Genetic determination of sexual differentiation

Each mature ovum contains 22 autosomal chromosomes and one X sex chromosome. A mature sperm contains 22 autosomal chromosomes and either one Y or one X sex chromosome. A fertilized egg, therefore, contains 22 pairs of autosomal chromosomes and one pair of sex chromosomes, either 46XX (female) or 46XY (male). At least 100 autosomal genes are present on the X chromosome and mutations of these genes cause a variety of sex-linked disorders including color blindness, hemophilia and Duchenne muscular dystrophy. In contrast, the Y chromosome has very few autosomal genes. One of the X chromosomes in females is inactivated, producing the Barr body. The inactivated chromosome must be reactivated when cells replicate and divide.

In the human, the default pattern of genital development is female. There are three important steps in sexual differentiation and the development of the normal male phenotype. The first is the differentiation of bipotential gonad primordia (identical in both XX and XY fetuses) into testes that secrete testosterone. The second is the development of the internal reproductive tract. In male fetuses, this requires the presence of anti-Müllerian hormone (AMH, see Box 6.47) that causes involution of the Müllerian ducts, the anlagen for the female type reproductive tract. The third is the development of the external genitalia that requires testosterone and, in some target tissues, its more potent metabolite 5α-dihydrotestosterone (DHT). Female differentiation occurs by default in the absence of these hormones. It is evident that the gonads are paired bilateral structures; in very rare circumstances the developmental processes may be different on either side.

Box 6.47

Structures of the inhibins, activins and anti-Müllerian hormone (AMH). *Shows strucyural homology to the β chains of activins and inhibins.

The genes involved in gonadal differentiation have been summarized in Box 6.2. To date, three genes have been identified in the formation of the bipotential gonad. WT1 and LIM1 code for zinc-finger DNA binding proteins and are expressed early in gonadal development. FTZ-F1 codes for an orphan nuclear hormone receptor, steroidogenic factor-1 (SF-1). Several functions have been ascribed to SF-1 including differentiation and maintenance of both gonadal and adrenal tissue, increasing the synthesis of testosterone and reducing its conversion to estradiol via the transcriptional regulation of the hydroxylases and P450 aromatase, respectively. It may also regulate transcription of the AMH gene.

Box 6.2

Summary of the genes that code for transcription factors that induce differentiation of a bipotential gonad into either a testis or an ovary and subsequent differentiation of the internal reproductive tracts as described in the text.

The importance of the Y chromosome in male development is that its short arm possesses a sex-determining region on the Y gene (SRY) that codes for a DNA-binding protein termed the ‘testis-determining factor’ (TDF). This protein is a transcription factor that regulates the expression of other genes or interacts with other transcription factors. Whilst the majority of XX males carry the SRY gene, only 15–20% human XY females have loss of function mutations in the SRY gene. This implies the involvement of other genes in sexual differentiation. Experimental studies have shown that SRY regulates the expression of genes coding for P450 aromatase and AMH, but a direct interaction between SRY protein and the AMH gene is not yet proven. Two other genes, also implicated in gonadal differentiation, are SOX9 and SOX3, so called because they have sequence homologies to a specific region (the HMG box) of the SRY gene - hence SRY-box-related.

The identification of genes that control male differentiation has progressed because mutations in such genes lead to the development of a female phenotype. This is not true for female differentiation that occurs by default. This is reflected in the fact that only one gene has so far been identified that appears to be required for ovarian, but not testicular, development. DAX-1 was originally identified on the short arm of the X chromosome and known as the dosage-sensitive sex (DSS) reversal locus. This was because duplications in this part of the chromosome were present in some XY females. The DAX-1 protein has been found to be a repressor of StAR expression. StAR transports cholesterol to the inner mitochondrial membrane where the first step in steroid synthesis occurs (see Box 4.4). Mutations of DAX-1 may lead to hypogonadism and adrenal hypoplasia although the precise action of DAX-1 in this respect is not known. Dosage-sensitive models have been proposed. For example, SRY protein could suppress DAX-1 expression but in duplication there may be insufficient SRY protein. Alternatively, SRY could out-compete DAX-1 binding to the StAR promoter leading to autorepression of DAX-1 and, hence, testis formation.

The male determining region of the Y chromosome is so powerful that individuals with 47 XXY or even 49 XXXXY are unequivocally male at birth. The inheritance of only one X chromosome (45 XO, termed Turner's syndrome) still leads to the development of a female phenotype even though there are additional phenotypic features (see Box 6.49). Clinical Case 6.1 is important because further detailed genetic analysis demonstrated the complete absence of the SRY gene, thus indicating the involvement of other genes in governing (albeit incomplete) male sexual differentiation. Clinical Case 6.2 demonstrates that, despite the importance of the Y chromosome, there are circumstances in which its defining actions are abrogated.

Box 6.49

Major clinical features of the more common genetic hypogonadal syndromes. Hereditary hemochromatosis - HFE gene (1 in 10 European Caucasians heterozygous, 1 in 200 homozygous) HFE gene mutations lead to increased iron absorption by unknown mechanisms

Sexual differentiation of the gonads and internal reproductive tracts

An understanding of this case requires further knowledge of the development of the Wolffian and Müllerian duct systems. The gonadal ridges originate from the intermediate mesoderm which gives rise to the mesonephros, the medial aspect of which forms the gonadal ridges (see Boxes 4.7 and 6.3). Around the 5th week of gestation, germ cells migrate from the yolk sac into the gonadal ridges, and this process is complete by about the 6th week. At this stage, the gonadal primordia look the same histologically in either genetic sex (Box 6.3). They consist of three cell types: the germ cells that develop into oogonia (primitive eggs) or spermatogonia (stem cell precursors of sperm); the supporting somatic cells that become either the Sertoli cells or the granulosa cells; and the stromal or interstitial cells. Considerable experimental work has shown that a number of autosomal genes is involved in the development of the primordial gonad.

Box 6.3

Schematic overview of the differentiation of the internal male and female reproductive tracts from the Wolffian and Müllerian ducts.

Differentiation of the testes begins around the 7th week of gestation when the somatic cells form sex cords (the future Sertoli cells) that incorporate the primitive germ cells. At this stage, the human fetus has both Müllerian (paramesonephric) and Wolffian (mesonephric) ducts that are the respective anlagen of the female and male reproductive tracts (Box 6.3). Sertoli cells begin to secrete AMH, a dimeric glycoprotein (molecular weight 70 000) that causes involution of the Müllerian ducts.

By the 8th fetal week, Leydig cells appear in the differentiating testis and begin to secrete androgens, the actions of which are essential to the masculinization of the fetus. With the regression of the Müllerian ducts and under the influence of testosterone, the Wolffian ducts develop into the epididymis, the vas deferens and the seminal vesicles (Box 6.3).

Testicular descent during sexual development occurs in two phases. The first is relative to development rather than actual movement and it reaches the internal inguinal ring by week 24. The second phase occurs in the last 2 months of fetal life with the testis passing through the inguinal canal to reach the scrotum. Androgens are an absolute requirement for this migration and in 97% of normal newborns the testes are in a scrotal position.

In the absence of AMH, the Müllerian ducts develop to form the Fallopian tubes, uterus, cervix and upper part of the vagina (Box 6.3). This differentiation begins around the 10th week of gestation and the Wolffian ducts begin to degenerate. Around the same time, the germ cells, that are now destined to become oogonia, enter their first meiotic division and are subsequently surrounded by a layer of granulosa cells to form primordial follicles. Such follicular development does not begin until about 15 weeks gestation (some 8 weeks later than the differentiation of the testes). At birth, each ovary contains about 2 million primordial follicles though this declines to about 200 000 primordial follicles by menarche. Each primordial follicle contains a primary oocyte half way through its first meiotic division. There is a vast over provision of potential oocytes and many become atretic well before the menopause. Thus, females are born with all the eggs they will ever have. This contrasts with the male germ cells or spermatogonia that, from puberty and throughout life, continue their ability to divide and to produce sperm.

Imaging performed by ultrasound on the patient in Clinical Case 6.2 showed that she had no Fallopian tubes or uterus and only the lower two-thirds of her vagina. She had male concentrations of circulating testosterone synthesized by the testes in her inguinal canals, and yet she showed no signs of masculinization. Her external genitalia were female and completely normal. Thus, during fetal development the Müllerian ducts regressed under the influence of SRY and other genes encoding for transcription factors that stimulated AMH production. Loss of Wolffian duct development was due to her complete insensitivity to the masculinizing actions of fetal testosterone and its metabolites. This also explains the lack of pubic and axillary hair, additional manifestations of androgen action, in her teens.

Surgical removal of her undescended testes was undertaken because of the high risk of malignant transformation. The diagnosis of androgen-insensitivity syndrome was confirmed by laboratory studies on cells obtained from a biopsy of genital skin taken at the same time. Fibroblasts grown from the biopsy demonstrated very low binding of radio-labeled testosterone, confirming an abnormality in the androgen receptor. Androgen insensitivity may not be complete and the phenotype depends crucially on the extent to which testosterone signalling is handicapped. In some cases, radio-labelled testosterone binding studies are normal because the loss of function mutation in the testosterone receptor affects DNA binding, not hormone binding.

Sexual differentiation of the external genitalia

Sexual differentiation of the male external genitalia is critically dependent on androgens and, as the next clinical case illustrates, defects in androgen signalling can result in ambiguous genitalia at birth.

Clinical Case 6.3

A man in his 50s was referred to the Endocrine Clinic from the Urology Clinic where he had attended for stress incontinence. He had been noted to have no palpable prostate gland and a wide bladder neck leading to the problems of incontinence, particularly when he coughed. This had been exacerbated by his asthma. The past medical history was noteworthy. He had been brought up as a girl until the age of 5 years and he remembered playing with dolls (and tea-sets) and had had no inkling of being a boy. At the age of 5 years, his hair had been cut short for his first term at school and he had been dressed in shorts. He vividly remembered the first time he tried to urinate in the standing position like other boys, an event that led to the soaking of his trousers and socks. A series of plastic surgical operations had been undertaken in his teens allowing the correction of the hypospadias, closure of the urogenital sinus and construction of a penis (Box 6.4). At the age of 16 years, his birth certificate had been changed from female to male. He was well aware of the implications of his anatomy. He found that sexual intercourse required ‘some innovation’ but had never been in any doubt that he was male. He had married but had no children. Examination in the Urology clinic revealed a small penis and two normal sized testes in a small scrotum (Box 6.4).

Box 6.4

Clinical photographs of the external genitalia of Clinical Case 6.3 before and after surgery. Prior to surgery. Pubic hair has been removed. The folds forming the labia minora have been retracted to show the urogenital sinus. A = anus; L = labial folds (more...)

Like the gonads, but unlike the internal reproductive tracts, the structures that develop into the external genitalia are initially identical in males and females. They develop from the same anlagen: the genital or labioscrotal swelling; the genital or urethral folds; the genital tubercle and the urogenital sinus (Box 6.5). The development of the external male phenotype requires the actions of testosterone. This, however, requires the conversion of testosterone to its active metabolite, 5α-dihydrotestosterone (DHT) within the cells of the anlagen for normal differentiation (Box 6.6).

