1. Ri-Cheng Chian, Ph.D. McGill Reproductive Center McGill University Health Center Department of Obstetrics and Gynecology McGill University, Montreal Canada Fertility cryopreservation with oocyte vitrification

2. Female fertility using cryopreservation 1.Embryos - generated from IVF cycles; 2.Ovarian tissues; 3.Eggs;

3. Egg freezing The development of an effective egg freezing system will have a significant impact on clinical practice of reproductive medicine;The development of an effective egg freezing system will have a significant impact on clinical practice of reproductive medicine; Fertility preservation for young women requiring sterilizing medical and surgical treatments;Fertility preservation for young women requiring sterilizing medical and surgical treatments; Cryobanking of eggs will benefit a large population of single women who wish to delay motherhood because of personal, professional and financial reason;Cryobanking of eggs will benefit a large population of single women who wish to delay motherhood because of personal, professional and financial reason;

4. Women without partners;Women without partners; Avoids ethical issues and legal restrictions related to embryo banking;Avoids ethical issues and legal restrictions related to embryo banking; Oocyte donation;Oocyte donation; Option for delayed motherhood;Option for delayed motherhood; Advantages of oocyte cryopreservation

5. The first live birth was reported by Chen (1986);The first live birth was reported by Chen (1986); Over two decades, very few live births were reported;Over two decades, very few live births were reported; Survival rate after thawing was approximately 50- 55%;Survival rate after thawing was approximately 50- 55%; Token together, less than 1,000 live births have been reported by the conventional slow-freezing method;Token together, less than 1,000 live births have been reported by the conventional slow-freezing method; % of live births per thawed egg ranges from 1-10% using this protocols;% of live births per thawed egg ranges from 1-10% using this protocols; Slow-freezing method for eggs:

6. Increased sucrose concentration in suspending solution (0.2M or 0.3M respectively) (Yang et al., 1998; Fabbri et al., 2001; Chen et al., 2002; 2005);Increased sucrose concentration in suspending solution (0.2M or 0.3M respectively) (Yang et al., 1998; Fabbri et al., 2001; Chen et al., 2002; 2005); Choline-based freezing medium to replace sodium (Stachecki et al., 1998; Quintans et al., 2002; Boldt et al., 2003);Choline-based freezing medium to replace sodium (Stachecki et al., 1998; Quintans et al., 2002; Boldt et al., 2003); The survival rate of the oocytes after thawing was increased to 65-70%;The survival rate of the oocytes after thawing was increased to 65-70%; Modified slow-freezing methods for eggs:

7. Vitrification method for eggs: Kuleshova et al. (1999): Open pulled straw (Hum. Reprod., 14: 3077-3079);Kuleshova et al. (1999): Open pulled straw (Hum. Reprod., 14: 3077-3079); Yoon et al. (2000; 2003): Electron microscope grid (Fertil. Steril., 74:180-181; Fertil. Steril., 79:1323- 1326.);Yoon et al. (2000; 2003): Electron microscope grid (Fertil. Steril., 74:180-181; Fertil. Steril., 79:1323- 1326.); Katayama et al. (2003): Cryotop (Fertil. Steril., 80: 223-224.);Katayama et al. (2003): Cryotop (Fertil. Steril., 80: 223-224.); The survival rate of the oocytes after thawing was approximately 85-90%;The survival rate of the oocytes after thawing was approximately 85-90%;

8. What is vitrification? Vitrification is a process which allows glasslike solidication of water without ice-crystal formation in the living cells.

9. History of vitrification Luyet B. (1937): Working hypotheses on the nature of life. Biodynamica, 1:1-7. Luyet B. (1937): The vitrification of organic colloids and protoplasm. Biodynamica, 1:7-14. Fahy GM. (1981): Prospect for vitrification whole organs. Cryobiology, 18:617-625. Rall WF and Fahy GM. (1985): Ice-free cryopreservation of mouse embryos at -196°C by vitrification. Nature, 313:573-575.

10. Nature of water The temperature below 0ºC will introduce formation of water ice-crystal; Below -130ºC is the glass transition temperature of water;

11. Theoretically, if the formation of intracellular and extracellular ice-crystal prevented and the glass transition occurred, the cells will be survival after freezing-thawing; However, the cells may have other injuries during freezing-thawing procedures; Cryobiology

12. Cryobiology (cont.) Chilling injury: The temperature between +30ºC and 0ºC may compromise cell membrane integrity, metabolism and cytoskeleton; Cryoprotectant may be required to add into freezing solution;

13. Cryoprotectants CH 3 –S–CH 3 || O Glycerol Dimethylsulphoxide (DMSO) Propylene glycerol (PROH) Ethylene glycol (EG)

14. Cryoprotectant (cont.) The mechanism of the protective action of cryoprotectants is the same, but their toxicities are different; Permeation ability is different with different cryoprotectants and temperatures; Therefore, the toxicity of cryoprotectants must be considered for freezing;

15. There are osmotic change before and after freezing in cryopreservation solution; These osmotic changes may cause the death of cells, normally it is referred to ‘osmotic injury’; Cryoprotectant (cont.)

