title: Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers.
abstract: Does the amount of mitochondrial DNA (mtDNA) in blastocyst biopsy specimens have the potential to serve as a biomarker of euploid embryo implantation ability, independent of morphology?
  
  
  
  The results of this study strongly suggest that elevated mtDNA levels, above a previously defined threshold, are strongly associated with blastocyst implantation failure and represent an independent biomarker of embryo viability.
  
  
  
  Improved methods of embryo selection are highly desirable in order to increase the efficiency of IVF treatment. At present, even the transfer of chromosomally normal embryos of high morphological grade cannot guarantee that a pregnancy will follow. Recently, it has been proposed that the quantity of mtDNA in embryonic cells may be an indicator of developmental potential, with higher levels of mtDNA associated with reduced implantation. However, thus far reported data sets have been relatively small and in some cases have lacked appropriate validation.
  
  
  
  This large, blinded, retrospective study involved the analysis of relative mtDNA levels in 1505 euploid blastocysts obtained from 490 couples undergoing preimplantation genetic testing for aneuploidy. Implantation outcomes were compared to mtDNA levels in order to determine the capacity of the method to predict viability and to assess the validity of previously established thresholds.
  
  
  
  DNA from blastocyst biopsy samples was amplified and then subjected to aneuploidy analysis using next generation sequencing or array comparative genomic hybridization. Only those embryos classified as chromosomally normal had their mtDNA levels assessed. This analysis was undertaken retrospectively using quantitative real-time PCR, without knowledge of the outcome of embryo transfer. Predictions of implantation failure, based upon mtDNA levels were subsequently compared to the observed clinical results. All cycles involved the transfer of a single embryo.
  
  
  
  Of all blastocysts analyzed, 9.2% (139/1505) contained mtDNA levels above a previously established viability threshold and were therefore predicted to have reduced chances of implantation. To the date of analysis, 282 euploid blastocysts had been transferred with an overall implantation rate of 65.6% (185/282). Of the transferred embryos, 249 contained levels of mtDNA in the normal range, 185 of which produced a pregnancy, giving an implantation rate of 74.3% for euploid embryos with 'normal' quantities of mtDNA. However, 33 of the transferred embryos were determined to have elevated mtDNA quantities. None of these led to a pregnancy. Therefore, the negative predictive value of mtDNA assessment in this cohort was 100% (33/33). The difference between the implantation rates for embryos with normal and elevated mtDNA levels was highly significant (P < 0.0001). The mtDNA thresholds, used for classification of embryos, were unaffected by female age or the clinic in which the IVF was undertaken. The probability of an embryo having elevated levels of mtDNA was not influenced by variation in embryo morphology.
  
  
  
  This study provides strong evidence that mtDNA quantification can serve as a valuable tool to assist the evaluation of blastocyst viability. However, to determine the true extent of any clinical benefits, other types of investigations, such as non-selection studies and randomized controlled trials, will also be necessary.
  
  
  
  The results of this study suggest that mtDNA quantity can serve as an independent biomarker for the prediction of euploid blastocyst implantation potential. Prospective studies should now be undertaken to confirm these results. Additionally, investigations into the underlying biological cause(s) of elevated mtDNA levels and an enhanced understanding of how they relate to diminished implantation potential would be invaluable.
  
  
  
  This study was supported by funding provided by Reprogenetics. None of the authors have any competing interests.
authors.affiliation: Reprogenetics, A Cooper Surgical Company, 3 Regent Street, Livingston, NJ 07039, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ravichandran
authors.affiliation: NYU Langone Medical Center, 333 E 38th Street, New York, NY 10016, USA.
authors.forename: C
authors.initials: C
authors.lastname: McCaffrey
authors.affiliation: NYU Langone Medical Center, 333 E 38th Street, New York, NY 10016, USA.
authors.forename: J
authors.initials: J
authors.lastname: Grifo
authors.affiliation: Fertility Specialists Medical Group, 8010 Frost Street, San Diego, CA 92123, USA.
authors.forename: A
authors.initials: A
authors.lastname: Morales
authors.affiliation: Georgia Reproductive Specialists, 5445 Meridian Mark Road, Atlanta, GA 30342, USA.
authors.forename: M
authors.initials: M
authors.lastname: Perloe
authors.affiliation: Reprogenetics, A Cooper Surgical Company, 3 Regent Street, Livingston, NJ 07039, USA.
authors.forename: S
authors.initials: S
authors.lastname: Munne
authors.affiliation: Reprogenetics UK, Institute for Reproductive Sciences, Oxford Business Park North, Oxford OX4 2HW, UK.|Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.
authors.forename: D
authors.initials: D
authors.lastname: Wells
authors.affiliation: Reprogenetics UK, Institute for Reproductive Sciences, Oxford Business Park North, Oxford OX4 2HW, UK.|Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.
authors.forename: E
authors.initials: E
authors.lastname: Fragouli
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D001755:Blastocyst / Q000378:metabolism*; D015331:Cohort Studies; D004272:DNA, Mitochondrial / Q000378:metabolism*; D015536:Down-Regulation; D004477:Ectogenesis; D005191:Family Characteristics; D005260:Female; D005307:Fertilization in Vitro; D047109:Fetal Development; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007247:Infertility, Female / Q000378:metabolism / Q000628:therapy*; D007248:Infertility, Male; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D011247:Pregnancy; D018873:Pregnancy Rate; D015203:Reproducibility of Results; D056826:Single Embryo Transfer; D014481:United States / Q000453:epidemiology
chemical_list: D015415:Biomarkers; D004272:DNA, Mitochondrial
keywords: biomarker; blastocyst; embryo viability; mitochondrial DNA; mitochondrion