Abnormalities of Sexual Determination & Differentiation
Felix A. Conte MD
Melvin M. Grumbach MD
Advances in molecular genetics, experimental embryology, steroid biochemistry, and methods of evaluation of the interaction between the hypothalamus, pituitary, and gonads have helped to 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.
NORMAL SEX DIFFERENTIATION
The normal human diploid cell contains 22 autosomal pairs of chromosomes and two sex chromosomes (two X, 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 (Figure 14-1) permit positive identification of each chromosome by its
unique “banding” pattern. Bands can be produced in the region of the centromere (C bands), with the fluorescent dye quinacrine (Q bands), and with Giemsa stain (G bands). Fluorescent banding (Figure 14-2) is particularly useful because the Y chromosome stains so brightly that it can be identified easily in both interphase and metaphase cells. A technique called fluorescence in situ hybridization has been particularly useful in identifying “marker” chromosomes, especially deleted sex chromosomes that are not readily identifiable by standard banding techniques. High-resolution chromosome banding and “painting techniques” provide precise identification of each chromosome. The standard nomenclature for describing the human karyotype is shown in Table 14-1. A complete clone map of the euchromatic region of the Y chromosome has been described. This is the first map of this type for a human chromosome, and it spans about 35 million base pairs.
Figure 14-1. A normal 46,XY karyotype stained with Giemsa's stain to produce G bands. Note that each chromosome has a specific banding pattern. (Reproduced, with permission, from Grumbach MM, Hughes IA, Conte FA: Disorders of sex differentiation. In: Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
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 carry 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 (and the genes for testicular organogenesis), lead to the formation of an ovary, whereas a Y chromosome or the presence of the male-determining region on the short arm of the Y chromosome—thetestis-determining factor—will lead 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 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. The result of this process is formation of an X chromatin body (Barr body) in the interphase cells of persons having two or more X chromosomes (Figure 14-3). A gene termed XIST (X inactive specific transcripts) is located in the region of the putative 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 facilitate inactivation of 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) segment homologous to a segment on the distal portion of the short arm of the Y chromosome. This segment is called the pseudoautosomal region; 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 seven genes have been localized to the pseudoautosomal region on the short arm of the X and Y chromosomes. Among these are MIC2, a gene coding for a cell surface antigen recognized by the monoclonal antibody, 12E7; the gene for the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor; the human interleukin-3 receptor; a gene whose deletion results in the neurocognitive defects observed in Turner's syndrome; and a gene for short stature, PHOG/SHOX. This gene is expressed in bone and is associated with idiopathic short stature as well as dyschondrosteosis (Leri-Weill syndrome) in heterozygotes. 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 (Figures 14-4 and 14-5).
Figure 14-2. Metaphase chromosomes stained with quinacrine and examined through a fluorescence microscope. Note the bright fluorescence of the distal arms of the Y chromosome, which can also be seen in interphase cells (“Y body” at right). (Reproduced, with permission, from Grumbach MM, Hughes IA, Conte FA: Disorders of sex differentiation. In: Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
In buccal mucosal smears of 46,XX females, a sex chromatin body is evident in 20–30% of the interphase nuclei examined, whereas in normal 46,XY males, a comparable sex chromatin body is absent. In patients with more than two X chromosomes, the maximum number of sex chromatin bodies in any diploid nucleus is one less than the total number of X chromosomes. Using sex chromatin and Y fluorescent staining, one can determine indirectly the sex chromosome complement of an individual (Table 14-2). FISH analysis for SRY (Y chromosome) and the pericentric region of the X can rapidly identify the sex chromosome constitution in interphase as well as metaphase cells (Figure 14-6).
Sex Determination (SRY Is the Testis-Determining Factor)
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 (Figure 14-5). This gene has been cloned, expressed, and named sex-determining region Y (SRY). Sry (the mouse analog of the human SRY gene) is expressed in the embryonic genital ridge of the mouse between days 10.5 and 12.5, just before and during the time at which testis differentiation first occurs. Furthermore, deletions or mutations of the human SRY gene occur in about 15–20% of 46,XY females with complete XY gonadal dysgenesis. The most compelling evidence that SRY is the testis-determining factor is that transfection of the Srygene into 46,XX mouse embryos results in transgenic 46,XX mice with testes and male sex differentiation.
Table 14-1. Nomenclature for describing the human karyotype pertinent to designating sex chromosome abnormalities.1
Figure 14-3. X chromatin (Barr) body in the nucleus of a buccal mucosal cell from a normal 46,XX female. (Reproduced, with permission, from Grumbach MM, Hughes IA, Conte FA: Disorders of sex differentiation. In: Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
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 is thus thought to facilitate interaction between DNA-bound proteins to affect the transcription of “downstream genes.” The mechanism of action of SRY and its downstream targets have not yet been defined, although SOX9 is a candidate downstream gene. At least four cellular roles for the SRY protein have been defined. They include induction of Sertoli cell differentiation, migration of mesonephric cells into the genital ridge, proliferation of cells in the genital ridge, and the development of male-specific vasculature in the gonad with recruitment of a large number of endothelial cells from the mesonephros. Most of the mutations thus far described in 46,XY females with gonadal dysgenesis have occurred in the nucleotides of theSRY gene encoding the DNA binding region (the HMG box) of the SRY protein.
A number of genes are involved in the testis-determining cascade. Heterozygous mutations and deletions of the Wilms tumor gene (WT1)located on 11p13 result in urogenital malformations as well as Wilms tumors. Knockout of the WT1 gene in mice results in apoptosis of the metanephric blastema with the resultant absence of the kidneys and gonads. Thus, WT1, a transcriptional regulator, appears to act on metanephric blastema early in urogenital development. Heterozygous mutations in humans result in the Denys-Drash and Frasier syndromes.
SF-1 (steroidogenic factor-1) is an orphan nuclear receptor involved in transcriptional regulation. It is expressed in both the female and male urogenital ridges as well as in steroidogenic tissues, where it is required for the synthesis of testosterone, and in Sertoli cells, where it regulates the antimüllerian hormone gene. SF-1 is encoded by the mammalian homolog of the drosophila gene Ftz-f1. Knockout of the gene encoding SF-1 in mice results in apoptosis of the cells of the genital ridge that give rise to the adrenals and gonads and thus lack of gonadal and adrenal gland morphogenesis in both males and females. This gene thus appears to play a critical role in the formation of all steroid-secreting glands, ie, the adrenals, testes, and ovaries. WT1 and SF1 are both active early in the development of the genital ridge and in the determination of both the ovaries and the testes.
XY gonadal dysgenesis with resulting female differentiation in 46,XY patients with intact SRY function has been reported in individuals with duplications of Xp21, a locus that contains the DAX1 gene. A mutation or deletion of DAX1, which encodes a transcriptional factor, results in X-linked congenital adrenal hypoplasia (AHC) and hypogonadotropic hypogonadism. Deletion or mutation of the DAX1 gene in 46,XY individuals has not resulted in an abnormality of testicular differentiation in humans. Similarly, duplication of the DAX1 gene appears not to affect ovarian morphogenesis and function in 46,XX females. Duplication or deletion of the Dax1 (mouse homolog) gene in 46,XY mice has caused sex reversal when tested against weak alleles of Sry. Thus, it appears that duplication of DAX1 is responsible for dosage-sensitive sex reversal in human XY individuals. It has been suggested that DAX1 is an “antitestis” factor rather than an ovary-determining gene. This hypothesis is supported by the finding that null mutations of the Dax1 locus in the female mouse do not affect ovarian differentiation or fertility (Figure 14-7).
Campomelic dysplasia is a skeletal dysplasia associated with sex reversal due to gonadal dysgenesis in 46,XY individuals. The gene for campomelic dysplasia (CMPD1) has been localized to 17q24.3-q25.1. Mutations
in one allele of the SOX9 gene, a gene related to SRY (called a SOX gene because it has an SRY HMG box that is more than 60% homologous to that of SRY), can result in both CMPD1 and XY gonadal dysgenesis with sex reversal. Duplication of the SOX9 gene both in humans and mice results in sex reversal in XX individuals who are SRY-negative. Hence, it appears that SOX9 is the only critical gene needed “downstream” of SRY for male sex determination. XY individuals with 9p- and 10q- deletions exhibit gonadal dysgenesis and male pseudohermaphroditism. Haploinsufficiency of DMRT1—a gene related to “double sex” in drosophila
and “Mab3” in C elegans— is a candidate for the abnormality in testis development noted in patients with 9p-. No gene has as yet been identified on 10q to account for sex reversal in these patients. Recently, an XY female with a duplication of chromosome region 1p31–35 was described containing a duplication of the WNT4 gene. Studies indicated that testis development was inhibited as a result of up-regulation ofDAX1 by the WNT4 duplication, resulting in inhibition of SF1 and SOX9 up-regulation in the developing testes and sex reversal. WNT4 is expressed in the ovary and is thought to be an essential signal for ovarian determination along with germ cells and unknown other genes (Figure 14-7A).
Figure 14-4. Diagrammatic representation of G-banded X chromosome. Selected X-linked genes are shown. (PHOG, pseudoautosomal homeobox osteogenic gene; SHOX, short-stature homeobox gene; MIC2, a cell surface antigen recognized by monoclonal antibody 12E7;PRKX, a member of the cAMP-dependent serine-threonine protein kinase gene family (illegitimate X-Y interchange occurs most frequently between PRKX and PRKY); DAX1, DSS-AHC-critical region on the X chromosome gene 1; GK, glycerol kinase; DMD, Duchenne's muscular dystrophy; USP9X, human x-linked homolog of the drosophila fat facets-related gene (DFFRX); RPS4X, ribosomal protein S4; XIST, Xi-specific transcripts; XIC, X-inactivation center; ATRX, α-thalassemia, X-linked mental retardation; DIAPH2, human homolog of the drosophila diaphanous gene.) (Reproduced with permission from Grumbach MM, Hughes IA, Conte FA. Disorders of sex differentiation. In Larsen PR et al (editors): Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
Figure 14-5. Diagrammatic representation of a G-banded Y chromosome. SHOX/PHOG, short stature homeobox gene/pseudoautosomal homeobox osteogenic gene; MIC2, gene for a cell surface antigen recognized by monoclonal antibody 12E7; SRY, sex-determining region Y; RPS4Y, ribosomal protein S4; ZFY, zinc finger Y; TSPYA,B, members of the testes-specific factor gene family; PRKY, a member of the cAMP-dependent serine-threonine protein kinase gene family; DAZ, deleted in azoospermia; AZF, azoospermic factor. (Reproduced with permission from Grumbach MM, Hughes IA, Conte FA. Disorders of sex differentiation. In Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
Table 14-2. Sex chromosome complement correlated with X chromatin and Y bodies in somatic interphase nuclei.1
TESTICULAR & OVARIAN DIFFERENTIATION
Until the 12-mm stage (approximately 42 days of gestation), the embryonic gonads of males and females are indistinguishable. By 42 days, 300–1300 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. Under the influence of SRY and other genes that encode male sex determination (Figure 14-7B), the gonad will begin to differentiate as a testis at 43–50 days of gestation. Leydig cells are apparent by about 60 days, and differentiation of male external genitalia occurs by 65–77 days of gestation.
Figure 14-6. FISH analyses for SRY in 46, XY interphase cells and metaphase chromosomes. The SRY probe (red) localizes to the distal short arm of the Y chromosome (Yp11.3). Another probe (blue) localizes the centromere region of the X chromosome. Both probes are visible in an interphase nucleus. (Courtesy of Dr. Philip Cotter, Oakland Children's Hospital; and Helen Jenks, U.C. Davis Medical Center. Reprinted with permission from Grumbach MM, Hughes IA, Conte IA: Disorders of sex differentiation. In Larsen PR et al. (editors)Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
In the gonad destined to be an ovary, the lack of differentiation persists. At 77–84 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. 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 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 ovaries (Figure 14-8).
