Ambiguous Genitalia

Urogenital Ridge and Bipotential Gonad Development

The urogenital ridge gives rise to the gonads, adrenal cortex, kidney, and reproductive tract. The gonads are derived from intermediate mesoderm and depend on the correct ingression, proliferation, and orientation of the coelomic epithelial cells ( Fig. 6.1 ). In humans, at 4 to 6 weeks of gestation, the urogenital ridges develop as paired outgrowths of coelomic epithelium (mesothelium) on the ventral side of the mesonephros. As the coelomic epithelium proliferates, its basement membrane disintegrates to allow ingression of coelomic cells to form the developing gonad. Notch signaling ensures proper polarization of these cells. In the developing testis, the ingressing cells initially form the Sertoli cells, whereas the later ingressing cells develop into the interstitial cells, including Leydig cells. In the ovary, ingressing cells give rise to both theca and granulosa cells.

The somatic cells of the bipotential gonad require expression of Wilms tumor ( WT1) , GATA binding protein 4 (GATA4), and steroidogeneic factor 1 (NR5A1/SF1 ) genes. The WT1 gene codes a zinc transcription factor, which is expressed in embryonic mesodermal tissues and appears to influence mesodermal-epithelial interactions. GATA4 is expressed in the somatic cells of the urogenital ridge and bipotential gonad before showing sex-specific expression. Chromobox homolog 2 ( CBX2 ) is expressed in the developing gonad at 7 weeks of gestation and plays a role in early gonadal development. CBX2 functions upstream of SRY , promotes transactivation of NR5A1 and SOX9, and represses forkhead transcription factor 2 (FOXL2) . NR5A1 is expressed in the urogenital ridge and appears to upregulate SRY expression, furthering testis development. FOXL2, a forkhead transcription factor expressed in the ovary, orchestrates cell fate decisions; FOXL2 favors ovarian development and represses testicular development. In addition to transcription factors and specific secreted factors (hormones), physical contact with the mesonephros appears to be important for subsequent gonadal differentiation.

Because of their origin within the developing urogenital system, ovaries, and testes are initially located high in the abdomen near the kidneys. Specific signaling molecules activate or repress gonad determination for both testes and ovaries. As noted earlier, ongoing mutual antagonism or competition between specific genes and proteins influences cell fate decisions in gonad development. Transcription factors modulate this “competition” by influencing gene expression. Specific examples include FOXL2 versus SRY-box 9 ( SOX9 ) and SOX9 versus Wingless-type MMMTV integration site family member 4 ( WNT4 )/β-catenin, which are discussed later. Hence, sex determination and differentiation reflect mutual antagonism between the testes determining factors , SRY-SOX9-FGF9 , and the ovary determining factors, roof plate-specific spondin-1 ( RSPO1)-WNT -β-catenin- FOXL2 . Curiously, the antagonism persists postnatally (see later). As would be anticipated, steroidogenic enzyme genes show higher expression in developing human testes compared with ovaries.

Human Testicular Development

Testicular differentiation begins earlier than ovarian development. The first evidence of testicular differentiation is the appearance of primitive Sertoli cells at 6 to 7 weeks’ gestation in the human fetal testis. Cells, mostly endothelial cells, migrate from the mesonephros and interact with the pre-Sertoli cells to promote development of the testicular cords, which is initiated at approximately 7 to 8 weeks of gestation with identifiable cords by 9 to 10 weeks of gestation. The testicular cords are precursors of the seminiferous tubules that will contain Sertoli and germ cells. Interactions between endothelial and mesenchymal cells appear to influence development of the testicular cords.

The binary switch responsible for testicular development is the SRY gene located on the short arm of the Y chromosome. The precise mechanism(s) responsible for triggering SRY expression are incompletely defined. In the human testis, SRY is expressed in supporting cells at approximately 6 weeks of gestation; low-level expression persists throughout gestation. The SRY protein contains a high-mobility group (HMG) domain and is encoded by a single exon gene. Two nuclear localization signals are located in the HMG domain. The SRY protein is expressed in pre-Sertoli cells, where it triggers a molecular switch to induce Sertoli cell differentiation, thus, initiating the process of male sexual differentiation. The HMG domain of the SRY protein binds to the minor DNA groove, where it functions as a transcription factor by bending DNA to presumably permit other proteins access to regulatory regions and to promote assembly of nucleoprotein transcription complexes. A threshold SRY level must be achieved at a critical time during gestation to establish male sexual differentiation. Otherwise, the ovarian differentiation pathway is activated.

SRY expression is independent of the presence of germ cells. SRY increases the expression of the SRY-related HMG box-containing-9 ( SOX9 ) gene. Phenotype-genotype studies of humans and mice demonstrate that SOX9 expression is a crucial step, downstream of SRY , in testis development. Upstream from the SOX9 transcription start site, at least three enhancers, eSR-A, eSR-B, and eALDI, synergize to promote SOX9 expression. Available data suggest that SRY and NR5A1 bind at eALDI to promote initial SOX9 expression. Once expression has been initiated, SOX9 positively autoregulates its own expression. Subsequently, SOX9 and NR5A1 bind to regions within all three enhancer loci to amplify testicular SOX9 expression. Using both direct and indirect mechanisms, SOX9 interferes with genes promoting ovarian differentiation. These data are consistent with the suggestions that mammalian gene regulation depends on multiple redundant enhancers and that copy number variants in this region may cause sex reversal.

