Journal of Assisted Reproduction and Genetics

, Volume 36, Issue 9, pp 1867–1875 | Cite as

mtDNA dynamics between cleavage-stage embryos and blastocysts

  • Neelke De MunckEmail author
  • Alberto Liñán
  • Ibrahim Elkhatib
  • Aşina Bayram
  • Ana Arnanz
  • Carmen Rubio
  • Nicolas Garrido
  • Barbara Lawrenz
  • Human M. Fatemi
Assisted Reproduction Technologies



The aim was to evaluate mtDNA content and its dynamics in euploid and aneuploid embryos from cleavage to blastocyst stage following consecutive biopsies. The effect of female age on mtDNA content was evaluated by comparing reproductively younger (≤ 37 years) with older (> 37 years) women.


A retrospective single-centre descriptive study was performed between August 2016 and January 2017. Forty patients, with 112 embryos, undergoing preimplantation genetic testing for aneuploidies (PGT-A) by next-generation sequencing (NGS) were included. Embryos that reached the blastocyst stage and were not selected for fresh embryo transfer were included following consecutive biopsies of a single blastomere on day 3 and trophectoderm biopsy of day 5 blastocysts.


Cleavage-stage mtDNA was significantly lower in fast cleaving embryos (p = 0.016). Based on the concordance between day 3 and day 5 biopsies, a difference was identified in blastocyst mtDNA content between groups (p = 0.019); true euploid blastocysts presented a lower mtDNA content. No association was identified between cleavage-stage mtDNA content and ploidy status (OR 1.008 [0.981–1.036], p = 0.565) nor between blastocyst mtDNA content and ploidy outcome (OR 0.954 [0.898–1.014], p = 0.129). No difference was found when comparing mtDNA content and ploidy outcome between the two reproductive age groups (p = 0.505 (cleavage stage) and p = 0.774 (blastocyst)).


Mitochondrial DNA content of cleavage-stage embryos and blastocysts is unable to predict ploidy status. Subgroup analysis based on ploidy concordance between day 3 and day 5 revealed a significantly lower mtDNA content for true euploid blastocysts. Reproductive ageing does not affect mtDNA content.


Cleavage-stage embryo Blastocyst mtDNA PGT-A NGS 


Compliance with ethical standards

Approval for this study was obtained from the local Ethics Committee of IVIRMA Middle East Fertility Clinic, Abu Dhabi, UAE (Research Ethics Committee IVI-MEREF010/2017/REFA009). All patients signed a consent form allowing additional trophectoderm biopsy of supernumerary euploid and aneuploid embryos not selected for transfer.

Supplementary material

10815_2019_1544_MOESM1_ESM.docx (13 kb)
ESM 1 (DOCX 12 kb)


