Journal of Assisted Reproduction and Genetics

, Volume 32, Issue 4, pp 597–606 | Cite as

Is transplantation of a few leukemic cells inside an artificial ovary able to induce leukemia in an experimental model?

  • Michelle Soares
  • Pascale Saussoy
  • Karima Sahrari
  • Christiani A. Amorim
  • Jacques Donnez
  • Marie-Madeleine Dolmans
Fertility Preservation



To evaluate the tumor-inducing ability of a few leukemic cells xenotransplanted inside an artificial ovary.


Ten and 100 BV-173 leukemic cells were embedded in a fibrin matrix along with 50,000 human ovarian stromal cells, and grafted to the peritoneal bursa of 5 and 5 SCID mice respectively. Four mice grafted with 3x106 leukemic cells in fibrin served as positive controls. At 20 weeks post-transplantation, the grafts, liver, spleen, blood and bone marrow were analyzed for the presence of leukemia by anti-CD79α IHC, flow cytometry (FC) and PCR.


All mice grafted with 3x106 cells developed peritoneal masses 4 weeks after xenotransplantation, and systemic disease was confirmed by IHC, PCR and FC. Among mice grafted with 10 or 100 leukemic cells, none showed any sign of leukemia after 20 weeks, and IHC, FC and PCR on the different recovered tissues all proved negative.


This study investigates the tumor-inducing potential of a few leukemic cells grafted inside an artificial ovary. Transplantation of 100 leukemic cells appears to be insufficient to induce leukemia after 20 weeks. These results in an immunodeficient xenografting model are quite reassuring. However, for clinical application, follicle suspensions must be purged of leukemic cells before grafting, as even the slightest risk should be avoided.


Artificial ovary Leukemia Ovarian tissue cryopreservation Ovarian follicles Malignant cell purging Minimal disseminated disease Follicle isolation 



The authors thank Mira Hryniuk for reviewing the English language of the manuscript and Olivier Van Kerk and Dolores Gonzalez for their technical assistance. Help from specialized technicians, namely Patricia Leveugle and Anne-Marie Mazzon for flow cytometry experiments and Béatrice Delepaut for PCR, was also greatly appreciated.

This work was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (5/4/150/5 and 7.4518.12F), Fonds Spéciaux de Recherche, Fondation Saint Luc, Foundation Against Cancer, and donations from Mr. Pietro Ferrero, Baron Frère, and Viscount Philippe de Spoelberch.

Supplementary material

10815_2015_438_Fig4_ESM.jpg (87 kb)
Supplementary Fig 1

(JPEG 86 kb)

10815_2015_438_MOESM1_ESM.doc (44 kb)
Supplementary Table 1 Flow cytometry results for leukemic cell detection expressed as the percentage of total events showing the BV-173 lineage phenotype. This table presents results obtained using panel 2 (CD44-FITC/CD10-PE/CD45-ECD/CD38-PC5/CD19-PC7), but percentages were similar with panel 1 (CD44-FITC/CD10-PE/CD45-ECD/CD38-PC5/CD19-PC7). Mice grafted with 10 or 100 leukemic cells showed no sign of leukemia in any of the different tissue samples by FC. Conversely, in all mice grafted with 3 million leukemic cells, the BV-173 cell phenotype was identified in peritoneal grafts and at least one other sample, showing the presence of systemic disease after 4 weeks of grafting. Neg = negative, Pos = positive, NP = analysis not possible due to small sample volume. (DOC 43 kb)


