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SEXUAL DIFFERENTIATION Sexual Differentiation 1 Normal and Abnormal Sexual Differentiation At conception, the embryo has bisexual potential. Each gonad can develop into either a testis or an ovary. Internal ductal primordia are present for both Mullerian and Wolffian systems and the external genitalia can either feminize or masculinize. An orderly sequence of embryonic events is required for normal sexual differentiation of the gonads, internal ductal system. and the external genitalia. The timetables of normal male and female sexual differentiation (I) are provided to Figs. 16.1 and 16.2. For recapitulation of the species, this is probably the most important embryologic process. Our genetic blueprint ensures that sexual differentiation in utero occurs flawlessly time and again such that abnormalities of sexual differentiation are only rarely noted at birth. A wide spectrum of intersex disorders has been described (Table 16.1), with separate defects occurring at nearly every step in the process depicted in Figs. 16.1 and 16.3 (1). These defects result in very predictable abnormalities of the gonads. internal ductal systems, and external genitalia. When faced with the clinical dilemma of a child with ambiguous genitalia. the clinician can work backward. By determining the nature and location of external genitalia, internal ductal systems, and gonads, the physician can often make an initial differential diagnosis with a minimum of ancillary testing. Despite the fact that most intersex disorders are associated with some degree of ambiguity and sterility, the psychosocial considerations involved have potentially far more deleterious ramifications than these physical changes. Delivering a child with ambiguous genitalia should be considered a medical emergency. Expeditious evaluation and concomitant counseling, early diagnosis and assignment of sex-of-rearing, and initiation of treatment during the birthing hospitalization is essential for meeting all of the emotional and medical needs of these individuals and families. The age of molecular medicine has allowed for rapid testing and decision making during the first 24 hours of life. Furthermore, prenatal ultrasound screening can identify at-risk fetuses even before delivery. However. none of this technology can substitute for the informed and experienced counseling that is necessary in the care of these infants from the time of identification through attainment of adulthood at the conclusion of puberty (2). NORMAL SEXUAL DIFFERENTIATION At the time of conception, the embryo has the potential to develop into either a male or a female. The gonads can differentiate to the medullary region along testicular lines or in the cortical areas as ovaries. Primordia for both the Wolffian and Mullerian systems are present from the beginning of sexual Sexual Differentiation 2 differentiation. The Mullerian system will continue to grow and develop unless it is ablated by the action of Mullerian-inhibiting substance (MIS). The Wolffian system, in contrast. will ultimately require androgen stimulation for completion of its development. The external genitalia of any fetus can either masculinize or feminize. Two principal differences exist between the development of male and female fetuses. First, testicular determinant genes must be present and active in order for testicular development to begin. In contrast, ovarian determinant genes need only be present at later stages of ovarian development. In the absence of testicular determinants and absence of other major ovarian genetic stimuli, ovarian development will begin. This occurs independently of the genetic sex of the embryo. The second principal difference between male and female sexual development is that the testis is necessarily an endocrine organ during sexual differentiation while the developing ovary is relatively endocrine quiescent. In the male the character of the internal and external genitalia depends on testicular steroid (i.e., testosterone and dihydrotestosterone [DHT]) and nonsteroid (e.g., MIS) hormone production and secretion. For the female. internal and external genital development occurs in the absence of ovarian hormones. Male sexual differentiation The process of male sexual differentiation m utero is well characterized (see Fig. 16.1). A cascade of molecular events muse occur to permit development of the testes, the Wolffian system, and the external genitalia. Genes controlling the major steps in male sexual differentiation and the ultimate fate of each of these systems in male reproductive morphogenesis are largely characterized. The initial switch for these events appears to be the Y -chromosomal SRY gene (3,4). A gathering body of data supports the idea that lust as has been characterized for invertebrates, a host of genes downstream m expression from SRY exist and act in concert for gonadal morphogenesis. That other genes exist is supported by the faces that most 46.XY sex-reversed females do not have SRY mutations. some 46,XX sex-reversed males do not have SRY present, and nearly all 46,XX true hermaphrodites lack SRY It appears that the protein produce of SRY is a transcription factor that acts as the initial switch for turning on a number of other genes (5). The protein products of these genes that likely act downstream in expression to SRY are required for formation of the testis and perhaps regulation of internal ductal development. Candidates for such downstream genes include MIS (6), dosage-sensitive sex-reversal gene (DSS) (7), SOX9 (8,9), and the Wilms' tumor or WT-I Sexual Differentiation 3 gene. The SRY protein has been observed to bind with the MIS promotor. DSS represents a gene locus on the short arm of the X chromosome which, when duplicated in 46,XY individuals. is associated with sex reversal. Mice with deletions of WT-1 do not form mesoderm for gonads and this may represent an upstream rather than a downstream gene in expression to SRY. Individuals with the 46,XY karyotype and autosomally inherited campomelic dysplasia present with sex reversal m addition to having a broad spectrum of bone abnormalities (8.9). The translocation breakpoints of three unrelated patients with this syndrome have all mapped to 17q24.3-q25.1, a locus referred to as the autosomal sex-reversal locus, SRA1. SOX9 is an SRY-related gene that has been mapped to this region of 17q. Patients with campomelic dysplasia harbor mutations in this locus (8,9). It appears that this gene is involved both m testicular formation and in bone morphogenesis. Concomitant with the initial genetic signaling for testicular morphogenesis, the primordial germ cells travel by amoeboid motion from the yolk sac over the dorsal mesentery to the genital ridge. These primordial germ cells increase m number by mitosis and become rapidly enveloped by the developing seminiferous tubules. At the base of these tubules. the Sertoli cells appear. They have many functions and, like the granulosa cells in the ovaries, assume the guardianship of the germ cells. They produce the first hormone of the testis. MIS. This protein has many functions, including arrest of the development of the Mullerian system, in part by apoptosis (10). Shortly thereafter, fetal Leydig cells appear and produce the second set of hormones, the androgens. Steroidogenesis in the fetal Leydig cells is initially stimulated by human chorionic gonadotropin (hCG) and then by fetal luteinizing hormone (LH). The androgen receptor-hormone dissociation constant is lower for DHT than for testosterone. This ensures a longer time spent on the androgen receptor for DHT and significantly greater potency as compared to testosterone. However, during early pregnancy, the human Wolffian system does not have activity for the steroidogenic enzyme, 5α-reductase, m contrast to the external genitalia (11). Testosterone stimulates completion of Wolffian development and DHT is responsible for masculinization of the external genitalia (13). The effects of MIS and testosterone on their respective Mullerian and Wolffian systems appear to be local and unilateral. In a set of classic fetal rabbit experiments, just et al. (13) demonstrated that unilateral removal of a testis from a developing male rabbit resulted in development of a Mullerian system on that side and a Wolffian system on the side of the remaining gonad. Similarly, transplantation of a fetal testis from a male to a position adjacent to a developing ovary in a fetal female rabbit caused regression of the Mullerian system and concomitant 4 Sexual Differentiation stimulation of the Wolffian system on that side. For humans. these same observations have been demonstrated by natural experiments of nature, that is, the existence of patients with discordant gonads such as those with 43,X/46,XY gonadal dysgenesis and some true hermaphrodites. External genital development probably begins by 9 weeks' gestation and is completed during the early part of the middle trimester. It involves labioscrotal fusion concomitant with placement of the urethral groove onto the genital tubercle and subsequent elongation for development of a penis. Testicular descent occurs around 32 weeks gestation and results from development of the gubernaculum and its developmental "pulling" of the testes toward the scrotum. In addition, testosterone appears to be a required hormonal stimulus for descent and some believe that MIS may be involved as well (10). Female sexual differentiation Ernbryogenesis for female sexual differentiation has been similarly well characterized (see Fig. 16.2). While molecular events must mediate growth and development of all female genitalia, it would appear that these genes are broad-spectrum developmental genes and not, as in the male, step-dependent loci. Ovarian development begins in the absence of testicular or ovarian determinant genes. Mullerian development begins and continues unless arrested by MIS, and the external genitalia will feminize in the absence of androgens. Ovarian function during development appears to be exocrine rather than endocrine. Hormone production is negligible and insignificant. More importantly, mitosis of germ cells occurs at a much greater