Differentiation of Genital Ducts (Figure 14-9)
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 antimüllerian hormone (AMH), a dimeric glycoprotein secreted by fetal Sertoli cells. This hormone acts “locally” to cause müllerian duct repression ipsilaterally.
The gene for AMH encodes a 560-amino-acid protein whose carboxyl terminal domain shows marked homology with transforming growth factor (TGF)-β and the B chain of inhibin and activin. The gene has been localized on the short arm of chromosome 19. AMH is secreted by human fetal and postnatal Sertoli cells until 8–10 years of age and can be used as a marker for the presence of these cells. SF-1, an orphan nuclear receptor, in combination with WT1, SOX9, and GATA4 (Figure 14-6), has been shown to regulate AMH gene expression. The human AMH receptor gene has been cloned and mapped to the q13 band of chromosome 12. The receptor has been identified as being similar to other type II receptors of the TGF-β family.
The differentiation of the wolffian duct is mediated by testosterone secretion from the testis. SF-1 regulates steroidogenesis by the Leydig cell in the testes by binding to the promoter of the genes encoding P450scc and 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.
Figure 14-7. A: Hypothetical diagrammatic representation of the cascade of genes involved in testes determination in humans shown as a linear pathway rather than a “combinational network.” WT1, Wilms tumor suppressor, SF1, steroidogenic factor-1; DAX1, DSS-AHC-critical region on the X chromosome gene 1; a double dose of DAX1 inhibits the up-regulation of SOX9 and SF1 and results in inhibition of testes determination; WNT4, human gene related to the drosophila “wingless gene.” Duplication of WNT4 up-regulates DAX1 and prevents testes determination. SOX9, autosomal gene containing an SRY-like HMG box; AMH, antimüllerian hormone; GATA4, a transcription factor. SRY-regulated homeobox gene 9; DMRT1, double sex (drosophila) Mab3 (C elegans)-related transcription factor. B:Hypothetical diagrammatic cascade of genes involved in sex differentiation. (Reproduced with permission from Grumbach MM, Hughes IA, Conte FA. Disorders of Sex Differentiation. In: Larsen PR [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
Figure 14-8. Schematic sequence of sexual differentiation in the human fetus. Note that testicular differentiation precedes all other forms of differentiation. (Reproduced, with permission, from Grumbach MM, Conte FA: Disorders of sex differentiation. In: Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
Differentiation of External Genitalia (Figure 14-10)
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 will occur in the presence of an ovary or streak gonads or if no gonad is present (Figure 14-11). Differentiation of the external genitalia along male lines depends on the action of testosterone and particularly dihydrotestosterone, the 5α-reduced metabolite of testosterone. In the male fetus, testosterone is secreted by the Leydig cells, perhaps autonomously at first and thereafter under the influence of human chorionic gonadotropin (hCG), and then by stimulation from fetal pituitary luteinizing hormone (LH). Masculinization of the external genitalia and urogenital sinus of the fetus results from the action of dihydrotestosterone, which is converted from testosterone in the target cells by the enzyme 5α-reductase. Dihydrotestosterone (as well as testosterone) is bound to a specific protein receptor in the nucleus of the target cell. 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 (Figure 14-5). Thus, an X-linked gene controls the androgen response of all somatic cell types by specifying the androgen receptor protein.
As in the case of the genital ducts, there is an inherent tendency for the external genitalia and urogenital sinus to develop along female lines. Differentiation of the external genitalia along male lines requires androgenic stimulation early in fetal life. The testosterone metabolite dihydrotestosterone and its specific nuclear receptor must be present to effect masculinization of the external genitalia of the fetus. Dihydrotestosterone stimulates growth of the genital tubercle, fusion of the urethral folds, and descent of the labioscrotal swellings to form the penis and scrotum. Androgens also inhibit descent and growth of the vesicovaginal septum and differentiation of the vagina. There is a critical period for action of the androgen. After about the 12th week of gestation, fusion of the labioscrotal folds will not occur even under intense androgen stimulation, though phallic growth can be induced. Incomplete masculinization of the male fetus results from (1) impairment in the synthesis or secretion of fetal testosterone or in its conversion to dihydrotestosterone, (2) deficient or defective androgen receptor activity, or (3) defective production
and local action of antimüllerian hormone. Exposure of the female fetus to abnormal amounts of androgens from either endogenous or exogenous sources, especially before the 12th week of gestation, can result in virilization of the external genitalia.
Figure 14-9. Embryonic differentiation of male and female genital ducts from wolffian and müllerian primordia. A: Indifferent stage showing large mesonephric body. B: Female ducts. Remnants of the mesonephros and wolffian ducts are now termed the epoophoron, paroophoron, and Gartner's duct. C: Male ducts before descent into the scrotum. The only müllerian remnant is the testicular appendix. The prostatic utricle (vagina masculina) is derived from the urogenital sinus. (Redrawn from Corning and Wilkins.)
Figure 14-10. Differentiation of male and female external genitalia from bipotential primordia. (Reproduced, with permission, from Grumbach MM, Hughes IA, Conte FA: Disorders of sex differentiation. In: Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
Figure 14-11. Diagrammatic summation of human sexual differentiation. DHT, dihydrotestosterone. (Reproduced, with permission, from Grumbach MM, Hughes IA, Conte FA: Disorders of sex differentiation. In: Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
Psychosexual differentiation may 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.
Over the past 30 years, the prevailing dogma has been that newborns are born psychosexually neutral and that gender identity is imprinted postnatally by words, attitudes, and comparisons of one's body with that of others. Recently, this hypothesis has been rigorously challenged by Diamond and Sigmundson as well as Reiner. Their studies, among others, suggest that prenatal exposure to androgen and the presence of genes on the Y chromosome can influence gender identity in the patient with ambiguous genitalia. Gender identity has been hypothesized to be established by 12-18 months of age; however, it appears to be 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-hydroxysteroid dehydrogenase-3 deficiency, have had doubts about their gender identity and have chosen to change their assigned sex from female to male. However, there are 46,XY individuals who have had normal testicular function and androgen responsiveness in fetal life and have been assigned female gender which has been well accepted over time. This suggests that androgens have a “facultative”and not a “deterministic” role in gender identity. Thus, it is obvious that both genes and hormones (Nature) and environment (Nurture) are critical factors in the development and maintenance of gender identity. Further studies on patients with ambiguous genitalia and, in particular, long-term outcomes of gender identity, gender role, and sexual activity in intersex patients are necessary before the relative roles of nature and nurture can be resolved.
ABNORMAL SEX DIFFERENTIATION
Classification of Errors in Sex Differentiation (Table 14-3)
Disorders of sexual differentiation 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 true hermaphrodite is defined as a person who possesses both ovarian and testicular tissue. A male pseudohermaphrodite is one whose gonads are exclusively testes but whose genital ducts or external genitalia (or both) exhibit incomplete masculinization. A female pseudohermaphrodite is a person whose gonadal tissue is exclusively ovarian but whose genital development exhibits ambiguous or male appearance.
SEMINIFEROUS TUBULE DYSGENESIS: CHROMATIN-POSITIVE KLINEFELTER'S SYNDROME & ITS VARIANTS
Klinefelter's syndrome is one of the most common forms of primary hypogonadism and infertility in males. The invariable clinical features in adults are a male phenotype, firm testes less than 3 cm in length, and azoospermia. Gynecomastia is common. Affected patients usually have a 47,XXY sex chromosome constitution and an X chromatin-positive buccal smear, though subjects with a variety of sex chromosome constitutions, including mosaicism, have been described. Virtually all of these variants have in common the presence of at least two X chromosomes and a Y chromosome, except for the rare group in which only an XX sex chromosome complement is found.
Surveys of the prevalence of 47,XXY fetuses by karyotype analysis of unselected newborn infants indicate an incidence of about 1:800 newborn males. Prepubertally, the disorder is characterized by small undescended testes, disproportionately long legs, personality and behavioral disorders, and a lower mean verbal IQ score when compared with that of control subjects but no significant difference in full-scale IQ. Severe mental retardation requiring special schooling is uncommon. Gynecomastia and other signs of androgen deficiency such as diminished facial and body hair, a small phallus, poor muscular development, and a eunuchoid body habitus occur postpubertally in affected patients. 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. Untreated adult males, especially those with subnormal sex steroid levels, are at increased risk for the development of osteoporosis. They also have an increased incidence of mild diabetes mellitus, varicose veins, stasis dermatitis, cerebrovascular disease, chronic pulmonary disease, and carcinoma of the breast; the incidence of breast carcinoma in patients with Klinefelter's syndrome is 20 times higher than that in normal men. Patients with Klinefelter's syndrome often have a delay in the onset of adolescence. There is an increased risk for developing malignant
extragonadal germ cell tumors, including central nervous system germinomas and mediastinal tumors, which may be hCG-secreting and cause sexual precocity in the prepubertal patient.
Table 14-3. Classification of anomalous sexual development.1
The testicular lesion is progressive and gonadotropin-dependent. It is 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, and patients who have been reported to be fertile invariably have been 46,XY/47,XXY mosaics. Recently, the technique of intracytoplasmic sperm injection (ICSI) has been utilized with some success for achieving fertility in patients with Klinefelter's syndrome. It is likely that success is achieved only in those patients who are mosaics with a 46,XY cell line in their gonads.
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.
The diagnosis of Klinefelter's syndrome is suggested by the classic phenotype and hormonal changes. It is confirmed by the finding of an X chromatin-positive buccal smear and 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 testosterone production rate, the total and free levels of testosterone, and the metabolic clearance rates of testosterone and estradiol tend to be low, while plasma estradiol levels are relatively normal or high. Testicular biopsy reveals the classic findings of hyalinization of the seminiferous tubules, severe deficiency of spermatogonia, and pseudoadenomatous clumping of Leydig cells.
Treatment of patients with Klinefelter's syndrome is directed toward androgen replacement, especially in patients in whom puberty is delayed or fails to progress or in those who have subnormal testosterone levels for age and developmental stage. Testosterone therapy may help to enhance secondary sexual characteristics and sexual performance, prevent osteoporosis, prevent or cause regression of gynecomastia, and improve general well-being in most patients. If testosterone deficiency and/or elevated gonadotropins are present, testosterone therapy early in adolescence should commence with 50 mg of testosterone enanthate in oil intramuscularly every 4 weeks, gradually increasing to the adult replacement dose of 200 mg every 2 weeks after a bone age of 14 1/2 years. Thereafter, transdermal testosterone therapy (testosterone patch) may be used for adult replacement. A marked decrease in gynecomastia may result from testosterone therapy; however, once advanced, gynecomastia may not be amenable to hormone therapy but can be surgically corrected if it is severe or psychologically disturbing to the patient. Early diagnosis, support, and appropriate counseling will improve the overall prognosis.
Variants of Chromatin-Positive Seminiferous Tubule Dysgenesis
- VARIANTS OF KLINEFELTER'S SYNDROME
Variants of Klinefelter's 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
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. XX males have normal body proportions and a mean final height that is shorter than that of patients with an XXY sex chromosome constitution or normal males but taller than that of normal females. As in XXY patients, testosterone levels are low or low normal, gonadotropins are elevated, and spermatogenesis is impaired postpubertally. Gynecomastia is present in approximately one-third of cases.
The presence of testes and male sexual differentiation in 46,XX individuals has been a perplexing problem. However, the paradox has been clarified by the use of recombinant DNA studies. Males with a 46,XX karyotype have been shown by genetic linkage studies and X chromosome restriction fragment length polymorphisms (RFLPs) to possess one X chromosome from each of their parents. Approximately 80% 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 heterologous in length but always includes the SRY gene, which encodes testis-determining factor as well as the pseudoautosomal region of the Y chromosome. The site of translocation in 30–40% of 46,XX males involves PRKX/PRKY, a novel protein kinase gene, with homologs on the X and Y chromosome. Thus, in 80% 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 20% of XX males tested have been shown to lack Y chromosome-specific DNA sequences, including the SRY gene and the pseudoautosomal region of the Y chromosome. These XX, Y DNA-negative males tend to have hypospadias and may have relatives with true hermaphroditism.
