ther coupled to microspheres or to somatostatin analogues such as lanreotide, is a newer technique which appears to have similar toler- ability to conventional TAE/​TACE. There appears to be a tumour re- sponse in less than 60% of patients and disease stabilization in 35%. Adverse effects and contraindications are similar to TAE/​TACE, but the radiation may also cause damage to the liver, the lungs, and the gastrointestinal tract if delivered off-​target. Treatment of specific tumour syndromes Insulinomas Treatment of insulinomas is discussed in Chapter 13.9.2. Gastrinomas Short-​ or long-​term treatment with high-​dose proton pump in- hibitors, which inhibit gastric acid secretion, is highly effective for symptomatic relief and tachyphylaxis does not occur. Somatostatin analogues may be superfluous if symptomatic relief occurs with high-​dose proton pump inhibitors, although these may be used in metastatic disease to limit tumour progression. As hypercalcaemia tends to aggravate gastrin release, consideration should be given to surgical resection of the hyperplastic parathyroid glands in patients with MEN-​1 that present with primary hyperparathyroidism and Zollinger–​Ellison syndrome. VIPomas VIPomas are usually exquisitely sensitive to SSTA, with small doses often significantly reducing diarrhoeal symptoms. During acute crises, patients require aggressive intravenous rehydration com- bined with potassium and bicarbonate replacement if necessary. Parenteral hydrocortisone is often useful also to control the diar- rhoea in the acute situation. Glucagonomas Octreotide is particularly useful as a prompt and effective treatment of necrolytic migratory erythema, providing improvement within 48 to 72 h of initiating treatment. Similarly, other symptoms such as diarrhoea and weight loss may also improve. Somatostatin ana- logues have a variable effect on glucose tolerance and adjuvant oral hypoglycaemic agents or insulin may be required. Most patients with glucagonoma syndrome are treated empirically with oral zinc sulphate supplementation, regardless of plasma zinc levels. Topical zinc sulphate paste is also useful for relieving the rash. Patients should be anticoagulated because of the high incidence of thrombo- embolic disease. Somatostatinomas There are a small number of cases reported demonstrating im- provements of symptoms by administration of somatostatin ana- logues. Pancreatic enzyme supplementation and insulin for diabetes mellitus may also be necessary. Ectopic ACTH syndrome Pancreatic neuroendocrine tumours secreting ACTH present with a very aggressive Cushing’s syndrome with marked oedema, rapid de- velopment of diabetes mellitus, and immunosuppression. Bilateral adrenalectomy, followed by steroid replacement therapy, is fre- quently necessary to control the hypercortisolaemia, and to enable patients to be treated using the other treatment modalities discussed earlier. PTHrP-​secreting tumours The humoral hypercalcaemia that follows from PTHrP secretion can be treated with intravenous bisphosphonates. Zoledronic acid is recommended for this purpose as it is markedly more effective and longer-​lasting in its effects compared to pamidronate. Where the hypercalcaemia is unresponsive to bisphosphonates, denosumab may be used, with some case studies suggesting that this is effective in controlling calcium levels. Multiple endocrine neoplasia Multiple endocrine neoplasia refers to rare hereditary cancer syn- dromes characterized by a predisposition to tumour development within two or more endocrine organs. The two major forms of MEN, namely MEN-​1 and MEN-​2, are caused by germline mutations which display an autosomal dominant pattern of inheritance and a high degree of penetrance. MEN-​1 is associated with mutations in the MEN1 gene, whereas MEN-​2 results from a RET (REarranged during Transfection) gene mutation. Recent advances in our under- standing of the molecular and clinical genetics of these syndromes have significantly altered the approach to diagnosis and manage- ment of these patients. Multiple endocrine neoplasia type 1 (MEN-​1) Clinical features and classification The major components of MEN-​1 are parathyroid adenomas, pancreatic neuroendocrine tumours, and pituitary adenomas (see Table 13.8.3). Underdahl first described the association of these tumours in 1953, and Wermer subsequently proposed their auto- somal dominant inheritance in 1954, with the latter providing the eponym for this syndrome. MEN-​1 occurs in approximately 1 in

13.8  Pancreatic endocrine disorders and multiple endocrine neoplasia 2459 30 000 individuals, with an equal sex distribution and may be de- fined as a case in which two of the three main MEN-​1-​related endo- crine tumours occur. Two different forms of MEN-​1, sporadic and familial, have been described. Familial MEN-​1 (OMIM 131100) is more prevalent, with an autosomal dominant pattern of inheritance, and is defined as an MEN-​1 case with at least one first-​degree rela- tive with one of these three characteristic endocrine tumours. More than 95% of MEN1 mutation carriers manifest clinical features of the syndrome by the age of 40 years. Parathyroid hyperplasia/​adenomas Primary hyperparathyroidism is the most common presenting fea- ture of MEN-​1, reaching almost 100% penetrance by age 50 years. The typical age of onset of primary hyperparathyroidism in MEN-​1 is 20 to 25 years, which is 30 years earlier than that of sporadic primary hyperparathyroidism. Patients present either with asymptomatic hypercalcaemia on biochemical screening or with features similar to those of sporadic primary hyperparathyroidism (see Chapter 13.4). In MEN-​1, hyperparathyroidism reflects hyperplasia of multiple parathyroid glands and supernumerary glands are common. There is a consensus that minimally invasive parathyroidectomy is not ad- visable, since it prevents the routine identification of all four glands. However, controversy exists regarding the most appropriate surgical approach. Subtotal parathyroidectomy with near total thymectomy (to remove ectopic parathyroid tissue in the thymus) is the most common approach. Some centres advocate total parathyroidectomy with autotransplantation of a fresh parathyroid gland into the forearm to avoid reoperative neck surgery if recurrent primary hyperpara- thyroidism in the transplanted hyperplastic gland occurs. An alter- native approach is a total parathyroidectomy followed by immediate replacement therapy with 1α-​hydroxycholecalciferol or calcitriol. Pancreatic neuroendocrine tumours Pancreatic neuroendocrine tumours are the second most common clinical manifestation of MEN-​1, occurring in about 30 to 75% of MEN-​1 patients, with gastrinomas accounting for 60% of cases. Insulinomas represent about 30% of MEN-​1-​associated pancre- atic neuroendocrine tumours and coexist with gastrinomas in 10% of cases. Other functional tumour types such as VIPomas, glucagonomas, and somatostatinomas are rare. MEN-​1 patients with pancreatic neuroendocrine tumours usually manifest with symptoms of hormone hypersecretion by the age of 40 years, al- though these tumours may be detected earlier if asymptomatic carriers are having routine biochemical or imaging screening. The pancreas characteristically contains numerous microadenomas, the majority of which are harmless but which have the potential to grow to clinically relevant lesions. Tumours arise in any part of the pancreas, although MEN-​1 associated gastrinomas frequently arise within the duodenal submucosa. Surgical management of MEN-​1 associated pancreatic neuroendocrine tumours is described earlier in this chapter. MEN-​1-​associated gastrinomas are associated with a high risk of recurrence after surgery and hence some centres ad- vocate medical management with high-​dose proton pump inhibi- tors in preference to surgery. Pancreatic neuroendocrine tumours associated with MEN-​1 are less malignant than sporadic tumours and carry a better prognosis, with a median survival of 15 years com- pared to 5 years in patients with sporadic tumours. This may reflect more indolent disease or—​since known MEN-​1 patients usually participate in a surveillance programme—​earlier diagnosis. Pituitary adenomas The incidence of pituitary adenomas in MEN-​1 patients varies from 10 to 60%. These adenomas are detected by screening in 30% of pa- tients, but are found at autopsy in more than 50% of patients. Most these tumours are microadenomas (diameter <1 cm). Prolactinomas are the most common type of pituitary adenoma in MEN-​1 (60%), although tumours secreting growth hormone or ACTH are not un- common. Double and even triple pituitary adenomas, which may secrete different pituitary hormones, have been described in MEN-​

  1. Imaging and treatment are the same as for sporadic pituitary tu- mours (see Chapter 13.2.1). Other manifestations of MEN-​1 Foregut neuroendocrine tumours occur in 3 to 4% of patients with MEN-​1. These are rarely associated with hypersecretion of hor- mones. MEN-​1 thymic neuroendocrine tumour is seen mainly in men, whereas bronchial neuroendocrine tumours are commoner in women. Gastric neuroendocrine tumours in MEN-​1 are small and multiple, and their malignant potential remains uncertain. Adrenal cortical adenomas are present in up to 40% of patients with MEN-​1. These are often non​functioning and bilateral, but can occasionally present with a non-ACTH dependent Cushing’s syndrome emanating from the adrenal adenomas. Other forms of adrenal pathology associated with MEN-​1 are diffuse adrenal hyper- plasia, adrenal nodular hyperplasia, and adrenocortical carcinoma. The presence of multiple facial angiofibromas, which consist of acneiform papules, is highly suggestive of MEN-​1. Collagenomas are another common feature and are multiple, skin-​coloured, or oc- casionally hypopigmented cutaneous nodules, on the trunk, neck, and upper limbs. Subcutaneous or, rarely, visceral lipomas occur in 10 to 30% of MEN-​1 patients. Furthermore, MEN-​1 gene mutations have been demonstrated in individuals with atypical familial endo- crine syndromes including phaeochromocytoma. Genetics of MEN-​1 The MEN1 gene was identified in 1997 by positional cloning and is a putative tumour suppressor gene, in keeping with the ‘two-​hit’ model of hereditary cancer as originally postulated by Knudson for retinoblastoma. Knudson proposed that affected family members inherited a ‘first hit’ as an inactivating germline mutation in one al- lele of a tumour suppressor, resulting in a predisposition to tumour development. The ‘second hit’ is acquired as a stochastic somatic event in a susceptible cell type. The inactivation of the remaining functional allele results in progression to neoplasia. Therefore, to Table 13.8.3  Clinical features of MEN1 (Wermer’s syndrome) with estimated prevalence in parentheses Endocrine features Associated non​endocrine features Parathyroid hyperplasia/​adenoma (>95%) Pancreatic tumour (30–​75%)   (gastrinoma most common) Anterior pituitary tumour (30%)   (prolactinoma most common) Adrenal cortical tumour (non​functioning) (25%) Foregut carcinoid (3–​4%) Facial angiofibromas (85%) Collagenomas (70%) Lipomas (10–​30%)

section 13  Endocrine disorders 2460 explain the apparent paradox of an autosomal dominant disease that is clearly recessive at the cellular level, there must be a relatively high frequency of such stochastic somatic events. Sporadic cases of MEN-​1 involve distinct first and second ‘hits’ in somatic cells. There is an enormous diversity of mutations occurring in the MEN1 gene, with over 400 different germlines or somatic muta- tions currently reported in MEN-​1 families and sporadic cases. These mutations are dispersed throughout the entire coding region. Unlike the RET gene in MEN-​2, there is no significant correlation between the nature or position of the mutation in the MEN1 gene and clin- ical status. Sequencing of the MEN1 gene detects a germline mutation in about 70–80% of index cases for familial MEN-​1. The remaining 20–30% are mostly false negatives, reflecting mutations in the MEN1 gene which are not detected by current gene sequencing techniques. More than 10% of MEN1 mutations arise de novo and may be trans- mitted to subsequent generations. Menin is usually located in the nu- cleus in non​dividing cells but becomes localized in the cytoplasm in dividing cells. It appears to have multiple interacting partners, and has been implicated in the following processes: 1. Transcription regulation (key interactions with JunD, NF-​ kappaB, Smads 1, 3, and 5); 2. DNA replication, recombination and repair, and genomic sta- bility (interactions with RPA2, FANCD2); 3. Cell division (NMMHC II-​A, GFAP, vimentin) and cell cycle control (NM23, ASK); 4. Epigenetic control via modulation of chromatin remodelling (MLL histone methyl-​transferase complex ER-α, HDAC). Multiple cellular pathways therefore become dysregulated by mu- tations in MEN1 but the detailed links between MEN1 mutation to the development of its characteristic tumours are still opaque. Certainly, the observation that patients who come from families with the same MEN1 mutation can have widely divergent pheno- types with some showing only hyperparathyroidism, and others showing the full panoply of tumours, suggests again that stochastic somatic events must play an important part in the development of MEN-​1 manifestations. Genetic screening and management Mutational analysis of the MEN1 gene is recommended in an index case with two or more MEN-​1-​associated endocrine tumours. In addition, mutational analysis should be considered in patients with parathyroid adenomas before the age of 30 years or multigland para- thyroid disease; gastrinoma or multiple pancreatic islet-​cell tumours at any age; or individuals who have two or more MEN1-​associated tumours, which are not part of the classical triad of parathyroid, pan- creatic islet and anterior pituitary tumours (e.g. parathyroid tumour plus adrenal tumour). It should be noted that between 5 and 25% patients with manifestations of MEN-​1 may not be shown to possess a mutation in MEN1, for various reasons including: (a) alternative causative mutations, for example in CDKN1B (also known as MEN4), CDC73 (associated with the hyperparathyroidism-​jaw tumour syn- drome), the calcium sensing receptor CaSR (responsible for familial hypocalciuric hypercalcaemia), and the aryl hydrocarbon receptor interacting protein AIP (responsible for familial isolated pituitary adenoma syndrome); (b) genetic analyses that do not include multi- plex ligation-​dependent amplification for deletions which are found in up to 33% of patients; (c) the occurrence of phenocopies, defined as the coincidental occurrence of MEN-​1-​associated tumours. Following identification of an MEN1 mutation in an index case, genetic analysis in first-​degree relatives allows identification of af- fected family members. Such early identification of MEN-​1 in asymptomatic carriers is particularly useful to allow subsequent periodic surveillance. This screening may detect the onset of the dis- ease about 10 years before symptoms develop and thus provide an opportunity for earlier treatment. This early detection and treatment of the potential malignant neuroendocrine tumours should reduce the morbidity and mortality associated with MEN-​1 syndrome. The manifestation of MEN-​1 tumours is rare before the age of 5 years, so screening does not need to occur before that age. Current guide- lines suggest that the minimal surveillance programme for individ- uals known to have MEN-​1 syndrome or to have a family-​specific mutation of the MEN-​1 gene should include annual measurement of: serum prolactin (from age 5 years), fasting serum calcium and PTH (from age 8 years), and fasting gut hormone concentrations (including chromogranin A, gastrin, insulin and glucose, glucagon, VIP, PP—​from age 20 years). Patients should undergo a baseline radiological screening (pituitary MRI, pancreatic protocol, and chest CT scan) and periodic rescreening every 1–​3 years. Where the screening investigations suggest that the patient has a manifestation of MEN-​1, further investigations including dynamic testing for acromegaly and Cushing’s syndrome, endoscopic ultra- sound, and somatostatin receptor imaging may be appropriate. Multiple endocrine neoplasia type 2 (MEN-​2) Clinical features and classification MEN-​2 has at least three distinct variants: MEN-​2A, MEN-​2B and familial medullary thyroid carcinoma, and their clinical features are outlined in Table 13.8.4. Although MEN-​2 can arise sporadically, familial cases are more common. Familial MEN-​2 is defined as an MEN-​2 case with at least one of the characteristic endocrine tu- mours in a first-​degree relative. MEN-​2 has an estimated prevalence Table 13.8.4  Clinical features of MEN-​2 with estimated prevalence in parentheses MEN-​2Aa MTC (99%) Phaeochromocytoma (>50%) Parathyroid hyperplasia/​adenoma (15–​30%) Hirschsprung’s disease (aganglionic megacolon—​7%) Cutaneous lichen amyloidosis (rare) MEN-​2B MTC (100%) Phaeochromocytoma (40–​50%) Intestinal ganglioneuromatosis and mucosal neuromas (40%) Marfanoid habitus Megacolon Familial MTC Medullary thyroid carcinoma is sole manifestation MEN, multiple endocrine neoplasia; MTC, medullary thyroid carcinoma. a MEN-​2A accounts for c.95% of all MEN-​2 cases.

13.8  Pancreatic endocrine disorders and multiple endocrine neoplasia 2461 of 1:30 000. Each variant of MEN-​2 is caused by germline mutations in the RET proto-​oncogene, which is located on chromosome 10. MEN-​2A (OMIM 171400) This may also be referred to as Sipple’s syndrome and is character- ized by medullary thyroid carcinoma, phaeochromocytoma and parathyroid hyperplasia/​adenomas. MEN-​2A accounts for c.95% of MEN-​2 cases. Medullary thyroid cancer is usually the first neo- plastic manifestation in MEN-​2, appearing between the ages of 5 and 25 years. Phenotypic variants of MEN-​2A include MEN-​2A plus aganglionic megacolon (MEN-​2A plus Hirschprung’s disease) and MEN-​2A plus cutaneous lichen amyloidosis, a pruritic rash located on the upper back, which usually arises before the onset of the thy- roid cancer and which is characteristically relieved by sun exposure. MEN-​2B (OMIM 162300) This variant represents about 5% of all cases of MEN-​2, and al- though MEN-​2B is often diagnosed earlier than MEN-​2A because of the characteristic associated physical features, it exhibits a higher morbidity and mortality than MEN-​2A. Approximately 75% of pa- tients with MEN-​2B are sporadic cases with de novo RET mutations, 25% cases are familial. The endocrine features of this subtype are medullary thyroid cancer and phaeochromocytoma, but not pri- mary hyperparathyroidism. The medullary thyroid cancer asso- ciated with MEN-​2B occurs about 10 years earlier than MEN-​2B. Patients with this syndrome have a marfanoid habitus, skeletal ab- normalities (kyphoscoliosis or lordosis), mucosal neuromas, intes- tinal ganglioneuromas (which may cause chronic megacolon) and myelinated corneal nerves (Fig. 13.8.5). Familial medullary thyroid carcinoma (OMIM 155240) Familial medullary thyroid carcinoma (FMTC) is considered a variant of MEN-​2A where medullary thyroid carcinoma (MTC) is the only clinical feature, although this may sometimes be associated with Hirschprung’s disease. Since MTC is usually the first neoplasm to manifest in MEN-​2, because of its earlier and overall higher pene- trance, it is essential to correctly classify FMTC. Misdiagnosis of fa- milial medullary thyroid carcinoma in situations where the correct diagnosis is actually MEN-​2A may unintentionally exclude future screening for phaeochromocytoma, which may have catastrophic consequences. FMTC may be diagnosed where there are at least 10 carriers in the kindred, multiple carriers or affected members above the age of 50 years, and a family history adequate to exclude hyper- parathyroidism and phaeochromocytoma. Clinical features Medullary thyroid carcinoma Medullary thyroid carcinoma originates from the parafollicular cells (C-​cells) of the thyroid (see Chapter 13.3.2). These cells se- crete calcitonin, which serves as the primary tumour marker, and sometimes carcinoembryonic antigen. In MEN-​2, familial medul- lary thyroid carcinoma behaves in a relatively benign and indolent manner, whereas medullary thyroid carcinoma in association with MEN-​2B represents the most malignant form of the disease. C-​cell hyperplasia is the precursor to hereditary MTC, with a variable pro- gression to nodular hyperplasia and finally, through clonal progres- sion, to malignancy. MTC occurs at a younger age in patients with MEN-​2 than does the sporadic form. Local invasion is common, with metastatic spread to lymph nodes in the neck and mediastinum occurring in up to 50% of cases. Distant metastases to liver, bone, and lung are seen in 15 to 25% of cases. The clinical presentation in MTC may be with a neck mass, or symptoms from metastases (diar- rhoea, flushing, weight loss, or bone pain). Rarely, ectopic ACTH secretion from MTC may cause Cushing’s syndrome. Calcitonin is elevated in all cases of clinically palpable MTC. In smaller tumours or cases of C-​cell hyperplasia, basal calcitonin levels may be normal and stimulation testing with a secretagogue such as pentagastrin or calcium gluconate may be necessary to con- firm the diagnosis. Genetic screening in MEN-​2-​associated MTC has now largely replaced biochemical screening for MTC using the pentagastrin-​stimulated calcitonin test. Cytological diagnosis using fine needle aspiration and staining for calcitonin may be useful where MTC manifests in previously unidentified MEN-​2 carriers as a neck mass. Cross-​sectional imaging of MTC using CT or MRI may be useful when planning surgery, and somatostatin receptor imaging is often valuable in detecting metastatic disease. As the tumour stage at presentation is the major prognostic factor, early diagnosis and surgical intervention before cervical lymph-​node metastases ap- pear is necessary to improve survival. Current practice involves total thyroidectomy with central node dissection. Postoperatively, in (a) (b) Fig. 13.8.5  (a) Characteristic phenotype of MEN-​2B showing facial appearance. (b) Characteristic phenotype of MEN-​2B showing mucosal neuromas on the tongue.

section 13  Endocrine disorders 2462 addition to a diagnostic role, calcitonin is used as a tumour marker for metastases or disease recurrence. Phaeochromocytoma Phaeochromocytomas are neuroendocrine neoplasms of neural crest origin. These occur in approximately 50% of patients with MEN-​2A or MEN-​2B, are usually benign, and are invariably confined to the ad- renal glands. The penetrance of phaeochromocytomas in MEN-​2A depends on the exact codon mutated in RET with codon 634 muta- tions being associated with a high penetrance of 88% by age 77 years, and exon 10 mutations (codons 609, 611, 618, 620) being associated with a lower penetrance of up to 25% or so. Phaeochromocytomas associated with MEN-​2A or MEN-​2B are bilateral in 50 to 80% of cases. The features and management of phaeochromocytomas are outlined in Chapter 16.17.3. Earlier detection and improved manage- ment with laparoscopic adrenalectomy have resulted in a significant reduction in the morbidity associated with phaeochromocytoma in MEN-​2. If MTC and phaeochromocytoma are diagnosed simultan- eously in MEN-​2A or MEN-​2B individuals, adrenalectomy should be performed before thyroidectomy. Parathyroid hyperplasia/​adenomas Primary hyperparathyroidism is a feature in 20–​30% of MEN-​2A patients and is usually asymptomatic. Compared to MEN-​1, para- thyroid disease in MEN-​2A is usually milder and has a later onset. Surgical management of primary hyperparathyroidism in MEN-​2A is similar to that in MEN-​1. During thyroidectomy in MEN-​2A, en- larged parathyroid glands in a normocalcaemic individual should be evaluated and removed if necessary. Genetics of MEN-​2 In contrast to MEN1, which is a tumour suppressor gene, RET is an oncogene. RET has 21 exons and encodes a membrane tyrosine kinase receptor protein called RET. Normal RET is expressed mainly in developing and adult neural ectoderm and comprises extracel- lular, transmembrane, and intracellular regions. The extracellular region contains four cadherin-​like domains and a juxtamembrane cysteine-​rich region. Two tyrosine kinase domains located in the intracellular region are involved in the activation of numerous intra- cellular signal transduction pathways. Mutations involving exons 8, 10, 11, 13, 14, 15, and 16 have been identified in patients with MEN-​2A, MEN-​2B, and familial medullary thyroid carcinoma. These exons should therefore be routinely screened for RET muta- tions. MEN-​2A and FMTC mutations usually affect the extracellular cysteine-​rich domain, whereas those associated with MEN-​2B most frequently involve the intracellular tyrosine kinase domains of RET. In less than 95% of cases of MEN-​2A, codons 609, 611, 618, 620, 634 are affected. More than 95% of cases of MEN-​2B result from the M918T mutation, with less than 5% due to A883F mutations. Generally, mutations associated with FMTC are distributed among the six cysteine codons. In contrast to MEN-​1, MEN-​2 displays a strong genotype–​phenotype correlation and mutations are identi- fied in more than 95% of patients. Genetic screening and management Early recognition of carriers of RET mutations can prevent and cure medullary thyroid cancer, by enabling prophylactic thyroidectomy before metastatic spread. Genetic testing for germline RET mutations is performed in blood leucocytes. Since there is a strong correlation between the specific RET codon mutation and the aggressiveness of the tumour, decisions regarding prophylactic thyroidectomy are based on the RET mutation identified. Genetic testing for germline RET mutations is recommended in all children with a parent known to have MEN-​2. Based on the location and nature of the muta- tion, these patients can be triaged into risk categories according to the American Thyroid Association’s classification, in order of highest to lowest risks: ATA-​HST, ATA-​H, ATA-​MOD. Those chil- dren carrying the most common MEN-​2B mutation M918T have the highest risk of aggressive MTC (risk category ATA-​HST) and total thyroidectomy with central node dissection is recommended within the first 6 months of life. This surgery should be performed by age 5 years in carriers of RET codon mutations 611, 618, 620, and 634. Those in the ATA-​H category (e.g. mutations in codons 634 and 883) should be considered for total thyroidectomy at or before 5 years or age. Those in the ATA-​MOD category (e.g. mutations af- fecting codons 533, 609, 611, 620, 631, 666, 768, 790, 804, 891, and 892) may be considered for prophylactic surgery later on in life or following first detection of abnormal stimulated calcitonin. Genetic screening may also be useful in the management of MEN-​2-​associated phaeochromocytoma. Individuals with RET mutations associated with a high risk of developing phaeochromo- cytoma should have annual measurement of urinary metanephrines or catecholamines. Other MEN syndromes In addition to MEN-​1 and MEN-​2, there are four other MEN syndromes. Carney complex (OMIM 160980)  is a rare syn- drome characterized by myxomas (cutaneous, mucosal, and car- diac), spotty skin pigmentation (lentiginosis), primary pigmented adrenocortical disease, and pituitary adenomas. A  diagnosis of McCune–​Albright syndrome (OMIM 174800)  requires at least two features of the triad of polyostotic fibrous dysplasia, café-​au-​ lait skin pigmentation, and autonomous endocrine hyperfunction. Patients with Carney complex or McCune–​Albright syndrome have mild hypersomatomammotropinemia (excess growth hor- mone secretion) starting in adolescence. In both disorders, pituitary hyperplasia appears to precede tumour development. Patients with neurofibromatosis type 1 (NF1; OMIM 162200) are predisposed to neuroendocrine tumours including phaeochromocytomas and duo- denal somatostatinomas. As previously described, VHL syndrome (OMIM 193300) is associated with phaeochromoytomas and pan- creatic neuroendocrine tumours. Finally, familial paraganglioma syndromes (OMIM 168000, 601650, 605373, 115310, 614165) are as- sociated with mutations in the succinate dehydrogenase or SDHAF2 genes, and present with phaeochromocytomas, paragangliomas, and occasionally renal cell carcinomas and gastrointestinal stromal tumours. FURTHER READING Herrera MF, et  al. (2015). AACE/​ACE disease state clinical re- view:  pancreatic neuroendocrine incidentalomas. Endocr Pract, 21, 546–​53.

13.8  Pancreatic endocrine disorders and multiple endocrine neoplasia 2463 Jensen RT, et  al.; Barcelona Consensus Conference participants (2012). ENETS Consensus Guidelines for the management of patients with digestive neuroendocrine neoplasms:  functional pancreatic endocrine tumor syndromes. Neuroendocrinology, 95, 98–119. Kaderli RM, et al. (2019). Therapeutic options for neuroendocrine tumours: a systematic review and network meta-analysis. JAMA Oncol, 5, 480–9. Kaltsas G, et al. (2010). Paraneoplastic syndromes secondary to neuro- endocrine tumours. Endocr Relat Cancer, 17, R173–​93. Kaltsas GA, et al. (2005). Treatment of advanced neuroendocrine tu- mours with radiolabelled somatostatin analogues. Endocr Relat Cancer, 12, 683–​99. Modlin IM, et al. (2008). Gastroenteropancreatic neuroendocrine tu- mours. Lancet Oncol, 9, 61–​72. Pavel M, et al.; Barcelona Consensus Conference participants (2012). ENETS Consensus Guidelines for the management of patients with liver and other distant metastases from neuroendocrine neoplasms of foregut, midgut, hindgut, and unknown primary. Neuroendocrinology, 95, 157–76. Ramachandran R, et al. (2015). Comparison of the utility of cocaine-​ and amphetamine-​regulated transcript (CART), chromogranin A, and chromogranin B in neuroendocrine tumor diagnosis and assess- ment of disease progression. J Clin Endocrinol Metab, 100, 1520–​8. Thakker RV (2014). Multiple endocrine neoplasia type 1 (MEN1) and type 4 (MEN4). Mol Cell Endocrinol, 386, 2–​15. Thakker RV, et al.; Endocrine Society (2012). Clinical practice guide- lines for multiple endocrine neoplasia type 1 (MEN1). J Clin Endocrinol Metab, 97, 2990–​3011. Toumpanakis C, Caplin ME (2013). Update on the role of somatostatin analogs for the treatment of patients with gastroenteropancreatic neuroendocrine tumors. Semin Oncol, 40, 56–​68. Wells SA, et  al.; American Thyroid Association Guidelines Task Force on Medullary Thyroid Carcinoma (2015). Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid, 25, 567–​610.

13.9 Diabetes and hypoglycaemia 2464

13.9 Diabetes and hypoglycaemia 2464

13.9.1 Diabetes 2464

13.9.1 Diabetes 2464

CONTENTS 13.9.1 Diabetes  2464 Colin Dayan and Julia Platts 13.9.2 Hypoglycaemia  2531 Mark Evans and Ben Challis 13.9.1  Diabetes Colin Dayan and Julia Platts ESSENTIALS Diabetes mellitus can be defined as a state of chronic hyperglycaemia sufficient to cause long-​term damage to specific tissues, notably the retina, kidney, nerves, and arteries. It is due to inadequate produc- tion of insulin and/​or ‘resistance’ to the glucose lowering and other actions of insulin, and is a significant and growing threat to global health, affecting more than 400 million people worldwide. Definitions—​normal fasting blood glucose concentration is in the range 3.5 to 5.5 mmol/​litre, and even large carbohydrate loads do not raise the concentration above 8 mmol/​litre. Widely accepted diag- nostic criteria for diabetes and other hyperglycaemic states are (1) dia- betes mellitus—​fasting glucose more than 7.0 mmol/​litre (126 mg/​dl) and/​or a value exceeding 11.1 mmol/​litre (199 mg/​dl), either at 2 h during a 75-​g oral glucose tolerance test or in a random sample; or an HbA1c value in a standardized assay of more than 48 mmol/​mol; (2) impaired glucose tolerance—​2-​h oral glucose tolerance test value between 7.8 and 11.1 mmol/​litre (140–​199 mg/​dl); (3) impaired fasting glucose—​fasting glucose 5.6 to 6.9 mmol/​litre (100–​125 mg/​dl). Impaired glucose tolerance is a not a stable state: within 5 years, about 25% of subjects deteriorate into type 2 diabetes, while a further 25% revert to normoglycaemia. Type 1 diabetes This condition, previously referred to as ‘juvenile-​onset’ or ‘insulin-​ dependent’ diabetes, most commonly develops in childhood, with highest incidence in northern European countries, and accounts for 5 to 15% of all cases of diabetes. Aetiology—​type 1 diabetes is caused by an autoimmune, pre- dominantly T-​cell-​mediated process that selectively destroys the pancreatic β cells. Genetic factors explain 30 to 40% of total sus- ceptibility: at least 59 loci are involved, with the HLA class II locus IDDM1 having by far the greatest effect. Environmental factors that have been implicated but not confirmed include viral infection (par- ticularly coxsackie B), bovine serum albumin from cow’s milk (by immunological cross-​reactivity), and other toxins. Notable β-​cell selective autoantibodies that are commonly found are those that recognize GAD65 (a heat shock protein), IA-​2 (a protein tyrosine phosphatase-​like molecule), ZnT8 (a zinc transporter molecule) and insulin itself, but these are clearly not the immediate cause of the disease. Several years of progressive autoimmune damage usually precede the clinical onset of diabetes. Pathogenesis—​in untreated type 1 diabetes at diagnosis, insulin concentrations are generally 10 to 50% of non​diabetic levels in the face of hyperglycaemia which would normally greatly increase insulin secretion. Such severe deficiency cannot sustain the normal anabolic effects of insulin and leads to runaway catabolism in carbohydrate, fat, and protein metabolism. A similar clinical picture of insulin de- pendence can be caused by other forms of severe pancreatic damage. Clinical features—​classical presentation of untreated or poorly controlled type 1 diabetes is with onset over days or a few weeks of polyuria (caused by osmotic diuresis due to hyperglycaemia), thirst, weight loss, and general tiredness/​malaise. Other features can include blurred vision (due to hyperglycaemia-​related refractive changes in the lens), infection (particularly genital candidiasis), and diabetic keto- acidosis. Chronic diabetic complications are not seen at presentation. Type 2 diabetes Type 2 diabetes (previously referred to as ‘non-​insulin-​dependent’ or ‘maturity-​onset’) is a heterogeneous condition, diagnosed em- pirically by the absence of features suggesting type 1 diabetes. It is most commonly diagnosed in those over 40 years of age, with peak incidence between 45 and 64 years, but it is increasingly being diag- nosed in children. Type 2 diabetes accounts for 85 to 90% of diabetes worldwide, but with striking geographical variation (prevalence <1% in rural China, 50% in Pima Indians of New Mexico). Currently more than three-​quarters of people with diabetes live in low-​ or middle-​ income countries. 13.9 Diabetes and hypoglycaemia

13.9.1  Diabetes 2465 Aetiology—​type 2 diabetes is due to the combination of insulin re- sistance and β-​cell failure. Genetic factors explain 60 to 90% of total susceptibility, with a polygenic pattern reflecting the inheritance of a critical mass of minor diabetogenic polymorphisms in genes that in- fluence insulin secretion, insulin resistance, pancreatic development, and obesity. An important specific risk factor for type 2 diabetes, which aggravates insulin resistance, is obesity—​particularly if this develops after the early twenties, and especially around the waist. Increasing rates of obesity and sedentary activity has led to a very marked (2–​3-​fold) increase in incidence of type 2 diabetes world- wide, especially in developing countries. The mechanism of β-​cell failure in human type 2 diabetes is not known. Clinical features—​in type 2 diabetes significant hyperglycaemia may have been present for several years at the time of diagnosis, hence cases are often discovered by screening or at routine health checks. Many cases present with classical symptoms of osmotic diuresis, blurred vision, and genital candidiasis. The hyperosmolar non​ketotic state can present with confusion or coma, but diabetic ketoacidosis is rare. Chronic diabetic complications may be a presenting feature. Monogenic and other types of diabetes Maturity-​onset diabetes of the young (MODY, now referred to as monogenic diabetes)—​is most often caused by mutations in the genes for glucokinase (MODY2) and HNF-​1α (MODY3). This diag- nosis should be considered if there is a family history of young-​ onset diabetes in more than one generation, with at least one family member diagnosed under the age of 25; affected members are not markedly obese; there is no evidence of insulin resistance; fasting C-​peptide is detectable and within the normal range; islet cell or anti-​GAD autoantibodies are absent; other associated features may be present (e.g. renal cysts in HNF-​1β, deafness in mitochondrial genetic diabetes). The likelihood of monogenic diabetes can be pre- dicted from an online risk calculator (https://​www.diabetesgenes. org). Individuals are often mistakenly treated with insulin when they would benefit from other treatments such as sulfonylureas (HNF-​1α) or do not require treatment (glucokinase). Other types of diabetes include those related to pancreatic disease (chronic pancreatitis, cystic fibrosis, haemochromatosis) and gesta- tional diabetes. Management of diabetes General aspects—​management requires tackling cardiovascular risk factors and obesity in addition to hyperglycaemia. Important issues include (1)  dietary modification—​reducing total energy in- take in patients who are overweight (body mass index >28 kg/​m2), improving dietary composition (fat <30% total energy intake, with saturated animal fat <10%; carbohydrates—​preferably pulses, root/​ leaf vegetables and fruit—​>55% total energy intake; sodium <6 g/​day); (2) increasing physical activity; (3) smoking cessation; and in some patients (4) antiobesity drugs and/​or bariatric surgery. Glucose-​lowering drugs—​these include (1)  insulin—​soluble (regular, or short-​acting) insulin injected subcutaneously begins to lower glucose within 30 min, has a peak effect between 1 and 2 h and lasts 3 to 5 h; long-​acting preparations (e.g. isophane and lente insulins) are used to cover basal insulin requirements; (2) in- sulin analogues—​have improved physicochemical characteristics for subcutaneous absorption and can be fast acting (e.g. insulin lispro and insulin aspart), or long acting (e.g. insulin glargine (Lantus), insulin detemir (Levemir) insulin degludec (Tresiba)); (3) oral hypo- glycaemic agents—​(a) sulphonylureas and meglitinides—​insulin secretagogues; (b) metformin—​a biguanide that acts primarily by inhibiting gluconeogenesis in the liver; (c) thiazolidinediones—​act to improve insulin sensitivity; (d) α-​glucosidase inhibitors—​partly block digestion of complex carbohydrates and so damp postprandial gly- caemic rises, but are of low efficacy and poorly tolerated; (e) incretin mimetics—​augment insulin secretion—​GLP-​1 analogues or inhibi- tors of the enzyme that breakdown GLP-​1, dipeptidyl peptidase (DPP IV)—​gliptins (f) renal glucose transporter (SGLT2) inhibitors—​ gliflozins—​that result in renal glucose wasting. Type 1 diabetes—​patients must be given insulin immediately and for life. Standard treatment involves giving a short-​acting insulin 20 to 30 min before eating or a fast-​acting insulin immediately before eating, and a twice (sometimes once) daily dose of a long-​acting insulin. Common practice is to commence with low dosages of long-​acting insulin (e.g. 8–​12 U in the morning and 4–​6 U at night), with short/​fast-​acting insulin then added to cover excessive pran- dial hyperglycaemia. Premixed insulins (e.g. 30% short-​acting with 70% long-​acting) can be given twice daily and are more convenient than giving short-​ and long-​acting insulins separately, but they lack flexibility. Administration is usually by conventional syringes or pen injection devices, but pumps can be used to administer continuous subcutaneous infusions of insulin with greatly enhanced flexibility. Type 2 diabetes—​remission of type 2 diabetes can be achieved by significant weight loss (e.g. >15 kg) and first line management is by appropriate diet and increased exercise. The first-​line oral hypogly- caemic agent for so-​called ‘dietary failure’ is metformin, with a (usually) sulphonylurea or (sometimes) thiazolidinedione added as second-​line treatment. A once-​daily dose of a long-​acting insulin can be combined effectively with metformin. Insulin therapy can range from once-​ or twice-​daily long-​acting insulin in subjects with residual insulin, to the more intensified basal and prandial regimens used in type 1 diabetes (>200 U/​day may be required in very obese, insulin-​resistant patients). However, DPPIV inhibitors (glitazones), GLP-1 analogues and SGLT2 inhibitors are increasingly being used second-​line as an alternative to insulin therapy to avoid weight gain and hypoglycaemia. Treatment targets for blood glucose—​these have been selected to reduce the risk of chronic diabetic complications. Avoiding acute episodes of hyper-​ and hypoglycaemia is also important. In most patients the treatment goal should be to reduce HbA1c to <53 mmol/mol (<7.0%), but a target of 42–48 mmol/litre (6.0–6.5%) can be employed in selected patients (e.g. short disease duration, long life expectany) if achievable without significant hypoglycaemia, and a target of <64 mmol/mol (<8.0%) may be appropriate for some (e.g. limited life expectancy). Multidisciplinary care—​diabetes is best managed by the combined efforts of a well-​trained primary care team and a team of special- ists with complementary and overlapping skills: physician, specialist diabetes nurse, dietitian, and chiropodist. Patients require education about diabetes, with key elements including (1) causes of hypergly- caemia and diabetic symptoms; (2) own treatment—​diet and lifestyle; drawing up and injecting insulin; oral agents; recognizing and treating hypoglycaemia ‘hypos’; (3) self-​monitoring technique—​targets and danger levels; how to respond to poor control; (4) ‘sick-​day’ rules—​ monitoring during intercurrent illness; how to adjust own treatment; when and how to call for help (never stop taking your insulin; check

section 13  Endocrine disorders 2466 your blood glucose every 4 h; test your urine for ketones; call for help if you start vomiting, have glucose over 15 mmol/​litre that does not come down after insulin, get hypos, get ketones in the urine, are wor- ried, and do not know what to do). Acute metabolic complications of diabetes Diabetic ketoacidosis—​uncontrolled hyperglycaemia with hyper­ ketonaemia severe enough to cause metabolic acidosis. Precipitating factors include new presentation of type 1 diabetes, omission or underdosing of insulin by patients known to have type 1 diabetes, the use of SGLT2 inhibitors and intercurrent illness (compounded by failure to monitor blood glucose and take appropriate action). Usual presentation is with classical hyperglycaemic symptoms together with acidotic (Kussmaul) breathing and ketotic foetor, vomiting, evidence of dehydration and hypovolaemia, and signs of any precipitating con- dition. Drowsiness and coma are late features. Diagnosis is confirmed with a finger-​prick blood glucose measurement and urine or blood analysis for ketones: other investigations should include a biochem- ical screen, full sepsis screen, arterial blood gas analysis, and ECG. Management requires (1) fluid replacement—​usually with 0.9% saline
(typically 1–​2 litres in 2 h, then 1 litre in 4 h, then 4 litres in next 24 h); (2) potassium replacement—​typically 40 mmol of KCl to each litre of intravenous fluid if K+ is normal (3.5–​5.0 mmol/​litre), but adjusted in response to frequent monitoring; (3) intravenous insulin—​initially
at a rate of 6 U/​h or 0.1 U/​kg, and continued (if necessary in combin- ation with 10% dextrose infusion) until ketosis has resolved (blood ketones <0.6  mmol/​litre); (4)  treatment, when possible, of any precipitating condition. Intravenous fluids and insulin can be discon- tinued when the patient can eat and drink, and they can be restarted on their usual insulin regimen (or a typical maintenance regimen can be introduced). Hyperosmolar Hyperglycaemic (formerly known as non​ketotic) state—​is distinguished from diabetic ketoacidosis by the absence (be- cause circulating insulin levels are high enough to suppress lipolysis and ketogenesis) of marked hyperketonaemia and metabolic acid- osis. Presentation is typically with classical hyperglycaemic symp- toms; confusion, drowsiness, and coma are more common than in diabetic ketoacidosis. Typical biochemical features include severe hyperglycaemia (>30 mmol/​litre) and hypernatraemia (sodium often

