Chapter 11 – 46,XY Disorders of Sexual Development

Sorahia Domenice, MD
Assistant Professor of Endocrinology, Division of Endocrinology, Hospital das Clinicas of the University of São Paulo School of Medicine, São Paulo, Brazil

Ivo J P Arnhold, MD
Associate Professor of Endocrinology, Division of Endocrinology, Hospital das Clinicas of the University of São Paulo School of Medicine, São Paulo, Brazil

Elaine M F Costa, MD
Assistant Professor of Endocrinology, Division of Endocrinology, Hospital das Clinicas of the University of São Paulo School of Medicine, São Paulo, Brazil

Berenice Bilharinho Mendonca, MD
Professor of Medicine, Head of the Division of Endocrinology, Hospital das Clinicas of the University of São Paulo School of Medicine, São Paulo, Brazil. Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular, LIM/42, Disciplina de Endocrinologia, Hospital da Clínicas da Universidade de São Paulo, Brasil, and Developmental Endocrinology Unit, Laboratory of Hormones and Molecular Genetics LIM/42, Division of Endocrinology, Clinical Hospital, Medical School, Sao Paulo University, Sao Paulo, Brazil

Updated January 2013

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INTRODUCTION

Male phenotypic development can be viewed as a 2-step process: 1) testis formation from the primitive gonad (sexual determination) and 2) internal and external genitalia differentiation by action of factors secreted by the fetal testis (sexual differentiation). The first step is very complex and involves interplay of several transcription factors and signaling cells [1, 2]. Dosage imbalances in genes involved in DSD (deletions or duplication) have been identified as a cause of these disorders (Fig. 1).

The second step, male sex differentiation, is a more straightforward process. Anti Müllerian hormone (AMH) secreted by the testicular Sertoli cells acts on its receptor in the Müllerian ducts to cause their regression. Testosterone secreted by the testicular Leydig cells acts on the androgen receptor in the Wolffian ducts to induce the formation of epididymis, deferent ducts and seminal vesicles (Fig. 2).

Testosterone is further reduced to dihydrotestosterone (DHT), which acts on the androgen receptor of the prostate and external genitalia leading to its masculinization (Fig. 3 and 4).

The term disorders of sex development (DSD) includes congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical. This nomenclature has been proposed to replace terms such as intersex, pseudohermaphroditism and sex reversal [3, 4]. These terms, previously used to describe the disorders of sex development, are potentially offensive to the patients and the consensus on the management of intersex disorders recommends a new nomenclature that will be followed in this chapter [3, 4]. The proposed changes in terminology aim to integrate upcoming advances in molecular genetics in new DSD classification [5].
The 46,XY disorders of sex development (46,XY DSD) are characterized by ambiguous or female external genitalia, caused by incomplete intrauterine masculinization with or without the presence of Müllerian structures. Male gonads are identified in the majority of 46,XY DSD patients, but in some of them no gonadal tissue is found. Complete absence of virilization results in normal female external genitalia and these patients generally seek medical attention at pubertal age, due to the absence of breast development and/or primary amenorrhea. 46,XY DSD can result either from decreased synthesis of testosterone or from impairment of androgen action [6] Our proposal classification of 46,XY DSD is displayed in Table 1 [5].

Table 1: CLASSIFICATION OF 46,XY DSD

Investigation of DSD patients

Optimal care of patients with disorders of sex development requires a multidisciplinary team and begins in the newborn period. The careful clinical evaluation of the neonate is fundamental because most DSD patients may be recognized in this period and precocious diagnosis allows a better therapeutic approach. Family and prenatal history, general physical examination and assessment of genital anatomy are the first steps for a correct diagnosis. The diagnostic evaluation of DSD includes hormone measurements, imaging, cytogenetic and molecular studies. In some cases, endoscopic and laparoscopic exploitation and gonadal biopsy are required [5, 7].

The endocrinological evaluation of 46,XY DSD infants includes assessment of testicular function by basal measurements of LH, FSH, inhibin B, anti-Mullerian hormone (AMH) and steroids.

AMH and inhibin B are useful markers of the Sertoli cells presence and their assessment could help in the diagnosis of testis determination disorders. In boys with bilateral cryptorchidism serum AMH and inhibin B correlate with the presence of testicular tissue and undetectable values are highly suggestive of absence of testicular tissue [8-10].

In postpubertal patients with testosterone synthesis defects, the diagnosis is made through basal steroid levels. Testosterone levels are low and steroids upstream from the enzymatic blockage are elevated. This pattern can be confirmed with an hCG stimulation test, which increases the accumulation of steroids before 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.

There are several hCG stimulation protocols and normative data have to be established for each of them. We established a normal testosterone response 72 and 96 hours after the last of 4 doses of hCG, 50-100 U/kg body weight, given via intramuscular every 4 days in boys with cryptorchidism but an otherwise normal external genitalia: testosterone peak levels reached 391 ± 129 ng/dL and we consider a subnormal response a value <130 ng/dL (equivalent to -2 SD) [11].

Imaging evaluation is indicated in the neonatal period when genital ambiguity is identified. If apparent female genitalia with clitoral hypertrophy, posterior labial fusion, foreshortened vulva with single opening or inguinal/labial mass is present, imaging study may also be performed. A family history of DSD and later presentations as abnormal puberty or primary amenorrhea, cyclic hematuria in a male, inguinal hernia in a female also require an imaging evaluation.

Ultrasonography is always the first and often the most valuable imaging modality in DSD patients’ investigation. Ultrasound shows the presence or absence of Müllerian structures at all ages and can locate the gonads and characterize their echo texture. This exam can also identify associated malformations such as kidney abnormalities [12].

Genitography and cystourethrography can display the type of urethra, the presence of vagina, cervix, and urogenital sinus. Although, the imaging features are non-specific for the cause of DSD, these diagnostic methods are important in gender assignment and specially for surgical planning.
The genetic evaluation includes karyotype, FISH and, more recently, specific molecular studies to screen for the presence of mutations or gene dosage imbalance.

Nevertheless, the attainment of molecular diagnosis is related to a properly established clinical and hormonal diagnosis.

46,XY DSD DUE TO ABNORMALITIES IN GONADAL DEVELOPMENT

Gonadal determination and differentiation: this process initiates with the organization of the early urogenital ridge that is controlled by a number of factors acting in concert such as the nuclear receptor proteins Wilms’ tumor suppressor (WT1) and steroidogenic factor 1 (SF1), which prepare the gonad for the sex determination step [13, 14]. Wt1 functions upstream of two orphan nuclear receptors: Sf1 and Dax1 (Dosage sensitive sex reversal, congenital adrenal hypoplasia, X chromosome) [15]. SF1 gene prepares the ground for SRY expression, cooperating to express AMH, the first marker of testis differentiation [16, 17]. SF1 will later regulate steroid production by Leydig cells, whose proper development depends on the previous and successful establishment of the Sertoli lineage.

The discovery of the sex-determining region of the Y chromosome (SRY) was the first crucial step towards a general understanding of sex determination [18, 19]. SRY gene, located in the distal region of the short arm of the Y chromosome (Yp11.3), encodes a protein containing a "high-mobility group" domain (HMG box), which enables it to bind and bend DNA [18, 20]. In the mammalian male embryo, the first molecular signal of sex determination is the expression of Sry within a subpopulation of somatic cells of the indifferent genital ridge. The transient expression of Sry drives the initial differentiation of pre-Sertoli cells that would otherwise follow a female pathway becoming granulosa cells. Once Sry expression begins, it initiates the cascade of gene interactions and cellular events that direct to the formation of a testis from the indifferent fetal gonad. So, pre-Sertoli cells proliferate, polarize and aggregate around the germ cells to define the testes cords. Migration of cells into the gonad from the mesonephros or the coelomic epithelium is subsequently induced by signals emanating from the pre-Sertoli cells. Peritubular myoid cells surround the testes cords and cooperate with pre-Sertoli cells to deposit the basal lamina and further define the testis cords. Signalling molecules produced by the pre-Sertoli cells promote the differentiation of somatic cells, found outside the cords, into fetal Leydig cells, thus ultimately allowing the production of testosterone. Endotelial cells are associated to form the coelomic vessel, which promotes efficient export of testosterone. The gene Sox9 is up-regulated immediately after Sry expression and is involved in the initiation and maintenance of Sertoli cell differentiation during early phases of testis differentiation [21]. The mechanism by which SF1 and SRY increase endogenous SOX9 expression was sophisticatedly demonstrated in human embryonal carcinoma cell line NT2/D1 [22]. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1 [22].

