Correlation of methylation status in MTHFR promoter region with recurrent pregnancy loss

Abstract Background DNA methylation is an epigenetic process for modifying transcription factors in various genes. Methylenetetrahydrofolate reductase (MTHFR) stimulates synthesis of methyl radical in the homocysteine cycle and delivers methyl groups needed in DNA methylation. Furthermore, numerous studies have linked gene polymorphisms of this enzyme with a larger risk of recurrent pregnancy loss (RPL), yet scarce information is available concerning the association between epigenetic deviations in this gene and RPL. Hypermethylation at precise DNA sequences can function as biomarkers for a diversity of diseases. We aimed by this study to evaluate the methylation status of the promoter region of MTHFR gene in women with RPL compared to healthy fertile women. It is a case–control study. Hundred RPL patients and hundred healthy fertile women with no history of RPL as controls were recruited. MTHFR C677T was assessed by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Quantitative evaluation of DNA methylation was performed by high-resolution melt analysis by real-time PCR. Results The median of percentage of MTHFR promoter methylation in RPL cases was 6.45 [0.74–100] vs. controls was 4.50 [0.60–91.7], P value < 0.001. In the case group, 57 hypermethylated and 43 normo-methylated among RPL patients vs. 40 hypermethylated and 60 normo-methylated among controls, P< 0.005. Frequency of T allele in C677T MTHFR gene among RPL patients was 29% vs. 23% among the control group; C allele vs. T allele: odds ratio (OR) = 1.367 (95% confidence interval (CI) 0.725–2.581). Conclusion Findings suggested a significant association between hypermethylation of the MTHFR promoter region in RPL patients compared to healthy fertile women.

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Association of M55L and Q192R polymorphisms of paraoxonase 1 gene (PON1) with recurrent pregnancy loss risk: A case–control study

International Journal of Reproductive BioMedicine ◽

10.18502/ijrm.v19i6.9377 ◽

2021 ◽

Author(s):

Mehdi Alizadeh ◽

Mahboobeh Nasiri ◽

Morteza Samadi ◽

Nasrin Ghasemi ◽

Ali Moradi

Keyword(s):

Statistical Analysis ◽

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Case Control Study ◽

Case Control ◽

Paraoxonase 1 ◽

Control Group ◽

Case Group ◽

Significant Difference ◽

Control Study

Background: Recurrent pregnancy loss (RPL) refers to the incidence of two or more abortions before the first half of pregnancy. Oxidative stress has been hypothesized to play a central role in RPL. Objective: To investigate the relationship between Q192R and L55M polymorphisms of PON1 as antioxidant enzyme and the risk of RPL. Materials and Methods: In this case–control study, 110 women with RPL (case) and 110 healthy fertile women (control) referred to the Research and Clinical Center for Infertility, Shiraz, Iran were enrolled. Genomic DNA was extracted from the peripheral blood in all participants. Polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism method. Results: Statistical analysis of Q192R polymorphism showed a significant difference for the RR genotype between the case and control group (OR = 11, CI = 1.39–86.87, p = 0.005) but none for the QR and QQ genotypes. No significant association was observed between the R and Q allelic frequency in the RPL participants compared to the control group (p = 0.53). Also, statistical analysis of the L55M polymorphism for MM genotype in the case group compared with the control group showed a significant difference (OR = 3.59, CI = 0.97–13.30, p = 0.042), but none for the LM and LL genotypes. Conclusion: The findings showed a significant correlation between the Q192R polymorphisms and the L55M PON1 enzyme and RPL in this study population. Key words: Pregnancy, Abortion, PON1, Polymorphism, Recurrent pregnancy loss.

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THE DNA METHYLATION STATUS IN PRESENT DAY POPULATIONS LIVING IN AREAS OF VIETNAM SUBJECTED TO AGENT ORANGE SPRAYING DURING THE WAR IN NAM DONG AND A LUOI, THUA THIEN HUE PROVINCE

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2018.2.10 ◽

2018 ◽

pp. 59-66

Author(s):

Thanh Tin Nguyen ◽

Phan Minh Triet Le ◽

Viet Nhan Nguyen ◽

Cristina Giuliani ◽

Donata Luiselli ◽

...