155 mmol/​litre). Management is largely as for diabetic ketoacidosis, excepting that (1) 0.45% saline is often given if plasma sodium is over 150 mmol/​litre or osmolality over 350 mosmol/​kg; (2) intravenous insulin infusion at low doses (0.05 IU/​kg/​hr) rapidly controls hyper- glycaemia in most cases—​indeed recent guidelines suggest initially treating with aggressive fluid replacement alone (3–​6 litres in the first 12 hours), and only introducing insulin if the glucose if falling at less than 5 mmol/​litre per hour; (3) the risk of thrombotic events is particularly high, hence prophylactic doses of low molecular weight heparin should be given. Hypoglycaemia—​an inevitable side effect of antidiabetic drugs that raise circulating insulin levels. Typical features include (1) auto- nomic symptoms—​pallor, sweating, tremor, and tachycardia, and (2) symptoms of neuroglycopenia—​commonly drowsiness, confu- sion, incoordination, and dysarthria, but also automatic or disinhib- ited behaviour and focal neurological deficits. Diagnosis is confirmed with a finger-​prick blood glucose measurement below 3.5 mmol/​litre in an appropriate clinical context. Treatment is with (1) oral glucose or sucrose or other carbohydrate—​if the patient can swallow safely; or (2) intravenous glucose (15–​20 g as 10% or 50% solution) or intra- muscular glucagon (1 mg)—​if the patient is not able to swallow safely. Chronic complications of diabetes Long-​term tissue damage is the major burden of diabetes, the greatest source of fear for people living with diabetes, and the most expensive item in the diabetes healthcare budget. Pathogenesis—​possible mechanisms for diabetic complications include glycation of proteins and macromolecules, overactivity of the polyol pathway, activation of protein kinase C and abnormal microvascular blood flow. Microvascular complications—​retinopathy, neuropathy, and nephropathy—​are specific to diabetes and reflect damage inflicted on the microcirculation throughout the body. Macrovascular disease is atherosclerosis, which behaves more ag- gressively than in non​diabetic people, and causes typical coronary heart disease, stroke, and peripheral arterial disease. Diabetic eye disease—​is the commonest cause of blindness in people of working age in most Westernized countries, although blind- ness rates are falling following the introduction of retinal screening programmes. Stages of diabetic retinopathy are (1)  background—​ microaneurysms, hard exudates, haemorrhages (flame, dot, blot), cotton wool spots (<5); (2)  preproliferative—​rapid increase in microaneurysms, intraretinal microvascular abnormalities, multiple deep haemorrhages, cotton wool spots (>5), venous beading/​loops/​ duplication; (3) proliferative—​new vessels on the disc or elsewhere, fibrous proliferation on the disc or elsewhere, preretinal or vitreous haemorrhages; (4) advanced eye disease—​retinal detachment, retinal tears, rubeosis iridis, neovascular glaucoma. Disease of the macula (maculopathy), serious enough to affect central vision, can accom- pany any stage of diabetic retinopathy including background, and may be present in newly diagnosed type 2 patients. Management requires (1) general preventive measures—​tight glycaemic control, control of hypertension, stopping smoking, regular (annual) eye screening; and (2) specific treatments—​laser photocoagulation can preserve useful vision in many cases of proliferative retinopathy and maculopathy. Intraocular (intravitreal) injections of vascular endo- thelial growth factor (VEGF) blocking agents have been shown to improve vision in diabetic maculopathy. Diabetic neuropathies—​recognized clinically distinct syndromes include (1) diffuse symmetrical polyneuropathy—​classically a distal ‘glove and stocking’ peripheral polyneuropathy that affects all sizes of sensory and motor fibres; (2) autonomic neuropathy—​manifest as sexual difficulties (erectile failure, ejaculatory failure), postural hypotension, disturbed gastrointestinal motility, abnormal sweating, neuropathic bladder, abnormal blood flow, sudden unexplained death; (3) acute mononeuropathy; (4) diabetic amyotrophy; (5) cra- nial and other nerve palsies. Management is difficult: specific treat- ments have so far been disappointing. Numb feet are at greatly increased risk of ulceration and require sensible shoes and good foot care. Poor glycaemic control should be corrected. Pain may be diffi- cult to treat: simple analgesics are generally ineffective but drugs for neuropathic pain including specific antidepressants and anticonvul- sants are of more value. Autonomic neuropathic symptoms may be treated as follows: (1) erectile failure with oral phosphodiesterase type 5 inhibitors (e.g. sildenafil); (2) postural hypotension with compres- sion stockings, fludrocortisone, and/​or midodrine; (3) gastroparesis with erythromycin, metoclopramide or domperidone; (4)  exces- sive sweating with oral clonidine or topical glycopyrrolate cream;

13.9.1  Diabetes 2467 (5) neuropathic bladder with regular bladder training, but intermit- tent self-​catheterization may be needed. Diabetic nephropathy—​see Chapter 21.10.1. Macrovascular disease—​(1) dyslipidaemia—​first-​line treatment is with statins, ideally aiming to reduce low-​density lipoprotein chol- esterol (LDL-C) to below 1.8 mmol/​litre (approximately equivalent to 2.5  mmol/​litre non-​high-​density lipoprotein-​c cholesterol), especially in those with overt cardiovascular disease or neph- ropathy (2)  hypertension—​clinic blood pressure should be re- duced to a target of less than 140/85–90 mmHg in most patients (130/80 mmHg may be appropriate in some), with angiotensin-​ converting enzyme (ACE) inhibitors often recommended as first line; (3) coronary heart disease—​there should be a low threshold for referring patients with diabetes presenting with typical or atypical chest pain suggestive of angina for further evaluation (as coronary ischaemia is often asymptomatic of with minimal chest pain in dia- betes); (4) stroke—​investigation and management are conventional; (5) peripheral vascular disease—​investigation and management are conventional. Diabetic foot disease—​ulceration and severe ischaemia leading to gangrene of the toes or forefoot are the commonest problems. Many problems can be avoided by teaching the patients basic foot care, by regularly checking their feet (especially the soles of the feet) and shoes, and by providing prophylactic podiatry and special foot- wear as appropriate. Typical manifestations include (1) neuropathic ulcers—​occur at high-​pressure sites (heel, metatarsal heads) and ap- pear cleanly punched out of the surrounding callus; (2) ischaemic ulcers—​tend to affect the edges of the foot and toes; (3) traumatic damage (e.g. symmetrical damage across the toes and margins of the feet from tight shoes); with (4) all lesions prone to be complicated by infection (including osteomyelitis). Management requires the prevention of further trauma, treatment of infection, and optimiza- tion of the circulation. Charcot’s arthropathy most commonly affects the ankle and joints in the mid-​ and forefoot, which in advanced cases degenerate (usually painlessly) into a ‘bag of bones’: treatment is often unsatisfactory—​offloading pressure with a plaster-​cast boot may temporarily halt bone destruction; bisphosphonate infusions may slow the disease process by inhibiting osteoclast activity but the evidence base is weak. Introduction Diabetes mellitus can be defined as a state of chronic hypergly- caemia sufficient to cause long-​term damage to specific tissues, notably the retina, kidney, nerves, and arteries, but this functional label gives little insight into the long and colourful history of this disease, its clinical and scientific importance, or its immense per- sonal and socioeconomic impact. Diabetes was recognized in an- tiquity, and its clinical features (with empirical treatment guidelines) were recorded over 3500 years ago in the Egyptian Ebers papyrus. Our understanding of the disease has advanced greatly, especially during the last two decades, but many aspects of its management remain imperfect. The American Diabetes Association has pro- posed a generally accepted classification of diabetes mellitus into four types: type 1 is associated with β-​cell destruction leading to ab- solute deficiency of insulin, is immune-​mediated and of unknown root cause; type 2 is associated with a relative insulin deficiency and insulin resistance—​a range of abnormalities occur, and in some pa- tients a secretory defect predominates; gestational diabetes; and the fourth type is ‘other specific types of diabetes’ is used to encompass diabetes caused by specific defects, other endocrine abnormalities, and drug-​induced diabetes, and accounts for about 5% of patients. The incidence of all types of diabetes is rising. Extensive studies have shown a strong relationship between glycaemic control, the fraction of glycated haemoglobin (HbA1c), and disease outcomes. Treatment of raised blood pressure and lowering blood lipids also contributes to improved outcomes for nephropathy, retinopathy, myocardial infarction, and stroke. In both type 1 and type 2 dia- betes, there are compelling data to show that outcomes are improved by intensive therapy: better glycaemic control appears to have bene- fits on macrovascular complications in very long-​term studies, and lowering blood pressure has significant and more immediate bene- fits on both small-​vessel (microvascular) and macrovascular disease. The use of SGLT2 inhibitor drugs reduces the risk of death from heart failure in those at high risk. Diabetes is a significant and growing threat to global health. Worldwide, diabetes affects more than 400  million people. This number was more than 10-​fold less in 1985 (30 million) and the world prevalence is predicted to reach 640 million by 2040, with 10% of all adults affected. Diagnosis of diabetes Blood glucose concentrations are normally tightly regulated: fasting values lie between 3.5 and 5.5 mmol/​litre and even large carbohy- drate loads do not raise the concentration more than 8 mmol/​litre. It is logical to define diabetes by the blood glucose concentrations which cause the chronic complications of the disease, but the choice of the diagnostic glucose levels has been contentious (and has stirred up much passion among epidemiologists). One difficulty is that some diabetic complications show a ‘threshold’ effect with the risk rising above a cut-​off level (e.g. fasting plasma glucose of >7 mmol/​ litre for retinopathy), whereas macrovascular disease (atheroma) and the complications of pregnancy do not (see later). Another problem is that even the current criteria are not self-​consistent (e.g. up to 30% of patients with a diagnostic raised fasting glucose will have a 2 h value in the glucose tolerance test that is below the diag- nostic cut-​off). The current diagnostic criteria for diabetes and other hypergly- caemic states (see Fig. 13.9.1.1) based on blood glucose levels have been approved by the World Health Organization (WHO) and most national diabetes associations. All values refer to venous plasma glu- cose concentrations (see next). Since 2011, the American Diabetes Association and the WHO have additionally proposed that HbA1c measurements can be used, which are more convenient as they can be measured at any time of day, and that measurement of fasting glucose is not required. • Diabetes mellitus:  fasting glucose greater than 7.0 mmol/​litre (126 mg/​dl) and/​or a value exceeding 11.1 mmol/​litre, either at 2 h during a 75 g oral glucose tolerance test or in a random sample. The corresponding levels in non-​SI units are 126 and 200 mg/​dl, respectively. The diagnostic fasting glucose level was lowered from the previous value of 7.8 mmol/​litre to reflect more accurately the risk of developing diabetic retinopathy. The HbA1c range used

section 13  Endocrine disorders 2468 to diagnose diabetes is HbA1c in excess of 48 mmol/​mol (6.5%). Note that values less than 48 mmol/​mol do not exclude diabetes if the glucose values indicate diabetes. • Impaired glucose tolerance (WHO): 2 h oral glucose tolerance test value between 7.8 and 11.1 mmol/​litre (140–​199 mg/​dl). • Impaired fasting glucose:  fasting glucose 5.6 to 6.9 mmol/​litre (100–​125 mg/​dl). The lower value for this range was reduced from 6.0 mmol/​litre to 5.6 mmol/​litre by the American Diabetes Association in 2003. The HbA1c range 39–​46  mmol/​mol (5.7—​6.4%) has been proposed to correspond to impaired fasting glucose. Impaired glucose tolerance (IGT) and the more recently distin- guished impaired fasting glucose (IFG) are intermediate categories of hyperglycaemia (sometimes referred to as ‘prediabetes’) that carry definite risks and so require follow-​up and risk-​factor management (see next). They are often transient stages and overlap to some ex- tent: about one-​third of subjects with impaired fasting glucose also have impaired glucose tolerance, while one-​quarter of those with impaired glucose tolerance also show impaired fasting glucose. Recent criteria put much emphasis on the fasting plasma glucose concentration. However, the time-​consuming oral glucose tolerance test is still required in some cases with borderline fasting hypergly- caemia, because the 2-​h oral glucose tolerance test value in such patients may be high enough to put them at risk of microvascular complications. Moreover, the oral glucose tolerance test remains the only way to define impaired glucose tolerance. However, the use of HbA1c measurements is being increasingly accepted as a con- venient method of screening for diabetes. At an HbA1c cut-​off level of 48 mmol/​mol (6.5%), up to 30% of cases of diabetes positive on fasting glucose would be missed, but it is argued that the increased practicality of the tests means that overall more people will be tested, and hence more patients will be correctly diagnosed. Practical screening and diagnostic procedures Figure 13.9.1.2 shows an algorithmic approach to screening for and diagnosis of diabetes and its associated hyperglycaemic states. Certain high-​risk groups need to be actively screened for type 2 dia- betes, which may be present (and causing complications) for several years before it is noticed. Risk factors include having a first degree Venous plasma glucose (mmol/litre) Diabetes 11.1 IGT Random 12 10 8 6 4 2 0 Fasting 7.8 7.0 IFG 2 h after OGTT Diabetes Diabetes 11.1 5.6 Fig. 13.9.1.1  Diagnostic thresholds for diabetes, impaired glucose tolerance (IGT), and impaired fasting glucose (IFG). For conversion to mg/​ dl, multiply values in mmol/​litre by 18. Source data from Genuth S, et al. (2003). Follow-​up report on the diagnosis of diabetes mellitus. Diabetes Care, 26, 3160–​67. Fig. 13.9.1.2  Screening algorithm for diagnosing diabetes, impaired glucose tolerance, and impaired fasting glucose. All glucose values relate to venous plasma (mmol/​litre). Adapted from data in Shaw JE, Zimmet P (2000). Do we know how to diagnose diabetes and do we need to screen for the disease? In: Gill GV, Pickup JC, Williams G, (eds) Difficult diabetes, pp. 3–​21. Blackwell Science, Oxford.

13.9.1  Diabetes 2469 relative with type 2 diabetes, body mass index (BMI) more than 25, racial origin (including south Asian and Afro-​Caribbean descent), high-​density lipoprotein (HDL) less than 0.9 mmol/​litre, triglyceride more than 2.8 mmol/​litre, and giving birth to a baby weighing more than 4 kg. The American Diabetes Association (ADA) recommend three-​yearly screening for everyone age more than 45  years and for people with one or more risk factors aged more than 25 years. However, this would be a huge challenge for healthcare systems and would still miss many young-​onset cases. At present no screening system has been universally adopted, and a ‘low threshold for oppor- tunistic screening’ approach is widely practised. Diabetes is not a trivial diagnosis, and certain practical points must be carefully observed: • Glucose should be measured in venous plasma using a quality-​ controlled laboratory method. Capillary (finger-​prick) samples contain higher glucose levels than venous blood, from which glu- cose has been extracted by the tissue bed; whole-​blood glucose levels are lower than in plasma, because red cells actively metab- olize glucose and so contain only low concentrations. These dif- ferences may reach 0.5 to 1.0 mmol/​litre. Portable glucose meters correlate well with laboratory glucose methods, but because of po- tential technical errors they should not be used to make or refute the diagnosis. • An oral glucose tolerance test is indicated for borderline hypergly- caemia (Fig. 13.9.1.2). After an overnight fast, the subject drinks 75 g of anhydrous glucose dissolved in 250 ml water (or 419 ml of a glucose drink such as Lucozade Energy Original—​73 kcal/​ 100 ml); venous blood is sampled at baseline and 2 h later. Food intake should be normal during the preceding few days: poor nu- trition can cause delayed hyperglycaemia with a raised 2 h value (the lag curve). • Abnormal values need confirmation. Postchallenge glucose levels in particular can vary considerably. Because of this and possible laboratory error, the diagnosis of diabetes should be verified using a further sample on another day unless there is a clear history of symptoms of hyperglycaemia confirming that this value is not a one-​off result. • HbA1c is now widely accepted as an alternative test for type 2 dia- betes diagnosis that does not require fasting or repeated samples. Values greater than 48  mmol/​mol (6.5%) are considered diag- nostic of diabetes and the range 38–​47  mmol/​mol (5.7–​6.5%) indicates increased risk of diabetes in the future (equivalent to impaired glucose tolerance). The limitations of this test should be borne in mind; the levels are affected by haemoglobinopathies and anaemia and in circumstances where the hyperglycaemia may be acute, such as type 1 diabetes and gestational diabetes, HbA1c should not be used as a test of exclusion of the diagnosis. Glycosuria depends on the renal threshold for glucose reabsorption and its presence does not necessarily indicate hyperglycaemia; con- versely, glucose may be absent from the urine in diabetic subjects who also have a high renal threshold. However, abnormal results with any of these tests suggest diabetes and indicate the need for formal blood glucose screening. Impaired glucose tolerance Impaired glucose tolerance is a not a stable state: within 5 years, about 25% of subjects with impaired glucose tolerance deteriorate into type 2 diabetes, while a further 25% revert to normoglycaemia. The degree of hyperglycaemia in impaired glucose tolerance falls, by definition, below the threshold for microvascular complications but is enough to predispose to cardiovascular disease (see later). Subjects found to have impaired glucose tolerance must be fol- lowed up because of the hazards of both diabetes and macrovascular disease. An oral glucose tolerance test or HbA1c should be repeated at least annually, and dietary and lifestyle advice given to decrease metabolic and cardiovascular risks; increased physical activity, a low-​fat diet and weight loss convincingly reduce both the pro- gression to type 2 diabetes (by 58%) and cardiovascular risk. Risk factors such as smoking, hypertension, dyslipidaemia, and obesity should be managed actively. Specific antihyperglycaemic treatments also reduce progression to type 2 diabetes—​metformin (24%), pioglitazone (effective in preventing type 2 diabetes following an episode of gestational diabetes)—​in addition to pharmacological (orlistat) or physical (bariatric surgery) weight-​loss interventions (see Chapter 11.6). These measures should be used in combination with lifestyle intervention, which is recommended for all subjects with impaired glucose tolerance. Impaired fasting glucose As with impaired glucose tolerance, the 5-​year risk of progressing to type 2 diabetes appears to be about 25%, and IFG predisposes to cardiovascular disease. Long-​term monitoring and management should therefore be as for impaired glucose tolerance. Metabolic basis of diabetes Diabetes is due to inadequate production of insulin and/​or ‘resistance’ to the glucose lowering and other actions of insulin. To put this in context, key aspects of normal metabolism will be briefly reviewed. The islets of Langerhans There are about 1  million islets of Langerhans in the normal adult: insulin is produced by the β cells, which make up the bulky core of each islet; β cells also synthesize the peptide known as amylin or islet-​associated polypeptide. The other islet cell types, mostly sur- rounding the β-​cell core, are the α cells that produce glucagon, the δ cells that produce somatostatin, and the pancreatic polypeptide (PP) cells that synthesize pancreatic polypeptide. All islet cells are derived embryologically from the buds of gut endoderm which also give rise to the exocrine pancreatic tissue. The various islet cell types communicate with each other through the hormones they secrete into the islet’s rich capillary plexus and probably by paracrine effects on adjacent cells; these inter- actions presumably regulate hormone secretion. Insulin inhibits release of glucagon, while glucagon powerfully stimulates insulin secretion—​an action exploited in the testing of β-​cell reserve (see next). Somatostatin suppresses the secretion of insulin and glu- cagon. Amylin can inhibit insulin and glucagon secretion as well as reduce appetite and gastric emptying. Its physiological role is uncer- tain but amylin analogues when used as pharmacotherapy have been shown to reduce weight as well as blood glucose levels. Amylin also polymerizes outside the β cell to produce fibrils of amyloid material, which have been implicated in the progressive β-​cell damage of type 2 diabetes.

section 13  Endocrine disorders 2470 Insulin Insulin is a 5800 Da protein made up of an A chain (21 amino acid residues) and a B chain (30 residues), joined covalently by two disul- phide bridges. The precursor molecule, proinsulin, consists of the A and B chains linked end-​to-​end through a connecting (C) peptide which is cleaved off during insulin processing. In the circulation, insulin is monomeric but in crystals and more concentrated solu- tions (e.g. in the insulin vial and the subcutaneous injection site), six insulin molecules self-​associate around a central Zn2+ ion. Self-​ association influences the pharmacokinetic properties of subcuta- neously injected insulin: the rate-​limiting dissociation of hexamers into monomers slows the absorption of even fast-​acting insulin. Insulin regulates metabolism in birds, fish, and reptiles as well as mammals, and its structure is remarkably well conserved across the phyla. Three species of insulin are used therapeutically; the human sequence differs from porcine at a single residue (B30) and from bovine at two others. These differences affect the pharmacokinetic and immunogenic characteristics of the insulins (see next). The physicochemical behaviour of insulin has been successfully ma- nipulated in synthetic ‘designer’ insulins that have improved absorp- tion profiles: modification of the C terminus of the B chain, a region crucial for self-​association, produces analogues that remain in the monomeric state and are therefore absorbed faster than the native soluble insulin (see later). Insulin biosynthesis and processing Insulin is a product of the INS gene, located on the short arm of chromosome 11, whose coding region contains three exons. Translation of INS mRNA in the rough endoplasmic reticulum pro- duces preproinsulin, which is successively cleaved during its passage through the Golgi vesicles and secretory vesicles to yield first pro- insulin and finally insulin and C-​peptide. Proinsulin is converted into insulin by the proteolytic excision of the C-​peptide chain; the two intermediate cleavage products (with either end of the C-​ peptide remaining attached to insulin) are called split products of proinsulin. Normally, almost all proinsulin is processed through this regulated pathway to yield equimolar amounts of insulin and C-​peptide. However, a constitutive pathway may predominate in dysfunctional β cells (e.g. in type 2 diabetes and insulinoma), when processing is not complete and large quantities of proinsulin and split products may be released into the circulation. C-​peptide is generally regarded as an inert by-​product of insulin production. However, its structure is also conserved across species and it may have vasoactive and other properties. Insulinopathies are point mutations in the INS gene which either produce a mutant insulin (e.g. insulin Chicago:  a phenylalanine for leucine substitution at residue B25) or interfere with one of the cleavage sites of proinsulin so that the mutant split product cannot be further processed (e.g. proinsulin Tokyo). These conditions are inherited as autosomal dominant traits; circulating insulin-​like or proinsulin-​like immunoreactivities may be extremely high, but glu- cose intolerance is often surprisingly mild. Insulin secretion Glucose is the main insulin secretagogue; this action of glucose is modulated by other ingested nutrients, by hormones released by the islets and the gut, and by the autonomic innervation of the islet. The process gives insight into the mode of action of the sulphonylureas and related drugs, and some of the causes of maturity-​onset diabetes of the young and neonatal diabetes (see next). Glucose-​stimulated insulin secretion The amount of insulin released by the normal β cell is tightly coupled to blood glucose levels and begins to increase immediately when blood glucose rises. The ability of the β cell to sense ambient glucose levels accurately and rapidly depends on the glucose transporter isoform GLUT-​2 and the glucose metabolizing enzyme glucokinase, while insulin release hinges on depolarization of the β-​cell mem- brane which is controlled by a specific ion channel, the ATP-​sensitive K+ channel. The characteristics of GLUT-​2 allow glucose at physio- logical concentrations to freely enter the β cell, where it is immedi- ately converted by glucokinase into glucose 6-​phosphate—​the point of entry into the glycolytic pathway which ultimately yields ATP; ATP production within the β cell is therefore proportionate to extra- cellular glucose. ATP binds to and closes the ATP-​dependent K+ channel; when open, this channel allows K+ ions to leave the β cell along their con- centration gradient and thus helps to maintain the negative charge inside the β-​cell membrane. ATP-​induced closure of the channel therefore causes K+ ions to accumulate within the cell and the mem- brane to depolarize, which triggers the opening of specific (voltage-​ gated) Ca2+ channels in the membrane. Ca2+ ions then flood into the β cell from the outside and activate the contractile proteins which drag the secretory vesicles containing insulin and C-​peptide to the cell surface. Here, the vesicles fuse with the cell membrane and re- lease their contents into the extracellular space (exocytosis), from where insulin and C-​peptide enter the islet capillaries. Other factors affecting insulin secretion Sulphonylureas induce insulin secretion by closing the same ATP-​ sensitive K+ channel as glucose: they bind to a specific sulphonylurea receptor (SUR1) linked to the K+ channel protein (called Kir 6.2). Repaglinide also closes this K+ channel, but binds to a different site from the sulphonylureas. By contrast, diazoxide locks the channel open, hyperpolarizing the β-​cell membrane and inhibiting insulin secretion—​hence its use in treating insulinoma. Glucagon and glucagon-​like peptide 1 7–​36 amide (GLP-​1; a gut peptide with insulin secretagogue (incretin) actions) both stimulate insulin secretion by raising cytosolic Ca2+ concentrations; binding to their receptors increases generation of cAMP which blocks re- moval of Ca2+ into intracellular organelles. Conversely, somatostatin and possibly amylin act to decrease production of cAMP and inhibit insulin secretion. Arginine stimulates insulin secretion, possibly by depolarizing the β-​cell membrane as it enters the cell (it is cationic). The autonomic nervous system is an important modulator of in- sulin secretion; it is stimulated by the parasympathetic (vagal) out- flow and inhibited by the sympathetic. Vagal stimulation is mediated by acetylcholine acting via muscarinic receptors, while the inhibi- tory sympathetic neurotransmitter is noradrenaline, interacting with α2-​adrenoceptors. Defects in insulin secretion due to mutations affecting glucokinase are responsible for 20% of cases of maturity-​onset dia- betes of the young (MODY), that is, glucokinase-​dependent MODY (MODY 2). This impairs ATP production from glucose, blunting

13.9.1  Diabetes 2471 the insulin response of the β cell to rising glucose and resulting in variable hyperglycaemia (see next). By contrast, familial neonatal hyperinsulinism is caused by inactivating mutations in ABCC8 (SUR1) or KCNJ11 (Kir6.2) that result in closure of the ATP-​sensitive K+ channel, leading to sustained insulin secretion and severe hypo- glycaemia soon after birth. Activating mutations of KCNJ11 (Kir6.2) cause impaired ATP-​sensitive K+ channel closure and have recently been shown to be a cause of persistent neonatal diabetes that can be treated with high-​dose sulphonylureas. Normal pattern of insulin secretion Insulin concentrations in peripheral blood show basal levels of about 10 mU/​litre (1 mU/​litre is approximately equivalent to 6.5 pmol/​ litre) that tend to fall overnight, on which are superimposed pran- dial peaks reaching 80 to 100 mU/​litre, roughly proportionate to the amount eaten. The prandial peaks are elicited by the insulin secreta- gogue effects of glucose and other nutrients, augmented by incretin gut peptides (such as GLP-​1) and the vagal outflow (the early ceph- alic phase of insulin release). Very frequent sampling (every minute) shows that ‘basal’ insulin secretion is in fact pulsatile, with clear but low-​amplitude peaks every 9 to 13 min. This may help to keep the target tissues sensitive to insulin; loss of this pulsatility is an early sign of β-​cell dysfunction in type 2 diabetes. An acute insulin secretagogue challenge (e.g. an intravenous glucose bolus) induces a sharp ‘first-​phase’ insulin peak, loss of which is another early abnormality in type 2 diabetes. The insulin response elicited by eating is larger than when an equivalent nutrient load is given intravenously. This is because glu- cose entering the gut stimulates neuroendocrine cells in the gut wall to release ‘incretin’ hormones which act on the β cell to enhance insulin secretion (the enteroinsular axis: see Chapter 15.9.1). An im- portant incretin appears to be GLP-​1, a product of alternative pro- cessing of the preproglucagon gene (glucagon itself is not produced, in contrast to in the islet α cell). GLP-​1 released from the small intes- tine augments insulin release in the presence of glucose, slows gas- tric emptying, and acts on the central nervous system to generate a feeling of satiety, effects currently being exploited in the treatment of type 2 diabetes by use of long-​acting GLP-​1 analogues or inhibitors of GLP-​1 breakdown (see next). Peripheral insulin levels are lower than those in the portal vein, into which the islets drain, because up to 30% of insulin is removed on its first pass through the liver—​one of the main targets for insulin action. The kidney also actively clears and degrades insulin; the cir- culating half-​life is only a few minutes. C-​peptide provides a robust measure of residual β-​cell function, because it is cleared more slowly than insulin and its plasma concen- trations are therefore more stable. C-​peptide is generally measured after intense β-​cell stimulation with the powerful insulin secreta- gogue glucagon; alternatives are a heavy oral load of carbohydrate, mixed meal stimulation including amino acids (such as Boost or Sustacal) or simply the measurement of 24-​h secretion of C-​peptide in urine (it is cleared largely intact through the kidneys). In normal subjects and most with type 2 diabetes, peak C-​peptide concentra- tions at 6 min after 1 mg of intravenous glucagon are 1 to 4 nmol/​litre, whereas type 1 diabetic individuals are typically C-​peptide negative, with peak levels less than 0.2 nmol/​litre after 5 years. At diagnosis of type 1 diabetes there may be overlap with levels in patients with type 2 diabetes and accordingly the test is not used diagnostically (see Fig. 13.9.1.6). However, measurement of C-​peptide concentrations, and spe- cifically urine C-​peptide:creatinine ratio after a mixed meal, has recently been proposed as a valuable method of distinguishing MODY from type 1 diabetes. The insulin receptor and signal transduction The insulin receptor belongs to the family that also includes the insulin-​like growth factor 1 (IGF-​1) receptor. Insulin receptors are found in the obvious insulin target tissues (fat, liver, and skeletal muscle) but also in unexpected sites, such as the brain and gonads, in which glucose uptake does not depend on insulin. The insulin receptor is a 400-​kDa heterotetramer composed of two α and two β glycoprotein subunits, interconnected by disulphide bridges (Fig. 13.9.1.3). Both α and β subunits are encoded within a complex gene (22 exons) on chromosome 19q. The α subunit (135 kDa) lies entirely extracellularly, while the β subunit (95 kDa) spans the cell membrane and extends into the cytoplasm. Part of the intracytoplasmic tail functions as a tyrosine kinase, attaching phosphate groups from ATP to tyrosine residues elsewhere on the receptor (autophosphorylation) and on other intracellular proteins. This tyrosine kinase activity is essential for insulin signalling and for insulin to exert its many effects on its target tissues. Insulin binds to a site on the extracellular α subunits, and binding triggers a conform- ational change in the receptor which activates the tyrosine kinase domain of the β subunits. P P P P P P P IRS-1 IRS-2 IRS-3 IRS-4 PI 3-kinase Akt mTOR GSK3 FOXO-1 Lipid synthesis Glucose/protein metabolism Cell growth differentiation Raf MEK MAP kinase SREBP-1c PKCλ Insulin receptor P85 P110 Ras Grb2 P62 GAB-1 Sos Shc Fig. 13.9.1.3  The insulin receptor and signal transduction pathways within insulin’s target cells. Binding of insulin to the extracellular α subunits of the receptor activates the tyrosine (Tyr) kinase domain of the intracellular β subunit. This results in phosphorylation of more than
10 substrate proteins including four structurally related proteins of the insulin receptor substrate (IRS) family which vary in their tissue distribution, as well as Shc, Cbl, p62dok, and Gab-​1. These activated proteins then trigger other reactions that result in the biological actions of insulin, including enhanced glucose uptake, anabolic effects, and cell growth. MAP kinase, mitogen-​activated protein kinase; PI3 kinase, phosphatidylinositol-​3-​kinase. Akt is also known as protein kinase B (PKB). Source data from Biddinger SN, Kahn CR (2006). From mice to men: insights into the insulin resistance syndromes. Annu Rev Physiol, 68, 123–​58.

section 13  Endocrine disorders 2472 Postreceptor mechanisms The activated receptor phosphorylates tyrosine residues on spe- cific intracellular proteins which initiate the signal transduction pathway within the target cell. One group of proteins is the insulin receptor substrate family (IRS 1–​4) that vary in their tissue distri- bution and subcellular localization. Additional substrates include Shc, Cbl, p62dok, and Gab-​1. All these substrates carry docking sites for proteins possessing specific Src homology region SH2 do- mains. Docking of these proteins by the IRS molecules and other substrates begins a cascade of intracellular reactions that lead ultim- ately to the effects of insulin on glucose, lipid, and protein metab- olism and its many other actions (see Fig. 13.9.1.3). A key element is the phosphatidylinositol 3-​kinase pathway which appears to me- diate almost all of insulin’s effects on glucose transport, lipogenesis, and glycogenesis. The mitogen-​activated protein kinase pathway, by contrast, is particularly relevant to insulin’s actions on cell growth, with less relevance to its metabolic effects (see Fig. 13.9.1.3). Receptor turnover Receptors that bind insulin are internalized (i.e. taken up into the target cell by an invagination of the cell membrane that is coated with the protein clathrin). Bound insulin is degraded in the lyso- somes, while most of the insulin receptors are carried back to the cell surface and reinserted into the membrane. The density of receptors on the cell surface is therefore a dynamic quantity, regulated partly by new receptor synthesis and partly by receptor recycling, which in turn is determined by insulin binding. Prolonged exposure to high insulin concentrations increases the proportion of internalized re- ceptors and so decreases the density of receptors available on the cell surface. This downregulation of receptors reduces the sensitivity of the target tissue to insulin. Disorders due to insulin receptor defects Many mutations have now been described in the insulin receptor, including point mutations that cause single-​residue substitutions or truncation of the α or β subunits. The most severe mutations af- fect the insulin-​binding extracellular domain and result in so-​called leprechaunism, while less severe mutations affect the tyrosine kinase domain and interfere with insulin signalling (Rabson–​Mendenhall syndrome). Both syndromes are associated with severe insulin re- sistance (type A) as well as serious mental and physical abnormal- ities, confirming the importance of insulin in fetal development. Antibodies may develop against the insulin receptor and usually cause insulin resistance with variable hyperglycaemia (the type B insulin resistance syndrome); rarely, hypoglycaemia results from antibodies that activate the receptor (analogous to thyrotoxicosis in- duced by antibodies to the thyroid-​stimulating hormone receptor in Graves’ disease). Metabolic actions of insulin Insulin functions as an anabolic hormone, favouring the uptake, utilization, and storage of glucose, the storage of lipids as trigly- ceride, and preventing the breakdown of protein. Effects on carbohydrate metabolism Insulin lowers blood glucose in two main ways (Fig. 13.9.1.4). At low basal concentrations (overnight and between meals) it shuts off the production of glucose by the liver, which is the main determinant of fasting glycaemia. Hepatic glucose output is fuelled by both glycogen breakdown (glycogenolysis) and gluconeogenesis (i.e. glucose synthesis from substrates including lactate, glycerol, and alanine and other amino acids); the rate-​limiting enzymes for these processes are powerfully inhibited by insulin. Conversely, insulin stimulates glycogen synthesis. At higher concentrations, such as after meals, insulin also stimu- lates glucose transport into skeletal muscle (where it is utilized to provide energy via glycolysis or stored as glycogen) and into fat (where it is used to synthesize triglycerides). In both these tissues, insulin enhances glucose uptake through a specific glucose trans- porter protein, GLUT-​4 (Fig. 13.9.1.4). Insulin causes GLUT-​4 units to be translocated rapidly to the cell surface and inserted into the membrane: there, GLUT-​4 units act as hydrophilic pores through which glucose can cross the otherwise impermeable membrane into the cell, following its concentration gradient. Insulin also stimulates GLUT-​4 synthesis. Overall, insulin acting via GLUT-​4 can increase glucose uptake into muscle and fat by up to 40-​fold over the basal, non-​insulin-​mediated, glucose uptake. Non-​insulin-​mediated glu- cose uptake occurs through other glucose transporter isoforms that operate in the absence of insulin, notably GLUT-​1 in peripheral tis- sues and erythrocytes and GLUT-​3 in brain. Effects on lipid metabolism Insulin inhibits triglyceride breakdown (lipolysis), while promoting its synthesis (lipogenesis). Lipolytic enzymes that split triglyceride into glycerol and free fatty acids are powerfully inhibited by insulin, even at low basal insulin concentrations. Profound insulin defi- ciency, such as in untreated type 1 diabetes, is therefore required before uncontrolled lipolysis occurs and generates enough free fatty acids to cause ketoacidosis (see next pargraphs). Effects on protein metabolism Insulin inhibits protein catabolism and thus reduces the gener- ation of amino acids which can act as gluconeogenic precursors to Peripheral glucose uptake + + + + + Other GLUTS CNS and other tissues Gluconeogenic precursors Lactate Glycerol Amino Fat Triglyceride Glucose Glucose Glucose Glycogen Muscle Glycolysis Glucose Glycerol Other GLUTS Other GLUTS GLUT4 Ins GLUT4 Ins Ins Ins Ins – – Ins acids Gluconeogenesis Liver Glucose Glycogenolysis Glycogen Hepatic glucose output Fig. 13.9.1.4  Effects of insulin on glucose homeostasis. Insulin inhibits gluconeogenesis and glycogen breakdown in the liver, thus decreasing hepatic glucose output. Blood glucose is also lowered by increased glucose uptake into fat and skeletal muscle, mediated by the insulin-​ stimulated glucose transporter, GLUT-​4. (Non-​insulin-​mediated glucose uptake is effected by other GLUT proteins.)

13.9.1  Diabetes 2473 enhance glucose production by the liver and kidney. Insulin also promotes protein synthesis and cellular and tissue growth. Other actions of insulin These include vasodilatation, mediated by endothelial production of nitric oxide; growth and differentiation of the fetal nervous system; and enhanced tubular reabsorption of Na+ ions by the kidneys. Measurements of insulin action Glucose lowering is the most easily tested biological action of insulin, and forms the basis for most measurements of insulin resistance. Several methods are used in the research setting; the- oretically, the simplest could be used in clinical diabetes care, to identify patients with marked insulin resistance who might benefit particularly from insulin-​sensitizing drugs such as the thiazolidinediones: • Homeostatic model assessment (HOMA) is an index derived by mathematical modelling of the relationship between the fasting glucose and insulin concentrations: with decreasing insulin sen- sitivity, insulin secretion increases in an attempt to maintain euglycaemia, resulting in compensatory hyperinsulinaemia. Homeostatic model assessment yields measures of both insulin resistance and β-​cell function; the test can be performed on a single fasting blood sample and the results compare well with the insulin–​glucose clamp. • Insulin–​glucose (hyperinsulinaemic–​euglycaemic) clamp. Insulin is infused intravenously to achieve constant high concentrations and a separate infusion of glucose is adjusted to maintain blood glucose ‘clamped’ at a normal value. The more glucose required, the greater is the insulin sensitivity. The clamp is generally re- garded as the gold standard method but demands blood glu- cose measurements every few minutes and takes some hours to perform. • Intravenous glucose tolerance test. An intravenous glucose bolus stimulates insulin release, and mathematical modelling of the re- lationship between the insulin peak and the decay in blood glu- cose levels can yield indices of both insulin secretion and insulin sensitivity. Insulin resistance Insulin resistance (or insensitivity) is a poorly defined term signi- fying decreased biological activity of insulin, and which is usually equated with impaired glucose lowering. There is no universal normal range for insulin sensitivity, be- cause the ability of insulin to lower glucose varies considerably between and within individuals—​it is influenced (e.g. by levels of physical activity and fitness). Subjects with ‘insulin-​resistant’ con- ditions such as type 2 diabetes or essential hypertension commonly show reductions of 40 to 60% in glucose disposal (measured by the clamp technique), as compared with matched healthy controls, yet many apparently normal subjects also have comparable decreases in insulin sensitivity. There is no argument about extreme examples of insulin resistance:  in some patients with leprechaunism, over 20 000 U/​day of insulin have failed to control hyperglycaemia and ketosis. A working definition of clinically relevant insulin resistance in insulin-​treated diabetic patients is a daily requirement of more than 1.5 U/​kg. Causes of insulin resistance Inherited causes Inherited causes include the very rare mutations affecting the insulin receptor or postreceptor signalling pathways which can lead to extreme insulin resistance (type A  insulin resistance syndrome); milder polygenic defects contribute to the insulin resistance of type 2 diabetes (see next). Insulin receptor muta- tions cause clinically distinct syndromes, often with acanthosis nigricans and, in women, features of polycystic ovary disease and masculinization; hyperglycaemia is variable. Specific syndromes include the speculatively named leprechaunism and various in- herited lipodystrophies in which fat is lost from subcutaneous and other depots in defined but unexplained anatomical patterns. Recently, mutations affecting the PPARG gene (the target for the thiazolidinedione drugs; see next) have been shown to modify insulin sensitivity. Several mutations in loci that predispose to obesity have recently been reported, but interestingly, only a subset of these also predispose to type 2 diabetes (e.g. LEP, FTO) suggesting that genetic influences in addition to obesity are re- quired for the generation of diabetes. Obesity Obesity induces insulin resistance, especially in skeletal muscle, and weight loss can improve insulin sensitivity in the obese. Insulin resistance is particularly associated with truncal (central) obesity, where fat is deposited in and around the abdomen; both the sub- cutaneous and intra-​abdominal (visceral) fat depots have been implicated to various degrees that may reflect ethnic and other dif- ferences. Visceral fat (and especially intrahepatic fat) appears to be particularly associated with insulin resistance and diabetes risk because it represents ‘ectopic’ fat deposition occurring when the larger, safer, and more metabolically inert subcutaneous fat stores are saturated. Consistent with this, individuals with lipodystrophy syndromes that are unable to store fat subcutaneously develop high levels of visceral fat and are frequently insulin resistant without have a particularly high BMI. It is still not clear at the molecular level how an increased fat mass can decrease whole-​body insulin sensitivity, but cir- culating fat-​derived products are presumed to be responsible. Intra-​abdominal fat depots can secrete potentially diabetogenic mediators into the portal circulation—​where they would be de- livered directly to the liver—​and this may explain how visceral adiposity causes insulin resistance. Possible candidates include free fatty acids and the cytokine tumour necrosis factor-​α (TNFα); both are secreted by adipocytes and, under experimental condi- tions at least, interfere with aspects of insulin action. Levels of free fatty acids are raised in obese subjects, apparently because lip- olysis is enhanced, and free fatty acids may cause hyperglycaemia by competing with glucose metabolism in liver and muscle. In liver, free fatty acids enhance gluconeogenesis by stimulating the rate-​limiting enzyme pyruvate carboxylase and so increase hep- atic glucose production. In muscle, free fatty acids inhibit gly- colysis at the level of phosphofructokinase and glucose oxidation via pyruvate dehydrogenase, causing a decrease in glucose utiliza- tion and a secondary reduction in glucose uptake (the glucose–​ fatty acid or Randle cycle). In vitro, TNFα inhibits the tyrosine kinase activity of the insulin receptor that is crucial for insulin

section 13  Endocrine disorders 2474 signalling. Production of TNFα by adipose tissue is increased in obesity but its role as a mediator of insulin resistance in human obesity is uncertain. Recently, an adipocyte product, adiponectin, has been shown to enhance insulin sensitivity in rodents; intri- guingly, circulating adiponectin concentrations are decreased in human obesity. Several other recently identified hormones re- leased by adipose tissue (adipokines) also have effects on insulin action (e.g. resistin, visfatin, interleukin-​6) but their role in type 2 diabetes is less well defined. Obesity is also accompanied by the ectopic deposition of triglyceride in liver and skeletal muscle, and the accumulation of triglyceride is correlated with impairment of insulin action in these tissues. Physical inactivity strongly predisposes to obesity and also pro- motes insulin resistance which can be reversed by regular exer- cise. The mechanism is unknown but physical training is known to stimulate translocation of GLUT-​4 glucose transporters to the sur- face of muscle cells independently of insulin. In addition, muscle contraction enhances expression of the enzyme AMP kinase which mediates improved glucose transport and fatty acid metabolism. AMP kinase has recently been shown to be a molecular target of two drugs known to reduce insulin resistance (metformin and thiazolidinediones). Other acquired causes There are several other acquired causes of insulin resistance. Intrauterine growth retardation may contribute (see the Barker–​ Hales hypothesis later). Physiological states of insulin resistance, due to the appropriate oversecretion of the counterregulatory hormones whose metabolic actions oppose those of insulin, are puberty and pregnancy (see gestational diabetes, Chapter 14.10). Endocrine diseases that induce insulin resistance and can cause glucose intolerance and overt diabetes through excessive pro- duction of anti-​insulin hormones include acromegaly (preva- lence of diabetes and impaired glucose tolerance each c.25%), Cushing’s disease (diabetes c.30%), thyrotoxicosis, and the very rare glucagonoma (diabetes in >90% of cases). In these disorders, diabetes is mostly non​ketotic, although insulin may be needed to control hyperglycaemia. Intercurrent illnesses (e.g. myocardial infarction, stroke, or severe infections) induce the secretion of counterregulatory stress hor- mones that can cause marked insulin resistance—​insulin-​treated diabetic patients may need twice their usual insulin dosages during such episodes. Many drugs decrease insulin sensitivity, including glucocorticoids, β2 adrenoceptor agonists (ritodrine, salbutamol), and certain oral contraceptive pills containing high-​dose oestrogen or levonorgestrel; glucocorticoid-​induced hyperglycaemia com- monly requires insulin treatment. Acquired lipodystrophies, most notably that induced by drugs used to treat HIV, especially protease inhibitors and nucleoside analogue inhibitors of viral reverse tran- scriptase (NRTIs), are also associated with insulin resistance and the development of diabetes. The type B insulin resistance syndrome is due to the development of autoantibodies against the insulin receptor which interfere with insulin binding and/​or signalling. Most patients are young women, usually with pre-​existing autoimmune diseases such as lupus erythematosus, and masculinization often occurs. ‘Immune insulin resistance’ describes insulin-​treated patients with very high insulin requirements (sometimes several thousand U/​day) because of high titres of insulin-​binding antibodies that bind and inactivate admin- istered insulin. This has become very rare since the introduction of highly purified human-​sequence insulin preparations with low im- munogenicity (see next). Metabolic and clinical features of insulin resistance The metabolic disturbance due to insulin-​resistant syndromes ranges from subclinical glucose intolerance to severely symptomatic hyperglycaemia, sometimes with ketosis. A crucial determinant is the capacity of the individual’s β cells to secrete insulin in response to the rises in blood glucose that are due to impaired insulin action. The resulting hyperinsulinaemia is extremely variable, with plasma insulin levels ranging from twice normal in many obese subjects to 500 times normal in patients with defects of insulin receptors. Near normoglycaemia can be maintained as long as hyperinsulinaemia can compensate for the underlying defect in insulin signalling; diabetes occurs when β-​cell failure supervenes and insulin secre- tion falls below a critical level. In the total absence of functional insulin receptors (e.g. in leprechaunism), massive endogenous hyperinsulinaemia or administration of industrial insulin dosages cannot prevent severe diabetes, although very high insulin concen- trations may exert some metabolic actions through ‘cross-​talk’ with the IGF-​1 receptor. Acanthosis nigricans, a characteristic skin manifestation of se- vere insulin resistance, may be due to high insulin concentrations activating growth factor receptors (perhaps the IGF1 receptor) that drive the proliferation of keratinocytes and melanocytes. Hyperplasia of these cells leads to a velvety thickening and variable darkening of the skin, especially in the axillae (often with prolifer- ation of skin tags), groin, and nape of the neck (see Chapter 23.8). Widespread acanthosis nigricans can also accompany gut tumours, which may also secrete dermal growth factors. Increased androgen concentrations may lead to hirsutism and oc- casionally virilization in women with severe insulin resistance; high insulin concentrations may stimulate androgen production by the ovaries, which often show a polycystic appearance. Insulin resist- ance is a feature of polycystic ovary syndrome, especially in obese patients (Chapter  13.6.1). Enhancing insulin sensitivity through weight loss or treatment with metformin or the thiazolidinediones can decrease androgen levels and improve hirsutism and menstrual dysfunction. The metabolic syndrome The metabolic syndrome (syndrome X) denotes the co-​occurrence of insulin resistance and glucose intolerance (ranging from mild to overt type 2 diabetes), with truncal obesity, dyslipidaemia (raised triglycerides and a high low-​density lipoprotein:high-​density lipo- protein (HDL/​LDL) ratio), and hypertension (see Fig. 13.9.1.5). These abnormalities are all common in most Westernized populations, and it is still not clear whether or not this constella- tion of cardiovascular risk factors represents a genuine syndrome with a common underlying cause. Reaven and others have argued that insulin resistance is the central abnormality, and that the key features can be explained either by loss of specific actions of in- sulin or by the effects of the compensatory hyperinsulinaemia on organs that remain relatively insulin sensitive. For example, raised

