Joint Institute for High Temperatures of the Russian Academy of Sciences, Izhorskaya st. 13 Bd.2, Moscow, Russian Federation, 125412.
Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, 12-1, Moscow, Russian Federation, 119234.
J Assist Reprod Genet. 2019 Jun;36(6):1251-1261. doi: 10.1007/s10815-019-01424-x. Epub 2019 May 30.
Our purpose was to study whether application of femtosecond laser pulses for alphanumeric code marking in the volume of zona pellucida (ZP) could be effective and reliable approach for direct tagging of preimplantation embryos.
Femtosecond laser pulses (wavelength of 514 nm, pulse duration of 280 fs, repetition rate of 2.5 kHz, pulse energy of 20 nJ) were applied for precise alphanumeric code engraving on the ZP of mouse embryos at the zygote stage for individual embryo marking and their accurate identification. Embryo quality assessment every 24 h post laser-assisted marking as well as immunofluorescence staining (for ICM/TE cell number ratio calculation) were performed.
Initial experiments have started with embryo marking in a single equatorial plane. The codes engraved could be clearly recognized until the thinning of the ZP prior to hatching. Since embryo may change its orientation during the ART cycle, multi-plane code engraving seems to be more practical for simplifying the process of code searching and embryo identification. We have marked the ZP in three planes, and no decrease in developmental rates as well as no morphological changes of embryos post laser-assisted engraving have been observed as compared to control group embryos.
Our results demonstrate the suitability of femtosecond laser as a novel tool for noninvasive embryo tagging, enabling embryo identification from day 0.5 post coitum to at least early blastocyst stage. Thus, the versatility and the potential use of femtosecond lasers in the field of developmental biology and assisted reproduction have been shown.
本研究旨在探讨飞秒激光脉冲在透明带（ZP）内标记字母数字代码是否可作为一种有效、可靠的方法，用于直接标记植入前胚胎。
将飞秒激光脉冲（波长 514nm，脉冲持续时间 280fs，重复频率 2.5kHz，脉冲能量 20nJ）应用于卵裂期小鼠胚胎的 ZP 上，用于进行个体化胚胎标记和精确定位。对激光辅助标记后每 24 小时的胚胎质量进行评估，并进行免疫荧光染色（用于计算 ICM/TE 细胞数量比）。
初始实验从胚胎的单一赤道平面开始进行标记。在孵化前 ZP 变薄之前，可清晰识别雕刻的代码。由于胚胎在 ART 周期中可能改变其方向，因此多平面标记似乎更适合简化代码搜索和胚胎识别过程。我们已经在三个平面上进行了 ZP 标记，与对照组胚胎相比，激光辅助标记后胚胎的发育率没有下降，也没有观察到胚胎形态发生变化。
我们的研究结果表明，飞秒激光可作为一种新型的非侵入性胚胎标记工具，适用于从受精后 0.5 天至早期囊胚阶段的胚胎鉴定。因此，飞秒激光在发育生物学和辅助生殖领域具有多功能性和潜在用途。
