Association Of AHR Signal Pathway Gene Differential Expression And 8-OHdG With Missed Abortion

Objective:1.To explore the relationship between AHR signaling pathway related genes and missed abortion,and further analyze its mediating effect between PAHs exposure and missed abortion.2.To analyze the difference in 8-OHd G levels between the missed abortion case group and the control group,and explore the relationship between 8-OHd G levels and PAHs exposure.3.To explore the influencing factors of missed abortion.Methods:95 cases of missed abortion diagnosed in obstetrics at the First Hospital of Shanxi Medical University from April 2021 to April 2022 were selected as the case group,and 190pregnant women of similar age（age difference≤3 years）,same ethnic group（Han nationality）,cohabitation（Taiyuan）,who participated in normal obstetric examination during the same period but did not have missed abortion but actively requested artificial abortion were matched according to the 1:2 matching principle as the control group.A face-to-face questionnaire survey was conducted on the subjects.The contents of the questionnaire included the general situation of the subjects（age,education level,body mass index,BMI,etc.）,behavior and lifestyle（smoking,drinking,exercise,etc.）,pregnancy and childbirth history（pregnancy,adverse pregnancy history,etc.）and the current pregnancy situation.The clinical obstetrician performed negative pressure abortion to attract the abortion tissue,collected 50～300mg villous tissue,washed the blood stains and impurities with normal saline,and transported the ice box back to the laboratory,and stored it in an ultra-low temperature refrigerator at-80℃for testing.Trizol method was used to extract RNA from villus tissue,and then reverse transcribed into c DNA.The expression level of AHR pathway related genes was detected by fluorescence quantitative PCR.The concentrations of PAH-DNA adduct and 8-OHd G in villi were detected by double-antibody one-step sandwich enzyme-linked immunosorbent assay（ELISA）.Use Epi Data 3.1 to establish the database.SPSS 22.0 software was used for t-test,chi-square test,Fisher exact probability method,Manny-Whitney U test,Spearman rank correlation analysis,ROC curve analysis and logistic regression analysis;Use Graph Pad Prism 8.0.1 and Origin 2022 to draw statistical charts;Use the R language Mediation package to test the mediating effect of the secondary dependent variable,bilateralα=0.05is the significance level.Results:1.Basic information of the subjects:the average age of the subjects was（29.38±6.16）years old.There was no significant difference between the case group and the control group in age,registered residence,per capita monthly income,smoking,drinking,body mass index and physical exercise（P>0.05）.The difference of education level between the two groups was statistically significant（χ²=8.59,P=0.035）。The gestational age of the case group was greater than that of the control group（t=12.31,P<0.001）,and the proportion of adverse pregnancy history was higher than that of the control group（χ²=15.49,P<0.001）。2.The relationship between PAH-DNA adduct and missed abortion:the concentration of PAH-DNA adduct in the case group was 479.68（442.31,534.29）pg/ml,which was higher than 446.82（394.49,502.45）pg/ml in the control group,with a statistically significant difference（Z=-4.120,P<0.001）.According to P₂₅ and P₇₅ of PAH-DNA adduct concentration,the subjects were divided into three levels:low exposure T₁(<410.14pg/ml;<P₂₅),medium exposure T₂(410.14～510.97 pg/ml;P₂₅～P₇₅)and high exposure T3(≥510.97 pg/ml;≥P₇₅).Univariate logistic regression analysis showed that the risk of missed abortion at T2 and T3 levels was 5.07 times（OR=5.07,95%CI:2.26-11.38,P<0.001）and 6.10 times（OR=6.10,95%CI:2.55-14.60,P<0.001）,respectively.After adjusting for covariates such as age,education level,gestational week and gestational