One-step warming of vitrified human cleavage and blastocyst stage embryos does not adversely impact embryo survivability and subsequent developmental potential
Authors: Shioya Masashi , Hashizume Ryoko , Okabe-Kinoshita Miki , Kojima Katsushi , Nishi Sumie , Nakano Shun , Koga Kaori , Fujita Maki , Takahashi Keiichi 
Published: December 2024

Abstract

STUDY QUESTION
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)?

SUMMARY ANSWER
OW showed no detrimental effects on survival and developmental potential compared to SW in cleavage and blastocyst stage embryos.

WHAT IS KNOWN ALREADY
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.

STUDY DESIGN, SIZE, DURATION
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.

PARTICIPANTS/MATERIALS, SETTING, METHODS
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.

MAIN RESULTS AND THE ROLE OF CHANCE
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 (56 vs 49%, P = 0.4056) compared to those warmed by SW. Time-lapse monitoring analysis revealed that the frequency of collapses was reduced in OW (30 vs 50%, P = 0.0410). Additionally, all other abnormal morphokinetics were equivalent between OW and SW (P &gt; 0.05); moreover, the time required for blastocyst formation (P &gt; 0.05) and the morphological quality after development into the blastocyst stage (P &gt; 0.05) were not significantly different between OW and SW. In warming of blastocyst stage embryos, the time required for full re-expansion was longer with OW (3.20 ± 3.03 h vs 2.14 ± 2.17 h, P = 0.0008), but there was no significant difference in the proportion of full re-expanded blastocysts at 3- (67 vs 75%, P = 0.2417) and 24-h (98 vs 97%, P = 1.0000) post-warming. The in vitro adhesion assay showed no significant differences in adhesion rate and outgrowth area at all observation points (P &gt; 0.05).

LIMITATIONS, REASONS FOR CAUTION
This study was carried out as a preliminary trial using discarded embryos, which limited the number of embryos analyzed. Additionally, the impact on embryo transfer outcomes, such as clinical pregnancy and livebirth rates, remains unclear.

WIDER IMPLICATIONS OF THE FINDINGS
Our findings suggest that OW is a safe and efficient alternative to SW protocols and may improve the efficiency of IVF laboratory workflow without impairing embryo potentials.

STUDY FUNDING/COMPETING INTEREST(S)
No funding was obtained for this study. The authors have no conflicts of interest to declare related to this study.

TRIAL REGISTRATION NUMBER
N/A.