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Normal and abnormal sexual differentiation 

Normal and abnormal sexual differentiation

Chapter:
Normal and abnormal sexual differentiation
Author(s):

I.A. Hughes

DOI:
10.1093/med/9780199204854.003.130903_update_001

Update:

Sex chromosome disorders of sex development (DSD)—SOX3 gene can be involved in testis determination and explains the paradox of testis development in some XX males lacking SRY. Description of newly discovered genes that explain the molecular basis of XY gonadal dysgenesis, including CBX2 and MAPK31.

Clinical features associated with variations in the androgen receptor.

Updated on 30 Nov 2011. The previous version of this content can be found here.
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Essentials

Human sex development follows an orderly sequence of embryological events coordinated by a cascade of gene expression and hormone production in a time- and concentration-dependent manner. Underpinning the entire process of fetal sex development is the simple mantra: sex chromosomes (XX or XY) dictate the gonadotype (ovary or testis), which then dictates the somatotype (female or male phenotype).

The constitutive sex in fetal development is female. Male development to form a testis from the indifferent gonad (sex determination) and the internal and external phenotype (sex differentiation) is due to (1) testis-determining genes, in particular SRY (sex chromosome related gene on the Y chromosome), which is first expressed in the XY gonad at 6 to 7 weeks of gestation; and (2) production by the testis of (a) antimullerian hormone—to repress mullerian ducts forming the uterus and fallopian tubes, (b) androgens (testosterone and dihydrotestosterone)—to stabilize the Wolffian ducts; and (c) insulin-like factor 3 (INSL3)—required for the migration of the testis from the urogenital ridge to its site at birth within the scrotum. An understanding of these basic principles is essential to formulate a logical approach to the diagnosis of disorders of sex development.

Disorders of sex development (DSD)—these can be classified into three broad categories based on the knowledge of the karyotype: (1) sex chromosome abnormality—e.g. XO/XY, mixed gonadal dysgenesis; (2) XX DSD—e.g. congenital adrenal hyperplasia (see Chapter 13.7.2); (3) XY DSD—e.g. partial androgen insensitivity syndrome.

Clinical features—the commonest presentations of DSD are (1) ambiguous genitalia of the newborn; and (2) development of secondary sexual characteristics at puberty discordant with the sex of rearing—generally signs of virilization occurring in a child hitherto assumed to be female.

Investigation and management—an extensive repertoire is available, but the choice of genetic, endocrine, and imaging tests should be based on the DSD classification (determined by sex chromosome analysis by fluorescent in situ hybridization (FISH) using X centromeric and SRY probes, followed by full karyotyping) and aimed at reaching a consensus about the choice of sex assignment. Any surgical procedure required to alter the external phenotype consonant with sex assignment need not be undertaken urgently. It is essential that families of children with DSD have the benefit of support and counselling by a multidisciplinary team that comprises at a minimum an endocrinologist, urologist/gynaecologist, geneticist, and psychologist.

Normal fetal sex development

The following key facts underpin the mechanism of dimorphic sex development:

  • The constitutive sex is female.

  • Male development requires the presence of a Y chromosome, a testis, and the production and action of testosterone during a critical time in early gestation.

  • Fetal experiments in mammals indicate that early castration leads to a female phenotype, despite the presence of the Y chromosome (Jost’s hypothesis).

  • Absence of an ovary does not affect the female phenotype at birth (e.g. Turner syndrome).

  • Oestrogen is not required for fetal female development, but androgen is essential for fetal male development.

The link between sex chromosomes, gonad determination and the expression of the phenotype (somatotype) is illustrated in simple configuration in Fig. 13.9.3.1. The events that occur during fetal male development are depicted in Fig. 13.9.3.2. The indifferent gonad develops in the urogenital ridge, where the kidney and adrenals also have their origins. This is germane to the frequent association of urinary tract anomalies with ambiguous genitalia and the occasional occurrence of nests of adrenal remnants found in the testis of males with congenital adrenal hyperplasia (see Chapter 13.7.2). Germ cells migrate from the yolk sac to take their position within the developing gonad. The testis is histologically defined initially by its seminiferous tubules and predates comparable maturation of the ovary by a few weeks. Three products of the somatic component of this testis are key to development of the male phenotype, i.e. sex differentiation. Anti-mullerian hormone (AMH), a product of Sertoli cells, acts on its type II receptor to repress mullerian ducts forming the uterus and fallopian tubes. This process is permitted to occur in the female because of the absence of AMH at this stage in gestation. Testosterone produced by Leydig cells under the control initially of placental human chorionic gonadotropin (hCG) acts locally in high concentration to stabilize the wolffian ducts. These form the vas deferens, epididymis, and seminal vesicles. A further product of the Leydig cells, insulin-like factor 3 (INSL3), is required for the transabdominal phase of migration of the testis from the urogenital ridge to its site at birth within the scrotum. The inguinoscrotal phase of testis descent in late gestation is under the control of androgens. All these events take place in a specific chronological order and are controlled by genes and hormones expressed at critical dosage thresholds and timing. The genetic and hormonal control of the events in male sex development is shown in Fig. 13.9.1.3. Not all the genes characterized for mammalian development are shown, but those identified in the human and relevant to disorders of sex development are emphasized. The formation of the urogenital ridge is dependent on factors such as WT1 and SF1, their role vividly illustrated by mouse gene knock-out studies (absence of gonads and kidneys or adrenals, respectively) and syndromes of urogenital anomalies (WAGR, Denys–Drash, Frasier syndromes) and combined gonadal dysgenesis/adrenal insufficiency in humans with inactivating mutations of WT1 and SF1 genes, respectively.

Fig. 13.9.3.1 The basic components of fetal sex development.

Fig. 13.9.3.1
The basic components of fetal sex development.

Fig. 13.9.3.2 The embryology of fetal sex development in the male. Mesoderm represents the tissue source for the somatic components of testis development. The solid line denotes the rise in fetal serum testosterone levels (nmol/litre).

Fig. 13.9.3.2
The embryology of fetal sex development in the male. Mesoderm represents the tissue source for the somatic components of testis development. The solid line denotes the rise in fetal serum testosterone levels (nmol/litre).

The master regulator of testis development (sex determination) is SRY (sex chromosome related gene on the Y chromosome) which is first expressed in the XY gonad at 6 to 7 weeks of gestation, just before the indifferent gonad differentiates as a testis. SRY is a 204 amino acid protein functioning as a high mobility group (HMG) box transcription factor. The HMG box of 79 amino acids is related to similar proteins such as SOX (SRY-like HMG box) which is also a key protein in the regulation of testis development. That SRY is essential for testis development is supported by the following observations: (1) translocation of SRY to the X chromosome during paternal meiosis is present in 90% of XX males (2) mutations in SRY in 15 to 20% of XY females with complete gonadal dysgenesis leads to complete sex reversal; (3) induction of a male phenotype occurs by transgenic insertion of Sry in XX mice. However, the observation that 10% of XX males lack SRY and the vast majority of XY gonadal dysgenic females have a normal SRY indicates that other genes must also be involved in testis determination. Candidates such as SOX9 and SF1 play some role, but their inactivation in humans leads to various syndromes of which gonadal dysgenesis is only one component. The role of clinicians recording detailed phenotypes in cases of disordered sex development is essential to continue the search for the multitude of genes that must be involved in testis determination. What is known in the human is summarized in Fig. 13.9.3.3. The ovary is devoid of known factors, although genes such as WNT4, RSPO1, and DAX1 may act in female development by suppressing testis-determining genes.

Fig. 13.9.3.3 Genetic and hormonal control of fetal sex development. Emphasis is placed on genes relevant to human development.

Fig. 13.9.3.3
Genetic and hormonal control of fetal sex development. Emphasis is placed on genes relevant to human development.

