Up-regulated MiR-218-5p In Extracellular Vesicles Derived From Endometrium Of Patients With Recurrent Implantation Failure Affect Pre-implantation Embryo Development

Objective: To explore the effect of miR-218-5p in extracellular vesicles（EVs）derived from endometrium of patients with recurrent implantation failure on preimplantation embryo development and investigate the potential mechanism.Methods: miR-218-5p,miR-1246,miR-1307-3p,miR-433-3p,and miR-6131 agomir were co-cultured with the mouse embryo in vitro respectively.The blastocyst rate and hatching rate were calculated at E4.5 and E5.5.The RNA-Seq library of the miR-218-5p treated morula was prepared following the Illumina library preparation protocol and then loaded on the Illumina Hiseq platform for PE150 sequencing.Then the Gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis were constructed based on the RNA seq data.The number of blastomeres from the miR-218-5p and miR-NC group was calculated based on the 4’,6-diamidino-2-phenylindole（DAPI）stained nucleus.The proliferation of embryos was detected by using an Ed U assay.The terminal deoxynucleotidyl transferase d UTP nick end labeling（TUNEL）assay was used to detect the apoptosis of embryos in miR-218-5p and miR-NC groups.The intracellular ROS levels and activity of mitochondria were detected by using ROS assay kit and Enhanced Mitochondrial Membrane Potential Assay Kit respectively.The expression Cdx2 was detected with immunofluorescence.The EVs derived from Ishikawa cells were extracted and miR-218-5p mimics subsequently engineered into EVs.The characteristics of all EVs were identified with Western Blot,nanoparticle tracking analysis（NTA）,and transmission electron microscopy（TEM）.Then the engineered EVs were co-cultured with mouse embryos in vitro.Results: The blastocyst rate and hatching rate of the embryos were significantly reduced by miR-218-5p,miR-1246,miR-1307-3p,or miR-433-3p,while no reduction was observed in miR-6131 group.The development of embryos was arrested at morula stage with miR-218-5p agomir treatment.The differential expressed genes（DEGs）were identified,and 445/474 genes were up/down-regulated respectively in the miR-218-5p group compared with the NC group.KEGG enrichment analysis suggested that the DEGs were enriched in Hippo signal pathway.The number of total or Ed U-positive blastomere were both reduced in miR-218-5p treated group.There were no significant differences in the level of apoptosis,ROS or mitochondrial membrane potential,between miR-218-5p and miR-NC treated groups.The percentage of Cdx2 positive nuclei in the morula was much lower in miR-218-5p treated group than that in miR-NC treated group（35.2% v.s.60.8%）.Engineered EVs loaded with miR-218-5p were presented with TSG101 positive and restricted particle size in114.8～162.8 nm with a visible lipid bilayer.The engineered EVs were uptaken by blastomeres,and impaired the blastocyst formation.Conclusions: The blastocyst rate and hatching rate of mouse embryos were reduced by miR-218-5p,miR-1246,miR-1307-3p,and miR-433-3p,which were considered as the effective ingredients of EVs derived from the endometrium of patients with recurrent implantation failure（RIF）.Among them,miR-218-5p led to an arrested development of embryos at morula stage.The expression of Cdx2 was decreased in the miR-218-5p treated embryos at 8-16 cell stage.Engineered EVs loaded with miR-218-5p could be uptake by the embryos and impair embryo development.Part Ⅰ: The micro RNA in extracellular vesicles derived from the endometrium of patients with recurrent implantation failure affects preimplantation embryo developmentObjective: To identify the effective micro RNAs in EVs derived from the endometrium of patients with recurrent implantation failure（RIF）,and explore the effect of these micro RNAs on preimplantation embryosMethods: The expression of micro RNAs in mouse embryos at different stages was detected with RT-PCR.The embryos were co-culture with miR-218-5p,miR-1246,miR-1307-3p,miR-433-3p,and miR-6131 agomir respectively in vitro,and the expression of micro RNA was detected at E4.5 by using q RT-PCR.The blastocyst rate and hatching rate were calculated by the Chi-square tests.Results: The expression patterns of micro RNAs in the different stages of preimplantation embryos were distinct.The expression of miR-218-5p was slightly increased at the 4-cell stage followed by the highest increase in transcript abundance at the 8-cell stage and then a continuous decline in the morula and blastocyst stage.The blastocyst rates were 40%,16.7%,1.55%,and 24% after miR-218-5p,miR-1246,miR-1307-3p,miR-433-3p agomir treatment,respectively,which were significantly decreased.While there was no difference in blastocyst rate and hatching rate between miR-6131 and miR-NC groups.the most optimal scheme of miR-218-5p agomir treatment was 5n M at E2.0.The delay of compaction was presented