The effect of MicroRNAs variants on idiopathic recurrent pregnancy loss

Abstract

Background

Although the importance of miRNA variants in female reproductive disorders has been frequently reported, the association between miRNA polymorphisms and recurrent pregnancy loss (RPL) has been poorly studied. In this study, we aimed to assess the correlation of four different miRNA variants to unexplained RPL.

Methods and results

The prevalence of four SNPs including miR-21 rs1292037, miR-155-5p rs767649, miR-218–2 rs11134527, and miR-605 rs2043556 in 280 cases with iRPL and 280 controls was performed. The DNA was extracted from all subjects and the SNPs were genotyped using RFLP-PCR methods. The data revealed that rs1292037 and rs767649 were significantly associated with higher rates of iRPL in patients compared with controls while rs11134527 and rs2043556 showed no association with increased rates of iRPL among patients. The haplotypes T-A-G-G and T-A-G-A were the most frequent in both cases and controls. Three haplotypes including T-T-G-A, C-T-G-G, and T-A-A-A showed significantly different frequencies in patients in comparison to healthy females.

Conclusion

This study suggests that rs1292037 and rs767649 could be risk factors for increased rates of iRPL.

Introduction

Recurrent pregnancy loss (RPL), is defined according to the American Society for Reproductive Medicine (ASRM) as two or more miscarriages; that is, pregnancies with the same partner, and affects about 1–2% of couples worldwide [1]. Different causes including immunological, hormonal, pathological, genetic, and environmental factors contribute to the increased rate of recurrent pregnancy loss [2]; however, half of the cases remain unexplained which is called idiopathic recurrent pregnancy loss (iRPL). Genetic factors have been shown to play a major role in female reproductive disorders such as infertility, recurrent miscarriage, and implantation failure [3–7]. While researchers have been mostly focused on investigating the effects of related genes on reproductive problems [8–11], the important role of microRNA variants has been neglected and only a few studies have been centered on the impacts of microRNAs on reproductive diseases such as idiopathic pregnancy loss [12–14], which constitutes 15% of all clinically recognized pregnancies losses [15].

MicroRNAs (miRNAs) are small noncoding RNAs that are essential components in post-transcriptional gene regulation and are responsible for controlling the expression of different genes by pairing with the complementary base sequence in mRNA molecules and inhibiting their translation, therefore, miRNAs play a crucial role in different physical cell processes and their dysregulation is the blame for the development of many diseases [16, 17]. Studies have demonstrated that specific gene mutations including single nucleotide polymorphisms (SNPs) in miRNAs may potentially interfere with miRNA-mediated regulation of cellular functions and lead to certain female reproductive dysfunctions [18–20]. Assessing the role of miRNAs in female reproductive disorders would further assist in clarifying the mechanisms involved in female infertility and result in finding more suitable and potential biomarkers and therapeutic agents for these diseases. Thus, in the present study, we have investigated the effects of four different miRNAs variants including miR-21 rs1292037, miR-155-5p rs767649, and miR-218–2 rs11134527 and miR-605 rs2043556 to further clarify the significance of miRNA variants in reproductive diseases. To the best of our knowledge, this is the first study to investigate the association of these SNPs with iRPL.

Methods

A total of 560 women including 280 patients with iRPL and 280 age-matched healthy individuals who had at least one successful pregnancy and a live child participated in this study. Healthy females in this study were chosen carefully and no female had a history of pregnancy complications including, abortions, stillbirth, pre-eclampsia, ectopic gestation, preterm birth, and other deformities. They all were healthy women with regular menstrual cycles, normal ovary, and uterus morphology (based on the pelvic ultrasound examination), and no anomalies in the reproductive system. The patient group included 280 women who all suffered from iRPL. Women who had at least one live birth or a child were excluded from the case group. Pregnancy loss was diagnosed by a gynecologist or reproductive endocrinology specialists before 20 weeks of gestational age according to the definitions of infertility and recurrent pregnancy loss by the American Society for Reproductive Medicine [21]. Blood tests for glucose levels, sex hormones including prolactin, thyroid-stimulating hormone, free T4, follicle-stimulating hormone, luteinizing hormone, and progesterone were done. Also, their husbands were healthy individuals and had no fertility problems which was confirmed through their medical records. Chromosome karyotypic analysis of peripheral blood was performed in both parents. In addition, environmental factors such as alcohol consumption, smoking, radiation exposure, and other diseases like cancer, autoimmune disorders, and illnesses that might affect reproductive functions were considered before conducting this research. Females who had cancer, immune disorders, genetic abnormalities, infectious diseases (STDs), or systemic diseases such as diabetes mellitus were removed from the study. As smoking and drinking alcohol are major risk factors and could negatively affect pregnancy and fetal development, not smoking cigarettes and not drinking alcohol were among the main criteria for selecting subjects for studying. Women who smoked and consumed alcohol were excluded from this investigation.

