Laser drilling is used clinically for assisted hatching and for artificial shrinkage of the blastocoel cavity prior to vitrification. However, there is limited research on the safety of the heat emitted from the laser or its impact on embryo development and viability. Therefore, the aim of this study was to establish the effect of laser assisted hatching and artificial shrinkage on embryo development and ROS production.
Mouse zygotes were collected from super-ovulated pre-pubertal females (CBAF1, n=1182) and cultured in G1/G2 sequential media at 37°C in 6%CO2:5%O2:89%N2 for 73h. Morula/blastocysts were lasered or artificial shrunk, by directly lasering the blastocyst, and cultured for a further 24h. Morphology, cell number, and ROS levels (MitoSOX Red) were assessed on day 5 (97h culture). Statistical analysis by univariate general linear modelling with LSD post-hoc test, performed in SPSS software.
There was no effect of laser drilling on blastocyst development on day 5 or cell number (P>0.05) between the groups, control (60.9±2.9, n=45), 5µm laser drilling (59.8±2.9, n=45), 5µm laser drilling + artificial shrinkage (58.0±2.9, n=45).
ROS levels (proportion of MitoSOX positive cells / total number of cells) were significantly elevated by 5µm laser drilling + artificial shrinkage (13.8%±1.3, n=45) compared to controls (9.4%±1.3, n=45) (P<0.05). Additionally, MitoSOX positive cells in artificial shrinkage treatment were localised to the site of laser pulse. Although there was an increase in ROS levels in the 5µm laser drilling treatment group (10.7%±1.3, n=45, P<0.08) this did not reach significance.
There appears to be no detrimental impact of laser drilling for assisted hatching on embryo development or ROS production. However, laser drilling for artificial shrinkage did increase ROS levels in the area of the laser pulse, suggesting cellular damage. Further research is underway to assess the impact of laser drilling on subsequent viability, particularly when the embryo is directly pulsed.