Chapter 6: Disorders of Sex Development

The nomenclature, Disorders of Sex Development (DSDs), replaces the term “intersex,”^1,2 to encompass various congenital conditions in which there is inconsistency between chromosomal, gonadal, and/or anatomical sex. Conditions with ambiguous external genitalia are included, encompassing atypical genital development. While this system (Table 6-1) allows the incorporation of new genetic and other information, the primary clinical advantage is a guideline for early diagnostic evaluation, approaching by phenotype and karyotype, to guide further etiological evaluation and management. This classification of diagnostic categories (Table 6-2) is limited because some conditions fail to fit into a single specific diagnostic category or fit into more than one category; for example, forms of gonadal dysgenesis may have a 46,XY, 46,XX, or 45,X/46,XY karyotype. This schema includes the genetic cause when known and allows for phenotypic variation within etiological categories. DSDs primarily include those conditions impacting reproductive or sexual function, while including variations of normal development such as mild hypospadias and idiopathic clitoromegaly. Even though a karyotype is used for initial categorization, the ideal current approach involves specific molecular etiologies, as genes are more specific, and would avoid the perception that sex is determined by chromosomes. For example, the presence of a Y chromosome may not be associated with consistent findings regarding factors important in determining sex assignment or assessing outcome, including fetal androgen exposure, genital masculinization, or germ cell development.

Accurate estimates regarding the incidence of DSDs are not available because of the multiple and rare ­etiologies. The most common cause of a DSD, classicadrenal hyperplasia (CAH) secondary to 21-hydroxylasedeficiency, occurs in 1:10,000-1:18,000 births as determined by newborn screening data, with considerable variation among ethnic/racial backgrounds related to mutation frequency. Deficiency of 11-hydroxylase is generally rare, although the incidence is high among Moroccan Jews (∼1 in 6000). The estimated incidence of 17-hydroxysteroid dehydrogenase deficiency is 1 in 147,000. The Smith-Lemli-Opitz syndrome, which is associated with multiple congenital abnormalities, including urogenital anomalies, occurs with an estimated incidence of 1 in 20,000 to 60,000 live births. There are no estimated incidences of most rare DSDs.

Differentiation of the reproductive/sexual system can be divided into the components outlined in Table 6-3.

Basics of Sexual Differentiation

Internal reproductive system differentiation

The paramesonephric/müllerian and mesonephric/wolffian ducts, primordial reproductive structures, develop in both sexes. In fetal testes, Sertoli cells begin to secrete anti-müllerian hormone (AMH) (also known as müllerian-inhibiting hormone [MIH] or müllerian-inhibiting substance [MIS]) by the seventh week of gestation. This hormone triggers side-specific regression of müllerian duct development. Subsequently, testosterone secreted by testicular Leydig cells, under the drive of placental human chorionic gonadotropin (hCG) before the fetal pituitary begins to secrete luteinizing hormone (LH) around the 12th week of gestation, promotes side-specific wolffian duct development into the epididymis, vas deferens, and seminal vesicle. The lack of both of these testicular hormones in the normal female fetus allows müllerian duct maturation into fallopian tubes, uterus, cervix, and upper vagina, without wolffian duct development.

External reproductive system differentiation

Primordial bipotential genital structures, the genital tubercle, urethral folds, and labioscrotal swellings, differentiate into male or female external genitalia. The enzyme, 5α-reductase type 2 (hereafter referred to as 5α-reductase), expressed in these structures in the male fetus, converts testosterone, secreted by the fetal testes, into dihydrotestosterone (DHT). DHT, produced in sufficient amounts from testosterone even from a single functioning testis, induces male external genital differentiation. In the normal human male fetus, a cylindrical phallus approximating 2 mm in length with genital swellings develops by 9 weeks of gestation. The developing penis is not larger than the clitoris at 12 weeks although sonography can differentiate the male from the female phallus based on its angle. By 14 weeks of gestation, the urethral folds fuse to form the cavernous urethra and corpus spongiosum. The genital tubercle develops into the corpora cavernosa of the penis, and the labioscrotal folds fuse from posterior to anterior to form the scrotum. By 14 weeks, the external genitalia are clearly masculine except for testicular descent. Fetal phallic growth after genital differentiation is a result of fetal pituitary LH stimulation of testosterone production by testicular Leydig cells. Anogenital distance (AGD), measured from the anus to the most posterior edge of the labia or scrotum, is an index of extent of androgen exposure occurring during a masculinization programming window, likely at the onset of fetal genital differentiation, based on data from rodents.³ While such a masculinization programming window suggests a regulated limit for genital growth, adequate androgen exposure subsequently is crucial for maximal growth. Hence, for the clinician, it is unclear to what extent treatment with androgen will increase penile length. Though the AGD may be useful when assessing male physical and physiologic development at any age because reduced AGD distance suggests diminished androgen exposure during the aforementioned masculinization programming window among males, the clinical application is limited. Initial assessment may still involve androgen stimulation to assess responsiveness. A diminished AGD for age has been associated with hypospadias or poorer sperm quality, or exposure to endocrine disruptor chemicals.

Deficient testosterone production, inadequate testosterone action, or other factors result in incomplete fusion of the genital components as illustrated in Figure 6-1. This figure depicts genitalia along the spectrum from female to male as may occur in a 46,XX fetus with excessive androgen stimulation or in a 46,XY fetus with deficient androgen exposure and/or response. However, the size and extent of fusion of phallic development do not necessarily correlate with the extent of labioscrotal fusion, so each should be described separately. The high prevalence of hypospadias in boys suggests that urethral fusion is a delicate and finely regulated process that may be impacted by numerous other factors in addition to androgen. The degree of fusion of the urethra may extend further toward the tip than fusion of the underlying spongiosum, creating a condition that makes surgical repair difficult.

The critical role of DHT to promote full differentiation of male external genitalia is illustrated by the incomplete differentiation of male external genitalia among boys with 5α-reductase deficiency. Affected boys have normally functioning testes and normal testosterone production, but they are unable to convert testosterone to DHT within external genital structures and the prostate during fetal life. However, increased androgen exposure at puberty typically results in further virilization. Such undervirilization at birth has led to a female sex of rearing, particularly if the diagnosis is not recognized, whereas virilization at puberty has led to gender reassignment. In the modern era, hopefully, the diagnosis is made in a timely fashion leading to a male sex assignment.

In the absence of androgens or androgen action, the urethral folds and labioscrotal swellings in the 46,XX (or 46,XY) fetus remain unfused and develop into the labia minora and labia majora, respectively. The genital tubercle forms the clitoris, and canalization of the vaginal plate creates the lower portion of the vagina. By 11 weeks of gestation, the clitoris is prominent and the lateral boundaries of the urogenital sulcus have separated. The separation of vagina and urethra, which is not hormone dependent, is completed by 12 weeks. However, high levels of androgen prevent this separation as evidenced by high insertion of the urethra into the vagina that may occur with CAH. At 20 weeks of gestation, normal female differentiation results in minimal clitoral growth and well-defined labia majora, but as yet relatively hypoplastic labia minora with separate vaginal and urethral perineal openings. Excessive androgen stimulation from any source in the female fetus during the period of genital differentiation prior to 12 weeks of gestation results in labial fusion and phallic urethral development, apparently related to extent of androgen exposure (see Figure 6-1). Later during gestation androgen excess cannot change differentiation, but it results in excessive clitoral growth and scrotalization of the unfused labial folds. The gestational timing of androgen excess can thus be estimated by the extent of masculinization (differentiation) and virilization (growth excess) of the genitalia.

Gonadal differentiation

The urogenital ridges, the primordial structures that form the gonads, reproductive tract, kidneys, and adrenal cortices, are present by 6 weeks of gestation, are outgrowths of coelomic epithelium (mesothelium). Proximity to the mesonephros, the source of endothelial cells, is necessary for gonadal differentiation. The primitive gonad in the urogenital ridge has a cortex and a medulla. The medullary portion begins to differentiate into testicular tissue around the seventh week of gestation, earlier than ovarian development from the primitive gonadal cortex.