  1. 1.
    Edwards RG. Causes of early pregnancy loss. Hum Reprod. 1986;1(3):185–98.CrossRefGoogle Scholar
  2. 2.
    Van Blerkom J. Mitochondrial function in the human oocyte and embryo and their role in developmental competence. Mitochondrion. 2011;11:797–813.CrossRefGoogle Scholar
  3. 3.
    Eichenlaub-Ritter U, Wieczorek M, Lüke S, Seidel T. Age related changes in mitochondrial function and new approaches to study redox regulation in mammalian oocytes in response to age or maturation conditions. Mitochondrion. 2011;11(5):783–96.CrossRefGoogle Scholar
  4. 4.
    Reynier P, May-Panloup P, Chretien MF, Morgan CJ, Jean M, Savagner F, et al. Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Rep. 2001;7:425–9.CrossRefGoogle Scholar
  5. 5.
    May-Panloup P, Chrétien MF, Jacques C, Vasseur C, Malthièry Y, Reynier P. Low oocyte mitochondrial DNA content in ovarian insufficiency. Hum Reprod. 2005;20(3):593–7.CrossRefGoogle Scholar
  6. 6.
    Seli E. Mitochondrial DNA as a biomarker for in-vitro fertilization outcome. Curr Opin Obstet Gynecol. 2016;28(3):158–63.CrossRefGoogle Scholar
  7. 7.
    Ogino M, Tsubamoto H, Sakata K, Oohama N, Hayakawa HM, Kojima T, et al. Mitochondrial DNA copy number in cumulus cells is a strong predictor of obtaining good-quality embryos after IVF. J Assist Reprod Genet. 2016;33(3):367–71.CrossRefGoogle Scholar
  8. 8.
    Desquiret-Dumas Desquiret-Dumas V, Clément A, Seegers V, Boucret L, Ferré-L’Hotellier V, Bouet PE, et al. The mitochondrial DNA content of cumulus granulosa cells is linked to embryo quality. Hum Reprod. 2017;32(3):607–14.Google Scholar
  9. 9.
    Santos TA, El Shourbagy S, St John JC. Mitochondrial content reflects oocyte variability and fertilization outcome. Fertil Steril. 2006;85(3):584–91.CrossRefGoogle Scholar
  10. 10.
    Cecchino GN, Garcia-Velasco JA. Mitochondrial DNA copy number as a predictor of embryo viability. Fertil Steril. 2018:0015–282.Google Scholar
  11. 11.
    Hashimoto S, Morimoto N, Yamanaka M, Matsumoto H, Yamochi T, Goto H, et al. Quantitative and qualitative changes of mitochondria in human preimplantation embryos. J Assist Reprod Genet. 2017;34(5):573–80.CrossRefGoogle Scholar
  12. 12.
    Lin DP, Huang CC, Wu HM, Cheng TC, Chen CI, Lee MS. Comparison of mitochondrial DNA contents in human embryos with good or poor morphology at the 8-cell stage. Fertil Steril. 2004;81(1):73–9.CrossRefGoogle Scholar
  13. 13.
    Shang W, Zhang Y, Shu M, Wang W, Ren L, Chen F, et al. Comprehensive chromosomal and mitochondrial copy number profiling in human IVF embryos. Reprod BioMed Online. 2018;36(1):67–74.CrossRefGoogle Scholar
  14. 14.
    Ho JR, Arrach N, Rhodes-Long K, Salem W, McGinnis LK, Chung K, et al. Blastulation timing is associated with differential mitochondrial content in euploid embryos. J Assist Reprod Genet. 2018;35(4):711–20.CrossRefGoogle Scholar
  15. 15.
    Diez-Juan A, Rubio C, Marin C, Martinez S, Al-Asmar N, Riboldi M, et al. Mitochondrial DNA content as a viability score in human euploid embryos: less is better. Fertil Steril. 2015;104(3):534–41.CrossRefGoogle Scholar
  16. 16.
    Van Blerkom J. Mitochondria as regulatory forces in oocytes, preimplantation embryos and stem cells. Reprod BioMed Online. 2008;16(4):553–69.CrossRefGoogle Scholar
  17. 17.
    St John JC, Facucho-Oliveira J, Jiang Y, et al. Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells. Hum Reprod Update. 2010;16:488–509.CrossRefGoogle Scholar
  18. 18.
    Fragouli E, Spath K, Alfarawati S, Kaper F, Craig A, Michel CE, 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:e1005241.CrossRefGoogle Scholar
  19. 19.
    de los Santos MJ, Diez-Juan A, 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. 2018;109(1):110–7.CrossRefGoogle Scholar
  20. 20.
    Victor AR, Brake AJ, Tyndall JC, Griffin DK, Zouves CG, Barnes FL, et al. Accurate quantitation of mitochondrial DNA reveals uniform levels in human blastocysts irrespective of ploidy, age, or implantation potential. Fertil Steril. 2017;107(1):34–42.CrossRefGoogle Scholar
  21. 21.
    Ravichandran 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(6):1282–92.CrossRefGoogle Scholar
  22. 22.
    Fragouli E, McCaffrey C, Ravichandran K, Spath K, Grifo J, Munne S, et al. Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study. Hum Reprod. 2017;32(11):2340–7.CrossRefGoogle Scholar
  23. 23.
    Klimczak AM, Pacheco LE, Lewis KE, Massahi N, Richards JP, Kearns WG, et al. Embryonal mitochondrial DNA: relationship to embryo quality and transfer outcomes. J Assist Reprod Genet. 2018;35(5):871–7.CrossRefGoogle Scholar
  24. 24.
