Molecular Investigation of Association Among Common IL-6 Polymorphism with Cytomegalovirus(CMV) Infection and Recurrent Miscarriage in Iranian Women

Background: Recurrent pregnancy loss (RPL) is described as two or more spontaneous abortions. Until now, although various factors such as genetic, endocrinology, anatomy, immunology, and microbiology have been distinguished that affect abortions, the precise basic etiology in up to 50% of RPL cases are not determined. Human cytomegalovirus (CMV) infection and host genetic background, like IL-6 SNP polymorphisms play important roles in RPL etiology. Objective: This study aimed to evaluate relationship among single nucleotide polymorphisms (-634C/G and -174 G/C) in the IL-6 gene with CMV infection and risk of RPL for early detection and treatment of RPL. Materials and methods: This case-control study was carried on 80 Iranian females with RPL and 80 healthy females as control group. The extraction of DNA from samples and detection of CMV and IL6 SNPs were determined by Tetra ARMS-PCR. Finally, the statistical analysis for detection CMV and two polymorphisms roles in RPL were analyzed by Epi Info TM software by X2 test. Results: Our results indicated an increased rate of CMV infection in RPL group (44%) versus the control group (25.45%). Also, the prevalence of IL-6 -634C/G genotype among RPL patients with CMV infection was 80%, while the frequency of this genotype among RPL patients without CMV infection was 50%. Furthermore, no substantial relation was found between IL-6 -174 G/C genotypes and RPL (P ≤ 0.0001). Conclusion: This study not only indicated a signicant role of CMV in RPL, but also showed CMV association with allele G in IL6 -634 among Iranian women. In addition, suggested the use of CMV and IL6 -634 GG genotypes in RPL as diagnostic and prognostic biomarkers in Iranian population.


Introduction
Miscarriage is a severe pregnancy disorder which described as the loss of a pregnancy before 20th week.
It usually occurs in 20 percent of all rst pregnancies. genetic, endocrine, anatomic, immunologic factors, and various infections can lead to RPL. There is substantial con rmed evidence presents that preventable infections can cause up to 15% of early spontaneous abortions and up to 5% of all RPL [1]. Congenital infections are common vital dangers to the fetus's wellbeing, and it is noticeable that most of them are caused by viruses. Rubella, cytomegalovirus (CMV), and Toxoplasma are prevalent cause of infections in the human's uterus and can lead to various problems such as premature infant, abortion, stillbirth, and congenital anomalies [2,3].
Human CMV, belongs to the viral family known as Herpesviridae, is one of the most prevalent causes of congenital infections [4,5]. Over the last few decades, several studies were carried out on the pathogenesis and epidemiology of CMV-related infections, the most prevalent reason of congenital viral infections, in pregnant females. Congenital CMV, can develop during pregnancy as a result of reactivation of formerly acquired CMV or primary infection [6,7]. The effects of primary infection of CMV in pregnant women which can lead to the loss of pregnancy is still under study and analysis, and basic mechanisms are unknown [8][9][10]. Some investigations discovered the high level of CMV-related antigens in tissue from abortion [11] and some ndings revealed higher seropositivity [12]. In addition, one study indicated an increased risk between pregnancy loss and CMV infection [13], though the ndings of other related studies did not compatible with it [14]. Although some of these studies demonstrated the role of CMVrelated infection in uncontrolled pregnancy loss, the effects of CMV infection on RPL is unknown.
Etiologic factor related with RPL are proposed as genetic, infectious, anatomical immunological, and environmental causes [15,16].
On the other hand, the commitments of immunogenetic variables to the etiopathology of RPL have been recommended. Throughout the early phases of ordinary pregnancies, pro-in ammatory T aide (Th) 1 cytokine such as IL-6, TNF-α, and IL-10 are fundamental for fortifying vasculogenesis which is fundamental for effective embryonic implantation. While, drawn out presentation to Th1 cytokines can lead to a cell-mediated resistant reaction that's inconvenient to the baby, driving to pregnancy misfortunes [17,18].
It has been shown that ordinary pregnancies are went with an in general higher level of Th2 cytokines whereas pregnancies with undesirable results are ordinarily related to an in general higher expression of Th1 cytokines such as interleukin 6 (IL-6) [19][20][21][22]. The IL-6 is a vital regulator of in ammation, has a remarkable role in the pro-in ammatory responses, and considers as an anti-in ammatory cytokine in the regulation of immune system. Several SNPs within the gene region of IL-6 could alter IL-6 expression and thence affect downstream target genes. Previous reports have suggested that -634C/G and -174 G/C SNPs may affect the IL-6 expression and is linked to the risk of RPL [23][24][25][26]. However, some studies display a negative a liation between IL-6 polymorphism and RPL risk.
The aim of this study is to evaluate the associations between IL-6 polymorphism at position 634 and 174, CMV infection, and incidence of RPL in Iranian population. In order to achieve this purpose, we conducted a study through Tetra-ARMS PCR, PCR-RFLP, and statistical analysis (SPSS) methods. Our results demonstrated a signi cant association among CMV infection, IL-6 -634 G/C polymorphism, and RPL patient. We hope the ndings of this study can shed light on molecular markers having signi cant roles in the ethology of the miscarriage.

