Cytokine gene polymorphisms and susceptibility to recurrent pregnancy loss in Iranian women

Recurrent pregnancy loss (RPL) is defined as three or more sequential abortions before the 20th week of gestation. There is increasing evidence to support an immunological mechanism for the occurrence of RPL. Defective production of T helper type 2 (Th2) and/or higher production of T helper type 1 (Th1) cytokines have been reported in RPL. As cytokine gene polymorphisms may be associated with different rates of cytokine production, the aim of the present study was to investigate the bi-allelic polymorphisms in TNF-alpha -308 G-->A, TNF-beta +252 G-->A, IFN-gamma +874 A-->T genes as Th1 or pro-inflammatory factors as well as IL-4 -590 C-->T, IL-10 -592 C-->A, -819 C-->T, -1082 A-->G genes as Th2 cytokines in women with RPL compared with healthy women. A total of 139 women with RPL and 143 control women with at least two successful pregnancies were included in the study. The allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) or PCR-RFLP (restriction fragment length polymorphism) methods were used for genotyping. Results indicated a significant association between the presence of CC genotype of IL-10 -592 C-->A polymorphism and the occurrence of RPL in Iranian women (63% in women with RPL and 46% in controls; OR=0.51, 95% CI: 0.3-0.85; p<0.01). There was no significant association with other positions. It may be concluded that IL-10 polymorphism at position -592 could be a genetic factor for RPL.     

Vascular endothelial growth factor-A gene polymorphisms in women with recurrent pregnancy loss

Vascular endothelial growth factor-A (VEGFA) is normally expressed at high levels in the human placenta, and lower levels have been observed in placental tissue of women with recurrent pregnancy loss. The objective of this study was to determine if genetic polymorphisms in the VEGFA gene associated with altered gene expression play a role in some cases of recurrent pregnancy loss (RPL). A case-control study of 99 women with RPL and 181 fertile controls was performed evaluating four common VEGFA polymorphisms associated with altered gene expression (-2578 C/A, -1154 G/A, -634 G/C, and +936 C/T). The allele frequency of the -2578 A allele was lower among women with RPL compared to fertile controls (0.39 vs. 0.48, p=0.049), while the allele frequency of the -634 C allele was higher among women with RPL compared to fertile controls (0.39 vs. 0.29, p=0.020). Women with RPL and controls had similar allele frequencies for the -1154 and +936 minor alleles. We conclude that some allelic polymorphisms associated with altered expression of VEGFA are more common among women with RPL compared to fertile controls.     

Associations between cytokine gene polymorphisms and recurrent pregnancy loss

Since certain cytokines may play a role in unexplained recurrent pregnancy loss (RPL) and also some cytokine gene polymorphisms may affect the level of cytokine production, the aim of the present study was to investigate the relationship between RPL and polymorphisms of the genes coding for TNF-alpha (-308 G-->A), IL-10 (-1082 G-->A), IL-6 (-174 G-->C), and IFN-gamma (+874 A-->T). Genotyping was performed in 48 RPL women and 108 ethnically matched healthy individuals. In addition, we performed a meta-analysis encompassing the present results and those from studies on the association of TNF-alpha, IL-10 and IFN-gamma polymorphisms with RPL published in the literature until December 2001. The results showed: (1) no evidence of association with IL-6 gene polymorphisms; (2) significant associations, revealed by the meta-analysis, with the high cytokine production genotypes of IFN-gamma (+874 T/T: odds ratio (OR)=1.92, P=0.04) and IL-10 (-1082 G/G: OR=1.75, P=0.03), and a trend for association with the high TNF-alpha production genotypes -308 A/A and A/G (OR=1.61; P=0.18). We believe that the associations of these genotypes with RPL are interesting not only as risk factors but also because they represent another piece of evidence that these cytokines might be important in the pathogenesis of RPL.     

