O-061 Single cell atlas of small RNAs in the human preimplantation embryo reveals the miRNA dynamics of lineage segregation

Abstract

Abstract Study question How does the small non-coding RNA (sncRNA) expression profile of the human embryo change from day three (E3) to seven (E7) of preimplantation development? Summary answer From E3 to E7, micro-RNA expression becomes more dynamic, targeting genes important for lineage segregation. What is known already The drivers of lineage specification [SP1] in the human embryo remain unknown. SncRNAs regulate gene expression in most biological systems studied to date, with critical roles in cell development, differentiation, and disease. The most well-studied class of sncRNA, micro-RNAs (miRNAs), are required for embryo development to the peri-implantation stage in animal models, and their expression regulates embryonic stem cell differentiation in vitro. Study design, size, duration Eighty-seven E3-E5 embryos donated for research to the CReATe Fertility Centre (Veritas IRB#16580) between 2002 and 2021 were thawed and cultured to E3-E7. A total of 1279 cells were profiled for sncRNAs. Of these, 463 had known gender and 189 cells were co-sequenced for their mRNA complement. Participants/materials, setting, methods Good-quality embryos by morphology were enzymatically and mechanically dissociated into single cells and subjected to small and large RNA sequencing, as outlined in Petropoulos et al. 2016 and Hagemann-Jensen et al. 2018. Gene expression was analyzed with the ‘Seurat’ package. MiRNA targeting and pathway analysis was performed with Mienturnet. Main results and the role of chance We identified the complete complement of small RNAs present in the human preimplantation embryo, with piRNAs being the most abundant in E3 and miRNAs increasing in diversity and abundance to E7. Uniform Manifold Approximation and Projection (UMAP) analysis revealed progression with developmental stage and lineage. Split-cell co-sequencing identified canonical transcriptional markers of preimplantation development, allowing the classification of the small RNA profiles of 16-cell, early blast, inner cell mass (ICM), mural TE, and polar TE populations. Enriched miRNAs in the E5 ICM included miR-302b-5p and miR-302c-3p, which have known functions in stem cell programming. Conversely, TE cells were enriched for miR-519b-3p, miR-519c-3p, and miR-516a-5p, all members of the imprinted primate-specific microRNA gene cluster (C19MC), which is required for placenta formation. Targeting analysis identified miRNA-gene networks involving the Hedgehog, Hippo, and FoxO signalling pathways, all of which are developmentally significant. Limitations, reasons for caution This study was performed on embryos from a single center – embryo handling and culture conditions may influence sncRNA expression. Furthermore, the ability of these embryos to implant was unknown, therefore we likely profiled some non-viable embryos. Wider implications of the findings The epigenomic changes which drive lineage segregation are poorly understood, and there are vast differences in embryonic development between conventional model systems and humans. We report the first sncRNA profile of single cells between human E3 and E7, providing a resource for further exploration of their role in preimplantation development. Trial registration number not applicable