Participants
This study carried on two different groups as case-control study. The control group contained 80 women who experienced at least two successful pregnancies and no background of abortion. The case group consisted of 80 Iranian women with a history of RPL. The case samples involved women referred to Khatam-Ol-Anbia hospital, Tehran, Iran from April 2017 to June 2018. Written consent was received from all the subjects to do molecular analyses. Detailed pedigree analyses and detailed assessment of the clinical reports was undertaken in all the subjects. Patients having acquired or hereditary thrombosis, anatomical abnormalities, hormonal diseases, such as abnormality in thyroid function, erythroblastosis fetalis (Rh disease), chromosomal aberrations, hyperprolactinemia, and toxoplasmosis. Other infections, Rubella, HSV infections were excepted from the molecular study. To DNA extraction, under sterile conditions 5ml of peripheral blood were took from all participants into tubes containing EDTA, consequently genomic DNA was extracted through standard guidelines, as previously explained [27]. The material of endocervical for CMV infection test was attained by a special swab. Then, care was taken with scraping and rotation to obtain adequate sample. Finally, the samples were kept frozen at -80 o until CMV screening.

Dna Extraction And Cmv Detection
Total genomic DNA was isolated from the endocervical sample by viral DNA extraction kit (CinnaGen, Iran). The DNA from endocervical specimens was ampli ed by CMV primers (Oligo7 software was used for primer design). The PCR primers are described in Table 1. For detection of CMV, PCR ampli cation was done using two primers. The PCR reaction mixture underwent initial denaturation process at 95°C for 7 min, then 35 cycles at 95°C for 55 secs, 55°C for 45 secs, and 74°C for 59 sec. The nal extension was performed at 74°C for 8 min in the thermocycler. The PCR fragments (280 bp) were run in 2% agarose gel and were visualized through cyber green staining. Table 1 Nucleotide sequences of the primer sets for CMV detection Name Primer sequence Length (bp) Il-6 -174 G/c Genotyping By Tetra-arms Pcr Method The genotypes of IL-6 -174 G/C polymorphism of the IL-6 gene were detected by the use of the Tetraprimer ampli cation refractory mutation system-polymerase chain (Tetra ARMS-PCR) procedure. The PCR was done with a total volume of 25 µl with 100 ng DNA template, 3.5 µl of 10X PCR buffer, 1 U of Taq DNA polymerase, 0.5 mM dNTPs and 0.5 µmol/l of each primer. The PCR conditions were 94°C for 7 minutes, 94°C for 58 secs, 60°C for 58 seconds, 72°C for 58 seconds, and 72°C for 10 minutes. Then, the obtained fragments were separated using electrophoresis on a 2.5% agarose gel and consequently visualized in three various patterns of fragments. The GG genotype made two fragments (326 and 205 bp), the CC homozygote made two fragments (326 and 176 bp) and the GC heterozygote produced distinct three fragments (326, 205 and 176 bp). The Tetra ARMS-PCR-primers are described in Table 2.

