Cryopreservation of whole ovaries with vascular pedicles: vitrification or conventional freezing?

  • Jian-Min Zhang
  • Yan Sheng
  • Yong-Zhi Cao
  • Hong-Yan Wang
  • Zi-Jiang Chen
Fertility Preservation



To compare the efficacy of vitrification and conventional freezing of whole ovaries.


Ovaries obtained from 5-year-old female bovines were cryopreserved by conventional freezing, rapid freezing and vitrification. The ovarian cortical strips were cryopreserved by conventional freezing. Follicular viability was assessed using the trypan blue test; the percentage of morphologically normal primordial follicles, hormones concentrations in the culture supernatants, and lactate dehyrogenase levels were measured.


The efficacy of cryopreservation of whole ovaries by vitrification was higher than those by conventional freezing and rapid freezing. Conventional freezing of ovarian cortical strips was more effective than cryopreservation of whole ovaries by conventional freezing, rapid freezing, and vitrification.


Vitrification seems to be more suitable than conventional freezing for cryopreservation of whole ovaries. However, further studies are required to improve the efficacy of vitrifying whole ovaries.


Conventional freezing Cryopreservation Rapid freezing Vitrification Whole ovaries 


  1. 1.
    Apperley JF, Reddy N. Mechanism and management of treatment-related gonadal failure in recipients of high dose chemoradiotherapy. Blood Rev. 1995;9:93–116.PubMedCrossRefGoogle Scholar
  2. 2.
    Baker TG. Radiosensitivity of mammalian oocytes with particular reference to the human female. Am J Obstet Gynecol. 1971;110:746–61.PubMedGoogle Scholar
  3. 3.
    Oktay K, Karlikay G. Ovarian function after transplantation of frozen, banked autologous ovarian tissue. N Engl J Med. 2000;342:1919.PubMedCrossRefGoogle Scholar
  4. 4.
    Oktay K, Buyuk E. Fertility preservation in women undergoing cancer treatment. Lancet. 2004;363:1830.PubMedCrossRefGoogle Scholar
  5. 5.
    Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russell SA, et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin’s lymphoma. Lancet. 2001;357:1172–5.PubMedCrossRefGoogle Scholar
  6. 6.
    Donnez J, Dolmas M, Demylle D. Live birth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet. 2004;364:1405–10.PubMedCrossRefGoogle Scholar
  7. 7.
    Meirow D, Levron J, Eldar-Geva T. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med. 2005;353:318–21.PubMedCrossRefGoogle Scholar
  8. 8.
    Schmidt KL, Andersen CY, Loft A, Byskov AG, Ernst E, Andersen AN. Follow-up of ovarian function post-chemotherapy following ovarian cryopreservation and transplantation. Hum Reprod. 2005;20:3539–46.PubMedCrossRefGoogle Scholar
  9. 9.
    Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at −196°C. Endocrinology. 1999;140:462–71.PubMedCrossRefGoogle Scholar
  10. 10.
    Wang X, Chen H, Yin H, Kim SS, Lin Tan S, Gosden RG. Fertility after intact ovary transplantation. Nature. 2002;415:385.PubMedCrossRefGoogle Scholar
  11. 11.
    Bedaiwy MA, Jeremias E, Gurunluoglu R, Hussein MR, Siemianow M, Charies B, et al. Restoration of ovarian function after autotransplantation of intact frozen-thawed sheep ovaries with microvascular anastomosis. Fertil Steril. 2003;79:594–602.PubMedCrossRefGoogle Scholar
  12. 12.
