| BackgroundRecurrent pregnancy loss (RPL) is a common obstetric disease which is challenging for both the patients and obstetricians. It is usually defined as three or more consecutive spontaneous miscarriages before 20 gestational weeks, however, it has been redefined as two or more consecutive spontaneous miscarriages by the American Society for Reproductive Medicine (ASRM). It is a complex disease; both genetic and environmental factors contribute to its development.Thrombophilia was identified as one of the major causes of RPL, especially in the first half of pregnancy. Mutations in hemostasis-related genes were considered as risk factors for hereditary thrombophilia and can act either in the first half of pregnancy or later in pregnancy,causing possible miscarriages. Hyperhomocysteinemia (HHcy) has been reported to be an independent risk factor for many pregnancy-related disorders. Folic acid is a cofactor in Hcy metabolism, it deficiencies lead to increased Hcy level. Abnormal folic acid metabolism may have an effect on Hcy metabolism-related enzyme activity and result in increasing the concentration of Hcy in the bloodstream,eventually leading to miscarriage. Therefore, we speculate polymorphisms in folic acid metabolism-related genes may be a risk factor of RPL.ObjectiveMeta-analysis to study the association of recurrent pregnancy loss with polymorphisms in haemostasis-related genes and folic acid metabolism -related genes, then by conducting case-control study to futher investigate the association of recurrent pregnancy Loss with polymorphisms in hemostasis-related genes and the association of recurrent early pregnancy loss (REPL) with polymorphisms in folic acid metabolism-related genes in chinese populationMethodsA systematic review of the literature was conducted with PubMed MEDLINE, EMBASE and CNKI databases searching for reports have been publicated in English and in Chinese. The meta-analysis was performed using the MIX 1.7 software, examined the association of genotypes or alleles(eNOS intron 4 VNTR and Asp298Glu (rs1799983),MTHFR C677T(rs1801133) and A1298C(rs1801131))and the risk of RPL. For all statistical analyses, two-sided P value<0.05 was considered to be statistically significant. The case-control study included 94 RPL women with at least two consecutive pregnancy losses before 20 week’gestational age as case and 169 ethnic-matched healthy controls with regular menstrual cycles, at least one naturally conceived pregnancy and no history of pregnancy loss or other pregnancy complication. Genomic DNA was abstracted from the peripheral blood cell. Six single nucleotide polymorphisms(SNP) in hemostasis-related genes(SERPINC1 G786A (rs2227589), THBD C1418T (rs1042579), TFPI T-33C (rs8176592), Factor V G1628A (rs6020), Factor Ⅱ G19911A (rs3136516), ANXA5 C76T (rs113588187)) and three SNPs in folic acid metabolism -related genes (MTRR A66G (rs1801394), SLC19A1 G80A (rs1051266) and C696T (rs12659)) were assayed by utilizing the Sequenom MassARRAY iPLEX Platform. The other two SNPs in folic acid metabolism -related genes (MTHFR C677T (rs1801133) and A1298 (rs1801131)) were detected by PCR for amplification followed by DNA sequencing. Differences in genotype or allele frequencies between case and control subjects were compared using the chi-square test by SPSS version 13.0. Significance levels were adjusted by Bonferroni correction to account for multiple testing.Results一:Meta-analysis1. The results showed that eNOS rs1799983 polymorphism was associated with RPL (OR was 1.67 (1.36-2.04), P<0.001). When analyses were separated by ethnic subgroups, the association between eNOS rs1799983 polymorphism and RPL was only observed in East Asians (OR=1.88 (1.52-2.33),P<0.001 under additive model), and there was no association between eNOS intron 4 VNTR polymorphism and RPL in Caucasians and East Asians.2. The integrated results showed that MTHFR rs1801133 polymorphism was associated with RPL. When analyses were separated by ethnic subgroups, the association between MTHFR rs1801133 and RPL in the East Asian subgroup (OR=2.11(1.40-3.19), P<0.001), but not in Caucasian subgroup. No significant association between MTHFR rs1801131 polymorphism and RPL was found.二:Case-control Study1. The distribution of THBD rs1042579 allele shown significant differences between RPL cases and healthy controls (OR:1.58,95% CI1.05-2.39, P=0.027).It is predicted that the T allele in THBD rs1042579 affect THBD splicing regulation, then, maybe have an effect on the expression of protein.2. Genotype and allele frequencies for SERPINC1 rs2227589, TFPI rs8176592, Factor V rs6020, Factor Ⅱ rs3136516, ANXA5 rs113588187 were similar between RPL case and healthy control groups.3. Significant differences in genotype distributions and allele frequencies between case and control group were observed in MTHFR rs1801131 (AC/AA, AOR=2.58, 95%CI:1.40-4.74, P=0.002; AC+CC/AA, AOR=2.53,95%CI:1.40-4.57 P=0.002; C/A, OR=2.13,95%CI:1.27-3.57, P=0.004 respectively). And the allele frequencies of MTHFR rs1801133 (T/C, P=0.246), SLC19A1 rs1051266 (A/G, P=0.165), SLC19A1 rs12659 (T/C, P=0.158) and MTRR rs1801394 (G/A, P=0.241) in REPL cases were similar to those in healthy controls.4. Among the four haplotypes of MTHFR rs1801133-rs1801131, the rs1801133C-rs1801131C haplotype was positively associated with REPL (P<0.001). No significant relation was found between haplotypes of SLC19A1 rs1051266-rs12659 and the risk of REPL. The rs1801133C-rs1801131A and rs1051266G-rs12659C haplotypes had lower frequencies in patients with REPL, whereas P values were>0.05 (P=0.093 and P=0.084 respectively).ConclusionThe results of meta-analysis showed that eNOS rs1799983 and MTHFR rs1801133 polymorphisms were associated with RPL in the East Asian subgroup, but not in Caucasian subgroup. The case-control study indicated that polymorphism in THBD rs1042579 may contribute to some RPL occurrences and polymorphism in rs1801131 was obviously associated with REPL; The MTHFR rs1801133C-rs1801131C haplotype was positively associated with REPL (P<0.001); The MTHFR rs1801133C-rs1801131A and SLC19A1 rs1051266G-rs12659C allele combinations had lower frequencies in patients with REPL (P=0.093 and P=0.084), suggesting a protective nature on REPL. Further replication studies with larger sample are vital for confirmation of these findings. The exact biological functions and the contribution of THBD rs1042579 to protein are still needed to be clarified. |
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