Box 6.5

Diagram of the development of the external genitalia. The genital tubercle elongates and forms the shaft and glans of the penis. The urogenital sinus becomes continuous with a groove that develops on the caudal face of the genital tubercle and this groove (more...)

Box 6.6

The roles of testosterone (T) and 5α-dihydrotestosterone (DHT) in the development of the male internal and external reproductive tract. The reduction of T to DHT is performed by the enzyme 5α-reductase expressed in the tissues shown. There (more...)

In a male fetus, between 7 and 13 weeks gestation: the genital swellings migrate and become the scrotum; the urogenital folds enlarge and enclose the penile urethra and corpus spongiosa; the genital tubercle becomes the glans penis; and the urogenital sinus forms the prostate gland (Box 6.5). Hypospadias arises when the urethra is not completely enclosed by the urogenital or urethral folds. At 15 weeks gestation, the size of the external genitalia is roughly the same in males and females and it is not until the latter two-thirds of pregnancy that growth of the male external genitalia takes place and descent of the testes into the scrotal sac is completed.

Female differentiation begins later and, in the absence of testosterone: the genital swellings form the labia majora; the genital folds remain unfused and form the labia minora; the genital tubercle forms the clitoris and the urogenital sinus the lower part of the vagina (Box 6.5). This is the ‘standard’ or default pattern of differentiation and appears to be independent of gonadal steroids. The patient in Clinical Case 6.2 was completely insensitive to testosterone and DHT (androgen insensitivity) due to a receptor defect and, thus, had normal female external genitalia including a blind ending vagina. She was, thus, capable of a sexually active life as a female but without the ability to be fertile.

In contrast, Clinical Case 6.3 showed clear evidence of masculinization of the external genitalia and, hence, of androgen actions, but this was incomplete. The patient had normal circulating blood concentrations of testosterone but low levels of its metabolite, DHT. A defect in 5α-reductase results in complete or partial failure in the masculinization of the external genitalia (depending on the degree of enzyme deficiency). The lack of the full effect of DHT during the fetal life of Clinical Case 6.3 led to his designation at birth as female.

It is, thus, clear that abnormalities of sexual differentiation occur when there is one of three situations. There may be virilization of a genetic female, incomplete virilization of a genetic male or the rarest situation of all when both testicular and ovarian tissue are present (true hermaphrodite). The causes of these abnormalities are summarized in Box 6.7.

Box 6.7

Causes of abnormalities of sexual differentiation. Fetal androgens - e.g. congenital adrenal hyperplasia, adrenal adenoma or hyperplasia Maternal androgens - e.g. ovarian or adrenal tumors

The presentation of ambiguous genitalia at birth is a medical emergency. Since ambigous sex is surrounded by a maelstrom of emotion and misunderstanding, its management requires the logical application of these embryological principles so that the situation can be resolved rapidly and with the minimum of investigations (Box 6.8). A clinically based approach is given in Box 6.9 and treatment will require appropriate endocrine induction of puberty, maintenance of adult sex steroid replacement and reconstructive surgery as dictated by the individual anatomy.

Box 6.8

Clinical investigation of ambiguous genitalia. Elicit family history and any consanguinity Examine for dysmorphic features

Box 6.9

Ambiguous genitalia - principles of management and treatment. Avoid the use of gender-specific pronouns or the term intersex (that promulgates the idea of the neonate being of neither sex) and simply make reference to ‘your baby’ Avoid (more...)

Control of steroid production in the fetal gonads

The major control of steroid synthesis in the fetal and adult gonad is through the actions of the gonadotrophins, LH and FSH, secreted by the pituitary gland (see Box 6.13). In the fetus, the hypothalamo-hypophyseal vascular connections responsible for LH release by hypothalamic GnRH are only established between 11 and 12 weeks after conception. This is some 3 weeks after the onset of testosterone production in the Leydig cells of the testis. Early sexual differentiation depends, therefore, on human chorionic gonadotrophin (hCG) secreted from the placenta in high concentrations during the first trimester of pregnancy. This hormone, the detection of which forms the basis of the commonly used pregnancy test, is homologous to LH and acts on the same G-protein linked receptor. There is a good correlation between the circulating concentrations of hCG and testosterone and it is clear that hCG is also important for the proliferation and differentiation of Leydig cells.

Box 6.13

Simplified diagram of the actions of gonadotrophins. GnRH stimulates transcription of the genes coding for the common α and specific β subunits of LH and FSH. Glycosylation of the proteins occurs in the pituitary and may be modified in (more...)

Estrogen formation in the fetal ovaries also begins early in development even though primordial follicles do not start to develop until the second trimester of pregnancy. At this early stage, the ovary does not contain all the enzymes required for the synthesis of estrogen and it may be produced from conversion of androgens. Its physiological role is uncertain.

Gradually, gonadotrophin secretion from the fetal pituitary takes over the role of hCG and peripheral blood concentrations of fetal pituitary LH and FSH peak around mid-gestation falling to low levels by the time of birth. This later reduction is thought to result from the maturation of the negative feedback effects of steroids on the hypothalamo-pituitary axis. During the latter two thirds of pregnancy, these steroids are required for further virilization (e.g. growth of the penis) and the final shaping and growth of the female external genitalia. A second, but smaller peak, of LH and FSH secretion occurs post-partum stimulating increased steroid secretion. This post-natal peak is thought to be due to the loss of the negative feedback effects of steroids from the feto-placental unit.

The post-natal period of relatively high steroid concentrations gives rise to an interesting phenomenon and two clinically important sequelae. The phenomenon is ‘witches' milk’ that occurs as a result of estrogen effects on the neonatal breast leading to milk production. The first clinical sequela is that it allows the investigation of the hypothalamo-pituitary-gonad axis. If this opportunity is missed it does not occur again for about a decade, at the onset of puberty. It also allows a therapeutic window of opportunity for male children born with small penis (micropenis) or undescended testes, both processes being dependent on testosterone. Depot injections of testosterone (e.g. 50 mg intramuscularly each month for 3 months) can be used to increase penis size or to encourage testicular descent. At this time, there is relatively little adverse effect of such therapy on bone development.

In the post-natal period, a new equilibrium is soon established and circulating androgen and estrogen concentrations decrease over several months to the very low levels that characterize childhood (when serum testosterone and estradiol are unmeasurable in the usual clinical assays). Then follows the organized mayhem of puberty.

Puberty

Puberty marks the transition from a non-reproductive state into a reproductive state and its name is derived from the Latin word pubes meaning hair. It is a time of widespread endocrine changes including the adolescent growth spurt, skeletal changes (e.g. increase in hip width, in females), increase in muscle and fat tissue and profound psychological changes. Other physical changes associated with puberty are development of pubic and axillary hair (pubarche) and development of breasts (thelarche).

The age at which puberty occurs has decreased dramatically during the last century and this is thought to be due to better nutrition and health. The ages at which pubertal changes occur, however, vary greatly among individuals and there are ethnic differences. In order to assess the speed and extent of development, Tanner and co-workers developed a set of stages for axillary and pubic hair development, and breast and male genital development. They are widely used and referred to as Tanner stages (Box 6.10) although it must be remembered that there is nothing stepwise about normal puberty. Unlike males, who have no noteworthy event by which its passage may be timed, females usually remember the year of the onset of menstruation (termed menarche). It is to be emphasized that normal puberty consists of a smooth ordered progression of processes often termed consonance.

Box 6.10

Tanner stages.

In terms of the endocrinology of human puberty this consists of two processes, adrenarche and gonadarche that are independently regulated. Adrenarche refers to the increase in androgen secretions from the zona reticularis of the adrenal cortex that occurs between the ages of about 6 and 8 years whilst gonadarche, occurring several years later, refers to the activation of gonadal sex steroid production. Disorders of sex steroid action cause the major clinical problems at puberty.

The onset of puberty is characterized by increasing secretions of LH and FSH that stimulate gonadal activity. These are induced by an increasing drive from the hypothalamic GnRH neurons. Thus, it is pertinent to discuss the hormones of the hypothalamo-pituitary-gonadal axis before embarking on the hormonal changes that occur in puberty and the endocrine disorders that can cause abnormal pubertal development.

GnRH and the control of gonadotrophin synthesis and secretion

GnRH is synthesized in about 1000–3000 neurons diffusely situated in the arcuate nucleus and other nuclei of the hypothalamus. Most of their axons terminate on the hypophyseal portal capillaries in the median eminence through which the GnRH is transported to the gonadotrophs of the anterior pituitary gland (see Box 7.3). Some axons project to other brain areas and animal studies suggest these may play a role in sexual behavior. GnRH is also synthesized in the placenta, gonad, breast, lymphocyte and pituitary gland where its exact physiological roles remain uncertain.

It is synthesized as a large pre-prohormone (Box 6.11) consisting of a 23-amino-acid signal sequence at the N-terminal, the 10 amino acids which form GnRH, a 3-amino-acid sequence used for molecular processing and a 56-amino-acid sequence at the carboxy-terminus known as GnRH associated peptide (GAP). GnRH and GAP are cleaved before secretion. In the adult, GnRH is secreted in a pulsatile manner with a single pulse occurring approximately hourly. Different patterns are seen in infants, and during puberty a diurnal rhythm of pulsatile GnRH secretion is seen. GnRH acts on the pituitary gonadotroph via typical G-protein-linked receptors that predominantly activate phospholipase C with the resulting hydrolysis of PIP₂ to IP₃ and increased intracellular calcium and generation of diacylglycerol and activation of protein kinase C (Box 6.11).

Box 6.11

Synthesis of GnRH and its actions on pituitary gonadotrophs. GnRH is synthesized as a large prohormone and released with gonadotrophin associated peptide (GAP). It acts on the gonadotroph via a G-protein (G) linked receptor that activates phospholipase (more...)

GnRH stimulates both the synthesis and release of LH and FSH and it is the pattern (amplitude and frequency) of GnRH secretory pulses that is thought to regulate these functions. Thus, low- amplitude, high frequency pulses, as seen in women in the follicular phase of the menstrual cycle, may preferentially stimulate synthesis and secretion of the FSH β-subunit. High-amplitude, low-frequency pulses typical of the luteal phase of the menstrual cycle may preferentially stimulate synthesis of the LH β-subunit.

The LH and FSH responses to a given pulse of GnRH are governed by two further factors, the feedback action of gonadal steroids on the pituitary gonadotrophs and the regulation on GnRH receptors on these cells. In the absence of regular GnRH pulses, pituitary gonadotrophs lose GnRH receptors and become less and less sensitive. As will be seen in Clinical Case 6.6, patients with hypothalamic hypo-gonadism have a small gonadotrophin response to an intravenous injection of synthetic GnRH. Repeated pulses of GnRH approximately 90 min apart up-regulate its receptors and the gonadotrophin response. In contrast, continuous (non-pulsatile) infusions of GnRH cause an initial stimulus to gonadotrophin secretion followed by a down-regulation of GnRH receptors and loss of responsiveness. This has important clinical consequences (Box 6.12).