The differentiation of the internal genital ducts and external genitalia into male structures is entirely androgen dependent. For this to occur, an intact pathway of gonadotrophin-induced steroidogenesis is required to produce the potent androgens testosterone and dihydrotestosterone (DHT), which in turn promote androgen signalling by ligand activating the nuclear androgen receptor (AR) in target cells. The pathways of testicular steroidogenesis and the ligand-activated AR signalling are shown in Figs. 13.9.3.4 and 13.9.3.5, respectively. The enzymatic steps in androgen production are encoded by genes, each of which, when mutated, results in syndromes of undermasculinization. Some of the more proximal enzyme defects also involve adrenal steroidogenesis and lead to syndromes that include adrenal insufficiency (see Chapter 13.7.1).

Fig. 13.9.3.4 Pathway of androgen biosynthesis, including aromatase conversion to estrogen. 3β‎HSD, 3β‎-hydroxysteroid dehydrogenase; 5α‎RD, 5α‎-reductase; 17β‎HSD, 17β‎-hydroxysteroid dehydrogenase; P450arom, P450 aromatase. The cognate genes are depicted in italics.

Fig. 13.9.3.4
Pathway of androgen biosynthesis, including aromatase conversion to estrogen. 3β‎HSD, 3β‎-hydroxysteroid dehydrogenase; 5α‎RD, 5α‎-reductase; 17β‎HSD, 17β‎-hydroxysteroid dehydrogenase; P450arom, P450 aromatase. The cognate genes are depicted in italics.

Fig. 13.9.3.5 Schematic of androgen action in a target cell. ARA70, an androgen receptor specific coactivator; GTA, general transcriptional apparatus; HSP, heat shock protein; P, phosphorylation; SHBG, sex hormone binding globulin.

Fig. 13.9.3.5
Schematic of androgen action in a target cell. ARA70, an androgen receptor specific coactivator; GTA, general transcriptional apparatus; HSP, heat shock protein; P, phosphorylation; SHBG, sex hormone binding globulin.

Androgen signalling is mediated by a single AR encoded by an X-linked gene at Xq11–12. The AR is a member of a large family of nuclear receptors that comprise four general functional domains: an N-terminal transactivation domain, a central DNA binding domain, a hinge region and a C-terminal domain to which the ligand binds (Fig. 13.9.3.6). Subdomains are involved in dimerization, nuclear localization and transcriptional regulation. Circulating androgens are bound to carrier proteins such as sex-hormone binding globulin (SHBG) and albumin but diffuse freely in to target cells where the ligand binds to cytoplasmic AR complexed to heat shock proteins. The hormone–receptor complex translocates to the nucleus where it binds to DNA response elements as a homodimer. An added refinement in androgen signalling is provided by interaction with a number of coactivator proteins to enhance up-regulation of androgen-responsive genes via the general transcriptional machinery. Androgens have pleiotropic effects beyond fetal male development. These include the development of secondary sexual characteristics at puberty, muscle and skeletal growth, stimulation of sebaceous glands, and elongation and thickening of the vocal cords that give rise to the ‘voice breaking’ characteristic of the later stages of male puberty. Prostate development and growth is also androgen dependent, a feature that is countered by antiandrogenic forms of treatment for prostate cancer. It is also clear that androgens also have effects on brain development, with prenatal influences being especially relevant to the sex dimorphism in gender role behaviour. Much of the evidence for the role of androgens in psychosocial functioning has come from studies of females exposed to excess androgens (e.g. congenital adrenal hyperplasia) and syndromes associated with defects in androgen signalling (e.g. complete androgen insensitivity syndrome).

Fig. 13.9.3.6 Distribution of mutations in androgen insensitivity syndrome (AIS) as recorded on the Cambridge DSD Database. The three functional domains of the AR are indicated, encoded by their respective exons (1–8). The numbers within the vertical bars are a summation of nonsense and missense mutations and a breakdown according to type of AIS is denoted above the AR. MAIS, minimal/mild androgen insensitivity syndrome.

Fig. 13.9.3.6
Distribution of mutations in androgen insensitivity syndrome (AIS) as recorded on the Cambridge DSD Database. The three functional domains of the AR are indicated, encoded by their respective exons (1–8). The numbers within the vertical bars are a summation of nonsense and missense mutations and a breakdown according to type of AIS is denoted above the AR. MAIS, minimal/mild androgen insensitivity syndrome.

Definitions, terminology, and nomenclature

Clarity of thought is required when faced with a newborn infant whose external genitalia are so ambiguous in appearance that sex assignment is not instantaneously possible. The problem does not need compounding by the use of unclear, ambiguous terms such as ‘true hermaphroditism’ and ‘pseudohermaphroditism’. The following definitions are relevant to the understanding of normal sex development, both somatic and psychosexual:

  • sex determination—transformation of the indifferent gonad into a testis or an ovary

  • sex differentiation—development of the phenotype (somatotype) as an expression of hormones produced by the gonad (in reality, testis only during fetal development)

  • gender (sex) assignment—allocation of male or female at birth, usually instantaneous

  • gender identity—the sense of self as being male or female

  • gender role—sex-typical behaviours and preferences in which males and females differ (e.g. toy preferences, aggression)

  • sexual orientation—refers to the target of sexual arousal

  • gender attribution—assigning as male or female on first encounter with an individual

‘Gender dysphoria’ is a gender identity disorder characterized by a mismatch between the body habitus (phenotype as male or female) and gender identity as perceived by the affected individual (‘I feel like a woman trapped in a man’s body’). The desire to ‘convert’ from male to female, or vice versa, is a state of transsexualism. There is no direct evidence of a genetic or endocrine explanation for gender dysphoria. However, individuals with a disorder of sex development such as an androgen biosynthetic defect may express feelings of transsexualism in later life.

The term ‘intersex’ has traditionally been applied to the clinical scenario of an infant born with ambiguous genitalia and in whom the sex is indeterminate. That allocation has often strayed beyond this defined scenario to include conditions such as severe hypospadias, milder forms of congenital adrenal hyperplasia, and the complete androgen insensitivity syndrome, where intersex is an inappropriate term. Indeed, affected individuals representing a range of anomalies of the reproductive tract consider the term as pejorative and have clamoured for a change in terminology. In response, a consensus statement produced by a faculty of experts in genetics, endocrinology, surgery, and psychology has redefined the terminology that should now become the lexicon in this branch of medicine (Table 13.9.3.1).

Table 13.9.3.1 A revised nomenclature relating to disorders of sex development

Previous

Proposed

Intersex

Disorders of sex development (DSD)

Male pseudohermaphrodite

Undervirilization of an XY male

Undermasculinization of an XY male

46,XY DSD

Female pseudohermaphrodite

Overvirilization of an XX female

Masculinization of an XX female

46,XX DSD

True hermaphrodite

Ovotesticular DSD

XX male or XX sex reversal

46,XX testicular DSD

XY sex reversal

46,XY complete gonadal dysgenesis

A ‘disorder of sex development’ (DSD) is a wide-ranging term defined as a congenital condition in which development of chromosomal, gonadal, or anatomic sex is atypical. This broad definition enables a wide range of conditions to be considered as DSD. This would include the prototypic ambiguous genitalia of the newborn (e.g. due to congenital adrenal hyperplasia), simple hypospadias, cloacal exstrophy, the XX male, complete XY gonadal dysgenesis (Swyer’s syndrome), undescended testes, and simple labial adhesions. Most of these examples could never be construed as intersex. Disorders of puberty are excluded by incorporating ‘congenital’ within the definition of DSD. Refining the terms to lead to specificity in definitions is achieved by subdividing according to knowledge of the sex chromosomes. Above all, the new consensus rids the medical literature of such confusing and ambiguous terminology as ‘female pseudohermaphroditism’ which purports to define a genetic female with virilized external genitalia at variance with the gonadal sex. Taking congenital adrenal hyperplasia as a common example, this becomes an example of XX DSD in which the sex chromosomes and gonads are congruent (female) but the virilized external genitalia are atypical. ‘True hermaphroditism’ had its adjectival preface to emphasize that the presence of both a testis and an ovary in the one individual was required to meet the definition of hermaphroditism. Why not precisely describe the morphology as being ovotesticular? The underlying karyotype may be 46,XX (the most frequent), 46,XY, or 46XX/XY.