Extracellular signaling pathways (Fgf9 and Igf1r/Irr/Ir) play a significant role in Sox9 expression. A model has been suggested in that the fate of the bipotential gonad is controlled by mutually antagonistic signals between Fgf9 and Wnt4/Rspo1. In this model Sox9 up-regulates Fgf9-Fgfr2 and Fgf9 maintains Sox9 expression, forming a positive feed-forward loop in XY gonads. The balance between Fgf9 and Wnt4/Rspo1 signals is shifted in favor of Fgf9, establishing the male pathway. In addition, Sry inhibits b-catenin-mediated Wnt signaling [23]. In the absence of this feed-forward loop between Sox9 and Fgf9, Wnt4/Rspo1, the activated b-catenin pathway, blocks Fgf9 and promotes the ovarian fate [1, 24].
Furthermore, SOX9 directly binds to the promoter of the Ptgds gene which encodes prostaglandin D synthase that mediates the production of PGD2 ([25], which, in turn, promotes nuclear translocation of SOX9, facilitating Sertoli cell differentiation [26]. Antagonism between Dmrt1 and Foxl2 comprises another step for sex-determining decision. Recently, Dmrt1 has been described as essential to maintain mammalian testis determination, preventing female reprogramming in the postnatal mammalian testis [27]

Abnormalities in the expression (underexpression or overexpression or timing of expression) of genes involved in the cascade of testis determination can cause anomalies of gonadal development and consequently, 46,XY DSD. The absence, regression or the presence of dysgenetic testes results in abnormal development of the genital ducts and/or external genitalia in these patients.

Gonadal agenesis

Total absence of gonadal tissue or gonadal streak confirmed by laparoscopy has rarely been described in XY subjects with female external and internal genitalia indicating the absence of testicular determination [28]. Mendonca et al described a pair of siblings, one XY and the other XX, born to a consanguineous marriage, with normal female external and internal genitalia associated to gonadal agenesis [29]. Mutations in SF1 and LHX9 were ruled out in these siblings [30, 31]. The origin of this disorder remains to be determined, but a defect in another gene essential for bipotential gonad development is the most likely cause of this disorder.

46,XY DSD due to gonadal dysgenesis

Complete and partial 46,XY gonadal dysgenesis

46,XY gonadal dysgenesis consists of a variety of clinical conditions in which the development of the fetal gonad is abnormal and encompasses both a complete and a partial form. The complete form of gonadal dysgenesis was first described by Swyer et al. [32] and is characterized by female external and internal genitalia, lack of secondary sexual characteristics, normal or tall stature without somatic stigmata of Turner syndrome, eunuchoid habitus and the presence of bilateral dysgenetic gonads in XY subjects. Mild clitoromegaly is present in some cases.
The partial form of this syndrome is characterized by impaired testicular development that results in patients with ambiguous external genitalia with or without Müllerian 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 predominates over LH serum 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 the X chromosome or mutations in autosomal genes. Most of the authors reported mutations in SRY gene in less than 20% of the patients with complete 46,XY gonadal dysgenesis [33-35]. 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 in 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 [36, 37]. In few of these cases, the father’s somatic mosaicism for the normal and mutant SRY gene have been proven [38]. This variable penetrance in familial mutations of SRY have been described in mutant SRY proteins with relatively well preserved in vitro activity [39].

An interesting 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 [40]. 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 [40].

The clinical condition named embryonic testicular regression syndrome (ETRS) has been considered part of the clinical spectrum of partial 46,XY gonadal dysgenesis [41]. 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 [42]. 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 [41].

Gonadal dysgenesis associated with syndromic phenotype

In humans, there are several syndromes associated with 46,XY gonadal dysgenesis caused by mutations in genes, which are involved in gonadal determination. They will be described according to the period 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 and mutations in this gene impair gonadal and urinary tract development. Three disorders are associated with WT1 mutations: WAGR syndrome, Denys-Drash syndrome and Frasier syndrome.

WAGR syndrome: is characterized by Wilms’ 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 [43]. Heterozygous deletions of WT1 and contiguous gene are the cause of this syndrome [44]. Deletions of PAX6 gene are related to the presence of aniridia in these patients. Severe obesity is present in some subjects with the WAGR syndrome and the acronym WAGRO has been suggested for this condition [45]. 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 of approximately 10 Mb encompassing WT1 and PAX6 was described [46]. This report demonstrated an overlap of clinical and molecular features in WAGR, Frasier and Denys-Drash syndromes that confirms 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 Wilms´ tumor development in the first decade of life [47]. 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 [48]. Gonadal development is impaired to variable degrees, resulting in a spectrum of 46,XY DSD [49].

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. We described a patient presenting an unusual DDS nephropathy progression, which reinforces that patients carrying WT1 mutations should have the renal function carefully monitored due to the possibility of late-onset nephropathy [50, 51]. However, the nephrotic syndrome may be evident early in life [52].

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 [53]. 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 [54]. The c.1432+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 [51]. 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 [47, 55]. Frasier syndrome is usually associated with the c.1432+4C>T mutation [56], although exonic mutations also cause Frasier syndrome [57]. We reported a patient presenting an overlapping of some typical characteristics of Frasier syndrome (end-stage renal failure in the second decade, gonadoblastoma and the c.1432+4C>T mutation, but with the gonadal and external genitalia development usually found in Denys-Drash syndrome [51].

The report of ambiguous external genitalia [57], the presence of Wilms’ tumor [58] and the description of exonic mutations in the DNA binding domain of WT1 gene [57] 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)

Steroidogenic Factor-1 (SF1) also known as NR5A1 and Ad4BP was originally identified as a master-regulator of steroidogenic enzymes in the early 1990s following the Keith L. Parker and Kenichirou Morohashi inspiring work [59-61]. SF1 has since been shown to control many aspects of adrenal and reproductive function [62-64]. SF1, together with several signaling molecules are also involved in adrenal stem cell maintenance, proliferation and differentiation inducing adrenal zonation, probably acting in the progenitor cells [65]. Homozygous 46,XY null mice (-/-) have adrenal agenesis, complete testicular dysgenesis, persistent Müllerian structures, partial hypogonadotropic hypogonadism, and other features such as late-onset obesity [66].

Therefore, it is clear that SF1 is an essential factor in sexual and adrenal differentiation (53, 54) and a key regulator of adrenal and gonadal steroidogenesis and also of the hypothalamic-pituitary-gonadal axis.

The first reported human case of NR5A1/SF1 mutation, the heterozygous p.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. This patient presented with salt-losing adrenal failure in early infancy and was thought to have a high block in steroidogenesis (e.g. in CYP11A1, STAR) affecting both adrenal and testicular functions. However, the identification of a streak-like gonad and Müllerian structures was consistent with testicular dysgenesis, thereby, a disruption of a common developmental regulator such as SF1 was hypothesized. The patient was found to have a de novo heterozygous p.G35E change in the P-box of SF1 which is important in dictating DNA binding specificity through its interaction with DNA response elements in the regulatory regions of target genes [67]. This case established that disruption of SF1 could be associated with severe gonadal defects and primary adrenal insufficiency also in humans.

The second report of SF1 defects in humans was described by Biason-Lauber and Schoenle, in a 14 month-old 46,XX girl who had presented primary adrenal insufficiency and seizures [68]. She had a de novo heterozygous SF1 change resulting in the p.R255L mutation into the proximal part of the ligand-like binding domain of the protein. The mutant SF1 protein was transcriptionally inactive, without a dominant negative effect. The ovaries were detected by MRI scan and Inhibin A levels was normal for her age, suggesting that SF1 change had not disrupted ovarian function.

