46,XY DSD due to gonadal dysgenesis

Complete and partial 46,XY gonadal dysgenesis

46,XY gonadal dysgenesis consist of a variety of clinical conditions in which the development of fetal gonad is abnormal and encompasses both complete and a partial forms. The complete form of gonadal dysgenesis was first described by Swyer et al (26) and it is characterized by female external and internal genitalia, lack of secondary sexual characteristics, normal or tall stature without somatic stigmata of Turner syndrome and the presence of bilateral dysgenetic gonads in XY subjects. Mild clitoromegaly is present in some cases. Affected individuals are unusually tall for females and present eunuchoid habitus.

The partial form of this syndrome is characterized by impaired testicular development that results in patients with ambiguous external genitalia with or without Mullerian structures. Similar phenotypes can also result from a 45,X/46,XY karyotype.

Serum gonadotropin levels are elevated in both the complete and partial forms, mainly FSH levels, which predominate over LH levels. Testosterone levels are at prepubertal range in the complete form and in the partial form, they can be elevated for a female, but rarely reach male pubertal levels.

46,XY gonadal dysgenesis is a heterogeneous disorder that results from deletions or point mutations of SRY gene, duplication of the DSS locus on X chromosome or mutations in autosomal genes. Most of the studies found mutations in SRY gene in less than 20% of the patients with complete 46,XY gonadal dysgenesis (27-29). In the partial form, the frequency of SRY mutation is even lower than in the complete form. To date, around 55 mutations have been identified within the SRY gene, and most of them are located into the HMG box, showing the critical role of this domain. Most of the mutations described in SRY are predominantly de novo mutations. However, some cases of fertile fathers and their XY affected children, sharing the same altered SRY sequence, have been reported (30, 31). In few of these cases, the father’s somatic mosaicism for the normal and mutant SRY gene have been demonstrated (32). This variable penetrance found in familial mutations of SRY have been described in mutant SRY proteins with relatively well preserved in vitro activity (33).

A recent study describes a remarkable family pedigree across four generations with multiple affected family members, of both sexes, with variable degrees of gonadal dysgenesis. The phenotypic mode of inheritance was strongly suggestive of X-linkage (34). In this report, a fertile woman had a 46,XY karyotype in peripheral lymphocytes, mosaicism in cultured skin fibroblasts (80% 46,XY and 20% 45,X) and a predominantly 46,XY karyotype in the ovary (93% 46,XY and 6% 45,X). She gave birth to a 46,XY daughter with complete gonadal dysgenesis. The range of phenotypes observed in this unique family suggests a new mechanism which predisposes to chromosomal mosaicism (34).

Embryonic testicular regression syndrome (ETRS)

ETRS has been considered part of the clinical spectrum of partial 46,XY gonadal dysgenesis (35). In this syndrome, most of the patients present ambiguous genitalia or severe micropenis associated with complete regression of testicular tissue in one or both sides. The variable degree of masculinization of the internal and external genitalia is a consequence of the duration of testicular function prior to its loss. The dysgenetic testes showed disorganized seminiferous tubules and ovarian stroma with occasional primitive sex cords devoid of germ cells; primordial follicles are sometimes observed in the streak gonad in the first years of life (36). The abnormal pattern of sex duct development in these subjects suggests that the gonadal tissue was intrinsically altered before the testicular regression took place. Familial cases have been reported with variable degrees of sexual ambiguity, but the nature of the underlying defect is still unknown (35).

Gonadal dysgenesis associated with syndromic phenotype

There are several syndromes associated with 46,XY gonadal dysgenesis in humans caused by mutations in genes involved in gonadal determination. They will be described according to the time of gene expression in gonadal determination.

46,XY DSD due to underexpression of WT1 gene

The Wilms’ tumor suppressor gene (WT1) encodes a zinc-finger transcription factor involved in the development of the kidneys and gonads and their subsequent normal function. WT1 gene is located on 11p13. Mutations in this gene impair gonadal and urinary tract development and 3 disorders are associated with WT1 mutations: WAGR syndrome, Denys-Drash syndrome and Frasier syndrome.

WAGR syndrome: is characterized by Wilm´s tumor, aniridia, genitourinary abnormalities and mental retardation. The genitourinary anomalies are renal agenesis or horseshoe kidney, urethral atresia, hypospadias, cryptorchidism and more rarely ambiguous genitalia (37). Heterozygous deletions of WT1 and contiguous gene are the cause of this syndrome (38). Deletions of PAX6 gene is related to the presence of aniridia in these patients. Severe obesity is present in some patients with the WAGR syndrome and the acronym WAGRO has been suggested this condition (39). The existence of a gene in the 11p14-p12 region responsible for obesity is proposed. A 46,XY patient with WAGR syndrome and female external and internal genitalia with an interstitial deletion at approximately 10 Mb encompassing WT1 and PAX6 was described (40). This report demonstrated an overlap of clinical and molecular features in WAGR, Frasier and Denys-Drash syndromes that confirm these conditions as a spectrum of disease due to WT1 alterations.

Denys-Drash syndrome: is characterized by dysgenetic 46,XY DSD associated with early-onset renal failure (diffuse mesangial sclerosis) and Wilm´s tumor developed in the first decade of life (41). Müllerian ducts differentiation varies according to the Sertoli cells function. The molecular defect of this syndrome is the presence of heterozygous missense mutations in the zinc finger encoding exons (DNA-binding domain) of WT1 gene (42). Gonadal development is impaired to variable degrees, resulting in a spectrum of 46,XY DSD (43).

Frasier syndrome: is characterized by a female to ambiguous external genitalia phenotype in 46,XY patients, streak gonads and high risk of gonadoblastoma development and renal failure in the second decade of life. However, the nephrotic syndrome may be evident as precocious as 6-months-old (44). The WT1 gene contains 10 exons, of which exons 1–6 encode a proline/glutamine-rich transcriptional-regulation region and exons 7–10 encode the four zinc fingers of the DNA-binding domain. There are four major species of RNA with conserved relative amounts, different binding specificities, and different subnuclear localizations, generated by two alternative splicing regions (45). Splicing at the first site results in either inclusion or exclusion of exon 5. The second alternative splicing site is in the 3’ end of exon 9 and allows the inclusion or exclusion of three amino acids lysine, threonine and serine (KTS) between the third and fourth zinc fingers, resulting in either KTS-positive or negative isoforms. Isoforms that only differ by the presence or absence of the KTS amino acids have different affinities for DNA and, therefore, possibly different regulatory functions. (46) The IVS9 + 4C>T mutation leads to a change in splicing resulting in deficiency of the usually more abundant KTS positive isoforms and reversal of the normal KTS positive to negative ratio, indicating that a precise balance between WT1 isoforms is necessary for normal WT1 function.(47) Constitutional heterozygous mutations of the WT1 gene, almost all located at intron 9, are found in patients with Frasier syndrome, leading to a change in splicing that results in reversal of the normal KTS positive/negative ratio from 2:1 to 1:2 (41, 48). Frasier syndrome is usually associated with IVS9 + 4C>T mutation (49) although exonic mutations also cause Frasier syndrome (50). We reported a patient presenting an overlapping of some typical characteristics of Frasier syndrome (end-stage renal failure in the second decade, gonadoblastoma and IVS9 +4C>T mutation) but with the gonadal and external genitalia development usually found in Denys-Drash syndrome (47). The report of ambiguous external genitalia (50), the presence of Wilms’ tumor (51) and the description of exonic mutations in the DNA binding domain of WT1 gene (50) in patients with Frasier syndrome indicate an overlap of clinical and molecular features in Denys Drash and Frasier syndromes.

46,XY DSD due to the underexpression of steroidogenic factor-1 (NR5A1/SF1)

The steroidogenic factor 1 (SF1) or Nuclear Receptor Subfamily 5, Group A, Member 1; (NR5A1) is a member of the nuclear hormone receptor superfamily of transcriptional factors (52). SF1 is a key reproduction regulator within the hypothalamic-pituitary-gonadal axis and adrenal and gonadal steroidogenesis; it is also an essential factor in sexual differentiation (53, 54).

