Journal of Assisted Reproduction and Genetics

, Volume 30, Issue 11, pp 1397–1406 | Cite as

In vitro development of human primordial follicles to preantral stage after vitrification

  • Fariba Khosravi
  • Robert L. Reid
  • Ashraf Moini
  • Farid Abolhassani
  • Mojtaba R. Valojerdi
  • Frederick W. K. KanEmail author
Technological Innovations



The aim was to culture primordial follicles in vitro to reach preantral stage in vitrified human ovarian tissue.


Ovarian tissue samples were obtained from six women. Tissue strips were vitrified by infiltration with a cryoprotectant followed by mounting on a stainless steel carrier. After culturing for 7 days the morphology and developmental stages of follicles enclosed in fresh and vitrified groups were analyzed.


High proportion of viable follicles in vitrified ovarian strips was obtained. After culturing for 7 days the percentage of secondary and preantral follicles increased significantly (P < 0.05) whereas primordial and transitory follicles showed a significant decrease (P < 0.05) compared to their respective counterparts at day 0 of culture.


Vitrification of ovarian strips with an improved carrier device and culturing of follicles in ovarian strips after warming yielded developed follicles with high viability and morphological integrity that may be suitable for use in fertility preservation among cancer patients.


Carrier device In vitro culture Preantral follicles Preservation of fertility Primordial follicles 



This research was supported, in part, by a grant from the Canadian Institutes of Health Research to F.W.K.K and by a research grant (No.17764) from the Tehran University of Medical Sciences to F.A.


