Not all Day-4 embryos are equal: developmental stage predicts outcomes after single embryo transfer

Evaluate outcomes of Day-4 embryo transfer according to embryonic developmental stage.

This retrospective single-center study included fresh IVF/ICSI cycles with day-4 single embryo transfer between January 2021 and July 2025. Cycles with double embryo transfer, rescue ICSI, or missing follow-up were excluded. Transfers were divided into three groups based on embryonic stage at transfer: cleavage, morula or blastocyst-stage. Demographic and cycle characteristics, as well as clinical outcomes, were compared. Clinical pregnancy was the primary outcome.

A total of 669 day-4 single fresh embryo transfer cycles were included: 130 cleavage-stage (19.4%), 449 morula-stage (67.1%), and 90 blastocyst-stage (13.5%). Compared to the morula stage, the cleavage-stage group was characterized by an older age, lower oocyte yield and fewer 2PN embryos, while the blastocyst-stage group was characterized by a younger age and a similar oocyte yield and 2PN embryos. Clinical pregnancy rates increased significantly with advancing embryonic stage (10.0%, 32.96%, and 48.89%, respectively; p < 0.05). In multivariable analysis, only increasing maternal age (OR 0.92, 95% CI 0.88-0.96) and cleavage-stage transfer (OR 0.37, 95% CI 0.18-0.78) remained negatively associated with clinical pregnancy.

Fresh day 4 embryo transfer outcomes are strongly influenced by embryonic developmental stage. Cleavage-stage embryos on day 4 are associated with poor pregnancy rates and may benefit from extended culture. In contrast, day 4 morula or blastocyst transfer represents a viable clinical option, particularly when logistical constraints preclude day 5 transfer.