Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome

Mol Hum Reprod. 2014 Dec;20(12):1238-46. doi: 10.1093/molehr/gau086. Epub 2014 Sep 17.


In assisted reproduction technology, embryo competence is routinely evaluated on morphological criteria. Over the last decade, efforts in improving non-invasive embryo assessment have looked into the secretome of embryos. Human embryos release genomic DNA (gDNA) and mitochondrial DNA (mtDNA) into the culture medium, and the mtDNA/gDNA ratio is significantly correlated with embryo fragmentation. Here, we investigate whether mtDNA/gDNA ratio in embryo spent medium is correlated with blastulation potential and implantation. The mtDNA/gDNA ratio was assessed in 699 Day 3 culture media by quantitative polymerase chain reaction (qPCR) to investigate its correlation with embryo morphology, blastocyst development and implantation. A logistic regression model evaluated whether mtDNA/gDNA ratio in the secretome may improve the prediction of blastulation. We found that embryos that successfully developed into blastocysts exhibited a significantly higher mtDNA/gDNA ratio in the culture medium compared with those that arrest (P = 0.0251), and mtDNA/gDNA, combined with morphological grading, has the potential to predict blastulation better than morphology alone (P = 0.02). Moreover, mtDNA/gDNA ratio was higher in the media from good-quality embryos that reached the full blastocyst stage on Day 5 compared with those that developed more slowly (P < 0.0001). With respect to blastocyst morphology, higher trophectoderm quality was associated with a higher mtDNA/gDNA ratio in the culture medium. Finally, a high mtDNA/gDNA ratio in spent medium was associated with successful implantation outcome (P = 0.0452) of good-quality embryos. In summary, the mtDNA/gDNA ratio in the Day 3 embryo secretome, in combination with morphological grading, may be a novel, non-invasive, early biomarker to improve identification of viable embryos with high developmental potential.

Keywords: blastocyst; cell-free DNA; human embryos; implantation; mtDNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blastocyst / metabolism*
  • Blastocyst / pathology
  • Cell Survival
  • Culture Media / metabolism*
  • DNA, Mitochondrial / genetics
  • DNA, Mitochondrial / metabolism*
  • Embryo Culture Techniques
  • Embryo Implantation*
  • Embryo Transfer
  • Genetic Markers
  • Humans
  • Logistic Models
  • Polymerase Chain Reaction
  • Predictive Value of Tests
  • Time Factors


  • Culture Media
  • DNA, Mitochondrial
  • Genetic Markers