Shen Jing, Yu Zixuan, Li Weijie, Zhou Xinli
Institute of Biothermal Science & Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China.
Shanghai Co-innovation Center for Energy Therapy of Tumors, Shanghai, 200093, China.
Ann Biomed Eng. 2025 Feb;53(2):471-480. doi: 10.1007/s10439-024-03623-9. Epub 2024 Sep 25.
Oocyte vitrification has a wide range of applications in assisted reproduction and fertility preservation. It requires precise cryoprotectant agents (CPAs) loading and removal sequences to alleviate osmotic shock, which requires manual manipulation by an embryologist. In this study, a microfluidic system was developed to facilitate the precise adjustment of the CPA concentration around the oocyte by linear loading and removal of CPA. In addition, the microfluidic-based automated vitrification (MAV) device combines CPA loading/removal process, with vitrification process, thereby achieving automated oocyte vitrification. Oocytes were vitrified by Cryotop/QC manual method and MAV method. The results showed that the survival, cleavage, and blastocyst rates of oocytes were 80.44, 54.17, and 32.95% for the MAV method, which were significantly higher than Cryotop manual method (73.35, 43.73, and 23.67%) (p < 0.05). In MAV, solution injection rate during CPA loading/removal process was designed as a 1-segment, 2-segment, and 4-segment function. Accordingly, three concave loading and convex removal protocols were adopted to vitrify oocytes. Oocytes vitrified using the 4-segment function group exhibited increased survival (86.18%), cleavage (63.29%), and blastocyst (45.58%) rates compared to those vitrified using the 1-segment and 2-segment groups. The oocytes vitrification with the highest concentration of CPA, denoted as VS1-TS1, exhibited the highest survival rate after rewarming (86.18%). In contrast, the VS3-TS3 group, characterized by a CPA concentration half that of VS1-TS1, exhibited lower survival (74.14%) and cleavage (59.31%) rates, but displayed the higher blastocyst rate (50.79%) following oocyte activation. Our study demonstrates potential of the MAV device for oocyte or embryo vitrification.
卵母细胞玻璃化冷冻在辅助生殖和生育力保存方面有广泛应用。它需要精确的冷冻保护剂（CPA）加载和去除顺序来减轻渗透休克，而这需要胚胎学家手动操作。在本研究中，开发了一种微流控系统，通过线性加载和去除CPA来促进卵母细胞周围CPA浓度的精确调节。此外，基于微流控的自动化玻璃化冷冻（MAV）装置将CPA加载/去除过程与玻璃化冷冻过程相结合，从而实现卵母细胞的自动化玻璃化冷冻。采用Cryotop/QC手动方法和MAV方法对卵母细胞进行玻璃化冷冻。结果显示，MAV方法的卵母细胞存活率、卵裂率和囊胚率分别为80.44%、54.17%和32.95%，显著高于Cryotop手动方法（73.35%、43.73%和23.67%）（p＜0.05）。在MAV中，将CPA加载/去除过程中的溶液注入速率设计为1段、2段和4段函数。相应地，采用了三种凹形加载和凸形去除方案对卵母细胞进行玻璃化冷冻。与采用1段和2段函数组玻璃化冷冻的卵母细胞相比，采用4段函数组玻璃化冷冻的卵母细胞存活率（86.18%）、卵裂率（63.29%）和囊胚率（45.58%）均有所提高。用最高浓度CPA（记为VS1-TS1）进行卵母细胞玻璃化冷冻，复温后的存活率最高（86.18%）。相比之下，以CPA浓度为VS1-TS1一半为特征的VS3-TS3组，存活率（74.14%）和卵裂率（59.31%）较低，但卵母细胞激活后的囊胚率较高（50.79%）。我们的研究证明了MAV装置在卵母细胞或胚胎玻璃化冷冻方面的潜力。
