Analysis of embryo intactness and developmental potential following slow freezing and vitrification

Abstract

The aim of this study was to identify the parameters that are related to intactness and developmental potential of a day 3 embryo after warming to improve the selection criteria used to cryopreserve and transfer embryos. We also sought to compare slow freezing and vitrification methods of cryopreservation and to evaluate the viability of non-intact embryos. Embryos warmed following slow freezing (n=220) or vitrification (n=522) were divided into 3 groups according to the proportion of surviving blastomeres (I<50%; II=50-99%; and III=100%). The developmental potential of embryos, including the mitosis resumption rate, blastocyst formation rate, and formation rate of grade A blastocysts (i.e., fully expanded blastocysts with an inner cell mass and grade A or B trophectoderm) were retrospectively assessed in embryos. Cleavage-stage embryos with <50% blastomere survival were analyzed using next-generation sequencing (NGS). Logistic regression analysis showed that vitrification and grade 1 were independent predictive factors of embryo intactness and developmental potential (all p<0.05). On day 3, embryos with 4-6 cells or blastomere damage had lower developmental potential than those with 7-9 cells or intact blastomeres (all p<0.05). NGS results showed that the chromosomal status was completely normal in 8 embryos that developed into expanded blastocysts, whereas 4 out of 5 embryos in which development was arrested were abnormal. The results of this study suggest that vitrification is a better choice than slow freezing for embryo cryopreservation. Embryos showing poor quality (fragmentation >30% and/or a non-stage-specific cell size) and lower cell numbers (4-6 cells) on day 3 should be cultured to the blastocyst stage and then vitrified if they develop into good-quality blastocysts. The developmental potential of non-intact embryos is lower than that of intact embryos; however, after they are cultured to the fully expanded blastocyst stage, embryos with <50% blastomere survival appear to be better candidates for transfer. Abbreviations ART: assisted reproductive technology; grade A blastocyst: fully expanded blastocyst with an inner cell mass and grade A or B trophectoderm; NGS: next-generation sequencing; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; FET: frozen-thawed embryo transfer.

Keywords: Blastocyst culture; cryopreservation; embryo; next-generation sequencing; non-intact embryo.