Pregnancy is a complex biological phenomenon. The whole process is an immunological anomaly, where the fetus lives by avoiding maternal immune response and is delivered after completing the period of gestation [1]. About 1 to 1.5% of women of reproductive age have recurrent pregnancy loss (RPL), a disease characterised by the recurring loss of two or more successive miscarriages before the 22nd week of gestation. About 10-15% of clinically recognised pregnancies are affected by this embarrassing reproductive problem, which presents a clinically unpleasant and emotionally upsetting challenge for the women experiencing the loss [2]. Numerous chromosomal and molecular abnormalities, anatomical disorders, immunological variations, hormonal abnormalities, environmental variables, and thrombophilic malformations are among the high-risk factors and potential causes of RPL [3, 4]. According to [5], there is no abnormality present in over 50% of RPL instances, and the precise aetiology is still unknown.

The ovary secretes oestrogen, a significant steroid hormone that affects female reproduction in a number of ways. It is essential for the development of pre-implantation, uteroplacental circulation, and the maintenance of fetoplacental maturation and function. The effects of biological function of estrogen are mediated by Estrogen Receptor Alpha (ERα) encoded by ESR1 gene located on chromosome 6 and Estrogen Receptor beta (ERβ) encoded by ESR2 gene on chromosome 14 showing unique functional features in determining cellular growth potential [6, 7, 8, 9]. Estrogen signaling is either elevated or inhibited depending upon a balance of ERα and ERβ in various target organs [10]. ESR1-α gene is extremely polymorphic gene and the two most extensively studied intronic polymorphisms are rs 2234693 (-397C/T) & rs 9340799 (-351A/G) identified by PvuII and XbaI [11, 12]. These intronic variants have been proposed as genetic markers for several ESR1 associated disorders because of linkage disequilibrium with other regulatory sequence variations that may affect the expression and function of the ESR1-α gene. These variations do not result in changes to the amino acids found in proteins. [13, 14]. Various studies have reported an association between RPL and these intronic genetic variations [15, 16; 17].

It may be inferred that the function of these variations varies significantly across various populations given the significance of ESR1-α genetic variations in RPL formation and the varying outcomes of correlation between ESR1-α genetic variations and RPL in different ethnic communities. It is crucial to conduct this study because no previous research of this kind has been done from our ethnically conserved population, and because understanding the underlying causes is crucial to improving future pregnancy outcomes for couples who have experienced repeated miscarriages and to advancing more effective treatment. Considering the above given data, it is clear that one of the most significant genes linked to RPL is ESR1-α. Therefore, we used the PCR-RFLP technique to analyse and determine the function of two common genetic variants of ESR1-α.  Moreover, we used Real-Time PCR to compare the expression of ESR1-α mRNA between patients with RPL and healthy persons.

Participants

In the present study, 100 Recurrent Pregnancy Loss patients and 100 healthy Control were enrolled, which were recruited from the departments of advanced centre for Human Genetics and Gynecology Sher-i-Kashmir Institute of Medical Sciences. Cases with no history of any other endocrinological /autoimmune/inflammatory or any other disorders were also included in the study. Data from all RPL patients was obtained from personal interviews with patients/guardians, and from clinical examination. The data collected included, age, dwelling, TORCH profile, immunological profile, endocrinological profile, consanguinity, ultrasound report and family history.  An informed consent was obtained from both patients and controls to participate in the study. The study was approved by the ethics committee of Sher-i-Kashmir Institute of Medical Sciences hospital.

Sample Collection and Molecular Analysis

Each RPL patient and healthy control provided four millilitres of peripheral blood in EDTA vials for the purpose of isolating genomic DNA. Using the conventional phenol/chloroform procedure, DNA was extracted. The polymerase chain reaction (PCR) was used to identify the genetic variations rs2234693 (C/T, PvuII) and rs9340799 (A/G, XbaI) in intron 1 of the ESR1-α gene, which were then subjected to restriction fragment length polymorphism (RFLP) analysis. 50 ng of genomic DNA, 1× PCR buffer (Biotools, B & M Labs, S.A. Madrid-Spain) containing 2mM MgCl2, 0.20mM dNTPs (Biotools, B & M Labs, S.A. Madrid-Spain), 0.4 μmol of each primer (Sigma-Aldrich Co. LLC·USA), and 1 U DNA polymerase were included in the 25 μl total volume of the polymerase chain reaction (PCR).The following were the conditions of the PCR: a first denaturation step of 95 °C for 5 min; 35 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 60 °C, and extension for 30 s at 72 °C); finally, a final elongation for 5 min at 72 °C [20]. Table 1 lists the primers that were used to amplify ESR1α-397C/T and ESR1α-351A/G. Using the enzymes PvuII (-397C/T) and XbaI (-351A/G) (Fermentas Thermo Fisher Scientific Inc., Massachusetts, USA) (1 U at 37°C for 16 h), 10 μl of the PCR products were digested for the RFLP analysis. The restriction digested products and the PCR products (Table 1) were visualised using a 3% agarose gel.

