Development of the Gonads

The gonadal blastema begins to form at 4 to weeks in the genital ridge, located bilaterally on the medial aspect of each mesonephros. The somatic component of the gonad is made up of cells from the coelomic epithelium and the underlying mesenchyme or adjacent mesonephros.⁷⁵ The germinal component comprises the primordial germ cells, which are mitotically active and migrate from the yolk sac endoderm at 24 days to reach the genital ridge at to 6 weeks, thereby forming the indifferent gonad (Figure 98-2).⁵³

Sex differentiation of the gonad begins at 7 weeks and denotes the onset of fetal sexual differentiation. The gonad differentiates as a testis if the SRY gene and its pathway are present and as an ovary if it is absent. The sex differences that appear in the developing gonad at this time include differentiation of cord somatic cells, changes in germ cell mitotic and meiotic activity, hormone production by the cord and interstitial (mesenchymal) cells, and development of the outer cortex. These differences are detailed in Table 98-2, and an overview is schematically represented in Figure 98-2.

TABLE 98-2

Descriptive Features of Testicular and Ovarian Differentiation at 7 Weeks of Gestation

Gonadal Cells	Testicular Differentiation	Ovarian Differentiation
Cord somatic cells	Differentiate as Sertoli cells	Differentiate as granulosa cells
	Synthesize antimüllerian hormone (AMH) at weeks	Do not synthesize AMH at this age
	Unable to aromatize androgens to estrogens	Able to aromatize androgens to estrogens
Germ cells	Reduced mitotic activity results in small number of spermatogonia	High mitotic activity results in large number of oogonia
	Meiosis is inhibited until puberty	Meiosis is promoted within several weeks
Interstitial cells	Differentiate as Leydig cells	Remain undifferentiated until 15 weeks
	Synthesize testosterone de novo at 8 weeks	Lack steroidogenic capability at this age
Outer cortex	Sex cords lose connection with surface epithelium	Sex cords retain connection with surface epithelium
	Mesenchymal tissue forms tunica albuginea that lacks germ cells	Thickened zone that contains germ cells

Testicular Differentiation

Testicular differentiation begins at 7 to 8 weeks, when the sex cords form loops in the cortex and differentiate as the seminiferous cords; in the hilum, they anastomose to form the rete cords. From 8 weeks on, the seminiferous cords become coiled and thickened and contain 8 to 10 layers of Sertoli cells, whereas the spermatogonia remain located near the basement membrane of the cords. Leydig cells appear in the interstitium between the seminiferous cords at to 8 weeks. They increase strikingly in number during the third month, occupy half the volume of the testis at 13 to 14 weeks, and then show a significant fall in number. Some Leydig cells are present postnatally, but histologically disappear after 3 to 6 months because of the physiologic lowering of gonadotropin stimulation. As the fetus elongates, the testis descends and occupies a more caudal position. In the sixth and seventh months, the cremaster muscle differentiates in the caudal testicular ligament to form the testicular gubernaculum, which penetrates the inguinal canal and is anchored to the connective tissue of the scrotum. The testis descends behind the peritoneum and reaches the scrotum by the eighth or ninth month; the inguinal canal closes after testicular descent is complete.³³

Ovarian Differentiation

Ovarian differentiation begins at 7 weeks, as outlined previously and in Table 98-2. The sex cords in the ovary show germ cells (oogonia) undergoing repeated mitotic divisions from 7 weeks through about the fifth month. Most oogonia differentiate into oocytes between about 10 and 24 weeks; in so doing, they enter meiosis, proceeding through the first meiotic prophase. After reaching the meiotic prophase, many oocytes become surrounded by a single layer of granulosa cells to form the primordial follicles from about 15 weeks through term. Two X chromosomes are needed for differentiation of the primordial follicle. Oocytes degenerate if they are not enclosed in a follicle. During ovarian differentiation, the number of germ cells greatly increases to several million oogonia and oocytes by the fifth month. Most germ cells degenerate thereafter either before or during the primordial follicle stage, leaving about 150,000 oocytes in each ovary at birth.⁸⁹

Development of the Genital Ducts

The internal genital ducts, unlike the gonads, develop from separate anlagen, from the mesonephric or wolffian ducts in the male and from the paramesonephric or müllerian ducts in the female. The wolffian ducts are formed in 26- to 32-day-old embryos; the müllerian ducts begin development at about 37 days in close association with the wolffian ducts, which serve as a guide to the caudal progression of the müllerian ducts. In the absence of the wolffian ducts, the müllerian ducts do not develop.

