Lee Jaewang, Kim Eun Jung, Kong Hyun Sun, Youm Hye Won, Kim Seul Ki, Lee Jung Ryeol, Suh Chang Suk, Kim Seok Hyun
Department of Biomedical Laboratory Sciences, Eulji University, Seongnam, South Korea; Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, South Korea.
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, South Korea; Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, South Korea.
Theriogenology. 2018 Nov;121:97-103. doi: 10.1016/j.theriogenology.2018.07.022. Epub 2018 Aug 3.
In vitro follicle growth (IVFG) is an emerging alternative option for fertility preservation in women instead of ovarian tissue cryopreservation and transplantation. To widen the application of this technique, follicle cryopreservation should be established prior to clinical use. In the present study, we tried to determine the optimal vitrification protocol of mouse ovarian follicle for in vitro culture and oocyte maturation by comparing four different compositions of cryoprotective agents (CPA). Secondary follicles were mechanically isolated from 2-week-old BDF-1 mice and randomly assigned to fresh control and four different groups by the composition of CPAs (ES, EDS, EFS and EPS groups; E: ethylene glycol, D: dimethyl sulfoxide, S: sucrose, F: ficoll, P: 1,2-propanediol (PROH)). After vitrification and warming procedures, the follicles were cultured in vitro for 10 days and then treated with human chorionic gonadotropin and epidermal growth factor to induce oocyte maturation. Fourteen to 16 h later, oocyte maturation and quality were assessed. Follicle viability was evaluated by Calcein-AM/ethidium homodimer-1 staining immediately after warming, and their survival and diameters were measured during follicle culture periods. Antral cavity formation was observed at the end of the culture period (on the 10th day of culture). Following oocyte maturation, its maturational ability and meiotic spindle formation were assessed to evaluate their competence. There was no significant difference in viability after warming among the vitrification groups. From the 8th day of culture, the survival rate of ES and EDS were significantly higher than those of other vitrification groups (EPS and EFS). The follicle diameter was largest in the fresh-control group from the 6th day, while smallest in the EFS with statistical significance. On the 10th day of culture, the antral-cavity formation rate of EDS was comparable to that of the fresh control group. However, the oocyte maturation was significantly decreased in all four vitrification groups when compared with control group; especially, the EFS showed a more marked reduction in the oocyte maturation. There were no significant differences in meiotic spindle formation among all of those groups. Our results suggest that EDS combination for mouse follicle vitrification are the most effective vitrification protocols for mouse follicle and evaluated by an in vitro culture and oocyte maturation after warming.
体外卵泡生长（IVFG）是一种新兴的女性生育力保存替代方法，可替代卵巢组织冷冻保存和移植。为了扩大该技术的应用，应在临床应用前建立卵泡冷冻保存方法。在本研究中，我们通过比较四种不同组成的冷冻保护剂（CPA），试图确定用于体外培养和卵母细胞成熟的小鼠卵巢卵泡的最佳玻璃化方案。从2周龄的BDF-1小鼠中机械分离次级卵泡，并根据CPA的组成将其随机分为新鲜对照组和四个不同组（ES、EDS、EFS和EPS组；E：乙二醇，D：二甲基亚砜，S：蔗糖，F：聚蔗糖，P：1,2-丙二醇（PROH））。经过玻璃化和复温程序后，将卵泡体外培养10天，然后用人绒毛膜促性腺激素和表皮生长因子处理以诱导卵母细胞成熟。14至16小时后，评估卵母细胞成熟度和质量。复温后立即通过钙黄绿素-AM/碘化丙啶同型二聚体-1染色评估卵泡活力，并在卵泡培养期间测量其存活率和直径。在培养期结束时（培养第10天）观察卵泡腔形成情况。卵母细胞成熟后，评估其成熟能力和减数分裂纺锤体形成以评估其能力。玻璃化组之间复温后的活力无显著差异。从培养第8天起，ES和EDS组的存活率显著高于其他玻璃化组（EPS和EFS）。从第6天起，新鲜对照组的卵泡直径最大，而EFS组最小，具有统计学意义。在培养第10天，EDS组的卵泡腔形成率与新鲜对照组相当。然而，与对照组相比，所有四个玻璃化组的卵母细胞成熟度均显著降低；尤其是，EFS组的卵母细胞成熟度下降更为明显。所有这些组之间减数分裂纺锤体形成无显著差异。我们的结果表明，用于小鼠卵泡玻璃化的EDS组合是小鼠卵泡最有效的玻璃化方案，并通过复温后的体外培养和卵母细胞成熟进行评估。