Box 6.12

Clinical uses of GnRH and its analogs. GnRH is not orally active because it is rapidly broken down in the gut; it has a short half life (<15 minutes) Many stable, long-acting GnRH agonists and antagonists have been developed

The gonadotrophins - LH and FSH - and their actions

The gonadotrophins, like TSH and hCG, are glycoproteins (molecular weights approximately 30 000) made up of a species-specific common α-subunit and a β-subunit that confers biological specificity to each hormone. LH and FSH are important regulators of steroidogenesis in the gonads and, like GnRH, their receptors are also typical G-linked proteins. Receptor activation by gonadotrophin binding stimulates adenylate cyclase and a consequent rise in cAMP. There is also evidence that LH/hCG can signal via the activation of phospholipase C and phosphoinositide hydrolysis (Box 6.13).

Just like the action of ACTH on adrenal cortical cells, LH and FSH increase intracellular concentrations of free cholesterol, its transport to the mitochondria and the transport of cholesterol to the inner mitochondrial membrane by the StAR protein. Here, cholesterol is converted to pregnenolone by side chain cleavage enzyme (CYP11A1, see Box 4.5) and subsequent synthetic processes depend on the target cell. The rate-limiting step in gonadotrophin-induced steroid synthesis is the regulation of StAR and cholesterol side-chain cleavage activity.

These mechanisms are common to both male and female but divergence arises in regard to the target cells and actions of gonadotrophins in the gonads. In the testis, LH is the exclusive steroidogenic hormone acting only on the interstitial or Leydig cells whilst FSH acts exclusively on the Sertoli cells. In the ovary, both LH and FSH are involved in the control of steroidogenesis and each acts on more than one cell type. There are numerous isoforms of circulating LH and FSH and biological potency depends on the degree and sites of glycosylation and the electrical charge of the molecule. Thus, different assays of LH and FSH concentrations may not always give a true index of biological activity (Box 6.14).

Box 6.14

Measurement of LH, FSH and hCG.

The human genes coding for the LH and FSH receptors are located on chromosome 2p21 and are very large containing 10 and 11 exons, respectively. Inactivating mutations of the β-chain of the LH receptor have been described and, in males, the loss of gonadotrophin stimulation on fetal Leydig cell proliferation and development results in Leydig cell agenesis or hypoplasia. The clinical features depend on the degree of compromise in receptor function. Loss of testosterone secretion may cause an ambiguous phenotype. Other cases present as fertile males (because FSH secretion is maintained) with eunuchoidal body proportions (‘fertile eunuch syndrome’). Similar inactivating mutations in females do not alter phenotype and folliculogenesis can still occur, though ovulation does not. Constitutive activating mutations of the LH receptor have been reported (see Clinical Case 6.4) and inactivating mutations of the FSH receptor, associated with ovarian dysgenesis and amenorrhea, have been identified.

Endocrine changes in puberty

The next two cases are graphic demonstrations of early (precocious) sexual development and late (delayed) puberty and are used to introduce the physiological processes controlling puberty.

Clinical Case 6.4

A 20-month-old boy was referred to the clinic with his mother who was concerned about his abnormal development. He had been normal at birth. His mother had noted pubic hair from an early age and thought that he had always been ‘much larger’ than the male babies of other mothers. When seen, he was indeed tall with advanced general physical development and sexual development compatible with Tanner stage 3 but associated with small testes of 3 ml volume (Box 6.15). He had frequent erections. The rest of his development had been normal. The family history was noteworthy for the fact that two maternal cousins had been treated in Thailand for precocious sexual development, though no other information was available. The bone age (estimated from an X-ray of the left hand and wrist, see Box 7.11) was advanced at 4.2 years.

Box 6.15

Clinical photographs of Clinical Case 6.4. Clinical photos of Clinical Case 6.4 demonstrate that the general body habitus is clearly much more mature than that expected from his chronological age of 20 months Tanner stage 3 - the penis is lengthened and (more...)

Prior to the onset of puberty, both LH and FSH are secreted in very small amounts (with low concentrations in peripheral blood) and there is no apparent stimulation of the gonads (Box 6.16). However, as puberty approaches, the amplitude of LH and FSH pulsatile secretion increases (hence, mean secretion rates) and the nocturnal rise in LH secretion is amplified. It is notable that this nocturnal rhythm is specific to puberty as it disappears in adulthood. The gonad itself does not seem necessary for the changes to occur and, thus, it must be concluded that the brain is in some way programmed to produce more GnRH and, hence, LH and FSH.

Box 6.16

Diagrammatic representation of the changes in the circulating concentrations of LH and FSH in males [•] and females [•] during various pubertal stages and a summary of the factors that increase gonadotrophin secretion and stimulate gonadal (more...)

Several theories have been put forward to account for this change in the secretion of GnRH. One is that there is an inherent maturation of these neurons. Another is that melatonin secretion from the pineal gland is reduced at puberty relative to increasing body mass and, thus, there is less inhibition of GnRH secretion by this hormone (see Box 7.33). A third idea is that the achievement of adequate body fat may be an important determinant of puberty. Finally, it has been suggested that the early pubertal rise in DHEA secretion from the adrenal cortex (adrenarche) aids maturation of the GnRH neurons. Whatever the mechanism, the increasing drive from the GnRH neurons and the increasing sensitivity of the pituitary gland to this neurohormone raises circulating concentrations of LH and FSH. This stimulates pubertal development of the gonads and, thus, an increase in the output of sex steroids. These, together with adrenal androgens, induce the physical changes that occur at this time.

Other hormones that may play a part in puberty are the metabolic hormones, somatotrophin and leptin. The former augments the pattern of sexual maturation once a pubertal pattern of gonadotrophin secretion is established. Leptin, on the other hand, may play a part in controlling body weight (fat mass). This may be important since the onset of puberty has been associated with the body reaching a critical mass. That said, the evidence and proposed mechanisms for the relationship between body fat and leptin in regulating puberty have yet to be established.

In males, the earliest sign of increasing LH and FSH secretions is an increase in testicular volume greater than 4 ml or 2.5 cm in length. In girls, thelarche (breast development) is a sensitive indicator of LH- and FSH-stimulated ovarian steroid secretions. Prior to menarche, however, estradiol secretion fluctuates widely probably reflecting waves of follicular development that fail to reach the ovulatory stage. The estrogen stimulates growth of the uterus but menarche does not occur until the estrogens have stimulated sufficient uterine growth such that their withdrawal causes the first menstruation. The onset of menstruation is such a milestone in the development of girls that primary amenorrhea can be relied upon to indicate underlying pathology. The growth of axillary and pubic hair in both sexes is a consequence not only of increasing gonadal steroids (gonadarche) but also adrenal steroids (adrenarche).

Precocious sexual development

Precocious sexual development results from the premature exposure of tissues to sex steroids whatever their source (Box 6.17). Such sexual development is not the same as precocious puberty since the latter maintains its underlying pattern or consonance. The 20-month-old boy in Clinical Case 6.4 was tall for his age and parental heights and had Tanner stage 3 genital development and pubic hair but no signs of gonadarche; i.e. no growth of testicular volume. Thus, he had precocious sexual development not precocious puberty. This conclusion was supported by the fact that LH and FSH were unmeasurable (<0.1 IU/l) in peripheral blood. The lack of FSH explained his small testicular volume since seminiferous tubules, forming the bulk of testicular volume, are primarily stimulated by FSH.

Investigations of Clinical Case 6.4 showed normal 17α-hydroxyprogesterone (6.2 nmol/l, NR <20 nmol/l) and androstenedione (2.1 nmol/l NR 3–8 nmol/l) and suppressed dehydroepiandrosterone sulfate (<0.5 mmol/l, NR 2.8–12 mmol/l). This indicated that the adrenal glands were not the source of androgens. Thus, his testes were producing testosterone independent of the hypothalamo-pituitary axis. This condition, sometimes called ‘testotoxicosis’, is often due to an activating mutation of the LH receptor (Box 6.13).

The investigation and treatment of precocious sexual development follows logically from the principles outlined in the text and are summarized in Boxes 6.17 and 6.18. Clinical Case 6.4 was treated with cyproterone acetate but testolactone and spironolactone were added to improve control of sexual development. There was little regression in the physical signs with which he presented to medical attention and his growth chart (see website) indicated continued poor control in part exacerbated by poor complicance with medication. True precocious pubertal development may be arrested by treatment with a long-acting synthetic analog of GnRH (see Box 6.12).

Box 6.18

Treatment of precocious sexual development *.

Delayed puberty

The definitions and causes of delayed puberty are given in Box 6.19. Just as precocious puberty prejudices final height, late puberty (in the presence of a normal GH and IGF-1 axis) leads to a final height that is above that predicted from the mid-parental value, with long legs and arms and a relatively short sitting height. This was illustrated in Clinical Case 6.5 with markedly delayed puberty. In this young man, the serum testosterone concentration was low (1.1 nmol/l, NR 9–25 nmol/l) as were the serum gonadotrophins (LH 0.5 IU/l, FSH 1IU/l). When these results became available, the only additional piece of clinical information was that he had markedly reduced sense of smell.

Box 6.19

Definitions and causes of delayed puberty. Definitions: Delayed puberty: no increase in testicular volume (i.e. <4 ml) by the age of 14 years in boys and no breast development by the age of 13.5 years in girls.

The low serum gonadotrophin concentrations coupled with low testosterone and lack of testicular development indicate that he had hypo-gonadotrophic hypogonadism. His growth and pubic hair development, however, suggested that he had undergone normal adrenarche. This patient has Kallman's syndrome, a genetically heterogenous condition in which congenital hypogonadotrophic hypogonadism is associated with reduced sense of smell. In the X-linked form there are mutations in the KAL gene on the short arm of the X chromosome coding for anosmin, an adhesion molecule. The defect leads to the inability of the GnRH neurons to migrate to the hypothalamus during development.

During embryonic development nerves grow from the nasal cavity to the olfactory bulbs at the base of the cerebral hemispheres. From there the olfactory tracts project to the hypothalamus and other brain areas, a process that is complete at about 25 days gestation. The embryonic cells of the GnRH neurons also originate in the nasal cavity and normally migrate along the olfactory tracts to the hypothalamus. However, with incomplete development of olfactory nerves, the GnRH neurons have no olfactory highway to guide them in their migration and they remain in the nose, hence, the association of anosmia and one cause of hypogonadotrophic hypogonadism. KAL is also expressed in other tissues and associated features of Kallman's include renal aplasia, ataxia, cleft palate. However, it is to be emphasized that the phenotype-genotype correlations are poor and, indeed, that X-linked is the least common form of inheritance. This indicates that a number of other genes or factors are operating in governing the expression of the condition.

Testicular development and normal testosterone secretion can be restored in Kallman's syndrome by treatment with pulsatile GnRH therapy (Box 6.12), a use that contrasts with the use of long-acting GnRH analogs to delay pubertal development. A clinical approach to the investigation and treatment based on the principles outlined in the text are given in Boxes 6.20 and 6.21.