In the male fetus, the müllerian ducts begin to regress at 7 to weeks, shortly after the Sertoli cells have differentiated and begun to produce anti-müllerian hormone (AMH), otherwise known as müllerian-inhibiting substance (MIS), and before the onset of local testosterone production by Leydig cells. Regression is complete by 9 weeks. Differentiation of the wolffian ducts is testosterone dependent and begins at about weeks. The wolffian ducts differentiate into the epididymis and vas deferens, and beginning at weeks, the seminal vesicles and their ejaculatory ducts develop at the caudal end from lateral outpouchings (Figure 98-3).

In the female fetus, müllerian duct differentiation occurs in the absence of AMH and does not require the presence of any ovarian hormone. The müllerian ducts form the fallopian tubes, uterus, and upper portion of the vagina beginning in the third month, stimulated in part by epidermal growth factor. The wolffian ducts degenerate in the absence of local testosterone and disappear by 13 weeks (see Figure 98-3). The fallopian tubes later descend with the ovaries and are included in a fold of peritoneum called the broad ligament. At birth, the position of the uterus is vertical, and uterine development is disproportionate in that the uterine cervix is twice as large as the fundus; it remains so until puberty. Maternal estrogens stimulate uterine growth in utero, so its size at birth is larger than it is at several months of age; endometrial hyperplasia may occasionally result in transient neonatal uterine bleeding. The development of the vagina depends on the caudal müllerian ducts’ contacting the endodermal epithelium of the urogenital sinus. At this junction, a multilayered, solid epithelial cord known as the vaginal plate is formed; it disintegrates beyond 4 months to form the vaginal lumen. It is not clear whether the lower portion of the vagina is derived from the urogenital sinus, with the upper portion of müllerian origin, or the vagina originates entirely from the urogenital sinus.

Development of the External Genitalia

The external genitalia in both sexes, like the gonads, are derived from common anlagen (Figure 98-4). Their inherent development is along female lines unless systemic androgens, specifically dihydrotestosterone (DHT), induce male differentiation. The genital tubercle forms early in the fourth week at the cranial end of the cloacal membrane, and on each side, the labioscrotal swellings and urogenital folds develop. Fusion of the urorectal septum with the cloacal membrane in the seventh week creates a dorsal anal membrane and a ventral urogenital membrane, which rupture in the eighth week to form the anus and urogenital orifice. Abnormal development of the urorectal septum results in anorectal malformations.

In the male fetus, the indifferent stage of the external genitalia lasts until the ninth week when, in the presence of systemic androgens, masculinization begins with lengthening of the anogenital distance (see Figure 98-4). The urogenital and labioscrotal folds fuse in the midline, beginning caudally and progressing anteriorly. The urethra develops with fusion of the urogenital folds, which form both the membranous urethra in the perineum and the penile urethra along the ventral surface of the phallus. Midline fusion of the labioscrotal folds forms the scrotal raphe, whereas the penile raphe represents the fused portions of the urogenital folds. These processes are complete in the 14-week fetus.

In the 9-week female fetus, the anogenital distance does not increase, and the urogenital folds and labioscrotal swellings do not fuse (see Figure 98-4). The labioscrotal swellings develop predominantly in their caudal portions but less so than in male fetuses and they remain unfused as they are transformed into the labia majora. The urogenital folds develop into the labia minora. The epithelium of the vaginal vestibule between the labia minora and the hymen is endodermal, being derived from the urogenital sinus, whereas the epithelium between the labia minora and majora is ectodermal in origin.⁵⁴

Fetal Gonadal Endocrine Function

Antimüllerian hormone is a large glycoprotein (molecular weight, 140,000 d) produced by the fetal Sertoli cells beginning at weeks that induces irreversible involution of the müllerian ducts. Regression of the müllerian ducts can be induced by AMH only during a fetal age of 44 to 70 days. The gene coding for AMH is located on chromosome 19p13.3, and for the AMH receptor on chromosome 12.

