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. 2021 Feb;38(2):517-529. doi: 10.1007/s10815-020-01998-x. Epub 2020 Nov 17.
To study whether the application of femtosecond laser pulses for zona pellucida (ZP) drilling of blastocysts at the embryonic or abembryonic poles can promote hatching to start immediately through the hole formed and ensure high hatching rates and embryo viability.
Mouse blastocyst (E3.5) ZP were microdissected with femtosecond laser pulses (514-nm wavelength, 280-fs pulse duration, 2.5-kHz repetition rate) close to the trophoblast or inner cell mass (ICM). The sizes of the holes formed were in the range of 4.5-8.5 μm. Additional longitudinal incisions (5-7-μm long) on either side of the hole were created to determine whether hatching had started at the correct position. Embryos post-laser-assisted ZP drilling and intact embryos were cultured under standard conditions for 2 days; embryo quality was assessed twice daily. The hatching rates and in vitro and in vivo implantation rates (only for embryos with ZP dissected close to the ICM) were estimated.
Femtosecond laser-assisted ZP drilling at the early blastocyst stage facilitated embryo hatching to start at the artificial opening with probability approaching 100%. Despite the artificial opening's small size, no embryo trapping during hatching was observed. Both experimental groups had higher hatching rates than the control groups (93.3-94.7% vs. 83.3-85.7%, respectively). The in vitro implantation rate was comparable with that of the control group (92.3% vs. 95.4%). No statistically significant differences were obtained in the in vivo implantation rates between the experimental and control groups.
Blastocyst-stage femtosecond laser microsurgery of ZP is fast and delicate and enables the hatching process to be initiated in a controlled manner through a relatively small opening, with no embryo trapping.
研究在胚胎或胚胎极的透明带（ZP）上应用飞秒激光脉冲进行爆破胚泡钻孔，是否可以通过形成的孔立即启动孵化，并确保高孵化率和胚胎活力。
使用飞秒激光脉冲（514nm 波长，280fs 脉冲持续时间，2.5kHz 重复率）在靠近滋养层或内细胞团（ICM）的位置对小鼠胚泡（E3.5）ZP 进行微解剖。形成的孔的大小在 4.5-8.5μm 范围内。在孔的任一侧创建额外的纵向切口（5-7μm 长），以确定孵化是否已在正确位置开始。激光辅助 ZP 钻孔后的胚胎和完整的胚胎在标准条件下培养 2 天；每天评估两次胚胎质量。评估孵化率以及体外和体内植入率（仅适用于在 ICM 附近解剖 ZP 的胚胎）。
在早期胚泡阶段，飞秒激光辅助 ZP 钻孔有助于胚胎在人工开口处开始孵化，成功率接近 100%。尽管人工开口很小，但在孵化过程中没有观察到胚胎被捕获。实验组的孵化率均高于对照组（93.3-94.7% vs. 83.3-85.7%）。体外植入率与对照组相当（92.3% vs. 95.4%）。实验组和对照组的体内植入率无统计学差异。
ZP 的胚泡期飞秒激光显微手术快速而精细，可以通过相对较小的开口以受控方式启动孵化过程，并且不会捕获胚胎。