Box 6.20

Investigation of delayed puberty. Elicit family history of delayed puberty, anosmia, color blindness Examine for dysmorphic features (e.g. Prader-Willi or Turner's syndrome, see Box 6.49) and in a boy the genitalia for features such as micropenis (suggestive (more...)

Box 6.21

Treatment of delayed puberty*.

Premature adrenarche

Premature adrenarche may occur between 4 and 8 years of age. Pubic hair growth in a child of, say, 5 years may cause parental concern that the child has progressive precocious sexual development. Most commonly, however there is no underlying medical problem and the most important evaluation is clinical assessment and careful auxology to verify that there is no progression to precocious puberty and no acceleration in growth rate. When investigations are performed, they confirm that gonadotrophin and gonadal steroid secretions are pre-pubertal and that the circulating concentrations of dehydroepiandrosterone and its sulfate (whilst elevated for age) are matched with appropriate bone age and pubic hair development. No treatment is required for premature adrenarche.

Acne, hair growth and hirsutism

Axillary and pubic hair growth are important aspects of puberty as are the problems of teenage spots. Problematical acne and excess hair growth in females (hirsutism) result from the action of androgens on pilo-sebaceous units (PSUs) of which there are four types, vellus, terminal, apo and sebaceous (Box 6.22).

Box 6.22

The four different classes of pilosebaceous units (PSUs) and the effects of excess androgens. Androgens stimulate sebum secretion and together with infection this can cause acne. Androgens can induce differentiation of vellus PSUs to terminal PSUs encouraging (more...)

It is the sebaceous PSUs that are the cause of much teenage angst. As cells of the sebaceous gland differentiate and move centrally they accumulate lipid droplets that eventually coalesce and burst the cell (it has given its all - holo in Greek - hence the term holocrine secretion). This sebum contains large amounts of glycerides, fatty acids and cholesterol and its production is stimulated by androgens and inhibited by estrogens. Retinoids are also involved in sebaceous gland development.

Acne is a multifactorial disorder and not simply the result of increased sebum secretion. Infection, abnormal keratinization and immune influences can all play a role. The role of adrenarche in acne, however, is supported by the fact that DHEAS concentrations are higher in pre-gonadarcheal girls with comedonal (non-scarring) or inflammatory acne than in those without. Its treatment includes antibiotics and local antiseptics to deal with the Propionobacterium acnes bacterial infection that occurs at puberty. Anti-androgens such as cyproterone are useful in female patients and severe disease may require 13-cis-retinoic acid that causes a marked reduction in sebaceous gland size.

With regard to hair growth, androgens can cause problems in two common circumstances. In females, they can cause the vellus PSUs to differen-tiate into terminal PSUs, though the sensitivity to androgens declines with distance from the androgen-dependent facial, pubic and axillary hairs. In the presence of excess androgens, mustache growth occurs first, followed by beard growth, extension of hair in the pubic area (e.g. up the linea alba or onto thighs) and finally terminal hair over the shoulders. The severity of this process is determined, to some extent, by family inheritance and/or ethnicity (indicating genetic factors). It can be measured by the scoring system of Ferriman and Galwey (Box 6.23). Masculinization manifest as clitoral hypertrophy and breast atrophy is only seen at the very highest concentrations of androgens.

Box 6.23

Diagram of the Ferriman & Galwey scoring system for assessing hirsutism. Individual scores are added to give a total.

Whilst androgens cause hair growth in females, in genetically at-risk males androgen exposure causes baldness with scalp terminal hair regressing to vellus hair with a large sebaceous gland. The patient in Clinical Case 6.3 with a deficiency of 5α-reductase had a full head of scalp hair, little beard growth, normal axillary hair and a female pattern of pubic hair. This has been seen in many patients with 5α-reductase deficiency, indicating that 5α-DHT is the active androgen in the beard and balding area and that testosterone is the active androgen in the axilla and female-distribution pubic hair.

The breast - premature development, hypoplasia and gynecomastia

Breast development normally occurs at puberty in response to estrogen, prolactin and local hormones, although excess prolactin cannot compensate for estrogen deficiency. Premature thelarche (Box 6.24) is characterized by isolated breast development in young girls that either regresses or remains static. Whilst circulating estrogen concentrations may still be low (and below the sensitivity of most radioimmunoassays), ultrasound data and cytological examination of the vaginal epithelium often indicate that estrogen concentrations are elevated relative to age-matched controls. In girls with premature thelarche, or indeed premature adrenache, the use of non-invasive pelvic ultrasound may allow discrimination from precocious puberty since the changes of puberty on the ovary and uterus have been quantified (Box 6.25). Lack of development of the breast (hypoplasia) is seen in delayed or arrested puberty, or when tissue is damaged e.g. local radiation (Box 6.26).

Box 6.24

Premature thelarche. An 18-month-old child with marked breast development Breast development <2 years age, cyclical changes in size ~6 weeks

Box 6.26

Breast hypoplasia. Clinical photograph of a young adult female treated in childhood with external beam radiotherapy for a tumor on the right chest wall. The radiation field included the right breast.

Gynecomastia refers to growth of mammary glands in males. This may result from changes in sex steroid production during sexual development or senescence, drugs that affect endogenous hormone production or action and genetic disorders linked with gonadal dysgenesis. Excess prolactin can also cause gynecomastia but most common is pseudo-gynecomastia due to the deposition of fat in the pectoral area. Causes of and treatment for gynecomastia are summarized in Boxes 6.27 and 6.28.

Box 6.27

Causes of male gynecomastia. Causes: Age related (non-pathological):

Box 6.28

Treatment of gynecomastia in men.

Testicular function

The importance of FSH in the development and maturation of the seminiferous tubules and of LH or maternal hCG on the proliferation, differentiation and testosterone production of the Leydig cells has been defined. Clinical Case 6.6 is the focus for discussion of their roles in controlling the adult testicular functions of spermatogenesis and steroid production.

The Sertoli cells and the germinal cells (the precursors of spermatocytes) are the major components of the seminiferous tubules (Box 6.29). These tubules, that are surrounded by a basement membrane and form the bulk of the testis, drain into the vas deferens. Sertoli cells span the thickness of the tubule wall and at their base they are connected to adjacent Sertoli cells through tight junctions. These tight junctions form a blood-testis barrier (somewhat analgous to the blood-brain barrier) that enables the Sertoli cells to maintain an extracellular environment within the tubule that is different from normal extracellular fluid. The blood-testis barrier also helps to protect maturing sperm from potential toxins and immune assault and leakage of sperm out of the tubule. Sertoli cells organize and nurture waves of spermatogenesis.

Box 6.29

Diagram of the structural organization of the human seminiferous tubule and the stages of spermatogenesis. Spermatogonia push their way through the tight junctions between adjacent Sertoli cells and become primary spermatocytes. In the first meiotic division (more...)

Leydig cells lie between the tubules (hence the term interstitial cells) and comprise less than 5% of the total testicular volume. Their prime function is to synthesize and release androgens including androstenedione, dehydroepiandrosterone and testosterone, the latter being the most important in potency and quantity (Box 6.30) Testosterone diffuses into the seminiferous tubules where it is essential for maintaining spermatogenesis. Some binds to an androgen-binding protein (ABP) that is produced by the Sertoli cells and is homologous to the sex-hormone binding globulin that transports testosterone in the general circulation. The ABP carries testosterone in the testicular fluid where it maintains the activity of the accessory sex glands and may also help to retain testosterone within the tubule and bind excess ‘free’ hormone. Some testosterone is converted to estradiol by Sertoli cell-derived aromatase enzyme.

Box 6.30

The major steroidogenic pathway in the human testis. Testosterone and smaller amounts of androstenedione not only enter the circulation but diffuse into the seminiferous tubules where they may bind directly to androgen receptors or first be converted (more...)

Control of testicular function

Leydig cell steroidogenesis is controlled primarily by LH with negative feedback of testosterone on the hypothalamic-pituitary axis (Box 6.31). FSH acts on Sertoli cells to stimulate protein synthesis, mobilization of energy resources, production of testicular fluid, and the output of Sertoli cell proteins such as inhibin and ABP. The actions of testosterone and FSH on Sertoli cells is synergistic allowing spermatogenesis to be completed. FSH stimulates production of an androgen receptor that makes the Sertoli cell responsive to androgens. Androgens stimulate the synthesis of FSH receptors.

Box 6.31

Diagram of the feedback control of the hypothalamo-pituitary-testicular axis.

In the human, the feedback control of FSH secretion is less clearly defined than that of LH. Administration of testosterone reduces LH secretion but has little, if any, effect on FSH secretion. Inhibin, secreted by Sertoli cells (see Box 6.47), binds to target cells in the anterior pituitary gland and has a selective negative feedback action on FSH secretion (Box 6.31). There is also evidence that the small amount of estrogen that is formed from the peripheral aromatization of testosterone can inhibit both FSH and LH secretion.

It is clear that testicular damage may result in loss of testosterone production or the loss of spermatogenesis or both. Loss of androgen production results in hypogonadism, the symptoms of which (Box 6.32) reflect the functions of testosterone and the causes of which will be essentially those discussed previously causing delayed male puberty (see Box 6.19).

Box 6.32

Clinical features of adult male hypogonadism. Finely wrinkled facial skin may be the only obvious feature of adult male hypogonadism

The requirement of spermatogenesis for high local concentrations of testosterone means that loss of androgen production is likely to be accompanied by loss of spermatogenesis. Indeed, if testicular androgen production is inhibited by the administration of exogenous androgens then spermatogenesis ceases. This is the basis of using exogenous testosterone as a male contraceptive. The patient in Clinical Case 6.6 had normal adult male secondary sexual characteristics and libido and his peripheral venous testosterone concentration was as expected in the normal range as was the serum LH concentration. His small testicular volume and high serum FSH concentration (a result of reduced inhibin secretion from the Sertoli cells) indicated that his Sertoli cells had been damaged resulting in loss of spermatogenesis and infertility.

Transport, metabolism and actions of androgens

The testis secretes 4–10 mg testosterone daily, over 95% of the circulating testosterone. The rest is derived from peripheral conversion of adrenal androgens (Box 6.33). The testis also secretes small amounts of the more potent DHT, the weak androgens DHEA and androstenedione and estradiol and progestagens. Most of the DHT and estradiol circulating in men is derived from peripheral conversion of testosterone.

Box 6.33

Gonadal versus adrenal androgen production or peripheral conversion as a percentage contribution to total circulating androgens in males and females.

Only about 2% of circulating testosterone is in the free form and able to enter cells. The rest is either bound to albumin (approximately 40%) or to sex-hormone-binding globulin (SHBG) and is in equilibrium with the free form. SHBG is synthesized in the liver and its circulating concentration is increased by estrogen or excess thyroid hormones and decreased by exogenous androgens, glucocorticoids or growth hormone and by hypothyroidism, acromegaly and obesity.

