CHAPTER 21

Disorders of puberty and sex development

S. Faisal Ahmed; Jane D. McNeilly

CHAPTER OUTLINE

INTRODUCTION

The clinician is most commonly faced with the investigation and management of abnormal sexual development at two age periods: birth and puberty. Abnormal sexual development at birth is the result of a disorder of sex development (DSD) presenting with genitalia that are either atypical for sex or ambiguous. Disorders of sex development can also present at adolescence as abnormalities of pubertal development. However, in most cases, abnormal pubertal development is due to an alteration in the regulation of puberty in an individual without a DSD. To understand these disorders there is a need to understand the normal processes of sex and pubertal development.

NORMAL SEX DEVELOPMENT

Male or female sex development is programmed at fertilization, depending on whether the zygote is heterogametic (46XY) or homogametic (46XX). A scheme summarizing the embryology and genetic control of sex development in the male and female fetus is shown in Figure 21.1. The indifferent, or primitive, gonad develops from a thickened ridge of coelomic epithelium that arises from the intermediate mesoderm. Cell precursors from this region give rise to the kidneys, adrenals and gonads. The adrenogenital primordium comprises a single population of steroidogenic factor-1 (SF-1) immunoreactive cells. The gonadal ridge becomes populated with primordial germ cells that have migrated from the wall of the yolk sac.

The first histological expression of fetal testicular development is the appearance of seminiferous or sex cords at between six and seven weeks of gestation. Surrounding the sex cords is the interstitial region, which contains the precursors of Leydig cells and also the peritubular myoid cells. Primitive interstitial cells differentiate later into Leydig cells, an event that coincides with the onset of testosterone synthesis by the fetal testis. Steroidogenesis is placental human chorionic gonadotrophin (hCG)-dependent and results in the production of fetal testosterone concentrations within the normal adult male range. The pivotal role for the Y chromosome in male sex differentiation has been recognized for more than 40 years, based on the phenotype of individuals with sex chromosome aneuploidies such as XXY and XO. The study of sex-reversed XX males and XY females led to the identification of the SRY gene (sex-determining region Y) on the short arm of the Y chromosome. That this is the prime testis-determining gene is illustrated by the introduction of the Sry homologue into a transgenic XX mouse and a resulting male phenotype. The majority of XX males are positive for the SRY gene, as a result of terminal exchange between the X and Y chromosomes during paternal meiosis. In the case of XY females, mutations have been identified that disrupt the function of the SRY protein, which acts as a transcription factor. However, these are found in only 15–20% of patients, indicating there are other genes also involved in testis determination. SRY acts to up-regulate the transcription of the related gene, Sox9 in the Sertoli cell lineage. High concentrations of SOX9 protein are maintained in these cells through a positive interaction with the secreted molecules fibroblast growth factor 9 (FGF9) and prostaglandin D2 (PGD2). These molecules are important in securing two other necessary steps in testis development: the recruitment of other somatic cells to differentiate into the Sertoli cell lineage and, in the case of FGF9, spread of the initial central testis-determining signal to the anterior and posterior poles of the gonad. Not only does SOX9 promote testis development, but it also antagonizes the Wnt signalling pathway and FOXL2, both of which are crucial for ovarian development and which can, themselves, antagonize SOX9 action.

With the committed development of the gonad as a testis, two trophic factors produced by the testis control subsequent differentiation of internal and external genitalia in the male. Anti-Müllerian hormone (AMH) is produced by the Sertoli cells and signals through two trans-membrane receptors expressed in the mesenchyme that gives rise to the Müllerian ducts. Anti-Müllerian hormone acts ipsilaterally to cause regression of the Müllerian ducts, the anlage (rudimentary precursor) of the uterus, fallopian tubes and upper two-thirds of the vagina. Circulating concentrations of AMH in males are high in early infancy and fall gradually throughout childhood, until they become undetectable after puberty; its measurement is useful for detecting testicular tissue, especially in early childhood. Testosterone is the other key trophic factor produced by the early developing testis. There is indirect evidence, based on luteinizing hormone (LH) receptor mouse knockout models and human LH β-chain mutations, that production is initially autonomous, but, thereafter, is under the control of hCG and then fetal pituitary gonadotrophin secretion. High local concentrations of testosterone stabilize Wolffian duct development in the male to differentiate into the vas deferens, epididymis and seminal vesicles. The external genitalia in both sexes develop from a common anlage, comprising the urogenital sinus, genital tubercle and swellings and urethral folds. In the male, androgens, again, play the key role in differentiation of these primordial structures, to develop as the penis, scrotum and opening of the urethra on the glans. Dihydrotestosterone (DHT) is necessary to provide an amplification of the androgenic effect. The evidence for DHT dependency is illustrated by the predominantly female external genital phenotype in 5α-reductase deficiency.

