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Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos

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Abstract

Purpose

The objective of this retrospective study was to evaluate the efficacy of vitrification and slow freezing for the cryopreservation of human cleavage stage embryos in terms of post-warming survival rate, post-warming embryo morphology and clinical outcomes.

Methods

The embryos of 305 patients at cleavage stages were cryopreserved either with vitrification (153 patients) or slow-freezing (152 patients) methods. After warming; the survival rate, post-warmed embryo morphology, clinical pregnancy and implantation rates were evaluated and compared between the two groups.

Result(s)

In the vitrification group versus slow freezing group, the survival rate (96.9% vs. 82.8%) and the post-warmed excellent morphology with all blastomeres intact (91.8% vs. 56.2%) were higher with an odds ratio of 6.607 (95% confidence interval; 4.184–10.434) and 8.769 (95% confidence interval; 6.460–11.904), respectively. In this group, the clinical pregnancy rate (40.5% vs. 21.4%) and the implantation rate (16.6% vs. 6.8%) were also higher with an odds ratio of 2.427 (95%confidence interval; 1.461–4.033) and 2.726 (95% confidence interval; 1.837–4.046), respectively.

Conclusion(s)

Vitrification in contrast to slow freezing is an efficient method for cryopreservation of human cleavage stage embryos. Vitrification provides a higher survival rate, minimal deleterious effects on post-warming embryo morphology and it can improve clinical outcomes.

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References

  1. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 1983;305:707–9. doi:10.1038/305707a0.

    Article  PubMed  CAS  Google Scholar 

  2. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1, 2 propanediol. Fertil Sterli. 1985;44:645–51.

    CAS  Google Scholar 

  3. Anderson AR, Wilkinson SS, Prince S, Crain JL. Reduction of high order multiples in frozen embryo transfers. Reprod Biomed Online. 2005;10:402–5.

    Article  PubMed  Google Scholar 

  4. Mazur P. Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. Cell Biophys. 1990;17:53–92.

    PubMed  CAS  Google Scholar 

  5. 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.

    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. 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. doi:10.1093/humrep/den222.

    Article  PubMed  CAS  Google Scholar 

  8. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online. 2005;11:608–14.

    PubMed  Google Scholar 

  9. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196°C by vitrification. Nature. 1985;313:573–5. doi:10.1038/313573a0.

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  12. Park SP, Kim EY, Oh JH, Nam HK, Leek S, Park Y, et al. Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids. Hum Reprod. 2000;15:1787–90. doi:10.1093/humrep/15.8.1787.

    Article  PubMed  CAS  Google Scholar 

  13. Son WY, Yoon SH, Park SJ, Yoon HJ, Lee WD, Lim JH. Ongoing twin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome: Case report. Hum Reprod. 2002;17:2963–6. doi:10.1093/humrep/17.11.2963.

    Article  PubMed  Google Scholar 

  14. El-Danasouri I, Selman H. Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos. Fertil Steril. 2001;76:400–2. doi:10.1016/S0015-0282(01)01907-0.

    Article  PubMed  CAS  Google Scholar 

  15. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, Van Roosendal E, Vandervorst M, 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. doi:10.1093/humrep/17.3.744.

    Article  PubMed  Google Scholar 

  16. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, Bollen N, Van Roosendal E, et al. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod. 2003;18:1504–11. doi:10.1093/humrep/deg298.

    Article  PubMed  CAS  Google Scholar 

  17. 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.

    PubMed  CAS  Google Scholar 

  18. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology. 2007;67:73–80. doi:10.1016/j.theriogenology.2006.09.014.

    Article  PubMed  CAS  Google Scholar 

  19. 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. doi:10.1016/S0015-0282(99)00418-5.

    Article  PubMed  CAS  Google Scholar 

  20. Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K. Successful birth after transfer of vitrified human blastocysts with use of a CryoLoop containerless technique. Fertil Steril. 2001;76:618–20. doi:10.1016/S0015-0282(01)01968-9.

    Article  PubMed  CAS  Google Scholar 

  21. Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M, et al. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod. 2003;18:384–91. doi:10.1093/humrep/deg047.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  23. Reed ML, Lane M, Gardner DK, Jensen NL, Thompson J, et al. Vitrification of human blastocysts using the CryoLoop method: successful clinical application and birth of offspring. J Assist Reprod Genet. 2002;19:304–6. doi:10.1023/A:1015789532736.

    Article  PubMed  Google Scholar 

  24. Yoon TK, Kim TJ, Park SE, Hong SW, Ko JJ, Chung HM, et al. Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril. 2003;79:1323–6. doi:10.1016/S0015-0282(03)00258-9.

    Article  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  26. Chian RC, Huang JY, Gilbert L, Son WY, Holzer H, Cuis J, et al. Obstetric outcomes following vitrification of in vitro and in vivo matured oocytes. Fertil Steril. 2008; June 23. Epub ahead of print.

  27. Chian RC, Gilbert L, Huang JY, Demirtas E, Holzer H, Benjamin A, et al. Live birth after vitrification on in vitro matured human oocytes. Fertil Steril. 2008;91:372–6. Epub ahead of print.

    Article  PubMed  CAS  Google Scholar 

  28. 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. doi:10.1007/s10815-005-0818-8.

    Article  PubMed  Google Scholar 

  29. Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod. 1998;13:2874–9.

    PubMed  CAS  Google Scholar 

  30. Saito H, Ishida GM, Kaneko T, Kawachiya S, Ohta N, Takahashi T, et al. Application of vitrification to human embryo freezing. Gynecol Obstet Invest. 2000;4:145–9. doi:10.1159/000010236.

