Skip to main content
SpringerLink
Log in

Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method

  • CRYOPRESERVATION
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos.

Methods

Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups.

Results

Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P > 0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P < 0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P < 0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P < 0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P > 0.05).

Conclusions

Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196°C by vitrification. Nature. 1985;313:573–5.

    Article  CAS  PubMed  Google Scholar 

  2. Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online. 2006;12:779–96.

    PubMed  Google Scholar 

  3. Gordts S, Roziers P, Campo R, Noto V. Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil Steril. 1990;53:469–72.

    CAS  PubMed  Google Scholar 

  4. Kuwayama M. Highly efficient vitrification for cryopreservationof human oocytes and embryos: the Cryotop method. Theriogenology. 2007;67:73–80.

    Article  CAS  PubMed  Google Scholar 

  5. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online. 2005;11:300–8.

    PubMed  Google Scholar 

  6. Al-Hasani S, Osmen B, Koutlaki N, Schoepper B, Diedrich K, Schultze-Mosgau A. Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing? Reprod Biomed Online. 2007;14:288–93.

    Article  PubMed  Google Scholar 

  7. Kumasako Y, Kumon M, Utsunomiya T, Araki Y. Successful pregnancy after the vitrification of zygotes using commercial vitrification solutions and conventional straws to protect against infections in liquid nitrogen. J Assist Reprod Genet. 2005;22:33–5.

    Article  PubMed  Google Scholar 

  8. Rama Raju GA, Haranath GB, Krishna KM, Prakash GJ, Madan K. Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates. Reprod Biomed Online. 2005;11:434–7.

    Article  PubMed  Google Scholar 

  9. Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, et al. A randomized controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod. 2008;23:1976–82.

    Article  CAS  PubMed  Google Scholar 

  10. Desai N, Blackmon H, Szeptycki J, Goldfarb J. Cryoloop vitrification of human day 3 cleavage stage embryos: post vitrification development, pregnancy outcomes and live births. Reprod Biomed Online. 2007;14:208–13.

    Article  PubMed  Google Scholar 

  11. Rama Raju GA, Jaya Prakash G, Murali Krishna K, Madan K. Neonatal outcome after vitrified day3 embryo transfer: a preliminary study. Fertile steril. 2009;92(1):143–8.

    Article  Google Scholar 

  12. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod. 2004;19:2884–8.

    Article  PubMed  Google Scholar 

  13. Zech NH, Lejeune B, Zech H, Vanderzwalmen P. Vitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification. Reprod Biomed Online. 2005;11:355–61.

    Article  CAS  PubMed  Google Scholar 

  14. Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. Current aspects of blastocyst cryopreservation. Reprod Biomed Online. 2008;16:311–20.

    Article  CAS  PubMed  Google Scholar 

  15. Park SP, Kim EY, Oh JH, Nam HK, Leek S, Park Y, et al. Ultra-rapid freezing of human multipronuclear zygotes using electronmicroscope grids. Hum Reprod. 2000;15:1787–90.

    Article  CAS  PubMed  Google Scholar 

  16. Son WY, Yoon SH, Park SJ, Yoon HJ, Lee WD, Lim JH. Ongoingtwin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycysticovary syndrome: Case report. Hum Reprod. 2002;17:2963–6.

    Article  PubMed  Google Scholar 

  17. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, Bollen N, Van Roosendal E, et al. Vitrification of human blastocysts with theHemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod. 2003;18:1504–11.

    Article  CAS  PubMed  Google Scholar 

  18. Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reprod Biomed Online. 2002;4:146–50.

    Article  CAS  PubMed  Google Scholar 

  19. Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M, et al. Vitrification of human blastocysts using cryoloops: clinicaloutcome of 223 cycles. Hum Reprod. 2003;18:384–91.

    Article  CAS  PubMed  Google Scholar 

  20. Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using CryoLoop technique. Reprod Biomed Online. 2003;6:221–5.

    Article  PubMed  Google Scholar 

  21. Kono T, Suzuki O, Tsunoda Y. Cryopreservation of rat blastocysts by vitrification. Cryobiology. 1988;25:170–3.

    Article  CAS  PubMed  Google Scholar 

  22. Anzai M, Nakagata N, Matsumoto K, Takahashi A, Takahash Y, Miyata K. Cryopreservation of in vitro fertilized embryos from transgenic rats by ultrarapid freezing. Exp Anim. 1994;43:247–50.

