1. Vitrification as a tool to preserve women fertility in cancer patients Safaa Al-Hasani Department of Gynecology and Obstetrics Reproductive Medicine University of Schleswig-Holstein, Campus Lübeck Germany

2. J. Liebermann, 2009

3. Woldwide 4.0 millions children born through ART 20% of the children born through cryopreservation procedure

4. Introduction During the last decades there have been tremendous improvements in cancer treatment with the survival rates for most types of cancer increasing considerably. The dramatic increase in survival after cancer treatment is indeed true for the types of cancer that girls and young females may acquire.

5. Introduction The most common cancer type in children is leukemia followed by cancer of the central nervous system, neuroblastoma, Hodgkin‘s, non-Hodgkin‘s lymphoma, and Wilm‘s tumour.

6. Introduction The most common type of malignancy among females during the reproductive age is breast cancer and 64% of the patients are younger than 40 years old. It was also shown that the 5 year survival rates for breast cancer patients increased from 75% in the mid 1970s to 88% in the late 1990s.

7. Introduction Nowadays vitrification procedure showed better results in preservation of women fertility compared to the slow freezing method.

8. In this presentation we will discuss the ultra-rapid method for the cryopreservation of human oocytes, embryos, ovarian tissue but not the whole ovary

9. Indications for fertility preservation a)Oncological b)Non-oncological 1.Premature ovarian failure 2.Autoimmune diseases 3.Infections

10. Indications for fertility preservation c)Environmental factors 1.Radiation d)Exposure to gonadotoxic agents e)Surgical menopause f)Women wishing to postpone motherhood

11. Healthy delivery of a twin after transfer of embryos resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue Safaa Al-Hasani Case report

12. Cryopreservation of human Oocytes  Slow Freezing Method  Ultrarapid Freezing Method (Vitrification)

13. 1.Low concentration of cryoprotectants 2.Slow controlled cooling rates 3.Slow process of dehaydration of the oocyte to reduce intracellular ice crystal formation and to reduce cell damage The principles of Slow Freezing procedure:

14. Meiotic spindle Microtubule system chain of tubulin polymer hold the chromosomes in the metaphase plate The polymer formation is temperature dependant: –Lowering the temperature decrease polymerization shortening of the chain –At 22˚C, the tubulin arm completely disappear –This process is reversible

15. Oocyte Freezing

16. Principles of cryopreservation Water in cell: Around 90% of water is free (water) while the remaining 10 % bounds to other molecular components of the cell (proteins, lipids, nucleic acids and other solutes). This water does not freeze and called hydrated water –Removal of water is necessary during freezing to avoid ice crystal formation, dehydration is limited to the free water –Removal of hydrated water could have adverse effect on the cell viability and the molecular function (freezing injuries)

17. Factors causing cell injuries Cryopreservation involves chilling and even freezing or vitrifying cells in order to put their life “on hold” For either freezing or vitrification to maintain vital function of the cells, cooling warming and solute concentration must be managed in a way to favor survival and to minimize injury Living cells can be injured by reduction in temperature, by ice crystals, by osmotic forces, and by chemical toxicity All are factors related to slow freezing procedure

18. Oocyte Freezing Experience A) From 1986 to 1996 Chen, 1986 Al-Hasani et al. 1987 Van Uem et al. 1987 Serafini et al. 1995

19. Experience B) from 1997 Porcu et al., 1997 Antinori et al., 1998 Borini et al., 1998 Polak de Fried et al., 1998 Porcu et al., 1988 Videli et al., 1998 Yang et al., 1998 Young et al., 1998 Porcu et al., 1999 Porcu et al., 1999a Porcu et al., 1999b Yang et al., 1999 Porcu et al., 2000 Fabbri et al., 2001 Porcu et al., 2002 Yang et al., 2002 Oocyte Freezing

20. Factors that have improved results The use of mature eggs Cryoprotective solutions Freezing speed: slow Thawing speed: rapid ICSI Vitrification Oocyte Freezing

