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Mitochondrial DNA, a new biomarker of embryonic implantation potential: fact or fiction?

2018; Elsevier BV; Volume: 109; Issue: 1 Linguagem: Inglês

10.1016/j.fertnstert.2017.10.017

1556-5653

Peter Humaidan, Stine Gry Kristensen, Kevin Coetzee,

Assisted Reproductive Technology and Twin Pregnancy

In their most recent publication, de los Santos et al. (1de los Santos M.J. Juan A.D. Mifsud A. Mercader A. Meseguer M. Rubio C. et al.Variables associated with mitochondrial copy number in human blastocysts: what can we learn from trophectoderm biopsies?.Fertil Steril. 2017; 109: 110-117Abstract Full Text Full Text PDF Scopus (43) Google Scholar) address the unfolding questions surrounding the biological significance or not of mitochondrial DNA (mtDNA) in trophectoderm cells of human blastocysts. Interestingly, the authors of the study focus on the relationship between patient characteristics, ovarian stimulation, blastocyst culture variables, and mtDNA rather than on the possible relationship between mtDNA and the reproductive outcome. A total of 1,641 biopsied blastocysts from 402 patients were included in the study, which is consistent with the numbers included in studies published to date. The authors report that maternal body mass index, serum P levels on the day of ovulation induction, and blastocyst aneuploidy/quality affect the mtDNA content. Although the authors have some difficulty in rationalizing the outcomes observed, recent studies have also been plagued by contradictory outcomes. Thus, maternal age, known to be significantly associated with reproductive outcomes, was found to be associated with mtDNA content in some studies (2Fragouli E. McCaffrey C. Ravichandran K. Spath K. Grifo J.A. Munné S. et al.Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study.Hum Reprod. 2017; 32: 2340-2347Crossref PubMed Scopus (72) Google Scholar, 3Ravichandran K. McCaffrey C. Grifo J. Morales A. Perloe M. Munne S. et al.Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers.Hum Reprod. 2017; 32: 1282-1292Crossref PubMed Scopus (80) Google Scholar, 4Treff N.R. Zhan Y. Tao X. Olcha M. Han M. Rajchel J. et al.Levels of trophectoderm mitochondrial DNA do not predict the reproductive potential of sibling embryos.Hum Reprod. 2017; 32: 954-962PubMed Google Scholar) but not in others (1de los Santos M.J. Juan A.D. Mifsud A. Mercader A. Meseguer M. Rubio C. et al.Variables associated with mitochondrial copy number in human blastocysts: what can we learn from trophectoderm biopsies?.Fertil Steril. 2017; 109: 110-117Abstract Full Text Full Text PDF Scopus (43) Google Scholar, 5Victor A.R. Brake A.J. Tyndall J.C. Griffin D.K. Zouves C.G. Barnes F.L. et al.Accurate quantitation of mitochondrial DNA reveals uniform levels in human blastocysts irrespective of ploidy, age, or implantation potential.Fertil Steril. 2017; 107: 34-42Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Moreover, outcomes related to blastocyst aneuploidy, quality (i.e., blastocyst expansion and grade), and viability were similarly found to be contradictory in these studies, suggesting fundamental discrepancies in inter-laboratory methodologies and patient selection differences, which casts some doubt on whether mtDNA at present is of any prognostic value for embryo selection in daily clinical practice. The relatively low implantation rates of cultured embryos during IVF treatment, as well as the inability to discriminate embryos with or without implantation potential, continues to concern. Static morphology-based scoring methods have remained the cornerstone of laboratory-based embryo selection, although there is only a modest association between embryo scoring and implantation. Despite the introduction of extended embryo culture, dynamic embryo selection using time-lapse monitoring technologies, and whole-genome aneuploidy screening using preimplantation genetic testing technologies, a significant number of high-quality and chromosomally normal embryos or blastocysts still do not implant. As a result of whole-genome preimplantation genetic testing innovation, the measurement of mtDNA of oocytes, blastomeres of embryos, and differentiated cells of blastocysts was recently proposed as a biomarker of implantation potential. However, conflicting results in the area have already challenged the potential significance of mtDNA in human implantation: some studies report a clear association between high mtDNA quantities and failure of blastocyst implantation (2Fragouli E. McCaffrey C. Ravichandran K. Spath K. Grifo J.A. Munné S. et al.Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study.Hum Reprod. 2017; 32: 2340-2347Crossref PubMed Scopus (72) Google Scholar, 3Ravichandran K. McCaffrey C. Grifo J. Morales A. Perloe M. Munne S. et al.Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers.Hum Reprod. 2017; 32: 1282-1292Crossref PubMed Scopus (80) Google Scholar), whereas others do not find such a relationship (4Treff N.R. Zhan Y. Tao X. Olcha M. Han M. Rajchel J. et al.Levels of trophectoderm mitochondrial DNA do not predict the reproductive potential of sibling embryos.Hum Reprod. 2017; 32: 954-962PubMed Google Scholar, 5Victor A.R. Brake A.J. Tyndall J.C. Griffin D.K. Zouves C.G. Barnes F.L. et al.Accurate quantitation of mitochondrial DNA reveals uniform levels in human blastocysts irrespective of ploidy, age, or implantation potential.Fertil Steril. 2017; 107: 34-42Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). One of the most obvious and simple explanations for the contradictory results was reported by Ravichandran et al. (3Ravichandran K. McCaffrey C. Grifo J. Morales A. Perloe M. Munne S. et al.Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers.Hum Reprod. 