Human embryo-derived extracellular vesicles can modify the endometrial cell transcriptomic profile upon incorporation: insights for embryo-maternal cross-talk.

Introduction: Successful pregnancy depends on efficient implantation, which relies on a productive embryo-endometrium cross-talk defined as a well-orchestrated dialogue between a 'competent' embryo and a receptive endometrium. A competent embryo releases specific paracrine effectors enhancing the differentiation of endometrial cells (ECs) into decidual cells. In 2017 Giacomini and colleagues, demonstrated for the first time that human embryos cultured in vitro release extracellular vesicles (EVs) with a specific molecular profile. There is plenty of evidence that EV-shuttled biomolecules can profoundly affect the phenotype and activity of the receiving cell. Objectives: The aim of our study was to validate the hypothesis that human preimplantation embryos are able to release EVs to supply endometrial cells with a functional transcriptomic package important for embryonic-maternal cross-talk during the window of implantation. Methods: RNA derived from extracellular vesicles of both day 3 embryos (D3-EVs) and day 5 embryos (D5EVs) was amplified and sequenced on an Illumina NextSeq 500 System. After EVs staining with Vybrant¿ DiO cell-labeling solution, a monolayer of primary endometrial cells (pECs) were co-cultured with labeled embryo-derived EVs to validate the uptake of human embryo-derived EVs by the endometrial counterpart. RNA-seq analysis was performed on pECs treated for 48 hours with embryo-derived EVs (n=3). The RNA-Seq library was generated and sequenced using an Illumina HiSeq 2000. Nanoparticle Tracking Analysis (NTA) was used to evaluate size (nm) and concentration (particles/ml) of EVs derived from high and low quality human embryos. Results: NLRP2 and PDSS1 were the top abundant RNAs in D3EVs and D5EVs respectively. Treatment of pECs with D3EVs had little impact on their transcription, while D5EVs up-regulated numerous critical genes including SEC63, ARNT2 and IGFBP7. D3 low-quality embryos (n=13) released significantly more (p=0.007) EVs in spent medium compared to D3 high-quality embryos (n=7). D5 low-quality embryos (n=15) released EVs with a greater size (p=0.0005) when compared to EVs released from D5 high-quality embryos (n=12). Conclusions: RNAs over-represented in embryo-derived EVs support an EV-mediated transcriptomic contribution to the mechanisms underlying maternal tolerance and endometrial receptivity. The abundance of NLRP2 in D3EVs may reflect its well-known roles in embryogenesis and maintenance of pregnancy. Delivery of D5EV-RNAs may regulate mitochondrial activity and cell division in pECs to establish polyploidy, a hallmark of decidualization. The top up-regulated genes in pECs treated with D5EVs suggest their possible contribution to the mechanisms of implantation. These findings confirm the existence of a novel functional embryo-endometrial cross-talk via EVs critical for the establishment of implantation, which warrants further investigation.