Journal of Assisted Reproduction and Genetics

, Volume 31, Issue 4, pp 435–442 | Cite as

Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method: A time-lapse study

  • Mette Warming JoergensenEmail author
  • Inge Agerholm
  • Johnny Hindkjaer
  • Lars Bolund
  • Lone Sunde
  • Hans Jakob Ingerslev
  • Kirstine Kirkegaard
Embryo Biology



To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method.


Time-lapse analysis.


Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p < 0.001), displayed longer duration of the 3-cell stage (p < 0.001), and arrested development from the compaction stage and onwards (p < 0.001). For the IVF embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0.001). More 3PN IVF than ICSI embryos displayed early cleavage into 3 cells (p < 0.001).


This study reports differences in cleavage patterns between 2PN and 3PN embryos and for the first time demonstrates differences in the cleavage pattern between 3PN IVF and ICSI embryos.


Cell division Embryo Humans Triploidy Time-lapse 



The authors wish to thank the clinical, paramedical, and laboratory team of the Fertility Clinic, Aarhus University Hospital. The study was supported by Aarhus University, University of Southern Denmark, Aase and Einar Danielsen Foundation, Lipperts Foundation, the Augustinus Foundation and the Toyota Foundation. Research at the Fertility Clinic is funded by an unrestricted grant from Ferring and MSD.

Conflict of interest

M.W.J., J.H., L.B., L.S., H.J.I, and K.K, declare no conflicts of interest. I.A. works part-time as a scientific consultant for Unisense FertiliTech and holds stocks in the company.

Supplementary material


(MPEG 13782 kb)

10815_2014_178_MOESM2_ESM.pdf (32 kb)
ESM 2 (PDF 31 kb)