ACE and MTHFR gene polymorphisms in unexplained recurrent pregnancy loss

AIM: To assess the association between polymorphisms in angiotensin converting enzyme and methylene tetrahydrofolate reductase genes and recurrent pregnancy loss by a case-control study in South Indian women. METHODS: DNA was extracted from peripheral blood leukocytes of 104 women with Recurrent Pregnancy Loss (RPL) and 120 controls. Genotyping of ACE Insertion Deletion and MTHFR C677T polymorphism were carried out by PCR and PCR-RFLP, respectively. RESULTS: No statistically significant difference was observed in the distribution of genotypes between cases and controls for ACE and MTHFR polymorphisms. Further, the combination of MTHFR and ACE genotypes failed to reveal an association. CONCLUSION: In conclusion, the present study reveals lack of association of MTHFR C677T and ACE I/D polymorphisms in RPL in South Indian women. However, we cannot exclude the possibility that other polymorphisms of ACE and MTHFR genes could be associated with the disease and might be clinically useful as a marker to assess risk for RPL.     

Cytokine polymorphisms in women with recurrent pregnancy loss: meta-analysis

BACKGROUND: Inflammatory cytokine cascades have been implicated in the pathogenesis of recurrent pregnancy loss (RPL). Polymorphisms in cytokine genes may affect the risk of RPL, but genetic association studies are often limited by small sample sizes. Meta-analysis of all available studies can increase the precision of these estimates. AIMS: To assess and synthesise the available data from association studies of inflammatory cytokine polymorphisms with RPL. METHODS: Systematic review and random effects meta-analysis of genetic association studies. RESULTS: Sixteen reports of genetic association studies of cytokine polymorphisms with RPL were identified. Meta-analyses did not identify any significant associations with tumour necrosis factor (-308A, or -238A), interferon-gamma (+874T), interleukin (IL)-1beta (-511T), IL-6 (-174G), or IL-10 (-1082A, or -819T, or -592A). Significant associations were found with IL-1B (-31T) (two studies: pooled odds ratio (OR) 2.12 (95% confidence interval (CI) 1.04 to 4.33)) and IL-6 (-634G) (one study: OR 0.22 (95% CI 0.09 to 0.57)). CONCLUSIONS: The available data are not consistent with more than modest associations between these candidate cytokine polymorphisms and RPL. Data from future association studies may be added to the meta-analyses to obtain more precise estimates of effect sizes.     

Candidate gene study of genetic thrombophilic polymorphisms in pre-eclampsia and recurrent pregnancy loss in Sinhalese women

Aim: Genetic thrombophilias are known to contribute to adverse pregnancy outcomes. Studies in Western populations show that 5, 10-methylenetetrahydrofolate reductase (MTHFR) 677C>T and Factor V (F5) 1691G>A (Leiden) polymorphisms are commonly associated with pre-eclampsia and recurrent spontaneous pregnancy loss. The objective of this study was to investigate the association of MTHFR 677C>T (rs1801133); 1298A>C (rs1801131) and F5 1691G>A (rs6025); 4070A>G (rs1800595) polymorphisms with pre-eclampsia and recurrent pregnancy loss among Sinhalese women in Sri Lanka. Material and Methods: Genotype and allele frequencies at each polymorphic site in the MTHFR and F5 genes and the haplotypes defined by them were determined in 175 Sinhalese women with pre-eclampsia, 171 normotensive controls, 200 Sinhalese women with two or more recurrent pregnancy losses and 200 controls with two or more living children and no pregnancy losses. Genotyping was done by polymerase chain reaction/restriction fragment length polymorphism. Odds ratios and χ(2) -testing were performed to compare genotype/haplotype frequencies at each polymorphic site for both cases and controls. Results: The genotype frequencies at each polymorphic site in the MTHFR 677C>T; 1298A>C; F5 1691G>A and 4070A>G genes and the haplotypes defined by them were not significantly associated with either pre-eclampsia or recurrent pregnancy loss. There was no significant association of genetic thrombophilia with either early or late pregnancy losses. Conclusions: The MTHFR and F5 polymorphisms and the haplotypes defined by them were not significantly associated with either pre-eclampsia or recurrent pregnancy loss in this group of Sinhalese women.     

Cytokine promoter gene polymorphisms and idiopathic recurrent pregnancy loss.

