miR-27a and miR-449b polymorphisms associated with a risk of idiopathic recurrent pregnancy loss

Objective MicroRNAs (miRNAs) regulate gene expression during the peri-implantation period. The purpose of this study was to investigate whether genetic polymorphisms in the four miRNAs associated with fetal or placental development play roles in the development of idiopathic recurrent pregnancy loss (RPL) in Korean females. Study design A case-control study involving 225 controls and 387 women with at least two consecutively recurrent pregnancy losses between 1999 and 2012 was performed. The genotypes of the four miRNA polymorphisms, including miR-27a rs895819, miR-423 rs6505162, miR-449b rs10061133, and miR-605 rs2043556, were analyzed by the polymerase chain reaction-restriction fragment length polymorphism assay. Odds ratios and 95% confidence intervals were estimated using multivariate analyses after maternal age adjustments. The relationships between each of the four microRNA genotypes and each of the six clinical parameters of the RPL patients (plasma homocysteine and folate levels, natural killer cell number, platelet count, prothrombin time, and, activated partial thromboplastin time) were analyzed using multiple linear regression analyses. Results Our results suggest that weak associations between decreased RPL risk and the genotypes of miR-27a (AG and AG+GG), combination genotype of miR-27a/miR-423 (AG/GC), and haplotypes of miR-27a/miR-423/miR-449b/miR-605 (G-C-A-G) and miR-27a/miR-449b/miR-605 (G-A-G), whereas weak associations between increased RPL risk and genotypes of miR-449b (GG and AG+GG), combination genotypes of miR-423/miR-449b (CC/GG and CA/AG), miR-449b/miR-605 (AG/AG), haplotypes of miR-27a/miR-423/miR-449b/miR-605 (A-C-G-A, A-A-A-G, and G-C-G-G), miR-27a/miR-423/miR-449b (A-C-G), miR-27a/miR-449b/miR-605 (A-A-G, A-G-A, and G-G-G), miR-423/miR-449b/miR-605 (C-G-G and A-A-G), and miR-423/miR-449b (C-G and A-A). The genotypes of miR-27a (AG and AG+GG) also showed significant contributions to the prediction of folate levels in RPL patients. Conclusions The study showed associations between miRNA polymorphisms (miR-27a rs895819 and miR-449b rs10061133) and RPL development, and between the miRNA polymorphism (miR-27a rs895819) and plasma folate levels.


Introduction
Recurrent pregnancy loss (RPL) or recurrent spontaneous abortion has been defined as the occurrence of at least two consecutive pregnancy losses prior to the 20th week of gestation [1,2]. RPL occurs in approximately 1% of all pregnancies; however, the etiology for more than half of the RPLs remains undetermined [3]. Genetic variation has been suggested one of the contributing factors leading to RPL and a number of single nucleotide polymorphisms (SNPs) have been reported to be associated with RPL [4]. MicroRNAs (miRNAs) are short (approximately 22 nt) noncoding RNA molecules regulating expression of target genes at the posttranscriptional level by translational repression or messenger RNA degradation [5]. Several studies recently reported the associations between miRNA polymorphisms and RPL [6][7][8][9]. One study identified two SNPs in miR-125a altering the production of miR-125a which was subsequently associated with an elevated risk for RPL in the Han Chinese women [8]. Another study reported an association between two pre-miRNA polymorphisms (miR-196a2 and miR-499) and the occurrence of RPL in Korean females [9], which was supported in Iranian women [6]. The most recent study identified a polymorphism in the coding region of miR-423 contributing to an increase in the expression of mature miR-423 associated with RPL in the Han Chinese population [7]. Several miRNAs that are considered important during pregnancy were chosen for this study because of their elevated expression (miR-27a), decreased expression in the endometrium and in trophoblasts (miR-423), lower expression during endometriosis (miR-449b), and involvement in pregnancy loss via the p53 network (miR-605) [10][11][12][13]. In this study, we determined the susceptibility to RPL associated with genetic variants of miR-NAs associated with placental or fetal development.

