Disorders of Sexual Differentiation

1. Disorders of Sexual Differentiation Vincenzo Galati, D.O. Stephen Confer, MD Ben O. Donovan, MD Brad Kropp, MD Dominic Frimberger, MD University of Oklahoma Department of Urology Section of Pediatric Urology

2. Normal Sexual Differentiation Jost paradigm: • Establishment of chromosomal sex at fertilization • Development of the undifferentiated gonads into testes or ovaries • Differentiation of the internal ducts and external genitalia

3. Chromosomal Sex • TDF was mapped to the most distal aspect of the Y-unique region of the short arm of the Y chromosome, adjacent to the pseudoautosomal boundary • Sry is localized to the smallest region of the Y chromosome capable of inducing testicular differentiation in humans and in mice • Sry appears to be capable of recognizing specific sites on DNA, and, by binding and producing bending of the DNA, it is able to activate downstream gene expression

4. TDF candidates • ZFY (zinc finger gene on Y chromosome) was excluded with certainty as a candidate for TDF when four individuals with testicular development were found to have inherited a fragment of the Y chromosome that did not include ZFY • H-Y gene: A number of women with 45,X gonadal dysgenesis were found to be H-Y antigen positive

5. Other Important Genes • WT-1 : originally isolated in experiments that identified an oncogene on chromosome 11 as being involved in the etiology of Wilms' tumor. Research on WT-1 in the mouse suggests that it exerts its effects upstream of SRY and is likely to be necessary for commitment and maintenance of gonadal tissue • SF-1: a nuclear receptor, is expressed in all steroidogenic tissue and appears to be a regulator of müllerian inhibiting substance (MIS) • SOX-9 gene: identified in patients with camptomelic dysplasia, a congenital disease of bone and cartilage formation that is often associated with XY sex reversal • SOX-9 HMG-box amino acid sequence has 71% similarity to that of SRY. • Expression of the gene in adults is greatest in the testes and is thought to be involved in gonadal differentiation

6. Other Important Genes • DSS (DAX-1)(dosage-sensitive sex reversal). Found in XY females with duplication of this gene • Suggests duplicated X chromosome causes XY sex reversal by expressing a double dose of the gene normally subject to X inactivation. Screening of XY females with a normal Sry gene detected a submicroscopic duplication designated DSS • Implicated in adrenal hypoplasia congenita • WNT4 (factor in ovarian pathway) • Thought to repress the biosynthesis of gonadal androgen in female mammals, therefore is suppresses male sexual differentiation (Hughes, NEJM, 351(8), Aug 19, 2004. 792-798)

7. Gonadal Stage of Differentiation • During the first 6 weeks of embryonic development structures are bipotential in both 46,XY and 46,XX embryos • Migration of the germ cells begins in the 5th week of gestation through the mesentery to the medial ventral aspect of the urogenital ridge • SRY initiates the switch that induces the indifferent gonad toward testicular organogenesis • In the absence of SRY, ovarian organogenesis results • The differentiation of Sertoli cells is associated with the production of MIS, a glycoprotein encoded by a gene on the short arm of chromosome 19 • Primordial cells of steroidogenic mesenchyme remain among the testicular cords and represent future Leydig cells, which differentiate at 8 to 9 weeks

8. Gonadal Stage of Differentiation • Duplicate copies of at least one X chromosomal locus is likely necessary for normal oviarian organogenesis • Dysgenetic ovaries in Turner's syndrome patients • In embryonal ovaries, germ cells undergo intense mitotic proliferation and in the process exhaust their entire mitotic potential prenatally • a maximum endowment of 20 million cells by 20 weeks gestation

9. Gonadal Function • The initial endocrine function of the fetal testes is the secretion of MIS by the Sertoli cells at 7 to 8 weeks' gestation • Testosterone secretion by the fetal testes is detectable shortly after the formation of Leydig cells in the interstitium at approximately 9 weeks' gestation • testosterone peaks at 13 weeks and then declines • testosterone enters target tissues by passive diffusion • DHT binds to the androgen receptor with greater affinity and stability than does testosterone • the gene encoding the androgen receptor has been cloned and mapped to the X chromosome between the centromere and q13 • Estrogen synthesis is detectable in the female embryo just after 8 weeks of gestation

