1955 Grumbach et al., originally introduced the term gonadal dysgenesis to designate vestigial gonads of patients with Turner syndrome (34). Currently it is used to describe a variety of clinical conditions in which the development of fetal gonad is abnormal. 46,XY gonadal dysgenesis encompasses both complete (pure) and a partial (mixed) forms. These two forms are well-characterized clinical forms whereas an unusual syndrome called embryonic testicular regression (ETRS) has been considered part of the clinical spectrum of 46,XY gonadal dysgenesis (35). In this syndrome, most of the patients present ambiguous genitalia and the degree of masculinization of the internal and external genitalia is a consequence of the duration of testicular function prior to its loss. The abnormal pattern of sex duct development in these subjects suggests that the gonadal tissue was intrinsically altered before the testicular regression took place. The presence of gonadal regression in more than one member of the same family, suggests a genetic basis for this disease (36).

The complete form of gonadal dysgenesis was first described by Swyer et al. (37) 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. Three patterns of inheritance have been described in Swyer syndrome: X-linked (38) sex-limited autosomal dominant (39) and autosomical recessive inheritance (40).

The partial form of this syndrome is characterized by partial testis development that results in patients with ambiguous external and internal genitalia.

Hormonal diagnosis: Serum gonadotropin levels are elevated in either the complete or the 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 do not reach male pubertal levels.

Etiology: 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 XY females with complete gonadal dysgenesis (41-43). In the partial form, the frequency of SRY mutation is even lower than in the complete form. Most of the mutations described in SRY are predominantly de novo mutations. However, some cases of fertile fathers and their XY children sharing the same altered SRY sequence have been reported (44, 45). In a few of these cases, the father's somatic mosaicism for the normal and mutant SRY gene have been demonstrated (46). In the other cases with inherited SRY mutations, the mechanism of sex reversal remains unclear (47).

Therefore, the great majority of gonadal determination disorders cannot be explained solely through SRY mutations. Patients without SRY alterations probably present mutations in the other genes involved in sex determination cascade. Recent data have shown that mutations in X chromosome and autosomal loci are also associated with sex reversal (48).

The existence of an X-specific gene involved in human sex determination was first postulated based on the history of a family with X-linked 46,XY gonadal dysgenesis (49). Male patients with female or ambiguous external and internal genitalia due to partial duplications of Xp and an intact SRY gene have also been described (48). These patients present with dysgenetic or absent gonads associated with, or without mental retardation, cleft palate and dysmorphic face. Bardoni et al. identified in these patients, a 160-kb region of Xp21 named dosage sensitive sex (DSS) locus which, when duplicated, resulted in male-to-female sex reversal (48). Transgenic mouse models for the study of the mammalian sex differentiation cascade showed that Dax1 functions as an anti-testis gene by acting antagonistically to Sry (2). These facts suggest that the dosage-sensitive sex reversal may be due to the duplication of DAX1. However, until now, there has not been a description of isolated DAX-1 duplication causing 46,XY sex reversal in humans suggesting that other genes, present in the DSS locus, should be involved in dosage-sensitive sex reversal. Although, DAX1 was considered an anti-testis gene, recent evidence of XY sex reversal in Dax1-deficient mice strongly supports a role for Dax1 as a pro-testis gene. Patients with deletions or mutations in the DAX1 presented adrenal hypoplasia congenita (AHC) and normal male genitalia. Although AHC patients develop testes, they present gonadal defects that include disorganized testis cords and hypogonadotropic hypogonadism similar to Dax-1 deficient mice (50).

The spectrum of genital anomalies resulting from interruption of testicular function between 8 and 14 weeks of gestation have been designated as XY gonadal agenesis, XY gonadal dysgenesis or rudimentary testes syndrome (51). These subjects have either no gonads or streak gonads (52) associated with variable degrees of genital ducts, urogenital sinus, and external genitalia differentiation. Familial occurrence has been noted with variable degrees of sexual ambiguity (one patient with micropenis and the other a phenotypic female with slight fusion of the genital folds and absent Müllerian ducts were observed (35). The report of several kindreds with multiple affected individuals suggests a genetic disorder but the nature of the underlying defect is still unknown.

Total absence of gonadal tissue or gonadal streak confirmed by laparoscopy has rarely been described in XY subjects with female external and internal genitalia suggesting the absence of testicular determination (53). Mendonca et al. described a pair of siblings, one XY and the other XX, born to a consanguineous marriage, both with gonadal agenesis and normal female external and internal genitalia (54). The origin of this disorder remains to be determined but a defect in a gene essential for gonadal determination in both sexes is the most likely cause of this disorder.