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 or duplication of a “downstream” autosomal gene involved in male sex determination, eg, SOX9; or (2) mutation, deletion, or aberrant inactivation of a gene sequence on the X chromosome, critical to testis determination and differentiation; or (3) circumscribed Y chromosome mosaicism (eg, occurring only in the gonads). Further studies will be necessary to elucidate the pathogenesis of male sex determination and differentiation in those 46,XX males who lack ascertainable Y-to-X chromosome translocations.
SYNDROME OF GONADAL DYSGENESIS: TURNER'S SYNDROME & ITS VARIANTS
Turner's Syndrome: 45,X Gonadal Dysgenesis
One in 5000 newborn females has a 45,X sex chromosome constitution. It has been estimated that 99% of 45,X fetuses do not survive beyond 28 weeks of gestation, and 15% of all first-trimester abortuses have a 45,X karyotype. In about 70–80% of instances, the origin of the normal X chromosome is maternal. Patients with a 45,X karyotype represent approximately 50% of all patients with X chromosome abnormalities. 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's syndrome include coarctation of the aorta (10%), hypertension, renal abnormalities (50%), pigmented nevi, cubitus valgus, a tendency to keloid formation, 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. Routine intravenous urography or renal sonography is indicated for all patients to rule out a surgically correctable renal abnormality. The most common renal anomalies are rotation of the kidney, duplication of the renal pelvis and ureter, and hydronephrosis secondary to uteropelvic obstruction. Complete absence of the kidney or gross renal ectopia has 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 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 133–153 cm. Short stature found in patients with the syndrome of gonadal dysgenesis is not due to a deficiency of growth hormone, insulin-like growth factor I, sex steroids, or thyroid hormone. It is related, at least in part, to haploinsufficiency of the PHOG/SHOX gene in the pseudoautosomal region of the X and Y chromosome. (See earlier section.) Nevertheless, administration of high-dose biosynthetic human growth hormone results in an increase in final height.
Gonadal dysgenesis is another feature of patients with a 45,X chromosome constitution. The gonads 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 children (Figure 14-12). Thus, plasma and urinary gonadotropin levels, particularly FSH levels, are high during early infancy and after 9–10 years of age. Since ovarian function is impaired, puberty does not usually ensue spontaneously; hence, sexual infantilism is a hallmark of this syndrome. Rarely, patients with a 45,X karyotype may undergo spontaneous pubertal maturation, menarche, and pregnancy.
Figure 14-12. Diphasic variation in basal levels of plasma follicle-stimulating hormone (FSH) (ng/mL-LER 869) in patients with a 45,X karyotype (solid triangles) and patients with structural abnormalities of the X chromosome and mosaics (solid circles). Note that mean basal levels of plasma FSH in patients with gonadal dysgenesis are in the castrate range before 4 years and after 10 years of age. (Reproduced, with permission, from Conte FA, Grumbach MM, Kaplan SL: A diphasic pattern of gonadotropin secretion in patients with the syndrome of gonadal dysgenesis. J Clin Endocrinol Metab 1975;40:670.)
A variety of disorders are associated with this syndrome, including obesity, osteoporosis, diabetes mellitus, Hashimoto's thyroiditis, rheumatoid arthritis, inflammatory bowel disease, intestinal telangiectasia with bleeding, chronic liver disease, sensorineural hearing loss, and anorexia nervosa. Because an increased prevalence of bicuspid aortic valve, coarctation of the aorta, and aortic dilation with aneurysm formation and rupture has been reported in patients with Turner's syndrome, screening and periodic echocardiography are indicated in all patients with a 45,X cell line.
Phenotypic females with the following features should have a karyotype analysis: (1) short stature (> 2.5 SD below the mean value for age); (2) somatic anomalies associated with the syndrome of gonadal dysgenesis; and (3) delayed adolescence with an increased level of plasma FSH.
Therapy should be directed toward maximizing final height and inducing secondary sexual characteristics and menarche at an age commensurate with that of normal peers. The results of clinical trials suggest that patients treated with recombinant growth hormone (0.375 mg/kg/wk divided into seven once-daily doses), with or without oxandrolone (0.0625 mg/kg/d by mouth), had an increase in growth rate that was sustained and resulted in a mean 8–10 cm increase in height after 3–7 years of therapy. However, beginning growth hormone therapy earlier (when the child's height is less than -2 SD from the mean) will result in a greater gain in height and allow for the administration of estrogen replacement at an age commensurate with normal puberty. Before initiation of growth hormone therapy, a thorough analysis of the costs, benefits, and possible side effects must be discussed with the parents and the child. Long-term studies with low-dose estrogen therapy have not demonstrated a positive effect on final height in girls with Turner's syndrome. No synergistic effect of combined estrogen and growth hormone therapy on final height has been found. In patients who have been treated with growth hormone and have achieved an acceptable height and in those who have refused growth hormone therapy, estrogen replacement therapy is usually initiated after 12–13 years of age. Conjugated estrogens (0.3 mg or less) or ethinyl estradiol (5 ľg) are 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 0.6–1.25 mg of conjugated estrogens or 10 ľg of ethinyl estradiol daily for the first 21 days of the month. The minimum dose of estrogen necessary to maintain secondary sexual characteristics and menses and prevent osteoporosis should be administered. After the first year 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.
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 (Table 14-4). 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 (Figure 14-4). In general, 45,X/46,XX mosaicism will modify the 45,X phenotype toward normal and can even result in normal gonadal function. Some patients with a 45,X/46,Xr(X) karyotype may manifest mental retardation and congenital anomalies not usually associated with Turner's syndrome. Recent data indicate that 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.
Table 14-4. Relationship of structural abnormalities of the X and Y to clinical manifestations of the syndrome of gonadal dysgenesis.1
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's syndrome through patients with ambiguous genitalia to completely virilized males with few stigmas of Turner's syndrome. The variations in gonadal differentiation range from bilateral streaks to bilateral dysgenetic testes to apparently “normal” testes, and there may be asymmetric development, ie, a streak on one side and a dysgenetic testicle 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, presumably, the capacity of the fetal testes to secrete antimüllerian hormone and testosterone.
The risk of development 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 scanning, or magnetic resonance imaging (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.
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 fluorescence in situ hybridization or molecular analyses with X- and Y-specific probes is indicated to definitively determine the origin of the marker chromosome, since gonadoblastomas have been reported in patients with deleted Y chromosomes—even those with deletions of theSRY gene. The decision regarding the sex of rearing should be based on the age at diagnosis and the potential for normal function of the external genitalia. Most 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.
In phenotypic female XO/XY patients assigned a female gender role, the dysgenetic gonads should be removed. Estrogen therapy should be initiated at the age of puberty, as in patients with a 45,X karyotype (see above). 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. Removal of the müllerian structures and repair of hypospadias are also indicated. At puberty, depending on the functional integrity of the retained gonads, androgen 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 is indicated postpubertally to rule out the possibility of carcinoma in situ, a premalignant lesion (see below).
In infants and children with 45,X/46,XY mosaicism who have normal genitalia and normal testicular integrity as assessed by gonadotropin levels and pelvic MRI, gonadal biopsy may be deferred until adolescence. If biopsy and sonography show no evidence of carcinoma in situ, 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 is still to be ascertained.
46,XX & 46,XY Gonadal Dysgenesis
The terms 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's 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.
46,XX Gonadal Dysgenesis
Familial and sporadic cases of XX gonadal dysgenesis have been reported with an 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 revealed that a locus on chromosome 2p was linked to XX gonadal dysgenesis in females. The gene for the FSH receptor has been localized to chromosome 2p. Analysis of this gene revealed a mutation in exon 7 of the FSH receptor that segregated with XX gonadal dysgenesis. This mutation affected the extracellular ligand-binding domain of the FSH receptor and reduced the binding capacity of the receptor and consequently signal transduction, resulting in variable ovarian function, including “streak ovaries” and hypergonadotropic hypogonadism in some XX females at puberty. Further studies in Western Europe and the United States of females with 46,XX gonadal dysgenesis have been negative for FSH receptor gene mutations, suggesting that a mutation in this gene is rare and that other causes for this phenotype are more common. Preliminary data suggest that males homozygous for this mutation are phenotypically normal, with spermatogenesis varying from normal to absent.
Studies of familial cohorts have revealed apparent marked heterogeneity in pathogenesis. 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. Rather, they may have a mutation in an autosomal recessive gene involved in gonadal determination. In one family, four affected women had an inherited interstitial deletion of the long arm of the X chromosome involving the q21-q27 region. This region seems to contain a gene or genes critical to ovarian development and function. In three families, XX gonadal dysgenesis was associated with deafness of the sensorineural type. In several affected groups of siblings, a spectrum of clinical findings occurred, eg, varying degrees of ovarian function, including breast development and menses followed by secondary amenorrhea. Recently, haploinsufficiency of FOXL2, a gene on chromosome 3q23, has been shown to cause autosomal dominant blepharophimosis-ptosis-epicanthus inversus syndrome (BPES type 1) and XX gonadal dysgenesis. In contrast to Turner's syndrome, stature is normal. The diagnosis of 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's syndrome). Karyotype analysis reveals only 46,XX cells. As in Turner's syndrome, gonadotropin levels are high, estrogen levels are low, and treatment consists of cyclic estrogen and progesterone replacement.
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 infections such as mumps, antibodies to gonadotropin receptors, biologically inactive FSH, gonadotropin-insensitive ovaries, and galactosemia as well as errors in steroid (estrogen) biosynthesis. In the latter group, ultrasound or MRI should reveal polycystic ovaries.
46,XY 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 quite common, and, in familial 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 due to the degree of differentiation of testicular tissue and the functional capacity of the fetal testis to produce testosterone and antimüllerian hormone. Early in infancy and after the age of puberty, plasma and urinary gonadotropin levels are markedly elevated.
Analysis of familial and sporadic cases of 46,XY gonadal dysgenesis indicates that about 15–20% of patients have a mutation in the HMG box of the SRY gene that affects DNA binding or bending by the SRY protein. So far, all patients in whom mutations have been detected have had “complete” gonadal dysgenesis. Patients with large deletions of the short arm of the Y chromosome may have, in addition to gonadal dysgenesis, stigmas of Turner's syndrome. Mutations outside the HMG box region of the SRY gene as well as in X-linked or autosomal genes may be responsible for those patients in whom no molecular abnormality has as yet been found. A mutation in the HMG box of the SRY gene has been described in normal 46,XY fathers and their “daughters” with 46,XY gonadal dysgenesis. These familial cohorts suggest that these mutations and 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.
More than 20 patients with 46,XY gonadal dysgenesis have been reported with a duplication of the Xp21.2 → p22.11 region of the X chromosome. This region contains a gene, DAX1. 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; duplicating DAX1, however, impairs testis differentiation. Thus, DAX1 appears to be an antitestis gene. (See earlier section.)
XY gonadal dysgenesis associated with campomelic dysplasia is due to a mutation of one allele of an SRY-related gene, SOX9 on chromosome 17. In addition, XY gonadal dysgenesis has been associated with 9p- (DMRT1) and 10q- deletions, as well as duplication of 1p32–36 (WNT4).
Therapy for patients with 46,XY gonadal dysgenesis who have female external genitalia involves prophylactic gonadectomy at diagnosis and estrogen substitution at puberty. In the incomplete form of XY gonadal dysgenesis, assignment of a male gender role and treatment with testosterone to augment phallic size in infancy must be considered. Prophylactic gonadectomy must be considered, since fertility is unlikely and there is an increased risk of malignant transformation of the dysgenetic gonads in these patients. Biopsy of all retained gonads should be done pre- and postpubertally in order to detect early malignant changes (carcinoma in situ). In affected individuals raised as males, prosthetic testes should be implanted at the time of gonadectomy, and androgen substitution therapy is instituted at the age of puberty. Testosterone enanthate in oil (or another long-acting testosterone ester) is used, beginning with 50 mg intramuscularly every 4 weeks and gradually increasing after a bone age of 14 1/2 years to a full replacement dose of 200 mg intramuscularly every 2 weeks.