    CAS  Google Scholar 

  23. Hirabayashi M, Chaya N. Low temperature storage of rat 2-cell embryos by vitrification. J Mamm Ova Res. 1990;7:72–7.

    Google Scholar 

  24. Tada N, Sato M, Mizorogi T, Kasai K, Ogawa S. Efficient cyropreservation of hairless mutant and normal Wistar rat embryos by vitrification. Lab Anim Sci. 1995;45:323–5.

    CAS  PubMed  Google Scholar 

  25. Leibo SP, Martino A, Kobayashi S, Pollard JW. Stage-dependent sensitivity of oocytes and embryos to low temperatures. Anim Reprod Sci. 1996;42:45–53.

    Article  Google Scholar 

  26. Han M-S, Niwa K, Kasai M. Vitrification of rat embryos at various developmental stages. Theriogenology. 2003;59:1851–63.

    Article  PubMed  Google Scholar 

  27. Zhou GB, Zhu SE, Hou YP, Jin F, Yang QE, Yang ZQ, et al. Vitrification of mouse embryos at various stages by Open-pulled Straw (OPS) method. Anim Biotechnol. 2005;16:153–63.

    Article  CAS  PubMed  Google Scholar 

  28. Chang-Liang YAN, Qi-En YANG, Guang-Bin ZHOU, Yun-Peng HOU, et al. Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages. Acta Vet Hung. 2008;56(2):245–53.

    Article  Google Scholar 

  29. Ling XF, Zhang JQ, Tong GQ, et al. Effect of cryotop vitrification on preimplantation developmental competence of murine morula and blastocyst stage embryos. Reprod Biomed Online 2009; 19(5):708–13.

    Google Scholar 

  30. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel CryoLoop container-less technique. Fertil Steril. 1999;72:1073–8.

    Article  CAS  PubMed  Google Scholar 

  31. Valojerdi MR, Eftekhari-Yazdi P, Karimian L, et al. Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet Online. 2009;10.

  32. Loutradi KE, Kolibianakis EM. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril. 2008;90(1):186–92.

    Article  PubMed  Google Scholar 

  33. Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Curr Opin Obstet Gynecol. 2009;21:270–4.

    Article  PubMed  Google Scholar 

  34. Lane M, Lyons EA, Bavister BD. Cryopreservation reduces the ability of hamster 2-cell embryos to regulate intracellular pH. Hum Reprod. 2000;15(2):389–94.

    Article  CAS  PubMed  Google Scholar 

  35. Graves-Herring JE, Boone WR. Blastocyst rate and live births from vitrification and slow-cooled two-cell mouse embryos. Fertil Steril. 2009;91(3):920–4.

    Article  PubMed  Google Scholar 

  36. Sheehan CB, Lane M, Gardner DK. The CryoLoop facilitates re-vitrification of embryos at four successive stages of development without impairing embryo growth. Hum Reprod. 2006;21(11):2978–84.

    Article  PubMed  Google Scholar 

  37. Pedro PB, Yokoyama E, Zhu SE, et al. Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants. J Reprod Dev. 2005;51(2):235–46.

    Article  CAS  PubMed  Google Scholar 

  38. Cuello C, Sanchez-Osorio J, Almiñana C, Gil MA, Perals ML, Lucas X, et al. Effect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts. Cryobiology. 2008;56:189–94.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank all our team members for giving technical support and valuable suggestions. This research is supported from grants of 07NMUZ026, ZKX08006 and 200801092.

Author information

Authors and Affiliations

  1. Department of Reproduction, Nanjing Maternity and Child Health Hospital, Nanjing Medical University, Nanjing, 210004, People’s Republic of China

    Junqiang Zhang, Ji Cui, Xiufeng Ling, Xiuling Li, Yuzhu Peng & Guo Qing Tong

  2. Departmant of Paediatrics, Nanjing Maternity and Child Health Hospital, Nanjing Medical University, Nanjing, 210004, People’s Republic of China

    Xirong Guo

  3. Ivymed International Pty Ltd., #03-02a, Serangoon Plaza, 320 Serangoon Road, Singapore, 218108, Singapore

    Boon Chin Heng

Authors
  1. Junqiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ji Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiufeng Ling
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiuling Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuzhu Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xirong Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Boon Chin Heng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guo Qing Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Guo Qing Tong.

Additional information

Capsule Vitrification of early cleavage stage mouse embryos results in better retention of developmental potential when applied to later (4-8 cell) rather than earlier (2-cell) stages of development suggesting that this method of cryopreservation may require stage-specific applications in a species-specific context.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, J., Cui, J., Ling, X. et al. Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method. J Assist Reprod Genet 26, 621–628 (2009). https://doi.org/10.1007/s10815-009-9370-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-009-9370-2

Keywords

Search

Navigation