Association Study Of Single Nucleotide Polymorphisms In The Genes Of Folate Metabolism And Idiopathic Male Infertility

The world's population is increasing at an alarming rate and is projected to reach nine billion by 2050. Despite this, 15% of couples world-wide experience some forms of infertility problem, in which male factors may account for up to 50%. Although several causes have been proposed for impaired male fertility, the majority of cases, which involve either inadequate spermatogenesis or defective sperm, remain to be elucidated. In recent years, the new reproductive technology of ISI(Intracytoplasmic Sperm Injection)can help some of these men to father their own children. However, there is a concern about passing on mutations and an infertility condition to future generations. Therefore, discovery of more genetic causes of male infertility besides the known Chromosomal abnormalities and Yq microdeletions may have therapeutic implications. Single Nucleotide Polymorphisms (SNP) is a good method to search for candidate genes for many diseases including male infertility .Although it is well known folate is important for normal reproductive function in women, little research on the role of folate in male reproductive function has been reported. Treatment with folate antagonists for various diseases have been shown to impair reproductive in men. A high-affinity folate-binding protein has been indentified in human semen. These findings suggest an association between folate status ans male reproductive function. Four enzymes are important in folate synthesis and metabolism including methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), methionine synthase reductase (MTRR) and thymidylate synthase (TYMS). In our case-control study, we evaluated whether four common polymorphisms in methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MS A27566G) methionine synthase reductase (MTRR A66G), thymidylate synthase (TYMS, a common 3'-UTR insertion /deletion polymorphism )genes may have a role in altering susceptibility to idiopathic male infertility by using PCR-RFLP(polymerase chain reation—restriction fragment length polymorphism).PART 1: Polymorphisms of MTHFR gene and idiopathic male infertility165 infertile patients and 132 healthy fertile males have been enrolled. Using themethod of PCR-RFLPF, two common polymorphisms (C677T and A1298C) in the MTHFR gene have been studied. For the C677T polymorphism, the heterozygous genotype (CT) was present at a statistically significance in infertile men. The combined genotypes (CT+TT) showed an association with infertility (P=0.031). For the A1298C polymorphism, the frequencies of allele C and the combined genotype (AC+CC) were significant different between the case and the control. Notably, while there was 1.520% CC homozygote in the control population, 9.090% infertile cases were CC homozygotes. Considering that CC genotype occurs in very low frequency in the control population, 1298CC is clearly a risk factor for male infertility in the Chinese population.We identified MTHFR haplotypes between fertile and infertile men. The majorhaplotypes in infertile men were CA (43.94%), followed by TA (28.78%) ,TC(18.19%) ,CC (9.09%) for 677c>t/1298a>c. The haplotypes for the control subjectswere CA(44.69%), and TA(36.74%),CC (14.39%) ,TC (4.18%) .The haplotypes TCshowed significant differences in frequency between infertile men and control subjects[OR 4.439,95%CI (2.233-8.825)].PART 2: Polymorphism of MS A2756G gene and idiopathic male infertilityUsing the method of PCR-RFLP, We studied the frequencies of allele and genotype of MS A2756G in the 165 infertile patients and 132 healthy fertile males. The allele frequency of A2756G was 10.99% and 17.27% in the control and cases, respectively. The difference was statistically significant [P = 0.03, OR 1.692, 95%CI (1.047-2.734)]. The prevalence of the three different genotypes for the MS A2756G polymorphism between the infertile men and controls was not significant different (P =0.079, P value for heterogeneity across three categories). The combined genotypes (AC+CC) showed an association with infertility [P=0.025, OR 1.861,95% CI (1.076-3.217)].PART 3: Polymorphism of MTRR A66G gene and idiopathic male infertilityWe enrolled 165 infertile patients and 132 healthy fertile males. The frequencies of the 66G allele were 41% and 55% in the control and the case, respectively. The difference was statistically significant (P=0.001). The prevalence of the three different genotypes for the A66G MTRR polymorphism between the infertile men and controls was also significant different (P =0.003, P value for heterogeneity across three categories). While genotype GG showed no difference between infertile men and fertile men (P=0.330), the genotype AG and the combined genotype (AG+GG) showed signicant different in the two groups, indicating genotypes AG and AG+GG may play roles for male infertility in a Chinese population.PART 4: Polymorphism of TYMS, a common insertion /deletion polymorphism (3'-UTR) and idiopathic male infertility We genotyped a common insertion /deletion polymorphism in a case-control study composed of 165 infertile patients and 132 healthy fertile males in a Chinese population. The allele frequencies of TYMS 3'-UTR del 6 were0.25 in the controls and 0.35 in the cases, indicating no statistically differences was founded (P = 0.09 for TYMS 3'-UTR del6). The distribution of homozygosity of del/del and the combied genotype ins/del+del/del in the control and the case showed significantly different (P =0.026 and 0.027 respectively). These findings suggest that the genotypes del/del and ins/del may play a role in the etiology of idiopathic male infertility.These data are in consistent with our hypothesis that polymorphisms in the genes involved in folate metabolism might play a important role in the occurrence of idiopathic male infertility by influencing DNA synthesis and DNA methylation.