The critical gene for male sex determination, SRY (sex-determining region Y), which is located on chromosome Yp11.3, initiates gonads to differentiate into testes, induces Leydig cells to secrete testosterone, develops Wolffian ducts, and forms male external genitalia. At the same time, Sertoli cells secrete Müllerian inhibiting factor that induces regression of Müllerian ducts that in females would differentiate into the uterus. These processes for sex determination do not occur in females in the absence of SRY. Sex differentiation related genes such as SOX9, FGF9, DAX1, WT1, RSPO1, and SOX10, which are located on either autosomes or the X chromosome, may have a role in gonad development and function. These genes were studied in XX testicular/ovotesticular disorders of sex development (DSDs) in the absence of the SRY gene.

46,XX male sex reversal (also known as testicular DSD) is reported in 1:20,000 to 1:25,000 of newborn males [1], and is categorized using clinical phenotypes or molecular genetic analysis depending on the presence or absence of the SRY gene. Clinical sexual phenotypes of individuals with 46,XX testicular DSD range from complete masculinization to true hermaphrodites (also known as ovotesticular DSD) and are comprised of three groups: 1) XX testicular DSD with normal genitalia of which 85% of cases are normal males with internal and external genitalia, and are usually diagnosed after puberty because of hypogonadism, gynecomastia, and/or infertility [2]; 2) patients with XX testicular DSD with ambiguous genitalia are identified at birth by external genital ambiguities such as micropenises, cryptorchidism, or hypospadias; and 3) patients with XX ovotesticular DSD have internal and external genital ambiguities that are detected at birth or histologically—these patients have either an XX karyotype (60%) or a 46,XX/46,XY or 46,XY karyotypes.

46,XX testicular DSD is also classified genetically based on the presence or absence of the SRY gene [3]. In 90% of cases in which the SRY gene is present, the disorder is the result of an aberrant Y to X chromosomal interchange during meiosis I in paternal gametogenesis. Affected males show a similar phenotype compared to males with Klinefelter syndrome, which is characterized by a 47,XXY karyotype and consists of an active X chromosome, an inactive X chromosome, and the SRY gene. Rarely, the terminal region of the Y chromosome, that includes the SRY gene, is cytogenetically detectable on the X chromosome. For the majority of cases in which the SRY gene is absent, patients with 46,XX testicular DSD have genital ambiguities such as a micropenis, hypospadias, and cryptorchidism. However, completely virilized 46,XX testicular DSD with a SRY deficiency has rarely been reported in the literature. Therefore, other autosomal or X-linked genes may have regulatory roles with SRY in the sex-determination process.

We present three rare cases of SRY-negative 46,XX testicular DSD found in the past 31 years at our center, and our review of the literature on previous cases of 46,XX testicular DSD with a focus on recently reported related genes since the characterization of SRY.

1. Clinical findings and cytogenetic analysis

We retrospectively investigated the clinical records of 8,386 males who were referred for cytogenetic analysis from 1983 to 2013 at the Cheil General Hospital, Seoul, Korea. We examined hormonal profiles, histological findings, and semen analysis. For the hormonal profile, we evaluated the levels of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17β-estradiol, testosterone (T), prolactin, and sex hormone-binding globulin.

Conventional cytogenetic analyses were performed according to standard techniques. High-resolution chromosomes of 700-band levels by GTL-banding were prepared using cultured peripheral blood cells. We analyzed 100 metaphases of each patient to exclude potential sex chromosome mosaicism.

Detection of the SRY gene in some patients was confirmed using fluorescence in-situ hybridization (FISH) analysis with the Y-specific probes: CEP Y Sat III Spectrum Green/CEP Y Alpha Spectrum Orange (Vysis Inc., Downers Grove, IL, USA) and the LSI SRY Spectrum Orange/CEP X Spectrum Green (Vysis Inc.) (data not shown).

2. Molecular genetic analysis

Three individuals with 46,XX testicular DSD were further investigated using azoospermia factor (AZF) microdeletion analysis with multiplex polymerase chain reaction (PCR). The oligonucleotides used for multiplex PCR are described in our previous study [4]. Briefly, PCR was performed using the following cycle conditions: an initial denaturation at 95°C for 10 minutes, followed by 35 cycles at 95°C for 10 minutes, 62°C for 90 seconds, and 65°C for 90 seconds with a final extension at 65°C for 10 minutes. Each round of PCR that was performed using patient’s DNA included normal male DNA and normal female DNA as a positive and a negative control, respectively. Amplified products were separated on 3% agarose gel by electrophoresis and visualized using ultraviolet illumination.

3. Ethical considerations

This study was approved by the Ethics Committee of Cheil General Hospital and Women’s Health Care Center (#CGH-IRB-2014-47) and consent was obtained from all patients.