10. Undifferentiated Urogenital Tract 8 wks 10 wks Undifferentiated External genitalia

11. Differentiation Timeline

12. Psychosexual Differentiation • gender identity:the identification of self as either male or female • gender role:aspects of behavior in which males and females appear to differ • gender orientation: choice of sexual partner (heterosexual, homosexual, or bisexual) • cognitive differences

13. Psychosexual Differentiation • Experience in patients with congenital adrenal hyperplasia (CAH) who were exposed prenatally to androgen and in patients reared in a sex opposite to their chromosomal or gonadal sex have provided evidence to indicate that gender identity is not merely a function of chromosomal complement or prenatal endocrine milieu • strong evidence has accumulated for the impact of prenatal hormonal influences on sexually dimorphic behavior or gender role • previously accepted dogma that children are psychosexually neutral at birth and capable of being environmentally oriented has been seriously challenged by those who support the concept of prenatal psychosexual differentiation

14. Disorders of Gonadal Differentiation and Development

15. Klinefelter's syndrome (Williams Textbook of Endocrinology, 10th ed, 2003)

16. Seminiferous Tubule Dysgenesis (Klinefelter's syndrome) • Syndrome characterized by eunuchoidism, gynecomastia, azoospermia, increased gonadotropin levels, and small, firm testes, 47,XXY karyotype • nondisjunction during meiosis • 1 of 1000 liveborn males • associated with 48,XXYY; 49,XXXYY; 48,XXXY; 49,XXXXY; 46,XY/47XXY • Gynecomastia can be quite marked at pubertal development • 8 X risk for breast carcinoma compared with normal males • Seminiferous tubules degenerate and are replaced with hyaline • Fertility, with the benefit of ICSI, has been reported in one patient • decreased androgens prevents normal secondary sexual development • poor muscle development, the fat distribution is more female than male. • Normal amounts of pubic and axillary hair, but facial hair is sparse. • Patients tend to be taller than average, due to disproportionately long legs • Predisposed to malignant neoplasms of extragonadal germ cell origin. • Androgen supplementation to improve libido & reduction mammoplasty • surveillance for breast carcinoma

17. 46,XX maleness • Occurs in 1 of every 20,000 males • Testicular development in subjects who have two X chromosomes and lack a normal Y chromosome. • Most of these subjects have normal male external genitalia, but 10% have hypospadias and all are infertile • 80% are Sry positive and rest are Sry negative • Sry -positive group rarely have genital abnormalities, but they have phenotypic features of Klinefelter's syndrome • Shorter (mean height, 168 cm) and have more normal skeletal proportions than Klinefelter’s patients • Due to translocation of Y chromosomal material, including SRY, to the X chromosome • Infertile  lack of germ cell elements

18. Turner’s Syndrome (45,XO) • No oocytes remain in the ovaries, which become streaks • Fertility = 60% pregnancy rate w/ART • Ovum donation for those with bilateral streaks • 1 in 2500 live births • 60% are 45,XO and 40% are mosaics • Y chromosomal material masculinization & gonadoblastoma (30%) • 33% - 60% have structural or positional abnormalities of the kidney • horseshoe kidney = 10%, • duplication or renal agenesis= 20% • malrotation= 15% • multiple renal arteries = 90% • Four classic features: • female phenotype • short stature • lack of secondary sexual characteristics • a variety of somatic abnormalities: Webbed neck Wide spaced nipples Broad chest (shield) Cubitus valgus Short stature peripheral edema at birth, short 4th metacarpal, hypoplastic nails, multiple pigmented nevi, coarctation of the aorta, and renal anomalies

