Preclinical validation of fast oocyte vitrification and warming protocols with comparable efficiencies to a standard method

Abstract

STUDY QUESTION Can fast vitrification (FV) and fast warming (FW) protocols effectively reduce oocyte exposure times to cryoprotectants while maintaining survival, developmental potential, and laboratory efficiency compared to a standard protocol? SUMMARY ANSWER FV and FW protocols significantly reduce exposure times compared to a standard protocol without compromising oocyte integrity or developmental potential in the animal models tested, offering a more efficient workflow for IVF laboratories. WHAT IS KNOWN ALREADY Vitrification is a widely used oocyte cryopreservation technique in IVF centers, but current protocols are time-consuming and labor-intensive. Additionally, prolonged exposure to cryoprotectants under non-physiological conditions, such as high osmolality and room temperature, raises concerns about oocyte viability and developmental potential. STUDY DESIGN, SIZE, DURATION This preclinical study involved oocytes from two animal models (mouse and rabbit) and a subset of discarded human oocytes. Experimental stages included: (a) evaluation of meiotic spindle integrity; (b) assessment of survival and developmental rates post-warming and ICSI; (c) embryo transfers in mice to evaluate full-term developmental potential; and (d) development and validation of an in silico model for human oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS Mouse and rabbit oocytes were randomly allocated to either a fast or standard vitrification (SV)/standard warming (SW) protocol. Survival and developmental rates were assessed post-warming. Meiotic spindle integrity and chromosomal alignment were analyzed using immunofluorescence. Full-term development was evaluated through embryo transfers in mice. Theoretical osmotic modeling was performed on human oocytes to predict their behavior under FV conditions, with empirical validation using discarded human oocytes. MAIN RESULTS AND THE ROLE OF CHANCE The FV/FW protocol significantly reduced the time required for vitrification compared to a SV/SW protocol, while maintaining comparable oocyte survival, meiotic spindle integrity, and developmental rates in animal models. In mouse oocytes, the FV/FW protocol achieved the highest survival rate (n = 249 oocytes; 97.2%) not statistically significantly different from the SV/SW protocol (n = 224 oocytes, 94.2%), but significantly higher (P = 0.008) than the SV/FW group (n = 229 oocytes, 91.7%). After ICSI, the SV/SW group reached 83.4% blastocyst rates, followed by the FV/FW group at 80.9%, and the SV/FW group at 75.9%, all comparable to the fresh oocyte control group (n = 123) with 86.4%. Embryo development after ICSI resulted in blastocyst formation rates of 80.9% for the FV/FW protocol compared to 83.4% in the SV/SW and 86.4% in the SV/FW group. Embryo transfer outcomes in mice demonstrated no statistically significant adverse effects on implantation or full-term development, with live birth rates of 38.7% (FV/FW) compared to 47.8% (SV/SW) and 43.2% (SV/FW). Survival rates of rabbit metaphase II oocytes ranged between 90% and 100% across all protocols, while blastocyst developmental rates were higher in the FV/FW group (28.6%) compared to the SV/SW group (22.2%) and the SV/FW group (13.6%). The mathematical in silico osmotic model predicted favorable responses for human oocytes, which were confirmed experimentally in discarded human oocytes with survival rates of 94.1% (n = 101) for the SV/FW protocol and 97.1% (n = 103) for the FV/FW protocol. LARGE SCALE DATA NA LIMITATIONS, REASONS FOR CAUTION This study was preclinical and involved animal models and discarded human oocytes. Further clinical trials are required to confirm the safety and efficacy of FV protocols in routine IVF clinical practice. WIDER IMPLICATIONS OF THE FINDINGS FV and FW protocols offer a promising alternative to conventional methods, enhancing laboratory workflow efficiency and reducing oocyte exposure to potentially harmful cryoprotectants. These findings lay the foundation for translational research and future clinical applications in clinical IVF settings. STUDY FUNDING/COMPETING INTEREST(S) This study was privately funded by Conceivable Life Sciences and Embryotools S.L. N.C.-B. reports stock with Embryotools and stock options with Conceivable Life Sciences and Fertility. N.C.-B. is also salaried by Embryotools. A.C.-B. received support for attending meetings from Conceivable Life Sciences and holds patents and stock with Conceivable Life Sciences, and stock with Hope IVF Mexico and IVF 2.0. A.F.-S.F. has received travel support from Conceivable Life Sciences and has patents with them as well as stock options. A.C. is salaried by IVIRMA. J.C. is salaried by Conceivable Life Sciences, holds stock options with Conceivable Life Sciences, IVF 2.0 and Athea Science and has other financial/non-financial interests with Reproduction Healthcare and TMRW Life Science. TRIAL REGISTRATION NUMBER NA