In the absence of the testis-determining factors, testosterone and AMH, and in the presence of the trophic proteins necessary for ovarian development, the gonad associated with the 46XX karyotype develops as an ovary and the Müllerian ducts differentiate normally into female internal genital ducts. The external genital anlage, in the absence of androgens, remains underdeveloped with respect to growth of the genital tubercle and the absence of midline fusion of the labioscrotal folds and swellings. The development of the lower part of the vagina from canalization of the vaginal plate is incompletely understood, but the process is probably influenced by oestrogens. Virilization of the female external genitalia from an extraneous source of androgens leaves the development of the female internal genitalia intact.

NORMAL PUBERTAL DEVELOPMENT

Endocrinology of normal puberty

The precise trigger that initiates the onset of puberty in the human remains an enigma. There is activation of pituitary gonadotrophin secretion both in early fetal life and, again, after birth for several months. During this latter period, plasma concentrations of gonadotrophins and sex steroids may reach values normally observed at puberty. Throughout later infancy and childhood, gonadotrophin concentrations remain low, although evidence of pulsatile LH secretion using more sensitive immunoassays can be detected in some children. Consequently, puberty represents the reactivation of gonadotrophin secretion that has been restrained during childhood.

The initial endocrine event at puberty is an increase in nocturnal pulsatile LH secretion in response to gonadotrophin releasing hormone (GnRH) released into the pituitary portal system. The neurons that release GnRH migrate to the hypothalamus, from the medial olfactory placode, during fetal development. A failure of such migration is considered to be the cause of some inherited forms of hypogonadotrophic hypogonadism that are associated with anosmia (Kallman syndrome). A GnRH pulse generator controls the onset of puberty by causing a progressive increase in amplitude and frequency of LH pulses, which occurs about one year before the onset of physical signs of puberty. The neuroendocrine mechanisms that cause activation of the GnRH generator at the appropriate age are extremely complex, but appear to involve excitatory amino acids, catecholamines, neuropeptide Y, leptin and acetylcholine, as well as external influences such as nutritional intake. Furthermore, an additional factor discovered as a result of studies in patients with hypogonadotrophic hypogonadism, is the kisspeptin/GPR54 system, which appears to be a gatekeeper of reproductive development. GPR54 is a G-protein-coupled receptor whose endogenous ligand is a 54-amino acid peptide, cleaved from the parent 145-amino acid peptide encoded by the KISS-1 gene. GPR54-deficient mice are infertile and have low circulating gonadotrophin concentrations. Mutations in the GPR54 gene cause hypogonadotrophic hypogonadism in humans. It appears that GPR54 is one of the key factors in the control of puberty. Administration of kisspeptin stimulates LH and follicle stimulating hormone (FSH) secretion, which can be blocked by GnRH antagonists, indicating that the kisspeptin/GPR54 system effect on the hypothalamic–pituitary–gonadal axis is mediated via GnRH release.

Increased gonadotrophin secretion leads to a gradual increase in the secretion of gonadal steroids, principally testosterone in males and oestradiol in females. Random, daytime sex steroid measurements are seldom predictive of pubertal events in an individual, although early morning testosterone concentration as a reflection of nocturnal secretion is more useful. Inhibin B, a marker of Sertoli cell function, increases early in puberty and reaches adult concentrations by mid-puberty. Spermatogenesis starts between 11 and 15 years of age and sperm can be detected in early morning urine specimens by 13 years of age.