The first reported human case of NR5A1/SF1 mutation, the heterozygous G35E in the DNA binding domain, was a 46,XY patient who presented female external genitalia and Müllerian duct derivatives, indicating the absence of male gonadal development, associated with adrenal insufficiency. These data suggest a loss-of-function effect, in which the absence of one allele of NR5A1/SF1 in human is enough to cause a severe clinical phenotype (55). Remarkably, R92Q mutation in a highly conserved residue of the A-box of NR5A1/SF1, was described in the homozygous state in a 46,XY baby with female external and internal genitalia who presented primary adrenal failure. This second mutation reinforce the dose-dependent action of NR5A1/SF1, affecting only subjects with both compromised alleles (56). We identified a heterozygous frameshift mutation resulting from the deletion of 8 nucleotides at position 2783 of NR5A1/SF1 gene in a 46,XY 31-year-old female patient with clitoromegaly, absence of Müllerian derivatives and gonadal tissue and normal adrenal function (57). This is the first described patient with a NR5A1/SF1 inactivating mutation with normal adrenal function, suggesting that SF-1 transcription might have tissue-specific effects in humans. In sequence, several cases with NR5A1/SF1 mutations in the heterozygous state were described in several 46,XY DSD patients with intact adrenal steroid biosynthesis (55, 56).

Mothers carriers of SF1 mutations present normal reproductive development and adrenal function (58, 59). In mice, ovarian development is relatively preserved after tissue-specific target deletion of Sf1, possibly due to compensatory role of the related nuclear receptor LRH1 (NR5A2). (60). The apparent sex-limited dominant transmission of some cases of SF1 mutations has implications for counseling the families about the risk of have future affected 46,XY fetuses and carriers daughters, similar to an X-linked pattern of inheritance

Most of the point mutations identified in NR5A1/SF1 are located in the DNA-binding domain of the protein. The L437Q mutation, the first located in the ligand-binding region, was identified in a patient with a mild phenotype, a penoscrotal hypospadias; this protein retained partial function in several SF1-expressing cell lines and its location points to the existence of a ligand for SF1, until now considered an orphan receptor (59). In 2007, sphingosine has been shown to be an endogenous ligand for SF1, antagonizing its capacity to increase CYP17 reporter gene activity (61).

A novel heterozygous missense mutation (V355M) in SF1 was identified in one boy with a micropenis and testicular regression syndrome (62).

Recently, a new frameshift mutation in SF1 (c536delC) was described in two 46,XY newborn infants who presented ambiguous genitalia associated to a hormonal phenotype mimicking androgen insensitivity syndrome (63).

In summary, these recent reports indicate that SF1 mutations should be considered in patients with 46,XY DSD due to several abnormalities of gonadal development with and mainly without adrenal failure.

46,XY DSD due to the underexpression of DMRT1 and DMRT2 genes

Raymond et al identified both DNA-binding Motif (DM) domain genes expressed in testis (DMRT1 and DMRT2) located in chromosome 9p24.3, a region associated with gonadal dysgenesis and 46,XY DSD (64-66). The human 9p deletion syndrome is characterized by variable degrees of 46,XY DSD (female genitalia to male external genitalia with cryptorchidism associated to agonadism, streak gonads or hypoplastic testes and internal genitalia disclosing normal Müllerian or Wolffian ducts) mental retardation and craniofacial abnormalities (67). However, smaller deletions may be associated with isolated 46,XY DSD.

Endocrine function varies from hypergonadotropic hypogonadism to near normal testicular function. The authors inferred that haploinsufficiency of the 9p sex-determining gene(s) primarily impedes the formation of the undiferentiated gonad, leading to various degrees of defective testis formation in males and of the ovary in females (67).

ATR-X syndrome (X-linked -thalassemia and mental retardation

ATR-X syndrome results from diverse mutations in the gene that encodes for X-linked helicase-2, implicating ATR-X in the development of the human testis (68). Genital anomalies that lead to a female sex of rearing were reported in several affected 46,XY patients with ATR-X syndrome (69).

ATR-X syndrome is characterized by severe mental retardation, alpha thalassemia and a range of genital abnormalities in 80% of cases ((70). In addition to these definitive phenotypes patients also present with typical facial anomalies comprising a carp-like mouth and a small triangular nose, skeletal deformities and a range of lung, kidney and digestive problems. A range of phenotypically overlapping conditions (Carpenter-Waziri syndrome, Holmes-Gang syndrome, Jubert-Marsidi syndrome, Smith-Fineman-Myers syndrome, Chudley-Lowry syndrome and X-linked mental retardation with spastic paraplegia) without thalassemia has been associated with ATRX mutations. Because ATRX lies on the X chromosome (Xq13) the disease has been confined to males; in females carriers with one X carrying the normal allele and one the mutant, the pattern of X-inactivating is skewed against the ATRX mutation.

Mutations in human ATRX cause urogenital abnormalities ranging from undescended testes to testicular dysgenesis and female or ambiguous genitalia. ATRX duplications leading to gene disruption were also described (71). There is a phenotypic spectrum of this syndrome. Although all cases of severe genital abnormality reported in ATRX syndrome have been associated with severe mental retardation, this is not so for alpha-thalassemia. Despite the role of ATRX in the sexual development cascade is poorly understood, it is suggested that ATRX could be involved in the normal Leydig cell development (72). There are two major functional domain in ATRX protein: at the N-terminus is the ATRX-DNMT3-DNMT3L (ADD) domain and at the C-terminal half of the protein lies the helicase/ATPase domain, both domains act as chromatin remodellers. Mutations in the ADD domain correlated with severe psychomotor impairment associated to urogenital abnormalities whereas mutations in the C-terminus region have been related with mild psychomotor impairment without severe urogenital abnormalities (73, 74).

46,XY DSD due to the overexpression of DAX1 (NR0B1) gene

Male patients with female or ambiguous external and internal genitalia due to partial duplications of Xp and an intact SRY gene have been described (75). These patients present with dysgenetic or absent gonads associated or not with mental retardation, cleft palate and dysmorphic face. Bardoni et al identified in these patients, a common 160-kb region of Xp2 containing DAX1 gene named dosage sensitive sex (DSS) locus which, when duplicated, resulted in 46,XY DSD (75). Recently two reports of 46,XY isolated gonadal dysgenesis because of a DAX1 locus duplication were described. Previously all the cases described with large duplications of Xp21 presented gonadal dysgenesis as part of a complex phenotype with dysmorphic features and/or mental retardation. The first report identified a 637kb tandem duplication on Xp21.2 that in addition to DAX1 includes the four MAGEB genes in two sisters with isolated 46,XY gonadal dysgenesis and gonadoblastomas (76). The second case presented a duplication with size of approximately 800kb and in addition to DAX1 contains the four MAGEB, Cxorf21 and GK. Analysis of healthy mother showed that she was a carrier of the duplication (77).

Smyk et al. described a 21-years-old 46,XY patient manifesting primary amenorrhea, a small immature uterus, gonadal dysgenesis and absent adrenal insufficiency with a submicroscopic (257 kb) deletion upstream of DAX1. The authors hypothesized that loss of regulatory sequences may have resulted in up-regulation of DAX1 expression, consistent with phenotypic consequences of DAX1 duplication (78).

However, until now, there has not been a direct proof that an isolated DAX1 duplication is sufficient to cause 46,XY gonadal dysgenesis in humans, suggesting that other contiguous genes such as MAGEB genes, present in the DSS locus, should be involved in dosage-sensitive 46,XY DSD.

46,XY DSD due to the overexpression of WNT4 gene

The Wnt4 (wingless-type mouse mammary tumour virus integration site member 4) gene family consists of structurally related genes that encode cysteine-rich secreted glycoproteins that act as extracellular signaling factors (79).

Overexpression of the WNT4 and RSPO1 may be a cause of 46,XY DSD. A 46,XY newborn infant, with multiple congenital anomalies including bilateral cleft lips and palate, intrauterine growth retardation, microcephaly, tetralogy of Fallot, ambiguous external and internal genitalia, and undescent gonadas consisted of rete testes and rudimentary seminiferous tubules, who carried a duplication of 1p31-p35, including both WNT4 and RSPO1 gene, was reported (80). In vitro studies suggest that Wnt4 up-regulates Dax1 in Sertoli cells, so that 46,XY DSD may result from an excess of Dax1 expression (81).

Dysgenetic 46,XY DSD associated with campomelic dysplasia (underexpression of the SOX9)

SRY-related HMG-box gene 9 (SOX9) is a transcription factor involved in chondrogenesis and sex determination. SOX9 gene, located on human chromosome 17 is a highly conserved HMG family member and it is also implicated in the sex-determination pathway (82, 83). In all subjects, SOX9 mutation was identified in heterozygous state indicating that this disorder is due to haploinsufficiency of SOX9 gene (82). This syndrome is characterized by severe skeletal malformations (campomelic dysplasia) associated to dysgenetic 46,XY DSD in three-quarters of the affected 46,XY patients. The external genitalia vary from that of normal males with cryptorchidism through ambiguous to female and internal genitalia can include vagina, uterus, and fallopian tubes (84).