21. Pregnancies and births from frozen human oocytes (slow cooling) Authors Year Cryoprotectant Oocyte stage Pregnancies/Births 1986, 1987 1987 1989 1995 1996 1998 1999 2000 2002 2003 Chen-Al-Hasani Diedrich -Van Uem Siebzehnrubl Serafini Tucker Tucker - Polak de Fried Young- Yang Naworth and Kissing Antinori-Borini Tucker Lanzerndof Porcu - Chia Chen - Quintans Albani Boldt - Fosas DM-PR/DMSO DMSO PR/DMSO PR MII GV MII 2/2 - 2/? 1/? - 1/1 1/? 2/0 3/0 5/2 - 1/1 1/? - 1/? 1/0 1/1 - 3/3 1/1 1/2 19/19 - 1/0 1/? - 6/2 1/? 4/4 - 4/4 1/? - 1/?

22. Pregnancies per cycle With frozen eggs:17.2% With frozen embryos:18.7% Porcu et al., 2002 Oocyte Freezing

23. Vitrification of human oocytes and embryos

24. In 1937, Luyet wrote that “crystallization is incompatible with living systems and should be avoided whenever possible” Luyet. Biodynamica 1937; 1: 1–14

25. Historical review It was described at the end of the 18 th Century Tammann, 1898 Vitrification of mouse embryos at –196°C Rall & Fahy, 1985; Ali & Shelton, 1993 Blastocyst development from bovine oocytes Martino et al., 1996 Blastocyst development, pregnancies, deliveries from human vitrified oocytes, zygotes, cleaved eggs and blastocyst Tammann. Z Phys Chem 1898; 25: 441-479 Rall & Fahy. Nature 1985; 313 (6003): 573–575 Ali & Shelton. J Reprod Fertil 1993; 98 (2): 459–465 Martino et al. Biol Reprod 1996; 54 (5): 1059–1069

26. Vitrification: Two droplets of different solutions plunged directly into liquid nitrogen: left droplet is pure Dulbecco’s phosphate-buffered saline (DPBS) with ice crystallization, in contrast to the right droplet containing an equimolar combination of 20% ethylene glycol and dimethyl sulphoxide with 0.4 M sucrose in DPBS without ice crystallization (glassy, vitrified state). DPBS EG+DMSO+0.4 M sucrose Ice crystallization Vitreous, glassy state

27. Vitrification Successful vitrification requires: –Minimum volume of holding media (<1  l) –Increased viscosity –High CPAs concentration –High cooling rate (-50 000 C and warming rate (+36 000 C) –Special vehicle device (carrier) (Cryotops) –Direct plunging in LN2

28. Risk of Contamination of Germplasm during Cryopreservation and Cryobanking in IVF Units Bielanski and Vajta: 2009 -It has to be stated that none of the reported infections after insemination or embryo transfer in humans and demostic animals can be clearly attributed to the applied cryopreservation and storage procedure. -To ensure rapid cooling in some vitrification techniques requiring direct contact, sterile LN2 should be used, then samples should be safely sealed into pre-cooled secondary containers. Human Reprod. 24, 2457-2467

29. Shortly before loading on Cryotop

30. Cryotop (Kuwayama)

31. Risk of Contamination of Germplasm during Cryopreservation and Cryobanking in IVF Units Bielanski and Vajta: 2009 -Unfortunately, the open system and direct contact might be indispensable to achieve the required cooling and warming rates for every sensitive samples: so far, commercially available closed analogues (Cryotips, CBS and SSV analogues) have failed to demonstrate the same efficiency for human oocytes when compared with their counterparts using the direct contact approach. Human Reprod. 24, 2457-2467

32. Slow cooling (langsames Einfrieren) - 0.3°C/min Vitrifikation (verlusrieren) - 50.000°C/min 1 sec. Vitrifikation (ultra-rapides Einfrieren)

33. Example of cooling rates -2500°C/min by using 0.25 mL straws –thick straws and large volumes of medium do not allow a high cooling rate and thawing rate -25.000 – -50.000°C/min by using a carrier that allows very small volumes –direct contact with LN 2

