Epidemiological Investigation Of Hypertensive Disorders In Pregnancy And Screening Of Plasma Biomarkers In Early Pregnancy For Predicting Pre-Eclampsia Based On Proteomics

[Objective]Hypertensive disorder in pregnancy（HDP）,as one of the most common serious complications during pregnancy,seriously threatens maternal and infant health.Studies have shown that women with HDP and their offspring have significantly higher risks of cardiovascular events in the future.HDP provides an opportunity to identify women with an increased risk of future cardiovascular events.Early and accurate identification of risk factors for HDP,early diagnosis,and early intervention of HDP may improve their long-term health.The vast majority of risk factors for HDP are irreversible,while pre-pregnancy obesity and gestational weight gain（GWG）are modifiable,but for the limitations of the methodology for assessing GWG,the association between GWG and the risk of HDP remains uncertain or insufficient.Preeclampsia（PE）,as one of the most important subtypes of HDP,is a multi-system dysfunction disease that mainly occurs after 20 weeks of pregnancy.Studies have found that intervention in early pregnancy can significantly reduce the incidence of PE in high-risk groups,but no effective biomarkers have been found currently in the early prediction of PE.Therefore,based on a prospective cohort study for singleton pregnant women in Tianjin with HDP as the main entry point,the present study evaluated the risk factors of HDP in the first trimester,explored the relationship between weight gain in the first half of pregnancy and HDP occurring in the second half of pregnancy,and through the proteomics analysis of the plasma samples in early pregnancy of pregnant women with severe preeclampsia（sPE）and healthy pregnant women,the study explored the ideal plasma markers in early pregnancy of PE.[Methods]First,this present study prospectively included normotensive singleton pregnant women undergoing prenatal examination in 19 community health service centers in Tianjin.The demographic characteristics and blood samples of pregnant women were obtained through questionnaires and routine prenatal examination,and maternal and infant outcomes were obtained through the medical record system of the hospitals for delivery.According to the pre-pregnancy BMI,pregnant women were divided into three groups:underweight,normal-weight,and overweight/obesity,and weekly weight gain rates at the three periods(0-13⁺⁶,14⁺⁰-20⁺⁶,and 0-20⁺⁶)were calculated.The generalized linear model was used to analyze the relationship between the weekly weight gain rates of the three periods and the risk of HDP in the whole cohort and three pre-pregnancy BMI subgroups respectively.The pre-pregnancy weight and early pregnancy weight were used as different starting points to explore the relationship between weight gain in the first half of pregnancy and the risk of HDP occurring in the second half of pregnancy.Secondly,based on the above cohort study,the study selected the first-trimester plasma samples of 10 pregnant women who eventually developed sPE and 10 women with uncomplicated pregnancies.A new data acquisition mode-Data Independent Acquisition（DIA）was used to conduct proteomic analysis,and the candidate differentially expressed proteins were identified through bioinformatics analysis and literature review to further study the pathogenesis of sPE and explore biological markers in early pregnancy for sPE.Finally,based on the results of proteomics analysis,The differential expression of some secreted proteins was verified by ELISA in the early-pregnancy plasma of pregnant women who finally progressed to sPE and healthy pregnant women.The differences in baseline characteristics and plasma biological indicators between the two groups were screen out through univariate analysis,and the predictive value of the above-mentioned difference indicators on sPE was analyzed by the binary Logistic regression model,and an early prediction model of sPE was established.[Results]1.A total of 11,195 singleton normotensive pregnant women were initially enrolled.During the follow-up,793 pregnant women were excluded due to termination of pregnancy,withdrawal from the follow-up,and other reasons;597pregnant women were excluded due to missing or abnormal data during data cleaning,and 9805 pregnant women with complete information were finally included for data analysis.Among them,508 pregnant women were diagnosed with HDP,included 178pregnant women with gestational hypertension,330 pregnant women with preeclampsia/eclampsia.The total population incidence of HDP was 5.2%,and the incidence of HDP in the underweight,normal weight and overweight/obese populations was 1.8%,3.9%,and 12.2%,respectively.By analyzing the baseline characteristics of pregnant women and the relationship between weight gain at three gestational periods before 20⁺⁶weeks and the risk of HDP,this study supported that pre-pregnancy BMI,primipara,and family history of hypertension were