Basic concepts of the embryology of sex differentiation

An essential concept is that the human embryo is bipotential until 40 days gestation. Regardless of whether they are derived from a 46,XX or a 46,XY zygote, the gonads and the internal (Müllerian and Wolffian ducts) and external genitalia (genital tubercle and urethral and genital folds) have a similar appearance. After 40 days, the gonads differentiate into either testes, which start producing anti‐Müllerian hormone (AMH), a glycoprotein, at about six to eight weeks, and then testosterone, or into ovaries. Recent studies have shown that the differentiation of both the ovaries and the testes requires activation of specific gene cascades and that the ovaries actively inhibit Wolffian duct stabilization, thus challenging the long‐held concept of ‘female development by default’. However, this concept still applies to the differentiation of the external genitalia and of the urogenital sinus (see section ‘Urogenital Sinus’).

Genetic control of gonadal sex determination

A detailed discussion of this complex process is beyond the scope of this chapter. With few exceptions, the presence of a Y chromosome containing a normal SRY (sex‐determining region of the Y chromosome) gene will result in the differentiation of the bipotential gonad into a testis. However, a number of other genes, acting upstream or downstream of SRY, are required for normal testicular development. As noted above, some genes are also actively involved in the differentiation of the ovary. The list of these genes constantly expanding (see Further Reading).

Internal genitalia

From six to eight weeks, in the presence of a testis, the Müllerian system involutes under the influence of AMH secreted by the Sertoli cells, while the Wolffian system is stabilized by testosterone secreted by the Leydig cells, which are stimulated by placental human chorionic gonadotrophin (hCG), and develop into the epididymis, vas deferens, seminal vesicles, and ejaculatory ducts (Figure 7.1). It is important to note that AMH exerts its effect in a paracrine fashion (i.e. a local effect near its site of production), while testosterone acts both locally (to stabilize the Wolffian ducts) and in a classical endocrine way (to masculinize the urogenital sinus and the external genitalia) after being transported through the bloodstream to its target tissues. In the absence of a testis, the Wolffian system involutes and the Müllerian system develops into the Fallopian tubes, uterus, and the upper third of the vagina.

Urogenital sinus

Under the influence of testosterone, the sinus narrows to form the posterior urethra, and the prostate and the glands of Cowper develop from outgrowths of the sinus. In the absence of testosterone, the sinus forms the lower two‐thirds of the vagina and the urethra, and its outgrowths form the para‐urethral glands of Skene and the vestibular glands of Bartholin that secrete mucus to lubricate the vagina.

External genitalia

From eight weeks onwards in the male, under the influence of dihydrotestosterone (DHT), converted from testosterone in genital skin by 5‐alpha reductase type 2, the genital tubercle will grow to form the penis, the urethral folds will develop into the corpus spongiosum surrounding the urethra and the genital folds will fuse in the midline to form the scrotum and ventral part of the penis (Figure 7.2). In the female, the same structures will form the clitoris, the labia minora, and the labia majora.

History

• Consanguinity.
  
• Pregnancy history:
  
  
  
  • increased body hair and acne (suggests aromatase or POR deficiency);
    
  • virilization (suggests a tumour).
  
  
• Age at menopause in the maternal grandmother (SF‐1 mutations).

Examination

• General examination to rule out dysmorphic syndrome ± congenital anomalies (25% of subjects with a DSD have extra‐genital malformations).
  
• Palpation of the abdomen and inguinal regions for gonads.
  
• Meticulous description ± diagram of abnormal genitalia, using neutral words (see description of the bipotential embryo above) and including labioscrotal folds; genital tubercle; nature and size of opening below genital tubercle; identification where possible of urethral meatus and vaginal opening; and presence and site of gonad(s).
  
• Examination of anus.
  
• Measurement of blood pressure (CAH due to 11‐β‐hydroxylase deficiency may lead to hypertension, but this is seldom present in neonates; conversely, cortisol and aldosterone deficiency may lead to hypotension and shock).
  
• Medical photograph with legs spread apart and ruler next to the genital tubercle.

Of note, premature girls, because of the lack of subcutaneous fat, may appear to have clitoral hypertrophy. In these cases, it is useful to estimate the ratio of the distance between the anus and the posterior labial fourchette to the distance between the anus and the base of the clitoris: a ratio less than 0.5, regardless of gestational age, reasonably excludes androgenization during the first trimester.

Further investigations and management

The following professionals should be informed as soon as possible:

• paediatric endocrinologist;
  
• radiologist, surgeon, and mental health professional with expertise in DSD;
  
• medical geneticist.

If access to the professionals listed above is difficult, then transfer to a specialist centre for specific tests or for general management may be necessary.

The following investigations should be carried out as quickly as possible:

• Karyotype: This takes a minimum of three days. In contrast, fluorescent in situ hybridization (FISH) to determine the presence of a Y chromosome or DNA amplification by polymerase chain reaction (PCR) to determine the presence of SRY sequences gives a ‘provisional karyotype’ within hours.
  
• Abdominal ultrasound: most importantly to determine if a uterus is present; may also be useful for imaging the gonads and adrenals.
  
• Plasma 17‐hydroxyprogesterone (17‐OHP).
  
• Genitography: This is usually only necessary prior to feminizing genitoplasty to define the point of confluence of the vagina and urethra.

Transition to adult care

At the end of puberty and after full disclosure, people with DSDs should be transferred to an endocrinologist or a gynaecologist with expertise in these disorders. How this is organized in practice will vary according to local circumstances, but ensuring that the transfer has actually happened is important. Especially in individuals who do not need chronic medication, it is not unusual that they do not show up at the appointment made with the physician in charge of overseeing their evolution and management in adult life. Even individuals on chronic replacement therapy may stop taking their glucocorticoids and present with acute adrenal insufficiency, while gonadectomized or hypogonadal individuals who stop their sex hormones are at risk of developing osteoporosis at an early age.

