De Munck N, Petrussa L, Verheyen G, Staessen C, Vandeskelde Y, Sterckx J, Bocken G, Jacobs K, Stoop D, De Rycke M, Van de Velde H
Reproduction and Genetics, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, Brussels, Belgium Centre for Reproductive Medicine, UZ Brussel, Laarbeeklaan 101, Brussels, Belgium
Reproduction and Genetics, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, Brussels, Belgium.
Mol Hum Reprod. 2015 Jun;21(6):535-44. doi: 10.1093/molehr/gav013. Epub 2015 Mar 31.
Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analysed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after intracytoplasmic sperm injection (ICSI) were compared in three different assays. Firstly, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at Day 1. Secondly, embryo development up to Day 3 was analysed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on Day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analysed by immunostaining on Day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between Day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older sub-fertile/infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.
卵母细胞玻璃化冷冻技术在未经过广泛的临床前安全性测试的情况下就已应用于临床。在本研究中，我们在一个高度安全的封闭系统中分析了人类卵母细胞玻璃化冷冻的主要安全方面：（i）染色体减数分裂分离，（ii）胚胎发育动力学，以及（iii）DNA（羟）甲基化状态。在三种不同的检测中比较了年轻供体卵母细胞在胞浆内单精子注射（ICSI）后新鲜的和玻璃化冷冻的同卵姐妹卵母细胞。首先，从第1天活检的两个极体获得的基因芯片比较基因组杂交结果推断受精卵的染色体组成。其次，通过延时成像分析至第3天的胚胎发育情况。记录了10个特定时间点、6个形态动力学时间间隔以及第3天的平均细胞数。第三，通过对第3天胚胎进行免疫染色分析整体DNA甲基化和羟甲基化模式。通过Volocity成像软件测量核荧光强度。对极体进行的全面染色体筛查表明，新鲜的和玻璃化冷冻的卵母细胞在ICSI后获得的受精卵中至少有一半是整倍体。延时分析表明，新鲜的和玻璃化冷冻的卵母细胞发育而来的胚胎在分裂时间、预测的形态动力学时间间隔以及平均细胞数方面没有显著差异。最后，新鲜的和玻璃化冷冻的卵母细胞来源的第3天胚胎之间的整体DNA（羟）甲基化模式没有显著差异。我们的数据进一步证实了卵母细胞玻璃化冷冻技术的安全性。然而，对年轻和年长的亚生育/不育患者进行额外检测以及对卵母细胞冷冻保存后出生的儿童进行完善的随访研究仍然是必要的。