Patients with campomelic dysplasia and XY gonadal dysgenesis with intact SOX9 were reported. In one patient a microdeletion of ~380 kb upstream of SOX9 was identified (85) and in the other case an apparently balanced chromosome translocation and breakpoints scattered up to 1 Mb upstream and even >1 Mb downstream of SOX9, leaving the gene itself intact (86).

Dysgenetic 46,XY DSD due to Desert hedgehog (DHH) underexpression

Desert hedgehog (Dhh), a member of the hedgehog family of signaling proteins, located in 12q12-q13.1 (87) is also a gene involved in the testis-determining pathway. To date, three missense mutations have been described in DHH gene. One of them, located at the initiation codon of exon 1 was found in a 46,XY patient with partial gonadal dysgenesis associated with polyneuropathy (88); the other two missense mutations located at exon 2 and exon 3 respectively, were identified in three patients with complete gonadal dysgenesis without neuropathy, two of whom harbored gonadal tumors (89). The homozygous mutation described anteriously in exon 3 of DHH gene by Canto et al. was identified by the same group in two patients with mixed gonadal dysgenesis in heterozygous condition (90).

A summary of the phenotypes of the disorders of sex determination are presented in Table 2

NR: non reported

46,XY DSD ASSOCIATED WITH Cholesterol Synthesis

Smith-Lemli-Opitz syndrome: This syndrome, caused by a deficiency of 7-dehydrocholesterol reductase, is the first true metabolic syndrome leading to multiple congenital malformations (91, 92). This disorder is caused by mutations in the sterol delta-7-reductase (DHCR7) gene, which maps to 11q12-q13. Typical facial appearance is characterized by short nose with anteverted nostrils, blepharoptosis, microcephaly, photosensitivity, mental retardation, syndactyly of toes 2 and 3, hypotonia and genital ambiguity. Adrenal insufficiency maybe be present or evolve with time. Ambiguity of the external genitalia is a frequent feature of males (71%) and ranges from hypospadias to female external genitalia despite normal 46,XY karyotype and SRY sequences. Müllerian derivative ducts can also be present (93, 94). The aetiology of masculinization failure in the SLO syndrome remains unclear. However, the description of patients with SLOS who present with hyponatremia, hyperkalemia, and decreased aldosterone-to-renin ratio suggest that the lack of substrate to produce adrenal and testicular steroids is the cause of adrenal insufficiency and genital ambiguity (95).

Affected children present with low plasma cholesterol and elevations of plasma 7-dehydrocholesterol. Considering the relative high frequency of Smith-Lemli-Opitz syndrome, approximately 1 in 20,000 to 60,000 births, we suggest that at least cholesterol levels should be routinely measured in patients with 46,XY DSD.

46,XY DSD due to testosterone synthesis defects

46,XY DSD due to Leydig cell hypoplasia (complete and partial forms)

In 46,XY DSD due to Leydig cell hypoplasia there is failure of intrauterine and pubertal virilization due to the inability of interstitial Leydig cells to secrete testosterone. Both hCG and LH act by stimulating a common seven transmembrane LH receptor, a G-protein coupled receptor and mutations in LHCGR gene are the cause of Leydig cells hypoplasia (96, 97). In 1976, Berthezene et al. (98) described the first patients with Leydig cell hypoplasia and subsequently other cases have been reported, (99-102). The study of 8 of our cases and review of the literature allowed us to delineate the characteristics of 46,XY DSD due to the complete form of Leydig cell hypoplasia as: 1) female external genitalia leading to female sex assignment 2) no development of sexual characteristics at puberty, 3) undescended testes slightly smaller than normal with relatively preserved seminiferous tubules and absence of mature Leydig cells 4) presence of rudimentary epidydimis and vas deferens and absence of uterus and fallopian tubes, 5) low testosterone levels despite elevated gonadotrophin levels, with elevated LH levels predominant over FSH levels, 6) testicular unresponsiveness to hCG stimulation, and 7) no abnormal step up in testosterone biosynthesis precursors (103-106). Several different mutations in the LH receptor gene were reported in patients with Leydig cell hypoplasia (96, 97, 107-112).

In contrast to the homogenous phenotype of the complete form of Leydig cell hypoplasia, the partial form can have a broad spectrum (96, 97, 104, 112-115). Most patients have predominantly male external genitalia with micropenis and or hypospadias. Testes are cryptorchidic or in the scrotum. During puberty, partial virilization occurs and testicular size is normal or only slightly reduced, while penile growth is significantly impaired. Spontaneous gynaecomastia does not occur. Before puberty the testosterone response to the hCG test is subnormal without accumulation of testosterone precursors. After puberty, LH levels are elevated and testosterone levels are intermediate between those of children and normal males.

Mutations in the LHCGR gene have also been identified in patients with the partial form of Leydig cell hypoplasia (96, 97, 112, 113). In vitro studies showed that cells transfected with LH receptor gene containing these mutations had an impaired hCG-stimulated cAMP production (97, 112). Latronico et al. (96) reported a homozygous mutation in the LH receptor (Ser616Tyr) in a boy with micropenis. Subsequently, mutations were identified in further patients with the partial form of Leydig cell hypoplasia (97, 112, 113)

Leydig cell hypoplasia was found to be a genetic heterogenous disorder since Zenteno et al, (116) ruled our molecular defects in the LHCG receptor as being responsible for Leydig cell hypoplasia in three siblings with 46,XY DSD, using segregation analysis of a known polymorphism in exon 11 of the LHCG receptor gene.

The identification and characterization of a novel, primate-specific bona fide exon (exon 6A) within the LHCGR determined a new regulatory element within the genomic organization of this receptor and a new potential mechanism of this disorder. Kossack et al studying exon 6A in 16 patients with 46,DSD due to Leydig cells hypoplasia without molecular diagnosis detected mutations in three patients. Functional studies revealed a dramatic increase in expression of the mutated internal exon 6A transcripts, resulting in the generation of predominantly nonfunctional isoforms of the LHCGR, thereby preventing its proper expression and functioning (117).

In addition, the absence of causative mutations in LHCGR several patients strongly suspected to have Leydig cell hypoplasia, supported the idea that other genes must be implicated in the molecular basis of this disorder

We observed that 46,XX sisters of patients with 46,XY DSD due to Leydig cell hypoplasia, with the same homozgous mutation in the LH receptor, have primary or secondary amenorrhaea, spontaneous breast development, infertility, normal or enlarged cystic ovaries with elevated LH and LH/FSH ratio, measurable estradiol levels and normal androgen levels (96, 104, 106, 118, 119). Our findings were subsequently confirmed by other authors who studied 46,XX sisters of 46,XY DSD with Leydig cell hypoplasia (115).

46,XY DSD due to testosterone synthesis defect

Five enzymatic defects that alter the normal synthesis of testosterone have been described to date (Fig 6).

Figure 6- Adrenal and testicular steroidogenesis displaying the genes in which mutations result in 46,XY DSD in humans.

Three of them are associated with defects in cortisol synthesis leading to congenital adrenal hyperplasia. All of them present an autosomal recessive mode of inheritance and genetic counseling is mandatory, since the chance of recurring synthesis defects among siblings is 25%.

Defect in Corticosteroid and Testosterone Synthesis

Adrenal hyperplasia syndromes are examples of hypoadrenocorticism or a mixed of hypo- and hyper corticoadrenal steroid secretion. Synthesis of cortisol only or both cortisol and aldosterone are impaired. When cortisol production is impaired there is a compensatory increase in ACTH secretion. If mineralocorticoid production is impeded, there is a compensatory increase in renin-angiotensin production. These compensatory mechanisms may return cortisol or aldosterone production to normal or near normal levels, but at the expense of excessive production of precursors that can cause undesirable hormonal effects.

Congenital lipoid adrenal hyperplasia

Deficiency of the acute steroidogenesis regulatory protein (StAR)

Deficiency of P45011A

The earliest step in the conversion of cholesterol to hormonal steroids is hydroxylation at carbon 20, with subsequent cleavage of the 20-22 side chain to form pregnenolone. In steroidogenic tissues, such as adrenal cortex, testis, ovary, and placenta, the initial and rate-limiting step in the pathway leading from cholesterol to steroid hormones is the cleavage of the side chain of cholesterol to yield pregnenolone. This reaction, known as cholesterol side-chain cleavage, is catalyzed by a specific cytochrome P450 called P450scc or P45011A and by the steroidogenic acute regulatory (StAR) protein, a mitochondrial phosphoprotein (120).

