Influence of laser microdisection of the zona pellucida of embryos on the its thinning dynamics under cryopreservation protocols

The authors studied the dynamics of thinning of the zona pellucida (ZP) in mouse embryo as a result of laser-assisted hatching performed at the blastocyst stage. Mouse embryos previously subjected to a freeze–thaw cycle (cryo-embryos) were selected as model embryos. For ZP microsurgery, femtosecond laser pulses were used (radiation wavelength $512$ nm, pulse duration $100$ fs, intensity $2.5$ TW/cm$^2$). The thickness of the ZP was measured before microsurgery at the blastocyst stage ($\sim \rm E3.5$, i.e., $3.5$ days of embryonic development) and at the hatching stage $(\sim \rm E5)$. It was found that the ZP of control group embryos (cryo-embryos not subjected to laser exposure) thinned more strongly (from $6.6~(\rm E3.5)$ to $4.9~\mu$m $(\rm E5))$ compared to experimental embryos after the laser-assisted hatching procedure $($from $7.1~(\rm E3.5)$ to $6.4~\mu$m $(\rm E5))$. In both the first and second cases, changes in ZP thickness were statistically significant. The results were compared with data for “fresh” embryos not subjected to cryopreservation. There was no pronounced effect of ZP hardening in cryo-embryos compared to fresh embryos. The use of the laser-assisted hatching procedure for cryo-embryos made it possible to increase the probability of successful hatching compared to embryos in the control group from $38.5$ to $52.5\%$.