The attainment of reproductive capacity in the female is dependent on cyclical gonadotrophin secretion to ensure ovulation. Follicle stimulating hormone-induced oestrogen secretion, by developing ovarian follicles, leads to a positive feedback on LH secretion and a midcycle surge that induces rupture of the mature follicle. After ovulation, the luteinized follicle secretes progesterone while oestradiol concentrations fall. Inhibin B secretion predominates during the follicular phase, while inhibin A secretion is dominant during the luteal phase. All these events are coordinated by pulsatile release of GnRH secretion and modulation of gonadotrophin and sex steroid production, by both negative and positive feedback loops. It is not surprising that there is generally a 1–2-year interval after menarche before the majority of girls are ovulating regularly. Ultrasonography can be used to assess the development of reproductive function in girls. Enlargement of the uterus and endometrial thickening are evident after birth because of the effect of maternally derived oestrogens. Multiple ovarian follicular cysts are often seen on ultrasound at this time. The effect of increasing secretion of oestradiol during puberty can be observed by appropriate morphological changes in the appearance of the uterus. The prepubertal uterus starts to increase in size from seven years of age onwards.

The onset of increased production of dehydroepiandrosterone (DHEA) and its sulphate (DHEAS) by the zona reticularis of the adrenals, between six and eight years of age, defines the phenomenon of adrenarche. Concentrations of DHEAS thereafter increase throughout puberty into early adult life in micromolar amounts and then start to decline gradually in older age (adrenopause). There is no concomitant increase in adrenal glucocorticoid and mineralocorticoid secretion, so ACTH is not the primary trophic factor controlling DHEA production. Intra-adrenal modulation of steroidogenesis, by post-translational regulation of the 17-hydroxylase enzyme (by phosphorylation on serine/threonine residues, and electron transfer by the P450-oxidoreductase enzyme), is the key factor in the production of C19 steroids such as DHEA. There is also a relative underexpression of the enzyme 3β-hydroxysteroid dehydrogenase enzyme in the zona reticularis that contributes to a preponderance of Δ⁵ steroids. Extra-adrenal factors postulated to play a role in adrenarche include prolactin, oestrogens, growth factors and cytokines. There is an association between leptin concentration and the timing of adrenarche in obese children.

Physical signs of normal puberty

Puberty is the transitional period between childhood and adulthood that spans adolescence and leads to the acquisition of reproductive capacity. The time span for the physical changes to take place is generally 4–5 years, but individual variations can result in a time interval of 2–6 years. This is referred to as the tempo of puberty. The age of onset of puberty also varies considerably, with epidemiological evidence suggesting that puberty may now be starting earlier.

The first sign of puberty in girls is breast development, starting, on average, at 11 years with an age range of 8–13 years (see Fig. 21.2A). This is termed ‘thelarche’ and starts as a small mound of tissue beneath the nipple manifest as a breast ‘bud’, which is usually distinguishable, on palpation, by its firmness compared with the softer and more diffuse texture of subcutaneous fat. Further development of the nipple, areola and underlying breast tissue takes place over the ensuing four years or so (Tanner stages B2–B5). Epidemiological studies conducted in the USA indicate that 25% of girls may have already started thelarche by eight years of age. However, it is unclear whether misinterpretation of adipose tissue as breast development may have skewed the data.

Coincident with breast development is the onset of growth of pubic hair which may precede thelarche in 10% of girls. Hair growth usually starts on the labia before spreading over the mons pubis and is quantified as Tanner stages PH2–5. Sometimes there may be further growth on the upper medial aspects of the thighs and along the linea alba (PH6). Axillary hair starts to appear at about 12.5 years of age and takes a further 18 months to reach adult distribution.

Menarche, the onset of menses, is a relatively late event in the pubertal process and occurs at around 12.5–13 years of age. This coincides with breast stage B4. A trend in the lowering of the age of menarche has occurred in the past, but the reduction has only been by a few months during the past 30 years. There are racial differences as well as geographical variations. The age of menarche tends to be slightly higher in Northern than Southern Europe.

Increased growth velocity is a measurable, indirect sign of puberty. In girls, peak height velocity (defined as the maximum rate of growth achieved during puberty, which is usually ~ 10 cm/year) is achieved relatively early in puberty (during breast stages B2–3) and before the onset of menarche. By the time of menarche, the majority of adult height has been achieved. Changes in body composition also occur, particularly with respect to fat distribution.

The first sign of puberty in boys is an increase in testis size (see Fig. 21.2B). Testicular size can be assessed clinically by the use of a series of standard ovoids (the Prader orchidometer). Testicular volume remains between 2–3 mL throughout infancy and childhood, an increase to 4 mL heralds the onset of puberty that occurs, on average, at 11.5 years with an age range of 9–14 years. Progressive enlargement of the testes takes place over three years with testicular volumes reaching up to 25 mL in adult life. Leydig cells constitute only a small part of testicular volume, the majority being the result of an increase in Sertoli cell and seminiferous tubule number and size.

