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Differentiation of the reproductive/sexual system can be divided into the components outlined in Table 6-3.
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Basics of Sexual Differentiation
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Internal reproductive system differentiation
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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.
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External reproductive system differentiation
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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.
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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.
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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.
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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.
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Gonadal differentiation
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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.
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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.
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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.
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Time Course of Sexual Differentiation
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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.
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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.
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Molecular/Genetic Overview
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Genes known to be involved in the differentiation of the gonads, external genitalia, and internal genitalia are depicted in Figure 6-3.
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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).
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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
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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]).
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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.
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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.
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Disorders Associated With Ambiguous Genitalia
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Steroidogenesis-metabolic pathways
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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.
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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).
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Virilizing disorders (defects in steroidogenesis) impacting 46,XX fetuses
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21-Hydroxylase deficiency.
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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).
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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.
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Box 6-1.
Clitoromegaly in a 46,XX infant usually results from excess androgen exposure as occurs in patients with:
21-Hydroxylase deficiency
11-Hydroxylase deficiency
3β-Hydroxysteroid dehydrogenase deficiency
Placental aromatase deficiency
Oxidoreductase deficiency
Ovotesticular DSD
Testicular DSD
Maternal androgen exposure
Also, rarely results from inclusion of other tissues, such as neurofibromatosis
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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.
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11β-Hydroxylase deficiency.
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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.
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3β-Hydroxysteroid dehydrogenase isozyme deficiencies.
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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.
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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.
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Placental aromatase deficiency.
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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.
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Oxidoreductase deficiency.
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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.
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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
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Defects in testosterone synthesis impacting the 46,XY fetus
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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.
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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.
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5α-Reductase deficiency.
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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
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Congenital lipoid adrenal hyperplasia (StAR mutations).
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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.
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Box 6-2. Congenital Lipoid Adrenal Hyperplasia
Consequence of a “two-hit mechanism”
Autosomal recessive disorder due to inactivating mutations in the steroidogenic acute regulatory protein (StAR) gene. The consequence is impaired transport of cholesterol into mitochondria leading to loss of ability to synthesize glucocorticoids, mineralocorticoids, and sex steroids.
Associated with accumulation of cholesterol esters and other cholesterol metabolites that alter cell cytostructure, leading to cell destruction.
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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
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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.
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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.
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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.
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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.
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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
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Luteinizing hormone defects
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Leydig cell hypoplasia.
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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.
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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
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Hypogonadotropic hypogonadism.
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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.
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Gonadal differentiation
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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
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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.
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Disorders of gonadal differentiation, both general categories and specific forms, resulting in gonadal dysgenesis are listed in Table 6-4.
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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
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Smith-Lemli-Opitz syndrome.
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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
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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
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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.
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Steroidogenic factor 1/NR5A1 gene.
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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
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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.
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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.
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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.
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46,XX gonadal dysgenesis
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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.
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46,XX sex reversal (46,XX males)
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Mutations in the R-spondin1 (RSPO1) gene.
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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
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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.
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46,XY gonadal dysgenesis.
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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.
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45,X/46,XY gonadal dysgenesis (formerly known as mixed or asymmetrical gonadal dysgenesis).
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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
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Environmental causes.
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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.
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Müllerian duct abnormalities.
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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.
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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.
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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.
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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.
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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