Patterns of cell exclusion in human embryos reveal a stage-dependent association with the detection of fetal heartbeat within fresh cycles

Cell exclusion (CE) is a morphological phenomenon observed and characterized relative to embryo morulation, yet its implications for reproductive competence seem heterogeneous. This study investigated whether variability in reproductive success among CE-containing (CE+) embryos could be attributed to differences in the timing and physical extent of cell exclusion, cleavage-stage morphology and morphokinetics. A total of 698 embryos cultured in time-lapse incubators, across two independent fertility clinics, were retrospectively analysed. Embryos were assessed for the presence of CE (CE+, n = 155) prior to fresh embryo transfer and stratified according to the cleavage stage at which excluded cell(s) first appeared. Reproductive success varied markedly across CE+ subgroups, where exclusions at the 9-cell stage (CE+ 9 ) resulted in the highest proportions of FHB and LB, comparable to embryos without CE (CE-, n = 543). Exclusions at the 6-cell (CE+ 6 ), 8-cell (CE+ 8 ), 3-cell (CE+ 3 ) stages, and combination (CE+ C ) exclusions were associated with the poorest outcomes following logistic regression analysis. Neither the number of excluded cells nor the relative surface area occupied by exclusions (%SA) was shown to predict reproductive success, however correlation analysis revealed associations between %SA and various morphokinetic parameters. Routine embryo morphology annotations did not vary between CE+ subgroups. These findings demonstrate that the developmental stage of CE, rather than the extent of exclusion, may represent the primary driver of heterogeneity in reproductive competence among CE+ embryos.