Developmental trajectories from day 2 to day 3 refine ranking and selection of cleavage-stage embryos

To determine whether day 2 to day 3 developmental trajectories improve day 3 embryo ranking and selection compared with day 3 cell number alone, thereby offering better prognostic information for cleavage-stage embryos, including fast-cleaving embryos whose developmental potential remains uncertain.

This retrospective multicenter study (2016-2024) developed a ranking framework using 27,002 embryos from one center and validated it in 1691 fresh day 3 single embryo transfers across three centers (maternal age ≤ 38 years; embryos derived from two pronuclei, with fragmentation ≤ 10%, symmetric, and no multinucleation for confounder control). Developmental trajectories combined day 2 and day 3 cell numbers. Mixed-effects logistic models assessed trajectory-blastulation associations; post hoc comparisons consolidated trajectories into four ranking categories. Validation used trend tests between ranking levels and pregnancy rates, multivariable mixed-effects logistic model to estimate adjusted odds ratios (aORs) between ranking levels, and AUC for pregnancy prediction.

Trajectory analysis reversed the advantage of day 3 8-cell over 9-16-cell embryos (30.7% vs. 24.2% high-quality blastulation; P < 0.001), with Normal_Fast (4 → 9-16) achieving the highest rate (40.7%), significantly higher than Normal_Normal (4 → 8; 35.1%; P < 0.001). Trajectories were consolidated into a four-category ranking: (1) Normal_Fast (4 → 9-16); (2) Normal_Normal (4 → 8); (3) Normal_Slow (4 → 6-7), Slow_Normal (< 4 → 8), Fast_Normal and Fast_Fast (> 4 → 8-16); and (4) all others. Ranking 1 had a higher likelihood of clinical pregnancy than ranking 2 (aOR = 2.06 [1.05-6.82]), although this validation was limited by sample size (n = 24). Trajectory outperformed day 3 cell number in predicting high-quality blastocyst (AUC 0.776 vs. 0.729; P < 0.001) and clinical pregnancy (AUC original 0.619 vs. 0.596, P = 0.02; down-sampled 0.682 vs. 0.618, P = 0.016).

For women ≤ 38 years undergoing fresh day 3 single embryo transfer, developmental trajectories from day 2 to day 3 improve embryo ranking and selection with other morphological features controlled (two pronuclei, fragmentation ≤ 10%, symmetry, no multinucleation). The 4 → 9-16 pathway emerges as potentially optimal, though requiring further validation. This dual-day framework treats both cell numbers as interdependent markers, where day 2 status redefines the priority of day 3 cell number, rather than serving only as a secondary criterion.