rate in the developing ovary than for the testis. A maximum ovarian endowment of nearly 6 million germ cells is attained by 20 weeks' gestation. Unlike the male in whom germ cell replenishment occurs throughout adult life, the germ cell mass in females undergoes only depletion, beginning during fetal life. The most critical step for ovarian morphogenesis that appears to depend on ovarian determinant genes is preservation of ,,arm cells within the developing ovary. This requires the surrounding of the primordial germ cells by a primitive granulosa cell mantle forming a primitive follicle and she initiation and arrest of meiosis I at prophase (14). These required ovarian determinant genes are located on both arms of the X chromosome and the autosomes as well. Fetuses with the 45,X karyotype of Turners syndrome have as many germ cells at midgestation as do 46,XX fetuses. Most of their germ cells are incompletely surrounded by- the granulosa cell mantle and some have been observed to be physically herniating through these defects of the follicular wall (14). It is also possible that atresia and germ cell loss may be accelerated to them by the lack of sufficient granulosa cell-produced meiosis inhibitor(s). Sisters with the 46,XX karyotype who are concordant for gonadal 5 Sexual Differentiation dysgenesis have been repeatedly reported, suggesting that other genes important to the preservation of the germ cell mass exist upstream or downstream in expression to the X-located ovarian determinants (2). In summary once the primordial germ cells arrive at the undifferentiated gonadal ridge, male sexual development requires 1) the presence and expression of SRY and related testicular genes. 2) the presence of bilateral MIS production. 3) the presence of bilateral testicular testosterone and DHT production, and 4) competent androgen receptors to translate the hormone signal to the end-organ effect of masculinization. Each of the major genes for these steps has been cloned and sequenced, and mutations producing predictable abnormalities have been found. These genes include SPY, MIS, the MIS receptor gene, all of the enzyme genes in steroid biosynthesis, and the androgen receptor (AR) gene. In contrast, once the primordial germ cells arrive at the genital ridge, female sexual differentiation requires only ovarian determinant gene action at a later time in development. Not one of the genes regulating female sexual differentiation has been identified. While male sexual differentiation appears to be a very dependent developmental event, female sexual differentiation occurs relatively independently. Nature has seemingly put into place a fail-safe mechanism such that failures of male sexual differentiation default to a female phenotype. CLASSIFICATION OF PATIENTS WITH ABNORMALITIES OF SEXUAL DIFFERENTIATION Abnormalities can occur at each of the steps involved in normal sexual differentiation (see Figs. 16.1 and 162) (1). Each of these abnormalities may present with predictable alterations of the gonads, internal and eternal genitalia, and pubertal development. An understanding of the molecular nature of many of these defects has broadened the spectrum of presenting phenotypic changes identified for them. Two classifications of patients having abnormalities of sexual differentiation are outlined in Table 16.1 (1). The standard classification system uses the terms pseudohermaphrodite and hermaphrodite. A pseudohermaphrodite is an individual whose external genitalia is contradictory to the karyotype. For example. a male pseudohermaphrodite is a 46,XY individual whose external genitalia is undermasculinized. A female pseudohermaphrodite has a 46.XX karyotype and masculinized external genitalia. A true hermaphrodite is an individual possessing both ovarian and testicular tissue and Sexual Differentiation 6 manifesting varying degrees of genital ambiguity. Gonadal tissue must demonstrate ovarian follicles and seminiferous tubules. Another classification presented in Table 16.1 demonstrates a degree of overlap between some of these syndromes and is based purely on the karyotype of the patient (1). The following summary of these abnormalities follows this newer classification. Deletion syndromes Patients with Turner's syndrome present with an abnormality of sexual differentiation because of a sex chromosomal deletion syndrome that results in abnormal gonadal development (see Table 16.1). A normal endowment of oocytes is initially present in the developing ovaries of these patients (14). However, they are missing X-chromosomal material with important ovarian determinant genes, which are necessary for preservation of ovarian follicles. These genes are probably located on both arms of both X chromosomes, as well as the autosomes. Privation of any or all ovarian determinant genes appears to be associated with incomplete formation of the primitive follicular mantle of granulosa cells that surrounds the oocyte (14). Unarrested meiotic activity results and the oocytes appear to be able to herniate through the poorly formed granulosa cell membrane; ovarian streaks relatively devoid of follicles are formed. The classic karyotype associated with Turners syndrome is a single 45,X cell line. Most patients with Turner's syndrome, however, have mosaicism with a 45,X cell line accompanied by another cell line in which the X or Y chromosomes may be structurally normal or abnormal (i.e., 45,X/46,XY; 45,X/46,XX; or 45,X/46,X,1[Xq]) (2). The most common form of mosaicism involves the 45,X/46,XY karyotype (2). Patients with Turner's syndrome classically present with bilateral streak gonads and normal Mullerian and external genital development. Turner's stigmata and associated cardiovascular and renal abnormalities (i.e., coarctation or dilatation of the aorta and horseshoe kidney) variably occur for them. A wide spectrum of phenotypic findings for Turner's stigmata and genitalia is associated with the 45,X/46,XY karyotype (2,15,16). Gonadal development for these individuals may range from bilateral streaks through unilateral streak/contralateral immature testis to bilateral immature testes. The testes may be located infra-abdominally or within the labioscrotum. Mixed gonadal dysgenesis or asymmetric gonadal dysgenesis refers to those 45,X/46.XY patients who have " mixed" gonadal structures: a unilateral streak gonad and contralateral testis (15,16). These patients are Jostian experiments of nature in that the accessory genital structures depend on the nature of the accompanying gonad. Sexual Differentiation 7 Streak gonads are accompanied by a Mullerian system. Immature testes are associated with Wolffian derivatives. The spectrum of phenotypic findings for these patients also includes the external genitalia. For patients with bilateral streak gonads, their external genitalia is unquestionably female (15,16). Varying degrees of external genital masculinization occur in utero and at puberty for those patients who harbor an XY cell line and at least one testis. Minimal masculinization (i.e., clitoromegaly) usually occurs for patients with a unilateral infra-abdominal testis. Ambiguity is usually found in individuals with a unilateral descended testis. A normal male phenotype may be associated with bilateral descended testes. These latter males are usually diagnosed during an infertility evaluation for azoospermia. Recent studies suggested that a number of 45.X/46,XY males not only have a normal phenotype, but also may have normal spermatogenesis (17). Gonadal tumor formation has been reported ` to occur in 15"/o to 25°/ of patients with -45.X,t46,XY ;gonadal dysgenesis and infra-abdominal ,gonads/streaks if they are left in place (18,19). The most common tumor is a gonadoblastoma. Any germ cell tumor. commonly dysgerminoma and rarely others including yolk sac tumors and choriocarcinoma. may be found (2.20). The risk for tumor formation in individuals with a normal male phenotype and descended bilateral testes, however, appears to be very small and potentially nonexistent (17). All patients with 45,Xi46,XY gonadal dysgenesis need gonadal extirpation, irrespective of the age at diagnosis. except for the few phenotypically normal men with apparently bilaterally descended testes. This latter group may be followed. Some individuals suggest testicular biopsy after puberty. Patients with ambiguity at birth and given a female sex-of-rearing should have corrective surgery as early as possible and preferably during the birthing hospitalization. Except for the phenotypically normal males with bilateral testes. a male sex-of-rearing should be avoided for these patients. Penile reconstructive surgery is often extensive and rarely satisfactory, the patients are infertile as men, and the risk for gonadal neoplasia exists. 46,XY Abnormalities of sexual differentiation Abnormalities of sexual differentiation can occur to individuals with single 46,XY cell lines and be manifested with varying degrees of undermasculinization (see Table 16.1). Abnormalities resulting in one of these syndromes have been identified at each of the steps for normal male sexual differentiation (see Fig. 16.1). This syndrome results from an error that occurs at one of the earliest steps in male gonadal morphogenesis (see Fig. 16.1) (21). It was previously theorized for some of these patients that the germ cells failed to migrate to the genital ridge. Recent studies indicated that for others, mutations may occur in one of the genes involved in testicular differentiation (23,24). Approximately 15°/u of women Sexual Differentiation 8 with Swyer’s syndrome harbor mutations in SPY (23-25). As most patients with Swyer's syndrome appear to have normal SPY sequences and some present with an X-linked form, mutations must also occur in autosomal or X-chromosomal genes that are downstream to SRY in the cascade of molecular events (2,22). No matter what this early defect is, the 46,XY germ cells are not directed appropriately to begin testicular differentiation. Streak gonads form. The absence of MIS production and androgen biosynthesis is associated with the development of a normal M611erian system and female external genitalia. 