In true hermaphroditism, both ovarian and testicular tissue 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 ambiguous. 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, though 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 true hermaphrodites (60%), followed by the ovary and, least commonly, by the 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 almost always dysgenetic.
Sixty percent of true hermaphrodites have been reported to have a 46,XX karyotype, 20% 46,XY, and about 20% have chromosome mosaicism or 46,XX/46,XY chimerism. 46,XX true hermaphroditism appears to be a genetically heterogeneous entity. A small proportion of 46,XX true hermaphrodites, including some in family cohorts with 46,XX males, have been reported to be SRY-positive. Hence, Y-to-X and Y-to-autosome translocations, hidden sex chromosome mosaicism, or chimerism can explain the pathogenesis in these patients. The majority of 46,XX true hermaphrodites, however, are SRY-negative. A number of families have been reported that had both SRY-negative 46,XX males and 46,XX true hermaphrodites. This latter observation suggests a common genetic pathogenesis in these patients. Possible genetic mechanisms to explain SRY-negative true hermaphroditism include (1) mutation of a downstream autosomal gene or modifier genes involved in testicular determination; (2) mutation, deletion, duplication, or anomalous inactivation of an X-linked locus involved in testis determination; or (3) circumscribed chimerism or mosaicism that occurred only in the gonads.
The diagnosis of true hermaphroditism should be considered in all patients with ambiguous genitalia. The finding of a 46,XX/46,XY karyotype or a bilobate gonad compatible with an ovotestis in the inguinal region or labioscrotal folds suggests the diagnosis. Basal plasma testosterone levels are elevated above 40 ng/dL in affected patients under 6 months of age, and testosterone levels increase after hCG stimulation. The estradiol response to human menopausal gonadotropins has been shown to be a reliable test for differentiating infants with true hermaphroditism from those with other disorders of sexual differentiation. If all other forms of male and female pseudohermaphroditism have been excluded, laparotomy and histologic confirmation of both ovarian and testicular tissue establish the diagnosis. The management of true hermaphroditism is contingent upon the age at diagnosis and a careful assessment of the functional capacity of the gonads, genital ducts, and external genitalia. In general, 46,XX true hermaphrodites should be raised as females, with the possible exception of the well-virilized patient in whom no uterus is found.
Gonadal Neoplasms in Dysgenetic Gonads
While gonadal tumors are rare in patients with 47,XXY Klinefelter's syndrome and 45,X gonadal dysgenesis, the prevalence of gonadal neoplasms is greatly increased in patients with certain types of dysgenetic gonads. The frequency is increased in 45,X/46,XY mosaicism, especially in those with female or ambiguous genitalia; in patients with a structurally abnormal Y chromosome;
and in those with XY gonadal dysgenesis, either with a female phenotype or with ambiguous genitalia. Gonadoblastomas, germinomas, seminomas, and teratomas are found most frequently. Prophylactic gonadectomy is advised in these patients as well as in those with Turner's syndrome who manifest signs of virilization, regardless of karyotype. The significance of hidden mosacism in patients with Turner's syndrome for Y chromosomal DNA determined by recombinant DNA technology is controversial at present with respect to the risk of gonadal neoplasms. Gonadoblastomas have been reported in patients with marker chromosomes of Y origin lacking the SRY gene. The testis should be preserved in patients who are to be raised as males only if it is histologically and functionally normal and 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, as seminomas tend to metastasize at an early stage before a mass is obvious. If a testis is preserved in the scrotum in a patient with 45,X/46,XY mosaicism or in rare cases of true hermaphroditism, it is prudent to follow the patient closely with sonography or pelvic MRI and a biopsy postpubertally in order to monitor for the development of a premalignant or malignant lesion.
Affected individuals have normal ovaries and müllerian derivatives associated with ambiguous external genitalia. In the absence of testes, a female fetus will be masculinized if subjected to increased circulating levels of androgens derived from a fetal or maternal source. The degree of masculinization depends upon the stage of differentiation at the time of exposure (Figure 14-13). After 12 weeks of gestation, androgens will produce only clitoral hypertrophy. Rarely, ambiguous genitalia that superficially resemble those produced by androgens are the result of other teratogenic factors.
Congenital Adrenal Hyperplasia (Figure 14-14)
Congenital adrenal hyperplasia is responsible for most cases of female pseudohermaphroditism and around 50% of all cases of ambiguous genitalia. There are five major types of congenital adrenal hyperplasia, 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 in adrenal hyperplasia. 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. Defects in 3β-hydroxysteroid dehydrogenase type II, 17α-hydroxylase (17,20-lyase), and StAR (steroidogenic acute regulatory protein) and P450scc (side chain cleavage) 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 14-5). Consequently, these will be discussed primarily as forms of male pseudohermaphroditism.
P450c21 Hydroxylase Deficiency
21-Hydroxylase activity is mediated by P450c21, a microsomal cytochrome P450 enzyme. 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. Over 95% of patients with congenital adrenal hyperplasia 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 and P450c21B, in this region in tandem with the two genes for complement, C4A and C4B. P450c21A is a nonfunctional “pseudogene,” ie, it is missing critical sequences and does not encode a functional 21-hydroxylase. Seventy-five percent of patients with “classic” P450c21 deficiency have point mutations that change a small 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 exon 3 to 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. The remainder have gene deletions and macrogene conversions. Recent work has demonstrated that classic salt-wasting 21-hydroxylase deficiency is associated with a mutation, deletion, or gene conversion that abolishes or severely reduces 21-hydroxylase activity. Most patients with 21-hydroxylase deficiency are compound heterozygotes, ie, they have a different genetic lesion in each of theirP450c21B allelic genes. The phenotypic spectrum observed—salt loss,
simple virilization, or late onset of virilization—is a consequence of the degree of enzymatic deficiency. The latter is determined by the functionally less severely mutated P450c21B allele. 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. These include Bw51 in the simple virilizing form, Bw47 in the salt-losing form, and B14 in the nonclassic form.
Figure 14-13. Female pseudohermaphroditism induced by prenatal exposure to androgens. Exposure after the 12th fetal week leads only to clitoral hypertrophy (diagram at left). Exposure at progressively earlier stages of differentiation (depicted from left to right in drawings) leads to retention of the urogenital sinus and labioscrotal fusion. If exposure occurs sufficiently early, the labia will fuse to form a penile urethra. (Reproduced, with permission, from Grumbach MM, Ducharme J: The effects of androgens on fetal sexual development: Androgen-induced female pseudohermaphroditism. Fertil Steril 1960;11:757.)
Figure 14-14. A diagrammatic representation of the steroid biosynthetic pathways in the adrenal and gonads. I-VI correspond to enzymes whose deficiency results in congenital adrenal hyperplasia. (OH, hydroxy or hydroxylase; 3β-HSD, 3β-hydroxysteroid dehydrogenase and Δ5-isomerase; 17β-HSD3, 17β-hydroxysteroid dehydrogenase 3; P450scc, cholesterol side-chain cleavage, previously termed 20,22 desmolase; P450c21, 21-hydroxylase; P450c17, 17-hydroxylase; P450arom, aromatase. P450c17 also mediates 17,20-lyase activity; P450c11B2 (aldosterone synthase) mediates 18-hydroxylase and 18-oxidase reactions; P450c11B1 mediates 11-hydroxylation of deoxycortisol to cortisol and DOC to corticosterone. The dashed arrow indicates that this reaction may not occur in humans. (Modified and reproduced, with permission, from Conte FA, Grumbach MM. Pathogenesis, classification, diagnosis, and treatment of anomalies of sex. In: DeGroot L [editor]: Endocrinology. Grune & Stratton, 1989.)
Table 14-5. Clinical manifestations of the various types of congenital adrenal hyperplasia.1
- P450C21 HYDROXYLASE DEFICIENCY WITH VIRILIZATION
This defect in P450c21 (21-hydroxylase) activity results in impaired cortisol synthesis, increased ACTH levels, and increased adrenal androgen precursor and androgen secretion. Its incidence is 1:50,000 persons, and it accounts for about 20% of individuals with P450c21 hydroxylase deficiency. Before 12 weeks of gestation, high fetal androgen levels lead to a varying degree of labioscrotal fusion and clitoral enlargement in the female fetus; exposure to androgen after 12 weeks induces clitoromegaly alone. 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, though they may have 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 peripubertal bone ages.
Mild defects in P450c21 (21-hydroxylase) activity 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. It has been postulated that “nonclassic” P450c21 hydroxylase deficiency is the most common autosomal recessive disorder, affecting about one in 100 persons of all ethnic groups but having an incidence two to three times higher in Hispanics and Ashkenazic Jews. Females with late-onset P450c21 hydroxylase deficiency have normal female genitalia at birth and do not have an electrolyte abnormality. Mild virilization occurs later in childhood and adolescence, resulting in the premature development of pubic or axillary hair, slight clitoral enlargement, menstrual irregularities,
acne, hirsutism, polycystic ovary syndrome, and an advanced bone age. Affected males have normal male genitalia at birth, rapid growth, and advanced skeletal maturation. Later in childhood, they exhibit premature growth of pubic or axillary hair, sexual precocity with inappropriately small testes, and increased muscular development. While tall as children, they end up as short adults due to advanced bone maturation and premature epiphysial fusion. Asymptomatic individuals who have the same biochemical abnormalities as patients with mild forms of P450c21 hydroxylase deficiency have been detected by hormonal testing of families in which there is at least one member with symptoms.
- P450C21 HYDROXYLASE DEFICIENCYWITH 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 more severe deficit of P450c21 hydroxylase, which leads 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 later at 6-12 weeks, usually associated with a concomitant physiologic stress. Masculinization of the external genitalia of affected females tends to be more severe than that found in patients with simple 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.
The diagnosis of P450c21 hydroxylase deficiency should always be considered (1) in patients with ambiguous genitalia who have a 46,XX karyotype (and are thus female pseudohermaphrodites); (2) in apparent cryptorchid males; (3) in any infant who presents with shock, hypoglycemia, and chemical findings compatible with adrenal insufficiency; and (4) in males or females with signs of virilization before puberty, including premature adrenarche. 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 still valid and useful, urinary steroid determinations have been replaced by the simpler and more cost-effective measurement of plasma 17-hydroxyprogesterone, androstenedione, and testosterone levels.
The concentration of plasma 17-hydroxyprogesterone is elevated in umbilical cord blood but rapidly decreases into the range of 100–200 ng/dL (3–6 nmol/L) by 24 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), depending on the age of the patient and the severity of P450c21 hydroxylase deficiency. Patients with mild P450c21 hydroxylase deficiency, ie, 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 demonstrated by New and coworkers.
Salt losers may be ascertained clinically or by chemical evidence of hyponatremia and hyperkalemia on a regular infant diet. In these patients, aldosterone levels in both plasma and urine are low in relation to the serum sodium concentration, while plasma renin activity is elevated. Breast milk and many infant formulas have a low concentration of sodium.
HLA typing, measurement of amniotic fluid 17-hydroxyprogesterone levels, and chorionic villus biopsy with HLA typing and gene analysis have been used in the prenatal diagnosis of affected fetuses. Data indicate that prenatal therapy with dexamethasone given to the mother early in pregnancy can lessen the genital ambiguity seen in affected newborn females; however, the use of this therapy is controversial. While the immediate effects of maternal dexamethasone therapy on reducing masculinization of the female external genitalia may be striking, long-term studies are needed to exclude late untoward effects. (See Consensus statement on 21-hydroxylase deficiency from the Lawson Wilkins Pediatric Endocrine Society and the European Society for Paediatric Endocrinology. J Clin Endocrinol Metab 2002;87:4048
Heterozygosity has been ascertained by HLA typing in informative families, by the use of ACTH-induced rises in plasma 17-hydroxyprogesterone levels and by genetic analysis. Measurement of plasma 17-hydroxyprogesterone levels using heel-stick capillary blood specimens blotted onto paper has been shown to be a useful and valid screening tool for the diagnosis of 21-hydroxylase deficiency in newborn infants.