    Article  Google Scholar 

  31. Zhu GJ, Jin L, Zhang HW, Liy F, Weiy L, Hu J. Vitrification of human cleaved embryos in vitro fertilization-embryo transfer. Zhonghua Fu Chan Ke Za Zhi. 2005;40:682–4.

    PubMed  Google Scholar 

  32. Zheng WT, Zhuang GL, Zhou CQ, Fang C, Ou JP, Li T, et al. Comparison of the survival of human biopsied embryos after cryopreservation with four different methods using non-transferable embryos. Hum Reprod. 2005;20:1615–8. doi:10.1093/humrep/deh808.

    Article  PubMed  CAS  Google Scholar 

  33. 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 

  34. Rama Raju GA, Jaya Prakash G, Murali Krishna K, Madan K. Neonatal outcome after vitrified day 3 embryo transfer: a preliminary study. Fertile steril 2008; 8 [Epub ahead of print].

  35. 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. doi:10.1093/humrep/deh504.

    Article  PubMed  Google Scholar 

  36. Huang CC, Lee TH, Chen SU, Chen HH, Cheng TC, Liu CH, et al. Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod. 2005;20:122–8. doi:10.1093/humrep/deh540.

    Article  PubMed  Google Scholar 

  37. Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, et al. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reprod Biomed Online. 2005;11:53–7.

    Article  PubMed  Google Scholar 

  38. Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transfer with vitrification using CryoLoop: a 4-year follow-up study. Fertil Steril. 2005;84:88–92. doi:10.1016/j.fertnstert.2004.12.051.

    Article  PubMed  Google Scholar 

  39. 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  PubMed  CAS  Google Scholar 

  40. Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil Steril. 2006;86:20–6. doi:10.1016/j.fertnstert.2006.01.029.

    Article  PubMed  Google Scholar 

  41. Stachecki JJ, Garris J, Sabino S, Caetano JP, Wiemer KE, Cohen J. A new safe, simple and successful vitrification method for bovine and human blastocyst. Reprod Biomed Online. 2008;17:860–7.

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  43. Porter RN, Smith W, Craft IL, Abdulwahid NA, Jacobs HS. Induction of ovulation for in- vitro fertilisation using buserelin and gonadotropins. Lancet. 1984;324:1284–5. doi:10.1016/S0140-6736(84)92840-X.

    Article  Google Scholar 

  44. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1, 2 propanediol. Fertil Steril. 1985;44:645–51.

    PubMed  CAS  Google Scholar 

  45. Prato LD, Borini A, Cattoli M, Bonu MA, Sciajno R, Flamigni C. Endometrial preparation for frozen-thawed embryo transfer with or without pretreatment with gonadotropin-releasing hormone agonist. Fertil Steril. 2002;77:956–60. doi:10.1016/S0015-0282(02)02960-6.

    Article  Google Scholar 

  46. Gardner DK, Pawelczynski M, Trounson AO. Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation. Mol Reprod Dev. 1996;44:472–5. doi:10.1002/(SICI)1098-2795(199608)44:4<472::AID-MRD6>3.0.CO;2-I.

    Article  PubMed  CAS  Google Scholar 

  47. Lane M, Gardner DK. Vitrification of mouse oocytes using a nylon loop. Mol Reprod Dev. 2001;58:342–7. doi:10.1002/1098-2795(200103)58:3<342::AID-MRD13>3.0.CO;2-X.

    Article  PubMed  CAS  Google Scholar 

  48. Lane M, Maybach JM, Gardner DK. Addition of ascorbate during cryopreservation stimulates subsequent embryo development. Hum Reprod. 2002;17:2686–93. doi:10.1093/humrep/17.10.2686.

    Article  PubMed  CAS  Google Scholar 

  49. 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;21:2978–84. doi:10.1093/humrep/del253.

    Article  PubMed  Google Scholar 

  50. Larman MG, Sheehan CB, Gardner DK. Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes. Reproduction. 2006;131:53–61. doi:10.1530/rep.1.00878.

    Article  PubMed  CAS  Google Scholar 

  51. 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. doi:10.1002/jcp.1041560202.

    Article  PubMed  CAS  Google Scholar 

  52. 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. doi:10.1634/stemcells.2005-0427.

    Article  PubMed  CAS  Google Scholar 

  53. 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. doi:10.1016/j.fertnstert.2007.06.010.

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Embryology Department, Reproductive Medicine Research Center, Royan Institute, ACECR, Tehran, Iran

    Mojtaba Rezazadeh Valojerdi, Poopak Eftekhari-Yazdi, Leila Karimian, Fatemeh Hassani & Bahar Movaghar

  2. Department of Anatomy, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran

    Mojtaba Rezazadeh Valojerdi

  3. Department of Embryology, Royan Institute, No.12, Hafez St., Banihashem St., Resalat St., P.O.Box: 19395-4644, Tehran, Iran

    Mojtaba Rezazadeh Valojerdi

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  1. Mojtaba Rezazadeh Valojerdi
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  2. Poopak Eftekhari-Yazdi
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  3. Leila Karimian
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Correspondence to Mojtaba Rezazadeh Valojerdi.

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Capsule Cryopreservation of human cleavage stage embryos with vitrification versus slow freezing provides better laboratory and clinical outcomes.

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Rezazadeh Valojerdi, M., 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 26, 347–354 (2009). https://doi.org/10.1007/s10815-009-9318-6

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  • DOI: https://doi.org/10.1007/s10815-009-9318-6

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