46,XY Gonadal dysgenesis (Swyer's syndrome) is usually identified because of delayed pubertal development (2). These women are otherwise phenotypically normal. They are usually taller than their peers because of the presence of probable Y-chromosomal statural genes. Also their epiphyses remain open longer during their adolescent years in the absence of sex steroid production. Gonadotropin levels are elevated and subsequent chromosomal analysts demonstrates a 46.XY cell line. These women are at the highest risk (i.e., 25%u-35%) for genital ridge tumors of any patients with dysgenetic testes and Y-cell lines (?,18). Rarely, these germ cell tumors are capable of steroid biosynthesis and may produce varying amounts of estrogen or testosterone. This has been reported for not only patients with Swyer's syndrome. but also others with -h.X/46,XY gonadal dysgenesis (26,27). It is for this reason that such patients have signs of masculinization or feminization at puberty. Gonadal extirpation is necessary at the time the diagnosis is made. Mullerian inhibiting substance deficiency There are 46,XY individuals to whom germ cell migration occurs and SRY is expressed. Testicular development is initiated. However, there appears to be absent MIS production or a lack of its effect (see Fig. 16.1) (10:38.29). Testicular development and androgen production are otherwise normal. Patients unable to produce or express MIS are usually identified as phenotypic males with unilateral or bilateral cryptorchidism. or an inguinal hernia (2&). At the time of surgery. the hernia or cryptorchid testes are found to contain Mullerian elements. Also called the persistent Mullerian duct syndrome, this disorder was previously hypothesized to result from mutations in either the MIS gene or the MIS receptor gene (28,29). The initial molecular studies of such males uncovered Mutations to the MIS gene (29,30). No doubt, a host of mutations that either prevent transcription or produce a protein with abnormal or absent function will be identified. The MIS receptor gene recently was cloned and a mutation identified for a male with this syndrome who was found to have normal MIS gene sequences. For these 46,XY patients, testicular development arid its steroid biosynthesis are normal. Mullerian regression does not occur because an abnormal MIS or MIS receptor protein is produced. Either or Sexual Differentiation 9 both of these otherwise normal testes may be infra-abdominal or inguinal in location and associated with bilateral fallopian tubes, a uterus, and an upper vagina. Androgen biosynthesis appears normal for these individuals both in utero and at puberty, with normal penile development resulting. Sterility may result from variable development of the vas deferens at the time of persistent Mullerian development or unavoidable damage to the Wolffian system during, surgical removal of the Mullerian system. Agonadia (testicular regression syndrome) Phenotypic female patients are rarely identified at puberty arid, like women with Swyer s syndrome, do not go through the normal pubertal changes. Similarly. they have elevated gonadotropins levels and a 46.XY karyotype. Unlike the patients with 46.XY gonadal dysgenesis, however, these women have a blind vaginal pouch and both gonads as well as all internal genitalia are absent. These 46,XY individuals typically have germ cell migration. normal expression of testicular determining factor (TDF) and testicular development, and normal MIS production and Mullerian regression. It is hypothesized that a defect in sexual differentiation occurs after the elaboration of MIS and before androgen biosynthesis (see Fig. 16.1). An environmental insult or potentially a vascular accident may be associated with the disappearance or regression of the developing testes after the Mullerian system has regressed. The Wolffian system is not stimulated to continue development because androgens are not subsequently produced. These patients have also been given the diagnosis of "the empty pelvis syndrome" because of the absence of all internal genitalia, as demonstrated by laparoscopy. A spectrum of this disorder exists and variable phenotypes have been described. Testicular regression can occur at any time during sexual differentiation (31,32). Internal and eternal genital development depends on the precise timing of the testicular regression. Defects in androgen biosynthesis Patients with the 46,XY karyotype and steroid enzyme deficiency have normal expression of TDF genes and normal MIS production. As a result, these individuals develop normal appearing testes and Mullerian regression appropriately occurs. However, as hCG stimulates steroidogenesis within the feral Leydig cells. sub optimal levels of testosterone or DHT, or both, are produced because of an enzyme deficiency at one of the steps in androgen biosynthesis (Figs. 16.1 and 16.3). Normally, Wolffian system development is stimulated by testosterone and not DHT The 5a-reduction of testosterone to DHT is absent m the Wolffian primordia during early embryogenesis. External genital development, however, depends on the presence of 5a-reductase activity and androgen receptor stimulation by DEFT Deficiencies of both testosterone and DHT adversely affect both Wolffian and external genital deSexual Differentiation 10 velopment. Deficiency of only DHT adversely affects only external genital development. External genital ambiguity to a 46,XY individual may include under masculinization of the genital tubercle or scrotum and abnormal testicular descent. Many of these patients have a blind vaginal pouch that represents the remnant of the under stimulated prostatic utricle. Some of these 46,XY patients with enzyme deficiencies produce negligible. if any, androgens, and therefore, eternal genital masculinization is totally absent. These are individuals with deficiencies of enzymes necessary for the very early steps of androgen biosynthesis such as cholesterol side-chain cleavage enzyme deficiency (33-38). 17α - hydroxylase deficiency (38-44), and 17,20-lyase deficiency (see Fig. 16.3) (45-50). Partial defects can exist for these enzyme-deficient states and produce under masculinization and ambiguity. A number of other 46,XY patients with enzyme-deficient states predictably have minimal androgen production associated with varying degrees of testicular descent and genital ambiguity. For example, 3β-hyroxysteroid dehydrogenase deficiency results in the production or accumulation of the weak △5 androgens (i.e., 5 dehydroepiandrosterone (DHEA) and in tremendous under masculinization and external genital ambiguity (see Fig. 16.3) (51-60). Patients with 17-hydroxysteroid dehydrogenase (17-ketoreductase) deficiency produce and accumulate both weak △5 and weak △4 androgens (i.e., DHEA and androstenedione),and similarly present with tremendous under masculinization and ambiguity (61-66). Sex-of-rearing has most commonly been female for these patients because of absent or minimal masculinization associated with such high blocks in the steroid pathway. In contradistinction, testosterone production may be normal or high for patients with 5α-reductase deficiency, whose block is at the end of the steroid pathway (i.e. the enzyme chat converts testosterone to DHT) (see Fig. 163) (67-79). Testosterone stimulates the same androgen receptor as does DHT However, its dissociation constant is much higher (i.e., is moves away from the receptor much more quickly). The high levels of testosterone produced in patients with 5a-reductase deficiency allow for normal Wolffian development. Despite minimal DHT production, these high testosterone levels stimulate external genital development. Although ambiguous, these infants are more masculinized than are infants with high steroid pathway blocks. Most of these patients have also been given a female sex-of-rearing at birth. Patients with enzyme deficiencies may have associated abnormalities that appear after the newborn period. Some of these conditions have been associated with adrenal failure and others with hypertension. Adrenal failure is an almost certain occurrence for the untreated newborn with cholesterol side-chain cleavage enzyme deficiency. It is very likely to occur, although with less consistency, for the untreated infant with 3β-hyroxysteroid dehydrogenase deficiency. Hypokalemic hypertension occurs in 11 Sexual Differentiation 17α-hydroxylase-deficient patients because of accumulation of mineralocorticoid precursors to 17α-hydroxyprogesterone (see Fig. 16.3). The risk of tumor formation exists for all of these 46,XY patterns with enzyme deficiencies, but is probably no higher for them than it is for otherwise normal males having only cryptorchidism. Finally, varying degrees of masculinization or feminization, or both, may occur at puberty for 46.XY patients with androgen-deficient states whose gonads have been left in place. The pubertal phenotype best characterized is that of 5a-reductase deficiency. A very large kindred has been studied and reported from the Dominican Republic (77.78). Affected individuals became known as "guevedoces” meaning penis at 12 (years of age). Characteristically given the female sex-of-rearing at birth, these patients had tremendous masculinization at puberty and many developed a male gender identity and changed sex-of-rearing. While they are unable to convert testosterone to DHT, the high and sustained testosterone levels at the androgen receptor are seemingly sufficient to stimulate the receptor-mediated masculinization. Additionally, such masculinization at puberty may be explained by the fact that two genes encode 5n-reductase (84-83). It is the 5α-reductase 2 gene that is responsible for genital production of DHT. Mutations in this gene have been found to be associated with the intersex syndrome. It has been suggested that in these individuals the 5a-reductase 1 enzyme may convert testosterone to DHT as an alternative pathway and at other locations mediating masculinization. Another associated abnormality of some androgen enzyme deficiencies includes gynecomastia. This occurs in 17 hydroxysteroid dehydrogenase deficiency because of conversion of weak androgen precursors (i.e., androstenedione) to estrogens (i.e., estrone). Patients with 17α-hydroxylase