Chapter 14. Disorders of Sex Determination and Differentiation

• ACTH Adrenocorticotropic hormone
• AKR1C2 3α-Hydroxysteroid dehydrogenase-3 (3α-HSD-3)
• AMH Anti-Müllerian hormone
• BPES Blepharophimosis-ptosis-epicanthus inversus syndrome
• CAH Congenital adrenal hyperplasia
• CIS Carcinoma in situ
• CBX2 Chromobox homolog 2, homolog of mouse m33
• CMPD1 Campomelic dysplasia
• DAX1 DSS-AHC-critical region on the X chromosome gene 1
• DHEA Dehydroepiandrosterone
• DHH Desert hedgehog
• DHT Dihydrotestosterone
• DMRT1 Double sex Mab3-related transcription factor gene 1
• DSS Dosage-sensitive sex reversal
• DSD Disorders of sexual development—congenital conditions in which development of chromosomal, gonadal, or anatomic sex is atypical
• 46,XX DSD Previously designated female pseudohermaphrodite
• 46,XY DSD Previously designated male pseudohermaphrodite
• FISH Fluorescent in situ hybridization
• FGF-9 Fibroblast growth factor-9
• FOXL2 Forkhead box L2
• FSH Follicle-stimulating hormone
• GATA-4 Member of the GATA family of zinc finger transcription factors
• GH Growth hormone
• GnRH Gonadotropin-releasing hormone
• hCG Human chorionic gonadotropin
• HMG High mobility group
• 17β-HSD-3 17β-Hydroxysteroid dehydrogenase-3
• ICSI Intracytoplasmic sperm injection
• IGF Insulin-like growth factor
• LH Luteinizing hormone
• MAMLD1 Mastermind-like domain containing 1
• MAP3K4 Mitogen-activated protein kinase kinase kinase 4
• Ovotesticular DSD Previously designated true hermaphrodism
• PAR Pseudoautosomal region
• POR P450 oxidoreductase
• PRKX Protein kinase X linked
• PRKY Protein kinase Y linked
• RFLP Testriction fragment length polymorphism
• RSPO1 R-spondin1
• SF-1 Steroidogenic factor-1
• SHOX Short stature homeobox gene on the X chromosome
• SOX3 SRY-like HMG box-3
• SOX9 SRY-like HMG box-9
• SRY Sex-determining region Y gene
• StAR Steroidogenic acute regulatory protein
• TSPY Testes-specific protein-Y encoded
• WAGR Wilms tumor-aniridia-genital anomalies-mental retardation syndrome
• WNT4 Human homolog of Drosophila wingless gene
• WT1 Wilms tumor gene
• XIST X-inactive specific transcript gene

Lowercase letters for genes, for example, Sry, Sox9, Fgf9 are used to indicate the mouse homologue of the human gene where a particular function has not yet been demonstrated in humans.

Advances in developmental and cell biology, molecular genetics, experimental embryology, steroid biochemistry, and methods of evaluation of the interaction between the hypothalamus, pituitary, and gonads as well as behavioral science have helped clarify problems of sexual determination and differentiation. Anomalies may occur at any stage of intrauterine development of the hypothalamus, pituitary, gonads, and genitalia and lead to gross ambisexual development or to subtle abnormalities that do not become manifest until sexual maturity is achieved.

Chromosomal Sex

The normal human diploid cell contains 22 autosomal pairs of chromosomes and two sex chromosomes (two Xs, or one X and one Y). When arranged serially and numbered according to size and centromeric position, they are known as a karyotype. Advances in the techniques of staining chromosomes permit positive identification of each chromosome by its unique banding pattern. A technique called fluorescence in situ hybridization (FISH) is particularly useful in identifying quickly both sex chromosomes, mosaicism and structural abnormalities involving the sex chromosomes, the presence or absence of SRY (the testes determining gene on the Y chromosome), and other deleted genes (Figure 14–1). High-resolution chromosome banding and painting techniques provide precise identification of each chromosome.

Studies in animals as well as humans with abnormalities of sexual differentiation indicate that the sex chromosomes (the X and Y chromosomes) and the autosomes harbor genes that influence sex determination and differentiation by causing the bipotential gonad to develop either as a testis or as an ovary. Two intact and normally functioning X chromosomes, in the absence of a Y chromosome or its SRY gene, lead to the formation of an ovary under normal circumstances, whereas a Y chromosome (whose SRY gene is intact) or the translocation of SRY, the testis-determining gene on the short arm of the Y chromosome, to an X chromosome or autosome leads to testicular organogenesis.

In humans, there is a marked discrepancy in size between the X and Y chromosomes. Gene dosage compensation is achieved in all persons with two or more X chromosomes in their genetic constitution by partial inactivation of all X chromosomes except one. This phenomenon is thought to be a random process (except when a structurally abnormal X chromosome is present) that occurs in each cell in the late blastocyst stage of embryonic development, during which either the maternally or the paternally derived X chromosome undergoes heterochromatinization. A result of this epigenetic process is formation of an X chromatin body (Barr body) in the interphase cells of persons having two or more X chromosomes. In patients with two or more X chromosomes, the maximum number of Barr bodies (partially inactivated X chromosomes) seen in interphase cells will be one less than the number of X chromosomes in the karyotype. A gene termed XIST (X-inactive specific transcripts) is located in the X inactivation center at Xq13.2 on the paracentromeric region of the long arm of the X chromosome. XIST is expressed only by the inactive X chromosome. The XIST gene encodes a large RNA that appears to coat the X chromosome and facilitates inactivation of selective genes on the X chromosome.

The distal portion of the short arm of the X chromosome escapes inactivation and has a short (2.5-megabase [mb]) segment homologous to a segment on the distal portion of the short arm of the Y chromosome (Figures 14–2 and 14–3). This segment is called the pseudoautosomal region (PAR); it is these two limited regions of the X and Y that pair during meiosis, undergo obligatory chiasm formation, and allow for exchange of DNA between these specific regions of the X and Y chromosomes. At least 10 genes have been localized to the pseudoautosomal region on the short arm of the X and Y chromosomes. Among these are a gene whose deletion results in the neurocognitive defects observed in Turner syndrome and a gene for short stature, SHOX (short stature homeobox gene), which is expressed in bone. A mutation or deletion of SHOX on either the X or Y chromosome is associated with "idiopathic" short stature as well as dyschondrosteosis (Leri-Weill syndrome). Homozygous mutations of this gene are associated with a more severe form of short stature, Langer mesomelic dwarfism. A pseudoautosomal region has also been described for the distal ends of the long arms of the X and Y chromosomes (see Figures 14–2 and 14–3). The pseudoautosomal region of the long arms of the X and Y chromosomes contains genes that are mostly growth factors and signaling molecules. The Y chromosome (see Figure 14–3) represents only 2% of the human genomic DNA and is about 60 mb in length. It is unique in that it contains few active genes compared to the X and autosomal chromosomes and has a large apparently noncoding heterochromatic region. It contains at least two genes affecting growth, the SHOX gene in the PAR and the growth control gene on the Y chromosome (GCY). The euchromatic region of the short arm of the Y chromosome contains the SRY gene (the male determining factor) distal to the PAR region at Yp11.3. The short arm of the Y chromosome contains genes that when deleted produce the phenotype stigma of Turner syndrome. The pericentromeric region of the Y is the locus for the TSPY (testes-specific protein Y encoded) genes which predispose to gonadoblastoma formation in the presence of dysgenetic testicular development. The PRKY gene is homologous to a gene PRKX (protein kinase X linked) on the X chromosome and is the locus for Y to X translocation observed in 46,XX SRY-positive males. The euchromatic region of the long arm of the Y chromosome has genes that when deleted result in azoospermia.

Genes Involved in Organogenesis of the Bipotential gonad

WT1

Heterozygous mutations and deletions of the Wilms tumor gene (WT1) located on 11p13 result in urogenital malformations as well as Wilms tumor. Knockout of the WT1 gene in mice results in apoptosis of the metanephric blastema and as a consequence, absence of the kidneys and gonads. Thus, WT1, a transcriptional regulator, appears to act on metanephric blastema early in urogenital development (Figure 14–4A). Dominant-negative point mutations of WT1 in human beings results in the Denys-Drash and Frasier syndromes, whereas a contiguous deletion of the gene and surrounding DNA results in Wilms tumor, aniridia, ambiguous genitalia, and mental retardation—the WAGR syndrome.

SF-1

Steroidogenic factor-1 (SF-1) is a nuclear receptor involved in transcriptional regulation of many genes, including those that are involved in gonadal development, adrenal development, steroid synthesis, and reproduction (see Figure 14–4A). SF-1 is located on 9q33 and is expressed in the urogenital ridge as well as in steroidogenic organs. SF-1 is required for the synthesis of testosterone in Leydig cells; in Sertoli cells it regulates the anti-Müllerian hormone (AMH) gene. Homozygous knockout of the gene encoding Sf-1 (Sf-1 is the mouse homologue of mammalian SF-1) in mice results in apoptosis of the cells of the genital ridge that give rise to the adrenals and gonads and thus absence of gonadal and adrenal gland morphogenesis in both males and females. This gene has a critical role in the formation of all steroid-secreting glands (ie, adrenals, testes, and ovaries). Initial studies in humans with SF-1 mutations identified individuals with adrenal insufficiency, a 46,XY karyotype, complete gonadal dysgenesis, and the presence of müllerian derivatives, similar to the mouse phenotype. Subsequently, the spectrum of affected patients has included a range of 46,XY disorders of sexual development without adrenal insufficiency including hypospadias, anorchia, micropenis, complete gonadal dysgenesis, infertility in otherwise normal males, and ovarian failure in 46,XX females.

DMRT1

Haploinsufficiency of DMRT1 (double sex Mab3-related transcription factor gene 1)—a gene related to double sex in drosophila and Mab3 in Caenorhabditis elegans is a candidate for the abnormality in testis organogenesis in patients with 9p deletions (see Figure 14–4A). 46,XY patients invariably have the stigmata of the 9p syndrome (mental retardation, trigonocephaly, upslanting palpebral fissures, etc), as well as female or ambiguous external genitalia and Müllerian structures associated with streak gonads or dysgenetic testes. Recent data suggest that ovarian function may either be compromised or normal in 46,XX females with the 9p syndrome.

Ambiguous genitalia has also been reported in 46,XY patients with deletions of 10q. However, as yet a specific mutant gene(s) causing this DSD has not been identified on the 10q region.

Genes Involved in Testicular Determination

(Figure 14–4B)

SRY

In studies of 46,XX males with very small Y-to-X translocations, a gene was localized to the region just proximal to the pseudoautosomal boundary of the Y chromosome (Yp11.3) (see Figure 14–3) which has been named SRY. Deletions or mutations of the human SRY gene occur in about 15% to 20% of 46,XY females with complete XY gonadal dysgenesis and 90% of 46,XX males have a Y to X translocation which includes the SRY gene. Compelling evidence that SRY is the testis-determining factor is the observation that transfection of the Sry gene into 46,XX mouse embryos results in transgenic 46,XX mice with testes and male sex differentiation.

The SRY gene encodes a DNA-binding protein that has an 80 amino acid domain similar to that found in high-mobility group (HMG) proteins. This domain binds to DNA in a sequence-specific manner (A/TAACAAT). It bends the DNA and thus is thought to facilitate interaction between DNA-bound proteins to affect the transcription of downstream genes. The human SRY gene has no intron; two nuclear localization sites, calmodulin and importin B are critical for the function of the gene. In mice, Sry facilitates the upregulation of Sox9 by binding to multiple enhancing elements in the Sox9 gene and along with Sf-1 establishes a feed-forward pathway resulting in upregulation of Sox9 and its continued expression in Sertoli cells. A feed–forward loop involving Sox9 and Fgf9 has also been demonstrated in the mouse but not yet in the human gonad. Both Sry and Sox9 in mice appear to inhibit the Rspo1 (R-spondin 1)-Wnt4-β-catenin- FOXL2 signaling pathway, facilitating Sertoli cell induction and testicular organogenesis and inhibiting ovarian development (see Figure 14–4B). In the absence of Sry, the Wnt4-β-catenin canonical pathway is stabilized and Sox9 is suppressed which results in ovarian development. Hence, it appears that Sry and Sox9 are repressors of a major ovarian determining pathway—Rspo1-Wnt4-β-catenin—which induces ovarian development along with the downstream gene—Fox L2 and other genes yet to be defined, as well as germ cells which are essential for ovarian development (but not testicular differentiation). Duplications of DAX1 or WNT4 in humans repress testicular development presumably by preventing the repression of β-catenin on its downstream gene FOXL2 by SRY and SOX9. Ovarian determination is a genetically controlled pathway and not, as previously thought, a default pathway.

Most of the mutations thus far described in 46,XY females with gonadal dysgenesis have occurred in the nucleotides of the SRY gene encoding the DNA-binding region (the HMG box) of the SRY protein. Mutations that affect DNA bending as well as nuclear transportation of the SRY protein have also been implicated in defective testicular development.

SOX9

SOX9 has an HMG box that is more than 60% homologous to that of SRY. It is localized to 17q24.3-q25.1 and is expressed in the developing sex cords and, thereafter, in the Sertoli cells (see Figure 14–4B). It is also expressed in cartilage. Mutations in one allele of the SOX9 gene can result in a bone abnormality called campomelic dysplasia (CMPD) and XY gonadal dysgenesis with ambiguous genitalia in affected 46,XY males. Duplication of the SOX9 gene both in humans and mice results in sex reversal in XX individuals who are SRY negative. It appears that SOX9 is the critical gene acting downstream of SRY for Sertoli cell differentiation and consequent testicular development.

DHH

Desert hedgehog (DHH), a gene that codes for a signaling molecule located on chromosome 12q13.1 in humans, is an important gene in mammalian testes organogenesis and function. Mutations in DHH have been reported in individuals with 46,XY gonadal dysgenesis.

GATA4 GATA4 is a transcription factor which interacts with other proteins including SF-1 and FOG2 to regulate the expression of SRY. Recently, a heterozygous missense mutation has been described affecting three male siblings and resulting in 46,XY DSD and congenital heart disease.

Genes Involved in Ovarian Determination

DAX1

A mutation or deletion of DAX1, which encodes a transcription factor, results in X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism in 46,XY males (see Figures 14–2, 14–4B). Deletion or mutation of the DAX1 gene in 46,XY individuals has not resulted in an abnormality of testicular differentiation in humans. However, XY gonadal dysgenesis has been reported in individuals with duplications of Xp21, a locus that contains the DAX1 gene on the X chromosome. Duplication of the DAX1 gene appears not to affect ovarian morphogenesis and function in 46,XX females. Thus, DAX1 dosage plays a critical role in testicular development and function (see Figure 14–4B).

WNT4

WNT4 is a signaling molecule encoded by a gene located on the short arm of chromosome region 1 (1p35). Duplication of WNT4 has been associated with dysgenetic testicular development and ambiguous genitalia in 46,XY, SRY, positive individuals. Testis development is inhibited likely as a result of upregulation of DAX1 and stabilization of β-catenin-FOXL2 which would inhibit SOX9 expression in the developing gonad. 46,XY patients who have duplications of WNT4 exhibit a heterogenous phenotype varying from cryptorchidism alone to female external genitalia. WNT4 is expressed in the primordial ovary and is an essential signal for ovarian determination (see Figure 14–4B). In mice, Wnt4 expression in the ovary prevents the migration of steroid (androgen)-producing cells into the ovary, restrains the development of a testis-specific blood vessel (the coelomic vessel), and is critical for the development of the Müllerian ducts and the maintenance of germ cells.

A 46,XX female with absent Müllerian structures (atypical Mayer-Rokitansky-Küster-Hauser syndrome) has been reported who had a loss of function mutation in the WNT4 gene. Her phenotype was similar to that described in Wnt4-mutated mice. She had unilateral renal agenesis as well as clinical signs of androgen excess as manifested by severe acne requiring antiandrogen therapy. Wolffian duct development was not ascertained. Two other similar patients have been reported.

RSPO1 (R-Spondin 1)

RSPO1 (R-spondin 1), a gene located in 1p34.3, appears to stabilize the expression of WNT4-β-catenin-FOXL2 and thus promotes ovarian determination (see Figure 14–4B). Mutations in RSPO1 cause 46,XX females to develop testes and male sexual development, as well as palmoplantar hyperkeratosis and a predisposition to squamous cell carcinoma of the skin. The fact that a mutation in RSPO1 can redirect gonadal development in a 46,XX individual from an ovary to a testes suggests that it is a critical determinant in ovarian determination in humans.

FOXL2 is a putative forkhead transcription factor, which does not cause a DSD in human. Heterozygous FOXL2 mutations in affected 46,XX human females causes premature ovarian failure with or without blepharophimosis. However, induced FoxL2 deletions in the ovary of the mouse lead to transdifferentiation of the ovary into testes.

A 46,XY phenotypic female with normal ovaries and a mutation in the CBX2 (chromobox homolog 2) gene, the human homolog of mouse m33, an ortholog of the Drosophila Polycomb gene, has recently been reported. It was postulated that this gene is upstream of SRY and that mutations in this gene prevent the repression of the ovarian gene pathway presumably by decreasing SRY/SOX9 expression enabling the expression of RSPO1-WNT4-β-catenin-FOXL2 and other ovarian determining genes.

Our lack of complete understanding of ovarian and testicular determination is illustrated in the study by Dumic et al who reported a familial cohort of 46,XY females. A 46,XY female was reported to have undergone spontaneous menarche and given birth to a 46,XY daughter with gonadal dysgenesis. Studies in this cohort have uncovered a mutation in a gene not previously known to be involved in the sex determination cascade—mitogen-activated protein kinase kinase kinase 4 (MAP3K4). Mutations in this gene involved in the mitogen-activated protein kinase signaling pathway cause a striking decrease in the expression of Sry and Sox9 in the developing mouse gonad resulting in sex reversal. Recently, mutations in MAP3K1 have been reported to 46,XY DSD leading to either partial or complete gonadal dysgenesis. Further studies in humans will be necessary to elucidate the exact mechanism which allows for repression of testicular development and the genesis of an apparently normal functioning ovary in SRY positive 46,XY individuals.

(Figure 14–5)

The mammalian gonad forms on the surface of the mesonephros within the genital ridge as a result of proliferation of the coelomic epithelium. Until the 12-mm stage (∼42 days of gestation), the embryonic gonads of males and females are indistinguishable. By 42 days, 300 to 1300 primordial germ cells have seeded the undifferentiated gonad from their extragonadal origin in the yolk sac dorsal endoderm. These large cells are the progenitors of oogonia and spermatogonia; lack of these cells is incompatible with further ovarian differentiation but not testicular differentiation. The undifferentiated gonad also contains supporting cells and steroidogenic cells. Under the influence of SRY/SOX9 and other genes that encode male sex determination, the gonad begins to differentiate as a testis at 43 to 50 days of gestation. The supporting cells differentiate into pre-Sertoli cells which organize into seminiferous tubules and surround the germ cells which undergo mitotic arrest. Leydig cells derived from steroidogenic cells are apparent by about 60 days, and differentiation of male external genitalia occurs by 65 to 77 days of gestation.

In the undifferentiated gonad destined to be an ovary, lack of further differentiation persists. At 77 to 84 days—long after differentiation of the testis in the male fetus—a significant number of germ cells enter meiotic prophase to characterize the transition of oogonia into oocytes, which marks the onset of ovarian differentiation from the undifferentiated gonads. The supporting cells become granulosa cells and steroidogenic cells become theca cells. Germ cell meiosis in the developing ovary is induced by retinoic acid signaling. Sry induces an enzyme which metabolizes retinoic acid to an inactive product and prevents meiosis of germ cells in the developing testis. Primordial follicles (small oocytes surrounded by a single layer of flat granulosa cells and a basement membrane) are evident after 90 days. Preantral follicles are seen after 6 months, and a population of fully developed oocytes with fluid-filled cavities and multiple layers of granulosa cells are present at birth. As opposed to the testes, there is little evidence of hormone production by the fetal ovary.

Differentiation of Genital Ducts

(Figure 14–6)

By the seventh week of intrauterine life, the fetus is equipped with the primordia of both male and female genital ducts. The Müllerian ducts, if allowed to persist, form the uterine (fallopian) tubes, the corpus and cervix of the uterus, and the upper third of the vagina. The Wolffian ducts, on the other hand, have the potential for differentiating into the epididymis, vas deferens, seminal vesicles, and ejaculatory ducts of the male. In the presence of a functional testis, the Müllerian ducts undergo apoptosis under the influence of AMH, a dimeric glycoprotein secreted by fetal Sertoli cells. AMH acts locally to cause Müllerian duct repression ipsilaterally. Müllerian duct regression is the earliest indicator of male sex differentiation.

