Association of selected polymorphisms in GPX4, COMT, pre-miR-125a, pre-miR-10a, and pre-miR-323b genes in Iranian women with idiopathic recurrent pregnancy loss: A case-control study

Abstract Background Recurrent pregnancy loss (RPL) is a major concern among women worldwide. However, the exact mechanisms underlying miscarriage are not well understood. Recent evidence suggests that single nucleotide polymorphisms in various genes, especially miRNAs, may be responsible for RPL. Objective We surveyed the association between polymorphisms in pre-miR-125a, pre-miR-10a, pre-miR-323b, GPX4, and GPX4 in Iranian women with idiopathic RPL. Materials and Methods DNA was extracted from blood samples of 116 women with idiopathic RPL and 89 healthy women as controls who had previously had at least two successful pregnancies. Polymerase chain reaction was used for the amplification of the genes. Genotype screening along with SNaPshot were performed to detect different polymorphisms. Finally, the polymorphisms and frequency of each genotype were compared between the two groups. Results The frequencies of polymorphisms in pre-miR-125a (p < 0.001) and pre-miR-10a (p = 0.04) were calculated among the case and control groups, which showed a statistical difference (p < 0.05), indicating an association between these polymorphisms and the symptoms of RPL. The frequencies of polymorphisms of genotypes in GPX4, COMT and pre-miR-323b did not demonstrate any difference between the two groups. Also, the amount of alleles in pre-miR-125a and pre-miR-10a were significantly different (p < 0.001 and p = 0.02, respectively) and the dominant inheritance model was proposed. Conclusion In conclusion, pre-miR-125a and pre-miR-10a can be associated with RPL in women. The SNaPshot technique is a valuable tool to evaluate possible associations between polymorphisms and health conditions.


Introduction
Recurrent pregnancy loss (RPL) is defined as "two or more abortions before 24 weeks of gestation" (1,2). According to previous studies (3) is involved in glutathione synthesis (5) and may be associated with RPL in women; however, the exact mechanism is not clear. Catechol-O-methyl transferase protein (COMT) is now increasingly reported to have a role in pregnancy. In particular, recent reports have noted its expression in fetal membranes. This enzyme is also active both in the placenta and in the decidua (6). Reports have demonstrated that hypertension in pregnancy can reduce the placental COMT activity (7). Recent studies have shown that women with severe preeclampsia have reduced placental COMT protein expression (8). Besides, micro RNAs translation by joining to untranslated regions . This protein has an essential role in the mother's immune system tolerating the fetus, and the down-regulation of this protein leads to RPL (9). miR-125 is a group of miRs which consists of two subgroups: miR-125a and miR-125b. The expression of these miRs occurs in different organs like the stomach, liver, lungs, rectum, breast, prostate, and ovaries. Research on these organs suggests that the malignant process may be controlled by preventing gene expression.
Embryo development may also be affected by low secretion of the mother's miR-125a polymorphism.
Previous studies have also shown that women with RPL have down-regulation of 41 miRs and overexpression of four miRs (10). Although the critical role of miR-125a in RPL is unclear, recent evidence has proposed that its down-regulation may induce RPL in multiple ways (11).
By considering the wide range of miR activities in messaging processes, especially in the female genital system, studies on these regulatory RNAs seem to be necessary. The importance of miscarriage necessitates more comprehensive studies on the role of miRs in RPL. Therefore, the current study aimed to use the SNaPshot method to investigate the association between polymorphisms of pre-miR-125a (rs41275794), pre-miR-10a (rs3809783), pre-miR-323b (rs56103835), GPX4 (rs4680) and GPX4 (rs713041), and RPL in Iranian women with idiopathic recurrent miscarriage.