34. “The physical definition of vitrification is the solidification of a solution (water is rapidly cooled and formed into a glassy, vitrified state from the liquid phase) at low temperature, not by ice crystallization but by extreme elevation in viscosity during cooling” Fahy, 1984 Fahy et al. Cryobiology 1984; 21: 407–426

35. In contrast to slow-rate freezing protocols, during vitrification the entire solution remains unchanged and water does not precipitate, so no ice crystals are formed

36. Slow freezing versus ultra-rapid freezing TraditionalVitrification CPA concentration1.5 M3.0–5.0 M Volume0.3–1.0 mL<1 µL Contact between N 2 and cell NoYes Cooling rate~0.5°C/min~25.000– 50.000°C/min FreezingSlowUltra-rapid Thawing / warmingSlowRapid Time consuming≥180 min1 sec DehydrationNot controlledControlled

37. TraditionalVitrification Reduced osmotic injuryNoYes Zona pellucida fracturePossibleNo Ice crystal formationYesNo SeedingYesNo need ProcedureComplicatedSimple DeviceYesNo need Costs Liquid nitrogen amount Duration out Incubator High > 4 Hrs Less Much less 10 min Slow freezing versus ultra-rapid freezing

38. Author Warmed Oocytes N° survived N° 2 PN cleaved embryos N° ET N° Preg Cha, 1999777--1 Kuleshova, 199917115331 Kuwayama, 2000--2211 Yoon, 20009057393273 Yoon, 2003474325142125286 Katayama, 20034642383462 Kim, 20051861395839104 Kim, 2005233 165 7749127 Chian, 200518016912654157 Ruvalcaba, 200560463834108 Kyono, 2005555111 Kuwayama 20051078677642912 Lucena, 2006159120105972313 Selman, 2006241814 62 Total %15881190 (75)733 (61.6)548 (74.7)15168 (45)

39. Vitrification Kuleshova (1999) Hong (1999) Katayama (2003) Yoon (2003) 11/17 (65) 30/33 (90) 42/46 (94) 325/474 (69) 45 69 91 72 1(33) 1 2/6 (33) 6/28 (21) Fertilized % Pregnancies/ transfer N (%) Oocytes AUTHORS Survived N (%)

40. Vitrification of Human Oocytes Kuwayama 2005 Vitrified oocytes64 Survived oocytes after warming58(91%) Fertilized oocytes52(89.6%) Embryo Transfer29 N° of clinical pregnancy12(41%) Deliveries7 Ongoing pregnancies3

41. Figs. 5 Human oocytes before and after vitrification, ICSI and IVC. Before Vitrification2hrs after CultureJust after Thawing 4-cell stage (Day2) Blastocyst (Day5)PN stage (Day 1)

42. Vitrified / warmed group Update up to July 2007 No. of cycles120270 No. of warmed oocytes330707 No. of oocytes survived (%) 328 (99.3%)699 (98.8%) No. of injected oocytes328699 No. of fertilised oocytes (2PN) 305 (92.9%)639 (91.4%) No. of cleaved oocytes295 (96.7%)624 (97.6%) Antinori et al. Reprod Biomed Online 2007; 14 (1): 72-79

43. Vitrified / warmed group Update up to July 2007 No. of transfers120270 No. of transferred embryo295624 No. of embryos per transfer2.452.31 No. of clinical pregnancies39 (32.5%)76 (28.1%), 5 twins No. of ongoing pregnancies–24 No. of abortions8 (20.5%)16 (21%) No. of deliveries3136, all singleton IR per transferred embryo*13.2 %12.9% IR per thawed oocyte**11.8%11.6 Antinori et al. Reprod Biomed Online 2007; 14 (1): 72-79