DSD classification

Using the new nomenclature as a catalyst, it has been possible to derive a classification system which is simple, reflects procedures followed during initial investigation and is flexible enough to be adapted as new conditions are recognized and defined. Subtending the entire process is knowledge of the karyotype, a starting point which is now routine for the investigation of DSD. Since fertilization of the ovum with an X- or Y-bearing spermatozoon and sex chromosome aneuploidy or mosaicism are so fundamental to normal fetal sex development, it is logical to consider the causes of DSD in three broad categories. Table 13.9.3.2 contains a fairly comprehensive list of the causes of DSD; Table 13.9.3.3 focuses on the causes of ambiguous genitalia from a functional standpoint.

Table 13.9.3.2 Classification of disorders of sex development (DSD)

Sex chromosome DSD

46,XY DSD

46,XX DSD

  • A: 47,XXY (Klinefelter’s syndrome and variants)

  • B: 45,X (Turner’s syndrome and variants)

  • C: 45,X/46,XY (mixed gonadal dysgenesis)

  • D: 46,XX/46,XY (chimerism)

  • A: Disorders of gonadal (testicular) development

    1. 1. Complete or partial gonadal dysgenesis (e.g. SRY, SOX9, SF1, WT1, DHH, etc.)

    2. 2. Ovotesticular DSD

    3. 3. Testis regression

  • A: Disorders of gonadal (ovary) development

    1. 1. Gonadal dysgenesis

    2. 2. Ovotesticular DSD

    3. 3. Testicular DSD (eg SRY+, dup SOX9, RSP01)

  • B: Disorders in androgen synthesis or action

    1. 1. Disorders of androgen synthesis

      • LH receptor mutations

      • Smith–Lemli–Opitz syndrome

      • Steroidogenic acute regulatory protein mutations

      • Cholesterol side chain cleavage (CYP11A1)

      • 3β‎-hydroxysteroid dehydrogenase 2 (HSD3B2)

      • 17α‎-hydroxylase/17,20-lyase (CYP17)

      • P450 oxidoreductase (POR)

      • 17β‎-OH steroid dehydrogenase (HSD17B3)

      • 5α‎-reductase 2 (SRD5A2)

    2. 2. Disorders of androgen action

      • Androgen insensitivity syndrome

      • Drugs and environmental modulators

  • B: Androgen excess

    1. 1. Fetal

      • 3β‎-hydroxysteroid dehydrogenase 2 (HSD3B2)

      • 21-hydroxylase (CYP21A2)

      • P450 oxidoreductase (POR)

      • 11β‎-hydroxylase (CYP11B1)

      • Glucocorticoid receptor mutations

    2. 2. Fetoplacental

      • Aromatase (CYP19) deficiency

      • Oxidoreductase (POR) deficiency

    3. 3. Maternal

      • Maternal virilizing tumours (e.g. luteomas)

      • Androgenic drugs

  • C: Other

    1. 1. Syndromic associations of male genital development (e.g. cloacal anomalies, Robinow, Aarskog, hand–foot–genital, popliteal pterygium)

    2. 2. Persistent mullerian duct syndrome

    3. 3. Vanishing testis syndrome

    4. 4. Isolated hypospadias (CXorf6, MAMDL1)

    5. 5. Congenital hypogonadotropic hypogonadism

    6. 6. Cryptorchidism (INSL3, GREAT)

    7. 7. Environmental influences

  • C: Other

    1. 1. Syndromic associations (e.g. cloacal anomalies)

    2. 2. Mullerian agenesis/hypoplasia (e.g. MURCS)

    3. 3. Uterine abnormalities (e.g. MODY5)

    4. 4. Vaginal atresias (e.g. McKusick–Kaufman)

    5. 5. Labial adhesions

Table 13.9.3.3 Causes of ambiguous genitalia: a functional classification

Type/cause

Illustrative examples

Masculinized female (46XX,DSD)

Fetal androgens

CAH, placental aromatase deficiency

Maternal androgens

Ovarian and adrenal tumours

Undermasculinized male (46XY,DSD)

Abnormal testis determination

Partial (XY) and mixed (XO/XY) gonadal dysgenesis

Androgen biosynthetic defects

  • LH receptor inactivating mutations

  • 17β‎-OH-steroid dehydrogenase deficiency

  • 5α‎-reductase deficiency

Resistance to androgens

Androgen insensitivity syndrome variants

Ovotesticular DSD

Presence of testicular and ovarian tissue

Karyotypes XX, XY, XX/XY

Syndromal

  • Denys–Drash, Frasier’s

  • Smith–Lemli–Opitz

Sex chromosome DSD

It can be argued that Klinefelter’s and Turner’s syndromes are not examples of DSD in the context of abnormalities of the external genitalia. Klinefelter syndrome (47,XXY) has a genital component that typically comprises small, soft testes with oligo- or azoospermia and infertility. The syndrome affects 1 in 500 to 1 in 1000 live births. The problem of infertility is not absolute now that advances in artificial reproductive technologies have enabled some men with Klinefelter’s syndrome to father children through testicular sperm extraction combined with intracytoplasmic sperm injection. Small testes are already evident in childhood and the penis may be small. Hypospadias may also occur and genitalia can be sufficiently undermasculinized to lead to ambiguity and even a sex-reversed phenotype. Klinefelter variants such as 48,XXXY and 49,XXXXY may have associated genital anomalies.

Turner’s syndrome without evidence of Y chromosomal material is seldom associated with external genital abnormalities. The DSD classification has included this syndrome since it is a congenital disorder characterized by gonadal (ovarian) dysgenesis and a sex chromosome aneuploidy. Turner’s syndrome has an incidence of 1 in 2500. The classic form is associated with a 45,XO karyotype, which accounts for more than 50% of cases. Genital anomalies are more evidenced in mosaic forms of Turner’s syndrome characterized by a 45,X/46,XY karyotype. The external genitalia can range in appearance from normal female or mild clitoromegaly, through overt ambiguous genitalia to a male spectrum of simple hypospadias or normal male genitalia. Indeed, most individuals with this karyotype appear to be normal males based on amniocentesis analyses that have revealed fetuses with a 45,X/46,XY karyotype. The minority with ambiguous genitalia are detected at birth and can pose difficulties for assignment of gender. A multitude of factors need to be considered, including the genital appearance and urogenital anatomy, risk of gonadal tumour, fertility and reproductive options, gender identity, and psychosexual function. Those infants who are severely undermasculinized and have a uterine remnant are likely to be assigned female and any dysgenetic gonad should be removed. Infants assigned male will require a number of hypospadias procedures and removal of any dysgenetic gonads. Long-term outcome studies are not available. Furthermore, finding a 45,X/46,XY karyotype during investigations for male infertility or in men presenting with a tumour of the testis is rare.

Ovotesticular DSD (true hermaphroditism) is a rare cause of abnormal genital development and is characterized by the presence of testicular and follicle-containing ovarian tissue. The external genitalia can be variable, comprising ambiguous genitalia or just severe hypospadias. The gonadal distribution may be a testis on one side and an ovary on the other, bilateral ovotestes, or, most frequently, an ovotestis on one side and a testis or ovary on the other. An ovary will be sited in its normal pelvic position whereas a testis or an ovotestis can be located anywhere along the migratory path to the scrotum, but usually in the inguinal region. The pattern of internal genital ducts can also be variable, but generally follows that of the ipsilateral gonad. A rudimentary uterus is often found adjacent to an ovary or ovotestis. The karyotype is 46,XX in most cases, but only in about 30% is the SRY gene X-translocated. Familial cases are reported, with evidence for both autosomal recessive and sex-limited autosomal dominant transmission.

