Single Cell Proteomics Profiling Revealing that Embryo Secreted TNF-α play Critical Role during Embryo Implantation to Endometrium

Abstract Problem: Although it has long been known that endometrium secreted cytokines play critical role during embryo implantation, whether cytokines secreted from embryo is relevant to embryo quality and is actively involved in embryo attachment remain unclear.Method of study: The concentration of cytokines in embryo culture medium were tested by a new developed high-sensitive single cell proteomic platform, compared with embryo quality and clinical outcome. The effect of TNF-α on embryo and endometrium Ishikawa cell was investigated using immunofluorescence staining, CCK- 8 assay, TUNEL staining, and RT-qPCR reaction.Results: Of the 10 cytokines measured, only TNF-α concentration is significantly higher in group of embryo implantation failure. Immunofluorescence staining showed that the expression of TNF-α was unevenly distributed in blastocysts, and the expression level was significantly correlated with blastocysts inner cell mass (ICM) quality score. Adding TNF-α caused significant increase of apoptotic cells, which could be inhibited by TNF-α receptor blocker entanecept (ETA). Gene profiling showed that adding TNF-α lead to increased expression of TNFR1 and apoptosis related genes, as well as ion channel genes, including CFTR, ENaCA, AQP3 and CRISP2, and the increase can be inhibited by ETA. Conclusion: In conclusion, our result showed that higher TNF-α level is associated with implantation failure through activation of TNF-α receptor, and TNF-α maybe an independent predictor for pre-transfer assessment of the embryo development potential in IVF patients.