The third report of SF1 defects in humans was found in an infant with a similar phenotype of the first case: primary adrenal failure and 46,XY DSD. However, this child had inherited the homozygous p.R92Q alteration in a recessive manner [69]. The change lies within the A-box of SF1, which interferes with monomeric DNA binding stability, but in vitro functional activity was in the order of 30–40% of the wild type [69-71]. Carrier parents showed normal adrenal function suggesting that the loss of both alleles is required for the phenotype development when disrupted protein keeps this level of functional activity. In addition, another family has been reported with a homozygous missense mutation (p.D293N) in the LBD of SF1 [72]. This change also showed partial loss-of-function (50%) in gene transcription assays.

In 2004, we reported the fourth SF1 mutation in humans which brought two novel variables to SF1 phenotype: it was the first frameshift mutation and it appeared in a 34 year old 46,XY DSD female with normal adrenal function [73]. Another interesting aspect in this patient was the absence of gonadal tissue at laparoscopy. Since she had ambiguous genitalia and absence of Müllerian derivatives we assumed that testicular tissue regressed completely in postnatal life.

However, some 46,XY DSD patients with NR5A1 mutations can produce sufficient testosterone for spontaneous virilization during puberty [74]
SF1 changes associated with 46,XY DSD are usually frameshift, nonsense or missense changes that affect DNA-binding and gene transcription [70]. Most of the point mutations identified in NR5A1/SF1 are located in the DNA-binding domain of the protein. The p.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, considered an orphan receptor so far [71].
In several cohort studies, SF1 changes have been reported in approximately 10–15% of the individuals with gonadal dysgenesis [70, 71, 75]. Although many of the heterozygous changes are de novo, about one-third of these changes have been shown to be inherited from the mother in a sex-limited dominant manner [70]. These women are at potential risk of primary ovarian insufficiency but while fertile they can pass SF1 heterozygous changes to their children. This mode of transmission can mimic X-linked inheritance [70]. The features in different affected family members can be variable.

A novel role of SF1 in human reproductive function was described by Bashamboo and co-workers [76]. They investigated whether changes in SF1 could be found in a cohort of 315 men with normal external genitalia and non-obstructive male factor infertility where the underlying cause was unknown [76] Analysis of SF1 in this cohort identified heterozygous changes in seven individuals; all of them were located within the hinge region of the SF1 protein. The men who harbored SF1 changes had more severe forms of infertility (azoospermia, severe oligozoospermia) and in several cases low testosterone and elevated gonadotropins were found. A serial decrease in sperm count was found in one-studied men raising the possibility that heterozygous changes in SF1 might be transmitted to offspring, especially if fatherhood occurs in young adulthood rather than later in life [77]. As progressive gonadal dysgenesis is likely, gonadal function should be monitored in adolescence and adulthood, and early sperm cryopreservation considered in male patients, if possible. In conclusion, this study shows that changes in SF1 may be found in a small subset of phenotypically normal men with non- obstructive male factor infertility where the cause is currently unknown. These individuals may be at risk of low testosterone in adult life and may represent part of the adult testicular dysgenesis syndrome [74, 76, 77].

A novel heterozygous missense mutation (p.V355M) in SF1 was identified in one boy with a micropenis and testicular regression syndrome [78].
SF1 mutations have also been identified in familial and sporadic forms of 46,XX primary ovarian insufficiency (POI) not associated with adrenal failure [72, 79] Most of these women harbored heterozygous alterations in SF1 and had been identified in families with history of 46,XY DSD and 46,XX POI. Heterozygous SF1 changes were also found in two girls with sporadic forms of POI [72]. In one large kindred a partial loss-of-function SF1 change (p.D293N) was inherited in an autosomal recessive manner. These 46,XX women with SF1 mutation presented with either primary or secondary amenorrhea and with a variable age of features onset. The detection of SF1 alterations in 46,XX ovarian failure shows that SF1 is also a key factor in ovarian development and function in humans. Thus, some 46,XX women with SF1 mutations have normal ovarian function and can transmit the mutation in a sex-limited dominant fashion. The inheritance patterns associated with SF1 changes can be autosomal dominant, autosomal recessive or sex-limited dominant.

SF1 defects can be found in association with a wide range of human reproductive phenotypes such as 46,XY disorders of sex development (DSD) associated or not with primary adrenal insufficiency, male infertility, or primary ovarian insufficiency (Figure 6).

Figure 6 : Spectrum of loss of function SF1 mutations in humans.

46,XY DSD due to the underexpression of DMRT1 gene

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 [80-82]. The human 9p deletion syndrome is characterized by variable degrees of 46,XY DSD, from 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 [83]. Genomic–wide copy number variation screening has revealed DMRT1 deletions associated with isolated 46,XY gonadal dysgenesis [84]. DMRT1 inactivating mutations are rarely described in isolated 46,XY gonadal dysgenesis [81, 85].

Gonadal function varies from hypergonadotropic hypogonadism to near normal testicular production. 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 [83]. In humans and mice, DMRT1 is required for the postnatal maintenance of Sertoli and germ cells. Recently, Matson et al. (2011) have shown in mouse that Dmrt1 and Foxl2 create another regulatory network necessary for maintenance of the testis during adulthood. Loss of Dmrt1 in mouse Sertoli cells induces the reprogramming of those into granulosa cells, due to Foxl2 upregulation. Consequently, theca cells are formed, estrogens is produced and germ cells appear feminized [27]

ATR-X syndrome (X-linked a-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 [86]. Genital anomalies leading to a female sex of rearing were reported in several affected 46,XY patients with ATR-X syndrome [87].

ATR-X syndrome is characterized by severe mental retardation, alpha thalassemia and a range of genital abnormalities in 80% of cases [88]. 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 variety 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) have also been associated with ATRX mutations. ATRX lies on the X chromosome (Xq13) and the disease has been confined to males; in female carriers of a ATRX mutation, the X-inactivating pattern is skewed against the.X carrying the mutant allele.

Urogenital abnormalities associated to mutations in human ATRX range from undescended testes to testicular dysgenesis with female or ambiguous genitalia. ATRX duplications leading to gene disruption were also described [89]. There are two major functional domains in ATRX protein: 1- the ATRX-DNMT3-DNMT3L (ADD) domain at the N-terminus and 2- the helicase/ATPase domain at the C-terminal half of the protein, both acting as chromatin remodeling. Mutations in the ADD domain have been related to severe psychomotor impairment associated to urogenital abnormalities. On the other hand, mutations in the C-terminus region have been related with mild psychomotor impairment without severe urogenital abnormalities [90, 91].

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. The role of ATRX in the sexual development cascade is poorly understood and it is suggested that it could be involved in the development of the Leydig cells [92].

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 [93]. 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 [93].

The large duplications of Xp21 reported prior to array-CGH and MLPA techniques were identified by conventional karyotyping. Patients carried large genomic rearrangements involving several genes. In these patients, the presence of XY gonadal dysgenesis was part of a more complex phenotype which also included dysmorphic features and/or mental retardation [94]
Interestingly, in all cases with isolated 46,XY gonadal dysgenesis, the IL1RAPL1 gene, located immediately telomeric to the duplication containing NR0B1, is not disrupted. Deletions or mutations of this gene have been identified in patients with mental retardation [20]. Disruption of this gene could explain the mental retardation previously described in patients with larger Xp21 duplications [95].

Several patients with isolated 46,XY gonadal dysgenesis and duplications of Xp21 have been described. The first report identified a 637 kb 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 [96]. The second case exhibited a duplication with approximately 800 kb in size and, in addition to DAX1, contains the four MAGEB, Cxorf21 and GK genes. The healthy mother was a carrier of the duplication [97].

Smyk et al. described a 21-years-old 46,XY patient manifesting primary amenorrhea, a small immature uterus, gonadal dysgenesis and absence of adrenal insufficiency with a submicroscopic deletion (257 kb) 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 [98].

By using array-CGH and MLPA techniques, additional NR0B1 locus duplications have been identified in patients with isolated 46,XY gonadal dysgenesis [84, 99, 100]. 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 located in the DSS locus, should be involved in dosage-sensitive 46,XY DSD.

