Investigation of the association between eNOS gene promoter polymorphism (-786 T>C) and idiopathic recurrent pregnancy loss in Iranian women

Background: Repeated Pregnancy Loss (RPL) is dened as 2 or more consecutive spontaneous losses of pregnancy before 20 weeks. Some genetic polymorphisms such as endothelial nitric oxide synthase (eNOS) gene, which lead to the synthesis of nitric oxide, could be the reasons for RPL. This case-control study was investigated the frequency of -786 T>C variant in eNOS gene promoter in Iranian women with RPL. Methods : Blood samples were obtained from 100 unrelated women affected by recurrent pregnancy loss and 100 unaffected women as controls. Genomic DNA was extracted and -786 T>C polymorphism in eNOS gene promoter investigated by PCR-RFLP method in all of the samples. Statistical analysis in the group patients and controls were performed by chi-square test and P-values of <0.05 were considered signicant. Results: Frequency of homozygous TT was 40% in cases and 46% in control group and frequency of CC was 6% in cases and 5% in the control group and frequency heterozygote TC was 54% in cases and 46% in control group. Genotype frequencies between the two groups showed no signicant differences (P>0/05). Conclusion: The result of this study showed that this polymorphism is not more frequent in recurrent pregnancy loss in this population.


Background
Recurrent pregnancy loss (RPL), is one of the most common complications of pregnancy, occurs in 2 to 5% of clinically recognized pregnancies.It de ned as 2 or more consecutive miscarriages before 20 weeks gestation, which is greater than expected by chance (0.34%) [1,2].Consequently, RPL is an extremely stressful condition for couples and physicians and is an important area of research.This is a multifactorial obstetric problem with a polygenic background [3].
The important and evident roles of nitric oxide (NO) within the body and in diseases have been highlighted.NO is produced by the NO synthase (NOS) family of enzymes.There are three separate enzymes each coded by a separate gene, with clear contrasts in location, regulation, catalytic properties, and sensitivity to inhibitors [4].The three NOS isoforms are named accordingly to their position within the human body; NOS found in the endothelium is known as endothelial nitric oxide synthase (eNOS) (also known as Type III, NOS3), NOS found in macrophages is known as inducible nitric oxide synthase (iNOS) (also known as Type II, NOS2) and the nal type is neuronal nitric oxide synthase (nNOS) (also known as Type I, NOS1) [5].
The structure of the eNOS gene was rst determined by Marsden et al. (1993) [6].It was discovered to contain 26 exons incorporating 21 kb of genomic DNA, encoding for 1,203 amino acids in the form of a 135-kD protein.Also, this gene is located in chromosome 7 (7q35-q36) [7].The eNOS gene has been shown to exhibit different variations.The in uences of these variations on eNOS function and consequently disease is still disputed.The hypothesis is that the presence of the variation within this gene may individually or collectively reduce eNOS function and subsequently NO synthesis.
Numerous studies imply that several gene polymorphisms could be proposed as a risk factor for RPL.One of these genes is eNOS which produces nitric oxide synthase in the vascular endothelium and causes vasodilation and smooth muscle relaxation [8].This enzyme catalyzes the biosynthesis of L-citrulline and Nitric oxide (NO) from L-arginine [9].The level of NO as a signaling molecule will increase during pregnancy.This results in fetal blood supply without hypertension.Therefore, decreasing serum nitric oxide levels at the beginning of pregnancy can interfere with oxygen supply and nutrition for the fetus and leads to abortion [10].In this study, we aimed to analyze the relationship of -786T > C polymorphism (rs2070744) in eNOS gene regulatory region and RPL among central and southern Iranian women.

Study participants
The present study was approved by the Human Ethics Research Committee of the Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences.
All of the controls and subjects who agreed to participate in this study have signed informed consent before the collection of peripheral blood samples.
One hundred women with idiopathic spontaneous repeated pregnancy loss who were attending the recurrent miscarriage clinic and 100 women without any miscarriage who had at least one successful pregnancy were recruited in this study.Control blood samples were collected in routine checkup test.

Blood collection and DNA extractions
Five ml peripheral blood samples were collected in blood collection tubes containing EDTA for anticoagulation.They stored at -20°c until further used for DNA extraction.Genomic DNA extraction was carried out using a commercially available kit (Qiagen, Germany.cat.No.51104) according to the manufacturer's protocol.
The quality of DNA was assessed using 1% agarose gel electrophoresis.Concentration and purity of DNA were estimated in absorbance at 260 nm by spectrophotometer (thermo scienti c).

