Assessment of the Amount of Cell-Free DNA in Spent Media from Mouse Embryo Culture

Abstract

Introduction: Preimplantation genetic test for aneuploidy（PGT-A）has already been clinically recognized as a method for examining the condition of in vitro fertilized embryos. Cell-free DNA（cfDNA）in the spent culture medium can be used for assessing the status of embryos as an alternative to PGT-A.

Method: We fertilized mice eggs in vitro and cultured them for 5 days. We performed ethanol precipitation on the spent culture medium that contained the embryos, which were in the post-morula stage. Quantitative real-time PCR with the calibration curve method was performed using the mouse brain tissue DNA. Ethanol precipitation was also performed on controls.

Result: We measured the amount of cfDNA present in the spent culture medium. cfDNA was detected in some of the samples but not in all the samples. In the samples in which cfDNA was detected, the amount of cfDNA was approximately 900 pg on an average.

Discussion: We were able to obtain cfDNA from the spent culture medium that contained mice embryo by performing ethanol precipitation. We speculate that the spent culture medium contains cfDNA from few cells, and it was difficult to detect cfDNA in all the cultures due to the presence of a negligible amount. For the recovery of cfDNA from spent culture medium, it was considered necessary to select a purification method based on the subsequent analysis method.