P-212 A comparison of multi-step versus single-step thawing protocols for vitrified blastocysts using thawing solution or culture media

Abstract

Study question Does one-step warming (OW) using thawing solution (TS) or culture media (CM) impact the survival rate of vitrified blastocysts compared to standard multi-step warming (SW)? Summary answer One-step warming using a thawing solution or culture medium is comparable to the multi-step warming protocol. Further clinical evidence is required to prove these findings. What is known already The multi-step warming protocol, which involves reducing non-permeable cryoprotectant concentrations, aims to mitigate osmotic shock caused by the rapid influx of water. Recent studies have shown that a simplified warming protocol using only thawing solution or culture media, yields comparable survival rates while increasing pregnancy rates and reducing the workload of embryologists. Study design, size, duration A total of 128 donated vitrified blastocysts, encompassing both good-quality and poor-quality blastocysts, were randomly allocated into three groups: 44 for TS protocol using Warm 1 Blast of RapidWarm Blast kit (VitroLife), 42 for the CM protocol using Sage one-step with HSA (Origio) and 42 blastocysts for the SW protocol using Cryotec RtU (Cryotech). The primary outcome was the survival rate of vitrified blastocyst post-warming. Secondary outcomes included full re-expansion rate, hatching and hatched rate. Participants/materials, setting, methods The study was conducted at a tertiary IVF center. In the TS and CM protocols, blastocysts were rinsed in thawing solution for 2 minutes and culture media for 1 minute, respectively. The SW protocol required 6.5 minutes. Post-warming blastocysts were cultured in a timelapse incubator and their survival rate was assessed based on the presence of blastocoel re-expansion after 2, 4, and 24 hours. Additionally, rates of full re-expansion, hatching, and hatched blastocyst were calculated. Main results and the role of chance The survival rates of vitrified blastocyst warmed using the TS and CM protocols were 97.73% (n = 43) and 97.62% (n = 41), respectively, which were comparable to the 100% survival rate (n = 42) observed in the SW control group. Timelapse monitoring indicated that the revived blastocysts were capable of re-expansion, similar to conventional vitrified-warming method. The hatching and hatched rates after 24 hours were highest in the SW group, but similar between TS and SW groups, as well as between the TS and CM groups. However, these rates were significantly lower at 2, 4, and 24 hours when comparing the CM group (7.14%, 19.05%, and 50% respectively) to the SW group (35.71%, 54.76%, and 76.19% respectively) (all p < 0.05). Similarly, blastocysts warmed by the SW protocol had the shortest time to full re-expansion, with a median time of 108.00 minutes [51.00; 164.25]. In contrast, the time was significantly longer in the CM group with a median time of 153.00 minutes [107.25; 295.50] (p = 0.024). The time for blastocyst recovery tended to be longer in the CM group compared to the other two groups. Limitations, reasons for caution This study was limited by the small sample size of blastocysts. Additionally, there was a lack of clinical data, such as clinical pregnancy and live birth rates, following the transfer of warmed blastocysts using the TS, CM, and SW protocols. Wider implications of the findings Direct warming using CM or an OW solution supports survival rates irrespective of blastocyst grading. One-step warming may be considered a potential alternative to SW protocols. However, further studies are necessary to elucidate factors influencing embryo recovery potential and to optimize the laboratory workflow for the TS and CM protocols. Trial registration number No