high-risk factors for HDP,especially for pregnant women with normal weight before pregnancy.In general,even adjusted for the high-risk factors of HDP,such as age,pre-pregnancy BMI,primipara,and family history of hypertension,weight gain in early pregnancy(0-13⁺⁶weeks)and/or in the first half of the pregnancy(0-20⁺⁶weeks),based on pre-pregnancy weight as the starting point of the study,were related to the increased risk of HDP in the second half of pregnancy,and this relationship was different in different BMI groups.In the entire cohort,compared to women with weight gain≤0.00 kg/w before 13⁺⁶weeks of pregnancy,women with weight gain≥0.18 kg/w had an increased risk of HDP by 28%（a RR:1.28,95%CI:1.04-1.55）,among overweight/obese pregnant women,women with weight gain≥0.18 kg/w had a 38%higher HDP risk than those with weight gain≤0.00 kg/w（a RR:1.39,95%CI:1.04-1.85）,while weight gain before 13⁺⁶weeks among underweight and normal-weight pregnant women did not increase the risk of HDP.In the entire cohort,compared with a weekly weight gain rate of≤0.14kg/w,a weekly weight gain rate of≥0.28kg/w before 20⁺⁶weeks increased HDP risk by 36%（a RR:1.36,95%CI:1.11-1.67）;among normal-weight pregnant women,compared with the lowest weekly weight gain rate distribution（≤0.15kg/w）,women with the highest weekly weight gain rate distribution（≥0.29kg/w）had a 46%increased risk of HDP（a RR:1.46,95%CI:1.11-1.93）;among overweight/obese women,weight gain rate before 20⁺⁶weeks seemed to increase the risk of HDP,but the difference was not statistically significant（P=0.059）,while the association was null among underweight women.However,weight gain during 14⁺⁰-20⁺⁶weeks of pregnancy in any group,based on early pregnancy weight(before 13⁺⁶weeks)as the starting point of the study,was not related to the risk of HDP.2.In this study,high-throughput proteomics analysis based on a new data acquisition mode（DIA）was carried out to analyze early-pregnancy plasma samples from women with sPE and healthy pregnant women,and a total of 84 differentially expressed proteins were identified.Cluster analysis could effectively distinguish the PE group from the healthy control group.Bioinformatics analysis showed that the differentially expressed proteins in plasma samples between women with sPE and normal pregnant women were mostly related to energy metabolism,immune response,cytoskeleton formation,etc.suggesting that there were abnormalities in the complement system,lipid metabolism,inflammatory response,and immune tolerance in the first trimester of pregnant women with sPE.3.Based on the results of differentially expressed protein obtained by proteomics analysis,the present study used the ELISA method to detect the concentration of FN,Apo F and C4b in early-pregnancy plasma of women who eventually progressed to sPE and healthy pregnant women.The plasma concentrations of FN and C4b of women with sPE were higher than those of healthy pregnant women,and the differences were statistically significant（P<0.001,P=0.013 respectively）,while the Apo F concentration of women with sPE had a decreasing trend,but there was no statistical difference between the two groups（P=0.096）.Univariate analysis screened out the different indicators between the two groups,including BMI,SBP,DBP at enrollment,and plasma FN and C4b levels.The binary Logistic regression model analysis indicated that plasma FN and C4b levels can improve the accuracy of predicting sPE of the basic model（BMI+SBP）.Finally,four variables including BMI,SDP,plasma FN and C4b levels were included,and the formula for predicting the risk of sPE was obtained:Logit（P）=-15.703+0.130*BMI+0.074*SBP+0.007*FN+0.003*C4b.[Conclusions]1.The present study supported that pre-pregnancy BMI,primipara,and family history of hypertension were high-risk factors for HDP.This study showed a positive relationship between weight gain during the first half of pregnancy and the risk of HDP in the second half of pregnancy among women of childbearing age（except for pre-pregnancy underweight women）.This study suggested that the inconsistent conclusions of previous studies,focusing on the relationship between weight gain in the first and/or second trimester and the risk of HDP,may be partly due to the different starting points of the maternal weight of these studies（pre-pregnancy weight and early pregnancy weight）.2.In this study,a total of 84 differentially expressed proteins were found through high-throughput proteomics analysis on the plasma during the first trimester of women with sPE and healthy pregnant women,suggesting that there were abnormalities in the complement system,lipid metabolism,inflammation,and immune tolerance in the early pregnancy of women with sPE.It was also confirmed by ELISA that the concentrations of plasma FN and C4b in early pregnancy of pregnant women with sPE were higher than those of healthy pregnant women.The study showed that combining BMI,SBP,plasma FN and C4b levels of pregnant women in early pregnancy can effectively predict the occurrence of sPE.