Beneficial effect of desialylated erythropoietin administration on the frozen-thawed canine ovarian xenotransplantation

  • Hiroshi SuzukiEmail author
  • Takako Ishijima
  • Saori Maruyama
  • Yoshiko Yanagimoto Ueta
  • Yasuyuki Abe
  • Hideki Saitoh



The main drawback of ovarian cryopreservation followed by transplantation is that a large proportion of follicles are lost after transplantation. Thus, effects of erythropoietin (EPO) and desialylated EPO administration on the frozen-thawed canine ovarian xenotransplantation were examined.


The protective and survival-promoting effects of EPO and desialylated EPO on the follicles of frozen-thawed canine ovaries after transplantation were examined using NOD-SCID mice. Frozen-thawed dog ovarian tissue with 400 U/kg of EPO or asialo EPO was placed into the ovarian bursa.


At 4 weeks after the transplantation, the ovaries were removed and subjected to histological examination. The survival rate of early primary follicles was 15.2% in the EPO group and 157.6% in the asialo EPO group, in contrast to 10.1% in the untreated group.


These results demonstrate that administration of asialo EPO could be effectively used to enhance the survival of the follicles of transplanted cryopreserved ovaries.


Cryopreservation Dog Erythropoietin Fertility Follicle Ovary Transplantation 



This study was supported by funds from the Ministry of Health, Labour and Welfare of Japan.