Analysis of ESR1α mRNA expression

One hundred newly diagnosed RPL patients were chosen based on the differential status of their ESR1α genotypes to test for mRNA expression. Using TRIZOL (Sigma Aldrich, USA), total RNA was isolated from 100 PBMCs of RPL patients and 100 PBMCs of healthy controls. On a 1% agarose gel, the mRNA's integrity was examined, and the 260/280 ratio was used to measure it. Using a first strand cDNA synthesis kit, RNA was transformed into cDNA in accordance with the manufacturer's instructions (Fermentas, USA). To get a consistent amount of cDNA in every sample, dilution of the cDNA was carried out. For ESR1αprimer sequence was as follows F: 5′-TGATTGGTCTCGTCTGGCG-3′ and reverse R: 5′-CATGCCCTCTACACATTTTCCC-3′ and for glyceraldehyde 3-phosphate dehydrogenase, the primers were as follows: forward F: 5?AGAAGGCTGGGGCTCATTTG-3' and reverse R: 5?-AGGGGCCATCCACAGTCTTC -3'.The identification of ESR1α mRNA was achieved using Quantitative real-time PCR (Agilent Biotechnologies, Germany) using Applied Bio systems Inc. Step One software version 2.0. Maxima® SYBR Green qPCR Master Mix (2X) was used for the PCR, and all samples—both unknown and standard—were run in triplicate with a non-template control (NTC) present. 40 cycles of thermal cycling were conducted at 95 °C for 15 s and 57.2 °C for 30 s each cycle. To identify authentic results and identify contamination from non-specific products and primer dimer, the melting curves of all final real-time PCR products were examined. To ensure the right amplification products, all real-time polymerase chain reaction amplified products were separated on 2% agarose gel electrophoresis. Delta CT (Δ^CT) method was used to check the ESR1αmRNA expression in patients with RPL and healthy individuals by normalization against GAPDH used as a reference gene.

Statistical Analysis

Using the χ2 test, the genotypic and allelic frequencies of RPL patients and controls were compared for prevalence.  When a cell's expected count was less than one or more than twenty percent of the cells had an expected count of less than five, the χ2 test was violated, and Fisher's exact test was employed. For each genotype and allele, odds ratios with 95% confidence intervals (CIs) were evaluated to determine how likely they were to cause the illness.  SPSS version 20 was used for the statistical analysis, and the findings were deemed statistically significant for (P < 0.05).

A total of 200 individuals were included in our study (100 cases and 100 controls). Among 100 RM cases 25 cases were positive for TORCH profile, APLA and ANA. Each were seen positive in 08 cases. Thyroid profile was normal in 83 patients and abnormal in 17 cases that consist of 13 hypothyroidism and 04 cases of hyperthyroidism and PCOS was seen in 17 RM cases. 

The distribution of ESR1α-397C/TCC, CT, TT genotypes in patients with RM was 33%, 50% and 17% respectively, while in healthy controls the genotypic distribution of ESR1α-397C/T CC, CT, TT was 50%, 40% and 10% (Table 2). The ESR1α-397 CT+TT genotype was associated with an increased risk of RM development compared withESR1α-397CC genotype [0.5(0.4-0.9), p<0.01].

The distribution of ESR1α-351A/G AA, AG, and GG genotype in patients with RM was 46%, 46% and 08% respectively, while in controls the distribution was 60%, 35% and 05%. The ESR1α-351A/G did not show any significant association with RM. The ESR1-α mRNA expression in peripheral blood of RPL patients and Healthy controls was done by qRT-PCR.

