Morphokinetic parameter S2 predicts early embryo developmental potential and associated metabolic pathways

The aim of this study was to determine the predictive value of the morphokinetic parameter S2 (the duration between cleavage from the 3-cell to 4-cell stage in the absence of direct cleavage) for early embryo developmental potential, establish its optimal cutoff value, and explore associated metabolic pathways.

This prospective study analyzed 662 embryos from 65 patients (September 2023-October 2024) which were cultured in time-lapse incubators. S2's predictive value for blastocyst formation/quality was assessed, with a cutoff value calculated through receiver operating characteristic (ROC) analysis. Subsequently, metabolomic profiling of spent culture media (SCM) from 20 patients was stratified by S2 cutoff using liquid chromatography-tandem mass spectrometry (LC-MS).

Among 662 embryos cultured in time-lapse incubators, 474 were analyzed to evaluate the association between S2 and developmental potential. Significant differences in S2 values were observed between the blastocyst formation vs. non-formation groups and high- vs. low-quality blastocyst groups (both median 0.5 vs. 0.8, P < 0.05). Logistic regression analysis showed that S2 predicted available blastocyst formation with an area under the curve (AUC) of 0.622 (95%CI = 0.559-0.685, P < 0.001) and blastocyst quality with an AUC of 0.645 (95% CI = 0.569-0.722), with an optimal cutoff of 0.7 h. Based on this cutoff, the day 3 (D3) SCM from 20 patients was divided into two groups (S2 < 0.7 and S2 ≥ 0.7) for metabolomic analysis. The results revealed 98 differentially expressed metabolites (59 upregulated and 39 downregulated), enriched in protein digestion/ absorption, aminoacyl-tRNA biosynthesis, fatty acid metabolism, central carbon metabolism in cancer, and glutamatergic synapse.

The S2 parameter (optimal cutoff = 0.70 h) shows moderate predictive capacity for embryo developmental potential (AUC = 0.64), with its regulation by specific metabolic pathways, supporting its inclusion in future multifactorial embryo evaluation models.