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 (55) The SF1 gene is considered an orphan nuclear receptor as its natural ligand remains unidentified. 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 (8, 56). SF1 expression is required at three different moments during testicular development: in the bipotential gonad prior to its determination, in the Sertoli cells to regulate AMH expression and later in Leydig cells to regulate the production of steroid hormones (5). Thus, this gene plays a role in sex determination and differentiation. Disruption of the Sf1 gene in mice causes adrenal and gonadal agenesis, XY sex reversal, persistence of Müllerian structures in males, near absence of the VMH hypothalamus and decreased levels of pituitary gonadotropins (8).

The first reported human case of SF1 mutation, the heterozygous G35E in the DNA binding domain, was a 46,XY patient who presented female external genitalia and Müllerian duct derivatives This indicated the absence of male gonadal development, associated with adrenal insufficiency. The clinical features were similar to the knockout mice, although a heterozygous mutation was detected. These data suggest a loss-of-function effect, in which the absence of one allele of SF1 in human is enough to cause a severe clinical phenotype (57).

Remarkably, the SF1 mutation (R92Q) in a highly conserved residue of the A-box, a region that functions as a secondary DNA-binding domain, was described in homozygous state in a 46,XY baby with female external and internal genitalia who presented primary adrenal failure. Analysis of the family showed that the normal first-cousin parents and one sister were heterozygous for this mutation indicating an autosomal recessive mode of inheritance for this mutation. In functional assays, the R92Q mutant exhibited partial loss of DNA binding and transcriptional activity when compared with the G35E mutation. This second mutation reinforced the dose-dependent action of SF1, affecting only subjects with both compromised alleles (58). We identified a heterozygous frameshift mutation resulting from the deletion of 8 nucleotides at position 2783 of SF1 gene in a 46,XY 31-year-old female patient with clitoromegaly, absence of Müllerian derivatives and gonadal tissue and normal adrenal function (59). This mutation leads to a premature termination at codon 378 resulting in a carboxy-terminal truncated SF1 protein without AF-2 motif, which is essential for transcriptional activation (60). In transfection experiments, the mutated protein possessed no intrinsic transcriptional activity but rather inhibited the function of the wild-type protein in most cell types. This was the first example of an apparent dominant negative effect of an SF1 mutation in humans. This is the first described patient with an SF1 inactivating mutation with normal adrenal function, suggesting that SF-1 transcription might have tissue-specific effects in humans (59).

Two others cases with SF1 mutations in heterozygous state were described in 46,XY patients with testicular dysgenesis and normal adrenal function. The nonsense mutation C16X, in exon 2, was reported in a 46,XY subject with gonadal dysgenesis without adrenal insufficiency, causing premature termination of translation (61). This study suggests that a heterozygous SF1 mutation impairs the adrenal function only if both the gene dosage and dominant negative effects occur.

The heterozygous single base pair deletion at exon 2, 18delC, causing a frameshift at the 6th codon and resulting in a termination at the 74th codon, was reported in a 46,XY patient with small dysgenetic testes, no mullerian derivatives and clitoromegaly. The function studies revealed no transcriptional activity of the mutated SF1 protein or dominant negative effect. These results suggest that SF1 haploinsufficiency selectively impairs testicular development, although the AMH and testosterone biosynthesis by dysgenetic testes and gonadotropins production are partially sustained (62).

In practical terms, these recent reports indicate that SF1 mutations should be considered in patients with XY gonadal dysgenesis with or without adrenal insufficiency (63). These cases highlight how different mutations in DNA-interacting regions of SF1 can produce distinct functional effects and variable phenotypic penetrance, thus emphasizing the importance of gene dosage and residual function of SF1 as a determinant of clinical phenotype.

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 which act as extracellular signaling factors (64). Six new members of the Wnt gene family in mice, including Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a and Wnt7b were identified (65). They present a homology and share between 50% and 85% amino acid identity and contain 83 absolutely conserved amino acid residues, including 21 cysteines.