13.9.1  Diabetes 2475 insulin levels could contribute to hypertension by enhancing re- tention of Na+ by the kidney; conversely, blood pressure could also be raised through loss of the direct vasodilator action of in- sulin. The pattern of abnormalities would therefore require in- sulin resistance to affect certain tissues and specific actions of insulin but not others. Other proatherogenic defects identified in subjects with various features of syndrome X include increased coagulability of the blood (e.g. increased levels of plasminogen activator inhibitor-​1) and impaired endothelial-​mediated vaso- dilatation. The relationship of these abnormalities to insulin re- sistance is uncertain. Obesity, dyslipidaemia, hypertension, and glucose intolerance are all independent cardiovascular risk fac- tors; any possible proatherogenic role of hyperinsulinaemia per se remains controversial. The aetiology of syndrome X is unresolved; indeed, it has been argued that it is not a distinct entity, but simply represents the vari- able association of several abnormalities that are relatively common in all populations, and especially those that generally overeat and are too sedentary. Adiposity, insulin sensitivity, and blood pres- sure show variable strengths of familial transmission that differ be- tween populations and generally suggest polygenic inheritance of multiple minor genes. On the other hand, Barker and Hales have suggested that fetal malnutrition programmes insulin resistance, hypertension, and dyslipidaemia in middle to late adult life. The underlying mechanisms remain elusive. Because obesity leads to insulin resistance and glucose intolerance, dyslipidaemia, hyper- tension, and atheroma, weight gain in middle age may be particu- larly hazardous in subjects who were underweight at birth. Clustering of these metabolic and cardiovascular risk factors is important clinically because it predisposes to atheroma for- mation and substantially increases the risk of dying prematurely from myocardial infarction or stroke. Treatment is currently based on correcting any factors (e.g. type 2 diabetes, hypertension, and dyslipidaemia) present in the individual patient. Lifestyle and dietary modification that achieves weight loss can improve most aspects of the syndrome. Several drugs have been shown to slow progression from impaired glucose tolerance to type 2 diabetes (e.g. metformin, pioglitazone, and weight-​loss drugs such as orlistat), although their role in treatment of the metabolic syn- drome remains controversial. Types and classification of diabetes mellitus The current WHO classification is based on aetiology (see Table 13.9.1.1). Type 1 and type 2 diabetes together account for 90 to 95% of cases and will be described in detail. Type 1 diabetes Type 1 diabetes is due to autoimmune destruction of the β cells (the type 1 process). A  similar clinical picture of insulin dependence can be caused by other forms of severe pancreatic damage such as pancreatitis. Epidemiology and demographic features Type 1 diabetes is considerably rarer than type 2, accounting for be- tween 5 and 15% of all diabetes and 30 to 50% of insulin-​treated cases in various populations. It appears predominantly in childhood, with a peak age at presentation of about 11 years in girls and 14 years in boys—​hence the old description of juvenile onset. However, it can develop at any age; up to 50% of all cases are diagnosed over the age of 18 and about 5% of newly diagnosed white diabetic patients over 65 years are considered to have type 1 diabetes. The prevalence of type 1 diabetes varies considerably throughout the world. Incidence is highest in northern European countries (about 30 to 35 cases per 100 000 children per year in Finland and Scotland) and declines progressively towards the equator; there are some isolated hot spots such as Sardinia, where the incidence is as high as in Finland. High susceptibility is found in European populations throughout the world, while African and East Asian populations are relatively spared (incidences of less than 1 per 100 000 per year). Superimposed on this geographical variation are time-​related changes in incidence that hint at the import- ance of the environment in causing the disease. Type 1 diabetes presents more frequently during the winter months, particularly in children aged 10 to 14 years. In many countries (e.g. Norway, Poland, Sweden, and the United Kingdom), there have been sharp 30 to 50% increases in incidence over 10-​ to 20-​year periods, al- though the explanation and significance of these secular trends are not clear. In particular, there has been a shift to diagnosis at a younger age with a particularly marked rise in cases being diag- nosed under the age of 5. Susceptibility to type 1 diabetes shows no gender bias in cases diagnosed before puberty, but in older individuals there is a male preponderance in new cases of 1.5:1 to 1.8:1 Aetiology Type 1 diabetes is an autoimmune, predominantly T-​cell-​mediated process that selectively destroys the β cells. Susceptibility is multi- factorial, resulting from the impact of environmental agents in a genetically disadvantaged subject. Of these two components, the en- vironment appears more important; genetic factors explain only 30 to 40% of total susceptibility. Genetic factors Over 50 genetic loci have been robustly associated with type 1 diabetes, most of which are believed to be causal. The genetic re- gions are tagged by around 100 single nucleotide polymorphisms, around half of which are shared with other autoimmune diseases Hypertension Atheroma Dyslipidaemia ( TG, HDL) Procoagulant tendency Glucose intolerance Truncal obesity Insulin resistance + Hyperinsulinaemia Fig. 13.9.1.5  Metabolic syndrome, a constellation of atherogenic risk factors which may each be related to insulin resistance and/​or the hyperinsulinaemia that accompanies insulin-​resistant states. ↓HDL, reduced high-​density lipoprotein cholesterol; ↑TG, hypertriglyceridaemia.

section 13  Endocrine disorders 2476 Table 13.9.1.1  Classification of diabetes mellitus according to aetiology Type 1 diabetes β-​Cell destruction, usually leading to absolute insulin deficiency (10–​15% of cases in Europe and United States): 1A-​Immune mediated 1B-​Idiopathic (e.g. fulminant type 1 diabetes) Type 2 diabetes May range from predominantly insulin resistance with relative insulin deficiency to a predominantly secretory defect with insulin resistance (80–​85% of cases in Europe and United States) Other specific types Other types with specific causes (5% of cases in Europe and United States)   A Genetic defects of β-​cell function MODY: see Table 13.9.1.4 Others, including mitochondrial DNA defects (MELAS syndrome) Neonatal diabetes: mutations in KCNJ11; imprinting abnormality in ZAC and HYMAI—​may be transient   B Genetic defects of insulin action Type A insulin resistance syndrome Leprechaunism Rabson–​Mendenhall syndrome Congenital lipodystrophies   C Diseases of the exocrine pancreas Pancreatitis, chronic and acute Carcinoma of the pancreas Haemochromatosis Cystic fibrosis Pancreatectomy, trauma Fibrocalculous pancreatopathy   D Endocrinopathies Acromegaly Cushing’s disease and syndrome Phaeochromocytoma Glucagonoma Hyperthyroidism Somatostatinoma Aldosteronoma   E Drug-​ or chemically-​induced Glucocorticoids β-​Blockers Thiazides Diazoxide Others—​phenytoin, pentamidine, nicotinic acid, interferon-​α   F Infections Congenital rubella Cytomegalovirus   G Uncommon forms of
  immune-​mediated diabetes Type B insulin resistance (insulin receptor antibodies) ‘Stiff man’ syndrome   H Other genetic syndromes Prader–​Willi syndrome Wolfram’s syndrome (DIDMOAD) Down’s syndrome Turner’s syndrome Klinefelter’s syndrome Others (e.g. Laurence–​Moon–​Biedl syndrome) Gestational diabetes Gestational diabetes or glucose intolerance DIDMOAD, diabetes insipidus, diabetes mellitus, optic atrophy, and deafness; MELAS, myopathy, encephalopathy, lactic acidosis, and stroke-​like episodes (associated with type 1 or type 2 diabetes); MODY, maturity-​onset diabetes of the young. Adapted from American Diabetes Association (2007). Diagnosis and classification of diabetes. Diabetes Care, 30, S42–​S47.

13.9.1  Diabetes 2477 (e.g. coeliac disease, RA, psoriasis, inflammatory bowel disease, or autoimmune thyroid disease). The best characterized are the HLA class II locus (HLA-​DQB1, IDDM1) and the insulin gene promoter region (IDDM2), of which the HLA class II locus has by far the greatest effect (odds ratio for diabetes is 7–​13:1 for sus- ceptible alleles). The HLA class II locus lies within the major histocompatibility complex region on chromosome 6, that encodes several proteins in- timately involved in immune responses. Of particular importance is HLA-​DQB1; this encodes the DQB1 peptide chain, which forms part of the cleft in the surface of the HLA class II molecule that is crucial in presenting peptide fragments of antigen to the T-​helper lymphocyte. Changes in the structure of the DQB1 molecule could therefore influence the coupling between the class  II molecule–​ peptide complex and the T-​lymphocyte receptor, and thus modulate the immune response against the (auto)antigenic peptide. Specific DQB1 polymorphisms have been shown to predispose to type 1 diabetes (e.g. DQB10302), whereas others (e.g. DQB10602) are protective—​at least in certain racial groups. The relationships of these polymorphisms to the long-​recognized influences of the DR3 and DR4 class II antigens (which increase several-​fold the risk of type 1 diabetes) and of the protective DR2 are discussed further in Chapter 13.12.2. The IDDM2 locus corresponds to the insulin gene (INS) whose uniqueness as a β-​cell product makes it an obvious candidate gene. The insulin coding sequence is unchanged in type 1 diabetes. However, variation is observed in a region upstream of the insulin gene in which there is a variable number of repeats of the consensus sequence, 5′-​ACAGGGGTGTGGGG-​3′ one after another, known as the variable number of tandem repeats (VNTR) minisatellite. The short class I VNTR alleles (26–​63 repeats) predispose to diabetes, while class III alleles (140–​210 repeats) have a dominant protective effect (odds ratio for type 1 diabetes with class I vs. class III alleles is 2.2:1). This protective effect appears to be mediated by a two-​ to threefold increased expression of insulin in the thymus. Insulin, like many other self proteins, is normally expressed at low level in the thymus as part of the process which promotes central tolerance to self antigens among T cells. The further increased levels associated with class III alleles thereby results in a relative reduction in the risk of autoimmunity. Additional loci confirmed to predispose to type 1 diabetes show a strong predominance of genes affecting the immune system, including PTPN22 and CTLA4, both negative regulatory molecules of the immune system, IL2RA (CD25), the high-​affinity interleukin-​ 2 receptor, interleukin-​27, and IFIH1, a cytoplasmic helicase that mediates induction of interferon in response to viral RNA. Work is in progress to define more precisely how disease predisposition is increased by the high-​risk alleles and should ultimately shed light on the pathogenesis of the disease. Environmental factors Viruses have long been popular candidates as an environmental trigger for diabetes. Some (e.g. mumps, Coxsackie, cytomegalo- virus, and rubella) infect the pancreas but normally damage the entire gland, particularly the exocrine tissue, rather than causing se- lective β-​cell injury. Certain viruses target the β cell in animals (e.g. the Kilham rat virus) and can cause insulin-​dependent diabetes, either through their direct cytolytic effects or by provoking a type 1-​like autoimmune process. Important contenders in humans are coxsackieviruses (especially B4), rubella, and rotaviruses. Serological studies indicate that recent coxsackie B infections and possibly other enteroviruses are relatively common among newly diagnosed patients with type 1 diabetes; these could represent the final insult in the disease’s long natural history, since the autoimmune process can be detected many years prior to this. Coxsackieviruses capable of damaging rodent β cells have also been isolated post-​ mortem from the islets of some type 1 diabetic subjects. About 20% of children who survive intrauterine rubella infection develop type 1 diabetes, with typical autoimmune markers. Endogenous retro- viruses were previously implicated as aetiological agents, but this has not been confirmed in further studies. For other viruses, the epi- demiological data are conflicting; for example, the eradication of ru- bella by vaccination has not reduced the incidence of type 1 diabetes in Finland while the prevalence of coxsackie infections is lower in Finland than in the adjacent Russian Karelian population which is genetically related but has a substantially lower type 1 diabetes risk. Viruses could trigger or maintain autoimmune β-​cell damage in various ways. Acute or persistent viral infection of β cells could re- lease β-​cell antigens that are normally sequestered beyond the reach of the immune cells. Certain viral proteins may elicit an immune re- sponse which cross-​reacts with specific β-​cell antigens that happen to be similar (molecular mimicry): e.g. peptide sequences of the P2-​ C capsid protein of coxsackie B viruses may cross-​react with glu- tamate decarboxylase-​65 (GAD65) in the β-​cell membrane. Other environmental factors are suggested to include bovine serum albumin from cow’s milk and various toxins. Bovine serum albumin contains a peptide sequence that may cross-​react with a β-​ cell surface protein (see next); this was suggested as an explanation for an apparent excess risk of type 1 diabetes among children fed with cow’s milk in the neonatal period, although a protective effect for breastfeeding remains controversial. Various toxins selectively damage β cells, including streptozotocin, a nitrosourea used to in- duce experimental diabetes in rodents. Related nitrosamine com- pounds have been blamed for the higher risk of type 1 diabetes in the children of women who eat fermented smoked mutton (a traditional delicacy in Iceland). To try to resolve the controversies in this complex area, an international consortium—​The Environmental Determinants of Diabetes in the Young (TEDDY; https://​teddy.epi.usf.edu/​)—​was established. This followed several thousand children with high-​risk HLA genotypes from birth until adolescence to identify infectious agents and dietary or other environmental factors that trigger β-​cell autoimmunity in genetically susceptible people. Autoimmune features Type 1 diabetes has strong associations with endocrine and other autoimmune diseases, including Schmidt’s syndrome (with hypo­ thyroidism and adrenocortical failure) and the autoimmune polyendocrinopathy–​candidiasis–​ectodermal dystrophy (APECED) syndrome caused by mutations in the AIRE gene which controls self-​ tolerance by influencing thymic expression of autoantigens. Type 1 dia- betes is also a feature of the IPEX syndrome (immunodysregulation, polyendocrinopathy, and enteropathy, X-​linked syndrome) caused by mutations in the key T-​cell regulatory gene, FOXP3. Most β-​cell damage is probably inflicted by T lymphocytes. Insulitis—​infiltration of the islets with immune cells, mostly

section 13  Endocrine disorders 2478 cytotoxic/​suppressor (CD8+) T lymphocytes—​is a pathognomonic feature of the disease, and circulating T-​helper lymphocytes can be identified that react against β-​cell antigens including proinsulin, GAD65, IA-​2, and IGRP (islet-​specific glucose-​6-​phosphatase catalytic subunit-​related protein). Various circulating autoanti- bodies also occur. Some target antigens are unique to the β cell, while other autoantigens are shared by other islet cell types. Notable β-​cell selective autoantibodies are those that recognize GAD65, a heat shock protein (hsp60), and insulin itself. GAD catalyses the conversion of glutamic acid to γ-​aminobutyric acid, whose role in the β cell is uncertain. Studies in rodents with type 1 diabetes suggest that the level of GAD65 expression influences the inten- sity of the autoimmune attack on the β cells. The GAD67 isoform of the enzyme is also expressed in the central nervous system, and autoimmune damage of GABAergic neurons is presumed to explain the association of type 1 diabetes with the rare ‘stiff man’ syndrome (Chapter 24.19.4). High frequencies of autoantibodies to the protein tyrosine phosphatase-​like molecule IA-​2 are also seen. Most recently, autoantibodies against the cation efflux transporter, zinc transporter 8 (ZnT8) and the transmembrane glycoprotein, tetraspanin-​7 have been identified. GAD65 antibodies are present in 70 to 90% of newly diagnosed type 1 patients, insulin antibodies in 40 to 70%, IA-​2 autoantibodies in around 50 to 60%, and ZnT8 antibodies in around 70%. Islet cell antibodies detected by immunofluorescence on tissue sections are present in 80 to 90% of newly diagnosed patients but are technic- ally difficult to measure. Recent studies suggest that automated combined testing for GAD and IA-​2 has equivalent sensitivity and specificity to islet cell antibodies (ICA) testing. This is increasingly replacing ICA assays, although undoubtedly ICA reactivity encom- passes more (as yet undetermined) antigens than insulin, GAD 65, IA-​2, and ZnT8 alone. These antibodies cannot explain the selective destruction of β cells; although some islet cell surface antibodies are complement-​fixing, most of the islet cell destruction is believed to be caused by T cells. High titres of each of these classes of antibodies have some value in predicting diabetes in high-​risk individuals—​the combination of high titres of three autoantibodies (GAD, IA-​2, and insulin or ICA) among family members of subjects with type 1 diabetes is 90% pre- dictive of disease, although hyperglycaemia may not develop for 20 years or more. However, they are clearly not the immediate cause of the disease: single autoantibody-​positive individuals rarely pro- gress to disease, suggesting that these autoantibodies are general markers of autoimmunity against the β cell, rather than evidence of β-​cell destruction, which is primarily cell mediated. Titres of all these antibodies tend to be high at presentation and (according to prospective studies of high-​risk subjects) during the months leading up to this. Thereafter, antibody levels decline progressively (espe- cially to insulin and ZnT8) and may even become undetectable, possibly through dwindling of the antigen load that perpetuates autoimmunity as any remaining β cells disappear. Natural history of type 1 diabetes The damage to β cells might be initiated by direct viral attack, en- vironmental toxins, and/​or a primary immune attack against specific β-​cell antigens such as GAD65, perhaps via molecular mimicry. T-​helper lymphocytes (CD4+) are activated by β-​cell antigens presented together with diabetogenic class II antigens by antigen-​presenting cells (dendritic cells). Activated T-​helper cells produce cytokines that attract T and B lymphocytes and encourage them to proliferate in the islet, leading to insulitis. B lymphocytes might then damage β cells by producing antibodies against released β-​cell antigens, while cytotoxic (CD8+) T lymphocytes directly at- tack β cells carrying the target autoantigens. Insulitis is a patchy and unpredictable process that might flare up after encounters with new environmental triggers such as viral infections, but which can also fade and abort for unknown reasons. Several years of progressive autoimmune damage usually precede the clinical onset of diabetes. This long prediabetic phase is asymp- tomatic, although careful testing (e.g. with the intravenous glucose tolerance test) reveals loss of the first phase, then increasingly obvious disturbances of insulin and C-​peptide secretion, and eventually glu- cose intolerance. Finally, when the β-​cell mass has been eroded to a critical level (probably 5 to 10% of normal), falling insulin secretion can no longer restrain hyperglycaemia and clinical diabetes develops. Residual β-​cell mass is variable at presentation of type 1 dia- betes: some newly diagnosed type 1 patients are C-​peptide positive, and β-​cell secretion may improve temporarily during the ‘honeymoon period’ that can follow the lowering of blood glucose when insulin treatment is started (see following section). As a result, it is not possible to absolutely distinguish type 1 and type 2 diabetes by measurement of C-​peptide at diagnosis although levels tend to be very much lower in type 1 diabetes (see Fig. 13.9.1.6, panel (a). With continuing β-​cell destruction, endogenous insulin production declines progressively, and more than 90% of type 1 patients become permanently C-​peptide negative within 5 years of presentation. The loss of C-​peptide is more rapid in individuals diagnosed in childhood than in new onset dis- ease in adults (see Fig. 13.9.1.6, panels (b) and (c). Ultimately, insulitis burns itself out and the immune cells retreat, leaving islet remnants that are devoid of β cells, but which still contain intact α, δ, and PP cells. Interestingly, there is a concomitant 50% reduction in the size of the exocrine pancreas in patients with long-​standing type 1 dia- betes: this appears not to result in clinically significant malabsorption and the mechanism by which it occurs is unknown. The protracted prediabetic phase provides an opportunity to prevent subjects with active insulitis from developing clinical disease. Progression is more rapid in subjects with circulating islet autoantibodies directed at more than one islet antigen (see Fig. 13.9.1.7); indeed, the risk of diabetes in subjects with three anti- bodies appears to be more than 90% although it may take 15 years or more to present. A combination of autoantibody titres and gen- etic markers (HLA haplotypes) can be used to predict the chances of the disease developing in high-​risk subjects, such as the siblings of children with type 1 diabetes; various immunosuppressive and immunomodulatory treatments are currently undergoing clinical trials as forms of early intervention or prevention. Preservation of residual insulin at diagnosis has potential to substantially improve metabolic control, including a 50% reduction in hypoglycaemia, 2–​3-​fold more individuals reaching glycaemic targets and reduced long-​term complications. Metabolic disturbances of type 1 diabetes In untreated type 1 diabetes, insulin concentrations are generally 10 to 50% of non​diabetic levels in the face of hyperglycaemia which would normally greatly increase insulin secretion. Such severe de- ficiency cannot sustain the normal anabolic effects of insulin and

13.9.1  Diabetes 2479 10.0 C-peptide (ng/ml) 5.0 4.0 3.0 2.0 T1DM T2DM *µ = 2.66 *µ = 0.38 Adults (N = 2432) N = 1694 1.0 0.9 0.8 0.7 0.6 0.5 Stimulated C-peptide (mmol/litre) Stimulated C-peptide (mmol/litre) 0.4 0.3 0.2 0.1 0.0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 2 3 4 5 6 7 8 Duration of T1DM (years) Duration of T1DM (years) Adoleseents (N = 1304) N = 838

N = 466 92% 6% 2% 15% 11% 0.4% 2.6% 97% 22% 67% 33% 52% 9 10 11 12 13 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 14 15 N = 738 *P<0.001 1.0 0.0 (a) (b) (c) Fig. 13.9.1.6  Loss of insulin production in type 1 diabetes. Panel (a) compares fasting insulin C-​peptide levels at diagnosis in children with type 1 vs. type 2 diabetes showing that although the levels are very different, there is still some degree of overlap; the lower panels show the peak C-​peptide during a mixed meal tolerance test in patients more than 18 years of age (panel (b), adults) or less than 18 years of age (panel (c)) at onset of diabetes and when tested after 1 to 15 years duration of diabetes, as part of screening for entry into the DCCT study. Note that C-​peptide levels fall faster in younger subjects so that after 5 years 8% of adults but only 3% of children have a peak C-​peptide level > 200 pmol/​litre. Panel (a) adapted from Katz LE, et al. (2007). Fasting C-​peptide and insulin-​like growth factor-​binding protein-​1 levels help to distinguish childhood type 1 and type 2 diabetes at diagnosis. Pediatr Diabetes, 8, 53–​9; panels (b) and (c) source data from Palmer JP, et al. (2004). C-​peptide is the appropriate outcome measure for type 1 diabetes clinical trials to preserve β-​cell function: report of an ADA workshop, 21–​22 October 2001. Diabetes, 53, 250–​64.

section 13  Endocrine disorders 2480 leads to runaway catabolism in carbohydrate, fat, and protein me- tabolism. Each of these processes accelerates hyperglycaemia, while the oxidation of excess free fatty acids generated by triglyceride breakdown can result in diabetic ketoacidosis. Carbohydrate metabolism Basal hyperglycaemia is due mainly to unrestrained production of glucose by the liver and is accentuated after eating by the failure of glucose to be cleared peripherally (see Fig. 13.9.1.4). Hepatic glu- cose output is boosted, especially by increased gluconeogenesis: the normal inhibition of the process by insulin is lost, while the supply of gluconeogenic precursors (glycerol from lipolysis, amino acids such as alanine from protein breakdown) is increased. Enhanced gluconeogenesis in the kidney may also contribute. Postprandial glu- cose uptake into muscle and fat, mediated by insulin and GLUT-​4, is greatly decreased; this is partly offset by increased non-​insulin-​ dependent glucose uptake into peripheral tissues, via glucose transporters that do not require insulin. The overall result is hyper- glycaemia, commonly in the range of 15 to 25 mmol/​litre and higher after meals. Glucose concentrations of over 40 mmol/​litre are not uncommon during intercurrent illness and especially when insulin treatment is omitted or not increased sufficiently. Fat metabolism Lipolysis is stimulated by severe insulin deficiency, generating gly- cerol (a gluconeogenic precursor) and free fatty acids, the substrate for ketone formation. Ketogenesis is particularly enhanced by con- comitant glucagon excess (see next). Mobilization of body fat con- tributes to the marked weight loss in untreated type 1 diabetes. Protein metabolism Loss of the net anabolic effect of insulin encourages catabolism of proteins (primarily through the proteasome-​mediated pathway), thus generating amino acids including gluconeogenic precursors such as alanine and glutamine. Muscle wasting may be prominent. Role of counterregulatory hormones The effects of hypoinsulinaemia are compounded by the counter­ regulatory hormones which are secreted in excess in response to stress (e.g. infections, myocardial infarction, trauma, surgery) and when circulating volume falls (e.g. in hyperglycaemic dehydrated patients). Insulin deficiency also leads to increased glucagon secre- tion, because insulin normally inhibits the α cells. Glucagon increases hepatic glucose production, both by driving glycogen breakdown and by increasing uptake of glucogenic amino acids by the liver and enhancing gluconeogenesis. It also stimu- lates ketogenesis by increasing entry of free fatty acids (as their fatty acyl-​CoA derivatives) into liver mitochondria (see Fig. 13.9.1.12). Glucagon excess is an important factor that promotes diabetic keto- acidosis, acting synergistically with insulin deficiency (see next). Cortisol and catecholamines enhance gluconeogenesis. Cortisol, catecholamines, and growth hormone oppose the lipogenic action of insulin and favour lipolysis, in the presence of hypoinsulinaemia. Cortisol is a powerful inducer of proteolysis, whereas growth hor- mone cooperates with insulin to stimulate protein synthesis. Clinical features of type 1 diabetes The classical presentation of untreated or poorly controlled type 1 diabetes reflects the consequences of catabolism and hypergly- caemia (see Table 13.9.1.2). These features usually develop progres- sively and quite rapidly over a period of a few days to a few weeks. Diuresis is due mainly to the osmotic effect of glucose remaining in the renal tubule, when its concentration exceeds the reabsorption threshold for glucose (corresponding generally to plasma glucose levels of about 10 mmol/​litre). The osmotic loads of urinary ketones and of electrolytes that are obligatorily lost with glucose also con- tribute. Urine output may reach several litres per day, causing poly- uria, nocturia, and in children, enuresis. Thirst generally parallels urine output and can be very intense; it is characteristically made worse by sugar-​rich drinks. Taking water to bed at night is a useful sign of pathological thirst. A high fluid in- take is an important homeostatic response to diuresis, and patients unable to drink (e.g. through nausea in ketoacidosis) can rapidly be- come dehydrated and hypovolaemic. Weight loss, due to loss of fat and muscle and later to dehydration, can be dramatic and reach several kilograms over a few weeks. The energy deficit caused by catabolism and urinary losses of glucose can amount to several hundred calories per day. Appetite is often increased; the mechanism in humans is not known; falls in circu- lating leptin and insulin, both of which act on the central nervous system to inhibit feeding, are probably responsible for hyperphagia in diabetic rodents. Systemic symptoms include tiredness, malaise, lack of energy, and muscular weakness. Blurred vision is commonly due to (reversible) changes in the shape of the lens due to osmotic shifts, typically causing long-​ sightedness. Rarely, acute ‘snowflake’ cataracts develop because of reversible refractile changes, rather than the permanent denatur- ation of lens proteins in senile cataract. Infections are often present because hyperglycaemia predisposes to infections and also because infections stimulate the secretion 100 80 60 40 20 0 0 5 None None Number of islet autoantibodies 1 islet 1 islet 2 islet 2 islet 3 islet 3 islet Number of events Age (Years) Proportion of patients without type 1 diabetes (%) 358 227 474 430 168 250 112 82 272 5253 20 19 118 1161 .. 1 9 44 12318 8875 10 15 20 Fig. 13.9.1.7  Proportion of patients without type 1 diabetes in relation to the number of islet autoantibodies after being followed up from birth. Source data from Ziegler AG, et al. (2013). Seroconversion to multiple islet autoantibodies and risk of progression to diabetes in children. JAMA, 309, 2473–​79.

13.9.1  Diabetes 2481 of stress hormones. Genital candida infections, causing recurrent pruritus vulvae in women and balanitis in men, are frequent and should always prompt testing for diabetes. Pyogenic skin infections and urinary tract infections, sometimes complicated by severe renal damage, are also common, and certain rare infections have a par- ticular predilection for diabetic people (see next). Diabetic ketoacidosis presents with hyperglycaemic symptoms, which are usually severe, together with nausea and vomiting, acidotic (Kussmaul) breathing, the smell of acetone on the breath, and, espe- cially in children, altered mood and clouding of consciousness that may progress to coma. Diabetic ketoacidosis is described in detail later. Unlike type 2 diabetes, which is often present for several years be- fore diagnosis, hyperglycaemia in newly presenting type 1 patients develops too acutely for chronic diabetic complications to appear. Because obvious symptoms appear quickly, very few cases are picked up fortuitously, although doctors who have forgotten to think of dia- betes in their differential diagnosis of weight loss or hyperventila- tion may be surprised when hyperglycaemia is detected by routine screening. With the rising incidence and awareness of diabetes in the general population (due to rising rates of type 2 diabetes), an increasing number of cases of type 1 diabetes are detected before ketosis develops—​giving rise, especially in adults, to confusion over whether the diagnosis is type 1 or type 2 diabetes. Prognosis of type 1 diabetes Before the introduction of insulin during the early 1920s, type 1 diabetes was invariably fatal, usually within months. With various semistarvation diets, hyperglycaemic symptoms could be improved somewhat and life extended by a few miserable months. Over the last 20 years the mortality rate from diabetic ketoacidosis has fallen from 8% to 0.67%, although one-​third of deaths in diabetic children and young adults are still due to metabolic emergencies, not- ably ketoacidosis. The main threat to survival with type 1 diabetes is now chronic tissue damage, particularly renal failure from nephropathy, and vascular disease, notably myocardial infarction and stroke. Throughout adult life, the overall risk of dying within 10 years is about fourfold higher for patients with type 1 diabetes than for their non​diabetic peers. Life expectancy There is encouraging evidence from Europe and the United States of America that the outlook for type 1 diabetes has improved over the last 10 to 20 years, with definite declines in the incidence of microvascular complications and extended survival—​at least in countries able to af- ford effective diabetes care. This is partly attributable to tighter control of hyperglycaemia, which can reduce by 30 to 40% the risks of nephrop- athy and retinopathy developing or progressing to a clinically signifi- cant degree (see next). Other measures have undoubtedly contributed, including better treatment of raised blood pressure and blood lipids. Tragically, however, in many parts of the world patients with type 1 diabetes still die today as they did a century ago, simply because insulin is not available or is not affordable. Type 2 diabetes Type 2 diabetes is a heterogeneous condition, diagnosed empirically by the absence of features suggesting type 1 diabetes (see Table 13.9.1.2) and of the many other conditions that cause hyperglycaemia (see Table 13.9.1.1). Diagnostic accuracy may depend on the thoroughness of investigation; for example, up to 10% of subjects with presumed type 2 diabetes show evidence of autoimmune β-​cell damage and thus prob- ably have slowly evolving type 1 diabetes (so-​called latent autoimmune diabetes in adults, LADA) and a further 1% will have monogenic diabetes. The term ‘type 2’ replaces ‘non-​insulin-​dependent’ and ‘maturity-​ onset’ which were both clumsy and misleading: many type 2 patients require insulin to control hyperglycaemia and increasingly type 2 diabetes is being diagnosed in (overweight) children. Epidemiology and demographic features Type 2 diabetes accounts for 85 to 90% of diabetes worldwide. It is very common, affecting at least 3 to 4% of the white populations in Table 13.9.1.2  Typical features of type 1 and type 2 diabetes, with some distinguishing characteristics Type 1 diabetes Type 2 diabetes Osmotic and glycosuric symptoms: polyuria, nocturia, enuresis; thirst, polydipsia; blurred vision;
genital candidiasis (pruritus vulvae, balanitis)

Clinical insulin dependence (weight loss and hyperglycaemia without insulin replacement) + –​ Special investigations: C-​peptide Low, especially 5 years after diagnosis Normal or raised HLA DR3 or DR4 ++ –​ Islet cell antibodies: ICA, GAD, IA-​2, ZNT8 ++ –​ GAD, glutamic acid decarboxylase; HLA, human leukocyte antigen; ICA, islet cell antibodies; IA-​2, insulinoma associated antigen-​2, ZnT8 –​zinc transporter-​8

section 13  Endocrine disorders 2482 most countries, with rates rising to between 8 and 11% in Eastern Europe and North America. The prevalence rises with age to well over 10% of those over 70 years. It is substantially more common in certain immigrant populations living in more affluent countries (e.g. 10–​15% of adults in some Asian or Afro-​Caribbean groups in the United Kingdom are affected, compared with a prevalence of 4% in the white population). Type 2 diabetes is most commonly diagnosed in those over 40 years of age and the incidence rises to a peak at 45 to 64 years. However, much younger people are now presenting with type 2 dia- betes, following the rapid rise in childhood obesity. Up to one-​third of North Americans diagnosed as diabetic under 20 years of age have type 2 diabetes, with Afro-​Caribbean and Hispanic populations being at particular risk. Monogenic diabetes or maturity-​onset dia- betes of the young (MODY) due to single-​gene defects, commonly presents before 25 years of age in more than one generation, and is now classified separately (read on for more details). The prevalence of type 2 diabetes shows striking geographical variation—​entirely different from that of type 1—​and ranges from less than 1% in rural China to 50% in the Pima Indians of New Mexico. Prevalence is also rising rapidly, especially in developing countries and, worldwide, will increase by at least 50% within 10 to 15 years. This pandemic can be largely explained by Westernization, and is following in the wake of the obesity that is spreading throughout the world. The Pima Indians illustrate this process especially viv- idly; most developed and developing countries are now showing the same phenomenon, albeit more slowly. Diabetes was rare while the Pima tribes led a frugal existence in desert conditions and were lean and physically active. Following urban resettlement and exposure to overnutrition and inactivity, there were rapid increases in the preva- lence of obesity (currently 80% of adult Pima Indians have a BMI of over 30 kg/​m2) and later of type 2 diabetes. There is a 3:2 male preponderance among subjects with type 2 diabetes in Western countries although worldwide there is a 10% excess of females. Aetiology Type 2 diabetes is due to the combination of insulin resistance and β-​ cell failure, the latter preventing sufficient insulin secretion to over- come insulin resistance. These two components vary in importance between different individuals, who may be clinically quite similar, and each has numerous possible causes. Susceptibility is determined by the interactions between genes and environment. The steeply rising prevalence of type 2 diabetes suggests that diabetogenic genes are common and are now enjoying an unparalleled opportunity to express themselves through the global spread of Westernized life- style and obesity. Genetic factors Overall genetic susceptibility to type 2 diabetes is probably 60 to 90%, rather less than was previously deduced from twin studies. Generally, transmission does not follow simple mendelian rules, and this polygenic pattern reflects the inheritance of a critical mass of minor diabetogenic polymorphisms which interfere with insulin action and/​or insulin secretion. Having a first-​order relative with the disease increases an individual’s chances of developing it fivefold, representing a lifetime risk in white people of about 40%. However, this figure will be strongly influenced by modifiable factors, notably the BMI and physical activity of the at-​risk individual. Much progress has been made recently in identifying the gene loci predisposing to type 2 diabetes by using genome-​wide scanning in large population databases. Importantly, these findings have been verified by repeat analyses in other data sets to exclude spurious statistical findings arising from the very large number of statistical comparisons performed. At least 30 loci have been confirmed, with predicted effects on insulin resistance (PPARG) and obesity (FTO), but interestingly, a greater number of confirmed loci seem to relate to pancreas development and/​or insulin secretion (TCF7L2, KCNJ11, HHEX–​IDE, CDKAL1, CDKN2, IGF2BP2, and SLC30A8)—​see Fig. 13.9.1.8. Although confirmed, the influence of each locus is relatively weak: the strongest association is with TCF7L2 (odds ratio for dia- betes of high-​risk polymorphism is 1.5) with the remaining loci con- ferring odds ratios of 1.1 to 1.25. Taken together, the known loci still only explain a small proportion of the inheritance of type 2 diabetes, indicating that there are many more minor loci to be identified. Interestingly, only three of the defined loci for common polygenic type 2 diabetes are the same as those identified to cause the much rarer monogenic diabetes syndromes of maturity-​onset diabetes of the young (MODY, see next—​glucokinase, HNF-α, HNF-​1 β). Environmental factors These clearly play a critical part, because obesity and type 2 dia- betes are spreading too rapidly to be explicable by changes in the genome; environmental factors are also important in practice be- cause they may be modified to treat and prevent the disease. Known environmental diabetogenic factors mostly induce insulin resist- ance (e.g. obesity, pregnancy, intercurrent illness, certain drugs). Hyperglycaemia per se can both impair insulin sensitivity and in- hibit insulin secretion (glucotoxicity). Specific risk factors for type 2 diabetes Obesity, itself determined by both genes and environment, is one of the most important risk factors, apparently due to aggravation of insulin resistance (see earlier). The diabetogenic properties of excess fat depend not only on its bulk but also on its anatomical distribution and the time of life at which it is laid down. The risks of developing type 2 diabetes begin to increase steeply once the BMI exceeds 28 kg/​ m2; some studies estimate the risk at a BMI over 35 kg/​m2 to be 80-​ fold higher than for individuals with a BMI of less than 22 kg/​m2—​a lifetime risk of about 50%. Fat in the truncal (central) distribution is more diabetogenic than that deposited around the hips and thighs, and the visceral (intra-​abdominal) depot is strongly associated with insulin resistance. Increasing adiposity after the early twenties, espe- cially around the waist, aggravates the risk of a high BMI. Physical inactivity, especially from the twenties onwards, is an in- dependent predictor of diabetes in middle age, the risk increasing by about threefold for sedentary people as compared with regular athletes. This is due to worsening insulin resistance, which can be improved by physical training and may in part be due to changes in activity of the enzyme AMP kinase in skeletal muscle. The Barker–​Hales hypothesis suggests that poor fetal growth can programme enduring metabolic and vascular abnormalities that are manifested in adult life, especially in people who were underweight at birth but then become obese. These abnormalities include key features of the metabolic syndrome (hyperglycaemia, hypertension, dyslipidaemia), resulting in atheroma formation, myocardial infarction, and stroke (see earlier). Evidence, mainly

