Investigating functional roles of conserved microRNAs in the endometrium of placental mammals

Early pregnancy events like implantation strategies are diverse amongst eutherian mammals. Some molecular signals between embryo and endometrium during the peri-implantation period of pregnancy are conserved e.g., progesterone, whilst others are species-specific, such as those involved in maternal recognition of pregnancy. MicroRNAs are one key set of molecules which may be involved in conservation or diversification of early pregnancy events. Recently, our lab identified a set of microRNAs which arose concurrent with eutherian mammals and were subsequently never lost. Three (miR-340-5p, -542-3p and -671-5p) are differentially expressed following progesterone treatment. We hypothesised that these miRNAs modify endometrial protein expression, facilitating receptivity to implantation. To investigate mechanistically, Ishikawa cells (n=3) were transfected with miRNA mimics, inhibitors, and controls for 48hrs. Protein was extracted and TMT mass spectrometry performed. Statistical significance was calculated using paired T- Tests and Benjamini-Hochberg correction. Differential expression of miRNAs significantly altered many endometrial epithelial proteins. Functions significantly enriched include metabolism, localisation, and endoplasmic reticulum stress - involved in decidualisation, important to implantation. Many altered proteins were associated with proliferation, which may be important for establishing receptivity. To investigate functionally, embryo-like spheroids (trophoblast cell line) were added to transfected Ishikawa cells to determine spheroid attachment. MiR-542-3p significantly reduced attachment, and some miRNA combinations also altered embryo attachment. We propose these miRNAs are involved in altering protein abundance, having implications for endometrial receptivity to embryo implantation. Finally, 2 novel 3D models of the endometrium were developed. A 3D multicellular human endometrial model using Alvetex scaffolds - where miRNAs of interest could be differentially expressed - showed reduced proliferation following overexpression of miR-340- 5p, miR-542-3p and miR-671-5p. Bovine endometrial organoids derived from endometrial glands were shown, by RNASeq, to be progesterone responsive. These models provide tools to investigate endometrial receptivity and implantation, allowing species comparison to understand the diversity in reproductive strategies.