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Veronica Bianchi declares no conflict of interest

Veronica Bianchi declares no conflict of interest. Pre & Post morphological selection of oocytes cryopreserved with slow freezing clinical outcomes. Veronica Bianchi Ph.D. ESHRE Senior Embryologist. Outlines. Indications Morphological evaluation (using slow freezing)

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Veronica Bianchi declares no conflict of interest

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  1. Veronica Bianchi declares no conflict of interest

  2. Pre & Post morphological selection of oocytes cryopreserved with slow freezing clinical outcomes Veronica Bianchi Ph.D. ESHRE Senior Embryologist

  3. Outlines • Indications • Morphological evaluation (using slow freezing) • Protocols and procedures available in the literature • Clinical outcome worldwide • Our results with slow cooling • Conclusions

  4. Indications • Restrictions to embryo storage • Ethical • Legal • Failure to produce semen sample and/or no sperm the day of egg retrieval • Fertility preservation in cancer patients • Preservation of women’s fertility • Egg donation

  5. Goals of Cryopreservation Minimize/prevent cryo-injuries from Ice crystals (internally and externally) Toxicity of cryoprotectants Solute effects Osmotic shock Chilling sensitivity Fracture damage Preserve structural integrity and biological viability of cells

  6. sucrose Pr-OH H2O H2O Slow freezing • Wash in PBS • 1.5 M Pr-OH, 10 min • 1.5 Pr-OH / 0.2 M sucrose • load • RT to -8°C, -2°C/min • Seed • Hold, 10 min • -8°C to -30°C,  -0.3°C/min • -30°C to -150°C,  -50°C/min • Plunge into LN2 Temperature °C Time (min)

  7. Strict adherence to protocol Handling Incubation times 10 min 5 min 1.5 M PROH, 0.1/0.3M sucrose in PBS (L) 1.5 M PROH in PBS (S) Oocyte(s) Loading in straws medium air

  8. Rapid thawing • RT, 30 sec • 30°C water, 40 sec • RT,1.0 M Pr-OH, 0.3 M sucrose, 5 min • RT, 0.5 M Pr-OH, 0.3 M sucrose, 5 min • RT, 0.2 M Pr-OH, 0.3 M sucrose, 10 min • RT, PBS, 10 min • N.B. All freezing/thawing solutions prepared with PBS supplemented with PPS 20% (v/v)

  9. Morphological Data:slow freezing

  10. Spindle and chromosome organization in human oocyte frozen after exposure to1.5 Pr-OH / 0.1 M sucrose or1.5 Pr-OH / 0.3 M Coticchio et al., Hum Rep 2006

  11. Nottola et al Hum Rep 2006

  12. 0.2M/0.3M Protocol Resulted in Less Ultrastructural Damage Title Protocols 0.1M/0.2M 0.3M/0.3M 0.2M/0.3M Ultrastructural damage (vacuolisation) + + + + Cortical granules loss + + + MII spindle damage + – – Coticchio et al., 2006; Nottola et al., 2007; 2008; 2009; De Santis et 2008; Cobo et al., 2008; Bromfield et al., 2009; Coticchio et al., 2009 Bianchi et al 2007

  13. Clinical Data:slow freezing

  14. Steady outcome improvement by slow freezing over time 1986 1997 2001-2006 2007 1980 1990 2000 70-75% 70-75% 40-50% Survival 20-30% 70-75% 70-75% 40-50% Fertilization <40% 10-15% 20-30% ??? Cleavage (4-cell) ??? Implantation per thawed oocyte 2.5-2.7% > 5% ??? ???

  15. PROH-Sucrose – SLOW FREEZING protocol 4 different protocols a) 1.5M PROH + 0.1M sucrose (La Salle, 1985; Gook, 1993) b) 1.5M PROH + 0.3M sucrose (Fabbri et al., 2001) c) 0.75 M PROH and 1.5M PROH + 0.2M sucrose (Borini et al. 2009) d) 1.5M PROH + 0.2M sucrose (Bianchi et al. 2007)

  16. 0.1M sucrose 0.2M sucrose at freezing at thawing I PROTOCOL (La Salle, 1985; Gook, 1993)

  17. Materials and methods: We started our program on January 1997 until December 2000. 68 couples which asked not to have supernumerary embryos were involved in the program. THREE of the recovered oocytes were inseminated, the remaining oocytes were cryopreserved. Enough oocytes were thawed in order to obtain 3-4 oocytes suitable for ICSI.

  18. Thawing cycles: oocyte cryopreservation data

  19. Thawing cycles: clinical data

  20. Thawing cycles: pregnancy outcome

  21. The Italian IVF Law 40 - 2004

  22. 0.3M sucrose 0.3M sucrose at freezing at thawing II PROTOCOL (Fabbri et al 2001)

  23. Results from Different Centres Using 0.3 M Protocol were Similar Title Borini Levi Setti La Sala De Santis 2006 2006 2007 2007 % Survived 74 69 73 71 % Fertilised 76 68 N.R. 80 % Implantation 5.2 5.8 3.4 5.7 % Implantation per thawed oocyte 2.6 1.9 2.5 2.6

  24. 2 steps PROH (0.75M - 1.5M) + 0.2M sucrose 0.3M sucrose at freezing at thawing III PROTOCOL (Borini et al., 2010)

  25. Slow cooling: multicentric study • Borini et al. 2009 Biological and clinical outcome:

  26. Slow cooling: multicentric study • Borini et al. 2009 Pregnancy outcome:

  27. 1 steps PROH (1.5M) + 0.2M sucrose 0.3M sucrose at freezing at thawing IV PROTOCOL (Bianchi et al 2007; submitted)

  28. Our updated results Unpublished data

  29. Overall survival, fertilization and cleavage rates Bianchi et al. submitted 2011

  30. Overall clinical data Bianchi et al. submitted 2011

  31. Survival, fertilization and cleavage rates Bianchi et al. submitted 2011

  32. Overall clinical data Bianchi et al. submitted 2011

  33. Impact of protocol: Performance in vitro of embryos from sibling fresh and frozen oocytes P < 0.001 for all protocols in comparison to control Borini et al., unpublished

  34. Thank you for your attention 37

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