Association Of Genetic Polymorphisms Related To Folate Metabolism And Serum Hcy And Folic Acid In Patients With Unexplained Recurrent Spontaneous Abortion

Background and ObjectiveRecurrent spontaneous abortion?RSA?is a complex disease.Although it is defined as a pregnancy loss of 3 times or more in the country,most experts believe that two spontaneous abortions should be given attention and evaluated,because the recurrence of spontaneous abortion may be similar to that of the third time^[1].And the American Society for Reproductive Medicine defines RSA as pregnancy failures that occur two or more within 20 weeks^[2,3],with early abortion occurring before 12weeks of gestation,late Abortion occurs between 12 and 20 weeks of gestation^[4].The incidence of women in childbearing age is 3%to 5%,while the incidence of spontaneous abortion in RSA patients is as high as 70%to 80%^[5].The etiology of RSA is complex and diverse.In addition to genetic,anatomical abnormalities,endocrine disorders,infection,autoimmunity,sperm quality,lifestyle,mental psychology and environment^[1,6-9],50%-75%of RSA remains unclear,known as unexplained recurrent spontaneous abortion?URSA?^[6].A large number of studies have shown that URSA is related to gene polymorphisms,such as:PAI-1,FVL,FII,MTHFR,MTRR,etc.^[10],and MTHFR and MTRR are more concerned.It has been suggested that mutations in the two polymorphisms,MTHFR A1298C and C677T,and MTRR A66G,may affect the activity of methylenetetrahydrofolate reductase and methionine synthetic reductase and affect the metabolism of homocysteine,resulting in hyperhomocysteinemia and thrombosis^[10].Hyperhomocysteinemia can cause vascular endothelial damage,leading to thrombosis^[11],and embryonic blood supply decrease and necrosis of villus due to placental artery embolization,can eventually lead to miscarriage^[12]The risk of recurrence of URSA increases with the number of abortions^[13],which has serious impacts on families and society,but there is currently no clinically effective indicator for predicting the occurrence of URSA.Many studies have concluded that the occurrence of URSA is associated with thrombophilia^[14-20],about50%-65%of patients with RSA are diagnosed as thrombophilia^[18],while elevated serum HCY can lead to thrombosis.Folic acid acts as an important raw material in HCY metabolism^[17],a deficiency in pregnant women can also lead to hyperhomocysteinemia,leading to miscarriage.Although studies have reported the relationship between MTHFR A1298C and C677T,MTRR A66G polymorphisms and URSA,the results are not consistent^[3,21-23].This study used a larger sample size to further study the relationship between MTHFR and MTRR gene polymorphism and URSA.It is expected to discover the genetic risk factors of URSA.By detecting the levels of HCY and folic acid in the serum of pregnant women with unexplained recurrent miscarriage and normal pregnant women,this study is aiming to explore the value of HCY and folic acid in pregnant women's serum in predicting the occurrence of URSA,and provide a new basis for the prevention and treatment of URSA.Materials and methods1 Patients and Groups1.1 A total of 218 women with URSA who received perinatal care at the hospital from September 2016 to February 2018 were selected,and 264 women were included in the control group.The general clinical data?ethnic,smoking,alcohol consumption,pre-pregnancy body mass index?were counted and the sequences of the C677T,A1298C sites and the ATRG gene A66G sites of the MTHFR gene were detected.1.1.1 The inclusion criteria for the control group were women who had at least one normal pregnancy with no history of spontaneous miscarriage.The URSA group comprised women who had a history of two or more spontaneous miscarriages with a diagnosis of recurrent spontaneous abortion^[2].1.1.2 The exclusion criteria were women with a definitive diagnosis of anatomic genital anomalies via gynecologic examination,ultrasound,or hysterosalpingography;women with a history of taking teratogenic drugs during early pregnancy;couples or embryos with chromosomal abnormalities;women with a history of gastrointestinal surgery,severe digestive diseases,or hepatic or renal insufficiency;women with a history of diabetes,coronary heart disease,malignancy,thyroid disease,or immune or hematologic disorders;and women with a pathogenic microbial infection in the genital tract.1.2 From January 2018 to December 2018,120 pregnant women with URSA?URSA group?,including an early?<12 weeks,60 cases?and late spontaneous abortion?12�20 weeks,60 cases?