19. 46,XX pure gonadal dysgenesis • Features: • normal female external genitalia • normal müllerian ducts with absence of wolffian duct structures • a normal height • bilateral streak gonads • sexual infantilism • normal 46,XX karyotype • streak gonads elevated serum gonadotropins • Management of 46,XX "pure" gonadal dysgenesis: • cyclic hormone replacement with estrogen and progesterone. • growth is basically normal so GH is not needed • possibly autosomal recessive trait

20. Gonadal dysgenesis (Williams Textbook of Endocrinology, 10th ed, 2003)

21. Mixed gonadal dysgenesis(MGD) • Characterized by a unilateral testis, often intra-abdominal • Contralateral streak gonad • Persistent müllerian structures with varying inadequate masculinization • Most are 45,XO/46,XY, the most common form of Y chromosome mosaicism • Second most common cause of ambiguous genitalia after CAH • Dysgenetic or streak gonad is associated with ipsilateral müllerian derivatives (uterus, fallopian tube) • Well-differentiated testis with functional Sertoli and Leydig cells will have ipsilateral wolffian but no müllerian ducts • no germ cells so infertility is the rule • Increased risk of developing gonadoblastoma or dysgerminoma of 15% to 20% • Also increased risk for Wilm’s tumor and association with Denys-Drash • Endocrine function of testis is normal post-pubertally • fetal testis dysfunction may account for ambiguous genitalia • 90% to 95% of 45,X/46,XY mosaicism have normal-appearing male genitalia

22. Dysgenetic Male Pseudohermaphroditism • Two dysgenetic testes rather than one dysgenetic testis and a streak gonad as in MGD • Typically are 45,X/46,XY or 46,XY • Present with a spectrum of external genital abnormalities • Dysgenetic testis is composed of immature hypoplastic seminiferous tubules and persistent stroma resembling that seen in the streak gonad • Incidence of gonadoblastoma or dysgerminoma is 46% by 40 years • At risk for Denys-Drash

23. 46,XY Complete Gonadal Dysgenesis • Characterized by : • normal female genitalia • well-developed müllerian structures • bilateral streak gonads • nonmosaic karyotype • Ambiguity of genitalia is not an issue • Sexual infantilism is the primary clinical problem • present in their teens with delayed puberty • An abnormality of the Sry gene function, or loss of another gene downstream from Sry that is necessary for SRY protein action • LH elevated  clitoromegaly • 30% risk of germ cell tumor development by age 30 years • gonadoblastoma is most common • embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, and immature teratoma have also been reported • Management  removal of both streak gonads and proper cyclic hormone replacement with estrogen and progesterone

24. Embryonic Testicular Regression and Bilateral Vanishing Testes Syndromes • 46,XY karyotype and absent testes but clear evidence of testicular function during embryogenesis • "embryonic testicular regression" = loss of testicular tissue within the first trimester and is associated with ambiguity of external genitalia • "bilateral vanishing testes syndrome" refers to individuals in whom male sexual differentiation of ducts and genitalia took place but loss of testicular tissue occurred subsequently in utero • Diagnosis can be made on the basis of a 46,XY karyotype and castrate levels of testosterone despite persistently elevated serum LH and FSH • bilateral vanishing testes syndrome, agonadal XY phenotypic males with fully developed wolffian structures, but an empty scrotum, absent prostate, and microphallus • intermediate point presentation is the 46,XY patient with absent gonads and internal ductal structures but with ambiguous genitalia  incomplete elaboration of androgen • most severe form, agonadism is discovered in a 46,XY phenotypic female with no internal genital structures;  the testis has elaborated MIS but vanishes at 60-70 days before elaboration of androgen Spectrum of presentation

25. True Hermaphroditism • Individuals who have both testicular tissue with well-developed seminiferous tubules and ovarian tissue with primordial follicles, which may take the form of one ovary and one testis or, more commonly, one or two ovotestes. • External genitalia and internal duct structures of true hermaphrodites display gradations between male and female (Williams Textbook of Endocrinology, 10th ed, 2003)

