Kinetics of cell shrinkage and developmental competence of mouse zygotes vitrified following conventional or automated (DaVitri) protocols.

Abstract

Objective: To test the developmental ability of murine zygotes vitrified using a novel vitrification device and microfluidic chip (DaVitri®, Overture Life).

Design: Murine zygotes were randomly allocated to two groups, one was vitrified using the vitrification device and the other following a conventional manual protocol.

Subjects: Murine zygotes obtained in vivo.

Exposure: Automatic vitrification was achieved by a linear exposure to cryoprotectants using DaVitri device. Manual vitrification was conducted using Kitazato® kit.

Main outcome measures: Morphokinetic behavior of the zygotes during the exposure to cryoprotectants analyzed by microscopy, developmental rates following thawing, lineage development at the blastocyst stage assessed by immunohistochemistry and light-structured fluorescent microscopy, and survival rates and pup weight following embryo transfer.

Results: Automated vitrification led to a gradual reduction in zygote volume during the equilibration steps preceding ultra-fast cooling in liquid nitrogen, as opposed to the conventional manual protocol where sharp changes in zygote volume were observed as a result of exposure to static concentrations of cryoprotectants. Survival rates of the automated procedure were comparable to those of the manual protocol, resulting in ∼95% blastocyst formation rates. Developmental analysis of the resulting blastocysts revealed comparable numbers of total, trophectoderm and inner cell mass numbers in blastocysts developed from zygotes vitrified under the manual and automated protocols. No differences were found in survival to term or pup weight a D1 or D21.

Conclusion: Automated vitrification using DaVitri device diminished the osmotic stress caused by exposure to CPAs during the equilibration steps and resulted in comparable developmental competence in terms of development to blastocysts, lineage segregation and survival to term.