Disorders of Sex Development

Overview of Sex Differentiation

In typical differentiation from the sexually undifferentiated early fetus, the final phenotype of the external and internal genitalia is consistent with a normal sex chromosome complement (either XX or XY). The process of sex differentiation and development follows a consistent timeline ( Fig. 26.4 ). A 46,XX complement of chromosomes as well as genetic factors, including DAX1, the signaling molecule WNT-4, CTNNB1, and R-spondin 1, are among the many factors needed for the development of normal ovaries and müllerian (paramesonephric) ducts (uterus, fallopian tubes, and upper vagina). Development of the male phenotype requires the product of a Y chromosome gene called SRY (Sex-determining Region on the Y chromosome), which, in concert with products of other genes such as SOX9 , SF1 , WT1 , and FGF9 , directs the undifferentiated gonad to become a testis. SRY acts as a transcriptional regulator to increase cellular proliferation, attract interstitial cells from adjacent mesonephros into the genital ridge, and stimulate testicular Sertoli cell differentiation. Sertoli cells act as an organizer of steroidogenic and germ cell lines and produce antimüllerian hormone that causes the female (paramesonephric) duct system to regress. Aberrant genetic recombinations may result in X chromosomes carrying SRY , resulting in XX males (46,XX testicular DSD), or Y chromosomes that have lost SRY , resulting in XY females (46,XY DSD due to gonadal dysgenesis). Epigenetic causes of abnormal sex differentiation have been shown in plants, invertebrates, and vertebrates and will likely be shown to contribute to human DSD as well.

Antimüllerian hormone (AMH) from the ipsilateral fetal testis causes the müllerian (paramesonephric) ducts to regress. In its absence, they persist as the uterus, fallopian tubes, cervix, and upper vagina. By about 8 weeks of gestation, the Leydig cells of the testis begin to produce testosterone. During this critical period of male differentiation, testosterone secretion is stimulated by placental human chorionic gonadotropin (hCG), which peaks at 8–12 weeks. In the latter half of pregnancy, lower levels of testosterone are maintained by luteinizing hormone (LH) secreted by the fetal pituitary. Testosterone produced locally initiates development of the ipsilateral wolffian (mesonephric) duct into the epididymis, vas deferens, and seminal vesicle. Development of the external genitalia also requires dihydrotestosterone (DHT) , the more active metabolite of testosterone. DHT is produced largely from circulating testosterone and is necessary for fusion of the genital folds to form the penis and scrotum. DHT is also produced via an alternative biosynthetic pathway from androstanediol, and this pathway must also be intact for normal and complete prenatal virilization to occur. A functional androgen receptor , produced by an X-linked gene, is required for testosterone and DHT to produce the androgen effects.

In the XX fetus with normal long and short arms of the X chromosomes, the bipotential gonad develops into an ovary by about the 10th–11th week. This occurs only in the absence of SRY, testosterone, and AMH and requires a normal gene in the DSS (Dosage Sensitive Sex reversal) locus of DAX1 (DSS Adrenal hypoplasia congenital region on X, also known as NROB1), WNT-4, and R-spondin 1. A female external phenotype will develop even in the absence of fetal gonads. Unlike development of the male external phenotype, which requires androgen production and its action, estrogen is unnecessary for normal female prenatal sex differentiation. This is demonstrated by 46,XX patients who lack estrogen due to a deficiency of aromatase, the enzyme required for conversion of androgen to estrogen. Development of the ovary was once thought to be a passive process in the absence of SRY. Although the morphologic changes in the developing ovary are less marked than in the testis, there are a number of sequentially expressed genes and pathways that are required for complete ovarian development as well as maintenance of ovarian integrity postnatally. One of these genes is R-spondin 1, which, if variants result in abnormal function, can result in testicular or ovotesticular development in 46,XX individuals. Once developed, the ovary requires FAX12 to preserve its differentiation and stability.

Several genes important to the pathoetiology of DSD are listed in Table 26.4 .