Normal and abnormal sexual differentiationThe syndrome of XX male is similar to Klinefelter’s syndrome where the external genitalia usually differentiate normally as male but the testes are small. Hypospadias may occur infrequently. The incidence is 1 in 20 000 male births and, unlike Klinefelter’s syndrome, height is below average for normal males. Infertility is invariable; around 90% of XX males are SRY positive. However, the coexistence of ovotesticular DSD and XX male within families and the reported occurrence of 46,XX SRY-negative monozygotic twins with genital anomalies suggests that these two forms of DSD are manifestations of the same underlying disorder in gonad determination. Mutations in RSPO1, an ovarian-specific determining gene, results in female-to-male XX sex reversal and hence may explain the phenotype in some SRY-negative XX males. The phenotype is also replicated in a Rspo1 (–/–) XX mouse model. SOX3 is another SRY-related HMG box-containing gene which is postulated to have evolved from SRY. When overexpressed in XX mice, it induces testis development and male sex reversal. Furthermore, genomic rearrangements of the SOX3 regulatory region were identifiedin three XX males who were SRY negative. It is likely that the mechanism of testis determination in the absence of SRY is via ectopic gonadal expression of SOX3 and subsequent activation of the male pathway of development. SOX3 rearrangements are quite frequent in XX males and the observation goes some way to explain the paradox of XX male development in the absence of a SRY gene.

46,XX DSD (the masculinized female; female pseudohermaphroditism)

In most instances, this broad category of DSD is characterized by a list of conditions where the effect of an abnormal excess of endogenous or exogenous androgens is superimposed on the constitutive female sex development. Thus internal genital development is normal (ovaries, uterus, and fallopian tubes; absence of wolffian ducts) whereas the external genitalia are virilized to a variable degree. The prime example of XX, DSD is congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH)

This autosomal recessive disorder of adrenal steroidogenesis is covered in detail in Chapter 13.7.2. CAH is the commonest cause of ambiguous genitalia of the newborn, and mutations affecting the CYP21A2 gene account for more than 90% of cases. The degree of external masculinization can be so extreme as to resemble a newborn male. However, routine newborn examination should reveal the absence of palpable testes, a sign that must prompt urgent investigation. The diagnosis is straightforward and must be undertaken promptly in view of the potential life-threatening consequences for the infant from glucocorticoid and mineralocorticoid deficiency. The much rarer enzyme deficiencies affecting early steps in adrenal steroidogenesis are also potentially life-threatening and are described in Chapter 13.7.2.

Other causes of endogenous fetal androgen excess

The fetal adrenals are unique in containing a large fetal zone which involutes after birth. Its peculiar role in producing large amounts of androgens does not become established again until late childhood when the zona reticularis differentiates functionally and is manifest as adrenarche. Large quantities of dehydroepiandrosterone (DHA) and its sulphated moiety (DHAS) are produced greatly in excess of the principle adrenal glucocorticoid, cortisol, yet the precise function of this steroid in both fetal and postnatal life remains a mystery.

DHEAS is 16-hydroxylated in the adrenals and liver before transfer to the placenta where the sulphate is cleaved by placental sulphatase. The substrates DHEA and 16-OH-DHEA are converted to more potent androgens such as androstenedione and testosterone and all three androgens are converted to oestrogens (oestrone, oestradiol, oestriol). This reaction is mediated by a single gene, CYP19, which encodes for placental aromatase enzyme via a tissue-specific promoter. Measurement of maternal serum or urinary oestriol was previously used as a rather nonspecific marker of placental dysfunction. However, the levels are specifically low in CAH, in placental sulphatase deficiency (as the sulphate moiety is uncleaved to allow substrate for aromatization), and as a marker of suppression of fetal adrenal steroidogenesis during prenatal treatment for CAH.

The effects of placental aromatase deficiency are profound on both the fetus and the mother. Exposing a female fetus to large amounts of adrenal-derived androgens leads to virilization of the external genitalia as severe as in CAH. Salt wasting is not an associated feature. The mother is also virilized during pregnancy, with evidence of hirsutism, acne, and sometimes clitoromegaly. These signs resolve postnatally but can recur in subsequent pregnancies. Affected girls in later life can present with delayed puberty due to primary gonadal failure where there are cystic ovarian changes and hypergonadotropism. A male fetus exposed to excess adrenal androgens, as with CAH, is not affected at birth. However, males with aromatase deficiency are very tall in young adulthood because of failure in oestrogen-induced closure of the growth plate. The aromatase enzyme has a massive capacity to convert androgens to oestrogens as the mother escapes virilization if only 1 to 2% activity of mutant enzyme remains. The mechanism of virilization in a 46,XX infant with P450 oxidoreductase (POR) deficiency may also be partly related to a disturbance in aromatase deficiency as POR is also an electron donor for P450 aromatase. It has also been suggested that an alternative fetal pathway to DHT synthesis is adopted secondary to POR deficiency (see Chapter 13.7.2).

Maternal androgen excess

The fetus is protected against excess androgens because of a highly efficient placental aromatase enzyme that converts androgens to oestrogens. Women with CAH who become pregnant have relatively high serum androgen levels during pregnancy, yet virilization of a female fetus has not been observed. However, the enzyme can become overwhelmed by excess androgens originating from maternal adrenal or ovarian androgen-secreting tumours. Ovarian luteomas can be recurrent in pregnancies and are most common in multiparous women of Afro-Caribbean descent. Other virilizing ovarian tumours include hyperreactio luteinalis, arrhenoblastoma, hilar cell tumour, and a Krukenberg tumour. Occasionally, polycystic ovarian syndrome may result in fetal virilization. Danazol, a synthetic derivative of 17β‎-ethinyltestosterone with androgenic, antioestrogenic and antiprogestogenic properties, readily crosses the placenta. It is used for a number of conditions as diverse as endometriosis, benign fibrocystic breast disease, for unexplained female infertility, and in hereditary angioedema. A female fetus can become virilized and the use of danazol is contraindicated in pregnancy.

46,XY DSD (the undermasculinized male; male pseudohermaphroditism)

A lengthy list of causes is included in this category of DSD and the frequency is quite high if more common conditions such as hypospadias and cryptorchidism are included. Although the problem of XY DSD is more complex than XX DSD in terms of diagnosis and management, knowledge of the normal process of male fetal sex development allows the causes to be subdivided into (1) gonadal dysgenesis (defects in testis determination), (2) defects in androgen biosynthesis, and (3) resistance to androgens.

Defects in testis determination (gonadal dysgenesis)

The pivotal role of SRY in human testis development is vividly illustrated by the phenotype of complex XY sex reversal as a manifestation of an inactivating mutation of the SRY gene. There is complete gonadal dysgenesis with the histological appearance of streak gonads and no discernible testis development in the form of seminiferous tubules or Leydig cells. Lack of Sertoli cells and hence no AMH production leads to a uterine remnant and the external genitalia are female as a result of no androgen production. Presentation does not usually occur until adolescence on account of delayed puberty and primary amenorrhoea. The syndrome (Swyer’s) may present in later adulthood because of a gonadal tumour, typically as a dysgerminoma. Other tumours include gonadoblastoma, teratoma, and embryonal carcinoma. The risk of gonadal tumours is in the range of 15 to 35%. Mullerian structures are preserved and the uterus increases in size when oestrogen replacement is started. Bone mineral density is reduced in the majority. Adult height in a United Kingdom series was 174 cm as compared with 164 cm in the normal female population. Successful pregnancies have been achieved following egg donation.