PCR-RFLP technique
Final assessing was performed by PCR-RFLP method.In other words, digestion of the PCR product with the restriction enzyme NgoMIV allowed detection of the alleles of -786T > C. this enzyme cleavage site on the gene is shown in Fig. 1.The digestion reaction was carried out in a total volume of 25 µl, containing 10 µl PCR product, 0/25 µl R buffer (10X), 0/25 µl NgoMIV enzyme, and 14/5 µl distilled water.The tubes were incubated at 37°C for 3 hours.After digestion, the samples were separated by gel electrophoresis (2% agarose gel) so as to visualize the different products.

Statistical analysis
All data were analyzed using the SPSS standard software (Version 21.0, IBM, Armonk, NY, USA) and chisquare test.Additionally, the results were reported as a graphical representation by GraphPad Prism 6 software.P-values of < 0.05 were considered statistically signi cant for all data.

Study characteristic
Present study was performed on 100 Iranian women who had spontaneous recurrent pregnancy loss and 100 women without history of abortion with one normal pregnancy as control for detection of the frequency of -786C > T variant in RPL.
Extracted DNA evaluated on 1% agarose gel (Fig. 2).PCR reaction was performed and after loading the PCR product on 1/5% agarose gel, the bands were seen at 180 bp (Fig. 3).Enzymatic digestion by NgoMIV was done to produce two smaller fragments (89 bp, 91 bp) and the sequences containing the T base in that position were not cut (Fig. 4).

Allele and genotype frequency distribution
The frequency of TT, TC, CC in cases were 40%, 54%, and 6%, respectively; whereas in control were matched with 46%, 49%, and 5%, in that order.Figure 5 shows that the differences of these genotypes in cases and control were not signi cant (P-value = 0/687).Figure 6 shows that there were no differences between the frequency of C and T alleles among case and control (P-value = 0/091) (Table 1).

Discussion
Recurrent pregnancy loss (RPL), present a critical clinical and stressful problem which affects about 5% of couples around the world [11].The extensive research had been carrying out about its causes and treatment.Although several reasons had been recognized for it, about 50% of RPL cases are still unexplained [12,13].
A normal pregnancy is a complex and dependent process on several essential factors such as cytokines, hormones, growth factors, angiogenesis agents and some signaling pathways.Any dysfunction of these, depending on the stage of pregnancy, can lead to disturbing in adjustments of the pathways involved in fetal development and consequently may cause miscarriage or the other fetus disorders [14].
Some researches disclosed the role of eNOS gene in RPL.Changes in physiological levels of NO could be related to eNOS variants [15].It had been shown that − 786T > C, 27 bp repeat 4b/4a, 894G > T in the eNOS gene supported an association with increasing the risk of RPL [16], but the role of some of these variants like − 786T > C, in RPL, is less studied and there is still a controversy about them.
The present study investigated the prevalence of alleles of the eNOS gene promoter − 786T > C polymorphism in women mostly from south and center of Iran with RPL in compared this with normal control of that population.The χ2 test results demonstrated that there were no signi cant differences between genotype distribution in controls and women with RPL as a whole group.According to -786T > C position in the gene promoter, studies have focused more on this gene expression level.The in vitro investigations asserted the substitution T allele by C in -786 position caused a fty-percent decline in transcription level and maybe this is the result of RPA1 (Replication protein A1) binding as a gene suppressor to this region in peoples with mutant allele [17,18].Also, some researches exposed the lower levels of serum nitrite in the patients with endothelial and vascular disease [19,20].Previous studies had investigated the distribution of -786T > C polymorphism in various groups.No association of this variant and RPL in a cohort of Tunisian had been reported [21].Some other studies among different ethnicities after that had also reported similar ndings [22][23][24][25].In contrast, in the study on the South Korean population had been discovered that − 786T-4b-894T haplotype signi cantly increased in the RPL group [23].Another study on Lebanon women suggested that eNOS gene promoter variation (-786T > C) sustained a substantial impact on RPL in their women population.[26].An investigation on a group of Egyptian women exposed a signi cantly association between RPL and this variant [27].A meta-analysis study reported that this variant has a signi cantly correlation with the RPL risk [28].Azani et al had analyzed the prevalence of this variant among Iranian women in Tabriz.They had revealed that there was a signi cant relationship between this variant and RPL [29].
Unlike the studies mentioned so far, one research evaluated the effect of eNOS gene polymorphisms on the aborted embryos and it displayed that CC and TC genotypes for − 786T > C variant in the eNOS gene in fetus had a signi cant relationship with spontaneous abortion.Indeed, it claimed this gene polymorphisms special − 786T > C promoter variant in the fetus is more likely to be related to RPL than women [30].

Conclusion
The results of this study suggested that the mentioned polymorphism is not related to RPL in the south and center of Iran population.As point out before that, the outcomes of various studies are contradictory.These results infer the association of this polymorphism with RPL among distinct ethnic groups from different countries can be inconsistent and it is because of their genetic variations.

Figure 3 Results
Figure 3

Figure 4 A
Figure 4

Table 1
Distribution of different alleles of eNOS gene in control and the case group of RPL.