The gene for AMH encodes a 560 amino acid protein, whose carboxyl terminal domain shows marked homology with transforming growth factor-β (TGF-β) and the B chain of inhibin and activin. The gene is localized to the short arm of chromosome 19. AMH is secreted by human fetal and postnatal Sertoli cells and is used as a marker for the presence of these cells. SF-1 (an orphan nuclear receptor) in combination with WT1, SOX9, and GATA4 (Figure 14–7), regulates AMH gene expression. The human AMH receptor gene maps to the q13 band of chromosome 12. The receptor is similar to other type II receptors of the TGF-β family.

The differentiation of the Wolffian duct is mediated by testosterone (T) secretion from the testis. SF-1 regulates steroidogenesis by the Leydig cell in the testes by binding to the promoter of the genes encoding P450 side chain cleavage enzyme (P450scc) and 17α-hydroxylase (P450c17). In the presence of an ovary or in the absence of a functional fetal testis, Müllerian duct differentiation occurs, and the Wolffian ducts involute (see Figure 14–6).

Differentiation of External Genitalia

(Figure 14–8)

Up to the eighth week of fetal life, the external genitalia of both sexes are identical and have the capacity to differentiate into the genitalia of either sex. Female sex differentiation occurs in the presence of an ovary or streak gonads or if no gonad is present (see Figure 14–8). The fetal ovary does not secrete sex steroids during gestation or AMH during the critical period of female differentiation of the genital ducts and external genitalia. Studies in the past have indicated that the fetal adrenal gland is 3β-HSD-2 (3 beta-hydroxysteroid dehydrogenase-2) deficient and secretes primarily DHEA-S (dehydroepiandrosterone sulfate) until late in gestation. DHEA-S is converted by the placenta to DHEA which is metabolized to T. T is aromatized to estrogen, thereby preventing the exposure of the female external genitalia to high levels of T/dihydrotestosterone (DHT). Placental aromatase activity is low from 8 to 12 weeks of gestation but increases markedly after the first trimester (Figure 14–9). Recent studies indicate that the normal female genitalia are protected from androgen exposure early in gestation (8-12 weeks), a stage when placental aromatase activity is relatively low. Sex differentiation of the external genitalia is occurring by the transient expression of 3β-HSD-2 in the fetal adrenal, resulting in cortisol secretion, and consequently relative suppression of both adrenocorticotropin (ACTH) and DHEA-S secretion with a resultant decrease in placental testosterone synthesis and secretion. Adrenally derived androgen levels appear to be the same during this period in both males and females. After 12 weeks of gestation, 3β-HSD-2 activity diminishes in the fetal adrenal which leads to an increase in ACTH and DHEA-S secretion. Testosterone is now converted by the increased placental aromatase to estrogen. In addition, androgen receptor activity diminishes in the labioscrotal folds, and excess androgen exposure after 12 to 14 weeks gestation results in only clitoromegaly, but not labioscrotal fusion. Thus, the transient expression of 3β-HSD-2 and the consequent ability of the fetal adrenal gland to transiently synthesize cortisol, as well as the decrease in androgen receptor activity in genital skin protects the female external genitalia from masculinization during the window of dimorphic differentiation of the external genitalia.

Differentiation of the external genitalia along male lines depends on the action of DHT, the 5α-reduced metabolite of T. In the male fetus, T is secreted by the Leydig cells, autonomously at first and later under the influence of human chorionic gonadotropin (hCG), and later fetal pituitary luteinizing hormone (LH). Masculinization of the external genitalia and urogenital sinus of the fetus results from the action of DHT, which is synthesized from T in the target cells by the enzyme 5α-reductase-2 as well as possibly secreted by the testes via the backdoor pathway (Figure 14–10). This pathway was first found in marsipials and then implicated in patients with P450 oxidoreductase (POR) deficiency. This backdoor pathway synthesizes DHT, a nonaromatized androgen, without utilizing T as a precursor. This pathway may play a signifcant role in male differentiation; however, further studies are necessary to define this.

DHT (as well as T) is bound to a specific protein receptor in the nucleus of the target cell (Figure 14–11). The transformed steroid-receptor complex dimerizes and binds with high affinity to specific DNA domains, initiating DNA-directed, RNA-mediated transcription. This results in androgen-induced proteins that lead to differentiation and growth of the cell. The gene that encodes the intracellular androgen-binding protein has been localized to the paracentromeric portion of the long arm of the X chromosome (see Figure 14–2). Of note, an X-linked gene controls the androgen response of all somatic cell types by specifying the androgen receptor protein. As noted previously, there is a decrease in androgen receptor activity in the labioscrotal folds after 12 to 14 weeks so that even with intense T/DHT stimulation, labioscrotal fusion does not occur although growth of the genital tubercle can continue resulting in clitoromegaly in females and penile enlargement in males.

Incomplete masculinization of the male fetus results from (1) impairment in the synthesis or secretion of fetal T and/or DHT (3α-HSD-3 deficiency) or the conversion of T to DHT (5α-reductase-2 deficiency, (2) deficient or defective androgen receptor activity, or (3) defective production and local action of AMH.

Psychosexual differentiation can be classified into four broad categories: (1) gender identity, defined as the identification of self as either male or female; (2) gender role (ie, those aspects of behavior in which males and females differ from one another in one's culture at this time); (3) gender orientation, the choice of sexual partner; and (4) cognitive differences.

For much of the past 50 years, the prevailing dogma has been that newborns are born psychosexually neutral and that gender identity is imprinted postnatally by words, attitudes, family dynamics, and comparisons of one's body with that of others. Gender identity had been hypothesized to be neutral at birth and not established until 12 to 18 months of age. During the last decade, this hypothesis has been rigorously challenged. It has become evident that prenatal exposure to T/DHT and possibly genes on the Y chromosome can influence gender identity in the individual with a DSD. A growing body of evidence indicates that gender identity is more plastic than previously thought. If, at puberty, discordant secondary sexual characteristics are allowed to mature, some individuals, especially those with 5α-reductase deficiency, 45,X/46,XY mosaicism, or 17β-HSD-3 deficiency, have had doubts about their gender identity and have chosen to change their assigned sex from female to male. However, in general, it has been demonstrated that most patients with DSD (except for those mentioned above) will accept their gender assignment even when it is not concordant with their sex chromosome constitution—a phenomenon which underscores both the complexity and plasticity of the processes that determine gender identity. This suggests that androgens have a facultative and not a deterministic role in gender identity. Androgens have a significant effect on childhood play behavior, maternal interest, and sexual orientation. It is apparent that both genes and hormones (nature) and environment and experience (nurture) are critical factors in the development and maintenance of gender identity. Further studies on patients with ambiguous genitalia, including long-term outcomes of the factors influencing gender identity, gender role, and sexual activity in DSD patients, are critical to furthering our understanding of the multifaceted complexities of the differentiation of gender identity and to support more evidence-based guidelines for decisions about sex assignment.

(Table 14–1)

An International Conference on Management of Intersex was held in Chicago, Illinois, in 2005 because of ethical issues and patient concerns about the venerable but antiquated terms intersex, pseudohermaphrodite, true hermaphrodite, and so forth, which were perceived by patients as pejorative. A new nomenclature was proposed and accepted. The term, disorders of sexual development (DSD), connoting all conditions in which chromosomal, gonadal, or anatomic sex are atypical, was coined. (See Lee PA et al for the complete consensus statement.)

DSDs are the result of abnormalities in complex processes that originate in genetic information on the X and Y chromosomes as well as on the autosomes. A 46,XY DSD (previously male hermaphrodite) is one whose gonads are exclusively testes in origin but whose genital ducts or external genitalia (or both) exhibit incomplete masculinization. A 46,XX DSD (previously female hermaphrodite) is a person whose gonadal tissue is exclusively ovarian in origin but whose genital development exhibits ambiguous or even a male appearance. Persons who possess both ovarian and testicular tissue were previously referred to as true hermaphrodites and now called ovotesticular DSD.

(See Also Chapter 12)

Klinefelter syndrome is one of the most common forms of primary hypogonadism and infertility in males. Surveys of the prevalence of 47,XXY fetuses by karyotype analysis of unselected newborn infants indicate a prevalence of about 1:700 newborn males. However, there is a striking disparity between the prevalence diagnosed at birth by karyotype screening and the frequency of Klinefelter syndrome identified in adults. It has been estimated that only 25% of individuals with Klinefelter syndrome are diagnosed during life and less than 10% in childhood. This most likely is due to the broad spectrum of phenotypes, testicular function, and psychosocial status found in patients with this syndrome. Ninety percent of affected patients have a 47,XXY sex chromosome constitution and an X chromatin-positive buccal smear, the rest demonstrate a variety of sex chromosome constitutions, including mosaicism. Virtually all of these variants have at least two X chromosomes and a Y chromosome in common, except for the rare group that has only an XX sex chromosome complement (3%-4%).

Hormonal levels are variable, and T levels may be in the low-normal to normal range. In addition, variability in phenotype can result in part from sex chromosome mosaicism with a normal XY cell line as well as variability in androgen sensitivity. The androgen receptor is a ligand-dependent transcription factor that has a polymorphism involving CAG (glutamine) trinucleotide repeats. The longer the length of the CAG repeat, the less the transactivation activity of the ligand-bound receptor. In a study of 35 patients with Klinefelter syndrome, the one genetic factor affecting phenotype was the CAG repeat length of the functional androgen receptor. The repeat lengths correlated inversely with penile length, a T-sensitive organ. Neither an effect of parental origin of the X chromosome (imprinting) nor evidence of skewed X chromosome inactivation affected the phenotype. In a study of 77 patients with Klinefelter syndrome, preferential inactivation of the shorter CAG allele was detected.

The most consistent feature of Klinefelter syndrome is small atrophic testes due to seminiferous tubule dysgenesis and consequent azoospermia. Tall stature is common in Klinefelter syndrome, reflecting the presence of three SHOX genes. Adult males with a 47, XXY karyotype tend to be taller than average, with adult height close to the 75th percentile, mainly because of the disproportionate length of their legs, which is present prepubertally. Untreated adult males, especially those with subnormal sex steroid levels, are at increased risk of osteoporosis. In addition, individuals with Klinefelter syndrome have an increased prevalence of mild diabetes mellitus, autoimmune thyroid disorders, early tooth decay, varicose veins, stasis dermatitis, cerebrovascular disease, chronic pulmonary disease, deep vein thrombosis, carcinoma of the breast, midline germ cell tumors, and Hodgkin lymphoma. The data on carcinoma of the breast are controversial. There were no cases in a cohort of 696 men with Klinefelter syndrome from the Danish cytogenetic register; however, 7 of 93 men with breast cancer in a Swedish study had Klinefelter syndrome. Life expectancy is reduced by about 2.1 years over controls, with a lower death rate than expected from ischemic heart disease and prostate cancer.

Prepubertally, Klinefelter syndrome is characterized by small undescended testes (> 25% of patients), relatively reduced penile length, clinodactyly, hypotonia, disproportionately long legs, personality and behavioral disorders, and a lower mean verbal IQ score. There is no significant difference in full-scale IQ. Deficits are found primarily in specific areas of cognition such as language and problem solving. Severe mental retardation requiring special schooling is uncommon. Gynecomastia and signs of androgen deficiency such as diminished facial and body hair, a small phallus, and poor muscular development occur postpubertally in affected patients.

Patients with Klinefelter syndrome may have delayed adolescence although a recent study suggested that the onset of puberty and early progression were relatively normal. There is an increased risk of malignant extragonadal germ cell tumors in the central nervous system, mediastinum, and sacral region. The germ cell tumors often secrete hCG and cause sexual precocity in the affected prepubertal individual. Accordingly, we recommend that boys with hCG-secreting midline germ cell tumors have a karyotype analysis.

The testicular lesion is progressive and gonadotropin dependent. Gonadal germ cells become depleted at an accelerated rate after the onset of puberty, and there appears to be a maturation arrest of spermatogenesis. The testes are characterized in the adult by extensive seminiferous tubular hyalinization and fibrosis, absent or severely deficient spermatogenesis, and pseudoadenomatous clumping of the Leydig cells. Although hyalinization of the tubules is usually extensive, it varies considerably from patient to patient and even between testes in the same patient. Azoospermia is the rule; patients reported to be spontaneously fertile invariably have been 46,XY/47,XXY mosaics. Using surgical techniques, sperm has been found in 50% of the nonmosaic patients with Klinefelter syndrome. In biopsied testes of patients with 47,XXY Klinefelter syndrome, only a minor fraction of tubules contain germ cells. FISH analysis revealed that the spermatocytes were euploid, suggesting that the spermatogonia that undergo mitosis have only one X chromosome thus allowing for progression in cell differentiation and the ability to engage in meiosis. Intracytoplasmic sperm injection (ICSI) with sperm retrieved surgically from testes (TESE) of patients with Klinefelter syndrome has been used with success for ovum fertilization. Using ICSI, most infants born to fathers with Klinefelter syndrome have had normal karyotypes. However, there is an increased incidence of sex chromosome and autosomal aneuploidy, possibly related to the assisted fertility technique (ART). Preimplantation genetic diagnosis (PGD) is recommended. However, the potential risk to the embryo of PGD must be weighed against the risk of chorionic villus biopsy or amniocentesis in the event of a successful pregnancy.

Nondisjunction during the first or second meiotic division of gametogenesis plays an important role in the genesis of a 47,XXY karyotype. Fifty-three percent of cases appear to result from paternal nondisjunction at the first meiotic division, 34% from meiotic nondisjunction during the first maternal meiotic division, and 9% from nondisjunction at the second meiotic division. Only 3% of patients appear to have arisen from postzygotic mitotic nondisjunction. Early studies suggested that parental age was a factor in the etiology of Klinefelter syndrome, but in a study of 228 patients, no association with advanced parental age was found.

Diagnosis: Klinefelter syndrome is suggested by the classic phenotype and hormonal changes. It is confirmed by the finding of an X chromatin-positive buccal smear or an FISH analysis which demonstrates an XXY constitution or the demonstration of a 47,XXY karyotype in blood, skin, or gonads. After puberty, levels of serum gonadotropins especially follicle-stimulating hormone (FSH) are raised. The T production rate, the total and free levels of T, and the metabolic clearance rates of T and estradiol tend to be low, whereas plasma estradiol levels are relatively normal or high for a male. Testicular biopsy reveals the classic findings of hyalinization of the seminiferous tubules, severe deficiency of spermatogonia, and pseudoadenomatous clumping of Leydig cells.

Hormonal evaluation in the newly diagnosed patient with Klinefelter syndrome should include plasma LH and FSH by immunochemiluminescence (ICMA). These levels are usually in the normal range prior to the onset of puberty. Thereafter, a rise in FSH and subsequently LH is usually observed. A plasma estradiol and T by liquid chromatography-tandem mass spectrometry (LCMSMS) should be obtained. In the adolescent or postadolescent patient, a bone density is indicated to determine whether the patient has osteopenia/osteoporosis and requires therapy for this.

Treatment: Treatment in adolescents and adults with Klinefelter syndrome should be directed toward health maintenance and the options for fertility (eg, cryopreservation of sperm from ejaculate in adolescence or TESE in older patients, T replacement if necessary, and prevention of osteopenia/osteoporosis and deep vein thrombosis). Klinefelter patients are born with spermatogonia, and testicular failure is progressive beginning at puberty. Obtaining ejaculated sperm in early adolescence for cryopreservation, if possible, might obviate the need for TESE at a later time. Although several studies have reported that sperm have been found in 8% of ejaculated serum sample fron nonmosaic 47,XXY patients, a recent study reported lack of sperm in samples from 13 patients less than 20 years of age. Because of the progressive nature of testicular failure which occurs after the onset of puberty, surgical sperm retrieval is less successful with advancing age. T therapy in the hypogonadal patient will enhance secondary sexual characteristics and sexual performance, increase muscle mass, prevent or help ameliorate osteopenia/osteoporosis and possibly prevent the development of gynecomastia. If T deficiency is evident in adolescence, therapy with T enanthate in oil at a dose of 50 mg intramuscularly monthly may be initiated. Alternatively, testosterone by patch or gel may be used. The plasma concentration of T can be assessed midway between injections, and the doses gradually increased to attain a level in the mid-normal range for stage of development and/or age.

In the adult patient, transdermal T by patch or gel can be used for replacement therapy. T levels can be assessed 1 week after the start of therapy and adjusted to maintain levels in the mid-normal range for age. Patients should be informed of the possibility of deep vein thrombosis and the T discontinued if the hematocrit rises above 54% because of the risk of hypercoagulability associated with elevated hematocrit levels. If gynecomastia is present and is not ameliorated by T therapy, surgical reduction is required if it is psychologically disturbing to the patient.

Early diagnosis with remedial therapy for speech and behavior problems, counseling for the patient and parents, as well as support group services appears to improve the well being and overall quality of life in patients diagnosed with Klinefelter syndrome.

Variants of Chromatin-Positive Seminiferous Tubule Dysgenesis

Variants of Klinefelter Syndrome

Variants of Klinefelter syndrome include 46,XY/47,XXY mosaics as well as patients with multiple X and Y chromosomes. With increasing numbers of X chromosomes in the genome, both mental retardation and other developmental anomalies such as radioulnar synostosis become prevalent.

46,XX Males (46,XX Testicular DSD)

Phenotypic males with a 46,XX karyotype have been described since 1964; the incidence of 46,XX males is approximately 1:20,000 births. In general, these individuals have a male phenotype, male psychosocial gender identity, and testes with histologic features similar to those observed in patients with a 47,XXY karyotype. At least 10% of patients have hypospadias or ambiguous external genitalia, primarily those patients who are SRY negative. 46,XX males have normal body proportions and a mean final height that is shorter than that of patients with a 47,XXY sex chromosome constitution or normal males but taller than that of normal females. There is a higher incidence of undescended testes in 46,XX males than in 47,XXY patients. As in 47,XXY individuals, T levels are low or low-normal, gonadotropins are elevated, and spermatogenesis is impaired postpubertally. Gynecomastia is more frequent than that observed in 47,XXY males.

Males with a 46,XX karyotype have acquired one X chromosome from each of their parents. Approximately 90% of XX males have a Y chromosome–specific DNA segment from the distal portion of the Y short arm translocated to the distal portion of the short arm of the paternal X chromosome. This translocated segment is variable in length but always includes the SRY gene, which encodes testis-determining factor as well as the pseudoautosomal region of the Y chromosome. Thus, in at least 90% of XX males, an abnormal X-Y terminal exchange during paternal meiosis has resulted in two products: an X chromosome with an SRY gene and a Y chromosome deficient in this gene (the latter would result in a female with XY gonadal dysgenesis). Fewer than 10% of XX males tested have lacked Y chromosome–specific DNA sequences (including the SRY gene) and the pseudoautosomal region of the Y chromosome. These XX, SRY-negative males tend to have hypospadias and may have relatives with ovotesticular DSD.

The finding of XX males who lack any evidence of Y chromosome–specific genes suggests that testicular determination—and, thus, male differentiation—can occur in the absence of a gene or genes from the Y chromosome. This could be a result of (1) mutation (upregulation) or duplication of a downstream autosomal gene involved in male sex determination (eg, SOX9 which if duplicated in a 46,XX individual causes sex reversal both in mice and humans); or (2) mutation, deletion, or aberrant inactivation of a gene sequence, which causes repression of testis determination. Two families with multiple 46,XX phenotypically normal males who had palmoplantar hyperkeratosis and a predisposition to squamous cell carcinomas have been reported. All affected males had a homozygous deletion of 2700 base pairs of the R-spondin1 gene. RSPO1, an ovary-determining gene, has no role in testes determination as evidenced by the description of a 46,XY male with a homozygous mutation of RSPO1 who had normal testicular function; or (3) circumscribed Y chromosome mosaicism (eg, occurring only in the gonads); (4) A 46,XX SRY negative male has recently been described with a mutation in SOX3. SOX3 is a member of the SRY HMG box family of genes and is found on the X chromosome at Xp26-27. This mutation is postulated to cause aberrant expression of SOX3 in gonadal primordia and thereby upregulating SOX9 expression resulting in testicular determination.

Turner Syndrome: 45,X Gonadal Dysgenesis

One in 2000 to 2500 newborn females have an absence of a second sex chromosome (either an X or Y), a structural rearrangement of the X chromosome, and/or mosaicism. Patients with a 45,X karyotype represent approximately 50% of all patients with X chromosome abnormalities. It has been estimated that 99% of 45,X fetuses do not survive beyond 28 weeks of gestation probably related to congenital heart anomalies, and 15% of all first-trimester abortuses have a 45,X karyotype. In about 70% to 80% of 45,X individuals, the X chromosome is of maternal origin.