  1. 1.
    Donnez J, Jadoul P, Squifflet J, Van Langendonckt A, Donnez O, Van Eyck AS, et al. Ovarian tissue cryopreservation and transplantation in cancer patients. Best Pract Res Clin Obstet Gynaecol. 2010;24:87–100.PubMedCrossRefGoogle Scholar
  2. 2.
    Abir R, Fisch B, Raz A, Nitke S, Ben-Rafael Z. Preservation of fertility in women undergoing chemotherapy: current approach and future prospects. J Assist Reprod Genet. 1998;15:469–77.PubMedCrossRefGoogle Scholar
  3. 3.
    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;99:1503–13.PubMedCrossRefGoogle Scholar
  4. 4.
    Silber S, Kagawa N, Kuwayama M, Gosden R. Duration of fertility after fresh and frozen ovary transplantation. Fertil Steril. 2010;94:2191–6.PubMedCrossRefGoogle Scholar
  5. 5.
    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
  6. 6.
    Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker MJ. Potential importance of vitrification in reproductive medicine. Biol Reprod. 2002;67:1671–80.PubMedCrossRefGoogle Scholar
  7. 7.
    Rahimi G, Isachenko E, Sauer H, Isachenko V, Wartenberg M, Hescheler J, et al. Effect of different vitrification protocols for human ovarian tissue on reactive oxygen species and apoptosis. Reprod Fertil Dev. 2003;15:343–9.PubMedCrossRefGoogle Scholar
  8. 8.
    Mazoochi T, Salehnia M, Valojerdi MR, Mowla SJ. Morphologic, ultrastructural, and biochemical identification of apoptosis in vitrified-warmed mouse ovarian tissue. Fertil Steril. 2008;90:1480–6.PubMedCrossRefGoogle Scholar
  9. 9.
    Silber SJ. Ovary cryopreservation and transplantation for fertility preservation. Mol Hum Reprod. 2012;18:59–67.PubMedCrossRefGoogle Scholar
  10. 10.
    Amorim CA, Curaba M, Van Langendonckt A, Dolmans MM, Donnez J. Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod Biomed Online. 2011;23:160–86.PubMedCrossRefGoogle Scholar
  11. 11.
    Hasegawa A, Hamada Y, Mehandjiev T, Koyama K. In vitro growth and maturation as well as fertilization of mouse preantral oocytes from vitrified ovaries. Fertil Steril. 2004;81 Suppl 1:824–30.PubMedCrossRefGoogle Scholar
  12. 12.
    Hasegawa A, Mochida N, Ogasawara T, Koyama K. Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization. Fertil Steril. 2006;86:1182–92.PubMedCrossRefGoogle Scholar
  13. 13.
    Wang X, Catt S, Pangestu M, Temple-Smith P. Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births. Reproduction. 2011;141:183–91.PubMedCrossRefGoogle Scholar
  14. 14.
    Santos RR, Tharasanit T, Van Haeften T, Figueiredo JR, Silva JR, Van den Hurk R. Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods. Cell Tissue Res. 2007;327:167–76.PubMedCrossRefGoogle Scholar
  15. 15.
    Carvalho AA, Faustino LR, Silva CM, Castro SV, Luz HK, Rossetto R, et al. Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue. Theriogenology. 2011;76:933–41.PubMedCrossRefGoogle Scholar
  16. 16.
    Bao RM, Yamasaka E, Moniruzzaman M, Hamawaki A, Yoshikawa M, Miyano T. Development of vitrified bovine secondary and primordial follicles in xenografts. Theriogenology. 2010;74:817–27.PubMedCrossRefGoogle Scholar
  17. 17.
    Hashimoto S, Suzuki N, Amo A, Yamochi T, Hosoi Y, Morimoto Y. Good thermally conducting material supports follicle morphologies of porcine ovaries cryopreserved with ultrarapid vitrification. J Reprod Dev. 2013;in press.Google Scholar
  18. 18.
    Yeoman RR, Wolf DP, Lee DM. Coculture of monkey ovarian tissue increases survival after vitrification and slow-rate freezing. Fertil Steril. 2005;83 Suppl 1:1248–54.PubMedCrossRefGoogle Scholar
  19. 19.
    Suzuki N, Hashimoto S, Igarashi S, Takae S, Yamanaka M, Yamochi T, et al. Assessment of long-term function of heterotopic transplants of vitrified ovarian tissue in cynomolgus monkeys. Hum Reprod. 2012;27:2420–9.PubMedCrossRefGoogle Scholar
  20. 20.
    Hashimoto S, Suzuki N, Yamanaka M, Hosoi Y, Ishizuka B, Morimoto Y. Effects of vitrification solutions and equilibration times on the morphology of cynomolgus ovarian tissues. Reprod Biomed Online. 2010;21:501–9.PubMedCrossRefGoogle Scholar
  21. 21.
    Kagawa N, Silber S, Kuwayama M. Successful vitrification of bovine and human ovarian tissue. Reprod Biomed Online. 2009;18:568–77.PubMedCrossRefGoogle Scholar
  22. 22.