26. True Hermaphroditism • In most patients, the external genitalia are ambiguous but masculinized to variable degrees, and 75% are raised as male • Internal ductal development are influenced by ipsilateral gonad • Fallopian tubes are consistently present on the side of the ovary • a vas deferens is always present adjacent to a testis • Fallopian tube is present with 66% of ovotestes, vas or both in 33% • Most have urogenital sinus and and uterus • 80% of those raised as male have hypospadias and chordee • Ovaries usually on left in normal position, testis usually on right and located anywhere along path of descent • 60% of gonads palpable in canal or labia are ovotestes

27. True Hermaphroditism • Ovarian portion of the ovotestis is frequently normal, whereas the testicular portion is typically dysgenetic • 66% of patients are 46 XX • Gonadal tumors is approximately 10% in 46,XY true hermaphroditism and 4% in 46,XX true hermaphroditism • Most important aspect of management in true hermaphroditism is gender assignment • Sex assignment should be based on the functional potential of external genitalia, internal ducts, and gonads, according to the findings at laparoscopy or laparotomy. • Unlike patients with most other forms of gonadal dysgenesis, true hermaphrodites have the potential for fertility if raised as female with the appropriate ductal structures • Males, remove ovaries and/or ovotestis and mullerian duct structures consider gonadectomy • Females remove all testicular and wolffian structures

28. Female Pseudohermaphroditism 46,XX individuals with ovaries have a partially masculinized phenotype and ambiguous genitalia CAH is most common cause Uncommon etiologies: Maternal ingestion of androgens Virilizing tumors in the mother clitoromegaly labioscrotal fusion Marked virilization with hypospadiac-appearing phallus

29. (Williams Textbook of Endocrinology, 10th ed, 2003)

30. Congenital Adrenal Hyperplasia • Error in cortisol biosynthesis pathway • The most commonly recognized syndromes result from a deficiency of one of the terminal two enzymes of glucocorticoid synthesis (21-hydroxylase or 11-hydroxylase) • Formation of hydrocortisone is impaired, causing a compensatory increase in the secretion ACTH enhances formation of adrenal steroids proximal to the enzymatic defect and a secondary increase in the formation of testosterone, the active androgen in CAH • 21-hydroxylase is responsible for 95% of cases of CAH • Incidence is 1 in 5,000 to 1 in 15,000 in the United States and Europe. The highest incidence, 1 in 490, in the Alaskan Eskimo

31. Congenital Adrenal Hyperplasia

32. CAH: 21-Hydroxylase Deficiency • Three categories: • (1) salt wasters (patients with virilization and aldosterone deficiency), • (2) simple virilizers (patients with virilization, but without salt wasting), • (3) nonclassic patients (those without evidence of virilization or salt wasting). • 21-hydroxylase gene ( CYP-21 ) is located on chromosome 6p, • transmitted in an autosomal recessive pattern • Mutations leading to conversion of the active CYP-21 gene into the inactive gene occur in 65% to 90% of cases of classic 21-hydroxylase deficiency (i.e., salt wasting and simple virilizing forms) and in all nonclassic cases • Gene deletions are responsible for 10% to 35% of the remainder of mutations that produce 21-hydroxylase deficiency • 75% present with salt wasting and 25% with simple virilization

33. Prader Classification of Virilization

34. CAH: 21-Hydroxylase Deficiency • Salt-losing variant of CAH  symptoms begin within the first few weeks after birth, with failure to regain birth weight, progressive weight loss, and dehydration • In severely affected infants, adrenal crises occur within the first 10 to 21 days of life • Vomiting is prominent and can be so extreme that a mistaken diagnosis of pyloric stenosis is made, particularly in the male. • Death ensues from hyperkalemia, dehydration, and shock • Masculinization of the untreated female; pubic and axillary hair develop prematurely, acne appears, and the voice deepens • Isosexual precocity (2-3 yo) is hallmark for non-salt wasting males “little Hercules”

35. CAH: Diagnosis of 21-Hydroxylase Deficiency • Plasma levels of progesterone and 17-hydroxyprogesterone are markedly elevated • Urinary 17-ketosteroids and pregnanetriol are elevated. • The diagnosis may be made biochemically with the use of radioimmunoassay of plasma 17-hydroxyprogesterone • Replaced the more cumbersome 24-hour urine collection of metabolites (e.g., pregnanetriol). • A pelvic ultrasound study demonstrating the presence of müllerian tissues is confirmatory.

