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Time-lapse video analysis demonstrates laser-assisted hatching of ∼33% of the zona-pellucida after blastocyst warming significantly improves an embryo’s ability to fully hatch

      Objective

      The human embryo is known to undergo Zona-Pellucida (ZP) hardening due both to extended culture to the blastocyst and as a consequence of vitrification. This may affect the blastocysts ability to escape the ZP post-warming, especially within an optimal window of endometrial receptivity. We set out to determine if the percentage of blastocysts that were able to fully escape the ZP post-warming was different if laser assisted hatching (LAH) of ∼33% of the ZP was performed immediately after warming compared to embryos that were not LAH.

      Design

      Prospective analysis of donated vitrified/warmed embryos placed in an Embryoscope with and without LAH.

      Materials and Methods

      Overall, 24 research good/fair quality blastocysts vitrified on Day 5 and 6 were used. The mean age was 38.3 with a range of 23-46. Warming of embryos was performed per established protocol on the same day, at the same time, under the same incubation conditions. Embryos were randomized to either a group that had ∼33% of the ZP ablated using LAH on a Zilos laser at 400 μs (Group 1) or no LAH (Group 2). All embryos were incubated for 48 hours in an Embryoscope time lapse incubator in GTL medium (Vitrolife). We recorded if the embryo escaped the ZP, the time it took to fully hatch from the ZP and the number of expansions each embryo underwent to escape the ZP.

      Results

      Assessment was performed to determine if and when embryos hatched out. In Group 1, 11 of 14 embryos (78.6%) achieved complete hatching within 24 hours, with an average of 17.0 hours elapsed to fully hatch and an average of 2.1 expansions. In Group 2, none of the 10 embryos achieved complete hatching within 24 hours, after having undergone an average of 2.6 expansions. Two of the Group 2 embryos (20%) achieved complete hatching, at 37 and 48 hours with an average of 5 expansions. Group 1 vs. Group 2 complete hatching within 24 hours was significantly different (p<0.01).

      Conclusions

      Time lapse video evidence provides compelling information regarding the hatching process of blastocysts post-warming. A significantly higher percentage of warmed embryos were capable of completing the hatching process when ∼33% of the ZP was ablated as compared to those that were not LAH. Importantly, the average time for the LAH embryos to completely escape the ZP was within the optimal time range of endometrial receptivity. The LAH embryos also demonstrated that the expenditure of energy to complete the hatching process, through repeated expansions, is minimized, thus ensuring the energy stores are not depleted prior to implantation. Conversely, an overwhelming percentage of warmed embryos that do not have LAH prior to embryo transfer are unsuccessful in completing the hatching process, even within 48 hours, which is outside optimal endometrial receptivity. Overall, LAH of ∼33% of the ZP post-warming is highly beneficial to the embryo’s escape and could significantly increase implantation and success rates.