Approximately one in 300 women experience recurrent pregnancy loss (RPL), the aetiology of which is unknown in at least 40% of cases. Previously, some studies have shown increased production of pro-inflammatory cytokines (tumour necrosis factor-alpha and interferon-gamma) and reduced production of anti-inflammatory cytokines (interleukin-10) by circulating blood lymphocytes isolated from these patients when compared with controls. The reasons for this are unclear. The production of these cytokines are partly under genetic control. This study investigated whether polymorphisms in these three cytokine genes known to be associated with either high or low production, are associated with idiopathic RPL. No association was found. It may be that genetic factors are not a major determinant of cytokine production during pregnancy, or alternatively it may be that the observed differences in cytokine production by peripheral lymphocytes do not accurately indicate what is occurring at the local maternofoetal interface during successful and abortive pregnancies.     

The association between thrombophilic gene mutations and recurrent pregnancy loss

Purpose

To determine whether the Factor V (1691G/A), Factor V HR2 (4070A/G), Prothrombin (20210G/A), PAI-1 (-675 I/D, 5G/4G), ACE (intron 16 I/D), Factor VII (Gln353Arg), Factor XIII (Val34Leu), β-fibrinogen (-455G/A), Glycoprotein Ia (807C/T), tPA (intron 8 D/I) gene mutations could be risk factors for recurrent pregnancy loss (RPL).

Methods

Genotyping of thrombophilic gene mutations were carried out by amplification Refractory Mutation System-PCR (ARMS-PCR) method after DNA extraction.

Results

We found that the mutant allele frequencies of Factor V (1691G/A), Factor V HR2 (4070A/G), Prothrombin (20210G/A), PAI-1 (-675 I/D, 5G/4G), Factor XIII (Val34Leu) and β-fibrinogen (-455G/A) were more seen in the case group compared with the healthy control; However, the difference between the two group is not statistically significant (p > 0.05). Whilst the mutant allele frequencies of other studied genes were lower in the case in comparison to the fertile control women (p > 0.05).

Conclusion

Taken together, our data has shown that the prevalence of thrombophilic gene mutations was similar in women with RPL and healthy controls. Therefore, it appears that further studies on large-scale population and other genetic variants will be needed to conclusively find candidate genes for RPL unknown etiology in the future.     

Lack of association between the TGF-beta1 gene polymorphisms and recurrent spontaneous abortion

Transforming growth factor-beta1 (TGF-beta1) is produced by T regulatory lymphocytes (Treg), which play an important role in the physiology of pregnancy. Several polymorphisms of the TGF-beta1 gene (TGFB1) have been reported, some with an important correlation with TGF-beta1 production and disease severity. We performed an association study between TGFB1 polymorphisms and recurrent spontaneous abortion (RSA). We first used a PCR-RFLP method to detect three known TGFB1 cSNPs (coding single nucleotide polymorphisms) among 111 RSA and 110 normal control women from Southern Iran, such as 29T-->C (Leu 10 Pro), 74G-->C (Arg 25 Pro) and 788C-->T (Thr 263Ile), and compared their frequencies between the two groups of subjects. To confirm results of the RFLP study and to identify new SNPs in the RSA women, we then sequenced their DNA samples for seven exons and adjacent intronic regions of TGFB1. Consequently, 10 SNPs were detected; one (-14G-->A) was located in the upstream region of exon 1, three in exons (two in exon 1 and one in exon 5) and six in intronic regions. Two (IVS5+18G-->C and IVS6+910G-->A) of the 10 SNPs were novel. Statistical analysis on the frequency of six most frequent SNPs, including the three cSNPs, as well as on the frequencies of genotypes and 13 haplotypes regarding the 6 SNPs, revealed no significant difference between RSA and control women. Therefore, this study concludes that there is no association between exonic and adjacent intronic polymorphisms of TGFB1 and RSA.     

The association between polymorphisms of genes related to inflammation and recurrent pregnancy loss

Aim: Recurrent pregnancy loss (RPL) occurs in 1–2% of pregnant women and about 50% of RPL cases are unexplained. Previous studies have shown that genetic variation in immune response genes can contribute to the risk in pregnancy maintenance during pregnancy. The aim of the present study was to evaluate the relationship between RPL and genes those have previously been associated with an inflammatory process on 107 RPL cases and 187 healthy controls.

Methods: In this work, the single-nucleotide polymorphisms was examined by utilizing the direct sequencing and the Sequenom MassARRAY system.