The cardinal features of 45,X gonadal dysgenesis are a variety of somatic anomalies, sexual infantilism at puberty secondary to gonadal dysgenesis, and short stature. Patients with a 45,X karyotype can be recognized in infancy, usually because of lymphedema of the extremities and loose skin folds over the nape of the neck. In later life, the typical patient is often recognizable by her distinctive facies in which micrognathia, epicanthal folds, prominent low-set ears, a fish-like mouth, and ptosis are present to varying degrees. The chest is shield-like, and the neck is short, broad, and webbed (40% of patients). Additional anomalies associated with Turner syndrome include renal abnormalities (30%), pigmented nevi, cubitus valgus, congenital hip subluxation, a tendency to keloid formation, lymphedema of the lower extremities or puffiness of the dorsum of the hands and feet, short fourth metacarpals and metatarsals, Madelung deformity of the wrist, scoliosis, and recurrent otitis media, which may lead to conductive hearing loss. The high prevalence of otitis media is a consequence of an abnormal relationship between the Eustachian tube and the middle ear. Sensorineural hearing loss increases with age and is present in 61% of women with Turner syndrome over 35 years of age. All patients should have routine periodic auditory screening.

Patients with Turner syndrome have an increased prevalence of cardiovascular anomalies. The incidence of bicuspid aortic valves diagnosed by echocardiography has varied between 9% and 34%. A bicuspid aortic valve is a risk factor for subacute bacterial endocarditis. With age there is an increased tendency for a stenotic and/or insufficient aortic valve. Coarctation of the aorta is present in 10% of patients, especially individuals with a webbed neck. Bicuspid aortic valves, coarctation, aortic dilation, and hypertension are critical risk factors for aneurysm formation and death due to aortic rupture. Thus, all patients with Turner syndrome should have a complete cardiac evaluation by a cardiologist at diagnosis as well as periodic follow-up echocardiography or magnetic resonance imaging (MRI) as necessary to monitor aortic diameter and cardiovascular status. Routine intravenous urography or renal sonography is indicated to detect a surgically correctable renal abnormality. The most common renal anomalies are a horseshoe kidney, duplication of the renal pelvis and ureter, and hydronephrosis secondary to uteropelvic obstruction. Complete absence of a kidney and gross renal ectopia have been reported. The internal ducts as well as the external genitalia of these patients are invariably female except in rare patients with a 45,X karyotype, in whom a Y-to-autosome or Y-to-X chromosome translocation or cryptic mosaicism for a Y-containing cell line has been found.

Short stature is an invariable feature of the syndrome of gonadal dysgenesis. Mean final height in 45,X patients is 143 cm, with a range of 133 to 153 cm. Short stature found in patients with the syndrome of gonadal dysgenesis is not due to a deficiency of growth hormone (GH), insulin-like growth factor I (IGF-I), sex steroids, or thyroid hormone. It is related, at least in part, to haploinsufficiency of the SHOX gene in the pseudoautosomal region of the X and Y chromosome (see earlier sections). The administration of high-dose recombinant human GH leads to an increase in final height.

Gonadal dysgenesis is a cardinal feature of patients with a 45,X chromosome constitution. The gonads at adolescence are typically streak-like and usually contain only fibrous stroma arranged in whorls. Longitudinal studies of both basal and gonadotropin-releasing hormone (GnRH)-evoked gonadotropin secretion in patients with gonadal dysgenesis indicate a lack of feedback inhibition of the hypothalamic-pituitary axis by the dysgenetic gonads in affected infants and adolescents (Figure 14–12). Plasma and urinary gonadotropin levels, particularly FSH levels, are high during early infancy and after 9 to 10 years of age. Because ovarian function is impaired, puberty does not usually ensue spontaneously; sexual infantilism is a hallmark of this syndrome. Rarely, patients with a 45,X karyotype may undergo spontaneous pubertal maturation, menarche, and even pregnancy. It has been suggested that measurable levels of inhibin B may indicate the presence of functional follicles and the possibility of spontaneous pubertal development while low levels of AMH might herald ovarian failure. A variety of other disorders are associated with this syndrome, including obesity, nonosteoporotic fractures in childhood and osteoporotic fractures in adulthood, congenital dislocation of the hip, type 2 diabetes mellitus, an increased number of melanocytic nevi but no increase in melanoma, orthodontic problems, Hashimoto thyroiditis, rheumatoid arthritis, inflammatory bowel disease, intestinal telangiectasia with bleeding, hypertension, coeliac disease, ischemic heart disease, stroke, and anorexia nervosa. Abnormal liver enzymes with elevation of transaminases, alkaline phosphatase, and gamma-glutaryl transferase have been reported in more than 50% of adults. Most patients with Turner syndrome have normal intelligence. Only a small percentage of patients have significant developmental delay. However, 70% have learning disabilities involving spatial relationships, nonverbal problem-solving, and attention. Early testing and remediation is recommended, as well as psychologic support.

Diagnosis: Phenotypic females with the following features should have a karyotype analysis: (1) short stature (>2.5 standard deviations [SDs] below the mean value for age) with or without somatic anomalies associated with the syndrome of gonadal dysgenesis; and (2) delayed adolescence with an increased concentration of plasma FSH; (3) patients with clinical findings suggestive of Turner syndrome (eg, lymphedema of hands or feet, left-sided cardiac anomalies, characteristic facies, short fourth metacarpals, etc).

The initial evaluation of a patient with Turner syndrome should include karyotype analysis as well as FISH of 200 cells to rule out mosaicism or the presence of a Y chromosome in individuals with a 45,X karyotype and in those in whom a small nonidentifiable marker chromosome is present. A renal ultrasound is indicated to rule out surgically correctable anomalies. A cardiac evaluation with echocardiogram or MRI should be done to evaluate aortic size and rule out coarctation of the aorta and an anomaly of the aortic valve. Besides routine chemistries, gonadotropins, AMH (provides an estimate of the number of antral follicles), and inhibin B should be assessed in order to determine the status of the gonads. Periodic assessment of the cardiac status, especially in those with bicuspid aortic valves and/or hypertension, is indicated throughout the lifetime of the patient. Other systems which need periodic evaluation include: (1) thyroid function studies, (2) assessment of tissue transglutaminase activity for celiac disease, (3) hearing evaluation, (4) orthodontics, and (5) psychosocial and school performance issues. In view of the increased incidence of specific learning defects involving spatial relationships, executive skills, and so forth, it is prudent to evaluate these functions at 4 to 5 years of age so that appropriate remedial therapy can be undertaken as early as possible if warranted.

At adolescence, in addition to the continuing above assessments, lipids, fasting glucose and insulin, and liver function should be assessed. Psychosocial and educational issues should continue to be addressed with the patient and the parents.

Treatment: Maximizing growth and hormone replacement at puberty in order to engender an orderly progressive development of secondary characteristics at a time concordant with peers are two of the main goals of therapy in patients with Turner syndrome.

Individuals with Turner syndrome treated with recombinant GH (0.375 mg/kg/wk divided into seven once-daily doses (maximum dose 15-18 mg/wk), with or without oxandrolone (0.0625 mg/kg/d by mouth), have an increase in the rate of growth that is sustained and results in a mean increase in height of 7 to 10 cm after 3 to 7 years of therapy. The serum IGF-1 level should be followed and the dose titered to maintain the IGF-1 level below the upper limit of normal for age. Beginning GH therapy earlier (as soon as growth failure is evident) in childhood allows for a longer duration of therapy which results in a greater gain in height and fosters initiation of estrogen replacement at an age commensurate with normal puberty. Before initiation of GH therapy, a thorough analysis of the costs, benefits, and possible side-effects must be discussed with the parents and the child. Significant side effects include: (1) the accelerated onset of diabetes mellitus in the prediabetic, (2) pseudotumor cerebri, (3) slipped femoral epiphysis, and (4) unknown long-term side effects. The influence of the gain in height on any of the quality of life parameters in individuals with Turner syndrome treated with GH has been questioned by some. There is no synergistic effect of combined estrogen replacement and GH therapy on final height in this disorder. However, in a recent study, percutaneous estrogen administration for pubertal induction was associated with a 2.1 cm greater adult height than oral estrogen administration. These data suggest that, as observed in adult women, oral estrogens decrease the plasma concentrations of IGF-1 and suppress IGF-1's metabolic effects. In patients who have been treated with GH and have achieved an acceptable height and in those who have refused GH therapy and have evidence of gonadal dysgenesis, estrogen replacement therapy is usually initiated after 11 to 13 years of age.

Increasingly, transcutaneous estrogen administration by patch or gel, which avoids first pass through the liver, is the treatment of choice to induce female secondary sexual characteristics in hypogonadal girls. In a study of 15 patients, an estradiol patch was placed nocturnally for 12 hours to simulate the diurnal pattern of estradiol secretion in early puberty. By cutting the 25-μg patch (Vivelle-Dot) when necessary and measuring serum estradiol concentrations, it was concluded that 0.08 to 0.12 μg/kg of body weight was an appropriate starting dose of estradiol transcutaneously. Plasma concentrations of estradiol were measured in the morning and maintained in a range appropriate for Tanner stage by adjusting the dose. The starting dose was usually maintained for 9 months before increasing the dose. The aim was to achieve midpuberty estradiol levels by 15 months. A progestin was added to the estradiol within 2 years after the start of estradiol.

Conjugated estrogens (0.3 mg or less) or ethinyl estradiol (3-5 μg) can be given orally for the first 21 days of the calendar month. Thereafter, the dose of estrogen is gradually increased over the next several years to 1.25 mg of conjugated estrogens or 10 μg of ethinyl estradiol daily for the first 21 days of the month. The minimum dose of estrogen necessary to initiate and maximize uterine development, secondary sexual characteristics, and menses, as well as to prevent osteoporosis should be administered. After the first 18 to 24 months of estrogen therapy, medroxyprogesterone acetate (5 mg) or a comparable progestin is given on the tenth to twenty-first days of the month to ensure physiologic menses and to reduce the risk of endometrial carcinoma, which is associated with unopposed estrogen stimulation.

We recommend a transition program when transferring adolescent or young adult patients to an adult caregiver. Ideally, this program will introduce the patient to the physicians who will be involved in her future care which may include an internist, endocrinologist, reproductive endocrinologist, ENT specialist, adult cardiologist, and psychologist.

Many of the morbidities that contribute to the decrease in lifespan are detectable with comprehensive medical surveillance and are amenable to medical and/or surgical therapy. Protocols for the management of the adult Turner syndrome patient have been published (see Gravholt et al; Bondy et al; Conway in reference list at the end of this chapter). Individuals with Turner syndrome by nature are very maternal. They should be reassured about their sexual function and informed about their potential for maternity with donor eggs and in vitro fertilization in an age-appropriate manner. The success rate of ovum donation is 40% per treatment cycle, and only 50% of pregnancies achieve a live birth due to a high miscarriage rate. In one study, 38% of patients had pregnancy associated hypertensive problems. There is an increased risk of aortic dissections during pregnancy. Thus, all women with Turner syndrome who desire a pregnancy should have a careful, complete cardiac evaluation prior to egg implantation and close continued monitoring if pregnancy ensues. We recommend caesarian delivery to avoid the increased stress to the cardiovascular system associated with labor and because of the risk of cephalopelvic dystocia in these women.

X Chromatin–Positive Variants of the Syndrome of Gonadal Dysgenesis

Patients with structural abnormalities of the X chromosome (deletions and additions) and sex chromosome mosaicism with a 45,X cell line may manifest the somatic as well as the gonadal features of the syndrome of gonadal dysgenesis . Evidence suggests that genes on both the long and short arms of the X chromosome control gonadal differentiation, whereas genes primarily on the short arms of the X prevent the short stature and somatic anomalies that are seen in 45,X patients (see Figure 14–2). In general, 45,X/46,XX mosaicism modifies the 45,X phenotype toward normal and can even result in normal gonadal function and fertility. Patients with duplication of the long arms of the X chromosome and deletion of the short arm—the so-called Xq isochromosome—appear to have an increased prevalence of autoimmune thyroiditis, type 2 diabetes, and inflammatory bowel disease in comparison to those with a 45,X karyotype. Patients with a 46,XrX (ring) cell line in their karyotype may manifest mental retardation and congenital anomalies not usually associated with Turner syndrome. These abnormalities are related to lack of inactivation of small ring X chromosomes and, hence, functional disomy for genes on the ring X chromosome and the normal X chromosome.

X Chromatin–Negative Variants of the Syndrome of Gonadal Dysgenesis

These patients usually have mosaicism with a 45,X and a Y-bearing cell line—45,X/46,XY; 45,X/47,XXY; 45,X/46,XY/47,XYY—or perhaps a structurally abnormal Y chromosome. They range from phenotypic females with the features of Turner syndrome to patients with ambiguous genitalia to completely normal-appearing males with few stigmas of Turner syndrome and short stature. The variations in gonadal differentiation range from bilateral streaks to bilateral dysgenetic testes to apparently normal testes, and there may be asymmetric development (eg, a streak on one side and a dysgenetic testis or, rarely, a normal testis on the other side)—sometimes called mixed gonadal dysgenesis. The development of the external genitalia and of the internal ducts correlates with the degree of testicular differentiation and the capacity of the fetal testes to secrete AMH and T.

The risk of gonadal tumors is greatly increased in patients with 45,X/46,XY mosaicism and streak or dysgenetic gonads; hence, prophylactic removal of streak gonads or dysgenetic undescended testes in this syndrome is indicated. Breast development at or after the age of puberty in these patients is commonly associated with a gonadal neoplasm, usually a gonadoblastoma. Pelvic sonography, computed tomography (CT), or MRI may be useful in screening for neoplasms in these patients. Gonadoblastomas are calcified and so may be visible even on a plain film of the abdomen.

Diagnosis: The diagnosis of 45,X/46,XY mosaicism can be established by the demonstration of both 45,X and 46,XY cells in blood, skin, or gonadal tissue. In some mosaics, a marker chromosome is found that is cytogenetically indistinguishable as X or Y. In these cases, either FISH or molecular analyses with X- and Y-specific probes is indicated to definitively determine the origin of the marker chromosome, because gonadoblastomas have been reported in patients with deleted Y chromosomes—even those with deletions of the SRY gene. Ninety percent of patients with 45,X/46,XY mosaicism ascertained by amniocentesis have normal male genitalia and normal testicular histology. Thus, the ambiguity of the genitalia invariably described in patients with 45,X/46,XY mosaicism is due to ascertainment bias. We have observed a short 30-year-old male with documented 45,X/46,XY mosaicism who has normal male genitalia and is fertile.

Treatment: The decision regarding the sex of rearing in this variant of the syndrome of gonadal dysgenesis should be based on the age at diagnosis and the potential for normal function of the external genitalia. In phenotypic females with 45,X/46,XY mosaicism assigned a female gender role, the dysgenetic gonads should be removed because of the risk of malignancy. Estrogen therapy should be initiated at the age of puberty, as in patients with a 45,X karyotype (see earlier). In affected infants who are assigned a male gender role, all gonadal tissue except that which appears functionally and histologically normal and is in the scrotum should be removed. An excellent phallic response to T can be anticipated. Repair of hypospadias is also indicated. At puberty, depending on the functional integrity of the retained gonads, T replacement therapy may be indicated in doses similar to those prescribed for patients with the incomplete form of XY gonadal dysgenesis. In patients with retained scrotal testes, frequent clinical examinations and ultrasonography is indicated. A gonadal biopsy postpubertally to rule out the possibility of carcinoma in situ (CIS), a premalignant lesion, should be performed. If CIS is present, low-dose radiation therapy or gonadectomy after sperm retrieval is indicated.

In infants and children with 45,X/46,XY mosaicism who have normal genitalia and normal testicular integrity as assessed by AMH, inhibin B, gonadotropin levels, and ultrasonography, the gonads should be retained and followed closely both hormonally and by ultrasound. In those 45,X/46,XY individuals with presumably "normal" testes, a gonadal biopsy should be performed, post-pubertally. If biopsy and sonography show no evidence of CIS, a second biopsy at 20 years of age is recommended. The risk of gonadal malignancies in males with 45,X/46,XY mosaicism, who have normal male genitalia and histologically and functionally normal testes in the scrotum, remains to be ascertained, although we hypothesize it may be no higher than that seen in 46,XY males.

Dysgenetic 46,XX and 46,XY DSD (46,XX and 46,XY Gonadal Dysgenesis)

The terms complete XX and XY gonadal dysgenesis have been applied to 46,XX or 46,XY patients who have bilateral streak gonads, a female phenotype, and no somatic stigmas of Turner syndrome. After the age of puberty, these patients exhibit sexual infantilism, castrate levels of plasma and urinary gonadotropins, normal or tall stature, and eunuchoid proportions.

Dysgenetic 46,XX DSD (46,XX Gonadal Dysgenesis)

Normal ovarian determination is dependent on X chromosomal genes as well as autosomal genes. Unlike the testes, the ovary requires the presence of germ cells in order to develop and function. Therefore, mutations in genes on the autosomes and X chromosome that affect gonadal determination (eg, DMRT1 and SF-1) as well as those that affect germ cell differentiation and development, migration into the gonadal ridge, and maturation into oocytes can cause XX gonadal dysgenesis. Expansion of the CAG repeat region of the fragile X locus can cause accelerated oocyte depletion and premature ovarian failure. Haploinsufficiency of FOXL2, a geneon chromosome 3q23, is a cause of autosomal dominantblepharophimosis-ptosis-epicanthus inversus syndrome (BPES type 1) and XX gonadal dysgenesis. A woman with a FOXL2 mutation, but without the BPES phenotype, has been described. Recently, SF-1 mutations in members of four families as well as 2/25 subjects with isolated ovarian insufficiency have extended the spectrum of phenotypes associated with SF-1 mutations. A multiplicity of genes involved in meiosis, DNA repair and folliculogenesis have recently been implicated as a cause of ovarian dysgenesis.

Familial and sporadic cases of XX gonadal dysgenesis are reported with an estimated incidence as high as 1:8300 females in Finland. Pedigree analysis of familial cases is consistent with autosomal recessive inheritance. Analysis of familial cases in Finland linked a locus on chromosome 2p to XX gonadal dysgenesis in females; the gene for the FSH receptor resides on chromosome 2p. A mutation in exon 7 of the gene encoding the FSH receptor segregates with XX gonadal dysgenesis. This mutation is in the extracellular ligand-binding domain of the FSH receptor and reduces the binding capacity and, consequently, signal transduction of the receptor. At puberty, this resulted in variable ovarian function, including streak ovaries and hypergonadotropic hypogonadism in some girls. Several studies of females with 46,XX gonadal dysgenesis in Western Europe and the United States were negative for FSH receptor gene mutations, suggesting that a mutation in this gene is a rare cause of XX gonadal dysgenesis outside of Finland. Males homozygous for this mutation are phenotypically normal, with spermatogenesis varying from normal to absent.

Studies of familial cohorts have supported heterogeneity in the pathogenesis of this syndrome. Siblings, one with a 46,XX karyotype and the other with a 46,XY karyotype, both with gonadal agenesis, have been reported, supporting the involvement of an autosomal gene in this family. However, in view of the normal phenotype in XY males observed with a mutation in the FSH receptor, it seems unlikely that these patients have an FSH receptor defect. In one family, four affected women had an inherited interstitial deletion of the long arm of the X chromosome involving the q21-q27 region (see Figure 14–2). This region apparently contains a gene or genes critical to ovarian development and function. In several affected families, XX gonadal dysgenesis was associated with deafness of the sensorineural type as well as other neurologic dysfunction.

Diagnosis: 46,XX gonadal dysgenesis should be suspected in phenotypic females with sexual infantilism and normal Müllerian structures who lack the somatic stigmas of the syndrome of gonadal dysgenesis (Turner syndrome). Karyotype analysis reveals only 46,XX cells. As in Turner syndrome, gonadotropin levels are high, inhibin A and B and AMH levels as well as estrogen levels are low. Ultrasound or MRI reveals a streak or dysgenetic ovary, and treatment consists of cyclic estrogen and progesterone replacement as previously discussed for patients with Turner syndrome.

Sporadic cases of XX gonadal dysgenesis, similar to familial cases, may represent a heterogeneous group of patients from a pathogenetic point of view. XX gonadal dysgenesis should be distinguished from ovarian failure due to radiation and chemotherapy for childhood malignancies, infections such as mumps, antibodies to gonadotropin receptors, biologically inactive FSH, gonadotropin-insensitive ovaries, and galactosemia.