    Martinez-Madrid B, Dolmans MM, Van Langendonckt A, Defrere S, Donnez J. Freeze-thawing intact human ovary with its vascular pedicle with a passive cooling device. Fertil Steril. 2004;82:1390–4.PubMedCrossRefGoogle Scholar
  13. 13.
    Revel A, Elami A, Bor A, Yavin S, Natan Y, Arav A. Whole sheep ovary cryopreservation and transplantation. Fertil Steril. 2004;82:1714–5.PubMedCrossRefGoogle Scholar
  14. 14.
    Migishima F, Suzuki-Migishima R, Song SY, Kuramochi T, Azuma S, Nishijima M, et al. Successful cryopreservation of mouse ovaries by vitrification. Biol Reprod. 2003;68:881–7.PubMedCrossRefGoogle Scholar
  15. 15.
    Yin H, Wang X, Kim SS, Chen H, Tan SL, Gosden RG. Transplantation of intact rat gonads using vascular anastomosis: effects of cryopreservation, ischemia and genotype. Hum Reprod. 2003;18:1165–72.PubMedCrossRefGoogle Scholar
  16. 16.
    Chen CH, Chen SG, Wu GJ, Wang J, Yu CP, Liu JY. Autologous heterotopic transplantation of intact rabbit ovary after frozen banking at −196°C. Fertil Steril. 2006;86(4 Suppl):1059–66.PubMedCrossRefGoogle Scholar
  17. 17.
    Imhof M, Bergmeiter H, Lipovac M, Rudas M, Hofstetter G, Huber J. Orthotopic microvascular reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy and live birth. Fertil Steril. 2006;85 Suppl 1:1208–15.PubMedCrossRefGoogle Scholar
  18. 18.
    Courbiere B, Caquant L, Mazoyer C, Franck M, Lornage J, Salle B. Difficulties improving ovarian functional recovery by microvascular transplantation and whole ovary vitrification. Fertil Steril. 2009;91:2697–706.PubMedCrossRefGoogle Scholar
  19. 19.
    Rall WF, Fahy GM. Ice free cryopreservation of mouse embryos at −196°C by vitrification. Nature. 1985;313:573–5.PubMedCrossRefGoogle Scholar
  20. 20.
    Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online. 2005;11:608–14.PubMedCrossRefGoogle Scholar
  21. 21.
    Huang JY, Chen HY, Tan SL, Chian RC. Effect of choline-supplemented sodium-depleted slow freezing versus vitrification on mouse oocyte meiotic spindles and chromosome abnormalities. Fertil Steril. 2007;88:1093–100.PubMedCrossRefGoogle Scholar
  22. 22.
    Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, et al. Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, an molecular biological evalution. Reproduction. 2009;138:319–27.PubMedCrossRefGoogle Scholar
  23. 23.
    Keros V, Xella S, Hultenby K, Pettersson K, Sheikhi M, Volpe A, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod. 2009;24:1670–83.PubMedCrossRefGoogle Scholar
  24. 24.
    Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at −196 degrees C. Hum Reprod. 1994;9:597–603.PubMedGoogle Scholar
  25. 25.
    Scott JE. carlsson IB, Bavister BD, Hovatta O. Human ovarian tissue cultures: extracellular matrix composition, coating density and tissue dimensions. Reprod Biomed Online. 2004;9:287–93.PubMedCrossRefGoogle Scholar
  26. 26.
    Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod. 1996;11:1487–91.PubMedGoogle Scholar
  27. 27.
    Dissen GA, Lara HE, Frahrenbach WH, Costa ME, Ojeda SR. Immature rat ovaries become revascularized rapidly after autotransplantation and show a gonadotropin-dependent increase in angiogenic factor gene expression. Endocrinology. 1994;134:1146–54.PubMedCrossRefGoogle Scholar
  28. 28.