All the women were asked to read and sign the consent form. This study was approved by the ethics review board of the Department of Obstetrics, Hengshui People’s Hospital.

The authors of this study were strict about the criteria and the subjects included in this study to minimize the effects of other risk factors. The 560 subjects included in this study were chosen carefully and they all had the same ethnicity. The cases and controls in this study all belonged to the Han ethnic group.

After taking blood samples from all cases and controls, the genomic DNA was extracted using the Blood and Cell Culture DNA kit (QIAGEN, China) according to the manufacturer’s protocol. Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) was used for genotyping. The details regarding the primers, annealing temp, and the restriction enzymes used for each miRNA variant are shown in Table 1. After the digestion, the resultant was separated on the agarose gel. The SNPAlyze software (ver.9, Dynacom, Japan) and also SPSS (ver.26) were used for statistical analysis. The genotype frequencies and the deviations from Hardy–Weinberg equilibrium (HWE) were assessed using a Pearson χ2 statistic and χ2 goodness-of-ft test, respectively. In addition, haplotype analysis of all variants was done according to the maximum likelihood method with an expectation–maximization algorithm. P value less than 0.05 was considered significant.

Table 1.

General information of selected SNPs

SNP	Position	Primer sequences	Annealing °C	Enzyme	Products length (bp)
miR-21 rs1292037	Chr17:59841547	5'-ACTGTCTGCTTGTTTTGCCTA-3' 5'- TGAAAGAGATGAACCACGACT-3'	57	TspRI	540, 338,202
miR-155-5p rs767649	Chr21:25572410	5’-CCT GTA TGA CAA GGT TGT GTT TG-3' 5’-GCT GGC ATA CTA TTC TAC CCA TAA-3’	60	TSP451	252, 158,94
miR-218–2 rs11134527	Chr5:168768351	5’-AGCGACTGGTCAGAGTCAAGG-3’ 5’-CCTGAAGCTCCCGAGTATGGG-3’	67	Hin1II	252, 144,108
miR-605 rs2043556	Chr10:51299646	5’-CGCCTCTTTTTGCTCATTCT-3’ 5’-AGAGCAGTTACGCCACATGA-3’	59	HinfI	284, 142, 142

Results

The DNA of 560 women comprising 280 cases and 280 controls was evaluated to assess the association of four different miRNA variants with iRPL.

The BMI (Kg/m2) of patients (29.42 ± 3.96) showed no statistically significant difference from the BMI of healthy subjects (28.85 ± 4.32) (p = 0.47). No difference was also seen in assessing the age of subjects (31.26 ± 3.87) (33.26 ± 4.10) (p = 0.16) (Table 2).

Table 2.

The demographic and biochemical characteristics of cases and controls

Variables	Controls (280)	Patients (280)	P-value
Age (mean ± SD)	33.26 ± 4.10	31.26 ± 3.87	0.16
BMI (Kg/m2)	28.85 ± 4.32	29.42 ± 3.96	0.47
Smoking	NA	NA
Alcohol consumption	NA	NA
At least one successful pregnancy (n)	1.5 ± 0.8	NA
History of pregnancy loss (n)	NA	3.11 ± 1.77
Mean gestational age (weeks)	39.13 ± 1.61	7.23 ± 1.30	< 0.001
FSH (mIU/mL, mean ± SD)	7.13 ± 2.44	5.92 ± 10.43	< 0.001
LH (mIU/mL, mean ± SD)	3.85 ± 1.74	8.41 ± 9.36	< 0.001
TSH (µIU/mL, mean ± SD)	NA	2.66 ± 1.49
Prolactin (µIU/mL, mean ± SD)	NA	16.45 ± 12.94

The numbers in bold is the statistical significance (p < 0.05)

The frequencies of SNP alleles in both cases and controls were in accordance with the Hardy–Weinberg equilibrium (Table 3).

