P-220 Two strategies for reducing cryoprotectant toxicity: recent trends in oocyte, embryo and tissue cryopreservation
Authors: Risco R 
Published: July 2024

Abstract

Study question
In this study, we demonstrate two methods to reduce the toxicity of cryoprotectants. Both have been tested in cells and tissues

Summary answer
Employing both the theoretical model of the incubation process and an ultra-rapid rewarming based on high-intensity focused ultrasound, it is possible to reduce this toxicity

What is known already
Cryoprotectants used in assisted reproduction for the cryopreservation of gametes, embryos, or ovarian or testicular tissue are based on DMSO, ethylene glycol, glycerol, etc. ... Minimizing exposure to these substances is always desirable. In this work, we show how, by modeling the hydraulic permeability of the embryo membrane and by the practical application of high-intensity focused ultrasound, it is possible to drastically reduce the biological material’s exposure to these agents. The former achieves a 90% reduction in incubation times in the cryoprotectant. The latter significantly increases the speed of rewarming, thereby reducing the amount of cryoprotectant needed for tissue recovery.

Study design, size, duration
The system was modeled using computer simulations based on MATLAB algorithms. It has been verified that in 1 minute, 90% of the cryoprotectant penetrates the embryo. The results have been experimentally validated in human embryos and oocytes. Regarding rewarming, High-Intensity Focused Ultrasound (HIFU) has achieved rewarming rates two orders of magnitude higher than standard rewarming. This has enabled the recovery of tissue samples that otherwise would not have been viable.

Participants/materials, setting, methods
For this study, donated oocytes and embryos were used. The MATLAB programming language was utilized for computer simulations. For ultrasonic rewarming, a piezoelectric excited at a frequency of 1.2 MHz and 60VDC was used.
Standard vitrification was compared with our toxicity reduced protocol in the case of oocytes and embryos,
Standard slow freezing was used in the case of rewarming with high intensity focused ultrasound.

Main results and the role of chance
The application of mathematical modeling, together with the experimental resultas on oocytes and embryos, demonstrate that is it possible to reduce in 90% the toxicity of the cryoprotectant in a typical vitrification protocol.
Also, the application of High Intensity Focused Ultrasound reaches warming rates that are two orders of magnitud higher than the conventional rewarming in a water bath. This finding represent a significative improvement of the present cryopreservation protocols.
The success of both strategies is demonstrated, both in reducing incubation time and the superiority of rewarming using ultrasound.
The study is generally applicable to oocytes, sperm, embryos, ovarian and testicular tissue.

Limitations, reasons for caution
No limitations.

Wider implications of the findings
We believe that these findings will soon be adopted by centers where the cryopreservation of biological material is necessary.

Trial registration number
NOT APPLICABLE