Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification
Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification
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DOI：
10.1093/molehr/gav013
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发表时间：
2015-06-01
影响因子：
4
通讯作者：
Van de Velde, H.
中科院分区：
文献类型：
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作者：
De Munck, N.;Petrussa, L.;Van de Velde, H.
卵母细胞玻璃化冷冻已被引入临床环境中，而没有进行广泛的临床前安全性测试。在这项研究中，我们分析了高安全性封闭系统中人卵母细胞玻璃化冷冻的主要安全性方面：（i）染色体减数分裂分离，（ii）胚胎发育动力学和（iii）DNA（羟基）甲基化状态。采用三种不同的方法对年轻供体的新鲜和玻璃化冷冻的同胞卵母细胞进行卵胞浆内单精子注射（ICSI）。首先，受精卵的染色体组成是从第1天两个极体活检获得的阵列比较基因组杂交结果推导出来的。其次，通过延时成像分析胚胎发育至第3天。记录第3天的10个特定时间点、6个形态动力学时间间隔和平均细胞数。第三，通过免疫染色分析第3天胚胎的整体DNA甲基化和羟甲基化模式。用Volocity成像软件测量细胞核荧光强度。对极体进行全面的染色体筛查表明，新鲜和玻璃化冷冻卵母细胞ICSI后获得的受精卵中至少有一半是整倍体。时间推移分析表明，有没有显着差异的卵裂时间，预测的形态动力学时间间隔，也没有从新鲜和玻璃化冷冻卵母细胞发育的胚胎之间的平均细胞数。最后，全球DNA（羟基）甲基化模式之间没有显着不同的第3天从新鲜和玻璃化冷冻卵母细胞获得的胚胎。我们的数据进一步巩固了卵母细胞玻璃化冷冻技术的安全性。尽管如此，在年轻和老年低生育力/不育患者中进行额外的检测以及对卵母细胞冷冻后出生的儿童进行良好的随访研究仍然是强制性的。
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.