Analysis of STAG3 variants in Chinese non-obstructive azoospermia patients with germ cell maturation arrest

STAG3 is essential for male meiosis and testis of male Stag3−/− mice shows the histopathological type of germ cell maturation arrest (MA). Whether variants of the STAG3 gene exist in Chinese idiopathic non-obstructive azoospermia (NOA) patients needs to be determined. We recruited 58 Chinese NOA men with MA who underwent testis biopsy and 192 fertile men as the control group. The 34 exons of the STAG3 gene were amplified using polymerase chain reaction (PCR) and sequenced. We identified eight novel single nucleotide polymorphisms (SNPs), including two missense SNPs (c.433T > C in exon2 and c.553A > G in exon3), three synonymous SNPs (c.539G > A, c.569C > T in exon3, and c.1176C > G in exon8), and three SNPs in introns. The allele and genotype frequencies of the novel and other SNPs have no significant differences between two groups. Our results indicated that variants in the coding sequence of the STAG3 gene were uncommon in NOA patients with MA in Chinese population. Future studies in large cohorts of different ethnic populations will be needed to determine the association between the STAG3 gene and NOA.

www.nature.com/scientificreports/ The stromal antigen 3 (STAG3) is involved in formation of cohesin core with three other proteins including SMC1β and two α-kleisins (RAD21L and REC8), and required for synaptonemal complex formation during meiosis 21 . Stag3 -/male mice showed no overt phenotype apart from sterility, which is due to azoospermia and meiotic arrest 21,22 . Notably, Stag3 -/spermatocytes only reached zygotene-like stage of prophase I, and apoptosis occurred 23 . These results suggest that STAG3 plays an essential role in meiosis and may be a candidate gene for NOA patients with MA 24 . In this study, we investigated whether perturbations of the STAG3 gene were present in Chinese idiopathic NOA patients with MA histopathology.

Methods
Participants. In this study, male patients newly diagnosed with idiopathic NOA were recruited from the Center for Reproductive Medicine, Shandong University, from January 2014 to December 2018. All NOA patients were diagnosed on the basis of an andrological examination that included medical history, ultrasound, physical examination, hormone analysis, semen analysis, karyotype testing, and Y chromosome microdeletion screening. Subjects with known reasons or any relevant history may account for their infertility, such as childhood disease, cryptorchidism, environmental exposure, radiation, heat and other negative environmental exposure, varicocele, chromosomal abnormalities, hypogonadotropic hypogonadism, obstructive azoospermia, repeated infections, iatrogenic infertility, testicular trauma, abnormal karyotype, or Y-chromosome microdeletions, epididymitis, epididymo-orchitis, orchitis and/or sexualiy transmitted infections [25][26][27] were excluded.
According to the WHO recommendations and standards 28 , after two or more inspections of semen, testicular biopsies were performed in patients without available sperm. Biopsy samples were immersed in Bouin's fluid and then sent for histopathology examination. MA histopathology in our study exhibited that spermatogenesis blocked at the spermatocyte stage (Supplemental Fig. S1). Participants include 58 Chinese MA patients, and their mean age was 28 ± 4.1 years. A total of 192 fertile men with normal sperm concentrations were used as control group, and their mean age was 29 AAA TAG GGG CGT GGT CTC C  AAG ATT CCA GAA AAG CGC GG  463   2  Exon2  GAG AAG TGC TGT GGT AGG AG  GGC CAC ACA ATG CAA CAT CT  433   3  Exon3  ATG GAG GGA ATA GGG TGG TT  GTT CAC GCC ATT CTC CTG C  427   4  Exon4  ACC AAG CGT TAA TGT CAC TGT TGG TAT CAA CAG AGG TGA GACA  437   5  Exon5  CCT CCC AGG GTT GCT ACT TA  GGC TGG GAA TTA GAA AGG GG  327   6  Exon6  GGT CTT CTC ATT CCC CAC CT  AGG ATC CTG GTC ATC TTC TTCC  Consent for publication. The publication consent was obtained from all participants. www.nature.com/scientificreports/

Results
We sequenced the STAG3 gene in 58 patients with idiopathic NOA with MA histopathology and the control 192 fertile men. As shown in Table 2, we found 12 single nucleotide polymorphisms (SNPs), including 4 known SNPs and 8 novel SNPs. The 8 novel SNPs included 2 missense SNPs (c.433T > C in exon2 and c.553A > G in exon3), 3 synonymous SNPs (c.539G > A, c.569C > T in exon3, and c.1176C > G in exon8), and 3 SNPs in introns region. The allele and genotype frequencies of all SNPs have no significant differences between the cases and control group. No plausible variants were identified.

Discussion
The development of male gametogenesis includes the differentiation of spermatogonia, the process of spermatocyte meiosis, and spermatogenesis 29,30 . Meiosis is a critical stage in gametogenesis, in which alignment and synapsis of chromosome pairs occur, allowing the recombination of the maternal and paternal genomes 31 . Many of the gene variants in this process could have profound effects on gametogenesis and lead to male infertility 22 . Many gene knockout mouse models showed meiotic arrest in infertility, suggesting that they are candidate genes for NOA with MA histopathology 32 .
The STAG3 gene encodes a critical subunit of the meiosis-specific cohesin complex, ensures sister chromatid cohesion and enables correct synapsis and segregation of homologous chromosomes during meiosis 19,27 . While variant in STAG3 was identified in premature ovarian failure and oocytes in Stag3 -/female mice were arrested at early prophase I, the knockout male mice were also infertile and showed meiotic arrest and azoospermia 33,34 . These findings indicated that STAG3 may be a potential candidate gene for NOA in human. In this study, we analyzed the STAG3 gene in 58 Chinese NOA patients with MA histopathology, which is coincided with the phenotype of the gene knockout mice 35 . Eight novel SNPs were identified, including two missense SNPs, three synonymous SNPs and three SNPs in intron region. Our findings suggest that variants in coding region of the STAG3 gene are uncommon in NOA patients with MA histopathology in China. However, it has been reported that two SNPs (rs1727130 and rs1052482) located in the 3'-UTR of the STAG3 gene were identified to be associated with NOA in Korean population 36 . Furthermore, homozygous or compound-heterozygous variants of the STAG3 gene have been identified in NOA patients from Germany, Spain, and Australia [37][38][39] . In this study, we did not identify the same variants which may be due to the small sample size and ethnic diversity. Consistently, whole-exome sequencing was performed in 314 Han Chinese patients with unrelated NOA and Severe Oligozoospermia, but no deleterious variants were found in STAG3 40 .

Conclusions
The present study investigated variants in STAG3 in a cohort of idiopathic NOA with MA histopathology, and found no pathogenic variants. Our results suggest that variants in the STAG3 gene may not be responsible for NOA with MA in Chinese population. However, due to ethnic diversity, the exact role of STAG3 in the pathogenesis of NOA needs to be explored in large samples and other populations in the future.

Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.