46,XX testicular disorder of sexual development with SRY-negative caused by some unidentified mechanisms: a case report and review of the literature

46,XX testicular disorder of sex development (DSD) is a rare genetic syndrome, which is characterized by a complete or partial mismatch between genetic sex and phenotypic sex [1]. In addition, 46,XX testicular DSD always presents as one of three phenotypes: (1) classic XX males, infertility with normal male internal and external genitalia; (2) XX males with ambiguous genitalia, which is usually apparent at birth by external genital ambiguities, such as hypospadias, micropenis, or hyperclitoridy, and (3) XX true hermaphrodites with internal or external genital ambiguities detected at birth [2‐4]. The sex-determining region Y (SRY) gene plays a major role in encoding a testis determining factor (TDF), which is located on the Y chromosome [5, 6]. Approximately 80% of patients with 46,XX testicular DSD are SRY-positive and usually have a normal male phenotype at birth. Other SRY-negative 46,XX males exhibit different degrees of masculinization [7]. However, even though 46,XX males carry different phenotypes, these patients are often infertile because of the absence of the AZFa, AZFb and AZFc regions [8].

Here, we analyzed the clinical characteristics, chromosomal karyotype, and related genes in a male with 46,XX testicular DSD, who referred to our hospital because of abnormal gender characteristics. The aim of the present study was to investigate the different clinical characteristics in different categories of sex reversed 46,XX individuals and the relationships with variable clinical phenotypes and expression levels of the SRY, RSPO1, DAX1, SOX9, SOX3, SOX10, ROCK1, and DMRT genes. We also reviewed the literature to identify proposed mechanisms to explain the etiology of SRY-negative 46,XX sex reversal (Table 1).

The patient was treated for congenital scrotal type hypospadias at the age of 4 years. On physical evaluation, the patient’s weight was 51 kg (age-appropriate mean weight, 53.37 ± 9.29 kg) and height was 155 cm (age-appropriate mean height, 165.90 ± 7.21 cm). The grade of gynecomastia was IA [12]. Pubic hair was normal, the length of stretched penile was 3 cm and bilateral testicular volume was 2 mL. The results of laboratory analysis were as follows: T = 5.8 nmol/L (normal range, 9.4–37 nmol/L), E2 = 0.142 nmol/L (normal range, 0.129–0.239 nmol/L), FSH = 24.4 IU/L (normal range, 1.5–11.5 IU/L), LH = 15.7 IU/L (normal range, 1.1–8.2 IU/L), and PRL = 174.2 mIU/L (normal range, 89.04–826.8 mIU/L). Semen analysis by centrifugation of the ejaculate indicated azoospermia and no spermatogenic cells were observed. The levels of seminal plasma fructose, α-glucosidase, and acid phosphatase were 2.08 g/L (normal range, 0.87–3.95 g/L), 51.93 U/mL (normal range, 35.1–87.7 U/mL), and 12.37 U (normal range, 48.8–208.6 U), respectively.

The gonadal biopsy results showed hyalinization of the seminiferous tubules, but without evidence of spermatogenic cells (Figure 1). Consistent with gonadal biopsy, immunohistochemical analysis also confirmed that the gonadal tissue was immunoreactivity positive to inhibin and vimentin, respectively (Figure 2), which indicated the presence of Leydig cells. Karyotype analysis of the patient confirmed a 46,XX karyotype and FISH analysis was negative for the SRY gene (Figure 3). Molecular analysis revealed locus deletions at SY84, SY86, SY127, SY134, SY254, and SY255 within the AZF sequence on chromosome Y, with the absence of the SRY gene (Figure 4). Sequencing of the coding regions and exon/intron boundaries of the RSPO1, DAX1, SOX9, SOX3, SOX10, ROCK1, and DMRT genes detected no mutations. No copy number variation in the whole genome sequence was found. In particular, there was no variation in copy number between positions 64000 kb and 72000 kb of chromosome 17q24.2-25.1. Duplication of this region, which codes for SOX9, can trigger sex reversal (Figure 5).

46,XX testicular DSD is a rare form of sex reversal in infertile men, which was first described by la Chapelle et al. in 1964 with a frequency of 1:20,000 of newborn males [1]. Most males have normal phenotypes at birth and are usually diagnosed in adolescence because of delayed puberty, gynecomastia, or infertility. Moreover, some XX males have hypospadias, cryptorchidism, or more severe genital ambiguity. However, all 46,XX males are infertile because of the absence of the azoospermia factor region in the long arm of the Y chromosome. 46,XX males can be classified into two subgroups, SRY-positive and SRY-negative, according to the presence or absence of the SRY gene, which is located in the Y chromosome and regulates testicular differentiation [10]. Patients with SRY-positive, which always translocates to the X chromosome or to an autosome, are usually more likely to have a normal male phenotype at birth and are referred for infertility treatment after puberty. SRY-negative patients include those with ovotesticular-DSD, which is characterized by the presence of testicular and ovarian tissue in the same individual. Testicular-DSD is characterized by full development of both gonads as testes without evidence of ovarian tissue [13].

