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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files.Funding: DW is supported by the NIHR Biomedical Research Centre Oxford. This study was funded by Reprogenetics UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: EF, SA and DW are employees of Reprogenetics UK. This does not alter our adherence to all PLOS Genetics policies in Author SummaryMitochondria are small membrane-enclosed structures and are found inside the cells of the body. Mitochondria actively participate in cellular life, and their main function is to generate energy which is used by the cell. For this reason mitochondria are considered as the powerhouses of cells. Unlike other cellular organelles, mitochondria contain their own DNA (mtDNA). MtDNA carries important genetic information concerning cellular metabolism and the gener...

Accurate Quantitation of Mitochondrial DNA Reveals Uniform Levels in Human Blastocysts Irrespective of Ploidy, Age, or Implantation Potential

“…Conditions that deviate from the steady state such as aneuploidy, advanced maternal age, or chemically induced-stress tend to associate with higher mtDNA content. Of clinical relevance, such reports suggested that embryos with mtDNA levels above the norm showed significantly poor frequency of pregnancy when transferred (4,5).…”

“…In human embryos, recent studies have investigated amounts of mtDNA, generally describing a correlation of high mtDNA lev (4)(5)(6). Conditions that deviate from the steady state such as aneuploidy, advanced maternal age, or chemically induced-stress tend to associate with higher mtDNA content.…”

“…Therefore, in the case of euploid embryos being chosen for transfer, sub-optimal intrinsic or environmental factors could elicit higher levels of mtDNA. Consequently, mtDNA content has been proposed as a biomarker for embryo viability (4,5).…”

Adjuncts in the IVF laboratory: where is the evidence for ‘add-on’ interventions?

“…It has been estimated that metaphase II oocytes contain about 10 5 mitochondrial DNA (mtDNA) copies, but since no replication of the mtDNA occurs until the blastocyst stage of embryonic development, the mtDNA molecules are divided over the cleaving cells (Fragouli and Wells, 2015). In 2015, two papers were published reporting an association between higher mtDNA level and lower implantation potential in blastocysts (Diez-Juan et al, 2015;Fragouli et al, 2015), pointing to disturbed energy provision and metabolic stress in embryos with a higher mtDNA content.…”

“…In 2015, two papers were published reporting an association between higher mtDNA level and lower implantation potential in blastocysts (Diez-Juan et al, 2015;Fragouli et al, 2015), pointing to disturbed energy provision and metabolic stress in embryos with a higher mtDNA content. While the paper of Diez-Juan et al focused on euploid, transferred blastocysts, the other report also showed a relationship between aneuploidy of the blastocyst and a higher mtDNA load.…”