grade,the logistic regression results showed that the risk of missed abortion at T₂ and T₃levels was 4.62 times（OR=4.62,95%CI:1.59-13.44,P=0.005）and 5.51 times（OR=5.51,95%CI:1.60-18.92,P=0.007）,respectively.3.AHR path-related gene expression:There were significant differences in AHR,ARNT,AHRR,CYP1A1 and GSTP1 gene expression between the case group and the control group（P<0.01）.Among them,the expression level of AHR,ARNT,CYP1A1 and GSTP1 genes in the case group was higher than that in the control group（Z=-7.818,P<0.001;Z=-4.704,P<0.001;Z=-3.405,P<0.001;Z=-3.532,P<0.001）,while the expression of AHRR was lower than that in the control group（Z=-6.855,P<0.001）.4.Gene correlation analysis of AHR pathway:Spearman correlation analysis showed that there was a significant positive correlation between AHR and gene expression of ARNT（r_s=0.38,P<0.001）,CYP1A1（r_s=0.36,P<0.001）and GSTP1（r_s=0.19,P=0.001）,while there was a negative correlation between AHR and AHRR expression（r_s=-0.11,P=0.073）,but there was no statistical significance.In addition,ARNT was positively correlated with AHRR（r_s=0.23,P<0.001）,CYP1A1（r_s=0.30,P<0.001）and GSTP1（r_s=0.48,P<0.001）;CYP1A1 was positively correlated with AHRR（r_s=0.34,P<0.001）and GSTP1 gene expression（r_s=0.24,P<0.001）.5.ROC analysis of AHR pathway gene:ROC analysis showed that AHR,ARNT,AHRR,CYP1A1 and GSTP1 could distinguish missed abortion and normal pregnant women in all subjects,AUC of AHR=0.784（95%CI:0.724～0.785,P<0.001）,AUC of ARNT=0.671（95%CI:0.606～0.736,P<0.001）,AUC of AHRR=0.749（95%CI:0.688～0.810,P<0.001）,AUC of CYP1A1=0.624（95%CI:0.554～0.694,P=0.001）,AUC of GSTP1=0.628（95%CI:0.564～0.693,P<0.001）.6.Logistic regression analysis of AHR pathway related genes and missed abortion:The expression of AHR,AHRR,and CYP1A1 were found to be predictive factors for missed abortion through univariate and multivariate conditional logistic regression analysis using each gene as a numerical variable.Among them,AHR and CYP1A1 are the risk factors of missed abortion,while AHRR is the protective factor.To understand the impact of various levels of AHR pathway genes on missed abortion,AHR,ARNT,AHRR,CYP1A1,and GSTP1 were divided into three groups（low expression,medium expression,and high expression）based on P₃₃ and P₆₇,and their relationship with missed abortion was analyzed.When the covariates were not adjusted,the high expression of AHR,medium and high expression of ARNT,medium and high expression of AHRR,high expression of CYP1A1 and medium and high expression of GSTP1 were all the influencing factors of missed abortion.After adjusting for age,education level,gestational week,gestational times,adverse pregnancy history,smoking and drinking,AHR was highly expressed（OR=5.30,95%CI:2.03～13.82,P=0.001）,AHRR was moderately expressed and highly expressed（OR=0.29,95%CI:0.12～0.73,P=0.009;OR=0.20,95%CI:0.08～0.52,P=0.001）,CYP1A1 was highly expressed（OR=3.47,95%CI:1.39～8.62,P=0.007）The expression of GSTP1（OR=4.51,95%CI:1.77～11.51,P=0.002）was the influencing factor of missed abortion.After adjusting age,education level,gestational week,gestational times,adverse pregnancy history,smoking,drinking PAH-DNA adduct,the results showed that AHR high expression（OR=4.68,95%CI:1.783～12.29,P=0.002）,CYP1A1 high expression（OR=2.95,95%CI:1.16～7.54,P=0.024）,GSTP1 high expression（OR=5.82,95%CI:2.14～15.87,P=0.001）were risk factors for missed abortion,The expression and high expression of AHRR（OR=0.26,95%CI:0.10～0.68,P=0.006;OR=0.20,95%CI:0.08～0.53,P=0.001）are the protective factors of missed abortion.7.The ROC curve is constructed based on the prediction probability of logit model of AHR,AHRR and CYP1A1 panel.The AUC of AHR-AHRR-CYP1A1 axis is 0.904（95%CI:0.867～0.941,P<0.001）,the sensitivity is 78.95%,and the specificity is 89.47%.8.Logistic analysis of PAH-DNA adduct and genes related to AHR pathway:AHR,ARNT,AHRR,CYP1A1,GSTP1 were divided into two groups