Results: The FAU rs769440?G allele had higher frequencies in patients with RPL (p?=?.019). No association was observed between other polymorphisms and RPL.

Conclusion: The results showed an association between FAU rs769440 polymorphism and RPL in Chinese Han population.     

反复妊娠丢失患者的亚甲基四氢叶酸还原酶的多态性

目的检测反复妊娠丢失(recurrent pregnancy loss,RPL)妇女亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)的多态性。方法选择不明原因的反复妊娠丢失二次以上的妇女71例,正常对照93例,无妊娠丢失及血栓病史,除外口服避孕药妇女。MALDI-TOF质谱检测技术检测MTHFR的多态性C677T、A1298C、T1317C和G1793A。结果RPL妇女MTHFR C677T、G1793A的杂合子和纯合子的突变率均明显高于对照组,差异有显著性(P<0.05)。MTHFR的A1298C在RPL组和正常对照组无明显差别。T1317C在两组中均未发现。RPL组连锁基因频率677CT/1793GA明显高于正常对照组(RR=4.92)。结论RPL与MTHFR多态性密切相关,MTHFR C677T、G1793A突变是RPL的危险因素。其连锁基因突变可使妊娠丢失的发生率增加4.92倍。     

Gene-gene interactions and gene polymorphisms of VEGFA and EG-VEGF gene systems in recurrent pregnancy loss

Purpose

Both vascular endothelial growth factor A (VEGFA) and endocrine gland-derived vascular endothelial growth factor (EG-VEGF) systems play major roles in angiogenesis. A body of evidence suggests VEGFs regulate critical processes during pregnancy and have been associated with recurrent pregnancy loss (RPL). However, little information is available regarding the interaction of these two major major angiogenesis-related systems in early human pregnancy. This study was conducted to investigate the association of gene polymorphisms and gene-gene interaction among genes in VEGFA and EG-VEGF systems and idiopathic RPL.

Methods

A total of 98 women with history of idiopathic RPL and 142 controls were included, and 5 functional SNPs selected from VEGFA, KDR, EG-VEGF (PROK1), PROKR1 and PROKR2 were genotyped. We used multifactor dimensionality reduction (MDR) analysis to choose a best model and evaluate gene-gene interactions. Ingenuity pathways analysis (IPA) was introduced to explore possible complex interactions.

Results

Two receptor gene polymorphisms [KDR (Q472H) and PROKR2 (V331M)] were significantly associated with idiopathic RPL (P < 0.01). The MDR test revealed that the KDR (Q472H) polymorphism was the best loci to be associated with RPL (P = 0.02). IPA revealed EG-VEGF and VEGFA systems shared several canonical signaling pathways that may contribute to gene-gene interactions, including the Akt, IL-8, EGFR, MAPK, SRC, VHL, HIF-1A and STAT3 signaling pathways.

Conclusion

Two receptor gene polymorphisms [KDR (Q472H) and PROKR2 (V331M)] were significantly associated with idiopathic RPL. EG-VEGF and VEGFA systems shared several canonical signaling pathways that may contribute to gene-gene interactions, including the Akt, IL-8, EGFR, MAPK, SRC, VHL, HIF-1A and STAT3.     

Fas and FasL genetic polymorphisms in women with recurrent pregnancy loss: a case-control study

Aberrant apoptosis at the trophoblast–maternal interface and abnormal expression of Fas and Fas ligand (FasL) have been reported in complicated pregnancies with recurrent pregnancy losses (RPL) and preeclampsia. We assessed the prevalence of Fas and FasL genetic polymorphisms in Korean women with RPL and in fertile controls. In total, 306 women with RPL and 298 fertile controls were enrolled. Genotype distributions of Fas and FasL in RPL patients versus fertile controls were examined under the Hardy–Weinberg equilibrium. Fas ?670 A/G genotype (AA versus AG versus GG, p?=?0.340) and allele frequencies (A versus G, p?=?0.412) were not different between the RPL and control groups. There was no difference in each Fas ?1377?G/A and FasL ?844 C/T genotype, and their allele frequencies. In addition, the unions of two zygosities of each genotype and their combined genotypes did not differ between two groups. No difference in the prevalence of Fas and FasL single-nucleotide polymorphisms (SNPs) was observed between women with RPL and fertile controls among Korean women. To determine the possibility of genetic polymorphisms in Fas and its ligand as risk factors for RPL, further studies in various races and a large study population are needed.     