In addition to SRY , SOX9 , and NR5A1 , expression of other genes is required for normal male sexual differentiation. Fibroblast growth factor (FGF9) acting through FGF receptors (FGFRs) promotes expression of SOX9 and repression of WNT4 and FOXL2 . Genes found to have expression pattern similar to SOX9 during testis development include CITED1, ANKRD18A, G6PD, SLC52A3, KEL, ZNF280B, PRPS2, and INHBB . In addition to activation of the testicular differentiation pathway, the developing testis expresses factors to actively repress ovarian development. Specifically, the developing testis represses the R-spondin-WNT signaling pathway. Transmember E3 ubiquitin ligases, ZNRF3 and RNF43, inhibit WNT signaling by targeting the Frizzled receptor for degradation.

By immunohistochemistry, NR5A1 and SOX9 proteins can be detected in human embryonic gonadal tissue at 6 to 7 weeks of gestation. At this time, SOX9 expression becomes limited to nuclei of Sertoli cells in a 46,XY fetus but remains cytosolic in a 46,XX fetus. Subsequently anti-Mullerian hormone (AMH) is expressed; with increased AMH protein expression Wilms tumor (WT1) and GATA-4 protein expression increase in the fetal testis. Male-limited expression of doublesex-related and Mab-3--related transcription factor 1 (DMRT1 ) was found in 6- and 7-week-old human fetuses. DMRT1 appears to be necessary to maintain testicular cords and prevent transdifferentiation to a female phenotype. The most rapid growth in Sertoli cell number appears to occur during the latter half of the first trimester and the second trimester. After Sertoli cells have developed, fetal Leydig cells appear around the seventh to eighth week of gestation and produce androgens to promote internal and external male genital structures.

Leydig cells are comprised of two distinct populations; fetal Leydig cells differentiate in utero and adult Leydig cells emerge after birth. Fetal Leydig cell differentiation depends on paracrine signals, such as platelet-derived growth factor receptor-alpha (PDGFR-α), Desert hedgehog (DHH), PTCH1, and Aristaless-related homeobox (ARX). NR5A1 is expressed in Leydig cells to promote steroidogenic enzyme gene expression. The number of fetal Leydig cells reflects gonadotropin stimulation because the number is decreased in anencephalic male fetuses and increased in 46,XY fetuses, with elevated gonadotropin concentrations secondary to complete androgen insensitivity.

Expression of genes associated with testicular steroidogenesis increases around 8 weeks of gestation. Placental human chorionic gonadotropin (hCG) stimulates early testicular androgen secretion. Later in gestation, pituitary LH secretion drives testicular androgen secretion. By 11 weeks of gestation, the testicular compartments, tubular and interstitial components, and the cell types of interest (Leydig, Sertoli, and germ cells) can be visualized. In human fetal testes, HSD17B3, CYP11A1 , and PTC1 messenger ribonucleic acid (mRNA) levels increased significantly through the second trimester without significant changes in CYP17A1, LHR, or INSL3 levels.

Human Ovary Development

Rather than ovarian differentiation being the default pathway occurring in the absence of SRY gene expression, it is clear that specific genes influence ovarian differentiation. Genes involved in ovarian differentiation include WNT4 , FOXL2 , follistatin ( FST ), bone morphogenic protein-2 ( BMP2 ), GATA4/FOG2 , and RSPO1 . RSPO1 is a secreted factor that activates the β-catenin WNT signaling pathway. FOXL2 represses male-specific genes, especially SOX9 beginning in the fetus; this active repression continues throughout adulthood. Hence FOXL2 and SOX9 expression are mutually exclusive in developing gonads, as well as postnatal ovaries and testes.

Normal ovarian development requires FOXL2, RSPO1, and canonical WNT/β-catenin signals. RSPO1 acting through LGR4/5 cell-surface receptors sequesters the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43, resulting in increased WNT signaling. RSPO1 and WNT4 stabilize and amplify β-catenin signaling to activate target gene transcription. WNT4 suppresses androgen-secreting interstitial cells, inhibits coelomic vascularization, and supports Mullerian derivatives.

Two genes that factor in germline development, alpha ( FIGLA ) and newborn ovary homeobox ( NOBOX ), are expressed in fetal or neonatal germ cells, where they recruit developing granulosa cells to form primordial and primary follicles. Additional transcription factors involved in ovarian development include SOHLH1, SOHLH2, NOBOX, LHX8, FIGLA, and LHX9. The second trimester human fetal ovary expresses proteins necessary to synthesize and respond to estrogenic, progestogenic, and androgenic signaling.

Germ Cell Development

Despite the prerequisite for germ cell development to perpetuate the species, germ cells are not required for the initial development of ovaries or testes. The primordial germ cells originate in the epiblast. At approximately 6 weeks of gestation in the human, the primordial germ cells proliferate and migrate from the hindgut along nerve fibers to colonize the genital ridges. SOX17 is a pluripotency factor that is critical for human primordial germ cell specification through its action to promote germ cell–specific gene expression. SOX17 appears to promote BLIMP expression, which appears to suppress endodermal and mesodermal genes.

Upon reaching the developing gonad, the local environment directs the fate of the primordial germ cells toward male or female development. Hence the terminal differentiation pathways for germ cells are sexually dimorphic. When this migration process goes awry, the gonadal germ cell population may be deficient. Aberrant migration can result in ectopic migration into other organs. Ectopic germ cells are primarily located in the CNS, but can also be found in the mediastinum, thorax, and pelvic region. Germ cell tumors may develop in ectopic germ cells when the germ cells are not eliminated by apoptosis.

