Total And Mitochondrial Cell Free DNA Quantification in Day 5 Embryos Culture Media Reflect Embryos Quality

Background: In vitro fertilization (IVF) technology still uses the morphological criteria as the main approach for selecting embryos of a certain quality, embryo fragmentation, blastomere size and cleavage rate. This group of tools is routinely used to grade cleavage stages of human embryos.
As a result of blastomere fragmentation, cell free mitochondrial DNA (cf mtDNA) is released into the embryo culture medium. Our study aims to confirm the presence of cell-free DNA (cfDNA) in embryos culture media by detecting a specific gene using PCR and to evaluate the correlation between two kinds of cf DNA (total cfDNA and cf mtDNA) content in blastocyst stage (Day 5 embryos) culture media and embryo grading.

 
Subject and Method: 40 spent culture media samples are collected; each blastocyst was morphologically graded. cfDNA is extracted from embryo culture media. Quality of cfDNA is checked by conventional PCR with specific primer then visualized by agarose gel. The cf mtDNA is profiled by isothermal PCR.
Results: Purified cfDNA from embryo culture media could be used to amplify specific genes by PCR. Further studies indicated that insignificant interdependent relationship is found when correlating the total cfDNA amount on day 5 and embryo grading. Similarly, the significant interdependent relationship is found when correlating cf mtDNA amount on day 5 and embryo grade. Notably, a significant correlation is noticed between cf mtDNA amount and blastocyst formation.
Conclusion: We confirmed the presence of cfDNA in embryo’s culture media and noticed a significant correlation between the quality and embryos secrotome cf mtDNA levels.