46,XY DSD (Complete or Partial Gonadal Dysgenesis)

46,XY gonadal dysgenesis occurs both sporadically and in familial aggregates. Patients with the complete form of this syndrome have female external genitalia, normal or tall stature, bilateral streak gonads, Müllerian duct development, sexual infantilism, eunuchoid habitus, and a 46,XY karyotype. Clitoromegaly is common and, in familial cases as well as sporadic cases, a continuum of involvement ranging from the complete syndrome to ambiguity of the external genitalia has been described. The phenotypic difference between the complete and incomplete forms of XY gonadal dysgenesis is related to the degree of differentiation of testicular tissue and the functional capacity of the fetal testis to produce T and AMH. Early in infancy and after the age of puberty, plasma gonadotropin levels are markedly elevated.

Analyses of familial and sporadic cases of 46,XY gonadal dysgenesis indicate that about 15% to 20% of patients have a mutation of the SRY gene that affects DNA binding or bending by the SRY protein or a mutation in the calmodulin or importin domains of the SRY gene that affects the nuclear localization signals. Most patients in whom mutations have been detected have had complete gonadal dysgenesis. Patients with deletions of the short arm of the Y chromosome may have, in addition to gonadal dysgenesis, stigmas of Turner syndrome. A mutation in the HMG box of the SRY gene has been described in normal 46,XY fathers of daughters who had 46,XY gonadal dysgenesis. These familial cohorts suggest the possibility of germ cell mosaicism in the father or that a mutation or modifier genes may affect either the level or the timing of SRY expression and in this manner result in either normal or abnormal testicular differentiation. Mutations outside the HMG box region of the SRY gene or in X-linked or autosomal genes involved in testicular determination and function may be responsible for those individuals in whom no molecular abnormality in the SRY gene has been found. Three of six 46,XY patients with complete gonadal dysgenesis in one study had homozygous mutations of the gene encoding DHH (Desert Hedgehog).

A number of patients with 46,XY gonadal dysgenesis has been reported with a duplication of the Xp21.2 → p22.11 region of the X chromosome. This region contains the DAX1 gene. Deletion or mutation of DAX1 in males causes adrenal hypoplasia congenita and hypogonadotropic hypogonadism. The finding that 46,XY males with adrenal hypoplasia and hypogonadotropic hypogonadism have normal sex differentiation suggests that DAX1 is not required for testicular differentiation in the human; duplication of DAX1, however, impairs testis differentiation possibly by upregulating B-catenin/WNT4, which downregulates SOX9 preventing testicular development.

XY gonadal dysgenesis associated with campomelic dysplasia is due to a mutation of one allele of an SRY-related gene, SOX9 on chromosome 17. Sometimes the mutation occurs in upstream regulatory elements of the gene. In addition, XY gonadal dysgenesis has been associated with 9p–(DMRT1) and 10q–deletions, mutations in, as well as duplication of, 1p31-35 (WNT4).

Steroidogenic factor-1 (SF-1) is a transcriptional regulator of adrenal and gonadal development, hormone synthesis, and functions with other genes in the sex determination and differentiation cascade. The phenotype is variable and depends on whether the individual has a heterozygous or homozygous mutation as well as the functional deficiency in SF-1 activity as a result of the specific mutation. The phenotype is also most likely affected by background genetic factors and cofactors. Mutations in the gene encoding SF-1 are associated with 46,XY DSD. Similar to the mouse knockout, the severely affected XY individual presents with gonadal streaks with female sex differentiation and neonatal onset adrenal insufficiency. Recently, 46,XY patients with ambiguous genitalia (and in one case, female external genitalia) with or without Müllerian ducts and normal adrenal function have been reported, which suggests that adrenal development and function are less sensitive to SF-1 dosage levels than gonadal development and function. The phenotypic spectrum of SF-1 deficiency includes 46,XY males with micropenis and in one case normal male external genitalia and anorchia as well as infertile males and 46,XX females with primary or secondary ovarian failure. The phenotypic spectrum was recently expanded to include a 46,XY newborn with hypospadias and micropenis who virilized normally at puberty but had evidence of progressive Sertoli cell deficiency as evidenced by elevated FSH levels and low inhibin B levels. Thus, as with other genes in the sex determination cascade, gene dosage and cofactors play a critical role in phenotypic expression. Both heterozygous and homozygous mutations in SF-1 have been described in 46,XX females with isolated primary ovarian insufficiency.

Mutations and deletions of the Wilms tumor repressor gene WT1 are a cause of XY gonadal dysgenesis and renal disease (eg, Denys-Drash syndrome, Frasier syndrome, and the WAGR syndrome). Denys-Drash syndrome is due to a mutation in a coding exon of the WT1 gene. These mutations act in a dominant-negative fashion to produce the Denys-Drash phenotype, which is characterized by testicular dysgenesis, early renal failure due to mesangial sclerosis, and a predisposition to the development of Wilms tumor. Müllerian ducts may be present or absent depending on the degree of Sertoli cell dysfunction. The external genitalia are undervirilized. Frasier syndrome is due to a mutation in the donor splice site of exon 9, resulting in loss of three amino acids—lysine, threonine, and serine (KTS), thus changing the ratio of KTS+ to KTS− isoforms of this protein. The resultant phenotype is characterized by streak gonads, the presence of Müllerian structures, female external genitalia, late-onset nephropathy due to segmental glomerulosclerosis, and an increased risk of gonadoblastoma. WAGR syndrome is due to a deletion of a segment of 11p13, which contains the WT1 gene and the PAX6 gene causing haploinsufficiency of these genes. The resultant phenotype includes Wilms tumor, aniridia genitourinary abnormalities, and mental retardation.

A 46,XY patient with normal female genitalia and Müllerian structures had a homozygous mutation of CBX2—the human homologue of m33, a gene that when mutated causes sex reversal in mice. This gene is postulated to be upstream of SRY in the ever-expanding gene cascade of sex development. The patient had elevated FSH levels in infancy and the presence of "bilateral ovaries" with sparse primordial follicles on biopsy at 4½ years of age suggesting gonadal dysgenesis.

Mutations in the Map3k1 have been recently demonstrated to cause sex reversal in mice as well as 46,XY humans. Recently, mutations in this gene have been described in patients with partial and complete gonadal dysgenesis. In two families, the mutation was inherited as a sex limited autosomal dominant. Interestingly, in these families the mutation resulted in activation of the MAPK pathway as opposed to inactivation as observed in the mouse and in a 46,XY fertile female. A resolution to this apparent discrepancy in action is still to be clarified.

Therapy: Therapy for phenotypic females with 46,XY gonadal dysgenesis involves prophylactic gonadectomy at diagnosis, due to the high risk of malignancy in these gonads, and estrogen substitution at puberty. In the incomplete form of XY gonadal dysgenesis, assignment of a male gender is possible and treatment with testosterone to augment phallic size in infancy is recommended. Prophylactic gonadectomy must be considered, because fertility is unlikely (gonadotropins are invariably elevated, and AMH and inhibin B levels are low or unmeasurable). In addition, there is an increased risk of malignant transformation of the dysgenetic gonads in these patients, especially those who are SRY-positive and have intra-abdominal dysgenetic gonads and those with Frasier and Denys-Drash syndrome. If a gonad is preserved in the scrotum because it is histologically normal and hormonally functional, it should be followed closely by physical examination and ultrasound and biopsied postpubertally to rule out CIS, a premalignant lesion. In affected individuals raised as males, we recommend that prosthetic testes be implanted at the time of gonadectomy and androgen substitution therapy instituted at the age of puberty. Testosterone enanthate in oil (or another long-acting T ester) is used, beginning with 50 mg intramuscularly every 4 weeks and gradually increasing after a bone age of 14½ years to a full replacement dose of 200 mg intramuscularly every 2 weeks. Alternatively T patches or gels may be used.

In these individuals, both ovarian and testicular tissues are present in one or both gonads. Differentiation of the internal and external genitalia is highly variable. The external genitalia may simulate those of a male or female, but most often they are atypical. Cryptorchidism and hypospadias are common. A testis or ovotestis, if present, is located in the labioscrotal folds in one-third of patients, in the inguinal canal in one-third, and in the abdomen in the remainder. A uterus is usually present, although it may be hypoplastic or unicornuate. The differentiation of the genital ducts usually follows that of the ipsilateral gonad. The ovotestis is the most common gonad found in these individuals (60%), followed by an ovary and, least commonly, by a testis. At puberty, breast development is usual in untreated patients, and menses occur in over 50% of cases. Whereas the ovary or the ovarian portion of an ovotestis may function normally, the testis or testicular portion of an ovotestis is invariably dysgenetic and should be removed in 46,XX patients raised as females.

Sixty percent have a 46,XX karyotype, 20% are 46,XY, and about 20% have sex chromosome mosaicism or 46,XX/46,XY chimerism. Those with a 46,XX karyotype are genetically heterogeneous. Only a small proportion, including some in family cohorts with 46,XX males, are SRY positive. In these individuals, Y-to-X and Y-to-autosome translocations, hidden sex chromosome mosaicism, or chimerism may explain the pathogenesis. A number of families have been reported in which both SRY-negative 46,XX males and 46,XX ovotesticular DSDs occurred. This latter observation suggests a common genetic pathogenesis in these patients. Possible genetic mechanisms to explain SRY-negative individuals include (1) mutation of a downstream autosomal gene or modifier gene involved in testicular determination; (2) mutation, deletion, or anomalous inactivation of an X- or autosome-linked locus involved in repression of testis determination. A 46,XX ovotesticular DSD with palmoplantar keratoderma, congenital bilateral corneal opacification, onychodystrophy, and hearing impairment has been reported. This patient had a homozygous splice site mutation in the RSPO1 gene. This is the first demonstration of a mutation in a single gene (in the ovarian determining pathway) causing ovotesticular DSD; or (3) circumscribed chimerism or mosaicism that occurred only in the gonads. Studies of eight ovotestes from 46,XX individuals detected low levels of the SRY gene. Immunohistochemistry has identified SRY protein localized predominantly in Sertoli and germ cells.

Diagnosis: Ovotesticular DSD should be considered in all patients with ambiguous genitalia. The finding of a 46,XX/46,XY karyotype or a bilobulate gonad compatible with an ovotestis in the inguinal region or labioscrotal folds suggests the diagnosis. Basal plasma T levels are elevated above 40 ng/dL in affected patients under 6 months of age, and T levels increase after hCG stimulation. The estradiol and inhibin A response to human menopausal gonadotropins or recombinant human FSH is a reliable test for the presence of ovarian tissue. This test, when coupled with the hCG-induced rise in T, differentiates infants with both ovarian and testicular tissue from those with other disorders of sexual differentiation.

If all other forms of 46,XY and 46,XX DSD have been excluded and hormonal studies are nondiagnostic, laparotomy and histologic confirmation of both ovarian and testicular tissue establish the diagnosis. The management of these individuals is contingent on both the age at diagnosis and of a careful assessment of the functional capacity of the gonads, the genital duct differentiation, and the appearance of the external genitalia. In general, 46,XX individuals are raised as females, with the possible exception of the well-virilized patient in whom no uterus is found. Preservation of the ovarian tissue and removal of all testicular tissue are sometimes possible in patients who have an ovary on one side or an ovotestis with distinct margins of separation (bilobate). Fertility has been reported in 46,XX individuals with functional ovarian gonadal tissue.

Gonadal Neoplasms in Dysgenetic Gonads

Gonadal tumors are rare in patients with 47,XXY Klinefelter syndrome and 45,X gonadal dysgenesis. However, the prevalence of gonadal neoplasms is greatly increased in patients with 46,XY DSDs with dysgenetic testes (Table 14–2). The frequency is increased in 45,X/46,XY mosaics who have female or ambiguous genitalia; in those with a structurally abnormal Y chromosome; and in those with XY gonadal dysgenesis, either with a female phenotype or with ambiguous genitalia (eg, Frasier [60%], Denys-Drash syndrome [40%]) (see Table 14–2). Gonadoblastomas, germinomas, seminomas, and teratomas are found most frequently. Prophylactic gonadectomy is indicated in these patients as well as in those with Turner syndrome who manifest signs of virilization, regardless of karyotype. The significance of hidden mosaicism for Y chromosomal DNA determined by polymerase chain reaction analysis in patients with 45,X Turner syndrome is controversial with respect to the risk of gonadal neoplasms. Registry studies in Denmark indicate that the incidence of gonadoblastoma in patients with Turner syndrome without a cytogenetically identified Y chromosome is rare. Thus, routine use of polymerase chain reaction studies of all patients with 45,X Turner syndrome is not indicated. Gonadoblastomas have been reported in patients with marker chromosomes of Y origin lacking the SRY gene. Thus, screening by FISH for Y chromosomal DNA in blood seems prudent in these patients, as well as other patients with Turner syndrome, as it enables one to count a large number of cells with a minimum of effort and time. The testis should be preserved in patients who are to be raised as males, only if it is histologically and functionally normal, and it is or can be situated in the scrotum. The fact that a testis is palpable in the scrotum does not preclude malignant degeneration and tumor dissemination, because seminomas tend to metastasize at an early stage before a mass is obvious.

Malignant testicular germ cell tumors including seminomas, embryonal carcinomas, teratomas, and yolk sac tumors appear to develop from CIS. These cells are thought to arise in utero from fetal gonocytes and to metamorphose with time in the dysgenetic environment into testicular malignancies. The incidence of CIS and seminomas is low in patients with complete androgen insensitivity but significantly higher in patients with partial androgen insensitivity with intra-abdominal gonads (see Table 14–2). Clues to the presence of CIS are the presence of an inhomogeneous testicular parenchyma and testicular microlithiasis, as well as a low inhibin B level. It has been suggested that immunohistochemistry for Oct 3/4 is mandatory when evaluating testicular biopsies for CIS, since Oct 3/4 is a gene that may be involved in germ cell tumors and is not normally expressed in postpubertal testes. An increased prevalence of low sperm counts, cryptorchidism, hypospadias, and testicular cancer has been reported in Denmark compared to Finland. This has been called the testicular dysgenesis syndrome and ascribed to both genetic and environmental factors such as exposure to endocrine disrupters.

Affected individuals have normal ovaries and Müllerian derivatives associated with ambiguous external genitalia. In the absence of testes, a female fetus is masculinized if subjected to increased circulating levels of androgens derived from a fetal or maternal source. The degree of masculinization depends on the stage of differentiation at the time of exposure (Figure 14–13). After 12 weeks of gestation, androgens produce only clitoral hypertrophy. Rarely, ambiguous genitalia that superficially resemble those produced by androgens are the result of other teratogenic factors.

Fetal Source: Congenital Adrenal Hyperplasia

(Figure 14–14, Table 14–3)

Congenital adrenal hyperplasia (CAH) is responsible for most cases of 46,XX DSD and about 50% of all cases of ambiguous genitalia. There are six major types of CAH, all transmitted as autosomal recessive disorders. The common denominator of all six types is a defect in the synthesis of cortisol that results in an increase in ACTH and consequently adrenal hyperplasia and the secretion of steroids synthesized prior to the enzymatic block. Both males and females can be affected, but males are rarely diagnosed at birth unless they have ambiguous genitalia, are salt-losers and manifest adrenal crises, are identified during newborn screening, or are known to be at risk because they have an affected sibling. Defects in 21-hydroxylation and 11β-hydroxylation are confined to the adrenal gland and produce virilization.

A new form of CAH has been described due to mutations in the gene encoding P450 oxidoreductase (POR), a single flavoprotein that donates electrons to all microsomal cytochrome P450 enzymes, including 17α-hydroxylase/17,20-lyase, 21-hydroxylase, and aromatase. The mothers of affected fetuses are at risk for virilization during pregnancy if aromatase activity is significantly affected by the mutation. 46,XX patients with this defect in adrenal and ovarian steroidogenesis have ambiguous genitalia, whereas those with a 46,XY karyotype have incomplete masculinization of the external genitalia.

P450c21 Hydroxylase Deficiency

21-Hydroxylase activity is mediated by P450c21, a microsomal cytochrome P450 enzyme that is expressed as an autosomal recessive. A deficiency of this enzyme results in the most common type of adrenal hyperplasia, with an overall prevalence of 1:14,000 live births in Caucasians. More than 90% of patients with CAH in North America and Europe have 21-hydroxylase deficiency. The locus for the gene that encodes 21-hydroxylation is on the short arm of chromosome 6, close to the locus for C4 (complement) between HLA-B and HLA-D. DNA analysis has detected two genes, designated P450c21A(CYP21p) and P450c21B (CYP21), in this region in tandem with the two genes for complement, C4A and C4B, as well as two genes Xa and Xb, which overlap one another on the opposite strands of DNA from P450c21A and P450c21B. Xa and Xb code for an extracellular matrix protein called Tenascin-X. P450c21A is a nonfunctional pseudogene (ie, it is missing critical structural sequences and does not encode a functional 21-hydroxylase). Seventy-five to eighty percent of patients with classic P450c21 deficiency have point mutations that change a portion of the P450c21B to a sequence similar to that in the nonfunctional P450c21A gene—hence a microgene conversion. Approximately 15% of severely affected 21-OH genes have a deletion extending from a position between exon 3 and exon 8 of the P450c21A pseudogene to a similar region of the 21-OHB gene, resulting in a nonfunctional fusion 21-OHA/21-OHB gene. This is the result of misalignment and unequal crossing over. The remainder have de novo point mutations and macrogene conversions.

Classic salt-wasting 21-hydroxylase deficiency is associated with point mutations, deletions, or gene conversions that abolish or severely reduce 21-hydroxylase activity. Most patients with 21-hydroxylase deficiency are compound heterozygotes (ie, they have a different genetic mutation on each of their P450c21B allelic genes) having inherited a mutant gene from each of their parents. A spontaneous mutation in the 21-hydroxylase gene occurs in 1% to 2% of patients.

The phenotypic spectrum—salt loss with severe virilization, simple virilization, or late onset of virilization—is a consequence of the degree of enzymatic deficiency. The phenotype is determined in patients with compound heterozygosity by the functionally less severely mutated P450c21B allele. In rare cases, there is variability between genotype and phenotype. The gene for P450c21 (21-hydroxylase) deficiency is not only closely linked to the HLA supergene complex, but certain specific HLA subtypes are found to be statistically increased in patients with 21-hydroxylase deficiency.

P450c21 Hydroxylase Deficiency with Virilization and Salt Loss

The salt-losing variant of P450c21 hydroxylase deficiency accounts for about 80% of patients with classic 21-hydroxylase deficiency and involves a severe reduction (98%) in P450c21-hydroxylase activity, leading to impaired secretion of both cortisol and aldosterone. This results in electrolyte and fluid losses after the fifth day of life and, as a consequence, hyponatremia, hyperkalemia, acidosis, dehydration, and vascular collapse. Rarely, this can occur as late as 6 to 12 weeks, usually associated with a concomitant physiologic stress. In the female fetus before 12 weeks' gestation, high fetal DHT levels result from increased T and DHEA-S secreted from the 21-hydroxylase-deficient adrenal gland which cannot synthesize cortisol when aromatase activity is relatively low. In addition, P450c21-21-hydroxylase deficiency results in an elevated level of 17OH progesterone which is not readily converted to androstenedione in humans but may be converted via a backdoor steroid pathway to DHT, a nonaromatizable androgen (see Figure 14–10). It has been suggested that DHT synthesis from elevated 17OH progesterone levels may contribute to the fetal masculinization of the external genitalia and the postnatal virilization of patients with P450c21-hydroxylase deficiency. Elevated DHT levels from 8 to 12 weeks of gestation results in varying degrees of labioscrotal fusion and clitoromegaly in affected 46,XX females. After 12 weeks, androgen receptor activity in the labioscrotal folds diminishes, and only clitoromegaly ensues from the elevated DHT levels.

Masculinization of the external genitalia of affected females who are salt-losers tends to be more severe than individuals with apparent non–salt-losing P450c21-hydroxylase deficiency. Affected males may have macrogenitosomia. Recently, patients with 21- hydroxylase and 11β-hydroxylase deficiency have been shown to have adrenomedullary hypofunction secondary to low intra-adrenal concentrations of cortisol and developmental defects in formation of the adrenal medulla. This defect in catecholamine synthesis may predispose these patients to hypoglycemia with stress in infancy.