Barbaro et al identified a relatively small NR0B1 locus duplication responsible for isolated complete 46,XY gonadal dysgenesis in a large English family [97]. The duplication extends from the MAGEB genes to part of the MAP3K7IP3 gene, including NR0B1, CXorf21, and GK genes. Unfortunately, the authors were unable to set up the rearrangement mechanism and distinguish between a nonallelic homologous recombination or a nonhomologous end joining mechanism. The authors also analyzed the X-inactivation patterns in fertile female carriers of each of the three small NR0B1 locus duplications previously identified by their group. They established that female carriers of macroscopic Xp21 duplications are thought to be healthy and fertile due to skewed X-inactivation, preferentially inactivating the duplicated chromosome and thereby protecting them from increased gene expression. Similarly, a skewed inactivation in carriers of small duplications would indicate a deleterious effect on increased NR0B1 (DAX1) gene expression on ovarian function [101].{Barbaro, 2012 #1601;Barbaro, 2012 #1602}

46,XY DSD due to the overexpression of WNT4 gene

The Wnt4 (wingless-type mouse mammary tumor virus integration site member 4) gene belongs to a family that consists of structurally related genes that encode cysteine-rich secreted glycoproteins that act as extracellular signaling factors [102].

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 undescended gonads consisted of rete testes and rudimentary seminiferous tubules, who carried a duplication of 1p31-p35, including both WNT4 and RSPO1 gene, was reported [103]. In vitro functional studies showed that Wnt4 up-regulates Dax1 in Sertoli cells, suggesting that Dax1 overexpression was the cause of 46,XY DSD in this infant.[104].

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-determining pathway [105, 106]. In all affected subjects, SOX9 mutation was identified in heterozygous state indicating that this disorder is due to haploinsufficiency of SOX9 gene [105]. 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 [107].

Patients with campomelic dysplasia and 46,XY gonadal dysgenesis with intact SOX9 were reported. In one patient a microdeletion of ~380 kb upstream of SOX9 was identified [108]. In the other patient an apparently balanced chromosome translocation with breakpoints scattered ~1.3 Mb downstream of SOX9 was found [109].

Dysgenetic 46,XY DSD due to DHH underexpression

Desert hedgehog (Dhh), a member of the hedgehog family of signaling proteins, is located in chromosome 12-q13.1 [110] and is one of the genes involved in the testis-determining pathway. Dhh seems to be necessary for Sf1 up-regulation in Leydig cells [111]. To date, five mutations have been described in DHH gene. The first one, a homozygous missense mutation, located at the initiation codon of exon 1 (p.M1T), was found in a 46,XY patient with partial gonadal dysgenesis associated with polyneuropathy [112]. Two other mutations located at exon 2 (p.L162P) and exon 3 (c.1086delG) were identified in three patients with complete gonadal dysgenesis without neuropathy, two of them harboring gonadal tumors [113]. One year later, the exon 3 mutation (c.1086delG), previously described in homozygous state was identified by the same group in heterozygous condition in two patients with mixed gonadal dysgenesis [114]. In addition, two novel homozygous mutations were described in two patients with complete 46,XY gonadal dysgenesis without clinically overt neuropathy: the c.271_273delGAC that resulted in deletion of one amino acid (p.D90del) and a duplication (c.57_60dupAGCC) that resulted in a premature termination of DHH protein [115]

Dysgenetic 46,XY DSD due to MAP3K1 underexpression

Mutations in MAP3K1 (Mitogen-Activated Protein 3 Kinase 1 gene) result in 46,XY DSD due to partial or complete gonadal dysgenesis implicating this pathway in normal human sex determination. To date four mutations in MAP3K1 gene have been described in 46,XY DSD patients [116]. The locus for an autosomal sex-determining gene was mapped via linkage analysis at the long arm of chromosome 5 in two families with 46,XY DSD. A splice-acceptor mutation (c.634-8T>A) in MAP3K1, which disrupted RNA splicing, segregated with the phenotype in the first family. Two novel mutations were demonstrated in a second family (p.Gly616Arg) and in two of 11 sporadic cases of dysgenetic 46,XY DSD (p.Leu189Pro and p.Leu189Arg). In primary lymphoblastoid cells in culture from family 1 and from the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced the binding of the Ras homolog gene family, member A (RHOA) to the MAP3K1 complex [116] . RhoA is a small GTPase protein encoded by the gene RHOA known to regulate the actin cytoskeleton in the formation of stress fibers. In human’s articular chondrocytes, it regulates SOX9 expression involving p38 MAPK activation and mRNA stabilization [117].

Dysgenetic 46,XY DSD due to GATA4 and FOG2 underexpression

Gata4 (GATA-binding factor 4 gene) cooperatively interacts with several proteins to regulate the expression of genes involved in testis determination and differentiation as SRY, SOX9, NR5A1, AMH, DMRT1, STAR, CYP19A1, and others [118].

In humans, GATA4 mutations were first described in patients with congenital heart defects without genital abnormalities [119]. However, genitourinary anomalies, as hypospadias and cryptorchidism, were described in 46,XY patients with 8p deletions [120].

A novel GATA4 mutation (p.Gly221Arg) was identified in three members of a French family, in both 46,XX and 46,XY members [121]. The affected 46,XX patients had heart abnormalities and apparently normal ovarian function. In 46,XY DSD patients with GATA4 mutation heart disease was present in 2 out of 3 patients. This mutation compromised the ability of the protein to transactivate the anti-Müllerian hormone promoter. The mutation does not interfere with the direct protein–protein interaction, but it disrupts the synergistic activation of the AMH promoter by GATA4 and NR5A1. This mutant protein also failed to bind to its protein partner FOG2 [121].

The role of FOG2 in human testis development was corroborated by the identification of a balanced translocation t(8;10) (q23.1;q21.1) in a patient with partial gonadal dysgenesis and congenital heart abnormalities [122].

Dysgenetic 46,XY DSD due to CBX2 underexpression

CBX2 (Chromobox homolog 2 gene) defects in SRY-positive mice cause male-to-female sex reversal with small or absent ovaries suggesting that CBX2 acts repressing ovarian development in XY gonads [123, 124]. A girl, with a 46,XY karyotype performed during prenatal life, was born with a completely normal female phenotype, including uterus and histologically normal ovaries. The gonads were evaluated at 4.5 years of age and at this time she had high FSH levels. Direct sequencing of the CBX2 gene revealed the presence of the variants (c.C293T) and (c.G1370C) both in exon 5 in heterozygous state, leading to P98L (inherited from the father) and R443P (inherited from the mother) mutations in the CBX2 protein [125].

46,XY DSD ASSOCIATED WITH Cholesterol Synthesis

Smith-Lemli-Opitz syndrome (SLOS)

This syndrome, caused by a deficiency of 7-dehydrocholesterol reductase, is the first true metabolic syndrome leading to multiple congenital malformations [126, 127]. 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 [128, 129]. The aetiology of masculinization failure in the SLOS 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 [130], although, a revision of HPA axis in these patients showed normal HPA axis function [131].
Affected children present elevations of 7-dehydrocholesterol (7DHC) in plasma or tissues. 7DHC is best assayed using Gas Chromatography/Mass Spectroscopy (GC/MS). 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. However, although frequently low, plasma cholesterol levels can be within normal limits in affected patients.

DHCR7 mutation analysis can confirm a diagnosis of SLOS. The human DHCR7 gene is localized on chromosome 11q13 and contains nine exons encoding a 425 amino-acid protein [122]. More than 130 different mutations of DHCR7 have been identified and the great majority of them are located at the exons 6 to 9 [132, 133] However, the genotype-phenotype correlation in SLOS is relatively poor [134].

Currently, most SLOS patients are treated with cholesterol supplementation that can be achieved by including high cholesterol foods and/or suspensions of pharmaceutical grade cholesterol. Data suggests that early intervention may be of benefit to SLOS patients [135]. Observational studies report improved growth and muscle tone and strength, increased socialization, decreased irritability and aggression in SLOS patients treated with cholesterol supplementation. However, in a group of SLOS patients’ treatment with a high cholesterol diet did not improve developmental scores [122, 136].

Treatment with sinvastatin, an HMG-CoA reductase inhibitor, aiming to block the cholesterol synthesis pathway avoiding the formation of large amounts of 7DHC/8DHC, in this manner limiting exposure to potentially toxic metabolites in SLOS patients, has been proposed. Simvastatin can also cross the blood–brain barrier and may provide a means to treat the biochemical defect present in the CNS of SLOS patients [137]. A major effect of statins therapy is the transcriptional upregulation of genes controlled by the transcriptional factor SREBP, as DHCR7. Thus, if any residual activity is present in the mutant DHCR7, its upregulation could increase intracellular cholesterol synthesis. Simvastatin use in SLOS patients resulted in a paradoxical increase in serum and cerebral spinal fluid cholesterol levels [137]. Determination of residual DHCR7 enzymatic activity may be helpful in selecting SLOS patients being considered for a beneficial response of statins [133].