SRY (sex-determining region on the Y chromosome) is a nuclear high-mobility group (HMG) domain protein expressed in pre-Sertoli cells where it triggers a molecular switch to initiate the process of male sexual differentiation. In fact, the first evidence of testicular differentiation, induced by the SRY protein, is the appearance of primitive Sertoli cells at 6 to 7 weeks of gestation. This is followed by migration of endothelial cells and the appearance of peritubular myoid cells, Leydig cells, and germ cells. Interaction of primitive Sertoli cells and endothelial cells results in testicular cord development, which become seminiferous tubules containing mature Sertoli and germ cells. Cell proliferation rates increase, and germ cells enter a state of mitotic arrest.

Germ cell meiosis in the ovary begins at 10 to 11 weeks of gestation. Over the next 10 weeks, after the appearance of oögonia, structured follicles are present. Primary follicles, derived from granulosa cell precursors, develop adjacent to the primary oöcyte, becoming evident at 20 weeks of gestation. As many as 7 million oöcytes present at mid-gestation begin to degenerate, so approximately 2 million are present at birth. Such follicular atresia appears to be accelerated in the streak gonads of fetuses with X monosomy.

Time Course of Sexual Differentiation

The sequence of the development of the components of the reproductive system development (Figure 6-2) involves differentiation of the bipotential primordial structures discussed earlier.

Differentiation of the testis with discernible ­Sertoli and Leydig cell lines beginning around the ­seventh to eighth week of gestation precedes ovarian differentiation in which oögonia do not appear until the 10th week of gestation. Secretion of testosterone by fetal Leydig cells begins concomitantly with differentiation of the wolffian ducts; müllerian duct regression in the male is evident slightly later. Male external genital differentiation begins around the eighth week of gestation and is completed by the 12th to 14th week. Virilization continues with penile growth and, in the presence of excessive androgen in the female fetus, clitoral enlargement. Vaginal development occurs during this period with the lower two-thirds arising from an invagination of the epidermal layers and merging with the upper third that develops from a downward extension of the müllerian ducts.

Molecular/Genetic Overview

Genes known to be involved in the differentiation of the gonads, external genitalia, and internal genitalia are depicted in Figure 6-3.

The SRY gene encodes a transcription factor that initiates the formation of the testes. Subsequently, the SOX9 gene, which is necessary for Sertoli-cell differentiation and type 2 collagen production, is expressed in the testes.⁴ Mutations of SOX9 are associated with sex reversal. Haploinsufficiency of SOX9 results in campomelic dysplasia, a disorder showing skeletal dysplasia that is associated with sex reversal in the majority of affected XY individuals (XY sex reversal). Familial campomelic dysplasia associated with a deletion upstream of SOX9 has been described in a mother and 46,XY child with female external genitalia, normal uterus, and streak gonads.⁵ Duplication of SOX9 is a cause of a male phenotype and an XX karyotype (XX sex reversal).

Other molecular factors involved in gonadal ­differentiation include steroidogenic factor-1 (SF1), a transcription factor necessary for steroidogenesis, male sexual differentiation, and fertility. Mutations of the SF1/NR5A1 gene have been identified in patients with agonadism, adrenal hypoplasia, hypogonadotropic hypogonadism, cryptorchidism, micropenis, and XY sex reversal.⁶

A gonad-specific transcription factor upregulated in the ovary called DAX1 (Dosage-sensitive Adrenal hypoplasia congenita region on the X chromosome) that is encoded by the DAX1/NROB1 gene is not required for normal testicular development, but it appears to function as an antitestis factor in the XX ovary (Figure 6-4). Duplication of DAX1 in the 46,XY fetus is thought to repress SRY resulting in XY sex reversal, and DAX1/NROB1 mutations are associated with a syndrome of adrenal hypoplasia and hypogonadotropic hypogonadism ­(adrenal hypoplasia congenita [AHC]).

Thus, a threshold level of SRY activity is necessary for male sexual differentiation. Sequential expression of several other genes, including SRY-related HMG box-containing-9 (SOX9), MIH, DAX1/NROB1, steroidogenic factor-1 (SF1/NR5A1), Wilms tumor 1 (WT1), GATA-binding-4 (GATA4), desert hedgehog (DHH), patched (PTC), wingless-related mouse mammary tumor virus (MMTV) integration site 4 (WNT4), and WNT7a, are required for normal male sexual differentiation. Cells, mostly endothelial in origin, migrate from the mesonephros and interact with the pre-Sertoli cells to promote development of the testicular cords.

Although it has generally been considered that ovarian differentiation is constitutive, evidence suggests that specific genes are required for normal ovarian ­development. Primordial germ cells migrate from their origin in the hindgut and colonize in the genital ridges under the influence of fragilis (interferon-induced transmembrane) proteins 1 and 3 (IFITM1 and IFITM3), ­stromal cell-derived factor 1 (SDF1 or CXCL12), and its receptor CXCR4.

Disorders Associated With Ambiguous Genitalia

Steroidogenesis-metabolic pathways

Steroidogenic enzyme gene mutations are a frequent cause of genital ambiguity resulting from alterations in sex steroid biosynthesis. These enzymes are expressed in the placenta, gonads, and adrenal cortex. In the 46,XY fetus, specific proteins necessary for testosterone and DHT biosynthesis include SF1, steroidogenic acute regulatory peptide (StAR), 17α-hydroxylase/17,20-lyase (CYP17), 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), 17α-hydroxysteroid dehydrogenase type 3 (HSD17B3), P450-oxidoreductase (hereafter referred to as oxidoreductase), and 5α-reductase type 2 (SRD5A2) (Figure 6-5). Defective testosterone biosynthesis can lead to ambiguous genitalia in the 46,XY fetus.

Steroidogenic pathways of the adrenal cortex, ovary, and testis are depicted in Figure 6-5. Differences in enzyme expression are indicated by the specific boxes showing androstenedione as the endpoint of sex steroid synthesis in the adrenal (zona reticularis), testosterone in the testis, and estradiol in the ovary. Other steroid endpoints in the adrenal cortex include aldosterone (zona glomerulosa) and cortisol (zona fasciculata).

Virilizing disorders (defects in steroidogenesis) impacting 46,XX fetuses

21-Hydroxylase deficiency.

21-Hydroxylase deficiency caused by mutations in the 21-hydroxylase (CYP21A2) gene is the most common cause of CAH. This autosomal recessive disorder is responsible for more than 95% of the cases of 46,XX genital ambiguity. Deficient 21-hydroxylase activity results in decreased cortisol biosynthesis, secondary to insufficient conversion of 17-hydroxyprogesterone (17-OHP) to 11-deoxycortisolin the zona fasciculata, and more variable degrees of decreased aldosterone biosynthesis from impaired conversion of progesterone to deoxycorticosterone in the zona glomerulosa. The loss of negative feedback inhibition from cortisol leads to increased adrenocorticotropic hormone (ACTH) secretion with subsequent increased adrenal androgen secretion (Figure 6-6).

46,XX patients with severe 21-hydroxylase deficiency manifest genital ambiguity at birth. Virilization of the external genitalia exists along a spectrum ranging from mild clitoromegaly and mild posterior labial fusion to, although rare, a genital appearance consistent with a normal male with bilateral cryptorchidism. Clitoromegaly results from any source of excessive fetal androgen, and hence the various causes are listed in Box 6-1. Despite the excessive prenatal androgen exposure, affected females have normal internal reproductive anatomy with a uterus, normally positioned ovaries, fallopian tubes, and upper vagina (müllerian derivatives). Because of glucocorticoid and mineralocorticoid deficiencies, severely affected untreated infants develop hyponatremia, hyperkalemia, and dehydration around 7 to 10 days of life. The serum 17-OHP concentrations are usually more than 10,000 ng/dL and are often considerably higher. Serum androstenedione and progesterone concentrations are also elevated, but to a lesser degree than are those of 17-OHP. Measurement of plasma renin activity (PRA) is necessary to assess mineralocorticoid status, although discernment in the neonatal period may be difficult because of normally high levels. Newborn screening programs in all 50 states measure whole-blood 17-OHP concentrations obtained on filter paper with the primary goal of permitting early detection in affected male infants. Newborn screening programs generally do not detect children with nonclassic adrenal hyperplasia (NCAH), which is not a form of DSD.