Table 3.

Allele and genotype distribution frequencies of SNPs in the case and control groups

SNP	Model	Case	Control	X2	OR (95% CI)	P-value
rs1292037	Allele T	399 (71.2)	429 (76.6)
	Allele C	161 (28.7)	131 (23.3)	4.31	1.32 (1.01–1.73)	0.03
	Co-dominant (TC v TT + CC)	109 (38.9)	103 (36.7)	4.94	1.09 (0.77–1.54)	0.08
	Dominant (TT v TC + CC)	145 (51.7)	163 (58.2)	2.46	1.30 (0.93–1.82)	0.11
	Recessive (CC v TT + TC)	26 (9) HWB = 0.40	14 (6.7) HWB = 0.65	3.92	1.95 (0.99–3.82)	0.04
rs767649	Allele A	378 (67.5)	441 (78.7)
	Allele T	178 (31.7)	119 (21.2)	18.35	1.79 (1.37–2.34)	< 0.0001
	Co-dominant (AT v AA + TT)	112(40)	91 (32.5)	17.19	1.40 (0.99–1.98)	< 0.0001
	Dominant (AA v AT + TT)	133 (47)	175 (62.5)	13.02	1.85 (1.32–2.60)	< 0.0001
	Recessive (TT v AA + AT)	33 (11.7) HWB = 0.21	14 (5) HWB = 0.62	9.94	2.72 (1.43–5.18)	0.002
rs11134527	Allele G	398 (71.0)	413 (73.7)
	Allele A	162 (28.9)	147 (26.2)	1.08	1.14 (0.88–1.49)	0.29
	Co-dominant (GA v GG + AA)	122 (43.5)	111 (39.6)	1.30	1.17 (0.84–1.64)	0.52
	Dominant (GG v GA + AA)	138 (49.2)	151 (53.9)	1.30	1.21 (0.87–1.69)	0.25
	Recessive (AA v GG + GA)	20 (7.1) HWB = 0.31	18 (6.4) HWB = 0.68	0.12	1.12 (0.58–2.17)	0.72
rs2043556	Allele G	415 (74.1)	418 (74.6)
	Allele A	145 (25.8)	142 (25.3)	0.05	1.03 (0.79–1.35)	0.81
	Co-dominant (GA v GG + AA)	95 (33.9)	112 (40)
	Dominant (GG v GA + AA)	160 (57.1)	153 (54.6)	4.01	0.77 (0.54–1.08)	0.13
	Recessive (AA v GG + GA)	25 (8.9) HWB = 0.06	15 (5.3) HWB = 0.34	0.31 2.73	0.90 (0.65–1.27) 1.73 (0.89–3.37)	0.57 0.09

The numbers in bold is the statistical significance (p < 0.05)

The sample power calculated for rs1292037 was 131 per group and 262 for both cases and controls. The calculation for rs767649 was 171 per group and 342 in total. It was 142 and 183 per group for rs11134527 and rs2043556, respectively.

The analysis of genotype and allele frequency was performed in all samples for four SNPs including rs1292037, rs767649, rs11134527, and rs2043556. The details regarding the allele and genotype distribution frequency for the mentioned SNPs in each group are shown in Table 3.

The data suggest that rs767649 was significantly associated with higher rates of iRPL in patients compared with controls under the dominant, recessive, co-dominant, and allelic model, whereas rs11134527 and rs2043556 demonstrated no statistically significant association with iRPL. It was also revealed that rs1292037 is related to increased rates of iRPL under recessive and allelic models.

Furthermore, the haplotype analysis was done for rs1292037, rs767649, rs11134527, and rs2043556. We found that the haplotypes T-A-G-G and T-A-G-A were the most frequent in both cases and controls and T-A-A-A was not found among the healthy individuals, whereas the haplotype C-T-G-A was not presented in cases. Further analysis revealed that three haplotypes including T-T-G-A, C-T-G-G, and T-A-A-A showed significantly different frequencies in patients compared to healthy females. The frequencies of estimated haplotypes between the two groups are shown in Table 4.