Overexpression of WNT4 may be a cause of XY sex reversal. A 46,XY new born infant with multiple congenital anomalies including bilateral cleft of lips and palate, intrauterine growth retardation, microcephaly, tetralogy of Fallot, ambiguous external genitalia and the presence of female and male internal genitalia carrying a duplication of 1p31-p35 was reported (66). Latterly, it was demonstrated that this duplication in tandem includes the WnT4 gene (67). In vitro studies suggest that Wnt4 up-regulates Dax1, a gene known to antagonize Sry, in testicular Sertoli and Leydig cells, so this XY sex reversal may result from an excess of Dax-1 expression (67). Thus, WnT4, a novel sex-determining gene, and DAX-1 play a shared role in the prevention of testis formation with the consequent female gonadal development. It seems that Wnt4 is involved in Müllerian ducts formation and in the suppression of Leydig cell formation in the developing gonad.

In the mouse, mutant Wnt-4 XX females are masculinized (persistence of Wolffian structures and no Müllerian ducts) while in the males the phenotype appears to be normal (68). A heterozygous mutation in the WNT4 was identified in a woman with features of Mayer-Rokitansky-Kuster-Hauser syndrome; this finding suggests a role of this gene in the development and maintenance of the female phenotype (69). On other hand, analysis of XY gonads from Wnt4 mutant embryos revealed a defect in early testis development. Sertoli cell differentiation was compromised in Wnt4 mutant testes. So, Wnt4 has also been implicated in initial testis development (70). These observations suggested that mammalian sex determination is sensitive to dosage at multiple steps in its pathway.

Raymond et al. identified both the DNA-binding Motif (DM) domain genes expressed in testis (DMRT1 and DMRT2) mapped to chromosome 9p24.3, a region of the genome associated with gonadal dysgenesis and XY sex reversal (17, 71, 72). The clinical and molecular findings of five XY males and one XX female with distal 9p monosomy were reported (73). The human 9p deletion syndrome is characterized by varying degrees of XY sex reversal, mental retardation and craniofacial abnormalities. However, smaller deletions may be associated with isolated sex reversal. The phenotype of male patients ranged from female genitalia to male external genitalia. Phenotypic examples include cryptorchidism associated with agonadism, streak gonads or hypoplastic testes and internal genitalia disclosing normal Müllerian or Wolffian ducts.

Endocrine studies showed from hypergonadotropic hypogonadism to almost normal testicular function. FISH and microsatellite analyses demonstrated that all cases had hemizygosity of the 9p sex-determining region with loss of DMRT1 and DMRT2 genes and normal SRY sequence. The authors inferred that haploinsufficiency of the 9p sex-determining gene(s) primarily impedes the formation of indifferent gonads, leading to various degrees of defective testis formation in males and of defective ovary formation in females (73). However, Ounap et al. have recently suggested that the 9p distal deletion, including DMTR1 and DMTR2 genes, can cause XY true hermaphroditism and normal puberty development in XX female (74).

Stayton et al. described the cloning and characterization of a new gene provisionally called X-linked helicase-2 (XH2), and later named ATR-X located in Xq13 (75). The gene undergoes X inactivation and encodes a putative NTP-binding nuclear protein homologous to several members of the helicase II superfamily. XH2 shares 6 conserved, collinear domains with other members of the family of proven and putative helicases. Type II helicases have been implicated in nucleotide excision repair and initiation of transcription.

The phenotype of this syndrome is characterized by severe psychomotor retardation, alpha-thalassemia, characteristic facial features (telecanthus, epicanthic folds, flat nasal bridge, midface hypoplasia, small triangular nose with anteverted nostrils, a carp-shaped mouth with full lips and a progressive coarsening of the facial appearance), and genital abnormalities (76, 77). All developmental milestones, especially walking, are delayed and speech is almost absent. The hematological findings may vary widely and in some cases the manifestation of alpha-thalassemia may be subtle or missed (77). Genital anomalies that lead to a female sex rearing were reported in several affected 46,XY patients with ATR-X syndrome (78). 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 (77).

Mutations in the ATR-X gene give rise to changes in the methylation pattern of several highly repeated sequences. In normal individuals, approximately 20% of rDNA repeats were methylated within most CpG-rich regions whereas in ATR-X patients, rDNA genes were substantially unmethylated (77). On the other hand, approximately 6% of DYZ2 repeats were unmethylated on the Y chromosomes of normal individuals, but almost all were methylated in ATR-X patients (77).

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 is characterized by Wilm´s tumor, aniridia, genitourinary abnormalities and mental retardation. The genitourinary anomalies are renal agenesis or horseshoe kidney, urethral atresia, hypospadias and cryptorchidism (79). Heterozygous deletions of WT1 and contiguous genes are the cause of this syndrome (80).