36. CAH: 11 b-Hydroxylase Deficiency • Accounts for about 5% of cases • mutations in the CYP-11B1 gene • Hypertension is common in patients with this type of CAH • due to increased serum levels of deoxycorticosterone (DOC). • The diagnosis can be confirmed by increased plasma levels of 11-deoxycortisol and 11-DOC. • Urinary 17-ketosteroids and 17-hydroxycorticoids are increased. • The treatment with glucocorticoid is identical to that of patients with 21-hydroxylase deficiency

37. CAH: 3b Hydroxysteroid Dehydrogenase (3b-HSD) Deficiency • Affects the early steroid biosynthesis in adrenals and gonads • inability to convert 3β-hydroxysteroids to 3-ketosteroids • females exhibit mild clitoromegaly and labial fusion with symptoms of aldosterone and cortisol deficiency • Autosomal recessive inheritance pattern • Increased serum levels of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) are diagnostic • Treatment is similar to that of patients with 21-hydroxylase deficiency

38. Congenital Adrenal Hyperplasia

39. CAH: Treatment • Early diagnosis could prompt prenatal treatment to prevent virilization • Prenatal diagnosis is made by amniotic fluid 17-hydroxyprogesterone • Diagnosed by chorionic villous cells at 8-10 weeks or amniotic cells at 16-17 weeks. • BUT treatment should be instituted at 5-6 weeks of gestation • Currently, it is not possible to confirm the diagnosis before therapy is initiated • Treat mother with dexamethasone which crosses placenta to prevent virilization • BUT the long-term effects of dexamethasone on unaffected fetuses undergoing treatment prenatally remain unknown

40. CAH: Treatment • Post-natally, after control of electrolytes and blood pressure has been achieved in the acute setting, maintenance therapy with fludrocortisone (0.05 to 2.5 mg daily) should be instituted • Children with the salt-losing form of the disease require increased salt intake and mineralocorticoid treatment in addition to hydrocortisone therapy • Genitoplasty at 3 to 6 months of age • Long-term fertility in males and feminization, menstruation, and fertility in females can be anticipated in the well-treated patient

41. Female Pseudohermaphroditism: Maternal Hormones & Tumors • Androgen or progestational agent affects the female fetus • Function of the strength of the agent, its maternal dosage, and timing and duration of administration • Masculinization occurred in 2% of female infants whose mothers were treated with progestins during pregnancy to prevent abortion (Ishizura et al, 1962 ) • Rarely, maternal ovarian or adrenal tumor has virilizing effects on a female fetus • arrhenoblastoma • hilar cell tumor • lipoid cell tumor • ovarian stromal cell tumor • luteoma of pregnancy • adrenocortical carcinoma and adenoma • Krukenberg's tumor • Management is confined to external genital reconstruction

42. Male Pseudohermaphroditism • 46,XY individuals with differentiated testes who exhibit varying degrees of feminization phenotypically. • Inadequate secretion of testosterone by the testes at the necessary period in development • Inability of target tissue to respond to androgen appropriately • Impaired production or action of MIS

43. Male Pseudohermaphroditism • Leydig Cell Aplasia (Luteinizing Hormone Receptor Abnormality) • 46,XY male karyotype, normal-appearing female phenotype • Typically, testes are palpable in the inguinal canals or labia majora • no rise in testosterone after HCG stimulation • spectrum  absent Leydig cells to Leydig cells with abnormal LH receptor • autosomal recessive trait • DDx = androgen insensitivity syndrome or a terminal defect in androgen synthesis. • testis histology = absent of Leydig cells in intratubular spaces, normal Sertoli cells