- P450C11 HYDROXYLASE DEFICIENCY
Classic P450c11 hydroxylase deficiency (virilization with hypertension) is rare; however, it is the second most common form of congenital adrenal hyperplasia, representing 5–8% of all cases. It occurs in 1:100,000 births in persons of European ancestry. However, in Middle Eastern people, it is much more common. In the classic patient, a defect in 11-hydroxylation leads to decreased cortisol levels with a consequent increase in ACTH and the hypersecretion of 11-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 11-deoxycorticosterone secretion. Plasma renin activity is either normal or suppressed. The hypertension is not invariable; it occurs in approximately two-thirds of patients and may be associated with hypokalemic alkalosis.
Two P450c11 hydroxylase genes have been localized to the long arm of chromosome 8: P450c11β1 and P450c11β2. Similar to 21-hydroxylase, these two genes are 95% homologous. P450c11β1 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 deoxycorticosterone (DOC) to corticosterone. It has about one-twelfth the capacity of P450c11β2 for 18-hydroxylation and does not oxidize 18-hydroxycorticosterone to aldosterone. P450c11β2 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 can produce a wide variety of clinical manifestations from virilization and hypertension (P450c11β1 deficiency) to isolated salt wasting (P450c11β2 [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 P450c11β1 and P450c11β2 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.
The diagnosis of P450c11β-hydroxylase deficiency can be confirmed by demonstration of elevated basal or ACTH-induced plasma levels of 11-deoxycortisol and 11-deoxycorticosterone, at least three times higher than the 95th percentile for age, and increased excretion of their metabolites in urine (mainly tetrahydro-11-deoxycortisol).
- TYPE IV—3β-HYDROXYSTEROID DEHYDROGENASE DEFICIENCY
Male or female pseudohermaphroditism and adrenal insufficiency are discussed below.
- TYPE V—P450C17 DEFICIENCY
Male pseudohermaphroditism, sexual infantilism, hypertension, and hypokalemic alkalosis are discussed below.
- TYPE VI—STAR DEFICIENCY
Congenital lipoid adrenal hyperplasia, male pseudohermaphroditism, sexual infantilism, and adrenal insufficiency are discussed below.
Treatment of patients with 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 50–100 mg/m2 added to the infusion fluid over the first 24 hours of therapy. If hyponatremia and hyperkalemia are present, 0.05–0.1 mg of fludrocortisone by mouth may be given along with the intravenous saline and hydrocortisone. Since 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.
Once the patient is stabilized and a definitive diagnosis has been arrived at by means of appropriate steroid studies, the patient should receive maintenance doses of glucocorticoids to permit normal growth, development, and bone maturation (hydrocortisone, approximately 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. 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 suggested. These include adrenalectomy in patients with null mutations of P450c21, growth hormone 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.
Patients with ambiguous external genitalia should have plastic repair. Clitoral recession or clitoroplasty—
not clitoridectomy!—is indicated. Of major importance to the family with an affected child is the assurance that the child will grow and develop into a normal adult. In patients with the most common form of adrenal hyperplasia—21-hydroxylase deficiency—fertility in males and feminization, menstruation, and fertility in females can be expected with adequate treatment. Long-term psychologic guidance and support by the physician for the patient and family are essential.
Adrenal rests in the testes of males with P450c21 hydroxylase deficiency (especially salt losers) may enlarge under the stimulus of ACTH and be mistaken for testicular neoplasms. These adrenal rests are often bilateral and are made up of cells that appear indistinguishable from Leydig cells histologically except that they lack Reinke crystalloids. The rests are usually seen in noncompliant or undertreated patients. To prevent this complication as well as the risk of adrenal crisis, pituitary basophil hyperplasia, and adrenal carcinoma, continuous treatment with a glucocorticoid (and, if indicated, a mineralocorticoid) is recommended even in adult males.
P450 AROMATASE DEFICIENCY
A new form of androgen-induced female pseudohermaphroditism has been defined that is due to aromatase deficiency. Mutations in the gene encoding P450arom result in defective placental conversion of C19 steroids to estrogens, leading to exposure of the fetus to excessive amounts of testosterone and masculinization of the external genitalia of the female fetus. Virilization of the mother during gestation can also occur. At puberty, defective aromatase activity in the gonads leads to pubertal failure, hypergonadotropic hypogonadism, polycystic ovaries, mild virilization, tall stature, and osteoporosis. A striking delay in bone age occurs despite increased concentrations of plasma testosterone, supporting the concept that estrogens rather than androgens are the major sex steroids affecting bone maturation, bone turnover, and epiphyseal fusion in females as well as males. The diagnosis of aromatase deficiency is suggested by the finding of the above clinical picture and elevated plasma androstenedione and testosterone levels in the face of low estrogen levels.
Glucocorticoid Receptor Gene Mutation
Glucocorticoid resistance due to a homozygous mutation in the gene encoding the glucocorticoid receptor has recently been reported to induce female pseudohermaphroditism. 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 female pseudohermaphroditism.
MATERNAL ANDROGENS & PROGESTOGENS
Masculinization of the external genitalia of a female infant can occur if the mother is given testosterone, other androgenic steroids, or certain synthetic progestational agents during pregnancy. After the 12th week of gestation, exposure results in clitoromegaly alone. Norethindrone, ethisterone, norethynodrel, and medroxyprogesterone acetate have all been implicated in masculinization of the female fetus. Nonadrenal female pseudohermaphroditism can occur as a consequence of maternal ingestion of danazol, the 2,3-d-isoxazol derivative of 17α-ethinyl testosterone. 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 androgen exposure by the ability of the fetal-placental unit to aromatize androgens to estrogens, especially after the first trimester.
The diagnosis of female pseudohermaphroditism arising from transplacental passage of androgenic steroids is based on exclusion of other forms of female pseudohermaphroditism and a history of drug exposure. Surgical correction of the genitalia, if needed, is the only therapy necessary.
Nonadrenal female pseudohermaphroditism can be associated with imperforate anus, renal anomalies, and other malformations of the lower intestine and urinary tract. Sporadic as well as familial cases have been reported.
Male pseudohermaphrodites have gonads that are testes, but the genital ducts or external genitalia, or both, are not completely masculinized. Male pseudohermaphroditism can result from deficient testosterone secretion as a consequence of (1) defective testicular differentiation (testicular dysgenesis), (2) impaired secretion of testosterone or antimüllerian hormone, (3) failure of target tissue response to testosterone and dihydrotestosterone or antimüllerian hormone, and (4) failure of conversion of testosterone to dihydrotestosterone.
Testicular Unresponsiveness to hCG & LH
Male sexual differentiation is dependent upon the production of testosterone by fetal Leydig cells. Leydig cell testosterone secretion is under the influence of placental hCG during the critical period of male sexual differentiation and, thereafter, fetal pituitary LH during gestation.
The finding of normal male sexual differentiation in XY males with anencephaly, apituitarism, or congenital hypothalamic hypopituitarism suggests that male sex differentiation in the human occurs independently of the secretion of fetal pituitary gonadotropins.
Absence, hypoplasia, or unresponsiveness of Leydig cells to hCG-LH results in deficient testosterone production and, consequently, male pseudohermaphroditism. The extent of the genital ambiguity is a function of the degree of testosterone deficiency, and the phenotype has ranged from extreme forms with female external genitalia to milder forms with micropenis and to males with normal male genitalia and hypergonadotropic hypogonadism at puberty. A small 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 as well as an animal model, the “vet” rat. In most of the patients thus far reported, the defect resulted in female-appearing genitalia and a short blind-ending vagina. Müllerian duct regression was complete. Basal gonadotropin levels as well as GnRH-evoked responses were elevated in postpubertal patients. Plasma 17α-hydroxyprogesterone, androstenedione, and testosterone levels were low, and hCG elicited little or no response in testosterone or its precursors. In two siblings with the extreme phenotype of this syndrome, a homozygous missense mutation in exon 11 of the LH receptor gene was found. This mutation resulted in an alanine-to-proline change in the sixth transmembrane domain of the LH receptor and a nonfunctional receptor. Other inactivating mutations of the LH receptor gene have been described in unrelated families with LH resistance. These mutations have resulted in a variable degree of hCG-LH resistance and a variable phenotype in the affected XY individual. Recent studies in patients with the clinical and chemical features of Leydig cell hypoplasia have demonstrated that the causes of the syndrome are heterogeneous, and it may result from mutations outside the coding region of the LH-hCG receptor or in other Leydig cell-specific genes. 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, testosterone will augment phallic development and virilize the patient at puberty.
Inborn Errors of Testosterone Biosynthesis
Figure 14-15 demonstrates the major pathways in testosterone biosynthesis in the gonads; each step is associated with an inherited defect that results in testosterone deficiency and, consequently, male pseudohermaphroditism. 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 testosterone. Thus, they represent forms of congenital adrenal hyperplasia.
- STAR DEFICIENCY AND CONGENITAL LIPOID ADRENAL HYPERPLASIA
Male pseudohermaphroditism, sexual infantilism, and adrenal insufficiency is a very early defect in the synthesis of all steroids affecting the conversion of cholesterol to Δ5-pregnenolone and results in severe adrenal and gonadal deficiency. The P450scc gene has been isolated, cloned, and localized to chromosome 15. However, thus far, molecular analysis of this gene has revealed only one patient in whom a heterozygous mutation (haploinsufficiency) was found. This patient was a hyperpigmented 4-year-old 46,XY male pseudohermaphrodite with clitoromegaly, no labial fusion, a blind vaginal pouch, and late-onset adrenal insufficiency. Mutations in a steroidogenic acute regulatory (StAR) protein that is necessary for the transport of cholesterol from the outer to the inner mitochondrial membrane, the site of P450scc, have been identified in all other patients with the clinical syndrome of congenital lipoid adrenal hyperplasia. StAR is expressed in the adrenals and gonads but not in the placenta; hence, placental synthesis of progesterone, which is required to maintain pregnancy in humans, is apparently not affected. A homozygous null mutation in the P450scc gene resulting in a deficit of side-chain cleavage enzymatic activity most likely would be lethal, as it is essential for progesterone synthesis by the human fetoplacental unit.
Affected males usually have female or, rarely, ambiguous external genitalia with a blind vaginal pouch and hypoplastic male genital ducts but no müllerian derivatives; the genitalia of affected females are normal, and ovarian function occurs at puberty but subsequently subsides, secondary to the accumulation of cholesterol in the functioning ovaries with subsequent organ failure. Large lipid-laden adrenals that displace the kidneys downward may be demonstrated by intravenous urography, abdominal ultrasonography, or computed tomography scan. Death in early infancy from adrenal insufficiency is not uncommon. 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. 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.
- 3β-HYDROXYSTEROID DEHYDROGENASE AND δ5-ISOMERASE 2 DEFICIENCY
3β-Hydroxysteroid dehydrogenase 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 male or female pseudohermaphroditism and adrenal insufficiency. This enzyme is encoded for by a gene on the short arm of chromosome number 1. Recent data indicate that there are two highly homologous genes encoding 3β-hydroxysteroid dehydrogenase on chromosome 1. The type 1 3β-hydroxysteroid dehydrogenase gene is expressed in the placenta and peripheral tissues, while type 2 is expressed in the adrenals and gonads. 3β-Hydroxysteroid dehydrogenase 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. This defect in its complete form results in a severe deficiency of aldosterone, cortisol, testosterone, and estradiol secretion. Males with this defect are incompletely masculinized, and females have normal female genitalia or mild clitoromegaly. Salt loss and adrenal crises usually occur in early infancy in affected patients. Affected males may experience normal male puberty but often have prominent gynecomastia. Patients with a mild non-salt-losing form of 3β-hydroxysteroid dehydrogenase type 2 deficiency have been described as well as late-onset patients presenting with only premature pubarche. These patients were shown to have elevated Δ5 steroids as well as mutations in the 3β-hydroxysteroid type 2 gene.
