P-156 Ultra-fast warming of blastocysts yields better survival rates for D5 non-PGT and similar survival rates for D5/D6 PGT blastocysts
Authors: De Gheselle S , Lierman S , De Vuyst L , Stoop D , Tilleman K 
Published: July 2024

Abstract

Study question
Can an ultra-fast single-step warming of vitrified blastocysts yield similar survival rates and post-warming development compared to the standard multi-step rehydration protocol?

Summary answer
Ultra-fast warming, shows significantly better rates of survival and post-warming development in vitro for non-PGT blastocysts and similar rates for PGT blastocysts.

What is known already
Excellent survival rates have been reported since the introduction of human blastocyst vitrification. Typically, most warming procedures involve three steps with decreasing concentrations of non-penetrating cryoprotectants. Recently, few studies have evaluated the effectiveness of simplifying the procedure to a single step for rehydration, while maintaining comparable survival and pregnancy rates. Adopting simpler procedures can optimize laboratory protocols, workflow, and safety, supporting further advancements in Assisted Reproduction Technologies (ART).

Study design, size, duration
This validation study ultra-fast warmed 246 vitrified blastocysts [129 non-PGT day5 (cryopreservation period 23/01/2017-24/04/2018 VIT-kit, Fujifilm), 57 PGT day5 (25/01/2023-31/03/2023 VIT-KitNX, Fujifilm), 60 PGTday6 (03/02/2023-22/08/2023 Vit-KitNX, Fujifilm)] and compared survival rates with 379 blastocysts [240 non-PGT day5, 97 PGT day5, 42 PGT day6] vitrified in the same time-periods and warmed for clinical application using the standard multi-step protocol. Ultra-fast warmed blastocysts were cultured for 24h in a time-lapse incubator and subjected to a viability staining.

Participants/materials, setting, methods
Blastocysts, originating from 111 patients that were donated to validation studies after the legal storage period(n = 129) or deemed non-transferable after PGT(n = 117), were ultra-fast warmed by immersing the straw in a 37 °C thawing solution (1M trehalose; Vit-kitwarmNX; Fujifilm) for 1 minute. Survival rates [total (≥50% intact) and fully (100% intact) ], transfer rates (2h post-warming, ≥50% intact), re-expansion time and the number of damaged cells (Live/Dead kit L-3224, Invitrogen) were analysed. Fisher’s Exact Test (p &lt; 0.05).

Main results and the role of chance
Overall, the immediate post-warming survival rates (≥50% intact) were comparable between ultra-fast and multi-step warming, 98.4% vs 96.8% respectively (p = 0.3041). However, the fully intact survival rate immediately post-warming was significantly higher with ultra-fast warming (85.4% vs 76.3%; p = 0.0058) as well as the percentage of blastocysts considered transferable (96.7% vs 91.2%; p = 0.0076). Sub-group analysis demonstrated significantly better results for non-PGT blastocysts following ultra-fast warming. Higher immediate survival post-warming (100% vs 96.6%; p = 0.036) and transfer rates (100% vs 90%; p = 0.0002) were achieved. Similar results were observed for PGT blastocysts on both day 5 and day 6, with no significant differences in total, fully intact, and transfer rates observed between the two warming procedures [PGT Day5: 96.5%, 80.7%, and 93.0% vs. 97.9%(p = 0.6270), 69.1%(p = 0.1334), and 93.8%(p = 1.0000)] [PGT Day6: 96.7%, 68.3%, and 93.3% vs. 95.2%(p = 1.0000), 64.3%(p = 0.6761), and 92.8%(p = 1.0000)]. Ultra-fast warmed blastocysts showed a total re-expansion rate of 93.3% in 3.3h±2.7h on average. Viability staining demonstrated very few cells damaged. Zero cell damage was observed in 31.5% of the blastocysts, 59.0% had 1-10 cells damaged, 9.5% &gt;10 cells damaged, and only 4 blastocysts were completely degenerated.

Limitations, reasons for caution
In order to limit the amount of embryos needed in this validation study, ultra-fast warming outcome parameters were compared with retrospective clinical results of blastocysts vitrified in the same time periods. Ultra-fast warming is a single-step process where precise temperature control(37 °C) of the warming medium is crucial for success.

Wider implications of the findings
Minimizing handling steps leads to an overall risk reduction in potential exposure to suboptimal warming conditions and operator/handling-related stress. This optimization brings huge efficiency to the busy work schedule that is characteristic for an IVF lab without dropping cryopreservation quality indicators.

Trial registration number
Not applicable