The degree of impairment of 21-hydroxylase activity varies depending on the compromise of enzyme function associated with specific mutations. In general, complete loss-of-function mutations are associated with more severe virilization of the external genitalia in affected females and clinically relevant salt wasting. The simple-virilizing form of CAH is associated with a 1% to 2% level of residual enzyme activity whereas that of NCAH generally has a 20% to 60% level of enzyme activity. Over 100 mutations of the 21-hydroxylase gene, CYP21A2, have been reported. A limited number of mutations account for the majority of the affected alleles, and there appears to be reasonable genotype-phenotype correlation.

11β-Hydroxylase deficiency.

CAH caused by 11β-hydroxylase deficiency is associated with glucocorticoid deficiency, hypertension (excessive concentrations of salt-retaining hormones), and excessive androgen secretion. Curiously, some infants with 11β-hydroxylase deficiency may manifest salt loss and hypotension in the neonatal period. Affected female infants may present with ambiguous genitalia. In contrast to patients with 21-hydroxylase deficiency, those with 11β-hydroxylase deficiency have a hormonal profile with elevated 11-deoxycortisol and relatively less severe elevations in 17-OHP, androstenedione, and testosterone. PRA concentrations are typically, but not invariably, suppressed. Mutations in the 11β-hydroxylase (CYP11B1) gene, located on chromosome 8q22, are causative.

3β-Hydroxysteroid dehydrogenase isozyme deficiencies.

CAH caused by deficiency of 3β-hydroxysteroid dehydrogenase, type 2, may result in genital ambiguity in both 46,XX and 46,XY fetuses. In this autosomal recessive disorder, increased dehydroepiandrosterone (DHEA) synthesis leads to virilization in the 46,XX fetus, whereas deficient testosterone production results in undervirilization in the 46,XY fetus. The condition is characterized by elevated serum concentrations of the Δ⁵ steroids (pregnenolone, 17-hydroxypregnenolone, and DHEA) because of a reduced ability to convert them to their corresponding Δ⁴ steroids (progesterone, 17-OHP, and androstenedione) (see Figure 6-5). Impaired biosynthesis of mineralocorticoids, glucocorticoids, and sex steroids is associated with acute adrenal insufficiency that may occur in the newborn period. Mutations in the HSD3B2 gene, located on chromosome 1p13.1 and expressed in the adrenal cortex and gonads, have been documented in affected individuals.

Swiss families previously reported to have isolated 17,20-lyase deficiency have been studied⁷ and found to have a normal CYP17A1 gene, but instead to harbor mutations in the 3α-hydroxysteroid dehydrogenase type III (AKR1C2) and another in the 3α-HSD gene (AKR1C4). This identifies alternative pathways for DHT synthesis (bypassing testosterone), in addition to the classically described pathways that work through testosterone, leading to the formation of the same end products. This finding suggests that both the classic and alternative “backdoor” pathways are necessary for normal male external genital development.

Placental aromatase deficiency.

The enzyme, aromatase, catalyzes the biosynthesis of estrogens from androgens. Aromatase deficiency is a rare autosomal recessive disorder in which inactivating mutations of the aromatase (CYP19A1) gene cause increased serum androgen levels. Because this enzyme promotes estrogen biosynthesis, there are distinct phenotypes in males and females.⁸ Aromatase is active in the placenta, where it normally protects the mother from the potential virilizing effects of the fetal androgens (predominantly DHEA and DHEAS). In placental aromatase deficiency, 46,XX fetuses are virilized and manifest variable degrees of labioscrotal fusion, clitoromegaly, and perineoscrotal hypospadias. Often, there is a history of progressive maternal virilization during pregnancycharacterized by acne, hirsutism, clitoral hypertrophy, and frontal balding. During the pregnancy, maternal concentrations of testosterone, DHT, and androstenedione are elevated and decline rapidly after delivery. Affected 46,XX children have delayed pubertal development secondary to hypergonadotropic hypogonadism with poor breast development, primary amenorrhea, and cystic ovaries. Affected 46,XY infants have normal internal and external genital development but, at the age of puberty, may have tall stature and continued growth (asymmetrical with relatively long legs) because estrogen is required to fuse the growth plates of the long bones. The estrogen deficiency found in men with aromatase deficiency is associated with abdominal obesity, insulin resistance, dyslipidemia, and relativeinfertility.

Oxidoreductase deficiency.

Another disorder affecting steroidogenesis is characterized by decreased 17α-hydroxylase and 21-hydroxylase activities. The phenotype includes genital ambiguity in males and females, craniosynostosis, mid-face hypoplasia, radiohumeral synostosis, and glucocorticoid deficiency. The skeletal manifestations are reminiscent of those seen in the Antley-Bixler syndrome, an autosomal dominant disorder caused by mutations in the fibroblast growth factor receptor-2 (FGFR2) gene. Affected males may be undervirilized whereas females may be virilized. Signs of androgen excess, including acne, hirsutism, and clitoromegaly, may develop during pregnancy in mothers carrying affected fetuses. Typically, postnatal virilization does not occur. The 17-OHP levels are elevated, sex steroid levels are low, and mineralocorticoid concentrations are normal.

This disorder is caused by loss-of-function mutations in the cytochrome P450 oxidoreductase (POR) gene that is located on chromosome 7q11-12. This gene encodes a protein that functions as a cofactor for several steroidogenic enzymes.⁹ It has been suggested that an alternative pathway of androgen biosynthesis, with conversion of 17-OHP to 5α-pregnane-3α,17α-dio-20-one to androstenedione, may be seen in this disorder.¹⁰

Defects in testosterone synthesis impacting the 46,XY fetus

17β-HSD deficiency.

The 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) gene is expressed in the Leydig cells of the testes where it catalyzes conversion of androstenedione to testosterone (Figure 6-5). Loss-of-function mutations in this gene are inherited in an autosomal recessive manner and result in testosterone deficiency and undervirilization of 46,XY fetuses. In some instances, affected males are thought to be females at birth. The external genital phenotype in the 46,XY infant with 17β-hydroxysteroid dehydrogenase deficiency may range from a completely female (see Figure 6-1) appearance with a blind-ending vaginal pouch to variable degrees of genital ambiguity with gonads palpable in the labioscrotal folds, labioscrotal fusion, and hypospadias.¹¹ Internal male reproductive structures derived from the wolffian ducts are usually present. Underproduction of androgen in females with this mutation appears to confer no obvious phenotype.

Elevated basal or hCG-stimulated androstenedione-to-testosterone ratios are consistent with this enzyme defect. When LH and FSH secretion increases during puberty, virilization may occur because of conversion of androstenedione to testosterone by the other unaffected isoforms of 17β-hydroxysteroid dehydrogenase present in extratesticular tissue. Affected children raised as females have been reported to change gender assignment at puberty. Thus, male gender assignment should be made in infancy for affected children. Breast development may result from increased conversion of androstenedione to estrone.

5α-Reductase deficiency.

The 5α-reductase type 2 (SRD5A2) gene is expressed in androgen target tissues such as the prostate and the primordial external genital tissue. This enzyme converts testosterone to DHT (see Figure 6-7), the hormone responsible for virilization of the external genitalia. The external genitalia of affected males are characterized by a small phallus, a urogenital sinus with perineal hypospadias, and a blind vaginal pouch. Internally, male (wolffian) structures are partially differentiated and female (müllerian) ducts are absent. Serum testosterone levels may be elevated, whereas DHT levels are typically low. Elevated basal or hCG-stimulated testosterone-to-DHT ratios (< 10 in normal and > 20 in affected) or diagnostic urinary Δ⁴/Δ⁵ steroid profiles are consistent with the diagnosis of 5α-reductase deficiency. In patients whose testicles are left intact, significant virilization often begins during puberty with development of penile growth, male physique, and voice change. In general, male ducts are inadequately developed to deliver mature sperm. Although this autosomal recessive condition is rare, there are clusters of affected individuals in the Dominican Republic, Papua New Guinea, Turkey, and the Middle East.¹²

Congenital lipoid adrenal hyperplasia (StAR ­mutations).

