An investigation of the effects of laser-assisted zona pellucida drilling on the preimplantation mouse embryo and the competency of embryo implantation

Objective: To investigate the impact of laser-assisted zona pellucida (ZP) drilling on the mouse embryo, with particular emphasis on molecular mechanisms, and the efficiency of embryo attachment capability using an in vitro model of implantation. Design: Experimental study. Setting: Academic research laboratory. Patients/Animals: C57BL/6JOlaHsd mouse embryos and B6C3F1 × B6D2F1 mouse embryos. Interventions: 8-cell stage mouse embryos were randomly assigned to a laser-assisted ZP drilling group (n=343), a ZP-partial drilling group (n=312), a ZP-quarter thinning group (n=289), and a control group (n=353). Embryos were cultured in vitro from E2.5 to E4.5 for 48 hours. To investigate the capacity to implant, E4.5 embryos (laser-assisted drilling group (n=46), ZP-partial drilling group (n=28), ZP-quarter thinning group (n=26), and control group (n=36)), were then transferred onto an attachment model based on Ishikawa cells and cultured for another 72 hours. Main Outcome Measures: Blastocyst formation, hatching status, and hatching morphology at E4.5. Blastocyst cell components, the extent of apoptosis in embryonic cells (DNA fragmentation, caspase-3 activation, and the expression of apoptosis-related genes), the expression of HSP70, and differentially expressed genes (DEGs) generated by RNA sequencing. Fully hatched embryo rate and stable attachment rate at E7.5 in the in vitro attachment model. Results: There were no significant differences between the laser-assisted ZP manipulation groups and the control group with respect to the formation of blastocysts, cell number, embryonic cell apoptosis, and cellular stress. All three of the laser-assisted ZP manipulations significantly increased the hatching rate at E4.5 when compared with control group, especially the ZP drilling group. However, only ZP drilling group was associated with a significantly higher proportion of ‘8’-shape hatching blastocysts. Furthermore, RNA-sequencing identified 48 DEGs between blastocysts from the laser-assisted drilling group and the control group; the metabolic pathways were significantly enriched in these DEGs. In addition, there were no significant differences between the laser-assisted ZP manipulation groups and the control group with respect to the rate of stable attachment at E7.5, although a significantly higher entrapment rate was observed in the ZP drilling group. Conclusions: Laser-assisted ZP manipulations did not induce cellular apoptosis or stress in mouse blastocysts. Nevertheless, for the first time, we found that laser-assisted ZP drilling could alter the embryonic transcriptome and may affect metabolic activity. Furthermore, although laser-assisted ZP manipulations can enhance the initiation of hatching, it is evident that ZP drilling comes with a potential risk of embryo entrapment.