Relationships On Gene Polymorphism Of Polycyclic Aromatic Hydrocarbon Metabolic Enzyme,Telomere Length And Missed Abortion

Objective:1.To analyze the correlations on PAHs intrauterine exposure,metabolic enzyme（CYP450,GST）gene polymorphisms and missed abortion in the first trimester.2.To analyze the impact of PAHs exposure on telomere length（TL）,and explore the relationship between telomere length and missed abortion.3.To explore the environmental,genetic factors and their interactions that affect missed abortion.Methods:An epidemiological method of case-control study was used in this study.Women with missed abortion were consecutively collected as the case group,who were diagnosed in the obstetric department of the First Hospital of Shanxi Medical University from March2019 to December 2019.Women with normal early pregnancy were selected as the control group in the same hospital at the same time,who voluntarily terminated their pregnancy.A total of 114 cases and 139 controls were included.A face-to-face questionnaire survey was used to collect the general information of the subjects,past disease history,adverse pregnancy history,and lifestyle during pregnancy.The villi tissue specimens of abortion were obtained by negative pressure aspiration,washed with normal saline to remove residual blood and impurities.Then the specimens were transported to the laboratory in an ice box and stored at-80°C.The villus tissue was selected quantitatively,and physiological saline was added according to the ratio（1:9）,and the tissue was homogenized using a homogenizer,and then centrifuged to obtain the supernatant.Double antibody sandwich enzyme-linked immunosorbent assay（ELISA）was used to detect the content of PAH-DNA adduct in tissue supernatant.Tissue DNA was extracted by phenol-chloroform method,and genotyping was performed by multiplex PCR amplification and high-throughput sequencing.Real-time fluorescent quantitative PCR（Q-PCR）method was used to detect the relative length of telomeres.The questionnaire data was formed by Epidata 3.1 software.Normally distributed data are represented by the mean±standard deviation（x±s）,and non-normally distributed data are represented by the median M(P₂₅^P₇₅).SPSS 23.0 was used for t-test,chi-square test,logistics regression analysis,Mann-Whitney U test and Kendall’s tau-b correlation analysis,etc..SAS 9.2 was used for Armitage trend test.R 3.6.3 software was used for Hardy-Weinberg balance test and haplotype analysis.GMDR 0.9 software was used to analyze gene-gene and gene-environment interactions.Results:1.Detection of PAH-DNA adduct:The PAH-DNA adduct content of the case group and the control group were 466.26（424.55～507.53）pg/ml and 446.51（384.14～506.19）pg/ml,respectively.The level of PAH-DNA adduct in the case group was higher than that in the control group（Z=-2.10,P=0.036）.2.Correlation between PAH-DNA adduct level and missed abortion:After adjusting the covariates,,compared with the PAH-DNA adduct low-level group（Q₁）,the risk of missed abortion in the Q₂,Q₃and Q₄groups were 3.79,3.59 and 3.05 times（adjusted OR=3.79,95%CI:1.67-8.58,P=0.001;adjusted OR=3.59,95%CI:1.59-8.12,P=0.002;adjusted OR=3.05,95%CI:1.33-7.00,P=0.008）.3.Hardy-Weinberg equilibrium test:The 11 gene loci included in the analysis were in line with Hardy-Weinberg genetic balance test（P>0.05）.4.Comparison of genotypes and allele frequencies between the two groups:The occurrence of missed abortion was associated with the genotype of the GSTM1 rs1065411locus（P=0.005）.The case group and the control group had different GSTM1 rs1065411locus allele frequency distributions（P<0.001）,the proportion of allele C was higher in the missed abortion group.The genotypes and alleles of the remaining loci were not statistically related to the occurrence of missed abortion（P>0.05）.5.Logistic regression analysis of each gene locus and susceptibility to missed abortion:After adjusting the covariates,the GSTM1 rs1065411 gene locus was associated with missed abortion（P<0.05）.The risks of GC and CC genotype pregnant women were4.82 times and 3.29 times higher than those of GG genotype pregnant women（adjusted OR=4.82,95%CI:1.74-13.42,P=0.003;adjusted OR=3.29,95%CI:1.55-7.00,P=0.002）.Carriers of TA genotype at GSTM1 rs449856 gene locus had a risk of missed abortion 0.21times that of TT genotype（adjusted OR=0.21,95%CI:0.06-0.71,P=0.044）.6.Haplotype analysis of CYP450,GST genes and missed abortion:After adjusting the covariates,the haplotypes created by GSTM1 rs1065411 and rs449856 gene locus showed that the haplotypes of G-T and G-A had a risk of missed abortion,respectively 1.50 and4.00 times that of C-T（adjusted OR=1.50,95%CI:1.10-2.10,P=0.010;adjusted OR=4.00,95%CI:1.40-11.00,P=0.008）.7.Gene-gene interaction analysis:After adjusting the covariates,there was no interaction between the measured CYP450 gene loci（P>0.05）.There was an interaction between GSTM1 rs1065411,GSTM3 rs7483 and GSTP1 rs1695 gene locus polymorphisms,the training balance accuracy was 0.6427,the test balance accuracy was0.6159,the cross-validation consistency was 10/10,P=0.011.There was no interaction between CYP450 and GST gene loci（P>0.05）.8.Gene-environment interaction analysis:After adjusting the covariates,there was no interaction between CYP450 gene locus polymorphism and PAH-DNA adduct.There was an interaction between GSTM1 rs1065411 gene polymorphism and PAH-DNA adduct,the training balance accuracy was 0.6620,the test balance accuracy was 0.6432,the cross validation consistency was 10/10,P=0.001.9.Detection of relative telomere length:The relative telomere length difference between the two groups was statistically significant（Z=-4.02,P<0.001）.The relative telomere length of the control group was greater than that of the case group.10.Analysis of the correlation between relative telomere length and the risk of missed abortion:According to the median relative length of telomeres,they were divided into two groups.After adjusting for covariates,the results of the two groups showed that the risk of missed abortion in the shorter telomere group（M₂）was 2.11 times that of the longer telomere group（M₁）（adjusted OR=2.11,95%CI:1.22-3.63,P=0.007）.11.Correlation analysis between PAHs exposure and telomere length:Kendall’s tau-b correlation analysis showed that there was no correlation between PAH-DNA adduct levels and telomere length（r=0.08,P=0.170）.12.Logistic regression analysis of the effect of PAHs exposure on telomere length:After adjusting for covariates,when the PAH-DNA adduct was the Q₃level,the risk of shortening the relative telomere length was 2.67 times that of the low-level Q₁group（OR=2.67,95%CI:1.28-5.58）,the difference was statistically significant（P=0.009）.13.Logistic regression analysis of multiple factors affecting missed abortion:Factors that were meaningful in the analysis results of Part 1 and Part 2 were included in Logistics regression analysis.The results showed that adverse pregnancy history（OR=3.08,95%CI:1.61-5.91）,PAH-DNA adduct Q₂and Q₃levels（OR=2.93,95%CI:1.32-6.52;OR=2.77,95%CI:1.23-6.25）,GC,CC genotype at rs1065411 site（OR=3.37,95%CI:1.46-7.80;OR=2.57,95%CI:1.40-4.69）and relatively short telomere length（OR=2.05,95%CI:1.19-3.56）were risk factors for missed abortion.Conclusions:1.High exposure to PAHs and mutations in GSTM1 rs1065411 gene may be risk factors for missed abortion.2.There were gene-gene and gene-environment interactions between PAHs exposure and GST gene polymorphism,which can jointly affect the occurrence of missed abortion.3.High exposure of PAHs may cause wear of telomere length,and shorter telomere length was related to missed abortion.