Congenital lipoid adrenal hyperplasia (Box 6-2) is an autosomal recessive disorder caused by loss-of-function mutations in the steroidogenic acute regulatory protein (StAR) gene. The StAR protein functions to move cholesterol from the outer to inner mitochondrial membrane,¹³ which, if absent or deficient, results in impaired conversion of cholesterol to pregnenolone, the first step in steroid synthesis.

Both 46,XY and 46,XX infants have essentially female external genitalia because external virilization is impaired as a result of decreased testosterone production. All adrenal and gonadal steroid hormone levels are low or undetectable, including 17-hydroxypregnenolone and pregnenolone, because steroidogenesis in both the adrenal cortex and in the gonads is deficient. Thus, affected infants also have mineralocorticoid and glucocorticoid deficiencies. In addition to the defective steroidogenesis, lipids accumulate in the steroidogenic cells, ultimately resulting in their destruction. Although congenital lipoid adrenal hyperplasia has been considered to be a disease affecting infants, later presentations have also been described.¹⁴

Steroid action

Androgen insensitivity is an X-linked recessive disorder caused by mutations in the androgen receptor (AR) gene which is located on the proximal long arm of the X chromosome.

Affected 46,XY children with complete androgen insensitivity syndrome (CAIS) present in infancy with inguinal or labial gonads, inguinal herniae, but otherwise normal external female genitalia. It has been estimated that 1% to 2% of girls with bilateral inguinal herniae have androgen insensitivity. Some affected individuals present in adolescence with primary amenorrhea. Findings in partial androgen insensitivity syndrome (PAIS) include ambiguous genitalia with perineoscrotal hypospadias, microphallus, bifid scrotum, and testes located in the scrotum or undescended. Müllerian derivatives such as the uterus are not present in androgen insensitivity because testicular MIH secretion results in regression of these structures.

Testosterone and LH levels may be elevated in infancy, although the usual LH surge and increased testosterone concentrations expected during the first few months of life may be absent. In affected children older than 10 years, elevated serum LH and testosterone concentrations become readily apparent. This loss-of-function mutation results in loss of hypothalamic-pituitary feedback inhibition leading to increased LH and testicular testosterone synthesis. In contrast, FSH concentrations may be normal or only mildly elevated because inhibin secretion by Sertoli cells is intact.

Infants with PAIS may require a trial of androgen administration to promote phallic enlargement. Over 300 AR gene mutations have been described in AIS. Generally, but not always, CAIS results from complete loss-of-function mutations such as deletions, insertions, or deletions associated with frameshifts and premature termination codons. In contrast, PAIS is associated with partial loss-of-function mutations.¹⁵ Although phenotype generally correlates with genotype, phenotypic heterogeneity can occur even among family members carrying the identical mutation. In addition to hormone determinations, DNA sequence analysis of the AR gene is commercially available (www.genetests.org and www.androgendb.mcgill.ca) and may be diagnostic.

Because of the presence of a Y chromosome, the risk for gonadal tumors, particularly carcinoma in situ (CIS) and seminomas, is increased. Thus, a decision must be made concerning the need for and timing of gonadectomy. For CAIS, but not PAIS, it can be argued that leaving the testes in place until pubertal feminization is complete is preferable. For those raised male, it can be argued that the testicles could be left intact as long as they are partially descended in the hopes of providing some fertility, function, and cosmetic appearance. Gonads should be carefully monitored for development of malignancy. Timing for gonadectomy remains controversial, with a tendency for earlier removal in the United States, but delayed removal in Europe. One series reported that only 2 of the 44 (< 5%) patients with CAIS who underwent gonadal biopsy had testicles with CIS.¹⁶

Luteinizing hormone defects

Leydig cell hypoplasia.

This autosomal recessive disorder arises from a loss-of-function mutation in the LH receptor gene (LHCGR). In the absence of effective LH stimulation because of LHCGR loss-of-function mutations, fetal Leydig cells fail to develop leading to deficient testosterone production. The phenotype ranges from complete male-to-female sex reversal or 46,XY undervirilization. Those with the female phenotype frequently have palpable labial gonads. Müllerian duct-derived structures are absent because MIH is normally secreted. Thus, the findings on physical examination are reminiscent of CAIS. Testes contain immature Sertoli cells, lack mature Leydig cells, but have rare spermatogonia.

Serum hormone levels include elevated LH, low testosterone, and normal FSH concentrations. There is no significant testosterone response to hCG stimulation. The LHCGR gene encodes for a seven-transmembrane domain G-protein–coupled receptor, which is expressed in fetal and adult Leydig cells. Sisters of affected 46,XY individuals having the same mutations have normal female genital differentiation and pubertal development with small- to normal-sized uteri and may develop ovarian cysts, but have amenorrhea secondary to inadequate estrogen secretion and infertility.¹⁷

Hypogonadotropic hypogonadism.

Male infants with hypogonadotropic hypogonadism (see Chapter 3) generally present with microphallus and/or cryptorchidism, but not genital ambiguity. Genetic etiologies of hypogonadotropic hypogonadism include, but are not limited to, inactivating mutations in the KAL1, FGFR1, FGF8, GPR54, PROKR2, PROK2, CHD, HS65T1, WDR11, GnRH receptor, GPR54, LHb, FSHb, TAC3R, TAC3, and DAX1/NROB1 genes.¹⁸ X-linked Kallmann syndrome (affecting mostly males) is often associated with disordered sense of smell and olfactory bulb ­hypoplasia on magnetic resonance imaging (MRI). Although affected individuals may show pubertal development and improvements in fertility in response to pulsatile/intermittent therapy with LH, hCG, or GnRH, most patients are treated with sex steroid replacement.

Gonadal differentiation

The karyotype of the primordial gonad directs development into a testis or an ovary. Subsequently, other factors such as transcription factors and hormones influence differentiation of the internal and external genital structures. Ovarian differentiation is no longer considered to be the “default” pathway since specific genetic factors contributing to ovarian differentiation have been described.¹⁹

Following migration from the hindgut to the developing gonadal ridges, local factors predominantly influence germ cell differentiation. Germ cell differentiation into male or female germ cells is also governed by cell cycle decisions between mitosis and meiosis.²⁰ Germ cell meiosis begins at approximately 10 to 11 weeks of gestation in the developing ovary. Following the first meiotic division, the primary oöcyte becomes associated with granulosa cells to constitute the primary follicle. In the developing testis, the developing germ cells undergo mitotic arrest.

Disorders of gonadal differentiation, both general categories and specific forms, resulting in gonadal dysgenesis are listed in Table 6-4.

Wnt4 gene.

The secreted protein encoded by this gene, located on chromosome 1p31-1p35, binds to members of the frizzled receptor family. Duplication of the Wnt4 gene in 46,XY patients results in ambiguous genitalia with hypospadias with remnants of both wolffian and müllerian ducts. Histological examination of the gonads shows differentiation of interstitial cell lineage, but suppression of Leydig cell development. Women carrying loss-of-function mutations may present in adolescence with primary amenorrhea secondary to müllerian duct abnormalities and hyperandrogenism.²¹

Smith-Lemli-Opitz syndrome.

