Altered Levels of Mitochondrial DNA Are Associated with Female Age, Aneuploidy, and Provide an Independent Measure of Embryonic Implantation Potential

Abstract

Mitochondria play a vital role in embryo development. They are the principal site of energy production and have various other critical cellular functions. Despite the importance of this organelle, little is known about the extent of variation in mitochondrial DNA (mtDNA) between individual human embryos prior to implantation. This study investigated the biological and clinical relevance of the quantity of mtDNA in 379 embryos. These were examined via a combination of microarray comparative genomic hybridisation (aCGH), quantitative PCR and next generation sequencing (NGS), providing information on chromosomal status, amount of mtDNA, and presence of mutations in the mitochondrial genome. The quantity of mtDNA was significantly higher in embryos from older women (P=0.003). Additionally, mtDNA levels were elevated in aneuploid embryos, independent of age (P=0.025). Assessment of clinical outcomes after transfer of euploid embryos to the uterus revealed that blastocysts that successfully implanted tended to contain lower mtDNA quantities than those failing to implant (P=0.007). Importantly, an mtDNA quantity threshold was established, above which implantation was never observed. Subsequently, the predictive value of this threshold was confirmed in an independent blinded prospective study, indicating that abnormal mtDNA levels are present in 30% of non-implanting euploid embryos, but are not seen in embryos forming a viable pregnancy. NGS did not reveal any increase in mutation in blastocysts with elevated mtDNA levels. The results of this study suggest that increased mtDNA may be related to elevated metabolism and are associated with reduced viability, a possibility consistent with the ‘quiet embryo’ hypothesis. Importantly, the findings suggest a potential role for mitochondria in female reproductive aging and the genesis of aneuploidy. Of clinical significance, we propose that mtDNA content represents a novel biomarker with potential value for in vitro fertilisation (IVF) treatment, revealing chromosomally normal blastocysts incapable of producing a viable pregnancy.

Mitochondrial unfolded protein response gene Clpp is required to maintain ovarian follicular reserve during aging, for oocyte competence, and development of pre‐implantation embryos

“…We first focused on parameters reflecting the efficiency of oocyte energy metabolism and found Clpp −/− mice oocytes to have higher amount of ROS, with decreased membrane potential and ATP production (Figure 2a–e), as well as lower expression of genes coding for ETC proteins (Figure 2g). Similar to that observed in human embryos that are aneuploid or fail to implant (Fragouli et al., 2015), Clpp −/− mice oocytes had significantly higher mtDNA copy number (Figure 2f). We then assessed whether the environment with impaired energy metabolism affects mitochondrial dynamics in Clpp −/− oocytes.…”

“…Mitochondrial replication, in turn, starts after implantation (Murakoshi et al., 2013; Pikó & Taylor, 1987). Mitochondrial DNA copy number is higher in aneuploid blastocysts (which contain an abnormal chromosome number) and in euploid blastocysts that fail to implant (Fragouli et al., 2015), suggesting that higher mtDNA copy number reflects embryonic stress and is associated with lower reproductive potential.…”

A putative role for anti-Müllerian hormone (AMH) in optimising ovarian reserve expenditure

“…Aneuploidy incidence rates increase with age in humans and mice, with evidence suggesting age-related degradation of cohesin proteins in the oocyte nucleus is involved (Chiang et al 2010, Lister et al 2010, Kuliev et al 2011, Duncan et al 2012. Oocyte mitochondrial counts also decline in older females, leading to reduced embryo viability (Wai et al 2010, Kushnir et al 2012, Fragouli et al 2015. Additional factors that contribute to the age-related decline in oocyte quality may yet be discovered, but there is no current evidence to suggest that AMH directly affects oocyte quality.…”

Theory about the Embryo Cryo‐Treatment

“…Thus, there are more “healthy” embryos and a higher implantation potential in women of advanced maternal age (Figure 4). In support of this, another study established that, in women of advanced reproductive age, there are statistically higher values of mtDNA present 37. Unfortunately, at this time, the results are not sufficient to definitely confirm or reject the theory.…”