1. The outcome of oocyte freezing as compared with embryo freezing Filippo Maria Ubaldi M.D. M.Sc. CLINICA VALLE GIULIA, Rome 3° Congress of Society of Reproductive Medicine Antalya 5-9 October 2011 www.generaroma.it

2. Oocyte cryopreservation “Need” or “no need”? www.generaroma.it

3. “No need” Oocyte cryopreservation www.generaroma.it

4. No need? Medical reason Malignant diseases Surgical ovary removal Polycystic ovary Hyperstimulation syndrome Premature menopause etc. Oocyte cryopreservation www.generaroma.it

5. No need? Logistic reasons Sperm collection problem Legal reasons Restrictions in embryo cryopreservation Fate of embryos of separated couples Social reasons Wish to delay motherhood Moral reasons Oocyte cryopreservation www.generaroma.it

6. No need? Oocyte donation Oocyte banks may result in - widespread availability - shortened, eliminated waiting list - safety (quarantine) - choice Oocyte cryopreservation www.generaroma.it

7. In Italy from march 2004 to may 2009 (Law 40/2004) we could only cryopreserve oocytes Oocyte cryopreservation www.generaroma.it

8. Lab outcomes: vitrification, infertile population www.generaroma.it

9. Laboratory outcomes www.generaroma.it

10. Cumulative ongoing pregnancy rates: vitrification www.generaroma.it

11. Study design o The study was design as a prospective longitudinal cohort study. o The baseline characteristics, embryological data, clinical and ongoing pregnancy rate were analyzed on a per cycle basis. o The cumulative pregnancy rate obtained with fresh and vitrified oocytes from the same stimulation cy- cle was analyzed on a per patient basis. www.generaroma.it

12. Material & methods o All consecutives patients undergoing ICSI treatment in the Centre for Reproductive Medicine GENERA between September 2nd 2008 and May 15th 2009 were considered for this study o Only patients with supernumerary oocytes available for cryopreservation were included. A single fresh attempt was included for each patient www.generaroma.it

13. 69/120 = 57,5% ongoing pregnancy rate Clinical results www.generaroma.it

14. Embryo freezing: cumulative pregnancy rates www.generaroma.it

15. Ubaldi et al., 2004 Embryo freezing: cumulative pregnancy rates 36% www.generaroma.it

16. Cumulative pregnancy rate: <38 y. (Ubaldi, 2004) Fresh ETFresh+frozen All patients (with frozen embryos) 232 232 Clinical preg. after fresh ET113 113 Clinical preg. after 1°thawed ET - 33 Clinical preg. after 2°thawed ET - 10 Total clinical pregnancies113 156 Cumulative clinical preg.rate/patient (%) 113/232 (48,7) 156/232 (67,2) www.generaroma.it

17. Oocyte vitrification: cumultive ongoing pregnancy rates 69/120 = 57,5% ongoing pregnancy rate VS VS <38 y: frozen embryos 156/232 = 67,2% clinical pregnancy rate www.generaroma.it

18. In Italy from may 2009 (Supreme Court 151/2009) we can also cryopreserve embryos Oocyte cryopreservation www.generaroma.it

19. Study design Retrospective observational study All consecutives patients undergoing fresh ICSI treatment where oocyte cryopreservation was performed (first period under the Law limitation: September 2008 – May 2009) and all consecutives patients undergoing fresh ICSI treatment where embryo cryopreservation was performed (second period without Law limitation: September 2009 – May 2010) Primary outcome: cumulative ongoing pregnancy rate obtained with fresh + vitrified oocytes (first period) or with fresh + vitrified embryos (second period). Warming cycles were included until December 2009 and December 2010, respectively www.generaroma.it

20. Cryopreservation method Vitrification protocol : Kuwayama method (2005,2007) Cryoprotectants: 15% DMSO 15% EG Tool: Cryotop (open system) Rienzi et al., Hum Reprod 2010 www.generaroma.it

21. Baseline patient’s characteristics Oocyte GroupEmbryo Group p No. patients 182156 % of the stimulated patients 182/428 (42.5)156/532 (29.3) P<0.0001 Mean age (mean+SD) 35.81±4.1936,10±3,67 ns Mean basal FSH (mean+SD) 6.5±2.26.75±2.6 ns Agonist protocol (%) 143/182 (78.6)108/156 (69.2) ns Antagonist protocol (%) 39/182 (21.4)48/156 (28.8) ns www.generaroma.it

22. Fresh and warming cycles laboratory outcomes Oocyte GroupEmbryo Group p Fresh cycle: laboratory outcome Inseminated MII (mean±SD) 2.9±0.46.2±2.0 P<0.000 2 PN (mean±SD)2.6±0.65.1±1.8 P<0.000 Top quality embryos (mean±SD)1.5±0.962.2±1.6 P<0.000 Embryo transferred (mean±SD)2.5±0.62.2±0.8 P<0.002 Oocyte/embryo vitrified (mean±SD)6.2±3.12.3±1.6 P<0.000 Warmed cycles: laboratory outcomes Oocyte/embryo warmed (mean±SD)4.2±1.22.1±0.8 P<0.000 Oocyte/embryo survived (mean±SD)3.8±0.92.0±0.7 P<0.000 Inseminated MII (mean±SD)2.9±0.2- 2 PN (mean±SD)2.5±0.6- Top quality embryos (mean±SD)1.5±0.9- Embryo transfer (mean±SD)2.4±0.82.0±0.7 P=0.03 www.generaroma.it

