Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

This morphometric protocol allows monitoring of the intensity of blastocyst shrinkage and re-expansion during prior vitrification interventions and post-warming recovery and provides insights into the effectiveness of virus vitrification methods. The main advantage of this technique is that it can be applied to in vitro fertilization laboratories equipped with time-lapse microscopes and computers with advanced image editing software. On day five or six of expansion, subject a blastocyst with at least a few inner cell mass cells and a cohesive trophectoderm to a 0.4 to 0.7 millisecond laser pulse with a hole diameter ranging from 4.3 to 9.4 micrometers tangentially directed at the zona pellucida and the junction of two adjacent trophectodermal cells.

Then, allow the blastocoel to fully or partially collapse for five minutes. Next, use a tiny pipette to aseptically add equilibration medium into the holes of the microdroplet area of a nine-well petri dish, specially designed for time-lapse microscopy and recording. When all the of the medium has been added, overlay the microdroplet area with approximately 30 microliters of equilibration medium.