Keyword(s):

Dna Methylation ◽

Methylation Status ◽

University Hospital ◽

Agent Orange ◽

Control Group ◽

Case Group ◽

Veterans Health ◽

Dna Hypomethylation ◽

Cross Sectional ◽

Cyp1a1 Gene

Introduction: Agent Orange was the most extensively used among herbicides sprayed on Vietnam territory during the Vietnam War. Its by-product, 2,3,7,8-tetrachlorodibenzo-paradioxin (Dioxin), is an extremely toxic and persistent chemical. The effects of this spraying on both Vietnamese and United States Veterans health has been reported in many publications. However, there wasn’t any study of the effects at the molecular level of the residual Dioxin in the environment on present Vietnamese civilians living in contaminated areas. Objective: To investigate the association between residual Agent Orange/Dioxin in the environment and the alterations of DNA methylation in the peripheral blood of the present day Vietnamese population living in spraying areas. Methods: Cross-sectional study. The subjects were 188 individuals who came to Hue University Hospital for health care: 94 individuals for case group from sprayed areas (A Luoi and Nam Dong, Thua Thien Hue Province), and 94 individuals for the control group from non-sprayed areas (Quang Binh to North Vietnam). MALDI-TOF MS technique was used to detect the alterations of DNA methylation of CYP1A1 gene. Results: Among 22 CpG position of CYP1A1 gene were investigated, there were the DNA hypomethylation at CpG_2.3.4, CpG_5, CpG_12.13 in case group compared to the control (p<0.05). After dividing case group into 2 subgroups, we found the significant DNA hypomethylation at CpG_2.3.4, CpG_5, CpG_9, CpG_10, CpG_11, CpG_12.13, CpG_17, CpG_18.19 in subgroup CASES_F_P compared to CASES_NON_F_P also control group (p< 0.05). Conclusions: Individuals living in A Luoi and Nam Dong– the Dioxin contaminated areas– had DNA hypomethylation in CYP1A1 gene. The DNA hypomethylation seem not due to the effects of residual Dioxin in the environment in present day, it was likely to be inherited by epigenetic way from the DNA methylation alterations on their parents who had directly exposure to that spraying. This theory should be verified through extensive studies with CASES_F_P family and more genes will be investigated. Key words: Agent Orange, Dioxin, DNA methylation, CYP1A1

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The Association Between Sperm DNA Fragmentation and Idiopathic Early Recurrent Pregnancy Loss

KnE Medicine ◽

10.18502/kme.v1i1.537 ◽

2016 ◽

Vol 1 (1) ◽

Author(s):

Binarwan Halim

Keyword(s):

Dna Fragmentation ◽

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Control Group ◽

Case Group ◽

Sperm Dna Fragmentation ◽

Male Factor ◽

Sperm Dna ◽

History Of ◽

Group 5

IntroductionVarious etiologies of recurrent pregnancy loss (RPL) have been extensively studied, but more than half of them still remain unknown. Male factor may play a role in incidence of idiopathic early recurrent pregnancy loss. Sperm DNA fragmentation as one of sperm test for male factor can be measured and expressed by a DNA Fragmentation Index (DFI). The aim of the study is to evaluate the association between sperm DNA fragmentation and the incidence of idiopathic early recurrent pregnancy loss.Material & MethodsA prospective study was done by recruiting 40 cases of male couple from patients with a history of idiopathic early recurrent pregnancy loss and 40 cases of control from normal fertile population from May 2010 to September 2011 in Halim Fertility Center. Sperm DNA fragmentation was detected by halosperm kit.Results Both of groups were comparable in terms of the age of male patients, body mass index, duration of infertility, history of miscarriage and sperm parameters. Sperm DFI in the case group was 34.12%. and in the control group was 16.02%. There was significantly higher sperm DFI in the case group than in the control group. Sperm DFI <30 was increased in control group (95%) compared with case group (40%). Sperm DFI ≥30 was increased in case group (60%) compared with control group (5%). There was a significant association between sperm DFI ≥30 and idiopathic early recurrent pregnancy loss (p<0,05).ConclusionThere is an association between higher sperm DNA fragmentation and incidence of idiopathic early recurrent pregnancy loss.