Deficiency of the acute steroidogenesis regulatory protein (StAR)

It is the most severe form of congenital adrenal hyperplasia (121). Lipoid adrenal hyperplasia is rare in Europe and America but it is thought to be the second most common form of adrenal hyperplasia in Japan. Affected subjects are phenotypic females irrespective of gonadal sex or sometimes have slightly virilized external genitalia with or without cryptorchidism, underdeveloped internal male organs and an enlarged adrenal cortex, engorged with cholesterol and cholesterol esters (122). Adrenal steroidogenesis deficiency leads to salt wasting, hyponatremia, hyperkalemia, hypovolemia, acidosis, and death in infancy, although patients can survive to adulthood with appropriate mineralocorticoid- and glucocorticoid-replacement therapy (123). Recently, a mild form of congenital lipoid adrenal hyperplasia was described in two families. The affected children presented with late primary adrenal insufficiency at 2-4 yr of age and the males had normal external genital. DNA sequencing identified homozygous StAR mutations in these two families and functional studies of StAR showed that these mutants retained approximately 20% of wild-type activity (124).

Hormonal diagnosis is based in high ACTH and renin levels and the presence of low levels of all glucocorticoids, mineralocorticoids and androgens.

The disease was firstly attributed to P450scc deficiency, but most of the cases studied through molecular analysis showed an intact P45011A gene and its RNA (125). Since StAR is also required for the conversion of cholesterol to pregnenolone molecular studies were performed in StAR gene and mutations were found in most of the affected patients (126). Congenital lipoid adrenal hyperplasia in most Palestinian cases is caused by a founder c.201_202delCT mutation causing premature termination of the StAR protein (127). Histopathological findings of excised XY gonads included accumulation of fat in Leydig cells and already at 1 yr of age, positive placental alkaline phosphatase and octamer binding transcription factor (OCT4) staining indicating neoplastic potential (127).

Deficiency of P450scc

It has been thought that CYP11A mutations are incompatible with human term gestation, because P450scc is needed for placental biosynthesis of progesterone, which is required to maintain pregnancy. However, a patient has been described with congenital lipoid adrenal hyperplasia with normal StAR and SF1 genes presenting a de novo heterozygous inactivating mutation in CYP11A. This patient was atypical for congenital lipoid adrenal hyperplasia, having survived for 4 yrs without hormonal replacement (128).

More recently, the study of infants with adrenal failure and disorder of sexual differentiation identified compound heterozygous mutations in CYP 11A1 recognizing that the disorder may be more frequent than originally thought. The phenotypic spectrum of P450scc deficiency ranges from severe loss-of-function mutations associated with prematurity, complete underandrogenization, and severe early-onset adrenal failure, to partial deficiencies found in children born at term with mild masculinization and later-onset adrenal failure. In contrast to congenital lipoid adrenal hyperplasia caused by StAR mutations, adrenal hyperplasia has not been reported in any of the six patients with P450scc deficiency (129).

3b-Hydroxysteroid Dehydrogenase type II Deficiency

3b-HSD converts 3b-hydroxy D5 steroids to 3-keto D4 steroids and is essential for the biosynthesis of mineralocorticoids, glucocorticoids and sex steroids (130). Two forms of the enzyme have been described in man: type I enzyme is expressed in placenta and skin, and type II in adrenals and gonads (131). The types I and II genes are known to be closely linked on chromosome 1p13.1. The two forms are very closely related in structure and substrate specificity, though the type I enzyme has higher substrate affinities and a 5-fold greater enzymatic activity than type II (132)

Male patients with 3b-HSD type II deficiency present with ambiguous external genitalia, characterized by micropenis, perineal hypospadias, bifid scrotum and a blind vaginal pouch associated or not with salt loss (130). Gynecomastia is common at pubertal stage.

Serum levels of D-5 steroids (pregnenolone, 17OHPregnenolone (17OHPreg), DHEA, DHEAS are elevated and basal levels of 17OHPreg and 17OHPreg/17OHP ratio are the best marker of this deficiency in both prepubertal and postpubertal stage. D-4 steroids are slightly increased due to the peripheral action of 3b-HSD type I enzyme but the ratio of D-5/D-4 steroids is elevated. Cortisol secretion is reduced but the response to exogenous ACTH stimulation varies from decreased (more severe deficiency) to normal. At adult age, affected males can reach normal or almost normal levels of testosterone due to the peripheral conversion of elevated D-5 steroids by 3b-HSD type I enzyme and also due to testicular stimulation by the high LH levels (133).

There are around 40 mutations in 3b-HSD type II gene already described. Mutations that lead to the abolition of 3b-HSD type II activity lead to congenital adrenal hyperplasia (CAH) with severe salt-loss (132, 134-136). Mutations that reduce but do not abolish type II activity lead to CAH with mild or no salt-loss, which in males is associated with 46,XY DSD due to the reduction in androgen synthesis (137, 138). Male subjects with 46,XY DSD due 3b-HSD type II deficiency without salt loss showed clinical features in common with the deficiencies of 17b-HSD 3 and 5a-reductase 2.

Most of the patients were raised as males and kept the male social sex at puberty. In one Brazilian family, two cousins with 46,XY DSD due to 3b-HSD type II deficiency were reared as females; one of them was castrated in childhood and kept the female social sex; the other was not castrated at childhood and changed to male social sex at puberty (133).

CYP17 (17-Hydroxylase and C-17-20 lyase deficiency)

CYP17 is a steroidogenic enzyme that has dual functions: hydroxylation and lyase and is located in the fasciculata and reticularis region/zone of the adrenal cortex and gonadal tissues. The first activity gives hydroxylation of pregnenolone and progesterone at the C (17) position to generate 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, while the second enzyme activity cleaves the C(17)-C(20) bond of 17α-hydroxypregnenolone and 17α-hydroxyprogesterone to form dehydroepiandrosterone and androstenedione, respectively. The modulation of these two activities occurs through cytochrome b5, necessary for lyase activity.

Deficiency of adrenal 17-hydroxylation activity was first demonstrated by Biglieri et al. (139). The phenotype of 17-hydroxylase deficiency in most of the male patients described is a female-like or slightly virilized external genitalia with blind vaginal pouch, cryptorchidism and high blood pressure, usually associated with hypokalaemia. New in 1970, reported the first affected patient with ambiguous genitalia which was assigned in the male sex (140).

At puberty, patients usually present sparse axillary and pubic hair. Male internal genitalia are hypoplastic and gynecomastia can appear at puberty. Most of the male patients were reared as female and sought treatment due to primary amenorrhea or lack of breast development. Female patients may also be affected and present normal development of internal and external genitalia at birth and hypergonadotropic hypogonadism and amenorrhea at post pubertal age; enlarged ovaries at adult age and infarction from twisting can occur (141, 142). These patients do not present signs of glucocorticoid insufficiency, due to the elevated levels of corticosterone, which has a glucocorticoid effect. The phenotype is similar to 46,XX or 46,XY complete gonadal dysgenesis and the presence of systemic hypertension and absence of pubic hair in post pubertal patients suggests the diagnosis of 17-hydroxylase deficiency (143).

Serum levels of progesterone, corticosterone, and 18-OH-corticosterone are elevated, while aldosterone, 17-OH-progesterone, cortisol, androgens and estrogens are decreased. Martin et al, (144) performed a clinical, hormonal, and molecular study of 11 patients from 6 Brazilian families with the combined 17-alpha-hydroxylase/17,20-lyase deficiency phenotype. All patients had elevated basal serum levels of progesterone and suppressed plasma renin activity. The authors concluded that basal progesterone measurement is a useful marker of P450c17 deficiency and that its use should reduce the misdiagnosis of this deficiency in patients presenting with male pseudohermaphroditism, primary or secondary amenorrhea, and mineralocorticoid excess syndrome.

Excessive production of deoxycorticosterone and corticosterone results in blood hypertension and suppression of renin levels and inhibition of aldosterone synthesis. The CYP17 gene, which encodes the enzymes 17-hydroxylase and 17-20 lyase, is a member of a gene family within the P450 supergene family and was mapped 10q24.3 (145). Several mutations in the CYP17 gene have been identified in patients with both 17-hydroxylase and 17,20 lyase deficiencies (141, 142, 144, 146). Four homozygote mutations, A302P, K327del, E331del and R416H, were recentely identified by direct sequencing of the CYP17A1 gene. Both P450c17 activities were abolished in all the mutant proteins but the mutant proteins were normally expressed, suggesting that the loss of enzymatic activity is not due to defects of synthesis, stability, or localization of P450c17 proteins (146).

