Abdelhady Abdallah W, Mittan-Moreau David W, Crane Patrick L, McLeod Matthew J, Cheong Soon Hon, Thorne Robert E
bioRxiv. 2023 Nov 17:2023.11.15.567270. doi: 10.1101/2023.11.15.567270.
Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction using cryopreserved oocytes and embryos. We use synchrotron-based time-resolved x-ray diffraction and tools from protein cryocrystallography to characterize ice formation within bovine oocytes after cooling at rates between ∼1000 °C/min and ∼600,000°C /min and during warming at rates between 20,000 and 150,000 °C /min. Maximum crystalline ice diffraction intensity, maximum ice volume, and maximum ice grain size are always observed during warming. All decrease with increasing CPA concentration, consistent with the decreasing free water fraction. With the cooling rates, warming rates and CPA concentrations of current practice, oocytes may show no ice after cooling but always develop substantial ice fractions on warming, and modestly reducing CPA concentrations causes substantial ice to form during cooling. With much larger cooling and warming rates achieved using cryocrystallography tools, oocytes soaked as in current practice remain essentially ice free during both cooling and warming, and when soaked in half-strength CPA solution oocytes remain ice free after cooling and develop small grain ice during warming. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes, establish the character of ice formed, and suggest that substantial further improvements in warming rates are feasible. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development, allowing other deleterious effects of the cryopreservation cycle to be studied, and osmotic stress and CPA toxicity reduced.
Cryopreservation of oocytes and embryos is critical in assisted reproduction of humans and domestic animals and in preservation of endangered species. Success rates are limited by damage from crystalline ice, toxicity of cryoprotective agents (CPAs), and damage from osmotic stress. Time-resolved x-ray diffraction of bovine oocytes shows that ice forms much more readily during warming than during cooling, that maximum ice fractions always occur during warming, and that the tools and large CPA concentrations of current protocols can at best only prevent ice formation during cooling. Using tools from cryocrystallography that give dramatically larger cooling and warming rates, ice formation can be completely eliminated and required CPA concentrations substantially reduced, expanding the scope for species-specific optimization of post-thaw reproductive outcomes.
在使用冷冻保存的卵母细胞和胚胎的辅助生殖中，冰造成的损伤以及抑制冰形成的冷冻保护剂（CPA）的潜在毒性是关键问题。我们使用基于同步加速器的时间分辨X射线衍射以及蛋白质低温晶体学工具，来表征牛卵母细胞在以约1000℃/分钟至约600,000℃/分钟的速率冷却以及以20,000至150,000℃/分钟的速率升温过程中的冰形成情况。在升温过程中总是观察到最大结晶冰衍射强度、最大冰体积和最大冰粒尺寸。随着CPA浓度的增加，所有这些参数均下降，这与自由水比例的降低一致。按照当前实践中的冷却速率、升温速率和CPA浓度，卵母细胞在冷却后可能不会出现冰，但在升温时总会形成大量的冰部分，并且适度降低CPA浓度会导致在冷却过程中形成大量的冰。使用低温晶体学工具实现更大的冷却和升温速率时，按照当前实践浸泡的卵母细胞在冷却和升温过程中基本上都不会结冰，而当浸泡在半强度CPA溶液中时，卵母细胞在冷却后不会结冰，在升温过程中会形成小颗粒冰。这些结果阐明了冷却、升温以及CPA浓度在卵母细胞中冰形成过程中的作用，确定了所形成冰的特征，并表明升温速率有进一步大幅提高的可行性。冰形成作为影响解冻后卵母细胞活力和发育的一个因素可以被消除，从而能够研究冷冻保存周期的其他有害影响，并降低渗透应激和CPA毒性。
卵母细胞和胚胎的冷冻保存在人类和家畜的辅助生殖以及濒危物种的保护中至关重要。成功率受到结晶冰造成的损伤、冷冻保护剂（CPA）的毒性以及渗透应激造成的损伤的限制。对牛卵母细胞的时间分辨X射线衍射表明，冰在升温过程中比在冷却过程中更容易形成，最大冰部分总是在升温过程中出现，并且当前方案中的工具和高浓度CPA充其量只能在冷却过程中防止冰的形成。使用能实现显著更大冷却和升温速率的低温晶体学工具，可以完全消除冰的形成，并大幅降低所需的CPA浓度，从而扩大了针对特定物种优化解冻后生殖结果的范围。
