O-120 Validation of a rapid vitrification protocol for human blastocysts

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Abstract Study question Can a novel rapid vitrification protocol for human blastocysts generate similar post-warming survival and re-expansion rates compared to a standard protocol? Summary answer The rapid vitrification protocol achieves comparable post-warming total survival and 24h re-expansion rates compared to the standard protocol. However, increased cellular damage is observed. What is known already Vitrification plays a pivotal role in successful cryopreservation of human embryos. It involves the transformation of intra- and extracellular fluids into a glass-like solid state, preventing ice crystal formation and preserving the embryo’s integrity. Vitrification protocols feature initial exposure to a non-vitrifying equilibration solution (ES), followed by a short exposure to a vitrification solution (VS). Currently, long equilibration times (8-15 min) make embryo vitrification a time-consuming procedure. Recent studies on human oocytes suggest the feasibility of a rapid vitrification protocol with reduced equilibration time, potentially offering a more time-efficient approach for daily routine practice. Study design, size, duration This single-center observational validation study compared post-warming total survival (fully intact + moderately damaged) and re-expansion rates of 200 human research blastocysts vitrified using a standard or rapid vitrification protocol between November 2024 and January 2025. For each arm, 100 day 5/6 blastocysts originating from IVF/ICSI cycles with (N = 50) or without preimplantation genetic testing (PGT; N = 50) were used. Survival was evaluated immediately after warming. Re-expansion and embryo quality were assessed at 4h and 24h post-warming. Participants/materials, setting, methods Blastocysts were warmed using a fast warming protocol and re-vitrified 4h later applying either a rapid (4’ ES – 1’ VS) or standard (10’ ES – 1’ VS) protocol, using two commercially available kits and CBS-HS-VIT straws. Both protocols were performed in 50 µl droplets. The blastocysts were subsequently re-warmed using a one-step one-minute protocol (0.5 M sucrose) and observed for 24h in the EmbryoScope+ time-lapse system (Vitrolife). Results were analyzed using Chi-square statistics. Main results and the role of chance Post-warming total survival rates were similar for the rapid versus standard protocol in both the non-PGT (98% vs. 100%, p > 0.05) and PGT group (88% vs. 98%, p = 0.117). In the non-PGT group, the proportion of fully intact blastocysts was significantly lower after rapid (64%) compared to standard vitrification (84%) (p = 0.04). In the PGT group, this difference was not statistically significant (50% vs. 70%, respectively; p = 0.066). Full re-expansion after 4h was significantly lower after rapid versus standard vitrification in both the non-PGT (78% vs. 96%, p = 0.017) and PGT group (46% vs. 72%, p = 0.015). After 24h, however, re-expansion rates were similar in both groups: 82% vs. 88% and 68% vs. 74%, respectively (p > 0.05). Despite similar hatching rates in both the non-PGT (82% vs. 80%, p > 0.05) and PGT group (68% vs. 70%, p > 0.05), a significantly lower proportion of top-quality blastocysts (≥BL3 AA, AB, BA) was obtained after rapid vitrification in both the non-PGT (32% vs. 64%, p = 0.003) and PGT group (14% vs. 38%, p = 0.01). Additionally, 24h post warming, patches of cell damage were more frequently observed after rapid versus standard vitrification in both the non-PGT and PGT group: 30% vs. 8% and 50% vs. 24%, respectively (p = 0.01). Limitations, reasons for caution Limitations of this study include the non-blinded assessment of embryo survival and re-expansion, and the use of previously vitrified-warmed rather than fresh blastocysts to compare both vitrification protocols. Wider implications of the findings While rapid vitrification offers a more time-efficient alternative to current standard protocols, clinical outcomes might be compromised due to reduced preservation of blastocyst integrity. This study highlights the need for further optimization of rapid vitrification protocols. Trial registration number No

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