Study participants
The study group consisted of 387 females, 33.21 ± 4.55 years of age [mean age ± standard deviation (SD)] and a body mass index (BMI) of 21.49 ± 3.84 (± SD) who were diagnosed as idiopathic RPL patients with at least two consecutive pregnancy losses prior to the 20th week of gestation according to the definitions of infertility and recurrent pregnancy loss by American Society for Reproductive Medicine [1]. These patients were enrolled in a study at the Infertility Medical Center of CHA Bundang Medical Center from March 1999 to February 2012. Among the RPL patients, none had a history of smoking or alcohol use. RPL patients due to anatomical, hormonal, chromosomal (patients or their spouses), infectious, autoimmune, or thrombotic causes are excluded from the study. The age-matched control group consisted of 225 females, 33.43 ± 5.89 years of age (mean age ± SD) and with a BMI of 21.68 ± 3.451 (± SD), each of whom had regular menstrual cycles, had a history of naturally conceived pregnancy at least once, had no history of pregnancy loss or karyotype 46,XX, and who were recruited from the CHA Bundang Medical Center. All patients and controls were Korean. The institutional review board of CHA Bundang Medical Center approved the study, and all patients provided written informed consent.
To determine the absolute number of NK cells, 200 mL of diluted blood was incubated for 20 minutes on ice with phycoerythrin-conjugated anti-CD56 and peridinin chlorophyll protein-conjugated anti-CD3 monoclonal antibodies (BD Biosciences, San Jose, CA, USA). Then, 20,000 fluorescein isothiocyanate conjugated beads were added, and the blood sample was subjected to erythrocyte lysis using FACS Lysing Solution (BD Biosciences). The samples were analyzed on a flow cytometer using the BD FACSCalibur (BD Biosciences). The NK cell number of the diluted blood samples was calculated as (NK cells/mL sample) = [(CD56 + /CD3cell count)/(bead count)] × 100. This FACS-based NK cell count required 0.1 mL of whole blood per tested condition.

Statistical analysis
The differences in four microRNAs (miR-27aA>G, miR-423C>A, miR-499bA>G, and miR-605A>G) genotype and haplotype frequencies between patients and normal controls were compared using Fisher's exact test and logistic regression analyses. Allele frequencies were estimated to identify deviations from the Hardy-Weinberg equilibrium (HWE). Adjusted odds ratios (AOR) and 95% confidence interval (CI) were estimated as a measure of the strength of association between genotypes and RPL risk. Multiple comparison tests were adjusted by using the false discovery rate (FDR) correction, and associations with an FDR-adjusted P value < .05 were considered statistically significant [15].
Gene-gene interactions among SNP loci were analyzed with multifactor dimensionality reduction (MDR) using MDR software, version 2.0 (www.epistasis.org) [16][17][18]. Based on the MDR identification of the most significant models using the best maximized cross validation value, the best multilocus combinations were determined. HAPSTAT software, version 3.0 (www.bios.unc.edu/Èlin/hapstat/) was used to estimate haplotype frequencies for polymorphisms that were determined by MDR analyses to have strong synergistic effects. Statistical analyses were performed using GraphPad Prism software, version 4.0 (GraphPad, San Diego, CA, USA) and StatsDirect software, version 2.4.4 (StatsDirect, Altrincham, UK). The statistical significance was set at P <0.05. The relationship between each of the four microRNA genotypes and each of the six clinical variables of the RPL patients (plasma homocysteine, folate, NK cell, PLT, PT, and aPTT which may contribute to an even balance of coagulation and fibrinolysis during pregnancy) was analyzed using multiple linear regression analyses. Regression models were examined for the six clinical variables with grades (10 levels) as dependent variables and the microRNA polymorphisms as independent variables. The differences in plasma homocysteine, folate, PLT, PT, aPTT, and NK cells as a function of the four microRNA genotypes and combination genotypes were evaluated by one-way analysis of variance and independent two-sample t-tests.

