Li Wenzhi, Liao Xiaoyu, Lin Kaibo, Cai Renfei, Guo Haiyan, Ma Meng, Wang Yao, Xie Yating, Zhang Shaozhen, Yan Zhiguang, Si Jiqiang, Gao Hongyuan, Zhao Leiwen, Chen Li, Yu Weina, Chen Chen, Wang Yun, Kuang Yanping, Lyu Qifeng
Department of Assisted Reproduction, Shanghai Ninth People's Hospital Shanghai Jiao Tong University School of Medicine Shanghai China.
MedComm (2020). 2023 May 8;4(3):e217. doi: 10.1002/mco2.217. eCollection 2023 Jun.
The second polar body (PB2) transfer in assisted reproductive technology is regarded as the most promising mitochondrial replacement scheme for preventing the mitochondrial disease inheritance owing to its less mitochondrial carryover and stronger operability. However, the mitochondrial carryover was still detectable in the reconstructed oocyte in conventional second polar body transfer scheme. Moreover, the delayed operating time would increase the second polar body DNA damage. In this study, we established a spindle-protrusion-retained second polar body separation technique, which allowed us to perform earlier second polar body transfer to avoid DNA damage accumulation. We could also locate the fusion site after the transfer through the spindle protrusion. Then, we further eliminated the mitochondrial carryover in the reconstructed oocytes through a physically based residue removal method. The results showed that our scheme could produce a nearly normal proportion of normal-karyotype blastocysts with further reduced mitochondrial carryover, both in mice and humans. Additionally, we also obtained mouse embryonic stem cells and healthy live-born mice with almost undetectable mitochondrial carryover. These findings indicate that our improvement in the second polar body transfer is conducive to the development and further mitochondria carryover elimination of reconstructed embryos, which provides a valuable choice for future clinical applications of mitochondrial replacement.
辅助生殖技术中的第二极体（PB2）移植被认为是预防线粒体疾病遗传最具前景的线粒体替代方案，因为其线粒体携带量较少且操作性更强。然而，在传统的第二极体移植方案中，重构卵母细胞中仍可检测到线粒体携带。此外，操作时间的延迟会增加第二极体DNA损伤。在本研究中，我们建立了一种保留纺锤体突出的第二极体分离技术，该技术使我们能够更早地进行第二极体移植，以避免DNA损伤积累。我们还可以通过纺锤体突出定位移植后的融合位点。然后，我们通过基于物理的残留去除方法进一步消除重构卵母细胞中的线粒体携带。结果表明，我们的方案在小鼠和人类中均能产生近正常比例的正常核型囊胚，同时线粒体携带进一步减少。此外，我们还获得了线粒体携带几乎不可检测的小鼠胚胎干细胞和健康活产小鼠。这些发现表明，我们对第二极体移植的改进有利于重构胚胎的发育和进一步消除线粒体携带，为未来线粒体替代的临床应用提供了有价值的选择。