Denys-Drash syndrome (DDS) is characterized by dysgenetic MPH associated with early-onset renal failure (diffuse mesangial sclerosis) and Wilm´s tumor developed in the first decade of life (81). Affected males usually present ambiguous genitalia and dysgenetic gonads. Müllerian ducts differentiation varies according to the Sertoli cells function. The molecular defect of DDS is the presence of heterozygous missense mutations in the zinc finger encoding exons (DNA-binding domain) of WT1 gene (82). These mutant forms of WT1 act as dominant negatives. As a result, WT1 levels in cells are probably reduced below 50% since WT1 protein is able to dimerize, resulting in non-functional homo- and heterodimers of mutant WT1 protein (83). Kidney function is severely impaired due to diffuse mesangial sclerosis, leading to nephrotic syndrome and kidney failure within the first 2 or 3 years of life (81). Gonadal development is impaired in variable degrees, resulting in a spectrum of male pseudohermaphroditism (84).

Frasier syndrome (FS): is characterized by female external genitalia in 46,XY patients, renal failure in the second decade of life, streak gonads and high risk of gonadoblastoma development. Normally, WT1 is spliced alternatively, resulting in a shorter isoform (-KTS), and in a longer isoform containing the additional C-terminal lysine, treonine and serine aminoacids. Constitutional heterozygous mutations of the WT1 gene are almost all located at intron 9. These mutations 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 (81, 85). This data suggests that a precise balance between WT1 isoforms is necessary for normal function of WT1 (86). Frasier syndrome is usually associated with IVS9 + 4C>T mutation (87) although exonic mutations also cause a Frasier syndrome (88). 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 (89). Our case provides new data concerning the heterogeneity of phenotypes associated with WT1 mutations by including predominantly male ambiguous genitalia and absence of gonadal dysgenesis, which is associated with the presence of a para-testicular leiomyoma, bilateral gonadoblastoma, germ cell neoplasia and delayed adrenarche, thus expanding the spectrum of the phenotypes associated with this syndrome (89). The report of ambiguous external genitalia in 4 patients (88), the presence of Wilms' tumor in one patient (90), and the description of exonic mutations in the DNA binding domain of WT1 gene (88) in patients with FS indicate an overlap of clinical and molecular features in DDS and FS suggesting that they are not distinct diseases but may represent two ends of a spectrum of disorders caused by alterations in WT1 gene and that genotype-phenotype correlation is not always possible.

This syndrome is characterized by severe skeletal malformations (campomelic dysplasia) associated with dysgenetic MPH, which occurs in three-quarters of the affected 46,XY patients. The phenotype includes macrocephaly, micrognathia, hypertelorism, hypoplastic scapula, 11 pairs of ribs in a small thoracic cage, bowed long bones, a deformed pelvis, a variety of cardiac and renal defects (91). Death from respiratory distress usually occurs in the neonatal age, although long-term survival has been reported (92). The external genitalia varies from that of normal males with cryptorchidism through ambiguous to female and internal genitalia can include vagina, uterus, and fallopian tubes (93). SRY-related HMG-box gene 9 (SOX9) is a transcription factor involved in chondrogenesis and sex determination. SOX9 gene, located on human chromosome 17, like SRY, is a highly conserved HMG family member and it is also implicated in the sex-determination pathway (94, 95). Mutations in a single allele of SOX9 are responsible for XY sex reversal and campomelic dysplasia (94). In all subjects, SOX9 mutation was identified in the heterozygous state indicating that this disorder is due to haploinsufficiency of SOX9 gene (94). SOX9 duplications in humans can result in XX males in the absence of SRY (96).

One of the candidate genes involved in the testis-determining pathway is Desert hedgehog (Dhh), a member of the hedgehog family of signaling proteins, located in 12q12-q13.1 (97).

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 (98). 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, suggesting that the localization of mutations influences the phenotype (99).

In this group of patients, ambiguous genitalia was found in the majority of the affected subjects. The degree of masculinization (of the internal and external genitalia) is considered to be a consequence of the amount of cells without the Y chromosome. In most of the cases, the patients present asymmetrical gonadal development, generally associated with asymmetrical Müllerian derivative development associated with Turner syndrome stigmata. They also present hypergonadtropic hypogonadism with elevated FSH and LH levels and low testosterone levels (34). Structural abnormalities of the Y chromosome affecting SRY gene, located at its short arm, also results in gonadal dysgenesis associated with Turner somatic stigmata (34).