Developmental phases for female germ cells include initiation of meiosis, formation and breakdown of germ cell nests, and assembly of single oocytes into primordial follicles. For male germ cells, the characteristic phases are migration, mitosis, and cell-cycle arrest. Oocytes undergo meiosis, whereas meiosis is actively inhibited in male germ cells. The RNA binding protein deleted in azoospermia-like (DAZL) appears to license entry into meiosis; DAZL expression increased between 9 and 14 weeks' gestation. Several RNA-binding proteins, such as LIN28, DAZL, and BOLL, are expressed during oogenesis. Another protein, meiosis specific with coiled-coil domain (MEIOC) stabilizes mRNAs that encode proteins relevant for meiosis; its expression appears to be independent of retinoic acid. DAZL inhibits SOX17 expression limiting germ cell pluripotency. Hence DAZL plays dual roles: it initiates meiosis in ovaries and represses pluripotency factors.

The fetal ovary is characterized by a developmental gradient with existence of multiple subpopulations of germ cells at different developmental stages; the more differentiated germ cells are located in the center of the developing ovary. In the developing ovary, the germ cells initially form oogonial clusters connected by intracellular bridges. Selected oogonia enter meiosis and progress through meiotic prophase I (MPI). Somatic cells surround individual oocytes to form follicles. Around 16 to 20 weeks’ gestation, the oogonial clusters break down to form primordial follicles. The oocytes within the primordial follicles undergo growth arrest at the diplotene stage, lasting until oocyte growth is reinitiated with the onset of puberty. The greatest number of follicles exists at midgestation. From a peak of 6.8 million oocytes at approximately 5 months of gestation, approximately 2 million are present at birth. Notch signaling mediates interactions between oocytes and granulosa cells, regulates oocyte survival, and promotes breakdown of the oogonial nests. The internal environment throughout fetal ovary development has the potential to directly influence the fertility of the developing female fetus (controlling the size of the ovarian reserve) and the quality of the oocytes that will eventually become her child (by influencing the extent of selection and apoptosis).

The primordial follicles constitute the ovarian reserve. Postnatally, recruitment and maturation of the primordial follicles is largely governed by AMH, androgen, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) secretion. Premature germ cell loss can lead to gonadal dysgenesis or premature ovarian insufficiency (POI). Accelerated follicular atresia contributes to the follicular depletion characteristic of streak gonads in X monosomy. Although not typically associated with genital ambiguity, mutations in genes governing oocyte and ovarian development cause DSDs characterized by delayed puberty or POI. These genes influence germ cell mitosis, germ cell meiosis, and DNA repair.

In contrast, testicular germ cell development is less dynamic. The Sertoli cells envelope the germ cells to form seminiferous cords at approximately 7 to 9 weeks of gestation in the human XY gonad. The germ cells in the developing testis enter a state of mitotic arrest until the onset of puberty.

Early during gestation, primordial germ cells undergo epigenetic reprogramming, which modifies chromatin architecture. In other words, inherited imprints are erased and DNA methylation is decreased at CpG islands, transcription start sites, genes, and intergenic regions. Demethylation affects almost the entire genome, including the inactive X chromosome by 10 to 11 weeks of gestation. During meiosis, both X chromosomes need to be active to enable efficient pairing. Subsequently, maternal and paternal alleles undergo sexually dimorphic imprinting, such that developing oocytes and sperm are differentially marked reflecting “parent-of-origin”-specific methylation patterns. This process occurs late in fetal development in the male and postnatally in female germ cells. Study of parent-of-origin-dependent disorders, such as Beckwith-Wiedemann, Prader-Willi, and Angelman syndromes and neonatal diabetes mellitus has elucidated the importance of this imprinting process.

Human Adrenal Development

By 33 days postconception, the human fetal adrenal cortex is distinct from the developing gonad. Because of its role as the source of dehydroepiandrosterone sulfate (DHEA-S) for placental estrogen biosynthesis, the fetal adrenal cortex grows rapidly. By 50 to 52 days postconception, expression of several steroidogenic enzymes, steroidogenic acute regulatory protein ( StAR ), 11β-hydroxylase ( CYP11B1 ), 17α-hydroxylase/ 17,20-lyase ( CYP17A1 ), and 21-hydroxylase ( CYP21A2 ) in the fetal adrenal cortex have been demonstrated immunohistochemically. Transitory fetal adrenal cortisol biosynthesis peaks at 8 to 9 weeks’ gestation, which coincides with transient adrenal expression of both nerve growth factor IB-like ( NGFI-B ) and 3β-hydroxysteroid dehydrogenase-2 ( HSD3B2 ). At the same time, adrenocorticotropin hormone (ACTH) can be detected in the anterior pituitary, suggesting the presence of negative feedback inhibition during the first trimester. During the time male sexual differentiation begins, this negative feedback inhibition may serve to prevent virilization of female fetuses to ensure normal female sexual differentiation. Distinct expression profiles for fetal adrenal steroidogenic enzymes have been described with differences between the first and second trimesters. Chromaffin cells derived from the sympathetic nervous system migrate into the adrenal cortex to form the adrenal medulla.

Development of Internal Genital Structures

The Wolffian duct originates as the excretory duct of the mesonephros and develops into the epididymis, vas deferens, ejaculatory duct, and seminal vesicle. The Mullerian or paramesonephric duct originates as an invagination of the coelomic epithelium and develops into the Fallopian tubes, uterus, and upper third of the vagina.

The Sertoli cells of the developing testis secrete AMH, also known as Mullerian inhibitory hormone ( MIH ). In human 46,XY fetuses, AMH expression can be detected by 7 weeks of gestation, is not dependent on the presence of germ cells within the testis, and promotes regression of the Mullerian ducts. AMH, a member of the transforming growth factor-β (TGF-β) family, undergoes proteolytic cleavage to become biologically active. AMH binds to its receptor, AMH-RII, on the surface of the Mullerian duct mesenchymal cells to induce increased matrix metalloproteinase 2 expression. The net result is degeneration and loss of basement membrane integrity of the epithelial and mesenchymal Mullerian cells, leading to regression of the Mullerian ducts.