Glucocorticoid replacement for hypertension management, gonadectomy and estrogen replacement at puberty for patients reared in the female social sex. In male patients, androgen replacement is usually necessary since they present very low levels of testosterone. These patients are very sensitive to glucocorticoids and low doses of dexamethasone (0.125-0.5 mg at night) are sufficient to control blood pressure. In some patients, however, estrogens might aggravate hypertension. The control of blood pressure can be initially achieved by salt restriction although mineralocorticoid antagonists might be necessary (146).

Defects in Testicular Steroidogenesis

Two defects in testosterone synthesis that are not associated with adrenal insufficiency have been described: CYP17 deficiency (17,20 lyase activity) and 17-b-HSD 3 deficiency.

CYP17 (17,20 lyase activity) Deficiency

Human male sexual differentiation requires production of fetal testicular testosterone, whose biosynthesis requires steroid 17,20-lyase activity. The existence of true isolated 17,20-lyase deficiency has been questioned because 17-a-hydroxylase and 17,20-lyase activities are catalyzed by a single enzyme and because combined deficiencies of both activities were found in functional studies of the mutation found in a patient thought to have had isolated 17,20-lyase deficiency (147). Later, clear molecular evidence of the existence of isolated 17,20 desmolase deficiency was demonstrated (142, 148).

The patients present ambiguous genitalia with micropenis, perineal hypospadias and cryptorchidism. Gynecomastia Tanner stage V can occur at puberty (148).

Elevated serum levels of 17-OHP and 17-OHPreg, with low levels of androstenedione, dehydroepiandrosterone and testosterone. The hCG stimulation test results in a slight stimulation in androstenedione and testosterone secretion with an accumulation of 17-OHP and 17-OHPreg.

The CYP17 gene of two Brazilian 46,XY DSD patients with clinical and hormonal findings indicative of isolated 17,20-lyase deficiency, since they produce cortisol normally, were studied. Both were homozygous for substitution mutations in CYP17 (148). When expressed in COS-1 cells, the mutants retained 17a-hydroxylase activity and had minimal 17,20-lyase activity. Both mutations alter the electrostatic charge distribution in the redox-partner binding site, so that the electron transfer for the 17,20-lyase reaction is selectively lost (148).

46,XY DSD due to 17b-HSD 3 Deficiency

This disorder consists in a defect in the last phase of steroidogenesis, when androstenedione is converted into testosterone and estrone into estradiol. This disorder was described by Saez and his colleagues (149) and it is the most common disorder of androgen synthesis, reported from several parts of the world (150).

There are 5 steroid 17b-HSD enzymes that catalyze this reaction (151) and 46,XY DSD results from mutations in the gene encoding the 17b-HSD3 isoenzyme (151, 152).

Patients present female-like or ambiguous genitalia at birth, with the presence of a blind vaginal pouch, intra-abdominal or inguinal testes and epididymides, vasa deferentia, seminal vesicles and ejaculatory ducts. Most affected males are raised as females (153, 154), but some have less severe defects in virilization and are raised as males (151). Virilization in subjects with 17b-HSD 3 deficiency occurs at the time of expected puberty. This late virilization is usually a consequence of the presence of testosterone in the circulation as a result of the conversion of androstenedione to testosterone by some other 17b-HSD isoenzyme (presumably 17b-HSD 5) in extra-gonadal tissue and, occasionally, of the secretion of testosterone by the testes when levels of LH are elevated in subjects with some residual 17b-HSD 3 function (151). However, the discrepancy between the failure of intrauterine masculinization and the virilization that occurs at the time of expected puberty is poorly understood. A limited capacity to convert androstenedione into testosterone in the fetal extragonadal tissues may explain the impairment of virilization of the external genitalia in the newborn. Bilateral orchiectomy resulted in a clear reduction of androstenedione levels indicating that the principle origin of this androgen is the testis (151, 154). 46,XY DSD phenotype is sufficiently variable in 17b-HSD3 deficiency to cause problems in accurate diagnosis, particularly in distinguishing it from partial androgen insensitivity syndrome (153, 155).

Laboratory diagnosis is based on elevated serum levels of androstenedione and estrone and low levels of testosterone and estradiol resulting in elevated androstenedione/testosterone and estrone/estradiol ratios indicating impairment in the conversion of 17-keto into 17-hydroxysteroids. At the time of expected puberty, serum LH and testosterone levels rise in all affected males and testosterone levels may be into the normal adult male range (154).

The disorder is due to homozygous or compound heterozygous mutations in the gene that encodes the 17b-HSD3 isoenzyme and several mutations have been reported (151, 156).

Most patients are raised as girls during childhood and change to male gender role behavior at puberty has been frequently described in individuals with this disorder who were reared as females (154, 157-159) including members of a large consanguineous family in the Gaza strip (160).

Altered steroidogenesis due to disrupted electron donor proteins

Two defects in steroid synthesis have been described: cytochrome P450 reductase (POR) deficiency and cytochrome b5 defect

Cytochrome P450 reductase (POR) deficiency

The apparent combined P450C17 and P450C21 deficiency is a rare variant of congenital adrenal hyperplasia, first reported by Peterson et al in 1985 (161). Affected girls and boys are born with ambiguous genitalia, indicating intrauterine androgen excess in females and androgen deficiency in males. Boys and girls can also present with bone malformations, which in some cases resemble a pattern seen in patients with Antley-Bixler syndrome. Findings of biochemical investigations of urinary steroid excretion in affected patients have shown accumulation of steroid metabolites, indicating impaired C17 and C21 hydroxylation, suggesting concurrent partial deficiencies of the 2 steroidogenic enzymes, P450C17 and P450C21. However, sequencing of the genes encoding these enzymes showed no mutations, suggesting a defect in a cofactor that interacts with both enzymes. POR is a flavoprotein that donates electrons to all microsomal P450 enzymes, including the steroidogenic enzymes P450c17, P450c21 and P450aro (122). Shephard et al. (1989) isolated and sequenced cDNA clones that encode the rat and human NADPH-dependent cytochrome P-450 reductase and located the human gene at 7q11.2 (162).

The underlying molecular basis of congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency was defined in 3 patients, who were compound heterozygotes for mutations in POR (25, 163). Antley-Bixler syndrome is characterized by craniosynostosis, severe midface hypoplasia, proptosis, choanal atresia/stenosis, frontal bossing, dysplastic ears, depressed nasal bridge, radiohumeral synostosis, long bone fractures, femoral bowing, and urogenital abnormalities (164). The occurrence of genital abnormalities in patients with Antley-Bixler syndrome, especially females was reported by Reardon et al. (2000) (165). In a recent large survey of patients with Antley-Bixler syndrome, it was demostrated that individuals with an Antley-Bixler-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and altered steroidogenesis have POR deficiency (166).

Methaemoglobinemia, type IV, with 46,XY DSD due to cytochrome b5 defect

A patient with type IV hereditary methaemoglobinemia and with 46,XY DSD was described by Hegesh et al. (167) The patient had a 16-bp deletion in the cytochrome b5 mRNA leading to a new in-frame termination codon and a truncated protein. The etiology of 46,XY DSD in this patient was attributed to the cytochrome b5 defect since cytochrome b5 has been shown to participate in 17-α hydroxylation in adrenal steroidogenesis by serving as an electron donor (168).

46,XY DSD due to defects in Testosterone Metabolism

5-Reductase type 2 Deficiency

In 1974 a rare autosomal form of 46,XY DSD was described in 2 families, one from Dallas (169) and one from the Dominican Republic (170), in which the underlying defect was shown to be a deficiency in the conversion of testosterone to its more active metabolite, dihydrotestosterone. There are 2 steroid 5-reductase enzymes that catalyze this reaction (171-173) and 46,XY DSD results from mutations in the gene encoding the steroid 5-reductase 2 isoenzyme (SRD5A2) (174-176). The gene that codifies 5-RD2 contains 5 exons and 4 introns and is located in chromosome 2 p23.

Affected patients present with ambiguous external genitalia, micropenis, normal internal male genitalia, prostate hypoplasia and testes with normal differentiation with normal or reduced spermatogenesis. The testes are usually located in the inguinal region, suggesting that dihydrotestosterone influences testis migration to the scrotum (152). Virilization and deep voice appear at puberty, along with penile enlargement, and muscle mass development without gynecomastia. These patients present scarce facial and body hair and absence of temporal male baldness, acne and prostate enlargement, since these features depend on dihydrotestosterone action. The main differential diagnosis of 5-RD2 deficiency is with 17-HSD3 deficiency and partial androgen insensitivity syndrome although in these two disorders it is common to observe the presence of gynecomastia.