23. Fresh cycles: overall results Oocyte GroupEmbryo Groupp No. of cycles182156 Mean age (M±SD) 35.81 ± 4.1936,10±3,67ns No. of ET172144 ET %172/182 (94,5)144/156 (92,3)ns Clinical pregnancy rate per cycle77/182 (42,3)78/156 (50,0)ns Clinical pregnancy rate per ET77/172 (44,8)78/144 (54,2)ns Implantation rate %101/435 (23,2)111/342(32,4)P<0,004 Abortion rate %9/77 (11,7)9/78 (11,5)ns N ongoing pregnancy fresh6869 Ongoing pregnancy rate per fresh cycle %68/182 (37,4)69/156 (44,2)ns Ongoing implantation rate90/435 (20,7)101/342 (29,5)P<0,005 www.generaroma.it

24. Warmed cycles: overall results Oocyte GroupEmbryo Group p No. of cycles 11578 No. of ET 11177 ET % 111/115 (96,5)77/78 (98,7)ns Clinical pregnancy rate per cycle 35/115 (30,4)21/78 (26,9)ns Clinical pregnancy rate per ET 35/111 (31,5)21/77 (27,2)ns Implantation rate % 43/266 (16,1)22/156 (14,1)ns Abortion rate % 6/35 (17,1)5/21 (23,8)ns N ongoing pregnancy vitrified 2916 Ongoing pregnancy rate per vitrified cycle % 29/115 (25,2)16/78 (20,5)ns Ongoing implantation rate 35/266 (13,2)16/156 (10,2)ns www.generaroma.it

25. Cumulative ongoing pregnancy rate after the fresh cycle, first warming cycle and second warming cycle. www.generaroma.it

26. Overall Oocyte Embryo p Fresh 68/182 (37.4)69/156 (44.2) ns I warming cycle 84/182 (51.6)85/156 (54.5) ns II warming cycle 97/182 (53.3)85/156 (54.5) ns Cumulative ongoing pregnancy rate after the fresh cycle, first and second warming cycle N° of cycles per patient1.631.50 509 oocytes and 178 embryos still available in the pregnant patient groups www.generaroma.it

27. Cumulative ongoing pregnancy rate after the fresh cycle, first and second warming cycle according to female age ≤34 yrs35-37 yrs38-40 yrs41-43 yrs OocyteEmbryoOocyteEmbryoOocyteEmbryoOocyteEmbryo Fresh cycle 29/72 (40.3)29/57 (50.9)20/48(41.7)25/44 (56.8)15/41(36.6)13/43 (30.2)4/21 (19.0)2/12 (16.7) I warming cycle 45/72 (62.5)34/57 (59.6)23/48 (47.9)29/44 (65.9)20/41(48.8)18/43 (41.9)6/21 (28.6)4/12 (33.3) II warming cycle 45/72 (62.5)34/57 (59.6)24/48 (50.0)29/44 (65.9)21/41 (51.2)18/43 (41.9)7/21 (33.3)4/12 (33.3) p = ns in any analyzed group www.generaroma.it

28. Overall Oocyte Embryo p Fresh 90/435 (20.7)101/342 (29.5) p = 0.005 Warming cycles 35/266 (13.1)16/156 (10.2) ns Fresh+ warming (cumulative) 128/701 (17.8)117/498 (23.5) p = 0.03 Cumulative ongoing implantation rate after fresh and warming cycle www.generaroma.it

29. Conclusion In this study we have compared two different policies applied on the general infertile population: - limited number of inseminated oocytes and oocyte cryopreser- vation - higher number of inseminated oocytes and embryo cryopreser- vation Same vitrification approach was used in both groups. According to our results when embryo selection is possible a significantly higher ongoing implantation rate can be achieved in the fresh cycle. www.generaroma.it

30. Conclusion The cumulative ongoing pregnancy rate is similar in both groups when including the warming cycles. The cumulative ongoing implantation rate is higher in the frozen embryo group Slightly more transfers are necessary in the oocyte group as compared to the embryo group to reach the same results With oocyte cryopreservation no supernumerary embryos are created (only oocytes with no related ethical and moral issues) www.generaroma.it

31. Conclusion Although the limits of the study design, with oocyte cryopreservation it is possible to achieve good cumulative clinical results in a standard infertility population www.generaroma.it A prospective randomized trial is needed to assess the efficacy of oocyte vs embryo cryopreservation

32. CLINICA VALLE GIULIA, Roma SALUS, Marostica (VI) GENERA, Umbertide (PG) www.generaroma.it Ginecologia: Filippo Ubaldi Elena Baroni Antonio Ciconte Silvia Colamaria Fabrizio Fiorini A. Giallonardo Madda Giuliani Fabio Sapienza Mauro Schimberni Silvia Venanzi Embriologia: Laura Rienzi Stefania Romano Roberta Maggiulli Laura Albricci Antonio Capalbo Nicoletta Barnocchi Benedetta Iussig Sara Fusco Federica Sanges Catello Scarica Elena Ievoli