Study of more than 600 euploid blastocysts finds mitochondrial DNA content does not correlate with female age and is not predictive of reproductive outcomes.
There was great interest – and hope – in a presentation at ESHRE’s 2014 annual meeting in Munich which suggested that levels of mitochondrial DNA in preimplantation embryos were associated with female age and aneuploidy, and could even reveal the implantation potential of chromosomally normal embryos. One of two studies described reported that embryos which went on to become viable pregnancies contained significantly lower levels of mitochondrial DNA than those which did not implant. Further analysis went so far as to allow the calculation of a threshold level of mitochondrial DNA above which no embryos would implant. The results were published a year later, but by then there was much speculation that mtDNA copy number might be the key to a new and accurate biomarker of embryo implantation potential – and a clue in the explanation of why some euploid and morphologically sound embryos fail to implant.(1)
Now, after five years of claim and counter claim, a new study from a group with a history in this debate has found that mtDNA copy number is not a predictive biomarker of euploid embryo competence, with results unable ‘to support the use of mitochondrial DNA copy number in clinical decision making when selecting which embryo to transfer’.(2)
Behind the conclusion lies a three-phase single-centre study designed to ‘approximate’ the design of the initial study which Fragouli and colleagues reported in 2015: first to validate the methodology; next to analyse mtDNA copy number in 615 euploid blastocysts used in single embryo transfers (at Reproductive Medicine Associates of New Jersey, USA, between July 2016 and June 2017); and finally to compare the outcome from paired sibling consecutive transfers (derived from the same oocyte retrieval) relative to respective mtDNA levels. The aim of this third-phase, reported the authors, was ‘to determine whether embryos within the same cohort with higher mtDNA copy number were less or more likely to implant’. All the embryos throughout the study had been previously tested for aneuploidy by next generation sequencing.
Results first showed that ongoing implantation was not associated with relative mtDNA copy number, nor with any threshold value above or below which ongoing implantation was more or less likely. The 2015 Fragouli study had found that mtDNA levels were significantly higher in embryos from older women and in aneuploid embryos independent of female age. Moreover, post-transfer assessment had shown that implanting blastocysts tended to contain lower mtDNA quantities than those failing to implant, indicative of a threshold above which implantation was never observed. Conversely, high mtDNA levels were found present in 30% of non-implanting euploid embryos, but were never evident in embryos forming a viable pregnancy. This, said Fragouli et al, thus described ‘a direct relationship between mtDNA quantity and the potential of an embryo to successfully become a baby’. This same conclusion was reaffirmed two years later in a blinded prospective study from the same group.(3)
Now, in this latest US study there was no correlation found between maternal age and relative mtDNA quantity, nor, in its third-phase study, between relative mtDNA levels in sibling embryos and subsequent implantation and delivery rates (in both successful and unsuccessful cycles). Indeed, looking at a catalogue of similar studies over the past few years, the US authors dismiss any association of female age with mtDNA copy number as ‘an inconsistent finding’.
So why the discrepancy? Why should mtDNA quantity in one study be closely – even causally – linked to embryo viability and implantation, yet in another reveal none of these associations? The US authors ask this same question and suggest inconsistencies in the ways in which mtDNA has been quantified. Indeed, in an earlier comment on such inconsistent results Wells et al noted first that ‘the incidence of blastocysts with elevated mtDNA varies widely between clinics and may be zero, or close to it, in some’, and second that ‘suboptimal techniques [of mtDNA quantification] have contributed to significant confusion concerning the relationship between blastocyst mtDNA levels, female age, aneuploidy and viability’.(4) The US authors of the latest study now apply a complex design of ‘targeted amplification’ followed by quantitative PCR for two mitochondrial loci to determine a precise mtDNA copy number.
Not beyond dispute, however, is the function of mitochondria in cellular metabolism, and particularly embryo viability.(5) However, with this latest author rejection of mtDNA quantity as a hopeful step in the ever contentious process of embryo selection, they propose that ‘more robust measures of embryonic mitochondrial function’ may now be necessary to unravel and explain these conflicting results.
1. Fragouli E, Spath K, Alfarawati S, et al. Altered levels of mitochondrial DNA are associated with female age, aneuploidy, and provide an independent measure of embryonic implantation potential. PLoS Genet 2015; 11(6): e1005241. https://doi.org/10.1371/journal.pgen.1005241
2. Scott RT, Sun L, Zhan Y, et al. Mitochondrial DNA content is not predictive of reproductive competence in euploid blastocysts. Reprod Biomed Online 2020; 41: 183-190.
3. Fragouli E, McCaffrey C, Ravichandran K, et al. Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study. Hum Reprod 2017; 32: 2340-2347.
4. Wells D, Ravichandran K, McCaffrey C, et al. Mitochondrial DNA quantification — the devil in the detail. Hum Reprod 2017: 32: 2150-2151.
5. See for example Perez-Sanchez M, Diez-Juan A, Beltran D, et al. Mitochondrial DNA content decreases during in vitro human embryo development: insights into mitochondrial DNA variation in preimplantation embryos donated for research. Fertil Steril Science 2020; 1: 36-45. DOI: https://doi.org/10.1016/j.xfss.2020.05.001