Accumulating evidence demonstrates that MMR plays a critical role in the maintenance of genetic integrity and participates in the meiotic recombination process [14
]. Although mutations in MMR genes are considered as potential risk factors for various cancers [28
], only limited data exist on the potential role of polymorphisms in the MMR genes on male infertility. To our knowledge, this study is the first to provide a comprehensive evaluation of the relationship between polymorphisms in MMR and susceptibility to male infertility in a relatively large sample size. On the basis of analysis of 480 controls and 524 infertility patients with azoospermia or oligozoospermia, we observed that one intronic SNP in MLH1
(rs4647269) and two non-synonymous SNPs in PMS2
(rs1059060, Ser775Asn) and MSH5
(rs2075789, Pro29Ser) were associated with increased susceptibility to poor sperm production.
As an important pathway in the DNA damage repair network, MMR also plays a critical role in the maintenance of genetic integrity. Thus, it would be expected that these three significant SNPs that affect sperm DNA integrity could also modify male infertility risk. Based on a case-control study consisting of 480 controls and 768 patients with normal sperm count, we found that PMS2
rs1059060 was significantly associated with male infertility with normal sperm count. Further analysis based on 450 infertile men revealed significant associations of MLH1
rs4647269 and PMS2
rs1059060 with sperm DNA fragmentation. However, we did not detect any association between MSH5
Pro29Ser polymorphisms and sperm DNA damage. This result is explained by the fact that MSH5 is a meiosis-specific protein crucial for reciprocal recombination, and it has no apparent mismatch repair activity because it is missing the appropriate amino acid residues [30
MLH1 and PMS2 form the MutLα heterodimer that leads to the repair of mismatched DNA through activation of exonuclease-mediated degradation of DNA [31
]. Guerrette et al.
localized the MLH1-PMS2 interaction region to amino acids 506-675 of MLH1 and amino acids 675-850 of PMS2 [32
]. It is conceivable that the PMS2
Ser775Asn polymorphism could directly impact the integrity of the interaction between MLH1 and PMS2. In the present study, we provided evidence, for the first time, that the PMS2
Ser775Asn variant attenuates the interaction of MLH1 and PMS2, as illustrated by FRET and co-immunoprecipitation assays.
rs2075789 polymorphism in the coding region of the human MSH5
gene leads to a proline to serine alteration and is located within the MSH4-MSH5 interacting domain. To address the effect of the Pro29Ser alteration on the interaction between MSH4 and MSH5, a quantitative yeast two-hybrid analysis has been performed [33
]. This alteration causes a moderate but significant reduction in the interactions between both proteins, which could affect the formation of the MSH4-MSH5 heterocomplex. These findings strongly support our molecular epidemiological observation that the MSH5 Pro29Ser polymorphism is associated with a significantly increased risk of azoospermia or oligozoospermia. Supporting evidence also comes from association studies by other investigators. In a recent study of a Chinese population with a small sample size, Xu et al.
observed a 2.89-fold increased risk of azoospermia or oligozoospermia among the MSH5
Pro29Ser allele carriers [34
]. In addition, a case-control study including 41 women with premature ovarian failure and 39 controls suggested that there is a correlation between the MSH5 Pro29Ser polymorphism and premature ovarian failure in women [35
Another SNP associated with risk in our study (rs4647269) is intronic. However, SNP rs4647269 tags SNP rs9852810 (r2
= 1, D
' = 1), which was associated with prostate cancer risk and prostate cancer recurrence [36
]. Because both of these two SNPs are located in the intron of the MLH1
gene, it is uncertain which one of these two variants causes increases in male infertility risk. To identify additional SNPs that could be associated with male infertility risk that may be in high linkage disequilibrium (LD) with these two sites, we screened all of the common variants (with MAF > 0.05) within an approximately 20 kb-long region surrounding these two sites (approximately 10 kb upstream and approximately 10 kb downstream of these loci) based on the CHB HapMap data resource. We found that rs4647269 is in complete LD with SNP rs1046512, which is located approximately 2.5 kb upstream of start codon of MLH1
. Therefore, it is highly likely that the rs1046512 SNP near the 5' region of the MLH1
gene may be the causal variant.
Another interesting finding was that smoking was associated with increased risk of male infertility. Although the effects of tobacco cigarette smoke on male reproduction are somewhat inconclusive, a number of studies have shown higher incidences of abnormal sperm morphology [37
] and decreased sperm motility concentration in men who smoke [39
]. A meta-analysis [41
], including 27 studies, indicated that cigarette smoking is associated with a 13% reduction in sperm concentration, a 10% reduction of sperm motility, and a 3% reduction of morphologically normal sperm. Furthermore, fluctuation in reproductive hormone levels have been documented in male smokers [42
]. However, the mechanism(s) of these changes, if any, remains unclear.
Of note, like all case-control studies, selection bias may exist and might influence interpretation of the results. However, we believe that potential confounding bias might have been minimized by matching the controls to the cases on age and by further adjustment for the confounding factors in statistical analyses. In addition, the fact that genotype frequencies of all SNPs in our controls fit Hardy-Weinberg equilibrium and were similar to those obtained from the HapMap Project further supports the randomness of our control selection. We believe that the selection bias, if any, is unlikely to be substantial.