Research On Clinical Factors And Related Mechanisms Affecting The Outcome Of Frozen-thawed Embryo Transfer

Part Ⅰ Main clinical factors affecting the live birth rate after the frozen-thawed embryo transfer of young femaleBackground:In recent years,the "freeze-all" strategy has been widely used and extended.However,factors that influence the success rate of transfer are still unclear except for age.Thus,recognizing and improving the factors that influence the live birth rate after the FET transfer is a good method to improve the rate of giving birth to a child after FET transfer.Objective:This study aimed to find out the factors that influence the live birth rate after FET transfer of young females with normal ovulation.Methods:This study is a secondary analysis of a multicenter RCT(ChiCTR-IOR-14005406)data that was published before,whose chief purpose was to make a comparison of the live birth rate(LBR)between fresh embryo transfer and FET of young females with normal ovulation.This study was conducted by using parts of data from former RCT,in which 917 young females accepted FET in cleavage stage were included,and sixteen clinical candidate variables that might influence the LBR after FET transfer were analyzed.The relation between predictive factors and pregnant live birth rate was assessed through both univariate and multivariate analysis so as to determine the independent risk factors that influenced the live birth rate after FET transfer.Results:The LBR in this study was 53%(486/917).Three independent predictive factors were confirmed that influenced the live birth rate after FET transfer.Duration of infertility(OR:0.933,95%CI:0.876-0.995,p=0.033),endometrial thickness(OR:3.375,95%CI:1.556-7.321 p=0.002),as well as transferred embryo numbers(OR:2.653,95%CI:1.226-5,743,p=0.013)were the main factors that influenced the live birth rate after FET transfer of young female.The section of infertility duration was 4.5 years,which that of endometrial thickness was 0.89cm.Conclusion:For females with normal ovulation,duration of infertility,endometrial thickness and transferred embryo numbers in cleavage stage are the main factors that influenced the pregnant live birth rate of young females.This result is beneficial to clinical decision and consultation for the improvement of live birth rate after FET of young female.Part Ⅱ Clinical prediction models of hypertensive disorders of pregnancy after frozen-thawed embryo transferBackground:Numerous studies have shown that pregnancies resulting from frozen-thawed embryo transfer(FET)have a higher incidence of hypertensive disorders of pregnancy(HDP)than those resulting from fresh embryo transfer or spontaneous conception.Early identification of patients with a high risk of HDP is vital to improving prognosis,especially in the high-risk frozen-thawed embryo transfer population.Finding clinical predictors and establishing a model for prediction are the most effeetive methods for prospective individualized prediction and targeted prevention.Objective:The goal of this study was to determine the associated risk factors and establish nomograms for HDP after frozen-thawed embryo transfer FET.Methods:This was a retrospective observational study consisting of 27948 patients undergoing FET,and data were collected between January 2011 and December 2019 at the Hospital for Reproductive Medicine Affiliated to Shandong University.The dataset was divided into a training(70%)and validation dataset(30%)by a credible random split-sample method.We used the least absolute shrinkage and selection operator(LASSO)method to select predictors and plot the nomogram.The training dataset was used to establish the model and plot the nomogram.The receiver operating characteristic(ROC)curves was used to assess the nomogram.We used the calibration curve to assess the accuracy of the nomogram.The decision curve analysis(DCA)was used to determine the clinical practicability of the nomogram.Results:The proportion of patients with HDP after FET was 6.81%in the training(n=19564)and validation(n=8384)datasets.Body mass index(BMI),endometrial preparation regimen,number of fetal hearts and mean arterial pressure(MAP)were identified as predictors and included in the nomogram.The area under the curve(AUC)of the ROC demonstrated adequate discrimination in the training dataset 0.726 and in the validation dataset 0.718.The nomogram was demonstrated to be well calibrated and clinically useful by calibration curve and DCA,respectively.Conclusions:The present study identified the associated risk factors and successfully established an effective predictive nomogram for HDP in patients with FET that included BMI,MAP,endometrial preparation regimen and the number of fetal hearts.This nomogram was assessed by internal validation exercises that confirmed the prediction power of the model.The nomogram is worthy of clinical promotion to identify people with a high risk of HDP,reduce the rates of HDP and improve pregnancy outcomes.Part Ⅲ Relationship between plasma exosomes in early pregnancy and high incidence of hypertensive disorders of pregnancy after frozen-thawed embryo transferBackground:Previous studies are found that compared to fresh embryo transfer,the incidence of hypertensive disorders of pregnancy(HDP)is significantly higher in patients with frozen-thawed embryo transfer(FET).The mechanism of HDP in patients with FET is unclear.It has been previously shown that the concentration of total exosomes presents in maternal plasma,placental exosomes and the microRNAs of exosomes content are altered in patients with HDP compared to women with normal pregnancies.Objective:The purpose of this study was to explore the relationship between the high incidence of HDP after FET and exosomes by detecting the concentration of plasma exosomes and exosome microRNAs expression in early pregnancy in patients with FET versus fresh embryo transfer.Method:In this study,plasma exosomes were extracted by differential centrifugation method.Compare the concentration of plasma exosomes in early pregnancy in patients with FET(29 cases)and fresh embryo transfer(30 cases)and perform plasma exosome miRNA sequencing(6 cases versus 6 cases)to obtain differently expressed miRNAs,then expand the sample size to 44 cases versus 44 cases of maternal plasma exosomes in early pregnancy in patients with fresh embryo transfer and FET separately for qRTPCR validation.Detect trophoblast cell proliferation by cell counting kit-8 assay and apoptosis by flow cytometry.Take the most distinctive phenotype of miR-409-3p as the target of the study.And target gene prediction for miR-miR-409-3p was performed with the aid of bioinformatics and further validated by qRT-PCR,protein electrophoresis,and dual luciferase reporter assay.The above experiments were repeated in trophoblast cells with down-regulation of target genes to test the proliferation and apoptosis.Result:Patients with FET had significantly higher concentration of total exosomes in maternal plasma in early pregnancy than that with fresh embryo transfer,with no significant differences in placental exosomes.Exosome miRNA sequencing showed that 21 differently expressed plasma exosome miRNAs in early pregnancy were identified in patients with fresh embryo transfer and FET,with 7 miRNAs showing upregulation and 14 showing down-regulation in women with FET.After expanding the sample,it was found that plasma exosome miR-409-3p expression was significantly down-regulated in early pregnancy in FET,and the down-regulation of miR-409-3p significantly prohibited trophoblast cell proliferation and promoted apoptosis.Tumor suppressor candidate 1(TUSC1)is the target gene of miR-409-3p.So miR-409-3p can affect trophoblast call proliferation and apoptosis through TUSC1 gene.Conclusion:Maternal plasma exosomes were significantly higher in total exosome concentration in early pregnancy in patients with FET compared to that with fresh embryo transfer,and placental exosome concentrations were not significantly different.The down-regulated miR-409-3p in the FET group’s exosomes may have an effect on trophoblast cell proliferation and apoptosis through TUSC1 gene.Plasma exosomes may play a role in the pathogenesis of HDP in patients with FET as carriers of pathogenic miRNAs,providing a new research direction for HDP of FET.