After hCG stimulation, affected children show lower DHT levels and elevated T/DHT ratio (152, 177). Post pubertal affected patients present normal or elevated testosterone levels, low DHT levels and elevated T/DHT ratio in basal conditions. Low DHT production after exogenous testosterone administration is also capable of identifying 5-reductase type 2 deficiency (178). Elevated 5/5 urinary metabolites ratio is also an accurate method to diagnose 5-reductase 2 even at prepubertal age and in orchiectomized adult patients (178, 179).

The mode of inheritance for 5a-reductase type 2 deficiency is autosomal recessive. There are more than 50 families with this disorder described in several parts of the world (170, 176, 180, 181). In a few cases of 46,XY DSD due to 5- reductase 2 deficiency diagnosed by clinical and hormonal findings no mutations were identified in 5-RD2 gene (170, 174, 176, 180, 181). Recently, Chavez et al. (2000) described different mode of transmission of 5- reductase type 2 deficiency in 2 unrelated patients due to due to uniparental disomy (182).

Most of the patients are reared in the female social sex due to the impairment of external genitalia virilization, but many patients who have not been submitted to orchiectomy in childhood undergo male social sex change at puberty (138,142,149,156,157). In our experience with 30 cases of 46,XY DSD due to 5--RD 2 deficiency from 18 families all subjects were registered in the female social sex except for two cases – one who has an affected uncle and the other who was diagnosed before being registered (5, 178). Fourteen patients changed to male gender role, two of them at prepubertal age, 9 at pubertal age and 2 at adult age. No correlation was observed between the mutation, T/DHT ratio and gender role in these families. In one family, the two siblings carry the same mutation but presented a different gender role (178). The patients treated at a later age referred severe social inadequacy, psychological anguish and suicidal ideas and all of them declared they would like to have been treated in childhood. Ten cases are adults now and nine of them are married. Three of them have divorced and re-married and in two cases the small penis size was considered the cause of separation. All patients refer male libido and sexual activity although the small penis size sometimes makes the intercourse difficult. Most of the patients have retrograde ejaculation highly viscous semen due to rudimentary prostate and underdeveloped seminal vesicles and needs in vitro fertilization to have children. Three cases adopted children and in two cases in vitro fertilization using the patient’s sperm cells resulted in twin siblings in one family and in a singleton pregnancy in the other (5, 178).

Fourteen patients maintained the female sexual identification. Three of them were castrated in childhood and the others, despite the virilization signs developed at puberty, kept the female social sex and sought medical treatment to correct absence of breast development and primary amenorrhrea. None of the 10 adult female patients, now aged 20 to 47 years are married and but 8 of them have satisfactory sexual activity.

In conclusion, the patients who change to male sex are more socially adapted them the ones that keep the female social sex, being their main problem the small penis size. New approaches as the use of donor-grafting tissue to elongate the urethra and penis to increase penis size will help them to be more integrated in life.

46,XY DSD DUE TO DEFECTS IN ANDROGEN ACTION

Androgen insensitivity syndrome - complete and partial forms

46,XY DSD due to androgen insensitivity syndrome (AIS) is characterized by 46,XY karyotype, normal testes, normal androgen secretion and impairment in androgen action with consequent impairment of the normal virilization in utero, during and after puberty. AIS is classified as complete form (CAIS) when there is an absolute absence of androgen action and partial form (PAIS) when there are variable degrees of impairment of androgen action. A milder form, mild androgen insensitivity syndrome (MAIS), with male genitalia and micropenis or only infertility has also been described (183). The human androgen receptor (AR) complementary DNA was cloned in 1988 and the AR gene located to the long arm of the X chromosome at Xq11-13, later refined to Xq11-12 (184).

Prenatal diagnosis of CAIS can be suspected based in the discordance between 46,XY karyotype on amniocentesis and female genitalia at prenatal ultrasound. In prepubertal age, an inguinal hernia in a girl can indicate the presence of testes. At puberty, CAIS patients have complete breast development and primary amenorrhea. Pubic and axillary regions remain covered with vellous hair only, or sparse pubic hair although some patients with CAIS can present almost normal pubic hair. Generally, mullerian ducts are absent in CAIS patients but some reports referred the presence of mullerian derivatives in these patients (185).

Whereas the clinical picture of CAIS is homogeneous, the phenotype of partial androgen insensitivity syndrome (PAIS) is quite variable and missdiagnosis with other causes of 46,XY DSD is more likely (153, 155). Patients with PAIS have ambiguous genitalia, ranging from predominantly female genitalia with mild clitoromegaly to predominantly male genitalia with micropenis and hypospadias and development of gynecomastia at puberty.

In a patient with the phenotype described above, after the age of puberty, hormonal diagnosis is performed by the demonstration of normal or elevated serum testosterone levels and slightly elevated LH levels. FSH levels can be slightly elevated due to the presence of cryptorchidism. Testosterone precursors are not elevated in relation to testosterone levels (186).

AR gene mutations are identified in most cases of CAIS and in several patients with PAIS. To date, more than 300 different AR gene mutations have been decribed and are listed in a database found in the web at http://androgendb.mcgill.ca/. Hemizygous deletions, frameshift and missense mutations in the AR gene were reported in patients with CAIS (187).

AR mutations are transmitted in an X-linked recessive manner in 70% of the cases, but in 30%, the mutations arise de novo. When de novo mutations occur after the zygotic stage, they result in somatic mosaicisms (188).

Additional AIS cases have been described with an unaltered DNA sequence of the coding region of the AR gene including all intron-exon boundaries supporting the concept that in a subset of AIS patients, particulary those with partial form, molecular alterations outside the coding region of the AR gene must be presumed. (189). Recently, a normal AR gene was found in patient with a CAIS phenotype although studies in genital skin fibroblasts revealed that transmission of the activation signal by the AF-1 region of the androgen receptor was disrupted, suggesting that a coactivator interacting with the AF-1 region of the AR was lacking in this patient (190).

Very recently a new mechanism for regulating steroid hormone receptor activity has been proposed by Cox et al. The authors suggest that the FKBP52, a cochaperone of the steroid receptor complex, phosphorilation can potentiate steroid receptor function (191). The physiological importance of FKBP52 in steroid receptor complexes is supported by the fact that male mice lacking the gene encoding FKBP52 have ambiguous external genitalia, dysgenetic prostate and seminal vesicles, features consistent with androgen insensitivity (192).

Patients with CAIS are raised as girls and have a female gender identity and role behavior (157, 186, 193). Gonadectomy should be performed because of the increased risk of testicular tumors, especially after puberty. We favor prepubertal gonadectomy, after diagnosis, and then induction of puberty with estrogens at the appropriate age. This approach diminishes the time that the girl has an inguinal mass and surgery is better handled psychologically by a young child than an adolescent. Female relatives on the maternal side of the patient can be studied for the mutation of an index case, and if the carrier status is identified genetic counseling should be performed.

Patients with PAIS have a broad spectrum of impairment in virilization. The external genitalia can vary from predominantly female with clitoromegaly and labial fusion to predominantly male with micropenis, hypospadias and gynecomastia. Testes are in the inguinal canal or labioscrotal folds or, less frequently, intraabdominal. At puberty, partial virilization and gynecomastia develop (186). Recently, Danilovic et al have been demonstrated that subjects with AIS had mean final height intermediate between mean normal male and female and decreased bone mineral density in the lumbar spine suggesting an important role for androgens in normal growth and bone density (194).

Serum LH levels are in the upper normal range or slightly elevated and testosterone levels are normal or also slightly elevated. Testosterone precursors are not increased in relation to testosterone. The testosterone/DHT ratio is not as high as in patients with 5 alpha-reductase type 2 deficiency or CAIS, but may be slightly higher than the normal population due to a secondary 5 alpha-reductase 2 deficiency. A definitive molecular diagnosis of PAIS is established by the identification of mutations in the AR gene of patients with PAIS.

Remarkable variations in the phenotypes of 3 patients within the same kindred due to M780I mutation, two female with CAIS and one male with PAIS (perineoscrotal hypospadias was reported (195). These distinct phenotypic variation can be attributed to differences in the availability of 5α-RD2 in activity in genital skin fibroblasts (196).

