Study On Endometrial Receptivity And Construction Of CeRNA Regulatory Network Of Patients With Repeated Implantation Failure

Repeated implantation failure(RIF)refers to the failure of more than 3 times of embryo transfer or cumulative transfer of 4～6 high-quality cleavage embryos or more than 3 high-quality blastocysts,which brings great mental pressure and economic burden to patients and their families.However,the etiology and mechanism of RIF are still unclear,and it is a hot and difficult point in the field of reproduction.Previous studies have shown that aneuploidy is one of the important causes of RIF.It is suggested that patients with RIF should apply the technique of pre-implantation genetic testing for aneuploidy(PGT-A)to improve embryo implantation rate.Another key factor affecting embryo implantation is endometrial receptivity(ER).In recent years,with the study of embryo-endometrium cross-talk and mechanism of embryo implantation,the differential expression and regulation of endometrial genes have been widely concerned,but the related research is still at the initial stage.RNA sequencing(RNA-seq)technology can comprehensively and quickly obtain all transcripts of specific tissues in this state,including messenger RNA(mRNA)and non-coding RNA(ncRNA).Competing endogenous RNA(ceRNA)is a brand-new mode of gene expression regulation,in which transcripts such as mRNA and ncRNA competitively combine the same miRNA with miRNA response elements to regulate their expression levels,thus affecting cell function and provides new clues for studying the mechanism of disease.In this study,we firstly observed whether PGT-A technique could improve the pregnancy outcome of RIF patients.And then,based on RNA-seq technique,taking patients with RIF and normal fertile women as research objects,the endometrium of them was sequenced,in order to obtain the whole expression transcriptome profile of them.And then we can construct ceRNA regulatory network and screen out the key genes that may lead to RIF.Verifying the regulatory relationship between RIF patients and their related ncRNA through experiments,so as to lay a theoretical foundation for elucidating the etiology and mechanism of RIF and provide potential intervention targets for improve the pregnancy outcome of RIF patients.Part Ⅰ The effect of PGT-A on pregnancy outcome in patients with repeated implantation failureObjectiveThe purpose of this study was to compare the pregnancy outcome of the first frozen-thawed embryo transfer(FET)cycle to select blastocyst between PGT-A technique based on next-generation sequencing(N GS)and only by blastocyst morphological score.As to explore the clinical application value of PGT-A in RIF patients.MethodsThe clinical data of RIF patients admitted to our hospital from January 2017 to December 2019 were retrospectively analyzed.A total of 29 RIF patients who received PGT-A based on NGS technique were selected as the PGT-A group.Besides,108 RIF patients who underwent in-vitro fertilization(IVF)during the same period were selected as the control group.After freezing all the embryos,one blastocyst with the best morphological score was selected for the first FET cycle and compared the pregnancy outcomes between two groups.Results1.There were no significant difference among average age,body mass index(BMI),antral follicle count(AFC),anti-Mullerian hormone(AMH),basic follicle-stimulating hormone(FSH),infertility type and other baseline data between two groups(P>0.05).2.A total of 120 blastocysts were biopsied in the PGT-A group,52.14%were aneuploidy blastocysts,27.35%were aneuploidy blastocysts and 20.51%mosaic blastocysts.The euploidy rate of the high-quality blastocyst group(78.95%)was significantly higher than that of the average-quality blastocyst group(41.67%)and the poor-quality blastocyst group(50.00%)(P=0.028).The results of fitting curve showed that the euploidy rate of PGT-A patients under 38 years old was not significantly decreased with age(P=0.149).3.The proportion of high-quality blastocyst transfer(55.17%vs.44.44%),endometrial preparation protocols were not significantly different between two groups(P>0.05).4.There were no significant differences in the clinical pregnancy rate(72.41%vs.52.78%),spontaneous abortion rate(19.05%vs.22.81%)and live birth rate(58.62%vs.40.74%)between the two groups(P>0.05).ConclusionPGT-A did not significantly improve pregnancy outcomes in RIF patients.Part Ⅱ Identifying key genes of endometrial receptivity based on RNA-seq analysisObjectiveThe purpose of this study was to evaluate endometrial receptivity in patients with RIF by ultrasound parameters and screen candidate genes that affect endometrial receptivity.In order to pave the way for further study of its regulatory network and the pathogenesis of RIF.MethodsA total of 15 RIF patients who failed to pregnancy after PGT-A from January 2017 to December 2019 were selected as the RIF group,and another 15 women with normal fertile during the same period were selected as the control group.1.The levels of serum estradiol(E2)and progesterone(P)during the window of implantation were measured by chemiluminescence method.2.Three-dimensional vaginal ultrasound examination was performed in the window of implantation to obtain endometrial ultrasound parameters.3.Endometrial biopsy was performed with endometrial sampler.And three cases were randomly selected from RIF group and control group to test endometrium by RNA-seq technique respectively,and to analyze the differential expression profile of mRNA.Then performing bioinformatics analysis that based on the high-throughput sequencing results