Results
The demographic characteristics and clinical profiles of RPL patients and control subjects are shown in Table 1. The two groups were matched for age and BMI. Platelet numbers were significantly higher in the patient group than in the control group. The genotype and allele frequencies of the four miRNA SNPs in females with RPL and controls are shown in Table 2. All genotypes in the study cases were in HWE. In Table 2, the miR-27a A>G polymorphism was significantly associated with a risk of RPL (AA vs. AG: AOR = 0.654; 95% CI = 0.456-0.937; AA vs. AG+GG: AOR = 0.682; 95% CI = 0.484-0.960); however, there was no association after adjustment for multiple tests using the FDR correction. When RPL patients were stratified according to the occurrence of consecutive recurrent pregnancy losses (RPL = 2 and ! 3 vs. all RPL patients with RPL ! 2), there was an association between the miR -27a A>G polymorphism and RPL risk in the RPL ! 3 subgroup alone (AA vs. AG: AOR = 0.611; 95% CI = 0.404-0.923; AA vs. AG+GG: AOR = 0.639; 95% CI = 0.432-0.945). However, the associations were not statistically significant after adjustment for multiple tests using the FDR correction. The miR-449b A>G polymorphism was significantly associated with RPL risk (AA vs. GG: AOR = 2.069; 95% CI = 1.033-.4.146; AA vs AG+GG: AOR = 1.406; 95% CI = 1.011-1.955). However, the association was not significant after adjustment for multiple tests using the FDR correction. Using combination analyses (Table 3), the AG/CC (AOR = 0.579; 95% CI = 0.366-0.917) combined genotype for miR-27a/miR-423 was associated with a lower RPL risk compared with reference genotypes when variant genotypes were located in the miR-27a loci. In addition, CC/GG (AOR = 2.888; 95% CI = 1.116-7.470), CA/AG (AOR = 1.925; 95% CI = 1.110-3.338) for the miR-423/miR-449b, and AG/AG (AOR = 1.804; 95% CI = 1.067-3.052) for the miR-449b/miR-605 were associated with an increased RPL risk compared with   reference genotypes when variant genotypes were located in the loci of miR-423, miR-449b, and miR-605. These results were consistent with associations between a RPL risk and individual microRNA genotypes; however, each association was not significant after the FDR correction for multiple comparisons, suggesting a weak association. Haplotype-based analyses of the four microRNA polymorphisms for gene-gene interactions are shown in S1 Table (all possible  allele Table 5. The AG and AG+GG genotypes of the miR-27a polymorphism showed significant contributions to the prediction of folate levels in RPL patients, with regression coefficients of 1.069 and 0.788, respectively. Although the differences in plasma homocysteine, folate, PLT, PT, aPTT, and NK cell number in relation to the four microRNA genotypes and haplotypes were evaluated by one-way analysis of variance and independent two sample ttests, no significant difference was found (S2 and S3 Tables).

