Background: Stepwise removal of cytotoxic cryoprotectant and rehydration in serial osmotic solutions to prevent osmotic shock have been the central dogmas in cryobiology for mammalian embryos. The theory behind is to gradually remove cytotoxic cryoprotectants and rehydrate the embryo to minimize the damages during the vitrified-thaw processes. The whole process is time consuming and laborious routine in the IVF laboratory. Here, we showed that direct thawing a vitrified blastocyst in a regular human embryo culture medium without any cryoprotectant support 100% thawing survival rate regardless to blastocysts' grading. Surprisingly, better implantation outcome was observed in our small cohort trial although not reached a significant different. There are more than million frozen embryo transfers around the world each year; a faster, safer and cheaper method can save significant amount of money for patients undergoing IVF treatment worldwide. Methods: A two-phase trial were conducted: Technical phase and clinical phase. In the Technical phase, 4 commercially available one-step media and PBS solution were used for thawing procedure on donated human vitrified blastocysts. The survival rate was determined by re-expansion under time-lapse imaging. All of the one-step media at a specific range of osmolality solution between 270+-10 (mOsm/KG) with human serum albumin were tested. PBS with human serum albumin at the same osmolality were used as control. We then investigated the technical comparability on routine blastocyst manipulation, we tested repeated direct thawing and repeated biopsies on the tested embryos in order to show the direct thawing method can be applied to any possible routine IVF procedures. In the clinical phase, 20 patients were recruited for the direct thawing compared to the conventional thawing of 96 patients during the same study period. Cost-effectiveness analysis was performed to estimate the different between two procedures. Results: In the technical phase, we showed that all of the one-step media supported human vitrified blastocyst thawing with 100% survival rate (N=52), even in the PBS control (N=11). And the revived blastocysts are capable to re-expend comparable to conventional vitrified-thawing method. We also demonstrate this single-direct rehydrated and thawed blastocyst in embryo culture medium survived from direct thawing, trophectoderm biopsy, second vitrified, second direct thawing with the second biopsy indicated that this new method is comparable to conventional IVF procedures (N=13). Pilot clinical study showed a higher implantation rate was also obtained from this direct thawing method compared to conventional warming (61.9% VS 37.2%) and leading to healthy live birth (45% VS 36.8%). Our cost-effectiveness analysis showed that the medium direct thawing could save 42% consumable cost for patients and 90% of labor time. Conclusions: We conclude that a regular embryo culture medium is comparable to the traditional thawing method that potentially can save billions of dollars and thousands of labour hours each year in IVF setting worldwide. We proved gradual cryoprotectant removal and gradual rehydration are not necessary for human blastocyst thawing survivals. Using a regular embryo culture medium for thawing blastocysts supported all clinical outcome equivalent to conventional thawing procedure. Our data also showed that the method can cut down the cost and labour time in all IVF clinics worldwide. Limitation: Only single Vitrification kit and embryo carrier were used in this study. The mechanism on how the human blastocysts survived from one-step thawing remain unknown and the actual clinical power at larger scale is yet to be resolved.

The authors have declared no competing interest.

NCT06322823

We would like to thank the Hong Kong Health and Medical Fund (Ref 18190321), from the Hong Kong government, The Hong Kong Research Matching Grant (RMG01-8601386) to support this clinical research.

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This study was approved by the Hospital research board of the Faculty of Medicine, Prince of Wales Hospital, The Chinese University of Hong Kong.

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Our data are available with a signed data use agreement; please contact drdcyl16@cuhk.edu.hk.