Mechanism Study Of Endometrial Scratching In Improving Endometrial Receptivity

The successful implantation of embryo mainly depends on embryo quality,endometrial receptivity and its synchronization.Clinically,some patients still experience repeated implantation failure(RIF)even after transferred high-quality embryos.Therefore,with high-quality embryos,how to improve endometrial receptivity and the clinical pregnancy rate is crucial.Whether endometrial scratching(ES)can improve endometrial receptivity has aroused researchers’ interests in the field of assisted reproduction technology.Small sample data of ES seem to improve clinical pregnancy rates.The mechanism of ES improve the endometrial receptivity is unknown,there is no relevant literature at the proteomic level.For the different ways,timing,and number of ES,the results of the research were also different,and no unified consensus was temporarily formed.This study was to explore the possible mechanisms of ES that improved endometrial receptivity by using proteomics analysis on patients experienced two or more implantation failures,which can better guide future clinical work and benefit more infertility patients.Part Ⅰ Effect of endometrial scratching during menstruation on clinical outcomes in frozen-thawed embryo transferObjective:To explore the effect of ES during menstruation on clinical outcomes before F-ET on patients experienced two or more implantation failures.Methods:A total of 200 F-ET patients who suffered at least two implantation failures between October 2017 and February 2018 in our center were randomly divided into two groups:endometrial scratching(ES)group and control group(n=100 in each group).Patients in the ES group received local endometrial scratching with a Pipelle catheter on the third day of the menstrual cycle before F-ET.Patients in the control group did not perform any treatment on the third day of the menstrual cycle.Primary outcomes were live birth,clinical pregnancy and implantation rates.Secondary outcomes were the rate of blood flow of endometrium,biochemical pregnancy,multiple pregnancy and ectopic pregnancy and abortion.Results:The rate of live birth in ES group was significantly higher than that of control group(51.00%VS 36.00%,P<0.05).Clinical pregnancy and implantation rates in ES group were significantly higher comparing to control group(64.00%VS 48.00%,P<0.05 and 46.74%VS 30.11%,P<0.05).The rate of biochemical pregnancyin ES group was significantly higher than that of control group(66.00%VS 49.00%,P<0.05).The proportion of blood flow of endometrium in ES group was significantly higher than that of control group(97.00%VS 88.00%,P<0.05).The rate of multiple pregnancy in ES group was significantly higher than that of control group(37.50%VS 18.75%,P<0.05).No significant difference in ectopic pregnancy rate and abortion rate was observed between ES group and control group(P>0.05).Conclusions:Applying ES to patients experienced two or more implantation failures during menstruation before F-ET can improve clinical outcomes.Part Ⅱ Proteomics analysis of endometrial scratching in improving endometrial receptivityObjective:Exploring the differentially expressed proteins(DEPs)of window of implantation(WOI)of endometrium after ES during menstruation who experienced two or more implantation failures provided data to support for further exploration of the mechanisms by which ES improves endometrial receptivity.Methods:18 patients who experienced two or more implantation failures with regular menstrual cycle were selected to voluntarily enter this study,ES group(9 cases)and Control group(9 cases):specific operation was shown in Part 1.Follicular development and ovulation of all patients were monitored through vaginal ultrasound from the 10th day of menstruation according to their conditions.Endometrial tissue samples were performed by using a curette and evenly scratching 5 days after ovulation.The samples were then immediately to put into disposable sterile freezing tubes and sealed before labeling and storage at-80℃ for later use.Using a combination of isobaric tags for relative and absolute quantification(iTRAQ)and liquid chromatography-mass spectrometry(LC-MS),we compared the DEPs of the endometrium of WOI between ES and control groups with a history of two or more implantation failures,followed by functional annotation and bioinformatics analysis.Afterwards,three DEPs were selected for further validated using Western blotting(WB)to explore whether their expressions were consistent with the result of iTRAQ.Results:This study identified 3275 endometrial proteins of WOI in patients who experienced two or more implantation failures.There were 270 DEPs(236 upregulated and 34 downregulated)between ES group and control group.GO analysis found that the biological processes of DEPs were mainly involved in molecular processes,metabolic processes,localization and establishment of localization,biological adhesion,locomotion,cell proliferation and reproductive and so on;the cellular components of DEPs were mainly distributed in cell and cell part,organelle and organelle part,extracellular region and extracellular region part,macromolecular complex,and so on.The molecular functions of DEPs were mainly binding and catalytic activity.KEGG pathway enrichment analysis found that the DEPs were mainly enriched to 153 signaling pathways,mainly related to glycolysis/gluconeogenesis,complement/coagulation cascade.PPI analysis of DEPs revealed that Caldesmon(CALD1)was associated with vinculin(VCL)and VCL was associated with β-catenin.CALD1 was also associated with Calponin-3(CNN3)in the PPI interaction of the 8 proteins of interested.WB identified the changes in the three DEPs,CALD1,Fascin(FSCN1)and CNN3,which were consistent with the results of iTRAQ technology,and further verified the proteomics findings of this subject.Conclusion:The three cytoskeleton-related proteins CALD1,FSCN1,CNN3 of DEPs might be associated with endometrial receptivity.Part Ⅲ Mechanism study of CALD1-β-catenin-FSCN1 in improving endometrial receptivityObjective:Based on the results of this study suggested that CALD1 was closely related to β-catenin and further explored whether CALD1-β-catenin-FSCN1 plays a role in embryo implantation and improving endometrial receptivity,and then obtained the possible mechanism of ES during menstruation in improving endometrial receptivity,and provided new data supporting for the application of ES in assisted reproductive technology.Methods:Human endometrial epithelial cells(iCell-0031a)was first selected,and siRNA-CALD1 was screened at the cell level using the siRNA interference technique to look for the best interference targets for subsequent animal experiments.Then constructed a pregnant mice model and injected siRNA-CALD1 dilution into the uterus of pregnant 3d mice as siRNA-CALD1 group and then injected 0.9%saline into the uterus of pregnant 3d mice as control group.Compared the effect of siRNA-CALD1 on the number of embryo implantation and littermate in mice.WB was used to compare the protein expression level of CALD1,β-catenin and FSCN1 of endometrial tissues of pregnant 21d mice between the two groups.Finally compared the protein expression level of endometrial receptivity indicators such as estrogen receptor(ER),progesterone receptor(PR),very late antigen-4(VLA-4),integrinβ3(ITGβ3),and homeboxA10(HOXA10)between the two groups to investigate the effect of CALD1-β-catenin-FSCN 1 on embryo implantation and endometrial receptivity in mice.Results:Of the three siRNA-CALD1 targets,Si-CALD1-3 interfered was the best.The number of embryo implantation was significantly decreased in the Si-CALD 1 group(5.00±2.37 VS 10.64±1.63,P<0.05).The number of littermate was significantly decreased in the Si-CALD1 group(5.57±2.99 VS 8.50±1.31,P<0.05).The protein expression level of CALD1,β-catenin and FSCN1 in endometrial tissue showed all significantly reduced in the Si-CALD1 group(P<0.05).The protein expression level of endometrial receptivity indicators(ER,PR,VLA-4,ITGβ3,HOXA10)in the Si-CALD1 group were all significantly reduced(P<0.05).Conclusion:CALDl-β-catenin-FSCN1 may be involved in improving endometrial receptivity.After CALD1 is interfered,it significantly reduces the protein expression levels of ER,PR,VLA-4,ITGβ3,HOXA10 in the endometrium,thereby reducing endometrial receptivity,which in turn affects the fertility of mice.