in miR-218-5p embryos until E4.5,and embryos were arrested at the morula stage with the failure of blastocyte formation during prolonged cultivation.Conclusions: The blastocyst rate and hatching rate were impaired by miR-218-5p,miR-1246,miR-1307-3p,and miR-433-3p.Among them,miR-218-5p arrested the development of embryos at morula stage.Part Ⅱ: miR-218-5p modulates the m RNA transcripts of morula in miceObjective: To clarify the major mechanism of morula arrest in miR-218-5p treated embryos and identify the differential expressed genes（DEGs）of compacted morula between miR-218-5p and miR-NC treated group.Methods: Seven to ten compacted embryos at E3.25 from at least 3 different mice were constituted as one sample of miR-218-5p or miR-NC groups respectively.The RNA-Seq library was prepared and loaded on Illumina Hiseq platform for Next-generation sequencing（NGS）.The expression of typical DEGs was detected by q RT-PCR.The Gene Ontology Consortium（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis were conducted.Transcription factors（TFs）were analyzed.The potential target genes of miR-218-5p were analyzed by using bioinformatics techniques.Results: Nine hundred and nineteen DEGs were identified,445 and 474 genes were up/down-regulated respectively in the miR-218-5p group morula compared with the NC agomir treatment group.The enrichment analysis indicated that the DEGs were enriched in Rap1 signal pathway,focal adhesion,regulation of actin cytoskeleton,NOD like receptor signaling pathway and Hippo signal pathway.Nineteen up-regulated and 26 down-regulated TFs were identified.The results of bioinformatics analysis of target genes identified 14 highly suspected miR-218-5p target genes,which were enriched in cell differentiation,epidermal Growth Factor/Epidermal Growth Factor Receptor（EGF/EGFR）,Interleukin-3（IL-3）,and EPH-Ephrin signaling pathway.Conclusions: The m RNA transcripts of miR-218-5p treated morula were significantly different from that of the control group.The bioinformatics analysis suggested several signaling pathway may contribute to the failure of blastocyte formation.Part III: miR-218-5p impaired the lineage specification of the arrested mouse embryosObjective: To explore the possible mechanism of morula arrest caused by miR-218-5p.Methods: The expression of Cyclin D1,Bax,Cdh1,Sox2,Nanog,Tead4,Cdx2,Gata3 and Yap1 m RNA in miR-218-5p treated morula were detected with q RT-PCR.The nuclei of blastomere were dyed with DAPI staining dye solution,and the total cell number of the miR-218-5p treated embryos were calculated.An Ed U and a TUNEL assay were used to test the proliferation and apoptosis of blastomere,respectively.A ROS and a JC-1 assay were used to measure the level of reactive oxygen and mitochondrial membrane potential.The expression of the Cdx2 protein was detected by immunofluorescence.Results: The m RNA expression of Sox2,Tead4,and Yap1 in morula decreased after miR-218-5p treatment.miR-218-5p inhibited the proliferation of blastomeres with no effect on apoptosis,ROS,or mitochondrial membrane potential.A ratio of 35.2% nuclei of miR-218-5p treated morula were Cdx2 positive,which was significantly lower than that in miR-NC group（60.8%）.Conclusions: miR-218-5p delayed the proliferation of embryos and impaired the lineage specification of the arrested embryos.Part IV: Effects of engineered extracellular vesicles on mouse preimplantation embryo developmentObjective: To explore the effect of engineered EVs on embryonic development and provide experimental support for the clinical transformation of EVs.Methods: The EVs derived from Ishikawa were extracted by using ultracentrifugation.The identification of EVs was conducted with Western Blot,nanoparticle tracking analysis（NTA）,and transmission electron microscopy（TEM）.The EVs were loaded with miR-218-5p by using Exo-Fect^TM Exosome Transfection Reagent,followed by ultracentrifugation.The engineered EVs were co-cultured with mouse embryos in vitro.q RT-PCR was used to detect the expression of miR-218-5p in the engineered EVs and the blastocyte.Results: The EVs from Ishikawa cells indicated TSG101 positive and CALNEXIN negative,which accord with the typical characteristics of EVs.The mean particle size of EVs was 114.8 nm in diameter and the concentration was1.6×10¹¹ Particles / m L.The structure of the lipid bilayer was visible.The engineered EVs also presented as typical EVs with an average particle size of 162.8nm in diameter and visible lipid bilayer.The expression of miR-218-5p was significantly up-regulated in the EVs after engineering.The FAM fluorescence was detected in lastomeres after co-culture embryos with the engineered EVs,which indicated that the engineered EVs could be absorbed by embryos,and impaired embryo development.Conclusions: The engineered extracellular vesicles loaded with miR-218-5p could be absorbed by the embryo and affected embryo development.