Patients with CAIS were raised as females and maintained female sex. Most of the patients with PAIS who were raised as females maintained a female social sex after postpubertal age, despite clitoral growth and partial virilization. In our experience all 5 cases with PAIS kept the female social sex. This is in distinct contrast to some other forms of 46,XY DSD such as 5α-reductase 2 deficiency and 17-hydroxysteroid dehydrogenase III deficiency in which several affected 46,XY individuals raised as females undergo a change to male social sex at puberty (14, 15). The impairment of androgen action in subjects with PAIS is probably similar during embryogenesis and puberty, whereas the action of androgens is stronger at puberty in subjects with enzymatic defects, because of alternate pathways and maturation of isoenzymes. There is an overlap in phallus length at the time of diagnosis in our postpubertal PAIS subjects with female and male social sex, suggesting that sex assignment at birth and sex of rearing were more important than phallus size for development of gender identity (47). In other study, either male or female sex of rearing lead to successful long-term outcome for the majority of the 39 subjects with 46,XY DSD, 14 of them with PAIS, 5 living as men and 9 as women (197).

PERSISTENT MÜLLERIAN DUCT SYNDROME

Defect in AMH synthesis

Defect in AMH receptor

The development of female internal genitalia in a male individual is due to the incapacity of Sertoli cells to synthesize or secrete anti-mullerian hormone (AMH) or to alterations in the hormone receptor. Persistent Müllerian duct syndrome (PMDS) phenotype can be produced by a mutation in the gene encoding anti-Müllerian hormone or by a mutation in the AMH receptor. These two forms result in the same phenotype and are referred to as type I and type II, respectively (198).

AMH is a 145,000 MW glycoprotein homodimer produced by Sertoli cells not only during the period when it is responsible for regression of the Müllerian ducts but also in late pregnancy, after birth, and even, albeit at a much reduced rate, in adulthood (198, 199). AMH is a small gene containing 5 exons, located in chromosome19p.13.3 (200) and its protein product acts though its specific receptor type 2 (AMHR2) a serine/threonine kinase, member of the family of type II receptors for TGF--related proteins (201).

Affected patients present a male phenotype, usually along with bilateral cryptorchidism and inguinal hernia (202). Leydig cell function is preserved, but azoospermia is common due to the malformation of ductus deferens or agenesis of epididymis. When the hernia is surgically corrected, the presence of a uterus, fallopian tubes and superior part of the vagina can be verified.

PMDS is a hetorogeneous disorder that is inherited in a sex-limited autossomal recessive manner. Mutations in AMH gene or AMH receptor 2 gene in similar proportions, are the cause of approximately 85% of the cases of PMDS (203, 204). In the remainig cases the cause of the persistent Mullerian duct syndrome is unknown (205).

Normally, AMH levels are measurable during childhood and decrease at puberty. Patients with AMH gene defects have low AMH levels since birth whereas patients with mutations in AMH receptor gene have elevated AMH levels.

Treatment is directed toward an attempt to assure fertility in males. Early orchiopecxy, proximal salpingectomy (preserving the epididymides), a complete hysterectomy with dissection of the vas deferens from the lateral walls of the uterus are indicated (206).

Congenital NON-GENETIC 46,XY DSD

Maternal intake of endocrine disruptors

The use of synthetic progesterone or its analogs during the gestational period has been implicated in the etiology of 46,XY DSD (207). Some hypothesis have been proposed to explain the effect of progesterone in the development of male external genitalia, such as reduction of testosterone synthesis by the fetal testes or a decrease in the conversion of testosterone into DHT due to competition with progesterone (also a substrate for 5-reductase 2 action). The effect of estrogen use during gestation in the etiology of 46,XY DSD has not been confirmed to date (208). Recently, a study in Japanese subjects support the hypothesis that homozygosity for the specific estrogen receptor alpha 'AGATA' haplotype may increase the susceptibility to the development of male genital abnormalities in response to estrogenic effects of environmental endocrine disruptors (209).

Daily exposure to a residues of a fungicide (vinclozolin), either alone or in association with a phytoestrogen genistein (present in soy products), induce hypospadias in 41% of mice, supporting the idea that exposure to environmental endocrine disruptors during gestation could contribute to the development of hypospadias (210).

Congenital non-genetic 46,XY DSD associated to impaired prenatal growth

Despite the multiple genetic causes of 46,XY DSD, around 30-40% of cases remain without diagnosis. Currently, there is a frequent, non-genetic variant of 46,XY DSD characterized by reduced prenatal growth and lack of evidence for any associated malformation or endocrinopathy (211, 212). Using the model of monozygotic twins, hypospadias has now been linked to low birth weight (211). We have identified a pair of monozygotic twins (46,XY; identical for 13 informative DNA loci) born at term after an uneventful pregnancy sustained by one placenta who were discordant for genital development (perineal hypospadias versus normal male genitalia) and postnatal growth (low birth weight versus normal birth weight). No evidence for uniparental dissomy was found (213). The most plausible cause of incomplete male differentiation associated with early-onset growth failure is a post-zygotic, micro-environmental factor since different DNA methylation patterns associated with silencing of genes important do sex differentiation has been shown.(214).

Additionally, three cohorts of undetermined 46,XY DSD report around 30% of cases as associated with low birth weight, indicating that adverse events in early pregnancy are frequent causes of congenital non-genetic 46,XY DSD (215-217).

46,XY ovotesticular DSD

There are rare decriptions of 46,XY DSD patients with well characterized ovarian tissue with primordial follicles and testicular tissue, a condition that histologically characterized 46, XY ovotesticular DSD. The diferential diagnosis of 46,XY ovotesticular DSD with partial 46,XY gonadal dysgenesis should be performed considering that in the latter condition there are descriptions of dysgenetic testes with disorganized seminiferous tubules and ovarian stroma with occasional primordial follicles in the first years of life (218). To our knowledge there are no descriptions of an adult patient with 46,XY ovotesticular DSD with functioning ovarian tissue, as occurs in all 46,XX ovotesticular DSD. Therefore the diagnosis of 46,XY ovotesticular DSD is debatable.

Non-Classified Forms

Hypospadias

Hypospadias is one of the most frequent genital malformation in the male newborn and 40% of the cases are associated with other defects of the urogenital system. Hypospadias results from an abnormal penile and urethral development that appears to be a consequence of various mechanisms including genetic and environmental factors. It is usually a sporadic phenomenon, but familial cases can be observed, with several affected members.

The presence of hypospadias indicates an intrauterus interference in the correct genetic programme of the complex tissue interactions and hormonal action through enzymatic activities or transduction signals. More recently, the mastermind-like domain-containing 1 gene (MAMLD1 or CXorf6).) have been reported to be involved in the etiology of hypospadias (219).

The activating transcription factor 3 (ATF3) expression was evidenced in the developing male urethra. Apparently ATF3 variants may influence the risk of hypospadias (220).

By definition, hypospadias is a form of 46,XY DSD and although most of the patients present fertility and masculinization at puberty, their testicular function should be assessed to rule out causes such as defects in testosterone synthesis and action, which require hormonal treatment and genetic counseling in addition to surgical treatment.

46,XY gender Identity disorders

Male to female transsexualism

Male to female transsexualism is characterized by the wish to live as member of the female sex with conviction and consistently and progressively works to achieve such state. 46,XY gender identity disorders is more frequent among the male sex, although it also occurs in the female sex. Its first manifestations usually start at childhood period. Its etiology remains unknown, although some hormonal alterations during intrauterus life and familial factors before and after the birth cannot be ruled out (221).

Management of patients with 46,XY DSD

It is important to stress that the treatment of 46,XY DSD patients requires an appropriately trained multi-disciplinary team. Early diagnosis is important for good outcome of the patients and should start with a careful examination of the newborn’s genitalia at birth (5, 222).

Psychological Evaluation: It is of crucial importance to treat DSD patients. Every couple that has a child with ambiguous genitalia must be assessed and receive counseling by an experienced psychologist, specialized in gender identity, who must be act as soon as the diagnosis is suspected, and them follow the family periodically, more frequently during the periods before and after genitoplasty (223, 224) .

Parents must be well informed by the physician and psychologist about normal sexual development. A simple, detailed and comprehensive explanation about what to expect regarding integration in social life, sexual activity, necessity of hormonal and surgical treatment and the possibility or not of fertility according to the sex of rearing, should also be discussed with the parents, before the attainment of final social sex.

The determination of social sex must take into account the ethiological diagnosis, penis size, ethnic traditions, sexual identity and the acceptance of the assigned social sex by the parents. In case parents and health care providers disagree over the sex of rearing, the parents’ choice must be respected. The affected child and his/her family must be followed throughout life to ascertain the patient’s adjustment to his/her social sex.