44. Embryo Development of Fresh „Versus“ Vitrified Metaphase II Oocytes after ICSI: A Prospective Randomised Sibling-Oocyte Study Conclussion: Our results indicate that oocyte vitrification procedure followed by ICSI is not inferior to fresh insemination procedure, with regard to fertilization and embryo developmental rates. Moreover, ongoing clinical pregnancy is comparable with this procedure, even with a restricted number of oocytes available for inseminat- ion. We believe that these results will help the spread of vitrification for human oocytes cryopreservation.The promising clinical results obtained, in a population of infertile patients, need to be confirmed on a larger scale. Rienzi et al., 2010 Human Reprod., 25, 66-73

45. Vitrified M II oocytes231 Survival96.9% Fertilization76.3% No. of transfers23 Mean number of embryos2.1 Ongoing pregnancy rate48% Cobo et al., 2008 Oocyte Donation & Vitrification

46. Obsteric and perinatal outcome in 200 infants conceived from vitrified oocytes Statement: These preliminary findings may provide reassuring evidence that pregnancies and infants conceived following oocyte vitrification are not associated with increased risk of adverse obstetric and perinatal outcomes. Chian et al, RBM online 16, May 2008

47. Comparison between fresh and frozen- thawed embryo transfer Vitrification of Zygotes (Luebeck) Fresh ETFrozen-Thawed ET No of patients5259 No of cycles5361 No of vitrified Zygotes/259 No of survived zygotes/250 (96.5) No of transferred embryos 114 (2.5)240 (2.6) No of embryo transfers5383 No of pregancies13 (24.5)29 (34.1)

48. Our Results in Avoiding Hyperstimulation Patients Triggered with GnRH-Agonist No. of Patients No. of Zygotes vitrif. No. of Zygotes re- warmed No. & (%) Zygotes survived No. & (%) Preg. No. Of Children born (%) of live birth 59433163 * 158 (97) 25 (42) 13 ** (25) * No. of Patients received warmed Zygotes 45 ** Two Twins

49. The blastocyst is characterized by early cavitation resulting in the formation of an eccentric and then expanded cavity lined by a distinct inner cell mass region and trophectoderm layer. The blastocele is less than half the volume of the embryo

50. Can fresh embryo transfers be replaced by cryo- preserved-thawed embryo transfers in assisted reproductive cycles? A prospective controlled trial. Fresh ET (n=191) FET (n=184)p value No. of oocytes retrievd14.214NS No. Of M II oocytes retrieved1110.8NS E2 day of hCG (pg/ml)2861.22793.4NS Fertilization rate72.773NS No. of embryos transferred2.2 + 0.42.1 + 0.3NS Implantation rate (%)14.123.00.004 Clinical pregnancy rate (%)24.636.40.013 Ongoing pregnancy rate (%)22.534.20.012 Multiple pregnancy rate (%)14.926.4NS Aflatoonian et al. 2009, Human Reprod. (submitted)

51. Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates Rama Raju et al. (2005) VitrificationSlow freezing Embryos, n436420 Embryos thawed, n127120 Embryos survival, n (%)121 (95.3)72 (60) Pregnancy, n (%)14 (35)4 (17.4) 40% ethylene glycol + 0.6 mol sucrose, nylon loop “Ethylene glycol is a good croyprotectant to preserve 8-cell embryos because of its low toxicity as shown by the high survival rate, and vitrification is a promising alternate to the conventional slow-freezing method.” Rama Raju et al. Reprod Biomed Online 2005; 11 (4): 434–437

52. Outcome of Blastocyst Cryopreservation by using the “Slow Freezing” Method Reference No. of thawed blastocysts Survival rate % Implantation rate % Pregnancy rate % Troup et al. 199034 38 (13/34) NA no pregnancy Menezo et al. 1992106NA 21.0 Kaufmann et al. 1995 1239 83 (1033/1239)13.421.7 Nakayama et al. 19956978 (54/69)18.8 1.7 Martin et al. 2003 624 8623.330.6 Andersen et al. 200420281 (164/202) 43.069.0 Veeck et al. 2004 628 76 (479/628)NA59.2 Kuwayama et al. 2005 15684 (131/156)NA51.0 Stehlik et al. 20057183 (59/71)6.816.7 Liebermann & Tucker 2006 254 91 29.642.8