46,XY DSD due to testosterone production defects

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

Inactivating mutations of human LHCG receptor (LHCGR) have been described in 46,XY individuals with a rare form of disorder of sex development, termed Leydig cell hypoplasia. These inactivating mutations in the LHCGR prevent LH and hCG signal transduction and thus testosterone production both pre- and postnatally in genetic males [138].

Both hCG and LH act by stimulating a common transmembrane receptor, the LHCGR [139, 140]. LHCGR is a member of G protein-coupled receptors, which were characterized by the canonical serpentine region, composed of seven transmembrane helices interconnected by three extracellular and three intracellular loops [141, 142]. The large amino-terminal extracellular domain, rich in leucine-repeats, mediates the high affinity binding of pituitary LH or placental human chorionic gonadotropin (hCG) [142].

LHCGR activates the Gs protein, which determines an increase in intracellular cAMP and a subsequent stimulation of steroidogenesis in gonadal cells such as testicular Leydig cells, ovarian theca cells and differentiated granulosa cells [141, 142]. A secondary mechanism of LHCGR stimulation is through G_q/11 protein activation and the inositol phosphate signaling pathway [142].

The LHCGR gene is located on the short arm of chromosome 2 (2p21). It spans nearly 80 kb and has been thought to be composed of 11 exons and 10 introns. Exon 11 of the LHCGR gene encodes the entire serpentine domain as well as the carboxy-terminal portion of the hinge region (NCBI GeneID 3973; http://www.ncbi.nlm.nih.gov). The amino-terminal portion of the hinge region is encoded by exon 10 and the signal peptide and remaining portion of the extracellular domain are encoded by exons 1-9 [138, 141]. A novel primate-specific exon (termed exon 6A) was identified within intron 6 of the LHCGR gene. This exon is not used by the wild-type full-length receptor. It displays composite characteristics of an internal/terminal exon and possesses stop codons triggering nonsense-mediated mRNA decay in LHCGR. When exon 6A is utilized, it results in a truncated LHCGR protein [143].

In 1976, Berthezene et al. [144] described the first patient with Leydig cell hypoplasia and subsequently several cases have been reported [145-148]. The clinical features are heterogeneous and result of a failure of intrauterine and pubertal virilization. The analysis of 8 of our cases and the 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 epididymis and vas deferens and absence of uterus and fallopian tubes, 5) low testosterone levels despite elevated gonadotropin 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 [138, 149-152].

Several different mutations in the LHCGR gene were reported in patients with Leydig cell hypoplasia [139, 140, 153-158].

In contrast to the homogenous phenotype of the complete form of Leydig cell hypoplasia, the partial form features a broad spectrum, ranging from incomplete male sexual differentiation characterized by micropenis and/or hypospadias to hypergonadotropic hypogonadism without ambiguity of the male external genitalia [139, 140, 150, 158-160]. Testes are cryptorchidic or in the scrotum and 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. Before puberty, the testosterone response to the hCG test is subnormal without accumulation of testosterone precursors. After puberty, LH levels are elevated as a result of insufficient negative feedback of gonadal steroid hormones on the anterior pituitary and testosterone levels are intermediate between those of children and normal males.
Several mutations in the LHCGR gene have also been identified in patients with the partial form of Leydig cell hypoplasia. Latronico et al. [139] first reported a homozygous mutation in the LHCGR (p.Ser616Tyr) in a boy with micropenis. Subsequently, other milder mutations were identified in further patients with the partial form of Leydig cell hypoplasia [140, 158, 159]. In vitro studies showed that cells transfected with LHCGR gene containing these mutations had an impaired hCG-stimulated cAMP production [140, 158].

Leydig cell hypoplasia was found to be a genetic heterogenous disorder since Zenteno et al. [161] ruled out, by segregation analysis of a known polymorphism in exon 11 of the LHCG receptor gene, molecular defects in the LHCG receptor as being responsible for Leydig cell hypoplasia in three siblings with 46,XY DSD. Most inactivating mutations of the LHCGR are missense mutations that result in a single amino acid substitution in the LHCGR. In addition, mutations causing amino acid deletions, amino acid insertions, splice acceptor mutation or premature truncations of the receptor have also been reported [142]. These mutations are usually located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction.
Although it is well known that hCG and LH act by stimulating a common receptor, a differential action of them in the LHCGR has been suggested. The identification of a deletion of exon 10 of the LHCGR in a patient with normal male genitalia at birth, but no pubertal development indicated that the mutant LHCGR was responsive to fetal hCG, but resistant to pituitary LH. The binding affinity of hCG for LHCGR was normal in vitro analysis, suggesting that exon 10 is necessary for LH, but not for hCG action [162].

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 analyzing the exon 6A in 16 patients with 46,XY DSD due to Leydig cells hypoplasia without molecular diagnosis, detected mutations (p.A557C or p.G558C) 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 [143].

A new compound heterozygous mutation of the LHCGR, constituted by a previously described missense mutation (p.Cys13Arg) and a large deletion of the paternal chromosome 2 was identified by array-Comparative Genomic Hybridization (array-CGH) in a 46,XY infant with sexual ambiguity and low hCG-stimulated testosterone levels associated with high LH and FSH levels [163].

In addition, causative mutations in LHCGR were absent in around 50% of the patients strongly suspected to have Leydig cell hypoplasia. These findings supported the idea that other genes must be implicated in the molecular basis of this disorder.

We observed that 46,XX sisters of the patients with 46,XY DSD due to Leydig cell hypoplasia, carrying the same homozygous mutation in the LHCGR, have primary or secondary amenorrhea, spontaneous breast development, infertility, normal or enlarged cystic ovaries with elevated LH and LH/FSH ratio, normal estradiol and progesterone levels for early to mid-follicular phase, but not for luteal phase levels, confirming lack of ovulation [139, 150, 152, 164, 165]. Our findings were subsequently confirmed by other authors who studied 46,XX sisters of 46,XY DSD patients with Leydig cell hypoplasia [166-168].
Subsequently, a novel homozygous missense mutation, p.N400S, has been identified by whole genome sequencing in two sisters with empty follicle syndrome [169].

46,XY DSD due to testosterone synthesis defect

Six enzymatic defects that alter the normal synthesis of testosterone have been described to date (Figures 7a and 7b). 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%.

Figure 7b- The alternative pathway to DHT. This pathway involves the conversion of 17OHP to P-diol via 5a-RD1 and AKR1C2/4. P-diol is cleaved to androsterone and reduced to androstanedione which is oxidized in peripheral tissues to DHT [170].

DEFECT IN CORTICOSTEROID AND TESTOSTERONE SYNTHESIS

Adrenal hyperplasia syndromes are examples of hypoadrenocorticism or mixed hypo- and hyper corticoadrenal steroid secretion. Synthesis of cortisol 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.

Deficiency of the acute steroidogenesis regulatory protein (StAR)

Congenital lipoid adrenal hyperplasia

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 [171].

It is the most severe form of congenital adrenal hyperplasia [172]. 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 [173]. 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 [174].

Hormonal diagnosis is based on 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 [175]. 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 [176]. Congenital lipoid adrenal hyperplasia (LCAH) in most Palestinian cases is caused by a founder c.201_202delCT mutation causing premature termination of the StAR protein [177]. Histopathological findings of excised XY gonads included accumulation of fat in Leydig cells since 1 yr of age, positive placental alkaline phosphatase and octamer binding transcription factor (OCT4) staining indicating a neoplastic potential [177].