The mechanisms of testicular tissue induction in SRY-negative patients remain unknown, although several hypotheses have been proposed, as follows: (1) hidden gonadal mosaicism for SRY; (2) mutations in some autosomal or X-linked genes that repress the male pathway can result in de-repression of the male pathway in XX gonads; and (3) altered expression of other sex determining genes downstream of the SRY.

True hermaphroditism (TH), also known as ovotesticular-DSD, is a rare form of intersexuality characterized by the presence of testicular and ovarian tissue in the same individual. Genetic heterogeneity has been proposed as a cause of dual gonadal development in some cases and recently, hidden mosaicism was reported to cause TH in some SRY-negative 46,XX patients [14]. However, Domenice et al. [15] reported the absence of the SRY sequence in DNA from blood samples of all true hermaphrodites and in testicular and ovarian tissues of a 46,XX true hermaphrodite case and a 46,XX male with ambiguous genitalia, which indicated that cryptic SRY mosaicism in gonadal tissues is not the usual mechanism responsible for testicular development in patients with 46,XX true hermaphroditism.

R-Spondin1 (RSPO1) is a novel regulator of ovary development through the up-regulation of the Wnt/β-catenin signaling pathway to oppose testis formation [16, 17]. Loss-of-function mutations in human RSPO1 lead to reduced β-catenin protein and WNT4 mRNA levels, consistent with downregulation of ovarian pathways, which cause testicular differentiation in 46,XX females [18].

Previous reports have identified gene dysregulation in 46,XX SRY-negative DSD. The SRY-box 9 (SOX9) gene is a widely expressed transcription factor that plays several relevant functions during development and essential for testes differentiation, which is considered to be the direct target gene of the SRY [19]. The essential role of SOX9 in male gonadal development was initially described in mice and subsequent reports stated that SOX9 duplications were a relevant cause of SRY-negative XX sex reversal in dogs [20]. SOX9 duplication was reported for the first time in Korea in a case involving a 4.2-year-old SRY-negative 46,XX boy with complete sex reversal, which indicated that SOX9 duplication was a rare cause of 46,XX testicular DSD in humans [21]. The genomic domain regulating SOX9 expression spans more than 1 Mb upstream of SOX9 [11]. Benko et al. [22] revealed that a non-coding regulatory region located far upstream of the SOX9 promoter was critical for gonadal SOX9 expression and subsequent normal sexual development and copy number variations were the genetic basis of isolated 46,XX testicular DSD of variable severity. Moreover, the minimum critical region associated with gonadal development, about a 67-kb region located 584–517 kb upstream of SOX9, was confirmed to induce SOX9 overexpression in female-to-male sex reversal [11]. Regulatory elements in the duplication region might enhance SOX9 expression through complex mechanisms. It has been suggested that changes in SOX9 expression resulting from disruptions in some regions upstream of SOX9 could be important in 46,XX testicular disorder of sexual development. However, other groups have suggested that duplication in the region of 17q that contains SOX9 is not a common cause of testis development in subjects with SRY-negative 46,XX testicular or ovotesticular DSD [23].

Consistent with SOX9, the DAX1 gene might also function downstream of the SRY gene in the sex-determination pathway. Overexpression of the DAX1 gene could cause female-to-male sex reversal [24]. ROCK1 (Rho-associated, coiled-coil protein kinase 1) phosphorylates and activates SOX9 in Sertoli cells to initiate testes formation [13]. SOX3 was recently shown to upregulate SOX9 expression via a similar mechanism to SRY and modulate XX male sex reversal in humans through gain-of-function mutations mediated by genomic rearrangements around SOX3, possibly leading to its altered regulation [25‐27]. Overexpression of the SOX10 gene at 22q13 might be the cause of sex reversal [28].

However, the above mechanisms were not applicable in this case, as any hidden gonadal mosaicism for SRY or mutations to autosomal or sex-linked genes that repress the male pathway and alter expression of other sex determining genes downstream of SRY were found in our case. These findings suggest that other unidentified genetic or environmental factors play significant roles in the regulation of sex determination and differentiation.

46,XX SRY-negative individuals with complete masculinization are rare and usually exhibit phenotypic differences. Our patient had incomplete masculinization, which was characterized by microorchidism and mild bilateral gynecomastia, and was diagnosed with abnormal gender characteristics. His T level was low and levels of FSH and LH were elevated. Furthermore, we detected locus deletions at SY84, SY86, SY127, SY134, SY254, and SY255 within the AZF sequence on chromosome Y, with the absence of the SRY gene. Further studies are needed to explain the mechanisms of 46,XX testicular disorder of sexual development characterized by the described phenotype with a lack of spermatogenesis.

Written informed consent was obtained from the parent of the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.