A high incidence of neoplasia (gonadoblastomas and germinomas) in dysgenetic gonads of patients with the XY karyotype has been described (34). Comparative studies of the frequency of gonadoblastoma in Turner mosaics with normal or rearranged Y chromosomes have suggested that the integrity of the Y chromosome, and in particular the presence of the distal fluorescent band Yqh, is required for the tumor to develop. No cases with distal deletions of the fluorescent band on Yq and gonadal tumors have been reported (100). More recently, the existence of a gene in chromosome Y (GBY gene) has been postulated, which would predispose dysgenetic gonads to undergo malignancy. The study of patients harboring a gonadoblastoma suggests that GYB gene is located near the centromeric region of the long arm of chromosome Y (101). Spontaneous breast development suggests the presence of an estrogen-secreting tumor (gonadoblastomas).

Additionally, Slowikowska-Hilczer et al. suggest that the incidence of neoplastic lesions is increased in less disturbed testicular organogenesis. This suggests that the testicular environment of a dysgenetic gonad plays an important role in germ cell neoplasia initiation, maintenance, or both (102).

Bilateral gonadectomy should be performed in XY patients at pubertal age to avoid degeneration of dysgenetic tissue, unless the gonad is functional and easily accessible to palpation and imaging studies, which should be conducted yearly.

In male pseudohermaphroditism due to Leydig cell hypoplasia, there is failure of intrauterine and pubertal virilization due to the inability of interstitial Leydig cells to secrete testosterone precursors and testosterone. Normally, testosterone secretion occurs because of stimulation of Leydig cells by hCG by the 9th week gestation, and by LH after the 15th week. Testosterone secretion in the first trimester and in the remaining trimesters of gestation is important for the masculinization of the external genitalia and Wollfian ducts for growth of male genitalia. Regression of Müllerian ducts depends on the secretion of anti-Müllerian hormone by the Sertoli cells. Since Sertoli cell function is not affected in Leydig cell hypoplasia, Müllerian ducts undergo involution as in normal males.

Both hCG and LH act by stimulating a common seven transmembrane LH receptor, a G-protein coupled receptor. Leydig cell hypoplasia can result from 1) an absence of mesenchymal precursor cells destined to become mature Leydig cells or 2) a defect in the CG/LH receptor in these cells.

In 1976, Berthezene et al. (103) described the first patients with Leydig cell hypoplasia and subsequently more cases have been reported, (104-107). The study of 8 of our cases and review of the literature has allowed us to delineate the characteristics of male pseudohermaphroditism due to Leydig cell hypoplasia: 1) a predominantly female external genitalia, leading to female sex assignment, 2) no development of either male or female sexual characteristics at puberty, 3) presence of rudimentary epidydimis and vas deferens and absence of uterus and fallopian tubes, 4) 46,XY karyotype, 5) low testosterone levels despite elevated LH values, 6) testicular unresponsiveness to hCG stimulation, 7) no abnormal step up in testosterone biosynthesis precursors, and 8) only slightly smaller than normal undescended testes with relatively preserved seminiferous tubules and absence of mature Leydig cells (108-111). Whereas complete Leydig cell failure leads to a female phenotype, a partial failure could lead to partial virilization. In fact, Toledo et al. (112) reported 3 brothers, born to consanguineous parents, who had male genitalia with micropenis, testes of normal adult size and variable degrees of hypogonadism. Gonadotropin levels were elevated and testosterone levels were intermediate between prepubertal and adult male levels. The testosterone response to hCG was subnormal. A testicular biopsy revealed arrest of spermatogenesis and rare Leydig cells (112)

In 1990, the human CG/LH receptor gene was cloned and sequenced and its involvement in Leydig cell hypoplasia could be investigated (113). The CG/LH receptor gene contains 11 exons and 10 introns. The first 10 exons encode the extracellular domain and exon 11 encodes the transmembrane and intracellular domains of the receptor (114). In 1995, Kremer et al. (115) studied DNA from 2 Brazilian 46,XY sisters with Leydig cell hypoplasia and identified a missense mutation (Ala593Pro) in the sixth transmembrane helix of the LH receptor. Transfection of the mutated LH receptor gene in human embryonic kidney 293 cells resulted in diminished expression of LH receptors in the cell surface and binding of 131I-hCG, which occurred with a normal affinity. When stimulated by hCG these cells were unable to produce cyclic AMP, when compared to cells transfected with the wild type. Subsequently, several different mutations in the LH receptor gene were reported in patients with Leydig cell hypoplasia (116). Latronico et al. (117) reported a homozygous mutation in the LH receptor (Ser616Tyr) in a boy with micropenis demonstrating its molecular etiology. Subsequently, mutations were identified in further patients with the mild form of Leydig cell hypoplasia (116, 118, 119)

