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. oRestrictions to embryo storage Ethical Legal oFailure to produce semen sample and/or no sperm the day of egg retrieval oFertility preservation in cancer patients oPreservation of women’s fertility oEgg donation Indications

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. Slow freezing Time (min) Temperature °C 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 LN 2 sucrose H2OH2O H2OH2O Pr-OH

7. Strict adherence to protocol Handling Incubation times Loading in straws 1.5 M PROH, 0.1/0.3M sucrose in PBS (L) 1.5 M PROH in PBS (S) 5 min10 min Oocyte(s) medium air

8. 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) Rapid thawing

9. Morphological Data: slow freezing

11. Spindle and chromosome organization in human oocyte frozen after exposure to 1.5 Pr-OH / 0.1 M sucrose or 1.5 Pr-OH / 0.3 M Coticchio et al., Hum Rep 2006

13. Nottola et al Hum Rep 2006

14. Title 0.2M/0.3M Protocol Resulted in Less Ultrastructural Damage ––+ MII spindle damage +++ Cortical granules loss ++ + Ultrastructural damage (vacuolisation) 0.2M/0.3M0.3M/0.3M0.1M/0.2M Protocols 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

15. Clinical Data: slow freezing

16. 1980 19902000 200719971986 Steady outcome improvement by slow freezing over time 2001-2006 Survival20-30% 40-50% 70-75% Fertilization<40% 40-50% 70-75% Cleavage (4-cell) ??? 10-15% 20-30% Implantation per thawed oocyte ??? 2.5-2.7% > 5%

17. 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)

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

19. 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.

20. Thawing cycles: oocyte cryopreservation data

21. Thawing cycles: clinical data

22. Thawing cycles: pregnancy outcome

23. The Italian IVF Law 40 - 2004

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

25. Title Results from Different Centres Using 0.3 M Protocol were Similar Borini 2006 Levi Setti 2006 La Sala 2007 De Santis 2007 % Survived74697371 % Fertilised7668N.R.80 % Implantation5.25.83.45.7 % Implantation per thawed oocyte2.61.92.52.6

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

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

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

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

31. Our updated results Unpublished data

32. Overall survival, fertilization and cleavage rates No patients (at least one thawing cycle)342 Mean age (  DS)35,4  4.28 No. of thawing cycles443 No. of oocytes Thawed2458 Survived (%)1766 (71.8) Microinjected1296 2PN (%)1010 (77.9) Cleaved (%)955 (94.5) G1 (%)339 (35.5) G2 (%)461 (48.3) Bianchi et al. submitted 2011

33. No. of embryo transfers394 No. of embryos transferred907 No. pregnancies90 Pregnancy rate/embryo transfer (%) 90/394 (22.8) Pregnancy rate/thawing cycle (%)90/443 (20.8) Pregnancy rate/patient (%)90/342 (26.3) Multiple pregnancy rate (%)26/90 (28.9) No. of gestational sacs122 No. of FHB93 Implantation rate13.5 Implantation/injected oocyte rate9.4 Abortion (%)32 (35.5) Overall clinical data Bianchi et al. submitted 2011

34. Survival, fertilization and cleavage rates Bianchi et al. submitted 2011 ≤3435-38  39 No patients ( at least one thawing cycle)13811490 Mean age (  DS)31.1  4.2836,4  4.2840.6  4.28 No. of thawing cycles177148118 No. of oocytes Thawed993815650 Survived (%)737 (74.2)570 (70.0)459 (70.6) Microinjected515435346 2PN (%)410 (79.6)347 (79.8)253 (73.1) Cleaved (%)382 (93.2)331 (95.4)242 (95.6) G1 (%)133 (34.8)105 (31.8)102 (42.2) G2 (%)186 (48.7)169 (51.0)106 (43.8)

35. ≤3435-38  39 No. of embryo transfers155131108 No. of embryos transferred365301241 No. pregnancies432819 Pregnancy rate/embryo transfer %27.721.417.6 Pregnancy rate/thawing cycle %24.318.916.1 Pregnancy rate/patient %31.224.621.1 Multiple pregnancies (%)16 (37.2)4 (14.3)6 (31.6) No. of gestational sacs613526 No. of FHB492420 Implantation rate %16.711.610.8 Implantation/injected oocyte %11.88.07.5 Abortion (%)10 (23.3)12 (42.8)10 (52.6) Overall clinical data Bianchi et al. submitted 2011

36. 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

37. 37 Thank you for your attention