AMH expression is highly regulated; inappropriate expression in a 46,XX fetus would lead to uterine agenesis. In the 46,XX fetus with absence of both AMH and testosterone, the Mullerian duct derivatives persist and the Wolffian ducts regress. When a female fetus is inappropriately exposed to AMH (as in freemartin cattle), Mullerian duct regression and ovarian masculinization occur. The paired Mullerian ducts arise as coelomic epithelial invaginations. Around the eighth week of gestation, the Mullerian ducts fuse, followed by degeneration of a midline epithelial septum. Variable persistence of this septum can lead to uterine malformations. Multiple factors influence the development of the uterus, cervix, and vagina. HOX genes specify the development program for distinct uterine regions; HOXA9 specifies the uterine tube, HOXA10 and HOXA11 specify the uterus, and HOXA13 specifies the vagina. The vaginal plate appears during the 11th week of gestation through occlusion of the Mullerian-derived uterovaginal canal and ultimately disappears as the vaginal plate lengthens and canalizes.

The fetal hypothalamic-pituitary-gonadal (HPG) axis is active by midgestation, with peak fetal testosterone concentrations occurring at approximately 15 to 16 weeks of gestation. Before this time, placental hCG stimulates testosterone production by the fetal Leydig cells. Secretion of testosterone by the fetal Leydig cells stabilizes the Wolffian ducts in 46,XY fetuses. Region-specific signaling molecules, such as BMPs, homeobox (HOXA10 and HOXA11), growth differentiation factor 7 (GDF7), relaxin, an orphan G-protein–coupled receptor (LGR4), platelet-derived growth factor A (PDGFA), and its cognate receptor (PDGFRA) influence the development of the epididymis and seminal vesicle.

The prostate, a male accessory sex gland, contributes to seminal fluid plasma and develops from the urogenital sinus. After the initial testosterone-dependent induction of prostate differentiation, subsequent development involves epithelial-mesenchymal interactions that lead to cell differentiation and branching morphogenesis. The requisite signaling molecules, FGFs, sonic hedgehog (SHH), BMPs, HOXA13, and HOXD13, are similar to those required for external genital differentiation.

Development of External Genital Structures

The genital tubercle, urethral folds, and labioscrotal swellings give rise to the external genitalia. Androgens play a time-dependent role in formation, differentiation, and growth of the fetal male external genitalia at approximately 8 to 14 weeks of gestation, during the masculinization programming window (MPW).

During this period, a cylindrical 2-mm phallus with genital swellings develops by 9 weeks of gestation. Within the developing penis, the urethra forms by canalization of the urethral plate with subsequent fusion to form the penile urethra by 12 to 14 weeks of gestation. The genital tubercle develops into the corpora cavernosa of the penis, and the labioscrotal folds fuse to form the scrotum. By 14 weeks, the external genitalia are clearly masculine, apart from testicular location. Thereafter, the penis grows at a rate of 0.7 mm/week from 14 weeks to term. Deficiency of androgen or androgen action during the MPW reduces penis length, an effect that cannot be fully rescued by postnatal T therapy. The MPW predetermines potential phallic size, whereas postnatal androgen action is required to realize normal potential.

In the 46,XX fetus, in the absence of androgens, the urethral folds and labioscrotal swellings do not fuse 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. Minimal clitoral growth, well-defined labia majora, hypoplastic labia minora, and separate vaginal and urethral perineal openings are present by 20 weeks of gestation.

Anogenital Distance

The anogenital distance (AGD), the distance from anus to the genital tubercle, indicates prenatal androgen action and, as anticipated, is sexually dimorphic. Investigators have used different anatomic landmarks to assess AGD in humans. Salazar-Martinez and colleagues defined AGD as being from the anus to the perineoscrotal junction (anoscrotal distance) in males and from the anus to the anterior fourchette (anofourchettal distance) in females; this definition appears to improve interobserver reliability. Salazar-Martinez and colleagues reported normative ranges for anoscrotal and anofourchettal distances to be 21 ± 3.0 mm in males and 11 ± 2 mm in females (Salazar). In both sexes, AGD increased up to 12 months and maintained the sex dimorphic pattern. AGD also showed positive correlation with penile length at birth and with the increase in AGD from birth to 3 months. AGD provides a biomarker of early fetal androgen exposure and masculinization. Measurement of AGD can be used as a diagnostic tool for in utero androgen exposure and assessing impact of potential environmental endocrine disruptors on external genital development. AGD at birth predicts adult anogenital distance, but correlations with adult male reproductive parameters are inconsistent.

Sexual Differentiation of the Brain

Clinical investigations suggest that the brain is sexually dimorphic and that testosterone is a masculinizing hormone in human. Males with aromatase deficiency manifest male psychosexual behavior and gender identity. Alternatively, 46,XY individuals with complete androgen insensitivity syndrome (CAIS) develop female gender identity. However, preliminary data implicate genetic differences, independent of sex steroid exposure, as the molecular basis for some aspects of sexual dimorphism of the brain and other nongonadal tissues. Androgens have organizational and activation effects on CNS function. Complex interactions between genetic factors and hormone contribute to human brain sex differences.

45,X/46,XY Mosaicism

Most sex chromosome disorders, such as 45,X and cytogenetic variants and 47,XXY and cytogenetic variants, are not associated with abnormal external genital development and will not be discussed in this chapter (see Table 6.1 ). In contrast, individuals with 45,X/46,XY and 46,XX/46,XY karyotypes manifest a broad range of phenotypes. Among individuals with 45,X/46,XY karyotype, internal and external genital structures range from normal male to ambiguous to female. Whereas the typical histological features consist of poorly developed seminiferous tubules surrounded by wavy ovarian stroma, gonadal differentiation can range from normal testis to streak gonads. At the time of puberty, virilization can occur.