Statistical Analysis
In order to statistical analysis of data, the statistical package for the social sciences (SPSS) software was used. Relations between genotypes, virulence markers, and clinical disorders were assessed.
Statistical analyses for detection of common IL-6 polymorphism and CMV roles in RPL were carried out by χ2 (Chi-square test) and p-values below 0.05 were statistically signi cant. Anticipated genotype prevalence were calculated from the allele frequencies according to the Hardy-Weinberg equilibrium.

Results Of Cervical Lesions
In total, there were two classi cations including RPL group and control group.

Detection Of Cmv
According a standard protocol, CMV was detected in all samples with PCR after DNA extraction. In patients, CMV positivity was found in 35 (44%) while it was present in only 20 (25%) of controls, which was detected to be signi cant (P-value = 0.002) ( g,1).

Il-6 -174 G/c Genotyping
In RPL, the frequency of GG, CG and CC genotypes from IL-6 -174 polymorphism were 0.64, 0.32 and 0.04, respectively, but in the control group, they were 0.58, 0.38 and 0.04, respectively (Table 4). Interestingly, no considerable relationship was observed between IL-6 -174 G/C genotypes and RPL (P ≤0.0001) (Fig. 2). The allele frequency of the IL-6 -634 G/C gene polymorphisms were also calculated. Results reveal substantial variations between cases and controls. In addition, the frequencies of G and C alleles were 81% and 19% in patients and 67% and 33% in controls, respectively. Both A and G allele of IL-6 -634 had a statistically noticeable association with the disease (P≤0.005) (Fig. 3).
Association Cmv With Il-6 -634c/g Polymorphism The frequency of IL-6 -634 GG genotypes among RPL with CMV infection was 80% and without CMV infection was 50% (P < 0.0005). Individuals carrying the IL-6 -634 GG homozygous genotype with CMV infection have a 4-times greater risk of presenting with RPL than an individual with the CC homozygous genotype (P≤0.0005). Additionally, the presence of RPL was signi cantly associated with family history and ages (P≤0.004). The Frequency of IL-6 -634 genotypes in RPL samples with CMV (+) and controls with CMV (-) are shown in (Table 4).