13.9.1  Diabetes 2483 from animals, suggests that maternal and therefore fetal malnutri- tion during a critical early phase of fetal development can reduce β-​cell mass and permanently impair insulin secretory reserve; de- ficiencies of sulphur-​containing amino acids may be responsible in experimental animals but the relevance to humans is unknown. Other studies suggest that insulin sensitivity may also be reduced into adult life. β-​Cell failure in type 2 diabetes β-​Cell failure is an obligatory defect in the pathogenesis of type 2 diabetes: near normoglycaemia can be maintained even in severe insulin resistance (e.g. due to mutations in the insulin receptor), as long as the β cell can respond to the challenge and secrete enough insulin to overcome the resistance. Subtle abnormalities of insulin secretion, including loss of the physiological pulses and of the first-​phase response to intravenous glucose injection, are seen in normoglycaemic subjects who later de- velop the disease. These defects presumably indicate that the β cell is already stressed in trying to produce enough insulin to overcome in- sulin resistance. Normoglycaemic first-​order relatives of type 2 dia- betic subjects also show loss of pulsatility of insulin secretion which might indicate an inherited tendency to β-​cell failure. The key role of β-​cell failure in predisposing to type 2 diabetes has recently been underlined by the finding that most of the confirmed genetic suscep- tibility loci for type 2 diabetes relate to islet cell function or devel- opment rather than insulin resistance (see earlier and Fig. 13.9.1.8). The mechanism of β-​cell failure in human type 2 diabetes is not known. Histologically, the islets in type 2 diabetes show no features of type 1 autoimmune insulitis, and β-​cell mass is not so dramatic- ally reduced. Animal models of the disease suggest various causes, including synchronized β-​cell apoptosis (possibly mediated by ni- tric oxide) in the Zucker diabetic fatty rat, and the deposition of amyloid fibrils (see earlier) in the rhesus monkey. Amyloid deposits are also prominent in the islets of some type 2 diabetic patients but may merely be due to dysfunctional β-​cell hypersecretion rather than the cause of β-​cell damage. Once hyperglycaemia is estab- lished, glucotoxicity per se may further worsen both insulin secre- tion and insulin resistance. Elevated free fatty acid levels resulting from insulin resistance have also been proposed to impair β-​cell function—​so-​called lipotoxicity—​but this remains controversial. In established type 2 diabetes, insulin secretion is unequivocally subnormal and tends to decline progressively with time, as illus- trated by the long-​term follow-​up data from the United Kingdom Prospective Diabetes Study. Initially, plasma insulin levels may be higher than in non​diabetic subjects but are still inappropriately low, as the normal pancreas would produce much higher insulin concentrations in response to diabetic levels of blood glucose. Conventional radioimmunoassays may overestimate insulin levels in type 2 diabetic patients because of cross-​reaction with incom- pletely processed insulin precursors (proinsulin and its split prod- ucts) released by the constitutive pathway which operates in the malfunctioning β cell (see earlier). Many type 2 patients ultimately need insulin replacement; this indicates relatively severe insulin deficiency, although still not as profound as in type 1 diabetes. Some type 2 patients who require insulin early have autoimmune markers characteristic of type 1 diabetes, suggesting that they in CDKALI, CDKN2A CDKN2B Reduced β-cell mass β-cell dysfunction Obesity Insulin resistance not due to obesity Reduced insulin secretion Insulin resistance Predisposition to type 2 diabetes MTNR1B, TCF7L2, KCNJ11 FTO IRS1, PPARG Fig. 13.9.1.8  Pathways to type 2 diabetes implicated by identified common variant associations. Type 2 diabetes results when pancreatic β cells are unable to secrete sufficient insulin to maintain normoglycemia, typically in the context of increasing peripheral insulin resistance. The β-​cell abnormalities fundamental to type 2 diabetes are thought to include both reduced β-​cell mass and disruptions of β-​cell function. Insulin resistance can be the consequence of obesity or of obesity-​ independent abnormalities in the responses of muscle, fat, or liver to insulin. Examples of susceptibility variants that, given current evidence, are likely to influence predisposition to type 2 diabetes by means of each of these mechanisms are shown. Reproduced from McCarthy MI (2010). Genomics, type 2 diabetes, and obesity. N Engl J Med, 363, 2339–​50. Copyright © 2010 Massachusetts Medical Society.

section 13  Endocrine disorders 2484 fact have an indolent variant of the disease. Although patients with type 1 diabetes have significantly lower insulin C-​peptide levels at diagnosis than in type 2, there remains overlap in the ranges (see Fig. 13.9.1.6) such that C-​peptide alone only has a sensitivity of 83% in diagnosing type 2 diabetes even in children. Natural history Longitudinal and cross-​sectional studies indicate that insulin re- sistance develops first and that compensatory increases in insulin secretion can initially maintain near normoglycaemia. Worsening insulin resistance is thought to drive the β cells towards maximal insulin output, a metastable stage that probably corresponds to impaired glucose tolerance (see earlier). Rescue is still possible if insulin resistance is decreased (e.g. through weight loss or insulin-​ sensitizing drugs): about 25% of subjects with impaired glucose tolerance return to normoglycaemia within 5 years. However, if insulin resistance persists or worsens, the β cells fail and insulin production falls. At this point, the brake-​limiting hyperglycaemia is released and blood glucose rises into the diabetic range. The bell-​ shaped response of insulin secretion, initially increasing to com- pensate but ultimately failing, has been termed the ‘Starling curve’ of the β cells because it recalls the classical plot of cardiac output against preload in heart failure. In common type 2 diabetes, these events usually take many years, and significant hyperglycaemia may have been present for several years at the time of diagnosis. The whole process can be greatly ac- celerated by acute increases in insulin resistance as those induced by steroid treatment or pregnancy, to give just two examples. Metabolic disturbances in type 2 diabetes Hyperglycaemia is the most obvious abnormality, the extreme case being the hyperosmolar non​ketotic state. Lipid metabolism is also disturbed but true ketoacidosis occurs only exceptionally and is usu- ally provoked by intercurrent events such as infections or myocar- dial infarction. Blood glucose concentrations are raised both in the basal (fasting) state and after eating. This reflects the impairment of insulin action in both liver and skeletal muscle, where insulin respectively shuts off hepatic glu- cose production and stimulates glucose uptake after meals. Hepatic glu- cose output is increased, due mainly to unsuppressed gluconeogenesis, and this is largely responsible for hyperglycaemia overnight and before meals. In muscle, GLUT-​4 activity and glycogen synthesis are especially decreased; this reduces insulin-​stimulated glucose uptake into muscle after meals, although basal glucose uptake (non-​insulin-​mediated glu- cose uptake; see earlier) is higher than in normal subjects because of the mass action effect of hyperglycaemia. The degree of hyperglycaemia varies widely: many patients have fasting plasma glucose levels of 8 to 13 mmol/​litre with postprandial peaks of up to 20 mmol/​litre, while values exceeding 60 mmol/​litre are not uncommon in the hyperosmolar non​ ketotic state. Insulin deficiency is less profound than in type 1 diabetes, so mo- bilization of triglyceride (loss of body fat, ketoacidosis) and catab- olism of protein (muscle breakdown) are not usually pronounced. Diabetic ketoacidosis may develop in patients with apparently typical type 2 diabetes who can subsequently be controlled by oral hypoglycaemic agents rather than insulin (see ‘Flatbush or ketone-​ prone diabetes’ next). Diabetic ketoacidosis is usually precipitated by severe intercurrent illness (e.g. myocardial infarction, stroke, or pneumonia) in which excessive secretion of counterregulatory stress hormones exacerbates the metabolic disturbance caused by relative insulin deficiency. Clinical features Many cases present with classical symptoms of osmotic diuresis, blurred vision due to hyperglycaemia-​related refractive changes in the lens, and genital candidiasis (see Table 13.9.1.2). Weight loss may occur but is generally less dramatic than with newly presenting type 1 diabetes and may not be obvious because many type 2 patients—​over two-​thirds in the United Kingdom—​are obese. Rapid or severe weight loss in patients who otherwise appear to have type 2 diabetes should be regarded with suspicion as it may point to an early need for insulin replacement (and possibly type 1 diabetes itself) or to coexisting illness: a well-​recognized but unex- plained association with recent onset type 2 diabetes is carcinoma of the pancreas. The hyperosmolar non​ketotic state can present with confusion or coma (see next); as mentioned earlier, diabetic ketoacidosis is rare. Chronic diabetic complications may be a presenting feature, be- cause hyperglycaemia severe enough to cause tissue damage may already have been present for several years. Extrapolating the num- bers of microaneurysms (which only develop at diabetic glucose concentrations) in type 2 patients at various intervals after diagnosis suggests that significant hyperglycaemia is present for an average of 5 to 7 years before diagnosis. Common problems are arterial disease (myocardial infarction, stroke, and peripheral vascular disease), cataracts—​which are especially common in the older population—​ and retinopathy, especially maculopathy, which can damage central vision, and foot ulceration. Increasing numbers of people with diabetes are detected by screening, either in high-​risk groups such as the obese and those with cardiovascular disease, or at routine health checks. Many of these are nominally asymptomatic but will admit to symptoms such as nocturia or perineal irritation if asked directly. Prognosis of type 2 diabetes A long-​held and prevalent misconception is that type 2 diabetes is mild. Some patients do have relatively unexciting or asymptomatic hyperglycaemia, but this can still be enough to cause complications which wreck the patient’s life just as much as in type 1 diabetes. Moreover, hyperglycaemia can be as hard to control (even with in- sulin) as in type 1 patients. Overall, life expectancy is shortened by up to a quarter in pa- tients with type 2 diabetes presenting in their forties, with vas- cular disease (myocardial infarction and stroke) being the main cause of premature death. Renal failure from diabetic nephrop- athy is becoming more common in type 2 patients as their survival from vascular complications improves, and the disease is now the most frequent pathology among people waiting for renal replace- ment therapy in the United States of America and some European countries. Type 2 diabetes is therefore an important threat to the patient’s health and survival, and must be taken seriously by patients and their medical attendants, even if the blood glucose concentrations are not dramatically raised. Accordingly, treatment guidelines for the disease are rigorous (see Table 13.9.1.3 and discussion later in this chapter).

13.9.1  Diabetes 2485 Monogenic diabetes: Maturity-​onset diabetes of the young (MODY) and neonatal diabetes Maturity-​onset diabetes of the young (MODY) While the vast majority of diabetes is polygenic in origin, there is now an expanding list of single-​gene loci that are associated with diabetes either in the neonatal period, in childhood, or in early adulthood. In 1974, Tattersall described a rare familial form of non-​insulin-​dependent diabetes that he distinguished from the generality of cases by its early age of onset, autosomal dominant inheritance, and an apparently low risk of microvascular compli- cations. The term ‘maturity-​onset diabetes of the young’ (MODY) came to be applied to individuals in which (1) a diagnosis of type 2 diabetes had been made under the age of 25; (2) there is evidence of autosomal dominant inheritance (diagnosis under the age of 25 in more than one generation); and (3) subjects can be managed without insulin. In 1992, the first conclusive evidence for the existence of mono- genic diabetes was provided when a subset of MODY (now known as MODY 2) was linked to the glucokinase gene locus. There are now at least eleven forms of MODY, accounting for around 1% of all cases of Table 13.9.1.3  Treatment targets for patients with diabetes Patient sub-group Source of recommendation Specific goal
of therapy Notes HbA1c Most EASD and ADA <53 mmol/mol (<7.0%) Regarded as ‘generally accepted’ (EASD) a ‘reasonable goal for many nonpregnant adults’ (ADA) Some EASD and ADA 42-48 mmol/mol (6.0-6.5%) Selected patients (e.g. short disease duration, long life expectancy, no ASCVD) if can be achieved without significant hypoglycaemia or other adverse effects Some ADA <64 mmol/mol (<8.0%) May be appropriate for patients with a history of severe hypogly­ caemia, also in circumstances including limited life expectancy, advanced complications, extensive comorbid conditions Blood pressure Most EASD <140/85 mm Hg Nephropathy with ‘overt proteinuria’ EASD <130/80 mm Hg If tolerated 10-year ASCVD risk <15% ADA <140/90 mm Hg Existing ASCVD or 10-year
ASCVD risk >15% ADA <130/80 mm Hg If can be attained safely Lipids T1DM and T2DM at ‘very high
risk’ (ASCVD, severe CKD, or
one or more CV risk factors
and/or target organ damage) EASD LDL-C <1.8 mmol/litre
(<70 mg/dL) Statin treatment recommended T1DM at ‘high risk’ (no other CV risk factors, no target organ damage) EASD Statin treatment may be considered irrespective of basal LDL-C T2DM at ‘high risk’ (no other CV risk factors, no target organ damage) EASD LDL-C <2.5 mmol/litre (<100 mg/dL) Statin treatment recommended Age <40 and no ASCVD or
10-year ASCVD risk <20% ADA Statin treatment not generally recommended, but moderate intensity statin may be considered based on presence of ASCVD risk factors and risk/benefit analysis Age <40 with ASCVD or
10-year ASCVD risk >20% ADA High intensity statin treatment recommended Age >40 and no ASCVD or
10-year ASCVD risk <20% ADA Moderate intensity statin treatment recommended Age >40 with ASCVD or
10-year ASCVD risk >20% ADA High intensity statin treatment recommended BMI T2DM - 25.0-26.9 kg/m2 ADA Advise diet, physical activity and behavioural therapy T2DM - 27.0-29.9 kg/m2 ADA

section 13  Endocrine disorders 2486 diabetes, in which the gene has been identified (see Table 13.9.1.4). Two forms predominate and have a distinctive clinical picture. MODY 2 (glucokinase mutations) is similar to the initial cases described by Tattersall with mild, non​progressive fasting hyperglycaemia and a very low risk of long-​term complications even without treatment. By contrast, MODY 3 (HNF1A mutations) is associated with progressive decline in glycaemic control. In addition, the renal threshold for glu- cose is low and there is a high risk of long-​term complications. Of par- ticular importance, MODY 3 is exquisitely sensitive to sulphonylureas and most patients wrongly diagnosed as having type 1 diabetes have been successfully transferred from insulin to sulphonylureas with im- provement in glycaemic control. Doses required may be as low as one-​ quarter of the normal adult starting dose. A diagnosis of MODY should be considered if: • There is a family history of young-​onset diabetes in more than one generation with at least one family member diagnosed under the age of 25. • Affected members are not markedly obese or of normal weight. • There is no evidence of insulin resistance—​no acanthosis nigricans, low insulin doses if insulin treated, high-​density lipoprotein greater than 1.2 mol/​litre. • Fasting serum C-​peptide (or urine C-​peptide:creatinine ratio) is detectable and within the normal range (not elevated). • Islet cell or anti-​GAD autoantibodies are absent. • Other associated features are present (see Table 13.9.1.4). None of these criteria are absolute and where doubt exists, advice from an expert centre should be sought before requesting genetic screening. Detection of MODY 3 is of particular importance be- cause of the excellent response to treatment with sulphonylureas. Neonatal diabetes Diabetes diagnosed under the age of 6 months is very unlikely to be type 1 (autoimmune) diabetes and alternative causes should be sought. Neonatal diabetes is insulin-​requiring diabetes, usually diagnosed with the first 3 months of life, and two subgroups have now been identified. Transient neonatal diabetes mellitus resolves around 3 months after birth although it can return in later life in up to 50% of cases. The most common cause is an imprinting ab- normality in the ZACN (ZAC) and HYMAI genes on chromosome 6 at the 6q24 locus. Macroglossia occurs in 23% of cases and is the only non​pancreatic feature. Presenting blood glucose levels are high (from 12 to >50 mmol/​litre) and insulin is required: if relapse oc- curs, this is normally not insulin requiring, at least in the initial stages. MODY 5 and KCNJ11 (Kir6.2) mutations (see next) occa- sionally also present as transient neonatal diabetes mellitus. Permanent neonatal diabetes mellitus requires continual insulin treatment from diagnosis. The most common cause is a mutation in the KCNJ11 gene, encoding the Kir6.2 subunit of the β-​cell KATP channel. Ninety per cent (90%) of cases are due to spontaneous (new) mutations so there is no family history. Affected individuals may have a range of neurological abnormalities that in the most se- vere form are referred to as DEND syndrome (developmental delay, epilepsy, and neonatal diabetes). Patients with Kir6.2 mutations be- have as insulin deficient, with a 30% risk of ketoacidosis and low or undetectable C-​peptide levels. However, most patients respond well to high doses of sulphonylureas, given at up to four times the normal Table 13.9.1.4  Maturity-​onset diabetes of the young (MODY) Type Genetic defect OMIM Frequency (% of MODY) Clinical features Sensitive to sulphonylureas MODY 1 HNF-​4α 125850 1% Rare. Similar to MODY 3 but renal threshold normal. Consider if MODY 3 screen negative May be sensitive MODY 2 Glucokinase 125851 20% Mild, non​progressive fasting hyperglycaemia (5.5–​8.5 mmol/​litre, HbA1c < 6%). Glucose increment < 3.5 mmol/​litre on OGTT. Complications rarely develop. Frequently do not response well to drug treatment and do not require it No MODY 3 HNF-​1α 600496 60% Young-​onset diabetes. Not particularly overweight and not insulin requiring (no ketosis) or surprisingly good control for several years on little insulin. Detectable C-​peptide beyond 3 years postdiagnosis. Low renal threshold. Large glucose increment (>5 mol/​litre) on OGTT. Progressive deterioration in glycaemic control and high risk of complications Extremely sensitive MODY 4 IPF-​1 606392 1% Rare. Possibly later-​onset disease. Some affected family members may not be diabetic Not determined MODY 5 HNF-​1β(TCF2) 137920 1% Renal cysts and diabetes. Renal, uterine, and/​or genital developmental abnormalities are typical initial presentation especially renal cysts. Gout, abnormal LFTs. Subclinical pancreatic exocrine insufficiency No MODY 6 NEUROD1 606394 <1% Rare Not determined MODY 7 KLF-​11 610508 <1% Rare Not determined MODY 8 CEL 609812 <1% Rare Not determined MODY 9 PAX-​4 612225 <1% Rare Not determined MOFY 10 INS 613370 <1% Rare Not determined MODY 11 BLK 613375 <1% Rare Not determined MODY X Unknown 15% Not defined Not determined HNF, hepatocyte nuclear factor; IPF-​1, insulin promoter factor 1; LFT, liver function test; NEUROD1, neurogenic differentiation 1 transcription factor; KLF-​11, Kruppel-​like Factor 11; CEL, carboxyl ester lipase; PAX-​4 (transcription factor); INS, insulin gene; BLK, B lymphocyte tyrosine kinase; OGTT, oral glucose tolerance test.

13.9.1  Diabetes 2487 adult therapeutic dosage (e.g. glibenclamide 0.5–​1 mg/​kg per day), with the restoration of insulin secretion. Occasionally, MODY 2 and 4 may also present as permanent neonatal diabetes mellitus as can other rare genetic syndromes (see next). Other types of diabetes (See Table 13.9.1.1.) Diabetes in pancreatic disease Chronic pancreatitis, most commonly due to alcohol abuse, causes diabetes that needs insulin in about one-​third of cases. Widespread flecks of fine to medium calcification are often scattered through the pancreas, outlining it on a plain abdominal radiograph. Concomitant destruction of the islet α cells means that glucagon secretion is lost as well as insulin; diabetic ketoacidosis is therefore rare, while hypo- glycaemia can be profound and prolonged—​a particular hazard in those who continue to drink alcohol. Acute pancreatitis causes acute hyperglycaemia in 50% of cases but few develop permanent diabetes. Carcinoma of the pancreas is associated with newly presenting type 2 diabetes, and should be suspected in older patients with weight loss (especially when accompanied by abdominal or back pain and jaundice). The mechanism is unknown but appears to be due to tu- mour products that cause insulin resistance rather than to β-​cell loss. Genetic diseases that cause diabetes through pancreatic damage in- clude haemochromatosis and cystic fibrosis. In one-​half of cases of haemochromatosis, heavy deposition of haemosiderin in the islets causes diabetes, usually requiring insulin; associated features are slate-​ grey skin pigmentation due to deposition of iron in the dermis (‘bronze diabetes’), cirrhosis, secondary gonadal failure, and pyrophosphate arthropathy. MRI shows abnormal signals in the liver and pancreas, while serum ferritin concentrations are greatly elevated; diagnosis is usually possible by means of molecular analysis of the HFE gene but Perls’ stain for iron deposition in a liver biopsy may be necessary (see Chapter 12.7.1). Diabetes due to excessive iron deposition in the pan- creas is also seen in children surviving thalassaemia major. Cystic fibrosis causes pancreatic exocrine failure, with an increasing risk of diabetes (often requiring insulin) that approaches 25% in subjects who survive beyond 20 years of age. Gestational diabetes This includes all degrees of hyperglycaemia (impaired glucose tolerance as well as overt diabetes) diagnosed during pregnancy in previously normoglycaemic women. The American Diabetes Association has re- cently removed type 2 diabetes diagnosed in the first trimester from the definition of gestational diabetes. It is covered in Chapter 14.10. Malnutrition-​related diabetes This controversial diagnostic category was omitted from the most recent WHO classification. It included ‘fibrocalculous pancreatic diabetes’ and ‘protein-​deficient diabetes mellitus’. Fibrocalculous pancreatic diabetes was identified by dense pancreatic fibrosis, the formation of discrete and often spectacularly large stones in the dilated pancreatic ducts, and recurrent abdominal pain; protein-​ deficient diabetes mellitus was a vaguer entity that lacked the pan- creatic stones. Patients conforming to these ‘syndromes’ were rare even in the tropical zones where they were described (<5% of all diabetes), and the current consensus is that they represent type 2 diabetes or chronic pancreatitis superimposed on malnutrition. Flatbush or ketone-​prone diabetes The term ‘Flatbush diabetes’ has been used to refer to diabetes in young Afro-​Caribbeans who present with profound diabetic keto- acidosis but later prove to be non​insulin dependent. It appears that at diagnosis they have both marked insulin resistance and impaired insulin secretion but the latter later recovers, sometimes sufficiently for them to go into prolonged remission. In at least one report there was an excess of HLA DR3 and DR4 alleles, but anti-​GAD autoanti- bodies are negative. More recently, the term ketone-​prone diabetes has been used for patients who experience an episode of ketoacidosis but have clinical features of type 2 diabetes (e.g. obesity). A distinc- tion is made between those with and without islet autoantibodies. This area remains to be more accurately classified. Fulminant type 1 diabetes This form of diabetes was first described in 2000 in Japan and refers to presentation with severe diabetic ketoacidosis but low HbA1c (<8.5%) relative to their initial marked hyperglycaemia, thus indicating an abrupt onset. Additional typical features include a short history of symptoms (2–​10 days), raised pancreatic enzyme levels, and negative anti-​GAD (and other) autoantibodies. Prevalence in Japanese and Korean populations may approach 20–​30% of cases of rapid onset diabetes with ketosis, especially where the presentation is in adult- hood and/​or in pregnancy. It is rare in other races including white populations. Pancreatic biopsy reveals T-​cell infiltrates in the exo- crine pancreas, but without insulitis or features of acute pancreatitis. Mitochondrial diabetes Maternal transmission of mutations in mitochondrial DNA (mtDNA), especially the A3243G substitution in the leucine tRNA gene, can result in maternal inheritance of diabetes. Typical clinical presentation includes a presentation age of 20 to 50 with associated sensorineural deafness and short stature as in MIDD syndrome (maternally inherited diabetes and deafness). There is progressive non-​autoimmune β-​cell failure which may progress rapidly to in- sulin dependence (40% are insulin-​dependent within 4 years) The same mutation occurs in MELAS syndrome (mitochondrial my- opathy, encephalopathy, lactic acidosis, and stroke-​like episodes) and both MIDD and MELAS can occur in the same family. The ratio of mutant to wild-​type mtDNA in the blood (i.e. the degree of heteroplasmy) at diagnosis does not correlate with disease pheno- type or severity, presumably because it does not reflect the degree of heteroplasmy in other tissues such as the pancreas. Management of diabetes The treatment of diabetes has traditionally concentrated on correcting hyperglycaemia with the aim of abolishing the symptoms of hypergly- caemia, preventing acute complications such as diabetic ketoacidosis and preventing long-​term complications. Intensive control of hyper- glycaemia particularly in the early years of diagnosis has been shown to reduce both microvascular and macrovascular complications in type 1 and type 2 diabetes. The principal cause of morbidity and pre- mature death is cardiovascular disease. Intensive control of hypergly- caemia in those with long-​standing diabetes does not confer the same cardiovascular protection and some studies have shown an increased mortality in this group. Control of hyperlipidaemia and hypertension

section 13  Endocrine disorders 2488 are the key factors in reducing cardiovascular disease in this group of patients however reducing hyperglycaemia remains important to reduce the risk of microvascular disease. The current treatment tar- gets for both type 1 and type 2 diabetes (see Table 13.9.1.3 - low risk column) are therefore more holistic, tackling cardiovascular risk fac- tors and obesity in addition to hyperglycaemia. This section describes the roles of lifestyle modification and antidiabetic drugs, followed by specific treatment strategies for type 1 and type 2 diabetes. Diet and lifestyle modification and
management of obesity About 80% of patients with type 2 diabetes are obese, as are at least 30% of those with type 1 disease. Obesity is arguably one of the greatest obstacles to successful management of diabetes: it worsens insulin resistance, dyslipidaemia, and hypertension, and is now rec- ognized in its own right as a risk factor for coronary heart disease. Proven benefits of 10% weight loss in type 2 patients with a BMI of 30 to 40 kg/​m2 include falls in fasting glucose of 2 to 4 mmol/​litre and a 1% decrease in HbA1c—​comparable with sulphonylureas or metformin—​and reduced dosages of antidiabetic drugs, including insulin. There may also be variable improvements in blood pressure and dyslipidaemia (decreased triglycerides and low-​density lipopro- tein cholesterol, increased high-​density lipoprotein). The traditional focus on obesity has been on type 2 diabetes, but there is no reason to assume that the cardiovascular hazards of obesity do not also apply to type 1 diabetes. Weight reduction is important in the management of people living with diabetes who are obese. The aetiology of obesity is often com- plex with psychological factors as important as physiological fac- tors and this may result in weight loss being surprisingly difficult to achieve. Modern approaches include peer support programmes, be- haviour modification techniques such as motivational interviewing and incorporating food labelling and shopping advice into structured education programmes and such measures have all shown benefit. It is important that all members of the diabetes team recognize the importance of lifestyle management so that consistent advice and ap- propriate emphasis may be placed on this. The notion of the ‘diabetic diet’ must now finally be laid to rest. Traditionally, carbohydrate intake was restricted because of the sim- plistic assumptions that sugar alone raised blood glucose and might even be diabetogenic; this strategy favoured a high fat intake that undoubtedly helped to sustain obesity and probably predisposed to atheroma. Current advice is close to the healthy eating recommenda- tions for the whole population and can therefore be suggested for the patient’s entire family, which will greatly increase the chances of com- pliance. It may also have benefit for the rest of the family as having a spouse with type 2 diabetes is a risk factor for type 2 diabetes. The following diet and activity recommendations apply to both type 1 and type 2 diabetes. The aims are to: • correct obesity, which worsens insulin resistance, reduces the ef- ficacy of glucose-​lowering, antihypertensive, and lipid-​modifying drugs, and is an independent risk factor for macrovascular disease (management of obesity is discussed in detail in Chapter 11.6); • reduce cardiovascular risk, by limiting fat, cholesterol, sodium, and alcohol intakes; • avoid hypoglycaemia in patients receiving insulin or sulphonylureas by optimizing the timing and content of meals. The steps in designing dietary advice for the individual patient are shown in Fig. 13.9.1.9. Reducing total energy intake This should be reduced by 500 to 600 kcal/​day (2100–​2520 kJ/​day) in patients who are overweight (BMI >28 kg/​m2). This energy deficit mobilizes fat preferentially, whereas protein, glycogen, and water are also lost with more aggressive energy restriction; initially, the rate of weight loss will be 0.5 to 1.0 kg/​week (adipose tissue contains c.7000 cal/​kg or 29 400 J/​kg). (unchanged) The desired energy intake should be calculated from standard for- mulae that employ the subject’s age, sex, weight, and level of physical activity to estimate energy expenditure, which must equal energy intake under steady state conditions. The standard dietary history is not useful for trying to assess energy intake, because overweight subjects consistently under-​report how much they eat. Specific ad- vice about how to cut energy intake is best left to the dietitian, but hinges on reducing fat intake—​a simple message that can be re- inforced by the entire diabetes care team. Fat-​rich foods not only have the highest energy density (9 cal/​g or 38 J/​g, compared with 4 cal/​g (17 J/​g) for carbohydrate and protein), but also have poor sa- tiating effects and so tend to encourage overeating. The initial target should be a 10% loss of starting weight, not the ‘ideal’ body weight or BMI, which is only rarely attained by obese diabetic patients. When energy intake is cut acutely, type 2 patients often show an immediate fall in blood glucose, due to a drop in hep- atic glucose output, even before weight loss begins. Weight loss during an energy deficit of 500 to 600 cal/​day (2100–​2520 J/​day) is a slow process: for a 100 kg patient, a 10% weight loss may take several months. Frequent contact and encouragement are the best predictors of success, and the patient should be re- assured that weight loss by a small but tolerable change in lifestyle is much more likely to be maintained than weight lost by a crash diet. As weight falls, resting energy expenditure also declines: it is proportional to lean body mass, which also decreases, although at a slower rate than fat. This means that greater reductions in energy intake (>600 cal/​day or 2520 J/​day) will be needed to maintain the same rate of weight loss. If the 10% target is met, further loss to- wards an ‘ideal’ BMI of around 23 kg/​m2 may be feasible. Total energy intake Calculate current Reduce by 500–600 cal/d if BMI>28 Protein (10–15%) Other recommendations Salt < 6g/d Alcohol < 3 U/d for men, < 2 U/d for women Avoid ‘diabetic’ foods Percentage of total energy intake Carbohydrate (> 55%) Complex, fibre-rich Sucrose < 50g/d Fat (< 30%) Saturated < 10% Cholesterol < 250 mg/d Fig. 13.9.1.9  Dietary recommendations for people with diabetes. These guidelines now reflect healthy eating for the general population, rather than a diabetic diet.

13.9.1  Diabetes 2489 Weight loss is harder to achieve in diabetic patients than in their non​diabetic counterparts; possible reasons include fears about sugar rather than fat, and the adipogenic effects of insulin, sulphonylureas, and thiazolidinediones. In practice, weight loss of even 10% is not commonly achieved by diet and lifestyle modifi- cation alone; only 15 to 30% of newly diagnosed type 2 diabetic patients can normalize glycaemia initially by this means, and fewer than 10% can sustain this for 5 years or more. The progressive β-​cell dysfunction in type 2 diabetes (see earlier) makes it inevitable that the proportion of ‘dietary failures’ will increase steadily. However, a recent trial suggested that 46% of patients on a meal-replacement diet could achieve remission of diabetes within 6 years of diag- nosis, especially if weight loss >15 kg was achieved. Improving dietary composition Intakes of fat, salt, and refined sugar are generally too high in Westernized populations. Current recommendations for healthy eating are based on evidence of beneficial effects on body weight, glycaemic control, lipids, and blood pressure (see Fig. 13.9.1.9). Fat should provide less than 30% of total energy intake (in most industrialized countries, it accounts for 40%). Polyunsaturated or monounsaturated fats (e.g. sunflower or olive oils, respectively) are preferred to saturated animal fats, which should comprise less than 10% of total energy intake. Patients may need to be reminded that ‘good’ unsaturated fats still contain 9 cal/​g (38 J/​g) and therefore sustain obesity just as effectively as the others. Cholesterol should be limited to less than 250 mg/​day (less if dyslipidaemia is present). Carbohydrates should account for more than 55% of total en- ergy intake, preferably in the form of foods rich in soluble fibre (e.g. pulses, root and leaf vegetables, and fruit); the current WHO recommendation for the general population is for the consump- tion of at least five portions of fruit or vegetables per day. Sugary drinks (especially fizzy glucose solutions that are supposed to give energy) should be avoided, except to treat hypoglycaemia. The pre- sent recommendation, which seems reasonable but is not based on evidence, is to limit added sucrose to less than 25 g/​day and total sucrose intake to less than 50 g/​day. Protein should contribute 10 to 15% of total energy—​close to current levels in the general population. Sodium intake should be less than 6 g/​day, and less in patients with hypertension. Alcohol contains 7 cal/​g (29 J/​g), and beers and wines in par- ticular can be fattening. Intake should not exceed three units (30 g) per day in men and two units (20 g) per day in women, and should be further limited or avoided in those with hyperten- sion or obesity. Alcohol can delay recovery from hypoglycaemia (see next); ‘diabetic’ beers (low in sugar, but strong in alcohol) and spirits with sugar-​free mixers are especially likely to provoke hypoglycaemia. Moderate amounts of sucrose are acceptable (see earlier), and non​caloric sweeteners (such as aspartame) have no adverse metabolic effects. Diabetic sweets and foods contain sorbitol or fructose instead of glucose, and are an expensive way to get diar- rhoea; they should be avoided by patients, and withdrawn by the manufacturers. Optimizing meal patterns Judging the size and content of meals so as to limit glycaemic excur- sions remains an art rather than a science, and a skill which some patients develop with experience. Dosages of glucose-​lowering drugs that act acutely to cover meals (short-​acting insulin and sulphonylureas) can be tailored reasonably accurately to meals of similar composition but may not be matched to other meals, even when the total weights of carbohydrate, fat, and protein are similar. There has been much interest in the ability of various foods to raise blood glucose, usually measured as the ‘glycaemic index’; that is, the area under the curve of the rise in plasma glucose after eating a standardized load (50 g) of the food, expressed as a per- centage of the area under the glucose curve after ingesting 50 g of glucose. Foods with a low glycaemic index include pulses and cereals, probably because of their high fibre and complex carbo- hydrate contents, while bread has a surprisingly high index. The glycaemic index of many foods such as potatoes and pasta varies widely according to the method of cooking (and even the shape of the pasta), and mixing different foods in a real-​life meal has unpre- dictable effects on the overall postprandial glucose rise. It may be sensible to base meals around components with a low glycaemic index, but it is clearly not feasible to use the index to adjust dosages of antidiabetic medication. Appropriate portion size in meals is also important in limiting overall calorie intake. Portion size has crept up inexorably in res- taurants in many countries and probably contributes to the observed association between excessive weight gain and eating outside the family home. Increasing physical activity Short-​term exercise and improved physical fitness both in- crease insulin sensitivity, partly through increased translocation of GLUT-​4 units to the surface of skeletal muscle cells resulting in increased glucose uptake; this effect is independent of insulin and can enhance glucose uptake (under clamp conditions) better than metformin or the thiazolidinediones. Physical training also improves muscle blood flow. Several studies, notably the Finnish and American Diabetes prevention trials, have demonstrated that regular physical exercise reduces by over 50% the risk of im- paired glucose tolerance progressing to type 2 diabetes. There is also evidence that it significantly decreases cardiovascular events. Exercise must therefore be encouraged in all diabetic patients, but the advice must be realistic, achievable, and safe. Brisk walking for 30 to 40 min every day is better physiologically than a hectic workout in the gym once or twice a week, and is within almost everyone’s reach. Potential hazards of exercise include hypoglycaemia in patients on sulphonylureas or insulin, which may be delayed by several hours (see next), and cardiac disease. Patients at risk should have an ECG, with consideration for an exercise tolerance test and echo- cardiography, and appropriate treatment for ischaemic heart disease or heart failure. Exercise remains beneficial and important in these cases but should be built up gradually. Specific advice on exercise may be needed for those with neuropathy or active foot disease to avoid precipitating or exacerbating a foot lesion. Antiobesity drugs and bariatric surgery in diabetes Antiobesity drugs may be indicated in selected obese diabetic pa- tients with a BMI over 28 kg/​m2 and who have demonstrated, by losing weight beforehand through diet and exercise alone, that they are prepared to make long-​term changes in their lifestyle. Without this commitment, clinically useful weight loss is unlikely to be achieved or maintained beyond the period of drug prescription; the

section 13  Endocrine disorders 2490 medical and pharmacoeconomic benefits of modest weight loss for a couple of years in the obese patient’s middle age are not known but are probably not dramatic. Therefore, these medications should al- ways be regarded as an adjunct to dietary modification and exercise rather than an alternative to these interventions. Until recently the only drug available in many countries was orlistat, a gastrointestinal lipase inhibitor. With this, up to 30% of obese type 2 patients lose 10% or more of body weight within 6 to 12 months, HbA1c can fall by 1% or more, and dosages of glucose-​ lowering drugs, including insulin, may be decreased. Previously, the selective type 1 cannabinoid (CB1) receptor antagonist rimonabant was introduced; this reduces insulin resistance and may have the additional benefit of promoting smoking cessation. However, the exacerbation of pre-​existing depression or anxiety has resulted in the drug being withdrawn by the manufacturer. Sibutramine, a com- bined serotonin/​noradrenaline reuptake inhibitor, has also been withdrawn in Europe because of evidence of increased cardiovas- cular events. Recently liraglutide and semaglutide, incretins which stimulate insulin secretion and are used to treat diabetes (see later in chapter) have received regulatory body approval for the treat- ment of obesity. This is an expensive treatment option and is likely to limit use. Surgical treatment (bariatric surgery) with gastric banding, sleeve gastrectomy, gastric bypass and duodenal switch operations is indi- cated in selected patients with a BMI over 40 kg/​m2 or a BMI over 35 kg/​m2 if the diabetes is of recent onset (Table 13.9.1.3, and see Chapter 11.5). The number of operations performed in the United Kingdom has risen from 470 in 2003/​4 to 6500 in 2009/​2010. These operations are generally safe when performed by an experienced team and can achieve dramatic weight loss (up to 70% of excess fat, maintained for several years). Remission of diabetes occurs in ap- proximately 60% of patients with type 2 diabetes undergoing gastric bypass, with significant improvement in glycaemic control in others. The improvement in glucose metabolism precedes the weight loss and this may be due to the increase in GLP-​1 secretion and the re- duced glucagon levels which occurs in gastric bypass procedures. The reduction in morbidity and mortality as a result of bariatric sur- gery is likely to result in an increase in this treatment modality. Smoking Smoking is at least as common among diabetic patients as in the general population. Smoking greatly amplifies macrovascular risk in diabetic subjects: 10-​year mortality (mainly from myocardial in- farction) is about 50% higher than in diabetic non​smokers and twice as high as in non​diabetic non​smokers. Smoking may also accelerate the progression of nephropathy and possibly retinopathy. Many people living with diabetes, especially young women, con- tinue to smoke as a means of keeping thin, and because they fear gaining weight if they stop. Nicotine reduces fondness for sweet, energy-​dense, foods and may also be mildly thermogenic. Weight gain after stopping smoking averages 3 kg but about 20% of cases gain more than 6 kg; much of this weight is often lost within the fol- lowing 1 to 2 years, and it can be limited or prevented by careful dietetic support beforehand and in the months after cessation. Moreover, the risks of continuing to smoke are much greater than this degree of weight gain, especially in people living with dia- betes. Pharmacological support to overcome nicotine dependence including the use of nicotine replacement, antidepressants (e.g. bupropion, nortriptyline), and the nicotine receptor partial agonist varenicline each increases the chance of quitting by around two-​ to threefold. Electronic cigarettes (e-​cigarettes) are battery-​powered devices with cartridges that contain nicotine, flavours, and other chemicals which are inhaled as a vapour. These are a popular substi- tute for smoking as they are cheaper, widely available, and probably less harmful than cigarette smoking. However, there are concerns about their long-​term safety as clinical trials to ascertain safety have not been performed; there are also concerns that the sweet flavours and bright colours may encourage children to use the products and develop a nicotine addiction. Glucose-​lowering drugs Insulin Insulin is the cornerstone of treatment for type 1 diabetes and many with type 2 diabetes will eventually require insulin. Unfortunately, subcutaneously injected insulin cannot match the physiological pro- file of normal insulin secretion (see Fig. 13.9.1.10) and is a poor substitute for the finely tuned β cell with its nearly instantaneous capacity for ‘in-​flight’ adjustment. Moreover, insulin given subcuta- neously is absorbed into the systemic circulation rather than se- creted into the portal system where an immediate effect on the liver, and first pass clearance by that organ, are important in regulating the metabolic actions of insulin. History of insulin Insulin was traditionally extracted from pork and beef pancreases in acid ethanol and purified by precipitation and recrystallization. Plasma insulin concentration Nondiabetic insulin profile 24 Insulin lispro Human soluble Premixed (30:70, soluble:NPH) NPH Insulin glargine Clock time (h) 06 24 17 12 B L D 08 06 17 12 08 06 06 Fig. 13.9.1.10  A time course of insulin preparations, compared
with the normal diurnal profile of plasma insulin concentrations in
non​diabetic subjects (top). Breakfast (B), lunch (L), and dinner (D) were given as shown. Fast-​acting analogues (such as lispro) act more rapidly than conventional soluble ones but are still sluggish compared with normal prandial insulin release. Premixed insulins injected in the early evening cover the evening meal adequately, but the long-​ acting component can cause hyperinsulinaemia and troublesome hypoglycaemia in the small hours. None of the conventional long-​acting insulins reliably lasts 24 h; new long-​acting analogues such as insulin glargine or insulin detemir may provide adequate background insulin levels with once-​daily injections.