^[4],and there are 90 and 30 cases of 2 and?3consecutive spontaneous abortions,respectively.140 concurrently pregnant women with normal perinatal care?control group?were selected.The general clinical data?ethnic,smoking,drinking,pre-pregnancy body mass index?were counted and serum HCY and folic acid levels were measured.The inclusion criteria and exclusion criteria are the same with 1.1.1 and 1.1.2.This study was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Zhengzhou University.All participants signed the informed consent form.2 Sampling and Detection Method2.1 After the subject gargles,the swab is repeatedly brushed several times on the buccal mucosa of one side of the mouth,and exfoliated cells of the oral mucosal epithelium were collected from 218 patients with URSA and 264 control women.Sequences of MTHFR C677T and A1298C,MTRR A66G were determined by the technology of fluorescence quantitative PCR.2.2 Early morning fasting venous blood was collected from 120 patients with unexplained spontaneous abortion and 140 control women.Chemiluminescence was used to determine serum FA level;cyclic enzymatic amplification was used to determine serum HCY levels.3 Statistical AnalysisSPSS version 21.0 software was used for the statistical analyses.Qualitative data?such as ethnic group frequency and rates of smoking and alcohol use?were analysed using the Pearson test,Fisher exact test.All normally distributed measurement data are expressed as x�s,and non-normally distributed data are expressed as median?M?and interquartile range?IQR?,and the t-test,corrected t-test,and rank-sum test were used for comparisons between independent samples in the groups.The frequencies of genotypes in the 482 samples were analysed and the Hardy-Weinberg genetic equilibrium test was used.The Pearson test?n?40 and expected frequencies?5?and Fisher exact test?n<40 or expected frequencies<5?were used to analyse the distribution of the genotypes and alleles at each locus in the samples.The odds ratio?OR?was calculated and presented within the 95%confidence interval?95%CI?.Specificity and sensitivity and were used to test the value of URSA prediction.The ROC curve and Youden index were used to evaluate the predictive value of serum indicators,and the corresponding prediction cut-off values were determined.Finally,the joint predictors were identified using the logistic equation based on the prediction cut-off values of each serum indicator.The joint predictors were used as test variables;the prediction of URSA was used as the state variable of the plotted ROC curves,and the Youden index was calculated.The difference was considered statistically significant when P<0.05.Results1 Relationship between URSA and genes related to folate metabolism1.1 The women with impaired metabolism of folate in URSA patients is significantly more than those in controls?P=0.041?,the difference was statistically significant,and the high-risk patients was the most in the risk patients.1.2 The frequency of T allele of MTHFR C677T in patients was significantly higher than that in controls?OR=1.324,95%CI:1.014-1.729,P=0.039?;the frequency of C allele MTHFR 1298 was significantly higher in patients than in controls?OR=1.557,95%CI:1.066-2.275,P=0.021?,and the AC and AC+CC genotype of patients are higher than in controls?OR=1.820,95%CI:1.169-2.834,P=0.008;OR=1.740,95%CI:1.137-2.661,P=0.010?,the difference was all statistically significant;the AG and AG+GG genotype frequency of MTRR A66G locus of patients was higher than that of the controls?OR=1.512,95%CI:1.047-2.185,P=0.027;OR=1.469,95%CI:1.024-2.107,P=0.036?,the difference were both statistically significant.1.3 The frequency of MTHFR 677CT/1298AC compound heterozygous genotype in patients was 6.589 times that of the controls?P=0.001?;those carrying the two mutations were the most in patients?69.3%?and significantly higher than the controls?OR=4.996,95%CI:1.650-15.129,P=0.002?,the difference was statistically significant.2 Combination of serum homocysteine and folic acid levels during pregnancy to predict unexplained recurrent spontaneous abortion.2.1 Compared with the control group,patients with 2 or?3 spontaneous abortions had statistically significant differences in age and serum HCY levels?p<0.05?,and serum HCY levels showed an increasing trend as the number of spontaneous abortions increased.Spontaneous abortion occurred earlier in patients with?3 spontaneous abortions compared with patients with 2 spontaneous abortions,and the difference between the groups was statistically significant?p=0.000?.2.2 Statistically significant differences were noted in age?33.50[31.00�36.00]vs.28.00[28.00�30.00]years;p=0.000?and serum HCY?7.50[6.90�8.50]vs.6.50[5.60�6.90]?mol/L;p=0.000?and FA?30.86[25.57�42.48]vs.23.14[15.81�24.00]nmol/L;p=0.000?levels between the early URSA and early control groups.Statistically significant differences were noted in age?32.10�5.60 vs.28.78�4.24years;p=0.000?and serum FA levels?19.46[15.45�33.40]vs.34.96[20.40�41.90]nmol/L,p=0.001?between the late URSA and late control groups.2.3 The joint predictors,HCY+FA,HCY+age,FA+age and HCY+FA+age,were used as test variables to plot the ROC curve and calculate the Youden index.At their respective prediction cut-off values,the areas under the curve?AUC?values were 0.672,0.783,0.770,0.782,respectively,and the Youden indices were 0.348,0.533,0.571 and 0.533,respectively.Conclusion1.The mutation of MTHFR C677T and A1298C,MTRR A66G locus is a risk factor for URSA.2.Maternal serum FA level in combination with age has a higher predictive value for URSA.