Most circulating testosterone is converted in the liver to metabolites such as androsterone and etiocholanolone that, after conjugation with glucuronide or sulfate are excreted in the form of 17-ketosteroids (Box 6.34). Note that the majority of urinary ketosteroids are of adrenal origin and, thus, determinations of ketosteroids do not reliably reflect testicular secretion.

Box 6.34

Major metabolic pathways of the three principal steroids secreted by the gonads. Most steroids are converted (-----) in the liver to various metabolites and then conjugated with glucuronide or sulfate before being excreted in the urine

In many target tissues, testosterone is rapidly converted to DHT by the 5α-reductase enzyme before it interacts with the androgen receptor, a protein that is encoded by a gene on the X chromosome. The binding of either testosterone or DHT with the receptor induces release of a heat shock protein, dimerization of two receptors and translocation to the nucleus where the dimer binds to an estrogen-like hormone response element (see Box 6.35) on DNA. Other transcription factors may be involved in modulating transcription of a number of genes. As with other steroid hormones there is evidence that testosterone may exert non-genomic effects on certain cells via cell surface molecules and also that SHBG may mediate some of these actions.

Box 6.35

Intracellular receptor mechanisms for the three major gonadal steroids. Receptors are mainly located in the cytoplasm attached to heat shock proteins (hsp). Upon ligand binding the hsp are released, receptors dimerize and are translocated to the nucleus. (more...)

Spermatogenesis

Spermatogenesis begins at puberty and involves mitotic proliferation, meiotic division and extensive cell modeling (Box 6.29). It starts with the spermatogonia or germinal cells dividing mitotically to produce a small clone of diploid (46XY) cells termed spermatocytes. The spermatocytes migrate through the tight junctions at the base of the Sertoli cells and, in their new environment cradled by the Sertoli cells, undergo two meiotic divisions. In the first division, pairs of chromosomes come together and exchange DNA (crossing over) and separate into two haploid cells (23X or 23Y) known as secondary spermatocytes. Almost immediately, a second meiotic division takes place in which the two chromatids that make up a single chromosome separate. These haploid cells, thus, contain 23 single half chromosomes and are called spermatids.

At this stage, they are still simple round cells but before they leave the nurturing of the Sertoli cells an acrosome (essential for fertilization) is formed at the head of the sperm and a tail develops. They are extruded from the Sertoli cells into the lumen of the seminiferous tubule. This whole process of spermatogenesis takes approximately 74 days and about 300–600 sperm/gram of testis are produced each second. Not all survive.

From the seminiferous tubules, the sperm are washed towards the rete testis which drains into the vasa efferentia and from there into the epididymis, a highly convoluted tube that finally drains into the vas deferens (Boxes 6.39 and 6.45). During the 12 day passage from the testis to the vas deferens, the sperm become motile and mature to reach full fertilizing ability. As a result of fluid absorption in the epididymis, sperm also become highly concentrated.

Box 6.39

Diagram of mid-sagittal section through the male pelvis and a testis and the major nervous pathways involved in penile erection. Psychogenic stimuli from the brain or from tactile receptors on the penis (via the pudendal nerve) activate efferent parasympathetic (more...)

Box 6.45

Diagram of the comparative anatomy of the male and female reproductive tracts.

Whilst spermatogenesis is dependent on the hormonal drive from the gonadotrophins and testosterone, temperature also plays a critical factor in this process. Normal spermatogenesis is impaired if the testis is maintained at normal body ‘core’ temperature (as occurs in cryptorchidism or through tight clothing). The temperature of the testes is normally maintained about 2°C lower than core body temperature because they lie outside the body, moving freely in the scrotal sac. Three layers of membranes enclose each testis and extensions of the fibrous tunica albuginea form septa that divide the gland into 200–300 lobes. In appearance, the testes are white with an oval shape (approximately 4 × 2 × 3 cm with a volume of about 25 ml and weighing between 10–14 g). Seminiferous tubules constitute 70–80% of this mass and each testis has approximately 200 m of seminiferous tubule. The blood supply comes from the testicular arteries that are branches of the internal spermatic arteries.

A fully functioning testis normally achieved by the age of 16 years has the capacity to produce over 200 million sperm each day. Only one is required for fertilization, but each tiny sperm (a few thousandths of a millimeter in length) must travel some 30–40 cm (100 000 times its own length) of the male and female reproductive tract before it reaches its final destination in the Fallopian tube. Less than 1 in a million ever completes this journey, though they are helped by the process of ejaculation.

Clinical Case 6.6 had normal Leydig cell function but clear evidence that his Sertoli cells had been damaged resulting in loss of spermatogenesis and infertility. There are a number of causes of this (Box 6.36) but it is highly likely given his past medical history that the cytotoxic drug cyclophosphamide was the cause. Clinical Case 6.6 underwent testing of his seminal fluid and it was found that he had azoospermia (Box 6.37). Treatment is possible in a number of causes of male infertility (Box 6.38) but currently there is none available for cyclophosphamide-induced azoospermia. Cryopreservation of ovary and testis of patients prior to undergoing such therapies is under active consideration (see website).

Box 6.36

Causes of male infertility. Unknown (~50%) Maldescent (~20%)

Box 6.37

Tests evaluating male fertility. Sperm number Sperm motility

Box 6.38

Treatment of male infertility. The majority of male infertility is untreatable because of irremediable damage to the germ cells Endocrine therapies are highly successful (e.g. GnRH pump or gonadotrophins) but are only applicable to a very small number (more...)

Erection and ejaculation

Sperm can be stored up to 5 weeks in the tail of the epididymis and vas deferens before they are released at ejaculation. In the absence of ejaculation sperm dribble into the urethra and are washed away in the urine. In men who have undergone a vasectomy (ligation of the vas deferens) sperm build up behind the ligation and are either removed by phagocytosis in the epididymis or leak through the epididymal wall.

The ejaculatory response is evoked by a complex series of reflexes and the physiological phases of this response have been defined as erection, emission and ejaculation. Failure to achieve penile erections has been termed impotence, but the word conjures up such images of functional incapacity that the term erectile dysfunction is recommended.

Erection is induced by tactile stimulation of the genital region, particularly the glans penis, or from visual cues or emotions that can stimulate descending pathways from the brain (Box 6.39). Such sensory stimulation induces dilatation of arterioles in the penis and the sinuses of the corpus spongiosum and cavernosa become engorged with blood. As these erectile bodies are surrounded by a strong fibrous coat the penis becomes rigid, elongated and increases in girth. This compresses venous outflow so that while inflow increases out-flow does not. At the same time, parasympathetic nerves stimulate the bulbo-urethral glands to produce a mucoid-like substance to aid lubrication.

Emission involves contractions of the smooth muscle in the walls of the vas deferens and genital ducts that push sperm into the upper part of the urethra. At the same time, the seminal vesicles and prostate gland (accessory sex glands) contract and seminal fluid is released into the urethra. At ejaculation, the semen (sperm plus seminal fluid) is expelled from the posterior urethra by contractions of the bulbo-cavernous and urethral muscles. The passage of semen from the upper part of the urethra back into the bladder is normally prevented by sympathetic contraction of the urethral sphincter. Failure of this sphincter can cause retrograde ejaculation.

In the resolution phase, all the physiological changes of sexual arousal (e.g. erection of the nipples, increased heart rate and blood pressure, flushing on the face neck and chest) are reversed. A man becomes refractive to any further sexual stimulation for a period from a few minutes to several hours.

Any interference with spinal reflexes or blood supply can cause erectile dysfunction although libido will be unaffected. Over 50% of cases of erectile dysfunction have a physical basis (Box 6.40). The complications of diabetes mellitus include both neuropathy and vascular damage and the disease is one of the most common causes of erectile dysfunction. Doppler blood flow studies of Clinical Case 6.7 showed normal vascular supply but tests of autonomic nerve function showed a marked dysfunction indicating a neurological cause. He was treated with oral sildenafil (better known as Viagra, Box 6.41) with some improvement.

Box 6.40

Causes of erectile dysfunction. performance anxiety depression

Box 6.41

Treatment of erectile dysfunction.

The possible role of testosterone in male sexual behavior is clouded by the variance between individuals and is also markedly influenced by learning. Thus, loss of androgens before puberty reduces the normal pubertal sex drive, but when androgens are deficient after puberty there is a gradual loss of sex interest and an increasing incidence of erectile dysfunction.

The sexual response may also be suppressed by the central nervous system either consciously or subconsciously leading to erectile dysfunction, loss of sexual interest, premature ejaculation, ejaculatory failure or a loss of generalized orgasm at erection. These are all common defects of this complex reflex response and may have a psychogenic basis in some patients. They are often amenable to behavioral therapy.

Given what is known of the embryology and anatomy of the male and female external genitalia, it could be asked whether there is a female equivalent of male erectile dysfunction. The recent experience with the drug sildenafil suggests that this drug is not only effective in treating erectile dysfunction in men but it can also help women to achieve orgasm. It seems highly likely that is works by improving clitoral erection and, thus, sensory sexual stimulation.

Ovarian control and the menstrual cycle

The ovary shows cyclical activity, unlike the testis that is maintained in a more or less constant state of activity. Hormone secretions vary according to the phase of the menstrual cycle (Box 6.42) and gonadal steroids have both negative and positive feedback effects on the control of gonadotrophin secretion (Box 6.43).

Box 6.42

The human menstrual cycle. The graph shows average serum concentrations of hormones during a typical 28 day menstrual cycle along with changes in the uterine endometrium. LH, FSH and GnRH, are secreted in a pulsatile pattern with the frequency and amplitude (more...)

Box 6.43

Feedback control of the hypothalamo-pituitary-ovarian axis.

The first day of the menstrual cycle is defined as the first day of menstruation when estrogen and progesterone secretions from the ovary are low and FSH secretion has increased as a result of the loss of negative feedback effects. The increased FSH stimulates the growth and differentiation of cohorts of preantral or antral follicles that are at different stages of development (see Box 6.46). As a consequence steroid production in the ovary increases.

Box 6.46

Diagram of the process of folliculogenesis. The development of a primordial follicle to a preovulatory follicle takes in excess of 120 days. After it has become a preantral follicle of about 0.2 mm diameter it takes about 65 days to develop into a preovulatory (more...)

The ovary requires both LH and FSH to produce sex steroids (Box 6.44). LH stimulates the thecal cells surrounding the follicle to produce progesterone and androgens. The androgens diffuse across the basement membrane to the granulosa cell layer, where, under the action of FSH, they are aromatized to estrogens, mainly estradiol. The developing follicle selected to become dominant (see Box 6.46) progresses to full maturity.

Box 6.44

Principal steroidogenic pathways in the ovarian follicle. Androstenedione, formed in thecal cells under the stimulatory effects of LH, diffuses across the basement membrane where, under the action of FSH, it is converted to estradiol. In the developing (more...)