  1. 1.
    Blatt J. Pregnancy outcome in long-term survivors of childhood cancer. Med Pediatr Oncol 1999;33:29–33. doi: 10.1002/(SICI)1096-911X(199907)33:1<29::AID-MPO6>3.0.CO;2-2.PubMedCrossRefGoogle Scholar
  2. 2.
    Donnez J, Matinez-Madrid B, Jadoul P, Van Langendonckt A, Demylle D, Dolmans MM. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update 2006;12:519–35. doi: 10.1093/humupd/dml032.PubMedCrossRefGoogle Scholar
  3. 3.
    Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196C. Hum Reprod 1994;9:597–603.PubMedGoogle Scholar
  4. 4.
    Donnez J, Bassil S. Indications for cryopreservation of ovarian tissue. Hum Reprod Update 1998;4:248–59. doi: 10.1093/humupd/4.3.248.PubMedCrossRefGoogle Scholar
  5. 5.
    Meirow D, Ben Yehuda D, Prus D, Poliak A, Schenker JG, Rachmilewitz EA, Lewin A. Ovarian tissue banking in patients with Hodgkin’s disease: is safe? Fertil Steril 1998;69:996–8. doi: 10.1016/S0015-0282(98)00093-4.PubMedCrossRefGoogle Scholar
  6. 6.
    Oktay K, Newton H, Aubard Y, Salha O, Gosden RG. Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology? Fertil Steril 1998;69:1–7. doi: 10.1016/S0015-0282(97)00207-0.PubMedCrossRefGoogle Scholar
  7. 7.
    Donnez J, Godin PA, Qu J, Nisolle M. Gonadal cryopreservation in young patient with gynaecological malignancy. Curr Opin Obstet Gynecol 2000;12:1–9. doi: 10.1097/00001703-200002000-00001.PubMedCrossRefGoogle Scholar
  8. 8.
    Donnez J, Dolmans MM, Matinez-Madrid B, Demylle D, Van Langendonckt A. The role of cryopreservation for women prior to treatment of malignancy. Curr Opin Obstet Gynecol 2005;17:333–8. doi: 10.1097/01.gco.0000175348.72566.47.PubMedCrossRefGoogle Scholar
  9. 9.
    Newton H, Aubard Y, Rutherford A, Sharma V, Gosden RG. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996;11:1487–91.PubMedGoogle Scholar
  10. 10.
    Candy CJ, Wood MJ, Whittingham DG. Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries. J Reprod Fertil 1997;110:11–9. doi: 10.1530/jrf.0.1100011.PubMedGoogle Scholar
  11. 11.
    Gunasena KT, Villines PM, Critser ES, Critser JK. Live births after autologous transplant of cryopreserved mouse ovaries. Hum Reprod 1997;12:101–6. doi: 10.1093/humrep/12.1.101.PubMedCrossRefGoogle Scholar
  12. 12.
    Aubard Y, Piver P, Cognie Y, Fermeaux V, Poulin N, Driancourt MA. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 1999;14:2149–54. doi: 10.1093/humrep/14.8.2149.PubMedCrossRefGoogle Scholar
  13. 13.
    Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196C. Endocrinology 1999;140:462–71. doi: 10.1210/en.140.1.462.PubMedCrossRefGoogle Scholar
  14. 14.
    Nisolle M, Godin PA, Casanas-Roux F, Qu J, Motta P, Donnez J. Histological and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice. Fertil Steril 2000;74:122–9. doi: 10.1016/S0015-0282(00)00548-3.PubMedCrossRefGoogle Scholar
  15. 15.
    Oktay K, Newton H, Gosden RG. Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice. Fertil Steril 2000;73:599–603. doi: 10.1016/S0015-0282(99)00548-8.PubMedCrossRefGoogle Scholar
  16. 16.
    Liu J, Van der Elst J, Van den Broecke R, Dhont M. Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod 2002;17:605–11. doi: 10.1093/humrep/17.3.605.PubMedCrossRefGoogle Scholar
  17. 17.
    Calvillo L, Latini R, Kajstura J, Leri A, Anversa P, Ghezzi P, Salio M, Cerami A, Brines M. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling. Proc Natl Acad Sci USA 2003;100:4802–6. doi: 10.1073/pnas.0630444100.PubMedCrossRefGoogle Scholar
  18. 18.
    Sakanata M, Wen TC, Matsuda S, Masuda S, Morishita E, Nagao M, Sasaki R. In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci USA 1998;95:4635–40. doi: 10.1073/pnas.95.8.4635.CrossRefGoogle Scholar
  19. 19.
    Brines M, Grasso G, Fiordaliso F, Sfacteria A, Ghezzi P, Fratelli M, Latini R, Xie QW, Smart J, Su-Rick C, Pobre E, Diaz D, Gomez D, Hand C, Coleman T, Cerami A. Erythropoietin mediates tissue protection through an erythropoietin and common β-subunit heteroreceptor. Proc Natl Acad Sci USA 2004;101:14907–12. doi: 10.1073/pnas.0406491101.PubMedCrossRefGoogle Scholar
  20. 20.