The majority of individuals identified by prenatal karyotype as being 45,X/46,XY appear to be normally androgenized males; however, individuals diagnosed postnatally tend to have more clinical signs. A normal peripheral blood lymphocyte karyotype in individuals with gonadal dysgenesis suggests the presence of sex chromosome mosaicism within the gonad(s). Individuals with sex chromosome DSDs because of gonadal dysgenesis have an increased risk of developing gonadal tumors, such as gonadoblastoma or dysgerminoma because a dysgenetic gonad carrying a Y chromosome has an increased risk for neoplastic changes. Although gonadal tumors typically do not develop until the second decade of life, they can occur at younger ages.

Among a series of 63 males with 45,X/46,XY, subjects were classified into two groups; one group presented with genital anomalies and the second group presented with other reasons, such as short stature or infertility. Individuals identified by genital anomalies tended to have lower rates of spontaneous pubertal development, shorter stature, and greater likelihood of germ cell neoplasia. Although most gonads were classified as dysgenetic testes, some testes appeared relatively normal with evidence of spermatogenesis. No follicles were observed, suggesting that gonads labeled as ovotestis were mislabeled and consisted of undifferentiated/streak-like tissue. Based on these histological findings, it has been suggested that the etiology of the 45,X/46,XY karyotype is the loss of a Y chromosome in some cells. Focal spermatogenesis was identified in approximately 25% of these subjects. Thus when gonadectomy is considered because of risk for neoplasia, fertility preservation should be considered.

ARX (X-Linked Lissencephaly 2)

X-linked lissencephaly 2 is characterized by genital ambiguity, lissencephaly, early-onset seizures, absence of the corpus callosum, intellectual disability, and hypothalamic dysfunction, and Aristaless-related homeobox ( ARX ) gene mutations. This gene is a member of the paired-type homeodomain transcription factor family and is mapped to Xp21.3. ARX contributes to many aspects of brain development including patterning, neuronal proliferation and migration, and cell maturation and differentiation, especially the generation and migration of GABAergic neurons. Findings in Arx knockout mice showed no expression in developing ovaries, but Leydig cell differentiation was impaired. Available data suggest that Arx is a positive regulator for the differentiation of fetal Leydig cells at the progenitor stage.

CDKN1C (IMAGe Syndrome)

The IMAGe syndrome is characterized by intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia, cryptorchidism, and micropenis. Genital anomalies appear to be limited to affected males. Both sporadic and familial cases have been described. Heterozygous missense mutations in the CDKN1C gene have been detected in some cases. This gene is located at chromosome 11p15.5, encodes a 316-amino-acid protein, p57 ^Kip2 , negatively regulates cellular proliferation, and inhibits cell cycle progression. The gene is located within a complex genetic locus containing several genes including, IGF2 and H19 , subject to imprinting modulation through cis-acting elements; CDKN1C is maternally expressed. Mutations associated with the IMAGe or Silver-Russell syndromes appear to be localized to the proliferating cell nuclear antigen–binding domain, resulting in gain of function with excessive inhibition of growth and differentiation. Curiously, loss-of-function mutations affecting the cyclin-dependent kinase-binding domain of this protein are associated with overgrowth syndromes, such as Beckwith-Wiedemann syndrome.

CHD7 (CHARGE Syndrome)

The CHARGE syndrome is associated with mutations in the chromodomain helicase DNA binding protein-7 (CHD7) gene. Features of this syndrome include eye coloboma, heart malformations, choanal atresia, short stature, genital anomalies, ear abnormalities, and hearing loss. Micropenis and cryptorchidism are found in males. Hypogonadotropic hypogonadism can occur. Although usually sporadic, autosomal dominant cases have been described. The CHD7 gene, located at chromosome 8q12.1-q12.2, codes for a large protein that participates in chromatin remodeling and transcription.

DHCR7 (Smith-Lemli-Opitz)

Several enzymes catalyze the conversion of lanosterol to cholesterol. Decreased activity of these enzymes leads to cholesterol deficiency. The enzyme, 7-dehydrocholesterol reductase, encoded by the 7-dehydrocholesterol reductase ( DHCR7 ) gene, located at chromosome 11q12-q13, catalyzes the last step in cholesterol biosynthesis. Smith-Lemli-Opitz (SLO) is an autosomal recessive disorder caused by DHCR7 loss-of-function mutations. As would be anticipated, elevated 7-dehydroxycholesterol concentrations are required to confirm the diagnosis.

Clinical features include urogenital anomalies, mental retardation, failure to thrive, facial abnormalities, developmental delay, behavioral abnormalities, and autism symptoms. The most common urogenital abnormalities include male-to-female sex reversal, hypospadias, and cryptorchidism. Facial abnormalities include microcephaly, broad nose, upturned nares, micrognathia, short neck, and cleft palate. Short thumbs, syndactyly of the second and third toes, and postaxial polydactyly are common. CNS malformations include septum pellucidum anomalies, agenesis of the corpus callosum, or holoprosencephaly in severe cases.

Infants often have hypotonia, feeding problems, and failure to thrive. Tube feedings may be necessary because of the poor weight gain. Impaired cholesterol biosynthesis may be associated with adrenal insufficiency. However, overt adrenal insufficiency is uncommon. Nevertheless, adrenal insufficiency, especially during stress, can occur. Thus the hypothalamic-pituitary-adrenal axis function should be assessed and glucocorticoid stress dosing may be beneficial. Although the efficacy remains unclear, dietary cholesterol supplementation is often prescribed.