    Onions VJ, Webb R, McNeilly AS, Campbell BK. Ovarian endocrine profile and long-term vascular patency following heterotopic autotransplantation of cryopreserved whole ovine ovaries. Hum Reprod. 2009;24:2845–55.PubMedCrossRefGoogle Scholar
  29. 29.
    Kim SS. Time to re-think: ovarian tissue transplantation versus whole ovary transplantation. Reprod Biomed Online. 2010;20:171–4.PubMedCrossRefGoogle Scholar
  30. 30.
    Gerritse R, Beerendonk CCM, Tijink MSL, Heetkamp A, Kremer JAM, Braat DDM, et al. Optimal perfusion of an intact ovary as a prerequisite for successful ovarian cryopreservation. Hum Reprod. 2008;23:329–35.PubMedCrossRefGoogle Scholar
  31. 31.
    Fabbri R, Venturoli S, D’Errico A, Iannascoli C, Gabusi E, Valeri B, et al. Ovarian tissue banking and fertility preservation in cancer patients: histological and immunohistochemical evaluation. Gynecol Oncol. 2003;89:259–66.PubMedCrossRefGoogle Scholar
  32. 32.
    Neto V, Buff S, Lornage J, Bottolier B, Guérin P, Joly T. Effects of different freezing parameters on the morphology and viability of preantral follicles after cryopreservation of doe rabbit ovarian tissue. Fertil Steril. 2008;89:1348–56.PubMedCrossRefGoogle Scholar
  33. 33.
    Sadeu JC, Cortvindt R, Ron-EI R, Kasterstein E, Smitz J. Morphological and ultrastructural evaluation of cultured frozen-thawed human fetal ovarian tissue. Fertil Steril. 2006;85:1130–41.PubMedCrossRefGoogle Scholar
  34. 34.
    Perdrix A, Macé B, Milazzo JP, Liard-Zmuda A, Baron M, Rives N. Ovarian tissue thawing: a comparison of two conditions. Fertil Steril. 2010;93:307–10.PubMedCrossRefGoogle Scholar
  35. 35.
    Wang Y. Reply: Human ovarian tissue: vitrification versus conventional freezing. Hum Reprod. 2009;24:1768–9.CrossRefGoogle Scholar
  36. 36.
    Fauque P, Amor AB, Joanne C, Agnani G, Bresson JL, Roux C. Use of trypan blue staining to assess the quality of ovarian cryopreservation. Fertil Steril. 2007;87:1200–7.PubMedCrossRefGoogle Scholar
  37. 37.
    Courbiere B, Massardier J, Salle B, Mazoyer C, Guerin JF, Lornage J. Follicular viability and histological assessment after cryopreservation of whole sheep ovaries with vascular pedicle by vitrification. Fertil Steril. 2005;84 Suppl 2:1065–71.PubMedCrossRefGoogle Scholar
  38. 38.
    Courbiere B, Odagescu V, Baudot A, Massardier J, Mazoyer C, Salle B, et al. Cryopreservation of the ovary by vitrification a an alternative to slow-cooling protocols. Fertil Steril. 2006;86(4 Suppl):1243–51.PubMedCrossRefGoogle Scholar
  39. 39.
    Pegg DE. The role of vitrification techniques of cryopreservation in reproductive medicine. Hum Fertil. 2005;8:231–9.CrossRefGoogle Scholar
  40. 40.
    Martinez-Madrid B, Camboni A, Dolmans MM, Nottola S, Van Langendonckt A, Donnez J. Apoptosis and ultrastructural assessment after cryopreservation of whole human ovaries with their vascular pedicle. Fertil Steril. 2007;87:1153–65.PubMedCrossRefGoogle Scholar
  41. 41.
    Bedaiwy MA, Hussein MR, Charles B, Falcone T. Cryopreservation of intact human ovary with its vascular pedicle. Hum Reprod. 2006;21:3258–69.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jian-Min Zhang
    • 1
  • Yan Sheng
    • 1
  • Yong-Zhi Cao
    • 1
  • Hong-Yan Wang
    • 1
  • Zi-Jiang Chen
    • 1
  1. 1.Shandong Provincial Key Laboratory of Reproductive Medicine, Center for Reproductive MedicineProvincial Hospital Affiliated to Shandong UniversityJinanChina

Personalised recommendations