AUTHOR=Wang Yuqiong , Wang Sheng , Qian Xilin , Kuai Yanrong , Xu Yang TITLE=The Inclusion Principles of Human Embryos in the WOW-Based Time-Lapse System: A Retrospective Cohort Study JOURNAL=Frontiers in Endocrinology VOLUME=Volume 12 - 2021 YEAR=2021 URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2021.549216 DOI=10.3389/fendo.2021.549216 ISSN=1664-2392 ABSTRACT=Time-lapse system (TLS) with well-of-the-well (WOW) dish, which allows individual identification and the possibility of autocrine and paracrine signaling between the group cultured embryos, has been widely used in clinic. However, there is a need to re-think the inclusion principles of human embryos in WOW-based TLS, especially for the Grade IV (G4) embryos, which are considered to potentially have detrimental effects on surrounding embryos. Here, we carried out a single-center, large-cohort, retrospective study, comprising 303 patients undergoing IVF (148 cases) and ICSI (155 cases), with a total of 3282 embryos, to compare embryonic development until the blastocyst stage in the group culture system with or without G4 embryos. Further, LC-MS/MS was used to analyze the G1-G4 embryo secretome to understand the influence of G4 embryos on the group culture microenvironment. We proved that the polyprokaryotic (PPN) embryos positively contribute to the development of the neighboring embryos through secretion of ILIAP, ITI-H4, and keratin. Existence of more than 1 G4 embryos showed a negative effect on the other embryos (p < 0.05). Moreover, G4 embryos were found to secrete KLKB1 and VTDB, which might harm the neighboring embryos. Thus, our study clarified that when embryos are subjected to group culture in the WOW-based TLS, the PPN-derived embryos need not be removed, and it is important to maintain the number of G4 embryos to no more than 1 to avoid negative effects on the neighboring embryos.