Table 4.

Distribution of haplotype blocks in endometriosis patients and controls

rs1292037	rs767649	rs11134527	rs2043556	Overall frequency	Case frequency	Control frequency	OR (95% CI)	P-value
T	A	G	G	0.29	0.32	0.26	0.81 (0.63–1.05)	0.12
T	A	G	A	0.14	0.15	0.13	0.77 (0.57–1.05)	0.10
C	A	G	G	0.11	0.12	0.10	0.88 (0.59–1.32)	0.55
T	A	A	G	0.10	0.09	0.10	0.86 (0.55–1.34)	0.51
T	T	G	G	0.09	0.08	0.10	1.00 (0.63–1.58)	0.98
T	T	A	G	0.06	0.05	0.06	1.40 (0.92–2.12)	0.11
C	A	A	G	0.05	0.04	0.06	0.97 (0.61–1.57)	0.91
T	T	G	A	0.04	0.03	0.05	3.06 (1.10–8.49)	0.02
C	T	G	G	0.04	0.02	0.05	1.80 (1.03–3.17)	0.03
T	A	A	A	0.02	0.04	0.00	0.11 (0.01–0.87)	0.02
C	A	G	A	0.02	0.01	0.02	2.01 (0.50–8.11)	0.50
C	T	G	A	0.01	0.00	0.02	4.05 (0.85–8.19)	0.11
C	T	A	G	0.01	0.01	0.01	1.05 (0.22–1.65)	0.47
C	T	A	A	0.01	0.01	0.01	1.83 (0.83–4.00)	0.12
C	A	A	A	0.01	0.01	0.01	2.02 (0.39–3.89)	0.61

The numbers in bold is the statistical significance (p < 0.05)

Discussion

Mutations and genetic variants in miRNA sequences and their target sites could negatively affect and disrupt their biological functions such as gene expression, leading to the progression of many diseases [22]; however, their essential roles have been underestimated by researchers and less attention has been drawn toward studying their significance in association with diseases. The result of the present study suggests that rs1292037 and rs767649 could be risk factors for increased rates of iRPL, while rs11134527 and rs2043556 showed no association with higher rates of iRPL among patients.

Over the last two decades, it was revealed that mutations within the sequence of the miR-21, miR-155-5p, miR-218–2, and miR-605 were the blame for a lot of human diseases including female reproductive diseases such as polycystic ovary syndrome (PCOS) [23], endometriosis [24], Recurrent implantation failure [25], gestational diabetes, and cervical cancer that would considerably impact reproductive health [26]. The most commonly studied SNPs rs1292037, rs767649, rs11134527, and rs2043556 are frequently found to be related to higher rates of different diseases [27–29]. Although the data suggested the critical role of these miRNAs and their expression in implantation, placental function, and the maintenance of pregnancy and how abnormalities in their sequence could lead to dysregulation and negatively affect the processes of implantation as well as placentation, ultimately resulting in miscarriage and different pregnancy complications, there is still a lack of data regarding the impacts of these variants on the female reproduction and pregnancy outcome. Therefore, we aimed to evaluate the effects of rs1292037, rs767649, rs11134527, and rs2043556 variants in relation to RPL.

Numerous studies have studied the vital roles of miRNAs in the prognosis and progression of pregnancy complications [30–34], highlighting the crucial roles miRNAs play in embryonic development and fetal growth by directly controlling the expression and translation of specific genes [35–38]. Scientists have frequently shown that aberrant expression of miRNAs is linked to several pregnancy disorders including, preeclampsia [39–41], abortion [42, 43], preterm delivery [44, 45], low birth weight [46, 47], ectopic pregnancy [48, 49], and gestational diabetes mellitus (GDM) [50–52], and have considered them potential biomarkers for monitoring the adverse pregnancy outcomes.

Genetic variants can alter tissue formation and remodeling during pregnancy [53]. A successful pregnancy necessitates structural and physiological alterations in maternal tissues during different stages including embryo implantation, placentation, blood vessel formation, maintenance of hemostasis, immune tolerance, and pregnancy maintenance [54]. That’s why changes in genes involved in implantation and embryonic development, as a result of mutations and SNPs, might be a potential risk factor for iRPL. Studies have frequently shown the significant roles of miRNA in every step of pregnancy and fetus development such as cell differentiation, adhesion, migration, apoptosis, and angiogenesis, and how the aberrant expression of miRNAs due to genetic variants has led to pregnancy-related complications [34, 55–57].

