Micrornas At The Embryo-Maternal Interface Have Effects On Endometrial Cell Proliferation

Abstract Study question Whether cell-free microRNAs are part of the embryo-maternal interactome with possible effects on processes related to implantation. Summary answer Specific microRNAs cause major transcriptomic changes in uterine cells and alter cellular proliferation which is pivotal for the implantation of the incoming embryo. What is known already A plethora of molecules present at the uterine luminal fluid including cytokines, growth factors, and adhesion proteins are involved in implantation. However little is known about the roles of extracellular microRNAs (miRNAs) at the embryo-maternal interface. MicroRNAs act mainly as gene regulators and a single miRNA can have thousands of gene targets. MiRNAs are released by blastocysts and uterine cells internalize miRNAs that are present in the extracellular environment. To date there is limited evidence on the molecular actions of these cell-free miRNAs and their effects on processes related to implantation. Study design, size, duration Human endometrial stromal cells (hESCs) were cultured in complete growth medium for 8 consecutive passages. A miRNA mimic experiment in 6 replications was carried out in which endometrial cells were transfected with miR–371a. Gene changes in the hESCs were studied with genome-wide microarray technology and the results were validated in vitro with PCR. Participants/materials, setting, methods The miR–371a mimic was transfected in hESCs using a Lipofectamine reagent. RNA was extracted and the samples were processed with microarray Clariom™ Human Assays using Affymetrix®. The transcriptomic profiles between transfected and control cells were compared using Partek®. Differentially expressed genes were considered significant when p-value was <0.05, false discovery rate, FDR ≤ 0.05 with Benjamini-Hochberg correction, and fold-change of > 1.5 or < –1.5. Functional enrichment analysis was carried out using WebGestalt and Enrichr. Main results and the role of chance MiR–371a altered the expression of 4.760 genes in endometrial cells (p < 0.05, fold-change 1.5). A total of 16 biological processes, 23 cellular components, and 24 molecular pathways were disrupted by this miRNA. WebGestalt analysis found 159 enriched categories including increase of negative cell cycle regulation, apoptosis signalling, and cycle arrest and decreased cell proliferation. Cell cycle was one of the most affected pathways in KEGG analysis with at least 54 genes dysregulated. Mammalian phenotype ontology analysis found 4.818 affected phenotypes, including decreased cell proliferation (58 genes), increased apoptosis (48 genes) and abnormal cell cycle (41 genes). Key-genes of endometrial proliferation at the window of implantation were significantly downregulated, including: CD44, PGR; IGFs, FGFs, and HAND2. Moreover, at least 25% decreased hESCs proliferation was verified in vitro after transfection. These negative effects of miR–371a in cell cycle could disturb implantation of the incoming embryo, since intense cellular proliferation is necessary for establishment of the implantation site. Limitations, reasons for caution These results are limited to miR–371a actions on human endometrial stromal cells. It is likely that miRNAs, cytokines, growth factors, and other molecules form complex regulatory networks that control uterine receptivity and embryo implantation. Wider implications of the findings: MiRNAs are important mediators of the embryo-maternal interactome. Their actions are likely involved in implantation-related processes including inter-cellular communication, decidualization, adhesion, invasion, and establishment of the implantation site. Embryo-secreted miRNAs change the transcriptome of the neighboring endometrial cells with effects on implantation-related pathways, serving thus secretory functions. Trial registration number N/A