2017; 32: 1282-1292Crossref PubMed Scopus (80) Google Scholar). The study comprised data provided by multiple clinics, and importantly, large deviations in mtDNA levels across clinics were seen. In fact, 17 of 35 of the clinics that contributed data did not have any blastocysts with elevated mtDNA content. Thus, clinics that do not produce embryos with high mtDNA levels will not detect an association to implantation failure, and thus the phenomenon of elevated mtDNA could potentially be a center-induced event rather than a universal biological occurrence. Embryos with high mtDNA levels, which are associated with cellular stress, might simply be influenced by center-specific factors, such as the choice of culture medium, embryo handling, or hormonal stimulation regimens. Sample storage is another issue which could potentially affect valid quantification of mtDNA. This point was recently raised by the group of D. Wells (6Wells D. Ravichandran K. McCaffrey C. Grifo J. Morales A. Perloe M. Munne S. Fragouli E. Reply: Mitochondrial DNA quantification-the devil in the detail.Hum Reprod. 2017; 32: 2150-2151Crossref PubMed Scopus (12) Google Scholar), suggesting that sample degradation could have had an impact on the lacking associations between mtDNA and embryo implantation in recent studies. According to their experience, genome amplification samples frequently fail to give valid mtDNA quantification results after 6 months of storage, partly owing to differential rates of degradation of sequences used for quantification of mtDNA and for normalization. In the study by Treff et al. (4Treff N.R. Zhan Y. Tao X. Olcha M. Han M. Rajchel J. et al.Levels of trophectoderm mitochondrial DNA do not predict the reproductive potential of sibling embryos.Hum Reprod. 2017; 32: 954-962PubMed Google Scholar), the samples had been stored up to 6.5 years, which could potentially have influenced their quantification of mtDNA; however, this in our opinion is yet another speculative explanation for the conflicting results in the mtDNA area. Current scientific evidence supports the notion that the mtDNA quantification methodology has some major technical limitations. Therefore it is essential that platforms with validated accuracy and reproducibility must be used, especially because mtDNA has been found to vary significantly even within and between mitochondria. As mentioned, the measurement of mtDNA levels produces a highly skewed distribution, with only a small proportion of euploid blastocysts having levels above a viability threshold (3Ravichandran K. McCaffrey C. Grifo J. Morales A. Perloe M. Munne S. et al.Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers.Hum Reprod. 2017; 32: 1282-1292Crossref PubMed Scopus (80) Google Scholar). Moreover, the measurement of mtDNA may be further complicated in that intra-laboratory factors, in terms of in vitro culture conditions and embryo manipulation techniques, may significantly affect mtDNA levels (2Fragouli E. McCaffrey C. Ravichandran K. Spath K. Grifo J.A. Munné S. et al.Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study.Hum Reprod. 2017; 32: 2340-2347Crossref PubMed Scopus (72) Google Scholar, 3Ravichandran K. McCaffrey C. Grifo J. Morales A. Perloe M. Munne S. et al.Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers.Hum Reprod. 2017; 32: 1282-1292Crossref PubMed Scopus (80) Google Scholar). Further research is, therefore, needed to determine the platform (i.e., array comparative genomic hybridization, next-generation sequencing, quantitative polymerase chain reaction), number and type of gene sequences, calculations (i.e., normalization and correction factors), and quality controls that most optimally quantify mtDNA levels—not only of embryos or blastocysts screened for aneuploidy but also of unscreened embryos or blastocysts. Furthermore, the optimal threshold to discriminate between normal and abnormal mtDNA levels needs to be validated. Until now, mtDNA quantification studies have only been performed on embryos and blastocysts undergoing single gene defect or aneuploidy screening, which could represent a significant bias. In conclusion, whether mtDNA measurement of cultured blastocysts provides value in the selection of blastocysts with a high implantation potential certainly deserves further research, especially research including a broad selection of infertile patients. Although the evidence of value seems encouraging, because the transfer of blastocysts with elevated levels of mtDNA resulted in no pregnancies (2Fragouli E. McCaffrey C. Ravichandran K. Spath K. Grifo J.A. Munné S. et al.Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study.Hum Reprod. 2017; 32: 2340-2347Crossref PubMed Scopus (72) Google Scholar, 3Ravichandran K. McCaffrey C. Grifo J. Morales A. Perloe M. Munne S. et al.Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers.Hum Reprod. 2017; 32: 1282-1292Crossref PubMed Scopus (80) Google Scholar), the value may still be limited: only 16% of nonviable blastocysts had elevated levels of mtDNA (2Fragouli E. McCaffrey C. Ravichandran K. Spath K. Grifo J.A. Munné S. et al.Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study.Hum Reprod. 2017; 32: 2340-2347Crossref PubMed Scopus (72) Google Scholar). Nonetheless, the inter-laboratory variation in mtDNA observed suggests that the measurement of mtDNA levels might be a significant key performance marker for IVF laboratories. Finally, further research is needed to investigate whether mtDNA quantification optimally discriminates between embryos of a single cohort or between the embryos of patients having the same infertility etiology. Improved selection of developmentally competent embryos and blastocysts will certainly reduce the number of transfers required to achieve a live birth and thus the time to pregnancy. Before routine use in the IVF laboratory the challenge at this point is standardization and validation of the methodology. Variables associated with mitochondrial copy number in human blastocysts: what can we learn from trophectoderm biopsies?Fertility and SterilityVol. 109Issue 1PreviewTo study the potential variables that affect the mitochondrial DNA (mtDNA) content of trophectoderm (TE) cells in blastocysts that have undergone TE biopsy. Full-Text PDF