The vital role of miR-21, and miR-155 during pregnancy has been shown in several studies. It’s been reported that both mir-21 and mir-155 are essential regulators of phosphatase and tensin homolog (PTEN). Mir-155 has suppressive impacts on PTEN in a first-trimester human trophoblast cell line, and can thus aid in the regulation of apoptosis through the AP-1/NF-κB pathway [58]. Likewise, mir-21 overexpression in a third-trimester human placental cell line downregulated PTEN mRNA and protein [59]. Similarly, mir-21 overexpression was negatively related to birthweight [60], formation of macrosomia [61], GDM [62], recurrent abortion [63], and preeclampsia [64, 65] by targeting RASA1 [66]. Mir-155 has also been related to increased rates of preeclampsia by down-regulating CYR61 [67] and inhibiting trophoblast cell migration [68].

Mir-218, another miRNA study in this research, was also reported to be significantly downregulated in severe cases of preeclampsia [69] and promote trophoblast invasion and differentiation through the TGF-β2 pathway [70]. Mir-218 by targeting LASP1 inhibited trophoblast invasion and migration [71, 72].

So, changes in the sequence of miRNAs responsible and involved in implantation and embryonic development, as a result of mutations and SNPs, might be a potential risk factor for iRPL. Based on our findings, miR-155 rs767649 was significantly associated with higher rates of iRPL in patients compared with controls under the dominant, recessive, co-dominant, and allelic model, suggesting A allele to be a prognostic factor in patients with iRPL. It was also revealed that miR-21 rs1292037 is related to increased rates of iRPL under recessive and allelic models, indicating the minor allele (C allele) to be a risk factor for iRPL.

The haplotype analysis also confirmed these results. The haplotypes beginning with TT, corresponding to rs1292037 and rs767649, were the most frequent haplotypes among healthy controls and TTGA showed significantly higher frequencies in healthy females compared to patients. Interestingly, the haplotypes containing the A allele at the rs767649 indicated higher prevalence in cases compared to controls. Furthermore, the haplotype TAAA was not found among healthy controls and demonstrated significantly high frequency in patients.

Several studies have shown miRNA variants’ fundamental role in susceptibility to female reproductive disorders. Farsimadan et al. reported that miR-146a, miR-149, and miR-499 variants may have a role in the pathogenesis of endometriosis [73]. The aforementioned miRNA SNPs were also connected to polycystic ovary syndrome and iRPL [74]. Also, in a study conducted on a group of 1041 individuals including 389 patients with RPL and 652 healthy controls, a SNP in mir-125 was found to be contributed to RPL [75]. Another study in China revealed the haplotype-based association of two SNPs in miR-423 with iRPL [11]. Similar to our findings, miR-605 rs2043556 was not linked to recurrent implantation failure in a survey in Korea [25]. Polymorphism rs2043556 has also been demonstrated to modulate the risk of developing gastric cancer in a Brazilian population [76]. The G allele at polymorphism of rs1292037 was suggested to be a prognostic factor for patients with cervical cancer [77].

Mir-21 variants have also shown an association with other diseases as well [78–80]. A significant association of mir-155 rs767649 with increased risk of preeclampsia [81], cervical cancer [82], lung cancer [27], multiple sclerosis [83], and type 1/2 diabetes mellitus [84] has also been confirmed.

In conclusion, the results of our study demonstrated the functional SNPs of two miRNAs including miR-21 rs1292037 and miR-155-5p rs767649 to be linked to iRPL among Chinese women. Understanding the vital role of miRNA in female reproductive disorders not only facilitates the understanding of their importance in a normal pregnancy but also helps find potential biomarkers to detect pregnancy-associated complications and assist those suffering from such disorders. The authors of the present study suggest studying the association of these miRNAs SNPs with female disorders in other populations.

Data availability

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Declarations

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. This study was confirmed by the ethics review board of the Department of Obstetrics, Hengshui People’s Hospital with a full ethical code (2020–0041-25).

Consent to publish

All the women were asked to read and sign the consent form.

Conflict of interest

None.