Lack of association between serum antibodies to Chlamydia trachomatis and a history of recurrent pregnancy loss.

OBJECTIVE: To study the relation between recurrent pregnancy loss (RPL) and infection with Chlamydia trachomatis, and to compare the prevalence of antibodies to C. trachomatis in women with primary and secondary RPL. DESIGN: Prospective comparative study. SETTING: University hospital and university student health center. PATIENT(S): Seventy patients with RPL were selected from women attending an RPL outpatient clinic; 40 normal parous women and 94 asymptomatic sexually active women served as controls. INTERVENTION(S): Blood samples were collected during the clinical examinations for RPL. MAIN OUTCOME MEASURE(S): Serum immunoglobulin (Ig) G and IgA antibodies were detected by two independent methods, a recombinant ELISA specific to the genus Chlamydia and microimmunofluorescence testing specific to the species C. trachomatis. RESULT(S): There was no statistically significant difference in the frequencies of IgG or IgA between the women with RPL and the controls. The antibody frequencies were similar in the women with primary and secondary RPL. CONCLUSION(S): The presence of serum antibodies to C. trachomatis is not associated with RPL. Women with primary and secondary RPL do not differ with respect to the prevalence of antichlamydial antibodies. Thus, women with RPL do not benefit from screening for chlamydial IgG or IgA antibodies.     

Single nucleotide polymorphisms in the promoter region of the interleukin-6 gene and the risk of recurrent pregnancy loss in Japanese women

OBJECTIVE: To investigate the relationships between recurrent pregnancy loss and single nucleotide polymorphisms (-634C-->G and -174G-->C genotypes) in the promoter region of the interleukin (IL)-6 gene in the Japanese population. DESIGN: A case-control study. SETTING: Obstetrics and gynecology department of a university hospital. PATIENT(S): Cases were 76 women with recurrent pregnancy loss; controls were 93 fertile women. INTERVENTION(S): Determination of IL-6 promoter gene polymorphisms performed by polymerase chain reaction and gel electrophoresis. MAIN OUTCOME MEASURE(S): Frequency and distribution of the promoter region of the IL-6 gene allele. RESULTS: There was a significant difference in the -634C-->G genotype frequency (CC vs. CG/GG) between women with recurrent pregnancy loss and controls. The risk of recurrent pregnancy loss was lower in the carriers of the G allele than in women with the wild type (CC) (odds ratio = 0.46; 95% confidence interval = 0.24-0.91). On the other hand, we did not detect any carrier of -174C among the 169 subjects. CONCLUSION(S): The results suggest that, in the Japanese population, women carrying the -634G allele of the IL-6 gene might have a decreased risk of recurrent pregnancy loss.     

Association of interleukin-10 gene promoter polymorphisms with recurrent pregnancy loss: a meta-analysis

Purpose

It has been reported single-nucleotide polymorphisms (SNPs) of the IL-10 promoter might be associated with the susceptibility to recurrent pregnancy loss (RPL). Owing to the inconclusive results, we conducted a meta-analysis to systematically summarize and clarify the association between the IL-10 promoter SNPs and RPL risk.

Methods

A systematic search of studies on the association of the three SNPs with RPL was conducted in PubMed and Embase. Odds ratios (ORs) and 95 % confidence intervals (95 % CIs) were used to pool the effect size.

Result

Eleven case–control studies on rs1800896, seven studies on rs1800871, and eight studies on rs1800872 were included. A significant association was identified between IL-10 rs1800896 with RPL risk (G versus A: OR?=?1.21, 95 % CI 1.09–1.35). No evidence of association was found between rs1800871 and RPL when restricted to those studies in Hardy–Weinberg equilibrium in controls (T versus C: OR?=?1.25, 95 % CI 0.76–2.06). No statistical association was demonstrated between rs1800872 and RPL (C versus A: OR?=?1.08, 95 % CI 0.83–1.42).

Conclusions

IL-10 rs1800896 significantly increases the risk of RPL, while rs1800872 is not correlated with RPL risk. No significant association is demonstrated between rs1800871 and RPL risk but this requires further investigation.