53. Outcome of Human Blastocyst Vitrification by using the “Hemi-straw” Method Reference No. of vitrif. blastocysts Survival rate % Implantation rate % Pregnancy rate % Vanderzwalmen et al. 2002 16758.5NA20.5 Vanderzwalmen et al. 2003 28160NA27 Zech et al. 2005177 82 NA 35

54. Outcome of Blastocyst Vitrification by using the “Cryoloop” Method Reference No. of vitrif. blastocysts Survival rate % Implantation rate % Pregnancy rate % Mukaida et al. 2001 6063NA31.5 Reed et al. 200215 100 15.425 Mukaida et al. 2003b 725 87NA 37

55. Outcome of Blastocyst Vitrification by using the “Cryotop” Method Reference No. of vitrif. blastocysts Survival rate % Implantation rate % Pregnancy rate % Hiraoka et al. 2003 49 98 33 50 Stehlik et al. 2005 41 100 NA 50 Kuwayama et al. 2005 6484 90NA 53

56. Post-thaw survival rates of human zygotes, embryos and blastocysts after vitrification n=5881n=897n=1175

57. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts Stehlik et al. (2005) Day 5Day 6 Slow freezing VitrificationSlow freezing Vitrification Embryos transferred, No. 24202715 Embryos survival, %83 100 89.5 100 Pregnancy, %16.7 50 18.5 33.3 Stehlik et al. Reprod Biomed Online 2005; 11 (1): 53–57 Day 5 vitrified blastocysts showed significantly increased survival and pregnancy rates compared with Day 5 slow-frozen blastocysts A similar trend was observed with Day 6 blastocysts

58. Comparison of vitrification and conventional cryopreservation of Day 5 and Day 6 blastocysts during clinical application Liebermann & Tucker (2006) VitrificationConventional Blastocysts warmed, n547570 Blastocysts survival, n (%)528 (96.5)525 (92) Blastocysts transferred, n523518 Implantation, n (%)160 (30.6)152 (28) Ongoing pregnancies, n (%) 117 (88.6)109 (79.6) Vitrification technique yields the same implantation and pregnancy rate as slow-frozen blastocyst transfer Liebermann & Tucker. Fertil Steril 2006; 86 (1): 20–26

59. “…We believe that vitrification shows much promise as a successful alternative to conventional freezing technology. Even without significant clinical improvement, the evident advantages of vitrification are that: 1.Cryosurvival seems more consistent, allowing greater case of patient management, with transfers being almost certain to occur 2.Vitrification is able to be undertaken on a more flexible basis by laboratory staff Liebermann & Tucker. Fertil Steril 2006; 86 (1): 20–26 Comparison of vitrification and conventional cryopreservation of Day 5 and Day 6 blastocysts during clinical application Liebermann & Tucker (2006)

60. ...and: 3.Vitrification allows for the potential reduction in personnel time needed during the entire vitrification process 4.It may enable more optimal timing of embryo cryopreservation, e.g., individual blastocysts may be cryopreserved at their optimal stage of development and expansion…” Liebermann & Tucker. Fertil Steril 2006; 86 (1): 20–26 Comparison of vitrification and conventional cryopreservation of Day 5 and Day 6 blastocysts during clinical application Liebermann & Tucker (2006)

61. ovarian biopsy and cryopreservation in vitro Growth ( „ IVG “ ) followed by In vitro Maturation ( „ IVM “ ) In the future Cryopreservation of „ Ovarian Cortex “ Oktay et al. 2004

62. Woldwide 10-12 children born through retransplantation of ovarian cortex The number of unsuccessful traials is unknown !!