In the present study, the analysis of total ESR1-αmRNA was detected and quantified in 100 RPL patients and 100 healthy control PBMCs. The fold change of ESR1-αmRNA expression in RPL compared to normal PBMCs was calculated by Livac method using the formula fold change= 2^–(ΔΔCt). The mRNA expression was checked by ΔC_T method with expression ratio of (amount of ESR1-αmRNA/amount of GAPDH mRNA) by applying the formula ΔC_T = (C_T GAPDH- C_TESR1-αmRNA). We found a decreased expression of ESR-α mRNA in PBMCs of RPL cases as compared to healthy controls with down regulated 4.369- fold change (Table 3). Furthermore, statistically significant difference in ΔC_T values of RPL patients with respect to ESR1-α 397 C/T SNP was found (Table 4). Genotype TT of ESR1-α 397 C/T genetic variant was found to be associated (p=0.039) imposing 5 fold risk (p=0.07) for RPL development (Table 5). However in ESR1-α 351 A/G SNP we found no statistically significant difference in ΔC_T   values of RPL patients (Table 6). Age and family history were the only parameters which had a significant association (p?0.05) where as other parameters were not significantly associated (p?0.05) with ESR1-α mRNA expression in RPL patients (Table 7).

Table 1: Primers used for amplification and restriction digested and PCR products of ESR1α-397C/T and ESR1α-351A/G.

AT*-Annealing Temperature

£-The same primer set was used to amplify for both polymorphisms variants.

Table 2: Genotypic frequencies of ESR1α- polymorphisms in RPL cases and controls.

Table 3: ESR1α mRNA expression in peripheral blood samples of RPL patients and healthy controls.

Table 4: ESR1αmRNA expression of RPL patients in relation to ESR1α-397C/T.

Table 5: ESR1αmRNA expression (Fold change) of RPL patients in relation to ESR1α-397C/T.

Table 6: ESR1α mRNA expression of RPL patients in relation to ESR1α-351A/G.

Table 7: Association of clinico-pathological variables and ESR1-α mRNA expression in RPL patients.

Furthermore, the ESE finder facilitates the identification of prospective ESE sites in order to investigate the impact of genetic variation utilising Exonic Splicing Enhancers (ESEs)/HSF. The motif scores are represented by the coloured bars' elevation, and the motif's length is shown by the bars' girth. Potential binding sites for the Serine-Arginine (SR) proteins SF2/ASF, SRp55, SC35, SF2/ASF (IgM-BRCA1), and SRp40 are shown by bars in red, yellow, blue, purple, and green, respectively. The bulk of the algorithms employed by the ESE/Hsf tool to identify enhancer/silencer motifs revealed that variations rs2234693 cause the breaking of Serine-Arginine (SR) proteins, SF2/ASF (IgM-BRCA1); however, rs9340799, as seen in figure 1, did not exhibit any change. 

Figure 1: Genetic variation's impact on Exonic Splicing Enhancers (ESEs) as determined by the ESE prediction programme.

Potential ESE locations can be identified with the help of ESE Finder. The motif scores are represented by the coloured bars' elevation, and the motif's length is shown by the bars' girth. Potential binding sites for the Serine-Arginine (SR) proteins SF2/ASF, SRp55, SC35, SF2/ASF (IgM-BRCA1), and SRp40 are shown by bars in red, yellow, blue, purple, and green, respectively. Panel I represents the ESE sequence in the population under investigation with the allele not posing any risk, while Panel II represents the ESE sequence in the group under study with the risk allele. Based on the figure, we may infer that there may be a shift in the possible splicing sites, shown by the bars, which could lead to an increase in the susceptibility to the disease.

Likewise, the putative functional role of the variants were also examined. Linkage disequilibrium plot shows the amount of correlation between a sentinel variant (blue coloured) and its surrounding variants (red coloured) and it was observed that the both genetic variants rs2234693 and rs9340799 of ESR1α gene signifies to have direct regulatory effect on transcript and putative effect on transcript respectively as shown in figure 2.

Figure 2: The degree of association between a sentinel variation (blue) and its surrounding variants (red) is displayed in a linkage disequilibrium graph.

The X-axis represents each SNP's chromosomal location, while the Y-axis represents the correlation coefficient (r2). Every variant's plot symbol indicates its functional findings.

Interaction analysis was also performed which demonstrates the interaction of ESR1α  with other genes via different process like genetic, physical, pathway, shared protein, predicted and co localization as in figure 3

Figure 3: shows the interaction of ESR1 with other genes via different processes.

