Increased number of cells in day 3 embryosis associated with higher live birth rate in D5 vitrified–thawed single blastocyst transfer cycles

Cell number in 3-day-old (D3) embryos influences pregnancy outcomes in patients undergoing cleavage-stage embryo transfer. However, the association between D3 cell number and pregnancy outcomes following 5-day-old (D5) vitrified–thawed single blastocyst transfer remains underexplored. This retrospective cohort follow-up study included 2035 cycles of D5 vitrified–thawed single blastocyst transfer. Patients were divided into six groups according to D3 embryo cell number: ≤5, 6, 7, 8, 9, and ≥ 10. Live birth rate (LBR), clinical pregnancy rate, miscarriage rate, and neonatal outcomes were compared across the groups. Statistical analyses were conducted using a multivariate logistic regression model. Furthermore, mediation analysis was performed to explore the association between D3 embryo cell number and LBR. LBR markedly increased with the D3 embryo cell number ( p < 0.001). Subgroup analysis of high- and low-quality blastocysts also revealed significant differences in LBR across the groups ( p < 0.05). Conversely, the miscarriage rate markedly decreased as the D3 embryo cell number increased ( p < 0.05). The D3 embryo cell number and perinatal outcomes did not show an association. Compared with the 8-cell group, the ≤ 5-cell group had significantly lower LBR [aOR: 0.681, 95% confidence intervals (CI): 0.468–0.991, p < 0.05], whereas the ≥ 10-cell group had a significantly higher LBR (aOR: 1.632, 95% CI: 1.214–2.159, p < 0.01). Mediation analysis revealed that D3 embryo cell number provides unique prognostic information for live birth outcomes beyond blastocyst quality metrics. Furthermore, the inclusion of D3 embryo cell number improved the predictive performance of the ROC model (ΔAUC = 0.012, 95% CI: 0.004–0.023, p = 0.007). D3 embryo cell number is a valuable supplementary indicator for selecting D5 blastocysts in vitrified–thawed single blastocyst transfer cycles. Among blastocysts with comparable morphological quality, those derived from fast-cleaving D3 embryos were associated with a lower miscarriage rate and a higher likelihood of live birth, specifically when transferring lower-quality blastocysts.