Abstract: Objective： To study the spatial expression of trophectoderm cells in human embryonic preantral blastocysts. Methods: The study used Gardner score 5AA blastocysts harvested on day 6 after fertilization from assisted reproductive technology. Microcapsules were used to separate trophectoderm cells from the epidermal cells. Single-cell sequencing was performed. P ＜ 0.05 was calculated by unpaired t test, and the difference was 2 times. Here we determined, for the first time, global gene expression patterns in the polar/mural trophectoderm isolated from human blastocysts. Unsupervised hierarchical clustering analysis and gene ontology (GO) functional classification were performed using bioinformatics software. Differentially expressed genes were annotated by the Database for Annotation, Visualization and Integrated Discovery. Functions of differentially expressed genes were further annotated using encyclopedia of genes and genomes. Results: The results showed that there were up to 306 genes in the trophoblast cells and up to 75 genes in the trophoblast cells. Unsupervised cluster analysis of polar trophoblast cells and mural trophoblast cells were divided into two groups, belonging to different types and biological functions. Differences in gene function indicated that the biological functions of GO gene uptake genes were mainly transcription, energy metabolism, protein synthesis, transport, oxidative stress, ion transport, protein synthesis and transport, cell cycle regulation, actin growth, etc. They were mainly involved in ubiquitin-mediated protein hydrolysis, oxidative phosphorylation, Wnt signaling pathway, estrogen androgen metabolism and other signal pathways; wall trophoblast cells up-regulated gene GO biological function, which was mainly proteolytic metabolism, cell cycle arrest, apoptosis, activation of MAPK, carbohydrate transport, synaptic regulation, cell growth, calcium channel activation, positive B cell differentiation, T cell apoptosis and other biological functions, which were mainly involved in B cell receptor, T cell receptor, white blood cells crossendothelial transplantation, VEGF expression, gap connection, GnRH secretion, apoptosis and other signaling pathways. Conclusion: The gene expression of blastocysts trophectoderm is revealed from the spatial dimension, indicating that differentiation of polar and mural trophectoderm of blastocysts is accompanied by differences between the two cell lineages, and the polar and mural trophectoderms are coordinated with each other and the blastocyst hatching and embryo implantation processes are finely adjusted. Further data analysis is expected to find the endogenous molecular specificity of the regulation of embryo implantation.

Key words: Blastocysts, Polar trophectoderm, Mural trophectoderm, Gene , Single cell sequencing