Chapter 11 – 46,XY Disorders of Sexual Development

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

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

Updated December 2008

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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 not rare cause of these disorders. The dosage effect on gonadal development is sex limited, deletions or duplications of such genes can therefore manifest only in 46,XY or 46,XX subjects (Fig 1).

Figure. 1 - Summary of the molecular events in sex determination indicating the genes in which molecular defects can cause gonadal disorders in animal models. Some of these disorders were confirmed in humans. Sf1, Wnt4 and Wt1 are expressed in the urogenital ridge whose development results in formation of the gonads, kidneys and adrenal cortex. Several genes, Wt1, Sf1, Lhx9, Lim1, Gata4, Dmrt1, Emx2, Dhh, Wnt4 and Fgf9, are expressed in bipotential gonad. Sf1 and Wt1 up-regulate Sry expression in pre-Sertoli cells and initiates the male gonad development. Sry strongly up-regulates Sox9 in Sertoli cells. Sox9 up-regulates Fgf9 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. If Wnt4/Rspo1 is over expressed activating the b-catenin pathway this system blocks Fgf9 and disrupt the feed-forward loop between Sox9 and Fgf9. Sry inhibits beta-catenin-mediated Wnt signaling. Overexpression in either DAX1 (locus DSS) or WNT4/RSPO1 antagonizes testis formation. On the other hand, Dax1 regulates the development of peritubular myoid cells and the formation of testicular cords. Dmrt1, Atrx and Dhh are also involved in testes determination.

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 epidydimis, deferent ducts and seminal vesicles (Fig. 2).

Figure 2 - Summary of the molecular events in sex differentiation indicating the genes in which molecular defects cause 46,XY DSD in humans. After testis determination, hormones produced by the male gonad induce the differentiation of internal and external genitalia acting on their specific receptor. The regulation of AMH gene requires cooperative interaction between SOX9 and SF1, WT1, GATA4 and HSP70 at the AMH promoter. Combinatorial expression of DHH and SF1 is required for Leydig cell development. SF1 regulates gonadal steroidogenesis. The Leydig cells also produce the INSL3, which causes the testes to descend to the scrotum. Testosterone is further reduced to dihydrotestosterone (DHT), which acts on the androgen receptor of the prostate and external genitalia to cause its masculinization (Fig. 3-4).

Fig 3 – The development of male internal genitalia in human embryo. The 6-wk-end embryo is equipped with both male and female genital ducts derived from mesonephrons

Fig 3 A – The development of male internal genitalia in human embryo. The regression of the Müllerian ducts is mediated by the action of AMH secreted by the fetal Sertoli cells.

Fig 3 B – The development of male internal genitalia in human embryo The stabilization and differentiation of the Wolffian ducts are mediated by testosterone synthesized by the fetal Leydig cells. The enzyme 5a-reductase 2 converts testosterone to dihydrotestosterone (DHT). The Wolffian ducts differentiate into epdidymis, vas deferens and seminal vesicles. DHT contributes to prostate differentiation.

Figure 4 -The development of male external genitalia in human embryo. At the 8-wk-end embryo the external genitalia of both sexes are identical and have the capacity to differentiate in both direction: male or female. The DHT stimulates growth of the genital tubercle and induces fusion of urethral folds and labioscrotal swellings. It also inhibits growth of vesicovaginal septum preventing the development of the vagina.

Figure 4A -The development of male external genitalia in human embryo. At the 12-week-end embryo the male external genitalia is entirely formed.

Figure 4B -Development of male internal and external genitalia in human embryo. At the 12-week-end embryo both internal and external genitalia are completely formed.

The term disorders of sex development (DSD) include congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical. This nomenclature has been recentely proposed to replace terms such as intersex, pseudohermafroditism 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 recommend a new nomenclature that will be followed in this chapter (3, 4). The proposed changes in terminology aim integrate upcoming advances in molecular genetics in the new classification of DSD (5).

The 46,XY disorders of sex development (46,XY DSD) are characterized by ambiguous or female external genitalia, caused by incomplete intrauterine masculinisation with or without the presence of Mullerian 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 the impairment of androgen action (6) Our proposal classification of 46,XY DSD is displayed in Table 1 (5).

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 evalution of the neonate is fundamental because most of DSD patients may be recognized in this period and precocious diagnosis allow a better therapeutic approach. Family and prenatal history, general physical examination, assessment of genital anatomy are the first steps for a correct diagnosis. The diagnostic evaluation of DSD includes hormone measuremts, imaging, citogenetic and molecular studies and in some cases endoscopic, laparoscopic and gonadal biopsy (5).

The endocrinological evaluation of 46,XY DSD infants includes assessment of testicular function by basal measurement of LH, FSH, inhibin B and steroids.

Anti-mullerian hormone (AMH) and inhibin B are useful markers of the presence of Sertoli cells 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 testicular tissue absent (7, 8).

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

There are several hCG stimulation protocols and normative data have to be established to each of them. We establish normal testosterone response 72 and 96 hours after the last of 4 doses of hCG, 50-100 U/kg body weight, given intramuscularly every 4 days in boys with cryptorchidism but an otherwise normal external genitalia. Peak testosterone levels reached 391 ± 129 ng/dL and we consider a subnormal response a value <130 ng/dL (equivalent to -2 SD) (9).

Imaging is indicated in neonatal period when genital ambiguity are identified. If apparent female genitalia with clitoral hypertrophy, posterior labial fusion, foreshortned 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 haematuria in a male, inguinal hernia in a female require a imaging evaluation.

The 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 its echo texture This exam also can identified associated malformations as kidneys abnormalities (10).

Genitografy 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 to the planning of surgery.

The genetic evalution includes karyotype, FISH and more recently specific molecular studies to screen the presence of mutations or gene dosage imbalance.

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

46,XY DSD DUE TO ABNORMALITIES IN GONADAL DEVELOPMENT

Gonadal determination and differentiation: this process iniciates 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 (11, 12). Wt1 functions upstream of two orphan nuclear receptors: Sf1 and Dax1 (Dosage sensitive sex reversal, congenital adrenal hypoplasia, X chromosome) (13). SF1 gene prepares the ground for SRY expression, cooperating to express AMH, the first marker of testis differentiation (14, 15). 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 (16, 17). 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 (16, 18). 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 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 associated to form the coelomic vessel which promotes efficient export of testosterone. The gene Sox9 is up-regulated immediately after Sry expression initiates and is involved in the initiation and maintenance of Sertoli cell differentiation during early phases of testis differentiation (19). Extracellular signaling pathways (Fgf9 and Igf1r/Irr/Ir) play a significant role in Sox9 expression. Recently, a new 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 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. Sry inhibits -catenin-mediated Wnt signaling by a novel nuclear function of Sry (20). In the absence of this feed-forward loop between Sox9 and Fgf9, Wnt4/Rspo1 the activated -catenin pathway blocks Fgf9 and promote the ovarian fate (1, 21).

Abnormalities in the expression (underexpression or overexpression or time 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 malformed testes induce to ambiguous development of the genital ducts and/or external genitalia in these patients.

46,XY DSD due to ABNORMALITIES OF GONADAL DEVELOPMENT

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 (22). 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 (23). Mutations in SF1 and LHX9 were ruled out in these siblings (24, 25). The origin of this disorder remains to be determined, but a defect in other gene essential for bipotential gonad development is the most likely cause of this disorder.