according to their P₇₅,and PAH-DNA adduct was used for logistic analysis of each gene.The results showed that before adjusting the covariates,the risk of AHR expression increased by the level of PAH-DNA adduct T₂ and T₃ was 2.96 and 6.31 times higher than that of T₁,respectively;The risk of increased CYP1A1 expression was 2.86 and 2.70 times of T₁ level,respectively.After adjusting the covariates,the risk of AHR expression increased by the level of PAH-DNA adduct T₃ was 4.66 times higher than that of T₁（OR=4.66,95%CI:1.72-12.66,P=0.003）;The risk of increased CYP1A1 expression caused by T₂ level was 2.38 times higher than that of T₁ level（OR=2.38,95%CI:1.06-5.33,P=0.036）.9.Intermediary analysis of AHR and CYP1A1 in the influence of PAH-DNA adduct on missed abortion:The results showed that the intermediary proportion of missed abortion related to PAH-DNA adduct mediated by AHR expression was 12.97%;The expression of CYP1A1 has no mediating role in the relationship between PAH-DNA adduct and missed abortion.10.Logistic regression analysis of 8-OHd G and missed abortion:the concentration of8-OHd G in the case group was 626.22（587.30,675.95）pg/ml,which was significantly higher than that in the control group 587.50（518.90,672.69）pg/ml（Z=-2.421,P=0.015）.According to the quartile of 8-OHd G concentration,they were divided into four groups:H₁(<535.33 pg/ml;<P₂₅),H₂(535.33.14～607.02 pg/ml;P₂₅～P₅₀),H₃(607.03～674.29pg/ml;P₅₀～P₇₅)and H₄(≥674.30 pg/ml;≥P₇₅).Before adjusting the covariates,the risk of missed abortion at the levels of H₂,H₃ and H₄ was 2.17 times,3.15 times and 2.21times higher than that of H₁,respectively.After adjusting the covariates,the risk of missed abortion at H₃ level is 5.00 times higher than that at H₁ level（OR=5.00,95%CI:1.68-14.82,P=0.004）.11.The relationship between PAH-DNA adduct and 8-OHd G:Spearman correlation analysis showed no correlation between the two variables（r_s=0.055,P=0.357）.8-OHd G[divided into high level group and low level group according to P₇₅（674.30 pg/ml）]as the dependent variable,and age,education level,gestational week,pregnancy times,adverse pregnancy history,smoking,drinking,and PAH-DNA adduct level as the independent variable,logistic regression analysis was performed.Results No correlation between PAH-DNA adducts and 8-OHd G was found.12.The relationship between AHR pathway gene expression and 8-OHd G:Spearman correlation analysis showed that there was a correlation between 8-OHd G and ARNT（r_s=0.162,P=0.006）;No correlation was found between 8-OHd G and AHR,AHRR,CYP1A1,GSTP1（P>0.05）.8-OHd G[divided into high level group and low level group according to P₇₅（674.30 pg/ml）]as the dependent variable,and age,education level,gestational week,gestational times,adverse pregnancy history,smoking,drinking,AHR pathway gene expression as the independent variable,logistic regression analysis was performed.Results No effect of AHR pathway gene on 8-OHd G was found.13.Multivariate analysis of missed abortion:after adjusting for age,smoking and alcohol consumption,the results showed that gestational weeks（OR=4.49,95%CI:2.65～7.60,P<0.001）,adverse pregnancy history（OR=1.14,95%CI:1.04～1.26,P=0.005）,PAH-DNA adduct T₂ level group（OR=5.26,95%CI:1.38～2.02,P=0.015）,AHR（OR=2.31,95%CI:1.35～3.97,P=0.002）,AHRR（OR=0.23,95%CI:0.13～0.43,P<0.001）,CYP1A1（OR=2.62,95%CI:1.57～4.36,P<0.001）,the level of H₃ in 8-OHd G（OR=12.88,95%CI:2.89～57.35,P=0.001）is the influencing factor of missed abortion.Conclusions:1.PAHs intrauterine exposure,high expression of AHR,high expression of CYP1A1,8-OHd G,gestational age,and adverse pregnancy history may be risk factors for missed abortion;High expression of AHRR may be a protective factor for missed abortion.2.AHR-AHRR-CYP1A1 axis RNA differential expression can be used as one of the diagnostic indicators for missed abortion.3.PAHs may cause missed abortion by influencing the expression of AHR.