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Frequency of the rs 14035 polymorphism of RAN gen in recurrent pregnancy loss: A case-control study

International Journal of Reproductive BioMedicine ◽

10.18502/ijrm.v13i5.7156 ◽

2020 ◽

Author(s):

Zahrasadat Mortazavifar ◽

Hamidreza Ashrafzadeh ◽

Seyed Morteza Seifati ◽

Nasrin Ghasemi

Keyword(s):

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Case Control Study ◽

Case Control ◽

Control Group ◽

Case Group ◽

Nuclear Pore ◽

Normal Delivery ◽

Significant Difference ◽

Control Study

Background: Genetic factors could account for recurrent pregnancy loss (RPL). The RAN gene is a member of the ”large RAS family” and a small GTPase that is essential for the translocation of Ribonucleic acid (RNA) and proteins through the nuclear pore. Mutation in the RAN constitutive gene could stop DNA synthesis and alter the expression of genes in the uterus, likely playing a role in recurrent miscarriage. Objective: The aim was to investigate the frequency of RAN (rs 14035) polymorphism in women with RPL compared with women without abortion history. Materials and Methods: In this case-control study, 100 women with at least two consecutive miscarriages before the 20th wk of gestation and having spouses with karyotype and normal sperm parameters as the case group and 100 women with no history of abortion and having at least one successful pregnancy and normal delivery as the control group. The groups were age matched (20-40 yr). The rs 14035 polymorphism of RAN gene was investigated by Polymerase Chain Reaction- Restriction Fragment Length poly morphism technique and the frequency of which was compared between the two groups. Results: The frequency of TT, TC, and CC genotypes of RAN gene polymorphism in the case group were 9%, 40%, and 51%, respectively, and in the control group were 11%, 38%, and 51%, respectively. There was no significant difference in the genotypes between two groups (p = 0.882). Conclusion: According to our results, it seems that RAN polymorphism (rs 14035) is not associated with the risk of RPL in this study population. Key words: RAN gene, Repeated abortion, Polymorphism, PCR-RFLP.

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High Fasting Insulin Levels and Insulin Resistance May Be Linked to Idiopathic Recurrent Pregnancy Loss: A Case-Control Study

International Journal of Endocrinology ◽

10.1155/2013/576926 ◽

2013 ◽

Vol 2013 ◽

pp. 1-4 ◽

Cited By ~ 7

Author(s):

Corina-Alina Ispasoiu ◽

Radu Chicea ◽

Florin Vasile Stamatian ◽

Florin Ispasoiu

Keyword(s):

Insulin Resistance ◽

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Case Control Study ◽

Fasting Glucose ◽

Case Control ◽

Control Group ◽

Case Group ◽

Fasting Insulin ◽

Control Study

Objective. Patients with more than two spontaneous pregnancy losses are diagnosed with recurrent pregnancy loss. The aim of this study was to evaluate the IR (insulin resistance) in patients with idiopathic recurrent pregnancy loss.Material and Method. A single center, case control study was performed on one hundred eighteen women divided into case group (with at least two pregnancy losses, earlier than 20 weeks of gestation, and negative for the recurrent pregnancy loss testing) and control group (with at least one live birth, no pregnancy loss). FG (fasting glucose) and FI (fasting insulin) were determined for all patients. IR was evaluated by HOMA-IR index.Results. There were not significant differences between the mean age and BMI in cases and controls (P>0.05). Fasting glucose was significantly higher in the control group (85.6 versus 79.8P<0.01), but fasting insulin (15.24 versus 12.83,P<0.001) and HOMA-IR (2.98 versus 2.69,P<0.05) were significantly higher in the case group.Conclusion. In women with idiopathic recurrent pregnancy loss FI and IR are higher than those in women without spontaneous abortion.

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High MTHFR promoter methylation levels in men confer protection against ischemic stroke

Bosnian Journal of Basic Medical Sciences ◽

10.17305/bjbms.2020.4636 ◽

2020 ◽

Author(s):

Shan Xu ◽

Qianping Shi ◽

Bo Li ◽

Liyuan Han ◽

Guodong Xu ◽

...

Keyword(s):