Ovulation requires that the follicle rises to the surface of the ovary and the granulosa cells develop LH receptors. At this stage, circulating concentrations of estradiol have reached a critical concentration (>750 pmol/l) and, 24–48 hours after peak production, its negative feedback effect on gonadotrophin secretion is switched to a positive feedback. Such positive feedback is a rare biological phenomenon and is thought to be the result of estrogen increasing the amplitude and frequency of GnRH pulses and a consequent up-regulation of GnRH receptors. Estrogen also increases the responsiveness of the pituitary gonadotrophs. Together, these effects induce a preovulatory peak of LH secretion and a smaller increase in FSH secretion. Ovulation occurs 9–12 hours after the LH surge and involves local paracrine mechanisms and induction of proteolytic enzymes.

After ovulation, the empty follicle is remodelled and plays an important role in the second half or luteal phase of the menstrual cycle. The granulosa and thecal cells remaining in the ruptured follicle proliferate rapidly and form the corpus luteum (from Latin, ‘yellow body’). This phase is dominated by progesterone and, to a lesser extent, 17β-estradiol secretion by the corpus luteum. Significant quantities of 17α-hydroxyprogesterone and inhibin are also secreted and these, together with other steroids, may help to modulate the actions of gonadotrophs in luteal development and maintenance. Small amounts of gonadotrophins, particularly LH, are required to maintain the function of the corpus luteum and in the luteal stage, high-amplitude, low-frequency LH pulses are observed compared to the high-frequency, low-amplitude pulses seen in the follicular stage of the cycle (Box 6.42).

In the absence of fertilization the corpus luteum breaks down by a process known as luteolysis. The precise mechanisms that induce this degeneration are unknown (although in some species it involves prostaglandins) but it may be that there is simply insufficient LH to maintain the corpus luteum. The consequences are that progesterone and estrogen secretions from the corpus luteum decline. The loss of the negative feedback of the steroids induces a selective rise in FSH secretion, more follicles are recruited and a new cycle begins.

Transport, metabolism and actions of ovarian steroids

Estradiol, the most important steroid secreted by the ovary because of its biologic potency and diverse actions, is transported bound to albumin (approximately 60%) and about 30% to SHBG. It is rapidly converted to estrone by 17β-hydroxy-steroid dehydrogenase in the liver and, whilst some estrone re-enters the circulation, most of it is further metabolized to estriol via 16α-hydroxyestrone or to 2- or 4-hydroxyestrone (catechol estrogens) by the action of catecho-O-methyltransferase. The latter metabolites can be formed in the brain and may compete with receptors for catecholamines. Metabolites are conjugated with sulfate or glucuronide before excretion by the kidney (Box 6.34).

Progesterone is mainly bound to albumin in the circulation and, to a lesser extent, cortisol-binding globulin. It is rapidly cleared from the circulation, being converted to pregnanediol and conjugated with glucuronic acid in the liver in which form it can be excreted. The ovaries also produce small amounts of testosterone, DHT, androstenedione and DHEAS and these are metabolized like the testicular hormones.

Estrogen and progesterone, like testosterone, also have multiple target cells and their actions are mediated by intracellular receptors which, upon ligand binding, release a heat shock protein, dimerize with another receptor and bind to a hormone response element or an AP-1 promoter regions on DNA (Box 6.35). Within the last few years two forms of the estrogen receptor have been identified - α and β. They have different distributions in estrogen target tissues and can form different dimers - α/β, α/α and β/β. The different dimers may have different biological effects. The progesterone receptor, unlike the estrogen receptor, can form heterodimers and, instead of binding with an estrogen response element on DNA, as does testosterone (see Box 1.12), progesterone-bound receptors bind to a response element similar to that of glucocorticoids. The specificity of the response is determined by which receptor is present in the cell as well as other cell-specific transcription factors.

The ovary - folliculogenesis and oogenesis

Each ovary nestles in a small depression of the posterior wall of the broad ligament on each side of the peritoneal cavity and just above the pelvic brim. They are connected to the fimbriated ends of the Fallopian tubes (Box 6.45). The ovaries are dull white in color, oval in shape (3 × 2 × 1 cm) and weigh about 5–8 g. They are enclosed in a tough fibrous capsule, the tunica albuginea, and consist of an outer cortex and an inner medulla. The cortex contains all the follicles and remains of ruptured follicles embedded in vascular fibrous tissue. The inner medulla is where the blood vessels, lymphatics and nerves enter the ovary. The appearance of the ovary varies with the age of the woman. Before puberty, the glands are smooth and rather solid in consistency and contain many primordial follicles. Between puberty and the menopause, their surfaces become more corrugated in appearance due to the activity of the gland during each ovarian cycle. After the menopause, they shrink and are covered with scar tissue, the result of monthly follicular rupture.

At birth, the cortex of the immature ovary contains about half a million primordial follicles consisting of flattened cells surrounding a primary oocyte that is halfway through its first meiotic division. At the time of puberty, the total number has been reduced to around 300–400 000 and <1.0% ever reach full maturity and ovulate. When primordial follicles enter their growth phase, granulosa cells begin to divide, the oocyte enlarges and becomes surrounded by a zona pellucida. Gradually the follicles become secondary follicles and, when fibroblast cells in the inner thecal layer differentiate, the secondary follicle is defined as a preantral follicle (Box 6.46). The early growth phase is considered to be independent of gonadotrophin stimulation despite the evidence for FSH receptors on these immature follicles. It is not known why a few primordial follicles begin to grow, nor how they are selected, but paracrine factors within the ovary such as cytokines and epidermal growth factor may be involved.

In the early luteal phase of each menstrual cycle, a cohort of preantral follicles undergo further growth into antral follicles (Box 6.46). At this time, the follicles enlarge, the thecal cells become richly supplied with blood vessels and a fluid-filled cavity (the antrum) forms. The oocyte becomes surrounded by several layers of granulosa cells known as the cumulus oophorus. During the basal growth phase follicle diameter increases from about 0.2 mm to 2 mm. By the late luteal phase of the third cycle those follicles that have not degenerated have reached the selectable phase. In other words they may be recruited for further development. Over a period of about 5 days (termed the selection window) the follicles continue to grow but only one is selected to undergo final maturation. The mechanisms responsible for the selection of the dominant follicle are not well understood but may be determined by the number of FSH receptors or the concentrations of steroids or growth factors within the follicle. Over 15 days, the dominant follicle increases in diameter from 5 mm to around 20 mm, secretes increasing concentrations of estradiol and becomes a fully mature Graafian follicle (Box 6.46). Ovulation occurs in the third cycle.

The LH surge induces the ovum to complete its first meiotic division prior to ovulation producing two haploid daughter cells containing 23 chromosomes, one of which is the X chromosome. One cell, however, retains most of the cytoplasm and the smaller one (the first polar body) sits cramped in a small space, the vitelline space, between the secondary oocyte and the zona pellucida. Unlike the process of spermatogenesis, the secondary oocyte does not undergo its second meiotic division until after fertilization has occurred. Like the process of spermatogenesis, folliculogenesis occurs in waves and at any one time the ovary contains follicles at all stages of development, apart from the dominant follicle that is only present during the follicular phase of a cycle.

Non-steroidal factors in the control of the hypothalamic-pituitary-gonadal axis

Whilst gonadotrophin-stimulated steroidogenesis is essential for the maintenance of gametogenesis, the ovary and testis elaborate a large variety of proteins and peptides that can act as local paracrine agents within the gland or can exert central feedback effects.

One of the first non-steroidal factors to be isolated was inhibin and subsequently other glycosylated proteins were identified. Inhibin is a glycoprotein made up of a common α unit combined with one of two β units: β_A (inhibin A) or β_B (inhibin B). Various combinations of the two different β subunits gives rise to three forms of activins; activin, A, β_Aβ_A, activin B, β_Aβ_B and activin C, β_Bβ_B (Box 6.47). A third glycosylated protein, follistatin, is a high-affinity monomeric binding protein for inhibin and activin.

Inhibin synthesized in the Sertoli cells and granulosa cells exerts negative feedback effects on the secretion of FSH via actions on the pituitary gonadotroph. Within the gonads, and along with its other associated peptides, there is evidence for a role for inhibin in modulating steroidogenesis, cellular proliferation and differentiation. Other factors located in the testis and/or ovary include transforming growth factor α, basic fibroblast growth factor, transforming growth factor β, insulin-like growth factor-1 and its binding proteins and a number of cytokines. A variety of functions have been ascribed to these non-steroidal factors and it would appear they help to orchestrate the complex processes of spermatogenesis, oogenesis and folliculogenesis.

Ovulation, menstruation and its problems

The average length of a menstrual cycle is approximately 28 days. Cycles do, however, vary considerably in length although in 95% of women each cycle lasts between 25 and 34 days. Most of the variation is due to differences in the length of the first (follicular) phase of the cycle; the luteal phase is more likely to be about 14 days in length.

Menstruation occurs because of the effects of steroids on the endometrial lining of the uterus. During the follicular phase of the cycle, endometrial cells proliferate, glands enlarge and the endometrium becomes richly supplied with blood vessels (Box 6.42). Hence, it is termed the proliferative phase of the uterine cycle. After ovulation, progesterone secretion from the corpus luteum stimulates a further increase in endometrial thickness and secretion of a fluid to nourish a fertilized egg and encourage implantation of the conceptus. This is the secretory phase of the endometrium. If fertilization occurs, the hCG secreted by the developing placenta maintains the corpus luteum and, hence, progesterone secretion. In the absence of fertilization, the steroid support of the endometrium is lost due to luteolysis. The spiral arteries that have grown up into the endometrium contract and cells, starved of their normal blood supply, break away and menstrual loss ensues.

There are several markers to indicate ovulation has occurred. Detection of the LH surge using commercial assays of the hormone in urine is both sensitive and robust. The increase in progesterone after ovulation results in a small rise in body temperature and whilst this small rise can be used to indicate when ovulation occurs, obtaining reliable temperature measurements is difficult. Some women feel mild pain in the abdomen, lasting from a few minutes to a couple of hours, around the time of ovulation. Known as Mittelschmerz (German for ‘midpain’) it is probably caused by irritation of the abdominal wall due to blood and fluid escaping from the ruptured follicle. Changes in the cervical mucus also occur around the time of ovulation.

The presence of cyclic menstruation does not necessarily indicate ovulation is occurring mid-cycle. In anovulatory cycles menstrual bleeding is due to estrogen withdrawal after the non-ovulatory follicles have degenerated. Measurement of circulating progesterone concentrations in the expected luteal phase of the cycle (usually day 21) is a reliable indicator of corpus luteum formation and, thus, previous ovulation. Total blood loss during each menstruation varies from cycle to cycle, and in different women at different stages of their reproductive life. However, the average blood loss is about 50–60 ml although this can vary from about 10 to 80 ml. Excessive loss of blood (menorrhagia) can lead to iron-deficiency anemia.

