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.

Fig 1. The relationship between mtDNA quantity, female age and embryo chromosome constitution.

a) Data obtained during quantitative real-time PCR analysis of TE samples removed from 302 blastocysts demonstrated a statistically significant increase (P = 0.003) in the level of mtDNA in relation to advancing female age. This phenomenon was evident for both euploid and aneuploid blastocysts. b) Real-time PCR analysis of 39 blastomeres showed that cleavage stage embryos from reproductively younger women contained significantly (P = 0.01) higher mtDNA levels, compared to those generated by reproductively older women. c) Real-time PCR analysis of TE samples also demonstrated that aneuploid blastocysts (n = 99) contained significantly (P = 0.025) larger quantities of mtDNA at all ages, compared to those that were euploid (n = 203). Statistical analysis of mtDNA values took place with the use of unpaired two-tailed t-tests.

Fig 2. mtDNA quantification via NGS analysis of chromosomally normal and abnormal blastocysts.

NGS analysis of TE samples biopsied from 38 embryos showed a statistically significant increase (P = 0.006) in the mtDNA levels occurring in the presence of chromosome errors.

Fig 3. The mtDNA content of chromosomally normal blastocysts in relation to clinical outcome.

On average, chromosomally normal blastocysts capable of establishing a clinical pregnancy contained significantly (P = 0.007) lower levels of mtDNA compared to chromosomally normal blastocysts that failed to do so.

Fig 4. Blastocyst mtDNA quantity threshold in relation to clinical outcome.

a) The mtDNA quantity viability threshold for euploid blastocysts, established via retrospective analysis of TE biopsies from transferred embryos with known outcomes. All blastocysts producing viable pregnancies contained mtDNA quantities below the 0.003 value (red line) whereas mtDNA quantities above this value were associated with failure to achieve an ongoing clinical pregnancy. b) Results of the prospective blinded study. The mtDNA threshold used was the same as that established in the retrospective study (4a). Validity was confirmed, since all blastocysts producing viable pregnancies contained mtDNA quantities below the cut-off (red line) and no blastocysts with mtDNA quantities above this value achieved an ongoing clinical pregnancy. c) NGS analysis of the mtDNA level in 23 euploid TE samples. The corresponding embryos were transferred during SET cycles, and clinical outcomes were known for 21 of them. As with the real-time PCR experiments, mtDNA levels were lower in the seven implanting embryos (note- the y-axis scale is different for NGS analyses and consequently cut-off values differ).