STUDY QUESTION Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? SUMMARY ANSWER LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. WHAT IS KNOWN ALREADY Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. STUDY DESIGN, SIZE, DURATION Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. PARTICIPANTS/MATERIALS, SETTING, METHODS Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. MAIN RESULTS AND THE ROLE OF CHANCE The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3m protein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. LIMITATIONS, REASONS FOR CAUTION The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. WIDER IMPLICATIONS OF THE FINDINGS LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. TRIAL REGISTRATION NUMBER N/A

中文翻译：

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添加LEA蛋白提高人体外成熟卵母细胞的玻璃化冷冻效率

研究问题 在体外成熟卵母细胞玻璃化冷冻保存过程中添加晚期胚胎发育丰富 (LEA) 蛋白作为冷冻保护剂是否可以增强其受精后的发育潜力？摘要答案 LEA 蛋白可提高冷冻保存后人类体外成熟卵母细胞的发育潜力，主要是通过下调 FOS 基因、减少氧化应激和抑制冰晶的形成。已知信息 玻璃化过程中的各种因素，包括冷冻保护剂毒性、渗透压和复温过程中冰晶的形成，可能对卵母细胞造成致命损伤，从而影响卵母细胞的发育潜力和随后的临床结果。最近的研究表明，LEA蛋白具有高亲水性和固有的应激耐受性，可以减轻低温损伤，但其发挥保护作用的分子机制仍不清楚。研究设计、规模、持续时间 将我们提取和纯化的两种 LEA 蛋白添加到浓度为 10、100 和 200 µg/mL 的卵母细胞玻璃化升温溶液中，以确定每种蛋白的最佳保护浓度。收集单个卵母细胞样本进行转录组分析，每组由三个样本重复组成。参与者/材料、背景、方法从接受联合体外受精（IVF）治疗且符合指定纳入和排除标准的患者中收集未成熟卵母细胞。这些卵母细胞经过体外成熟（IVM）培养以进行实验研究。采用荧光显微镜检测不同浓度LEA蛋白处理的玻璃化温热人卵母细胞线粒体膜电位（MMP）、活性氧（ROS）和钙的水平，以及蛋白的保护作用。评估了线粒体功能。通过冷冻显微镜表征人卵母细胞玻璃化加温后细胞内冰重结晶抑制（IRI）的水平，以确定LEA蛋白对重结晶的抑制作用。通过分析转录组测序数据来研究LEA蛋白发挥冷冻保护作用的潜在机制。主要结果和机会的作用 AfrLEA2 和 AfrLEA3m 蛋白的二级结构显示由大量 α-螺旋组成，并且该蛋白显示出高度亲水性，与之前的报道一致。共聚焦显微镜结果显示，与异硫氰酸荧光素（FITC）对照组相比，AfrLEA2-FITC和AfrLEA3m-FITC标记蛋白的免疫荧光似乎位于细胞外，并且没有穿透细胞膜，表明AfrLEA2和AfrLEA3m蛋白均位于细胞外。与10和200μg/mL组相比，用100μg/mL AfrLEA2或AfrLEA3m蛋白处理的组具有更均匀的细胞质颗粒和更少的空泡，并且最接近新鲜组。在100 µg/mL组中，MMP显着高于对照组，而ROS和钙水平显着低于对照组，并且更接近新鲜卵母细胞中观察到的水平。同时，与用二甲亚砜 (DMSO) 和乙二醇 (EG) 处理的对照组相比，100 µg/mL 的 AfrLEA2 或 AfrLEA3m 蛋白在 IRI 测定中引起更小的冰晶形成；因此，重结晶抑制优于传统冷冻保护剂 DMSO 和 EG。进一步的结果表明，这些蛋白质可能通过下调 FOS 基因和减少氧化应激来提高冷冻保存后人类卵母细胞的发育潜力。局限性和注意事项 由于伦理限制，研究中使用的体外成熟中期 II (IVM-MII) 卵母细胞可能无法准确反映 MII 卵母细胞的总体状况。 AfrLEA2和AfrLEA3m蛋白是重组蛋白，其合成稳定性需要进一步探索。研究结果的更广泛意义 LEA 蛋白作为一种无毒且有效的冷冻保护剂，可以减少冷冻保存过程中卵母细胞的冷冻损伤。它为各种细胞类型的冷冻保存提供了一种新的有前途的方法。研究经费/竞争兴趣这项工作得到了国家重点研发计划（2022YFC2703000）和国家自然科学基金（52206064）的支持。作者声明没有竞争利益。试用注册号 不适用可减少冷冻保存过程中卵母细胞的冷冻损伤。它为各种细胞类型的冷冻保存提供了一种新的有前途的方法。研究经费/竞争兴趣这项工作得到了国家重点研发计划（2022YFC2703000）和国家自然科学基金（52206064）的支持。作者声明没有竞争利益。试用注册号 不适用可减少冷冻保存过程中卵母细胞的冷冻损伤。它为各种细胞类型的冷冻保存提供了一种新的有前途的方法。研究经费/竞争兴趣这项工作得到了国家重点研发计划（2022YFC2703000）和国家自然科学基金（52206064）的支持。作者声明没有竞争利益。试用注册号 不适用