deficiency also may develop gynecomastia. Breasts develop for them because of the lack of the usual androgenic inhibition of breast maturation. It should be remembered that because these same enzyme deficiencies are usually the result of autosomal recessive mutations, they also occur in 46,XX individuals (49,50). Since normal female sexual differentiation is not dependent on in utero sex steroid production, these patients have a normal female phenotype at birth. The abnormality of sexual differentiation becomes apparent at birth only for newborns with an adrenal crisis or salt wasting, and cholesterol side-chain cleavage enzyme or 3βhyroxysteroid dehydrogenase deficiency. It may become apparent at puberty for patients with an enzyme deficiency that precludes estrogen production (i.e., commonly 17a-hydroxylase deficiency). Diagnosis may also become apparent because of the development of hypokalemic hypertension in the latter patients. All of the steroid enzyme genes have been cloned. As of this writing. gene mutations have not been identified within the cholesterol side-chain cleavage gene for deficient patients. Two genes have been 12 Sexual Differentiation identified to encode 3β-hyroxysteroid dehydrogenase: type I and type II genes. Mutations have been identified to the 3β-hyroxysteroid dehydrogenase type ll gene for patients with this deficiency (38). A single gene chat mediates both 17α-hydroxylation and 17,20-lyase functions has been cloned and sequenced (84). Mutations that seemingly cause either 17α-hydroxylase deficiency or 17,20-(lyase deficiency have been identified within this single gene (38.85-88). Dependent on the type of mutation, these patients produce varying amounts of testosterone and DHT The 17-hydroxysteroid dehydrogenase gene has been cloned. However. gene defects have not yet been identified to explain the phenotypic findings in patients with this deficiency state. Two genes exist with 5α-reductase activity: 5α-reductase 1 gene and 5a-reductase 2 gene (83.89-93). The 5α-reductase 2 gene codes for the major isoenzyme present in genital skin` Deletion of this gene has been found in males with 5α-reductase deficiency and a number of unique point mutations have been identified in other kindred (83,92,93). Androgen receptor defects (androgen insensitivity syndrome) Patients with the 46,XY karyotype who have androgen resistance syndromes have normal expression of TDF genes and testicular development. normal MIS production and Mullerian regression, and normal testicular androgen biosynthesis (94,95). Mutations of the X-located AR gene cause an absent or abnormally functioning androgen receptor and prevent the end-organ effect of the androgen signal for masculinization to utero and at puberty This abnormality of sexual differentiation occurs at the final step in male sexual morphogenesis (,see Fig. 16.1). The syndromes of androgen resistance have presented with a wide spectrum of phenotypic findings in 46.XY individuals (Fig. 16.4). Heterozygote carriers (46,XX individuals) may also have findings related to androgen resistance, although very subtle (i.e., sparse pubic and axillary hair). Complete androgen insensitivity syndrome (CAIS) has been associated with absent masculinization in utero, the presence of a vaginal pouch, and spontaneous breast development at puberty associated with absent or diminished pubic hair (94,95). All women have androgen receptors in breast tissue, and during normal pubertal development, ovarian and adrenal androgens will limit their ultimate breast size. However, patients `with CAPS often develop very abundant breasts, characteristically composed of more adipose than glandular tissue, because of their own resistance to androgens. Additionally, some of their testicular androgens ate converted to estrogens to aid to this breast development. The 13 presence of Y-chromosomal statural genes results in a tall linear body habitus for CAPS patients. Many of these women have modeling careers because of the abundant breasts and tall stature. Sexual Differentiation Patients with CAIS have normal-appearing testes, except for incomplete or absent spermatogenesis. The testes may be intra-abdominal, inguinal, or labioscrotal in location (9496). A relatively high incidence of neoplasia, ranging from 2%to 22%, is reported to occur in these testes if left in place after puberty. The occurrence of these rumors, usually seminomas, is probably more related to their cryptorchid location than to a molecular predisposition (96,97). Testicular rumors rarely develop prior to puberty. Because of the abundant breast development that these patients experience spontaneously, the testes are best left in place until such development is completed unless the testes are located in the inguinal or labioscrotal regions. It has been said that patients with androgen insensitivity syndrome whose testes are removed prior to puberty do not achieve the same abundant breast development from exogenously given steroids. In retrospect. this information comes from patients who likely had inguinal or labioscrotal gonads removed prepubertally and incomplete forms of androgen insensitivity syndrome associated with some degree of androgen effect. They. therefore, do nor have complete androgen resistance and after gonadectomy, their adrenal androgens limit breast development during exogenous estrogen supplementation similar to normal women. Incomplete androgen insensitivity (IAIS) is far cater than the complete form. Except for the presence of partial fusion of the labioscrotal folds and usually some degrees of clitoromegaly at birth, this syndrome is very similar to the complete form and infants have usually been given a female sex-of-rearing (9.1,95). Pubic hair and clitoral enlargement occur at puberty as does breast development, although typically not as abundant as in CAIS. A vaginal pouch is usually present and associated with underdevelopment of the Wolffian system. The spectrum of androgen insensitivity includes phenotypic males as well (see Fig. 16.-1). Reifenstein’s syndrome is presently used to describe most of the syndromes that previously included Reifenstein’s, Lubbs, and Gilbert-Dreyfus syndromes. The spectrum of phenotypic findings for these syndromes is extremely variable and wide ranging. Usually these syndromes present with a male phenotype. However, both phenotypic male and females have been affected in the same sibship. The spectrum ranges from undermasculinized men with varying degrees of hypospadias to near-normal phenotypic men with gynecomastia or infertility Axillary and pubic hair is often normal for these individuals, while chest and facial hair is minimal. Recent evidence 14 suggests that the spectrum for androgen-resistant syndromes also includes the infertile, normal-appearing male (i.e., normal male phenotype associated with azoospermia),as well as the under virilized fertile male (94,96,98).The latter syndrome is described in males with a normal but small Sexual Differentiation penis, decreased male-pattern hair, gynecomastia, and normal semen (99). Furthermore, the spectrum of androgen insensitivity syndrome may include heterozygote carriers often AR gene mutation. Females with the -46,XX karyotype who have a normal gene on one X chromosome and a mutant gene on the other chromosome may present with decreased axillary and pubic hair as their only finding (see Fig. 16.-1.) Previous studies of these individuals were limited to androgen receptor analysis. Androgen receptor studies demonstrated absent androgen binding, qualitatively abnormal androgen binding, decreased androgen binding, and normal androgen binding for patients with these various syndromes (94,95). Most patients with CAIS have absent androgen binding. Nearly 50% of all families with androgen insensitivity syndrome have qualitatively abnormal androgen binding. Only 10% of androgen-resistant states present with normal androgen binding. Molecular studies of the AR gene have been extremely informative to our understanding of this spectrum of syndromes (94,95,100-1Od).The AR gene has eight exons. Mutations have been isolated to all eight exons, and except for two unrelated families with the same point mutations. all of the mutations identified to date have been unique to a single family (94,95,104).This makes simple screening for mutations impossible. Large gene deletions are rare. Point mutations, however, comprise 95% of mutations identified. These mutations are largely located in the steroid binding domain of the gene (i.e., the region that codes for the portion of the receptor responsible for binding to the androgen molecule) and rarely in the DNA binding domain. Absent or nearly absent androgen binding has been associated with large-scale mutations and single nucleotide substitutions in the coding sequences. Patients with normal androgen binding have the most consistent DNA findings because their AR gene mutations are all located in the DNA binding domain. For these patients the results of androgen receptor studies are normal because the steroid binding domain is normal and androgens can bind to the receptor. However, the androgen-androgen receptor complex cannot bind to the DNA to elicit a response. Several mutations have been identified in patients with qualitatively abnormal androgen binding and chose with decreased androgen binding. Non-endocrine genital ambiguity Isolated defects of the genitalia that do not appear to be related to interruption of the usual mechanisms 15 for sexual differentiation rarely occur. Rather, these defects demonstrate isolated absence of one of the units of the reproductive system (e.g., absence of gonads or absence of penis), disorganized growth of Sexual Differentiation one of these units (e.g. duplicate phallus, epispadias, and cloaca] formation), or abnormal placement (e.g., scrotum located above the phallus) in the 46,XY infant. Environmental insults, major chromosomal defects, and developmental gene abnormalities have all been hypothesized as potential etiologies. Amniotic band syndrome could potentially cause isolated absence of the penis. Congenital rubella, for example. has been associated with 46,XY genital ambiguity. This suggests that viral