Hormonal Therapy

Female social sex: The purpose of the hormonal therapy is the development of female sexual characteristics and menses in the patients with uterus. The treatment must simulate normal puberty, by introducing low doses estrogen of at 9-11 years to avoid excessive bone maturation in short children. Estrogen therapy should be initiated at a low dose (one sixth to one quarter of the adult dose) and increased gradually at intervals of 6 months. Doses can then be adjusted to the response (Tanner stage, bone age), with the aim of completing feminization gradually over a period of 2–3 yr. In tall 46,XY females, adult estrogen dosage is recommended to avoid high final stature. The initial dosage of conjugate estrogens (0.07 to 0.15 mg/day orally) or oral or topic 17β-estradiol (0.5 mg daily) is kept as the patient presents progressive breast development. If breast development is not progressive, the estrogen dose is double. Low-dose transdermal hormone therapy is also a viable alternative estrogen replacement, offering lipid protection and preserve bone mass. After the breast development is complete, the estrogen dose is maintained at (0.625 mg/day of conjugate estrogen) or 1 mg twice a day of oral or topic 17-estradiol) continuously and medroxyprogesterone acetate (5 to 10 mg/day) or micronized progesterone 50 mg/day, from the 1st to the 12th day of the month) is added to induce menses. In patients without uterus only estrogen is indicated. The dilation of the blind vaginal pouch with acrylic molds (225) or surgical neovagina promote development of a vagina adequate for sexual intercourse after 6-10 months of treatment whem patients desire to initiate sexual activity (226) .
Male social sex: Testosterone replacement is started between 10 and 11 yrs, simulating normal puberty according to the child’s psychological evaluation and height. Intramuscular depot injections of testosterone esters are commonly used; another option is oral testosterone undecanoate and transdermal preparations.(227) The initial dose of depot injections of testosterone esters is 25 to 50 mg/month administered IM. The maintenance dose in an adult patient is 200 to 250 mg every 2 weeks or 1000 mg each 3 months. In male patients with androgen insensitivity, higher doses of testosterone esters (250-500 mg twice a week) are used to increase penis size and male secondary characteristics. Maximum penis enlargement is obtained after 6 months of high doses and after that, the normal dosage is re-instituted (176, 178).

The use of topic DHT gel is also useful to increase penis size with the advantage of not causing gynecomastia and promoting faster increase of penis size as it is 50 times more active than testosterone. Considering that DHT is not aromatized, one would expect it to have no effect on bone maturation, allowing the use of higher doses than testosterone and consequently attaining a higher degree of virilization.

Surgical Treatment

The aim of the surgical treatment is to allow development of adequate external genitalia and removal internal structures that are inappropriate for the social sex. Patients must undergo surgical treatment preferably before 2 years of age, which is the time when the child becomes aware of his/her genitals and social sex. Only skilled surgeons with specific training in the surgery of DSD should perform these procedures (3, 4)

Laparoscopy is the ideal method of surgical treatment of the internal genital organs in patients with 46,XY DSD (228). In these patients, the indications for laparoscopy are the removal of normal gonads and ductal structures that are contrary to the assigned gender and the removal of dysgenetic gonads which are nonfunctional and present potential for malignancy. In addition to being a minimally invasive surgery, one of the main advantages of this method is the lack of scars.

The aims of surgical treatment are to adequate external genitalia and remove internal structures that are inadequate for the social sex. Patients must undergo surgical treatment preferably before 2 year of age, which is the time when the child becomes aware of his/her genitals and social sex. Only skilled surgeons with specific training in the surgery of DSD should perform these surgeries (3, 4).

For those 46,XY DSD children been assigned female, laparoscopy is the ideal method for performing gonadectomy and for resection of internal organs if appropriate (228).

Feminizing genitoplasty should provide an adequate vaginal opening into the perineum, create a normal-looking vaginal introitus, fully separate the urethral from the vaginal orifice, remove phallic erectile tissue preserving glandular enervation and blood supply, and prevent urinary tract complications (229). The most reasonable procedure to perform clitoroplasty is based on the concept of maintaining the clitoral glans and sensory input, which facilitates orgasm. The use of an adequate size of tissue flap is mandatory in Y-V vaginoplasty, to avoid introital stenosis. Failure to interpose an adequate flap will result in persistent introital stenosis, requiring later revision. Vaginal dilation with acrylic molds in patients with introitus stenosis showed to be a good treatment choice when these patients wished to start sexual intercourse, resulting in good outcomes (225). In our experience, the single-stage feminizing genitoplasty consisting of clitoroplasty with the preservation of dorsal nerves and vessels and ventral mucosa, vulvoplasty and Y-V perineal flap, followed by vaginal dilation with acrylic molds, allowed good cosmetic and functional results (229).

For those raised as males, surgery consists in orthophaloplasty, scrotumplasty with resection of vaginal pouch, proximal and distal urethroplasty and orchidopexy when necessary. Surgeries were performed in 2 or 3 steps in the patients with perineal hypospadias. The most frequent complication is urethral fistula in the penoscrotal angle and urethral stenosis that can occur several years after surgery. The results of surgical correction are good, from both the aesthetical and functional points of view in our series as well as in others (3, 4, 197, 230).

Most of our patients present satisfactory sexual performance as long as they present a penis size of at least 6 cm. New approaches, such as the use of donor-grafting tissue to elongate the urethra and penis may help these patients in the future.

Dysgenetic or undescended gonads and tumor development

Specific variants of DSD (especially in patients with gonadal dysgenesis and hypovirilization) have a significant risk factor for type II germ cell tumors besides cryptorchidism, familial predisposition and birth weight.

A high risk of gonadoblastoma is found when sex determination is disrupted in an early stage of Sertoli cell differentiation (due to abnormalities in SRY, WT1, SOX9) Early Sertoli cell development is also disturbed patients with 45X/46,XY mosaicism. The same is true to for patients with 9p deletions, likely related to the loss of DMRT1. It must be remembered that GB can only be formed in the presence of GBY region of the Y chromosome. GB is found in patients that lack a certain level of Sertoli cell development. Careful histological analysis of gonadal tissue of DSD patients revealed that undifferentiated gonadal tissue (UGT) of DSD is the most likely precursor stage of GB.

Defects occurring later in gonadal development , like 17b –HSD insufficiency and AR mutants (predominantly PAIS) results in enhanced risk of CIS as precursor as can be found in males without any form of DSD albeit with a much lower incidence

Neoplastic transformation of germ cells in dysgenetic gonads (gonadoblastomas and/or an invasive germ cell tumor) occurs in 20-30% of 46,XY DSD patients (231) and is associated with the presence of Y chromosome or part of it. The presence of a well-defined part of the Y chromosome, known as the gonadoblastoma Y locus (GBY), is a prerequisite for malignant transformation. Among the genes located on GBY region the testis-specific protein Y (TSPY) seems to be the most significant candidate gene for tumor-promoting process (231).

Recently, the presence of undifferentiated gonadal tissue containing germ cells, that abundantly express TSPY and OCT4 has also been identified as a gonadal differentiation pattern bearing a high risk for the development of gonadoblastoma (231).

Spontaneous breast development suggests the presence of an estrogen-secreting tumor (gonadoblastomas). Bilateral gonadectomy should be performed in 46,XY patients before pubertal age to avoid degeneration of dysgenetic tissue, unless the gonad is functional and easily accessible to palpation and imaging studies, which should be performed yearly. A gonadal biopsy showing the presence of undifferentiated gonadal tissue or testicular tissue with OCT4-positive cells on the basal lamina suggests a high risk for germ cells tumors whereas testicular tissue displaying maturation delay of germ cells and stroma ovarian tissue can be safely be left in situ (231).

The risk for germ cell tumors is increased in patients with undescended testes, including all other 46,XY DSD syndromes (231). Although data are limited, in the androgen insensibility syndrome the risk seems to be markedly higher in the partial form than in the complete form and tumor prevalence in AIS is markedly increased after puberty. On the other hand, series reporting other causes of 46,XY undervirilized patients and gonadal tumors are too small and do not allow any conclusions.

The use of a uniform classification system of the various forms of DSD will hopefully shed light on the actual risk for malignant transformation of germ cells in the different DSD subgroups, which might result in a more conservative approach of gonadectomy in some patients. The benefits may include physiological induction of puberty and even fertility.

Fertility in patients with 46,XY DSD

Infertility is almost always present in 46,XY DSD patients due to impaired spermatogenesis secondary to gonadal dysgenesis, testosterone deficiency or action, cryptorchidism or retrograde ejaculation, frequently found in patients with perineal hypospadias. Currently, in vitro fertilization techniques has enabled 46,XY DSD patients to produce offspring.(178, 232). Successful pregnancy and delivery following in vitro fertilization using donor oocytes and embryo transfer in a patient with complete 46,XY gonadal dysgenesis was reported (233).