1. Clinical findings and cytogenetic analysis

Of 8,386 male individuals, we identified 1,172 cases with an abnormal karyotype, and 19 cases with 46,XX testicular DSDs. Of these 19 cases, three SRY-negative 46,XX testicular DSDs were confirmed experimentally, and all three cases showed azoospermia with small testes volumes. We found that these 19 patients with 46,XX testicular DSDs have serum concentrations that were partially out of range of normal hormonal profiles. Of those cases of XX testicular DSDs with the SRY gene, we found increased levels of LH, FSH, and T, whereas in cases of XX testicular DSDs without the SRY gene, we found increased levels of LH and FSH with or without decreased levels of T. Patient height ranged from 156 to 172 cm, and we found from our urological assessment that there were no cases of ovotesticular DSD or ambiguous genitalia. With regard to the three cases with 46,XX testicular DSDs without the SRY gene, all three patients showed masculinized external genitalia and small testes with azoospermia. Detailed clinical data is summarized in Table 1.

We analyzed 100 metaphase karyotypes of cultured lymphocytes from peripheral blood of the 19 cases with 46,XX testicular DSDs. All individuals showed a complete 46,XX karyotype. Regardless of the presence or absence of the SRY gene, we found that 0.23% (19/8,386 cases) of the male participants referred for cytogenetic analysis in our study had XX testicular DSDs. Furthermore, we found that 15.8% (3/19 cases) of cases with 46,XX testicular DSDs were SRY-negative or, alternatively, 0.26% (3/1,172 cases) of males with an abnormal karyotype in our cohort of 8,386 participants.

2. Molecular genetic analysis

Of the 16 sequence-tagged site (STS) markers tested, amplification products of 15 STS loci including the SRY gene (sY14) on the Y chromosome were not detected with the exception of ZFX (zinc finger protein, X-linked) located on chromosome Xp21.3, which indicates the complete absence of the Y chromosome (Fig. 1).

Through our study of 8,386 cases and a literature review (summarized in Table 2 [2,5-29]), we found that the major characteristics of male sex reversal for DSD were showed the clinical phenotypes of short stature without a T-dependent pubertal growth spurt and the Y-specific growth gene, azoospermia, internal or external genital abnormalities, gynecomastia, and high levels of FSH and LH, and a low level of T. However, a limitation of our retrospective study was that no gonadal biopsy samples were available for analysis and thus, gonadal mosaicism cannot be ruled out. Reports of familial cases of DSDs in the literature demonstrate that the various clinical phenotypes from ovotesticular DSD to masculinization may be because of incomplete penetrance of one or more genes.

The SRY gene has a critical role in the process of sex differentiation. However, the SOX9, FGF9, and MAP3K1 genes also have roles in the development of testis, the testis-androgen-genital tract to masculinization, anti-Müllerian duct regression, and the duct systems of external genitalia. In addition, RSPO1 and WNT4 function in gonads and have roles in ovary development, the duct system including fallopian tubes, the uterus, and external genitalia without the SRY gene [30,31]. These findings strongly indicate that DSDs with sex reversal involve the balanced regulation of genes in addition to the critical SRY gene, in the cascade of events that lead to development and sex differentiation.

McElreavey et al. [5] proposed the concept of a Z gene product that is a negative regulator of male sex determination. Therefore, homozygosity for loss-of-function and heterozygous mutants of Z alleles induce male sex reversal from SRY-negative complete masculinization to ovotesticular DSD.

Several familial studies have demonstrated that the varying clinical presentation of sexual development such as ovotesticular DSD or XX testicular DSD, may be a result of incomplete penetrance of paternal and maternal inheritance, while dosage-sensitive sex reversal may be a result of autosomal or X-linked genes in the sex-determining cascade [6,7,32].

DAX1 (also known as NROB1), an ‘anti-testis’ factor located on chromosome Xp21 that when duplicated was found to cause XY sex reversal. It has a role in ovarian development and/or in testis formation dependent on dosage-sensitivity during sex determination or sex differentiation pathways in mammals [32,33]. It was found that Dax1 deficiency caused varying degrees of sex reversal in mice [34,35]. In humans, it was found that duplication of the Xp21.3 region containing the DAX1 gene causes testicular regression in the presence of SRY, while the deletion of this region had no effect on male sex determination [36].

Haploinsufficiency of SOX9 mutations have been proposed to cause sex reversal in patients with XY sex reversal, skeletal malformation syndrome, and campomelic dysplasia [37,38]. Huang et al. [8] reported that XX sex reversal was with campomelic dysplasia is caused by duplication of the SOX9 gene, which is located on chromosome 17q24.3. Vernole et al. [9] reported XX testicular DSD with Huriez syndrome and palmoplantar keratoderma, and suggested that a gene related to sex determination that cause Huriez syndrome or tylosis, may be located at the SOX9 locus. Radi et al. [10] reported that male sex reversal with palmoplantar keratoderma/squamous cell carcinoma that was paternally inherited in the same family, would be homozygous for a single gene mutation or contiguous genes. Moreover, increased expression of SOX9 and reduced expression of Ad4BP/SF-1 (also known as NR5A1), DAX1 and anti-Müllerian hormone (AMH, also known as Müllerian inhibiting substance) in SRY-negative XX males, indicates that SOX9 has a key role in sex determination in SRY-negative XX males, and that Ad4BP/SF-1, DAX1 and AMH may contribute to their clinical features [11].