  1. 1.
    Edwards BK, Noone A-M, Mariotto AB, Simard EP, Boscoe FP, Henley SJ, et al. Annual Report to the Nation on the status of cancer, 1975–2010, featuring prevalence of comorbidity and impact on survival among persons with lung, colorectal, breast, or prostate cancer. Cancer. 2013:n/a-n/a.Google Scholar
  2. 2.
    Ward E, Desantis C, Robbins A, Kohler B, Jemal A. Childhood and adolescent cancer statistics, 2014. CA Cancer J Clin. 2014;64(2):83–103.CrossRefPubMedGoogle Scholar
  3. 3.
    Meirow D, Nugent D. The effects of radiotherapy and chemotherapy on female reproduction. Hum Reprod Update. 2001;7(6):535–43.CrossRefPubMedGoogle Scholar
  4. 4.
    Wallace WH, Anderson RA, Irvine DS. Fertility preservation for young patients with cancer: who is at risk and what can be offered? Lancet Oncol. 2005;6(4):209–18.CrossRefPubMedGoogle Scholar
  5. 5.
    Donnez J, Martinez-Madrid B, Jadoul P, Van Langendonckt A, Demylle D, Dolmans MM. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update. 2006;12(5):519–35.CrossRefPubMedGoogle Scholar
  6. 6.
    Byrne J, Fears TR, Gail MH, Pee D, Connelly RR, Austin DF, et al. Early menopause in long-term survivors of cancer during adolescence. Am J Obstet Gynecol. 1992;166(3):788–93.CrossRefPubMedGoogle Scholar
  7. 7.
    Larsen EC, Muller J, Schmiegelow K, Rechnitzer C, Andersen AN. Reduced ovarian function in long-term survivors of radiation- and chemotherapy-treated childhood cancer. J Clin Endocrinol Metab. 2003;88(11):5307–14.CrossRefPubMedGoogle Scholar
  8. 8.
    Green DM, Kawashima T, Stovall M, Leisenring W, Sklar CA, Mertens AC, et al. Fertility of female survivors of childhood cancer: a report from the childhood cancer survivor study. J Clin Oncol. 2009;27(16):2677–85.CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Anderson RA, Wallace WH. Antimullerian hormone, the assessment of the ovarian reserve, and the reproductive outcome of the young patient with cancer. Fertil Steril. 2013;99(6):1469–75.CrossRefPubMedGoogle Scholar
  10. 10.
    Jadoul P, Dolmans MM, Donnez J. Fertility preservation in girls during childhood: is it feasible, efficient and safe and to whom should it be proposed? Hum Reprod Update. 2010;16(6):617–30.CrossRefPubMedGoogle Scholar
  11. 11.
    Cancer in Children and Adolescents. Belgian Cancer Registry; 2013.Google Scholar
  12. 12.
    Dolmans MM, Jadoul P, Gilliaux S, Amorim CA, Luyckx V, Squifflet J, et al. A review of 15 years of ovarian tissue bank activities. J Assist Reprod Genet. 2013;30(3):305–14.CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Dolmans MM, Luyckx V, Donnez J, Andersen CY, Greve T. Risk of transferring malignant cells with transplanted frozen-thawed ovarian tissue. Fertil Steril. 2013;99(6):1514–22.CrossRefPubMedGoogle Scholar
  14. 14.
    Donnez J, Dolmans MM. Fertility preservation in women. Nat Rev Endocrinol. 2013;9(12):735–49.CrossRefPubMedGoogle Scholar
  15. 15.
    Cobo A, Garcia-Velasco JA, Domingo J, Remohi J, Pellicer A. Is vitrification of oocytes useful for fertility preservation for age-related fertility decline and in cancer patients? Fertil Steril. 2013;99(6):1485–95.CrossRefPubMedGoogle Scholar
  16. 16.
    Cakmak H, Rosen MP. Ovarian stimulation in cancer patients. Fertil Steril. 2013;99(6):1476–84.CrossRefPubMedGoogle Scholar
  17. 17.
    Meirow D, Hardan I, Dor J, Fridman E, Elizur S, Ra’anani H, et al. Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients. Hum Reprod. 2008;23(5):1007–13.CrossRefPubMedGoogle Scholar
  18. 18.
