Analyzing Cell-free Genomic DNA in Spent Culture Media: Noninvasive Insight into the Blastocysts.

ABSTRACT

A commonly accepted standard protocol for noninvasive techniques for the genetic evaluation of an embryo remains elusive due to inconclusiveness regarding the volume of spent media to be acquired and the possibility of acquiring the same for subsequent analysis. Single embryo culture is imperative for standardizing noninvasive preimplantation testing using cell-free DNA (cf-DNA) released by individual developing embryos. This study aims to compare the development dynamics of single-drop embryonic culture against with group embryonic culture to establish a standardized protocol for noninvasive Preimplantation Genetic Testing (PGT) in bovine. A total of 239 cumulus-oocyte complexes were aspirated and subjected to in vitro maturation and fertilization. Among these, 120 embryos of day 3 were transferred to single-drop culture until the blastocyst stage. The single-drop culture drops were prepared using microdrops of 30 µL. At the blastocyst stage, spent media from all single-drop embryos were utilized for extracting cell-free genomic DNA to standardize the protocol. The blastocyst rate indicates no significant difference between the two culture methods, suggesting that single-drop culture is suitable for the process. Additionally, the extracted spent media yielded sufficient quantities of cf-DNA, supporting its potential use for PGT ( p < 0.05). These findings support the hypothesis that single-drop embryo culture is a viable method for cf-DNA extraction and confirm the potential of using DNA fragments from spent media as a reliable source for noninvasive PGT.