Figure 14-15. Enzymatic defects in the biosynthetic pathway for testosterone. All five of the enzymatic defects cause male pseudohermaphroditism in affected males. Although all of the blocks affect gonadal steroidogenesis, those at steps 1, 2, and 3 are associated with major abnormalities in the biosynthesis of glucocorticoids and mineralocorticoids in the adrenal. All reported patients except one with apparent P450scc deficiency have a mutation in StAR (steroidogenic acute regulatory protein), a protein necessary for the transport of cholesterol from the outer to the inner mitochondrial membrane, where P450scc resides. OH, hydroxy; 3β-HSD, 3β-hydroxysteroid dehydrogenase; 17β-HSD, 17β-hydroxysteroid dehydrogenase-3. Chemical names for enzymes are shown with traditional names in parentheses. (Modified and reproduced, with permission, from Conte FA, Grumbach MM. Pathogenesis, classification, diagnosis, and treatment of anomalies of sex. In: DeGroot L [editor]: Endocrinology. Grune & Stratton, 1989.)
The diagnosis of 3β-hydroxysteroid dehydrogenase deficiency is based on finding elevated concentrations of Δ5-pregnenolone, Δ5-17α-hydroxypregnenolone, dehydroepiandrosterone (DHEA) and its sulfate, and other 3β-hydroxy-Δ5 steroids in the plasma and urine of patients with a consistent clinical picture. 3-keto-Δ4 steroids, ie, 17-hydroxyprogesterone and androstenedione, may be elevated owing 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-hydroxysteroid dehydrogenase 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 0.25 mg of synthetic ACTH. After ACTH stimulation, 17-hydroxypregnenolone levels are > 5.3 SD above the mean for unaffected individuals in affected infants, > 35 SD above the mean in prepubertal children, and > 21 SD above the mean in adults. The 17-hydroxypregnenolone/cortisol ratio is > 6.4 SD above the mean in infants, > 23 SD above the mean in prepubertal children, and > 221 SD above the mean in adults (see Lutfallah et al). It can be confirmed by detecting a mutation in the type II β-hydroxysteroid dehydrogenase Δ5 isomerase gene. Suppression of the increased plasma and urinary 3β-hydroxy-Δ5 steroids by the administration of dexamethasone distinguishes 3β-hydroxysteroid dehydrogenase deficiency from a virilizing adrenal tumor. Treatment of this condition is similar to that of other forms of adrenal hyperplasia (see above).
- P450C17 DEFICIENCY, 17α-HYDROXYLASE DEFICIENCY
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. Increased DOC secretion with resultant hypertension produces suppression of renin and, consequently, decreased aldosterone secretion. Clinically, this results in male pseudohermaphroditism, sexual infantilism, hypertension, and hypokalemic alkalosis.
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—dehydroepiandrosterone—in the adrenal cortex and gonads. Mutations affecting 17-hydroxylase activity have included stop codons, frame shifts, deletions, and missense substitutions.
The clinical manifestations result from the adrenal and gonadal defect. Affected XX females have normal development of the internal ducts and external genitalia but manifest sexual infantilism with elevated gonadotropin concentrations at puberty. Affected males with less than 25% 17α-hydroxylase activity have impaired testosterone synthesis by the fetal testes, which results in female or ambiguous genitalia. At adolescence, sexual infantilism, low renin hypertension, and often hypokalemia are the hallmarks of this defect.
The diagnosis of 17-hydroxylase deficiency should be suspected in XY males with female or ambiguous genitalia or XX females with sexual infantilism who also manifest hypertension associated with hypokalemic alkalosis. High levels of progesterone, Δ5-pregnenolone, DOC, corticosterone, and 18-hydroxycorticosterone in plasma and increased excretion of their urinary metabolites establish the diagnosis. Plasma renin activity and aldosterone secretion are diminished in these patients.
- 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 sterol Δ-7-reductase, DHCR7, causing deficiency of cholesterol. The diagnosis is made by the clinical features and confirmed by demonstration of low levels of cholesterol and increased levels of 7-dehydrocholesterol.
Note: The following errors affect testosterone and estrogen biosynthesis in the gonads primarily.
- P450C17 DEFICIENCY (17,20-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 human, the scission of 17-hydroxyprogesterone to androstenedione occurs at a low level. Rare patients are reported to have a defect primarily in the scission of the C21 steroids to C19 steroids, which results in a defect in testosterone synthesis and subsequently pseudohermaphroditism in the male and impaired sex steroid synthesis and secretion in the affected 46,XX female. Two male pseudohermaphrodites from consanguineous marriages who had micropenis, perineal hypospadias, bifid scrotum, a blind vaginal pouch, and cryptorchidism have recently been studied. The administration of hCG resulted in a marked rise in plasma 17-hydroxyprogesterone with a paucity of response in plasma DHEA, androstenedione, and testosterone consistent with a diagnosis of isolated 17,20-lyase deficiency. Analyses of the P450c17 gene in
these patients demonstrated one to be homozygous for an Arg 347 → His mutation and the other to have an Arg358 → Gln mutation. Both of these mutations result in a specific decrease in 17,20-lyase activity of the product encoded by the P450c17 gene.
Patients with 17,20-lyase deficiency have low circulating levels of testosterone, androstenedione, DHEA, and estradiol. The diagnosis can be confirmed by demonstration of an increased ratio of 17-hydroxy C21 steroids to C19 steroids (testosterone, DHEA, Δ5-androstenediol, and androstenedione) after stimulation with ACTH or hCG and by DNA analysis of the P450c17 gene.
- 17β-HYDROXYSTEROID DEHYDROGENASE-3 DEFICIENCY
There are at least six isoenzymes that mediate the 17β-hydroxysteroid dehydrogenase reaction in humans. The last step in testosterone and estradiol biosynthesis by the gonads involves the reduction of androstenedione to testosterone and estrone to estradiol by 17-hydroxysteroid oxidoreductase-3, an NADPH-dependent microsomal enzyme. This gene is located on chromosome 9q22 and is expressed primarily in the testes. Mutations in this gene have been described in male pseudohermaphrodites. At birth, males with a deficiency of the enzyme 17-hydroxysteroid dehydrogenase-3 have predominantly female or mildly ambiguous external genitalia resulting from testosterone deficiency during male differentiation. They have male duct development, absent müllerian structures with a blind vaginal pouch, and inguinal or intra-abdominal testes. The finding of wolffian ducts associated with female external genitalia in these patients is as yet not fully explained. At puberty, progressive virilization with clitoral hypertrophy occurs as a result of peripheral extraglandular conversion of androstenedione to testosterone by 17β-hydroxysteroid dehydrogenase-5. This is often associated with the concurrent development of gynecomastia. Plasma gonadotropin, androstenedione, and estrone levels are elevated, whereas testosterone and estradiol concentration are relatively low. A putative late-onset form of 17-hydroxysteroid oxidoreductase-3 deficiency has been reported in a small number of postadolescent males with gynecomastia and normal male genitalia.
Analysis of 17 patients with classic 17β-hydroxysteroid dehydrogenase-3 deficiency, including four from San Francisco, has revealed 14 mutations in the 17β-hydroxysteroid oxidoreductase-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.
17-Hydroxysteroid dehyrogenase-3 deficiency should be included in the differential diagnosis of (1) male pseudohermaphrodites with absent müllerian derivatives who have no abnormality in glucocorticoid or mineralocorticoid synthesis; and (2) male pseudohermaphrodites who virilize at puberty, especially if they also exhibit gynecomastia. The diagnosis of 17-hydroxysteroid dehydrogenase-3 deficiency is confirmed by the demonstration of inappropriately high plasma levels of estrone and androstenedione and increased ratios of plasma androstenedione to testosterone and estrone to estradiol before and after stimulation with hCG.
Management of the patients, as of those with other forms of male pseudohermaphroditism, depends on the age at diagnosis and the degree of ambiguity of the external genitalia. In the patient assigned a male gender identity, plastic repair of the genitalia and testosterone augmentation of phallic growth prepubertally as well as testosterone replacement therapy at puberty are indicated. In patients reared as females (the usual case), the appropriate treatment is castration, followed by estrogen replacement therapy at puberty. Affected females have no abnormalities in phenotype or gonadal function, since 17β-hydroxysteroid dehydrogenase-3 is not expressed in the ovary.
Defects in Androgen-Dependent Target Tissues
The complex mechanism of action of steroid hormones at the cellular level has recently been clarified (Figure 14-16; see also Chapter 12and Figure 12-3).
Free testosterone enters the target cells and undergoes 5α reduction to dihydrotestosterone. Dihydrotestosterone binds to the intracellular androgen receptor, inducing a conformational change that facilitates the release of heat shock protein, nuclear transport, dimerization, and binding to the specific hormone response elements of DNA. It initiates transcription, translation, and protein synthesis that leads to androgenic actions. A lack of androgen effect at the end organ and, consequently, male pseudohermaphroditism may result from abnormalities in 5α-reductase activity, transformation of the steroid-receptor complex, receptor binding of dihydrotestosterone, receptor-ligand complex binding to DNA, transcription, exportation, or translation.
End-Organ Resistance to Androgenic Hormones (Androgen Receptor Defects)
- SYNDROME OF COMPLETE ANDROGEN RESISTANCE (INSENSITIVITY) AND ITS VARIANTS (TESTICULAR FEMINIZATION)
The syndrome of complete androgen resistance (testicular feminization) 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 (33%). 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.
Figure 14-16. Diagrammatic representation of the putative mechanism of action of testosterone on target cells. Testosterone (T) enters the cells, where it is either 5α-reduced to dihydrotestosterone (DHT) or aromatized to estradiol (E2). Dihydrotestosterone binds to the androgen receptor (AR) in the cytoplasm and“activates” it with the release of heat shock proteins (HSP). The activated AR complex is then translocated to the nucleus, where it binds as a dimer to specific hormone response elements of the DNA and along with coactivators initiates transcription, translation, and protein synthesis, with consequent androgenic effects. (Redrawn and modified from Feldman D: The development of androgen-independent prostate cancer. Nat Rev Cancer 2001;1:34.)
Androgen resistance during embryogenesis prevents masculinization of the external genitalia and differentiation of the wolffian ducts. Secretion of antimüllerian hormone by the fetal Sertoli cells leads to regression of the müllerian ducts. Thus, affected patients are born with female external genitalia and a blind vaginal pouch. At puberty, androgen resistance results in augmented LH secretion with subsequent increases in testosterone and estradiol. Estradiol arises from peripheral conversion of testosterone and androstenedione as well as from direct secretion by the testes. Androgen resistance coupled with increased testicular estradiol secretion and conversion of androgens to estrogens result in the development of female secondary sexual characteristics at puberty. The timing of the pubertal growth spurt is similar to that in unaffected girls.
The androgen receptor gene is located on the X chromosome between Xq11 and Xq13. The gene is composed of eight exons, numbered 1 through 8. Exon 1 encodes the amino terminal end of the androgen receptor protein and is thought to play a role in transcription. Exons 2 and 3 encode the DNA-binding zinc finger of the androgen receptor protein. The 5′ portion of exon 4 is called the hinge region and plays a role in nuclear targeting. Exons 5–8 specify the carboxyl terminal portion of the androgen receptor, which is the androgen binding domain (Figure 14-17).
Patients with complete androgen resistance have been found to be heterogeneous with respect to dihydrotestosterone 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 have been described. Analysis of the androgen receptor gene has shed light on the pathogenesis of the heterogeneity in receptor studies found in patients with complete androgen resistance. Patients with the receptor-negative form of complete androgen resistance have been found to have primarily point mutations or substitutions in exons 5–8, which encode the androgen-binding domain of the receptor. Most of the mutations are familial in nature. Other defects such as deletions, mutations in a splice donor site, and point mutations causing premature termination codons are less common in this group of patients. Mutations in exon 3 (which encodes the DNA-binding segment of the androgen receptor) are associated with normal binding of androgen to the receptor but inability of the ligand-receptor complex to bind to DNA and thus to initiate mRNA transcription. These mutations result in receptor-positive complete androgen resistance. The phenotype of the affected patient does not correlate as well with the receptor studies as it does with the transcriptional activity of the ligand-androgen receptor complex. Other factors that play a role in genotype-phenotype variations include somatic mosaicism, coregulator proteins, and other “modifier” genes.
