Evaluation of the impact of laser-assisted hatching techniques on the hatching process of mouse blastocysts using time-lapse microscopy

Embryo Biology

VOLUME 2, ISSUE 1, P43-49, FEBRUARY 01, 2021


Maria García-Jiménez, M.Sc., Klaus Rink, Ph.D., Enric Mestres, M.Sc., Ivette Vanrell, M.Sc., Gloria Calderón, Ph.D., Nuno Costa-Borges, Ph.D. 



To study the effect of zona opening (ZO) and 2 zona thinning (ZT) techniques on the hatching process of mouse embryos using a last-generation laser system and time-lapse microscopy (TLM).


Prospective randomized study.


Private research center.


A total of 267 F1 hybrid (B6/CBA) mice embryos were included.


Morulae were randomly selected and the zona pellucida (ZP) manipulated using a laser system according to 4 experimental groups: control (ZP intact, n = 59), ZO (25 μm hole, n = 70), ZT25 (25% perimeter thinned, n = 71), and ZT35 (35% perimeter thinned, n = 67). Embryo development was monitored by TLM until day 6.

Main Outcome Measure(s)

Time to first breach the ZP, hatching time, time to complete hatching, multiple breaching, multiple hatching, loss of cells, hole size, and embryo quality were analyzed.


No significant differences in the proportion of completely hatched embryos were found among groups. However, the time (average hours ± SD) to complete hatching was significantly delayed in the control group compared with all laser-treated groups: 118.3 ± 9.5 hours in the ZT25 group, 116.6 ± 8.7 hours in the ZT35 group, and 120.4 ± 9.9 hours in the ZO group. The applied laser techniques did not interfere with the quality of the blastocysts at day 5/6 of culture.


ZO, ZT25, and ZT35 embryos hatched significantly earlier than the zona intact group without increasing the multiple hatching rates, suggesting an improvement of the hatching process. This study found that the pattern of the hatching process after ZT and ZO differs.