The other major hormone produced by the fetal testis is testosterone (Figure 98-5). It is synthesized by the Leydig cells beginning at 8 weeks, and testicular production achieves peak serum testosterone levels at 10 to 15 weeks. In the second half of gestation, males show a gradual decline and females a rise in serum testosterone levels, so by the third trimester, males have similar or only slightly higher levels than females.⁷⁹

The control of the fetal testicular synthesis of testosterone between 8 and 14 weeks is under human chorionic gonadotropin (hCG) of placental or fetal origin, which binds to fetal testes beginning at 8 weeks. In the second and third trimesters, fetal serum luteinizing hormone (LH) levels correlate with the maintenance and subsequent decline of serum testosterone.⁶ Follicle-stimulating hormone (FSH) receptors are present in fetal testes from 8 weeks on and may play a role in Sertoli cell or seminiferous tubule development.

The fetal ovary in the first trimester is hormonally quiescent, lacking hCG and FSH binding and the enzymes necessary for steroid synthesis. Starting at 8 weeks, the fetal ovary has aromatase activity and is capable of converting androstenedione or testosterone to estrone or estradiol, but actual production has not been demonstrated. Most estrogen that is present in the fetus is produced by placental aromatase. Amniotic fluid levels of estradiol at 12 to 16 weeks are slightly higher in females, raising the possibility of ovarian synthesis of estrogen. If it is synthesized, estradiol could play a local role in ovarian differentiation.³

Control of Genital Differentiation: Roles of Antimüllerian Hormone and Testosterone

In the absence of testes, female sex differentiation occurs regardless of whether an ovary or no gonad is present; a male fetus castrated early also undergoes female sex differentiation. This activity is in keeping with the inherent tendency of the fetus to develop along female lines.

Male sex differentiation requires bilateral testes that produce AMH from the Sertoli cell and testosterone from the Leydig cells. Antimüllerian hormone acts locally to cause regression of only the ipsilateral müllerian duct. If only one testis is present, müllerian duct development occurs on the contralateral side. Testosterone does not cause müllerian duct regression. A 46,XX female with müllerian duct regression resulting from a WNT4 mutation suggests that müllerian duct regression can be controlled by proteins other than AMH.³²

Testosterone produced by the fetal testes is secreted both locally, where it stimulates differentiation of the ipsilateral wolffian duct, and systemically, where it serves as a prohormone in the masculinization of the external genitalia. If only one testis is present, the contralateral wolffian duct fails to differentiate, indicating that the systemically circulating testosterone levels are inadequate for the stimulation of wolffian differentiation. Therefore, testosterone derived from maternal sources or the fetal adrenals, as in congenital adrenal hyperplasia (CAH), does not induce wolffian development.

Although the wolffian ducts are stabilized through the effects of testosterone, the external genitalia and urogenital sinus are stimulated to undergo male differentiation by DHT, which is formed in peripheral tissues (external genitalia, liver, kidney, and bone marrow) through the conversion of testosterone by 5α-reductase. Testosterone is a weaker androgen than DHT and does not stimulate male external genital differentiation, serving instead as a prohormone for DHT in this tissue. Systemic testosterone derived from the fetal adrenals, as in CAH, or from maternal sources can induce variable masculinization of the external genitalia when it is metabolized to DHT.

Congenital absence of one testis (anorchia) is usually associated with incomplete masculinization of the external genitalia, suggesting that two testes are generally required to provide adequate systemic levels of testosterone and thus adequate DHT. The period in which DHT can induce male differentiation extends up to 12 to 14 weeks. A deficiency in androgen receptor binding results in androgen insensitivity, which may be partial or complete. In terms of fetal sex differentiation, the androgen-mediated events of wolffian duct differentiation, masculinization of the external genitalia, and growth of genital structures are either impaired or completely fail to occur, depending on the extent of the receptor-binding deficiency.