and using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)to perform function enrichment analysis for candidate genes that associated with endometrial receptivity.Finally,we perform quantitative polymerase chain reaction(qPCR)to confirm in 15 RIF patients and 15 control patients.4.Immunohistochemical staining was used to observe the expression of candidate genes that may affect endometrial blood flow in the window of implantation of endometrium between two groups.Results1.The levels of estradiol(E2)and progesterone(P)in the window of implantation were significantly in accord with the endometrial secretory stage(P>0.05).2.The S/D of bilateral uterine arteries in RIF group was significantly higher than that in control group(11.28 ± 2.59 vs.9.06 ± 2.22)(P=0.018).The proportion of"good" endometrial blood flow distribution in RIF group(13.33%)was significantly lower than that in the control group(66.67%)(P=0.008).3.There were 820 differentially expressed mRNAs(338 up-regulated and 482 down-regulated).The genes are enriched in the HIF-1 signaling pathway,neuroactive ligand receptor interaction(NLRI),Hippo signaling pathway and cell adhesion molecules(CAM).Through GO enrichment and KEGG pathway analysis,obtain 12 differential candidate genes affecting endometrial receptivity:SDC1,LGR4,IL15,TLR4,TIMP2,WNT4,GSTM5,MSX2,UPK3BL1,MMP11,ECM1,and HOXA11.The candidate genes were verified by qPCR with enlarged samples,and the results were consistent with the results of sequencing samples.4.By consulting the literatures,the gene SDC1 which may affect endometrial blood flow.The expression of Syndecan 1(SDC1)in RIF group was significantly higher than that in the control group(P<0.05).5.Pearson linear correlation analysis showed that the expression of SDC1 was positively correlated with the sum of S/D of bilateral uterine arteries in RIF group(r=0.859,P<0.001).ConclusionThere were significantly differentially expressed mRNA in the endometrium of RIF patients compared with normal fertile women,SDC1 may lead to decreased receptivity by affecting endometrial blood flow in patients with RIF.Part Ⅲ Construction of ceRNA regulatory networks related to endometrial receptivity and study on the interaction mechanismObjectiveRNA-seq technique was used to test the ncRNA expression profile that regulatory target genes and to construct the ceRNA regulatory network of endometrial receptivity in RIF patients,and its targeted regulation was verified by endometrial cell experiments.In order to explore the molecular mechanism that influence endometrial receptivity in RIF patients and improve their pregnancy outcome.Methods1.Same as the sequencing samples in part II,using RNA-seq results,to screen the differential expression profiles of ncRNA including long non-coding RNA(lncRNA),circular RNA(circRNA)and miRNA.Then performing bioinformatics analysis that based on the high-throughput sequencing results and using gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)to perform function enrichment analysis for differentially expressed ncRNA.2.Some differentially expressed ncRNA were verified by qPCR in 15 RIF patients and 15 control patients.3.According to the transcriptome sequencing results,the ceRNA regulatory network was constructed.4.Human endometrial epithelium cells(hEECs)were cultured in vitro,was used silencing and overexpression techniques,qPCR and Western blotting(Western blot)experiments were used to verify the regulatory relationship between circRNAmiRNA-mRNA.5.The targeting relationship between circ1621 and miR-210-5p and between miR-210-5p and SDC1 in the regulatory network were further verified by the dual luciferase system.Results1.Compared with the control group,36 miRNAs were found to be differentially expressed(25 up-regulated and 11 down-regulated),1542 lncRNA were found to be differentially expressed(702 up-regulated and 840 down-regulated),131 circRNA were found to be differentially expressed(75 up-regulated and 56 down-regulated).Most of the target genes are enriched in cellular metabolism,protein kinase activities,intracellular transport biological processes and are mainly involved in RAP1 signaling pathway,RAS signaling pathway,and focal adhesion signaling pathways and pinocytosis signaling pathways.2.Differentially expressed ncRNAs were verified by qPCR with enlarged samples,and the results were consistent with the results of sequencing samples.3.The ceRNA regulatory network analysis showed that miR-210-5p was a central regulator and predicted its target genes SDC1.RPL4 and circ1621 might be"sponges" to absorb miR-210-5p and to participate in the regulation of SDC1 transcription or expression.4.qPCR and Western Blot results showed that overexpression of circ1621 in HEECs significantly decrease the expression of miR-210-5p,while silencing circ1621 significantly increased its expression.At the same time,miR-210-5p mimics transfected by HEECs could significantly inhibit the transcription and protein expression of SDC1.While the transcription and protein expression level of SDC1 were significantly increased after miR-210-5p inhibitor transfected into HESCs.5.Dual-luciferase experiment showed that miR-210-5p could bind target sequences of circ1621 and SDC1 3’UTR.Overexpression of miR-210-5p significantly reduced the activity of firefly luciferase of circ1621 and SDC1 3’UTR in dual-luciferase systems.Conclusioncirc1621,as a sponge adsorber of miR-210-5p,indirectly regulates the transcription or expression of SDC1 in endometrial epithelial cells,thus forming a ceRNA regulatory network to influence endometrial receptivity and participate in the pathogenesis of RIF.