Although the genotype of a fetus is determined at conception, sexual differentiation does not occur until 6 to 7 weeks of gestation (McGillivray, 1992). Before that point, the embryo has bilateral undifferentiated gonads and both müllerian and wolffian duct systems. Fetuses with a Y chromosome containing the gene for the testis-determining factor (SRY) will convert the undifferentiated gonad to a testis, which involves the formation of seminiferous tubules that surround the primitive germ cells. As shown in Figure 84-1, Leydig cells begin to produce testosterone, which acts on the wolffian ducts to produce male internal genitalia. Sertoli cells produce anti-müllerian hormone (AMH), also known as müllerian duct inhibiting substance (MIS), which causes regression of the mullerian system (McGillivray, 1992).

At 6–7 weeks of gestation, the external genitalia are undifferentiated and consist of a genital tubercle, genital folds, and swellings. During male embryogenesis, masculinization of the common primordia is induced by the activity of dihydrotestosterone (DHT). DHT is formed from testosterone via the enzyme 5α-reductase. The urogenital tubercle then differentiates into the glans. The urogenital folds differentiate into the shaft of the penis and a male type of urethra by 12 to 14 weeks of gestation. The urogenital swellings fuse at the midline into the scrotum. Without testosterone, the mullerian duct develops into the fallopian tubes, uterus, and upper vagina. The genital tubercle becomes the clitoris, and the genital folds and swellings become the labia. Two X chromosomes are needed to maintain the ovaries and germ cells.

Most fetuses with genital ambiguity are otherwise well formed, although malformations of the sex organs sometimes combine with malformations of the urinary tract (Aarskog, 1992). Abnormalities of sexual differentiation can be classified into disorders of chromosomal sex, gonadal sex, or phenotypic sex. One system of classification is based on the actual gonad found at surgical exploration. This classification divides infants with ambiguous genitalia into five subgroups:

1. 
   

   Only an ovary is present (female pseudohermaphroditism).

   
   
2. 
   

   Ovary and testis are present (true hermaphroditism).

   
   
3. 
   

   Only a testis is present (male pseudohermaphroditism).

   
   
4. 
   

   Testis and gonadal streak are present (mixed gonadal dysgenesis).

   
   
5. 
   

   Only a gonadal streak is present (pure gonadal dysgenesis).

Unfortunately, this system is not practical to use in the prenatal context.

The more practical classification to use prenatally is based on cause. Three categories can be delineated:

1. 
   

   True hermaphrodite—these fetuses have both ovarian and testicular tissue present.

   
   
2. 
   

   Female pseudohermaphrodites—these are virilized females.

   
   
3. 
   

   Male pseudohermaphrodites with and without mullerian structures—these are undervirilized males.

Female pseudohermaphrodites have a normal female karyotype (46, XX) and have ovarian gonadal tissue present. They have normal mullerian duct structures (uterus, fallopian tubes, and upper vagina) and no wolffian duct structures (epididymis, vas deferens, seminal vesicles). The female fetus becomes virilized because of exposure to androgens in utero. The causes for this include congenital adrenal hyperplasia (CAH), ingestion of androgens by the mother, and maternal virilizing tumors. Male pseudohermaphrodites with a normal 46, XY karyotype have only testicular tissue present. The Sertoli cells produce anti-mullerian hormone, but the under-virilization is due to inadequate synthesis of testosterone or dihydrotestosterone (see Figure 84-1), or the presence of an androgen receptor defect. Male pseudohermaphrodites with mullerian structures usually have a mixture of cell lines, with at least one cell line having a Y chromosome present, but no cells having two X chromosomes. The gonadal tissue in these cases is either normal testis or a streak gonad.

The incidence of ambiguous genitalia is 1 in 5000 livebirths (Kutteh et al., 1995). Hypospadias occurs in 2.5 to 8.2 per 1000 live male births (Bronshtein et al., 1995). Ambiguous genitalia due to CAH occurs in 1 in 14,000 newborns (Hurtig, 1992).

Axial scans often provide optimal visualization of the perineum and external genitalia when the fetal lower extremities are held in flexion. Sagittal scans of the perineum may be helpful to demonstrate the penis. When the fetus is male, testes can be identified within the scrotum after 28 weeks of gestation. de Elejalde et al. (1985) described the sonographic determination of fetal gender in 722 fetuses between 13 and 35 weeks of gestation. They compared their prediction with fetal karyotype, and all of these patients were studied prior to amniocentesis. These authors stated that the fetal genitalia could be imaged in 60.5% of fetuses examined before 18 weeks of gestation and 100% of those examined later than 20 weeks of gestation. In 3% of the cases, an error in sex assignment occurred, but these fetuses were all at less than 24 weeks of gestation. In female fetuses, the labia majora, labia minora, and occasionally the clitoris were visualized. In male fetuses, the scrotum was visualized in coronal and transverse planes. The transverse scan was useful for recognizing the penis. These authors stated that a fetal erection could be seen in utero by 20 weeks of gestation (de Elejalde et al., 1985). In an even larger study, Natsuyama (1984) determined fetal sex in 1879 pregnant women between 12 and 40 weeks of gestation. These authors used transverse, frontal, and sagittal scans of the fetal lower pelvic region. They were able to image 97.1% of cases and stated 99.9% accuracy in fetal gender assignment. These authors confirmed their findings postnatally. The most helpful findings were that between 12 and 23 weeks of gestation, the penis was pointed anteriorly toward the amniotic cavity but the clitoris was turned inferiorly. Another difference was the distance between the anal and genital regions. Males were noted to have a longer distance than females at later than 10 weeks of gestation (Natsuyama, 1984). Other authors described the “dome” sign, which was the sonographic visualization of the fetal scrotum and cranially directed phallus. In females, the diagnostic criterion is the presence of two or four parallel lines representing labial folds and a caudally directed clitoris. Some authors use the length of the phallus as a diagnostic criterion for gender assignment and some do not (Bronshtein et al., 1995). Benacerraf et al. (1989) have stated that sonographic visualization of the fetal genitalia is accomplished 80% to 90% of the time, with a correct sex assignment given 92% to 100% of the time. Fetal cephalic presentation is more favorable than breech for gender determination (Ali, 1992). Subsequent studies have established normal reference ranges for both penile length (Johnson and Maxwell, 2000; Zalel et al., 2001) and uterine development (Soriano et al., 1999).