Studies of subjects with DSD in adulthood are few. The best studied, most numerous and relatively homogeneous group is that of women with CAH. Clearly, the surgical techniques used in the past for feminizing genitoplasty have led to sexual difficulties in many women. When adherence to treatment is good, the clitoral hypertrophy should not progress after birth and clitoral surgery can be avoided. Separating the urethral and vaginal orifices is still performed based on the rationale that a urogenital sinus is a risk factor for ascending urinary infections but even that rationale is questionable.

People with 46,XY DSDs born with ambiguous genitalia are much more heterogeneous. Reports of female‐to‐male self‐reassignment at adolescence of 46,XY subjects with 5‐α reductase deficiency whose testes had not been removed in infancy are not generalizable. Importantly, almost half of 46,XY DSD subjects (mostly PAIS), reared male or female, reported that they were neither well informed about their medical and surgical history nor satisfied with their knowledge. This emphasizes the importance of the philosophy of full disclosure that is outlined above and that should now be adopted by all interdisciplinary teams following individuals with DSD.

Fused labia

This benign problem in infant girls is likely due to their low oestrogen milieu. Indeed, twice daily application of oestrogen‐containing creams for one to two weeks usually relieves the problem. Longer periods of application of oestrogen creams may lead to breast budding. Referral to a paediatric gynaecologist is only exceptionally needed. If the vaginal opening remains completely obstructed in spite of treatment with topical oestrogen, a pelvic ultrasound will rule out the Mayer‐Rokitansky‐Küster‐Hauser syndrome (congenital absence of the vagina and uterus). However, to avoid undue anxiety, this should be performed by a radiologist with paediatric expertise, given the small size of the normal prepubertal uterus.

Isolated clitoromegaly

As described above, this is a common finding in premature infants and tends to become less obvious with the development of subcutaneous fat. On the other hand, if it is first noted after birth but is isolated (i.e. not associated with accelerated linear growth), the probability that the girl has a ‘simple virilizing’ form of CAH (i.e. without salt wasting) is low. A short period of watchful waiting may be in order, but if clitoromegaly increases, an androgen‐producing tumour of the adrenals or ovaries should be considered.

Micropenis

The length of the penis should be evaluated by pressing firmly on the prepubic fat and should be compared with the existing growth curves. These show that the 10th centile for penile length in term newborns is 2.5 cm. In otherwise normal newborns with true micropenis, the presence of testes should be ascertained. If normal testes are felt with certainty, isolated gonadotrophin deficiency remains possible but can be easily ruled out by measuring plasma testosterone at about two months of age; indeed, the ‘minipuberty of infancy’ creates a critical window of opportunity to assess the integrity of the hypothalamo–pituitary–testicular axis. If no testes are felt, bilateral anorchia (which results from bilateral testicular torsion some time after sex differentiation is complete) can be ruled out by measuring plasma AMH levels as early as three days after birth (when FSH may still be normal due to inhibition by maternal oestrogens). In infant boys with micropenis due to either gonadotrophin deficiency or anorchia, sensitivity to androgens is normal and treatment with testosterone enanthate (25 mg im monthly for three months) will result in a doubling of penile size.

Penile length increases until about four years of age and then remains stable until puberty. Therefore, by far the most common reason for referral for ‘micropenis’ at school age is for obese boys with a penis that is buried in prepubic fat. Anosmia may suggest an associated gonadotrophin deficiency, but in its absence these boys can be reassured by a simple physical examination as described above.

Cryptorchidism

The best time to accurately assess the presence and position of the testes is the first year of life, before the cremasteric reflex sets in. Although testicular descent may not be complete at birth in some normal term newborns (in which case one may take advantage of the ‘minipuberty’ to exclude gonadotrophin deficiency as outlined above), the testes should have reached a scrotal position by the end of the first year. Parents should be informed that their son has two normally descended testes and this should be noted in the health record.

If this is not done, undue concern will arise at a later age and sometimes lead to superfluous investigations and even unnecessary surgeries. Indeed, even by careful examination with warm hands, many school‐aged boys will have testicles that are difficult to palpate. The vast majority of these boys have retractile testes and their testes will descend permanently in the scrotum at the beginning of puberty.

If true cryptorchidism is established after repeated examinations, the extent of investigations will depend on its unilateral or bilateral nature and on whether the testes are palpable or not. In the latter case, plasma AMH will establish whether there are intra‐abdominal testes (in mid‐childhood, plasma FSH may be normal even in the absence of functional gonads). How these intra‐abdominal testes can be located by imaging is beyond the scope of this chapter; suffice it to say that ultrasound examinations are useless except if the testes are in the inguinal canal. Otherwise normal boys with incompletely descended but palpable testes, either unilaterally or bilaterally, can be referred for surgery without investigation.

Hypospadias

Hypospadias is defined as incomplete fusion of the urethra with the meatus on the ventral aspect of the penis – the glans, corona, shaft of penis, and (most severe) the perineum. This abnormality is commonly accompanied by chordee – curvature of the penis – caused by fibrosis of the corpus spongiosum.

Isolated hypospadias (hypospadias without micropenis and with normally descended testes in a normal scrotum) is rarely associated with an endocrine disorder. Surgeons should be asked to refer the following conditions for further investigation, including karyotype:

• perineal hypospadias;
  
• hypospadias with micropenis;
  
• hypospadias with cryptorchidism ± scrotal abnormality.

However, even extensive investigation of such individuals does not always lead to a specific diagnosis.