13.9.1  Diabetes 2491 Soluble (or ‘crystalline’) insulin prepared in this way was contam- inated with other islet proteins, including glucagon and pancreatic polypeptide, which had an adjuvant-​like effect and enhanced the im- munogenicity of the injected insulin; immune reactions were rela- tively common with the ‘dirty’ animal insulins in use until the 1970s (see next). More sophisticated purification techniques including gel filtration yield ‘highly purified’ or ‘monocomponent’ insulins which only rarely provoke immune reactions. Biosynthetic human-​sequence insulin, produced by recombinant DNA technology, entered clinical practice in the early 1980s and was the first genetically engineered protein to be used therapeutic- ally. The current approach is to introduce a synthetic gene for recom- binant proinsulin or a novel insulin precursor into yeast; the secreted product is then cleaved enzymatically to yield insulin and C-​peptide. There are some clinically relevant differences between the three species used therapeutically, although the shortcomings of insulin therapy relate mainly to the general pharmacokinetic misbehaviour of injected insulin. Human insulin is more lipophilic than porcine and bovine insulins and is slightly more rapidly absorbed: human soluble insulin especially may lower glucose faster and patients being transferred from other species should be warned of this and prandial doses reduced initially by one-​third. Human ultralente has a shorter and steeper action profile than its animal counterparts, particularly the bovine preparation; in real life, human ultralente behaves similarly to lente or isophane insulins and does not provide adequate basal levels for a full 24 h. Human insulin has been sug- gested to interfere with awareness of hypoglycaemia, but the balance of evidence does not support this view (see next). In many countries, animal insulins are no longer available and in most countries, it is very unusual to initiate animal insulin in a patient naïve to insulin. Typical users of animal insulin were started on it many years ago and have either experienced adverse effects of switching to human insulin or fear these effects. It important that a supply of animal in- sulin is available for such patients. Insulin absorption Absorption of insulin injected subcutaneously is slow and unpredict- able. Individual day-​to-​day variability in the amount absorbed within a few hours can exceed 50%. This means that small changes (<10%) in insulin dosage are unlikely to influence glycaemic control, and that insulin treatment should generally not be adjusted on a daily basis. Insulin absorption is influenced by the physical state of the insulin (soluble or delayed action), its speed of dissociation into monomers, the lipophilicity of the insulin species, and by blood flow and other characteristics of the injection site. Absorption is accelerated and may lead to noticeably faster falls in blood glucose, by stimulating general or local blood flow through exercise, hot climate, saunas, and/​or massaging the injection site. Conversely, absorption is slowed when subcutaneous blood flow is reduced (e.g. in cold conditions or hypovolaemic states). Lipohypertrophy, which may develop at frequently used injection sites, can significantly delay absorption—​ another reason for avoiding such areas. The anatomical site of injection also influences the rate of sub- cutaneous absorption. It is fastest in the abdomen (also a good site to limit any effects of exercise) and arm, and slower in the leg. These differences are often eclipsed by the overall variability in absorption. Absorption from muscle is faster, presumably because of its higher blood flow, and this route is preferred for the emergency treatment of hyperglycaemia or ketoacidosis if the best option, controlled intravenous infusion, is not practicable. Insulin preparations Soluble (regular or short-​acting) insulin injected subcutaneously begins to lower glucose within 30 min, has a peak effect between 1 and 2 h and lasts 3 to 5 h (see Fig. 13.9.1.9). This action profile is suit- able for covering meals or hyperglycaemic emergencies and for use in insulin pumps or infusions. However, it would have to be injected several times per day to control hyperglycaemia around the clock, at the cost of frequent hypoglycaemia. Long-​acting preparations are therefore used to cover basal insulin requirements. Various approaches have been used to slow and prolong insulin absorption, especially the chemical combination of insulin into complexes that release it slowly. More recently, synthetic analogues have been designed whose structure promotes precipitation when injected subcutaneously (see Fig. 13.9.1.9). Isophane insulins are also known as NPH (neutral protamine Hagedorn, from the director of the Danish laboratory where they were developed). They consist of a microcrystalline complex of insulin and the highly basic protein protamine (intriguingly isolated from fish sperm), together with trace amounts of Zn2+. Isophanes were derived from protamine–​zinc insulin which has a longer but highly unpredictable action profile. Isophanes produce peak plasma insulin levels at variable intervals between 4 and 8 h after injection, and their glucose-​lowering action wears off rapidly after 10 to 12 h. Insulin–​zinc suspensions (lente insulins) employ higher Zn2+ concentrations which encourage insulin to form crystalline lattices. Varying the reaction pH can produce either larger crystals which are particularly slow to dissolve (ultralente) or the amorphous semilente which releases insulin faster; the familiar lente is a 70:30 mixture of ultralente and semilente. Ultralente made with bovine insulin has a long, relatively flat action profile that can last 24 h or more, while human ultralente and the lente insulins of all three species have glucose-​lowering profiles similar to that of isophane. These long-​ acting insulins have a cloudy appearance and need to be shaken be- fore use to bring the insulin into suspension; visibly large particles or discoloration indicates that the insulin has become denatured and will have lost activity. Both lente and isophane insulins can be in- jected alone or mixed with soluble insulin. Premixed insulins contain a short-​acting soluble component to- gether with a longer-​acting lente or isophane. The aim is to provide prandial cover and then basal levels for several hours thereafter. A  variety of proportions of short-​acting insulin were previously available however a mixture with a 30:70 ratio is the only insulin generally available as most insulin manufacturers have reduced the available range in favour of analogue premixed insulins. All these insulin types have been produced with porcine-​, bovine-​ and human-​sequence insulins, and are available in cartridges for pen injection devices and some in disposable pen devices. Insulin analogues The pharmacokinetic properties of native insulins of any species are poorly suited to subcutaneous injection: soluble insulins (des- pite their high-​speed trade names) are too slow and prolonged in duration, while long-​acting insulins do not provide reliable enough 24-​h basal levels to be given once daily. Various synthetic

section 13  Endocrine disorders 2492 insulin analogues, designed by molecular modelling, have improved physicochemical characteristics. Fast-​acting analogues are modified at the C-​terminal end of the B chain, an area crucial in the self-​association of insulin molecules, so as to resist dimerization and hexamerization. Insulin hexamers formed in the subcutaneous injection site, dissociate slowly into absorbable monomers, and this is a rate-​limiting step in insulin absorption. Faster-​acting analogues include insulin lispro (interchanging the B28 lysine and B29 proline residues of the normal human sequence), in- sulin aspart, which carries aspartic acid at position B28 instead of the usual proline and insulin glulisine, which substitutes two amino acids. They have an appreciably faster and shorter action profile (see Fig. 13.9.1.9), and day-​to-​day variability in absorption and glycaemic responses may also be decreased. They can therefore reduce both prandial hyperglycaemia and the risk of postprandial hypoglycaemia. A newer formulation with vitamin B3 and L-arginine has even faster absorption. Despite theoretical advantages, meta-​analyses show only very modest reductions in HbA1c (c.0.1%) and reductions in hypo- glycaemic episodes when a fast-​acting analogue is substituted for soluble insulin, but there are significant improvements in quality of life generally attributable to the convenience of injecting immediately before or after meals rather than 30 min beforehand. Long-​acting insulin analogues have also been developed and have become the most commonly prescribed insulin in many countries. These are designed to give a smoother 24-​h profile than isophane (‘peakless’ insulin). At present three forms are available. Insulin glargine (A21 glycine, with two extra arginine residues extending the C-​terminal of the B chain) has an altered isoelectric point such that it is soluble in the vial or cartridge at pH 4 but precipitates under the skin at pH 7. Insulin detemir has a delayed action due to the addition of a fatty acyl chain that binds to plasma proteins such as albumin. It has a slightly shorter half-​life than glargine and can be given once or twice daily. Insulin degludec has a duration of action up to 42 hours (the addition of hexadecanedioic acid to lysine at B29 results in hexamer formation which creates a subcutaneous depot) which results in a basal insulin with less variation and greater flexibility of timing of injection. These analogues are clear in the vial or cartridge—​potentially a source of confusion with rapidly acting insulin. Claims have been made for improved HbA1c levels and less daytime hypoglycaemia, as well as weight loss or neutrality for detemir in type 2 diabetes, but the most robust finding appears to be a reduction in nocturnal hypoglycaemia. Side effects of insulin Hypoglycaemia is the most common complication of insulin treatment and can be unpleasant, debilitating, and occasionally life-​threatening. Mild hypoglycaemia is common—​many insulin-​treated patients have at least one episode most weeks—​but serious attacks causing unconsciousness or requiring the assistance of others are rare, about once every 3 patient-​years. Predictably, the frequency of both mild and severe attacks rises progressively when mean blood glu- cose levels are lowered by intensive insulin therapy; hypoglycaemia was three times more frequent in the tightly controlled group of the Diabetes Control and Complications Trial than in conventionally treated patients (see next). The manifestations and treatment of hypoglycaemia are covered in detail later. As discussed there, there is no convincing evidence that the use of human as opposed to animal insulins specifically interferes with awareness of hypoglycaemic symptoms. Weight gain is due to the anabolic effects of insulin, compounded by energy saved from glycosuria and sometimes by overeating after hypoglycaemia. Fear of weight gain discourages some patients, es- pecially young women, from taking their full insulin dosages; sur- prisingly often, deliberate omission or underdosing of insulin may be used by patients wishing to stay thin. Lipohypertrophy is the local thickening of subcutaneous tissue at frequently used injection sites, and is probably due to the lipogenic effects of high local insulin concentrations. Lipohypertrophy can be unsightly and can significantly delay insulin absorption. It can be prevented by rotating injections around several sites, and large le- sions can be removed by liposuction. Insulin allergy, now very rare with highly purified (especially human) insulins, can include local IgE-​mediated erythematous re- actions or even anaphylaxis. The commonest manifestation is re- peated pain at the site of injection. Lipoatrophy (localized pitting of the skin due to loss of subcutaneous fat) is apparently related to a chronic immune response generated around insulin crystals. Immune insulin resistance was seen with impure animal and espe- cially bovine insulins; high titres of insulin-​binding antibodies mop up free insulin from the circulation, resulting in very high insulin re- quirements (occasionally more than 10 000 U/​day), sometimes with unpredictable hypoglycaemia following the release of antibody-​ bound insulin. Insulin oedema is rare, and is usually seen in patients recovering from ketoacidosis who have been deprived of insulin for long periods. Fluid retention is probably due to the sodium-​conserving effects of insulin on the renal tubule, and may cause ankle or gener- alized oedema. It usually resolves within a few days, although treat- ment with diuretics or ephedrine may be required. Insulin neuritis refers to severe, persistent neuropathy following the use of insulin in individuals with very poor control glycaemic control; however, this is a consequence of the sudden improvement in metabolic state rather than a side effect of the insulin itself. Usually the pain resolves after some months. Insulin regimens Different individuals may need quite different insulin regimens, depending on their residual insulin reserve and severity of insulin resistance, as well as the desired tightness of control and the incon- venience that the patient will accept. Specific insulin schedules used in type 1 and type 2 diabetes are described later. Insulin dosage The healthy pancreas secretes about 40 to 60 U of insulin daily. Therapeutic insulin requirements range from less than this in thin type 1 patients (notably during the ‘honeymoon period’) to more than 200 U/​day in very obese, insulin-​resistant type 2 patients. High insulin requirements are often due to insulin resistance (see earlier), whereas low or falling dosages may be caused by weight loss (including anorexia nervosa), coeliac dis- ease, or loss of counterregulatory hormones in Addison’s dis- ease or hypothyroidism—​all these conditions being associated with type 1 diabetes. Changing dosages, especially in previously stable subjects, should prompt investigation of these possibilities. Some patients with ‘brittle’ diabetes or psychological maladapta- tion to life with diabetes may pretend to take very high or very low dosages (see later). Interestingly, insulin requirements via

13.9.1  Diabetes 2493 continuous subcutaneous infusion are typically 30% less than by intermittent injections. Types of insulin Formularies contain a bewildering assortment of insulins, many distinguished by imaginative claims about their action profile. Practically, prescribers should become familiar with regimens based on one or two preparations from the following broad classes: • Fast-​acting insulin:  either a soluble (regular) insulin such as Humulin S or Actrapid, injected 20 to 30 min before eating, or a faster-​acting analogue (e.g. lispro or aspart) which can be given immediately before or even shortly after eating. Fast-​acting in- sulin is either given as a fixed dose or as a ratio to the ingested carbohydrate. • Long-​acting insulin: either a lente insulin (e.g. Humulin Zn or Insulatard) or an isophane (e.g. Humulin I or Monotard). With either, circulating insulin falls to below useful levels after 10 to 14 h; they therefore need to be given twice daily in C-​peptide negative patients, although those with residual insulin secretion (or who are given three premeal injections of soluble insulin) may be able to maintain good glycaemic control with a single bedtime injection. Bovine (but not human) ultralente can last a full 24 h, but its absorption is erratic and it is rarely used. The long-​acting analogues currently available (such as insulin glargine, detemir, or degludec) have flat, steady action profiles that can provide basal insulin levels with a single daily injection. The timing of long-​acting insulin injections does not have to be yoked to meal- times as tightly as for soluble insulin. It is convenient to inject the dose at bedtime rather than together with the before-​supper sol- uble dose. This is because the action profile of long-​acting insulin clashes with the physiological changes in insulin sensitivity that occur overnight. Growth hormone is normally secreted in large spikes on entering deep sleep, typically between 24.00 and 02.00 h; this induces delayed insulin resistance which raises blood glucose during the hours leading up to breakfast. This ‘dawn phenomenon’ is accentuated if insulin levels are falling simultaneously—​as hap- pens if long-​acting insulin is injected in the early evening. Another hazard with this timing is potentially dangerous nocturnal hypo- glycaemia when insulin levels peak during the early morning (typ- ically 02.00–​04.00). Both problems can be reduced by delaying the long-​acting injection until bedtime (22.00–​23.00), when the risk of nocturnal hypoglycaemia is lower, and insulin levels gen- erally persist long enough to counteract the insulin resistance of the dawn phenomenon. If a second injection is required, this can be given with the before-​breakfast soluble insulin. Note that the long-​acting analogue insulins (glargine and detemir) should not be mixed in the same syringe as short-​acting insulin. • Premixed insulins (e.g. 30% short-​acting with 70% long-​acting) are obviously more convenient than giving short-​ and long-​acting insulins separately, but they lack flexibility. Premixed insulin in- jected 30 to 40 min before breakfast can achieve good glycaemic control through the morning and afternoon, but timing the evening dose is problematic: giving it before supper will tend to cause both early morning hypoglycaemia and fasting hypergly- caemia because of the time course of the long-​acting component, and simply increasing the evening dosage often makes nocturnal hypoglycaemia worse while failing to lower the before-​breakfast glucose. Premixed preparations including rapidly acting ana- logues such as insulin aspart or lispro and isophane are also avail- able and may be of some advantage. Premixed analogue insulin which has 50% short-​acting analogue and 50% isophane is some- times given three times a day with the main meals to those who are insulin resistant and this dosing regime has shown benefit. Insulin strengths Until recently all insulins in the United Kingdom were available at a U100 strength (100 units per ml). Recently insulins of higher strength have been introduced such as Tresiba U200 (insulin degludec U200) and Lantus U300 (insulin glargine U300). The rationale for the introduction of these insulins is to allow a smaller volume injection to be given. These insulins may have altered properties compared to the U100 formulation, for example Lantus U300 is a longer-​acting insulin compared to the U100 form and has shown a lower incidence of nocturnal hypoglycaemia during initiation in trials, although a higher dose may be required to achieve targets. These insulins come in pens which display the actual dose given however they should not be given via a standard U100 insulin syringe as this may result in double or triple the dose administered. Biosimilar insulins As the patents expire on some of the analogue insulins, less ex- pensive generic formulations have been created referred to as biosimilar insulins. These insulins contain the same molecule as the original insulin however differences in manufacturing pro- cesses may alter some of the properties so that the biosimilar in- sulin is not identical. The main advantage is that these insulins are less expensive. Insulin injections Most insulin formulations are now available for both conventional syringes or pen injection devices. Pen injectors are compact, con- venient, and easy to use: the required dose is ‘dialled up’ and injected by pressing the plunger; the ratchet mechanism of most pens gives an audible click that can help blind patients to count dosages. Syringes and pens carry very fine (28–​31 G) needles that allow in- sulin to be injected almost painlessly. The needle should be pushed in vertically and the insulin injected over a few seconds. A needle does not need to be longer than 4 mm in all patients to reach the subcutaneous space. If using a needle longer than 8 mm then it is advisable to inject into a pinched-​up fold of skin to avoid intramus- cular injection in places where there is limited subcutaneous tissue. Backtracking of insulin to the skin surface, which can occasionally cause loss of several units of insulin, may be reduced by leaving the needle in place for a short while. A spot of bleeding may occur; very rarely, sudden hypoglycaemia may be due to direct injection of in- sulin into a subcutaneous vein. Injections can be given into any site that is accessible and well-​ padded with adipose tissue, especially the abdomen, thighs, buttocks, and upper arms. The abdomen has the advantage (theor- etically at least) of relatively faster absorption that is less influenced by exercise, as compared with the limbs. Rotating injection sites (e.g. between the abdomen and leg, or around the quadrants of the ab- domen) helps to avoid local reactions, especially lipohypertrophy which can make insulin absorption slow and erratic.

section 13  Endocrine disorders 2494 Jet injectors fire a metered dose of insulin as a high-​pressure aerosol that penetrates the skin. These have obvious appeal to pa- tients with needle phobia, although there may be bruising and de- layed discomfort at the injection site. Jet injectors are bulky and expensive and do not offer any pharmacokinetic advantages over conventional injections. Inhaled insulin Several companies have developed an aerosol formulation of in- sulin that can be inhaled into the lower airways (insulin is not ab- sorbed from the nasal passages). Inhaled insulin has almost identical pharmokinetic characteristics to subcutaneously injected soluble in- sulin and so its use might be considered to be predominantly a matter of convenience to avoid injections, especially in those with injection site problems or needle phobia. Sophisticated pharmaceutical prep- aration and delivery devices are required to ensure accurate dosing. It cannot be used by current smokers (as absorption is variably en- hanced to an unpredictable degree) or subjects with chronic airways disease, including asthma and chronic obstructive pulmonary dis- ease. Transient cough may occur. Regular lung function testing is advised, as there is a progressive fall in lung function although in most people this is no more rapid than the reduction with age. An increase in insulin autoantibodies has been noted although the sig- nificance is uncertain. Inhaled insulin can be used in both type 1 and type 2 diabetes, although in type 1 diabetes a subcutaneous in- jection of intermediate acting insulin is still required. The long-​term risks of inhaling insulin over many years are not known and there is a theoretical concern of an increased risk of lung neoplasia. A pre- vious preparation of inhaled insulin was withdrawn after poor sales; however, a new product is now available with a smaller device for inhalation. Insulin pumps Portable insulin pumps that administer continuous subcutaneous insulin infusion were developed by Pickup and colleagues in the late 1970s. Modern pumps are compact and light and worn in a belt or holster. Soluble insulin in a special cartridge is delivered through a fine-​bore butterfly-​type cannula, which is inserted subcutaneously in the anterior abdominal wall or other suitable site and generally left in place for 2 to 4 days; the pump can be safely removed for up to 60 min for bathing or other activities. Different basal rates can be preprogrammed, and mealtime boluses are selected and given by pressing a button. Typical basal rates are 0.5 to 1.5 U/​h during the day and 0.5 to 1 U/​h overnight, with mealtime boluses (given imme- diately before meals or snacks) amounting to about 50% of the total daily dose. Most centres use rapid acting analogues in pumps and there is trial evidence to support this. Continuous subcutaneous insulin infusion (CSII) CSII can achieve relatively steady insulin levels under laboratory conditions and can partly overcome the variability of subcuta- neous insulin absorption seen with intermittent injections of larger doses. When used carefully by highly motivated patients who are supported by an experienced diabetes care team, continuous sub- cutaneous insulin infusion can achieve glycaemic control which is better than that achieved with multiple injections; the two were used side by side in the Diabetes Control and Complications Trial. Insulin pumps are expensive (£2600–​£3500 or (US) $5000–​6000) as are consumables (another £1800 per year); medical backup can also be costly to provide. Continuous subcutaneous insulin infusion is indicated for well-​informed patients with type 1 diabetes who are prepared to monitor their blood glucose frequently, learn carbo- hydrate counting, and take responsibility for adjusting the pump. It provides more flexibility for varied lifestyles than multiple daily doses. Randomized trials suggest modest reductions in HbA1c and reduced hypoglycaemia. Although not all randomized trials confirm this, with careful patient selection these benefits are frequently seen in clinical practice and many patients refuse to return to convention- ally delivered insulin. CSII appears to be most beneficial in patients striving hard to improve glycaemic control who are limited by recur- rent hypoglycaemia. It is widely used in the United States of America and many European countries as well as other parts of the world. Infections at the infusion site with pyogenic skin commensals or unusual organisms (e.g. atypical mycobacteria) are uncommon but can be troublesome and cause rapid deterioration in glycaemic con- trol. An increased rate of diabetic ketoacidosis was reported with earlier and less reliable pumps. With CSII, the subcutaneous insulin depot is only a few units, and any interruption of insulin delivery (e.g. with pump failure or cannula blockage) can lead to rapid rises in blood glucose and especially ketone levels. However, modern pumps carry no excess risk of diabetic ketoacidosis as compared with inten- sified injection therapy. Similarly, the risk of hypoglycaemia due to the pump overrunning is now very low. Continuous glucose monitors measure interstitial fluid glucose; this has a good correlation with capillary glucose when the glucose level is stable however may lag behind capillary glucose when this is changing rapidly. Insulin pumps may be used simultaneously with continuous glucose monitoring systems, this form of therapy known as sensor augmented pump therapy. In some systems the glucose reading is continuously displayed on the pump, with indicators that show if the glucose is rising or falling and alarms set to alert the user to low or high glucose levels. This may be particularly useful for those who have no awareness of hypoglycaemia. The use of sensor aug- mented pump therapy may also improve overall HbA1c level if used regularly. Closed loop systems using insulin pumps and continuous glucose monitoring systems are now being introduced. A limiting factor has been accommodating food into the algorithms, however trials have demonstrated benefits in particular circumstances such as in pregnancy. The current systems of sensor augmented pump therapy include devices that may automatically suspend insulin de- livery when the glucose falls below a certain parameter and automat- ically restarts delivery as the glucose rises. Continuous intraperitoneal infusion The peritoneum is a good route for insulin administration: absorp- tion is very rapid across its large surface area and insulin enters the portal circulation. Continuous intraperitoneal insulin infusion has been used in some cases, mostly employing a pump and reservoir implanted subcutaneously in the abdomen and delivering insulin through a flexible cannula sewn into the peritoneal cavity. The reser- voir is filled with soluble insulin through an injection port lying just beneath the skin and is emptied by a liquid/​gas compression system at a rate that can be varied by an external electromagnetic control. Continuous intraperitoneal insulin infusion can provide basal in- sulin; meals need to be covered by additional insulin, either injected subcutaneously or triggered by an external control device.

13.9.1  Diabetes 2495 Intraperitoneal pumps are expensive, and convincing indications for their use are rare. They have been successful in some patients with apparently very high subcutaneous insulin dosages but surpris- ingly normal intravenous requirements. It is now clear that this situ- ation is not due to a mysterious syndrome of ‘subcutaneous insulin resistance’, and that most, if not all, of these patients are interfering with their own treatment (see next). In this setting, continuous intraperitoneal insulin infusion is probably effective because these pumps are difficult to sabotage. Pramlintide Amylin is a 37 amino acid peptide which is cosecreted from the β cell with insulin and is deficient in type 1 diabetes and relatively defi- cient in type 2 diabetes. Amylin slows gastric emptying and regulates postprandial glucagon release. Pramlintide is an amylin analogue which may be injected at mealtimes. This has demonstrated an im- provement in postprandial glucose excursions and HbA1c without an increase in weight or hypoglycaemia; however, it is not widely available outside the United States. Oral hypoglycaemic agents Sulphonylureas and meglitinides The sulphonylureas were the first orally active glucose-​lowering drugs to be used and were discovered in the 1930s when early sul- phonamide antibiotics were found to cause hypoglycaemia. The first generation (chlorpropamide, tolbutamide) have since been super- seded by the second generation (e.g. gliclazide and glibenclamide) and by newer agents such as glimepiride. Repaglinide, a meglitinide, acts in a similar way to the sulphonylureas. Mode of action  Sulphonylureas are insulin secretagogues but in- sulin synthesis is not stimulated. Insulin levels peak within 1 to 2 h and decline within 4 to 6 h for the short-​acting drugs (such as gliclazide) but may remain elevated for much longer with chlorpropamide and glibenclamide, which therefore carry a greater risk of hypoglycaemia. An extrapancreatic action has also been at- tributed to sulphonylureas (i.e. improving insulin sensitivity). This effect is small and is probably explained by the non​specific decrease in insulin resistance (glucotoxicity) when hyperglycaemia is cor- rected by any means. Repaglinide acts in a similar way to the sulphonylureas but is structurally different. It is derived from the non​sulphonylurea part of the glibenclamide molecule (called meglitinide), which was found fortuitously to have glucose-​lowering activity of its own. Nateglinide behaves in a similar fashion and both of these drugs are particularly effective at increasing insulin levels after meals. Meglitinides may be useful as a substitute for sulphonylureas if a patient experiences hypoglycaemia with sulphonylureas particularly with exertion. Efficacy and potency  The ability of these agents to lower glycaemia depends on how much insulin is available for release from the β
cells (which are already stimulated by hyperglycaemia) and by the severity of insulin resistance. In practice, all sulphonylureas lower basal and postprandial glucose levels by no more than 2 to 4 mmol/​litre and HbA1c by 1 to 2%; mild hyperglycaemia may therefore be corrected but patients with fasting glucose in excess of 13 mmol/​litre are very unlikely to achieve normoglycaemia (pri- mary failure). Moreover, as β-​cell function declines progressively in type 2 diabetes, many patients who initially respond well to sulphonylureas will subsequently need additional glucose-​ lowering drugs; this secondary failure overtakes 5 to 10% of pa- tients per year, in a cumulative fashion. These limitations apply to all sulphonylureas and repaglinide: the more potent drugs have lower therapeutic dosages than the earlier agents but cannot lower glycaemia any further. Pharmacokinetics  Most are taken twice daily with meals; glimepiride is taken once daily and repaglinide with each meal. Chlorpropamide has a very long action profile, while glibenclamide shows variable and sometimes prolonged hypoglycaemic activity. Sulphonylureas and repaglinide bind to circulating proteins and may be displaced by other strongly protein-​bound drugs, causing hypoglycaemia (see next). All these drugs are cleared through the kidneys and can accumulate in renal failure, causing frequent hypo- glycaemia and other side effects. Gliquidone and tolbutamide are metabolized mainly in the liver and may be slightly less hazardous in patients with renal impairment, although insulin is usually indi- cated in these cases. Side effects  Weight gain is due to the anabolic effects of hyper­ insulinaemia, compounded by reduced losses of energy through glycosuria. Weight gain is typically 2 to 3 kg greater than with diet alone or metformin. Hypoglycaemia is rarer than with insulin, but the risk is greater with longer-​acting sulphonylureas (glibenclamide, chlorpropamide), in renal failure, and especially in older people. Sulphonylurea-​ induced hypoglycaemia may be more protracted than that caused by insulin and is more likely to result in hospital admission. All patients taking sulphonylureas should be aware of this side effect and should have the means to check their capillary glucose, particularly if they are drivers. Sulphonylureas can cause allergic reactions including skin rashes (notably Stevens–​Johnson syndrome) and marrow dyscrasias, and can precipitate acute intermittent porphyria. Side effects exclusive to chlorpropamide include the syndrome of inappropriate secretion of antidiuretic hormone (see Chapter  21.2.1) and acetaldehyde-​ mediated facial flushing on drinking alcohol. The cardiovascular safety of sulphonylureas has remained under a cloud since tolbutamide was associated with an excess of cardio- vascular deaths during an essentially uninterpretable study (the University Group Diabetes Program or UGDP) conducted in the 1970s; the presence of the ABCC9 (SUR2) receptor on cardiomyocytes has recently reinforced suspicions that these drugs may trigger is- chaemia and arrhythmias (by preventing preconditioning). However, the long-​term United Kingdom Prospective Diabetes Study found no evidence that patients treated with sulphonylureas suffered cardiovas- cular events more often than those treated with insulin. Glimepiride is highly selective for ABCC8 (SUR1). Indications and contraindications  These drugs can be used as first-​line therapy for non​obese subjects with type 2 diabetes in whom lifestyle and dietetic measures have failed to control hyper- glycaemia. However, because of their tendency to increase weight, in the overweight majority of type 2 diabetes patients, sulphonylureas are used as second-​line agents, typically combined with metformin, which may partly offset the weight gain. Sulphonylureas also have a less durable effect than other agents for treating type 2 diabetes.

section 13  Endocrine disorders 2496 Insulin secretagogues are inappropriate for severely insulin-​ deficient patients or during intercurrent illness, when insulin is needed, and are unlikely to be effective if fasting glucose ex- ceeds 13 mmol/​litre. Sulphonylureas are contraindicated in renal failure: all should be stopped, and insulin started if serum creatinine exceeds 250 µmol/​litre. Most sulphonylureas cross the placenta and are contraindicated in pregnancy; however, glibenclamide does not cross the placenta. Studies show that it may be used to control hyperglycaemia in pregnancy in type 2 diabetes and gestational diabetes but not as effectively as insulin. Therefore, it is usually used as a substitute for insulin in pregnancy if insulin is not an acceptable therapy to the patient or if insulin is not available or affordable (see Chapter 14.10). Sulphonylureas are the therapy of first choice in patients with HNF1α MODY, since these subjects are exquisitely sensitive to these agents, and in patients with the Kir6.2 mutation, who may require very high doses (see earlier). Many drugs interact with sulphonylureas, the most common out- come being hypoglycaemia due to displacement and/​or decreased clearance of protein-​bound sulphonylureas (e.g. by sulphonamides, fibrates, salicylates, and probenecid). Potential interactions must al- ways be checked for any drug being contemplated in patients re- ceiving sulphonylureas. Choice of drug  There is little to choose between the newer agents; chlorpropamide is now obsolete. Glibenclamide should be avoided in older people because of its unpredictable tendency to cause hypoglycaemia. Metformin Metformin and phenformin are biguanides, the class of compounds responsible for the mild hypoglycaemic action of goat’s rue Galega officinalis (an otherwise undistinguished weed). Phenformin is no longer available in many countries because it carries a 10-​fold greater risk of lactic acidosis, and metformin has only fairly recently entered clinical use in the United States of America. Mode of action  Metformin acts primarily by inhibiting gluconeo­ genesis in the liver, thus reducing the raised hepatic glucose output which underpins basal and overnight hyperglycaemia; this effect- ively enhances the action of insulin on the liver. AMP kinase, a key enzyme that balances anabolic and catabolic processes in the liver and other tissues, is an important target for metformin action. Peripheral glucose uptake may also be increased, while gastrointes- tinal side effects may help to reduce fondness for food. Metformin does not stimulate insulin secretion. Overall, metformin lowers blood glucose (especially postpran- dial) by 2 to 4 mmol/​litre and HbA1c by 1 to 2%, which is compar- able to the effect of sulphonylureas. On its own, metformin does not cause hypoglycaemia, although this can obviously occur when it is combined with either a sulphonylurea or insulin. Weight does not usually increase with metformin, and may fall. Metformin may have beneficial cardiovascular effects, as the United Kingdom Prospective Diabetes Study found a reduction in vascular events in the metformin-​treated group only (see next). It is not clear whether this is related to the specific metabolic effects of metformin (improved insulin sensitivity), to its mild antiobesity properties, or to other actions such as reported reductions in blood pressure and coagulability. Reduced cancer risk is also reported with metformin. Pharmacokinetics  Metformin is given twice or three times daily with meals. It is cleared mainly through the kidneys, and the in- crease in plasma levels in renal failure is a major risk factor for lactic acidosis. A slow-​release preparation is also available, which is taken once daily and appears to produce fewer gastrointestinal side effects. Side effects  Gastrointestinal symptoms (30% of cases) include al- tered taste, loss of appetite, heartburn, abdominal discomfort and bloating, and diarrhoea (metformin is the most common cause of this in the diabetic clinic). These problems are mostly mild, but may discourage the patient from taking the drug; they can be reduced by starting with a low dosage and increasing it slowly. Lactic acidosis is very rare with metformin (about three cases per 100 000 patient-​years) if it is carefully prescribed. This stems from the mode of action of metformin, namely the inhibition of hepatic gluconeogenesis—​a process that constantly consumes the lactate produced by glycolysis. Blood lactate levels are modestly raised in patients receiving biguanides, and can escalate rapidly and cause life-​ threatening acidosis if lactate is overproduced (e.g. in respiratory or cardiac failure), or is not cleared by the liver (hepatic failure), or if metformin accumulates in renal failure. The risk is also increased in the presence of excessive amounts of alcohol. Lactic acidosis is described in detail later. Megaloblastic anaemia can occur due to impaired absorption of vitamin B12 and 5-​yearly vitamin B12 estima- tions have been recommended. Indications and contraindications  Metformin is now considered the first-​line treatment in type 2 diabetes patients whose hypergly- caemia does not respond adequately to modification of diet and life- style; as it does not tend to cause weight gain, and may even reduce weight, it is especially valuable in obese patients. Recent American Diabetes Association guidelines propose starting metformin con- currently with lifestyle interventions, but this is not universally accepted. The addition of metformin can also be helpful in obese patients who are poorly controlled by sulphonylureas or insulin. Metformin has also proved beneficial in other insulin-​resistant conditions such as polycystic ovary syndrome (resulting in im- proved fertility, reduced hirsutism, and oligomenorrhoea) and im- paired glucose tolerance where it reduces progression to diabetes by around 25%. Contraindications include all the major organ failures—​renal, hepatic, cardiac, and respiratory. It should not be used when serum creatinine concentration exceeds 150 µmol/​litre or the estimated glomerular filtration rate (GFR) is less than 30 ml/​min. Studies have reported beneficial outcomes when patients with stable cardiac failure are treated with metformin. It must also be discontinued for 2 days after receiving radiographic contrast media to reduce the risk of lactic acidosis if contrast mediated nephropathy occurs. Thiazolidinediones Thiazolidinediones are a class of glucose-​lowering drugs which improve insulin sensitivity. There are distinct differences between individual thiazolidinediones which influence their therapeutic spectrum and safety. Pioglitazone is currently available in many countries; troglitazone has been withdrawn because it caused rare

13.9.1  Diabetes 2497 but life-​threatening hepatic damage, and rosiglitazone because of concerns of cardiovascular effects. Mode of action and pharmacokinetics  Thiazolidinediones bind to specific receptors in the nucleus which have the cumbersome title of peroxisome proliferator activating receptor-​γ (PPAR​γ). PPAR​γ and the related PPAR​α (the target for the fibrate class of lipid-​lowering drugs) are ligand-​activated transcription factors whose natural lig- ands appear to be fatty acid derivatives. PPAR​γ that has bound a thiazolidinedione forms a heterodimeric complex with another nu- clear receptor, retinoid X receptor, bound to its own endogenous ligand, retinoic acid. The heterodimer then binds to specific recog- nition motifs found in the promoter sequences upstream of many genes, notably those involved in adipocyte and lipid metabolism. The affinity of individual thiazolidinediones at PPAR​γ par- allels their glucose-​lowering ability in animal models of type 2 diabetes, but their precise mode of action remains uncertain. Thiazolidinediones exert concerted effects that encourage the storage of triglyceride in mature adipocytes, including the differen- tiation of preadipocytes into adipocytes and enhanced expression of lipogenic enzymes; overall, circulating levels of free fatty acids fall and this may reduce hepatic glucose production and increase glucose uptake into muscle as described earlier. The net effect is to enhance the action of insulin—​hence their description as insulin sensitizers. Thiazolidinediones have negligible glucose-​lowering action unless insulin resistance and hyperglycaemia are present. As with metformin, they do not cause hypoglycaemia when used alone, but can exaggerate the hypoglycaemic effects of insulin or sulphonylureas. Efficacy and potency  Alone, all thiazolidinediones lower glu- cose by 2 to 3 mmol/​litre and HbA1c by 1%, somewhat less than the sulphonylureas. However, in some individuals they can result in marked falls in HbA1c, up to 4%. For unknown reasons, blood glucose declines slowly during thiazolidinedione treatment, and a maximal effect may not be reached for up to 6 months. Pharmacokinetics  All are metabolized in the liver and cleared chiefly through the kidney. They are highly protein bound. Side effects  Weight gain, averaging 1 to 4 kg, is due mainly to sub- cutaneous fat deposition. This appears to spare the visceral depot associated with insulin resistance and does not negate the glucose-​ lowering action. Fluid retention of unknown aetiology may cause a mild dilutional anaemia (haemoglobin typically falls by 1–​2 g/​dl) and ankle oedema (in 5–​10% of cases); heart failure may also be precipitated in patients with pre-​existing myocardial dysfunction, especially if they are also treated with insulin. Meta-​analyses have suggested that rosiglitazone is associated with an increased risk of myocardial ischaemic events, and this has resulted in its withdrawal in most countries. Hepatic damage, ranging from subclinical elevations of hepatic enzymes to fulminant and fatal hepatic necrosis (about one case per 1000 patient-​years), has been reported with troglitazone but does not appear to be a risk with rosiglitazone or pioglitazone. Indeed, early indications suggest that thiazolidinediones may be helpful in reducing and possibly reversing steatosis (fat deposition) in the liver that is associated with obesity and insulin resistance and can pro- gress to cirrhosis. An unexpected class side effect of the thiazolidinediones in clin- ical trials is an increase in fractures in the limbs rather than the axial skeleton. This is especially a concern in postmenopausal women. Mechanisms appear to include increased bone resorption and sup- pression of osteoblast formation from mesenchymal progenitors. There is concern about a small increased risk of bladder cancer with pioglitazone and this should be avoided in those with previous bladder cancer or haematuria. Indications and contraindications  Thiazolidinediones are gener- ally regarded as second-​ or third-​line drugs for treating type 2 dia- betes when sulphonylureas or metformin (or the combination of the two) are ineffective or unsuitable. They can be combined with either a sulphonylurea or metformin, when HbA1c may fall by more than 1%; if HbA1c has not fallen by more than 1% within 6 months of adding a thiazolidinedione, it should be discontinued especially in view of the recent concerns over heart failure and fractures. When used alone, they have a lower rate of failure than metformin or sulphonylureas alone, but cost and potential side effect concerns argue against using them as monotherapy. When pioglitazone is used with insulin, in- sulin dosage can be reduced but weight gain may be problematic; rarely, heart failure may be precipitated. Subjects with impaired glu- cose tolerance treated with a thiazolidinedione have a lower risk of progressing to overt type 2 diabetes, and the drugs can improve hir- sutism and menstrual dysfunction (sometimes inducing ovulation) in women with polycystic ovary syndrome. Contraindications include congestive heart failure. α-​Glucosidase inhibitors Acarbose (and the related miglitol and voglibose) are inhibitors of α-​glucosidase, an enzyme of the brush border of the small intestine essential for the breakdown of dietary starch to disaccharides, which are then hydrolysed to the absorbable monosaccharides. They partly block digestion of complex carbohydrates and so damp postpran- dial glycaemic rises, but the therapeutic effect is small: postprandial glucose may fall by 1 to 2 mmol/​litre, with predictably little impact on overnight glucose, and HbA1c by 0.5% or less. Side effects due to carbohydrate malabsorption (flatus, abdominal bloating, gassy diar- rhoea) are common and probably damage compliance. Incretin mimetics These drugs mimic or enhance the action of the incretin hormones that augment insulin secretion. GLP-​1 is an incretin that stimu- lates insulin secretion and may also induce satiety, particularly by delaying gastric emptying. Blood glucose can be lowered compar- ably to sulphonylureas with GLP-​1 infused intravenously. Exenatide (exendin-​4) is an analogue of GLP-​1, first identified in the saliva and concentrated in the tail of the American venomous lizard, the Gila monster, which by an interesting coincidence lives alongside the diabetes-​prone Pima Indians of Arizona. Exenatide shares 50% homology with GLP-​1 but has a considerably longer half-​life in vivo and is now available as a twice daily subcutaneous injection at a dose of 5 or 10 μg and can be used in combination with metformin, a sulphonylurea or insulin. When used alone or in combination with metformin the risk of hypoglycaemia is low. Mean falls in HbA1c of 0.8 to 1% are seen with the higher dose and direct comparison sug- gested that these were similar to the results of addition of insulin with less associated hypoglycaemia. In contrast to the weight gain

section 13  Endocrine disorders 2498 seen with insulin, exenatide is associated with a modest weight loss of around 4 kg, due in part to direct inhibition of appetite. The main side effect is nausea, which occurs in more than 50% of patients, and precludes continuing therapy in around 10% of patients. Pancreatitis has been reported rarely and the use of these drugs is contraindicated in people who have had a previous history of pancreatitis. Animal studies show that exenatide is trophic for β cells; confirmation of this very valuable effect in humans is awaited. Other once daily GLP-​1 analogues, liraglutide, and lixisenatide are available and appear to produce less nausea and equal if not greater glucose lowering. Once weekly exenatide is available; this takes some weeks to develop max- imum efficacy, tends to be better tolerated than the shorter acting pre- parations and can be given up to 3 days after the dose is due. However, the injection requires a more complicated procedure than the shorter acting preparation. Recently more convenient and effective once weekly preparations have been introduced including dulaglitide and semaglutide and an oral version of semaglutide is now available. The GLP-​1 agonists are appropriate for patients who are obese and for whom additional weight gain would have a deleterious effect on their health. They are expensive and should only be continued if cer- tain targets are achieved. In the United Kingdom it is suggested they should be discontinued if the HbA1c is not reduced by 1% or if the patient does not lose 3% of body weight. They should be used in caution with severe renal dysfunction. Increasingly GLP-​1 agonists are used in combination with a basal insulin. Combination products containing liraglutide and insulin degludec are available and trials have shown improved control, weight loss, and reduced hypogly- caemia when compared to multiple-​dose insulin regimes. The gliptin class of drugs (including sitagliptin, vildagliptin saxagliptin, linagliptin, and alogliptin) are oral selective inhibitors of dipeptidyl peptidase IV (DPP IV), the enzyme that causes the break- down of circulating GLP-​1. They therefore prolong the survival and enhance the action of endogenous GLP-​1. These drugs are better tolerated than GLP-​1 agonists but do not result in weight loss and have less impact on HbA1c levels. An unexpected side effect is an increase in infections, notably sinusitis which is linked to the expres- sion of DPPIV on the surface of lymphocytes (CD26). Generally, they are well tolerated and several are licensed at reduced doses to be used at low eGFR values. Linagliptin is excreted hepatically and therefore may be used without a change in dose at all levels of eGFR. Saxagliptin has demonstrated reassuring cardiovascular outcome data with the exception of increased hospital admissions with heart failure; however, recent sitagliptin data have shown this is not a class effect. As a result of the low incidence of hypoglycaemia and side effects, these agents have become a popular choice for treating older people although outcome data are limited in this group. Sodium-​glucose cotransporter 2 inhibitors (SGLT2 inhibi- tors)  Ninety per cent of reabsorption of filtered glucose occurs in the proximal tubule of the kidney and is mediated by SGLT2. The re- maining 10% is reabsorbed in the distal tubule mediated by SGLT1. SGLT2 inhibitors prevent the reabsorption of glucose and result in glycosuria and subsequently an osmotic diuresis. Several SGLT2 inhibitors are licensed (dapagliflozin, canagliflozin, and empagliflozin) to treat type 2 diabetes in combination with various oral agents (although the licensed combinations vary between agents) and insulin. These agents lower the HbA1c by approximately 0.8%. The loss of 70 g of glucose in the urine each day results in a gradual and sustained weight loss. The therapeutic glycosuria has a consequence of genital candida infections for around 5% of patients although stopping the SGLT2 inhibitor is not usually necessary. A small drop in blood pressure is often seen. The SGLT2 inhibitors should not be used together with loop diuretics as the resulting reduction of plasma volume may result in a decline in renal function. There have been re- ports of ketoacidosis with the SGLT2 inhibitors and patients should be informed of this. Recent data published on all SGLT2 inhibitors have demonstrated improved cardiovascular outcomes compared to other agents routinely used to treat type 2 diabetes, with especially dramatic reductions in death from heart failure. This has prompted their use in patients with heart failure without diabetes. There is also evidence that this class of drugs reduced progression from microalbuminuria to end stage renal failure, although currently their use is contra-indicated with an eGFR<45 ml/min. Bromocriptine  A quick release formulation of bromocriptine is licensed for the treatment of type 2 diabetes in some countries. It modulates central glucose and metabolism pathways which results in reduced hepatic glucose production and a reduction of post- prandial glucose. HbA1c typically is reduced by 0.5%. The doses of bromocriptine used to treat diabetes are very much lower than those used to treat Parkinson’s disease. Colesevelam  Bile acid sequestrants are primarily lipid-​lowering drugs however also have a glucose-​lowering effect and are licensed for the treatment of type 2 diabetes. The HbA1c is lowered by ap- proximately 0.5% although the exact mechanism is unclear. Practical management of hyperglycaemia Most newly diagnosed diabetic patients are relatively easily allocated to either type 1 or type 2 on clinical criteria (see Table 13.9.1.2) and treatment is started accordingly. However, initial impressions may be misleading: a thin young patient may not need insulin because he has monogenic diabetes, whereas a person who has classical features of type 2 diabetes may lose weight rapidly and develop ketoacidosis because he has type 1 diabetes. Continuing monitoring and vigi- lance are therefore essential. The diagnostic pitfalls of Flatbush and fulminant type 1 diabetes have been mentioned earlier. Type 1 diabetes These patients must be given insulin immediately and for life. The insulin regimen will depend particularly on any remaining en- dogenous insulin, the patient’s body weight, lifestyle, and motiv- ation. Patients with residual insulin secretion, especially newly presenting and particularly during the ‘honeymoon period’ (see next), can often fill in gaps in insulin replacement and enjoy good glycaemic control with few injections and low insulin dosages. However, C-​peptide negative patients will require exogenous insulin to cover both basal and prandial needs (see Fig. 13.9.1.9) to achieve good control. Regimens include: • Basal insulin given once a day (insulin glargine or degludec) or twice a day (insulin detemir or isophane) with short-​acting in- sulin (human soluble insulin) or short-​acting analogue (insulin aspart, lispro, or glulisine) given with meals. In type 1 diabetes the use of analogue insulins is associated with a reduction of hypogly- caemia and is the preferred option. An intensified insulin regime early in the course of type 1 diabetes has been shown to be asso- ciated with short-​term and long-​term reduction of microvascular and macrovascular complications and should be considered the