    Fragouli E, Wells D. Mitochondrial DNA assessment to determine oocyte and embryo viability. Semin Reprod Med. 2015;33:401–9.CrossRefGoogle Scholar
  25. 25.
    Cree LM, Hammond ER, Shelling AN, Berg MC, Peek JC, Green MP. Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones. Hum Reprod. 2015;30(6):1410–20.CrossRefGoogle Scholar
  26. 26.
    Boucret L, de la Barca JM C, Morinière C, Desquiret V, Ferré-L’Hôttelier V, Descamps P, et al. Relationship between diminished ovarian reserve and mitochondrial biogenesis in cumulus cells. Hum Reprod. 2015;30(7):1653–64.CrossRefGoogle Scholar
  27. 27.
    Babayev E, Seli E. Oocyte mitochondrial function and reproduction. Curr Opin Obstet Gynecol. 2015;27(3):175–81.CrossRefGoogle Scholar
  28. 28.
    Treff NR, 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(4):954–62.Google Scholar
  29. 29.
    La Marca A, Sunkara SK. Individualization of controlled ovarian stimulation in IVF using ovarian reserve markers: from theory to practice. Hum Reprod Update. 2014;20(1):124–40.CrossRefGoogle Scholar
  30. 30.
    Steirteghem V, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod. 1993;8(7):1061–6.CrossRefGoogle Scholar
  31. 31.
    Wells D, Kaur K, Grifo J, Glassner M, Taylor JC, Fragouli E, et al. Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J Med Genet. 2014;51(8):553–62.CrossRefGoogle Scholar
  32. 32.
    Kung A, Munné S, Bankowski B, Coates A, Wells D. Validation of next-generation sequencing for comprehensive chromosome screening of embryos. Reprod BioMed Online. 2015;31(6):760–9.CrossRefGoogle Scholar
  33. 33.
    Phillips NR, Sprouse ML, Roby RK. Simultaneous quantification of mitochondrial DNA copy number deletion ratio: a multiplex real-time PCR assay. SciRep. 2014;4:3887.Google Scholar
  34. 34.
    Stigliani S, Persico L, Lagazio C, Anserini P, Venturini PL, Scaruffi P. Mitochondrial DNA in day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome. Mol Hum Reprod. 2014;20(12):1238–46.CrossRefGoogle Scholar
  35. 35.
    Van Blerkom J, Davis P, Alexander S. Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organisation, ATP content and competence. Hum Reprod. 2000;15(12):2621–33.CrossRefGoogle Scholar
  36. 36.
    Murakoshi Y, Sueoka K, Takahashi K, Sato S, Sakurai T, Tajima H, et al. Embryo developmental capability and pregnancy outcome are related to the mitochondrial DNA copy number and ooplasmic volume. J Assist Reprod Genet. 2013;30(10):1367–75.CrossRefGoogle Scholar
  37. 37.
    Wells D. Mitochondria DNA quantity as a biomarker for blastocyst implantation potential. Fertil Steril. 2017;108(5):742–7.CrossRefGoogle Scholar
  38. 38.
    Cimadomo D, Capalbo A, Ubalid FM, Scarica C, Palagiano A, Cnaipari R, et al. The impact of biopsy on human embryo developmental potential during preimplantation genetic diagnosis. Biomed Res Int. 2016;2016:7193075.CrossRefGoogle Scholar
  39. 39.
    Capalbo A, Rienzi L. Mosaicism between trophectoderm and inner cell mass. Fertil Steril. 2017;107(5):1098–106.CrossRefGoogle Scholar
  40. 40.
    Munné S, Wells D. Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing. Fertil Steril. 2017;107:1085–91.CrossRefGoogle Scholar
  41. 41.
    Gleicher N, Metzger J, Croft G, Kushnir VA, Albertini DF, Barad DH. A single trophectoderm biopsy at blastocyst stage is mathematically unable to determine embryo ploidy accurately enough for clinical use. Reprod Biol Endocrinol. 2017;15(1):33.CrossRefGoogle Scholar
  42. 42.
    Northrop LE, Treff NR, Levy B, Scott RT Jr. SNP microarray based 24 chromosome aneuploidy screening demonstrates that cleavage stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts. Mol Hum Reprod. 2010;16:590–600.CrossRefGoogle Scholar
  43. 43.
    Capalbo A, Wright G, Elliott T, Ubaldi FM, Rienzi L, Nagy ZP. FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage. Hum Reprod. 2013;28:2298–307.CrossRefGoogle Scholar
  44. 44.
    Fragouli E, Munne S, Wells D. The cytogenetic constitution of human blastocysts: insights from comprehensive chromosome screening strategies. Hum Reprod Update. 2019;25(1):15–33.CrossRefGoogle Scholar
  45. 45.
    Viotti M, Victor AR, Zouves CG, Barnes FL. Is mitochondrial DNA quantitation in blastocyst trophectoderm cells predictive of developmental competence and outcome in clinical IVF? J Assist Reprod Genet. 2017;34(12):1581–5.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.IVIRMA Middle East Fertility ClinicAbu DhabiUnited Arab Emirates
  2. 2.IVIRMA Middle East Fertility ClinicMuscatSultanate of Oman
  3. 3.IgenomixValenciaSpain
  4. 4.IVI Foundation, Health Research Institute La FeValenciaSpain
  5. 5.Obstetrical DepartmentWomen’s University Hospital TuebingenTuebingenGermany

Personalised recommendations