Differential Expression And Identification Of MiRNAs In Plasma Of Patients With Biochemical Pregnancy After In Vitro Fertilization-Embryo Transfer

BackgroundBiochemical Pregnancy,a common early pregnant loss,occurs in about 8-30%of infertile women undergoing in vitro fertilization-embryo transfer(IVF-ET),is a big factor in preventing success in Assisted Reproductive Technology(ART).MicroRNAs(miRNAs)are non-coding miRNAs of 20-25 nt in length.MiRNAs are secreted by almost all kinds of cells in the human body.MiRNAs regulate the post-transcriptional level of gene expression by cutting or repressing targeted mRNAs.Although a large number of studies have reported that miRNAs can affect the embryo implantation from three important aspects:endometrial receptivity,embryo quality and endometrial-embryo synchronous development,however few researches have studied the predictive effect of miRNAs in peripheral blood on the outcome of embryo implantation during ART.Therefore,our study focus on whether miRNAs can be used as biomarkers for predicting the outcome of embryo implantation and Biochemical Pregnancy after IVF-ET.Part one:Differential expression and identification of miRNAs in plasma of patients with different pregnant outcomes after IVF-ETObjective:To investigate the differential expression of miRNAs in plasma during the window of implantation of infertile women with different pregnant outcomes after IVF-ET.Methods and subjects:In this part of the study,we selected 3 infertile patients who had failed embryo implantation after IVF-ET treatment in our center as the experimental group,the control group consisted of 3 infertile women with successful clinical pregnancies in the same period.RNA from all samples was extracted from plasma and exosomes collected during WOI,and miRNA sequencing was used to quantify the expression of multiple miRNAs.Then we validate the differential expressed miRNAs using GEO dataset.Furthermore,the enrichment signaling pathways of differential expressed miRNAs were identified by Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.Finally,we use Weighted Gene Co-Expression Network Analysis(WGCNA)to obtain a miRNA coexpression Network which is closely related to embryo implantation.Results:In the experimental group,13 miRNAs in plasma were differentially expressed during WOI compared with the control group,5 miRNAs(miR-150-5p,miR-150-3p,miR-149-5p,miR-146b-3p and miR-342-3p)were verified by GEO datasets.The expression of miRNAs in plasma of the experimental group was lower than that of the control group.KEGG pathway enrichment analysis showed that the pathways of differential miRNAs were closely related to embryo implantation.Finally,a miRNA co-expression network was constructed by WGCNA analysis.Conclusions:Failure of embryo implantation in infertile women after IVF-ET is associated with several miRNAs differentially expressed in plasma and exosome during WOI.Part Two:Time-dependent expression and identification of miRNAs in plasma of patients with Biochemical Pregnancy after IVF-ETObjective:To investigate the differential expressed miRNAs in peripheral plasma of infertile women with Biochemical Pregnancy after IVF-ET at different implanting time points.Methods and subjects:In this part of the study,we selected 20 patients who developed biochemical and clinical pregnancies after IVF-ET,peripheral plasma was collected at different planting time points(D0,D11)in each patient,and set up before and after control groups.RNA from all samples was extracted from the plasma collected by D0 and D11 in each patient.MiRNA sequencing was used to quantify miRNAs expression in all samples.We collected peripheral plasma from 24 patients with biochemical and clinical pregnancies by using the propensity score matching method at different implanting time points(D0,D11),RT-qPCR was used to quantify the differential miRNAs.Then,Gene Ontology and KEGG were used to analyze the biological enrichment function and related pathways of differentially expressed miRNAs.Results:There were significant differences in the expression of miRNAs in plasma at different planting time points in biochemical pregnant group and clinical pregnant group.In biochemical pregnant group,the expression level of 18 miRNAs and 25 miRNAs on the 11th day of embryo transfer was significantly lower and higher than that on the first day of embryo transfer.In clinical pregnant group,the expression level of 9 miRNAs and 8 miRNAs on the 11th day of embryo transfer was significantly lower and higher than that on the first day of embryo transfer.We screened 5 miRNAs(miR-9-5p,miR-133a-3p,miR-150-5p,miR-146b-5p and miR-342-3p)which were closely related to embryo implantation and expressed differentially only at different planting time points in patients with Biochemical Pregnancy,and the differential expressions were confirmed by RT-qPCR in another 24 samples.The expression levels of miR-9-5p,miR-150-5p,miR-146b-5p and miR-342-3p on the 11th day of embryo transfer were significantly higher than that on the first day,and the expression level of miR-133a-3p was significantly lower than that on the first day.GO and KEGG pathway enrichment analysis showed that 5 differential miRNAs target genes were closely related to embryo implantation.Conclusion:Biochemical Pregnancy in infertile women after IVF-ET was associated with 5 miRNAs(miR-9-5p,miR-133a-3p,miR-150-5p,miR-146b-5p and miR-342-3p)which were up-regulated in plasma on the 11th day of embryo transfer.Part Three:The effects of miR-9-5p on proliferation,migration and invasion of trophoblastObjective:To investigate the effects of miR-9-5p on the proliferation,migration and invasion of trophoblast.Methods and subjects:In this part of the study,we constructed over-expressed and lower-expressed miR-9-5p trophoblast.The effects of miR-9-5p on proliferation,migration and invasion of HTR-8/Svneo cells were observed by CCK-8,wound-healing and Transwell assays.Results:The abilities of proliferation,migration and invasion of trophoblast in miR-9-5p over-expressed group and lower-expressed group were significantly different from those in control groups.MiR-9-5p over-expressed group significantly inhibited the abilities of proliferation,migration and invasion of HTR-8/Svneo cells,miR-9-5p lower-expressed group significantly enhanced the abilities of proliferation,migration and invasion of HTR-8/Svneo cells.Conclusion:MiR-9-5p might affect the outcome of embryo implantation by regulating the abilities of proliferation,migration and invasion of trophoblast.Part Four:MiR-9-5p inhibits the proliferation,migration and invasion of trophoblast by targeting SHHObjective:To investigate the role of SHH in miR-9-5p inhibiting the abilities of proliferation,migration and invasion of trophoblast.Methods and subjects:In this part of the study,we chose HTR-8/Svneo cells as the subject,dual luciferase reporter assay was used to detect whether miR-9-5p could inhibit the expression of SHH.Western Blot and RT-qPCR assays were used to verify the results.Subsequently,we constructed HTR-8/Svneo cells with low expression of miR-9-5p and SHH.The effects of SHH in miR-9-5p inhibiting the abilities of proliferation,migration and invasion of trophoblast were observed by CCK-8,wound-healing and Transwell assays.Results:When miR-9-5p was over-expressed and lower-expressed,there was a significant difference in SHH expression in HTR-8/Svneo cells.MiR-9-5p could inhibit SHH expression in HTR-8/Svneo cells.The abilities of proliferation,migration and invasion of trophoblast in SHH lower-expressed group was significantly different from that in control group.When the expression of SHH was inhibited,miR-9-5p lower-expressed group could no longer promote the proliferation,migration and invasion of trophoblast.Conclusion:MiR-9-5p inhibits the abilities of proliferation,migration and invasion of trophoblast by targeting SHH.