In contrast, Rajender et al. [12] found that there was no evidence of a relationship between SOX9, duplication of 22q, and DAX1 in complete XX testicular DSD without SRY. Temel et al. [13] reported that SOX9 was not duplicated, and no common SOX9 haplotype was shared in nine familial cases with the absence of SRY, and a mutation at the SOX9 locus; the authors proposed that SRY-negative XX testicular DSD may be caused by a monogenic impairment. Maciel-Guerra et al. [14] found no mutations in the SOX9 and DAX1 genes in case with SRY-negative XX maleness (testicular DSD) and monozygotic twins with XX ovotesticular DSD, but showed varying expression of incomplete penetrance of either an autosomal or a X-linked mutation. Seeherunvong et al. [15] investigated 30 subjects without SRY-negative XX testicular or ovotesticular DSD and did not detect any mutations or duplications in the region of chromosome 17q that contains the SOX9 gene using three short tandem repeat (STR) markers.

However, specific critical regions within the SOX9 gene have been recently identified using microarray techniques. Cox et al. [16] found that three family members with 46,XX testicular DSD have a 178-kb microduplication of a gene desert region located 600-kb upstream of SOX9 using SNP-microarray. Vetro et al. [17] found that two brothers who had the same paternal haplotype at the SOX9 region had a 96-kb triplication of a region 500-kb upstream of SOX9 at chromosome 17q24.3 using oligonucleotide array-comparative genomic hybridization. Benko et al. [18] proposed that a 78-kb minimal non-coding region found in a gene desert 517- to 595-kb upstream of the SOX9 promoter included regions suggested by Cox et al. [16] and Vetro et al. [17], and that this region may have one or more gonad-specific SOX9 transcriptional enhancers to induce activation or inactivation of SOX9 gonadal expression in a tissue-specific manner. Xiao et al. [19] narrowed the candidate region to a 74-kb duplication in a 510- to 584-kb region upstream of SOX9 in SRY-negative 46,XX testicular DSD, and proposed based on previous studies, that the candidate region related to gonadal development is a 67-kb region located 584- to 517-kb upstream of SOX9. Recently, there was a report of a SOX9 duplication (~1.45 times) found in a Korean boy with XX testicular DSD [20].

Chiang et al. [21] investigated the FGF9, WT1, NR5A1, and SPRY2 genes in cases with SRY-negative 46,XX testicular DSD and found that FGF9 (located at chromosome 13q12.11) copy number was duplicated compared to that found in normal female controls and was significantly lower that of the normal male controls. Mizuno et al. [22] found that Leydig and Sertoli germ cells have increased expression levels of the Rho-associated, coiled-coil protein kinase 1 (ROCK1) protein, and proposed that testis formation may be regulated by an alternative ROCK1 signaling pathway.

Similar to SOX9, gain of function of the SOX3 (SRY-box 3) gene showed it may be regulated by SRY in sex determination of transgenic mice, and in patients with XX male sex reversal [39]. More recently, Mizuno et al. [23] found copy number gain in the upstream region of the SOX3 gene at chromosome Xq27.1.

A mutation in the RSPO1 (R-spondin 1) gene in patients with 46,XX male sex reversal in an Italian family has demonstrated that sex reversal and palmoplantar hyperkeratosis/squamous cell carcinoma are regulated under via RSPO1 stimulation of keratinocytes and a reduction of β-catenin in the affected keratinocytes [40].

At other autosomal loci, deletion of the short arm of chromosome 11 led to the suggestion that the WT1 (Wilms tumor 1) gene has a role in testis determination [41], while the SF-1 gene has roles in primary adrenal failure and 46,XY gonadal dysgenesis [42]. Aleck et al. [24] reported a partial duplication of 22q13.1 (SOX10) in ovotesticular DSD without SRY. Jiménez et al. [43] proposed ‘vanishing mosaicism’ with 46,XX ovotesticular DSD without SRY; partial deletion of the SRY gene forms a testicular structure, and that an inactivated X chromosome carrying the SRY gene results in the development of ovarian tissue. Mustafa and Mehmet [25] reported an SRY-negative man with complete masculinization and autoimmune thyroiditis, but did not test for any specific genes.

In summary, we report three rare SRY-negative 46,XX testicular DSD cases with complete masculinization and provide a literature review on XX testicular DSD. Although SRY has a master role in sex determination and differentiation, there is a tightly coordinated expression between related genes under SRY for sex development from bipotential gonads. Therefore, the development of maleness in the absence of the SRY gene may be a result of a disruption of this balanced gene expression and gene mutation. Over the past two decades, several genes such as SOX9, SOX3, DAX1, and RSPO1, and chromosomal regions have been reported as potentially critical candidate genes for sex reversal. However, a more comprehensive understanding of other genes that are involved in the underlying networking mechanisms of the sex-determination cascade is necessary.