Normal and abnormal sexual differentiationMutations in the SRY gene are found in only 15 to 20% of cases of complete XY gonadal dysgenesis. The majority are located within the HMG box, a DNA binding domain which appears to function by modulating local chromatin structure in order to transcribe adjacent target genes. The majority of mutations are de novo, yet there is a curious subset of cases where the mutation is familial and found in the phenotypically normal and fertile father. Explanations for this paradox include expression of the gene to a sufficient threshold against a particular genetic background that is not present in the 46,XY daughter. Alternatively, paternal gonadal mosaicism may be an explanation for familial cases. Partial gonadal dysgenesis refers to evidence of some virilization in the form of clitoromegaly and partial labial fusion or a more male pattern of external genital development with severe hypospadias, bifid scrotum, and undescended gonads. This phenotype is common to many other causes of 46,XY DSD. Mutations in SRY are not identified in this form of gonadal dysgenesis but mutations in SF1 are increasingly being found in partial gonadal dysgenesis without adrenal insufficiency as well as in phenotypes as diverse as hypospadias, anorchia, and male infertility.

A number of syndromes are described in association with XY gonadal dysgenesis where the genital anomalies can constitute complete sex reversal or varying degrees of external genital ambiguity. Denys–Drash syndrome (OMIM 194080) is characterized by gonadal dysgenesis, an early-onset nephropathy due to diffuse mesangial sclerosis and a high risk of Wilms’ tumour. A related condition is Frasier’s syndrome (OMIM 136680) which is also characterized by gonadal dysgenesis but of a greater severity (streak gonads), a later-onset nephropathy due to focal segmental glomerulosclerosis, and a high risk of gonadal tumours. The two syndromes may represent a continuum of phenotypes. Both have in common mutations in the Wilms’ tumour-related gene, WT1, as the underlying cause. The gene encodes for a four-zinc-finger transcription factor expressed in the developing urogenital ridge, kidney, and gonads. A macro- deletion affecting the region on chromosome 11p13 where WT1 resides causes the WAGR syndrome (Wilms’ tumour, aniridia, genitourinary abnormalities and mental retardation; OMIM 194072). Denys–Drash and Frasier’s syndromes are caused by heterozygous point mutations in WT1 that have a dominant-negative effect on the wild-type protein. In the former syndrome, these affect the DNA-binding zinc-finger region of WT1. Most mutations causing Frasier’s syndrome involve the donor splice site of exon 9. The use of an alternative splice donor site for exon 9 results in the addition of three amino acids—lysine (K), threonine (T), and serine (S)—between the third and fourth zinc fingers. The +KTS and –KTS isoforms are thought to have differential effects on gonad and renal development and an imbalance in the ratio of these isoforms may be the explanation for the phenotype of Frasier’s syndrome. It has been proposed that all cases of XY DSD with external genital anomalies should have routine urinalysis for proteinuria and renal ultrasound to exclude a Wilms’ tumour. Rarely, a WT1 mutation has been identified in a case of isolated hypospadias but this does not justify routine WT1 screening for this common genital anomaly.

Another syndrome associated with gonadal dysgenesis and genital anomalies is caused by mutations in SOX9 which encodes for an SRY-related HMG box protein of 509 amino acids. The protein is expressed in the developing testis shortly after SRY expression. The protein is also expressed in cartilage; heterozygous mutations in SOX9 (chromosome 17q24–25) cause campomelic dysplasia (long bone bowing, hypoplastic scapula and rib cage, deformed pelvis, cleft palate, macrocephaly, cardiac and renal defects) as well as the associated genital anomalies. SOX9 mutations do not occur without the skeletal abnormalities. There are rare examples of gonadal dysgenesis occurring in association with mutations in desert hedgehog (DHH) and testis-specific protein-like-1 (TSPYL1) genes and with chromosomal deletions at 9p24-pter, 10q25-pter, and Xq13.3.

Normal and abnormal sexual differentiationAnother gene now found to be associated with XY sex reversal is CBX2, the human homologue of M33, which, when ablated in mice, leads to XY sex reversal. A loss-of-function CBX mutation in a girl whose prenatal karyotype was 46,XY was shown to be the cause of her sex reversal based on detailed functional studies. This gene appears to actively repress ovarian development in XY gonads. Remarkably, a uterus and histologically normal ovaries were present in this girl. A linkage analysis study successfully identified mutations in MAP3K1 in a familial form of XY gonadal dysgenesis as well as in some sporadic cases. The mutations cause alterations downstream in the MAP kinase signalling pathway, possibly by altering SOX9 or β‎-catenin activities.

Normal and abnormal sexual differentiationAnalysis of rare mutations in candidate gonad-determining genes is chipping away at the current low frequency of determination of the molecular mechanism of XY gonadal dysgenesis. This will be further enhanced by the use of array-CGH analysis which is already proving a useful diagnostic tool to search for new candidate loci.

Defects in androgen biosynthesis

The steps in androgen production from LH-induced steroidogenesis via cholesterol through to testosterone and DHT are illustrated in Fig. 13.9.3.4. Many of the early steps in androgen biosynthesis are also essential for adrenal steroid biosynthesis and are described in Chapter 13.7. The same G-protein-coupled gonadotrophin receptor (LHR) on Leydig cells binds both placental hCG and later, fetal pituitary luteinizing hormone (LH) for the initiation of androgen biosynthesis. Inactivating mutations in the LHR gene in 46,XY DSD lead to a wide range of phenotypes that include complete sex reversal, ambiguous genitalia, severe hypospadias, or even just isolated micropenis. Why the phenotype should be so heterogeneous is not entirely clear, although partial loss-of-function mutations that result in a milder phenotype such as micropenis tend to localize within the seventh transmembrane domain of the receptor. The biochemical profile comprises elevated LHRH-stimulated LH and FSH levels and low androgen concentrations which do not respond to prolonged hCG stimulation. Leydig cells are absent or decreased in number on histological examination. Sertoli cells and seminiferous tubules are present but spermatogenic arrest attests to the importance of intracellular androgen concentrations in mediating the final stages of spermatogenesis. A range of homozygous or compound heterozygous mutations of the LHR gene are reported in this syndrome of Leydig cell hypoplasia. Their pathogenicity can be confirmed in vitro by demonstrating impaired hCG stimulation of intracellular cAMP due to disturbances in hCG binding, receptor stability or receptor trafficking. The extracellular N-terminal ligand binding domain of LHR consists of nine leucine-rich repeats flanked by cysteine-rich regions. The C-terminal cysteine-rich region is referred to as a hinge region of the receptor within which amino acid residues Asp330 and Tyr331 are key components of LH/hCG signalling. An XX female has been reported with an LHR-inactivating mutation. The phenotype comprised normal onset of puberty but associated primary amenorrhoea and elevated LH levels. Studies on a rare gene mutation in this context provides information to indicate that while the LHR is not required for oestrogen synthesis, it is necessary for the induction of ovulation and fertility, although some affected females may still have regular cycles.

Two penultimate steps in androgen biosynthesis essential for normal male sex differentiation are shown in Fig. 13.9.3.4. Both conversion steps are characterized by the respective substrates being the subject of catalysis by different isoenzymes and not involving steroid biosynthesis in the adrenals. The forms of XY DSD resulting from deficiencies in the two enzymatic steps have in common a severe degree of undermasculinization at birth but profound virilization at puberty. Thus, if unrecognized at birth and the affected infant is assigned female, the clinical presentation occurs at puberty with distressing signs of clitoromegaly, hirsutism, and deepening of the voice in a pubertal girl.