Delivery Room or Nursery Examination of all Newborns

Every newborn must have a careful genital examination in the delivery room or nursery. The purposes of the examination are to verify the gender assignment; to avoid missing the diagnosis of a DSD, particularly in females with CAH; and to recognize mild abnormalities that are not likely to affect gender assignment but that require follow-up, such as mild hypospadias or unilateral cryptorchidism. Neonates with overtly ambiguous genitalia should have gender assignment deferred. The parents of the neonate should be informed that most of the diagnostic evaluation can be performed in 2 to 3 days, at which time the appropriate gender assignment can usually be made.

Gonadal Descent and Size

Gonads should be carefully palpated in the scrotum or labial area and along the inguinal canal. Only a gonad-containing testicular tissue (testis or ovotestis) can descend to a position where it is palpable (an ovary almost never descends). A small gonad (longest diameter <8 mm) may be dysgenetic, rudimentary, or result from lack of gonadotropin stimulation. The ability to detect gonads in the inguinal area can be enhanced by applying soap or oil to the skin and the examiner’s fingers to decrease friction. With the infant supine, glide two or three fingers with gentle to firm pressure along the inguinal canal toward the scrotum, repeating this action numerous times if necessary. The gonad will be felt “popping” under the fingers. With this technique, less accessible or small gonads can be palpated that would otherwise be missed. Additionally, sit the infant in a frog-leg position and, particularly with crying or increased intra-abdominal pressure, the testis may descend into the lower inguinal or scrotal region.

Penis Size

It is important to assess both the length and the amount of erectile tissue of the penis. A penis that appears small must be measured fully stretched, pressing the ruler down against the pubic ramus, depressing the suprapubic fat pad completely, and measuring to the tip of the glans only, ignoring any excess foreskin (Figure 98-8). An excellent ruler can be made by placing marks 0.5 cm apart on a wooden stick such as a tongue blade or a flexible strip. The width is measured at the midshaft of the stretched penis. A micropenis is arbitrarily defined as a penis with a normally formed urethra that opens at the tip of the glans in which the stretched length is less than 2.5 cm in the full-term neonate. The length criteria must be adjusted to take into account the smaller phallus in the premature neonate (Figure 98-9).²⁵ The definition of a micropenis can also be used to describe a rare condition in which the penile corpora cavernosa are absent or severely deficient in size, resulting in an abnormally thin penis (Table 98-5).⁴¹

Clitoris Size

A clitoris that appears enlarged is best assessed by measurement of the width (diameter) of the paired corpora cavernosa that compose the erectile shaft of the clitoris. With clitoral enlargement caused by edema or birth trauma, a normal corporal width (<6 mm) is present, whereas clitoral enlargement caused by androgen stimulation (occurring in the second and third trimesters) results in increased corporal growth (>6 mm). The width of the clitoris is measured by gently but firmly pressing the shaft of the clitoris between the thumb and forefinger to exclude excess skin and subcutaneous tissue, thereby measuring predominantly the width of the corpora cavernosa.⁶¹

Urethral Opening

The male fetal urethral development is a hormone-dependent process that occurs at 8 to 14 weeks of gestation. During this stage, testosterone secretion is stimulated solely by the placental hCG. The main hormone involved in urethral development is DHT, which is produced from testosterone in a process catalyzed by 5α-reductase. If the foreskin is fully formed, the urethra is almost always at the tip of the glans; on rare occasions, the foreskin covers hypospadias on the glans. Hypospadias can vary from mild (off the center of the glans) to severe (at the base of the penis or on the perineum) (Figure 98-10). The prepuce in the hypospadiac penis is usually deficient ventrally and thus appears hooded. Chordee (ventral curvature of the penis) is caused by fibrotic contracture in the area of failed urethral development. The presence of severe hypospadias in a male infant indicates deficient testosterone or DHT action in the first trimester. In girls, the urethral meatus is normally a 1-mm pinhole-like or flat opening located just ventral to the vagina. Androgen exposure at 8 to 14 weeks of fetal age moves the urethral meatus ventrally on the perineum or the shaft of the phallic structure.

