Dr. Andrea Borini Clinical and Scientific Director Tecnobios Procreazione Bologna, Italy

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1 Oocyte Cryopreservation: Beating the Clock Dr. Andrea Borini Clinical and Scientific Director Tecnobios Procreazione Bologna, Italy

2 Question Why do we need to cryopreserve oocytes?

3 Answers For women suffering from cancer before undergoing radio/chemotherapy For women wishing to delay motherhood For women undergoing an ART cycle in a country where embryo freezing is forbidden by law or for personal reasons

4 Question Why is there so much research going into oocyte cryopreservation in Italy?

5 Title The Italian IVF Law

6 The Italian IVF Law How successful was oocyte cryopreservation in 2004?

7 Up to 2004 Few Centres Worldwide Practiced Oocyte Cryopreservation and Success Rates Were Low Oktay et al 2006

8 Tecnobios Procreazione Results Up Until M Sucrose Survival 37.0 Fertilisation 45.4 Cleavage 86.3 Implantation rate per transferred embryo 16.4 Implantation rate per thawed oocyte 2.3 Borini et al 2004

9 Increasing Sucrose Concentration Improved Oocyte Survival % survival *** *** 0.1 M 0.2 M 0.3 M Naked Naked Partial Cumulus Removal * Partial cumulus removal ** * Sucrose concentration in freezing solution Fabbri et al 2001 Fabbri et al., 2001

10 Title Our Journey Today Slow Freezing Protocol reproducibility Cryodamage Tecnobios Procreazione experience Vitrification Pros and cons Relative/objective efficiency Efficiency of oocyte cryopreservation Cumulative pregnancy rate from embryos and cryopreserved oocytes Tecnobios Procreazione data Children follow up Babies born Malformations

11 Slow Freezing

12 Results Title from Different Centres Using 0.3 M Protocol were Similar Borini Levi Setti La Sala De Santis % Survived % Fertilised N.R. 80 % Implantation % Implantation per thawed oocyte

13 By Title Using HP Confocal Microscopy We Can Study Spindle Damage From unpublished collaborative work with Albertini et al

14 0.2M/0.3M Protocol Resulted in Less Ultrastructural Title Damage Protocols 0.1M/0.2M 0.3M/0.3M 0.2M/0.3M Ultrastructural damage (vacuolisation) Cortical granules loss MII spindle damage +

15 0.2 M/0.3 M Protocol Resulted in Lower Osmotic Stress Dehydration Risk of ice formation Osmotic stress 0.1 M 0.2 M 0.3 M 0.1 M freezing 0.2 M thawing 0.3 M freezing 0.3 M thawing 0.2 M freezing 0.3 M thawing

16 Summary of Tecnobios Procreazione Experience Title with Different Protocols Protocol 0.1 M / 0.2 M Borini et al M / 0.3 M Borini et al M / 0.3 M Bianchi et al 2007 % Survival % Fertilisation % Implantation per transferred embryo % Implantation per thawed oocyte

17 Further Title Research to be Presented at ESHRE 2008 Human meiotic spindle dynamics following slow-cool cryopreservation A. Borini, J. Bromfield, K. Hutt, G. Coticchio, R. Sciajno, D.F. Albertini Meiotic spindle organization, chromosome alignment and meiotic checkpoint elements are preserved in frozen-thawed oocytes L. De Santis, G. Coticchio, I. Cino, V.Distratis, G. Rossi, A. Borini, S. Cecconi Ultrastructural changes in human metaphase II oocytes cryopreserved by vitrification S.A. Nottola, G. Coticchio, R. Sciajno, A. Gamnardella, M. Maione, G. Macchiarelli, A. Borini Confocal microscopy reveals that the Polscope fails to discriminate diverse configurations and measure accurately the meiotic spindle of human oocytes R. Sciajno, G. Coticchio, K. Hutt, J. Bromfield, D.F. Albertini, A. Borini Survival and meiotic spindle analysis through Polscope in human oocytes vitrified with either cryoleaf or cryoloop A. Gambardella, R. Sciajno, G. Coticchio, A. Borini