Posteriorly, Leydig cell hypoplasia was found to be a genetic heterogenous disorder. Zenteno et al, (120) ruled our molecular defects in the LH receptor as being responsible for Leydig cell hypoplasia in three siblings with male pseudohermaphroditism, using segregation analysis of a known polymorphism in exon 11 of the LH receptor gene. In addition, the absence of causative mutations in only 2 of 9 patients strongly suspected to have Leydig cell hypoplasia, has supported the idea that other genes must be implicated in the molecular basis of this disorder (121).

We observed that 46,XX sisters of patients with male pseudohermaphroditism due to Leydig cell hypoplasia, with the same homozgous mutation in the LH receptor, have primary or secondary amenorrhea, spontaneous breast development, infertility, normal or enlarged cystic ovaries with elevated LH and LH/FSH ratio, measurable estradiol levels and normal androgen levels (109, 111, 117, 122, 123). Our findings were subsequently confirmed by other authors who studied 46,XX sisters of male pseudohermafrodites with Leydig cell hypoplasia (124).

Depending on the nature and functional consequences of the mutation in the LH receptor patients have the complete or partial forms of Leydig cell hypoplasia, which will be discussed separately below.

Phenotype: Patients with Leydig cell hypoplasia are born with normal female external genitalia and are almost always raised as girls. If amniocentesis and ultrasound are performed during gestation a discrepancy between the 46,XY karyotype and female external genitalia may be noted. Otherwise, the diagnosis is made when inguinal masses (testes) are noted or when the patient fails to enter puberty and complains of primary amenorrhea. Less frequently, the testes are intra-abdominal. Testicular size is only slightly smaller than expected for age because seminiferous tubules, which represent the bulk of testicular size, are not affected. At puberty, patients with Leydig cell hypoplasia do not develop male or female secondary sexual characteristics.

Diagnosis: In addition to the clinical findings above, the diagnosis is made through hormonal measurements. In patients of postpubertal age, serum LH levels are elevated but testosterone and all testosterone precursors are very low. FSH levels are normal or only slightly elevated. Low testosterone levels permits differential diagnosis with complete androgen insensitivity syndrome and absence of high FSH levels excludes 46,XY gonadal dysgenesis; two conditions with a similar phenotype. In prepubertal patients, an hCG test is necessary to study responsiveness of testosterone and its precursors. We have used a stimulation protocol based on the long half-life of hCG and the necessity of a prolonged stimulation of the quiescent Leydig cells. It consists of administering hCG, 50-100 U/kg body weight, intramuscularly, every 4 days in a total of 4 doses. Testosterone and its precursors are measured 48 and 72 hr after the fourth hCG administration. A measurement of β-hCG in serum helps insure that hCG has been administered. Testicular biopsy shows relatively preserved seminiferous tubules and absence of mature Leydig cells. Sertoli cells are present and germ cells with arrest of spermatogenesis. Advancing age and cryptorchidic testes contribute for a thickening of the basal membrane and, eventually, hyalinization of seminiferous tubules. In prepubertal patients, testicular histology has to be performed after prolonged hCG stimulation to be informative. Usually a testicular biopsy is not done for diagnostic purposes but histology is analyzed after gonadectomy. Epidydimis and vas deferens have been identified in these patients (110).

Genetic studies: Leydig cell hypoplasia has an autosomal recessive pattern of inheritance. Homozygous or compound heterozygous missense, nonsense, deletion, and in-frame insertion mutations of LHR have been identified in patients with Leydig cell hypoplasia (110, 115, 117-119, 124-127). These mutations are not localized in any particular region of the gene. Patients with the complete form of Leydig cell hypoplasia have mutations that cause a severe loss of receptor activity. Transfection of these mutations in in vitro studies, have shown absence of receptor expression on the cell surface and absence of cyclic AMP production after stimulation with hCG. In 3 patients with typical clinical and hormonal features of Leydig cell hypoplasia, however, the involvement of LHR gene was excluded by discrepant LHR alleles in siblings with Leydig cell hypoplasia (120).

Gender identity/role behavior: Patients with the complete form of Leydig cell hypoplasia are unambiguously raised in the female sex and have a female gender identity and role behavior. Sexual intercourse is normal following vaginal dilation with acrylic molds. The patients married and adopted children.

