Ice formation and its elimination in cryopreservation of oocytes

TL;DR

This study investigates how ice forms in oocytes during freezing and thawing, showing that ice can be eliminated with optimized cooling and warming rates, improving cryopreservation outcomes.

Contribution

The study demonstrates that ice formation during warming can be eliminated using higher cooling rates and reduced cryoprotective agent concentrations.

Findings

Oocytes remain ice-free during cooling but develop large ice fractions during warming.

Higher cooling rates allow oocytes to stay ice-free during both cooling and warming.

Larger convective warming rates enable ice-free cryopreservation with lower CPA concentrations.

Abstract

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming were examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions — consistent with crystallization of most free water — during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

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