Sample collection
In the present case-control study, 116 women with idiopathic RPL and 89 healthy women as controls who had previously had at least two successful pregnancies (a total of 205 individuals) were included and referred to Baqiyatallah Hospital

DNA extraction
In the first examination, after an overnight fast, 5 ml peripheral blood samples were collected and placed in the EDTA-containing tubes. Then, the rapid genomic DNA extraction (RGDE) method was used to extract genomic DNA (12
Oligo analyzer software was used to consider or predict the formation of dimer or secondary structures of primer. Primer sequences and their amplicon size are shown in table I. Gene runner software was used to design appropriate singlebase extension (SBE) primers, as mentioned in table II. All individuals were analyzed for the presence of these polymorphisms (13).

Multiplex PCR amplification
To conduct the multiplex PCR, the following chemicals were prepared: 1 μM of each primer, 0.17 mM of dNTPs, 1.5 mM of MgCl 2 , one unit of Taq DNA polymerase, and 20 ng of template DNA in a 20 μl volume flask containing 10 X PCR buffer. The three conditions applied to the thermal cycle were: 1) 10 min at 95°C; 2) 30 sec at 95°C for 35 cycles; 3) 30 sec at 60°C, one min at 72°C and a final extension of 10 min at 72°C. Then, 3 μl of PCR product was run in 12% polyacrylamide gel to check for the quality and yield of the multiplex PCR. Next, the excess dNTPs and primers were removed by treating the 3 μl of remaining PCR products with one unit (1 μl) of exonuclease I. Then, three units of shrimp alkaline phosphatase and two units of exonuclease I (ExoSAP/USB, Cleveland, OH, USA) were used to purify the remaining PCR products, which was followed by incubation for 15 min at 37°C and 15 min at 80°C to remove excess dNTPs and primers, and to deactivate the Taq enzyme (14).

SNaPshot method
The SNaPshot assay applied five SBE primers which simultaneously annealed adjacent to SNP variant in our reaction. As shown in table II, poly (dC) tails of different lengths were attached to the SBE primers.
The SNaPshot solutions for multiplex PCR contained PCR product (1 μl), master mix SNaPshot (1 μl), each SBE primer (1 μl) and DDW (2 μl) in a final volume of 5 μl. The three thermal protocols applied to the SNaPshot were: two min at 96°C; 10 sec at 96°C; five sec at 50°C and 30 sec at 60°C. dNTPs were removed from the second PCR product by treating the remaining PCR products with three units (1 μl) of shrimp alkaline phosphatase and then the following two thermal protocols: 45 min at 37°C and 15 min at 75°C. An ABI PRISM 3130 Genetic analyzer was used to conduct capillary electrophoresis after which Gene Mapper idx, Ver 4.0 (Applied Biosystems, Foster City, CA, USA) was used to analyze the results (14).

Statistical analysis
Statistical analysis was conducted using the SPSS v. 21 statistics software. Data were shown as percentages of the mean or allele frequency. Pearson's Chi-square test was used to calculate inter-group significance. The homozygous and heterozygous genotypes of each group were unified as carriers and the odds ratios (OR) and 95% confidence intervals (CI) were obtained. Statistical significance was set at p < 0.05.