There are 14 known 17β‎-hydroxysteroid dehydrogenase (17HSD) isoenzymes, of which 12 are present in humans. They belong to a family of oxidoreductases involved in the metabolism of steroids, prostaglandins, and retinols. Of most relevance to XY DSD is 17HSD type 3, which is predominantly expressed in the testis and converts androstenedione to testosterone. The reaction is reversible and utilizes NADPH as a cofactor. The cognate gene, HSD17B3, is located on chromosome 9q22. A spectrum of mutations in this gene generally results in complete XY sex reversal at birth and can be mistaken for complete androgen insensitivity syndrome. Presentation in infancy may be in the form of an inguinal hernia or labial swelling where investigation reveals the presence of a testis. If sex assigned female, gonadectomy must be undertaken before puberty to avoid a pubertal girl becoming virilized. The mechanism of such profound androgenic effects is postulated to be the result of extraglandular conversion to androgens utilizing other isoenzymes such as types 1, 2, and 5. Some 17HSDB3 mutations are associated with retention of 15 to 20% of normal 17β‎HSD3 activity that leads to sufficient virilization of the external genitalia at puberty for sex reassignment. The biochemical profile shows elevated androstenedione and decreased testosterone levels so that the ratio of testosterone to androstenedione is typically 0.8 or less in this disorder. Wolffian ducts are stabilized to form the vas deferens, epididymis, and seminal vesicle which is presumably the result of high local concentrations of androstenedione. About 20 mutations in the 17HSDB3 gene are now reported, most being homozygous or compound heterozygous missense mutations. Females with 17β‎HSD type 3 deficiency are asymptomatic.

Testosterone is converted irreversibly to dihydrotestosterone (DHT) by the 5α‎-reductase type 2 enzyme which is expressed in the primordium of the prostate and external genitalia, but not in the wolffian ducts until after their differentiation to male internal genital ducts. As with 17β‎HSD deficiency, the male internal genital ducts develop normally in 5α‎-reductase deficiency. The phenotype is associated with some external virilization so that presentation is more frequent at birth because of ambiguous genitalia or severe hypospadias. This cause of XY DSD became well characterized through detailed descriptions of a genetic isolate in the Dominican Republic where males were born with severely undermasculinized external genitalia but then virilized to varying degrees at puberty. The testes enlarge appropriately at this stage, but the prostate gland remains hypoplastic, indicative of the DHT-dependent growth of this organ. Histology of the testes shows Leydig cell hyperplasia and decreased spermatogenesis due to maldescent of the testes. However, there are reports of male fertility occurring either following artificial reproductive techniques or even spontaneously after hypospadias repairs had been completed. Gender role changes occur frequently in this condition.

The biochemical profile is classically an elevated ratio of serum testosterone to DHT of more than 25:1 after puberty (or following hCG stimulation in a prepubertal child) and a reduced ratio of urinary 5α‎- to 5β‎-reduced C19 steroids. The 5α‎-reductase enzyme is also utilized in the metabolism of glucocorticoids, so C21 5α‎/5β‎ steroids can usefully be analysed even when gonadectomy has already taken place. There are two isoenzymes of 5α‎-reductase, the type 2 enzyme being affected in this condition. SRD5A2 is located on chromosome 2p23 and encodes for a 254 amino acid protein. The type 1 enzyme is expressed in skin and may contribute to the virilization which takes place at puberty. More than 40 mutations have been detected in the SRD5A2 gene. The majority are missense mutations, including the Gly183Ser substitution observed in the Dominican Republic population. A complete gene deletion is found in an affected New Guinea population.

Defects in androgen action

Androgen resistance is defined as a failure in complete male sex differentiation despite the presence of a normal 46,XY karyotype in association with testes that produce age-appropriate circulating concentrations of androgens. The androgen insensitivity syndromes (AIS) are subdivided into complete (CAIS) and partial (PAIS) forms as defined by complete XY sex reversal (female phenotype) and partial virilization of the external genitalia. The degree of virilization in the latter category can vary from mild, isolated clitoromegaly to normal male development with oligospermia.

Total resistance to androgens leading to CAIS is the sine qua non of a hormone resistance syndrome and was previously labelled as the testicular feminization syndrome. Typical presentation is in adolescence with primary amenorrhoea. There is normal breast development as male-typical androgen levels are aromatized to oestrogens, but there is absent or scanty pubic and axillary hair growth. The external genitalia are female and a shortened vagina is blind-ending. The upper part of the vagina, together with the uterus and fallopian tubes, are structures derived from the mullerian duct, hence these are absent in CAIS as a result of normal AMH action by the testes. CAIS may also present in infancy because of the appearance of inguinal herniae which, at surgical repair, are found to contain testes. It is now recommended that the karyotype is checked in all female infants with an inguinal hernia. The increasing trend towards prenatal tests that reveal the karyotype is also a mode of presentation when the phenotype at birth is realized to be a mismatch with the prenatal genotype. Reference to Fig. 13.9.3.5 indicates that a defect in any one of the steps in androgen signalling may underlie the pathophysiology of CAIS. The problem is generally located with the AR where numerous mutations have been identified that cause CAIS or PAIS. These are recorded on an international database (http://androgendb.mcgill.ca/) and also by the author via the Cambridge DSD database (Fig. 13.9.3.5). Mutations are distributed throughout the coding region of the AR gene and include deletions, insertions, premature stop codons, and splice-site as well as missense mutations. The majority are located within the ligand-binding domain and codons such as Arg840 and Arge855 appear to be relative ‘hotspots’ for mutagenesis. Mutations that are novel generally need re-creating by site-directed mutagenesis to determine their pathogenicity using reporter gene-based transactivation assays in vitro. Additional structural modelling studies can be used to predict the effect amino acid substitutions may have on the ligand-binding pocket. CAIS is an X-linked disorder and approximately 30% of AR gene mutations are spontaneous. The same mutation may manifest as different phenotypes, between and within affected families and to the extent of different sex assignments. The reasons for phenotypic variability are unclear but may include somatic mosaicism and differences in the lengths of the two AR trinucleotide repeats in the N-terminal domain, glutamine and glycine. The normal glutamine repeat range, (CAG)n, is approximately 10 to 31 and variations within this range are associated with a pleitrophic range of disorders, some of which are listed in Table 13.9.3.4. Studies in vitro indicate a less transcriptionally active AR containing a longer CAG repeat. Hyperexpansion of the triplet repeat (>50) underlies the pathogenesis of spinal and bulbar muscular atrophy (Kennedy’s disease). Males affected with this neurological disorder display signs of mild androgen insensitivity. A number of associations are also described for the glycine (GGN) repeat, either alone or in combination with variations in the CAG repeat.

Normal and abnormal sexual differentiationTable 13.9.3.4 Disease associations with variations in the AR glutamine repeat

Shortened (CAG)n

Increased (CAG)n

Prostate cancer

Above normal range

Ovarian hyperandrogenism

SBMA (Kennedy’s disease)

Androgenetic alopecia

Hypospadias (one reported case)

Aspects of Klinefelter phenotype

Within normal range

Response to androgen treatment

Male infertility

Central obesity

Gynaecomastia

Mental retardation

Hypospadias

Endometrial cancer

Aspects of Klinefelter phenotype

Coronary artery disease severity

Bone density

Endocrine disrupter exposure

Stress fractures

Seminoma

Fat mass

Childhood growth in boys

Premature ovarian failure

Muscle bulk

Breast cancer

Men’s mating behaviours

Pre-eclampsia risk

Metabolic syndrome risk

Gender assignment and sex of rearing in CAIS is female, as is later gender identity. Gonadectomy is recommended because of an approximately 5% risk of gonadal tumours. A precursor lesion to tumour development is intratubular germ cell neoplasia unclassified (ITGNU), also referred to as carcinoma in situ (CIS). This may subsequently lead to a gonadoblastoma, an occurrence which is rare before puberty. The timing of gonadectomy is variable but there is merit in delaying until young adulthood to enable spontaneous puberty to occur. There is no evidence that the slightly reduced bone mineral density in CAIS is ameliorated by this management approach, suggesting that androgens have a direct role in normal bone architecture. Oestrogen replacement needs to be started at about 11 years of age when gonadectomy is performed early. Ethinyl oestradiol starting at 2 μ‎g/day is increased gradually, reaching 20 μ‎g/day by 15 years of age. Final height rests between the average height of adult males and females.

