The Expression Patterns Of MiRNAs In Sheep Conceptus And Endometrium And Its Role In Maternal-fetal Crosstalk During Peri-implantation Stage

Crosstalk between sheep conceptus and endometrium during the implantation window is a complex and not well-understood process. The aims of this study were （ⅰ） to indentify novel miRNAs in sheep conceptus and endometrium with the transcriptomic profile of small RNA data, （ⅱ） to evaluate and compare the global gene expression profile in conceptus and endometrium at Day 17 and （ⅲ） try to identify potential microRNA roles in the early dialogues between maternal and embryonic cells during the implantation window. （Ⅳ） to select potential critical marker genes of sucessful implantation by comparing with failed implantation.Conceptus and endometrial biopsies from normal responder were performed on the day 17. Digital gene expression （DGE） analysis was used to identify specific small RNA and gene expression profiles during implantation window in both endometrium and conceptus. MiRDeep2 detected 110 known miRNAs and predicted 405 potential novel miRNAs that passed the relatively stringent score cut-off of 4. Removal of loci matching other RNA genes or genomic repeats reduced this list to 376 candidate novel miRNAs. Moreover, editing rate of all miRNAs were compared between conceptus and endometrium. Results show that editing rate at position 9 of endometrium is significantly higher than conceptus and editing rate of position 12 have reverse concequece. However, editing rate of sepicific miRNAs have various forms and editing rate of intercaruncle always higher than other groups.Then we clustered and compared the conceptus genes profiles with the endometrial （C and IC） expression pattern both displaying different potentials for implantation. Up-regulated genes in conceptus are significantly enriched in cell cycle, energy metablism and transport and so on. While up regulated genes in caruncle are enriched in cell adhesion, immue response, and cell development etc. Plus, up regulated genes in intercaruncle invovles in GO terms like cytokine production, cell migration and so on. In addition, several adhesion molecules and extracellular matrix genes （Mcam, Itgae and Lama1） were also over-expressed in the conceptus, while others （Alcam, Ceacaml, Pecaml, Itgb8 and Lama2） were restricted to the receptive endometrium.Moreover, integration analysis between small RNAome and transcripome were conducted. Comparison with mRNA expression profiles reveals potential mRNA targets of these regulated miRNAs, which indicates these regulated miRNAs might play roles during peri-implantation period. miRBase、miranda and RNAhybrid were used to predict targets of differentially expressed MicroRNA. Then overlapping genes in targets set and DEGs set were chosen to enrich functional analysis. A mount of candidate miRNA were selected which may involves in the embryo-endometrium interaction. For example, OAR-MIR-432 were down regulated in endomtrium. and up regulated targets like Fermt3、Pard6a、Stat2 and Tspan31, which involves in cell adhesion, migration and proliferation.. Furthermore, differentially expressed miRNA targets and differentially expressed protein were analyzed together. Some miRNA were found only inhibiting protein expression and do not affects mRNA. For instance, OAR1₁2343 were up regulated in conceptus and its targets COL7A1’s mRNA showed no change. But its protein level were down regulated, which indicates OAR1₁2343 might directly inhibits protein translated process.Finally, An increasing number of reports indicate that in vitro fertilization （IVF） is highly associated with long term side effects on embryonic and postnatal development, and can sometimes result in embryonic implant failure. While high throughput gene expression analysis has been used to explore the mechanisms of IVF-induced side effects on embryonic development, little is known about the effects of IVF on the conceptus and endometrium interaction during the peri-implantation period. Using the sheep as a model, we performed a comparative transcriptome analysis between in vivo （IVO; in vivo fertilized followed by further development in the uterus） and in vitro produced （IVP; IVF with further culture in the incubator） conceptuses, and the caruncular and intercaruncular areas of the ovine endometrium. We identified several differentially expressed genes （DEGs） between the IVO and IVP groups on day 17, when the adhesion between the trophoblast and the uterine luminal epithelium begins in sheep. By performing gene ontology enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analysis, we found that in the conceptus, DEGs were associated mainly with functions relating to cell binding and cell cycle. In the endometrial caruncular area, DEGs were involved in cell adhesion/migration and apoptosis; in the intercaruncular area, DEGs were significantly enriched in pathways of signal transduction and transport. These DEGs are potential candidates for further exploring the mechanism of IVF/IVP-induced embryonic implant failure that occurs due to a loss of interaction between the conceptus and endometrium during the peri-implantation period. In addition, in order to select potential critical marker genes of sucessful implantation, we compared it with failed implantation under IVF and IVO condition. Common most up-regulated and down-regulated genes were chosen to be potential marker genes.The present study revealed many novel miRNAs expressed in conceptus and endometrium. Our study also shows that integrins and adhesion molecules are expressed in the understanding of the mechanisms involved in the early dialogue between conceptus and endometrium during implantation. Potential roles of miRNAs in implantation were also indicated. Such results should be confirmed by further studies.