1. © 2008 Universitair Ziekenhuis Gent1 State of the ART of the vitrification of human oocytes … an embryologist view Antalya 2011 Etienne Van den Abbeel Department of Reproductive Medicine, University Hospital Gent, Belgium

2. 22 © 2008 Universitair Ziekenhuis Gent Cryopreservation of human oocytes: why? 1. Fertility preservation for medical reasons 2. Fertility preservation for social reasons 3. Use of cryo-banked oocytes for egg donation 4. Avoids the production of supernumerary embryos in IVF 5. Accumulation of excess oocytes in IUI cycles (Rienzi L ESHRE Stockholm 2011)

3. 33 © 2008 Universitair Ziekenhuis Gent Cryopreservation and cryoprotectants and influence on oocyte constituents: Zona pellucida Cortical granules Meiotic spindle Chromosomes Ca homeostasis Mitochondria SER Transcriptome Genome Proteoom Plasmamembrane Cryopreservation of mammalian oocytes is a challenge

4. 44 © 2008 Universitair Ziekenhuis Gent Efficient cryopreservation programmes? Avoiding injuries Dilemma Which strategy is better for our patients: freezing or vitrification? Vitrification is the best strategy?

5. 55 © 2008 Universitair Ziekenhuis Gent Oktay et al., 2006/2008 (abstract) VariableSlow Freezing (2006)Vitrification (2006) Age, mean33.732.3 Fertilization rate64.9 (2,478/3,818)74.2 (637/859) Clinical pregnancies per thawed oocyte 0.023 (153/6720)0.045 (61/1354) SLOW FREEZING VERSUS VITRIFICATION - oocytes Slow freezing/Vitrification (2008) Clinical pregnancies per thawed oocyte 0.022 (314/14215)/0.058 (212/3672)

6. 66 © 2008 Universitair Ziekenhuis Gent Outline of the presentation State of the ART of the technology State of the ART of the efficiency Slow freezing versus vitrification Fresh oocytes versus vitrified oocytes Open vitrification versus closed vitrification

7. 77 © 2008 Universitair Ziekenhuis Gent Vitrification State of the ART of the technology Claims made for vitrification Reduces the time of the cryopreservation procedure? Flexibility Eliminates the cost of expensive programmable freezing equipment? No ice crystallization? Very simple procedure?

8. 88 © 2008 Universitair Ziekenhuis Gent Claims made for vitrification No ice crystal formation? Is vitrification a simple procedure?

9. 99 © 2008 Universitair Ziekenhuis Gent T° Concentration of solute Th Tg Equilibrium Freezing Curve Liquid phase Glass phase Molecular organization as in a crystal structure Phase diagram Glass transition curve Ice phase molecular structure of a viscous liquid and is not crystalline

10. 10 © 2008 Universitair Ziekenhuis Gent 1997 Vajta et al  Minimal Volume Vitrification: Challenge = avoiding IIF Succesfull vitrification of human oocytes, embryos and blastocysts depends on a correct interplay between, “sufficient” permeation of a “sufficient” high concentration of penetrating cryoprotectant (equilibration step), “sufficient” dehydration by a non- penetrating cryoprotectant (vitrification step), a “sufficient” high cooling rate direct contact with LN2 and small volumes) and a “sufficient” high warming rate

11. 11 © 2008 Universitair Ziekenhuis Gent Basic principles of vitrification Principle variables of vitrification The effect of cooling and warming rates Permeability of cells to water and CPA CPA toxicity

12. 12 © 2008 Universitair Ziekenhuis Gent Vitrification State of the ART of the efficiency Slow freezing versus vitrification Fresh oocytes versus vitrified Open versus closed vitrification

13. 13 © 2008 Universitair Ziekenhuis Gent Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. (FS 2011) Cobo ACobo A, Diaz C.Diaz C OBJECTIVE: To perform a systematic review of the literature to identify randomized controlled trials assessing the efficacy of oocyte vitrification in terms of oocyte survival, fertilization, embryo development, and pregnancy rates. DESIGN: Systematic review and meta-analysis of randomized controlled trials (>2500 papers and abstracts). Five eligible studies were finally included. They involved 4,282 vitrified oocytes, 3,524 fresh oocytes, and 361 slow- frozen oocytes between 2005 and 2009.

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16. 16 © 2008 Universitair Ziekenhuis Gent Conclusions More studies should be done! The oocyte survival rate was higher in vitrified vs. slow-frozen oocytes (odds ratio [OR] 2.46, 95% confidence interval [CI] 1.82-3.32), although heterogeneity between studies was observed. The fertilization rate was higher in vitrified vs. slow-frozen oocytes (OR 1.50, 95% CI 1.07-2.11). Vitrification also resulted in a higher rate top-quality embryo (22.4% vs. 8.0%, OR 3.32, 95% CI 1.37-8.02) and embryo cleavage rate (day 2: 64.6% vs. 47.7%, OR 2.00, 95% CI 1.33-3.00; day 3: 53.0% vs. 33.3%, OR 2.25, 95% CI 1.32-3.85) as compared with slow freezing The rates of ongoing pregnancy, top-quality embryo, embryo cleavage, and fertilization did not differ between the vitrification and the fresh oocyte groups. A cautionnary note however

17. 17 © 2008 Universitair Ziekenhuis Gent Results some caution! – Substantial heterogeneity Patient selection (good responders, Age effects (Ubaldi et al 2010), oocyte donors) Warming and transfer policy No uniform reporting of data and (or) study endpoints Commercial bias? Different devices and different media formulations used Superiority of open devices?