Discussion
RPL is a frequent disorder among women and shows a remarkable concern for reproductive problem cause approximately one in 300 pregnancies [1,28]. Until now, while various criteria such as anatomic, genetic, endocrine, immunologic, and microbiologic have been acknowledged that affect miscarriage, the exact etiology of RPL cases in up to 50% is unknown [2,3,29]. The occurrence of RPL can be controlled by genetic-related factors such as genetic polymorphisms that have been connected with poor pregnancy. Some studies have led to the consciousness that cytokines were considered as key factors in the preservation of pregnancy by regulation the immune responses [17]. It is also found that embryo loss can be caused by improved uterine expression of some pro-in ammatory cytokines like (tumor necrosis factor (TNF)-α, interferon-gamma (IFN-γ), and interleukin (IL)-1β) [18][19][20]30]. In addition, anti-in ammatory cytokines such as IL-6 and IL-10 considered as crucial factors for maintaining a normal pregnancy. Among potential co-factors, functional SNPs in which alter the expression of IL-6 play a decisive role at various stages of RPL development. The main ndings of this study were an important positive association between IL-6 SNP with RPL risk. The IL-6 -634C/G polymorphism is located at the 634 base pair upstream of the IL-6 gene promoter. It might be able to control IL-6 transcription or regulates posttranscriptional modi cation. Genetic predisposing factors may in uence the likelihood of, sensitivity to or persistence of CMV infection, as well as the rate of RPL development [31,32].  [24]. Besides, several studies and investigations failed to discover the connection between the prevalent polymorphisms in the IL-6 SNP and RPL risk [23,24,32,34].
However, our results present a substantial association between RPL risk and G allele in the IL-6 -634 among Iranian women. This is in line with Nasiri et al. study that indicated a remarkable association between allele G and the risk of RPL among Iranian population [35]. We obtained favorable results from our cases, as the allele G is substantially linked to incidence of RPL in Iranian women. However, it is in contrast to Saijo et al. [33] and Ma et al. [36] who reported a relation between the decreased risk of RPL and allele G, in Japanese and Chines women, respectively. It is likely that the variation of results among these studies were attributed to ethnic differences or patients enrollment criteria impact. Besides, based on several studies we suggested that this polymorphism (IL-6 -634C/G) has different affect in RPL among different populations.
In this study, we also indicated that the IL-6 -174 G/C polymorphism has no association with RPL risk. This is in line with other studies. To evaluate the effect of the IL-6 174 on incidence of RPL, Daher et al. reviewed all ve research reported in Europe [37]. In none of the studies, no variations in IL-6 genotype frequencies were found between controls and RPL patients, even in Japanese women not a allele was identi es neither in control nor in the patients [37]. Since the race had a signi cant impact on ndings, they could not be applied to all people, because they were not comprehensive. In comparison to other populations, the Caucasian Northern Ireland people is the only one with a lower frequency of IL-6 174 allele [38]. One possible scienti c reason that can explain these ndings could be that -634C/G polymorphism is more effective than -174 G/C on IL-6 gene expression and translation. As a result, -634C/G polymorphism conceivably contributing to RPL risk among Iranian women.
On the other hand, the cytokine IL-6 has a protective role in immune responses against infections that caused by different bacteria and viruses. To cause an in ammatory reaction, pathogenic agents need access the intrauterine surroundings of the fetal tissues or endometrium, which can result in miscarriage [3,39]. Prasad et al. investigated the role of Th1/Th2/Th17 cytokines in the immunopathogenesis process of miscarriage in Chlamydia trachomatis (Ct)-positive rst-trimester aborters [19,40]. They found that some cytokines such as IL-6, IFN-γ, IL-2, TNF-α, and IL-17A were noticeably increased in spontaneous abortion (SA) group and recurrent miscarriage(RM) group (Ct-infected) versus controls. Moreover, TNF-α, IL-17A, IL-6, and IFN-γ cytokines were considerably elevated in Ct-positive RM group compared to Chlamydia-infected SA group. Williams et al. demonstrated the TNF-alpha was produced in vivo during C. trachomatis infection and has a prime role in host defense [41].
In this study, our ndings indicated a remarkable association between RPL with CMV positive patients and allele G in IL-6 -634 among Iranian women. Besides, the individuals with allele G in IL-6 -634 and CMV infection have greater risk of RPL in comparison with C allele. These ndings are compatible with Edmunds et al. study, reported that the anti HCMV-IgM was identi ed in 94% of the Iranian women with the abortion background [29,42]. Also, in the other study which conducted by Janan et al. on Iranian women with spontaneous abortion, results indicated a remarkable variation in the frequency of anti-Rubella, Cytomegalovirus, and Toxoplasma antibodies between RPL cases and healthy women [43]. They detected that the frequency of antibodies against Cytomegalovirus (CMV-IgM and CMV-IgG) in the patient group was noticeably higher than in the control group, and found that the high titer of CMV-IgM and CMV-IgG play the remarkable role in RPL [43]. In addition, Ebadi et al. discovered an important association between abortion and CMV infection in Iranian population [44]. According to these results we suggested that allele G in IL-6 -634 has both directly and indirectly effects on RPL in Iranian women. Directly, it can signi cantly increase risk of RPL, and indirectly it can increase CMV infection, which in turn might play effective roles in the incidence of RPL among Iranian women.

Conclusion
This study showed signi cant associations among IL-6 -634C/G polymorphism with RPL risk and CMV infection, may contribute to susceptibility of RPL, in Iranian patients. In particular, IL-6 -634GG genotypes and CMV infection are associated with increased RPL risk. Detection of CMV and IL-6 -634GG genotypes can play important roles for early detection and early treatment of RPL in Iranian women. As IL-6 -634GG frequencies vary among ethnic groups, large and different population-based genetic studies are required to approve our results and suggestions.

Declarations
Authors' contributions Parisa Pourroostaei Ardakani Methodology, Conceptualization, and Writing original draft. Bahareh Rahimi and Mohammad Panahi methodology, editing, and writing some sections of draft. Hamzeh Rahimi supervision, project administration, review and edit the manuscript. All authors read and approved the nal draft.

Data availability statement
Input data for the analyses are available from the corresponding authors on request. Figure 1 CMV PCR products. well 1 was positive control, well 2, 4,5,6 were positive CMV, wells 3 and 7 were negative CMV, wells 8 was negative control. add product size based on size marker