P450c21 Hydroxylase Deficiency with Virilization Alone

The simple virilizing form of P450c21 (21-hydroxylase) deficiency results in impaired cortisol synthesis, increased ACTH levels, and increased adrenal androgen precursor and androgen secretion. It accounts for about 20% of individuals with classic P450c21 hydroxylase deficiency and is primarily due to missense mutations such as I172N, which retain enough P450c21-hydroxylase activity to produce sufficient aldosterone to prevent hyponatremia/hyperkalemia under normal circumstances. In general, affected females have a less severe degree of masculinization of their external genitalia than those with the salt-wasting form of P450c21 hydroxylase deficiency. In the male fetus, no abnormalities in the external genitalia are evident at birth, but the phallus may be enlarged. These patients produce sufficient amounts of aldosterone to prevent the signs and symptoms of mineralocorticoid deficiency except when they are salt-deprived. They may also manifest a defect in mineralocorticoid synthesis as evidenced by an elevated plasma renin level. Virilization continues after birth in untreated patients. This results in rapid growth and bone maturation as well as the physical signs of excess androgen secretion (eg, acne, seborrhea, increased muscular development, premature development of pubic or axillary hair, and phallic enlargement). True (central) precocious puberty can occur following initiation of glucocorticoid therapy in affected children with advanced bone ages.

Nonclassic P450c21 Hydroxylase Deficiency

Mild defects in P450c21 (21-hydroxylase) activity not causing a DSD have been reported. Patients can be symptomatic (late-onset or nonclassic) or asymptomatic (cryptic form). These mild forms of P450c21 hydroxylase deficiency are HLA-linked, as is classic P450c21 hydroxylase deficiency; however, they occur much more frequently than the classic form of the disease in certain ethnic populations. It has been postulated that nonclassic P450c21 hydroxylase deficiency is the most common autosomal recessive disorder. The frequency of nonclassic P450c21 hydroxylase deficiency is thought to be as high as 1 in 27 for Ashkenazi Jews, 1 in 53 for Hispanics, 1 in 333 for Italians, and 1 in 1000 for other Caucasians. The prevalence of heterozygosity for a nonclassic allele has varied from 1.2 to 6%. Affected patients with nonclassical 21- hydroxylase deficiency are either homozygous for a mild mutation (eg, V281L) or are compound heterozygotes with the other allele harboring a severe mutation (27%-76% of the time). Thus, it is prudent to genotype the partners of a nonclassic patient who is carrying a severe mutation, in order to offer genetic counseling and, if necessary, prenatal diagnosis. The clinical expression of nonclassical congenital adrenal hyperplasia is very variable in individuals with the same genotypes within families with some being asymptomatic (cryptic). Females with late-onset P450c21 hydroxylase deficiency have normal female genitalia at birth. Mild virilization occurs later in childhood and adolescence manifesting primarily as hirsutism. Other signs of excess androgen secretion, such as the premature development of pubic or axillary hair (premature pubarche), slight clitoral enlargement, advanced bone age, menstrual irregularities acne, polycystic ovary syndrome, decreased fertility, may also occur. Affected males have normal male genitalia at birth. Later in childhood, some become symptomatic and exhibit premature growth of pubic or axillary hair, and advancement of their bone age. Although they are tall as children, they end up short as adults due to advanced bone maturation and premature epiphysial fusion unless they are treated.

Diagnosis: The diagnosis of P450c21 hydroxylase deficiency should always be considered in the following: (1) patients with ambiguous genitalia who have a 46,XX karyotype with a uterus and ovaries on ultrasonography; (2) apparent cryptorchid males; (3) infants who present with shock, hypoglycemia, and serum chemistries compatible with adrenal insufficiency; and (4) males or females with signs of virilization before puberty, including premature pubarche. In the past, the diagnosis of P450c21 hydroxylase deficiency was based on the finding of elevated levels of 17-ketosteroids and pregnanetriol in the urine. Although urinary steroid determinations are still useful, they have been replaced by the simpler and more cost-effective measurement of plasma 17-hydroxyprogesterone, androstenedione, and T levels by liquid chromatography and tandem mass spectroscopy (LCMSMS).

The concentration of plasma 17-hydroxyprogesterone is elevated in umbilical cord blood but rapidly decreases into the range of 100 to 200 ng/dL (3-6 nmol/L) by 48 hours after delivery. In premature infants and in stressed full-term newborns, the levels of 17-hydroxyprogesterone are higher than those observed in nonstressed full-term infants. In patients with P450c21 hydroxylase deficiency, the 17-hydroxyprogesterone values usually are greater than 5000 ng/dL (150 nmol/L) on baseline samples drawn after 48 hours, depending on the age of the patient and the severity of P450c21 hydroxylase deficiency. Patients with mild P450c21 hydroxylase deficiency (eg, late-onset and cryptic forms) may have borderline basal 17-hydroxyprogesterone values, but they can be distinguished from heterozygotes by the magnitude of the 17-hydroxyprogesterone response to the parenteral administration of ACTH as well as by genotyping. Patients with baseline 17-OH progesterone levels over 10,000 μg/dL after 2 days of age or those with ACTH-induced responses that are over 10,000 μg/dL have classic 21-hydroxylase deficiency. Those who have levels at baseline which are above the normal range and who respond to ACTH with levels at 1500 to 10,000 ng/dL probably have nonclassic 21-hydroxylase deficiency. Those who are unaffected or heterozygotes respond to ACTH with a rise to less than 1500 ng/dL. Patients with borderline 17-OH progesterone responses can be genotyped to ascertain their status. As 17-OH progesterone levels are elevated in patients with 11-hydroxylase deficiency, 3-beta-dehydrogenase, 17,20-lyase, and P450 oxidoreductase deficiencies, it is prudent to do an ACTH stimulation test measuring 17-OH progesterone, 11-deoxycortisol, 17-OH pregnenolone, DOC, progesterone, T, and cortisol at baseline and at 1 hour before reaching a definitive diagnosis. Elevated 21-deoxycortisol levels are a specific and sensitive indicator of 21-hydroxylase deficiency. 21-deoxycortisol measurements can be used to differentiate between patients with elevated 17-hydroxyprogesterone levels, who have 21-hydroxylase deficiency and sick newborns and premature infants who have false positive 17-hydroxyprogesterone levels on screening.

In patients with the salt-losing form of 21-hydroxylase deficiency, aldosterone levels in both plasma and urine are low in relation to the serum sodium concentration, while plasma renin activity is markedly elevated. The plasma aldosterone-renin ratio will be low. Breast milk and infant formulas have a low concentration of sodium which usually unmasks the diagnosis of salt loss by 7 to 14 days. The salt-losing episode is heralded by a rise in serum potassium with a subsequent fall in sodium and bicarbonate. The diagnosis of nonclassic (late-onset 21-hydroxylase deficiency) can be made in the symptomatic patient (eg, one with early development of pubic hair, acne, and other signs of androgen excess) by measuring a plasma 17-OH progesterone level between 7 to 9 am. Levels below 200 ng/dL rule out late-onset CAH. Higher levels should be evaluated by an ACTH test which will distinguish 21-hydroxylase deficiency from other causes of elevated 17-OH progesterone levels.

The diagnosis of 21-hydroxylase deficiency in newborn males has been facilitated by routine neonatal screening or 17-OH progesterone by filter paper (Guthrie cards) at 2 to 3 days of life. Screening reduces the morbidity and mortality associated with late or nondiagnosis especially in male infants with the salt-losing form of 21-hydroxylase deficiency. Males with simple virilization may not be diagnosed until later in infancy when virilization and advanced bone maturation associated with prepubertal testes are evident. Prematurity and illness are confounding factors since both are associated with elevated levels of 17-OH progesterone. If the patient is reported positive by the laboratory and the levels are borderline or moderately elevated, it is prudent to examine the patient, review the history (especially confounders such as prematurity, concomitant stress, day of life specimen obtained, etc.), and repeat the 17-OH progesterone screen by LCMSMS (liquid chromatography, tandem mass spectrometry) from a venous sample. The parents should be counseled about possible symptomatology until the results return. If the screening value is a lot higher than the cutoffs, then examination, electrolytes, plasma 17-OH progesterone, androstenedione, and T levels by LCMSMS should be obtained. Depending on the age and clinical status of the infant, hospitalization, an ACTH test and therapy should be considered. A karyotype and pelvic ultrasound are always indicated in any infant with atypical genitalia and in apparent males with undescended testes especially those with evidence of salt loss.

P450c11 Hydroxylase Deficiency

Classic P450c11 hydroxylase deficiency (virilization with hypertension) is rare. It occurs in 1:100,000 births in persons of European ancestry. However, in the Middle Eastern population, it is much more common. In the patient with classic disease, a defect in 11-hydroxylation leads to decreased cortisol levels with a consequent increase in ACTH and the hypersecretion of DOC (deoxycorticosterone) and 11-deoxycortisol in addition to adrenal androgens. Marked heterogeneity in the clinical and hormonal manifestations of this defect has been described, including mild, late-onset, and even cryptic forms. Patients with this form of adrenal hyperplasia classically exhibit virilization, secondary to increased androgen production, and hypertension, related to increased DOC secretion. Plasma renin activity is either normal or suppressed. The hypertension is variable; it occurs in approximately two-thirds of patients and may be associated with hypokalemic alkalosis. Newborns with 11-hydroxylase deficiency may manifest transient salt loss probably related to their salt-restricted diet and relative resistance to mineralocorticoids.

Two P450c11 hydroxylase genes have been localized to the long arm of chromosome 8: P450c11b1 and P450c11b2. Similar to 21-hydroxylase, these two genes are 95% homologous. P450c11b1 encodes the enzyme for 11-hydroxylation and is expressed in the zona fasciculata and zona reticularis and is ACTH dependent. It primarily mediates 11-hydroxylation of 11-deoxycortisol to cortisol and DOC to corticosterone. It has about one-twelfth the capacity of P450c11b2 for 18-hydroxylation and does not oxidize 18-hydroxycorticosterone to aldosterone. P450c11b2 encodes the angiotensin-dependent isozyme aldosterone synthase and is only expressed in the zona glomerulosa, where it mediates 11-hydroxylation, 18-hydroxylation, and 18-oxidation. Mutations, deletions, and gene duplications of these genes can produce a wide variety of clinical manifestations from virilization and hypertension (P450c11b1 deficiency) to isolated salt-wasting (P450c11b2 [aldosterone synthase] deficiency) to glucocorticoid-remedial hypertension (due to fusion of the ACTH-dependent regulatory region of the 11-hydroxylase gene with the coding region of aldosterone synthase). Both genes encoding P450c11b1 and P450c11b2 are located on chromosome 8 and thus are not linked to HLA. ACTH stimulation tests have thus far failed to demonstrate a consistent biochemical abnormality in obligate heterozygotes.

Diagnosis: The diagnosis of P450c11b1-hydroxylase deficiency can be confirmed by demonstration of elevated basal or ACTH-induced plasma levels of 11-deoxycortisol and DOC, at least three times higher than the 95th percentile for age, and increased excretion of their metabolites in urine (mainly tetrahydro-11-deoxycortisol). As previously noted, these patients have elevated 17-OH progesterone levels and need to be distinguished from those with other forms of CAH by measuring the steroid levels at baseline or after ACTH stimulation.

Star, P450scc, 17α-Hydroxylase (17,20-Lyase), 3β-Hsd

Defects in steroidogenic acute regulatory protein (StAR), P450scc (side chain cleavage), 17α-hydroxylase (17,20-lyase), and 3β-HSD type II have in common blocks in cortisol and sex steroid synthesis in both the adrenals and the gonads. The latter types produce chiefly incomplete masculinization in the male and little or no virilization in the female (Table 14–3). Consequently, these are discussed primarily as forms of 46,XY DSD (disorders of androgen synthesis).

Treatment

Treatment of patients with salt-losing adrenal hyperplasia may be divided into acute and chronic phases. In acute adrenal crises, a deficiency of both cortisol and aldosterone results in hypoglycemia, hyponatremia, hyperkalemia, hypovolemia, acidosis, and shock. If the patient is hypoglycemic, an intravenous bolus of glucose (0.25-0.5 g/kg; maximum 25 g), should be administered. If the patient is in shock, an infusion of normal saline (20 mL/kg) may be given over the first hour; thereafter, replacement of glucose, fluid, and electrolytes is calculated on the basis of deficits and standard maintenance requirements. Hydrocortisone sodium succinate (50 mg/m2) should be given as a bolus and another 50 to 100 mg/m2 over 24 hours, divided into every 6 hour doses. If hyponatremia and hyperkalemia are present, fludrocortisone (0.05-0.1 mg) by mouth may be given along with the intravenous saline and hydrocortisone. Because hydrocortisone has mineralocorticoid activity, it may suffice to correct the electrolyte abnormality along with the saline. In extreme cases of hyponatremia, hyperkalemia, and acidosis, sodium bicarbonate and a cation exchange resin (eg, sodium polystyrene sulfonate) may be needed. In patients with presumed chronic hyponatremia, it is always prudent to raise the sodium cautiously and slowly to avoid the possibility of central pontine myelinolysis. Initially, in newly diagnosed patients and newborns in the first few days of treatment, hydrocortisone in doses of 25 to 50 mg/m2/d are commonly used to facilitate suppression of the CRH-ACTH-adrenal axis. This dose should be reduced as soon as the steroid levels are suppressed to the desired range, usually after 5 to 7 days.

Once the patient is stabilized and a definitive diagnosis has been arrived at by means of appropriate steroid studies, the patient should receive suppressive doses of glucocorticoids to permit normal growth, development, and bone maturation (hydrocortisone, ∼10-15 mg/m2/d by mouth in three divided doses). The dose of hydrocortisone must be titrated in each patient depending on steroid hormone levels in plasma and urine, linear growth, bone maturation, and clinical signs of steroid overdose or of virilization. Therapy can be monitored by consistently timed (in relation to dose administration) steroid determinations, for example, the measurement of 17-OH progesterone, androstenedione, and T at 8 am before the morning dose of hydrocortisone. A target concentration for 17-OH progesterone (400-1200 ng/dL) with androstenedione and T levels in the normal range has been suggested. However, these levels will vary depending on when the evening dose of hydrocortisone is administered. Salt-losers need treatment with mineralocorticoid (fludrocortisone, 0.05-0.2 mg/d by mouth) and added dietary salt (1-3 g/d) in infancy. The dose of mineralocorticoid should be adjusted so that the electrolytes and blood pressure, as well as the plasma renin activity, are in the normal range.

Recently, as a result of the difficulty of optimally treating these patients, several novel therapies have been proposed. These include adrenalectomy in patients with null mutations of P450c21, GH to augment final height, and the use of physiologic doses of hydrocortisone (8 mg/m2), fludrocortisone, flutamide (an androgen receptor blocker), and an aromatase inhibitor in combination.

The parents need to be presented the whole picture diagnosis, natural history, possible medical and surgical therapy, risks, complications, and unknowns. It is critical that they have the knowledge and time to make an informed decision about any surgical procedures on their child. Only patients with markedly ambiguous external genitalia (Prader III-V) should be considered for surgery. Vaginoplasty and clitoral recession or clitoroplasty for markedly enlarged clitori—not clitoridectomy—is indicated. Surgical reconstruction done in infancy may need refinement at puberty. Surgery, if done, must emphasize function rather than cosmesis. It should be performed by a surgeon who is experienced in the operative procedure and is cognizant of the importance of preserving the functional integrity of the genital area. The variability of normalcy in relationship to size and appearance of the external genitalia of adult women has recently been quantified. These data can serve as a reference with respect to the need for surgery in older patients with CAH. The timing of any surgery remains controversial. This is a decision that needs to be made after careful discussion with the parents. It should be noted that there are no outcome data for delaying functionally cosmetic surgery on an infant with ambiguous genitalia until an age of consent as suggested by some psychologists and patient advocates.

The dose of glucocorticoid needs to be increased in response to stress, for example, temperature above 100°F orally, gastroenteritis with dehydration, trauma, and surgery. The daily maintenance hydrocortisone dose should be tripled and divided into every 6 hour doses around the clock until the fever or gastroenteritis has abated for 12 hours. Thereafter, the child may resume his/her maintenance dose. In the event that the oral dose cannot be administered, hydrocortisone (50 mg/m2) or its equivalent can be given intramuscularly and the child maintained on a dose of 50 to 100 mg/m2/d in divided doses until he/she is able to take medication by mouth. It is prudent to check electrolytes in any patient who is a salt-loser, manifests vomiting, and is a candidate for parenteral hydrocortisone. Even if electrolytes are normal, patients who are unable to take medication and fluid orally for longer than 6 to 8 hours usually require hospitalization for parenteral fluids, especially if they are salt-losers. All caregivers should be prepared to administer hydrocortisone in an emergency, and all patients should have a medi-alert notification with them.

Prenatal therapy of pregnant women who are at risk of carrying a female fetus with atypical genitalia due to 21-hydroxylase deficiency is controversial. Therapy with oral dexamethasone given to the mother beginning at 6 to 7 weeks of gestation has been advocated to reduce or eliminate virilization of the affected female genitalia. Statistically, only one in eight fetuses will be a female with CAH. Although this therapy is effective, in diminishing genital ambiguity, more long-term studies are needed to ascertain the long-term morbidities, both physical and psychological.

In postpubertal patients who have reached their final height, glucocorticoid replacement can be implemented with more potent glucocorticoids that have longer half-lives than hydrocortisone such as prednisone (5-7 mg/d divided into two doses) or methylprednisolone (4-6 mg/d in two doses). As with children, the lowest dose of medication required to optimally suppress adrenal androgen secretion and prevent glucocorticoid side-effects should be used. The dose needs to be titrated to the plasma steroid values, or better yet, the urinary 17 ketosteroids—an integrated 24-hour assessment of androgen secretion. Similar to hydrocortisone, the dose is a function of absorption, metabolism, and sensitivity and varies from patient to patient.

A host of studies indicate that the vast majority of 46,XX females with 21-hydroxylase deficiency and consequent virilization of their external genitalia have a female gender identity. Gender dysphoria, although rare, is more prevalent in these patients than in the general population. Behavior characterized as masculinization/defeminization occurs in affected females who play more with traditionally boys' toys, are more aggressive, and show less interest in infants than their unaffected female siblings.

Treatment of adults with CAH should be directed toward preventing and remediating the morbidities of long-term glucocorticoid therapy and optimizing sexual, reproductive, and bone health (see Auchus PMID 20613954). Continued monitoring of serum 17-OH progesterone, androstenedione, and T levels (in females) and/or 24-hour urinary 17-ketosteroids is indicated in order to prevent further virilization or the morbidity of excess glucocorticoid therapy. While many salt-losers appear not to need mineralocorticoid due to increased salt intake or extra adrenal 21-hydroxylation, it is prudent to follow plasma renin activity, since elevated plasma renin levels are a marker of mineralocorticoid deficiency and are associated with the need for higher doses of glucocorticoids to suppress adrenal androgen secretion. Multiple factors play a role in the decreased fertility rate seen in women with CAH. These include decreased sexual activity due to vaginal stenosis which results in painful intercourse, poor surgical repairs resulting in lack of sensation, lack of desire for maternity, and poor self-image. In males, spermatogenesis and Leydig cell function may be compromised by poor hormonal control or testicular adrenal rest tumors which can be readily identified by ultrasound.

Dual energy x-ray absorptiometry (DXA) scans have shown that osteopenia is present in 40% to 50% of patients less than 30 years and 70% of patients over 30 years old. The severity of the osteopenia/osteoporosis is related to the severity of the CAH, the age of the patient, and dose of glucocorticoid given. Thus, periodic DXA scans and vitamin D and calcium supplementation are indicated, both prophylactically and therapeutically. Quality of life issues need to be addressed. These include physical and psychological status as well as social and sexual relationships.

Glucocorticoid Receptor Gene Mutation

Glucocorticoid resistance due to a homozygous mutation in the gene encoding the glucocorticoid receptor reportedly induces 46,XX DSD. Glucocorticoid resistance results in an increase in ACTH levels with a consequent increase in cortisol, mineralocorticoids, and adrenal androgens. The latter steroids induce virilization, which in the female infant results in 46,XX DSD.

Feto-Placental Source

P450 Oxidoreductase

P450 oxidoreductase (POR) is a flavoprotein that transfers electrons from nicotinamide adenine dinucleotide to all microsomal P450s. These include P450c21, P450 17-hydroxylase/17,20-lyase and P450 aromatase which are involved in steroidogenesis, lanosterol 14-alpha-demethylase (P45014DM), and squalene monooxidase (SQLE), which are involved in cholesterol synthesis. POR deficiency is inherited as an autosomal recessive, and affected individuals are either homozygous or compound heterozygotes, predominantly for an A287P mutation in Caucasians and an R487H mutation in Japanese. More than 50 different mutations have been described. A broad range of phenotypes has been reported related to the degree of enzymatic function determined by the mutation and the specificity of the mutation for each one of the enzymes involved. Most, but not all patients reported with this condition, have skeletal abnormalities such as craniosynostosis, midfacial hypoplasia, and radioulnar synostosis. The phenotype of the skeletal dysmorphism has been termed the Antley-Bixler syndrome and appears to correlate with the degree of decrease in enzyme activity resulting from the mutations. The skeletal abnormalities of Antley-Bixler syndrome are digenic in origin. Individuals with normal genitalia and the Antley-Bixler phenotype have a mutation in the gene encoding fibroblast growth factor receptor-2 (FGFR-2) and no abnormality in steroidogenesis, whereas those with one or more of the above-mentioned skeletal abnormalities and ambiguous genitalia are found to have mutations in POR.