Results
The present study was performed on 116 cases with RPL and 89 healthy women without any history of RPL. There was no significant difference between their ages (38.93 ± 10.45 yr vs. 41.31 ± 10.74 yr, respectively; p = 0.40). After collection of blood samples, the DNA was extracted with the rapid genomic DNA extraction method, as shown in figure 1.
After multiplex PCR, the SNaPshot kit and specific SBE primers were used to detect rs713041, rs4680, rs41275794, rs3809783, and rs56103835 SNPs with the genetic analyzer machine, as shown in figure 4.
The comparison of SNP genotype distribution for all of the studied genes in the control and case groups is summarized in table III. The frequencies of AG, GG and AA genotypes in the polymorphism rs41275794 of pre-miR-125a (p < 0.001) and of TT, AT and AA genotypes in the polymorphism rs3809783 of pre-miR-10a (p = 0.04) were calculated for the women in the case and control groups. The findings showed significant differences and an association of these polymorphisms with the symptoms of recurrent miscarriage (p < 0.05). There was no significant difference (p = 0.09) in the frequency of TT, AT and AA genotypes in the GPX4 gene (rs713041 polymorphism) between the case and control groups. The frequency of TT, AT and AA genotypes in the miR-323b gene (rs56103835 polymorphism) showed no significant difference (p = 0.14) between the case and control groups. Also, no significant difference (p = 0.21) was observed in the frequency of GG, AG or AA genotypes in the COMT gene (rs4680 polymorphism) between the two groups (Table III). In the allele frequency study for alleles A and G of polymorphism rs41275794, a significant difference was found between the case and control groups (p < 0.001). Similarly, there was a significant difference (p = 0.02) in the frequency of alleles A and T of polymorphism rs3809783 between the two groups of cases and controls (Table IV). The assessment of risk at a 95% confidence level for allele A relative to G in the rs41275794 polymorphism showed that by changing the G allele to A, the risk of the RPL phenotype was increased by 1.912 times. Similarly, for the rs3809783 polymorphism, the changing of allele A to T increased the risk of the RPL phenotype by 1.704 times, showing a greater association of risk. To evaluate the genotypic inheritance of significant polymorphisms, we studied and calculated the risk of RPL in three models: dominant, recessive, and obvious models. The results showed that the dominant hereditary model had a significant correlation based on the genotypic frequency of the variant (Table V).   Chi-square test was used. P-value < 0.05 was considered statistically significant

Discussion
RPL is a clinical challenge that affects approximately 1-5% of women of childbearing age (11). The underlying mechanism of RPL in 37-79% of cases is unclear. Recent evidence has revealed that molecular and genetic disorders, including various polymorphisms, may be the main cause of RPL in these cases (15).
In this research, we compared for the first time five polymorphisms of pre-miR-125a (rs41275794), pre-miR-10a (rs3809783), pre-miR-323b (rs56103835), GPX4 (rs4680) and GPX4 (rs713041) between healthy women and women with RPL using the SNaPshot genotyping method. Our findings showed a significant difference in the miR-125a gene polymorphism rs41275794 between the control and case groups. GG was found to be the most common genotype in the control subjects (60.7%), while GA (50.9%) was the most common genotype in the case group. These data indicated that the polymorphism in miR-125a may be a risk factor for RPL among the Iranian female population. In our study, allele A was significantly associated with an increased risk of RPL indicating that this variant allele in pre-miR125a can change precursor and mature miR-125a expression.
Our data revealed that the dominant genotype was significantly associated with an increased risk of RPL. In another study, expression of mature miR-125a in the CC/AA genotype was ∼2.1-fold higher than that in the TT/AA genotype (16). They found that the A>T haplotype in rs41275794 affected miR-125a expression and caused RPL in women. In another study, Li and colleagues compared the expression pattern of six miRs, including miR-125a, miR-125b, miR-34a, miR-155, miR-24 and miR-141 between healthy women and those with RPL. They found that miR-125a, miR-125b, miR-34a, miR-155 and miR-141 were significantly overexpressed in women with RPL while miR-24 was significantly downregulated in these cases, compared to the healthy group. Additionally, they showed that the A>T polymorphism in pri-miR-10a (rs3809783) was significantly associated with an increased risk of RPL in women.
More studies are being conducted nowadays to find relationships between SNPs and the miRNA system's critical components, biological functions and disease (4,16,17  Bioinformatics studies on the secondary and tertiary protein structures of selected genes using related databases can provide more comprehensive information on recurrent miscarriages.

Conclusion
In conclusion, our data illustrated that a polymorphism in pre-miR-125a (rs41275794) and in pre-miR-10a (rs3809783) can be associated with RPL in women. SNaPshot was shown to be a suitable method for investigation of the role of polymorphisms in the incidence of health conditions.