18. 18 © 2008 Universitair Ziekenhuis Gent Vitrification of mature human oocytes: 2008-2010 (Open VIT, open storage, open warming = cryo-TOP/cryo Leaf) Kuwayama et al; Antinori et al; Nagy et al; Chian et al; Rienzi et al; Cobo et al Morphological survival: > 95% fully intact Fertilisation: >90% 2PN Pregnancy rates 40 to 65 % Neonatal outcome reassuring sofar (Noyes et al, Wennerholm et al, Chian et al) Long term consequences? CPA’sare not neutral

19. 19 © 2008 Universitair Ziekenhuis Gent Vitrification of oocytes Is open vitrification a safe procedure? Open devices direct contact between samples and LN2 Long term LN2 storage (vapour storage) of apparently vitrified, minimal-volume (<1µl) samples Spontaneous devitrification possible Open versus closed vitrification and storage?

20. 20 © 2008 Universitair Ziekenhuis Gent Prospective randomised controlled trial (morphological survival): open (cryo-TOP) versus closed (closed VIT, closed storage, open warming =CBS HSS) vitrification Brussels: unpublished observations Number of donors: 9 (144 oocytes) Open Closed Oocytes VIT 71 73 Oocytes warmed 64 73 Survival 83% 92% NS

21. 21 © 2008 Universitair Ziekenhuis Gent Prospective randomised controlled trial open (cryo TOP) versus closed (CBS HSS) vitrification Number of recipients: 23 Open Closed Transfers 11 12 Embryos transferred 15 21 HCG 1 6 Embryos VIT 2 6

22. 22 © 2008 Universitair Ziekenhuis Gent Closed (CBS HSS) vitrification of oocytes: clinical results Number of donors 14 Number of recipients: 20 Number of oocytes warmed: 123 Number of oocytes survived (%): 111 (90.2) % 2PN: 77.5 % GQ Embryos on Day 3: 61.6 Transfers: 20 Embryos transferred: 36 Clinical pregnancies (%): 10 (50) Implantations (%): 12 (33.3) Implantation rate per oocyte warmed: 13/123 (10.6%)

23. 23 © 2008 Universitair Ziekenhuis Gent Closed (vitrisafe) vitrification of oocytes: clinical results Pierre Van der Zwalmen (2010) Gynecol Obstet Fertil 38 (541-546) Number of oocytes warmed: 146 Number of oocytes survived (%): 137 (94%) Patients: 22 Embryos transferred: 63 Clinical pregnancies (%): 9 (41) % Implantations : 14% Implantation rate per oocyte warmed: 6%

24. 24 © 2008 Universitair Ziekenhuis Gent Vitrification of oocytes Closed vitrification is an efficient method for the vitrification of human oocytes suporting the domininance of the warming rate over the cooling rate in vitrification

25. 25 © 2008 Universitair Ziekenhuis Gent General conclusions Vitrification will it replace conventional freezing techniques? Recent published data of the vitrification of human oocytes, indicate that vitrification works and produces better results than conventional freezing. The efficiency of vitrification (FHB/oocyte warmed) is 5-10% for oocytes, It has replaced conventional freezing

26. 26 © 2008 Universitair Ziekenhuis Gent General conclusions Vitrification routine or experimental? Technical challenges/technical proficiency Development of more robust closed “true”vitrification procedures Media and devices for vitrification: knowing and understanding what is important Interactions between cryoprotectants, cooling rates and warming rates on survival and developmental potential Costs

27. 27 © 2008 Universitair Ziekenhuis Gent Vitrification of mature human oocytes 2011 Rienzi L, ESHRE 2011 Vitrification is an efficient method for cryopreservation of human MII phase oocytes. The overall efficiency was found to be consistently high in different centers. Female age and number of available oocytes are the most important predictive factors of success. In any case however, oocyte cryopreservation cannot be considered a guarantee of life-long fertility. Equal to fresh oocytes? ICSI required! ICSI done in fresh oocytes!

28. 28 © 2008 Universitair Ziekenhuis Gent Vitrification of mature human oocytes 2011 Noyes et al RBM online (RBM Online 19, 275-279)...“Perhaps one hesitation regarding oocyte cryopreservation partially lies in the commercial interests of companies promoting premature elective fertility preservation. In addition, competing interests exist for those who manufacture oocyte storage containersand/ or oocyte cryopreservation culture media. Clearly, clinicians and embryologists need to be cognizant of these latter conflicts as more publications appear in the literature”...