POR deficiency can cause ambiguous genitalia in both males and females, and micropenis, hypospadias, and infertility in males with mild mutations. The spectrum of patients reported includes females with normal genitalia who present with PCOS-like symptoms and have primary amenorrhea. There is evidence to suggest that virilization of the external genitalia in females with POR deficiency results from the synthesis of DHT in utero via the "backdoor" pathway (see Figure 14–10). Androgen excess can also result from a decrease in P450 aromatase activity in the placenta, which can cause maternal and fetal virilization during pregnancy. In patients with mild mutations, maternal virilization is not present, and males may have normal genitalia and rarely micropenis. The virilization in females is not progressive. After birth, however, in a Japanese cohort, pubertal maturation was definitely affected in females.

Diagnosis: POR deficiency should be suspected in males and females with ambiguous genitalia and the Antley-Bixler phenotype, in females whose virilization does not progress postnatally, and in patients with modestly elevated 17-OH progesterone levels. The diagnosis can be suspected from ACTH-induced steroid levels. Cortisol levels are normal at baseline, but there is a blunted response to ACTH. Both serum progesterone and 17-OH progesterone values in response to ACTH are higher than normal suggesting a defect in both 17- and 21-hydroxylation, whereas DHEA and androstenedione levels may be low. Genotyping will verify the diagnosis. Depending on the baseline concentration and ACTH-induced cortisol level, cortisol replacement may be indicated in some patients. In view of the blunted cortisol responses observed, it is prudent to give stress steroid coverage for illness, surgical procedures, and severe trauma.

Another form of androgen-induced 46,XX DSD is due to aromatase deficiency. Mutations in the gene CYP19, encoding P450 aromatase, result in defective placental conversion of C19 steroids to estrogens (C18), leading to exposure of the fetus to excessive amounts of T and to masculinization of the external genitalia of the female fetus. Virilization of the mother during gestation occurs commonly. The female fetus is virilized, due to the inability of the placenta to aromatize testosterone produced by the placenta from the conversion of fetal adrenal DHEA. At puberty, defective aromatase activity in the ovary leads to pubertal failure, hypergonadotropic hypogonadism, polycystic ovaries, mild virilization, tall stature, and osteopenia. A striking delay in bone age with continuing growth occurs in both affected males and females, despite increased concentrations of plasma T, supporting the concept that estrogens, rather than androgens, are the major sex steroids affecting bone maturation, bone turnover, and epiphyseal fusion in males as well as females. A nonclassic phenotype has been described in two females with sufficient aromatase activity to result in the development of Tanner stage 2 and 4 breast development.

Diagnosis: The diagnosis of aromatase deficiency is suggested by the above clinical picture as well as elevated plasma androstenedione and T levels in the face of low estrogen levels, elevated gonadotropins, and ovarian cysts in females. Gonadotropin levels are elevated due to lack of estrogen feedback. There is striking osteopenia. Low plasma concentrations of maternal estriol during pregnancy associated with maternal virilization can facilitate the prenatal diagnosis. Affected males have normal genitalia, normal pubertal development, osteoporosis, and tall stature due to lack of epiphyseal fusion as a result of estrogen deficiency.

Masculinization of the external genitalia of a female infant can occur, if the mother is given T, other androgenic steroids, or certain synthetic progestational agents during pregnancy. Norethindrone, ethisterone, norethynodrel, and medroxyprogesterone acetate have all been implicated in masculinization of the female fetus. These drugs were given in the past in an effort to control threatened or habitual abortions. Nonadrenal 46,XX DSD can occur as a consequence of maternal ingestion of danazol, the 2,3-d-isoxazol derivative of 17α-ethinyl T. In rare instances, masculinization of a female fetus is due to a virilizing maternal ovarian or adrenal tumor, congenital virilizing adrenal hyperplasia in the mother, or a luteoma of pregnancy. The fetus is protected from excess T exposure by the ability of the fetal-placental unit to aromatize T to estrogens, especially after the first trimester. The diagnosis of 46,XX DSD arising from transplacental passage of androgenic steroids is based on exclusion of other forms of female pseudohermaphrodism and a history of drug exposure. Surgical correction of the genitalia, if needed, is the only therapy necessary.

Nonadrenal 46,XX DSD can be associated with imperforate anus, renal anomalies, and other malformations of the lower intestine and urinary tract and in some affected individuals. There may be anomalies of the ovaries and Müllerian structures. Sporadic as well as familial cases have been reported.

46,XY DSD (Male Pseudohermaphrodism)

Patients with 46,XY DSD have gonads that are testes, but the genital ducts or external genitalia, or both, that are not completely masculinized. 46,XY DSD can result from deficient T secretion as a consequence of (1) defective testicular differentiation (testicular dysgenesis); (2) failure of T secretion by Leydig cell due to a mutation in the Leydig cell hCG/LH receptor which is associated with Leydig cell hypoplasia as well as an inability to respond to hCG/LH stimulation; (3) impaired secretion of T; (4) failure of conversion of T to DHT; (5) failure of target tissue response to T and DHT; (6) impaired secretion of AMH or defective AMH receptors; and (7) environmental influences.

Testicular Unresponsiveness to hCG and LH (Leydig Cell Hypoplasia)

Male sexual differentiation is dependent on the production of T by fetal Leydig cells. Leydig cell T secretion is initially autonomous and then later hCG dependent during the critical period of male sexual development in the latter part of the first trimester. After the first trimester, Leydig cell T secretion is dependent on stimulation by pituitary LH, as evidenced by the finding of micropenis in 46,XY patients associated with anencephaly, apituitarism, or congenital hypothalamic hypopituitarism.

Absence, hypoplasia, or unresponsiveness of Leydig cells to hCG-LH results in deficient T production and, consequently, ambiguous male genitalia. The extent of the genital ambiguity is a function of the degree of T deficiency. The phenotype ranges from complete forms with female external genitalia, undescended testes, absent Müllerian ducts, rudimentary Wolffian ducts, and hypergonadotropic hypogonadism to milder forms with micropenis or males with normal male genitalia and hypergonadotropic hypogonadism at puberty. Testes are small postpubertally and have decreased or absent Leydig cells, normal-appearing Sertoli cells, and seminiferous tubules with spermatogenic arrest. A number of patients with absent, hypoplastic, or unresponsive Leydig cells due to a mutation in the gene encoding the LH-hCG receptor have been reported. Studies in patients with the clinical and chemical features of Leydig cell hypoplasia indicate that 50% of the patients with this phenotype have had no identifiable mutation in the LH-hCG receptor. Recently a mutation in a previously unrecognized exon has been described in some of these patients. 46,XX females with mutations in the LH beta subunit of LH receptor have female external genitalia, feminization at puberty, oligo-amenorrhea, and infertility.

Diagnosis: Plasma 17α-hydroxyprogesterone, androstenedione, and T levels are low, and hCG elicits little or no response in T or its precursors in the complete form and a blunted T response in the partial form, in which partial virilization can occur at puberty. Adrenal steroidogenesis is normal in these patients.

Treatment depends on the age at diagnosis and the extent of masculinization. A female sex assignment has usually been chosen in patients with female external genitalia. In patients with predominantly male external genitalia, T augments penile development in infancy and childhood and virilizes the patient at puberty.

Inborn Errors of Testosterone Biosynthesis

Figure 14–15 demonstrates the major pathway in T biosynthesis in the testes; each step is associated with an inherited defect that results in T deficiency and, consequently, 46,XY DSD. Steps 1, 2, and 3 are enzymatic deficiencies that occur in both the adrenals and gonads and result in defective synthesis of both corticosteroids and T. Thus, they represent forms of CAH, whereas steps 4 and 5 in T synthesis are confined to the testes.

Star Deficiency (Congenital Lipoid Adrenal Hyperplasia), P450scc

46,XY DSD with female or ambiguous genitalia, sexual infantilism, and adrenal insufficiency are consequences of very early defects in the synthesis of all steroids affecting the conversion of cholesterol to Δ5-pregnenolone and result in severe adrenal and gonadal deficiency. Recessive mutations in the gene encoding StAR, a protein that effects transport of cholesterol from the outer to the inner mitochondrial membrane (where it is converted to pregnenolone), have been identified in almost all patients with the clinical syndrome of congenital lipoid adrenal hyperplasia. Mutations in this gene located on chromosome 8 are prevalent in Japan and Korea where patients with lipoid adrenal hyperplasia represent the second most common form of CAH after 21-hydroxylase deficiency. StAR is expressed in the adrenals and gonads but not in the placenta. Hence, placental synthesis of progesterone, which is required to maintain pregnancy after midgestation, is not affected by mutations in StAR. Affected males usually have female or, less commonly, ambiguous external genitalia with a blind vaginal pouch and hypoplastic male genital ducts, no Müllerian derivatives, adrenal insufficiency, and hypergonadoropic hypogonadism. Many infants with StAR deficiency do not manifest evidence of salt wasting until after several weeks of life. The accumulation of cholesterol in steroidogenic organs that are functioning in utero (adrenals and testes) destroys the function of these organs resulting in the phenotype, for example, adrenal insufficiency and impaired differentiation of the genitalia in males. Death in early infancy from adrenal insufficiency is not uncommon. Affected females have normal genitalia and go into puberty as the ovaries are relatively quiescent until puberty and roughly 14% of steroidogenesis is not StAR-dependent, apparently allowing for estrogen production until cholesterol engorgement destroys the function of the ovary and results in hypergonadotropic hypogonadism and large polycystic ovaries filled with cholesterol. The absence of Müllerian anlage distinguishes StAR/P450scc deficiency from patients with a similar phenotype due to SF-1 deficiency. As in other genetic defects, phenotypic variability results from the magnitude of the functional activity of the mutation. A nonclassic late-onset form of this condition has been described. Affected males have normal external genitalia; they develop, usually in the first decade of life, cortisol deficiency with or without mineralocorticoid deficiency. Those individuals with only cortisol deficiency need to be distinguished from those with isolated familial glucocorticoid deficiency due to mutations in the ACTH receptor (MC2R) and the melanocortin-2 receptor accessory protein (MRAP).

Diagnosis: The diagnosis is confirmed by the lack of or low levels of all C21, C19, and C18 steroids in plasma and urine and an absent response to ACTH and hCG stimulation. Large lipid-laden adrenals, that displace the kidneys downward, may be demonstrated by intravenous urography, abdominal ultrasonography, or CT scan. Treatment involves replacement with appropriate doses of glucocorticoids and mineralocorticoids, prophylactic orchiectomy in affected 46,XY patients raised as females, and estrogen replacement at puberty. Since the adrenals are not functionally hyperplastic, the glucocorticoid dose needed in these patients is less than is the case of P450c21-hydroxylase deficiency. A nonclassic late-onset form of this condition has been described.

P450scc

The P450scc gene has been cloned and localized to chromosome 15. It codes for the enzyme scc, which is the first step in the conversion of cholesterol to pregnenolone in all steroidogenic organs. Seven patients with mutations in the P450scc gene(s) have been reported. All seven cases reported have had a 46,XY karyotype. Female genitalia were present in five of seven, clitoromegaly in one patient, and midshaft hypospadias in another. The onset of adrenal insufficiency varied from the neonatal period to as late as 9 years of age in the patient with hypospadias. The latter patient and two more recently described patients with compound heterozygosity for P450scc mutations had residual P450scc activity. These patients had later onset of adrenal insufficiency than seen in classic patients and the males had hypospadias. These patients are similar to the non-classic form of StAR deficiency, both clinically and hormonally, except none had normal or enlarged adrenals on MRI or ultrasound. It has been suggested that patients with P450scc deficiency survive gestation due to either residual P450scc activity or due to continuing progesterone secretion beyond mid-gestation by the maternal corpus luteum.

Diagnosis: P450scc deficiency is suggested by the finding of a 46,XY DSD with undervirilization of the external genitalia, absent Müllerian ducts, adrenal insufficiency, and low baseline and deficient steroid responses to ACTH and hCG. The absence of adrenal enlargement on MRI or ultrasound distinguishes P450scc deficiency from a StAR deficiency.

3β-Hydroxysteroid Dehydrogenase Type 2 δ5-Isomerase Deficiency

3β-HSD type 2 Δ5-isomerase deficiency is an early defect in steroid synthesis that results in inability of the adrenals and gonads to convert 3β-hydroxy-Δ5 steroids to 3-keto-Δ4 steroids. Deficiency leads to 46,XY or 46,XX DSD and adrenal insufficiency. This enzyme is encoded for by a recessive gene on the short arm of chromosome number 1. There are two highly homologous genes encoding 3β-HSDs on chromosome 1. The type 1 3β-HSD gene is expressed in the placenta, liver, skin, and other peripheral tissues, whereas type 2 is expressed in the adrenals and gonads. 3β-HSD is not a cytochrome P450 enzyme, and it requires NAD+ as a cofactor.

Mutations causing frame-shifts, stops, and missense have been reported in the type 2 gene in affected patients. Mutations which decrease enzymatic activity of the type 2 enzyme significantly result in a severe deficiency of aldosterone, cortisol, T, and estradiol secretion (see Figure 14–15). Males with this defect are incompletely masculinized due to the defect in 3β-hydroxysteroid dehydrogenase in the testes. They have a small penis, hypospadias, a blind vaginal pouch, and absent Müllerian structures. Females have normal female genitalia or mild clitoromegaly.

The undervirilization in males is readily explainable by the block in T synthesis in the testes during the critical period of male genital differentiation. However, the mild virilization observed in some females is still not readily explained. It has been suggested that the conversion of Δ5-17-OH pregnenolone to 17-hydroxyprogesterone in utero by peripheral 3β-HSD-1 facilitates the production of low levels of DHT via the backdoor pathway (see Figure 14–10). Alternatively, the conversion of excess DHEA-S to T by 3β-HSD-1 in the placenta during the critical window of sex differentiation, when aromatase activity is relatively low, may be implicated. Further studies of steroidogenesis in affected patients with null mutations may shed light on this question.

Adrenal crises with salt loss usually occurs in early infancy in affected patients. Affected males may experience normal male puberty but often have prominent gynecomastia. Rare cases with normal puberty and fertility have been reported. Individuals with a mild non-salt-losing form of 3β-HSD type 2 deficiency are reported as well as a late-onset form in individuals presenting with premature pubarche alone. These patients had elevated Δ5 steroids as well as mutations in the 3β-HSD type 2 gene.

Diagnosis: The diagnosis of 3β-HSD deficiency type 2 is based on finding elevated concentrations of Δ5-pregnenolone, Δ5-17α-hydroxypregnenolone, DHEA and its sulfate, and other 3β-hydroxy-Δ5 steroids in the plasma and urine. 3-Keto-Δ4 steroids (ie, 17-hydroxyprogesterone and androstenedione) may be elevated due to peripheral conversion of 3β-hydroxy-Δ5 to 3-keto-Δ4 steroids by the enzyme encoded by the type 1 gene. The diagnosis of 3β-HSD-2 deficiency may be facilitated by detecting abnormal levels of serum Δ5-17α-hydroxypregnenolone and DHEA and its sulfates as well as abnormal ratios of Δ5 to Δ4 steroids after intravenous administration of ACTH. For example, after ACTH stimulation, 17-hydroxypregnenolone levels are more than 5.3 SD above the mean for normal individuals in affected infants, more than 35 SD above the mean in prepubertal children, and more than 21 SD above the mean in adults. The 17-hydroxypregnenolone-cortisol ratio is more than 6.4 SD above the mean in infants, more than 23 SD above the mean in prepubertal children, and more than 221 SD above the mean in adults. The diagnosis is confirmed by detecting mutations in the type 2 3β-HSD-Δ5 isomerase gene. Suppression of the increased plasma and urinary 3β-hydroxy-Δ5 steroids by the administration of dexamethasone distinguishes 3β-HSD deficiency from a virilizing adrenal tumor.

Treatment of this condition is similar to that of other forms of adrenal hyperplasia.

P450c17 Deficiency, 17α-Hydroxylase Deficiency

A single gene on chromosome 10 encodes both adrenal and testicular P450c17 hydroxylase as well as 17,20-lyase activity. This enzyme catalyzes the 17-hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone as well as the scission (lyase) of 17-hydroxypregnenolone to the C19 steroid (DHEA) in the adrenal cortex and gonads. Mutations affecting 17-hydroxylase activity have included stop codons, frame-shifts, deletions, and missense substitutions. P450c17 deficiency is transmitted in a manner similar to other enzymatic defects in steroidogenesis as an autosomal recessive trait.

A defect in 17α-hydroxylation in the zona fasciculata of the adrenal and in the gonads results in impaired synthesis of 17–hydroxyprogesterone and 17-hydroxypregnenolone and, consequently, cortisol and sex steroids. The secretion of large amounts of corticosterone and DOC leads to hypertension, hypokalemia, and alkalosis. Although these patients are cortisol-deficient, the excess mineralocorticoid secretion, as well as elevated levels of corticosterone, prevents symptomatic adrenal insufficiency except during stress. Increased DOC secretion from the zona fasciculata results in hypertension and hypokalemia, and suppression of renin (low renin hypertension) and, decreased aldosterone secretion. Affected 46,XY individuals with less than 25% enzymatic activity present with female genitalia with a blind vaginal pouch or hypoplastic male genitalia, hypertension, and hypokalemic alkalosis with hypergonadotropic hypogonadism at puberty. Müllerian structures are absent; Wolffian structures are hypoplastic. Partial forms of 17-hydroxylase deficiency have been described with hypospadias, with or without hypertension.

Affected 46,XX females have normal development of the internal ducts and external genitalia but manifest sexual infantilism with elevated gonadotropin concentrations at puberty and low renin hypertension. Affected females with 5% or greater activity of the enzyme can make estrogen at puberty and feminize, although menses are usually irregular.

Diagnosis: 17α-Hydroxylase deficiency should be suspected in a 46,XY DSD with hypokalemic alkalosis and hypertension, as well as a 46,XX female with the above findings and sexual infantilism at puberty. ACTH stimulation testing will reveal elevated levels of DOC, corticosterone, and 18-hydroxycorticosterone as well as progesterone and Δ5 pregnenolone, but low levels of 17-hydroxyprogesterone, 17-hydroxypregnenolone, sex steroids, and cortisol. Glucocorticoid therapy, as given for 21-hydroxylase deficiency, suppresses ACTH and hence deoxycorticosterone production. This decreases the blood pressure and returns the potassium and aldosterone levels to normal. These patients usually require sex steroid replacement at puberty.

Smith-Lemli-Opitz Syndrome

The phenotypic spectrum of this syndrome typically includes microcephaly, mental retardation, ptosis, micrognathia, severe hypospadias, micropenis, growth failure, and, rarely, adrenal insufficiency. The genitalia in affected 46,XY males may range from normal male to female. The syndrome is caused by a mutation in the gene encoding sterol Δ-7-reductase, DHCR7, causing defective cholesterol synthesis. The diagnosis is made by the clinical features and confirmed by demonstration of low levels of cholesterol and increased levels of 7-dehydrocholesterol.

Enzyme Defects Primarily Affecting Testosterone Biosynthesis by the Testes

7,20-Lyase Deficiency

The enzyme encoded by the P450c17 gene mediates both the 17-hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone and the scission of the C17,20 bond of 17-hydroxypregnenolone to yield DHEA in the adrenals and gonads (see Figure 14–15). In humans, the scission of 17-hydroxyprogesterone to androstenedione occurs at a low level. In this rare autosomal recessive disorder, the defect involves only the scission of the C21 steroids to C19 steroids. This results in a defect in T synthesis and, as a consequence, 46,XY DSD in the male and affected females with estradiol deficiency, hypergonadotropic hypogonadism, and giant ovarian cysts. The initially reported 46,XY DSD individuals both had micropenis, perineal hypospadias, a bifid scrotum, a blind vaginal pouch, and cryptorchidism. The administration of hCG resulted in a marked rise in plasma 17-hydroxyprogesterone with a poor response in plasma DHEA, androstenedione, and T consistent with a diagnosis of isolated 17,20-lyase deficiency. Analyses of the P450c17 gene in affected individuals has revealed mutations that result in a specific decrease in 17,20-lyase activity encoded by the P450c17 gene, but not 17-hydroxylase activity.