Treatment: Patients with Leydig cell hypoplasia are raised as girls and therefore a gonadectomy is indicated when the presence of testes is discovered. The cryptorchidic testes have no function and are subject to malignant degeneration. At puberty, estrogens are administered to induce female sexual characteristics and a normal bone mass. Since these patients do not have a uterus, administration of progestins is not required.

Phenotype: In contrast to the homogenous phenotype of the severe form of Leydig cell hypoplasia, the mild form can have a broad spectrum (109, 112, 116-119). Most patients have predominantly male external genitalia with micropenis and or hypospadias. Testes are cryptorchidic or topic. During puberty, partial virilization occurs and testicular size is normal or only slightly reduced, while penile growth is significantly impaired. Spontaneous gynecomastia does not occur.

Diagnosis: Before puberty the testosterone response to the hCG test is subnormal without accumulation of testosterone precursors. After puberty, LH levels are elevated but testosterone levels are intermediate between those of children and normal males.

Genetic Studies: Mutations in the LHR gene have been identified in patients with the mild form of Leydig cell hypoplasia (116-119). In vitro studies showed that cells transfected with LH receptor gene containing these mutations had an impaired hCG-stimulated cAMP production. A correlation between the clinical phenotype and the overall receptor signal capacity was observed (116, 119).

Gender identity/role behavior: Patients described to date have been raised as males and have a male gender role.

Treatment: When present, hypospadias have to be repaired surgically. Micropenis is treated by the administration of testosterone or DHT. At and after puberty, testosterone supplementation is necessary to maintain normal androgen levels.

Adrenal hyperplasia syndromes are examples of hypoadrenocorticism or a mixed of hypo- and hyperadrenocorticism. Synthesis of cortisol only or both cortisol and aldosterone are impaired. When cortisol production is hindered, 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.

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. This process is essential to the formation of all adrenal and gonadal steroids. 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 form of cytochrome P450 called P450scc or P45011A, which is located at the inner mitochondrial membrane. The conversion of cholesterol to pregnenolone includes the steps 20-hydroxylation, 22-hydroxylation, and cleavage of the C20-C22 bond to produce pregnenolone, all mediated by P450scc and the mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, which is an essential component in the regulation of steroid biosynthesis through cAMP-dependent pathways (128).

Prader and colegues described lipoid congenital adrenal hyperplasia in 1955 (129). It is the most severe form of congenital adrenal hyperplasia. No steroid hormones are synthesized in affected individuals and patients of both sexes are phenotypic females with a severe salt-losing syndrome that is fatal if not treated in early infancy. 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. The underdeveloped internal male organs and enlarged adrenal cortex are engorged with cholesterol and cholesterol esters (130). 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 (131).

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

Genetic studies: The disorder has an autosomal recessive inheritance. The disease was firstly attributed to P450scc deficiency, but most of the cases studied through molecular analysis showed an intact P45011A gene and RNA in several of the patients studied (132). 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 (133).

Phenotypic female infants with a 46,XX karyotype and identified as having lipoid CAH as newborns can present spontaneous thelarche, pubarche and menarche and periodic menstrual bleeding and can subsequently develop polycystic ovaries (134). These findings demonstrated that ovarian steroidogenesis can be spared to some extent throughout puberty. This is in marked contrast to the early onset of severe defects in testicular and adrenocortical steroidogenesis that characterize lipoid CAH. Bose et al. (1996) concluded that the congenital lipoid adrenal hyperplasia phenotype is the result of 2 separate events: the primary defect is genetic loss of STAR-dependent steroidogenesis and the secondary, a subsequent loss of StAR-independent steroidogenesis that is due to cellular damage from accumulated cholesterol esters (133).

It has been thought that P450scc (CYP11A) mutations are incompatible with human term gestation, because P450scc is needed for placental biosynthesis of progesterone, which is required to maintain pregnancy. A patient with congenital lipoid adrenal hyperplasia with normal StAR and SF-1 genes presenting a de novo heterozygous inactivating mutation in CYP11A was recently described. This patient was atypical for congenital lipoid adrenal hyperplasia, having survived for 4 yrs without hormonal replacement (135). More recently, an inherited CYP11A deficiency was described in a patient with congenital adrenal insufficiency born to healthy parents. The maternally inherited R353W mutation resulted in markedly reduced P450scc activity indicating that Arg(353) is a crucial amino acid residue for P450scc activity. The other mutation, a de novo A189V mutation in the paternal allele, is a splicing mutation, which created a novel alternative splice-donor site which resulted in a deletion of 61 nucleotides in the open reading frame and thus partially inactivating CYP11A. These experimental data are consistent with clinical findings that indicate the patient had partially preserved ability to synthesize adrenal steroid hormones. This is the first report of a CYP11A mutation in a compound heterozygote with congenital lipoid adrenal hyperplasia (136).