infection in utero may cause significant disorganization of testicular or external genital growth. Autosomal anomalies also have been associated with numerous somatic abnormalities including the external genitalia. D/G Chromosome groups are most commonly involved. In particular, deletion of the long arm of chromosome 13 (e.g., 46,XYdel[13][q]) produces the most consistent eternal genital abnormalities (Fig. 16.5). True hermaphroditism The karyotypes of true hermaphrodite patients are usually 46,XX, less frequently 46,XX/46,XY, and rarely 46,XY (see Table 16.1) (105.106). The latter two of these groups of patients present with similar findings, in that bilateral gonadal hermaphroditism is most frequent: unilateral ovary and contralateral testes (106). Patients with 46,XX/46,XY true hermaphroditism are chimeras: the presence of two or more cell lines originating from different embryos. Several mechanisms have been proposed to explain the presence of the two separate 46,XX and 46,XY cell lines m these individuals: 1) whole body fusion of separate 46.XX and 46,XY fetuses, and 2) fertilization of the secondary oocyte or the ovum concurrently with fertilization of the first or second polar body-, respectively (107). The opposing nature of the gonads (i.e., ovary and testis) is understandable given the opposing nature of 46,XX and 46,XY cell lines. Because the 46,XY true hermaphrodites present with phenotypes similar to those of the 46,XX/46.XY true hermaphrodites, it has been suggested that they may well have undetected 46,XX cell lines. Recently, one 46,XY true hermaphrodite was described with a mutation in SRY of gonad only: a somatic cell mutation rather than a germ line mutation (108). While the diagnosis for this patient is questionable and the second gonad was not described, it would seem possible that an otherwise normal male with an SPY mutation in one gonad could result in the development of ovotestis on that side only, suboptimal total androgen production. and ambiguous genitalia. The 46,XK true hermaphrodites usually present with unilateral gonadal hermaphroditism: a unilateral ovotestis with contralateral ovary- or testis (106). It has been proposed that these patients may have 16 gene mutations in one of the TDF genes. Unlike the 46,XX~sex-reversed males, interchange of Y material onto the X chromosome is the exception rather than the rule for the 46,XX true hermaphrodite Sexual Differentiation (109.110). In fact, very few of these true hermaphrodites harbor Y-DNA and the description of these patients is subject to skepticism. The fact that most, if not all, of them have testicular tissue in the absence of the SRY gene is further evidence that other genes intimately involved in testicular morphogenesis must exist. The ovarian component of true hermaphrodite gonads is typically better developed histologically and is endocrinologicallv and exocrinologically more functional than is the testicular component (106). The location of the testes and ovotestes varies for these patients and may be scrotal. inguinal, or abdominal. The nature of the internal ductal systems (i.e.. Mullerian versus Wolffian) depends on the associated gonad and its degree of differentiation. Most true hermaphrodites have at least a unilateral Mullerian system accompanying an ovary or ovotestes, which, if left intact. will function at puberty. The Wolffian system may accompany a testis or ovotestis and occasionally is on the same side as the Mullerian system. The development of Mullerian and Wolffian derivatives on the side of an ovotestis depends on the amount of MIS and testosterone produced. The external genitalia are ambiguous and usually markedly under masculinized because of suboptimal testosterone production. In previous years, these infants were reared as males because the masculinization, although poor, was often associated with a descended gonad. However, pubertal masculinization secondary to endogenously produced androgens or exogenously given testosterone was also poor and often resulted in gender identity problems. In tact, true hermaphrodites diagnosed aver puberty demonstrate more feminization than virilization. Breast development is common. Cyclic menstruation is usual. While only a few normal germ cells are identified in the testicular component of these gonads. numerous primordial follicles usually are present. The ovarian exocrine activity is often associated with normal gametogenesis. At least Six true hermaphrodites raised as females have achieved pregnancy and have been reported in the world literature. As a result, the infant diagnosed at birth with true hermaphroditism should be raised as a female and every effort should be made to preserve ovarian and Mullerian systems if normal. 46,XX abnormalities of sexual differentiation A number of disorders of sexual differentiation occur in individuals .,with a normal 46,XX karyotype (see Table 16.1). For these patients, varying degrees of masculinization occur, ranging from minimal clitoral enlargement to a normal male phenotype. Such abnormalities are the result of the superimposition of endogenously produced or exogenously given androgens on normal female sexual differentiation. In 17 some of these patients the anomalous expression of TDF genes and subsequent testicular development result in a male phenotype that is contradictory to the 46,XX karyotype. Sexual Differentiation True hermaphroditism As has been previously described, true hermaphrodites usually have a normal 46,XX karyotype. Almost all of these patients studied to date were devoid critic SKY gene or other Y-chromosomal DNA sequences studied (111,113). This provides further evidence that genes other than the SPY gene must be involved in testicular morphogenesis. The end result of anomalous expression of these yet to be identified testicular determinants is the organization of undifferentiated genital ridges along both testicular and ovarian lines with suboptimal testicular formation. Sex-reversed male 46,XX Sex reversal is an intersex disorder in which male sexual differentiation occurs in an individual with a contradictory 46,XX karyotype. The X and Y chromosomes normally pair during meiosis, and interchange of this material rarely occurs. The presence of Y DNA sequences of varying lengths translocated to the X chromosome appears to be the most common cause of 46,XX sec reversal. At least 60% of these patients have SRY present (110,113). The undifferentiated genital ridge develops along testicular lines and testicular endocrine function is more normal for these males than it is for the 46.XX true hermaphrodites (114-116). Accessory structures are Wolffian and external male genital development is complete or nearly so. However, subsequent spermatogenesis is absent. During the Y to X chromosome interchange, only SKY was translocated and not the other Y -located genes controlling sperm production. Phenotypically. these men are unquestionably stale and the diagnosis is usually nor made until after puberty or at the time of infertility evaluation because secondary sexual characteristic abnormalities may be subtle. The adult testes are usually small and firm and occasionally cryptorchid. The penis may be small with hypospadias present in fewer than 10% of these men. Pubic hair may have a female distribution. Congenital adrenal hyperplasia The most common and potentially serious abnormality of sexual differentiation is manifested in the 46,XX individual with an enzyme defect for cortisol biosynthesis (38, I 17). The most frequent of these deficiencies is 21-hydroxylase (21-OH) deficiency. with an incidence in Europe and the United States ranging between 1 in 5000 and 1 in 15,000 population (I 1 7). The greatest incidence is noted in the Alaskan Yupik Eskimos. at 1 in 684 (117). Less commonly 11β-hydroxylase, and 3β-hydroxysteroid 18 dehydrogenase deficiencies are reported. For patients with CAH, cortisol production and secretion are limited or prevented. Chronic cortisol deficiency results in a chronic state of adrenocoticotropin Sexual Differentiation hormone (ACTH) overproduction and secretion. because of the negative feedback control between cortisol and ACTH production and secretion (117-120). Constant adrenal stimulation results in an accumulation of cortisol precursors and androgens (see Fig. 16.3). The fetal adrenal begins to function by the third month of development. By the time adrenal androgen levels are significantly elevated. the ovaries are undergoing normal exocrine activity: the Wolffian system has already regressed, and the Mullerian system is completing its development. High fetal adrenal androgen levels adversely influence development of the external genitalia in the female fetus but not the otherwise normally developing male fetus with CAH. Labioscrotal fusion begins posteriorly to anteriorly, covering over the vaginal vestibule and forming a urogenital sinus (Fig. 16.6). The extent of virilization of the 46,XX fetus is variable. even within the same family, and probably depends on the androgen levels achieved and the embryologic time of exposure. A spectrum of anatomic changes may be present, ranging From minimal clitoromegaly and mild labioscrotal fusion, through varying degrees of hypospadias (117-1?0). In patients with extreme androgen overproduction, marked clitoral hypertrophy- occurs. In some the urethral orifice is displaced onto the genital tubercle and the clitoris looks like a normal penis. Any newborn identified with bilateral undescended gonads associated either with a normal-appearing penis or with varying degrees of penile underdevelopment must be considered to have CAH until proved otherwise. In contradistinction, the ambiguous infant with one or two gonads in their respective labioscrotal pouches does not have CAH. While both -46.XX and -46.XY infants may be affected with CAH. only the 46,XX CAH infants swill present with ambiguity Normal ovaries may herniate, but do not descend into the labioscrotum. Descended gonads have at least some androgen producing testicular elements present and when accompanied by ambiguity denote one of the other forms of intersex. Both 46,XX and 46,XY patients who are diagnosed with CAH and untreated will continue to