13.9.1  Diabetes 2499 first-​line treatment option unless insulin pump therapy is imme- diately available. The short-​acting analogue insulin may be given as per an insulin to carbohydrate ratio rather than as a fixed dose and this may improve overall control. • Premixed insulins injected before breakfast and before the evening meal may be preferred by some patients however the limitations of this therapy should be discussed. Intensive glucose control may be achieved with insulin pump therapy which has demonstrated reduced HbA1c without an in- crease in hypoglycaemia. It is necessary that staff involved should have particular expertise in insulin pump therapy and thorough pa- tient education should be available for this therapy to be safe and successful. Insulin dosages should be titrated according to blood glucose and HbA1c monitoring (see Table 13.9.1.3). Metformin and SGLT2 inhibitors in type 1 diabetes The addition of metformin to insulin is valuable in patients with type 1 diabetes who are overweight as this may help to reduce insulin resistance, lower insulin dose, and prevent weight gain. Recently, an SGLT2 inhibitor has been licensed for use in type 1 diabetes fol- lowing data showing reduced glycaemic variability, lower HbA1c without increased hypoglycaemia. However here is an increased risk of ketoacidosis, especially euglycaemic ketoacidosis and hence it should only be initiated by specialist teams in patients compliant with insulin therapy. Starting insulin therapy Patients at risk of ketoacidosis may need hospital admission, but most patients are clinically well and can start insulin as an outpatient, supervised by a specialist diabetes nurse. Basal insulin is usually started once or twice a day and short-​acting insulin is added to cover prandial hyperglycaemia. Wherever practicable, patients should be encouraged to give their own injections as soon as possible. Newly diagnosed patients starting insulin need to be warned about a possible ‘honeymoon period’ of good glycaemic control, when the fall in glucose levels allows partial though temporary recovery of the remaining β cells. Blood glucose can often be easily controlled with low insulin dosages (and exceptionally, without exogenous insulin) but the honeymoon ultimately ends usually within a few months: blood sugar levels and insulin requirements then escalate, because of the progressive loss of remaining β cells over the next 1–​5 years. Poor diabetic control and ‘brittle’ diabetes In real life, relatively few type 1 patients approach the high-​quality glycaemic control aspired to in Table 13.9.1.3. This largely reflects the pharmacokinetic shortcomings of current insulin preparations and the unpredictable nature of subcutaneous absorption. The patient’s compliance is a crucial determinant of overall diabetic con- trol; teenagers are notoriously resistant to advice about diabetes, as with other matters, and many have markedly elevated HbA1c con- centrations. This clearly increases the risk of future diabetic compli- cations. However, it should be noted that compliance with complex insulin regimes is very demanding and less than 20% of people with type 1 diabetes achieve levels of glycaemic control (HbA1c <53 mmol/​ml) that prevent long-​term complications. A few patients have such poor metabolic control that they cannot live a normal life. Most have chronically high blood glucose and suffer recurrent hospital admissions with ketoacidosis; some suffer frequent hypoglycaemia, while others have an unstable or ‘brittle’ blood glucose profile that can swing rapidly between hyper-​ and hypoglycaemia. Occasionally, endocrine or intercurrent illnesses are found to be responsible (see Table 13.9.1.5), but most cases remain idiopathic after even intensive investigation. It is now clear that poor compliance, often aggravated by deliberate inter- ference with treatment, is responsible in many of these patients. Most are young women who are generally hyperglycaemic despite apparently high insulin dosages; when tested under controlled conditions, however, their intravenous and subcutaneous insulin requirements are unremarkable. Many are probably omitting in- sulin or taking only small doses: common motives include escape from difficulties at school or home, or wanting to stay thin (dis- turbances of body image are common in this group). Coexistent eating disorders, such as anorexia and bulimia nervosa, are com- monly seen in these individuals. Initially, such patients may ap- pear to lead charmed lives despite frequent hospital admissions but many die prematurely (especially from ketoacidosis or hypo- glycaemia); significant diabetic complications frequently develop during their twenties or thirties. Management can be extremely difficult. Patients with sustained poor control should be admitted selectively for intensive educa- tion, observation, and exclusion of other possible causes (see Table 13.9.1.3). In some cases, it may be necessary to confirm that insulin is effective at conventional doses (for more information see the paper by Schade and Duckworth listed in ‘Further reading’). Even close super- vision in hospital does not exclude ingenious interference with insulin Table 13.9.1.5  Causes of poor glycaemic control in type 1 diabetic patients Characteristics Cause High insulin requirements, chronic hyperglycaemia ± recurrent ketoacidosis Obesity Puberty Endocrine diseases: Cushing’s syndrome, thyrotoxicosis Drugs: especially glucocorticoids Immune insulin resistance Low insulin requirements, recurrent hypoglycaemia Weight loss Loss of hypoglycaemia awareness Other endocrine diseases: adrenocortical failure, hypothyroidism, growth hormone deficiency, hypopituitarism Gastroparesis Coeliac disease Liver disease Erratic glycaemic profile, frequent hyper-​ and hypoglycaemia (‘brittle’ diabetes) Compliance issues Pancreatic damage Overtreating hypoglycaemia Gastroparesis Injection site problems (lipohypertrophy) Recurrent or chronic infections: tuberculosis, sinusitis For all three characteristics, always consider: unsuitable insulin regime; poor diabetes education; deliberate noncompliance; appetite disorders (anorexia nervosa, food bingeing).

section 13  Endocrine disorders 2500 treatment or glucose monitoring. Intensified insulin schedules or con- tinuous subcutaneous insulin infusion may help in some cases and increasingly whole pancreas transplantation is being considered as an option if patients are willing to take the associated risks (see next). Experimental and future treatments for type 1 diabetes Whole pancreatic transplantation, usually performed in conjunc- tion with renal transplantation for patients with diabetic nephrop- athy, can achieve good results including long-​term withdrawal of exogenous insulin (> 5 years) in up to 70% of cases. The whole gland or a segment is transplanted into the pelvis and anastomosed to the iliac vessels; to avoid damage from pancreatic exocrine secretions, the pancreatic duct is drained either into the gut or into the bladder (when urinary amylase excretion can indicate the health of the graft). Outcomes for both the pancreas and the kidney are better when sim- ultaneous transplantation is performed as the early treatment of re- jection, which is easier to identify in the kidney by serum creatinine and or biopsy, preserves both organs, and the improved glycaemic control from the pancreas is beneficial to the kidney. Problems in- clude the need for lifelong immunosuppression (required anyway for renal transplantation) and the global shortage of donor organs. An increasing number of pancreas transplants alone are being per- formed in type 1 diabetes but the balance of risks (especially of ma- lignancy and infection from the immunosuppression) and benefits (from improved glycaemic control) requires careful assessment in each individual patient. The optimal indications for this procedure, where available, remain poorly defined but it is generally performed for persistent poor metabolic control with or without recurrent ketoacidosis or recurrent, intractable hypoglycaemia. Relentless progression of complications despite good glycaemic control is a further indication as generally the progression of complications is halted but not reversed. Although there are attendant risks from the surgery and immunosuppression, recurrent ketoacidosis and poor metabolic control itself carries a not insignificant risk of death. Introduction of an improved immunosuppressive regimen (which omits glucocorticoids) by Shapiro and colleagues reported in 2000 has led to a resurgence in pancreatic islet transplantation. The most widely used method is by transcutaneous injection into the portal vein of islets isolated from a donor pancreas; these col- onize and function well in the liver, the first stop for insulin secreted physiologically. Even with less toxic (to the β cell) immunosuppres- sion, two or three donor pancreases are currently needed for each re- cipient, and only 10% of patients are insulin independent at 5 years, but newer immunosuppressive regimes may improve this further. Nevertheless, up to 90% of patients report significant reductions in the rate of hypoglycaemia; hence recurrent severe hypoglycaemia unresponsive to changes in insulin therapy or the use of CSII re- mains the main indication for this procedure. Healthcare professionals should always remain sensitive to the burden of diabetes for the individual and aware that glycaemic control may vary depending on the various physical, psychological, and emo- tional stresses of life. Often diabetes control will decline in the face of adversity. Healthcare professionals should avoid blaming a patient for such deterioration in control and remain supportive and helpful. The transition from paediatric services to adult services occurs at a particularly vulnerable time in life when a young person has competing physical and emotional issues, is developing a fragile in- dependence from their family, often participates in high-​risk behav- iour and has a need for peer group conformity. The removal of a familiar team who take a holistic, family approach and replacing this with an unfamiliar team that treats the young person as an autono- mous adult often results in disengagement from services and very poor glycaemic control. Transition services should be designed to prevent this and improve outcomes for young people. Prevention of type 1 diabetes by aborting insulitis during the long prediabetic phase by immunosuppression in high-​risk subjects, or preserving islet cell function in newly diagnosed patients, is a major goal of current research. Trials in the 1980s demonstrated that ciclosporin can achieve this, but the cost in terms of side effects of continuous therapy is too high. Newer immunomodulatory agents, such as anti-​CD20 (rituximab) or CTLA4-​Ig (abatacept), anti-​CD2 (alefacept) or treatments, that regulate rather than suppress immune responses such as non​depleting anti-​CD3, significantly improve the risk–​benefit ratio to the point of acceptability but are not currently available, and trials of further agents are underway. Observational studies suggest that preservation of even small amounts of en- dogenous insulin (stimulated C-​peptide >200 omol/​litre) is associ- ated with a 50% reduction or more in hypoglycaemia, 50% or more individuals achieving optimal glycaemic control and reduced long-​ term complications. Much effort is also being invested in promoting the regeneration of β cells either from pancreatic tissue or more generic stem cells. These studies remain at a preliminary stage, but potentially offer a renewable therapy. Although they may not require immunosup- pression for allograft rejection, it remains to be seen whether such new cells would be retargeted by the autoimmune process in subjects with type 1 diabetes. Management of type 2 diabetes Dietary and lifestyle measures form an essential foundation for the management of type 2 diabetes and must be maintained throughout, even though fewer than 10% of patients can be controlled satisfac- torily for more than a year by these means alone. Recent evidence from the Early ACTID study suggests that intensive dietary support may be as effective as promoting diet and exercise together in the early stages after diagnosis. Patients who fail to meet the glycaemic targets set out in Table 13.9.1.3 should generally follow the steps outlined next, although compromises may be more appropriate in older people or those at risk of hypoglycaemia. Progress should be reviewed every 3 months or less if blood glucose is unacceptably high. The mainstay of treating type 2 diabetes over the last 50 years has been to introduce agents to manage the inexorable deterioration in β cell function in type 2 diabetes for example using metformin followed by a sulphonylurea followed by insulin. Over the last 10 years there has been the introduction of agents that have a more durable effect and preserve the β cell function such as thiazolidinediones, GLP-​1 agonists, and SGLT2 inhibitors. It seems logical to use such agents early in the course of the disease; however, this optimism needs to be balanced by a caution that long-​term effects of these agents are not known, and they are generally more expensive. However, out- come data are becoming available due to increased vigilance from the regulatory bodies, for example the reduced mortality seen with empagliflozin therapy in recent trials. The first-​line oral hypoglycaemic agent for dietary failure is metformin. Metformin has some of the most robust outcome data for benefit, is weight neutral, and has an extremely low risk of hypoglycaemia.

13.9.1  Diabetes 2501 Several guidelines advocate beginning metformin immediately at diagnosis, but the effect this has in undermining commitment to lifestyle measures has not been defined. The addition to metformin of a sulphonylurea represents standard second-​line treatment in many guidelines. However, there is general acceptance that therapy should be individualized taking into account factors such as the risk of hypoglycaemia, cardiovascular/heart failure risk, the adverse effects of further weight gain, the need to monitor capillary blood glucose, and the loss of β-​cell function. In these cir- cumstances, the use of DPP IV inhibitors, GLP-1 agonists and SGLT2 inhibitors should be considered and treatment algorithms are evolving. Around 40% of people living with type 2 diabetes manage their diabetes with insulin, although this is decreasing with the increasing use of new non-insulin therapies. The standard method of commen- cing insulin is to start basal insulin in the form of isophane or a basal analogue such as insulin Lantus at a dose of 10 units or 0.1–​0.2 units per kg at night. Most will continue with oral therapy alongside this regime. The insulin is then titrated by 2 units every 3 to 5 days until the fasting glucose is persistently below 7 mmol/​litre. Self-​titration by patients using standard algorithms may be more effective than that done by healthcare professionals. Short-​acting insulin or short-​ acting analogue may be added at mealtimes if needed. At this point it is usual to stop the sulphonylurea, however, metformin is continued if possible and there may be benefits to continuing other agents such as gliptins, GLP-​1 agonists, SGLT 2 inhibitors, and thiazolidinediones. Premixed insulins may be used rather than a multiple-​dose injection regime; advantages to this may be convenience in those who have a regular lifestyle. Generally, the more effective the regime at HbA1c reduction, the greater the weight gain and the risk of hypoglycaemia. The evidence for hypoglycaemia reduction using analogues rather than human insulin is less convincing in type 2 diabetes compared to type 1 diabetes. Many health providers are suggesting human insulin as a first-​line insulin option in type 2 diabetes reserving the analogues for the small proportion who may develop problems with hypogly- caemia, as substantial cost saving may be made. Intensive insulin therapy at the time of diagnosis for 2 to 3 weeks has been associated with remission of diabetes, presumably sec- ondary to a reduction of glucotoxicity. The usual indications for early insulin initiation are marked hyperglycaemia or the features of weight loss or ketosis. Obesity (and therefore insulin resistance) may worsen when in- sulin treatment is started. The average weight gain is around 6 kg; possible reasons include reduced loss of energy through glycosuria, a tendency to relax dietary restriction when a more effective means of lowering glycaemia is introduced, and sometimes overeating during hypoglycaemic episodes. Increasing insulin resistance may lead to escalating insulin dosages. The addition of GLP-​1 agonists or SGLT2 inhibitors may help to achieve targets while preventing weight gain or allowing weight loss. Monitoring diabetic control Treatment targets for blood glucose in type 1 and type 2 diabetes (see Table 13.9.1.3) have been selected to reduce the risk of chronic diabetic complications. Avoiding acute episodes of hyper-​ and hypo- glycaemia is also important. Blood glucose monitoring Blood glucose concentration can be easily and quickly measured in small drops of blood (a few microlitres or less), using various test strips; the ability to perform such measurements is an essential skill for all professionals delivering diabetes care and for most diabetic patients. Test strips contain glucose oxidase (which catalyses the oxi- dation of glucose to gluconic acid) together with a detection system to measure specific reaction products, either electrochemically or colorimetrically (using dyes sensitive to hydrogen peroxide). The signal is read by a reflectance meter or electrically, and converted into the glucose concentration in the sample. Colour-​based test strips can also be read by eye against a printed standard scale, al- though this may be difficult for partially sighted or colour-​blind patients. A drop of blood is obtained by pricking the sides of the fingertip, avoiding the sensitive pads; various lancets and automatic finger-​ pricking devices are available. Blood must cover the reaction area completely and be left in contact for exactly the period stipulated; modern meters read out automatically at this point, whereas older strips must be wiped dry and left for the colour to develop. Failure to follow the manufacturer’s instructions is the main cause of in- accurate readings, which are disturbingly frequent. With attention to detail, readings correspond closely to laboratory measurements of glucose (which also employ the glucose oxidase reaction) but are not reliable enough to be used for diagnosing diabetes. Monitoring schedules Monitoring is not essential in patients treated with diet alone or a single oral agent. Generally, those treated with diet or medications which have a very low risk of hypoglycaemia (such as metformin, glitazones, gliptins,) do not need to routinely monitor their capillary blood glu- cose. The exception would be when significant hyperglycaemia is suspected and it would be inappropriate to wait to see the response of the HbA1c blood test. Those receiving sulphonylureas are suscep- tible to hypoglycaemia and therefore should have the means to test their blood glucose levels if they feel unwell or related to driving. Insulin-​treated patients need more frequent monitoring to ad- just insulin dosages. Bedtime and premeal testing (4-​point) as well as ideally 2-​hour postprandial (7-​point) testing is recommended. Fasting glucose is determined by the previous evening’s long-​acting insulin. Prandial short-​acting insulin dosages can be titrated from the glucose rise 90 to 120 min after eating. Readings can be scat- tered across these time points on different days; most patients can be persuaded to check their glucose levels once or twice per day but to achieve tight glycaemic control targets without hypoglycaemia, more frequent blood glucose testing is required. Written records help to bring out general patterns in glucose con- trol and many modern meters can be downloaded to display the pat- tern in different formats. Patients must also be encouraged to check their glucose if they feel unwell and, crucially, at frequent intervals during intercurrent illness. Occasional tests during the night (es- pecially between 02.00 and 04.00) are useful in patients at risk of nocturnal hypoglycaemia, including those injecting long-​acting or premixed insulins in the early evening. Checking the self-​monitoring technique and the patient’s action plan when glucose levels fall outside the target range is a core part of the patient’s diabetic education. The Freestyle Libre glucose monitor consists of a sensor inserted into the subcutaneous tissue of the upper arm that may stay in place for up to 2 weeks. The user can scan the sensor with the hand-​held device as frequently as they wish to obtain the current glucose result

section 13  Endocrine disorders 2502 and the last 8 hours of data. There is no need to do any calibration capillary blood tests. This is the first such system available for direct purchase from the manufacturer and it has proved very popular. Continuous glucose monitoring systems have a sensor which lasts up to a week, may store data for a greater period however they re- quire calibration with capillary blood tests four times a day and are more expensive. They have demonstrated an improvement in HbA1c when used regularly and in particular circumstances such as pregnancy. HbA1c and fructosamine These tests measure the non​enzymatic reaction of glucose with circulating proteins (see next), and therefore reflect longer-​term blood glucose levels. Glycated (glycosylated) haemoglobin (HbA1) results from the combination of glucose with the N-​terminal valine residue of the B chain of adult Hb (HbA), and can be separated from unaltered HbA by electrophoretic and other methods. HbA1 includes the stable HbA1c fraction, which is most closely related to average blood glucose levels over the preceding 6 to 8 weeks. The various assay methods for HbA1c were initially matched to a standard used in the Diabetes Control and Complications Trial (DCCT), giving a result in percentage terms which defined the long-​term risks of diabetic microvascular complications (see next). More recently, values have been matched to a standard from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and are now expressed as mmol HbA1c per mol of haemoglobin (mmol/​mol) to define the fact that they are refer- enced to a different standard. Table 13.9.1.6 gives the conversion of common values and the conversion formula. For assays con- forming to IFCC standards, non​diabetic HbA1c ranges from 14.8 to 36.6 mmol/​mol Hb (3.5–​5.5% in DCCT units), with good con- trol defined as values less than 53 mmol/​mol (7%) and poor control as more than 64 mmol/​mol (8%); some poorly compliant patients have HbA1c concentrations of 130–​150 mmol/​mol (14–​16%). HbA1c measurements are a useful index of medium-​term glycaemic con- trol, but may be invalidated by abnormal red cell turnover (values are spuriously low in haemolysis, bleeding, and pregnancy), in renal failure (carbamylated HbA coelutes with HbA1c, falsely raising levels), and with abnormal haemoglobins such as hetero-​ or homo- zygous sickle cell disease (HbF also comigrates with HbA1). Modern analytical methods for HbA1c detect the presence of abnormal haemoglobins and hence spurious results are usually highlighted by the laboratory. Conversion tips:

  1. For whole numbers of %HbA1c:  ‘minus 2 minus 2 rule’, e.g. 6.0% = (6 –​ 2), (6 –​ 2 –​ 2) (i.e. 42).
  2. Remember 6.0% = 42, then add 11 for each 1%.
  3. Normal less than 42, target less than 53 (7.0%).
  4. Formula:  IFCC–​HbA1c (mmol/​mol)  =  [DCCT–​HbA1c (%) –​2.15] × 10.929. Serum albumin also undergoes glycation, which is measured by the fructosamine reaction. As albumin turns over faster than haemoglobin, the fructosamine concentration reflects mean blood glucose over the previous 1 to 2 weeks. Assays are cheap but not standardized between laboratories, and are generally less reliable and reproducible than measurements of HbA1c. Measurements of urinary glucose and ketones Urinary glucose concentrations can be measured easily using glu- cose oxidase test strips, but are of limited use: urinary glucose con- centration depends on the renal threshold (which can lie between 7 and 13 mmol/​litre), urine output, and the time since the bladder was last emptied. Crucially, hypoglycaemia cannot be detected. Urinary glucose measurements are acceptable in type 2 diabetic patients with a normal renal threshold who are not receiving hypoglycaemic medication (insulin or sulphonylureas) and in patients who decline to prick their fingers. Urinary ketone measurements can be useful for predicting impending ketoacidosis, particularly during intercurrent illness when blood glucose is high. Moderate ketonuria can be caused by fasting or undereating, including during infections. Modern blood testing meters can measure blood ketones with appropriate testing strips. Values less than 0.3 mmol/​litre are normal; levels in excess of 3 mmol/​litre indicate ketoacidosis. Structures for diabetes care Diabetes is best managed by the combined efforts of a well-​trained primary care team and a team of specialists with complementary and overlapping skills: physician, specialist diabetes nurse, diet- itian, and podiatrist. The specialist diabetes nurse has a crucial role in educating patients about diabetes and its practical man- agement, and in starting and adjusting therapy. Many patients are more receptive and responsive to information given by pri- mary care teams and specialist nurses than by doctors. For com- plex cases, there must be frequent contact with and easy access to other specialists (ophthalmologist, vascular surgeon, renal phys- ician, obstetrician, and clinical psychologist), ideally in the setting of combined clinics. Each member of the team has a particular niche, but all must agree common strategies (such as dietary ad- vice for obesity) to avoid giving the patients conflicting or incon- sistent information. Diabetes care can be delivered effectively by well-​informed general practitioners or practice nurses, hospital-​based clinics, community mini-​clinics, or shared care schemes that bridge the primary and secondary sectors. Because of the unpredictable course and potential complications of diabetes, all patients must be thoroughly reviewed each year and be rapidly referred for Table 13.9.1.6  Conversion between DCCT aligned and IFFC values for HbA1c DCCT HbA1c (%) IFFC HbA1c (mmol/​mol) 6.0 42 6.5 48 7.0 53 7.5 59 8.0 64 9.0 75 10 86 DCCT, Diabetes Control and Complications Trial; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine.

13.9.1  Diabetes 2503 specialist help if the need arises. A check list for the annual review is suggested in Table 13.9.1.7. Diabetes education Living and coping with diabetes is a considerable burden that is poorly appreciated by many doctors and nurses. Careful education about diabetes, its complications, and its practical management can provide great reassurance to patients and also reduce emergency hospital admissions and complications such as foot ulceration and amputation. Diabetes education is most effectively provided by a trained practice nurse or specialist diabetes nurse, but all members of the diabetes care team should understand the key messages, and check and reinforce these whenever possible. Evidence suggests Table 13.9.1.7  Routine annual review of a diabetic patient: key points include those specific to patients taking insulin History and discussion Examination Investigations Diabetic treatment Diet, physical activity Weight, height, BMI Weight and change Waist–​hip ratio Glucose-​lowering drugs Insulin injection sites Diabetic control Self-​monitoring results (± check technique) Hyperglycaemic symptoms HbA1c Hypoglycaemia frequency and awareness of symptoms Liver function tests in type 2 diabetes if known fatty liver disease Diabetes education and skills (± family or associates) General knowledge Treatment targets ‘Sick-​day’ rules, driving rules, pregnancy plans Hypoglycaemia treatment Insulin injection technique Diabetic complications: Macrovascular Ischaemic heart disease (angina, MI, failure, arrhythmias) Examine heart, including signs of failure. Blood pressure, lying and standing Fasting lipid screen (total, HDL, and LDL cholesterol; triglycerides). ECG (if other risk factors or age >40) Peripheral vascular disease (claudication, stroke, TIA) Peripheral pulses, strength, and bruits Smoking history Other risk factors (hypertension, dyslipidaemia, family history) Eyes (retinopathy and cataract) Altered acuity, loss of vision Visual acuity (± corrected). Retinal examination (digital imaging, fundoscopy through dilated pupils) Nephropathy Blood electrolytes, urea, creatinine. Microalbuminuria screen (e.g. albumin:creatinine ratio) or timed urinary albumin excretion Neuropathy Altered or reduced sensation Sensory testing screen as appropriate (feet: see next) Pain Postural blood pressure drop Weakness in limbs Autonomic symptoms (sweating, postural dizziness, gastrointestinal) Feet Pain, numbness General condition: posture, callus, footwear appropriate Ulceration: current and previous Pulses and perfusion Footwear (sensible?) Oedema Foot care Sensory deficits (vibration sense, pin prick, monofilament) Sexual function Erectile and ejaculatory problems (men) Other illnesses Other medication (possible effects on glycaemic control and interactions with antidiabetic drugs) BMI, body mass index; MI, myocardial infarction; HDL, high-​density lipoprotein; LDL, low-​density lipoprotein; TIA, transient ischaemic attack.

section 13  Endocrine disorders 2504 that education in a group setting is often more effective than on a one to one basis and may promote informal support networks. Key elements of the education programme include: • causes of hyperglycaemia and diabetic symptoms • own treatment: diet and lifestyle; drawing up and injecting in- sulin; oral agents; recognizing and treating hypoglycaemia • self-​monitoring technique; targets and danger levels; how to re- spond to poor control • ‘sick-​day’ rules: monitoring during intercurrent illness; how to ad- just own treatment; when and how to call for help (Box 13.9.1.1) Structured education This term refers to programmes to facilitate self-​management of diabetes. Examples of these programmes are the Dose Adjustment For Normal Eating (DAFNE) programme, which is a programme for people with type 1 diabetes which teaches management of in- sulin depending on the ingested carbohydrate, activity, and illness and X-​PERT, which is a self-​management programme for people living with type 2 diabetes. There are various requirements of a structured education programme; there should be defined goals and policies, input from stakeholders and experts to ensure the quality of the programme, the needs of the population to be educated should be considered, educated staff, a curriculum, individualization of the programme as needed, ongoing support and measurement of progress, as well as quality assurance. Structured education pro- grammes are generally considered to be the gold standard of patient education. Employment, driving, and insurance Until recently people on insulin therapy were barred from holding heavy goods vehicle and public service vehicle driving licences. Similarly, certain occupations were restricted for those on insulin such as the armed forces, police service, fire service, and working on aircrafts. Following legislation preventing discrimination against people with disabilities including those with diabetes, each case must be reviewed individually. This has had particular impact for those with type 2 diabetes who previously delayed insulin treatment with the subsequent threat to their health to prevent losing their em- ployment. Licensing for taxi drivers varies between local authorities. Specific diabetic complications, notably sight-​threatening retinop- athy, may preclude particular jobs or pastimes. Patients must inform the driving licence authorities and their driving insurer that they have diabetes, and those receiving in- sulin or with clinically significant retinopathy may require peri- odic medical confirmation of fitness to drive. Hypoglycaemia with loss of awareness of symptoms is a bar to driving. Currently the use of GLP-​1 agonists carries no specific restrictions in the United Kingdom except for heavy goods vehicle or public service vehicle drivers taking these agents in conjunction with sulphonylureas, in which case the driving authority will make an assessment on individual basis. Special life insurance policies are available from companies en- dorsed by patient-​centred organizations such as Diabetes UK and the American Diabetes Association. Many patients find it valuable to join these organizations. Intercurrent events in diabetes and their management Infections People with diabetes probably have increased susceptibility to pyo- genic bacterial infections, especially when diabetes is poorly con- trolled. Hyperglycaemia can impair the killing of microorganisms by neutrophils and macrophages and may also interfere with the function of T lymphocytes. Some infections particularly associated with poorly controlled diabetes include: • recurrent and sometimes invasive candidiasis • tuberculosis, often widespread and cavitating • necrotizing fasciitis, rapidly spreading necrosis of subcutaneous tissues down to muscle, usually due to β-​haemolytic streptococci with staphylococci and often anaerobes • gas-​forming infections with anaerobes and clostridia, including emphysematous pyelonephritis, cholecystitis, cystitis, and foot in- fections; plain radiography shows gas in the affected tissues • diabetic foot ulcers (see next), which are often infected, with the risk of osteomyelitis and deep soft tissue spread • recurrent oral and genital candida infections • urinary tract infections, which may be complicated by ascending infections with pyelonephritis and renal or perinephric abscess (sometimes with gas), and occasionally acute papillary necrosis; severe loin pain and systemic symptoms, with deteriorating renal function, should suggest these possibilities and the need for ur- gent imaging • ‘malignant’ or necrotizing otitis externa, due to pseudomonas in- fection, which can invade the skull and facial nerve • periodontal infections, sometimes causing tooth loss—​these are common • rhinocerebral mucormycosis, a highly invasive fungal infection that originates in the sinuses but often spreads into the orbit and cranial cavity; mortality is about 50%, even with debridement and high-​dose intravenous amphotericin B The bacterial infections often require aggressive intravenous anti- biotic treatment with cover against anaerobes. Fastidious and rare organisms should be considered when standard antibiotic regimens are ineffective. Box 13.9.1.1  ‘Sick-​day’ rules for patients with type 1 diabetes If you feel unwell, and even if you think it is a minor infection: • Never stop taking your insulin—​you often need more when you are unwell • Check your blood glucose every 4 h—​glucose levels can rise very fast during infections • Test your urine or blood ketones if your glucose is greater than 14 mmol/​litre or you are feeling unwell with vomiting • Contact your doctor at once if you: — start vomiting — get high glucose levels (>15) that do not come down after insulin — get hypos (glucose <3) — get ketones in the urine or blood ketones greater than 1.0 mmol/​litre — are worried and do not know what to do

13.9.1  Diabetes 2505 Diabetic control during infections Minor viral infections rarely disturb diabetic control, but increased secretion of counterregulatory stress hormones during severe infec- tions, especially with fever, can rapidly worsen insulin resistance in both type 1 and 2 diabetes. Type 1 patients may need twice as much insulin as usual, even if they are unable to eat. Failure to increase the insulin dosage will therefore allow glucose to rise, sometimes dramatically fast, and risk precipitating ketoacidosis. It is therefore essential to continue taking insulin, to monitor blood glucose frequently, and to increase insulin if sustained hyperglycaemia develops. An increase of 30 to 50% in long-​acting insulin is often enough, but requirements will be deter- mined by blood glucose levels and should be decided in consultation with the diabetes care team. Avoidable deaths still occur every year because poorly educated patients (sometimes advised by ignorant doctors) reduce or even stop taking insulin because they feel ill, are not eating, and are worried about becoming hypoglycaemic. Clear ‘sick-​day’ rules (see Box 13.9.1.1) are a crucial part of diabetes edu- cation, which must be regularly checked and reinforced. During severe infections, insulin requirements may fluctuate rap- idly and the safest way to give insulin is by continuous intravenous infusion, backed up by frequent (hourly) blood glucose measure- ments (see next). Type 2 patients may similarly lose glycaemic con- trol, and are often best transferred temporarily to subcutaneous or intravenous insulin. It seems best to maintain blood glucose be- tween 5 and 10 mmol/​litre during intercurrent infections, although this is not firmly evidence-​based. Myocardial infarction This is discussed in detail later in this chapter. Surgery Guidelines for the United Kingdom can be found at: http://​ www.diabetologists-​abcd.org.uk/​JBDS/​JBDS_​IP_​Surgery_​Adults_​ Full.pdf Surgery can be hazardous to patients with diabetes:  the counter­regulatory stress response to surgical trauma can rapidly lead to hyperglycaemia and ketoacidosis, especially in insulin-​ deficient patients, while poorly controlled diabetes accelerates catabolism and delays wound healing. Moreover, insulin and the sulphonylureas can cause severe hypoglycaemia in fasted or anor- exic patients, which can be particularly dangerous during general anaesthesia. Glycaemic control must therefore be meticulous throughout the perioperative period. A  routine management policy should be agreed between the diabetes care team, surgeons, anaesthetists, and ward staff, and this will greatly reduce the risks of operating on diabetic people. Fitness for surgery should be carefully assessed, in view of cardiovascular or other complications. Patients may need to be admitted some days before operation to optimize their treatment. For type 2 patients who are well controlled by diet or oral agents and undergoing minor surgery only (anticipated to miss only one meal), long-​acting sulphonylureas (glibenclamide) should be changed to short-​acting ones (e.g. gliclazide) some days before sur- gery to reduce the risk of hypoglycaemia. Oral agents and breakfast should be omitted on the morning of operation and blood glucose should be monitored closely. Persistent hyperglycaemia should be treated with the intravenous insulin and fluids described next. For all other patients with diabetes, insulin should be given as a variable-​rate intravenous infusion (VRII, previously referred to as sliding scale insulin) adjusted according to hourly blood glucose measurements, which provides greater flexibility. If the patient is in steady state, this will both maintain satisfactory glycaemic control and prevent hypokalaemia (insulin enhances potassium entry into skeletal muscle). This regimen should be started on the morning of surgery and continued until the patient is able to eat and drink normally, when the usual treatment can be resumed. Recent guide- lines advise that the patient’s usual long-​acting insulin should be continued during the period of VRII. Adjustments to the insulin infusion rate are made aiming to keep blood sugars between 6 and 10  mmol/​litre (acceptable levels 4–​12  mmol/​litre). When using a variable-​rate intravenous insulin infusion the capillary glucose should be checked hourly and the serum electrolytes measured daily. A typical infusion scale is shown in Table 13.9.1.8, but in- sulin infusion rates can be increased for a given glucose range if glucose levels are not falling. Acute metabolic complications of diabetes
and their treatment Diabetic ketoacidosis This is uncontrolled hyperglycaemia with hyperketonaemia severe enough to cause metabolic acidosis. It remains a major cause of death in patients with type 1 diabetes under 20 years of age, although the mortality has fallen from 8% to 0.67% with modern treatment (higher in older patients with diabetic ketoacidosis precipitated by infection or myocardial infarction). Prompt diagnosis and careful management can prevent many deaths. Causes Diabetic ketoacidosis only develops when severe insulin defi- ciency, compounded by an excess of glucagon, stimulates lipolysis and a massive increase in ketogenesis (see earlier). It therefore Table 13.9.1.8  Starting insulin infusion scale for perioperative patients on variable-​rate insulin infusion Bedside capillary blood
glucose (mmol/​litre) Initial rate of insulin infusion
(units per hour) <4.0 0.5 (0.0 if a long-​acting background insulin has been continued) 4.1–​7.0 1 7.1–​9.0 2 9.1–​11.0 3 11.1–​14.0 4 14.1–​17.0 5 17.1–​20 6

20 Seek diabetes team or medical advice Reproduced, with permission from NHS Diabetes, from K Dhatariya et al., Management of adults with diabetes undergoing surgery and elective procedures: improving standards (2011), http://​www.diabetes.nhs.uk/​document.php?o=2178

section 13  Endocrine disorders 2506 almost always occurs in untreated or poorly treated type 1 dia- betes and is generally regarded as the hallmark of that disease. However, diabetic ketoacidosis can occur in subjects with type 2 diabetes who are relatively insulin deficient, especially when the secretion of counterregulatory hormones (especially glucagon) is increased by severe intercurrent illness. Precipitating factors include: • newly presenting type 1 diabetes • omission or underdosing of insulin by established type 1 diabetic patients, which may be deliberate in patients with disturbances of body image • intercurrent illness, such as infections, myocardial infarction, stroke, trauma, surgery, and burns; many patients (and their doc- tors) fail to increase insulin dosages or monitor blood glucose during such events About 30 to 40% of episodes are unexplained; omitted or inad- equate insulin treatment should always be suspected if no obvious infective or other cause is found. Pathophysiology Diabetic ketoacidosis is due to the accumulation of ketones, that is, acetoacetate and its derivatives, 3-​hydroxybutyrate (or β-​hydroxybutyrate) and acetone (see Fig. 13.9.1.11). They are gen- erated by β-​oxidation of free fatty acids within the mitochondria of the liver. Free fatty acids enter the cytoplasm of hepatocytes and combine with coenzyme A (CoA) to form their fatty acyl-​CoA de- rivatives. These are then transported into the mitochondria by the carnitine shuttle, a complex of two linked enzymes, carnitine palmitoyltransferase I (CPT-​I) on the outer mitochondrial mem- brane and carnitine palmitoyltransferase II (CPT-​II) on the inner. CPT-​I, and the overall activity of the shuttle, is powerfully inhibited by insulin and stimulated by glucagon. Once inside the mitochon- dria, free fatty acids undergo β-​oxidation to yield ATP (the process of oxidative phosphorylation) and acetyl-​CoA. The latter is con- verted to acetoacetate, which may be oxidized to 3-​hydroxybutyrate or undergo condensation to produce acetone. Ketones are transported out of the liver and are used as metabolic fuels by various tissues including the brain; they supply a few% of total energy needs after an overnight fast, but the proportion rises to over one-​third during prolonged fasting. When produced in ex- cess, they can accumulate rapidly, especially if plasma levels exceed 5 mmol/​litre (about 10 times normal), when tissue uptake mechan- isms become saturated. Ketogenesis is greatly enhanced in uncon- trolled type 1 diabetes because of the combination of low insulin with increased glucagon concentrations: lipolysis is unrestrained, and the uptake into liver mitochondria of the increased amounts of fatty acyl-​CoA is stimulated by the synergistic effects on CPT-​I of high glucagon and low insulin. The main consequences of raised cir- culating ketone levels are shown in Fig. 13.9.1.11 and listed next: • Acidosis:  acetoacetate and 3-​hydroxybutyrate are both moder- ately strong organic acids and lower the extracellular pH when the buffering capacity of plasma proteins is exceeded. Ion exchange across cell membranes leads to intracellular acidosis which com- promises cellular metabolism because many crucial enzymes operate within a narrow pH range. Clinical measurements of acid-​ base status are confined to the extracellular fluid and may under- estimate the severity of intracellular acidosis. • Diuresis: ketones are filtered in the urine and are osmotically ac- tive. They therefore exacerbate the osmotic diuresis caused by glycosuria and the resulting polyuria, electrolyte losses, dehydra- tion, and hypovolaemia. • Nausea: through direct stimulation of the chemoreceptor trigger zone in the medulla. Clinical features Diabetic ketoacidosis usually presents with classical hyperglycaemic symptoms (see Table 13.9.1.2), together with features of acidosis and hyperketonaemia: • Acidotic (Kussmaul) breathing is deep, sighing hyperventilation which has been mistaken for panic attacks, pulmonary embolism, and left ventricular failure. • Nausea and vomiting are ominous signs, because dehydration de- velops quickly in polyuric patients unable to drink. • Drowsiness and coma occur late and may indicate early cerebral oedema. The patient generally looks ill and may show postural hypoten- sion and other signs of dehydration and hypovolaemia. Acetone is volatile and may be smelled on the breath (ketotic foetor; ‘nail var- nish remover’ odour). Some patients are hypothermic due to heat loss from peripheral vasodilation, and this may mask the pyrexia of infection. Children with diabetic ketoacidosis often complain of abdominal pain, sometimes mimicking acute appendicitis or other surgical emergencies. A full examination is essential to identify any intercurrent illness. Investigations and diagnosis Once suspected, the diagnosis can be confirmed on the spot with a finger-​prick blood glucose measurement and urine or blood analysis for ketones. Recent guidelines place emphasis on the value of bed- side finger-​prick testing for blood ketones both in diagnosis (plasma ketone level >3.0 mmol/​litre, glucose <11.0 mmol/​litre or previous diabetes diagnosis, bicarbonate <15  mmol/​litre, and/​or pH <7.3) and in subsequent management. Treatment with intravenous saline and insulin should begin immediately, and baseline investigations Insulin deficiency + glucagon excess Cellular dysfunction Cerebral oedema Shock ? ? Vomiting Osmotic diuresis Blood ketones Fluid and electrolyte depletion Acidosis Blood glucose Fig. 13.9.1.11  Pathophysiological changes in diabetic ketoacidosis. Cellular dysfunction induced by intracellular acidosis, as well as cerebral oedema and shock are potentially life-​threatening.