Mutations are also distributed throughout the AR gene in PAIS, but are predominantly missense in nature. The partial androgenic effect can be verified by functional assays in vitro which demonstrate reporter gene responses close to the normal AR, but usually only after induction with very high concentrations of androgens. Such information can be valuable for predicting outcome at puberty in PAIS patients assigned male. Establishing a precise diagnosis in PAIS can be difficult as so many other disorders can be associated with the typical phenotype of severe hypospadias, micropenis, bifid scrotum, and undescended testes. These include androgen biosynthetic defects, partial gonadal dysgenesis, and mixed gonadal dysgenesis. In many instances, no single genetic cause can be found for a PAIS-like phenotype: external genitalia as described and normal androgen production. There is a strong association with low birth weight for gestational age in PAIS cases that have no AR gene mutation. This suggests placental dysfunction being a common link between early fetal growth restriction and inadequate placental hCG-induced early Leydig cell steroidogenesis. The infant with PAIS assigned male may require a number of surgical procedures to correct hypospadias, orchidopexy for undescended testes, and high supplemental androgen treatment to induce puberty. The risk of gonadal tumours is probably higher than in CAIS, but once in the scrotum, the testes can be monitored by self-examination and periodic testicular ultrasonography. Outcome data in adult males with PAIS are sparse but sexual function is reported to be impaired; fertility is rare. Those assigned female require genitoplasty procedures in infancy, gonadectomy before puberty, and oestrogen treatment to induce female secondary characteristics.

Other conditions within the XY DSD category

A number of disorders are associated with incomplete male development but do not raise any doubt that sex assignment at birth should be male. These include hypospadias, undescended testes, and the persistent mullerian duct syndrome (PMDS).

Isolated hypospadias has a birth prevalence of 3 to 4 per 1000 live births. The cause in the majority of cases is unknown, despite extensive analysis of the known genes involved in male reproductive tract development. Familial cases occur with a 7% incidence of one or more additional family members being affected with hypospadias. There is an association with increased maternal age, paternal subfertility, maternal vegetarian diet, maternal smoking, assisted reproductive techniques, exposure to pesticides, and twinning. The aforementioned low birth weight is also a further association, which is strong. Hypospadias is generally classified as mild to severe based on the site of the urethral meatus being subglanular, penile, or perineoscrotal (severe). There is often an associated chordee in the severe form. Numerous surgical techniques are described to resite the urethral opening on to the glans penis and may require several procedures. The initial procedure is usually undertaken in infancy. Complications include fistulas, meatal stenosis, and urethral strictures. Function is generally satisfactory in terms of urination and sexual intercourse, even though the cosmetic appearance may not be adequate.

Undescended testes or cryptorchidism is the commonest birth defect in boys, affecting 2 to 9% of male live births. Again, there is a strong association with low birth weight as well as disorders that affect pituitary–gonadal function and androgen action. These observations emphasize the importance of androgens in mediating complete descent of the testes into the scrotum by their action during the inguinoscrotal phase of descent. Other associations include maternal smoking or use of nicotine substitutes, alcohol use, and gestational diabetes. There is an association with intrauterine insemination, but not with other forms of artificial reproductive technology. Genetic factors also play a part, particularly for first-degree relatives among brothers and maternal half-brothers. Cryptorchidism can be unilateral or bilateral with the testis sited in the abdomen (nonpalpable), inguinal canal, suprascrotal, or high scrotal (where it is not possible to manipulate the testis to the bottom of the scrotum). Undescended testis must be distinguished from a retractile testis which ascends in response to a pronounced cremasteric reflex but can be manipulated completely into the scrotum. A testis may be descended at birth but found to be undescended at a later age. This has been termed the ‘ascending’ testis or an acquired form of cryptorchidism. Studies indicate that the phenomenon is more likely with a history of retractile testis, the processus vaginalis may be patent, and the testis is usually located in the inguinal region. Ascending testis accounts for nearly one-half of the cases of undescended testis and mostly explains why late orchidopexies occur around 7 years of age. It is recommended that orchidopexy for congenital cryptorchidism is undertaken between 6 to 12 months of age. Early surgery is associated with improved growth of the testis, less evidence of abnormal germ-cell development and a lower risk of developing a seminoma in adulthood. Hormonal treatment has low efficacy and stimulation with repeated injections of hCG may actually be harmful to future spermatogenesis by inducing apoptosis of germ cells. Despite evidence for the role of INSL3 and its receptor in testis descent, mutations in the genes that encode these proteins are found only in a minority of boys with cryptorchidism.

The components of a quartet of male reproductive tract disorders—hypospadias, cryptorchidism, abnormal spermatogenesis, testis cancer—are each interlinked, for which there is some epidemiological evidence to suggest an increase in frequency. Environmental factors have been proposed to explain the observation through the development of a testicular dysgenesis syndrome which has its origin in fetal life. Humans are exposed to more than 80 000 chemicals in the environment with any adverse effects assumed to be more profound on the developing fetus. Evidence that chemicals such as pesticides and phthalates can disrupt the androgen/oestrogen balance critical for normal fetal sex development is present in wildlife and in animal experiments. It is more difficult to prove similar effects in humans. However, such chemicals labelled as endocrine disruptors are reported to be present in higher concentrations in cord blood, placentas, and breast milk samples of mothers having male offspring with hypospadias or cryptorchidism, compared with normal control offspring. Furthermore, the anogenital distance, which is a sensitive index of androgen action used in rodent reproductive studies, is reduced in male infants of mothers who had higher prenatal exposure to phthalates.

Bilateral anorchia, also referred to as the vanishing testis syndrome, in an otherwise normal male infant indicates that testes were present and functioning normally in early gestation in order to programme normal male sex differentiation. It is hypothesized that interruption of the vascular supply to the testes must have occurred in later gestation (akin to bilateral torsion). This is supported by surgical findings which show a preserved vas deferens entering the internal inguinal ring at the end of which is only a nubbin of fibrous tissue containing haemosiderin-laden macrophages and dystrophic calcification. The diagnosis is confirmed by demonstrating elevated LH and FSH concentrations, no testosterone response to hCG stimulation, and an undetectable serum AMH. Even with this endocrine scenario, surgeons generally still perform a laparoscopy to ensure that any gonadal remnant is removed to avoid the risk of malignancy.

PMDS is also associated with testis maldescent but in this instance normal testes are prevented from descending to the scrotum because of being attached to a fallopian tube. The uterus and tubes in this syndrome are retained from early fetal development because of the lack of AMH action. This can either be the result of a mutation in the AMH gene with low or undetectable serum AMH, or serum AMH concentrations may be normal but the protein is unable to bind to its receptor because of a mutation in the gene coding for the AMH type II receptor. A mutation is found in the majority of cases with equal distribution between the two causative mutant genes. The phenotypes are identical. The external genitalia are otherwise normal; both testes may be descended to one hemiscrotum. Such transverse testicular ectopia is diagnostic of PMDS. The diagnosis is usually made at orchidopexy or for an inguinal hernia repair where the sac is found to contain a uterus or a fallopian tube. Care must be taken to re-site the testis to its normal position as such mobilization may damage the vas deferens. The uterus is often left in place.

