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Cryopreservation: slow freezing or vitrification?

Cryopreservation: slow freezing or vitrification?. Outline. Introduction Effects of cryopreservation on physiology Slow freezing versus vitrification: clinical results Open versus closed carrier system Cryoprotectant mixtures Summary. Introduction. History of embryo cryopreservation.

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Cryopreservation: slow freezing or vitrification?

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  1. Cryopreservation: slow freezing or vitrification?

  2. Outline • Introduction • Effects of cryopreservation on physiology • Slow freezing versus vitrification: clinical results • Open versus closed carrier system • Cryoprotectant mixtures • Summary

  3. Introduction

  4. History of embryo cryopreservation 2005 Cryoleaf and Cryotip 1986 First birth from slow frozen human oocyte 2004 Cryotop 1985 Vitrification of mouse embryos Dinnyes et al., 2000 Solid surface 1984 First birth from slow frozen human embryo Lane et al., 1999 CryoLoop 1983 Slow freezing of human embryos Vajta et al., 1998 Open-pulled straws 1972 Slow freezing of mouse embryos Martino et al., 1996 EM grids 1992 Advent of ICSI 1997 ICSI implemented with slow frozen human oocytes 1998 First birth from vitrified human cleavage-stage embryos 1999 First birth from vitrified human oocyte 2001 First birth from vitrified human blastocyst

  5. OOCYTE / EMBRYO H2O CRYOPROTECTANT SLOW COOLING RAPID COOLING SLOW FREEZING VITRIFICATION ICE CRYSTALS FORM IN THE EXTERNAL MEDIUM NO ICE CRYSTALS FORM IN THE EXTERNAL MEDIUM Slow freezing versus vitrification

  6. Slow freezing versus vitrification • Slow-freezing • low levels of cryoprotectants • slow controlled rates of cooling (0.1-0.3°C/min) • slow dehydration of cells to minimize ice crystal formation and damage • freezing machine required (calibration, expenses) • takes hours • Vitrification • high levels of cryoprotectants • extremely fast rates of cooling (>20,000°C/min) • no ice crystal formation or damage; straight to a glass-like structure • no freezing machine required • takes seconds

  7. Base medium + Cryoprotectant Base medium Vitrification • Vitrify/Vitrification from Latin vitrum (glass)

  8. Effects of Cryopreservation on physiology

  9. Outcome of cryopreservation USA 2006 Europe 2005 http://www.cdc.gov http://www.ESHRE.com

  10. Outcome of cryopreservation

  11. 100 * 80 60 ** ** 40 ** 20 0 Slow-Freeze Control Vitrification Effect of cryopreservation on development Survival (%) Fertilization (%) Blastocyst / oocyte (%) Blastocyst cell number *: p<0.05 **: p<0.01 Lane and Gardner, (2001) Mol. Reprod. & Dev 58, 342-7

  12. MII In vivo Oocyte MII Vitrified Oocyte MII Slow Frozen Oocyte MII In vivo Oocyte MIIVitrified Oocyte MII Slow Frozen Oocyte Oocyte Protein Profiles Da Da Line Plot Larman et al., (2007) Human Reproduction 22, 250-259 Larman et al. (2007) Human Reproduction 22, 250-259

  13. Vitrification Slow Freezing Vitrification Maintenance of the spindle after vitrification Before After Larman et al., (2007) RBM Online 15, 692-700

  14. 2 1.5 1 0.5 0 Maintenance of spindle retardance after vitrification Mouse oocytes Human oocytes 2.5 2 1.5 Spindle Retardance (nm) Spindle Retardance (nm) 1 0.5 0 Vitrification Slow Freezing BEFORE VITRIFICATION AFTER VITRIFICATION Larman et al., (2007) RBM Online 15, 692-700

  15. Protein Leakage by 2-cell Mouse Embryos following Cryopreservation LDH Leakage (pmol NADH oxidized/h) Slow freezing Vitrification 50 a 40 30 Different letters; P<0.05 b 20 c c 10 0 0 0.1 0 0.1 Ascorbate (mM) Lane et al. (2002) Human Reproduction 17, 2686-2693

