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

Abstract

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.

Segregation of Naturally Occurring Mitochondrial DNA Variants in a Mini-Pig Model

“…This process is aided by the shedding of mtDNA during preimplantation development (Stigliani et al 2014). This suggests that these variants provide a positive functional role during early development but, as they may be potentially lethal later in life, they are subsequently reduced.…”

The embryonic stress response to in vitro culture: insight from genomic analysis

“…The replication of mitochondrial DNA is under dynamic regulation during pre-implantation development (Cagnone et al 2016) and stress responses could trigger the release of this DNA into the culture medium (due to cell death or clearance of damaged mitochondria). Recent studies have shown that an increased copy number of the mtDNA genome in embryonic cells and in spent media is a sign of a metabolic response to developmental stress (Stigliani et al 2013(Stigliani et al , 2014.…”

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization

“…Palini et al (23) reported the observation of the existence of DNA in the blastocoele fluid, Gianaroli et al (24) performed a pilot study on chromosome screening using blastocentesis, and Stigliani et al (25,26) observed genomic and mitochondria DNA contents in the culture medium, which were correlated with embryo quality. Wu et al (27) reported the PCR detection of the secreted genomic DNA in the culture medium for preimplantation…”