To the best of our knowledge, this is the first investigation on the relationship between RPL propensity and the ESR1α genetic variant from our region. The relationship between RPL and two intronic genetic variants of the oestrogen receptor α gene (ESR1 −397C/T: [rs2234693] and −351A/G: [rs9340799]) was examined in this study. The primary outcome of this investigation shown that the CT+TT genotype of the genetic variation (-397C/T) is a risk factor that contributes to RPL, while the other genetic variation (−351A/G) in the ethnic Kashmiri community had no effect on RPL. Genetic differences in the genes encoding the oestrogen receptors may impact several pathways dependent on oestrogen, which in turn may impact vascular tone and flow and alter the maintenance of pregnancy [18, 19, and 20]. According to [2], ESR1 is crucial for maintaining systemic and uteroplacental circulation throughout pregnancy. The pathophysiological abnormalities of spontaneous abortions have previously been linked to reduced ESR1 expression brought on by genetic polymorphisms in the ESR1 gene [21]. In the ESR1 gene, many single nucleotide polymorphisms have been reported. Breast cancer risk is raised by SNPs G/T (rs 2881766) and T/C (rs 3798577) [22, 23]. Our research aligns with a Spanish study (Pineda et al., 2010) that established a link between the ESR1−397C.T polymorphism and an increase in miscarriages. Another investigation of [24], demonstrated no relationship between ESR1α−351 A/G SNP and RPL patients which was also in accordance with our results.

Different insilico techniques listed above have proven that distinct processes can cause phenotypic consequences through intronic variation. As seen in figure 2, it may purposefully modify RNA splicing to either increase or decrease gene transcription. This results in an alternative mRNA variation with notable changes in gene function [25, 26, and 27]. Furthermore, intronic polymorphism could be connected to a different sequence variant that is actually functional and should be a genetic marker for a different polymorphism. According to [3]. The C allele of the ESR1 α −397C/T gene polymorphism works as an intragenic enhancer and contains a portion of a B-myb transcription factor binding site, rather than the T allele. According to [28], the effects of oestrogen can be mediated by the enzyme 17β-Hydroxy sterioddehy drogenases (HSD), which is expressed less when the T allele is present. This causes a decrease in the expression of the ESR1α gene and, ultimately, a relative oestrogen deficit. The function of -351A/G polymorphism remains unascertained even though having functional implications [14]. A number of environmental and genetic factors are responsible for the development of RPL; however the ways in which they merge with each other remain poorly understood. The genetic pool is not the confined source of information; moderately its effect on phenotype is continually being impeded by various other factors. Relationship between genetic and non-genetic factors needs to be fully explicated to understand the progression of elements leading to RPL. A small number of SNPs selected for the ESR1-α gene examined in this study provide information on the site's evaluation and whether or not it is in linkage disequilibrium with it. They do not, however, fully explain all of the genetic variants seen in those genes. As a result, it is impossible to overlook the significant contributions that some uncommon mutations and SNPs make to the risk of RPL. It can be challenging to adequately characterise the synergistic effect of SNP pairings, particularly in polymorphic genes, in association studies. In-depth analyses and comprehensive explanations of genetic variants within the ESR1-α gene are crucial, as are well planned research including a larger sample cohort to detect RPL. None the less, we propose that further extensive population-based research is necessary to validate our results and to prospectively evaluate the contribution of ESR1-α (397C/T) and (351A/G) polymorphisms to the onset and progression of recurrent pregnancy losses.

We observed that the ESR1-αmRNA expression was significantly decreased with down regulated 4.369- fold change (Table 2) in RPL PBMCs as compared to those of healthy controls and can act as a promising marker in the early diagnosis of RPL. However, we found significant association of ESR1-α (397C/T)in ΔC_T values of RPL patients as TT genotype imposing 5 fold risk for RPL development (Table 3 and 4 ). However in ESR1-α−351A/G SNP no statistically significant difference was found in ΔC_T values of RPL patients (Table 5). Study done by [9] also found decreased expression levels of ESR- α mRNA levels in PBMCs in the Osteoporosis patient group than in menopausal control groups where expression levels were significantly higher.

No previous studies have aimed to focus on the expression levels of ESR1-α in RPL patients. Our data from RPL women signify that the expression of ESR1-α mRNA is decreased in PBMCs from patients with RPL as compared to healthy controls. The diminished ESR1-α mRNA expression in PBMCs in patients with RPL could emulate either predisposing factor or a repercussion of the disease.

The study will help in understanding the genetic landscape of recurrent pregnancy loss in genetically less explored population of Jammu and Kashmir and the findings of the study will act as a predictive or prognostic biomarkers and will be a step towards a personalized medicine.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are grateful to the Department of Advanced centre for Human Genetics SKIMS, who helped us in the sample procurement. Authors are grateful to all the patients for their cooperation.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institution and/ or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed Consent

Informed consent was obtained from all the individual participants included in the study.

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