Pubic hair growth, which may start as a few scrotal hairs, follows closely on testicular development and is quantified as Tanner stages PH2–5. Spread of hair up the abdominal wall is characteristic in many men and is classified as stage PH6. Growth of the penis, first in length and then in breadth, occurs concomitantly and is rated G1–G5. Growth of axillary and facial hair, are both later events in puberty, ranging in age from 14 to 16 years. ‘Breaking’ of the voice is due to enlargement of the larynx and elongation of the vocal cords and does not occur until stages G3–G4.

Peak height velocity in boys corresponds with stages G4 and PH4 and testicular volumes of 12–15 mL. This occurs, on average, at a chronological age of 14 years, as opposed to 12 years in girls. The adult male is, on average, taller than the adult female by 13 cm. This is partly due to the fact that boys start the growth spurt later and are, therefore, taller at its onset. In addition, the magnitude of growth achieved during the growth spurt is greater in boys (28 cm in males and 20 cm in females). Body composition alters in favour of increased muscle bulk and relatively less subcutaneous fat.

DISORDERS OF SEX DEVELOPMENT

Terminology of disorders of sex development

It is important to define terms used in relation to the investigation and management of disorders of sex development that present at birth or in childhood. Sex assignment (often used interchangeably with gender assignment), is the sexing of an infant at birth as being male or female. This is straightforward in the vast majority; indeed, sex assignment is increasingly an activity that occurs before birth. Gender identity refers to how individuals perceive themselves as being male or female, while gender role describes characteristics that are sexually dimorphic within a normal population. In childhood, for example, male-typical behaviour is attributed to toy preferences being vehicles and soldiers as opposed to playing with dolls, generally preferred by girls. This rather simplistic illustration of sexual dimorphism has some physiological underpinning from observations of girls’ play behaviour following exposure to androgens in utero. Thus, gender role is more male-typical in girls with congenital adrenal hyperplasia. There is less convincing evidence that prenatal hormones affect gender identity. Sexual orientation refers to the subject of erotic arousal, which may be heterosexual, homosexual or bisexual. Gender dysphoria or gender identity disorder are terms that describe gender dissatisfaction. The phenomenon appears to be more common in individuals with disorders of sex development than in the general population, but it is difficult to identify predisposing factors related to the specific disorder.

The use of terminology that is clear and easy to use and understand by all health professionals, patients and their families is fundamental to the understanding, investigation and management of affected newborns and children. In addition, terminology should respect the individual and avoid terms that might cause offence. The term ‘intersex’ has had variable connotations even among professionals; some employed it as a term that covered all affected newborns while at the other end of the spectrum, some believed that the term should only apply to those where there is complete mismatch between chromosomal and anatomic sex. The consensus reached in Chicago in 2005 on the management of these patients, stressed the importance of terminology and recommended a substitution for the term ‘intersex’ for ‘disorder of sex development (DSD)’, which is defined as any congenital condition in which development of chromosomal, gonadal or anatomic sex is atypical. It also recommended the abandonment of terms such as ‘pseudohermaphroditism’ and ‘true hermaphroditism’. While this new nomenclature (Table 21.1) is easier to use and understand, it will nevertheless evolve over time as our understanding of long-term outcome and molecular aetiology improves. Given that genital anomalies may occur as commonly as 1 in 300 births and may not always be associated with a functional abnormality, some have advocated the use of ‘differences’ in preference to the term ‘disorder’. The strength of the abbreviation ‘DSD’ is that it can be used to cover both differences and disorders of sex development. However, the likelihood of this difference existing as a disorder will depend on the functional implications of the condition, which may be heavily influenced by the social and cultural framework within which the child exists.

