Clinical, molecular and cytogenetic analysis of 46, XX testicular disorder of sex development with SRY-positive

The 46, XX disorder of sex development (DSD) is a rare form of sex reversal in infertile men, that was first described by la Chapelle et al. in 1964 and occurred 1:20000 in newborn subjects [1]. By 1996, 150 patients with classical XX male syndrome had been reported, and more than 100 cases of this disorder have been discribed between 1996 and 2006 worldwide [2]. Clinical phenotypes about 46, XX DSD have been identified to three groups, including males with normal phenotype, males with genital ambiguities and males with true hermaphrodites [3]. Ovotesticular DSD, which is characterized by the presence of both testicular and ovarian tissue in the gonads of the same individual, and testicular DSD characterized by a full development of both gonads as testes without any evidence of ovarian tissue [4]. Approximately 80% of individuals with 46,XX testicular DSD present after puberty with normal pubic hair and normal penile size, but small testes, and sterility resulting from azoospermia [5].

The sex-determining region Y gene (SRY) locating in Y chromosome, plays a major role in encoding a testis determining factor (TDF) [6, 7]. About 90% of these patients have Y chromosomal material including the SRY gene, that are usually translocated to the distal tip of the short arm of X chromosome or autosomal chromosomes. About 10% 46, XX males are negative for SRY gene, which could carry different degrees of masculinization [8, 9].

There are several pathogenic mechanisms explaining 46, XX testicular DSD patients: 1. translocation of Y sequences, including the SRY gene, to an X chromosome or to an autosome; 2. a mutation in a gene in the testis-determining pathway triggering testis differentiation in SRY negative XX males; and 3. a hidden Y chromosome mosaicism limited to the gonad [10].

This study aimed to describing five 46, XX male DSD with SRY-positive, investigating the clinical characteristics and their relationships with chromosomal karyotype and the SRY gene.

Our five patients had a lower body height. Physical examination revealed that their testes were small in volume, soft in texture and normal penis. No potential gynecomastia and congenital hypospadias were seen. And they all described that they had normal sexual function. Semen analyses showed azoospermia. Endocrinological data indicated that the patients had a higher FSH, LH level, lower free testosterone, testosterone level and normal Estradiol, Prolactin level. General characteristics and endocrine hormone levels are shown in Table 1.

Karyotype analysis of all patients confirmed 46, XX karyotype, and FISH analysis showed that SRY gene were positive and translocated to Xp (Figure 1). Molecular analysis revealed that the SRY gene was present, and the AZFa, AZFb and AZFc region were absent (Figure 2).

46, XX male syndrome is a rare sex reversal syndrome characterized by a female karyotype in discordance with a male phenotype. 90% of 46, XX testicular DSD usually have a normal male phenotypic heterogeneity at birth and are diagnosed after puberty on genital ambiguities, or infertility [8]. Our research reported that five patients had a female karyotype but were phenotypically male (46, XX males). They had normal external genitalia and masculinization, but showed azoospermia. That might be that all males were SRY-positive, which translocated on the short arm of X chromosome, and absent of the spermatogenic factors encoding gene on Yq, such as AZFa, AZFb and AZFc region in Y chromosome.

SRY gene is located in the Y chromosome and encodes a high mobility group(HMG) domain, a conserved motif present in many DNA-binding proteins, which could regulate testicular differentiation [11, 12]. SRY protein is expressed in the genital ridge before testis formation, and in the testis during the period of testicular formation early in fetal life, until the development of adult testis [13]. Molecular genetics analysis demonstrated that most 46, XX testicular DSD patients carry SRY gene which translocated to X chromosome [14‐16]. There was a report that an SRY gene fragment translocated from Y chromosome to autosomal chromosome [17]. Some patients showed SRY negative, who always had external genital ambiguities and gynecomastia. Despite the fact that SRY gene is considered to be the main regulatory factor for testis determination, phenotypic variability showed in 46, XX sex reversed cases cannot be explained only by whether SRY gene is presented. And a number of other genes such as SOX9, DAX-1, WT1, WNT4, FGF9 and RSPO1 have been involved in the process of gonadal differentiation [8].

The phenotype of the XX male observed in SRY positive 46,XX individuals varies greatly, from normal internal and external male gonads to abnormal secondary sexual characteristics, small testes and hypospadias, to a true hermaphrodite. It has been suggested that the variation in phenotype is primarily dependent on two mechanisms: X chromosome inactivation(XCI) pattern and the amount of Y material including SRY gene that has been translocated to the X chromosome [18]. Reviewing the literature, 46,XX males with true hermaphrodites or gonadal ambiguity have a small portion of the Y chromosome material translocated to the X, presumably allowing for XCI spreading and inactivating the SRY gene [19]. A normal male phenotype is expected to result from a larger Yp SRY bearing fragment being translocated to the X chromosome, where the length of the Yp fragment may protect the SRY gene from silencing by the spread of XCI [19]. In our study, all five cases have normal external genitalia and masculinization, which is expected that more Y chromosome material is present on the X, presumably protecting the SRY gene from the spread of inactivation. Because of the unavailable in specimens from the five cases to further study, we cannot do more molecular analysis to confirm the above point. Till now, both random and non-random XCI patterns have been reported in 46, XX males with a normal male phenotype [18, 20]. It is indicated that the XCI pattern may be not associated with the XX male phenotype.

However, another mechaniam, known as the position effect, has been reported to explain the observed phenotypic differences. The phenotypic differences are dependent on the proximity of the breakpoint to the SRY gene as well as the presence or absence of cryptic rearrangements affecting the expression of the SRY gene [21]. The rearrangements, may result in transcriptional repression, probably by removing essential regulatory elements or alterations of local chromatin structure [22].

Additionly, phenotypic variability might be associated with variations in genetic polymorphisms and copy number variation of specific genes on the X chromosome, such as NROB1 and TAF7L [23, 24].

Classical 46, XX male have normal testosterone level and free testosterone level during adolescence, but may decrease in adulthood, leading to hypergonadotropic hypogonadism [25]. Our cases had normal genitalia and were diagnosed for infertility after puberty. The level of testosterone and free testosterone is deficiency in five patients. In addition, high levels of FSH and LH are observed. This might explain that even though the 46, XX male have a normal external genitalia and masculinization, and they are lack of spermatogenesis.

The body heights of the patients we reported were all under 169 cm (the average height of Chinese male) and close to that of normal females. There are some phenotypic similarities between 46,XX men and those with Klinefelter syndrome, but 46,XX men tend to be shorter than men with KS [9]. Kirsch et al. indicated that the Y chromosome growth-control gene(GCY) which next to the centromere had a possible impact on growth [26]. And there were some papers indicating that SHOX gene(short stature homeobox) expression and SHOX enhancer regions played a role in the growth [27]. It has been suggested that specific growth genes in the Y chromosome cannot switched to the patients, which might make them to show a female stature. GH (growth hormone) therapy may have some statural effects in the SHOX haploinsufficiency and may be insufficient to prevent the development of skeletal lesions after puberty [28].