This autosomal recessive disorder is characterized by multiple malformations that include urogenital anomalies, mental retardation, failure to thrive, facial anomalies including cleft palate, developmental delay, and behavioral abnormalities. The urogenital defects include ­hypospadias, cryptorchidism, and male-to-female sex reversal. These anomalies are a consequence of mutations in the 7-dehydro-cholesterol reductase (DHCR7) gene, resulting in low cholesterol and high serum concentrations of 7-dehydrocholesterol and other sterol intermediates that accumulate as a result of defective enzyme activity. Decreased cholesterol concentrations, the precursor for steroid synthesis, result in reduced steroid synthesis (glucocorticoid, mineralocorticoid, and sex steroids).²²

IMAGe syndrome.

In 46,XY infants, the IMAGe syndrome is characterized by urogenital anomalies (including micropenis and cryptorchidism), adrenal hypoplasia, intrauterine growth retardation, and other congenital anomalies including metaphyseal dysplasia.²³

46,XY sex reversal

WT1 gene.

Denys-Drash and Frasier syndromes are both associated with mutations in the WT1 gene. Denys-Drash syndrome is characterized by 46,XY sex reversal, Wilms tumor, and renal failure caused by focal or diffuse mesangial sclerosis. Genetic females with Denys-Drash syndrome generally have normal female external genitalia. Frasier syndrome is characterized by 46,XY sex reversal, gonadal dysgenesis, gonadoblastoma, and renal failure caused by glomerular sclerosis. The majority of cases of Frasier syndrome are caused by a specific mutation in intron 9 of the WT1 gene.

Steroidogenic factor 1/NR5A1 gene.

The SF1 protein plays a role in gonadal differentiation, development of the ventromedial hypothalamus, steroidogenesis, and expression of the AMH gene. The phenotype of 46,XY patients with loss-of-function SF1/NR5A1 gene mutations is female internal and external genitalia, and streak gonads. The condition may be lethal in the neonatal period owing to profound adrenal insufficiency. There is also impaired expression of LH, FSH, and GnRH receptors, and absence of the ventromedial hypothalamic nucleus. Normal ovarian morphology has been reported in 46,XXindividuals with adrenal hypoplasia. Undervirilization of 46,XY fetuses without overt adrenal insufficiency has been reported in newborns with haploinsufficiency of SF1/NR5A1.²⁴ Heterozygous SF1/NR5A1 mutations were identified in 5/27 (18.5%) of 46,XY DSD patients; none of these patients had adrenal insufficiency.²⁵

DAX1/NROB1 gene.

The DAX1/NROB1 gene is located on the short arm of the X chromosome and encodes for an orphan nuclear receptor. Loss-of-function mutations in 46,XY individuals are associated with the syndrome of AHC and hypogonadotropic hypogonadism (see Figure 6-4). AHC can occur as a component of a contiguous gene deletion disorder in association with glycerol kinase deficiency, Duchenne muscular dystrophy, ornithine transcarbamylase deficiency, and mental retardation.

In 46,XY fetuses, duplication results in XY sex reversal. The presumed mechanism is that the increased dosage of DAX-1 expression represses SRY expression.

SOX9 gene.

SOX9 is an HMG box protein expressed in high levels in the fetal testis. In addition to 46,XY male-to-female sex reversal, haploinsufficiency of SOX9 caused by heterozygous mutations is associated with campomelic dwarfism. Features of campomelic dwarfism include congenital bowing of the long bones, hypoplastic scapulae, 11 pairs of ribs, clubfeet, micrognathia, and cleft palate. Respiratory insufficiency often results in neonatal death.

46,XX gonadal dysgenesis

FOXL2 gene mutation.

Mutations in FOXL2, a gene located on chromosome 3q23, are associated with the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) (see Chapter 3). Premature ovarian failure is the most common phenotype for this autosomal dominant condition. Data obtained from the investigation of transgenic mouse models suggest that the ovarian failure associated with BPES is caused by aberrant granulosa cell function during follicle formation.²⁶ Mutations in this gene have also been found in association with XX female-to-male sex reversal.

46,XX sex reversal (46,XX males)

Mutations in the R-spondin1 (RSPO1) gene.

In this autosomal recessive disorder, the phenotype includes absence of müllerian duct derivatives, masculinization of internal and external genitalia, and palmoplantar keratoderma. The dermatological features include a predisposition for squamous cell carcinoma.²⁷

Ovotesticular DSD.

Ovotesticular DSD (formerly known as true hermaphroditism) is defined as presence of ovarian tissue with follicles and testicular tissue with seminiferous tubules in the same individual. The most commonly identified gonad is an ovotestis, but there can be an ovary on one side and a testis on the other. In most ovotestes, ovarian and testicular tissues show distinct separation in an end-to-end arrangement. The majority of affected individuals have a 46,XX karyotype.

46,XY gonadal dysgenesis.

A substantial frequency, that is, 10 of 29 patients with hypergonadotropic hypogonadism, streak gonads, external and internal female genitalia and either a 46,XX or 46,XY karyotype, were reported to have 46,XY complete gonadal dysgenesis.²⁸ Mean age of diagnosis was late in adolescence, 7 of 10 had SRY mutations, and 4 of 9 were found to have gonadal tumors.

45,X/46,XY gonadal dysgenesis (formerly known as mixed or asymmetrical gonadal dysgenesis).

Individuals with sex chromosome DSDs caused by “mixed gonadal dysgenesis” generally present with asymmetrical genital ambiguity. Short stature, webbed neck, cubitus valgus, gonadal failure, and other features of Turner syndrome may be present. Although the most common karyotype is 45,X/46,XY, mosaic karyotypes with multiple cell lines, including a monosomic X cell line, may be detected. There is much phenotypic heterogeneity in that internal and external genital differentiation can range from ambiguous to normal male or female.²⁹

Environmental causes.

Although it has been hypothesized that synthetic and natural compounds in the environment can be “endocrine disruptors,” such molecules have not been clearly shown to cause DSDs. Genital ambiguity in three 46,XY infants born in heavily agricultural areas has been reported and attributed to fetal exposure to endocrine disruptors, as no other cause or known mutations were found.³⁰ Exposure to endocrine disruptors in the environment has also been implicated as a cause of the reported increased frequency of both cryptorchidism and poor semen quality for boys born during the decade beginning in 1983.³¹ Hence, whereas causality has yet to be verified, it should be recognized that some compounds have estrogenic or antiandrogenic properties, such as organochlorine pesticides, polychlorinated biphenyls (PCBs), and alkylpolyethoxylates. A possible mechanism by which hydroxylated PCB metabolites cause ambiguity is that they may bind to estrogen sulfotransferase with greater affinity than does estradiol. It is well known that prenatal exposure to the nonsteroidal synthetic estrogen, diethylstilbestrol, is associated with urogenital abnormalities in both sexes, including cryptorchidism in the male fetus. Further, some pesticides may inhibit placental aromatase activity.

Müllerian duct abnormalities.

AMH, a member of the TGF-β family, signals through two different interacting membrane-bound serine/threonine receptors. Abnormalities are associated with either a mutation in the AMH or in the AMH receptor (AMH-RII) gene³² and a common phenotype with autosomal recessive inheritance. Patients with mutations in the AMH gene have low serum concentrations of AMH, whereas levels are elevated or normal among those with AMH-RII gene mutations.

Features of the persistent müllerian duct syndrome (PMDS) usually include cryptorchidism, ectopic testes associated with inguinal hernia, and hernia uterus inguinalis.³³ Although testicular differentiation occurs normally, the male excretory ducts may be incompletely developed and embedded within remnants of the ­müllerian ducts. Because the poorly developed vasa deferentia are trapped within the uterine wall and the excretory ducts are not adequately connected to the testes, natural fertilization may be precluded. When diagnosed in childhood, it may be discovered during either an inguinal herniorrhaphy or orchiopexy when a uterine cervix is identified within the hernia sac. Testicular torsion may also occur when the testes are not fully attached to the processus vaginalis. Females who carry mutations on both AMH alleles have normal fertility.