The diagnosis of complete androgen resistance can be suspected from the clinical features. Before puberty, the presence of testis-like masses in the inguinal canal or labia in a phenotypic female suggests the diagnosis. 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 (Reifenstein's syndrome) of androgen resistance must be distinguished from other forms of male pseudohermaphroditism due to androgen deficiency or to 5α-reductase deficiency. Unfortunately, there is no readily available, rapid in vivo or in vitro assay for androgen sensitivity. The diagnosis is suggested by the clinical picture, the family history, and the presence of elevated basal and hCG-induced testosterone levels with normal levels of dihydrotestosterone. A lack of decrease in sex hormone-binding globulin levels after a short course of the anabolic steroid stanozolol has been suggested as a biologic test for androgen resistance. However, few or no confirmatory data on this assay have yet been reported. Also, one would expect that some patients with incomplete androgen resistance might respond to this test in somewhat the same way as normal individuals. It has been suggested that an elevated antimüllerian hormone level is a marker of androgen resistance and androgen deficiency. Abnormalities in androgen binding and mutational analysis as well as studies of transactivation are all diagnostic, but they are time-consuming, labor-intensive, and not universally available. In the infant with ambiguous genitalia in whom the sex of rearing is in question, we have used a trial of testosterone enanthate in oil, 25 mg intramuscularly every four weeks for 3 months, as a predictive test of androgen responsiveness and future phallic growth before assigning a sex of rearing.
Therapy of patients with complete androgen resistance involves affirmation and reinforcement of their female gender identity. Castration, either before or after
puberty, is indicated because of the increased risk of gonadal neoplasms with age. Estrogen replacement therapy is required at the age of puberty in orchidectomized patients. In most cases, vaginal reconstructive surgery is not required.
Figure 14-17. A: Diagrammatic representation of the androgen receptor gene divided into its eight exons. Exon A encodes the amino terminal domain and regulates transcription. Exons B and C encode two zinc fingers. Exons E-H encode the androgen-binding domain of the receptor. B: The organization of a steroid-responsive gene. Ligand binding activates the receptor, and it binds to the steroid response elements of the gene (as a dimer; not shown). Steroid-independent enhancers as well as a CAAT and a TATA box are present. Gene transcription begins 19-27 base pairs downstream of the TATA box. (Reproduced, with permission, from Grumbach MM, Conte FA: Disorders of sex differentiation. In: Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
- SYNDROME OF INCOMPLETE ANDROGEN RESISTANCE (INSENSITIVITY) AND ITS VARIANTS (REIFENSTEIN'S SYNDROME)
Patients with incomplete androgen resistance manifest a wide spectrum of phenotypes as far as the degree of masculinization is concerned. The external genitalia at birth can range from ambiguous, with a blind vaginal pouch, to hypoplastic male genitalia. 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 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 with perineoscrotal hypospadias and gynecomastia. Axillary and pubic hair are normal. The testes remain small and exhibit azoospermia as a consequence of germinal cell arrest. As in the case of patients with complete androgen resistance, there are elevated levels of plasma LH, testosterone, and estradiol. However, the degree of feminization in these patients despite high estradiol levels is less than that found in the syndrome of complete androgen resistance.
Androgen receptor studies in these patients have usually shown quantitative or qualitative abnormalities in androgen binding. It would be expected that mutations which lead to partial reduction of androgen action would result in incomplete virilization. As previously noted, the best correlation to phenotype is the degree of impairment of transcriptional activity of the ligand-androgen receptor complex. A wide variety of androgen receptor gene mutations can result in the same phenotype, and a specific mutation may not always be associated with the same phenotype in all affected patients. In general, point mutations that result
in more conservative amino acid substitutions are more likely to result in partial rather than complete androgen resistance.
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 testosterone levels. Infertility in otherwise normal men may be the only clinical manifestation of androgen resistance. However, infertility may not always be associated with androgen resistance. A family has been described in which there were five males with gynecomastia, all of them with a small phallus. Plasma testosterone levels were elevated, and a subtle qualitative abnormality in ligand binding was noted. Fertility was documented in four of the five males. These patients represent the mildest form of androgen resistance presently documented.
Defects in Testosterone Metabolism by Peripheral Tissues; 5α-Reductase-2 Deficiency (Pseudovaginal Perineoscrotal Hypospadias)
The defective conversion of testosterone to dihydrotestosterone produces a unique form of male pseudohermaphroditism (Figure 14-18). Phenotypically, these patients may vary from those with a microphallus to patients with pseudovaginal perineoscrotal hypospadias. At birth, in the most severely affected patients, ambiguous external genitalia are manifested 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 the urogenital sinus or onto the urethra, immediately behind the urethral orifice. 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 and the presence of temporal hair recession and hirsutism. A remarkable feature of this form of male pseudohermaphroditism in some cultural isolates has been the reported change in gender identity from female to male at puberty.
After the onset of puberty, patients with 5α-reductase-2 deficiency have normal to elevated testosterone levels and slightly elevated plasma concentrations of LH. As expected, plasma dihydrotestosterone is low, and the testosterone-dihydrotestosterone ratio is abnormally high. Apparently, lack of 5α reduction of testosterone to dihydrotestosterone in utero during the critical phases of male sex differentiation results in incomplete masculinization of the urogenital sinus and external genitalia, while testosterone-dependent wolffian structures are normally developed. Partial and mild forms of 5α-reductase deficiency have been described. These patients can present with hypospadias or microphallus (or both). Three male siblings in a Swedish kindred who were compound heterozygotes for 5α-reductase-2 deficiency had hypospadias repair in infancy, and two were demonstrably fertile.
5α-Reductase-2 deficiency is transmitted as an autosomal recessive trait, and the enzymatic defect exhibits genetic heterogeneity. There are two classes of affected individuals: those with absent enzyme activity and those with a measurable but unstable enzyme. Two genes catalyze the conversion of testosterone to dihydrotestosterone, and they are termed type 1 and type 2. The type I enzyme is not expressed in the fetus but is expressed in skin, especially from puberty onward. The type 2 isoenzyme is the enzyme found in fetal genital skin, male accessory glands, and the prostate. 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 localized to 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, while the remainder are compound heterozygotes. It has been suggested that the marked virilization noted at puberty as opposed to its absence in utero may be the result of the expression and function of the type 1 gene at puberty and, consequently, the generation of sufficient amounts of dihydrotestosterone by peripheral conversion to induce phallic growth and other signs of masculinization.
Figure 14-18. Metabolism of testosterone.
5α-Reductase-2 deficiency should be suspected in male pseudohermaphrodites with a blind vaginal pouch and in males with hypospadias or microphallus. The diagnosis can be confirmed by demonstration of an abnormally high plasma testosterone-dihydrotestosterone ratio, either under basal conditions or after hCG stimulation. Other confirmatory findings, especially in newborns, include an increased 5β:5α ratio of urinary C19 and C21 steroid metabolites. One can also examine the level of 5α-reductase activity in cultures of genital skin and the degree of conversion of infused labeled testosterone to dihydrotestosterone in vivo.
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 dihydrotestosterone (if available) or high-dose testosterone 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 puberty, genitoplasty, prophylactic orchiectomy and estrogen substitution therapy is still the treatment of choice.
Dysgenetic Male Pseudohermaphroditism (Ambiguous Genitalia Due to Dysgenetic Gonads)
Defective gonadogenesis of the testes results in ambiguous development of the genital ducts, urogenital sinus, and external genitalia. Patients with 45,X/46,XY mosaicism, structural abnormalities of the Y chromosome, and forms of XY gonadal dysgenesis manifest defective gonadogenesis and thus defective virilization. These disorders are classified under disorders of gonadal differentiation but are included also as a subgroup of male pseudohermaphroditism. 46,XY gonadal dysgenesis has been associated with deletion and mutation of the SRY gene on the Y chromosome, duplication of the DAX1 (AHC) gene of the X chromosome, and chromosome 9p- (DMRT1, 2) or 10q- deletions. In addition SOX9, DHCR7, and XH2 mutations and WNT4 duplications result in dysgenetic male pseudohermaphroditism (see pp. 566–569).
Male pseudohermaphroditism can occur in association with early-onset degenerative renal disease and hypertension as well as with Wilms' tumor (Denys-Drash syndrome) and late-onset renal disease and an increased incidence of gonadoblastoma formation in streak gonads (Frasier syndrome). In Denys-Drash syndrome, both the kidneys and the testes are dysgenetic, and a predisposition for renal neoplasms exists. Patients with the Wilms tumor-aniridia-genital anomalies-mental retardation (WAGR) syndrome have been described. These patients exhibit various forms of ambiguous or hypoplastic male genitalia, including bifid scrotum, hypospadias, and cryptorchidism. Recent data indicate that the Denys-Drash, Frasier, and WAGR syndromes are due to heterozygous mutations in coding exons—mostly exon 9 (Denys-Drash)—or heterozygous mutations in the donor splice site of intron 9, leading to a reversal of the +KTS/-KTS (lysine-threonine-serine) ratio of WT1 proteins (Denys-Drash) or deletions (WAGR) involving the Wilms tumor repressor gene WT1 on chromosome 11.
A mutation in the gene encoding SF-1 has recently been reported in a 46,XY patient. Similar to the mouse “knockout,” the patient had female sex differentiation including müllerian derivatives and severe, neonatal onset adrenal insufficiency. In the human, however, the mutation was heterozygous (present in only one allele of the SF-1 gene) and gonadotropin secretion was preserved (as opposed to the deficiency of gonadotropins observed in the homozygous Sf-1 “knockout” mouse). This illuminating patient demonstrates that SF-1 plays a critical role in adrenal and gonadal development and function in humans. Subsequently, a female heterozygote and a male with a homozygous SF-1mutation have been described. The heterozygote parents and siblings of the latter case had normal adrenal and gonadal function.
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. These patients have perfectly normal male differentiation of their internal and external structures, but gonadal tissue is absent. The diagnosis of anorchia should be considered in all cryptorchid males. Administration of chorionic gonadotropin, 1000–2000 units/m2 injected intramuscularly every other day for 2 weeks (total of seven injections), is a useful test of Leydig cell function. In the presence of normal Leydig cell function, there is a rise in serum testosterone from concentrations of less than 20 ng/dL (0.69 nmol/L) to over 200 ng/dL (6.9 nmol/L) in prepubertal males. In infants under 4 years of age and children over 10 years of age, plasma FSH levels are a sensitive index of gonadal integrity. The gonadotropin response to a 100 ľg intravenous injection of GnRH can also be used to diagnose the absence of gonadal feedback on the hypothalamus and pituitary. In agonadal children, GnRH elicits a rise in LH and FSH levels that is greater than that achieved in prepubertal children with normal gonadal function. Patients with high gonadotropin levels and no testosterone response to chorionic gonadotropin usually lack recognizable testicular tissue at surgery. Recent data indicate that both antimüllerian hormone and inhibin levels are useful in ascertaining the absence of functioning Sertoli cells and, hence, presumed anorchia.
Persistent Müllerian Duct Syndrome (Defects in the Synthesis, Secretion, or Response to Antimüllerian Hormone)
Patients have been described in whom normal male development of the external genitalia has occurred but in whom the müllerian ducts persist. The retention of müllerian structures can be ascribed to failure of the Sertoli cells to synthesize antimüllerian hormone and to an end-organ defect in the response of the duct to antimüllerian hormone. This condition is transmitted as an autosomal recessive trait. The gene for antimüllerian hormone has been cloned and mapped to chromosome 19, and mutations in the antimüllerian gene have been reported. More recently, the gene encoding the antimüllerian receptor has been isolated, and patients with mutations in the AMH receptor have been described. In these patients müllerian ducts are present despite the presence of normal to high levels of AMH in plasma. Therapy involves removal of the müllerian structures.