A two-hit model has been proposed by Bose et al. [178] as the pathophysiological explanation for LCAH. In response to a stimulus (e.g. ACTH), the normal steroidogenic cell recruits cholesterol from endogenous synthesis, stored lipid droplets or low-density lipoprotein-receptor mediated endocytosis. Subsequently StAR promotes the cholesterol transport from the outer to the inner mitochondrial membrane in which cholesterol is further processed to pregnenolone. In cells with mutant StAR (first hit), there is no rapid steroid synthesis, but still some StAR-independent cholesterol flows into the mitochondria, resulting in a low level of steroidogenesis. Due to increased steroidogenic stimuli in response to inadequately low steroid levels, additional cholesterol accumulates. Massive cholesterol storage and resulting biochemical reactions eventually destroy all steroidogenic capacity (second hit) [178]. This two-hit model has been confirmed by clinical studies [179, 180] as well as StAR knockout mice research [181].

The human STAR gene is localized on chromosome 8p11.2 and consists of seven exons [182]. It is translated as a 285-amino acid protein including a mitochondrial target sequence (N terminal 62 amino acids), which guides StAR to the outer mitochondrial membrane and a cholesterol binding site, which is located at the C-terminal region. In vitro studies revealed that StAR protein lacking the N terminal targeting sequence (N-62 StAR) can still stimulate steroidogenesis in transfected COS-1 cells, whereas mutations in the C-terminal region lead to severely diminished or absent function [183-185]. Most of the STAR gene mutations associated with LCAH are located in the C-terminal coding region between exon 5 and 7 StAR related lipid transfer (START) domain [186]. Mild phenotype of lipoid CAH was a recognized disorder caused by StAR mutations that retain partial activity [187]. Affected males can present with adrenal insufficiency resembling to autoimmune Addison disease with micropenis or normal development with hypergonadotropic hypogonadism [187, 188]. More than 40 StAR mutations causing classic lipoid CAH have been described [178, 186, 189, 190], but very few partial loss-of-function mutations have been reported [187-189]. Therefore, there is a broad clinical spectrum of StAR mutations, however, the StAR activities in vitro correlate well with clinical phenotypes [191].

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 essential to maintain pregnancy. In rodents and some other animals, the mother’s corpus luteum of pregnancy produces progesterone throughout gestation, consequently, Cyp11a1 knockout mice reach term without difficulty [185]. However, in humans, pregnancy is characterized by a second-trimester “luteo-placental shift” wherein the mother’s corpus luteum involutes and placental progesterone biosynthesis takes over. Thus this statement would predict that mutations in P450scc would be incompatible with term gestation [186].

Nevertheless, a number of patients with CYP11A1 mutations have now been described [187-191], including late-onset non-classical forms secondary to mutations that retain partial enzyme activity [191-194]. Clinically, these patients are indistinguishable from those with lipoid CAH, but none of them present enlarged adrenals that characterize lipoid CAH. Once the majority of these patients have born prematurely following unsuppressible labor, it appears that the maternal corpus luteum may simply survive longer in these pregnancies, but this hypothesis remains unproven [186].

Analyzing infants with adrenal failure and disorder of sexual differentiation compound heterozygous mutations in CYP 11A1 have been identified, recognizing that this 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. [191].

3b-Hydroxysteroid Dehydrogenase type II Deficiency

3b-HSD converts 3b-hydroxy 5steroids to 3-keto 4steroids and is essential for the biosynthesis of mineralocorticoids, glucocorticoids and sex steroids Two forms of the enzyme have been described in man: the type I enzyme which is expressed in placenta and peripheral tissues such as the liver and skin, and type II that is the major form expressed in the adrenals and gonads [192]. 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 [193].
Male patients with 3b-HSD type II deficiency present with ambiguous external genitalia, characterized by micropenis, proximal hypospadias, bifid scrotum and a blind vaginal pouch associated or not with salt loss [194]. Gynecomastia is common at pubertal stage.

Serum levels of D-5 steroids such as pregnenolone, 17OHpregnenolone (17OHPreg), DHEA, DHEAS are elevated and basal levels of 17OHPreg and 17OHPreg/17OHP ratio are the best markers 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 [195].
The human genome encodes two functional 3HSD genes on chromosome 1p13.1. The HSD3B2 gene is expressed in adrenal and gonads and consists of four exons coding for a 372 aminoacid protein [196]. To date, around 40 mutations in HSD3B2 gene have been described. Most of them are base substitutions, and they are located especially at the N-terminal region of the protein. The amino acids A10, A82, P222 and T259 could be considered as mutational hotspots since different mutations were reported in these HSD3B2 positions.

Mutations abolishing 3b-HSD type II activity lead to congenital adrenal hyperplasia (CAH) with severe salt-loss [171, 193, 197, 198]. 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 [199, 200]. 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-HSD3 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 underwent orchiectomy in childhood and kept the female social sex; the other did not undergo orchiectomy at childhood and changed to male social sex at puberty [195].

Combined 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 zone of the adrenal cortex and gonadal tissues. The first activity results in hydroxylation of pregnenolone and progesterone at the C(17) position to generate 17a-hydroxypregnenolone and 17a-hydroxyprogesterone, while the second enzyme activity cleaves the C(17)-C(20) bond of 17a-hydroxypregnenolone and 17a-hydroxyprogesterone to form dehydroepiandrosterone and androstenedione, respectively. The modulation of these two activities occurs through cytochrome b5, necessary for lyase activity [201].

Deficiency of adrenal 17-hydroxylation activity was first demonstrated by Biglieri et al. [202]. 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 hypokalemia. New in 1970, reported the first affected patient with ambiguous genitalia which was assigned to the male sex [203].

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 females and sought treatment due to primary amenorrhea or lack of breast development. Genetic 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 [204, 205]. 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 [206].

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, 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 [207]. 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 suggest that its use should reduce the misdiagnosis of this deficiency in patients presenting with male DSD, primary or secondary amenorrhea, and mineralocorticoid excess syndrome.

Excessive production of deoxycorticosterone and corticosterone results in systemic hypertension, 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 is mapped at 10q24.3 [208]. Several mutations in the CYP17 gene have been identified in patients with both 17-hydroxylase and 17,20 lyase deficiencies [204, 205, 207, 209]. Four homozygote mutations, p.A302P, p.K327del, p.E331del and p.R416H, were 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 [209].

Glucocorticoid replacement for hypertension management, gonadectomy and estrogen replacement at puberty for patients reared in the female social sex are indicated. 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 [209].

Cytochrome P450 reductase (POR) deficiency (electron transfer disruption)

The apparent combined P450C17 and P450C21 deficiency is a rare variant of congenital adrenal hyperplasia, first reported by Peterson et al in 1985 [210]. 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 skeletal 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 [173]. 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 [211].
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 [31, 212]. 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, phalangeal malformation (arachno-/campto-/clinodactilyly, brachytelephalangia, rocker bottom feet) and urogenital abnormalities [213]. The occurrence of genital abnormalities in patients with Antley-Bixler syndrome, especially females was reported in 2000 [214]. In a recent large survey of patients with Antley-Bixler syndrome, it was demonstrated that individuals with an Antley-Bixler-like phenotype and normal steroidogenesis have FGFR2 mutations, whereas those with ambiguous genitalia and altered steroidogenesis have POR deficiency [215]. The skeletal malformations observed in many, but not all patients with POR deficiency, are thought to be due to disruption of enzymes involved in sterol synthesis, 14a-lanosterol demethylase (CYP51A1) and squalene epoxidase, and disruption of retinoic acid metabolism catalyzed by CYP26 isoenzymes that depend on electron transfer from POR [216].

Pubertal presentation in seven patients with congenital POR deficiency described clinical and biochemical characteristics of patients during puberty. Incomplete pubertal development and large ovarian cysts prone to spontaneous rupture were the predominant findings in females. The ovarian cysts may be driven not only by high gonadotropins but possibly also by impaired CYP51A1-mediated production of meiosis-activating sterols due to mutant POR. In the two boys evaluated, pubertal development was more mildly affected, with some spontaneous progression. These findings may suggest that testicular steroidogenesis may be less dependent on POR than adrenal and ovarian steroidogenesis [217].

DEFECTS IN TESTICULAR STEROIDOGENESIS

There defects in testosterone synthesis that are not associated with adrenal insufficiency have been described: CYP17 deficiency (17,20 lyase activity), cytochrome B5 deficiency 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 [218]. Later, clear molecular evidence of the existence of isolated 17,20 desmolase deficiency was demonstrated [201, 205, 219].

The patients present ambiguous genitalia with micropenis, proximal hypospadias and cryptorchidism. Gynecomastia Tanner stage V can occur at puberty [219].