Assessment of a DSD

Ambiguous genitalia of the newborn and development of secondary sexual characteristics at puberty discordant with the sex of rearing are the two key stages in life when a problem of DSD requires careful assessment based on clinical examination followed by a focused and logical investigation plan. It must also be recognized that while a definitive diagnosis may not be possible in some cases, this must not delay a decision on sex assignment unduly and lessen the importance for a management plan.

Examination

For the infant with ambiguous genitalia, the following details need to be recorded: the size of the phallus, presence of chordee, and whether the appearance is indicative of clitoromegaly or a micropenis; site of urethral opening; single or dual openings on the perineum; development of labioscrotal folds or a bifid scrotum; whether gonads are palpable and their site. Allied to the examination are salient points in the clinical history such as family history and exposure to potential reproductive tract teratogens. Problems arising only at the time of puberty are generally signs of virilization occurring in a child hitherto assumed to be female. These can include deepening of the voice, hirsutism, acne, and clitoromegaly. Delayed pubertal development and primary amenorrhoea are manifestations of gonadal dysgenesis, whereas primary amenorrhoea but normal breast development is more consistent with CAIS.

Investigations

Box 13.9.3.1 lists clinical problems presenting in infancy that merit further investigation. The tests to be performed and which protocols to use vary according to centres, but Box 13.9.3.2 lists the categories according to the relevant specialist areas. These investigations should enable a functional diagnosis to be achieved within days of birth for the infant with ambiguous genitalia. The leading causes are likely to be CAH, PAIS and XO/XY mixed gonadal dysgenesis. A provisional result on the sex chromosomes is now rapidly available using FISH analysis. The karyotype in turn will steer subsequent investigations in the appropriate direction. For example, a FISH analysis suggesting XX chromosomes and confirmed on full karyotype analysis, an ultrasound showing a uterus, and a markedly elevated serum 17-OH progesterone concentration clinches a diagnosis of CAH in an infant with ambiguous genitalia. For the XY or XO/XY infant with DSD, the hCG simulation test and AMH measurement will provide information about the presence of testes and whether they produce normal concentrations of testosterone. Imaging studies (ultrasonography and MRI) may locate the site of gonads but often laparoscopy is the only reliable method to identify gonads. This also provides the opportunity to obtain biopsies for histology, the only sure way to establish a diagnosis of ovotesticular DSD (true hermaphroditism).

Management

Only the principles of DSD management can be described since each cause of DSD has specific requirements, some of which have been covered for CAH in Chapter 13.7.2. The greatest challenges for the endocrinologist are to manage the newborn with ambiguous genitalia and the pubertal child who develops physical signs incongruent with the sex of rearing. It is axiomatic that management should only be undertaken by a multidisciplinary team that comprises, at a minimum, an endocrinologist, urologist, gynaecologist, a geneticist, and a clinical psychologist. There is consensus that all infants with DSD should have a gender assignment, but this may have to be delayed until the results of relevant investigations are available. Surgery required to make the genitalia concordant with gender assigned may be deferred, even to an age where the child is of sufficient cognitive development to be involved with the discussions. Psychological support is required for the family from the outset, as misinformation given early can impact adversely in the longer term. As the child grows older preparations for disclosure must be carefully planned. In a female with XY DSD (CAIS, for example), this will entail explanation concerning the nature of the gonads, the presence of a Y chromosome, absence of a uterus, lack of menses, and future infertility. Such disclosure requires skilled counselling delivered as appropriate to the child’s development. Transitional care from adolescence to young adulthood is a further level of complexity that requires the recruitment of adult specialists relevant to whether sex assignment has remained male or female. Longer-term studies in women with CAH are now being conducted and provide valuable information on surgical, endocrine, and psychosexual outcomes. In terms of XY DSD, outcome data are reasonably robust for miscellaneous conditions such as cryptorchidism, hypospadias, mixed gonadal dysgenesis (XO/XY), and CAIS. In contrast, data remain sparse in PAIS and some androgen biosynthetic defects, conditions where sex reassignment may arise in later childhood and adolescence.

Further reading

Ahmed SF, Rodie M (2010). Investigation and management of ambiguous genitalia. Best Pract Res Clin Endocrinol Metab, 24, 197–218.Find this resource:

Audi L, et al. (2010). Novel (60%) and recurrent (40%) oncogene receptor gene mutations in a series of 59 patients with a 46,XY disorder of sex development. J Clin Endocrinol Metab, 95, 1876–88.Find this resource:

Baskin LS, Ebbers MB (2006). Hypospadias: anatomy, etiology, and technique. J Pediatr Surg, 41, 463–72.Find this resource:

Blackless M, et al. (2000). How sexually dimorphic are we? Review and synthesis. Am J Hum Biol, 12, 151–66.Find this resource:

Bouvattier C, et al. (2006). Impaired sexual activity in male adults with partial androgern insensitivity. J Clin Endocrinol Metab, 91, 3310–5.Find this resource:

Brain CE, et al. (2010). Holistic management of DSD. Best Pract Res Clin Endocrinol Metab, 24, 335–54.Find this resource:

Cohen-Kettenis PT (2010). Psychosocial and psychosexual aspects of disorders of sex development. Best Pract Res Clin Endocrinol Metab, 24, 325–34.Find this resource:

de Clemente N, Belville C (2006). Anti-Müllerian hormone receptor defect. Best Pract Res Clin Endocrinol Metab, 20, 599–610.Find this resource:

Deeb A, et al. (2005). Correlation between genotype, phenotype and sex of rearing in 111 patients with partial androgen insensitivity syndrome. Clin Endocrinol (Oxf), 63, 56–62.Find this resource:

Faisal AS, et al. (2011). UK guidance on the initial evaluation of an infant or an adolescent with a suspected disorder of sex development. Clin Endocrinol, 75, 12–26.Find this resource:

Finsterer J (2009). Bulbar and spinal muscular atrophy (Kennedy’s disease): a review. Eur J Neurol, 16, 556–61.Find this resource:

Han TS, et al. (2008). Comparison of bone mineral density and body proportions between women with complete androgen insensitivity syndrome and women with gonadal dysgenesis. Eur J Endocrinol, 159, 179–85.Find this resource:

Hannema SE, Hughes IA (2008). Neoplasia and intersex states. In: Hay I, Wass J (eds) Clinical Endocrine Oncology, 2nd edition, pp. 86–96. Blackwell Publishing, Oxford.Find this resource:

    Hughes IA (2010). The quiet revolution. Best Pract Res Clin Endocrinol Metab, 24, 159–62.Find this resource:

    Hughes IA, Acerini C (2008). Factors controlling testis descent. Eur J Endocrinol, 159 Suppl 1, S75–82.Find this resource:

    Hughes IA, Achermann JC (2011). Disorders of sex differentiation. In: Kronenberg H, et al. (eds) Williams’ Textbook of Endocrinology, 12th edition, Saunders Elsevier, Philadelphia.Find this resource:

    Hughes IA, et al. (2011). Androgen insensitivity syndrome. Seminar. Lancet (submitted).Find this resource:

      Hughes IA, Houk C, Ahmed SF, Lee PA (2006). Consensus statement on management of intersex disorders. Arch Dis Child, 91, 554–63.Find this resource:

      Johannsen TH, et al. (2006). Quality of life in 70 women with disorders of sex development. Eur J Endocrinol, 155, 877–85.Find this resource:

      Lee YS, et al. (2007). Phenotypic variability in 17β‎-hydroxysteroid dehydrogenase-3 deficiency and diagnostic pitfalls. Clin Endocrinol, 67, 20–8.Find this resource:

      Lin L, et al. (2007). Heterozygous missense mutations in steroidogenic factor 1 (SF1/Ad4BP, NR5A1) are associated with 46,XY disorders of sex development with normal adrenal function. J Clin Endocrinol Metab, 92, 991–9.Find this resource:

      Looijenga LH, et al. (2011). Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance. Int J Androl, 34(4 Pt 2), e234–51.Find this resource:

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