DHCR7 mutations are associated with decreased cholesterol and accumulation of sterol intermediates proximal to the defective enzyme. Decreased cholesterol concentrations lead to decreased steroid concentrations, with subsequent decreases in glucocorticoid, mineralocorticoid, and sex steroid biosynthesis. In addition to serving as the precursor for vitamin D and bile acids, cholesterol is involved in SHH signaling and cell membranes. Available data indicate that accumulation of sterol intermediates rather than cholesterol deficiency leads to some of the clinical findings. Impaired SHH signaling contributes to malformations, such as agenesis of the corpus callosum, holoprosencephaly, and postaxial polydactyly. Using induced pluripotent stem cells from subjects with SLOS, data suggest that accumulation of 7-dehydrocholesterol and its isomer, 8-dehydrocholesterol, rather than cholesterol deficiency, likely contributes to the neurological manifestations.

Prenatal diagnosis can be performed by measurement of amniotic fluid 7-dehydrocholesterol concentrations. Women pregnant with affected fetuses have low plasma estriol; elevated blood 16α-hydroxyestrogens concentrations; and elevated urinary Δ7 and Δ8 unsaturated C ₁₈ , C ₁₉ , and C ₂₁ dehydrosteroid concentrations, presumably because of impaired fetal cholesterol production. The incidence of biochemically confirmed SLO is estimated at 1/20,000 to 1/60,000 live births. The rate of heterozygosity for DHCR7 mutations is surprisingly high. Because the prevalence of SLO at 16 weeks of gestation is comparable to the prevalence at birth, early fetal loss and/or reduced fertility of carrier couples is likely occurring.

EMX2 Gene

One male patient presented with 46,XY DSD, a single kidney, and intellectual disability; he had a small microdeletion involving EMX2 .

GLI3 (Pallister-Hall Syndrome)

Pallister-Hall syndrome is characterized by genital anomalies, hypothalamic hamartoma, postaxial polydactyly, and imperforate anus. Hypospadias, micropenis, and bifid or hypoplastic scrotum are described in XY patients whereas hydrometrocolpos and/or vaginal atresia are described in XX patients. This autosomal dominant disorder is caused by mutations in the Gli-Kruppel Family Member 3 ( GLI3) gene, which is located at chromosome 7p14.1. The protein acts as both a transcriptional activator and a transcriptional repressor of downstream targets in the SHH pathway. Pallister-Hall is typically caused by frameshift/nonsense and splicing mutations located in the middle third of the gene, which are predicted to generate a truncated functional repressor protein. Curiously, mutations outside of this region are associated with Greigs cephalopolysyndactyly syndrome. Preclinical data using mouse knockout models showed aberrant ureteric budding, hydroureter, hydronephrosis, and renal hypoplasia.

HHAT (Nivelon-Nivelon-Mabille Syndrome)

The Nivelon-Nivelon-Mabille syndrome is an extremely rare disorder characterized by primordial short stature, generalized chondrodysplasia, microcephaly, brachydactyly, facial dysmorphism, and XY sex reversal. This syndrome is caused by autosomal recessive loss-of-function mutations in the O-acyl-transferase hedgehog acyl-transferase ( HHAT ) gene mapped to chromosome 1q32.2. The HHAT protein palmitoylates the N-terminal domain of the hedgehog proteins. The hedgehog proteins, SHH, DHH, and Indian hedgehog (IHH), play major roles in embryo patterning and differentiation. Based on the phenotype of the knockout mouse, loss of Hhat leads to testicular dysgenesis characterized by decreased testicular size, decreased testis cord, and almost complete absence of fetal Leydig cells. Curiously, the phenotype described for XY patients with DHH mutations and Dhh knockout mice resembles the phenotype reported for HHAT patients and Hhat knockout mice.

HOXA13 (Hand-Foot-Genital Syndrome)

HOX genes are a group of transcription factors involved in embryonic development and patterning. These genes are organized into clusters characterized by specific spatiotemporal expression patterns. The hand–foot–genital syndrome (HFGS) (OMIM #140000) is a rare, autosomal dominant disorder associated with mutations or deletions of the HOXA13 gene on chromosome 7p15. This syndrome is characterized by congenital genitourinary anomalies and variable skeletal anomalies. The skeletal anomalies mainly affect the hands and feet, such as short thumbs and halluces, shortening of the middle phalanges, fifth finger clinodactyly, and the fusion of distal and middle phalanges of the toes. Although approximately 50% of affected individuals have urogenital anomalies, many experience normal fertility. Males may have hypospadias or cryptorchidism. Females may have incomplete Mullerian fusion or bicornuate uterus associated with increased risks for spontaneous abortion, preterm delivery, or stillbirth. In some instances, the skeletal phenotype can be mild. Additional comorbidities include chronic urinary tract infections, ectopic ureteric orifices, chronic pyelonephritis, and renal failure.

KAT6B (Genitopatellar Syndrome)

The genitopatellar syndrome is a rare disorder characterized by skeletal dysplasia, flexion contractures, genital anomalies, craniofacial defects, and developmental delay associated with lysine acetyltransferase 6 ( KAT6B ) gene mutations. The skeletal features include hypoplastic or absent patellae, flat temporal bones, and brachydactyly. Genital anomalies include hypoplastic labia, clitoromegaly, scrotal hypoplasia, and cryptorchidism. Cardiac anomalies, hydronephrosis, and developmental delay have been described. The gene is located at chromosome 10q22.2 and contains 20 exons. The KAT6B protein is a component of the histone H3 acetyltransferase complex and functions as a histone acetyltransferase. The KAT6B protein functions as a chromatin modifier by catalyzing acetylation of specific lysines. Most mutations are de novo mutations. The Say-Barber-Biececker-Young-Simpson syndrome is also associated with KAT6B mutations; specific facial features consisting of blepharphimosis/ptosis and an immobile mask-like face distinguish these syndromes.