  1. 1.
    Grossmann M, Calafell JM, Brandy N, Vanrell JA, Rubio C, Pellicer A, et al. Origin of tripronucleate zygotes after intracytoplasmic sperm injection. Hum Reprod. 1997;12:2762–5.PubMedCrossRefGoogle Scholar
  2. 2.
    Staessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum Reprod. 1997;12:321–7.PubMedCrossRefGoogle Scholar
  3. 3.
    Yu SL, Lee RK, Su JT, Chih YF, Tsai YC, Lin MH, et al. Distinction between paternal and maternal contributions to the tripronucleus in human zygotes obtained after in vitro fertilization. Taiwan J Obstet Gynecol. 2006;45:313–6.PubMedCrossRefGoogle Scholar
  4. 4.
    Jacobs PA, Szulman AE, Funkhouser J, Matsuura JS, Wilson CC. Human triploidy: relationship between parental origin of the additional haploid complement and development of partial hydatidiform mole. Ann Hum Genet. 1982;46:223–31.PubMedCrossRefGoogle Scholar
  5. 5.
    Jauniaux E, Brown R, Rodeck C, Nicolaides KH. Prenatal diagnosis of triploidy during the second trimester of pregnancy. Obstet Gynecol. 1996;88:983–9.PubMedCrossRefGoogle Scholar
  6. 6.
    Macas E, Imthurn B, Rosselli M, Keller PJ. The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection. Hum Reprod. 1996;11:2496–501.PubMedCrossRefGoogle Scholar
  7. 7.
    Plachot M, Crozet N. Fertilization abnormalities in human in-vitro fertilization. Hum Reprod. 1992;7 Suppl 1:89–94.PubMedCrossRefGoogle Scholar
  8. 8.
    Angell RR, Templeton AA, Messinis IE. Consequences of polyspermy in man. Cytogenet Cell Genet. 1986;42:1–7.PubMedCrossRefGoogle Scholar
  9. 9.
    Rosenbusch B, Schneider M, Sterzik K. The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization. Hum Reprod. 1997;12:2257–62.PubMedCrossRefGoogle Scholar
  10. 10.
    Coonen E, Harper JC, Ramaekers FC, Delhanty JD, Hopman AH, Geraedts JP, et al. Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation. Hum Genet. 1994;94:609–15.PubMedCrossRefGoogle Scholar
  11. 11.
    Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod. 1987;37:395–401.PubMedCrossRefGoogle Scholar
  12. 12.
    Lim AS, Goh VH, Su CL, Yu SL. Microscopic assessment of pronuclear embryos is not definitive. Hum Genet. 2000;107:62–8.PubMedCrossRefGoogle Scholar
  13. 13.
    Pieters MH, Dumoulin JC, Ignoul-Vanvuchelen RC, Bras M, Evers JL, Geraedts JP. Triploidy after in vitro fertilization: cytogenetic analysis of human zygotes and embryos. J Assist Reprod Genet. 1992;9:68–76.PubMedCrossRefGoogle Scholar
  14. 14.
    Pang MG, Jee BC, Kim SH, Ryu BY, Oh SK, Suh CS, et al. Chromosomal constitution of embryos derived from tripronuclear zygotes studied by fluorescence in situ hybridization using probes for chromosomes 4, 13, 18, 21, X, and Y. Gynecol Obstet Invest. 2005;59:14–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Wong CC, Loewke KE, Bossert NL, Behr B, De Jonge CJ, Baer TM, et al. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat Biotechnol. 2010;28:1115–21.PubMedCrossRefGoogle Scholar
  16. 16.
    Palermo G, Munne S, Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum Reprod. 1994;9:1220–5.PubMedGoogle Scholar
  17. 17.
    Sathananthan AH, Kola I, Osborne J, Trounson A, Ng SC, Bongso A, et al. Centrioles in the beginning of human development. Proc Natl Acad Sci U S A. 1991;88:4806–10.PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Kirkegaard K, Hindkjaer JJ, Grondahl ML, Kesmodel US, Ingerslev HJ. A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator. J Assist Reprod Genet. 2012;29:565–72.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Kirkegaard K, Agerholm IE, Ingerslev HJ. Time-lapse monitoring as a tool for clinical embryo assessment. Hum Reprod. 2012;27:1277–85.PubMedCrossRefGoogle Scholar
  20. 20.
    Sundvall L, Ingerslev HJ, Breth Knudsen U. Kirkegaard K. Hum Reprod: Inter- and intra-observer variability of time-lapse annotations; 2013.Google Scholar
  21. 21.
    Gonzales DS, Pinheiro JC, Bavister BD. Prediction of the developmental potential of hamster embryos in vitro by precise timing of the third cell cycle. J Reprod Fertil. 1995;105:1–8.PubMedCrossRefGoogle Scholar
  22. 22.
    Grisart B, Massip A, Dessy F. Cinematographic analysis of bovine embryo development in serum-free oviduct-conditioned medium. J Reprod Fertil. 1994;101:257–64.PubMedCrossRefGoogle Scholar
  23. 23.
    Oh SJ, Gong SP, Lee ST, Lee EJ, Lim JM. Light intensity and wavelength during embryo manipulation are important factors for maintaining viability of preimplantation embryos in vitro. Fertil Steril. 2007;88:1150–7.PubMedCrossRefGoogle Scholar
  24. 24.
    Azimzadeh J, Bornens M. Structure and duplication of the centrosome. J Cell Sci. 2007;120:2139–42.PubMedCrossRefGoogle Scholar
  25. 25.
    Balakier H. Tripronuclear human zygotes: the first cell cycle and subsequent development. Hum Reprod. 1993;8:1892–7.PubMedGoogle Scholar
  26. 26.
    Schatten H, Sun QY. The role of centrosomes in mammalian fertilization and its significance for ICSI. Mol Hum Reprod. 2009;15:531–8.PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Rubio I, Kuhlmann R, Agerholm I, Kirk J, Herrero J, Escriba MJ, et al. Limited implantation success of direct-cleaved human zygotes: a time-lapse study. Fertil Steril. 2012;98:1458–63.PubMedCrossRefGoogle Scholar
  28. 28.
    Gu YF, Lin G, Lu CF, Lu GX. Analysis of the first mitotic spindles in human in vitro fertilized tripronuclear zygotes after pronuclear removal. Reprod Biomed Online. 2009;19:745–54.PubMedCrossRefGoogle Scholar
  29. 29.
    Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature. 1988;332:459–61.PubMedCrossRefGoogle Scholar
  30. 30.
    McFadden DE, Jiang R, Langlois S, Robinson WP. Dispermy–origin of diandric triploidy: brief communication. Hum Reprod. 2002;17:3037–8.PubMedCrossRefGoogle Scholar
  31. 31.
    Zaragoza MV, Surti U, Redline RW, Millie E, Chakravarti A, Hassold TJ. Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole. Am J Hum Genet. 2000;66:1807–20.PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Kirkegaard K, Hindkjaer JJ, Ingerslev HJ. Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring. Fertil Steril 2013; 99:738–744 e734.Google Scholar
  33. 33.
    Wale PL, Gardner DK. Time-lapse analysis of mouse embryo development in oxygen gradients. Reprod Biomed Online. 2010;21:402–10.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mette Warming Joergensen
    • 1
    • 2
    Email author
  • Inge Agerholm
    • 3
  • Johnny Hindkjaer
    • 4
  • Lars Bolund
    • 5
  • Lone Sunde
    • 5
    • 6
  • Hans Jakob Ingerslev
    • 4
  • Kirstine Kirkegaard
    • 4
    • 7
  1. 1.Department of Clinical GeneticsVejle HospitalVejleDenmark
  2. 2.Institute of Regional Health ResearchUniversity of Southern DenmarkOdenseDenmark
  3. 3.The Fertility ClinicHorsens HospitalHorsensDenmark
  4. 4.The Fertility Clinic and Centre for Preimplantation Genetic DiagnosisAarhus University HospitalSkejbyDenmark
  5. 5.Department of BiomedicineAarhus UniversityAarhusDenmark
  6. 6.Department of Clinical GeneticsAarhus University HospitalSkejbyDenmark
  7. 7.Department of Clinical BiochemistryAarhus University HospitalSkejbyDenmark

Personalised recommendations