Vaginal Opening

The vaginal orifice, a 3- to 4-mm slit or stellate opening surrounded by heaped-up mucosa, is normally visible when the examiner lifts up the labia majora. The presence and direct visualization of the vaginal opening indicate the absence of androgen effects and the presence of a distal vagina (of urogenital sinus origin); whether a uterus (of müllerian duct origin) is also present cannot be inferred from this finding. Conversely, at 8 to 14 weeks of fetal age, exposure of the female fetus to androgens or incomplete androgen stimulation of the male fetus results in variable masculinization. This process can be mild to moderate, with posterior midline fusion of the labia majora that partially or completely covers the vaginal opening, thereby preventing its direct visualization. With severe masculinization, there is formation of a common urogenital sinus (resulting from the internal junction of the vagina and urethra) that is seen as a single 1- to 2-mm flat orifice on the perineum or shaft of the phallus.

Labioscrotal Development

The labia majora are normally unfused in the female. Partial or complete midline fusion indicates androgen exposure at 8 to 14 weeks of fetal age and predicts ventral placement of the urethral meatus on the perineum or phallus. The scrotum in the male is normally completely fused, with a midline raphe. A compact or “unlived-in” scrotum indicates the lack of testes or undescended testes. In the male, incomplete or absent midline fusion indicates deficient or absent androgen effects at 8 to 14 weeks of fetal age and predicts a perineal location of the urethral meatus.

Associated Dysmorphology

A thorough physical examination should be done to identify any other dysmorphic features. Genital abnormalities are often part of dysmorphic syndromes and are frequently associated with other midline defects. Examples include the presence of the Turner phenotype in gonadal dysgenesis and camptomelic dwarfism (bowing and angulation of the lower limbs, flat facies, shortened vertebrae) in XY gonadal dysgenesis. Some DSD patients appear phenotypically normal at birth, including many (but not all) cases of gonadal dysgenesis, XX male, 46,XY male with persistent müllerian ducts, and complete androgen insensitivity syndrome (cAIS).

Chromosomes

Fluorescence in situ hybridization (FISH) or QF-PCR for X and Y markers may be available within 24 to 48 hours. Results reveal the number of X and Y chromosomes present. Most cytogenetic laboratories can provide a peripheral blood karyotype result within 2 or 3 days for cases of DSD. In cases in which the gender assignment is not clear, the laboratory should be personally contacted and asked to expedite the chromosome result.

Biochemistry

Interpretation of biochemistry tests is dependent on the timing of the sample and understanding of the normal levels of hormones at that time. A blood sample should be obtained after 24 to 48 hours of life, after the time of the neonatal surge of androgens, for measurement of testosterone and 17-hydroxyprogesterone (17-OHP). It is desirable to draw extra blood so that the laboratory can save the serum for analysis of other hormones that may be indicated as the evaluation continues. Increased serum levels of testosterone occur physiologically in neonates with functioning testes at 12 to 36 hours and at 2 weeks to 4 to 6 months of age.⁸⁸ The level may also increase pathologically at any time from the adrenal secretion of androgens in cases of CAH. Decreased levels of testosterone occur if Leydig cells are deficient or absent, LH activity is impaired, or there is a testosterone biosynthetic defect. Blood samples are also diagnostically useful at 1 or 2 months of age to assess the peak of hypothalamic-pituitary-testicular axis function in infancy.

An increased level of 17-OHP suggests 21- or 11β-hydroxylase deficiency and implies that any increased levels of testosterone were of adrenal rather than testicular origin in a 46,XX patient. The 17-OHP levels in premature infants are higher than those in full-term infants.⁴⁰ In CAH with 21-hydroxylase deficiency, the levels of 17-OHP are often 10 to 50 times the upper limit of the normal level, making the test virtually diagnostic.

The measurement of serum AMH has been shown to correlate with Sertoli cell function.⁸ Similarly, inhibin B levels have been shown to rise in males in the first week of life. Its measurement is therefore useful for identifying the presence of Sertoli cells and of functional testes in newborn boys with nonpalpable gonads.⁸ Other tests that may be useful, depending on the clinical presentation, are measurements of LH, FSH, estradiol, precursors of testosterone, and DHT.