13.9.1  Diabetes 2507 carried out. Venous blood is taken for biochemical screening and can also be used to assess acid-​base status as the difference between arterial and venous pH is 0.02 to 0.15 units and for bicarbonate it is 1.88 mmol/​litre, differences that are small enough not to influence management, particularly in view of the much greater convenience of venous sampling. High ketone concentrations cause a large anion gap (i.e. plasma [Na+ + K+] exceeds [HCO3–​ + Cl–​] by more than 17 mmol/​litre). Additional tests to identify the cause of the episode should include a full blood count, urine and blood culture, chest radiograph, and, especially in older patients, ECG and cardiac enzymes or troponin levels. Typical values and some diagnostic pitfalls in diabetic ketoacid- osis are shown in Fig. 13.9.1.12. Management Diabetic ketoacidosis is a potentially life-​threatening medical emer- gency that requires urgent treatment with scrupulous clinical and biochemical monitoring: many avoidable and serious accidents still happen because the patient is abandoned once treatment has been started. Severe diabetic ketoacidosis is best managed initially on a high-​dependency or intensive care unit. The highest priority is to correct hypovolaemia and dehydration, which will often improve acidosis and hyperglycaemia. Insulin re- placement must also be started urgently. However, it now appears likely that the high mortality of diabetic ketoacidosis has been partly due to overenergetic replacement of intravenous fluids (especially bicarbonate) and perhaps insulin, which may predispose to the de- velopment of cerebral oedema. The treatment guidelines (see Fig. 13.9.1.12) are based on large studies that have reported very low mortality and morbidity. Fluid replacement Good intravenous access is crucial: a large peripheral vein may be used but a central venous cannula is safest for severely hypovol- aemic patients and for older people or those at risk of heart failure, in whom monitoring of central venous pressure is essential. Most patients recover rapidly with slower fluid replacement than was previously recommended. For those who are not shocked give: • 1 to 2 litres in 2 h, then • 1 litre over the next 4 h, then • 4 litres over the next 24 h Fluid losses in urine or vomit should be added to these volumes. Shocked or oliguric patients may require faster fluid repletion, pos- sibly with plasma expanders rather than saline, while slower re- placement is safer in those with signs of fluid overload, myocardial infarction, heart failure, or any suspicion of cerebral oedema. Urine output must be monitored closely, as must blood pressure, central venous filling, and signs of pulmonary or peripheral oedema. Saline containing potassium is the logical fluid to replace the losses of Na+, K+, and Cl–​ induced by the osmotic diuresis of diabetic ketoacidosis. The use of intravenous bicarbonate to try to correct acidosis is contentious, both in terms of biochemistry and clinical outcome (see next). Isotonic (0.9%) saline is used initially. Half isotonic (0.45%) sa- line has been suggested to empirically replace 1 or 2 litres of iso- tonic saline, if severe hyperosmolarity (>350 mosmol/​kg) and/​or hypernatraemia (>150 mmol/​litre) are present. However, the ra- tionale may be flawed: 0.9% (normal) saline is already hypotonic with respect to the patient’s hypertonic plasma, and the use of even more hypotonic solutions would seem likely to exacerbate the intra- cellular movement of water which may lead to cerebral oedema. It is now recommended that 10% dextrose is added (125 ml/​h) as an add- itional infusion alongside the rehydration with saline when plasma glucose has fallen to below 14 mmol/​litre to prevent hypoglycaemia (insulin is still required to prevent ketogenesis and promote glucose utilization in the tissues). Intravenous sodium bicarbonate was previously recommended for severe acidosis. However, the hope that adding alkali will cor- rect acidosis may be oversimplistic. HCO3–​ and H+ ions (from 3-​ hydroxybutyric and acetoacetic acids) combine extracellularly to produce H2CO3, which dissociates to produce water and CO2; this may reduce extracellular acidosis, but as cell membranes are im- permeable to HCO3–​ ions, the all-​important intracellular acidosis is not improved. Indeed, CO2 can enter cells where it can combine with water to produce H2CO3, itself a weak organic acid that can dissociate into H+ and HCO3–​ ions. Paradoxically, therefore, intra- venous bicarbonate administration could worsen intracellular acid- osis and there is evidence from animal models of acidosis that this occurs. Worryingly, a recent study identified bicarbonate admin- istration as the most important independent predictor of cerebral oedema in children with moderately severe diabetic ketoacidosis. Another problem with high-​strength (8.4%) sodium bicarbonate so- lution is the intense thrombophlebitis it causes when given intraven- ously, which can obliterate even large central veins. Extravasation can also cause severe tissue necrosis. The current consensus is that bicarbonate is unlikely to be benefi- cial but runs the risk of doing harm, and that it should not be used in the treatment of diabetic ketoacidosis Potassium replacement Hyperkalaemia and hypokalaemia are the main causes of death in diabetic ketoacidosis (DKA) in adults. Diabetic ketoacidosis always Fig. 13.9.1.12  Guidelines for the management of diabetic ketoacidosis.

section 13  Endocrine disorders 2508 depletes total body K+ stores to a variable degree because of elec- trolyte losses through osmotic diuresis, but H+/​K+ exchange across the plasma membrane encourages K+ to leak out of cells in acidosis. Plasma K+ levels can therefore be low, normal, or high, and dan- gerous hyperkalaemia can be present, especially if severe hypovol- aemia causes prerenal failure. During insulin replacement, K+ is carried intracellularly with glucose, and plasma K+ levels can fall rap- idly. Frequent monitoring of K+ (every 2 hours) is therefore essential in the safe management of diabetic ketoacidosis, and patients with marked K+ disturbances should have continuous ECG monitoring. Potassium replacement should be determined by current plasma K+ levels: • Add 40 mmol of KCl to each litre of intravenous fluid if K+ is normal (3.5–​5.0 mmol/​litre). • Add 40 mmol/​litre of KCl to each litre if plasma K+ is less than 3.5 mmol/​litre and review in 1 h. • Omit KCl if plasma K+ is more than 5.0 mmol/​litre, because of the risk of precipitating arrhythmias. Repeat K+ in 2 hours and re- place if fallen below 5.0 mmol/​litre. Insulin replacement Continuous fixed rate intravenous infusion is the best way to give insulin in diabetic ketoacidosis; subcutaneous and intramuscular absorption are too erratic to be safe and the rate of fall of glucose (one of the factors implicated in cerebral oedema) cannot be easily controlled. A dose of 50 U soluble insulin should be added to 50 ml isotonic saline (i.e. 1 U/​ml) and delivered by a syringe driver pump, either into a separate vein or piggy-​backed into the intravenous fluids line only if a one-​way valve is present. Because the half-​life of insulin in the circulation is only a few minutes, blood glucose and ketone levels will rise rapidly if insulin delivery is interrupted; hourly monitoring of blood glucose is there- fore mandatory during intravenous insulin. Failure of glucose to fall usually means that the pump has been turned off or that the infusion cannula is blocked. It is now recommended that a fixed rate infusion at 0.1 U/​kg/​hr is used (to take account of the fact that some over- weight individuals will be insulin resistant), rather than a variable rate or ‘sliding scale’. The rationale is to promote as rapid resolution of ketosis as possible. When blood glucose falls to below 14 mmol/​ litre an infusion of 10% dextrose 1 litre per 8 hours is then recom- mended, along with any saline rehydration, to prevent hypogly- caemia and to allow the same rate of insulin infusion to correct the ketosis. Adequate response to insulin is judged by a fall in blood ke- tones by more than 0.5 mmol/​litre per hour, or a fall in blood glucose by more than 3 mmol/​h. If these parameters are not being met the infusion system should be carefully checked and, if working prop- erly, increases in the fixed rate insulin infusion by 1 U/​h are made until an adequate response is achieved. Resolution of ketosis is said to have occurred when the following criteria are satisfied: plasma ketones less than 0.6 mmol/​litre and venous pH greater than 7.3. The urine may misleadingly still test positively for ketones at this stage due to excretion of the previous ketone load. Once resolution of ketosis is confirmed, the patient can be converted back to their usual subcutaneous insulin regimen (or begun on a regimen if this is a new diagnosis of diabetes), assuming they are able to eat and drink normally. If it is impossible to give a controlled intravenous infusion, then intramuscular soluble insulin can be injected every 4 h or so, starting with 20 U and attempting to titrate subsequent dosages (e.g. 5–​10 U hourly). Other complications Intercurrent illness must be treated energetically. Broad-​spectrum antibiotics are often given prospectively. Myocardial infarction (see next) has a poor prognosis if it causes diabetic ketoacidosis. Shock may lead to prerenal failure and sometimes acute tubular necrosis. Plasma expanders and inotropes may occasionally be re- quired for severe hypotension, although rehydration, as already mentioned, is usually adequate. Cerebral oedema still accounts for 50% of fatalities in diabetic ketoacidosis, especially in children, although modern management protocols with slower fluid replacement and low-​dose intravenous insulin infusion can markedly reduce its incidence. The cause is thought to be shifts of ions and water into the brain, particularly the movement of water into dehydrated, hypertonic cells when relatively hypotonic fluids reach the extracellular space. Such shifts would be predicted with the administration of isotonic and particularly with hypotonic fluids. Risk factors for cerebral oedema include over-​ rapid falls in blood glucose, excessive fluid replacement, and high insulin dosages. Insulin can affect various ion transport mechanisms in the brain, but its role remains mysterious and may simply reflect changes in extracellular osmolarity. Interestingly, CT scanning be- fore fluid and insulin replacement has demonstrated subclinical cerebral oedema in children with diabetic ketoacidosis. Swelling of the brain within the cranium causes coning, leading to cardiorespiratory arrest. It presents as a decline in consciousness, usually rapid, and often when the patient’s metabolic state has been stabilized. Papilloedema may be present, and CT or MRI will show characteristic swelling, with loss of cortical features and squashing of the ventricular system (Fig. 13.9.1.13). It is usually fatal (in >90% of established cases), but intravenous mannitol (0.2 g/​kg over 30 min, repeated hourly if there is no improvement) may help by raising the Fig. 13.9.1.13  Cerebral oedema in a patient recovering from diabetic ketoacidosis. The CT shows generalized swelling and loss of cortical detail with squashing of the cerebral ventricles.

13.9.1  Diabetes 2509 osmolality of extracellular fluid and drawing free water out of the brain; there is no firm evidence to support the use of dexamethasone. Adult respiratory distress syndrome is due to accumulation of fluid in the alveoli, perhaps due to ionic and water shifts or to ex- cessive leakiness of the pulmonary capillaries. Hypoxia is severe, and chest radiography shows an appearance like left ventricular failure but with a normal heart size. Risk factors include rapid fluid replacement. It carries a poor prognosis, but ventilation with high-​ concentration oxygen may be useful supportive treatment. Acute gastric dilatation (gastroparesis) presents with vomiting and may produce a succussion splash and a ground-​glass appear- ance on abdominal radiograph. Nasogastric drainage may be needed to prevent aspiration, especially in the unconscious patient. Hypotension may persist or develop during treatment and gen- erally reflects inadequate fluid replacement. Alternative causes of hypotension including septic shock and cardiogenic shock should also be considered. Polyuria secondary to continuing high glucose levels may occasionally give false reassurance that the patient’s fluid replacement status is adequate. Persisting acidosis despite correction of blood sugar levels and a fall in plasma (and later urine) ketones raises the possibility of lactic acidosis secondary to sepsis or metformin use (see next). Blood lactate levels should be measured. Plasma sodium levels may rise despite fluid replacement as the initial high glucose levels may have resulted in an erroneously low initial reading. Falling sodium levels may reflect the need for more saline and less dextrose-​based fluid replacement. Hypothermia indicates a poor outcome. It may respond to rewarming with a space blanket. Subsequent management When the patient can eat and drink, intravenous fluids and insulin can be discontinued. There is no need for an insulin infusion; in- stead, the patient can be restarted on their usual subcutaneous insulin regimen (or a new regimen, if newly diagnosed). The intra- venous insulin infusion should be maintained until the first injec- tion containing soluble or short-​acting analogue insulin has had time to act (the intravenous infusion should not be stopped if only basal insulin has been given). The causes of the episode must be determined if possible, and efforts made to prevent a recurrence. from happening again. The patient’s understanding of diabetes, including the ‘sick-​day’ rules (Box 13.9.1.1), must be checked and reinforced if necessary. Recurrent diabetic ketoacidosis is a feature of brittle diabetes, and these patients need careful monitoring and counselling. Hyperglycaemic hyperosmolar state (HHS)—​formerly hyperosmolar non​ketotic state (HONK) HHS is distinguished from diabetic ketoacidosis by the absence of marked hyperketonaemia (<3.0  mmol/​litre) and metabolic acidosis (pH >7.3). Hyperglycaemia can be greater than in dia- betic ketoacidosis (typically >30 mmol/​litre) and, together with a rise in urea due to dehydration and prerenal failure, may elevate the plasma osmolality to well over 350 mosmol/​kg (usually >320 mosmol/​kg). HHS may be the first presentation of type 2 dia- betes. (Guidelines for treatment can be found at https://​abcd.care/​ joint-​british-​diabetes-​societies-​jbds-​inpatient-​care-​group.) Ketosis does not develop because circulating insulin levels are high enough to suppress lipolysis and ketogenesis; these pa- tients are therefore C-​peptide positive, with type 2 diabetes which is often previously undiagnosed. It is more common in people of Afro-​Caribbean origin. Precipitating factors include myocardial infarction, stroke, infection, and diabetogenic drugs such as gluco- corticoids and thiazide diuretics; fizzy glucose drinks may also contribute. Presentation is typically with classical hyperglycaemic symptoms (polyuria, intense thirst, weight loss, blurred vision), without the features of ketoacidosis. Confusion, drowsiness, and coma are more common than in diabetic ketoacidosis. At blood glucose levels over 30 mmol/​litre, drowsiness can lead to a cycle of deterioration as fluid loss continues due to the osmotic diuresis, but the patient is increas- ingly too lethargic to drink adequate replacement fluids. Progressive dehydration then leads to even higher glucose levels, more lethargy, and a further decline in oral intake. Complications include thrombotic events such as stroke and per- ipheral arterial occlusion, and deep venous thrombosis and pul- monary embolism, these being due apparently to increased blood viscosity. Mortality is 15 to 20%, partly because these patients are old and often have a serious precipitating illness. Biochemical features of HHS are: • hyperglycaemia:  often over 50 mmol/​litre, sometimes over 90 mmol/​litre • hypernatraemia: often over 155 mmol/​litre (may be artefactually depressed by high glucose levels) • uraemia due to dehydration, with or without renal failure • hyperosmolality: over 320 mosmol/​kg • blood and ketone levels are normal or only slightly raised (usually through anorexia) • arterial pH, venous bicarbonate, and anion gap show no features of severe acidosis Management of HHS • Saline replacement must be particularly cautious in older pa- tients, in whom cardiac disease is common; 0.9% saline should be used rather than the previously used half isotonic (0.45%) sa- line. Rehydration will lower the blood glucose level, which will lower osmolality and fluid will shift into the intracellular space. The falling glucose will cause a rise in sodium levels which is not an indication for hypotonic saline; a rising sodium is only cause for concern if the osmolality is not falling. Osmolality should be measured hourly. • Potassium levels must be carefully monitored and replaced as described. • Rehydration alone will lower glucose. A safe glucose fall is by 4 to 6 mmol/​l per hour, aiming for a glucose of 10–​15 mmol/​litre. If significant ketones are present intravenous insulin should be started immediately however if not insulin should not be com- menced immediately as it may drop osmolality dramatically and precipitate cardiovascular collapse. Once rehydration is adequate and glucose is still not falling then intravenous insulin may be started at a rate of 0.05 unit/​kg/​hour. • low-​dose heparin (5000 U subcutaneously 8-​hourly or low mo- lecular weight heparin once daily) should be given prophylactically,

section 13  Endocrine disorders 2510 but full anticoagulation should be reserved for proven thrombo- embolism as the risks of fatal gastrointestinal bleeding are high. Intercurrent illness, such as infection, must be sought and treated appropriately. After recovery, many of these patients should be converted to in- sulin however after weeks to months can be successfully weaned off insulin. Drugs and other precipitating factors must be identified and avoided if possible. Lactic acidosis Lactate is generated by glycolysis and its levels rise rapidly during tissue anoxia (e.g. during shock, cardiac failure, or pneumonia) or when the liver is prevented from utilizing it as a gluconeogenic substrate (e.g. in hepatic impairment). Lactic acidosis is best known in diabetic patients as a rare but often fatal complication of the biguanides, phenformin and metformin, which act mainly by inhibiting hepatic gluconeogenesis. The risk is about 10 times higher with phenformin than with metformin, and it is very rare during metformin treatment as long as other predisposing factors (the major organ failures) are avoided. Lactic acidosis presents as coma with metabolic acidosis (reduced arterial pH and venous bicarbonate) and a wide anion gap due to hyperlactataemia. Blood glucose levels are usually raised. Treatment is still unsatisfactory. Intravenous sodium bicar- bonate may paradoxically aggravate intracellular acidosis, although forced ventilation to blow off carbon dioxide may help (see earlier). Haemodialysis may both clear lactate and hydrogen ions, and cor- rect any sodium overload following bicarbonate administration. Sodium dichloroacetate, which stimulates pyruvate dehydrogenase to metabolize lactate, is undergoing evaluation. Mortality remains high (>30%), partly because of the organ fail- ures that commonly coexist. Hypoglycaemia Hypoglycaemia is an inevitable side effect of antidiabetic drugs that raise circulating insulin levels, namely insulin itself and sulphonylureas; it does not occur with metformin or thiazolidinediones alone, or with dietary restriction. Common contributory factors are: • accelerated insulin absorption (e.g. due to exercise or hot surroundings) • unfavourable timing of insulin injection: injecting too soon be- fore eating can cause late postprandial hypoglycaemia, while long-​ acting insulins injected in the early evening often cause nocturnal hypoglycaemia • too much insulin injected: dosage errors are quite common, par- ticularly in older people • inadequate food intake: missed, delayed, or small meals; vomiting, including gastroparesis • exercise:  this hastens insulin absorption while enhancing in- sulin action; delayed hypoglycaemia may occur many hours later because muscle continues to take up glucose to replenish glycogen • alcohol:  this inhibits hepatic gluconeogenesis, preventing the increase in hepatic glucose output that is crucial for restoring euglycaemia • impaired awareness of early warning symptoms (see next) Progressively more frequent or severe attacks may be caused by various conditions, which should always be considered: • weight loss, including anorexia nervosa and appetite disorders (relatively common in young women with type 1 diabetes) • loss of counterregulatory hormones:  Addison’s disease, hypo­ thyroidism, hypopituitarism, blunted glucagon secretion in long-​ standing type 1 diabetes • intestinal malabsorption, notably coeliac disease (more common in type 1 diabetes) • renal failure, which impairs the clearance of insulin • deliberate inappropriate injection of insulin, often in the context of ‘brittle’ diabetes Manifestations Clinical features of hypoglycaemia are due to an autonomic dis- charge, predominantly sympathetic, together with the cerebral effects of neuroglycopenia. Falling glucose levels are sensed by glucose-​sensitive neurons, which are found in the periphery (vagal sensory endings in the portal vein) and medulla as well as the hypo- thalamus. This triggers a powerful sympathetic discharge that re- leases adrenaline from the adrenal medulla and noradrenaline from sympathetic nerve endings, causing the familiar ‘flight or fight’ response. Features include pallor (cutaneous vasoconstriction), sweating (which can be very profuse), tremor (a β2-​adrenergic effect on skeletal muscle), and tachycardia; systolic blood pressure rises due to increased cardiac output while pulse pressure widens—​giving the typical bounding pulse—​because β2-​mediated vasodilatation in skeletal muscle causes peripheral resistance to fall. Hypoglycaemia also triggers the secretion of counterregulatory hormones, namely glucagon and adrenaline (both crucial to re- storing euglycaemia), growth hormone, and cortisol. Collectively, these inhibit insulin secretion and raise blood glucose by enhancing hepatic glycogenolysis and gluconeogenesis, causing glucose to pour out of the liver. Defects in glucagon or adrenaline release (which occur in long-​standing type 1 diabetes, for example), or in the ability of the liver to produce glucose (e.g. the presence of ethanol which inhibits gluconeogenesis, or a recent glucagon in- jection which depletes liver glycogen) will delay recovery of blood glucose. The physiological and neurological features of hypoglycaemia usually develop in a fixed sequence when blood glucose is lowered in a controlled fashion in the laboratory. However, this hierarchy may not be apparent in real life, and some patients specifically lose their awareness of the early warning symptoms (see next). Key events as glucose falls are: • at c.3.8 mmol/​litre: increased glucagon and adrenaline secretion • at c.3.0 mmol/​litre: onset of hypoglycaemic symptoms • at c.2.8 mmol/​litre: neuroglycopenia and cognitive impairment • less than 2 mmol/​litre: coma Symptoms of hypoglycaemia The symptom complex can be extremely variable, and hypogly- caemia should be suspected as the cause of any ‘funny turn’ in patients treated with insulin or sulphonylureas. Autonomic mani- festations include sweating, tremor, tachycardia, and hunger, while neuroglycopenia can cause drowsiness, confusion, incoordination,

13.9.1  Diabetes 2511 dysarthria, and automatic or disinhibited behaviour; distinct neurological deficits include aphasia, diplopia, and hemiparesis. Non​specific malaise and headache afterwards are also common. Nocturnal episodes may pass completely unnoticed by the patient, or may cause sweating and restlessness (often obvious to the patient’s partner), vivid nightmares, nocturnal epilepsy, or a hungover feeling the following morning. Awareness of hypoglycaemic symptoms Diabetic patients rely on the early autonomic symptoms (sweating, shaking, and hunger) to warn them of an impending hypoglycaemic attack, when corrective action can be taken. In some patients, the early warning symptoms are attenuated or not noticed at all; this clumsily named ‘hypoglycaemia unawareness’ is potentially dan- gerous because severe neuroglycopenia (confusion, fitting, ir- rational behaviour, coma) may suddenly incapacitate the patient. Reduced awareness of hypoglycaemia occurs particularly in two set- tings, which may coexist: • Long-​standing type 1 diabetes. Some 30 to 50% of patients with diabetes of more than 20 years’ duration have decreased awareness of symptoms, and many also show a flat glucagon and adrenaline response to hypoglycaemia. Blunted recognition of hypogly- caemia by the central nervous system may be responsible. • Excessively tight glycaemic control impairs awareness of hypogly- caemia; for unknown reasons, even a single episode can blunt per- ception of symptoms and counterregulatory hormone responses for some days. Conversely, relaxing control and avoiding hypogly- caemia completely for several weeks can partially restore aware- ness of warning symptoms. The use of human insulin has been suggested to impair aware- ness of hypoglycaemic symptoms. Human insulin is relatively lipophilic—​hence its faster subcutaneous absorption—​which could theoretically promote its entry into the brain. Insulin may act directly on the brain to affect various autonomic processes, but detailed comparisons of human and animal insulins, both in the laboratory and in real life, have not shown any species differences in counterregulatory responses or the intensity of hypoglycaemic symptoms. Sequelae of hypoglycaemia Even the most dramatic neurological manifestations of acute hypoglycaemia—​including aphasia, hemiparesis, fitting, and unconsciousness—​usually resolve rapidly when blood glucose is normalized. Recovery from profound coma may take many hours or even days, and this is probably due to cerebral oedema. Patients who survive severe and prolonged hypoglycaemic coma may show permanent neurological damage, including memory loss, aphasia, and a vegetative state. There are concerns that repeated mild attacks, especially in children and perhaps particularly at night, can cause cumulative intellectual impairment, but this is not yet proven. Severe hypoglycaemia has been implicated in precipitating myo- cardial infarction or stroke, particularly in older people; rises in blood pressure and increased coagulability of the blood following sympathetic stimulation may contribute. Like any convulsions, hypoglycaemic fits may cause injury, including limb and vertebral crush fractures. Prolonged severe hypoglycaemia can be fatal and is one of the most common causes of death in young type 1 patients. Post-​ mortem studies show neuronal damage and necrosis in the hippo- campus and cerebral cortex. Hypoglycaemia has been suspected as a cause of death in patients found unexpectedly dead in bed; however, it is thought arrhythmias secondary to autonomic dysfunction may be responsible. Diagnosis and detection of hypoglycaemia Hypoglycaemia is easy to diagnose but is also easily missed; dif- ferential diagnoses include transient ischaemic attacks, psychosis, drunkenness, epilepsy, and migraine. Symptoms may be instantly recognizable to some patients, but may present atypically. If sus- pected, the blood glucose levels should be checked, taking care to avoid under-​reading artefacts with reagent test strips. Urinalysis is obviously of no use—​hence all patients receiving insulin or sulphonylureas must be able to check their blood glucose. The patient’s close associates should also know how to diagnose and treat hypoglycaemia. Various experimental hypoglycaemia detectors are undergoing development including subcutaneous sensors and transcutaneous near-​infrared spectroscopy. Continuous glucose monitoring sys- tems are able to give a real-​time display of blood glucose levels every few minutes and may prove especially valuable in recurrent hypoglycaemia. Current limitations include cost (sensors require replacing every 3–​6 days) and the fact that there is a delay of ap- proximately 20 min before interstitial fluid and blood glucose levels equilibrate, which means that the sensor can underestimate the severity of hypoglycaemia when blood glucose levels are falling rapidly. Prevention and treatment of hypoglycaemia It has been shown that insulin-​treated patients fear hypoglycaemia as much as blindness or renal failure; this may prevent them from tightening their diabetic control as much as their doctors would prefer. Many doctors underestimate the impact of hypoglycaemia; asking about it and trying actively to prevent it are an essential part of diabetes care. It should be emphasized that a ‘good’ HbA1c level can be misleading in this context, as it can be the re- sult of a combination of high blood glucose levels and recurrent hypoglycaemia. Attention to the factors listed earlier should help to reduce the frequency and severity of attacks. Advice about exercise, moderating alcohol intake, and timing of insulin injections and meals are par- ticularly important. Nocturnal hypoglycaemia can be reduced by checking the blood glucose at bedtime, and by taking long-​acting carbohydrate (e.g. bread or cereal) if the level is less than 6 mmol/​ litre. Long-​acting analogue insulin and insulin pump therapy may also be of value in this situation (see earlier). Blood glucose levels of less than 3 mmol/​litre should be treated immediately (see Table 13.9.1.9). Oral glucose or sucrose or other carbohydrate should be given if the patient can swallow safely. Give 20 to 30 g (e.g. 3–​6 Dextrosol tablets or 75–​150 ml of a glucose drink such as Lucozade) initially; if possible, check the blood glu- cose 15 min later and repeat glucose administration if this has not risen. Taking too much carbohydrate—​which is an understandable reaction, given the unpleasantness of hypoglycaemia—​can cause marked rebound hyperglycaemia.

section 13  Endocrine disorders 2512 If the patient is unconscious, give either: • glucagon 1 mg (0.5 mg in children), subcutaneously or intramus- cularly; with either route, glucose should rise within 10 to 15 min. Side effects of glucagon include malaise, nausea, and abdominal discomfort. Importantly, as glucagon acts primarily by breaking down hepatic glycogen (a limited resource), a second injection may be ineffective • intravenous glucose: 15 to 20 g intravenously, as a 10% solution. Formerly, 50% glucose was used however this is associated with a painful thrombophlebitis. Glucose gels or jam can be smeared inside the mouth and cheeks in the unconscious patient, but these alone are unlikely to correct serious hypoglycaemia. On recovery, blood glucose should be checked and oral glucose given as earlier. Slow recovery from coma may be due to cerebral oe- dema, which has a high mortality (c.10%) but may respond to intra- venous mannitol and forced ventilation with high inspired oxygen concentration. Once the episode is treated, its cause must be iden- tified if possible and corrective action taken to prevent it from hap- pening again. Since it takes some time to recover cognitive function after an episode of hypoglycaemia, recent driving guidelines in the United Kingdom recommend not driving for at least 45 min after correction of hypoglycaemia. Options for intractable, recurrent hypoglycaemia Recurrent, disabling hypoglycaemia often develops in patients who have maintained very tight glycaemic control over many years. The frequent hypoglycaemic episodes that often occur with tight control themselves result in loss of hypoglycaemia awareness and physiological defence mechanisms (e.g. glucagon and adrenaline). The result is more frequent hypoglycaemia—​‘hypoglycaemia be- gets hypoglycaemia’. Options for reversing this situation include careful inspection of injection sites and avoidance of areas of lipohypertrophy or potential for inadvertent intramuscular injec- tion, attempting less tight glycaemic control, the use of analogue in- sulins and carbohydrate counting as part of intensive multiple-​dose insulin therapy, and insulin pump therapy (with or without real-​time glucose monitoring). If these measures fail and recurrent hypogly- caemia continues to impact very significantly on the patient’s quality of life, islet transplantation remains an option as it generally achieves marked improvement in hypoglycaemia with a lower risk procedure than whole pancreas transplantation (see earlier). The patients are nonetheless exposed to immunosuppressive risks which need to be taken into account in weighing the risks and benefits. Chronic complications of diabetes Long-​term tissue damage is now the major burden of the disease, the greatest source of fear for diabetic people, and the most expensive item in the diabetes healthcare budget. The list of possible compli- cations is depressingly long but fortunately at least 40% of diabetic patients escape clinically significant complications, and improved diabetes care should reduce the risks even further. Microvascular complications—​retinopathy, nephropathy, and neuropathy—​are specific to diabetes and reflect the damage in- flicted on the microcirculation throughout the body. Retinopathy and nephropathy are obviously microvascular disorders; the micro- circulation of nerves (vasa nervorum) is also damaged in diabetic neuropathy, although other functional and structural abnormalities in the nerves themselves probably contribute. Macrovascular dis- ease is simply atherosclerosis. This causes typical coronary heart dis- ease, stroke, and peripheral arterial disease, but often behaves more aggressively than in people without diabetes. Other complications are due to irreversible biochemical and structural changes in tissues chronically exposed to hyperglycaemia. These include cataracts, whose formation during normal ageing is accelerated by diabetes, and specific soft tissue disorders such as limited joint mobility (diabetic cheiroarthropathy). Causes of chronic diabetic complications Role of hyperglycaemia Tissue lesions are identical in all types of diabetes, indicating that hyperglycaemia (or a closely related metabolic abnormality) is likely to be responsible. Microvascular disease in the retina, kidneys, and nerves is generally determined by the severity and duration of hyperglycaemia, although individual susceptibility varies consid- erably. By contrast, macrovascular disease does not display a clear dose–​response relationship with hyperglycaemia: instead, the risk is increased above glucose values that lie below the diabetic range (see earlier). Recent intervention studies have confirmed that improving gly- caemic control is rewarded by partial protection against micro- vascular complications but not atheroma. This principle is valid for both type 1 and type 2 diabetes, and is now embodied in their treat- ment targets (see Table 13.9.1.3). Two landmark studies are gener- ally cited, although several smaller ones have also reached the same conclusion. Type 1 diabetes The Diabetes Control and Complications Trial (DCCT) was a 12-​ year North American study of over 1400 patients that compared intensive insulin treatment (aiming for an HbA1c of 6% (42 mmol/​ mol)) with conventional (i.e. bad) regimens of once or twice-​daily injections (HbA1c about 9% (75 mmol/​mol)). Intensive treatment consisted of at least three daily injections or an insulin pump (CSII), and achieved a mean HbA1c of 7% (53 mmol/​mol). Table 13.9.1.9  Management of hypoglycaemia Immediate Patient conscious Oral glucose (20–​30 g) or sucrose Patient unconscious Intravenous glucose or Intramuscular or subcutaneous glucagon (1 mg; 0.5 mg in children)a Then Check blood glucose after 15–​20 min Confirm recovery (glucose > 5 mmol/​litre) On recovery Give long-​acting carbohydrate (e.g. sandwich, meal) Identify cause Re-​educate patient to avoid future episodes If recovery is delayed Patient unconscious Set up infusion of 10% dextrose; transfer to hospital Patient conscious Take more oral glucose a Caution with glucagon: it often causes nausea and malaise; depletes liver glycogen—​a second injection may therefore be ineffective; contraindicated in hypoglycaemia caused by sulphonylureas (glucagon stimulates insulin secretion).

13.9.1  Diabetes 2513 The trial concluded that improved glycaemic control reduced the risks of microvascular complications. In subjects who were initially free of complications, intensified treatment for 9 years de- creased the prevalence of a defined degree of background retinop- athy by 70% (i.e. from 55% with conventional treatment to 15% see Fig. 13.9.1.14), while the risks of developing microalbuminuria or clinical neuropathy fell by 33% and 70%, respectively. In subjects who already had background retinopathy at baseline, intensified treatment reduced the overall progression of retinopathy by 50%; more importantly the risks of suffering sight-​threatening retin- opathy or requiring laser treatment were reduced by a similar degree. The development of clinical nephropathy (overt albumin- uria) and neuropathy were each decreased by about 60%. By con- trast, intensified insulin treatment did not reduce the prevalence of macrovascular disease. However, an 11-​year follow-​up of subjects after the end of the trial (the Epidemiology of Diabetes Interventions and Complications study, EDIC) showed a reduction in cardiovas- cular events in the subjects who had previously been in the inten- sive treatment arm, indicating that a prolonged period of good glycaemic control does confer a lasting cardiovascular protective ef- fect. Persistent reductions in microvascular complications were also seen in those originally in the intensive therapy group, despite the fact that HbA1c levels were similar in the extended follow period, indicating that good glycaemic control in the early years can have very long-​lasting benefits. Type 2 diabetes The United Kingdom Prospective Diabetes Study (UKPDS) was guided through its 20-​year course by the late Robert Turner, who died shortly after it was completed. This very large trial followed the outcome of over 5000 patients treated with diet and lifestyle alone (termed ‘conventional’ treatment), or together with sulphonylureas, metformin, or insulin; confusingly, sulphonylureas and insulin treatments were both described as ‘intensive’ treatment. The trial confirmed the real-​life difficulty of achieving good glycaemic con- trol, especially against the progressive deterioration of type 2 dia- betes:  very few patients achieved and maintained the intensive target fasting plasma glucose of 6 mmol/​litre. The trial has been criticized for its convoluted design (which diluted its statistical power) and both the lumping and splitting of data for outcome analysis. Nevertheless, it yielded useful messages about the import- ance of treating both hyperglycaemia and hypertension and about the natural history of the disease itself. Its conclusions were broadly similar to those of the DCCT study: improved glycaemic control de- creased the risk of microvascular complications. Lowering HbA1c from 7.9% (63 mmol/​mol, conventional) to 7.0% (53 mmol/​mol, in- tensive) decreased the lumped rate of microvascular events by 25% (see Fig. 13.9.1.15), including sight-​threatening retinopathy (20%) and the development of microalbuminuria (33%). Across a reason- ably wide variety of HbA1c, lowering HbA1c by 1% reduced the risk of microvascular disease by about one-​third. Improved glycaemic control had no overall effect on macrovascular disease, although metformin treatment significantly decreased cardiovascular events (see earlier). By contrast, blood pressure lowering had very signifi- cant benefits in terms of both micro-​ and macrovascular disease (see Fig. 13.9.1.14). Extended follow-​up results of the UKPDS study have now been reported. These show that although the difference in HbA1c and blood pressure control between conventional and inten- sively treated groups was lost during this period, intensively treated subjects had a lower myocardial infarction and death rate (for treat- ment with metformin, suphonylureas, or insulin) and continued to have lower microvascular complications rates (for subjects treated with sulphonylureas or insulin) 10 years beyond the end of the study. This extended benefit from early tight control has been referred to as a ‘legacy effect’ and similar benefits are seen in type 1 diabetes (see earlier). Note that by contrast no ‘legacy effect’ was seen with blood pressure lowering—​within 1–​2 years of blood pressures equating be- tween the conventional and intensively treated groups any benefit from earlier blood-​pressure lowering was lost. Possible mechanisms of hyperglycaemic tissue damage High glucose levels can damage the function and structure of many tissues. The mechanisms currently thought most relevant to human diabetic complications probably operate to different degrees in dif- ferent tissues. Glycation of proteins and macromolecules Glycation begins with the non​enzymatic combination of glucose and other reactive sugars with amino groups of proteins, and with acceptor groups of other long-​lived macromolecules such as nucleic acids. Glycation is initially reversible, yielding a Schiff base which undergoes molecular rearrangement to form an Amadori product. Amadori products then undergo further reactions, including cova- lent cross-​linking with the sugar groups in other glycated proteins. Onset of retinopathy 0 Conventional p < 0.001 Intensive Progression of retinopathy p < 0.01 Intensive Conventional 40 20 Frequency of retinopathy (percentage of patients) 60 50 30 10 Time (years) 0 1 2 3 4 5 6 7 8 9 40 20 Frequency of retinopathy (percentage of patients) 60 50 30 10 0 Fig. 13.9.1.14  Intensive insulin therapy and improved diabetic control in type 1 diabetes reduces the risks of developing retinopathy (upper panel) and of established retinopathy progressing (lower panel). Data from the Diabetic Control and Complications Trial (DCCT).

13.9.2 Hypoglycaemia 2531

13.9.2 Hypoglycaemia 2531

13.9.2  Hypoglycaemia 2531 long-​term survival after acute myocardial infarction in patients with diabetes mellitus. BMJ, 314, 1512–​15. Malmberg K, et al. (2005). Intense metabolic control by means of in- sulin in patients with diabetes mellitus and acute myocardial infarc- tion (DIGAMI 2): effects on mortality and morbidity. Eur Heart J, 26, 650–​61. Nathan DM, et al. (2005). Intensive diabetes treatment and cardiovas- cular disease in patients with type 1 diabetes. N Engl J Med, 353, 2643–​53. Davies MJ, et al. (2018). Management of hyperglycaemia in type 2 diabetes, 2018. A consensus report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetologia, 61, 2461–98. Nathan DM, et al. (2009). Medical management of hyperglycemia in type 2 diabetes: a consensus algorithm for the initiation and adjust- ment of therapy: a consensus statement of the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care 32, 193–​203. Nathan DM, et al. (2009). Modern-​day clinical course of type 1 dia- betes mellitus after 30  years’ duration:  the Diabetes Control and Complications Trial/​epidemiology of diabetes interventions and complications and Pittsburgh epidemiology of diabetes complica- tions experience (1983–​2005). Arch Intern Med, 169, 1307–​16. Pickup JC, Williams G (eds) (2002). Textbook of diabetes, 3rd edition. Blackwell Science, Oxford. Ryden L, et al. (2013). ESC Guidelines on diabetes, pre-​diabetes, and cardiovascular diseases developed in collaboration with the EASD. Eur Heart J, 34, 3035–87. Schade DS, Duckworth WC (1986). In search of the subcutaneous in- sulin resistance syndrome. N Engl J Med, 315, 147–​53. Shapiro A, et al. (2000). Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-​free immunosup- pressive regimen. N Engl J Med, 343, 230–​8. Tuomilehto J, et al. (2001). Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. N Engl J Med, 344, 1343–​50. Turnbull FM, et al. (2009). Intensive glucose control and macrovascular outcomes in type 2 diabetes. Diabetologia, 52, 2288–​98. UK Prospective Diabetes Study Group (1998). Intensive blood glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet, 352, 837–​53. UK Prospective Diabetes Study Group (1998). Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes. BMJ, 317, 703–​13. Unwin N, et  al. (2002). Impaired glucose tolerance and impaired fasting glycaemia: the current status on definition and intervention. Diabet Med, 19, 708–​23. Useful websites American Diabetes Association Home Page. http://​www.diabetes.org Diabetes Research Department and Centre for Molecular Genetics, Peninsula Medical School and Royal Devon and Exeter Hospital. Genetic Types of Diabetes Including Maturity-​Onset Diabetes of the Young (MODY). http://​www.diabetesgenes.org Diabetes UK Home Page. http://​www.diabetes.org.uk English National Screening Committee for Diabetic Retinopathy. http://​www.retinalscreening.nhs.uk/​pages/​ International Diabetes Federation Home Page. http://​www.idf.org Mendosa, D. Online Diabetes Resources. http://​www.mendosa.com/​ faq.htm NHS Diabetes. https://www.diabetes.org.uk​ World Health Organization. Diabetes Programme. https://www.who. int/diabetes/en/​ 13.9.2  Hypoglycaemia Mark Evans and Ben Challis ESSENTIALS Hypoglycaemia is a low blood glucose concentration, clinically im- portant because glucose is the main fuel supporting brain metab- olism and function. The commonest causes are as a consequence of insulin or sulphonylurea drugs used to treat diabetes, but there are many rarer causes including insulinoma, toxins (alcohol), organ failure (hepatic), endocrine diseases (adrenal insufficiency, pituitary insufficiency), non​islet cell tumour hypoglycaemia (large mesen- chymal tumours and other malignancies), post bariatric surgery (non​insulinoma pancreatogenous hypoglycaemia syndrome), auto- immune insulin syndrome, factitious or felonious administration of insulin/​sulphonylureas, and infections (malaria). Clinical features—​typical features include ‘autonomic’ symptoms (e.g. tachycardia, tremor, and sweating), hunger and neuroglycopenic symptoms related to brain glucose deprivation (e.g. impaired cogni- tive function, blurred vision, drowsiness, and irritation). If blood glu- cose falls sufficiently low, patients may become sleepy, comatose, and/​or suffer seizures, cardiac arrhythmias, irreversible cognitive damage, and even death. Recurrent hypoglycaemia can lead to a situation where symptomatic awareness of hypoglycaemia may be lost so that patients are unaware of their falling glucose until already cognitively impaired. Diagnosis—​diagnosis in diabetes is usually clinical, with or without confirmation by a point-​of-​care blood glucose meter. With sus- pected non​diabetic hypoglycaemia, symptoms compatible with hypoglycaemia should occur at a time when a low plasma glucose concentration is documented, and these should be ameliorated fol- lowing correction of hypoglycaemia. Measurement of blood total in- sulin immunoreactivity, C-​peptide, and proinsulin, and in some cases β-​hydroxybutyrate, alcohol, sulphonylureas, and other hormone as- says may be required for diagnosis. Management—​where conscious level is not impaired, the best treatment is oral intake of fast acting carbohydrates, but if the subject is drowsy or unconscious, injection rescue will be needed with intra- venous glucose or, if venous access is not available, intramuscular glucagon. Introduction—​the clinical approach to hypoglycaemia The most common clinical scenario where physicians may generally encounter hypoglycaemia is as a consequence of the treatment of

section 13  Endocrine disorders 2532 diabetes with insulin and/​or other blood glucose-​lowering therapies. Hypoglycaemia is uncommon in otherwise healthy non​diabetic in- dividuals due to existence of a robust counterregulatory hormonal response to falling plasma glucose concentrations. As described next though, there is a gamut of non​diabetes conditions which can result in hypoglycaemia that general physicians need to be aware of. The clinical approaches in these two situations are very different. With diabetes, there is usually less uncertainty about ‘diagnosis’ (i.e. whether this is hypoglycaemia or not, but the clinical strategy is directed towards minimizing consequences and/​or risk of recur- rence). In non​diabetes, the focus is different with the initial goal being to determine whether the patient is truly suffering from hypoglycaemia or not and, if so, then to determine the cause and management needed. In this chapter we first describe hypoglycaemia in diabetes then describe how this is approached systematically in a non​diabetic pa- tient with suspected hypoglycaemia. Hypoglycaemia in diabetes Defining hypoglycaemia in diabetes Despite its undoubted importance, there are no universal criteria for defining hypoglycaemia in diabetes. In general though, hypogly- caemia can be defined either ‘biochemically’ (i.e. using a glycaemic threshold) or described by the functional consequences of a low blood glucose. For the former, although there is no single agreed blood glucose threshold used to define hypoglycaemia, it is useful in clinical prac- tice to give advice about glucose levels to guide action. For example, those with insulin-​treated diabetes may be told to avoid results of below 4 mM (72 mg/​dl) with the intention of reducing the chances of dropping to levels at which cognitive function starts to become impaired (typically below 3 mM or 54 mg/​dl). An important point to be aware of is that home devices using capillary (‘finger-​prick’) samples may be inaccurate, particularly in the hypoglycaemic range even when used optimally. In diabetes research, clinical trials in dia- betes will often use biochemical definitions of hypoglycaemia; for example, many studies use a definition of below 3.9 mM (70 mg/​dl) although this particular level may be less useful in clinical practice (see description of pathophysiology below). A more pragmatic approach is to use a functional classification of the effects of hypoglycaemia in diabetes, for example, as severe (requiring external assistance for rescue—​usually family, friends, or work colleagues but may require paramedic attention) or not. There are limitations; for example, this cannot be used for those who are not independent (e.g. children or patients requiring long-​ term care) and it may also be difficult to determine whether help was needed or rather was offered and accepted (e.g. by a concerned spouse). This becomes particularly challenging where the definition has medico-​legal consequences (e.g. the ability to hold a driving li- cence may depend on whether an episode of hypoglycaemia was judged severe or not). Severe hypoglycaemia even in those at risk is relatively uncommon so an additional definition providing information about the burden of hypoglycaemia might be to ask about moderate hypoglycaemia, defined as events requiring patients to interrupt their activity and treat immediately to avoid severe hypoglycaemia. Aetiology of hypoglycaemia in diabetes Hypoglycaemia is the commonest adverse effect of insulin therapy in diabetes, but it also occurs with other therapies such as treatment with sulphonylurea drugs (which act by increasing insulin secretion from the endogenous pancreas) and/​or where insulin is used in combination with other blood glucose-​lowering therapies. As a consequence, use of sulphonylurea drugs in type 2 diabetes therapy is starting to fall in many countries. Insulin-​treated patients fear hypoglycaemia as much as other complications of diabetes such as amputation or blindness. This means that some will choose to accept worse (higher) overall con- trol of glycaemia to try to minimize hypoglycaemia risk, thus exposing themselves to increased risk of long-​term complications of diabetes. Epidemiology of hypoglycaemia in diabetes With increasing rates of type 2 diabetes treated with insulin, the most common presentations of hypoglycaemia to emergency services in developed countries are older patients (over 65 years) treated with insulin. The demography means that there are often coexisting comorbidities other than diabetes. Several observational studies have shown that severe hypoglycaemia in this age group is associated with increased risk of mortality within the following 12 months, although it is unclear whether this is directly related to hypoglycaemia or to the other comorbidities. Hypoglycaemia is also common in hospitalized patients with dia- betes, occurring in 5 to 10%. This may be attributed to multiple fac- tors such as coexisting illness, altered meals and schedule but also sometimes removing the locus of control from a patient who effect- ively self-​manages diabetes outside hospital. Increasingly, such pa- tients may be allowed where appropriate (conscious and alert) to continue managing their own diabetes while an inpatient. Independent of age, there is an increased risk of hypoglycaemia seen with increasing duration of diabetes, whether type 1 or type 2 diabetes. The cause is unclear although one possibility for the latter at least is that this reflects the progressive loss of endogenous (glucose-​responsive) insulin secretion that typifies type 2 diabetes. Hypoglycaemia is also a particular concern for very young children under the age of 5. This carries multiple additional challenges, for example with dosing small amounts of insulin accurately and unpredictable food intake. Pathophysiology of hypoglycaemia in diabetes Ultimately, hypoglycaemia in diabetes is caused by excess insulin ac- tion in real or absolute terms, whether this is exogenous insulin by injection or endogenous insulin stimulated by secretagogue drugs such as sulphonylureas. Excess insulin action may occur either be- cause of a systematic overtreatment of diabetes with therapy and/​or because of a change in circumstances occurring at the time of the event. A useful clinical strategy is to decide whether an event is a ‘one-​off’ with or without an identifiable precipitant or whether part of a recurring pattern needing a systematic change in approach. Common identifiable causes for episodes of hypoglycaemia are • Insulin errors (e.g. accidentally giving a duplicate insulin injec- tion, injecting the wrong insulin, or miscalculating a dose in those who adjust insulin doses). • Less food, particularly carbohydrate intake than anticipated from missed or inadequate meals or snacks. • Exercise or activity which can cause hypoglycaemia either at the time and/​or exert a delayed effect in the evening or night following activity.