Müllerian duct abnormalities may also occur in 46,XX patients, usually associated with other abnormalities. The Mayer-Rokitansky-Küster-Hauser syndrome involves congenital absence of the vagina and uterine hypoplasia or aplasia. It often is not diagnosed until the age of puberty when a nonpatent vagina or primary amenorrhea is evaluated. Associated renal anomalies, including unilateral agenesis, and skeletal malformations are also identified in affected individuals. Hence, evaluation for skeletal malformations and renal sonography are components of the workup of women with abnormal development of the müllerian duct system.³⁴ The association of müllerian duct aplasia with renal aplasia and cervicothoracic somite dysplasia is called the MURCS syndrome, and the association of müllerian duct hypoplasia with facioauriculovertebral anomalies constitutes the Goldenhar syndrome. Though not specifically a müllerian duct abnormality, transverse vaginal septae may occur, sometimes in association with the McKusick-Kaufman syndrome.

Testicular descent.

Testicular descent occurs in two phases: intra-abdominal and inguinoscrotal. The cranial suspensory ligament and the gubernaculum develop on either side of the developing gonad. The former becomes the suspensory ligament of the ovary whereas, in the male, it regresses in response to testosterone secretion. The latter connects the testis and the internal inguinal ring by 14 weeks of fetal life.³⁵ The intra-abdominal phase of descent appears to be a result of the testis staying near the pelvis as the abdomen elongates so that, in a relative manner, it becomes located much more distally in the abdomen. Factors important during this first phase of testicular descent include insulin-like factor 3 (INSL3) and its receptor, LGR8. The second phase of descent through the inguinal canal, also related to the gubernaculum, occurs at the beginning of the third trimester of pregnancy with descent into the scrotum usually completed by the middle of the third trimester. Androgens play a role in influencing the second or inguinoscrotal phase of testicular descent. Usually, testicular descent is completed by the 35th week of gestation. Increased androgen levels in the 46,XX fetus that occur with virilizing CAH are not associated with ovarian descent.

Cryptorchidism (undescended testes) is associated with many syndromes and other endocrine disorders. Approximately 3% of male infants have cryptorchidism at birth. The prevalence decreases by 6 months of age because of spontaneous late testicular descent. Incomplete testicular descent occurs in some instances of DSD, as in CAIS in which the intra-abdominal phase is usually completed, but not the inguinoscrotal phase that is presumably related to lack of androgen action and underdevelopment of a scrotal sac. Mutations in the INSL3 and LGR8 (also known as RXFP2 or the INSL3 receptor) genes have been identified in a small number of boys with cryptorchidism.³⁶

An algorithm for the differential diagnosis of DSDs based on palpable or nonpalpable gonads and external genital symmetry is shown in Figure 6-7.

Initial Evaluation

The initial evaluation involves verifying whether there is a medical emergency, such as adrenal insufficiency, renal failure, or other systemic life-threatening illness. Adrenal insufficiency, associated with CAH secondary to 21-hydroxylase deficiency, is the most common emergency. Hence, when a neonate presents with symmetrically ambiguous genitalia without palpable gonads, the initial testing should include karyotype and measurement of serum levels of 17-OHP, androstenedione, testosterone, 11-deoxycortisol, and cortisol, and, until these results are available, monitoring serum sodium and potassium levels after 48 hours of life, although availability of newborn screening with early institution of treatment may preclude the need for such rigorous laboratory testing of electrolyte concentrations.

CAH is the most common DSD with genital ambiguity so the child manifesting genital ambiguity without palpable testes may be at risk for adrenal insufficiency. However, the infant with genital ambiguity and one or two palpable gonads is unlikely to have CAH or adrenal insufficiency. Male infants with 21-hydroxylase deficiency have normal genital anatomy including descended testes, in the backdrop of hyperpigmentation of the genitals and nipples, and may present with adrenal insufficiency after several days of age. As noted previously, newborn screening for this form of CAH should minimize this risk through earlier disease detection and initiation of treatment.

Prenatal diagnosis of the fetus at risk for CAH is now possible (see Chapter 5). Suppression of fetal ACTH and adrenal androgens using dexamethasone treatment of pregnant mothers can reduce the extent of genital ambiguity in female fetuses with 21-hydroxylase ­deficiency. However, therapy must be begun before the diagnosis can be confirmed or refuted, thus exposing most fetuses to unnecessary therapy. Further, there are limited long-term safety data of this prenatal therapy and concerns about risks and concerns primarily related to impaired cognitive function in CAH-affected and nonaffected offspring.³⁷

The initial assessment (Box 6-3) is aimed at the identification of the etiology of the child’s genital ambiguity. This assessment should include immediate consultation with specialists in the fields of pediatric endocrinology, urology, psychology, and social services. When available, a team of health care providers representing these subspecialties and experienced with DSD management issues should delegate a leader to supervise the evaluation. Pertinent findings should be carefully recorded.

The steps of the initial evaluation, to be performed as soon as possible, involve a medical history with emphasis on familial disorders, a physical examination including a careful description of the genitalia, a karyotype or more rapid assessment for sex chromosome complement, hormone determinations, and a pelvic ultrasound.

Based on the findings from the history and physical examination, appropriate serum hormone levels should be obtained, which include adrenal and gonadal steroid hormones, gonadotropins, and other peptide hormones.

History

Pertinent facts from history should include items listed in Box 6-4.

A detailed family history should include siblings with ambiguous genitalia, deaths during infancy, and the presence of infertile aunts or partially virilized uncles in the maternal family.

Physical examination

A basic guide to items to be included in the genital examination is outlined in Box 6-5.

A thorough physical examination should be performed. Particular attention should be paid to the genitalia, including symmetry (as outlined in Box 6-6), with measurements as outlined in Table 6-5. Examples of symmetrical and asymmetrical genitalia are shown in Figures 6-8 and 6-9, respectively. The extent of skin pigmentation of the genitalia and nipples should be noted as dark pigmentation suggests excessive ACTH stimulation. If transabdominal ultrasound is readily available, it should be done to provide a reliable assessment of the presence of a uterus.

The extent of fusion (masculinization) of the labioscrotal and labiourethral folds should be carefully described, in comparison with the standards of Prader (see Figure 6-1), noting that relative phallic size may not be consistent with the extent of labial fusion. It should be documented whether a single or double opening can be visualized and whether the single opening is a consequence of the presence of a urogenital sinus or whether it appears as a urethral meatus. Partial fusion of the labiourethral folds results in the urethral opening being at various points along the underside of the phallus, with lack of fusion of the opening is on the perineum. With the presence of a urogenital sinus resulting from partial fusion of the labioscrotal folds, two separate openings, urethral and vaginal, may be present, but not visible by inspection. With virilization of the female fetus, as occurs with CAH, the vagina may open into this sinus or into the urethra at various points extending as high as the base of the bladder (low vs high confluence). The internal anatomy cannot be confirmed by physical examination. Hence, the urinary and reproductive system tract anatomy must be visualized using available radiographic, ultrasonographic, and cystoscopic modalities.

Phallic size should be carefully measured. The extended length, in the nonerect state, should be measured along with the dorsal surface from the tip of the glans, excluding the prepuce, to as close to the underlying pubic bone as possible and compared with published standards, with recognition that there are some minor racial differences. In general, a stretched length of less than 2 to 2.5 cm is considered abnormal for a term infant. The criteria for premature children are listed Table 6-5.

It is important to realize that the clitoris is prominent in the premature or very lean infant girl because of the lack of surrounding subcutaneous tissue. Both length and width can be reliably estimated. The length is measured from the palpable base to the tip of the glans; normal length ranges from as low as 9 mm, that is, less than 1 cm.³⁸ The width is measured by spreading surrounding tissues; usual dimensions are generally less than 6 mm. Published measurements are listed in Table 6-5.