Dna Methylation ◽

Ischemic Stroke ◽

Environmental Factors ◽

Promoter Methylation ◽

Protective Factor ◽

Methylenetetrahydrofolate Reductase ◽

Methylation Status ◽

Clinical Information ◽

Control Group ◽

Case Group

The MTHFR gene encodes methylenetetrahydrofolate reductase required for the metabolism of homocysteine (Hcy) – a previously reported independent risk factor for ischemic stroke (IS). In this study, we first aimed to clarify the association between DNA methylation levels in the MTHFR promoter and the risk of IS, followed by the analysis of potential interactions between environmental factors and DNA methylation levels that affect IS risk. We recruited 164 patients with hypertension and IS (case group) and 345 age- and sex-matched patients with hypertension only (control group). Demographic and clinical information was obtained using questionnaires, and blood samples were collected for biochemical analyses. Fluorescence quantitative methylation-specific PCR (qMSP) was used to detect MTHFR promoter methylation levels. A logistic regression analysis was performed to determine the relationship between environmental factors, MTHFR promoter methylation levels, and IS risk. We finally generated a receiver operating characteristic (ROC) curve to determine whether MTHFR promoter methylation levels can predict IS. The mean MTHFR methylation levels in case group (8.10 ± 6.14) were significantly lower than those in control group (17.44 ± 3.16; p < 0.05). MTHFR promoter methylation levels were also lower in patients with plasma Hcy levels ≥15 μmol/L (10.65 ± 4.05) than in those with Hcy levels <15 μmol/L (16.74 ± 4.26, p < 0.001). Finally, we found that MTHFR hypermethylation is a protective factor for IS, particular in men (OR in men: 0.07; 95% CI: 0.02–0.16; p < 0.001). Further, sex and MTHFR promoter methylation levels exhibited a preliminary interaction effect on IS risk. These results indicate that MTHFR promoter methylation status might have diagnostic value in IS.

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Effects of interleukin-1 receptor antagonist (IL-1Ra) gene 86 bp VNTR polymorphism on recurrent pregnancy loss: a case-control study

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2017-0010 ◽

2017 ◽

Vol 30 (3) ◽

Cited By ~ 1

Author(s):

Yasamin Sayed Hajizadeh ◽

Elina Emami ◽

Marina Nottagh ◽

Zahra Amini ◽

Nazila Fathi Maroufi ◽

...

Keyword(s):

Receptor Antagonist ◽

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Case Control Study ◽

Interleukin 1 ◽

Case Control ◽

Control Group ◽

Case Group ◽

Vntr Polymorphism ◽

Control Study

AbstractObjectiveRecurrent pregnancy loss (RPL) is a heterogeneous disease which is defined as two or more consecutive fetal losses during early pregnancy. Interleukin-1 receptor antagonist (Materials and methodsIn this case control study, genetic polymorphism was studied in 140 RPL patients and 140 healthy women as controls. Genomic DNA was extracted from the blood samples and polymorphism analysis was performed using the polymerase chain reaction (PCR) method. Finally, the data obtained were analyzed by statistical software.ResultsWe found an increased frequency of the IL-1Ra 1/1 genotype in the case group compared to the control group. Whereas, the frequency of IL-1Ra genotype 1/2 was higher in control group than in the case group. However, we did not observe an association betweenConclusionVNTR polymorphism may not be a genetic factor for RPL. However, investigation of

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Lack of Association between Factor V Leiden G1691A, Prothrombin G20210A, MTHFC677T Mutations, and Early Recurrent Pregnancy Loss in a Group of Sudanese Women

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2020.4384 ◽

2020 ◽

Vol 8 (B) ◽

pp. 553-557

Author(s):

Asaad Ma. Babker ◽

Itedal Abdelraheem Mohamed Ahmed ◽

Marwan Ismail ◽

Fathelrahman Mahdi Hassan ◽

Ahmed L. Osman ◽

...

Keyword(s):

