One-step warming of vitrified human cleavage and blastocyst stage embryos does not adversely impact embryo survivability and subsequent developmental potential

• Published in: Human reproduction (Oxford) 2024-12
• Main Authors: Shioya, Masashi, Hashizume, Ryoko, Okabe-Kinoshita, Miki, Kojima, Katsushi, Nishi, Sumie, Nakano, Shun, Koga, Kaori, Fujita, Maki, Takahashi, Keiichi
• Format: Article
• Language: English
• ISSN: 0268-1161; 1460-2350; 1460-2350
• DOI: 10.1093/humrep/deae283

Does one-step warming (OW), a simplified embryo warming protocol, adversely affect survival and developmental potential in vitrified cleavage or blastocyst stage embryos compared to standard multi-step warming (SW)? OW showed no detrimental effects on survival and developmental potential compared to SW in cleavage and blastocyst stage embryos. While standard embryo warming protocols involve a multi-step procedure using a stepwise osmotic solution to avoid a rapid influx of water into the embryo, recent studies suggest that eliminating the stepwise warming process does not reduce embryo survival and embryo transfer outcomes. However, previous reports have focused primarily on pregnancy rates, and a more detailed analysis of the effects of rapid osmotic pressure changes on embryos is necessary to standardize the protocol. This preliminary study includes donated 377 vitrified human embryos (177 cleavage and 200 blastocyst stage) from 210 patients approved for discard at the patient's consent. The embryos were randomly allocated and warmed using either SW or OW protocols. In the SW protocol, embryos were rinsed with a stepwise osmotic solution (thawing, dilution, and washing solutions), and the process was completed with a 13-min warming period. In the OW protocol, embryos were only rinsed in a single solution (thawing solution) for 1 min. Post-warming embryos were cultured using a time-lapse incubator. Survival rate and developmental potential, including the occurrence of abnormal morphokinetics and the time required for blastocyst formation after warming of cleavage stage embryos, were compared between SW and OW. Embryos that developed into the blastocyst stage were morphologically evaluated. In the warming of blastocyst stage embryos, the survival rate was determined by the presence of blastocoel expansion, and the proportion of full re-expanded blastocysts was observed at 3- and 24-h post-warming. An in vitro adhesion assay was also performed on blastocysts after culture, and adhesion rate and outgrowth area were measured 24, 48, and 72 h after culture with fibronectin-precoated dishes. OW did not negatively impact survival rates in either cleavage (100% in both OW and SW groups) or blastocyst stage embryos (99% in both groups). Cleavage stage embryos warmed by OW had superior or comparable rates of morulation (96 vs 85%, P = 0.0387), blastulation (78 vs 73%, P = 0.4044), full-blastocyst formation (60 vs 53%, P = 0.3196), and expanded-blastocyst formation