Skip to main content
SpringerLink
Log in

Slow freezing and vitrification of mouse morula and early blastocysts

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

Abstract

Purpose

To assess the relative success of morula and early blastocyst slow freezing and vitrification in regards to survival and implantation rates utilising protocols which could be clinically implemented as a viable alternative to expanded blastocyst stage freezing.

Methods

Mouse morula and early blastocysts were either slow frozen/thawed or vitrified/warmed. Their subsequent survival, blastocyst development and blastocyst cell number and allocation to either the inner cell mass, trophectoderm or epiblast was assessed. In addition blastocysts were also transferred to pseudopregnant recipients and implantation and fetal development was determined.

Results

Vitrification of both morula and early blastocysts resulted in significantly higher rates of survival and blastocyst development compared to slow freezing. In addition slow frozen early blastocysts had significantly reduced blastocyst cell number compared to control however vitrified morula and early blasocyts and slow frozen morula had equivocal blastocyst cell numbers. Transfer of blastocysts from both methods of cryopreservation resulted in similar implantation rates however the placentas created from slow frozen early blastocysts were significantly lighter than control (95.5 g ± 5.4 vs. 122.0 g ± 4.2 respectively).

Conclusions

Vitrification resulted in significantly higher rates of morula and early blastocyst survival and blastocyst development compared to slow freezing. In addition this study has validated the use of a closed DMSO free vitrification protocol which could then be investigated for use in the clinical setting as an alternative to expanded blastocyst freezing.

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

Similar content being viewed by others

References

  1. Blake DA, Farquhar CM, Johnson N, Proctor M. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Syst Rev. 2007;CD002118.

  2. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability. Fertil Steril. 2003;79:407–10.

    Article  PubMed  Google Scholar 

  3. Lane M, Gardner DK, Hasler MJ, Hasler JF. Use of G1.2/G2.2 media for commercial bovine embryo culture: equivalent development and pregnancy rates compared to co-culture. Theriogenology. 2003;60:407–19.

    Article  PubMed  Google Scholar 

  4. Breheny S, Healy D, Halliday J, Jaques A, Rushford D, Garrett C, et al. Fresh is not always best. An analysis of birth outcomes for women having fresh and frozen embryo transfers. The Australian and New Zealand J Obstet Gynaecol (ANZJOG). 2010;50:23.

    Google Scholar 

  5. Kansal Kalra S, Ratcliffe SJ, Milman L, Gracia CR, Coutifaris C, Barnhart KT. Perinatal morbidity after in vitro fertilization is lower with frozen embryo transfer. Fertil Steril. 2011;95:548–53.

    Article  PubMed  Google Scholar 

  6. Maheshwari A, Pandey S, Shetty A, Hamilton M, Bhattacharya S. Obstetric and perinatal outcomes in singleton pregnancies resulting from the transfer of frozen thawed versus fresh embryos generated through in vitro fertilization treatment: a systematic review and meta-analysis. Fertil Steril. 2012;98(368–77):e1–9.

    PubMed  Google Scholar 

  7. Kader A, Falcone T, Sharma RK, Mangrola D, Agarwal A. Slow and ultrarapid cryopreservation of biopsied mouse blastocysts and its effect on DNA integrity index. J Assist Reprod Genet. 2010;27:509–15.

    Article  PubMed  Google Scholar 

  8. Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction. 2011;141:1–19.

    Article  PubMed  CAS  Google Scholar 

  9. Vanderzwalmen P, Bertin G, et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod. 2002;17:744–51.

    Article  PubMed  Google Scholar 

  10. Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod. 2003;18:137–9.

    Article  PubMed  CAS  Google Scholar 

  11. Koustas G, Shaw L, Sjoblom C. The effect of vitrification on imprinted genes H19 and IGF2 in pre-implantation mouse embryos. Hum Reprod. 2010;25:i35–7.

    Google Scholar 

  12. Feil D, Henshaw RC, Lane M. Day 4 embryo selection is equal to Day 5 using a new embryo scoring system validated in single embryo transfers. Hum Reprod. 2008;23:1505–10.

    Article  PubMed  Google Scholar 

  13. Lane M, Mitchell M, Cashman KS, Feil D, Wakefield S, Zander-Fox DL. To QC or not to QC: the key to a consistent laboratory? Reprod Fertil Dev. 2008;20:23–32.

    Article  PubMed  Google Scholar 

  14. National-Research-Council. Guide for the care and use of laboratory animals. National research council of the national academies. Washington, DC: The National Academies Press; 2011.

    Google Scholar 

  15. Tao J, Craig RH, Johnson M, Williams B, Lewis W, White J, et al. Cryopreservation of human embryos at the morula stage and outcomes after transfer. Fertil Steril. 2004;82:108–18.

    Article  PubMed  Google Scholar 

  16. Tao J, Tamis R, Fink K. Cryopreservation of mouse embryos at morula/compact stage. J Assist Reprod Genet. 2001;18:235–43.