General principles of management

The management of a child with an abnormality of genital development can often be difficult, particularly in patients for whom the sex of rearing is uncertain. The initial contact with the parents of a child with a DSD is important as first impressions from these encounters often persist. A key point to emphasize is that a child with a DSD has the potential to become a well-adjusted, functional member of society. Establishing a dialogue and building rapport with the affected child and the parents, evaluating the child and then developing a logical, as well as pragmatic, plan for investigations are central to the initial approach and ongoing management. It is paramount that any child or adolescent with a suspected DSD is assessed by an expert with adequate knowledge about the range of variation in the physical appearance of genitalia. If there is any doubt, the patient should be discussed with the regional team. For most patients, particularly in the case of the newborn, the paediatric endocrinologist within the regional DSD team acts as the first point of contact. The underlying pathophysiology of DSD and the strengths and weaknesses of the tests that can be performed, should be discussed with the parents and child, as appropriate, and tests undertaken in a timely fashion. With babies in whom there is true genital ambiguity, it should be explained to the parents that the best course of action may not initially be clear, but the healthcare team will work with the family to reach the best possible set of decisions in the circumstances. Finally, in the field of rare conditions, it is imperative that the clinician shares the experience with others through national and international clinical and research collaboration.

General examination of a newborn with suspected DSD

The physical examination should determine whether there are any dysmorphic features and the general health of the baby. Affected infants, particularly those who have XY DSD, are more likely to be small for gestational age and may display other developmental abnormalities. In addition, the affected infant should be examined for midline defects which may point towards an abnormality of the hypothalamo-pituitary axis. The state of hydration and blood pressure should be assessed, as adrenal steroid biosynthetic defects can be associated with a variable extent of salt loss, masculinization and hypertension. In congenital adrenal hyperplasia (CAH), cardiovascular collapse with salt loss and hyperkalaemia does not usually occur until the second week of life (with salt loss usually evident from day four) and so will not be apparent at birth in the well neonate, but should be anticipated if the diagnosis is suspected. Jaundice (both conjugated and unconjugated) may be observed in babies with hypopituitarism or cortisol deficiency. Urine should be checked for protein as a screen for any associated renal anomaly (e.g. Denys–Drash or Frasier syndromes) and a pre-feed blood glucose concentration should be measured to exclude hypoglycaemia (suggestive of hypopituitarism, or occasionally CAH, e.g. 3β-hydroxysteroid dehydrogenase deficiency). Renal tract anomalies, such as ureteropelvic junction obstruction, vesicoureteric reflux, pelvic or horse-shoe kidney, crossed renal ectopia and renal agenesis, are reported to be more common in children with DSD.

Evaluation of the external genitalia

If the appearance of the external genitalia is sufficiently ambiguous to render sex assignment impossible, or the phenotype is not consistent with prenatal genetic tests, then investigations are clearly required. However, the ability to evaluate external genitalia fully may depend on the expertise of the observer and, before presentation to a specialist, the label of ambiguous genitalia has often already been assigned to newborns where the most appropriate sex of rearing was not clear to those present at the child’s birth. The birth prevalence of genital anomalies may be as high as 1 in 300 births but the birth prevalence of complex anomalies that may lead to true genital ambiguity on expert examination may be as low as 1 in 5000 births.

Apart from those whose genitalia are truly ambiguous, infants can often be divided into those who overall seem to have largely male or female genitalia but with some unusual features. However, it is very important to bear in mind that a 46XX newborn infant with congenital adrenal hyperplasia can either present as a girl with clitoromegaly or as a boy with bilateral undescended testes. When evaluating these infants, the clinical features of the external genitalia that require examination include the presence of gonads in the labioscrotal folds, the fusion of the labioscrotal folds, the size of the phallus and the site of the urinary meatus on the phallus, although the real site of the urinary meatus may, sometimes, only become clear on surgical exploration. These external features can be individually scored to provide an aggregate score, the external masculinization score (EMS) (see Fig. 21.3A), or they can be graded according to their overall description as classically described by Prader staging (see Fig. 21.3B).

Infants with suspected DSD who require further clinical evaluation and need to be considered for investigation by a specialist, should include those with isolated perineal hypospadias, isolated micropenis, isolated clitoromegaly, any form of familial hypospadias and those who have a combination of genital anomalies with an EMS of less than 11. This will avoid unnecessary detailed investigations of boys with isolated glandular or mid-shaft hypospadias and boys with unilateral inguinal testis. The coexistence of a systemic metabolic disorder, associated malformations or dysmorphic features would lower the threshold for investigation as would a family history of consanguinity, stillbirths, multiple miscarriages, fertility problems, genital abnormalities, hernias, delayed puberty, genital surgery, unexplained deaths and the need for steroid replacement. In addition, maternal health and drug exposure during pregnancy, and the pregnancy history itself, may hold key information.

Evaluation of the internal anatomy

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