In a pregnant woman with a positive family history of prior affected children with CAH, ambiguous genitalia were documented by the presence of two angulated lines representing labia and the presence of a central, domelike structure representing an apparent “scrotum” (Sivan et al., 1995) (Figure 84-2). In a follow-up study of 17 fetuses with genital malformations, 12 were raised as males, 4 were raised as females, and 1 genetic male was raised as a female due to congenital absence of a penis. In all cases an extremely small fetal phallus was observed, with undescended gonads (Figure 84-3). Four of the 17 fetuses had a sonographically abnormal phallus. The underlying diagnosis in 3 of the 4 fetuses with an abnormal phallus was congenital adrenal hyperplasia; 1 case was due to panhypopituitarism. The other underlying diagnoses in the remaining 13 cases included 2 cases of male pseudohermaphroditism, 3 cases of severe hypospadias with chordee, 2 cases of microphallus, 2 cases of cloacal anomaly, 2 cases of penoscrotal transposition, 1 case of intra-abdominal testes, 1 case of megaureter, and 1 case of cloacal exstrophy (Mandell et al., 1995).

The accuracy of the sonographic prenatal diagnosis of ambiguous genitalia was addressed by Cheikhelard et al. (2000). In this study, 34/43 cases were accurately diagnosed prenatally. The diagnoses were 100% correct when the fetus was diagnosed as an undervirilized male, but only 46% correct when the fetus was diagnosed as a virilized female. The authors diagnosed clitoromegaly when the clitoris measured more that 5 mm beyond the labia majora. This finding was clearly over diagnosed.

The use of three dimensional (3-D) sonography has been reported to improve visualization of a male fetus with ambiguous genitalia (Naylor et al., 2001). There have been no reports of prenatal use of MRI to diagnose ambiguous genitalia. However, a postnatal study compared 10 children with intersex disorders and found MRI and sonography to be equally sensitive (Biswas et al., 2004). For external detection of the gonads, MRI was marginally more sensitive.

Pinhas-Hamiel et al. (2002) identified 16 fetuses with ambiguous genitalia amongst their practice experience of over 10,000 fetuses. These affected fetuses underwent a full evaluation, including (1) repeated sonographic studies, (2) metaphase karyotype and FISH for SRY, and (3) hormonal assays of amniotic fluid. Twelve of the sixteen fetuses were referred because of an abnormal sonogram and 4 had a karyotype discrepancy. The underlying diagnoses were the following: 5 were virilized female fetuses (3 cases of CAH, 2 of urorectal septum malformation), 4 were undervirilized male fetuses (1 steroid sulfatase deficiency, 1 campomelic dysplasia, 2 unknown cause), 5 had chromosome abnormalities, and 2 were 46, XX, SRY+ males. These authors concluded that a screening ultrasound examination at 13 to 15 weeks was not sufficiently sensitive or specific to rule out ambiguous genitalia, and that the size and structures of the reproductive tract evolve throughout gestation. Importantly, after 19 weeks the sonographic presence of a normal uterus was strongly predictive of a virilized genetic female.

Ambiguous genitalia are either noted as a primary finding on sonographic examination, or as a secondary finding when karyotype and external genitalia are discordant. The key considerations in the differential diagnosis include determining whether the fetus is a virilized female or an undervirilized male (Table 84-1). The important diagnostic considerations include an inborn error of metabolism due to a single gene (mendelian) defect, an underlying chromosomal disorder, or a multiple malformation syndrome. Furthermore, the umbilical cord can be misinterpreted as a penis. Careful attention should be paid to distinguishing between the umbilical cord on longitudinal section and the genitalia (Ali, 1992). The most common cause of virilization of a female fetus is CAH, due primarily to the enzyme 21-hydroxylase deficiency (see Figures 84-2 and 84-3). In this disorder, 17-hydroxyprogesterone is not converted to 11-deoxycortisol. Cortisol is not synthesized and ACTH levels rise due to the lack of negative feedback. Subsequently, the levels of androgenic precursors rise, exposing the female embryo to androgen excess. In the severe form, limited secretion of cortisol as well as aldosterone leads to increased plasma renin activity and ultimately to hyponatremic dehydration and vascular collapse after birth. Other, rarer causes of CAH include 11β-hydroxylase deficiency and 3β-hydroxysteroid dehydrogenase deficiency.