Single-cell Transcriptome Analysis Uncovers The Molecular And Cellular Characteristics Of Thin Endometrium

Objective: Investigate the application of single-cell RNA sequencing to explore the differences in microenvironment associated with development and receptivity between thin endometrium(TE)and normal endometrium.Differential expression genes(DEGs)were analyzed involved in the biological processes and signaling pathways.Method: A pair of volunteers had been recruited.An infertility patient volunteer with thin endometrium and recurrent implantation failure served as the TE specimen source.Another volunteer who able to give natural birth with normal thick endometrium served as normal endometrium specimen source.We collected the endometrium of two women at late proliferative phase(1-2 days before ovulation)and middle secretory phase(6-7 days after ovulation),then to perform single-cell RNA sequencing.Sex hormones on the day of endometrial extraction were measured to confirm the endometrial phase,the thickness of endometrium was measured by ultrasound.Meanwhile,the endometrium samples were checked by conventional pathological examination and electron microscopic ultrastructure examination too.These results of two volunteers were compared and analyed,and the single-cell transcriptome data from the endometrium samples were filtered,dimensionality reduced,clustering and difference analyzed.Results: Compared with normal endometrium,TE patient has the following characteristics: 1.The thickness of endometrium measured by ultrasound is <6mm at both late proferative phase and middle secretory phase.2.Pathological examination showed that the number and shape of glands was reduced and distorted in the late proliferative phase,but the number and shape of glands was normal in middle secretory.3.In middle secretory phase,scanning electron microscope showed that the quantity and quality of mature pinopods were obviously decreased;transmission electron microscope showed that the secretion function of TE sample was poor.Analysis of single-cell RNA sequencing data obtained the following results: 1.The endometrium was identified to contain seven cell types: epithelial cells,stromal cells,endothelial cells,lymphocytes,monocytes,ciliated epithelial cells and mix cells.2.Compared with normal endometrium,TE presented higher proportion of epithelial cells and lower proportion of stromal cells at middle secretory phase.3.According to screening criteria,in late proliferative phase,TE epithelial cells have 97 up-regulated DEGs,102 down-regulated DEGs,stromal cells have122 up-regulated DEGs,96 down-regulated DEGs,in middle secretory phase,TE epithelial cells have 386 up-regulated DEGs,475 down-regulated DEGs,stromal cells have 96 up-regulated DEGs,110 down-regulated DEGs.Genes PAX8,DPP4 involved epithelial cells proliferation were up-regulated,genes COL1A1,COL1A2,COL3A1,LUM,IGF1 related to stromal cells proliferation were down-regualted.The expression of estrogen receptors 1(ESR1)and progesterone receptors(PGR)in stromal and epithelial cells declined in TE’s mid-secretory phase were found.4.Biological process of differential gene: down-regulated DEGs enriched in fibrillar collagen trimer and banded collagen fibril,which suggested the synthesis of collagen fibers was affected in stromal cells of TE.5.Differential genes of signaling pathway: some down-regulated genes enriched in ECM-receptor interaction and WNT signaling pathway associated with endometrium receptivity in middle secretory phase.Conclusion:1.In this study,we provided the single cell transcriptome atlas of TE for the first time.these atlas provide single cell m RNA information at two key point phases of proliferation and secretion.Seven different cell types had been identified in the atlas for further study.2.Compared with normal endometrium,in middle secretory phase,the proportion of epithelial cells increased significantly while that of stromal cells decreased in TE patients.GO analysis showed that DEGs was enriched in ribosomal protein,energy synthesis and metabolism mechanism,and biological process of fibrocollagen,some down-regulated DEGs enriched in ECM-receptor interactions and WNT signaling pathways,which can regulate endometrial receptivity.The estrogen receptor and progesterone receptor and genes related to TE receptivity were down-regulated.3.These findings were potentially valuable for understanding the critical molecular mechanism of TE on its pathgenesis and receptivity injury.However,this is one typical case control study.The data generalization is limited,and further repeat and validation are needed.