Disturbances in the menstrual cycle are not uncommon. Returning to Clinical Case 6.2, it can be seen that failure to start menstruating (primary amenorrhea) by the age of 16 years is rare and highly likely to have an organic cause (Box 6.48). Clearly, from the foregoing, it will occur as a result of any insult to the hypothalamo-pituitary-ovarian axis. It will also be seen if there is failure of Müllerian duct development or when there are more minor vaginal abnormalities (such as an imperforate hymen) preventing menstrual flow. A list of the causes of secondary amenorrhea will be very similar to that for primary amenorrhea except that the structural lesions of the Müllerian duct system will be excluded for the obvious reason that for menses to have occurred at all the system must be present. A number of syndromes of hypogonadism occur clinically; the commoner ones are detailed in Box 6.49.

Box 6.48

Causes of primary amenorrhea. dysgenesis - e.g. Turner's syndrome damage - e.g. radiation

Much more common than the cessation of menstruation is its irregularity. This is discussed in the context of Clinical Case 6.8.

Polycystic ovary syndrome (PCOS)

PCOS is conventionally described as a combination of hyperandrogenism (often manifest as acne and hirsutism, anovulation (which may manifest, as in this case, with menstrual infrequency termed oligomenorrhea) and the appearances of polycystic ovaries on ultrasound scanning. It is very common, affecting some 20% of Caucasian women and being even more common in some ethnic groups e.g. Hispanic Americans or UK Asians. It is considered to arise during puberty and in 40% or so of cases it is associated with obesity. The high prevalence and variation in clinical features suggest that it is not a single disease entity and this may account for the variable endocrine findings in the condition.

Study of this condition is confounded because there is no animal model for PCOS, there is no male equivalent and ultrasound features are only present between the menarche and the menopause making multigenerational genetic studies very difficult. Additionally the ultrasound appearances of polycystic ovaries are found in any condition in which there is hyperandrogenism because androgen excess appears to prevent the final development of follicles (that subsequently form cysts). Thus, PCOS is seen universally in congenital adrenal hyperplasia (Clinical Case 4.3).

There are four main theories to explain the causation of PCOS - the adrenal, the ovarian, the pituitary and the metabolic. A role of the adrenal in the etiology of PCOS has been invoked because of the association of PCOS with increased DHEAS (a steroid secreted almost exclusively from the adrenal cortex). In a minority of patients, there is evidence for adrenal hyperandrogenism alone. More often, it is found in combination with excess androgens of ovarian origin. When patients with this syndrome are given tetracosactrin (a synthetic ACTH analog, see Box 4.20) or metyrapone (an inhibitor of 11β-hydroxylase which inhibits the final step in cortisol synthesis and hence causes a secondary increase in ACTH secretion, see Box 4.19) the increase in DHEAS and androstenedione secretion is higher than that in control subjects. In addition, there is evidence of increased activity of 11β-hydroxy-steroid dehydrogenase (the enzyme that breaks down active cortisol into inactive cortisone). This would tend to reduce negative feedback effects, increase ACTH secretion and lead to increased production of adrenal androgens.

The ovarian and pituitary theories are difficult to disentangle because the two glands are functionally linked and because prospective studies of girls developing PCOS at puberty have not been done. It is not known which abnormality develops first. The ovarian hypothesis states that the prime etiology for PCOS is due to an increased capacity of the thecal cells to synthesize androgens. This is seen in ovaries of PCOS patients and thecal cells appear hyperplastic.

There are also clear abnormalities in gonado-trophin secretion. Serum LH concentration and the LH to FSH ratio are both elevated in PCOS patients and these are associated with an increase in the amplitude and frequency of LH pulses and loss of the nocturnal decrease in LH pulse frequency. This may be caused both by an increase in GnRH pulsatility from the hypothalamus and an increased sensitivity of the pituitary gonadotrophs to this neurohormone. Indeed, PCOS patients show an exaggerated LH response to a bolus injection of GnRH. Whilst reduction of endogenous gonadotrophin secretion by pituitary desensitization with a GnRH superagonist lowers circulating testosterone concentrations in patients with PCOS, it is not certain whether abnormalities in gonadotrophin secretion are the primary abnormality.

Many PCOS patients demonstrate insulin resistance that is exacerbated by obesity and the metabolic hypothesis suggests that the hyperandro- genism and the ovarian features of PCOS are the result of this insulin resistance. Indeed insulin resistance and hyperandrogenism are seen in a number of clinical situations, not just PCOS. The fasting serum insulin concentration is inversely related to SHBG concentrations and, thus, the higher the insulin concentration (as occurs in insulin resistance) the higher the free concentrations of circulating androgens. Additionally, in hyperandrogenic women, an acute increase in insulin leads to a rise in circulating testosterone concentrations.

Studies performed on cultured tissue have shown that insulin and the structurally similar insulin-like growth factor (IGF-1) act synergistically with LH to stimulate androgen production by theca-interstitial cells and with FSH to increase aromatase activity and induce LH receptors on granulosa cells. Thus, the pituitary and metabolic theories overlap in the ovary where the effects of insulin and gonadotrophins interact. Within the ovary, it is possible that a vicious cycle occurs in which atretic follicles produce more androgens (because they lack aromatase) and androgens favor further atresia. In the granulosa cells, insulin may sensitize the cells to LH leading to premature luteinization of small follicles and large follicles that are subject to down-regulation by LH.

Clinical Case 6.9 did not have signs of hirsutism but she had oligomenorrhea and confirmed polycystic ovaries. She had a family history of type 2 diabetes and further investigations showed that she was markedly insulin resistant. Her serum progesterone concentrations in the luteal phase of her cycle showed that she was anovulatory. Over 70% women with PCOS are infertile and over 60% are hyperandrogenic and hirsute.

Hirsutism may be treated by suppressing androgen production or blocking androgen action (Box 6.51). Other treatments for PCOS are given in Box 6.52. Infertility is the other major problem facing women with PCOS and who, like Clinical Case 6.9, wish to conceive. Treatment is aimed at stimulating follicular development.

Box 6.51

Treatment of hirsutism *.

Box 6.52

Treatment of PCOS*.

Contraception

The fertile couple is not a medical problem but, in global terms, contraception has important clinical and social implications not only regarding unwanted pregnancies but also in limiting the birth rate in overcrowded, socially deprived areas throughout the world. In this respect hormonal contraception, first marketed in the early 1960s, has had an important medical impact.

Box 6.53

Orally active estrogens and progestagens (highlighted) used in many contraceptive agents. The addition of an ethinyl group on C₁₇ makes estradiol orally active. Mestranol incudes an etherification on C₃. Removal of the C₁₉ methyl group and addition of (more...)

A large number of hormonal contraceptives containing synthetic estrogens and/or progestagens are now available and some of the most widely used long-acting and orally active derivatives of estrogen and progesterone are shown in Box 6.54. Essentially there are two types of hormonal contraceptive preparations: combination pills in which estrogen and progesterone are either given sequentially, or as biphasic or triphasic combinations and progesterone only preparations which may be administered orally or as depot injections. Most popular today are the biphasic and triphasic oral contraceptives in which a combination of estrogens and progestagens are taken together for 21 days at differential doses which are most likely to resemble hormonal changes throughout the menstrual cycle. During the 7 steroid-free days a withdrawal bleed occurs.

Box 6.54

Selective estrogen receptor modulators (SERMS). The estrogen receptor has two regions that have a transcriptional activating function (TAF). One in the A/B domain is independent of ligand binding and the other in the E domain is dependent on ligand binding. (more...)

The underlying principle of the combined estrogen/progesterone contraceptives is to suppress ovulation by negative feedback. Additionally, progesterone can exert direct anti-fertility effects on the female genital tract hence inhibiting implantation. Progesterone-only pills, which are taken continuously, or subcutaneous depot injections that release the steroid over a period of 8 weeks to 5 years, work principally by their effect on the cervical mucus and perhaps the endometrium preventing fertilization and implantation. Ovulation is also suppressed in about 20% women taking progesterone-only contraception. Compliance is important for women taking oral progesterone-only contraception because its effects on the cervical mucus only last for about 22–26 hours, after which time fertility returns.

Finally there are the post-coital contraceptives (inappropriately named ‘the morning after pill’) in which high doses of estrogenic contraceptives in combination with a progestagen, are given within 72 hours of unprotected sex and in two doses 12 hours apart. Their action is to interfere with the transport and implantation of a possible conceptus.

Infertility

Couples are considered to be infertile if they have not conceived after a year of unprotected sex. Infertility is related to age. About 4% of couples in their early 20s are infertile with the percentage rising to nearly 20% by their late 30s. In about 35% of these couples the problem is with the female, 35% the male, 20% both partners whilst about 10% have unexplained infertility.

Endocrine causes of infertility are frequently associated with menstrual irregularities. Thus, abnormalities in the basic pattern of menstrual bleeding in the post-pubertal adult is most likely to be due to hormonal disturbances and a consequent loss of ovulation. This can occur at all levels of the hypothalamo-pituitary-ovarian axis. Central disturbances can be induced by environmental changes, physical and emotional stress and extreme weight loss. For example, in anorexia nervosa LH pulsatility reverts to a prepubertal pattern and the LH response to an injection of GnRH is abnormally low. This is indicative of low levels of endogenous GnRH secretion.

High circulating prolactin concentrations e.g. from a pituitary adenoma or during lactation can also suppress ovarian activity as may all causes of hypopituitarism (see Box 7.9). There may be insufficient FSH secretion to produce adequate growth and maturation of follicles whilst continuous secretion of LH in the absence of maturing follicles may result in the over-production of androgens leading to amenhorrhea and hirsutism. PCOS is the most common causes of anovulatory infertility.

Ovulation induction and assisted conception

Treatments for infertility caused by endocrine abnormalities are based on simple application of the physiological principles inherent in the control of the hypothalamo-pituitary-gonadal axis. In anovulatory patients with functional ovaries, clomiphene citrate can be used to induce ovulation. The action of this partial estrogen agonist in reducing the negative feedback of the more potent endogenous estradiol increases LH and FSH secretion. It is of no use in the treatment of primary ovarian failure or pituitary failure. An alternative therapy is with gonadotrophins but these are more complicated and expensive therapies compared with clomiphene citrate and have an increased risk of multiple pregnancies.

Until the 1980s, gonadotrophins were exclusively extracted from the urine of menopausal women. First marketed in the 1960s, they contained both LH and FSH activity but subsequently the LH was removed and a ‘pure’, more effective, FSH product became available. However, demand for gonadotrophin therapy increased and by the 1980s literally hundreds of thousands of liters of urine a day were required to meet demand. Human recombinant FSH is now available for induction of follicular development and recombinant LH is in the final stages of clinical trials.

For the treatment of hypogonadotrophic hygonadism resulting from hypothalamic dysfunction, pulsatile GnRH therapy using a programmable infusion pump is a more physiologic way of inducing ovulation. Though somewhat cumbersome, patients require less monitoring and ovarian hyperstimulation is less likely to occur than during gonadotrophin therapy.