16. Hypertonic solution is required, i.e. sucrose is added to prevent swelling and shrinkage of the cells;

17. Chilling injury; Cryoprotectant (toxicity and temperature); Osmotic injury; Speed of freezing and thawing; Factors affect successful frozen-thawing

18. Is it difficult to freeze oocytes? Mammalian oocytes have proven to be more difficult to cryopreserve than cleavage-stage embryos because it is relatively large cell and contains more water in the cell; Mature oocytes with its special structures, like metaphase spindle is fragile for freezing;

19. Vitrification procedure EM (5 min) VM (1 min) Loading onto McGill Cryoleaf Plunge into LN 2 (-196ºC) 7.5% EG+PROH 15.0%EG+PROH 0.5 sucrose

20. Morphological change in EM and VM Before in EM 1 min in EM2 min in EM 3 min in EM4 min in EM5 min in EM 10 sec in VM 30 sec in VM1 min in VM

21. McGill Cryoleaf

22. Thawing procedure TM (37ºC)DM-I (3 min)DM-II (3 min)WM (3 min) 1.0M sucrose0.5M sucrose0.25M sucroseCulture medium

23. Morphological changes in thawing media 0.5 min in TM3.0 min in DM-13.0 min in DM-2 3.0 min in WM-13.0 min in WM-2 Transfer to culture

24. Initial data for survival rates of human oocytes StageNo. of oocytesSurvived (%) GV3232 (100) M-I3030 (100) M-II1919 (100) Total8181 (100)

25. Immuno-fluorescent staining of meiotic spindles and chromosomes

26. Aneuploidy screening of mouse oocytes following vitrification and slow-freezing

27. Slow-freezing of oocytes results in more spindle and chromosome abnormalities than vitrification; However, incidence of aneuploidy is similar between vitrification and slow freezing. Interpretation

29. Viability and pregnancy outcome of vitrified in- vivo oocytes following thawing and ICSI at McGill Reproductive Center Patients (cycles) 38 (38) Age 31.5 ± 0.5 No. of oocytes thawed 463 No. of oocytes survived (%) 383 (82.7) No. of oocytes fertilized (%) 287 (74.9) No. of embryos transferred 133 (3.5±1.1) No. of clinical pregnancies (%) 17 (44.7) No. of implantation (%) 25 (18.8) Chian et al (Fertil & Steril., In press)

30. Clinical pregnancy outcome of vitrified in-vivo oocytes following thawing and ICSI at McGill Reproductive Center Patients (cycles) 38 (38) No. of clinical pregnancies (%) 17 (44.7) No. of live birth (%) 15 (39.5) No. of miscarriages 2 No. of singleton 9 No. of Twins 5 No. of triplets 1 No. of newborn 22 Chian et al (Fertil & Steril., In press)

31. Patients20 Mean age (years)30.8 ±0.9 Mature oocytes retrieved6 Immature oocytes retrieved290 Mean oocyte maturation rate (%)67.3±4.9 Oocytes vitrified and thawed215 Oocytes survived (mean %±SEM)148 (67.5 ±5.8) Oocytes fertilized (mean %±SEM)96 (64.2 ±4.5) Embryos transferred (median; range)64 (4; range 1-6) Implantation (mean %±SEM)4 (9.6±5.4) Pregnancies per cycle (%)4 (20.0) Clinical pregnancies per cycle (%)4 (20.0) Ongoing pregnancies (%)0 Live births4 Mean birth weight (grams)4,049±413.7 IVM-Vitrification trial at MRC Chian et al (Fertil & Steril., In press)

33. Pregnancies conceived following oocyte vitrification are not associated with adverse obstetric and perinatal outcomes. Interpretation

37. Conclusions Vitrification of human oocytes is associated with acceptable pregnancy rate and normal obstetrical and neonatal outcomes; The offspring derived from vitrified oocytes are healthy; Vitrification of oocytes can be used safely for human reproductive medicine; Oocyte vitrification may offer cancer patients for fertility preservation.

38. Staff at McGill Reproductive Center; Dr. Ruvalcaba Castellon, L.A. at Instituto Mexicano de Infertilidad, Jalisco, Mexico; Dr. Lucena, E. at CECOLFES, Bogota, Colombia. Acknowledgements

39. Thank you! ri-cheng.chian@muhc.mcgill.ca