13.9.2  Hypoglycaemia 2533 • Alcohol—​typically exerting a delayed effect to reduce hepatic glu- cose production during the night or even the following morning after consumption. This can occur sometimes even with relatively modest intake. • Hot weather increasing insulin absorption. • Breastfeeding can markedly increase the metabolic demands on mother and many with insulin-​treated diabetes have to reduce insulin doses significantly at this time to avoid hypoglycaemia. In addition to these factors which may help explain individual epi- sodes of hypoglycaemia, there may be other risk factors that gener- ally increase the risk of hypoglycaemia: • Insulin injection site problems—​overuse of the same insulin injec- tion site can result in a build-​up of local fat deposits, lipohypertrophy or, more rarely, loss of fat (lipoatrophy). This can alter the predict- ability of insulin absorption so that blood glucose levels can swing widely without an obvious cause. This is an important and often overlooked cause of glycaemic instability and hypoglycaemia and patients should ‘rotate’ injection sites so as not to overuse an area and/​or avoid injecting into lumpy areas. • Use of inappropriately long needles for insulin injection resulting in deeper insulin delivery into more vascular muscle thus increasing absorption speed. Needle lengths of 4 to 6 mm are appropriate for most patients. • Impaired counterregulation and symptomatic unawareness (see next). • ‘Tight’ glycaemic control with overly-​aggressive attempts to lower average blood glucose levels. An inverse relationship between overall control measured by HbA1c and rates of severe hypogly- caemia was identified in the large and pivotal DCCT (Diabetes Control and Complications Study). In the real world of type 1 diabetes, more recent data suggest that this is not seen with no association between HbA1c and hypoglycaemic risk, perhaps illustrating the differences between clinical studies and everyday clinical practice where patients may be reluctant to intensify gly- caemic control if they start to suffer from hypoglycaemia. • Other medical conditions can predispose such as renal/​liver im- pairment and (rarely) coexisting adrenal/​pituitary insufficiency. Counterregulatory defences against hypoglycaemia In non​diabetes, a robust series of counterregulatory defences nor- mally prevent hypoglycaemia from occurring (Fig. 13.9.2.1). An early and potent defence is cessation of insulin secretion from the non​diabetic pancreas (typically at a plasma glucose of around 4 mM) to reduce circulating insulin, something that cannot occur in those treated with exogenous insulin or insulin secretagogues such as sulphonylureas. If plasma glucose concentrations continue to fall (for example under experimental conditions in non​diabetes Normal Counter-regulatory Defences against Hypoglycaemia Counter-regulatory Defences against Hypoglycaemia in Type 1 Diabetes Impaired Counter-regulatory Defences against Hypoglycaemia in Subset of Type 1 Diabetes Switch-off endogenous insulin secretion 4 3 2 1 Release of glucagon Release of other counter-regulatory hormones Warning symptoms Cognitive Impairment Lethargy, risk of coma/seizures Glucose (mM) (a) Release of other counter-regulatory hormones Warning symptoms 4 3 2 1 Cognitive Impairment Lethargy, risk of coma/seizures Glucose (mM) (b) (c) Release of counter-regulatory hormones/warning symptoms 4 3 2 1 Cognitive Impairment Lethargy, risk of coma/seizures Glucose (mM) Fig. 13.9.2.1  Counterregulatory responses to hypoglycaemia. (a) Normal responses with protective defences shown in blue and consequences of brain nueroglycopenic in red. (b) Altered responses in type 1 diabetes. (c) Impaired counterregulatory responses in a subset of patients with type 1 diabetes.

section 13  Endocrine disorders 2534 with insulin infusion), at a plasma glucose level of between 3 and 4 mM, a well-​orchestrated release of counterregulatory hor- mones occurs. Glucagon is released from α-cells of the pancreatic islets, acting predominantly to oppose insulin action at the liver. Neurohumoral sympathoadrenal responses with increased sympa- thetic nerve activation and release of adrenaline from the adrenal medulla also exert anti-​insulin actions in periphery and at the liver. Cortisol and growth hormone also rise and help counteract the effects of insulin. Associated with this neurohumoral response is the generation of symptoms alerting the patient of a falling glucose. These ‘autonomic’ symptoms include sympatho-​adrenal symptoms such as tachy- cardia, palpitations, pallor, anxiety, and tremulousness through ac- tivation of β-​adrenergic receptors, whereas cholinergic activation results in sweating and paraesthesia. Hunger is also generated, an important symptom in its own right as it prompts corrective feeding to restore glucose. A major rationale for the existence of such robust counter­ regulatory defences protecting against hypoglycaemia is because of the reliance of brain on blood glucose to fuel metabolism and support function. Brain has minimal local stores of glycogen and is extremely metabolically active. In adults, brain represents about 2% of body weight yet consumes at least 20% of glucose. As plasma glucose concentrations fall, ‘neuroglycopenic’ symptoms are ac- tivated because of brain glucose deprivation—​such as blurred vision, drowsiness, irritation, slurred speech, and behavioural changes. Cognitive function becomes impaired with slowing of re- action times, increased tendency to make errors and loss of judge- ment. This is important as this may mean that patients lose the ability to make appropriate and judicious decisions about treating their hypoglycaemia. Eventually if blood glucose falls sufficiently low for long enough, patients may become sleepy, comatose and/​ or suffer seizures. With prolonged deep hypoglycaemia, cardiac arrhythmias, myocardial infarction, stroke, irreversible cognitive damage, and even death may ensue. Impaired counterregulatory defences against hypoglycaemia in diabetes In diabetes, the protective counterregulatory defences described earlier may be altered (Fig. 13.9.2.1). The ability to switch off en- dogenous insulin secretion is lost in those treated with exogenous insulin or secretagogue drugs such as sulphonylureas. Glucagon re- sponses to hypoglycaemia are lost during the first few years of type 1 diabetes and probably also become blunted in those with insulin-​ treated type 2 diabetes. A subset of people with diabetes develop abnormalities in other counterregulatory defences, particularly catecholamine responses, associated with a loss of symptomatic awareness of hypoglycaemia so that defensive responses become diminished and delayed, occurring after the onset of cognitive dysfunction. This pattern is associated with a markedly increased risk of suffering from severe hypoglycaemia (as described earlier requiring assistance to correct). The mechanisms underpinning this reduction in counterregulatory responses and loss of symptomatic awareness remain unclear, but antecedent hypoglycaemia itself is a major contributor, resulting in blunted responses to subsequent hypoglycaemia (effectively a type of ‘stress desensitization’ where exposure to a repeated stress—​ here hypoglycaemia—​results in reduced responses). The label ‘hypoglycaemia-​associated autonomic failure’ (HAAF) is sometimes attached to the loss of counterregulatory neurohumoral responses that follows antecedent hypoglycaemia. Duration of diabetes is also a risk factor for HAAF/​hypoglycaemia unawareness although it is not clear whether this is because of hypoglycaemia exposure or an independent effect of diabetes. Clinical features of hypoglycaemia in diabetes Typical symptoms are as already described, with sympathoadrenal and neuroglycopenic symptoms and also hunger. In those with impaired counterregulation/​unawareness, hypoglycaemia may be more apparent to others—​family or work colleagues—​and may con- sist of the consequences of neuroglycopenia such as irritability and abnormal behaviour or increase in errors at work. Overnight hypoglycaemia is a particular concern for many pa- tients with insulin-​treated diabetes (and their carers/​parents/​rela- tives). During sleep, patients may be slower to recognize symptoms but also counterregulatory defences may be blunted—​perhaps related to supine posture. In addition to reduced defences, many people have circadian changes in insulin sensitivity during the hours of darkness. For example, a common pattern is for a relative insulin resistance at the end of the sleep period, sometimes called the ‘dawn phenomenon’. This means that patients may wake with high blood glucose values and lead to the temptation to increase background insulin doses which can increase risk at other times of night. A formerly common description of high morning glu- cose values being caused by rebound after silent overnight hypo- glycaemia (eponymously named as the Somogyi effect) has been largely discredited with modern methods for continuously moni- toring overnight glycaemia. Acute management of hypoglycaemia in diabetes Acute management is aimed at recognizing and taking early cor- rective action to restore blood glucose. Most episodes are self-​ managed with oral rapid-​acting carbohydrates with 15–​20 g glucose (e.g. 150–​200 ml orange juice, 150 ml cola, four dextrose tablets), repeated if need be. Good practice is to follow up this ini- tial rapid-​acting therapy with a more starchy or mixed snack or meal to sustain the restoration in glucose. Highly concentrated glucose gels are commercially available, administered orally by smearing into the inside of the cheek cavity. Although conven- tional teaching was that these were absorbed through the buccal mucosa, current thinking is that the glucose is swallowed and ab- sorbed from the stomach. These should not be used therefore in unconscious patients. Where the level of consciousness is reduced, rescue needs to be by injection of either glucose intravenously or 1 mg glucagon given intravenously or intramuscular/​subcutaneous delivery (with future options perhaps including nasal glucagon delivery). Although 50% glucose has often been used, many current protocols suggest that the maximum concentration used is 20% because of the risks of tissue necrosis if there is extravasation. Glucagon carries the potential ad- vantage of not requiring an intravenous cannula for administration. It is available as a kit for emergency use (e.g. to be given at home by partners/​parents). Glucagon acts predominantly by mobilizing hep- atic glycogen stores so is likely to be ineffective where these are low (for example after overnight fasting or in the few hours following a previous glucagon injection).

13.9.2  Hypoglycaemia 2535 Longer term management of hypoglycaemia
in diabetes In addition to acute restoration of glucose, patients and/​or their clinical teams should also reflect on the causes for an episode and whether any changes are needed to treatment and/​or self-​ management. Causes to consider were described earlier and listed in Table 13.9.2.1. In type 2 diabetes, sulphonylureas were widely used for many years as an effective therapy for diabetes but use has declined now with the availability of newer agents carrying less risk of hypogly- caemia. For example, therapies acting through the glucagon-​like peptide-​1 (GLP-​1) axis consist of either synthetic GLP-​1 agonists or agents which inhibit the breakdown of endogenous GLP-​1 by the en- zyme dipeptidyl peptidase IV (DPPIV inhibitors). These mimic the internal GLP-​1 signal that primes pancreatic β cells to release insulin but importantly do so in a glucose-​responsive fashion, thus redu- cing the potential for inappropriate insulin release during hypogly- caemia which can occur with non​glucose-​responsive therapy (e.g. sulphonylureas). In those using insulin therapy, there may also be adjustments needed to reduce risk of hypoglycaemia. Increasingly insulin re- gimens (in both type 1 and type 2 diabetes) are designed to try to tailor insulin doses to daily variations in diet/​activity etc rather than using fixed dosing. Modern insulin analogues are designed to have more rapid onset/​offset of action (to be used around mealtimes or for blood glucose corrections) or longer acting analogues for use as background cover. Probably most important is the training and support given to patients to allow them to gauge accurately insulin dosing. Developed from a successful German model, the DAFNE (Dose Adjustment for Normal Eating) programme in the United Kingdom, Ireland, and Australia successfully educates patients in op- timizing insulin dosing and can reduce problematic hypoglycaemia. For those struggling with hypoglycaemia despite optimizing therapy with injections, changing insulin delivery to continuous subcutaneous insulin infusion (CSII or ‘insulin pump’ therapy) may allow blood glucose to be controlled without hypoglycaemia. Other technologies available, albeit with limited access to date, are devices allowing continuous or semi-​continuous glucose monitoring (CGM) from subcutaneously inserted sensors measuring interstitial fluid glucose as a surrogate for blood glucose. CGM can be linked to alarms to warn patients of hypoglycaemia—​actual or anticipated if glucose is dropping. CSII can be linked to CGM so that insulin de- livery is automated, including being reduced or suspended if glucose levels are falling or low with the aim of reducing hypoglycaemia risk. Finally, small numbers of patients with recalcitrant and serious/​ potentially life-​threatening hypoglycaemia may be considered for transplantation of either a whole pancreas or isolated islets. Numbers are limited currently by the availability of donors and the require- ment for potentially toxic long-​term immunosuppressive therapy. Consequences of hypoglycaemia in diabetes Acute episodes of profound hypoglycaemia may result in seizures, sudden death, or structural brain damage, although these are un- common. More commonly, acute hypoglycaemia can increase risk of accidents; falls and road accidents for example. Patients with insulin-​treated diabetes fear hypoglycaemia and some may respond by deliberately running blood glucose levels high, thus exposing themselves to risks of long-​term complications of diabetes. Finding the correct balance between risk of hypoglycaemia and long-​term exposure to hyperglycaemia is challenging. Recent large studies examining the benefits of very tight blood glucose control in patients with type 2 diabetes who are at high risk of cardiovascular disease have shown an apparent increase in morbidity with lower glycaemia. The mechanisms are unclear but one possibility is hypoglycaemia. Over the long term, the possible cumulative effects of recurrent hypoglycaemia, particularly on brain and cognitive functioning, including memory, are unclear. Neuropsychological data suggest that in young children, the developing brain may be particularly sensitive to hypoglycaemia exposure. Future approaches to minimizing hypoglycaemia in diabetes Glycaemic management in diabetes is a trade-​off between the de- sire to lower average glycaemia to reduce long-​term complications and the acute hazard of hypoglycaemia. New therapies for type 1 and type 2 diabetes may allow the former without the latter. For ex- ample, new insulins with more rapid onset/​offset, relative hepato-​ selectivity, or even ‘smart’ glucose-​responsive insulins. Technology described briefly here earlier is advancing rapidly with automated insulin delivery from linked CGM and CSII opening up the real possibility of ‘artificial pancreas’ technology. This might also allow glucagon as well as insulin to be administered by a pump with delivery targeted to avoid or treat hypoglycaemia. Hypoglycaemia in non​diabetes As outlined previously, the approach to hypoglycaemia or suspected hypoglycaemia in non​diabetes is initially that of confirmation and diagnosis. A diagnosis of hypoglycaemia based on clinical symptoms alone is challenging and often erroneous due to lack of specificity. Moreover, it is not possible to biochemically define a single glycaemic threshold below which symptoms of hypoglycaemia develop in all individuals. Table 13.9.2.1  Causes of hypoglycaemia in diabetes Acute causes for individual episodes of hypoglycaemia Increased insulin action Insulin dose too large (relative to carbohydrates/​blood glucose) Increased absorption (hot weather, sauna) Injection site lipodystrophy Injections too deep (wrong needles) Insulin error (e.g. duplicate injection or incorrect insulin) Decreased carbohydrates Smaller or low-​carbohydrate meal (relative to insulin dose) Missed snack Increased glucose demand Exercise/​activity Breastfeeding Alcohol Factors associated with increased background risk of hypoglycaemia Previous problematic hypoglycaemia Long duration of diabetes Hypoglycaemia unawareness Adrenal/​pituitary insufficiency

section 13  Endocrine disorders 2536 For example, in healthy individuals hypoglycaemic symptoms may develop at plasma glucose concentrations approximating 3.0 mmol/L whereas in patients with antecedent hypoglycaemia, symptoms may occur at lower glycaemic thresholds due to an attenuated or absent physiologic response to hypoglycaemia. Therefore, a diagnosis of hypoglycaemia depends on satisfying Whipple’s triad. This man- dates that symptoms compatible with hypoglycaemia occur at the time a low plasma glucose concentration is documented and are ameliorated following correction of hypoglycaemia. Only after these criteria are fulfilled should further investigations to determine the aetiology of a hypoglycaemic disorder be embarked upon. Initial work-​up of a patient suspected of having a hypoglycaemic disorder involves detailed history, clinical examination, and scru- tiny of biochemical data. A history of hypoglycaemia should be interrogated for timing, duration, and nature of specific symptoms with special attention given to the temporal association of precipi- tants such as fasting, exercise, and medications, or relieving fac- tors including carbohydrate ingestion. Establishing the presence of comorbid conditions is critical as many diseases may manifest as hypoglycaemia including disorders of the liver and kidney, sepsis, non​islet cell tumours, and endocrine deficiency (cortisol and growth hormone). Eliciting a medication history is vital as many drugs, in addition to insulin and insulin secretagogues, cause drug-​induced hypoglycaemia. Moreover, ascertaining accessibility to diabetic medications may identify surreptitious, accidental, or rarely, malicious causes of hypoglycaemia. Finally, establishing a family history of hypoglycaemia is relevant for identifying con- genital hyperinsulinism syndromes, inborn errors of metab- olism, or inherited tumour syndromes that may predispose to hypoglycaemia. Aetiology of non​diabetic hypoglycaemia The principal causes of non​diabetic hypoglycaemia are presented in Table 13.9.2.2. Hypoglycaemic disorders may be classified into those that produce hypoglycaemia due to inappropriately elevated plasma insulin levels or those that result in symptomatic hypogly- caemia in association with appropriately suppressed plasma insulin levels (Table 13.9.2.3). Although many hypoglycaemic disorders in neonates and infants are recognized, the remainder of this chapter focuses primarily on hypoglycaemic disorders encountered in adults with reference to paediatric syndromes that may present for the first time in adulthood. Drug-​induced hypoglycaemia Several pharmacological agents and toxins predispose to hypogly- caemia. Unsurprisingly, the most common cause of drug-​induced hypoglycaemia is insulin followed by insulin secretagogues such as sulfonylureas and meglitinides. Few drugs cause hypoglycaemia in the absence of concomitant antidiabetic therapy usage and of these, ethanol, β-​adrenergic antagonists, sulphonamides, somatostatin analogues, and salicylates have been reported. Ethanol is a common cause of non​iatrogenic hypoglycaemia and frequent causative agent in hypoglycaemia-​related deaths. Ethanol induces hypoglycaemia through inhibition of gluconeogenesis due to increased hepatic alcohol dehydrogenase activity and subsequent depletion of NADH. As a consequence of a reduced NADH/​NAD+ ratio conversion of lactate to pyruvate, the main gluconeogenic substrate, is minimized resulting in impaired hepatic glucose output. In a fasted or malnourished patient con- suming alcohol, fasting hypoglycaemia may occur following de- pletion of hepatic glycogen stores. Biochemically, hypoglycaemia is associated with increased plasma β-​hydroxybutyrate levels and suppressed insulin, C-​peptide, and proinsulin together with de- tectable levels of blood ethanol. Because glycogen stores have been depleted and gluconeogenesis inhibited, glucagon administration is not an effective therapy and oral or intravenous glucose are the treatments of choice. Non​cardioselective β-​blockers, such as propranolol, increase the risk of fasting hypoglycaemia due to their ability to impair hepatic and renal gluconeogenesis, reduce glucagon secretion, and mask autonomic symptoms of hypoglycaemia. Cholinergic symptoms such as hunger and sweating are not affected, however, and in an unaware patient may serve as clinical indicators of hypoglycaemia. Through undefined mechanisms, sulphonamides stimulate insulin secretion while salicylates both inhibit hepatic glucose production and promote insulin secretion thereby inducing hypoglycaemia. Insulinoma Insulinomas are insulin-​secreting tumours of the pancreatic islets of Langerhans. Although rare, insulinoma is the most common cause of spontaneous fasting hypoglycaemia in an otherwise healthy adult and occurs with an annual incidence of 1–​2 per million population. Insulinomas may appear at any age, although they most typically present in the fourth to sixth decades with a slight female prepon- derance reported in some case series. Table 13.9.2.2  Causes of non​diabetic hypoglycaemia Hypoglycaemia with suppressed endogenous insulin Drugs Insulin Ethanol β-​adrenoceptor antagonists, salicyclates, others Critical illness Sepsis Chronic kidney disease Cirrhosis Congestive cardiac failure Malaria Endocrine deficiencies Cortisol deficiency Growth hormone deficiency Non​islet cell tumour-​associated hypoglycaemia (NICTH) Surreptitious, accidental, or malicious hypoglycaemia Inborn errors of metabolism Hypoglycaemia with inappropriately elevated endogenous insulin Drugs Insulin secretagogues Neuroendocrine tumours Insulinoma GLP-​1oma Postgastric bypass surgery Non​insulinoma pancreatogenous hypoglycaemia syndrome Postprandial (reactive) hypoglycaemia Autoimmune Hirata disease (anti-​insulin antibody) Type B insulin resistance (anti-​insulin receptor antibody) Congenital hyperinsulinism syndromes

13.9.2  Hypoglycaemia 2537 Signs and symptoms Insulinoma may present, initially, with fatigue, weight gain, altered mental state, and/​or autonomic symptoms that rapidly improve fol- lowing carbohydrate ingestion. Because of the non​specific and subtle nature of these symptoms the interval between symptom onset and diagnosis is often 3–​5 years with a delayed diagnosis of more than 20 years reported in some instances. With frequent hypoglycaemia patients with insulinoma may develop hypoglycaemia unawareness. Thus, for many patients, neuroglycopenic symptoms such as loss of consciousness, disorientation, behavioural changes and/​or seizure may dominate the clinical presentation and occur at times of ex- ercise or fasting with a minority of patients reporting postprandial symptoms alone. Pathology Most insulinoma are sporadic, small (<1  cm), solitary and be- nign tumours with less than 10% exhibiting malignant potential. Multiple insulinomas occur in less than 10% of cases, and most often in association with multiple endocrine neoplasia (MEN)-​ 1, an inherited tumour syndrome characterized by pancreatic neuroendocrine tumours in conjunction with primary hyper- parathyroidism and pituitary tumours. Tumours occurring in the setting of MEN1 are associated with loss of function of menin, the protein product of the tumour suppressor gene MEN1. Loss of heterozygosity at the MEN1 locus has been demonstrated in MEN1-​associated insulinomas; however, the mechanism(s) by which menin loss promotes tumourigenesis remain unclear. Although single-​copy deletion and somatic mutations in MEN1 have been identified in small subset of sporadic (non​familial) insulinoma, understanding the genetic events that underlie these tumours has, until recently, been less forthcoming. Through the use of whole exome sequencing several investigators have recently identified a recurrent somatic mutation in the transcription factor Ying Yang 1 (YY1) in one-​third of sporadic insulinomas. The re- current YY1 mutation (YY1T372R) alters the DNA binding speci- ficity of the transcription factor resulting in neomorphic activity and marked changes in tumoural gene expression which collect- ively contribute to disease pathogenesis by promoting constitutive insulin secretion from tumour cells. The main pathophysiological feature of insulinoma is the in- ability of tumour cells to adequately suppress insulin secretion as plasma glucose concentrations fall to hypoglycaemic levels. Hypoglycaemia, therefore, results from reduced rates of glucose production due to relative insulin excess for a given plasma glucose concentration. That 95% of patients with insulinoma have elevated fasting proinsulin plasma concentrations and tumour cells contain an elevated proinsulin-​to-​insulin ratio as well as reduced insulin content compared with normal β-​cells suggests that dysregulated insulin biosynthesis and secretion also contribute to the dysfunc- tion of tumourous islets. Diagnosis A 72-​hour fast is the investigation of choice to establish diag- nosis of insulinoma. During the fast, biochemical diagnosis is es- tablished when hypoglycaemia (<3 mmol/​litre), in the presence of Whipple’s triad, is associated with inadequately suppressed concentrations of plasma insulin (≥18 pmol/​litre), C-​peptide (≥0.2 nmol/​l), proinsulin (≥5 pmol/​litre), reduced plasma con- centrations of β-​hydroxybutyrate (≤2.7  mmol/​litre) and nega- tive plasma or urine sulfonylurea screen. An increase in plasma glucose concentration of at least 1.4 mmol/​litre following intra- venous glucagon indicates maintenance of glycogen stores and provides further evidence for excess insulin-​like activity. Using these criteria two-​thirds of patients with insulinoma will be diag- nosed within 24 hours of fasting, 95% of patients within 48 hours, and 99% by 72 hours. Conventional non​invasive imaging modalities such as com- puted tomography (CT), magnetic resonance imaging (MRI) and endoscopic ultrasound will detect up to 80%, 85%, and 90% of insulinomas, respectively (Fig. 13.9.2.2). However, a small number of insulinomas remain elusive following conventional imaging. To facilitate localization of these smaller tumours, se- lective arterial calcium stimulation with hepatic venous sam- pling for insulin quantification regionalizes insulinoma with high sensitivity (>90%); however, this method is invasive, technically challenging, and poses a risk for complications. Due to reduced expression of somatostatin receptor 2 (SSTR2) in insulinoma somatostatin receptor scintigraphy detects only 20–​50% of be- nign tumours. In contrast, GLP1 receptors are highly expressed in insulinomas leading to development of GLP1-​like radioligands that, when used in combination with whole planar body imaging and single photon emission CT (SPECT), may provide an alter- native, non​invasive method for safely and successfully localizing occult insulinomas. Table 13.9.2.3  Biochemical interpretation of a 72-​hour fast Diagnosis Glucose (mmol/​litre) Insulin (pmol/​litre) C-​peptide (nmol/​litre) Proinsulin (pmol/​litre) β-​hydroxybutyrate (mmol/​litre) IGF2:IGF1 ratio Oral agent screen Anti-​insulin receptor antibody Normal <3 <18 <0.2 <5

2.7 <10 –​ –​ Exogenous insulin <3 18 <0.2 <5 <2.7 <10 –​ –​ Sulfonylurea <3 18 0.2 5 <2.7 <10

–​ Insulinoma <3

18 0.2 5 <2.7 <10 –​ –​ Non​islet cell tumour <3 <18 <0.2 <5 <2.7 10 –​ –​ Autoimmune disorder <3 18 0.2 5 <2.7 <10 –​

section 13  Endocrine disorders 2538 Treatment Surgical resection remains the treatment of choice for insulinomas and is curative for benign and solitary lesions. Prevention of recur- rent hypoglycaemia is the goal of medical therapy and following dietary modification, diazoxide, a potent inhibitor of insulin se- cretion, is first-​line treatment for patients with inoperable, meta- static disease, or those who are poor surgical candidates. Adverse effects of diazoxide include hirsutism, oedema, or gastric irritation, which may prove intolerable and necessitate transition to an alter- native therapy. Owing to their inhibitory effect on insulin secretion, calcium channel blockers, such as nifedipine or verapamil, may be tried. Insulinomas are largely insensitive to traditional somatostatin analogues, such as octreotide, due to reduced expression of SSTR2 on insulinoma cells. In patients with metastatic disease, emboliza- tion, or surgical debulking of hepatic metastases to reduce disease volume may produce remission. Malignant insulinomas generally respond poorly to traditional cytotoxic chemotherapeutic agents al- though rapamycin and everolimus, oral inhibitors of the mamma- lian target of rapamycin (mTOR), have proved beneficial in some patients with metastatic insulinoma and refractory hypoglycaemia. Hypoglycaemia and gastric bypass surgery Roux-​en-​Y gastric bypass (RYGB) surgery is the most effective and commonly employed method for long-​term weight loss. An increas- ingly recognized complication of RYGB surgery is non​insulinoma pancreatogenous hypoglycaemia syndrome (NIPHS), a rare dis- order of hyperinsulinaemic hypoglycaemia that typically presents with severe neuroglycopaenia 1 to 3 hours following a meal. NIPHS occurs with an estimated postoperative prevalence of less than 1% with a median time of 2.7 years reported from surgery to first occur- rence of inpatient treatment for hypoglycaemia. Diagnosis In accordance with Whipple’s triad, at the time of hypoglycaemia patients have elevated insulin, C-​peptide, and proinsulin levels and symptoms rapidly improve following correction of a low plasma glucose level. Conventional imaging modalities are not helpful; however, selective arterial calcium stimulation tests may identify pancreatic regions responsible for hyperinsulinism if partial or sub- total pancreatic resection is being considered. Pathophysiology Initially, NIPHS was thought to be due to nesidioblastosis, a con- dition largely limited to newborns and rarely found in adults, based on histopathological findings of β-​cell hypertrophy, islet hyperplasia and increased β-​cell mass, following examination of pancreata resected from affected individuals. However, subse- quent studies have suggested alternative pathologic causes due to the absence of nesidioblastosis in some patients with postoperative hyperinsulinism. Enhanced levels of incretin hormones such as glucagon-​like peptide 1 (GLP-​1) and, to a lesser extent, gastric in- hibitory peptide potentiate postprandial insulin secretion following RYGB surgery, may increase β-​cell mass and have been implicated in the aetiology of hyperinsulinaemic hypoglycaemia. Treatment Carbohydrate restriction or postoperative delivery of nutrients to the bypassed proximal intestine by gastrostomy tube may be used to alleviate postprandial hypoglycaemia. When dietary strategies fail, medical therapy with diazoxide, somatostatin analogues, or α-​glucosidase inhibitors may be considered. Partial pancreatectomy has been advocated for some patients with refractory hypoglycaemia and life-​threatening neuroglycopenia. Non​islet cell induced hypoglycaemia Persistent hypoinsulinaemic hypoglycaemia occurs in the very rare circumstance of non​islet cell tumour-​associated hypoglycaemia (NICTH). NICTH is most commonly a late manifestation of large mesenchymal tumours, such as fibrosarcomas or mesotheliomas, that are capable of secreting large amounts of incompletely pro- cessed insulin-​like growth factor-​II (IGF-​II) precursor proteins (so-​called big proIGF-​II). Other tumour types reported to produce IGF-​II include hepatocellular carcinoma, gastrointestinal stromal tumours (GISTs), adrenocortical carcinoma, germ-​cell tumour, and renal cell carcinoma. Pathophysiology Elevated IGF-​II activity suppresses hepatic glucose production and increases glucose uptake by skeletal muscle. Attenuated secretion of counterregulatory hormones including growth hormone (GH) and glucagon is also a consequence of increased plasma IGF-​II (a) (c) (b) Fig. 13.9.2.2  Radiological localization of insulinoma by (a) computed tomography (CT), (b) magnetic resonance imaging (MRI) and
(c) endoscopic ultrasound (EUS). (a) 13 mm insulinoma localized to head of pancreas. (b) 12 mm hypervascular insulinoma localized to head
of pancreas. (c) 10 mm well circumscribed homogenous insulinoma.

13.9.2  Hypoglycaemia 2539 concentrations, thereby further increasing susceptibility to hypogly- caemia. Resultant GH suppression results in low plasma IGF-​1 and reduced binding of IGF-​II by high-​molecular weight protein com- plexes allowing for elevated free plasma IGF-​II levels. Diagnosis A biochemical diagnosis of NICTH is established by confirming hypoglycaemia in association with suppressed insulin, proinsulin, C-​peptide, β-​hydroxybutyrate and abnormally elevated IGF-​II to IGF-​1 ratio (>10). Once confirmed biochemically, conventional cross-​sectional imaging will localize most tumours. Treatment Initial therapy should focus on immediate correction of hypogly- caemia with further treatment directed against the underlying tu- mour. Surgical resection is the mainstay of treatment for benign tumours and may be curative for hypoglycaemia if complete re- section is achieved. In selected cases, reduction of tumour burden by subtotal resection, selective ablation of hepatic metastases, radiotherapy, or systemic chemotherapy may improve NICTH. In unresectable or metastatic disease, administration of oral or intra- venous glucose and/​or artificial nutrition may be sufficient in re- lieving hypoglycaemia. Often, however, additional medical therapy is required and may include high-​dose glucocorticoids (i.e. prednis- olone 30–​60 mg/​day), recombinant human growth hormone, and/​ or intravenous glucagon. There is no role for diazoxide or somato- statin analogues in the management of NICTH. Autoimmune causes of hypoglycaemia Autoimmune insulin syndrome Insulin autoimmune syndrome (IAS), or Hirata disease, is a rare cause of spontaneous hypoglycaemia with over 90% of cases re- ported among individuals of Japanese ethnicity. IAS is associ- ated with specific HLA class  II alleles and commonly occurs following exposure to sulphydryl-​containing medications including methimazole, carbimazole, and penicillamine. Association with other autoimmune disorders such as systemic lupus erythematous, rheumatoid arthritis, polymyositis as well as plasma cell dyscrasias and multiple myeloma is also recognized. Hypoglycaemia typically occurs 3–​4 hours after a meal subse- quent to a period of early postprandial hyperglycaemia. Insulin secreted early in response to a meal is sequestered by insulin auto- antibodies, usually of IgG isotype, that react with endogenous in- sulin to render it temporarily inactive. Subsequent dissociation of the insulin-​antibody complex produces inappropriately elevated free plasma insulin level resulting in hypoglycaemia. In most cases, hypoglycaemia remits spontaneously within 3–​ 6 months of diagnosis. Dietary modification is the most effective im- mediate treatment strategy with frequent, low-​carbohydrate meals encouraged to avoid excessive postprandial insulin secretion. In a limited number of cases prednisolone has been successfully used to lower insulin antibody titres. Insulin receptor autoantibodies Hypoglycaemia may occur as part of the type B insulin resistance syndrome in which circulating antibodies to the insulin receptor are present. Hypoglycaemia in this setting is rare, with most reported cases occurring in patients with a background of autoimmune illness or as a paraneoplastic manifestation of malignancy. Since in- sulin degradation is normally receptor-​mediated, circulating plasma insulin concentrations may be elevated during hypoglycaemia. Patients may demonstrate severe insulin resistance with acanthosis nigricans, alternating episodes of hyperglycaemia with postpran- dial hypoglycaemia or rarely, severe fasting hypoglycaemia due to insulin-​mimetic effects of stimulatory autoantibodies on insulin re- ceptors. Demonstrating the presence of anti-​insulin receptor anti- bodies confirms the diagnosis. Remission occurs in most patients with time, however severe hypoglycaemia requires immediate treatment with variable success reported with generalized immunosuppressant agents (glucocortic- oids, rituximab, cyclophosphamide, azathioprine, cyclosporine) or plasmapheresis. Factitious hypoglycaemia In factitious hypoglycaemia individuals surreptitiously induce hypoglycaemia with exogenous glucose-​lowering therapies. Self-​induced hypoglycaemia may occur for several reasons with underlying psychiatric disturbances at the forefront. A diagnosis of factitious hypoglycaemia should be considered in any individual with access to glucose-​lowering therapy and recognition of this differential may avoid unnecessary investigation for an insulin-​ producing tumour. Biochemically, factitious insulin-​induced hypoglycaemia dem- onstrates high plasma insulin concentrations in association with low C-​peptide and proinsulin concentrations at the time of hypo- glycaemia. Synthetic insulin analogues are undetectable with con- ventional insulin immunoassays and next generation insulin ELISA immunoassays capable of detecting insulin analogues may be re- quired to confirm the diagnosis. Plasma or urine samples should be screened for sulfonylureas if surreptitious use of these medications is suspected. The diagnosis is made by confirming the presence of hypoglycaemia in association with elevated plasma insulin and C-​peptide levels in the presence of a sulfonylurea. Congenital hyperinsulinism Persistent congenital hyperinsulinism (CHI) is rare, occurring in 1:50 000 births and increasing to 1:2500 births in areas with high rates of consanguinity. CHI is a heterogeneous group of genetic disorders characterized by inappropriate insulin secretion from pancreatic β-​cells for a given plasma blood glucose concentration. CHI presents with a spectrum of clinical phenotypes, typically in neonates but, as discussed next, rare subtypes may manifest in late child-​ or adulthood. Adults with undiagnosed CHI are most com- monly identified through predictive genetic screening following identification of an affected neonate although some may present as the index case. To date, CHI has been attributed to genetic mutations in nine different genes which may be broadly divided into defects affecting the pancreatic β-​cell ATP sensitive potassium channel or those af- fecting intracellular metabolic pathways. Of these, recessive mu- tations in the KATP channel genes (ABCC8 and KCNJ11) are most common. Relative to other forms of CHI, these ‘channelopathies’ present in early life with severe hypoglycaemia that is unresponsive

section 13  Endocrine disorders 2540 to diazoxide therapy and pancreatectomy is often required for nor- malization of plasma glucose levels. Glucokinase (GCK) is an important regulator of glucose homeostasis that serves as both the glucose sensor in pancreatic β-​cells and rate-​limiting enzyme regulating hepatic glycolysis. Heterozygous inactivating GCK mutations result in a subtype of maturity onset diabetes of the young (GCK-​MODY), while rare, dominant activating mutations cause familial hyperinsulinaemic hypoglycaemia (GCK-​HH). GCK-​HH is characterized by a spec- trum of clinical phenotypes that most commonly present in neo- nates but may also go unrecognized until later child-​ or adulthood. Adults with undiagnosed GCK-​HH may meet diagnostic bio- chemical criteria for endogenous hyperinsulinism prompting unsuccessful and sometimes prolonged searches for an insulin-​ secreting tumour. Suggestive clinical features of GCK-​HH are stability of hypoglycaemia during fasting and exacerbation of hypoglycaemia by an oral glucose load. Importantly, absence of family history does not necessarily exclude a diagnosis of GCK-​ HH given the associated phenotypic heterogeneity and potential for de novo mutations. Exercise-​induced hyperinsulinism (EIHI) is a rare hypogly- caemic disorder due to autosomal dominant mutations in the SLC16A1 gene that encodes for the monocarboxylate transporter subtype 1 (MCT1). MCT1 expression is normally low or absence in pancreatic β cells and gain-​of-​function genetic mutations within the SLC16A1 gene promoter induce ectopic MCT1 expres- sion. This permits pyruvate uptake into β-​cells, most commonly during aerobic exercise or in response to an intravenously admin- istered pyruvate load, resulting in pyruvate-​stimulated insulin secretion. EIHI may present in child-​ or adulthood. Affected pa- tients are diazoxide-​responsive and avoidance of strenuous exer- cise is advised. FURTHER READING Amiel SA (2014). Banting memorial lecture 2013. A life in balance: wandering the pathways of control. Diabet Med, 31, 382–​92. Bodnar TW, Acevedo MJ, Pietropaolo M (2014). Management of non-​ islet-​cell tumor hypoglycemia: a clinical review. J Clin Endocrinol Metab, 99, 713–​22. Brown E, et al. (2018). Multidisciplinary management of refractory insulinomas. Clin Endocrinol (Oxf), 88, 615–24. Cryer PE, et al. (2009). Evaluation and management of adult hypogly- cemic disorders: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 94, 709–​28. Elliott J, et al. (2014). Substantial reductions in the number of diabetic ketoacidosis and severe hypoglycaemia episodes requiring emer- gency treatment lead to reduced costs after structured education in adults with type 1 diabetes. Diabet Med, 31, 847–​53. Elwen FR, et al. (2015). An observational study of patient characteris- tics and mortality following hypoglycemia in the community. BMJ Open Diabetes Res Care, 3, e000094. Placzkowski KA, et  al. (2009). Secular trends in the presentation
and management of functioning insulinoma at the Mayo Clinic, 1987–​2007. J Clin Endocrinol Metab, 94, 1069–​73. Rahman SA, Nessa A, Hussain K (2015). Molecular mechanisms of congenital hyperinsulinism. J Mol Endocrinol, 54, R119–​29. Service GJ, et al. (2005). Hyperinsulinemic hypoglycemia with nesidio­ blastosis after gastric-​bypass surgery. N Engl J Med, 353, 249–​54. Thabit H, et al. (2014). Home use of closed-​loop insulin delivery for overnight glucose control in adults with type 1 diabetes: a 4-​week, multicentre, randomised crossover study. Lancet Diabetes Endocrinol, 2, 701–​9. Villani M, de Courten B, Zoungas S (2017). Emergency treatment of hypoglycaemia: a guideline and evidence review. Diabet Med, 34, 1205–11.