    Dolmans MM, Marinescu C, Saussoy P, Van Langendonckt A, Amorim C, Donnez J. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe. Blood. 2010;116(16):2908–14.CrossRefPubMedGoogle Scholar
  19. 19.
    Rosendahl M, Andersen MT, Ralfkiaer E, Kjeldsen L, Andersen MK, Andersen CY. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. Fertil Steril. 2010;94(6):2186–90.CrossRefPubMedGoogle Scholar
  20. 20.
    Amiot C, Angelot-Delettre F, Zver T, Alvergnas-Vieille M, Saas P, Garnache-Ottou F, et al. Minimal residual disease detection of leukemic cells in ovarian cortex by eight-color flow cytometry. Hum Reprod. 2013;28(8):2157–67.CrossRefPubMedGoogle Scholar
  21. 21.
    Greve T, Clasen-Linde E, Andersen MT, Andersen MK, Sorensen SD, Rosendahl M, et al. Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells. Blood. 2012;120(22):4311–6.CrossRefPubMedGoogle Scholar
  22. 22.
    Bastings L, Beerendonk CC, Westphal JR, Massuger LF, Kaal SE, van Leeuwen FE, et al. Autotransplantation of cryopreserved ovarian tissue in cancer survivors and the risk of reintroducing malignancy: a systematic review. Hum Reprod Update. 2013;19(5):483–506.CrossRefPubMedGoogle Scholar
  23. 23.
    Rosendahl M, Greve T, Andersen CY. The safety of transplanting cryopreserved ovarian tissue in cancer patients: a review of the literature. J Assist Reprod Genet. 2013;30(1):11–24.CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Dolmans MM, Martinez-Madrid B, Gadisseux E, Guiot Y, Yuan WY, Torre A, et al. Short-term transplantation of isolated human ovarian follicles and cortical tissue into nude mice. Reproduction. 2007;134(2):253–62.CrossRefPubMedGoogle Scholar
  25. 25.
    Dolmans MM, Yuan WY, Camboni A, Torre A, Van Langendonckt A, Martinez-Madrid B, et al. Development of antral follicles after xenografting of isolated small human preantral follicles. Reprod BioMed Online. 2008;16(5):705–11.CrossRefPubMedGoogle Scholar
  26. 26.
    Vanacker J, Luyckx V, Dolmans MM, Des Rieux A, Jaeger J, Van Langendonckt A, et al. Transplantation of an alginate-matrigel matrix containing isolated ovarian cells: first step in developing a biodegradable scaffold to transplant isolated preantral follicles and ovarian cells. Biomaterials. 2012;33(26):6079–85.CrossRefPubMedGoogle Scholar
  27. 27.
    Luyckx V, Dolmans MM, Vanacker J, Scalercio SR, Donnez J, Amorim CA. First step in developing a 3D biodegradable fibrin scaffold for an artificial ovary. J Ovarian Res. 2013;6(1):83.CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Luyckx V, Dolmans MM, Vanacker J, Legat C, Fortuno Moya C, Donnez J, et al. A new step toward the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold. Fertil Steril. 2014;101(4):1149–56.CrossRefPubMedGoogle Scholar
  29. 29.
    Dath C, Dethy A, Van Langendonckt A, Van Eyck AS, Amorim CA, Luyckx V, et al. Endothelial cells are essential for ovarian stromal tissue restructuring after xenotransplantation of isolated ovarian stromal cells. Hum Reprod. 2011;26(6):1431–9.CrossRefPubMedGoogle Scholar
  30. 30.
    Vanacker J, Camboni A, Dath C, Van Langendonckt A, Dolmans MM, Donnez J, et al. Enzymatic isolation of human primordial and primary ovarian follicles with Liberase DH: protocol for application in a clinical setting. Fertil Steril. 2011;96(2):379–83.e3.CrossRefPubMedGoogle Scholar
  31. 31.
    Dolmans MM, Michaux N, Camboni A, Martinez-Madrid B, Van Langendonckt A, Nottola SA, et al. Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles. Hum Reprod. 2006;21(2):413–20.CrossRefPubMedGoogle Scholar
  32. 32.