44. Male Pseudohermaphroditism

45. Male Pseudohermaphroditism • Disorders of Testosterone Biosynthesis • Defect in any of the five enzymes  incomplete (or absent) virilization of the male fetus during embryogenesis • Inheritance is autosomal recessive • Cholesterol Side Chain Cleavage Deficiency(StAR Deficiency) • a defect in cholesterol transport prevents conversion of cholesterol to pregnenolone • 46,XY individuals have female or ambiguous external genitalia • a blind-ending vaginal pouch • intra-abdominal, inguinal, or labial testes • absence of müllerian structures & Wolffian ducts are present but rudimentary • severe adrenal insufficiency and salt wasting • suspect this if nonvirilized female external genitalia with: • cortisol and aldosterone deficiency • hyponatremia, hyperkalemia, and metabolic acidosis. • Abdominal CT scanning demonstrates large, lipid-laden adrenal glands

46. Male Pseudohermaphroditism • 3β-Hydroxysteroid Dehydrogenase Deficiency • incomplete masculinization with salt-wasting  impaired aldosterone and cortisol synthesis • a small phallus, hypospadias with labioscrotal fusion, a urogenital sinus, and a blind-ending vaginal pouch. Testes are often scrotal, and wolffian ducts develop normally • diagnosis: increased levels of 3β-hydroxysteroids(pregnenolone, 17-hydroxypregnenolone, and DHEA) • 17α-Hydroxylase Deficiency • conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone • impaired cortisol production ACTH hypersecretion  increased DOC, corticosterone, and 18-hydroxycorticosterone in the adrenals (check levels) • These mineralocorticoids  salt and water retention, HTN, and hypokalemia • Fertility has not been reported and inadequate testosterone production makes androgen imprinting a less significant issue for these patients • Phenotype may dictate gender assignment

47. Male Pseudohermaphroditism • 17,20-Lyase Deficiency • cortisol and ACTH secretion are normal aldosterone normal  no HTN • ambiguous rather than totally female genitalia at birth • suspect this dx if absent müllerian derivatives and no defect in glucocorticoid or mineralocorticoid synthesis. • 17β-Hydroxysteroid Oxidoreductase Deficiency • similar to 5α-reductase deficiency  normal female phenotype, no significant virilization • well-differentiated testes located intra-abdominally, inguinally, or in the labia and no müllerian structures. • At puberty phallic growth and male secondary sexual characteristics • Androstenedione  increased to 10 to 15x normal • type III 17β-hydroxysteroid dehydrogenase isozyme mutationmale pseudohermaphroditism

48. Androgen Receptor & Post-Receptor Defects • Most common definable cause of male pseudohermaphroditism • All are 46,XY karyotype and have testes • Three classifications exist that describe the spectrum of phenotypes • Complete androgen insensitivity • female-appearing external genitalia, and absence of müllerian derivatives • Blind ending vagina, reduced pubic hair • 1 in 20,000 to 1 in 60,000 males • 2% of female with hernia  so vaginoscopy prudent • X-linked trait, chromosome Xq11–12, point mutation • unequivocal female gender identity androgen resistance of brain tissue • No reported female  male gender conversion at puberty • gonadectomy is key  wait until after puberty • 2% to 5% risk of seminoma or gonadoblastoma • Testis produces estradiol  feminization

49. Androgen Receptor & Post-Receptor Defects • Partial androgen insensitivity (Reifenstein's syndrome) • ambiguity of the external genitalia to varying degrees • male with perineoscrotal hypospadias, cryptorchidism, rudimentary Wolffian duct structures, gynecomastia, and infertility • the phenotypic spectrum can range from hypospadias and a pseudovagina to gynecomastia and azoospermia • etiology: • (1) a reduced number of normally functioning androgen receptors • (2) a normal receptor number but decreased binding affinity • gender assignment is often dictated by phenotype and degree of virilization • Infertile male syndrome • normal male phenotype but are azoospermic or severely oligospermic • normal to elevated serum testosterone • normal to elevated LH • decreased androgen receptor binding to DHT in genital skin fibroblasts

50. 5α-Reductase Deficiency (Williams Textbook of Endocrinology, 10th ed, 2003)