NR2F2 Gene

This gene encodes chicken oval albumin upstream promoter transcription factor (COUP-TF2), a nuclear receptor. This protein appears to function as a proovary and antitestis factor. Mutations in this gene are associated with a syndromic 46,XX DSD characterized by genital virilization, congenital heart disease, and varying somatic findings. Frameshift mutations have been reported in three 46,XX patients who were identified by genital ambiguity. All three patients had congenital heart disease. One infant died of hypoplastic left heart; no gonadal histology was available. The other two patients carried de novo c.97_103delCCGCCCG predicted to generate frameshift mutation, p.Pro33Alafs*77; they also had blepharophimosis ptosis epicanthus inversus syndromes (BPES) and uterus. One patient had ovotesticular disorder; gonadal histology was not available for the other patient.

Immunohistochemistry studies performed on human ovaries showed positive COUP-TF2 staining in FOXL2-negative stromal areas consistent with COUP-TF2 functioning as a proovary and antitestis factor in developing female gonads. Preclinical studies in mice showed that female embryos lacking Coup-tfII possess both Wolffian and Mullerian ducts; maintenance of the Wolffian ducts is androgen independent and is likely caused by enhanced phosphorylated extracellular signal-regulated kinase signaling in Wolffian duct epithelium. These data suggest that COUP-TFII actively promotes Wolffian duct regression in female embryos.

RSPO1

The R-spondin 1 (RSPO1 ) gene codes for a secreted furin-like domain protein that stabilizes β-catenin in the Wnt-signaling pathway. Mutations in this gene, located at chromosome 1p34, are associated with 46, XX sex reversal. This gene was initially identified by investigating in individuals with palmoplantar hyperkeratosis with squamous cell carcinoma of skin and sex reversal in one family. Affected XX, sex-reversed individuals lack Mullerian structures. A 46,XY individual, homozygous for RSPO1 mutations, fathered two children. An XX individual with ovotesticular disorder and palmoplantar keratoderma was found to be homozygous for a splicing mutation in the RSPO1 gene. In addition to sex reversal and palmoplantar keratosis, reported features include microphthalmia and nail dystrophy.

SAMD9 (MIRAGE Syndrome)

Mirage syndrome is characterized by myelodysplasia, infections, intrauterine growth restriction (IUGR), adrenal hypoplasia, XY male-to-female sex reversal, and enteropathy. Pulmonary disease, severe infections, and myelodysplasia contribute to deaths during the first 2 years of life. This disorder has been associated with heterozygous mutations in the sterile α motif domain–containing protein 9 ( SAMD9 ) gene located at chromosome 7q21.2. The 1589-amino-acid protein is highly expressed in the fetal adrenal; immunohistochemistry showed SAMD9 localization in the cytoplasm of adrenal definitive and fetal zone cells colocalized with cells positive for NR5A1 expression. SAMD9 is considered to be a tumor suppressor gene; mutations are gain-of-function mutations, leading to IUGR and consequences for adrenal gland and testes differentiation. Curiously, second mutations, such as acquired monosomy for chromosome 7, modify the phenotype and prolong survival. Buonocore and colleagues suggest that SAMD9 is essential to control the balance between cell proliferation and cell differentiation; activating SAMD9 mutations interfere with cell proliferation resulting in hypoplastic tissues.

SOX9

SOX9 is a member of the SRY-related HMG domain gene family located at chromosome 17q24.3-17q25.1 that encodes a 508-amino-acid protein. Heterozygous loss-of-function SOX9 mutations are associated with autosomal-dominant campomelic dwarfism and male-to-female sex reversal. Features of campomelic dwarfism include congenital bowing of long bones, hypoplastic scapulae, 11 pairs of ribs, narrow chest, congenital dislocated hips, and clubfeet. Facial features include micrognathia, cleft palate, large head, flat nasal bridge, and low-set malformed ears. When bone malformations are severe, postnatal survival may be limited. Affected 46,XY fetuses manifest sex reversal, with external genital differentiation, ranging from ambiguous to female. Gonadal dysgenesis and persistence of Mullerian duct derivatives are typical. Phenotypic heterogeneity with differing phenotypes, including ovotesticular DSD and complete sex reversal, has been described in affected siblings. Acampomelic dysplasia has been described in which the typical limb anomalies are absent. Patients with acampomelic dwarfism and sex reversal have been reported. 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.

SOX9 mutations can affect DNA-binding affinity, DNA bending ability, nuclear import, transactivation, and nuclear export. Haploinsufficiency is the mechanism responsible for many of the consequences of SOX9 mutations. Somatic cell mosaicism, de novo germline mutations, and mitotic gene conversion events have been described. Mutations located upstream of SOX9 in the regulatory regions have been identified in patients with varying degrees of sex reversal. Duplications of the eSR-A locus were associated with 46,XX testicular disorder and 46,XX ovotesticular disorder, whereas deletions have been identified in patients with 46,XY sex reversal.

TBX3 (Ulnar-Mammary syndrome)

The Ulnar-Mammary syndrome is characterized by cryptorchidism, micropenis, breast hypoplasia/aplasia, nipple hypoplasia/aplasia, delayed puberty, absent axillary hair, and skeletal anomalies. Skeletal anomalies include hypoplasia of humerus and ulna, oligodactyly, polydactyly, and finger anomalies. The TBX3 gene plays a role in development of the dorsoventral limb axis. Additional endocrine features include hypogonadotropic hypogonadism, short stature, growth hormone deficiency, and obesity. Neuroimaging studies have identified pituitary and brain malformations.