    Eppig JJ, O’Brien MJ. Development in vitro of mouse oocytes from primordial follicles. Biol Reprod. 1996;54(1):197–207.CrossRefPubMedGoogle Scholar
  33. 33.
    O’Brien MJ, Pendola JK, Eppig JJ. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod. 2003;68(5):1682–6.CrossRefPubMedGoogle Scholar
  34. 34.
    Spears N, Boland NI, Murray AA, Gosden RG. Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile. Hum Reprod. 1994;9(3):527–32.PubMedGoogle Scholar
  35. 35.
    Telfer EE, Zelinski MB. Ovarian follicle culture: advances and challenges for human and nonhuman primates. Fertil Steril. 2013;99(6):1523–33.CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    Hermann BP, Sukhwani M, Salati J, Sheng Y, Chu T, Orwig KE. Separating spermatogonia from cancer cells in contaminated prepubertal primate testis cell suspensions. Hum Reprod. 2011;26(12):3222–31.CrossRefPubMedCentralPubMedGoogle Scholar
  37. 37.
    Fujita K, Ohta H, Tsujimura A, Takao T, Miyagawa Y, Takada S, et al. Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukemia. J Clin Invest. 2005;115(7):1855–61.CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    Jahnukainen K, Morris I, Roe S, Salmi TT, Makipernaa A, Pollanen P. A rodent model for testicular involvement in acute lymphoblastic leukaemia. Br J Cancer. 1993;67(5):885–92.CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Hou M, Andersson M, Zheng C, Sundblad A, Soder O, Jahnukainen K. Decontamination of leukemic cells and enrichment of germ cells from testicular samples from rats with Roser’s T-cell leukemia by flow cytometric sorting. Reproduction. 2007;134(6):767–79.CrossRefPubMedGoogle Scholar
  40. 40.
    Fujita K, Tsujimura A, Hirai T, Ohta H, Matsuoka Y, Miyagawa Y, et al. Effect of human leukemia cells in testicular tissues grafted into immunodeficient mice. Int J Urol. 2008;15(8):733–8.CrossRefPubMedGoogle Scholar
  41. 41.
    Donnez J, Jadoul P, Pirard C, Hutchings G, Demylle D, Squifflet J, et al. Live birth after transplantation of frozen-thawed ovarian tissue after bilateral oophorectomy for benign disease. Fertil Steril. 2012;98(3):720–5.CrossRefPubMedGoogle Scholar
  42. 42.
    Donnez J, Dolmans MM, Pellicer A, Diaz-Garcia C, Sanchez Serrano M, Schmidt KT, et al. Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation. Fertil Steril. 2013;99(6):1503–13.CrossRefPubMedGoogle Scholar
  43. 43.
    Fingert HJ, Chen Z, Mizrahi N, Gajewski WH, Bamberg MP, Kradin RL. Rapid growth of human cancer cells in a mouse model with fibrin clot subrenal capsule assay. Cancer Res. 1987;47(14):3824–9.PubMedGoogle Scholar
  44. 44.
    Jahnukainen K, Hou M, Petersen C, Setchell B, Soder O. Intratesticular transplantation of testicular cells from leukemic rats causes transmission of leukemia. Cancer Res. 2001;61(2):706–10.PubMedGoogle Scholar
  45. 45.
    Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ, et al. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature. 2001;410(6832):1107–11.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Michelle Soares
    • 1
    • 4
  • Pascale Saussoy
    • 2
  • Karima Sahrari
    • 1
  • Christiani A. Amorim
    • 1
  • Jacques Donnez
    • 3
  • Marie-Madeleine Dolmans
    • 1
    • 4
  1. 1.Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et CliniqueUniversité Catholique de LouvainBrusselsBelgium
  2. 2.Département de Biologie CliniqueUniversité Catholique de Louvain et Cliniques universitaires Saint-LucBrusselsBelgium
  3. 3.Société de Recherche pour l’InfertilitéBrusselsBelgium
  4. 4.Département de GynécologieCliniques Universitaires Saint-Luc BrusselsBelgium

Personalised recommendations