masculinize after birth. The excess androgens produced will be converted to estrogens and accelerate childhood growth and cause premature epiphyseal fusion. While tall as children, these individuals will be short adults. Treated CAH patients are potentially fertile. Skin hyperpigmentation is commonly- associated with these defects and the degree of pigmentation may reflect the severity of the enzyme deficiency. The negative feedback stimulation of the hypothalamus and pituitary by cortisol deficiency, produces increased levels of pro-opiomelanocortin. This precursor peptide is subsequently split into the ACTH and β-lipoprotein fragments. α-Melanocyte-stimulating hormone (MSH) is a by-product of the ACTH 19 intermediate and (3-MSH is a (3-lipotropin subfragment. Levels of MSH are equally as high as are those of ACTH in patients with cortisol deficiency, and hyperpigmentation results. Two forms of classic 21-OH deficiency exist: salt-wasting and simple virilization (117-120). Non-classic CAH also occurs. Both types of classic 21-OH deficiency are manifested at birth or in the newborn. In Sexual Differentiation females, classic CAH results in ambiguity of the eternal genitalia. In males, external genital development is normal. However, pubertal-type changes occur during early childhood. Seventy-five percent of classic 21-OH deficiency patients have the salt-wasting varieties. These infants develop hypernatremia. hyperkalemia, inappropriately high urinary sodium concentrations, low serum and urinary aldosterone levels, and a resultant high plasma reran activity (PRA) at approximately 7 to 10 days. if untreated. The practitioner usually has a grace period during these first 2 weeks of life to make the diagnosis before a catastrophic event swill occur. Male infants with salt-wasting CAH are snore likely to be undiagnosed, and unsuspectingly develop a salt-wasting crisis than are their female ambiguous counterparts, because the males have a normal phenotype at birth. Infants with salt-wasting first demonstrate poor feeding and may develop diarrhea and lethargy prior to the full-blown salt-wasting crisis and death. No-classic 21-OH deficiency, is almost universally manifested in females only and is not a cause of intersex disorders. It presents during the peripubertal time and adult menstrual years with signs of androgen excess, menstrual abnormalities, or both (118). Precocious adrenarche and the peripubertal development of an atypical polycystic ovarian disease syndrome are the most common presentations. Non-classic CAH has been reported for 21-OH deficiency, 11β-hydroxylase deficiency, and 3β-hydroxysteroid dehydrogenase deficiency (118). Two forms of non-classic 21-OH deficiency also exist: symptomatic (adult onset) and asymptomatic (cryptic). Asymptomatic patients with non-classic CAH appear phenotypically normal (117-120). However, hormonally they are identical to patients with symptomatic disease: levels of cortisol precursors (i.e., 17-hydroxvprogesterone) and androgens arc not different for these two groups. Some symptomatic patients occasionally become asymptomatic and some asymptomatic patients occasionally develop menstrual abnormalities accompanied by signs of androgen excess. Nonclassic CAH appears to only rarely have an adverse effect on males with these saint mutations (121,122). Less than a half dozen men with nonclassic disease and oligospermia corrected by steroid treatment have been reported in the literature (121.122). Linkage disequilibrium seas found to exist with 21-OH deficiency and the HLA-B and DR antigens (123,124). Specific HLA-B antigens occur with increased frequency in association with various forms of 21-OH deficiency (e.g., HLA-Bw47 and classic CAH; HLA-B14 and nonclassic CAH). In some families. 20 different forms of 21-OH deficiency-salt-wasting classic, simple virilizing classic, and nonclassic 21-OH deficiency-have occurred in different members of the same family. Hormonal, HLA, and DNA studies of these family members demonstrated that patients can harbor a number of different 21-OH mutations (i.e., one on each chromosome) and that they manifest the least deleterious mutation that they harbor Sexual Differentiation (125,126). A patient with a classic salt-wasting mutation on one chromosome and a classic simple virilizing mutation on the other chromosome would express the simple virilizing form. A patient with a classic salt-wasting mutation on one chromosome and a nonclassic mutation on the other chromosome would express the nonclassic mutation. This finding, has serious implications for individuals who may be at risk of delivering an infant with ambiguity or salt-wasting potential. For example, women with nonclassic CAH may harbor a classic 21-OH mutation on one chromosome (126.127). The variability of 21-OH deficiency within the same family prompted investigations into the molecular nature of these different 21-OH-deficient states. Two genes for 21-OH have been identified and sequenced (119,127-129). These genes, CYP21 (previously known as 21-hydroxylase B) and CYP21P (previously known as 21-hydroxvlase A), encode a cytochrome P-450 specific for 21-hydroxylation. The CYP21 and CYP21P genes lie in tandem with the genes encoding the fourth components of complement, C4A and C4B, respectively. They are located on chromosome 6 to close association with the HLA-B and DR loci. While C4A and C-4B genes both encode protein products, only the CYP21 (21hydroxyiase B) gene encodes a specific gene product that is responsible for the 21-hydroxylation step in steroid biosynthesis. CYP21P (21-hvdroxylase A gene) is a pseudogene (128,129). It harbors three small mutations in its coding region and other mutations in introns that render it inactive. Mutations have been found in the CYP21 gene, and render it inactive and cause various forms of 2l-OH deficiency (119). Approximately 25°/ of these gene mutations are easily identified by Southern blot techniques rising the 21-OH gene probe. Half of these mutations involve large gene deletions of both the C-4B and the CYP21 genes (I 19,130). The other half of these easily identifiable gene mutations involve gene conversions: Any or all of the mutations causing inactivity of the pseudogene are transferred during chromosomal pairing at meiosis by abnormal crossing over to the active gene, rendering it also inactive. Seventy-five percent of the mutations identified in patients with classic 21-OH deficiency are point mutations and not easily identified by routine Southern blot techniques. Diagnosis for these latter patients has previously involved DNA studies of closely linked HLA-DR gene polymorphisms (119,131,132). Newer techniques such as denaturing gradient gel electrophoresis are being utilized to rapidly and more easily identify these point mutations, obviating the need for linkage studies and potential misdiagnosis (133). Recent studies demonstrated that 95% of mutations are now known and can be easily identified. Amplification of the CYP21 (active) gene alone by polymerase chain reaction (PCP,) will allow examination for mutations in it. most of which commonly occur also in the 21 CYP21P (inactive/pseudogene). Southern blotting will identify the large gene deletions or conversions and oligonucleotide probing of the amplified active gene sequences for common nitrogenous base changes will find the point mutations (134,135). Sexual Differentiation Androgen excess syndromes In-utero exposure to endogenously produced or exogenously given maternal androgen has been responsible for varying degrees of masculinization of the external genitalia in 46,XX females with otherwise normal sexual differentiation. The placenta has the ability to aromatize native androgens. except DHT, to estrogens and prevent masculinization of a female fetus. In fact, during the end of pregnancy the fetal adrenal gland makes nearly 200 mg of steroids a day, most of which is DHEA sulfate (DHEAS). In comparison, at any one time of the menstrual cycle. the regularly menstruating female never makes more than a total of 511 mg of all the steroids. most of which are estrogens. Despite the extremely high levels of androgens produced by the normal fetus and high levels of testosterone produced abnormally by some women during pregnancy, the female fetus does not masculinize because of placental aromatization. Several conditions exist in which this "neutralization" of androgens either does not occur or is bypassed and female fetuses are masculinized. The maternal androgen producing tumor is one such instance in which androgens may rarely bypass placental aromatization. The likely reason for such masculinization associated with these tumors is the presence of excessive 5α-reductase activity in the tumor. Testosterone is converted to DHT, a nonaromatizable androgen, prior to passage across the placenta. Conceivably, although unlikely, if tumor production rates for testosterone exceed the placenta's ability to aromatize it to estradiol, then native testosterone could get to the female fetus and cause masculinizing effects as well. The luteoma of pregnancy is the most common tumor associated with in utero masculinization of a female fetus and has 5α-reductase activity. This solid tumor appears to be hCG-dependent and usually disappears after pregnancy. Most cases of genital ambiguity previously labeled as being idiopathic probably resulted from a luteoma of pregnancy. Very rarely- ovarian and occasionally adrenal neoplasms have been associated with masculinization of a female fetus. A few rare testosterone-producing ovarian tumors include arrhenoblastomas, Leydig cell tumors, and occasionally epithelial tumors that develop within the ovary as a secondary metastasis. Exogenously given testosterone for such conditions as endometriosis and inadvertent continuation of this drug during pregnancy rarely masculinize a female fetus. Synthetic hormone derivatives of 22 testosterone such as ethisterone, norethindrone. and danazol (Danocrine) have been more commonly implicated in masculinization of the female fetus, as they are nonaromatizable. Sexual Differentiation Aromatase deficiency was not described until molecular mutations were identified. Aromatase deficiency recently was