    Gook DA, Edgar DH, Borg J, Archer J, McBain JC. Diagnostic assessment of developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting. Hum Reprod 2005;20:72–8. doi: 10.1093/humrep/deh550.PubMedCrossRefGoogle Scholar
  21. 21.
    Ishijima T, Kobayashi Y, Lee DS, Ueta YY, Matsui M, Lee JY, Suwa Y, Miyahara K, Suzuki H. Cryopreservation of canine ovaries by vitrification. J Reprod Dev 2006;52:293–9. doi: 10.1262/jrd.17080.PubMedCrossRefGoogle Scholar
  22. 22.
    Imai N, Higuchi M, Kawamura A, Tomonoh K, Oh-Eda M, Fujiwara M, Shimonaka Y, Ochi N. Physicochemical and biological characterization of asialoerythropoietin. Suppressive effects of sialic acid in the expression of biological activity of human erythropoietin in vitro. Eur J Biochem 1990;12:457–62. doi: 10.1111/j.1432-1033.1990.tb15639.x.CrossRefGoogle Scholar
  23. 23.
    Nakagata N. Survival of mouse morulae and blastocysts derived from in vitro fertilization after ultra rapid freezing. Jikken Dobutsu 1993;42:229–31.PubMedGoogle Scholar
  24. 24.
    Whittingham DG. Embryo banks in the future of developmental genetics. Genetics 1974;78:395–402.PubMedGoogle Scholar
  25. 25.
    Oktay K, Schenken RS, Nelson JF. Proliferating cell nuclear antigen marks the initiation of follicular growth in the rat. Biol Reprod 1995;53:295–301. doi: 10.1095/biolreprod53.2.295.PubMedCrossRefGoogle Scholar
  26. 26.
    Seibzehnrubl E, Kohl J, Dittrich R, Wildt L. Freezing of human ovarian tissue-not the oocytes but the granulose is the problem. Mol Cell Endocrinol 2000;169:109–11. doi: 10.1016/S0303-7207(00)00362-2.CrossRefGoogle Scholar
  27. 27.
    Martinez-Madrid B, Dolmans MM, Van Langendonckt A, Defrere S, Donnez J. Freezing-thawing intact human ovary with its vascular pedicle with a passive cooling device. Fertil Steril 2004;82:1390–4. doi: 10.1016/j.fertnstert.2004.06.036.PubMedCrossRefGoogle Scholar
  28. 28.
    Maltaris T, Beckmann MW, Binder H, Mueller A, Hoffmann I, Koelbl H, Dittrich R. The effect of a GnRH agonist on cryopreserved human ovarian graft in severe combined immunodeficient mice. Reproduction 2007;133:503–9. doi: 10.1530/REP-06-0061.PubMedCrossRefGoogle Scholar
  29. 29.
    Maltaris T, Dimmler A, Muller A, Binder H, Hoffmann I, Kohl I, Siebzehnrubl E, Beckmann MW, Dittrich R. The use of an open-freezing system with self-seeding for cryopreservation of mouse ovarian tissue. Reprod Domest Anim 2005;40:250–4. doi: 10.1111/j.1439-0531.2005.00595.x.PubMedCrossRefGoogle Scholar
  30. 30.
    Israely T, Dafni H, Nevo N, Tsafriri A, Neeman M. Angiogenesis in ectopic xenotransplantation: multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI. Magn Reson Med 2004;52:741–50. doi: 10.1002/mrm.20203.PubMedCrossRefGoogle Scholar
  31. 31.
    Kim SS, Yang HW, Kang HG, Lee HH, Lee HC, Ko DS, Gosden RG. Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment. Fertil Steril 2004;82:679–85. doi: 10.1016/j.fertnstert.2004.05.022.PubMedCrossRefGoogle Scholar
  32. 32.
    Nugent D, Newton H, Gallivan L, Gosden RG. Protective effect of vitamin E on ischemia-reperfusion injury in ovarian grafts. J Reprod Fertil 1998;114:341–6. doi: 10.1530/jrf.0.1140341.PubMedCrossRefGoogle Scholar
  33. 33.
    Maltaris T, Beckmann MW, Mueller A, Hoffmann I, Kohl J, Dittrich R. Significant loss of primordial follicles after prolonged gonadotropin stimulation in xenografts of cryopreserved human ovarian tissue in severe combined immunodeficient mice. Fertil Steril 2007;87:195–7. doi: 10.1016/j.fertnstert.2006.05.058.PubMedCrossRefGoogle Scholar
  34. 34.
    Yasuda Y, Fujita Y, Musha T, Tanaka H, Shiokawa S, Nakamatsu K, Mori S, Matsuo T, Nakamura Y. Expression of erythropoietin in human female reproductive organs. Ital J Anat Embryol 2001;106:215–22.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Hiroshi Suzuki
    • 1
    Email author
  • Takako Ishijima
    • 1
  • Saori Maruyama
    • 1
  • Yoshiko Yanagimoto Ueta
    • 1
  • Yasuyuki Abe
    • 1
  • Hideki Saitoh
    • 2
  1. 1.Research Unit for Functional Genomics, National Research Center of Protozoan DiseasesObihiro University of Agriculture and Veterinary MedicineObihiroJapan
  2. 2.Chugai Pharmaceutical Co., Ltd.TokyoJapan

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