Affected 46,XY males with 17,20-lyase deficiency may present with female external genitalia. Patients with 17,20-lyase deficiency have low circulating levels of T, androstenedione, DHEA, and estradiol in the face of markedly elevated levels of progesterone and 17-hydroxyprogesterone. At puberty, poor virilization occurs with elevated gonadotropins in affected 46,XY patients.

Cytochrome b5 reductase is a cofactor for 17,20-lyase. Mutations in this gene cause methemoglobinemia as well as a defect on 17,20-lyase. Two patients have been reported with mutations in b5 reductase causing 46,XY DSD.

Diagnosis: The diagnosis of 17,20-lyase deficiency can be confirmed by demonstration of an increased ratio of 17-hydroxy C21 steroids to C19 steroids (T, DHEA, Δ5-androstenediol, and androstenedione) after stimulation with ACTH or hCG and by DNA analysis of the P450c17 gene. Patients require sex steroid replacement at puberty.

17β-Hydroxysteroid Dehydrogenase-3 (17β-Hsd-3) Deficiency

At least six isoenzymes of 17β-HSD mediate steroidogenesis in humans. The last step in T biosynthesis by the testes involves the reduction of androstenedione to T, and estrone to estradiol by 17-hydroxysteroid dehydrogenase-3, an NADPH-dependent microsomal enzyme. This gene is located on chromosome 9q22 and is expressed primarily in the testes. As with other genes which code for enzymes, it is autosomal recessive. 17β-HSD-3 is not expressed in the ovary. The conversion of estrone to estradiol is mediated by 17β-HSD-1 expressed in the granulosa cells of the ovary.

Mutations in 17β-HSD-3 are a cause of 46,XY DSD. At birth, males with a deficiency of the enzyme 17β-HSD-3 have predominantly female or mildly ambiguous external genitalia resulting from T deficiency during the critical period of male differentiation. They have male duct development, absent Müllerian structures with a blind vaginal pouch, and inguinal or intra-abdominal testes. Affected infants with female genitalia are invariably raised as females, and the phenotypic appearance of the genitalia, along with the absence of Müllerian duct derivatives, can be confused with complete androgen insensitivity. The finding of Wolffian ducts, associated with female external genitalia in these patients, is not yet well explained. At puberty, progressive virilization with clitoral hypertrophy occurs as a result of peripheral extraglandular conversion of androstenedione to T by 17β-HSD-5. This is often associated with the concurrent development of gynecomastia related to increased estrone levels. Plasma gonadotropin, androstenedione, and estrone levels are elevated, whereas T and estradiol concentration are relatively low.

Analysis of a cohort of 17 patients with classic 17β-HSD-3 deficiency revealed 14 mutations in the 17 β-HSD-3 gene. Twelve patients had homozygous mutations, four were compound heterozygotes, and one was a presumed heterozygote. In a large cohort from the Gaza Strip, an Arg80 → Gln mutation was found with partial (15%-20%) enzymatic activity. In this isolate, virilization at puberty generally resulted in a change of gender identity from female to male.

Diagnosis: 17-HSD-3 deficiency should be included in the differential diagnosis of (1) 46,XY DSDs with absent Müllerian derivatives who have no abnormality in glucocorticoid or mineralocorticoid synthesis; and (2) 46,XY DSDs who virilize at puberty, especially if they also exhibit gynecomastia. The diagnosis of 17β-HSD-3 deficiency is confirmed by the demonstration of inappropriately high plasma levels of estrone and androstenedione and increased ratios of plasma androstenedione to T and estrone to estradiol after prolonged stimulation with hCG in infants and children.

Management of affected individuals depends on the age at diagnosis, the degree of ambiguity of the external genitalia, surgical options, and family and cultural views, as it does with those with other forms of 46,XY DSD. In the patient assigned a male sex identity, plastic repair of the genitalia and T augmentation of phallic growth prepubertally, as well as T replacement therapy at puberty, is necessary to suppress the pituitary gonadotropin levels and, consequently, the elevated estrone levels that result in gynecomastia in untreated pubertal patients. Retained testes must be observed closely by palpation and ultrasound because an increased incidence of malignancy has been reported in a small cohort of patients. In patients reared as females, the appropriate treatment is castration or prevention of virilization with GnRH agonist at puberty until the individual's gender identity can be confirmed. Affected 46,XX females have no abnormalities in phenotype or gonadal function because 17β-HSD-3 is not expressed in the ovary.

The prevalence of a female-to-male gender role change in patients with 17β-HSD-3 deficiency appears to approximate that found in patients with 5α-reductase-2 deficiency. Both these conditions are associated with spontaneous virilization at puberty in patients whose testes were not removed. However, a number of the large cohorts of patients with 17β-HSD-3 deficiency summarized in the literature are from areas where a primacy is placed on a male role by the cultural advantages of the society. In individuals diagnosed in infancy, the information on the change in sex makes discussion of this issue with the parents mandatory before they make an informed decision about sex assignment.

Enzyme Defects Affecting DHT Synthesis; 5α-Reductase-2 Deficiency (Pseudovaginal Perineoscrotal Hypospadias, Peripheral)

The products of two genes catalyze the conversion of T to DHT. They are termed type 1 and type 2 5α-reductase. The type 1 enzyme is expressed in skin postnatally until 2 to 3 years of age and thereafter remains low until puberty. No instances of mutation in the type 1 gene as yet have been reported. The type 2 isoenzyme is found in fetal genital skin, male accessory glands, and the prostate. 5α-Reductase-2 deficiency is transmitted as an autosomal recessive trait, and the enzymatic defect exhibits genetic heterogeneity. In patients with 5α-reductase deficiency, the isozyme with a pH 5.5 optimum is deficient (type 2). The gene encoding this enzyme contains five exons and is located on chromosome 2, band p23. A variety of mutations are reported, including deletions, nonsense, splicing defects, and the more common missense mutations. Two-thirds of patients are homozygous for a single mutation, and the remainder are compound heterozygotes.

The defective conversion of T to DHT produces a unique form of 46,XY DSD. Phenotypically, these individuals may vary from those with a microphallus to individuals with pseudovaginal perineoscrotal hypospadias. At birth, in the most severely affected individuals, ambiguous external genitalia are characterized by a small hypospadiac phallus bound down in chordee, a bifid scrotum, and a urogenital sinus that opens onto the perineum. A blind vaginal pouch is present, opening either into a urogenital sinus or directly into the urethra. The testes are either inguinal or labial. Müllerian structures are absent, and the Wolffian structures are well differentiated. At puberty, affected males virilize; the voice deepens, muscle mass increases, and the phallus enlarges. The bifid scrotum becomes rugose and pigmented. The testes enlarge and descend into the labioscrotal folds, and spermatogenesis may ensue. Gynecomastia is notably absent in these patients. Of note is the absence of acne, temporal hair recession, and hirsutism. The striking virilization noted at puberty, as opposed to its absence in utero, is attributed to the expression and function of the type 1 gene at puberty and, as a consequence, the generation of sufficient amounts of DHT by peripheral conversion to induce phallic growth and other signs of masculinization. A remarkable feature of this form of 46,XY DSD is the change in gender identity by many from female to male at puberty.

In the first 1 to 3 months of life after the onset of puberty, patients with 5α-reductase-2 deficiency have normal to elevated T concentrations and slightly elevated plasma concentrations of LH. As expected, plasma DHT is low, and the T/DHT ratio is abnormally high. Apparently, lack of 5α reduction of T to DHT in utero, during the critical phases of male sex differentiation, results in incomplete masculinization of the urogenital sinus and external genitalia. However, T-dependent Wolffian structures are normally developed. Partial and mild forms of 5α-reductase deficiency have been described, in which affected individuals can present with hypospadias and/or microphallus. Compound heterozygotes for 5α-reductase-2 deficiency are described who had a hypospadias repair in infancy and, as adults, were fertile.

Diagnosis: 5α-Reductase-2 deficiency should be suspected in 46,XY DSDs with a blind vaginal pouch and in males with hypospadias and microphallus. The diagnosis can be confirmed by demonstration of an abnormally high plasma T/DHT ratio, either under basal conditions (>10.5) or after hCG stimulation (>8.5). The T-DHT levels need to be measured accurately, preferably by LCMSMS. Other confirmatory findings, especially in newborns, include an increased 5β/5α ratio of urinary C19 and C21 steroid metabolites. 5α-reductase deficiency should be ruled out in all 46,XY DSDs who have a normal or elevated T response to hCG. In the event that the T/DHT ratio in blood or the 5β/5α ratio of urinary steroids is not diagnostic, genotyping should be performed.

The early diagnosis of this condition is particularly critical. In view of the natural history of this disorder, a male gender assignment is indicated, and DHT or high-dose T therapy should be initiated in order to augment phallic size. Repair of hypospadias should be performed in infancy or early childhood. In patients who are diagnosed after infancy in whom gender identity is unequivocally female after the age of informed consent, genitoplasty, prophylactic orchiectomy, and estrogen substitution therapy are still the treatments of choice.

Disorders of Androgen Action (Androgen Receptor and Postreceptor Defects)

Syndrome of Complete Androgen Resistance (Insensitivity) and Its Variants

The androgen receptor gene is located on the X chromosome between Xq11 and Xq13. Androgen resistance is transmitted as an X-linked recessive trait. Of note about 30% of mutations occur spontaneously. The gene is composed of eight exons. Exon 1 encodes the amino terminal end of the androgen receptor protein and is involved in the regulation of transcription. Exons 2 and 3 encode the DNA-binding zinc finger. The 5′ portion of exon 4 is called the hinge region and plays a role in nuclear targeting. Exons 5 to 8 specify the carboxyl terminal portion of the androgen receptor—the androgen-binding domain. The gene has eight serine residues that can be phosphorylated, three lysine residues that can be acetylated, and two lysine residues that can be sumoylated. More than 600 mutations in the androgen receptor are described.

The syndrome of complete androgen resistance (insensitivity) is characterized by a 46,XY karyotype, bilateral testes, absent or hypoplastic Wolffian ducts, female-appearing external genitalia with a hypoplastic clitoris and labia minora, a blind vaginal pouch, and absent or rudimentary Müllerian derivatives. Testes are present in the labial folds, inguinal canal or intra-abdominally. At puberty, female secondary sexual characteristics develop, but menarche does not ensue. Pubic and axillary hair is usually sparse and in one-third of patients is totally absent. Affected patients are taller than average females (mean height 162.3 cm). Some patients have a variant form of this syndrome and exhibit slight clitoral enlargement. These patients may exhibit mild virilization in addition to the development of breasts and a female habitus at puberty.

Androgen resistance during embryogenesis prevents masculinization of the external genitalia and complete differentiation of the Wolffian ducts. The finding of Wolffian duct derivatives in some patients with apparent complete androgen resistance has been attributed to mutant receptors that have some residual activity and can respond to high local levels of T in vivo or to cofactors which modulate androgen sensitivity. Secretion of AMH by the fetal Sertoli cells leads to regression of the Müllerian derivatives—the ducts, uterus, and upper one-third of the vagina. At puberty, androgen resistance results in augmented LH secretion with increases in T secretion and estradiol production due to lack of T feedback on the hypothalamus. FSH levels are normal because of inhibin B and estradiol feedback. Estradiol arises mainly from peripheral conversion of T and androstenedione as well as from direct secretion by the testes. Androgen resistance, coupled with increased testicular estradiol secretion and conversion of T to estradiol, results in the development of female secondary sexual characteristics at puberty. The timing of the onset of the pubertal growth spurt is similar to that in normal females.

Patients with complete androgen resistance exhibit heterogeneity in DHT binding to the androgen receptor. Receptor-negative and receptor-positive individuals with qualitative defects such as thermolability, instability, and impaired binding affinity as well as individuals with presumed normal binding are reported. Analysis of the androgen receptor gene in individuals with androgen resistance has shed light on the heterogeneity of DHT binding. Patients with the receptor-negative form of complete androgen resistance usually have point mutations or substitutions in exons 5 to 8, which encode the androgen-binding domain of the receptor; a striking number of missense mutations occur in the ligand-binding region of the androgen receptor.

Other genetic defects such as deletions, mutations in a splice donor site, and nonsense mutations causing premature termination codons are less common in this group of patients. Mutations in exon 1 commonly cause complete androgen resistance; almost all code for a premature stop codon that results in a truncated nonfunctional androgen receptor. Mutations in exon 3 (which encodes the DNA-binding segment of the androgen receptor) are associated with normal binding of DHT to the receptor but the inability of the ligand-receptor complex to bind to DNA and initiate mRNA transcription. These mutations result in receptor-positive complete androgen resistance. The phenotype of the affected patient correlates better with the transcriptional activity of the ligand-androgen receptor complex than with in vitro studies of the androgen receptor–binding activity.

Other factors that play a role in genotype-phenotype variations include somatic mosaicism, transcriptional coregulatory proteins, and other modifier genes. Somatic mutations—mutations that occur after zygote formation—result in the presence of both wild-type and mutant androgen receptor. Individuals with a somatic mutation may exhibit virilization at puberty owing to the presence of the wild-type receptor, even though some of these patients may present in infancy with the clinical picture of apparent complete androgen resistance.

Diagnosis: In the absence of a family history, the presence of a testis-like mass in the inguinal canal or labia majora may be the only clue to the diagnosis. Approximately 1% to 2% of females with inguinal herniae have androgen resistance. Postpubertally, the patients present with primary amenorrhea, normal breast development, and absent or sparse pubic or axillary hair. Pelvic examination or ultrasound confirms the absence of a cervix and uterus.

The complete and incomplete forms of androgen resistance must be distinguished from other forms of 46,XY DSD due to androgen deficiency, such as biosynthetic defects in T synthesis and 5α-reductase deficiency. There is no readily available, rapid, in vivo or in vitro assay to determine androgen sensitivity. The diagnosis is suggested by the clinical picture, the family history, and the presence of elevated basal and hCG-induced T levels with normal levels of DHT. Neonates with complete androgen resistance do not have elevated basal LH and T levels and do not experience a postnatal rise in these values, as is observed in normal infants and in those with incomplete androgen resistance. It has been suggested that an elevated AMH concentration is a marker of androgen resistance and androgen deficiency. It is not specific, as it may be elevated in other causes of 46,XY DSD. Defective or absent androgen receptor expression in Sertoli cells is responsible for the absence of AMH suppression. Determination of abnormalities in androgen binding as well as transactivation studies are diagnostic, but they are labor intensive and not universally available, whereas mutational analysis of the androgen receptor gene is commercially available.

Management: The patients with complete androgen resistance, as with most other patients with DSD, is complex. It requires a team of experienced physicians—an endocrinologist, psychologist and/or psychiatrist, a surgeon, and a social worker, as well as a primary care physician. When the diagnosis is definitive, the parents need to be fully informed about the karyotype, genetics, pathophysiology, medical and surgical options, and the natural history of complete androgen resistance. They should be informed that so far, except for very rare patients, gender identity, role, and behavior are female. Inguinal herniae, if present, need to be repaired. A gonadal malignancy rate of 1% to 2% is present after 14 years of age. Thus, it is prudent to allow these girls to feminize spontaneously at puberty and after full disclosure, discuss the question of prophylactic gonadectomy with them and their parents (who have been previously informed of this possibility). Spontaneous feminization at puberty may help to reinforce their sense of being a normal although infertile female. Allowing them to make an informed decision about gonadectomy reinforces their sense of autonomy. Hormonal replacement will be necessary postgonadectomy.

Postpubertally, the functional adequacy of the vagina should be assessed. Vaginoplasty is rarely necessary. Vaginal dilation with plastic molds is invariably successful in augmenting vaginal size and should be initiated if necessary postpubertally with the patient's informed consent.

The continuing involvement of the psychologist or psychiatrist will facilitate the question of when, how, and by whom the patients should be fully informed of their karyotype, infertility, and therapeutic options. There is no mandated guideline for this, and the approach needs to be individualized for each patient and family. In general, information is given in an age-appropriate way, taking into consideration the patient's age, cognitive abilities, and psychologic development. It is a gradual process, done in an understandable, caring way, which culminates in full disclosure. Continuing psychologic/psychiatric counseling should be available concerning sexual relationships and psychosocial issues. The Androgen Insensitivity Society (http://www.aissg.org) is a valuable support group for patients as well as parents and provides information, experiences from older, affected patients and parents, and the ability to meet and talk with females who have similar DSDs.

Syndrome of Partial Androgen Resistance (Insensitivity) and Its Variants (Reifenstein Syndrome)

Patients with partial androgen resistance manifest a wide spectrum of phenotypes as far as the degree of masculinization is concerned. The external genitalia at birth are classically characterized by perinoscrotal hypospadias, micropenis, and a bifid scrotum. The testes may be undescended. Isolated hypospadias may occur in the mildest form of partial androgen resistance. There is variability of masculinization of affected males even within kinships. Müllerian duct derivatives are absent and Wolffian duct derivatives are present, but they are usually hypoplastic. At puberty, virilization usually recapitulates that seen in utero and is generally poor; pubic and axillary hair as well as gynecomastia are usually present. The most common phenotype postpubertally is the male posthypospadial repair with a small phallus and gynecomastia. Axillary and pubic hairs are normal. The testes remain small and exhibit azoospermia as a consequence of germinal cell arrest. Unlike patients with complete androgen resistance, in infancy the levels of plasma LH and T are elevated. Postpubertally, as in individuals with complete androgen resistance, LH, T, and estradiol are elevated. However, the degree of feminization in these individuals, despite increased estradiol levels, is less than that found in those with the syndrome of complete androgen resistance.

Androgen receptor studies in these patients have usually shown quantitative or qualitative abnormalities in androgen binding. Mutations leading to partial reduction of androgen action result in incomplete virilization. As previously noted, the best correlation with the phenotype is the degree of impairment of transcriptional activity of the ligand-androgen receptor complex. A wide variety of androgen receptor gene mutations may result in the same phenotype, and a specific mutation is not always associated with the same phenotype. In general, point mutations that result in more conservative amino acid substitutions are more likely to result in partial rather than complete androgen resistance. In 111 individuals with partial androgen resistance in which DNA was sequenced, only 27 (24%) had an identifiable mutation. The majority of the patients with mutations had abnormal DHT binding to the androgen receptor.

Diagnosis: The differential diagnosis of patients with ambiguous genitalia and absent Müllerian structures includes partial androgen resistance, all the forms of dysgenetic 46,XY DSD, Leydig cells unresponsive to hCG/LH, biosynthetic errors in T synthesis, 5α-reductase deficiency, and SF-1 mutations. These entities may be parsed by steroid analysis in response to ACTH and hCG stimulation. A protocol frequently used for hCG stimulation is as follows: (1) a baseline LH, FSH by ICMA, AMH, inhibin B, androstenedione, T/DHT; (2) 2000 U/m2 hCG × 3 days; (3) serum androstenedione, T/DHT, and urine for steroid analysis 24 hours after the third dose of hCG. hCG will elicit an augmented T response in patients with partial androgen resistance and 5α-reductase deficiency. 5α-Reductase deficiency can be generally ruled out by a T/DHT ratio <8.5 after hCG or by the ratio of 5β/5α steroid metabolites in urine. However, in some reported cases with mild mutations, only genotyping was diagnostic. All the above entities, except for partial androgen resistance, will result in a "normal" penile response to a trial of administration of T enanthate in oil, 25 mg intramuscularly × 3 months in infants. We have utilized this test to diagnose partial androgen insensitivity syndrome (PAIS) before assigning a definitive sex of rearing.

Follow-up data have indicated dissatisfaction in many patients with PAIS, whether they are raised as males or females. The prevalence of gender dysphoria in partial androgen resistance is estimated at 10%. In individuals reared as males, a major decrease in all aspects of sexual function has been documented. If a decision is made to raise the infant in a female sex role, then gonadectomy prior to puberty or GnRH therapy pending definitive ascertainment of gender identity is indicated to avoid the possible risk of signs of virilization at puberty. In view of the incidence of gender dysphoria and the apparent unhappiness about their assigned sex in individuals with partial androgen resistance, a male sex of rearing is a judicious choice in individuals with Prader III or greater ambiguous genitalia or hypoplastic male genitalia because it preserves a choice. In some patients, high doses of T has engendered penile enlargement. Psychologic/psychiatric consultation is important in these individuals whether raised as males or females, since available follow-up studies indicate dissatisfaction with the outcome when they are assigned a male or female sex of rearing.

