O-295 Fast vitrification and warming protocols demonstrate similar efficiencies to a standard method and a substantial reduction in execution times
Authors: Costa-Borges N , Coelo A , Cohen J , Gallardo M , Flores-Saiffe Farias A , Mestres E , Acacio M , Matia Q , Castello C , Calderón G , Chavez-Badiola A , Cobo A 
Published: July 2024

Abstract

Study question
Can vitrification and warming procedures be executed efficiently with reduced exposure times to cryopreservation solutions?

Summary answer
Fast vitrification and warming protocols demonstrated efficiency rates similar to a standard protocol in all pre-clinical models tested and a significant reduction in execution times.

What is known already
Vitrification is an advanced technique with well established protocols. However, these require a considerable time and workload in the daily routine of IVF laboratories. Recent studies on permeability models in human oocytes have shown that shorter times of exposure to cryopreservation solutions could be feasible. This study explored new vitrification and warming protocols based on reduced exposure times and compare their efficiency with a standard method. In a first series of experiments, mouse oocytes and blastocysts were used, followed by discarded human oocytes, with the ultimate goal of confirming the efficiency of the protocols before starting their clinical validation.

Study design, size, duration
Hybrid B6CBAF1 mouse oocytes were used to compare survival, meiotic spindle morphology, and in-vitro development post-ICSI. Blastocysts derived from these experiments were transferred to synchronized CD1 recipients to assess further development and birth. Once validated in the mouse model, protocols were tested with discarded human donor oocytes that were in-vitro matured and randomly assigned to experimental groups. Their survival was evaluated at 2h and 24h post-warming.

Participants/materials, setting, methods
Standard volumes, solutions and devices (Cryotop) were used as indicated by the manufacturer for standard protocols (Kitazato). Experimental groups included a control with standard vitrification/warming (Kitazato), standard vitrification with fast warming (1min in TS), and fast vitrification (1min in ES and 1min in VS) with fast warming (1min in TS). The study received IRB (2310-VLC-181-AC) approval and informed consents for the use of discarded human oocytes. Results were analyzed by Chi-square and Fisher’s exact test.

Main results and the role of chance
In mouse oocytes, the tested protocols showed no impact on meiotic spindle morphology. Survival rates after vitrification and warming were statistically similar between the control group with standard vitrification and warming (n = 154, 91.6%), the standard vitrification with fast warming (n = 153, 94.1%), and the fast vitrification with fast warming (n = 159, 96.9%) groups. Similarly, once inseminated by ICSI, blastocyst formation rates of vitrified oocytes were comparable between these groups, reaching 92%, 91.2%, and 88.6%, respectively. When the protocols were tested in blastocysts (n = 672), survival rates were 100% in all groups and, once transferred, the rates of development to term were statistically similar between the control group (n = 92, 47.8%), standard vitrification with fast warming (n = 95, 48.2%), and fast vitrification with fast warming (n = 93, 38.7%) groups. In human oocytes, survival at 2h and 24h with standard vitrification and fast warming (n = 101, 97% and 94.1%, respectively) was statistically similar to that obtained with the fast vitrification and fast warming (n = 103, 100% and 97.1%, respectively) protocol. A pilot study with sibling donor oocytes is being conducted to explore the clinical efficiency of the new protocols.

Limitations, reasons for caution
While this pre-clinical study underscores the potential of the fast vitrification and warming protocols, their efficacy awaits confirmation in clinical cases with sibling oocytes. Caution is warranted until such validation is achieved to ensure their reliability and effectiveness in clinical cases.

Wider implications of the findings
The pre-clinical study showcases the potential of short vitrification and warming protocols, maintaining efficiency comparable to standard methods. This promising efficiency translates to substantial time savings in the laboratory, with a reduction of 86.7% and 90%, respectively, in execution times.

Trial registration number
Not applicable