WT1

The Denys-Drash syndrome is characterized by genitourinary anomalies, Wilms tumor, nephropathy, and Wilms tumor suppressor (WT1) gene mutations. Among affected 46,XY individuals, the external genitalia can range from ambiguous to normal female. Internal genital differentiation varies because of inconsistent Wolffian and/or Mullerian structure development and regression. Typically, the nephropathy begins during the first few years of life, manifests with proteinuria, and results in end-stage renal failure because of focal or diffuse mesangial sclerosis. Typically, the gonads are usually dysgenetic in 46,XY individuals. Affected 46,XX individuals typically have normal female external genital development.

However, a novel WT1 mutation was reported in an XX individual identified at birth because of clitoromegaly and a single perineal opening. She was found to have an immature right uterine tube; gonadal histology revealed bilateral testes with seminiferous tubules containing predominantly Sertoli cells and rare germ cells. The karyotype of both gonads was XX. Genetic analysis revealed a de novo frameshift variant in exon 10, which encodes the fourth DNA finger. This variant is predicted to generate a deleterious mutation p.Arg485Glyfs*14, which appears to be a gain-of-function mutation increasing DNA binding affinity, promoting NR5A1 overexpression and SOX9 upregulation, and culminating in testicular development.

The Wilms tumor suppressor ( WT1 ) gene, located at chromosome 11p13, plays an important role in both kidney and gonadal differentiation. Through alternative splicing, multiple translation start sites, and posttranslational RNA editing, over 30 isoforms are derived from this one gene. The carboxyl terminal domain of the WT1 protein contains four zinc fingers that serve as the nucleic acid binding domain.

The two major isoforms differ by the inclusion or exclusion of three amino acids, lysine, threonine, and serine (KTS), between the third and fourth zinc-finger domains. Subnuclear localization studies have shown that the –KTS form colocalizes predominantly with transcription factors and preferentially binds to DNA, whereas the + KTS form colocalizes mainly with splicing factors and plays a role in RNA processing. The ratio of the + KTS/–KTS isoforms appears to be tightly regulated. Hence depending on cell context, WT1 can function as a transcriptional activator, a transcriptional repressor, or tumor suppressor. WT1 plays a role in the balance between mesenchymal-epithelial transitions.

Patients with WT1 mutations manifest phenotypic heterogeneity. Although Wilms tumor and genitourinary abnormalities can be associated with heterozygous WT1 deletions, only 6% to 15% of sporadic Wilms tumors are associated with WT1 mutations. Heterozygous deletions at chromosome 11p13 can be part of a contiguous gene deletion syndrome known as WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, gonadoblastoma, and mental retardation). In general, missense mutations in exons 6–9 are associated with severe gonadal dysgenesis and early-onset nephropathy. The (–) KTS form appears to be protective from development of Wilms tumor, whereas mutations located in the N-terminal repression domain are associated with development of Wilms tumor.

Frasier syndrome is characterized by gonadal dysgenesis, progressive glomerulopathy, and an increased risk for gonadoblastoma. Wilms tumor is extremely rare in Frasier syndrome. The typical renal lesion is focal glomerular sclerosis. The majority of cases are associated with a specific WT1 point mutation in intron 9, associated with altered splicing and decreased amounts of the + KTS isoform.

Meacham syndrome is characterized by genital anomalies, cyanotic congenital heart defects, and pulmonary hypoplasia secondary to diaphragmatic abnormalities. In 46,XY infants, the spectrum of internal genital anomalies extends from presence of a uterus to a blind ending vaginal pouch. External genitalia have been described as ranging from normal female to ambiguous.

XH2 (ATRX Syndrome)

ATRX (α-thalassemia, mental retardation, X-linked protein) syndrome is an X-linked disorder characterized by mild alpha-thalassemia, severe mental retardation, and genital abnormalities because of mutations in the XH2 gene located at chromosome Xq13.3. Urogenital anomalies, including ambiguous genitalia, cryptorchidism, hypoplastic scrotum, hypospadias, shawl scrotum, and small penis occur in approximately 80% of patients. Additional features include short stature, psychomotor retardation, microcephaly, seizures, talipes equinovarus, and gastrointestinal problems. The facies are described as coarse with midface hypoplasia, short nose, and widely spaced incisors. The hemoglobin H inclusions associated with alpha-thalassemia can be demonstrated on brilliant cresyl blue stained peripheral blood smears. Typically, Wolffian duct structures are present, whereas Mullerian duct structures and germ cells are absent. Phenotype-genotype correlations are inconsistent. Most cases are inherited from carrier mothers and most, but not all, carrier females show preferential inactivation of the X chromosome carrying ATRX mutations.

This disorder, also known as Carpenter-Waziri syndrome, is caused by mutations in the ATRX (also known as XH2 or XHP ) gene located at Xq13.3. The ATRX protein is a member of the SWI/SNF DNA helicase family and consists of three domains: an N-terminal containing a plant-homeodomain-like (PHD) domain, a coiled-coil motif, and C-terminal helicase domain. The urogenital anomalies are typically associated with mutations located in the PHD domain and predicted to generate a truncated protein. During meiosis, ATRX maintains genomic integrity by modulating DNA damage response mechanisms. ATRX mutations are associated with abnormal methylation of repetitive DNA sequences, failure to establish necessary epigenetic modification for X chromosome inactivation, and impaired silencing of imprinted genes.

Sertoli cell ATRX knockout mice had small testes, decreased seminiferous tubule volume, decreased Sertoli cell number, and increased Sertoli cell apoptosis associated with prolonged G2/M phase during fetal life. This finding is consistent with the concept that ATRX protein expression impacts on G2-M progression with ultimate consequences on cell survival. Tissue-specific consequences of the epigenetic regulation and disrupted cell cycle progression may explain the disparate features associated with ATRX mutations.