found to result from a homozygous recessive inherited aromatase gene mutation (137-139). Like placental sulfatase deficiency; such pregnant women have inappropriatelylow- levels of estrogens for gestational age in the absence of fetal distress. However, unlike placental sulfatase deficiency, both the mother and the female fetus are masculinized or androgenized significantly. The extremely high fetal levels of adrenal DHEAS. which is usually converted to estrogens by the placenta. remain high and the DHEAS is converted to the more potent androgens, testosterone and DFIT For male infants with aromatase deficiency. the mothers, but not their sons, are "over androgenized" at birth. The only phenotypic abnormality described for affected males with estrogen deficiency is the absence of normal epiphyseal closure and significantly taller suture than would be predicted by mean parental height estimates (1-10). Idiopathic genital ambiguity For a number of 46,XX infants. the cause of genital ambiguity has not been identified. Except for varying degrees of virilization, these infants are otherwise normal at birth. Endocrine function at puberty is also normal. While a diagnosis of idiopathic genital ambiguity is given to these infants, it is very likely that` they were exposed to DHT from a luteoma of pregnancy. In a number of other 46.XX infants. however, unexplained external genital masculinization .,vas associated with multiple somatic anomalies. Such anomalies Include the genitourinary, gastrointestinal, and non-reproductive endocrine systems. These disorders are usually sporadic and may be caused by unknown teratogens or de novo gene mutations. An autosomal recessive etiology has been implicated in several consanguineous pedigrees in which two such affected infants were delivered. IDENTIFICATION AND EVALUATION OF INTERSEX DISORDERS Few- birth defects require the degree of expertise for early diagnosis and treatment as well as for concomitant psychosocial counseling as is necessary for those that involve the genitalia. The parents of these children must deal with routine issues surrounding any birth defect: 1) the delivery of an imperfect baby: 2) guilt and self blame: and 3) the requirements of "traumatic" testing and potential surgery, for their newborn. Additionally, these parents are burdened by issues surrounding the sex and future 23 reproduction of their child: 1) the sex-of-rearing decision: 2) subsequent development of gender identity; and, 3) potential sterility. Sex-of-rearing refers to the label of male or female given to an infant at birth. Gender identity refers to ones own feelings of sexuality. Children reared with normal Sexual Differentiation environmental influences that reinforce sex-of-rearing develop a concordant gender identity. This is true even for individuals with intersex disorders despite the fact that at birth their karyotype, gonads, or external genitalia may have been more consistent with the opposite sex-of-rearing. Confusion or discordant feelings about sex-of-rearing have occurred in intersex patients who have not had expeditious and long-term medical, surgical, and psychological treatment. These disorders should be considered medical and surgical emergencies, especially if identified in the newborn, in order to avoid potentially disastrous outcomes. Delivery room identification, reaction, and initial evaluation The delivers- of a child with anything other than normal genitalia calls on the immediate good judgment of the obstetrician. The physician should understand that ambiguous genitalia potentially caused by an intersex disorder may include minimal clitoromegalic isolated cryptorchidism (especially if bilateral), and hypospadias as well as overt ambiguity. It is important to be able to recognize a potential intersex disorder and to choose the most appropriate words for discussing this issue with the couple before addressing the sex of the child. When the sex of an infant is in question, it is best to withhold the decision on sex-of-rearing. The health care provider should explain that sexual development of this infant is incomplete and that after a few studies it will be easy to determine whether the baby is a boy or a girl. After addressing the immediate usual delivery concerns (e.g., delivery, of the placenta and suturing the episiotomy), the physician should enter into a more detailed discussion involving causes of ambiguity, concerns, and actions to be taken. It should be pointed out to the parents that the external genitalia of both male and female infants appear nearly identical until midgestation. For this reason arrested development in a male infant may make his genitalia appear underdeveloped; surgery will complete this development. "Overdevelopment" of an otherwise normal female infant by exposure to male hormones may make her genitalia appear more masculine. Most of these female infants will appear normal after surgery, and many are fertile. Spot decisions about sea-of-rearing may be comfortable in the delivery room but are usually risks-. They often result in the change of sex-of-rearing at least one more time and the potential development of adverse psychosocial affects for the infant and family. There are two exceptions for which it is usually 24 safe to give the sex-of-rearing immediately. First, an infant with minimal clitoral hypertrophy and normal-appearing urethral and vaginal orifices can usually be safely given the female sex-of-rearing. It would be impossible to make a normal functioning penis for this infant. Second. it is usually appropriate Sexual Differentiation to give a male set-of-rearing to an infant with a normal-size but hypospadic penis associated with unilateral cryptorchidism. It is, however, unwise to give spot diagnoses for all other forms of ambiguity. It would be very- tempting, but very wrong, to give a male sex-of-rearm, to an infant with a normal-appearing penis and scrotum and bilateral cryptorchidism. This child may be a normal fertile female with CAH! Changing sex-of-rearing should be avoided at all cost! It provides the parents and family members with questions as to whether the final decision for sex-of-rearing is correct. Gender identity and the eventual emotional well-being of the patient and fancily members largely depend on the comments made during the first critical moments of life. Delivery room clues The delivery of twins concordant or discordant for genital ambiguity is a very rare occurrence bur provides clues for diagnosis. Concordant ambiguity could occur in identical twins with an enzyme deficiency and should be suspect if neither of the infants have descended gonads (e.g.. 46,XX CAH) or if both infants have bilaterally descended testicles (e.g., 46,XY biosynthetic defect for testosterone). By far the most common cause of ambiguity reported in one or both infants of a set of twins is 45,X/46,XY gonadal dysgenesis. (111).45,X/46,XY Gonadal dysgenesis has been documented in male-male, female-female, and male-female identical twins. One or both of these twins usually has one or both testicles descended and one or both of them may have 45,X/46,XY karyotypes. This disorder is considered to represent an abnormal attempt at twinning; in the singleton pregnancy. the -46,XY fetus imperfectly attempts to twin and forms a mosaic ferns instead. In the twin pregnancy, the twinning process may occur but second attempts at twinning may result in mosaicism. Evaluation should begin in the delivery room A review of the maternal history might include questions concerning maternal ingestion of androgenic compounds (e.g., danazol) and the development during pregnancy of signs or symptoms related to maternal androgen overproduction (e.g., acne, hirsutism). Family history might reveal similarly affected 25 infants or relatives (e.g.. CAH, AIS). Previous unexplained neonatal deaths of other infants right provide a clue for the diagnosis of CAH. Maternal blood should be drawn in the delivery room for testosterone, DHEAS, and estrogen measurements to rule out the presence of an androgen-producing tumor (e.g., luteoma, ovarian neoplasm, or adrenal tumor) or placental aromatase deficiency. Similar studies on cord blood right be helpful. Sexual Differentiation Neonatal evaluation The evaluation of the neonate should be designed to make a diagnosis and determine sex-of-rearing as expeditiously as possible. The reaction and adjustment of the family depend on timely and informed counseling. The diagnostic evaluation should answer the following four questions: 1) Is the ambiguity a part of a syndrome ? 2) Does the infant have CAH ? 3) Is a testis or are testes present ? 4) Can an exact diagnosis be made ? A number of syndromes described above have been associated with abnormal genital development. For most of them ambiguity is the result of hormonally induced over masculinization or under masculinization. For a few syndromes, total disorganization of external genital development may be accompanied by gross detects of other bode parts such as the Iimbs or face, and the result of viral-induced or chromosomally related teratogenesis. Congenital rubella syndrome and the phenotype of unbalanced autosomal translocation, respectively, are examples of the latter two problems. The infant with an identifiable testis (or testes) and genital ambiguity does not have life-threatening, salt-wasting 21-OH deficiency; this syndrome causes ambiguity in affected females, not affected males. Rather, the infant with as identifiable testis (or testes) may have 45X/46,XY gonadal dysgenesis, one of the 4l6XY syndromes (see Table 16.1). or may be a true hermaphrodite. Finally, understanding the exact diagnosis will help the clinicians involved to give the most informed counseling about expectations for the family members. 