ObjectiveThis study aimed to determine whether the day of blastocyst expansion affects pregnancy outcomes in frozen-thawed blastocyst transfer (FBT) cycles.MethodsA retrospective match-cohort study was conducted. Patients who underwent blastocyst transfer in frozen-thawed cycles at day 5 or 6 were matched for potential confounding factors. A total of 2207 matched pairs of FBT cycles were included from January 2016 to December 2019 in our Reproductive Medicine Center.ResultsThe clinical pregnancy rate (CPR) and live birth rate (LBR) were significantly increased in day 5 blastocyst transfers when compared to day 6 blastocyst transfers, in terms of the same embryo quality. For FBT cycles with good-quality embryo, the CPR at day 5 and 6 was 61.30% and 57.56%, respectively (P=0.045), and the LBR was 44.79% and 36.16%, respectively (P<0.001). For FBT cycles with poor-quality embryo, the CPR at day 5 and 6 was 48.61% and 40.89%, respectively (P=0.006), and the LBR was 31.71% and 25.74%, respectively (P=0.019). The CPR for FBT cycles with good-quality embryo was statistically higher at day 6 than that at day 5 with poor-quality embryo transferred (57.56% vs. 48.61%, P=0.001). Maternal age, anti-Mullerian hormone (AMH), endometrial thickness, embryo quality, and the day of blastocyst expansion were independently correlated with the CPR and LBR. The FBT cycles at day 5 had significantly higher CPR (adjusted odds ratio [OR]=1.246, 95% confidence intervals [CI]: 1.097-1.415, P=0.001) and LBR (adjusted OR=1.435, 95% CI: 1.258-1.637, P<0.001) than those at day 6.ConclusionThe embryo quality is the primary indicator for FBT cycles. Day 5 blastocysts should be preferred when the quality of embryo at day 5 is the same as that at day 6.