The development of assisted conception techniques for infertile couples (where considered appropriate) required the developments in embryology of the 1970s. The first baby produced by in vitro fertilization was Louise Brown who was born on 25th July, l978. Today there are a variety of techniques but these procedures are not always available and are not cheap for the patients. Endocrinologically, to obtain eggs for fertilization in vitro and subsequent placement into the womb for implantation, the follicular phase of the menstrual cycle must be controlled. The initial stage of treatment is to suppress endogenous gonadotrophin secretion by administering a long-acting GnRH agonist (see Box 6.11). As a result of receptor down-regulation, the pituitary gland becomes desensitized and endogenous production of LH and FSH minimal. Treatment with gonadotrophins (either a mixture of LH and FSH, or, more commonly, ‘pure’ FSH) induces multiple follicular development that is monitored by ultrasound scanning. When the follicles have reached an appropriate stage of maturation (judged by size), hCG is administered (to simulate a natural pre-ovulatory LH surge) and the eggs retrieved for fertilization by aspiration under ultrasound guidance.

Ovarian failure, the menopause and andropause

The loss of menstruation has usually signalled pregnancy or the loss of reproductive capability and has been welcomed or hated accordingly. Clinical Case 6.9 was aware of the potential diagnoses and had sought confirmation. The circulating concentration of estradiol was very low (<37 pmol/l, NR 130–500 pmol/l) and the FSH and LH concentrations were high 67 IU/l (NR 2–9 IU/l) and 58 IU/l (NR 2–9 IU/l) respectively. These results indicated hypergonadotrophic hypogonadism due to ovarian failure and blood was taken for autoantibody studies in view of her pre-existing diagnoses of autoimmune Addison's and Hash-imoto's diseases. This confirmed that she had autoantibodies to ovarian tissue (see website). The diagnosis in this patient was autoimmune ovarian failure and a premature menopause. She was treated with sex steroid replacement. Unlike the situation for Clinical Case 6.6 (cyclophosphamide induced permanent azoospermia), the implications for fertility for Clinical Case 6.9 are not so final because of the potential availability of assisted conception and oocyte donation.

The menopause is defined as the cessation of menstruation at the end of a woman's reproductive life. Since menstrual periods may become very irregular peri-menopausally, the menopause is operationally defined as the absence of menstruation for a year. A variety of terms is associated with that of the menopause. It has been recommended that the term climacteric be avoided and the term peri-menopause be used for the time both prior to and just after the menopause. The term menopausal transition is used to refer to the period of menstrual cycle irregularity prior to the menopause.

Currently, the mean age of the menopause in the UK is about 53 years and average female life expectancy is 81 years. A woman may thus spend nearly 40% of her life in an estrogen-deficient state. Occasionally women may undergo a premature menopause (defined as <40 years) as seen in Clinical Case 6.9. It occurs because there are no ovarian follicles to develop and secrete estrogen under the influence of gonadotrophins. As a result, there is also no formation of a corpus luteum to secrete progesterone. The lack of female sex steroid hormones normally produced by the ovaries results in a loss of negative feedback and high circulating concentrations of gonadotrophins. The only source of female sex hormones after the menopause comes from the adrenal androgens converted peripherally to estrogens.

The loss of estrogen has several profound effects, not only physically but also psychologically. Some of these symptoms are limited to the peri-menopausal period when a woman is adjusting to the loss of her hormones. Others may become manifest at the menopause but can have serious consequences in the long term. Common symptoms associated with the peri-menopausal period are hot flushes and night sweats, vaginal dryness and depressive episodes. Hot flushes and night sweats affect about 70% of all menopausal women with about 25% seeking medical help due to the severity of symptoms. There is now increasing evidence that estrogens affect blood flow and dilatation of arterioles and thus symptoms linked with altered control of blood flow during estrogen withdrawal are not surprising. Loss of estrogen also leads to the thinning of the vaginal walls and loss of vaginal secretions. This causes vaginal dryness and sexual intercourse may become painful.

Psychological symptoms are often linked with the menopausal years, particular in those women who have a history of depression. To what extent these are due to social changes (e.g. children leaving home, marriage becoming dull) and negative cultural influences (aging and loss of reproductive status and sexuality) are difficult to determine. Nevertheless women do suffer from tiredness, lack of concentration, anxiety, tearfulness and loss of interest in sex.

Bone is severely affected in post-menopausal women so that during 2–5 years after the menopause there is an increased loss of bone mass (see Box 5.35). In both men and women, peak bone mass is usually achieved between the ages of 20 and 30 years. Thereafter there is a gradual age-related loss in both sexes. After the withdrawal of estrogens at the menopause, this bone loss is accelerated for several years, thereafter continuing at a similar rate to men. The result of this accelerated bone loss means women are more likely than men to suffer from fractures related to osteoporosis. Common fractures are those of the wrist, hip and spine (vertebrae). Compression fractures of vertebrae can occur without trauma and may cause the loss of several inches in height of an individual. Sex steroid replacement therapy can not only stop this accelerated bone loss but lead to an increase in bone density. When therapy is withdrawn, however, bone loss occurs again at an accelerated rate.

The other major long-term adverse affect of the menopause is on the cardiovascular system. This has been attributed to the metabolic effect of the loss of sex steroids increasing the atherogenic potential of circulating lipids leading to coronary artery disease and stroke. Epidemiologically, before the menopause women appear to be protected against cardiovascular disease by their sex hormones. Observational studies indicate the same is true for women taking HRT. The results of large-scale prospective studies of HRT in the primary prevention of coronary disease are awaited. A recent large prospective study on the effect of combined estrogen and progesterone in secondary prevention of cardiac events in older women with established coronary disease failed to show benefit. After the menopause or after withdrawal of HRT, their risk of developing cardiovascular disease becomes the same as that of men.

Hormone therapy at the menopause has also been suggested in epidemiological studies to reduce cognitive decline, Alzheimer's disease, cataract formation and colon cancer.

Whilst loss of female sex hormones, notably estrogens, do have profound physiological effects, there are clearly cultural influences on the way in which women experience and cope with menopausal symptoms. In Western cultures, social influences on the menopause are largely negative and women are left with the choice of being ‘saved’ by HRT or becoming old, sexless and a useless member of society. In contrast, in cultures where menopausal women achieve status and social advantages, the reported incidence of menopausal symptoms are often negligible or even absent. For example amongst the Rajput of Northern India, women who are past their menopause are no longer in purdah and are able to move freely within their community. Similarly the New Zealand Maoris view their post-menopausal years as a relief from child-bearing and thus the menopause is an attribute. Japanese women report a lower frequency of menopausal symptoms compared with American and Canadian women and the same is true for the Navaho Indians. Thus, whilst the menopause can be considered as the beginning of an estrogen-deficient state and will become an increasing health problem as longevity increases, cultural influences can affect the way women experience their menopause.

There is a question as to whether there is an equivalent ‘andropause’ in men. Testicular function and sperm production continues throughout life although loss of libido, impotence and failure to achieve orgasm do occur with higher frequency at increasing age. Whilst there is some evidence for a decrease in circulating concentrations of free testosterone, there is no evidence of a consequent rise in LH (or FSH) to suggest that this is sensed as abnormal by the hypothalamo-pituitary feedback loop. As a result, although the use of adjunctive androgen replacement therapy has been recommended by some, the ‘andropause’ cannot be seen in any way as analogous to the cataclysmic fall in female sex steroids that occurs with ovarian failure in all women.

Hormonal replacement therapy (HRT) and selective estrogen receptor modulators (SERMS)

Hormone replacement therapy may either be taken over a relatively short period of time to alleviate menopausal symptoms, such as hot flushes, or be taken prophylactically for several years or more to offset changes in bone density and cardiovascular risk. It must be noted, however, that when HRT is stopped the same physiological changes that accompany the untreated menopause still occur, but later in life. The time at which HRT should be started to prevent osteoporotic fractures is not known.

The usual estrogenic preparations are conjugated estrogens (glucuronides and sulfates), that are extracted from pregnant mares’ urine or synthetic estrogens such as estradiol or estradiol valerate. For women who have not undergone a hysterectomy, these estrogens need to be taken in combination with a progestagen because unopposed estrogen action causes excessive proliferation of the uterine lining and so increase the risk of developing endometrial cancer. The C-21 progesterone derivatives (e.g. medroxyprogesterone) or the C-19 derivatives of nortestosterone (i.e. norethisterone) may be used though they differ slightly in metabolic and androgenic effects. In cyclic HRT preparations, estrogen is given for about 25 days with a progestagen added for the last 10–14 days. A withdrawal bleed occurs.

For those women who object to the cyclic bleeding, continuous estrogens plus a progestagen (e.g. medroxyprogesterone) or a progestagen-impregnated intrauterine coil (Mirena coil containing levonorgestrel) can be used as an alternative. Others may use preparations such as tibolone, a synthetic steroid that has weak estrogenic, androgenic and progestagenic effects. It relieves menopausal symptoms, maintains skeletal integrity and does not cause endometrial hyperplasia.

Estrogens may also be given transdermally (as patches) to avoid the metabolic effects on the liver as they are absorbed from the gut or topically as intravaginal pessary, ring or cream. Progestagens are generally not given if a woman has had a hysterectomy.

Over the last few years there has been debate about HRT safety particularly with regard to the risk of developing breast cancer. In this regard, it has to be borne in mind that, although breast cancer is an emotive subject, the life time risk for the average Caucasian woman is 12% whilst the life time risk of coronary death is 35% and the risk for any osteoporotic fracture is 40%. Statistically, the increased risk is small and it has to be balanced against the adverse cardiovascular and skeletal effects of estrogen deficiency. Absolute contraindications to HRT include undiagnosed vaginal bleeding, active thromboembolic disease and active breast or endometrial cancer. Its use in cured breast cancer patients with marked menopausal symptoms remains under discussion. Estrogen use increases thromboembolic disease some 3-fold.

Drugs active at the estrogen receptor such as tamoxifen have been used for some time in the treatment of breast cancer. Around the menopause, this partial estrogen agonist/antagonist stabilizes bone loss and produces estrogen-like changes in plasma lipid profiles but has little, if any, action on breast tissue or the endometrium. It may, however, make vasomotor effects worse in the perimenopausal period. The large trials that established tamoxifen benefit in breast cancer failed to show any cardiovascular benefits. Whilst it has been in use for many years, its mode of action has only recently been understood. It binds the estrogen receptor but prevents the activity of one of the transcription domains on the receptor (Box 6.54). Thus, it has partial agonist/antagonist properties and these depend on the target tissue.

There is now intense investigation to develop SERMS, like tamoxifen, that exert selective estrogenic effects (e.g. on bone and plasma lipids) and anti-estrogenic or no effect on tissues where estrogen stimulation may be undesirable (e.g. breast and endometrium). One important current contender is raloxifene that has been shown to reduce fracture risk. The results of large-scale trials in the primary prevention of coronary disease are ongoing.

Clinical case questions

Clinical Case Study Q6.2

An otherwise healthy neonate has the external genitalia shown (Box Q6.3).

Genitalia of neonate in Clinical Case Study Q6.2

Question 1: What are the possible causes of these appearances and how would you investigate the child?

Question 2: No masses are palpable in the groins. What is the most likely diagnosis and what investigations would you perform?