The effects of DICER1 and DROSHA polymorphisms on susceptibility to recurrent spontaneous abortion

Abstract Background Recurrent spontaneous abortion (RSA) is a serious problem in pregnancy. The exact etiology of RSA is unknown in more than 50% of all the patients. However, genetic variations are known as susceptibility factors for idiopathic RSA. Considering the role of miRNA biosynthesis machinery in the miRNA production and effect of miRNAs on various diseases, this study aimed to evaluate the effects of DICER1 rs3742330 and DROSHA rs6877842 polymorphisms on RSA risk. Methods In this case‐control study, 150 RSA patients and 195 age‐matched healthy female controls were recruited. Both polymorphisms were genotyped using PCR‐RFLP method. Results The frequency of DICER1 rs3742330AG genotype was higher in the control group (P = .022). There was a statistically significant association between rs3742330 polymorphism and a reduced RSA risk in dominant and allelic models (P = .013 and P = .007, respectively). No statistically significant association was found between DROSHA rs6877842 variant and RSA risk. The combination of AG and GC genotypes and G‐G alleles of DICER1 rs3742330 and DROSHA rs6877842 polymorphisms led to a decreased RSA risk. However, the synergic effect of rs3742330A and rs6877842G alleles (A‐G) and AA‐GG genotypes was associated with an increased RSA risk. Conclusion the DICER1 rs3742330AG genotype and combination of AG and GC genotypes and G‐G alleles of DICER1 rs3742330 and DROSHA rs6877842 polymorphisms were associated with a reduced RSA risk.

problems, abnormal inflammation responses, thrombophilia, and infections are identified as other risk factors for the pathogenesis of RSA. 3,4 However, the exact cause of RSA remains unknown in more than 50% of RSA cases classified as idiopathic unexplained RSA (URSA). 5 Genetic variations are described as other risk factors for susceptibility to idiopathic RSA. 6 Higher frequency of RSA in siblings of patients confirmed the genetic etiology for idiopathic RSA. In addition, similar gestational age of miscarriage in a mother is another reason for this theory. 7 Recently, the role of noncoding RNAs like microRNAs (miRNA) in the different cellular processes, such as proliferation, differentiation, cell death, immunity, and metabolism, has been established. miRNAs are ~22 nucleotides noncoding RNAs which can affect gene expression at the post-transcriptional level by translational suppression or mRNA degradation. These short RNAs are generated from primary transcripts by nuclease activity of various enzymes in several steps, called miRNA biosynthesis machinery. 8,9 In the first step, Drosha enzyme catalyzes the conversion of first transcript to precursor miRNA (pre-miRNA) hairpins. In the second step, the transfer of pre-miRNAs to the cytoplasm is done by Ran-GTPase (Ran) and exportin 5 (Xpo5). Then, they are converted into small interfering RNA and microRNA by a Dicer1 enzyme. Therefore, insufficient function or level of these enzymes can lead to altered concentration of miRNAs and subsequently their target genes. 8,9 The altered levels of several miRNAs have been reported in RSA patients. 10,11 There is also evidence that enzymes of this process are involved in cell proliferation, differentiation, and apoptosis. 12 Indeed, several studies have shown that that Dicer and other components in miRNA biogenesis play key roles in sex-related activities for both females and males, including follicular development, ovulation, luteinization, sex hormone synthesis, and the regulation of the functions of the fallopian tube and endometrial receptivity in female reproduction. 13 Considering the role of genetic variants in pathogenesis of multifactorial diseases, numerous studies have evaluated the effects of miRNA and miRNA biosynthesis machinery polymorphisms on their pathogenesis. 14-16 DROSHA and DICER1 genes are located on chromosomes 5 and 14, respectively. 17,18 There are several single nucleotide polymorphisms (SNPs) in DROSHA and DICER1 genes, and their effects on various diseases have been investigated. 16,19 Two other studies demonstrated that the combination of DROSHA and DICER1 polymorphisms could increase RSA risk. 20,21 To date, very few studies have been conducted on the effects of DROSHA and DICER1 polymorphisms on RSA. Since no study in this regard has been done in Iran, the present study was designed to examine the effects of DROSHA and DICER1 polymorphisms on RSA susceptibility in an Iranian population.