64. Vitrifcation procedure: the Cryotissue method. The ovarian tissue slicer was developed, with a plate to produce 1 × 10 × 10 mm slices. (1) The tissue slicer was put on the surface of ovary. (2) Then another plate was put on the tissue slicer, the ovary was cut between the slicer and the surface of ovary by using a sharp edge. (3) The ovarian tissue was cut into 1 × 10 × 10 mm slices. Successful vitrification of bovine and human ovarian tissue Kagawa et al., RBMOnline 2009

65. Gross morphology of vitrifed human ovarian tissue using the Cryotissue method. Vitrifed human ovarian tissue was translucent in liquid nitrogen (–196°C). Scale bar represents 10 mm. Successful vitrification of bovine and human ovarian tissue Kagawa et al., RBMOnline 2009

66. All oocytes (arrows) were located in the cortical area of the human ovarian tissue. Note that they were all located within 0.75 mm of the surface, allowing much thinner slices to be made than can be obtained by hand, or than have been used in previous studies. Scale bar represents 1 µm. Successful vitrification of bovine and human ovarian tissue Kagawa et al., RBMOnline 2009

67. Morphologically normal oocyte in a pre-antral follicle from vitrifed-warmed human ovarian tissue. (A) Normal oocyte was surrounded by one or two layers of somatic cells in normal interstitial tissue of vitrifed ovarian tissue. Haematoxylin– eosin staining. (B) Normal oocyte was surrounded by three or four layers of somatic cells in normal interstitial tissue of vitrifed ovarian tissue. Scale bar represents 50 µm. Successful vitrification of bovine and human ovarian tissue Kagawa et al., RBMOnline 2009

68. Histological section of vitrifed human ovarian tissue. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), a marker protein for proliferating cells. Abundant PCNA-positive proliferating cells were demonstrated in the interstitial tissue cells and a few proliferating cells were noted in granulose cells (arrows) of vitrifed human ovarian tissue. Scale bar represents 50 µm. Successful vitrification of bovine and human ovarian tissue Kagawa et al., RBMOnline 2009

69. Surviving oocytes (arrows) of pre-antral follicles of vitrifed–thawed ovarian tissue in human (Hoechst/propidium iodide stain). Nuclei of living oocytes were blue. Scale bar represents 50 µm. Successful vitrification of bovine and human ovarian tissue Kagawa et al., RBMOnline 2009

70. No. of samples Number of Oocytes (%) CollectedSurviving Vitrified7954 (100)855 (89.6) Survival of oocytes in vitrified-warmed human ovarian tissue Successful vitrification of bovine and human ovarian tissue Kagawa et al., RBMOnline 2009

71. Abstract: […] In addition, human ovarian tissue from cancer patients, and from ovary transplant donors was also vitrifed by the Cryotissue method. After warming, high oocyte survival in human tissue (similar to bovine tissue) was obtained. These results indicate that an ultra-rapid cooling vitrifcation method has the potential for clinical use in human ovarian tissue cryopreservation. […]

72. Successful vitrification –High cooling rate (> –50.000°C) –Fast cooling period (<1 sec.) –Low volume (<1 µL) –High concentration of cryoprotectants => This will avoid crystal formation

73. Why do we prefer the vitrification procedure now? No mechanical injury (extracellular crystal formation) Less osmotic stress to cells No intracellular crystal formation Less labour in laboratory daily work Simple protocol Useful for oocytes and blastocysts, which have less success with slow freezing No need for expensive devices

74. Future Aspects Avoiding hyperstimulation syndrome in patients with PCOS by vitrification of all 2PN and replaced in a programmed cycle Cancelling of fresh ET in case of more than 10 Follicles Vitrification of all zygotes resulted from IVM programme An option for cancer patients to vitrify the oocytes instead of ovarian tissue In oocytes donation programme Vitrification of the oocytes to postpone fertility Mantains viability of specimens during long term storage

76. Quality is evolution Vitrification is a revolution Professor Josiane Van der Elst 19 January 2007

77. Conclusion Easy to perform Low cost Future first choice procedure It was shown to be superior to slow freezing procedure Very high survival rates of oocytes and embryos at all stages of development It seems that the cryotop method is the most efficient procedure Revitrification is possible Ovarian Cortex is now also possible

78. Thank you for your attention!