Anogenital distance (AGD), the distance from the posterior aspect of the scrotum in males or posterior fourchette in females to the anal verge, provides an index of fetal androgen exposure and masculinization. At birth, AGD in males approximates 20 ± 6 mm and in females 9 ± 3 mm. AGD is positively correlated with penile length at birth, while adult AGD correlates with serum testosterone levels, sperm density, and paternity.^39,40 Animal studies suggest that AGD is related to a masculinization programming window and concomitant androgen action. While AGD correlates with penis size in the newborn and potential adult size, postnatal androgen action is required to realize normal potential.⁴¹ The usefulness of AGD as a diagnostic tool may be to indicate an early defect or to assess the impact of potential environmental endocrine disruptors on external genital development,⁴² but it adds little to therapeutic planning because treatment is aimed at maximizing penile growth even though it is not ­adequate. The value may be to create an expectation ofless than desired future growth.

To ascertain the presence, size, and location of gonads, the inguinal area and the partially or fully developed scrotum and labia majora should be carefully palpated. Mobility, size, consistency, and location of the gonads should be carefully recorded. If gonads are present within the labioscrotal structures rather than within a hernia sac, they are probably testes. When there is asymmetry of labioscrotal development, the more virilized half (hemiscrotum) is more likely to contain a palpable gonad, either a testis or, rarely, an ovotestis. The contralateral gonad is usually nonpalpable and likely to be a dysgenetic testis, an ovary, or an ovotestis. When there is symmetry of the labioscrotal structure, at any Prader stage, and no palpable gonads, the most frequent diagnosis is virilizing CAH in a 46,XX virilized female. The fully virilized patient with an empty scrotum, however, is more commonly a 46,XY male with bilateral cryptorchidism or anorchia (vanishingtestis/testicular regression syndrome). In this latter situation, testicular function is obviously adequate to result in full male differentiation, with gradual or abrupt loss of function thereafter apparently resulting from a variety of insults.

Differential diagnosis.

Disorders have been discussed earlier in this chapter, which constitute the various DSD conditions that present with genital ambiguity. They are listed in Box 6-7.

Genital ambiguity more often results from excess androgen production in genetic females than from inadequate androgen production in genetic males. Excess production most commonly is a consequence of deficient enzyme activity in the adrenal glands. Diminished androgen production can be a result of inhibited production along the sex steroid biosynthetic pathway or of decreased action. Figure 6-10 provides a reference to visualize the pattern of steroid levels in relation to compromised conversion resulting in impaired steroidogenesis. In general, steroids proximal to the enzyme block (depicted with arrows) are produced in excessive amounts whereas those distal to the enzyme block are produced in diminished concentrations. Hence, profiles of steroid intermediates commonly suggest the sites of compromised synthesis. Steroid profile panels, from newer microliquid chromatography tandem mass spectrometry (LC/MSMS) methods that specifically pinpoint the location of enzyme defects, are available from the major contract laboratories. In addition, other methods currently used to determine defects are discussed in Chapter 5.

Diagnostic tests

General principles.

Before the transfer of the patient, even if expected imminently, it is helpful to obtain a blood sample for a karyotype for sex determination and hormone levels. The karyotype and/or a Y chromosome fluorescent in situ hybridization (FISH) should be ordered as soon as possible, using peripheral blood leukocytes for the initial sample. Further testing may be necessary, as mosaic patterns may be present, and karyotyping of gonadal tissue may differ from that of circulating leukocytes. An abdominal/pelvic ultrasound determines the presence or absence of a uterus, and sometimes the presence, size, and location of the gonads and adrenal glands. If surgery is being considered, a genitourethrogram or venography is useful in determining the interconnection of the genitaland urinary tract cavities and ducts, but it can be delayed. Cystoscopy is invasive, but it generally provides more information. Specific genetic testing may also be helpful in establishing a diagnosis that is not clear based on basic clinical, biochemical, and radiologic assessments.

Specific testing.

The choice of initial hormone tests depends on the physical findings present:

1. For symmetrical partially virilized external genitalia with no palpable gonads, particularly if a normal uterus is present, additional studies should be directed toward causes of virilization of a female infant, the most likely diagnosis being CAH secondary to 21-hydroxylase deficiency (Figure 6-11). Serum 17-OHP, androstenedione, testosterone, 11-deoxycortisol, and cortisol levels should be obtained. Even though adrenal insufficiency with hyponatremia and hyperkalemia may be associated with this diagnosis and may be life-threatening, severe symptomology does not usually develop until approximately 7 to 10 days of life. Monitoring of sodium and potassium may begin at 2 days of life or until the diagnosis is established or excluded. Plasma renin levels are normally relatively high in the newborn so measurements can be delayed until electrolytes become abnormal or toward the end of the first week in life. While 17-OHP concentrations greater than 5000 ng/dL are highly suggestive of CAH caused by 21-hydroxylase deficiency, mar­kedly elevated values greater than 10,000 ng/dL (∼300 nmol/L) are diagnostic. 17-OHP concentrations are also elevated, but generally not to the same degree, in the much less common form of CAH, 11β-hydroxylase deficiency. In contrast to CAH caused by 21-hydroxylase deficiency, that resulting from 11β-hydroxylase deficiency is diagnosed based on elevated 11-deoxycortisol concentrations. Although hypertension is a classic finding in this form of CAH, it may not be present in the neonatal period, and, in fact, salt loss may be present. Children with another rare type of CAH, 3β-hydroxysteroid dehydrogenase deficiency, typically have elevated pregnenolone, 17-hydroxypregnenolone, and DHEA concentrations. As stated previously, the exact timing and frequency of testing in this scenario will likely be influenced by the results of newborn screening.

2. When one or both gonads are palpable, screening tests should be first focused on gonadotropin and steroid hormone concentrations to assess androgen synthesis and action. Serum LH, FSH, testosterone, and DHT concentrations should be obtained to assess the hypothalamic-pituitary-gonadal axis and testicular function recognizing that these values may change remarkably just after parturition because hCG possesses LH-like stimulating properties. The relative levels of steroid hormone and gonadotrophin concentrations provide a basis to assess a functional hypothalamic-pituitary-gonadal axis and specific defects in steroidogenesis (see Figure 6-5).

Referral of all infants born with genital ambiguity to a center that can offer a team approach to DSDs is highly recommended and should be made during the first day of life, before sex of rearing is discussed in detail. It is appropriate to inform the parents that there has been a developmental problem involving the reproductive organs and that their child needs to be evaluated by a team of experts to determine the cause as it relates to future sexual and reproductive function before they make any decision about further evaluation, treatment, and sex assignment.

Referral should not be delayed pending the results of initial testing blood samples and discussions with the parents have been completed (Box 6-8). This should generally be accomplished during the first 24 hours of life. Obtaining blood for karyotype and hormone levels, with careful notation of age when the samples are obtained, can often be done without delay and will accelerate key decision making by the referral team. Ultrasonography and invasive radiologic examinations should be done only by an individual experienced in studying infants.

Treatment

Algorithm of treatment plan (includes multiple disciplines).

The current approach highly recommends a specialized team that includes endocrinologists, geneticists, and surgeons from pediatric, urologic, or gynecologic subspecialties, with psychologists, social workers, and specialized nurses (Figure 6-12). The team aims to verify the phenotype, determining as specific a diagnosis as possible, including, when appropriate, a recommendation for sex assignment. One or more team members should provide counseling to the parents, providing background information and treatment options, including, but not limited to, surgery for genital reconstruction and, when appropriate, biopsy to make a more specific diagnosis and gonadectomy to preclude malignant change as appropriate.

Information impacting the approach to management of the infant with genital ambiguity.

The Victoria State Government in Australia has recently published⁴³ a document presenting decision-making principles for the care of infants, children, and adults with DSDs, including support of patients and parents, management, human rights, and ethical and legal principles including an algorithm for application. Management and patient/parental interactions require verification of consistency with human rights, as well as ethical and legal principles. These principles are incorporated as follows.

Interactions with parents.