Overview of Gonadal Function

Testes

Levels of placental hCG peak at 8–12 weeks of gestation, and in males hCG stimulates the fetal Leydig cells to secrete testosterone, the main hormonal product of the testis. In the classical androgen biosynthetic pathway ( Fig. 26.5 ), testosterone is then converted by the enzyme 5α-reductase to its more potent metabolite, DHT. This early period is critical for virilization of the XY fetus including fusion of the midline to form the scrotum and extension of the urethral meatus to distal penile opening (see Fig. 26.1 ). Defects in this process lead to various deviations from typical male development. After virilization, fetal levels of testosterone decrease but are maintained at lower levels in the latter half of pregnancy by LH secreted by the fetal pituitary. This LH-mediated testosterone secretion is required for continued penile growth and, to some degree, for testicular descent.

As part of the normal transition from intrauterine to extrauterine life, perhaps related to the sudden withdrawal of maternal and placental hormones, newborns and young infants experience a transient surge of gonadotropins and sex steroids. This is the so-called minipuberty .

In males, LH and testosterone peak at 1–2 months of age and then decline to reach prepubertal levels by 4–6 months of age. Follicle-stimulating hormone (FSH), along with inhibin B, peaks at 3 months and declines to prepubertal levels by 9 and 15 months, respectively. The LH rise is more dominant than that of FSH.

The neonatal surge may be important for postnatal maturation of the gonads, for stabilization of male external genitalia, and perhaps also for gender identity and sexual behaviors. The postnatal surge in LH and testosterone is absent or blunted in infants with hypopituitarism, cryptorchidism, and complete androgen insensitivity syndrome (CAIS). The development of nocturnal pulsatile secretion of LH marks the advent of puberty.

AMH, inhibin, and activin are members of the transforming growth factor-β (TGF-β) superfamily of growth factors. AMH is the earliest secreted product of the Sertoli cells of the fetal testis. The AMH receptor is expressed in Sertoli cells. In the female it is present in fetal müllerian duct cells and in granulosa cells (fetal and postnatal). During sex differentiation in males, AMH causes involution of the müllerian ducts. AMH is secreted in males by Sertoli cells during both fetal and postnatal life. In females, it is secreted by ovarian granulosa cells from 36 weeks of gestation to menopause, but at lower levels. The serum concentration of AMH in males is highest at birth, whereas in females it is highest at puberty. After puberty, both sexes have similar serum concentrations of AMH. Its role in postnatal life is not yet fully characterized.

Inhibin is another glycoprotein hormone secreted by testicular Sertoli cells and ovarian granulosa and theca cells. Inhibin A consists of an α subunit disulfide linked to the β-A subunit, whereas inhibin B consists of the same α subunit linked to the β-B subunit. Activins are dimers of the B subunits, either homodimers (BA/BA, BB/BB) or heterodimers (BA/BB). Inhibins selectively inhibit, whereas activins stimulate pituitary FSH secretion. Inhibin A is absent in males and is present mostly in the luteal phase in women. Inhibin B is the principal form of inhibin in males, and in females during the follicular phase. Inhibin B is useful as a marker of Sertoli cell function in males . FSH stimulates inhibin B secretion in females and males, but only in males is there also evidence for gonadotropin-independent regulation. In males with delayed puberty, inhibin B may be a useful screening test to differentiate between constitutional delay of puberty and hypogonadotropic hypogonadism (HH). In HH the serum inhibin B level has been shown to be very low to undetectable.

Like inhibin and activin, follistatin (a single-chain glycosylated protein) is produced by gonads and other tissues such as the hypothalamus, kidney, adrenal gland, and placenta. Follistatin inhibits FSH secretion principally by binding activins, thereby blocking the effects of activins at the level of both the ovary and pituitary.

Many additional peptides act as mediators of the development and function of the testis. They include neurohormones such as growth hormone–releasing hormone, gonadotropin-releasing hormone, corticotropin-releasing hormone, oxytocin, arginine vasopressin, somatostatin, substance P, and neuropeptide Y; growth factors such as insulin-like growth factors (IGFs) and IGF-binding proteins, TGF-β, and fibroblast, platelet-derived, and nerve growth factors; vasoactive peptides; and immune-derived cytokines such as tumor necrosis factor and interleukins IL-1, IL-2, IL-4, and IL-6.