18 Vitrification

-100-120 -140-160 0 15 30 45 60 75 90 105 120 Time

19 Techniques for Storing Oocytes: Title Vitrification and Slow Freezing Temperature Time Glass Ice crystals Adapted from Liebermann et al, 2003

20 Oocyte Vitrification

21 Title Oocyte Vitrification Pros No ice formation and solution effect Less dependent on technology (freezing devices) Shorter operational times Very high survival rates Cons Risk of severe osmotic stress (chemical toxicity) Difficult to standardise Risk of accidental devitrification Risk of cross contamination

22 Superiority Title of Vitrification vs Slow Freezing: Actual or Presumed? Bianchi et al, 2007 Slow freezing Cobo et al, 2007 Vitrification Patients 62 (general) 30 (donors) Patient s age 33.7 ± ± 3.6 % Survival % Pregnancies per ET % Implantation per transferred embryo % Implantation per thawed oocyte

23 Efficiency of Oocyte Cryopreservation

24 Cumulative Title Pregnancy Rate as a Standard of Efficiency Oocyte cryopreservation relies on essentially unselected biological material. The true clinical efficiency of oocyte cryopreservation can only be accurately assessed when all oocytes from a cycle have been used (Edgar, 2007).

25 The Title Added Value of Frozen Oocytes, Zygotes and Embryos has been Clearly Demonstrated Frozen embryos, Tiitinen et al, 2004 Frozen embryos, Le Lannou et al, 2006 Frozen zygotes, Germond et al, 2004 Frozen embryos, Borini et al, 2008 Frozen oocytes, Borini et al, 2006 Fresh delivery rate Cumulative delivery rate

26 What is our Cumulative Pregnancy Rate? Title Tecnobios Procreazione Data Period: March 10, 2004 to March 31, Patients included: Three or more cryopreserved oocytes Pregnancy achieved in fresh or frozen cycles All cryopreserved oocytes used in thawed cycles

27 TitleThe Italian IVF law 40/2004

28 Title Outcome Results from Fresh Transfer Cycles <= >=43 TOT Ovum pick-up Clinical pregnancies % per pick-up % per embryo transfer % Singleton % Twins % High order

29 Title Outcome Results from Oocyte Thawed Cycles <= >=43 TOT Cycles ,206 Mean % Survival

30 Title Outcome Results from Thawed Cycles <= >=43 TOT Clinical pregnancies % per cycles % per embryo transfer % implantation per transferred embryos % implantation per injected oocytes

31 Cumulative Title Pregnancy Rates after Fresh and Thawed Cycles <= >=43 TOT Cumulative pregnancies % Cumulative pregnancy rate ,

32 Cumulative Pregnancy Rates are Similar Before and After Introduction of Italian Law <= >=43 TOT Cumulative pregnancy rates with frozen embryos * Cumulative pregnancy rates with frozen oocytes *Borini et al, 2008

33 Children Follow Up

34 Title Tecnobios Procreazione Births Babies born Singletons Twins Triplets Males (%) Females (%) (46.5%) 78 (53.4%) Four cases of malformations: Rubinstein-Taiby Syndrome (mental retardation) 47 xxx 19 Hypometilation Choanal atresia

35 A Worldwide Registry Would Help to Promote Safety and Safety Advance the Field Livebirth Outcomes >596 live births have been reported worldwide 421 singleton / 111 multiple 214 female / 250 male No increase in spontaneous miscarriage, chromosomal anomaly or birth defects Data compiled by Nicole Noyes M.D

36 Title Take Home Messages Oocyte cryopreservation today is a viable technique Not only for infertile women But also for women who wish to preserve their fertility - Oncology patients - Women wishing to delay motherhood

Cryopreservation of human oocytes with slow freezing techniques

ESHRE Campus Symposium Cryobiology and cryopreservation of human gametes and embryos Athens, Greece 25-26 September 2009 Cryopreservation of human oocytes with slow freezing techniques Giovanni Coticchio