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The aim of the study was to compare retrospectively the extent of blastulation timing (Day 5 or later) and expansion grade to predict the ability of blastocysts to give rise to a pregnancy. Blastocysts frozen on day 5 with a lower expansion grade (group D5) or day 6 with a higher expansion grade (group D6) were included. A single embryo was thawed and transferred on day 5 after ovulation or progesterone supplementation. Differences in patient baseline characteristics, endometrial preparation and pregnancy outcomes between groups were stratified by patient age and anti-Müllerian hormone (AMH) levels. Logistic regression was used to analyse the results. A total of 617 blastocysts in group D5 and 1134 blastocysts in group D6 were assessed. Stratified analyses showed higher biochemical pregnancy, clinical pregnancy and live birth rates for patients aged less than 30 years old, and higher ongoing pregnancy rate for patients with AMH ≥ 1.1 ng/ml. For patients aged less than 30 years old, the biochemical pregnancy, clinical pregnancy and live birth rates in group D5 were higher than those in group D6. [ABSTRACT FROM AUTHOR]