Testicular development is marked by major maturational changes at puberty (see Chapter 55 ). Clinical patterns of pubertal changes vary widely. In 95% of boys, enlargement of the genitals, which is typically the first sign of puberty, begins between 9.5 and 13.5 years, reaching maturity at 13–17 years. In a minority of normal boys, puberty begins after 15 years of age. In some boys, pubertal development is completed in less than 2 years, but in others it may take longer than 4.5 years. Pubertal development and the adolescent growth spurt occur at an older age in boys than in girls.

The median age of sperm production (spermarche) is 14 years. This event occurs in mid-puberty as judged by pubic hair, testis size, evidence of growth spurt, and testosterone levels. Nighttime levels of FSH are in the adult male range at the time of spermarche; the first conscious ejaculation occurs at about the same time.

Diagnostic Approach to the Patient with Atypical or Ambiguous Genitalia

Infants with ambiguous or atypical genitalia should be evaluated and treated at a center with multidisciplinary experience in DSD. The appearance of the external genitalia is rarely diagnostic of a particular disorder, and thus does not often allow distinction among the various forms of DSD. Some clues are noted in Table 26.5 . The most common causes of 46,XX DSD are virilizing forms of congenital adrenal hyperplasia (CAH) . It is important to note that in 46,XY DSD, the specific diagnosis is not found in up to 50% of cases. At one experienced center, the six most common diagnoses accounted for 50% of the cases. These included virilizing CAH (14%), androgen insensitivity syndrome (10%), mixed gonadal dysgenesis (8%), clitoral/labial anomalies (7%), hypogonadotropic hypogonadism (6%), and 46,XY small-for-gestational age males with hypospadias (6%). The etiology in cases of 46,XY DSD without a known diagnosis can be further delineated using exome sequencing technologies.

The difficulty of establishing a diagnosis in 46,XY DSD and the resulting lack of specific management emphasizes the need for thorough diagnostic evaluations ( Fig. 26.6 ). These include biochemical characterization of possible steroidogenic enzymatic defects, imaging studies to characterize internal genitalia, and determination of genetic sex as well as other genetic studies as determined by each individual patient with atypical genitalia. The parents need counseling about the potentially complex nature of the baby’s condition, and guidance as to how to deal with the curiosity of their well-meaning friends and family members. The evaluation and management should be carried out by a multidisciplinary team of experts that include practitioners in pediatric endocrinology, pediatric surgery/urology, pediatric radiology, newborn medicine/neonatology, genetics, and psychology. On occasion, the sex of rearing will need to be uncommitted until the diagnostic evaluation is completed . Once the sex of rearing has been agreed on by the family and team, treatment can be organized. Genetic counseling should be offered as part of routine work-up.

After a complete history and physical exam, the common diagnostic approach includes multiple steps that are usually performed at the same time rather than waiting for results of one test prior to performing another, in order to expedite the diagnosis. At many centers, the initial evaluation may include a broad genetic screening panel that simultaneously examines for multiple potential disease-associated genetic variants. Careful attention to the presence of physical features other than the genitalia is crucial to determine if a diagnosis of a particular multisystem syndrome is possible ( Table 26.6 and Fig. 26.7 ). A summary of many features of commonly encountered causes of DSD is provided in Table 26.7 .

Diagnostic tests include the following:

• 1.

  Blood karyotype, with rapid determination of sex chromosomes (in many centers this is available within 24–48 hours) (see Fig. 26.7 ).

• 2.

  Other blood tests (see Table 26.6 )

  • a.

    Screen for congenital adrenal hyperplasia: cortisol biosynthetic precursors and adrenal androgens, particularly serum levels of 17-hydroxyprogesterone and androstenedione for 21-hydroxylase deficiency, the most common form. In the United States, all 50 states have a newborn screen for 21-hydroxylase deficiency.

  • b.

    Screen for androgen biosynthetic defects with serum levels of androgens and their precursors.

  • c.

    Assess for gonadal responsiveness to gonadotropin to screen for testicular gonadal tissue: measure serum levels of testosterone and dihydrotestosterone before and after intramuscular injections of hCG.

  • d.

    Molecular genetic analyses for SRY (sex-determining region of the Y chromosome), other Y-specific loci, and, when appropriate, broad screening for other known single-gene defects associated with DSD (see Table 26.4 ).

