Abstract
BACKGROUND: Clarifying the impact of day-3 cell number on blastulation and subsequent pregnancy outcomes is essential for optimizing blastocyst selection criteria and refining embryo assessment protocols. While slow cleavage on day 3 is well-recognized as detrimental, the prognostic value of rapid cleavage (>8 cells) remains ambiguous. METHODS: This retrospective cohort study (January 2015-April 2024) included 64,853 embryos undergoing blastocyst culture (Cohort 1) and 2,669 single-blastocyst frozen embryo transfer (FET) cycles (Cohort 2) at a large tertiary assisted reproduction center. Cohort 1 examined the association between day-3 cell number and blastulation potential. Cohort 2 evaluated clinical pregnancy, live birth, and miscarriage rates following single-blastocyst transfer using multivariable logistic regression adjusted for confounders. Embryos were stratified by maternal age (<35 or ≥35 years) and blastocyst grade (top-quality [≥4BB on day-5] or non-top-quality [≥3BC on day-5/6, excluding ≥4BB on day-5]). RESULTS: In Cohort 1, compared to 8-cell embryos, 9- and 10-cell embryos had lower usable blastocyst rates (aORs [95% CI]: 0.77 [0.72-0.82] and 0.84 [0.77-0.91]); 11- and 12-cell embryos had comparable usable rates (0.96 [0.85-1.09] and 1.08 [0.93-1.24]) but higher top-quality rates (1.59 [1.37-1.85] and 2.17 [1.85-2.54]); and embryos with ≥13 cells had higher rates for both usable and top-quality blastocysts (all aORs > 1.4; 95% CIs excluded 1). This pattern was consistent across female age subgroups. In Cohort 2, however, the advantage of 11-16-cell embryos translated into superior pregnancy and live birth rates only in younger women receiving top-quality blastocysts versus 8-cell embryos (76.5% vs. 63.0%, P = 0.002, aOR = 1.95 [1.30-2.96]; 61.2% vs. 51.2%, P = 0.034, aOR = 1.58 [1.10-2.30]). Conversely, in older women with non-top-quality blastocysts, 11-16-cell embryos predicted lower pregnancy and live birth rates (26.5% vs. 51.0%, P = 0.023, aOR = 0.40 [0.15-0.97]; 14.7% vs. 38.5%, P = 0.019, aOR = 0.32 [0.10-0.89]). The 9-10-cell embryos generally showed outcomes comparable to 8-cell embryos, except for a reduced live birth rate in the older, non-top-quality blastocyst subgroup (23.9% vs. 38.5%, P = 0.047, aOR = 0.51 [0.26-0.98]). CONCLUSION: Day-3 cell number serves as a context-dependent prognostic indicator for optimizing blastocyst selection. For young women with top-quality blastocysts, ≥11-cell embryos are the strongest candidates; conversely, 8-cell embryos appear optimal for older women with non-top-quality blastocysts.