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Statistical Significance ◽

Factor V Leiden ◽

Reproductive Age ◽

Prothrombin G20210a ◽

Control Group ◽

Case Group ◽

Factor V ◽

Low Prevalence

BACKGROUND: Recurrent pregnancy loss is classically defined as the occurrence of three or more consecutive pregnancy loss. Recurrent pregnancy loss affects from 1-5% of the reproductive age couples. This diagnosis is both emotionally challenging and confusing for most couples, as the definitive diagnosis using conventional evaluations is found in fewer than half of the couples experiencing repeated loss. AIM: The purpose of this study was to define the association between Factor V Leiden G1691A, Prothrombin G20210A, MTHFC677T mutations and recurrent pregnancy loss in a group of Sudanese women. MATERIALS AND METHODS: This a retrospective analytical case control study was carried out at Omdurman Maternal Hospital, Sudan between July 2013 to July 2015. Consent was obtained from the ethical committee of the Faculty Research Board and Hospital of Omdurman Maternity Hospital (Sudan). The study included a hundred pregnant females with a history of recurrent spontaneous abortion as the (case group) and ninety-five healthy reproductive Sudanese women as the (control group). The data was collected with the help of a structured questionnaire and direct interview to collect information. Identification of point mutation in factor V Leiden G1691A, prothrombin G20210A and MTHF C677T gene by polymerase chain reaction was performed. The odds ratio and the 95% confidence interval (95%CI) were calculated for the presence of mutation case group and the control group and analyzed by SPSS program, version 17.0. RESULTS: The frequency of prothrombin G20210A, MTHFC677T, was low overall, except for the Factor V Leiden G1691A. The differences between patients and controls had no statistical significance (P- Value>0.05). CONCLUSION: Our study confirms the low prevalence of inherited thrombophilias in Sudanese populations and it is unlikely that the tested thrombophilias play a role in the pathogenesis of recurrent pregnancy loss in the Sudanse population.Therefore, we conclude that the low prevalence of Factor V Leiden, prothrombin G20210A and MTHFC677T in Sudanese women with RPL and does not play a role in the pathogenesis of recurrent pregnancy loss among our population.

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DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss

Human Reproduction ◽

10.1093/humrep/deaa278 ◽

2020 ◽

Author(s):

Kushaan Khambata ◽

Sanketa Raut ◽

Sharvari Deshpande ◽

Sweta Mohan ◽

Shobha Sonawane ◽

...

Keyword(s):

Dna Methylation ◽

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Methylation Status ◽

Differentially Methylated Region ◽

Imprinted Genes ◽

Epigenetic Factors ◽

Cpg Sites ◽

Sperm Dna ◽

Sperm Dna Methylation

Abstract STUDY QUESTION What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? SUMMARY ANSWER Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST), zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. WHAT IS KNOWN ALREADY RPL is defined as loss of two or more pregnancies, affecting 1–2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. STUDY DESIGN, SIZE, DURATION In this case–control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. MAIN RESULTS AND THE ROLE OF CHANCE In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. LIMITATIONS, REASONS FOR CAUTION In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. WIDER IMPLICATIONS OF THE FINDINGS Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

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Association of Tumor Necrosis Factor-α -308G>A, -238G>A and -376G>A polymorphisms with recurrent pregnancy loss risk in the Greek population

Fertility Research and Practice ◽

10.1186/s40738-021-00101-x ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Sofoklis Stavros ◽

Despoina Mavrogianni ◽

Myrto Papamentzelopoulou ◽

Evaggelos Basamakis ◽

Hend Khudeir ◽

...

Keyword(s):

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Pcr Amplification ◽

Greek Population ◽

Control Group ◽

Increased Risk ◽

Tnf Α ◽

Caucasian Women ◽

Factor Α ◽

And Control

Abstract Background Promoter region SNPs in TNF-α have been studied in association with Recurrent Pregnancy Loss (RPL) occurrence in various populations. Among them, −238G > A, −308G > A and − 376G > A have been frequently investigated for their potential role in recurrent abortions. The aim of the present study is to evaluate the correlation among TNF-α 238, TNF-α 308 and TNF-α 376 polymorphisms and recurrent pregnancy loss risk in Greek women. Methods This study included 94 Caucasian women with at least two miscarriages of unexplained aetiology, before the 20th week of gestation. The control group consisted of 89 Caucasian women of proven fertility, with no history of pregnancy loss. DNA samples were subjected to PCR amplification using specific primers. Sanger sequencing was applied to investigate the presence of TNF-α 238, TNF-α 308, TNF-α 376 polymorphisms in all samples. Results The TNF-α 238 and TNF-α 308 variants were both detected in RPL and control groups (7.45% vs 4.49 and 45.16% vs 36.73%, respectively), but with no statistically significant association (p-value 0.396 and 0.374, respectively). The TNF-α 376 variant was not detected at all in both control and RPL groups. When TNF-α 238 and TNF-α 308 genotypes were combined no association with RPL was detected (p-value = 0.694). In subgroup analysis by parity, RPL patients carrying the A allele reported less previous births. Conclusions This is the first study demonstrating TNF-α 238 and TNF-α 308 gene expression and the absence of TNF-α 376 variant in Greek women with RPL. However, no association emerged between each polymorphism studied and the occurrence of recurrent pregnancy loss. Accordingly, TNF-α -308G > A, −238G > A and -376G > A variants are not considered genetic markers for identifying women at increased risk of recurrent pregnancy loss in the Greek population.

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