  16. Pyruvate uptake in human day 3 embryos **: p<0.01 Balaban et al. Hum Reprod. 2008, 23 (9); 1976-82

  17. Slow freezing versus vitrification: clinical results

  18. Scientific evaluation • Is vitrification superior? • Randomized controled trials • Sample size • Correct statistical evaluation • Meta-analysis • Summary of RCT’s

  19. Meta-analysis • Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. • Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, Tarlatzis BC. • Fertil Steril. 2008 Jul;90(1):186-93. Epub 2007 Nov 5. Review. • Pubmed search: 873 titles • Number included: 4 • Increased survival after vitrification

  20. Meta-analysis • Cryopreservation of human embryos by vitrification or slow freezing: which one is better? • Kolibianakis EM, Venetis CA, Tarlatzis BC. • Curr Opin Obstet Gynecol. 2009 Jun;21(3):270-4. Review. • 3 additional studies included • Survival superior after vitrification • No difference in pregnancy rates

  21. Oocyte vitrification

  22. Oocyte vitrification Fadini et al. RBM Onl. 2009, 19 (2); 171-80

  23. Health of children born • Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study.Takahashi K, Mukaida T, Goto T, Oka C. Fertil Steril. 2005 Jul;84(1):88-92. • Neonatal outcome after vitrified day 3 embryo transfers: a preliminary study. Rama Raju GA, Jaya Prakash G, Murali Krishna K, Madan K. Fertil Steril. 2009 Jul;92(1):143-8. • Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A, Ruvalcaba Castellón LA, García Amador MI, Montoya Sarmiento JE. Reprod Biomed Online. 2008 May;16(5):608-10.

  24. Open versus closed carrier systems

  25. Open versus closed carrier systems Youssry et al. RBM Onl. 2008, 16 (2); 311-20

  26. Open versus closed carrier systems • Open systems mostly used so far • Cross-contamination risk • Some reports with evidence of cross-contamination • Regulatory requirements • FDA • EU

  27. Open versus closed carrier systems

  28. Development of closed carrier system • Rapid-i™ Rapid-i™ loaded with beads and 1st vitrification solution Solution has frozen Rapid-i™ loaded with Vitri-3 and beads and inserted into pre-cooled straw. Solution has vitrified

  29. Rapid-i™ • Temperature changes Cooling/warming rates around ice nucleation temperature sufficient to obtain proper vitrification and warming Yury A. Tarakanov, Björn O. J. Johansson, Hans J. Lehmann and S. Peter Apell, "Numerical Simulations Demonstrate Safe Vitrification and Warming of Embryos Using the Rapid-i™ Device", Proc. European COMSOL conference 2009.

  30. Rapid-i™ • Good survival rates and development after vitrification of mouse embryos as well as poor quality human embryos • Clinical testing initiated

  31. Cryoprotectant solutions for vitrification

  32. Cryoprotectant solutions • Mixture of penetrating cryoprotectants used to reduce possible toxic effects • Mixture used in combination with • Non-penetrating cryoprotectants (sucrose – trehalose) • Molecules increasing viscosity (Ficoll, hyaluronan...) • Most commonly used mixture: DMSO-EG • Has it been proven that DMSO-EG is superior?

  33. Cryoprotectant solutions • Cryoprotectants most commonly used for slow freezing • Oocytes: PrOH • Zygotes: PrOH • Multicellular embryos: PrOH, DMSO • Blastocysts: Glycerol • Argumentations on potential toxicity of DMSO • Why using DMSO for vitrification?

  34. DMSO-free vitrification Balaban et al. Hum Reprod. 2008, 23 (9); 1976-82

  35. DMSO-free vitrification Balaban et al. Hum Reprod. 2008, 23 (9); 1976-82

  36. DMSO-free vitrification Updated clinical results American hospital, Istanbul 1/1/2009

  37. Summary • Cryopreservation affects oocyte/embryo physiology • Vitrification affects less compared to slow freezing • Meta-analysis comparing slow-freezing and vitrification show increased survival rates for vitrification • Results on outcome of children born after vitrification available so far do not indicate negative effects of the vitrification procedure • Further comparison of efficacy of closed carrier systems as well as different cryoprotectant solutions will help to further optimize vitrification procedures

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