Elevated serum levels of 17-OHP and 17-OHPreg, with low levels of androstenedione, dehydroepiandrosterone and testosterone are found. 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 [219]. 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 [219].

Cytochrome B5 deficiency (allosteric factor for P450c17 and POR interaction)

In 1994, Hegesh et al described a 46,XY DSD patient with type IV hereditary methaemoglobinemia [220]. 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 cytocrome b5, acts as an allosteric factor, promoting the interaction of. P450c17 and POR favoring 17,20 lyase reaction [201].

Two homozygous mutations in CYB5 in 46,XY DSD patients with elevated methaemoglobinemia levels but without clinical phenotype of methaemoglobinemia were reported [221, 222].

46,XY DSD due to 17-HSD 3 Deficiency

This disorder consists in a defect in the last phase of steroidogenesis, when androstenedione is converted to testosterone and estrone to estradiol. This disorder was described by Saez and his colleagues [223] and is the most common disorder of androgen synthesis, reported from several parts of the world [224, 225].

There are 5 steroid 17b-HSD enzymes that catalyze this reaction [226] and 46,XY DSD results from mutations in the gene encoding the 17b-HSD3 isoenzyme [7, 226]. 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 [227, 228], but some have less severe defects in virilization and are raised as males [226]. 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 [226]. 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 main origin of this androgen is the testis [226, 228]. 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 [227, 229].
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 reach the normal adult male range [228].
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 [226, 230].

Most patients are raised as girls during childhood. Change to male gender role behavior at puberty has been frequently described in individuals with this disorder who were reared as females [228, 231-233] including members of a large consanguineous family in the Gaza strip [234].
A higher risk of tumor development (28%) has been reported in 46,XY DSD patients due to 17b-HSD3 deficiency [4]. However, this high frequency was based on the gonadal tissue analysis of only 7 patients with 17b-HSD3 deficiency [3].

In fact, there are 34 reports of histological analysis of testicular tissue stained with hematoxylin-eosin in patients with 17b-HSD3 deficiency and the prevalence of germ cell tumor is actually 5.8% [227, 228, 235-237]. Therefore, the evidence to support the statement not to encourage patients to assume male gender role due to the risk of gonadal malignancy, is not defendable [170, 225] and we consider that the maintenance of the testes in patients with male social sex is safe when the testes can be positioned into the scrotum.

46,XY DSD due to 3a-hydroxysteroid dehydrogenase deficiency (AKR1C2 and AKR1C4 defects)

Molecular analysis of the patients initially described, in 1972, as having 46,XY DSD due to isolated 17,20-lyase deficiency failed to find mutations in CYP17A1 [218]. The hormonal data were inconsistent with other enzymatic deficiencies, then the alternative or backdoor pathway was considered to explain the etiology of the DSD in these patients. The backdoor pathway was firstly described in marsupials and is remarkable for having both reductive and oxidative 3a-HSD steps: the reductive reaction converts 17-OH dihydroprogesterone (17OH-DHP) to 17OH-allopregnanolone (17OH-Allo), and the oxidative reaction converts androstanediol to DHT [170, 238, 239] (Figure 7). Therefore, synthesis of dihydrotestosterone (DHT) occurs without the intermediacy of DHEA, androstenedione or testosterone [239]. All the human genes participating in the backdoor pathway have not been identified, however it has been thought that the reductive 3a-HSD activity can be catalyzed by an aldo-keto reductase called AKR1C2) [240], and possibly by other enzymes as the oxidative 3a-HSD activity by 17-HSD6, also called as RoDH [241] and possibly by AKR1C4 [242].

The initially reported cases with isolated 17,20 lyase deficiency from 1972 [218] were found to carry mutations in two aldo-keto reductases, AKR1C2 and AKR1C4 which catalyze 3a-hydroxysteroid dehydrogenase activity. The two affected 46,XY females were compound heterozygotes for AKR1C2 mutations, the p.I79V/H90Q and p.I79V/N300T. However the mutant AKR1C2 enzymes retained 22-82% of wild-type activity in vitro analysis suggesting that another gene was probably involved. Analysis of AKR1C cDNA found that AKR1C4 was spliced incorrectly and gene sequencing displayed an intronic mutation 106 bases upstream from exon 2 that caused this exon to be skipped. So, in this family, a digenetic inheritance was found to impair testicular synthesis of DHT during prenatal life [243].

AKR1C2 is abundantly expressed in the fetal testis, but minimally expressed in the adult testis; on the other hand, the AKR1C4 was found in fetal and adult testes at lower levels. Therefore, it appears that both AKR1C2 and AKR1C4 participate in the backdoor pathway to DHT in the fetal testis, and that their defects appears to cause incomplete male genital development. However, the relative roles of these two AKR1C enzymes remain unclear and testosterone levels at adult age are not available in these patients.

The finding described above, which substantially advanced our understanding of the mechanisms by which male sexual differentiation occurs, illustrates the importance of detailed studies of rare patients who appear to have 17,20 lyase deficiency [201].

46,XY DSD DUE TO DEFECTS IN TESTOSTERONE METABOLISM

5a-Reductase type 2 Deficiency
In 1974 a rare autosomal form of 46,XY DSD was described in 2 families, one from Dallas [244] and one from the Dominican Republic [245], 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 5a-reductase enzymes that catalyze this reaction [246-248] and 46,XY DSD results from mutations in the gene encoding the steroid 5a-reductase 2 isoenzyme (SRD5A2) [249-251]. The gene that codifies 5a-RD2 contains 5 exons and 4 introns and is located in chromosome 2p23.

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 [7]. 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 5a-RD2 deficiency is with 17b-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 [7, 252]. 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 5a-reductase type 2 deficiency [253]. Elevated 5b/5a urinary metabolites ratio is also an accurate method to diagnose 5a-reductase 2, even at prepubertal age or in orchiectomized adult patients [253, 254].

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 [245, 251, 255, 256]. In a few cases of 46,XY DSD due to 5-a reductase 2 deficiency diagnosed by clinical and hormonal findings no mutations were identified in 5a-RD2 gene [245, 249, 251, 255, 256]. Recently, Chavez et al. (2000) described a different mode of transmission of 5-a reductase type 2 deficiency in 2 unrelated patients due to uniparental disomy [257].

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, 257). In our experience with 30 cases of 46,XY DSD due to 5-a-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, 253]. 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 [253]. The patients treated at a later age referred severe social inadequacy, psychological anguish and suicidal ideas and all of them declared they would have liked 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 of highly viscous semen due to a rudimentary prostate and underdeveloped seminal vesicles and need 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, 253, 258].

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, but 8 of them have satisfactory sexual activity. The patients who change to male sex are more socially adapted than 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.

Steroid 5a-RD2 deficiency should be included in the differential diagnosis of all newborns with 46,XY DSD with normal testosterone production before gender assignment or any surgical intervention because these patients should be considered males at birth [259].

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 [260].

The human androgen receptor (AR) complementary DNA was cloned in 1988 and the AR gene was located on the long arm of the X chromosome at Xq11-12 [261]. AR gene is composed of eight exons and seven introns and encodes a protein of 920 amino acids. Like other members of the nuclear receptor superfamily, the AR, is modular in structure and is composed of four major functional domains: the N-terminal transactivation domain (TAD), a central DNA-binding domain (DBD), a C-terminal ligand-binding domain (LBD), and a hinge region connecting the DBD and LBD [262]. Two autonomous transactivation functions, a constitutively active activation function (AF-1) originating in the N-terminal and a ligand-dependent activation function (AF-2) arising in the LBD, are responsible for the transcriptional activity of nuclear receptors [263].

The NTD is encoded by the first exon and this domain harbours a polyglutamine repeat of 9-36 residues and a polyglycine repeat of 10-27 residues. The variability in repeat lengths within the normal range may affect transcriptional activity [264].

The DBD is encoded by exons 2 and 3 and consists of two zinc protein modules (P-box and D-box) followed by a C-terminal extension. The hinge region, encoded partially by exons 3 and 4, contains the bipartite nuclear localisation signal (NLS). The C-terminal extension of the second zinc module is also part of the hinge region and is involved in the specification of the selection of androgen response elements (AREs) [265].