Discussion
The effects of miRNA polymorphisms on pregnancy loss have been reported in a limited number of studies [6][7][8][9]. These studies investigated the roles of miR-125, miR-196a2, miR-499 and miR-423 polymorphisms that were supported by functional evidence showing disruption of mature microRNA production and its downstream target gene [6][7][8][9]19]. Because more evidence in support of the functional importance of miRNAs in pregnancy has been reported, we determined whether the four miRNA SNPs associated with placental or fetal development played a role in pregnancy loss [14,[20][21][22][23][24][25][26][27]. Our results showed an association between the miR-27a variant G allele and a lower RPL risk, and an association between the miR-449b variant G allele and a higher RPL risk. The effects of variant alleles of miR-27a and miR-449b were also suggested using combination and haplotype-based analyses. Multiple linear regression analyses of clinical variables in Korean RPL patients revealed statistically significant relationships between the miR-27a genotypes and plasma folate levels. Functional analyses indicated that the variant genotypes of miR-27a, AG, and GG might be responsible for the elevated miR-   27a levels [14], and it has been speculated that the elevated miR-27a levels contribute to the increased folate concentration that is protective against RPL [20]. However, the possible effects of mir-27a on RPL mediated by folate require further investigation to confirm this hypothesis. The miR-449b was chosen for the study because it is one of the four miRNAs that was downregulated in hatched blastocysts [21]. However, the effect of rs10061133 A>G of miR-449b has rarely been reported, and there have been contradictory reports on its effects, depending on the cell type and gene expression pattern [24]. In our study, we found that the GG and AG +GG genotypes of miR-449b were associated with an increased risk of RPL. We, therefore, hypothesize that the GG and AG+GG genotypes of miR-449b rs10061133 affect the risk of RPL risk by modulating the expression of mature miR-449b [24,27].
The miR-423 rs6505162 polymorphism was chosen for the present study because it was reported to be differentially regulated in placental injury [25], and rs6505162 in pre-miR-423 affects the mature miRNA expression by increasing miRNA expression with the variant A allele [26]. However, in our study, we did not find a statistically significant association between miR-423 genotypes and RPL risk. Notably, a recent study in the Han Chinese population also failed to identify an association between the miR-423 genotypes and RPL risk; however, the study found an association between miR-423 alleles and RPL, with functional evidence showing that the minor A allele contributed to an increased expression of mature miR-423 [7]. An explanation for the different results may be related to the different allele frequencies. The miR-605 rs2043556 A>G polymorphism was included in the study because the variant G allele of miR-605 caused a 2.6-fold reduction in the processing levels of miR-605 [23], and miR-605 was significantly dysregulated in placentas after exposure to Bisphenol A, which may disrupt endocrine levels [22]. However, we did not find a significant association between the miR-605 polymorphism and a risk of RPL. A miR-125a polymorphism involving one nucleotide mutation in the pri-miR-125a coding region related to RPL was not included in the study because the location and nomenclature of the polymorphism were not clear, although the A>G mutation reduced the expression of mature miR-125a [8].
There were some limitations in our study. First, this case-control study identified an association between SNPs in miRNAs in RPL development, but it did not establish a cause-andeffect relationship. What we identified, therefore, warrants functional studies to elucidate the pathogenesis related to RPL. Second, we reported a significant relationship between miR-27a genotypes and plasma folate levels in Korean RPL patients, and we speculated that elevated miR-27a levels contributed to the increased folate levels. However, further functional studies are necessary to directly investigate the effect of mir-27a on RPL when mediated by folate levels. Third, we did not determine the effect of miRNA polymorphisms on miRNA expression in the placenta, which might have contributed to the potential roles of miRNA during the periimplantation and early pregnancy periods.
Accumulation of findings on post-transcriptional regulation of gene expression by miRNAs and their involvements in trophoblast differentiation, proliferation, and angiogenesis during the developments of placenta or fetus have broaden our perspectives on the roles of miRNA and microRNA machinery gene polymorphisms during the pathogenesis of idiopathic RPL since three recent reports on the association of miRNA polymorphisms with RPL in Han-Chinese and Korean women [6-9, 12, 19, 28-32]. In the present study, we identified associations between miRNA polymorphisms (miR-27aA>G and miR-449bA>G) and RPL risk in Korean females, and identified a relationship between miRNA polymorphism (miR-27aA>G) and plasma folate levels. However, what we identified in our study warrants further investigation to elucidate the underlying mechanism in which miRNA polymorphisms modulate folate levels and RPL development.
Supporting information S1 Table. Haplotype-based analyses of miR-27aA>G, miR-423C>A, miR-499bA>G, and miR-605A>G polymorphisms in Korean RPL patients and control subjects for all possible allele combinations (combinations of four sites, three sites, two sites are listed respectively).