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 “estrogens” 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. 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 ascribed to these agents.
UNCLASSIFIED FORMS OF ABNORMAL SEXUAL DEVELOPMENT IN MALES
Hypospadias occurs as an isolated finding in 1:300 newborn males. It is often associated with ventral contraction and bowing of the penis, called chordee. Deficient virilization of the external genitalia of the male fetus implies subnormal Leydig cell function in utero, end-organ resistance, or an inappropriate temporal correlation of the rise in fetal plasma testosterone and the critical period for tissue response. Although in most patients there is little reason to suspect these mechanisms, recent reports in a small number of patients have suggested that simple hypospadias can be associated with an abnormality (or competitive inhibition) of the androgen receptor, the nuclear localization of the ligand-receptor complex, an aberration in the maturation of the hypothalamic-pituitary-gonadal axis, and 5α-reductase deficiency. Further studies are necessary to determine the prevalence and role of these abnormalities in the pathogenesis of simple hypospadias. Nonendocrine factors that affect differentiation of the primordia may be found in a variety of genetic syndromes. A study of 100 patients with hypospadias reported one patient to be an XX female with congenital adrenal hyperplasia; five had sex chromosome abnormalities; and one had the incomplete form of XY gonadal dysgenesis. Nine affected males were the product of pregnancies in which the mother had taken progestational compounds during the first trimester. Thus, a presumed pathogenetic mechanism was found in 15% of patients.
Microphallus without hypospadias—micropenis—can result from a heterogeneous group of disorders, but by far the most common cause is fetal testosterone deficiency; more rarely, 5α-reductase deficiency or mild defects in the androgen receptor are implicated (Table 14-6).
In the human male fetus, testosterone synthesis by the fetal Leydig cell during the critical period of male differentiation (8–12 weeks) is under the influence of placental hCG. After midgestation, fetal pituitary LH modulates fetal testosterone synthesis by the Leydig cell and, consequently, affects the growth of the differentiated penis. Thus, males with congenital hypopituitarism as well as isolated gonadotropin deficiency and “late” fetal testicular failure can present with normal male differentiation and micropenis at birth (penis < 2.5 cm in length) (Table 14-7). Patients with hypothalamic hypopituitarism or pituitary aplasia may also have midline craniofacial defects, hypoglycemia, and giant cell hepatitis. After appropriate evaluation of anterior pituitary function (ie, determination of the plasma concentration of growth hormone, ACTH, cortisol, thyroid-stimulating hormone, thyroxine, and gonadotropins), stabilization of the patient with hormone replacement should be achieved. Thereafter, all patients with micropenis should receive a trial of testosterone therapy before definitive gender assignment is made. Patients with fetal testosterone deficiency as a cause of micropenis—whether due to gonadotropin deficiency or to a primary testicular disorder—respond to 25-50 mg of testosterone enanthate intramuscularly monthly for 3 months with a mean increase of 2 cm in penile length (Figure 14-19). 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 testosterone did not prevent the penis from responding to testosterone 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 testosterone in infancy or childhood and with replacement doses of testosterone in adolescence was in the normal adult range. Furthermore, these patients had a male gender identity, erections, ejaculation, and orgasm (Table 14-8). We found no clinical, psychologic, or physiologic indications to support conversion of males with micropenis secondary to diminished testosterone secretion during gestation to females.
Table 14-6. Etiology of micropenis.1
Table 14-7. Normal values for stretched penile length.
Complete absence of the phallus is a rare anomaly. The urethra may open on the perineum or into the rectum. Assignment of a female gender, castration, and plastic repair of the genitalia and urethra has been the approach followed in the past; however, this course is being seriously questioned by some investigators.
Figure 14-19. The response in phallic length to a 3-month course of testosterone in six patients with microphallus. Patients were under 2 years of age. Each patient was given 25 mg of testosterone enanthate in oil intramuscularly monthly for 3 months. Lines set off with solid triangles and open circles indicate two patients who subsequently underwent a second course of testosterone therapy. (Reproduced, with permission, from Burstein S, Grumbach MM, Kaplan SL: Early determination of androgen-responsiveness is important in the management of microphallus. Lancet 1979;2:983.)
UNCLASSIFIED FORMS OF ABNORMAL SEXUAL DEVELOPMENT IN FEMALES
Congenital absence of the vagina occurs in 1:5000 female births. It can be associated with müllerian derivatives that vary from normal to absent. Ovarian function is usually normal. Therapy may involve plastic repair of the vagina.
Müllerian agenesis may be associated with renal aplasia (an absent kidney) and cervicothoracic somite dysplasia (“MURCS”).
MANAGEMENT OF PATIENTS WITH INTERSEX PROBLEMS
The evaluation and management of the patient with ambiguous genitalia are best undertaken by a team consisting of an endocrinologist, a psychiatrist or psychologist, a pediatrician or internist, a surgeon, a urologist, and a social worker. The goal of management of patients with ambiguous genitalia is to establish an etiologic diagnosis promptly and, with the informed consent of the parents, assign a sex of rearing that is most compatible with the prospect for a well-adjusted life and sexual adequacy. Steps in the diagnosis of intersexuality are set forth in Figures 14-20 and 14-21.
Repeated, lucid, simple, comprehensive discussions with the parents about the cause of their child's “atypical” genitalia, the natural history of other patients with
similar pathophysiology, prognosis, and the possible hormonal and surgical options available is critical to their coming to an informed decision on the sex rearing of their child. This discussion must take into account the parental anxieties, religious views, social mores, cultural factors, and, most important, the level of understanding of the parents. Parents of intersexed children as well as the patient need the ongoing support of psychiatrists or psychologists who have knowledge of the pathogenesis of abnormalities of sex differentiation and who understand the complexities of intersexuality, gender behavior, and gender identity.
Table 14-8. Treatment of eight males with micropenis secondary to congenital hypogonadotropic hypogonadism, followed from infancy or childhood to maturity (ages 18–27 years).1
Figure 14-20. Steps in the differential diagnosis of ambiguous genitalia.
There is a great deal of discussion and controversy involving the management of infants with intersexuality. Advances in biochemistry, genetics, and endocrinology have increased our knowledge and understanding of the pathogenesis of abnormalities of sex determination and differentiation. It is now possible to make a specific diagnosis in the majority of patients with ambiguous genitalia. This information, coupled with phallic response to testosterone, advances in surgical reconstruction, strikes a note of optimism. Further, we now recognize that genes, hormones, and the environment are critical factors determining gender identity. Androgens in utero are facultative but not determinative in their effect on gender identity in patients with intersexuality. However, there are large gaps in long-term outcome data in many of these disorders. All of these considerations lead us to the following recommendations for newborns with abnormalities of sex determination and differentiation.
We recommend male sex assignment in 46,XY male pseudohermaphrodites except for those with complete androgen resistance or those who have completely female external genitalia due to Leydig cell unresponsiveness to hCG and LH and rare errors in testosterone biosynthesis, where extensive discussion with the family is especially warranted. In some societies, the social, cultural, and economic benefits of a male gender identity are more compelling than phallic adequacy and are a prevailing—if not the most important—factor in the parental decision about the sex of rearing. All female pseudohermaphrodites, including those affected infants with complete masculinization of the external genitalia, should be reared as females.
Reassignment of sex in infancy and childhood is always a difficult psychosocial problem for the patient, the parents, and the physicians involved. While easier in infancy than after 1 year of age, it should only be undertaken after deliberation and with provision for long-term medical and psychiatric supervision and counseling.
It is desirable to initiate plastic repair of the external genitalia by 6 months of age. In children raised as females, the clitoris should be salvaged by clitoroplasty. All surgical procedures should strive to preserve the functional capacity of all genital structures. This consideration should outweigh cosmetic appearance. Reconstruction of a vagina, if necessary, can be deferred until adolescence if a surgeon experienced in genitoplasty is not available. Hypospadias repair is best performed at 6 months to 1 year of age.
Removal of rudimentary gonads in children with Y chromosome material and gonadal dysgenesis should be performed at the time of initial repair of the external genitalia, because gonadoblastomas, seminomas, and germinomas can occur during the first decade. However, histologically and presumably functionally “normal” scrotal testes should be retained in male pseudohermaphrodites assigned a male identity—especially those with 45,X/46,XY mosaicism.
In a patient with complete androgen resistance, the gonads may be left in situ (provided they are not situated in the labia majora) to provide estrogen until late adolescence. The patient may then undergo prophylactic castration, having had her female identity reinforced by normal feminization at puberty. However, it is reasonable to remove the gonads prepubertally, especially if herniorrhaphy is necessary. In this circumstance, sex steroid replacement therapy at the time of puberty is indicated.
In patients with incomplete androgen resistance reared as females or in patients with errors of testosterone biosynthesis in whom some degree of masculinization occurs at puberty, gonadectomy should be performed before puberty.
Cyclic estrogen and progestin are used in individuals reared as females in whom a uterus is present. In males, virilization is achieved by the administration of a repository preparation of testosterone.
In all patients, continuing endocrinologic and psychologic support are critical aspects of follow-up and should be available throughout infancy, childhood, and adolescence. Patients should have progressive, step-by-step, age-appropriate discussion about their diagnosis, its pathophysiology, their quality of life, and their potential for fertility. Disclosure is critical. However, in those mature adult intersex patients who are well-adjusted and happy with their lives and their “assigned” gender identity, disclosure should be on a “need to know” basis. The patient (when possible) and the parents should be involved in decisions about surgery and sex hormone replacement.
In sum, the physician and the consortium concerned with the diagnosis, selection of sex of rearing, and management of the infant with intersexuality must be prepared to address the complex ethical, cultural, social, religious, clinical, and surgical issues presented by the intersex patient in order to maximize the patient's potential for a well-adjusted, normal life. This task is complicated now by the lack of complete outcome data.
Figure 14-21. Steps in the diagnosis of male pseudohermaphroditism in infancy and childhood. Step 1 involves initial workup and provisional diagnosis (see Figure 14-20). Step 2 (this figure) is utilized in selected cases. (Reproduced, with permission, from Grumbach MM, Hughes IA, Conte FA: Disorders of sex differentiation. In: Larsen PR et al [editors]: Williams Textbook of Endocrinology, 10th ed. Saunders, 2002.)
*Patients with dysgenetic male pseudohermaphroditism may manifest varying degrees of testicular dysgenesis with consequent testosterone/DHT or AMH deficiency (or both). Therefore, not all patients may manifest either ambiguous genitalia or the presence of müllerian ducts.
**CYP17 (P450c17) catalyzes the 17-hydroxylation of progesterone and pregnenolone to 17-hydroxyprogesterone and Δ5-17-hydroxypregnenolone as well as the scission (lyase) of 17-hydroxypregnenolone to DHEA. Patients with 17,20-lyase deficiency have elevated levels of 17-hydroxyprogesterone and Δ5-17-hydroxypregnenolone in relation to androstenedione and DHEA either before or after hCG stimulation.
***The StAR (steroidogenic acute regulatory) protein is involved in the transport of cholesterol from the outer to the inner mitochondrial membrane, where the enzyme P450scc resides. Patients with a mutation in the gene for this protein have a markedly diminished ability to convert cholesterol to Δ5-17-hydroxypregnenolone, though their P450scc enzymatic activity is intact, and they manifest congenital lipoid adrenal hyperplasia. WAGR, Wilms' tumor, aniridia, genital anomalies, and mental retardation; SF-1, steroidogenic factor-1; CYP17, 17α-hydroxylase/17,20-lyase; 3β-HSD 2, 3β-hydroxysteroid dehydrogenase/Δ5-isomerase; 17β-HSD 3, 17β-hydroxysteroid dehydrogenase (oxidoreductase); T, testosterone; DHT, dihydrotestosterone; AMH, antimüllerian hormone; SHBG, sex hormone-binding globulin; DHEA, dehydroepiandrosterone.
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