| Genotyping
Five hundred microliters of EDTA-treated blood samples was collected for DNA extraction by salting out method. The restriction fragment length polymorphism (RFLP) was used for the genotyping of both polymorphisms. 22,23 The protocol for PCR digestion was performed as previously described. 14,24

| Statistical analysis
Hardy-Weinberg equilibrium was analyzed by chi-square test in both groups. The odds ratio (OR) and 95% confidence interval (CI) were calculated using logistic regression analysis in order to assess the association between polymorphisms and RSA P < .05 was considered as statistically significant. Student's t test and Fisher's exact test were used for the comparison of two groups for quantitative and qualitative parameters. Statistical analysis was performed with SPSS version 23. In addition, Figure 1 shows the results of agarose gel electrophoresis for DICER1 rs3742330 and DROSHA rs6877842 polymorphisms using PCR-RFLP method. The frequency of DICER1 rs3742330 AG genotype in control group was higher than that in RSA women (26.7% vs 16.7%), associated with a 0.5-fold increased risk of RSA (P = .022). Moreover, this variant might decrease RSA risk in dominant and allelic models. The frequencies of DROSHA rs6877842 GC and CC genotypes were higher in control group, but the differences were not significant ( Table 2).

| RE SULTS
When we analyzed the effects of the combination of DICER1 rs3742330 and DROSHA rs6877842 polymorphisms on RSA susceptibility, we found higher frequencies of AA/GG combined genotypes in RSA cases and all other combined genotypes in controls (P = .008, Table 3). Moreover, the AG/GC combined genotype was associated with a decreased risk of RSA (P = .03).
In addition, the analysis of synergic effects of DICER1 rs3742330 and DROSHA rs6877842 alleles showed that the synergic presence of rs3742330A and rs6877842G alleles (A-G) was more frequent in RSA patients, associated with a 1.7-fold increased risk of RSA. But the G-G condition could lead to a decreased RSA risk (Table 4).

| D ISCUSS I ON
miRNAs as a class of noncoding RNAs can regulate several processes in the placentas, such as placental immune activation and trophoblast invasion; therefore, they can affect gestational process. 25 Aberrant expression and dysregulated pregnancy associated with miRNAs have been proven in several pregnancy complications, including preeclampsia and RSA. 25,26 It is well known that the disor- muscle cells showed that this defect could lead to developmental delay, extensive hemorrhage, and finally embryonic death. 29 There are single nucleotide polymorphisms (SNPs) in DICER1 and DROSHA genes which can affect their function or concentration.
The location of rs3742330 SNP is in the 3ˊ-untranslated region (UTR) of DICER1 gene, which can affect the DICER1 gene stability and expression. 30 Our previous in silico study revealed that rs3742330C or G allele generated the novel binding sites for three miRNAs, that could trigger the mRNA degradation. 14 In addition, the DROSHA rs6877842 polymorphism is located in the promoter region, which can affect the gene expression.
Several studies have found the relationship between DICER1 and DROSHA polymorphisms and several diseases, but the published reports on their impacts on RSA are rare. 16,19,24 Contrary to the results of current study, in their first investiga-  This study has some possible limitations which need to be pointed out. First, our data from placental pathology or immunologic parameters were not complete to analyze in association with the variants. Second, the relative small sample size could affect our results, especially for those with marginal P-values. Third, if we could analyze these variants in aborted fetus as well as the mRNA and protein expression levels of Dicer1 and Drosha, our findings become more valuable.
In conclusion, the results of present study showed the protective role of DICER1 rs3742330 polymorphism in RSA risk. However, there was no relationship between DROSHA rs6877842 polymorphism and RSA. The combination of DICER1 rs3742330 AG and DROSHA rs6877842 GC genotypes and G-G haplotype was associated with a reduced RSA risk; however, AA-GG combined genotypes and A-G haplotype could lead to an increased RSA risk.

ACK N OWLED G M ENTS
The study was supported by the Research Deputy in Zahedan University of Medical Sciences (IR. ZAUMS. REC.1396.261). We wish to acknowledge all the participants (patients and controls) who contributed to this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.