Parents play obligatory and vital roles in medical decisions after they have been fully informed. They must be made aware of all available information even though there are often inadequateoutcome data to provide absolute guidelines for sex assignment and for decisions for and timing and complexity of surgery. Suggestions for how to discuss the situation with parents are outlined in Box 6-9. Parents must be provided available data concerning surgical outcome, even though such data are not entirely based on results of current surgical techniques. For example, for almost a decade, efforts have been made to spare the neurovascular bundle supplying the clitoris during modern feminizing surgery. Though early outcomes of current procedures suggest more favorable cosmetic and functional status than with past approaches, verification will require at least another decade. In the meantime, sharing the current knowledge of psychological and biological factors, including reproductive potential and sexual responsiveness related to gender and sexual development, provides parents with information on which to base their decisions. The ultimate responsibility of the child’s care falls to the parents. With input from the DSD team and understanding of factors involved concerning the evaluation of their child, parents should be equipped to make the best possible decisions. Providing them with an overall understanding of the complex interplay between biological, social, and psychological/psychosexual factors, based on clear scientific data, provides them with a basis by which to form their own decisions, including both sex assignment, when unclear, and whether to and when to have surgery.

Ethical and legal principles.

The medical and surgical care of DSD patients with ambiguous genitalia has been criticized by some patient and parent advocacy groups with aggressive confrontations concerning the overall need for and timing of surgery, and who should make the decision for surgery (physician, parent, or patient when older). However, needed ethical guidelines have not been established, including the legal rights and responsibilities as parents including providing permission for surgery. Ethical guidelines should address minimizing physical and psychological risk, preserving reproductive and sexual function potential, leaving options open for the future, and considering parents’ wishes. Legal questions involve parental consent, whether treatment is major, minor, and/or reversible, and consequences of wrong decisions. A recent publication⁴⁴ summarizes the recommendations of a working group of Bioethics and Intersex of the German Network of Intersex/DSD. Three primary ethical principles were identified to (1) foster the well-being of the child and future adult, (2) uphold rights of children and adolescents to participate in and/or self-determine decisions that affect them now or later, and (3) respect family and parent-child relationships. Regarding genital surgery, support of the child and parents is more important than creation of biological normalcy, while, for surgery and surgical timing, delineation between functionally meaningful interventions and esthetic reasons for genitoplasty, but not vaginal reconstruction, may be unclear. The delineation of cosmetic versus functional indications for clitoral surgery is less clear, because, except for repositioning into the female location, there are no strong arguments for clitoroplasty, beyond that done as part of the overall genitoplasty.

Outcome data application to sex assignment.

Health care providers need to be cognizant that available outcome data to assist in the decision-making processes are limited. Currently accessible information in published reports is largely based on retrospective studies obtained using diverse methodologies.⁴⁵ For most conditions causing DSD with genital ambiguity, adult gender identity cannot be predicted so gender assignment should be guided by the estimated optimal outcome.⁴⁶ The inability to predict relative contributions of biological factors related to genes, prenatal hormone exposure, and environmental influences involving social and cultural factors, including parental attitudes and peer influences, precludes any accurate prediction of sexual identity generally or gender identity specifically. Specific diagnoses for which sex assignment can be recommended are listed in Box 6-10 while for most DSD cases adequate outcome data are unavailable. When gender identity is not consistent with sex assignment, assessment and care are similar to those of biologically normal patients with gender dysphoria. However, experience with gender dysphoria, not involving genital developmental issues, has as yet provided little information to guide sex assignment issues. For DSD patients, considerations should be based on factors including the following:

1. Gonadal development regarding projected potential for reproduction and spontaneous hormone secretion to stimulate pubertal development—Evaluation of potential gonadal function should be assessed by hormone levels during infancy including gonadotropins, AMH, inhibin, and sex steroids; documentation of the presence and volume of gonads; and, when indicated, hormonal response to hCG stimulation. Ability to secrete sex steroids, particularly T and DHT (basally and/or after hCG) during infancy, is predictive of, but does not guarantee adequate pubertal secretion. Because intragonadal germ cell retrieval potentially requires only a minimal number of cells, it is not possible to predict potential for assisted fertility unless there is clear evidence of no gonadal tissue.

2. Possibility that surgery will be able to construct genitalia with potential for sexual activity while preserving responsiveness—Though a position has been advocated never to do surgery until the patient is old enough to make an informed decision, and also that all surgery is for cosmetic purposes, the majority of parents do not agree with either statement when their child has genitalia that are not within the spectrum of variation of either male or female and may/will physically preclude intercourse. Parents should be aware that surgery should spare the neurovascular bundle supplying the clitoral hood, and that, because the introital border of the labia minora is highly innervated compared with the external border,⁴⁷ removal will likely impact sensory sexual arousal.

3. What the expected gender identity will most likely be based on current insight into biological, psychological, and social factors—Consideration of psychosexual development as it relates to gender assignment must emphasize the unknown role of the following factors in establishing gender identity: greater than typical female levels of androgen exposure during fetal life, family and social environment, and perception of genital anatomy. The effect of androgen exposure on the central nervous system (CNS) during fetal life, which has a clear impact on behaviors and cognitive function, but unclear impact on gender identity, occurs later than that which impacts genital development. Hence, physical evidence of androgen stimulation does not necessarily correlate with extent of androgen exposure to the CNS.

Reconsideration of former sex assignment guidelines.

In the past, a female gender assignment may have been chosen for a 46,XY individual if the penis were judged to be inadequate. However, because available evidence suggests an overall equivalent or better outcome when raised male even with a small or absent penis, currently most such individuals who have a 46,XY karyotype are assigned a male sex of rearing, particularly if there is evidence of functioning testes and androgen responsiveness. Evidence suggests that such a child can grow up with a male gender identity and be content even with physical limitations of sexual activity. Nevertheless, it is not clear that such persons will be free of significant psychological problems related to underdeveloped genitalia and inability to participate in usual male sexual activities. This may be particularly true for the person having surgery during or after puberty who previously experienced the associated pleasurable genital sensations. Further, there have been impressive advances in phalloplasty enabling creation of a cosmetically acceptable penis that is physically capable of intercourse.⁴⁸

There are also additional data concerning the outcomes of 46,XX CAH males who were born with Prader 4 or 5 genitalia.⁴⁹ While some have psychological problems because of the multifaceted ramifications resulting from confusion likely related to late diagnosis and attempted therapy, all known males were found to have a clear male gender identity, with many having long-term commitments to heterosexual relationships. While there will be no potential for fertility if male assignment is considered, male rearing should not be automatically excluded from consideration and known outcome data should be provided to parents. When this has been done, several parents have chosen a male gender assignment for their infants. Time will provide further outcome information.

Recent long-term outcome data

46,XX CAH women.

Increased incidence of sexual dysfunction in 46,XX CAH females results from genital insensitivity, orgasmic difficulty, and atypical genital appearance.⁵⁰ Outcome, related to both the underlying severity and surgery, was better after nerve-sparing clitoroplasty, while orgasmic ability was neither correlated with sensitivity nor different than control women. Sexual satisfaction did not differ from controls nor did it correlate with ability to have an orgasm, although severity of virilization was related to genital appearance and sexual function.⁵¹ Genital cosmetic outcome of 36 women, as assessed by both the patient and physician, was significantly impacted only by the level of confluence of the vagina and urethra. For the subset who had experienced intercourse, satisfaction was less than the reference group and was impacted by dyspareunia and urinary incontinence. Overall, CAH women have less sexual experience, more sexual anxiety, behavior avoidance, and passivity.

46,XY DSD women.

About one-third of 46,XY DSD women indicated dissatisfaction with overall sex life and almost half had sexual anxieties.⁵² Among those who had had reconstructive surgery, almost half felt that surgery did not enhance sexual function and were dissatisfied with clitoral arousal. Compared to controls, partially androgenized females were more likely to have dyspareunia and have either homosexual or bisexual preferences. Among those with complete gonadal dysgenesis or CAIS, the majority indicated problems with sexual desire, arousal, and dyspareunia. These outcome data highlight the known problems among virilized 46,XY females, but also have uncovered significant problems among those with a female phenotype, including CAIS.

Information concerning causes, assessment, and outcome of patients with DSDs must be based on verified data, and its application to the clinical setting should follow a primary principle of respect of each individual as a human and the goal of the best possible quality of life.