    Article  PubMed  CAS  Google Scholar 

  17. Mitchell M, Schulz SL, Armstrong DT, Lane M. Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention. Biol Reprod. 2009;80:622–30.

    Article  PubMed  CAS  Google Scholar 

  18. Zander DL, Thompson JG, Lane M. Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages. Biol Reprod. 2006;74:288–94.

    Article  PubMed  CAS  Google Scholar 

  19. Campbell JM, Nottle MB, Vassiliev I, Mitchell M, Lane M. Insulin increases Epiblast cell number of in vitro cultured mouse embryos via the PI3K/GSK3/p53 pathway. Stem Cells Dev. 2012;21:2430–41.

    Article  PubMed  CAS  Google Scholar 

  20. Rulicke T, Haenggli A, Rappold K, Moehrlen U, Stallmach T. No transuterine migration of fertilised ova after unilateral embryo transfer in mice. Reprod Fertil Dev. 2006;18:885–91.

    Article  PubMed  Google Scholar 

  21. Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, et al. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril. 2008;90:186–93.

    Article  PubMed  Google Scholar 

  22. 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  PubMed  CAS  Google Scholar 

  23. Larman MG, Katz-Jaffe MG, McCallie B, Filipovits JA, Gardner DK. Analysis of global gene expression following mouse blastocyst cryopreservation. Hum Reprod. 2011;26:2672–80.

    Article  PubMed  CAS  Google Scholar 

  24. Cockburn K, Rossant J. Making the blastocyst: lessons from the mouse. J Clin Invest. 2010;120:995–1003.

    Article  PubMed  CAS  Google Scholar 

  25. Lane M, Gardner DK. Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil. 1997;109:153–64.

    Article  PubMed  CAS  Google Scholar 

  26. Lane M, Gardner DK. Ammonium induces aberrant blastocyst differentiation, metabolism, pH regulation, gene expression and subsequently alters fetal development in the mouse. Biol Reprod. 2003;69:1109–17.

    Article  PubMed  CAS  Google Scholar 

  27. Mitchell M, Cashman KS, Gardner DK, Thompson JG, Lane M. Disruption of mitochondrial malate-aspartate shuttle activity in mouse blastocysts impairs viability and fetal growth. Biol Reprod. 2009;80:295–301.

    Article  PubMed  CAS  Google Scholar 

  28. Campbell JM, Mitchell M, Nottle MB, Lane M. Development of a mouse model for studying the effect of embryo culture on embryonic stem cell derivation. Stem Cells Dev. 2011;20:1577–86.

    Article  PubMed  Google Scholar 

  29. Massip A, Van der Zwalmen P, Leroy F. Effect of stage of development on survival of mouse embryos frozen–thawed rapidly. Cryobiology. 1984;21:574–7.

    Article  PubMed  CAS  Google Scholar 

  30. Raju GA, Prakash GJ, Krishna KM, Madan K. Vitrification of human early cavitating and deflated expanded blastocysts: clinical outcome of 474 cycles. J Assist Reprod Genet. 2009;26:523–9.

    Article  PubMed  Google Scholar 

  31. Edgar DH, Karani J, Gook DA. Increasing dehydration of human cleavage-stage embryos prior to slow cooling significantly increases cryosurvival. Reprod Biomed Online. 2009;19:521–5.

    Article  PubMed  Google Scholar 

  32. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology. 2000;40:110–6.

    Article  PubMed  CAS  Google Scholar 

  33. Van Landuyt L, Stoop D, Verheyen G, Verpoest W, Camus M, Van de Velde H, et al. Outcome of closed blastocyst vitrification in relation to blastocyst quality: evaluation of 759 warming cycles in a single-embryo transfer policy. Hum Reprod. 2011;26:527–34.

    Article  PubMed  Google Scholar 

  34. Iwatani M, Ikegami K, Kremenska Y, Hattori N, Tanaka S, Yagi S, et al. Dimethyl sulfoxide has an impact on epigenetic profile in mouse embryoid body. Stem Cells. 2006;24:2549–56.

    Article  PubMed  CAS  Google Scholar 

  35. Morley P, Whitfield JF. The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types. J Cell Physiol. 1993;156:219–25.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Repromed Department of R&D, 180 Fullarton Road, Dulwich, South Australia, Australia

    Deirdre Zander-Fox, Michelle Lane & Hamish Hamilton

  2. Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, South Australia, Australia

    Deirdre Zander-Fox & Michelle Lane

Authors
  1. Deirdre Zander-Fox
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michelle Lane
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hamish Hamilton
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Deirdre Zander-Fox.

Additional information

Capsule Vitrification of morula/early blastocysts results in increased survival rates compared to slow freezing.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zander-Fox, D., Lane, M. & Hamilton, H. Slow freezing and vitrification of mouse morula and early blastocysts. J Assist Reprod Genet 30, 1091–1098 (2013). https://doi.org/10.1007/s10815-013-0056-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-013-0056-4

Keywords

Search

Navigation