  • e.

    Gonadotropin levels (LH and FSH).

  • f.

    AMH and inhibin B levels.

• 3.

  The internal anatomy of patients with ambiguous genitalia can be defined with one or more of the following studies:

  • a.

    Pelvic ultrasound; renal and adrenal ultrasound.

  • b.

    Pelvic MRI.

  • c.

    Genitourethrograms.

  • d.

    Endoscopic examination of the genitourinary tract.

  • e.

    Exploratory laparoscopy to locate and characterize/biopsy the gonads.

Basic Approaches to the Diagnosis and Management of Disorders of Sex Development

In the neonate, the presence of atypical genitalia requires immediate attention to determine the etiology and then, if necessary, decide on the sex of rearing. In some cases, DSD is associated with other abnormalities that adversely impact the child’s health, particularly the salt-losing forms of CAH, adrenal insufficiency related to SF1 defects, and renal abnormalities associated with WT-1 defects.

The family of the infant needs to be informed of the child’s condition as early, completely, compassionately, and honestly as possible. Caution must be used to avoid feelings of guilt, shame, and confusion. Guidance needs to be provided to alleviate both short-term and long-term concerns and to allow the child to grow up in a completely supportive environment. The initial care is best provided by a team of professionals who remain focused foremost on the needs of the child. Management of the potential emotional and psychologic effects that these disorders can generate in the child and the family is of paramount importance and requires the involvement of physicians, psychologists, and other health care professionals with sensitivity, training, and experience in this field.

While awaiting the results of blood tests, imaging with pelvic ultrasonography and/or MRI is used to determine the presence of a uterus and ovaries. Presence of a uterus and absence of external palpable gonads often suggest a virilized XX female. A search for the source of virilization includes studies of adrenal hormones to rule out varieties of CAH, and studies of androgens and estrogens may be necessary to rule out aromatase deficiency. Virilized XX females with CAH are generally (but not always) reared as females even when the genitalia are Prader stage 4 or 5 (see Fig. 26.3 ).

The absence of a uterus, with or without palpable external gonads, may indicate an undervirilized XY male. Measurements of blood levels of gonadotropins, testosterone, AMH, and DHT are necessary to determine whether testicular production of androgen is present and is normal. Undervirilized males who are totally feminized may be reared as females. Certain significantly undervirilized infants, such as those with 5α-reductase deficiency, may be reared as males because these children virilize normally at puberty. Sixty percent of individuals with 5α-reductase deficiency assigned as female in infancy will identify as males as adults. An infant with a comparable degree of undervirilization resulting from an androgen receptor defect, such as androgen insensitivity syndrome (AIS), may be successfully reared as a female, depending on androgen responsiveness.

In some mammals, the female exposed to androgens prenatally or in early postnatal life exhibits nontraditional sexual behavior in adult life. Most, but not all, females who have undergone fetal masculinization from CAH have female sexual identity, although during childhood they may appear to prefer male typical play activities over female typical play activities.

In the past it was thought that surgical treatment of ambiguous genitalia to create a female appearance, particularly when a vagina was present, was more successful than construction of male genitalia. Considerable controversy has developed regarding these decisions. Sexual functioning is to a large extent more dependent on neurohormonal and behavioral factors than the physical appearance and functional capacity of the genitalia. Similarly, controversy exists regarding the timing of the performance of invasive and definitive procedures, such as surgery. Whenever possible, without endangering the physical or psychologic health of the child, an expert multidisciplinary team should consider deferring elective surgical procedures and gonadectomies until the child can participate in the informed consent for the procedure.

For all patients with DSD who have Y-chromosome material and intraabdominal gonads, gonadectomy is generally recommended due to the risk of gonadal tumors developing with increasing age, many of which are malignant. The risk is highest in children with gonadal dysgenesis, partial androgen insensitivity, or Frasier or Denys-Drash syndrome.

The pediatrician, pediatric endocrinologist, and psychologist, along with the appropriate additional specialists, should provide ongoing compassionate, supportive care to the patient and the patient’s family throughout childhood, adolescence, and adulthood. Active support groups are available for families and patients with many of the conditions discussed.

Specific Types of Disorders of Sex Development