The LBD is encoded by exons 4-8 and contains 11 a helices but without helix 2. The hydrophobic cleft formed by the helices 3, 4, 5 and 12 is referred to as AF2.

The AR is located in the cytoplasm and is associated with heat-shock proteins (hsp) and co-chaperones [266]. Binding of the ligand to AR induces an AR conformational change, the dissociation of the hsp and promotes AR nuclear translocation. The AR enters the nucleus via an intrinsic nuclear localization signal and the AR binds as homodimer to specific DNA elements present as enhancers in promoter sequences of androgen target genes. The direct and indirect communication of the AR complex with several components of the transcription machinery results in nuclear signaling and specific activation or repression of target gene transcription [267].

More than 800 mutations in the AR gene have been reported in AIS patients (www.androgendb.mcgill.ca/). They are distributed throughout the gene with a preponderance located in the ligand binding domain.

Complete Androgen Insensitivity Syndrome

Prenatal diagnosis of CAIS can be suspected based on 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 [268].

Whereas the clinical picture of CAIS is homogeneous, the phenotype of partial androgen insensitivity syndrome (PAIS) is quite variable and misdiagnosis with other causes of 46,XY DSD is more likely [227, 229]. 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 [269].

AR gene mutations are identified in most cases of CAIS and in several patients with PAIS. To date, more than 800 different AR gene mutations have been decribed and are listed in a database found in the web at http://androgendb.mcgill.ca/. Nonsense mutations as well as small deletions or insertions lead to a disruption of the reading frame, which lead to the generation of truncated, non-functional receptor protein [270, 271]. In frame deletions have been rarely identified. Missense mutations in the DNA binding domain may lead to a complete disruption of AR binding to AREs or only to an altered affinity and selectivity of AR-ARE interactions [272] associated respectively with CAIS and with PAIS or MAIS.

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 [273].

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 [274]. A specific role of certain coregulators in the pathophysiology of AIS is not established yet, although some mouse knock-out models of coactivators showed various degrees of androgen resistance [275].

A normal AR gene was found in a 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 [276].

Cox et al. have proposed a new mechanism for regulating steroid hormone receptor activity. The authors suggested that the FKBP52, a cochaperone of the steroid receptor complex phosphorylation can potentiate steroid receptor function [277]. 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 [278-280] This observation led to the screening for mutants in FKBP52 as a candidate gene for hypospadias in men, but no sequence variation could be associated with isolated hypospadias [281].

Patients with CAIS are raised as girls and have a female gender identity and role behavior [231, 269, 282]. 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.

Partial Androgen Insensitivity Syndrome (PAIS)

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 [269]. Danilovic et al 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 [283].

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 AIS in the same kindred was reported for p.M780I mutation in 3 patients, two females with CAIS and one male with PAIS with perineoscrotal hypospadias [284]. This distinct phenotypic variation can be attributed to differences in the availability of 5a-RD2 activity in genital skin fibroblasts [285]. The mutation p.Q798E was found in several patients with PAIS, who have been raised in the female sex [266, 286], and was also associated with infertility consisting the mild form of androgen insensitivity [287, 288]. A high variability of PAIS phenotypes was observed for the mutations p.L712F, p.I737T and p.F725L in the same family [289, 290]. Interestingly, these three mutations do not affect the ligand binding but disrupt AF2 and N/C-terminal interaction. It is not clear whether the phenotypic variability of PAIS mutations is related to a small set of mutations affecting key residues of AR function or if it could be applied to all mutations.

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 [291]. 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 5a-reductase 2 deficiency and 17b-hydroxysteroid dehydrogenase 3 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 [51, 269]. In another 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 [292].

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 [293].

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 [8, 293-295].

AMH is a small gene containing 5 exons, located in chromosome19p.13.3 [296] and its protein product acts through its specific receptor type 2 (AMHR2) a serine/threonine kinase, member of the family of type II receptors for TGF-b-related proteins [297].

Affected patients present a male phenotype, usually along with bilateral cryptorchidism and inguinal hernia [298]. 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 heterogeneous disorder that is inherited in a sex-limited autosomal 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 [299, 300]. In the remaining cases the cause of the persistent Mullerian duct syndrome is unknown [295].

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 orchiopexy, proximal salpingectomy (preserving the epididymis), and a complete hysterectomy with dissection of the vas deferens from the lateral walls of the uterus are indicated [301].

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 [302]. 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 to DHT due to competition with progesterone (also a substrate for 5a-reductase 2 action). The effect of estrogen use during gestation in the etiology of 46,XY DSD has not been confirmed to date [303]. Recently, a study in Japanese subjects supports the hypothesis that homozygosis 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 [304].

Environmental chemicals that display anti-androgenic activity via multiple mechanisms of action have been identified. They are: pesticides, fungicides, insecticides, plasticizers and herbicides. They can work as androgen receptor antagonists like pesticides, or they can decrease mRNA expression of key steroidogenic enzymes and also the peptide hormone insl3 from the foetal Leydig cells, like plasticizers and fungicides [305].
Daily exposure to 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 [306].

Supporting the idea that exposure to a mixture of chemicals can produce greater incidences of genital malformations, Rider et al examined the effects of exposure to a mixture of two chemicals that act as androgen receptor antagonists. They observed that the exposure to vinclozolin (fungicide) alone resulted in a 10% incidence of hypospadias and no vaginal pouch development in male rats, whereas procymidone, another fungicide exposure failed to generate malformations. However, the combined exposure resulted in a 96% incidence of hypospadias and 54% incidence of vaginal pouch in treated animals. Similar results were observed in phthalate (plasticizer) mixture studies [305].

Given that severe alterations of sexual differentiation can be produced in animal laboratory studies, the question arises of what would be expected in exposed humans given that humans are exposed to mixtures of compounds in their environment.

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 [307, 308]. Using the model of monozygotic twins, hypospadias has now been linked to low birth weight [307]. 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 [309]. 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 for sex differentiation has been shown [310].
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 [311-313].

46,XY ovotesticular DSD

There are rare descriptions 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 [314]. 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. MAMLD1 (mastermind-like domain containing 1) has been reported to be a causative gene for hypospadias [315]. It appears to play a supportive role in testosterone production around the critical period for sex development. To date, microdeletions involving MAMLD1 and nonsense and frameshift mutations in the gene have been found in 46, XY DSD patients, suggesting that MAMLD1 mutations cause 46,XY DSD primarily because of compromised fetal testosterone production, however, its role in the molecular network involved in fetal testosterone production is not known so far [316]

The activating transcription factor 3 (ATF3) expression was evidenced in the developing male urethra. Apparently ATF3 variants may influence the risk of hypospadias [281].

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 efforts to achieve such state. 46,XY gender identity disorders are more frequent among the male sex, although it also occurs in the female sex. Its first manifestations usually start during childhood. Its etiology remains unknown, although some hormonal alterations during intrauterus life and familial factors before and after birth cannot be ruled out [317].

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, 318, 319].

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 then follow the family periodically, more frequently during the periods before and after genitoplasty [320, 321] .

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, need 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 etiological 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 of estrogen 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 17-estradiol (0.5 mg daily) is kept as the patient presents progressive breast development. If breast development is not progressive, the estrogen dose is doubled. Low-dose transdermal hormone therapy is also a viable alternative estrogen replacement, offering lipid protection and preservation of bone mass. After 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 17b-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 [258] or surgical neovagina promote development of a vagina adequate for sexual intercourse after 6-10 months of treatment when patients desire to initiate sexual activity [322].

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 [323] 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 [251, 253].

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 surgical treatment is to allow development of adequate external genitalia and remove 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 [324]. 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.

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 [325]. 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 [258]. 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 [325].

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, 292, 326, 327].

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 (GB) 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 in 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 17-HSD3 deficiency and AR mutants (predominantly PAIS) results in enhanced risk of carcinoma-in-situ (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 [235] 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 [235].

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 [235].

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 cell tumors whereas testicular tissue displaying maturation delay of germ cells and stroma ovarian tissue can be safely be left in situ [235].

The risk for germ cell tumors is increased in patients with undescended testes, including all other 46,XY DSD syndromes [235]. Although data are limited, in the androgen insensitivity 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 have enabled 46,XY DSD patients to produce offspring [253, 328]. 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 [329].