26 Sexual Differentiation This set of Notes Contributed by Dr. Dorothy Villee PhD (Who gave these lectures for many years) What is sex? The Different Kinds of Sex The most important clinically is the genital sex and the brain sex. Decisions of sex assignment depend upon the appearance of the external genitalia. Sex Determination The processes of morphogenesis and cellular differentiation are dependent upon the activity of sets of genes normally in complex interacting networks or pathways. However, within these pathways specific genes occasionally appear to act as switches. One case in which this is very apparent is in the process of sex determination. The primary event in sex determination is the differentiation of the gonad. THUS, SEX DETERMINATION IS THE EQUIVALENT OF TESTIS DETERMINATION IN MAMMALS. In humans, the Y chromosome acts as a dominant male determinant. If the Y chromosome is present an embryo will develop as a male independent of the number of X chromosomes. The activity of the Y chromosome responsible for this has been referred to as Testis Determining Factor (TDF) in humans. 1. it should be in the minimum portion of the Y chromosome that is shown to be maledetermining 2. it should be conserved on the chromosome 3. there should be a molecular basis for any mutation known to be within the gene 4. it should be present in the somatic portion of the genital ridge, immediately prior to overt testis differentiation 5. it would be likely to have a structure consistent with a regulatory function 27 The most likely candidate for TDF is SRY found in four XX males who had some Y-unique sequences. It is located within a 35 kb region adjacent to the pseudoautosomal boundary of the Y-unique portion. It has homology to a number of genes known to bind to DNA in a nonspecific manner. In mouse SRY is expressed just prior to testis differentiation (10.5 days) and is switched off by 12.5 days, suggesting that it is not required for maintenance of any differentiated cell type or Sexual Differentiation function of the testis. Thus, it is expressed only to trigger the process of overt testis differentiation. The only site of SRY expression in the embryo is the genital ridge and the expression is tied to the somatic portion of the gonad and not the germ cell component. Germ cells are not necessary for testis development. Transgenic experiments Fragments of DNA encoding SRY were introduced into early mouse embryos to determine whether SRY is capable of changing the sex of an XX female embryo. Fourteen days after the injection the embryos were examined for testis or ovary. Two XX embryos with developing testes were found. Southern blot analysis showed that the two embryos carried multiple copies of SRY as a transgene. THIS EXPERIMENT PROVED THAT SRY CAN INITIATE TESTIS DEVELOPMENT. XX transgenlc male mice look and behave as males except that their testes are smaller and they are infertile, because XX is incompatible with spermatogenesis. The rest of the reproductive tract was normally male, indicated that the Sertoli cells in developing embryos were functionally normal in terms of MIF (Mullerian inhibiting factor) and that the Leydig cells were also producing testosterone normally. Gonadal Differentiation The formation of the gonadal blastema is completed by weeks 5 to 6 of gestation in human embryos. At this time the primitive ("indifferent") gonad is composed of three distinct cell types: (1) germ cells (migrated from yolk sac), (2) supporting cells of the coelomic epithelium of the gonadal ridge that give rise to the Sertoli cells of the testis and the granulosa cells of the ovary, and (3) stromal ("interstitial") cells derived from the original mesenchyme of the gonadal ridge. The first morphological sign of sexual dimorphism in the gonads is the development of the primordial Sertoli cells and their aggregation into spermatogenic cords in the fetal testis. In the human this occurs between weeks 6 and 7 of gestational development. In contrast to the early development of the fetal testis, the fetal ovary shows no characteristic development until months later in embryogenesis and initially is identified histologically early only by exclusion. Neither selective destruction of germ cells with drugs nor surgical excision of primordial germ cells in the anterior germinal crest before they reach the gonadal primordium inhibits gonadal development Thus, somatic cells can organize into an ovary or a testis irrespective of the presence or absence of germ cells. Hormonal Sex Two substances from the fetal testes are essential for male development: a nonsteroidal hormone that acts ipsilaterally to cause regression of the mullerian duct 28 (Mullerian Inhibiting Factor, MIF) and (2) an androgenic steroid (testosterone) responsible for virilization of the wolffian duct, urogenital sinus, and urogenital tubercle. Development of the female urogenital tract occurs in the absence of gonads and does not seem to require secretions from the fetal ovaries. Mullerian Inhibiting Factor MIF is a large (140,000 daltons) dimeric glycoprotein formed by the Sertoli cells of Sexual Differentiation the fetal and newborn testis. It acts locally to Suppress mullerian duct development. Persistence of the Mullerian ducts is usually accompanied by failure of the testes it descend. Mullerian-inhibiting activity is Tower in biopsied testicular cells from newborn boys with cryptorchidism than from normal newborns. Therefore, MIF may play a role in the descent of the testes, particularly in the transabdominal migration. Androgens Testosterone formation in the testis begins shortly after the onset of differentiation of the spermatogenic cords and is concomitant with the histological differentiation of the Leydig cells. In the rabbit the onset of testosterone synthesis and the resulting differentiation of the male urogenital tract are independent of gonadotropin control. It is uncertain whether a similar situation exists in embryonic sexual differentiation in humans. LH receptors are present in human fetal testes as early as the twelfth week of gestation and human fetal testes respond to human chorionic gonadotropin stimulation by exhibiting increased testosterone synthesis at this time. It is not known what happens between weeks 8 and 11, when the major portion of male phenotypic differentiation takes place. Testosterone synthesis is gonadotropin dependent during the latter two-thirds of gestation. Genital Sex Internal genitalia Prior to the eighth week of human development, the urogenital tract is identical in the two sexes. The internal accessory organs of reproduction develop from a dual duct system (wolffian and mullerian) that forms within the mesonephric kidney early in embryogenesis (Figure .2). Within the substance of the mesonephros, tubules connect primitive capillary networks with a longitudinal mesonephric (wolffian duct). The wolffian duct extends caudally to the primitive urogenital sinus. At about six weeks the development of the paramesonephric (mullerian) ducts begins in embryos of both sexes as an evagination in the coelomic epithelium, just lateral to the wolffian ducts. Mullerian duct development cannot take place in the absence of the wolffian duct. The first sign of male differentiation of the urogenital tract is degeneration of the mullerian ducts adjacent to the testes, a process that begins just after formation of the spermatogenic cords in the testes. Eventually, the mullerian ducts of the male undergo almost complete regression. The transformation of the wolffian ducts into the male reproductive tract begins subsequent to the onset of mullerian duct regression. The portion of the wolffian duct adjacent to the testis becomes convoluted to form the epididymis; the central portion of the duct becomes the vas deferens. The differentiation of the Wolffian duct is dependent upon local high concentrations of testosterone. The regression of the mullerian ducts is dependent upon local high 29 concentrations of MIF. External genitalia Prior to week six of gestation, the anlagen of the external genitalia are indistinguishable in the two sexes. The genital eminence is a rounded mass between the umbilicus and the tail and is composed of a genital tubercle flanked by prominent genital swellings. The opening of the urogenital sinus between the genital swellings (the urethral Sexual Differentiation groove) is surrounded by genital folds. At week 7 of gestation in the human, the genital tubercle begins to elongate; a shallow, circular depression defines the glans of the tubercle. At this stage of development, there are no differences between the external genitalia of male and female embryos. The external genitalia of the male begin to develop shortly after the onset of virilization of the wolffian ducts and urogenital sinus. The genital tuberde elongates, and the urethral folds begin to fuse over the urethral groove from posterior to anterior. The two urogenital swellings move posterior to the genital tubercle and eventually fuse to form the scrotum. The elongated urogenital cleft becomes closed to form the penile urethra. Male differentiation of the external genitalia depend upon the androgen dihydrotestosterone, which is formed from testosterone by 5 alpha reduction. dihydrotestosterone binds to the androgen receptor with greater affinity than does testosterone and the dihydrotestosterone-receptor complex is more readily transformed to the DNA-binding state than is the testosterone-receptor complex. The net consequence is that dihydrotestosterone formation generally amplifies the androgenic signal Brain Sex Animal experiments have shown that androgens and estrogens can affect cyclicity to the hypothalamus and sexual behavior if administered during the critical period of brain sexual differentiation. In humans patients who are 46XX and have adrenocortical hyperplasia with excessive production of adrenal androgens demonstrate an increased incidence of tomboy behavior. There is a higher incidence of homosexuality in such patients compared with normal females. Homosexual men have been shown to have size differences in sexually dimorphic areas of the brain compared to non-homosexual men. Such data suggests that sexual differentiation of the brain is dependent upon hormonal influences and that anatomical differences in these sexually dimorphic areas of the brain maybe responsible for sexual identity and behavior. Present evidence suggests that testosterone must be converted to estradiol in brain cells for these changes to occur. In the human brain sexual differentiation is thought to occur in the latter half of pregnancy. 30 Sexual Differentiation 31