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Vitrification as a tool to preserve women fertility in cancer patients

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. J. Liebermann, 2009. Woldwide 4.0 millions children born through ART

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Vitrification as a tool to preserve women fertility in cancer patients

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  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 • Oncological • Non-oncological • Premature ovarian failure • Autoimmune diseases • Infections

  10. Indications for fertility preservation • Environmental factors • Radiation • Exposure to gonadotoxic agents • Surgical menopause • 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. The principles of Slow Freezing procedure: • Low concentration of cryoprotectants • Slow controlled cooling rates • Slow process of dehaydration of the oocyte to reduce intracellular ice crystal formation and to reduce cell damage

  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. Oocyte Freezing • 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

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

  21. 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 PR PR PR PR PR PR PR PR PR PR PR PR MII MII MII MII MII MII MII MII MII GV MII MII MII MII MII 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/? 1986, 1987 1987 1989 1995 1996 1998 1998 1998 1998 1998 1999 2000 2002 2002 2003 2003 Pregnancies and births from frozen human oocytes (slow cooling) Authors Year Cryoprotectant Oocyte stage Pregnancies/Births

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

  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 18th 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). Vitreous, glassy state Ice crystallization EG+DMSO+0.4M sucrose DPBS

  27. Vitrification Successful vitrification requires: • Minimum volume of holding media (<1ml) • 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 UnitsBielanski and Vajta: 2009

  29. Shortly before loading on Cryotop

  30. Cryotop (Kuwayama)

  31. Risk of Contamination of Germplasm during Cryopreservation and Cryobanking in IVF UnitsBielanski and Vajta: 2009

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

  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 LN2

  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

  37. Slow freezing versus ultra-rapid freezing

  38. Vitrification Oocytes Pregnancies/ transfer N (%) AUTHORS 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) Survived N (%) Fertilized %

  39. Vitrification of Human Oocytes

  40. Before Vitrification Just after Thawing 2hrs after Culture PN stage (Day 1) 4-cell stage (Day2) Blastocyst (Day5) Figs. 5 Human oocytes before and after vitrification, ICSI and IVC.

  41. Antinori et al. Reprod Biomed Online 2007; 14 (1): 72-79

  42. Antinori et al. Reprod Biomed Online 2007; 14 (1): 72-79

  43. 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

  44. Oocyte Donation & Vitrification Cobo et al., 2008

  45. 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

  46. Comparison between fresh and frozen-thawed embryo transferVitrification of Zygotes (Luebeck)

  47. OurResults in Avoiding Hyperstimulation PatientsTriggeredwithGnRH-Agonist * No. of Patients received warmed Zygotes 45 ** Two Twins

  48. 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

  49. Can fresh embryo transfers be replaced by cryo-preserved-thawed embryo transfers in assisted reproductive cycles? A prospective controlled trial. Aflatoonian et al. 2009, Human Reprod. (submitted)

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