Androgen Resistance in Men with Normal Male Genitalia

Partial androgen resistance has been described in a group of infertile men who have a normal male phenotype but may exhibit gynecomastia. Unlike other patients with androgen resistance, some of these patients have normal plasma LH and T levels. Infertility in otherwise normal men may be the only clinical manifestation of androgen resistance. Infertility, however, is always associated with androgen resistance. Five phenotypic males in one affected family had gynecomastia and a small penis. Plasma LH and T levels were elevated, and a subtle qualitative abnormality in ligand binding was found. Fertility was documented in four of the five males. This is the mildest form of androgen resistance.

Dysgenetic 46,XY DSD (Ambiguous Genitalia Due to Dysgenetic Gonads)

See previous discussions of sex determination, XY gonadal dysgenesis, XO/XY mosaicism, Denys Drash/Frasier/WAGR syndromes, etc.

Testicular Regression Syndrome (Vanishing Testes Syndrome; XY Agonadism; Rudimentary Testes Syndrome; Congenital Anorchia)

Cessation of testicular function during the critical phases of male sex differentiation can lead to various clinical syndromes depending on when testicular function ceases. At one end of the clinical spectrum of these heterogeneous conditions are the XY patients in whom testicular deficiency occurred before 8 weeks of gestation, which results in female differentiation of the internal and external genitalia—so-called XY gonadal dysgenesis. At the other end of the spectrum are the patients with anorchia or vanishing testes in which the testes are lost later in gestation after the critical period of male genital differentiation from 8 to 12 weeks. These patients have normal male differentiation of their internal and external structures, but gonadal tissue is absent. The phallus varies in size dependent upon when Leydig cell failure occurs. Testicular regression may occur unilaterally especially on the left side or bilaterally, and the condition is seen in family cohorts. The latter suggests a genetic etiology, but only one individual with a mutation in the gene (SF-1) has been reported.

Diagnosis: Bilateral anorchia should be suspected in all 46,XY phenotypic males with undescended testes especially those with micropenis. MRI usually reveals absence of the gonads in the inguinal canal and no Müllerian duct derivatives. Plasma gonadotropins, especially FSH are elevated under 4 years of age and after 10 years of age. Inhibin B and AMH levels, measures of Sertoli cell integrity, are unmeasurable and a lack of rise in T at 1 to 3 months—"the window of opportunity"—will be evident. The absence of Leydig cell function is documented by the failure of the administration of hCG to elicit a rise in T. Finally, laparoscopic exploration, if necessary, indicates that 90% of patients lack testicular tissue, and in 10%, a testicular remnant without germ cells is present. Treatment of these males includes T therapy in childhood for microphallus, and at 12 to 13 years administration of T to induce male secondary sex characteristics and, thereafter, as replacement therapy.

Persistent Müllerian Duct Syndrome (Defects in the Synthesis, Secretion, or Response to Antimüllerian Hormone)

This male-limited autosomal recessive syndrome is associated with normal male development of the external genitalia but with Müllerian duct derivatives. The retention of Müllerian structures is either due to failure of the Sertoli cells to synthesize and secrete AMH or to an end-organ defect in the response of the Müllerian duct to AMH. The gene for AMH maps to chromosome 19; a variety of mutations in the AMH gene cause the syndrome as well as mutations in the gene encoding the AMH receptor. Individuals with mutations in the AMH receptor have Müllerian ducts, despite the normal to high concentrations of plasma AMH. Therapy involves removal of the Müllerian structures.

Environmental Chemicals

An increase in disorders of development and function of the urogenital tract in males has been noted over the past 50 years. It has been hypothesized that this increased incidence of reproductive abnormalities observed in human males is related to increasing exposure in utero to endocrine disrupters especially estrogens and androgen receptor blockers found in the diet both naturally and as a result of chemical contamination. It has been demonstrated that p,p′-DDE (dichlorodiphenyldichloroethylene)—the major and persistent DDT metabolite—binds to the androgen receptor and inhibits androgen action in developing rodents. Other chemicals such as phthalates, herbicides, and fungicides inhibit androgen actions. The term testicular dysgenesis syndrome describes a phenotype that has been attributed to endocrine disrupters. Further studies on the levels as well as the risks to humans of environmental chemicals and other endocrine disruptors are necessary before abnormalities of the reproductive tract can be confidently ascribed to these agents.

Hypospadias

Hypospadias is one of the most common genital abnormalities. It has an incidence of 1 in 125 live male births in the United States. It is often associated with ventral curvature of the penis, a condition called chordee. The aberrant urethral meatus can open anywhere along the shaft of the penis, penoscrotally (at the base of the shaft), or even on the perineum.

The etiology of hypospadias is multifactorial, and both genetic and environmental factors appear to play a role in its pathogenesis. Deficient virilization of the male genitalia implies subnormal fetal testosterone production or action at the end organ. Hence, mutations in genes such as WT1, SF-1, SRY, SOX9, and DHH can result in dysgenetic 46,XY DSD with varying degrees of hypospadias and other genital abnormalities. Mutations in genes that encode steroidogenic enzymes (eg, 3β-HSD, 17α-hydroxylase, 17,20-lyase, POR, and SF-1) or those that affect androgen action and lead to partial androgen resistance as well as 5α-reductase-2 deficiency are all uncommon causes of hypospadias. In the majority of cases of isolated hypospadias with no other genital abnormality, the etiology is uncertain. Hypospadias is also a component of a number of dysmorphic syndromes, including Smith-Lemli-Opitz syndrome (a defect in cholesterol synthesis), as well as hand, foot, and genital syndrome due to a mutation in HOX13.

Several paternal and maternal risk factors have been associated with the occurrence of hypospadias in a fetus. These include subfertility and/or genital anomalies in the father, maternal age, IUGR, and fetal exposure to oral progestins or combined progestins and estrogens. These all are associated with an increased risk of hypospadias. Ten percent of hypospadias cases occur in familial cohorts. An increase in hypospadias has also been noted in infants conceived by in vitro fertilization. However, advanced maternal age and subfertility, as well as the administration of progestational agents to the mother, may play a role in these patients.

There is a 15% risk of hypospadias in the sibling of a child with hypospadias and a 7% risk if the father has hypospadias, suggesting a genetic etiology. A mutation in a gene called MAMLD1 (mastermind-like domain containing 1, previously known as CXorf6 (chromosome X open reading frame 6), which appears to impair T secretion during the critical period (8-12 weeks) of male external genital differentiation, results in hypospadias.

Analysis of international demographic trends for hypospadias indicates that it is highly variable in different countries worldwide. The prevalence of hypospadias in Denmark is 1.1%, which is significantly higher than the prevalence in other Scandinavian countries, especially Finland. Hypospadias was associated with fetal growth retardation (small for gestational age), lower placental weight, and elevated plasma FSH levels at 3 months. Androgen receptor mutations were not found, and the CAG repeats in the androgen receptors were normal for the Danish population. It was suggested that environmental exposure is a likely explanation, but data of environmental exposure including demographic differences in endocrine disruptors (eg, organochloride, phalathes, xenoestrogens) are lacking.

Patients with severe hypospadias (perineoscrotal) especially those with micropenis and/or cryptorchidism should be evaluated. The evaluation should include gonadotropin, AMH, inhibin B, and an assessment of testosterone secretion either during the "window of opportunity" at 1-3 months of age or in response to hCG stimulation.

Micropenis

Micropenis without hypospadias results from a heterogeneous group of disorders; the most common cause is fetal T deficiency. Micropenis is rarely associated with 5α-reductase deficiency or mild defects in the androgen receptor (Table 14–4). In the human male fetus, T synthesis by the fetal Leydig cell during the critical period of male differentiation (8-12 weeks) appears to be autonomous at first, then influenced by hCG. After midgestation, fetal pituitary LH modulates fetal T synthesis by the Leydig cell and, consequently, affects the growth of the differentiated penis. Thus, males with congenital hypopituitarism or isolated gonadotropin deficiency and late fetal testicular failure (after 8-12 weeks) present with normal male differentiation and micropenis at term birth (by definition a penis length <2.5 cm) (Table 14–5).

Evaluation of the infant with micropenis should include anterior pituitary functions (ie, determination of the plasma concentration of GH, prolactin, ACTH, cortisol, thyroid-stimulating hormone, thyroxine, and gonadotropins), to rule out multiple pituitary hormone deficiencies. In infancy, there is a "window of opportunity" at 1 to 3 months of age when the perinatal rise in gonadotropin and T/DHT occurs. A lack of rise in T/DHT and LH and FSH suggests hypogonadotropic hypogonadism, while an abnormally high (>10.5 on unstimulated sample) T/DHT ratio suggests 5α-reductase-type 2 deficiency. All patients with micropenis should receive a trial of T therapy to ascertain the phallic response and to augment its length into the normal range for age. Infants with fetal T deficiency as a cause of micropenis—whether due to gonadotropin deficiency or to a primary testicular disorder—respond to 25 mg of T enanthate in oil intramuscularly monthly for 3 months with a mean increase of 2 cm in penile length. Older children require higher doses—50 mg intramuscularly monthly × 3 doses. A long-term study of eight males with micropenis, due to congenital hypogonadotropic hypogonadism who were followed in our clinic, revealed that fetal deficiency of gonadotropins and T did not prevent the penis from responding to T in infancy and at the age of puberty. Final penile length for all patients who were treated with one or more short courses of repository T in infancy or childhood and with replacement doses of T in adolescence was in the normal adult range. Furthermore, these patients had a male gender identity, erections, ejaculation, and orgasm. No clinical, psychologic, or physiologic indication supports conversion of males with micropenis, due to diminished fetal T secretion, to females.

Aphallia

Complete absence of the phallus is a rare anomaly. The urethra may open on the perineum or into the rectum. Assignment of a female sex of rearing, castration, and plastic repair of the genitalia and urethra has been the approach followed in the past; however, this policy is currently questioned by some investigators because of concern about the action of normal male fetal levels of circulating T on the fetal brain and its potential effect on postnatal psychosexual development and gender identity. There are no long-term follow-up studies of gender identity in patients with aphallia. A study investigated 46,XY patients with cloacal extrophy who were castrated in the neonatal period and assigned a female gender role. Fifty percent of this cohort announced their gender identity as male in childhood or adolescence. These data suggest that prenatal exposure to normal amounts of T during gestation is probabilistic but not deterministic in the establishment of gender identity.

Müllerian duct agenesis is characterized by congenital absence of the uterus and the upper one-third of the vagina and is termed the Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Ovarian function is usually normal. If Müllerian agenesis is associated with renal abnormalities and cervicothoracic dysplasia, it is referred to as the MURCS syndrome. Patients with the MRKH syndrome or the MURCS syndrome have not as yet had genetic mutations identified, although familial cases have been reported. Recently, an atypical form of the MRKH syndrome has been described in three patients who had primary amenorrhea with normal breasts and pubic hair development, lack of Müllerian duct development, and clinical and biochemical evidence of androgen excess. All three patients had a heterozygous mutation of the WNT4 gene. These patients illustrate the role of WNT4 in Müllerian duct development in humans as well as in inhibiting Leydig cell development and androgen synthesis by the ovary.

Features Which Suggest a DSD

A DSD should be suspected in apparent females with clitoromegaly (>0.9 cm) at birth in those with a mass in the labia, inguinal canal or with an inguinal herniae, and in those with edema of the distal extremities and/or loose skin folds over the nape of the neck. A DSD should be suspected in infants with truly ambiguous genitalia, in putative males with cryptorchidism, micropenis (<2.5 cm), and hypospadias, especially perineal, siblings or relatives of patients with DSDs, and in infants whose prenatal genital appearance by sonography is not congruent with their karyotype on amniocentesis.

The evaluation and management of the patient with atypical genitalia is complex and best undertaken by a team consisting of a pediatric endocrinologist, psychiatrist or psychologist, pediatric surgeon or urologist, social worker, religious counselor if appropriate, and an informed primary care physician at a center of excellence. The goal of management of patients with atypical genitalia is to establish an etiologic diagnosis promptly and with the informed consent of the parents, assign a sex of rearing which is most compatible with the prospect for a stable gender identity, well-adjusted life, sexual adequacy, and fertility if possible. Steps in the diagnosis of the infant with a DSD are set forth in Figures 14–16 and 14–17.

Before any decision about sex assignment is made, a thorough, comprehensive evaluation must be undertaken. The diagnostic evaluation should include a karyotype, pelvic sonography and determination of gonadotropins, and adrenal and gonadal steroids, AMH, and inhibin B levels. Depending on the karyotype and ultrasound, further analysis of steroids after hCG and ACTH stimulation may be indicated (see Figure 14–17). In all 46,XY DSDs with a micropenis (Tables 14–4 and 14–5), it is prudent to assess the phallic response to T to exclude partial androgen resistance before the decision about definitive sex assignment is made by the parents. Clitoral enlargement (Table 14–6) in 46,XX females suggests either an endogenous or exogenous source of androgens.

Gender identity—the identification of oneself as either male or female—is a complex and incompletely understood dimorphic phenomenon. During the last 20 years, it has been increasingly evident that both nature (eg, prenatal exposure to T) as well as genes on the X and Y chromosome, and nurture (sex assignment, psychosocial and experiential factors) play a role in determining one's gender identity. In any given infant, one can never be sure whether nature or nurture will be deterministic or prevail. Thus, physicians are faced with the daunting task of helping parents to make a critical decision about the sex of rearing of their child in the face of incomplete understanding of the complexity of gender identity. The task is further complicated by the limited ability to make a definitive etiologic diagnosis in many 46,XY DSDs, as well as by the lack of outcome data that incorporate modern guidelines, recent surgical advances, and advances in psychologic/psychiatric support for both the child and the parents.

It is the physician's and consultant's tasks to evaluate the patient thoroughly and to fully inform the parents. The ultimate decision of sex of rearing is made by the completely well-informed parents. It must be clearly stated which anatomic abnormalities can be surgically repaired, that hormone therapy can be given at an appropriate time and that continuing psychosocial support will be available both from the physicians involved and from community-based support groups. Repeated, lucid, simple, comprehensive discussions about the cause of their child's atypical genitalia and what is known about the long-term follow-up of similar patients with DSD, as well as the results of reconstructive surgery in terms of sexual gratification and fertility, must be discussed. The discussion needs to take into account parental concerns, religious views, cultural factors, social mores, and the parents' level of understanding. The parents need to be assured that an expeditous, definitive diagnosis can be made, but that naming the baby, sending out birth announcements, and filing the birth certificate may need to be delayed. They will need support, reassurance, and counseling as to how to deal with relatives and others.

In the individual with atypical genitalia, the option of assigning a sex of rearing but deferring surgery unless it is medically necessary until the patient has the opportunity to give his or her informed consent, has been proposed by advocacy groups and espoused by some DSD clinics. However, it should be noted that there are no data on patients with DSD and atypical genitalia to support the efficacy of this approach. On the other hand, a long-term study of 41 patients with DSD suggested that early surgical intervention accompanied by endocrinologic and psychologic/psychiatric support for the patient and parents resulted in positive outcomes in terms of cosmesis, minimal impairment in quality of life and appropriate gender identity. We concur with this position and feel that it is desirable to initiate plastic repair of the external genitalia as soon as is feasible with the informed consent of the parents. Surgeons experienced in genital repair recommend a one-stage repair by 4 to 6 months of age. In children raised as females, clitoroplasty should be performed only by experienced surgeons with the emphasis being on function rather than cosmesis. We do not recommend routine clitoroplasty for mild to moderate clitoromegaly (Prader II or III). Vaginoplasty can be deferred until adolescence when the patient may participate in the decision for surgery. Each patient is unique and must be treated as such. There is no rubric that can be applied to the management of all patients. However, the following are a few guidelines.

Male sex assignment is recommended in 46,XY DSDs, with the exception of those with complete androgen resistance syndrome; those who have completely female external genitalia; those with partial androgen resistance, who have less than Prader III ambiguity; or those who have a compelling reason for female sex assignment, including the parents' informed decision. In the latter case, extensive discussion with the family is especially warranted, particularly in the 46,XY infant whose ambiguous genitalia is evidence of significant fetal T exposure. The parents need to be aware of the effect of T during gestation on gender identity and counseled about the possibility of gender dysphoria if the child is assigned a female sex of rearing. In this case, it may be prudent to do no surgery unless medically indicated, until the child's gender identity can be verified, and informed consent for surgery obtained from the patient. In patients with XO/XY mosaicism, 17β-HSD-3 deficiency, and 5α-reductase deficiency diagnosed in infancy, the significant occurrence of female to male gender role change should be discussed with the parents. In some societies, the social, cultural, and economic benefits of a male sex assignment are more compelling than phallic adequacy and are a prevailing—if not the most important—factor in the parents' sex of rearing decision. Male sex assignment in 46,XY DSD individuals, especially those who can respond to T allows for a choice later in adolescence, by not removing functional genital structures which cannot be replaced.

All 46,XX DSDs with Prader I to IV (see Figure 14–13) external genitalia should be raised as females. 46,XX DSDs with Prader V genitalia (penile urethra) represent a management dilemma. As with other forms of DSD, there are at present insufficient long-term outcome data on these patients. Raising the child as male would require gonadectomy of a potentially fertile female. These infants are raised as females if diagnosed in infancy. If diagnosed after infancy and a male gender assignment has been made, a full discussion with the parents and psychologic/psychiatric evaluation of the child should ensue before any definitive decisions are made regarding gender assignment. Reassignment of sex in childhood is always a difficult psychosocial problem for the patient, the parents, and the physicians involved. Although easier in the infant under 1 year of age, it should only be undertaken after much deliberation and with provision for long-term medical and psychiatric supervision and counseling. 46,XX males and 46,XX ovotesticular DSDs with male genitalia and absent Müllerian ducts are exceptions to the suggestion that a female sex assignment be considered in 46,XX DSDs.

Psychologic studies in 46,XX DSD secondary to CAH suggests that the vast majority of 46,XX DSDs with CAH have a female gender identity. In some individuals, sex-specific behavior may be more masculine. The incidence of bisexuality and homosexuality is increased. In one study, the prevalence of bisexuality and homosexuality correlated modestly with the degree of masculinization of nonsexual behavior and the genitalia.

Removal of rudimentary gonads in children with Y-containing cell lines and gonadal dysgenesis should be performed at the time of initial repair of the external genitalia; gonadoblastomas, seminomas, and germinomas can occur during the first decade (see Table 14–2). Histologically and functionally normal scrotal testes can be retained in 45,X/46,XY males and others with regular monitoring as previously discussed.

In a patient with complete androgen resistance, the testes may be left in situ (provided they are not situated in the labia majora) to provide endogenous estradiol at puberty. There is one possible exception that of the rare somatic mosaics for the androgen receptor who are at risk of virilization at puberty, due to the presence of a population of cells with normal androgen receptors. Having had the female identity reinforced by normal feminization at puberty, prophylactic castration can then be performed, if informed consent by the patient and parents is obtained.

In patients with partial androgen resistance reared as females or in patients with errors of T biosynthesis reared as females in whom some degree of masculinization occurs at puberty, orchidectomy should be performed before the age of puberty or later if doubt exists about the gender identity. GnRH agonists can be given to prevent androgen secretion and virilization until the gender identity is confirmed. Cyclic estrogen and progestin replacement therapy are used in individuals reared as females in whom a uterus is present. In males, masculinization is achieved by the administration of repository or topical preparations of T.

Continuing endocrinologic and psychologic support are critical aspects of follow-up and should be available throughout infancy, childhood, adolescence, and adulthood for the patient as well as for the parents. Patients should have progressive, step-by-step, age-appropriate discussions about their diagnosis, its pathophysiology, their quality of life, and the potential for fertility. Disclosure is critical! However, in those mature adult DSD patients who are well-adjusted and happy with their lives and their assigned sex and who in the past have not had the opportunity of having full disclosure, disclosure may be on a need to know basis. Patients have access to their medical records. It is infinitely better to proceed with full disclosure if the patient has expressed a desire to know more about their diagnosis and management. Full disclosure should be approached in a similar manner as that carried out with parents and with the cooperation and help of a psychologist or psychiatrist familiar with the patient and issues of gender identity. The patient (when possible) and the parents should always be involved in decisions about surgery and sex hormone replacement.

In sum, the physician and the medical team concerned with the diagnosis, selection of sex of rearing, and management of the infant with DSD must be prepared to address the complex ethical, cultural, social, religious, clinical, legal, and surgical issues presented by the DSD patient in order to maximize the patient's potential for a well-adjusted, normal life. This task remains complicated by deficiencies in the availability of long-term outcome data as well as the inability to diagnose specifically many patients with a DSD especially those with a 46,XY karyotype.