Prevalent false positives of azoospermia factor a （AZFa） microdeletions caused by single-nucleotide polymorphism rs72609647 in the sY84 screening of male infertility

Multiplex polymerase chain reaction （PCR） has been widely used to detect Y-chromosome micredeletions, which is one of the major causes of male infertility. Both the European Academy of Andrology （EAA） and the European Molecular Genetics Quality Network （EMQN） have recommended the use of sY84 and sY86 markers for the detection of azoospermia factor a （AZFa） microdeletion during DNA testing for male infertility. In this study, a large-scale analysis of AZF microdeletion in a total of 630 Chinese males, including healthy semen donors （n=200）, infertile males with normal sperm count （n=226） and patients with either nonobstructive azoospermia or severe oligozoospermia （n=204）, was performed. A series of nine sequence-tagged site （STS） markers from the AZF region of the Y chromosome was used to detect microdeletions. All primers were designed based on the recommendations of the National Center for Biotechnology Information. An unusually high incidence （73/630, 11.6%） of sY84-absent but sY86-present genotypes was observed in the AZFa microdeletion screening. Sequencing the sY84-flanking region revealed a total of 73 patients with sY84-absent but sY86-present genotypes have a T-to-G transversion at the fifth base from the 5＇ end of the reverse sY84 primer. These prevalent false positives, which were not only observed in infertile men, but also observed in donors, resulted from a single-nucleotide polymorphism （SNP） named rs72609647 in the targeting sequence of the reverse sY84 primer. Our study suggests that a pre-screening of existence of rs72609647 polymorphism can prevent the frequent false positive results of AZFa microdeletions detection in the infertile Chinese males. Given the SNP rs72609647 was recently found in a deep sequencing of a Chinese individual, the current EAA and EMQN standards may need to be scrutinized among different populations to avoid the potential genetic variations in the primer binding sequences.

c.822＋126T〉G/C：a novel triallelic polymorphism of the TSSK6 gene associated with spermatogenic impairment in a Chinese population

TSSK6 is a member of the testis-specific serine/threonine kinase family. Male Tssk6 knockout mice are infertile owing to sperrnatogenic impairment,including sperm count reduction,a decrease in motile sperm number and motility rates,and an increase in the number of sperms with abnormal morphology. We investigated the possible association between variations oftbe TSSK6 gene and spermatogenic impairment in humans. Mutation screening of TSSK6 was carried out in 519 patients with azoospermia （n = 273） or severe oligozoospermia （n = 246） and in 359 controls with normozoospermia by denaturing high-performance liquid chromatography and DNA sequencing. The frequencies of alleles and genotypes of gene polymorphism were compared between patients and controls. A novel triallelic polymorphism in TSSK6,c.822＋126T〉G/C,was identified. The frequencies of genotype TT and allele T were increased dramatically in infertile patients compared with controls,whereas genotype TG,allele G and allele C frequencies were significantly higher in controls than in patients. Further study revealed that the allele C frequency of controls was remarkably higher than that of patients with oligospermia. Our findings,for the first time,suggested an association of c.822＋I26T〉G/C in TSSK6 with spermatogenic impairment in humans in which allele T may be a risk factor for male infertility,while alleles C and G may decrease susceptibility to male infertility.