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 2 groups; one was vitrified using the vitrification device, and the other was following a conventional manual protocol.

Subjects

Murine zygotes obtained in vivo.

Exposure

Automatic vitrification was achieved by a linear exposure to cryoprotectants (CPAs) using the DaVitri device. Manual vitrification was conducted using Kitazato kit.

Main Outcome Measures

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

Results

Automated vitrification led to a gradual reduction in zygote volume during the equilibration steps preceding ultrafast 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 CPAs. 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.