Gender role: Most patients with congenital lipoid hyperplasia were raised as females and kept the female social sex at puberty.

3b-HSD converts 3b-hydroxy D5 steroids to 3-keto D4 steroids and is essential for the biosynthesis of mineralocorticoids, glucocorticoids and sex steroids (137). Two forms of the enzyme have been described in the male: type I enzyme is expressed in placenta and skin, and type II in adrenals and gonads (138). The type 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 (139).

Phenotype: Male patients with 3b-HSD type II deficiency present with ambiguous external genitalia. This is characterized by micropenis, perineal hypospadias, bifid scrotum and blind vaginal pouch associated or not to salt loss (137). Gynecomastia is common at pubertal stage.

Diagnosis: Plasma 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.

Genetic studies: The disorder has an autosomal recessive inheritance. There are around 40 mutations in the 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 (128, 139-141). 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 pseudohermaphroditism due to the reduction in androgen synthesis (142, 143). Male subjects with pseudohermaphroditism due to 3b-HSD type II deficiency without salt loss showed clinical features in common with the deficiencies of 17b-HSD 3 and 5a-reductase 2.

Gender role: Most of the patients were raised as males and kept the male social sex at puberty. In one Brazilian family, two cousins with MPH 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 (144).

This is the last of the three enzymatic deficiencies in steroidogenesis which affects the testes and the adrenals. 17-hydroxylation does not enter the mineralocorticoid pathway and thus, only glucocorticoid and sexual steroid are affected.

Deficiency of adrenal 17-hydroxylation activity was first demonstrated by Biglieri et al. (145). New reported the first affected male (146). The clinical features in most of the male patients described are female-like or slightly virilized external genitalia with blind vaginal pouch, cryptorchidism, and high blood pressure, which is usually associated with hypokalemia. 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, hypergonadotropic hypogonadism, amenorrhea at post pubertal age; enlarged ovaries at adult age and infarction from twisting can occur (147, 148). 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 (149).

Diagnosis: Plasma levels of progesterone, corticosterone, and 18-OH-corticosterone are elevated, while aldosterone, 17-OH-progesterone, cortisol, androgens and estrogens are decreased. Martin et al, (150) 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.

Genetic studies: The disorder has an autosomal recessive inheritance. The gene CYP17, 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 (151). CYP17 gene was cloned and sequenced by Picado-Leonard et al in 1987 and showed similarities to the CYP21 gene (151, 152). Several mutations in the CYP17 gene have been identified in patients with both 17-hydroxylase and 17,20 lyase deficiencies (147, 148, 150)

Gender role: Most of the patients are reared as females and keep female social sex at puberty (149, 150).

Treatment: 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.

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.

In patients with testosterone synthesis defects post pubertal diagnosis is made through basal steroid levels. Testosterone levels are low and steroids past the enzymatic blockage are elevated. This pattern can be confirmed with an hCG stimulation test, which increases the accumulation of steroids past the enzymatic blockage with a slight elevation of testosterone. In pre-pubertal individuals, hCG stimulation test is essential for the diagnosis since basal levels are not altered.

Human male sexual differentiation requires production of fetal testicular testosterone, who's 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 (153). Later, clear molecular evidence of the existence of isolated 17,20 desmolase deficiency was demonstrated (148, 154).

Phenotype: Patients present ambiguous genitalia with micropenis, perineal hypospadias and cryptorchidism. Gynecomastia Tanner stage V can occur at puberty (154).

Diagnosis: 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.

Genetic studies: The CYP17 gene of two Brazilian male pseudohermaphrodites 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 (154). When expressed in COS-1 cells, the mutants retained 17a-hydroxylase activity and had minimal 17,20-lyase activity. Geller et al concluded that these 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. The authors stated that these were the first proven cases of isolated 17,20-lyase deficiency, and represented a novel mechanism for enzymatic activity loss (154).

Treatment: For male social sex patients, treatment consists of surgical masculinization of external genitalia and testosterone replacement at puberty. For female social sex patients, treatment consists of orchiectomy and estrogen replacement at puberty.

Gender Role: most of the patient were reared as male and kept the male sex at puberty.

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 colleague