The first level of sexual differentiation is the establishment of chromosomal sex. Most infants are 46,XX females or 46,XY males. Genetic sex determines gonadal sex. Gonadal structures differentiate from the “bipotential,” or primordial, gonadal ridge. The Y chromosome contains an area known as the sex-determining region, or SRY. The SRY gene product initiates the differentiation of the bipotential gonad into a testis. In its absence, the gonad becomes an ovary.

The next level of sex determination involves the genital duct structures. The genital duct structures are initially identical in the male and female. In the normal male, testicular Leydig cells produce testosterone, which is necessary to maintain ipsilateral wolffian duct structures (e.g., vas deferens, epididymis, seminal vesicles). The Sertoli cells of the testis produce müllerian-inhibiting factor (MIF), which acts ipsilaterally to cause regression of müllerian duct structures (fallopian tubes, uterus, upper third of the vagina). In the absence of testosterone and MIF, müllerian duct structures are preserved, and wolffian duct structures regress.

Male and female external genitalia arise from the same embryologic structures. In the absence of androgen stimulation, these structures remain in the female pattern, whereas the presence of androgens causes male differentiation (virilization). For complete virilization, testosterone must be converted to dihydrotestosterone (DHT) by the enzyme 5-alpha-reductase, and androgen receptors must be functional. Excessive androgens virilize a female. Inadequate androgen production, inability to convert testosterone to DHT, or inability to respond to androgens, as in androgen receptor defects, results in undervirilization of a male.

The TDF promotes differentiation of the bipotential gonad into a testis; SRY was eventually characterized as the TDF. SRY belongs to a family of DNA binding proteins. Specific manipulations have shown that the introduction of SRY results in sex reversal of XX mice, and site-directed mutagenesis of the SRY gene in XY mice yields an XY female. The activation of SRY is influenced by the Wilms’ tumor suppressor gene, WT1. Other genes that play a role downstream of SRY include SOX9, SF-1, DAX1,WNT4, DMRT1, ATRX, DHH, and GATA4.

Dr. Mary Lyon addressed the question of the extra X chromosomal material in females. Simply put, if two X chromosomes are necessary in each cell, how can males be developmentally normal? Lyon suggested that in each cell, one of the two X chromosomes is inactive, and in any given cell line, which X is active is randomly determined. In fact, the inactive X may be identified in many cells as a clump of chromatin at the nuclear membrane (Barr body). The important exception is in the ovary, where two functional X chromosomes are necessary for normal sustained ovarian development. Without two X chromosomes per cell (as in 45 XO Turner syndrome), the ovary involutes and leaves only fibrous tissue.

The fetus is sexually bipotential. Figure 42-1 shows schematically how male development is accomplished. The undifferentiated gonad is derived from coelomic epithelium, mesenchyme, and germ cells, which, in the presence of SRY, give rise to Leydig cells, Sertoli cells, seminiferous tubules, and spermatogonia. Testes are formed at 7 weeks. Testicular production of testosterone (Leydig cells) leads to wolffian duct development, whereas MIF (Sertoli cells) leads to müllerian duct regression. Masculinization of the external genitalia is mediated by DHT, which is produced from testosterone by the action of the enzyme 5-alpha-reductase.

In the absence of SRY, the undifferentiated gonad gives rise to follicles, granulosa cells, theca cells, and ova. Ovarian development occurs in the thirteenth to sixteenth week of gestation. Lack of testosterone and MIF allows regression of the wolffian ducts and maintenance of the müllerian ducts, respectively. Lack of DHT results in the maintenance of female external genitalia.

The external genitalia arise from the urogenital tubercle, urogenital swelling, and urogenital folds. In females, these become the clitoris, labia majora, and labia minora, respectively. In males, under the influence of DHT, the genital tubercle becomes the glans of the penis, the urogenital folds elongate and fuse to form the shaft of the penis, and the genital swellings fuse to form the scrotum. Fusion is completed by 70 days of gestation, and penile growth continues to term.

Female differentiation does not require ovaries or hormonal influence, whereas normal development of male genitalia requires normal testosterone synthesis, conversion to DHT by 5-alpha-reductase, and normal androgen receptors (Fig. 42-2).

There are three large categories of ambiguity (Table 42-1):

 Sex chromosome DSD

 46,XY DSD

 46,XX DSD

A virilized female (previously called female pseudohermaphroditism) is characterized by a 46,XX karyotype, ovaries, normal müllerian duct structures, absent wolffian duct structures, and virilized genitalia resulting from exposure to androgens during the first trimester. See Table 42-2.

The most common cause is congenital adrenal hyperplasia (CAH) resulting from 21-hydroxylase deficiency. In fact, this disorder is the single most common cause of sexual ambiguity. In this condition, the gene responsible for encoding the 21-hydroxylase enzyme is inactive. This enzyme blockage occurs along the pathway to cortisol and aldosterone. Because of low or absent levels of cortisol, the feedback mechanism produces increased adrenocorticotropic hormone (ACTH), which drives the pathway further and results in accumulation of precursor hormones, the measurement of which is useful for making a diagnosis. Increased ACTH also drives the production of excess adrenal androgens, which result in virilization. Virilization may also be caused by maternal ingestion of androgens or synthetic progesterones during the first trimester of pregnancy.

Affected infants may present with a wide spectrum of ambiguity, ranging from clitoromegaly alone to complete fusion of the labial swellings to form a scrotum and large phallus. (Beware the infant with bilaterally undescended testes.) Even in the most virilized girls, a penile urethra is rare.

An undervirilized male (previously called male pseudohermaphroditism) refers to a 46,XY male who has ambiguous or female external genitalia. The abnormality may range from hypospadias to a completely female phenotype. Such disorders result from deficient androgen stimulation of genital development and most often are secondary to Leydig cell agenesis, testosterone biosynthetic defects, 5-alpha-reductase deficiency, and partial or total androgen resistance (androgen receptor defects).