Fertility cryopreservation with oocyte vitrification

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

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

3. Egg freezing • 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; • Cryobanking of eggs will benefit a large population of single women who wish to delay motherhood because of personal, professional and financial reason;

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

5. Slow-freezing method for eggs: • The first live birth was reported by Chen (1986); • Over two decades, very few live births were reported; • 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; • % of live births per thawed egg ranges from 1-10% using this protocols;

6. Modified slow-freezing methods for eggs: • 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); • The survival rate of the oocytes after thawing was increased to 65-70%;

7. Vitrification method for eggs: • 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.); • Katayama et al. (2003): Cryotop (Fertil. Steril., 80: 223-224.); • 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. Cryobiology • 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;

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. CH3–S–CH3||O Cryoprotectants 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. Cryoprotectant (cont.) • 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’;

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

17. Factors affect successful frozen-thawing • Chilling injury; • Cryoprotectant (toxicity and temperature); • Osmotic injury; • Speed of freezing and 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 7.5% EG+PROH 15.0%EG+PROH 0.5 sucrose EM (5 min) Loading onto McGill Cryoleaf Plunge into LN2(-196ºC) VM (1 min)

20. Morphological change in EM and VM 1 min in EM 2 min in EM Before in EM 3 min in EM 4 min in EM 5 min in EM 10 sec in VM 30 sec in VM 1 min in VM

21. McGill Cryoleaf

22. Thawing procedure 1.0M sucrose 0.5M sucrose 0.25M sucrose Culture medium TM (37ºC) DM-I (3 min) DM-II (3 min) WM (3 min)

23. Morphological changes in thawing media 0.5 min in TM 3.0 min in DM-1 3.0 min in DM-2 3.0 min in WM-1 3.0 min in WM-2 Transfer to culture

24. Initial data for survival rates of human oocytes

25. Immuno-fluorescent staining of meiotic spindles and chromosomes

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

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

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. IVM-Vitrification trial at MRC Chian et al (Fertil & Steril., In press)

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

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

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