    Amorim CA, Dolmans MM, David A, Jaeger J, Vanacker J, Camboni A, et al. Vitrification and xenografting of human ovarian tissue. Fertil Steril. 2012;98:1291–8.Google Scholar
  23. 23.
    Sheikhi M, Hultenby K, Niklasson B, Lundqvist M, Hovatta O. Clinical grade vitrification of human ovarian tissue: an ultrastructural analysis of follicles and stroma in vitrified tissue. Hum Reprod. 2011;26:594–603.PubMedCrossRefGoogle Scholar
  24. 24.
    Bian J, Li T, Ding C, Xin W, Zhu B, Zhou C. Vitreous cryopreservation of human preantral follicles encapsulated in alginate beads with mini mesh cups. J Reprod Dev. 2013;59:288–95.PubMedCrossRefGoogle Scholar
  25. 25.
    Huang L, Mo Y, Wang W, Li Y, Zhang Q, Yang D. Cryopreservation of human ovarian tissue by solid-surface vitrification. Eur J Obstet Gynecol Reprod Biol. 2008;139:193–8.PubMedCrossRefGoogle Scholar
  26. 26.
    Li YB, Zhou CQ, Yang GF, Wang Q, Dong Y. Modified vitrification method for cryopreservation of human ovarian tissues. Chin Med J (Engl). 2007;120:110–4.Google Scholar
  27. 27.
    Isachenko E, Isachenko V, Rahimi G, Nawroth F. Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen. Eur J Obstet Gynecol Reprod Biol. 2003;108:186–93.PubMedCrossRefGoogle Scholar
  28. 28.
    Salehnia M, Abbasian Moghadam E, Rezazadeh Velojerdi M. Ultrastructure of follicles after vitrification of mouse ovarian tissue. Fertil Steril. 2002;78:644–5.PubMedCrossRefGoogle Scholar
  29. 29.
    Wang Y, Xiao Z, Li L, Fan W, Li SW. Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation. Hum Reprod. 2008;23:2256–65.PubMedCrossRefGoogle Scholar
  30. 30.
    Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev. 1996;17:121–55.PubMedGoogle Scholar
  31. 31.
    Depalo R, Loverro G, Selvaggi L. In vitro maturation of primordial follicles after cryopreservation of human ovarian tissue: problems remain. Med Pediatr Oncol. 2002;38:153–7.PubMedCrossRefGoogle Scholar
  32. 32.
    Dolmans MM, Luyckx V, Donnez J, Andersen CY, Greve T. Risk of transferring malignant cells with transplanted frozen-thawed ovarian tissue. Fertil Steril. 2013;99:1514–22.PubMedCrossRefGoogle Scholar
  33. 33.
    Poirot C, Vacher-Lavenu MC, Helardot P, Guibert J, Brugieres L, Jouannet P. Human ovarian tissue cryopreservation: indications and feasibility. Hum Reprod. 2002;17:1447–52.PubMedCrossRefGoogle Scholar
  34. 34.
    Hovatta O. Cryopreservation and culture of human ovarian cortical tissue containing early follicles. Eur J Obstet Gynecol Reprod Biol. 2004;113 Suppl 1:S50–4.PubMedCrossRefGoogle Scholar
  35. 35.
    Ting AY, Yeoman RR, Lawson MS, Zelinski MB. In vitro development of secondary follicles from cryopreserved rhesus macaque ovarian tissue after slow-rate freeze or vitrification. Hum Reprod. 2011;26:2461–72.PubMedCrossRefGoogle Scholar
  36. 36.
    Gook DA, Edgar DH, Borg J, Archer J, McBain JC. Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting. Hum Reprod. 2005;20:72–8.PubMedCrossRefGoogle Scholar
  37. 37.
    Martinez-Madrid B, Donnez J, Van Eyck AS, Veiga-Lopez A, Dolmans MM, Van Langendonckt A. Chick embryo chorioallantoic membrane (CAM) model: a useful tool to study short-term transplantation of cryopreserved human ovarian tissue. Fertil Steril. 2009;91:285–92.PubMedCrossRefGoogle Scholar
  38. 38.
    Isachenko V, Mallmann P, Petrunkina AM, Rahimi G, Nawroth F, Hancke K, et al. Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue. PLoS One. 2012;7:e32549.PubMedCrossRefGoogle Scholar
  39. 39.
    Amorim CA, David A, Dolmans MM, Camboni A, Donnez J, Van Langendonckt A. Impact of freezing and thawing of human ovarian tissue on follicular growth after long-term xenotransplantation. J Assist Reprod Genet. 2011;28:1157–65.PubMedCrossRefGoogle Scholar
  40. 40.
    Rahimi G, Isachenko V, Kreienberg R, Sauer H, Todorov P, Tawadros S, et al. Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. Eur J Obstet Gynecol Reprod Biol. 2010;149:63–7.PubMedCrossRefGoogle Scholar
  41. 41.
    Nisolle M, Casanas-Roux F, Qu J, Motta P, Donnez J. Histologic and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice. Fertil Steril. 2000;74:122–9.PubMedCrossRefGoogle Scholar
  42. 42.
    Van den Broecke R, Liu J, Handyside A, Van der Elst JC, Krausz T, Dhont M, et al. Follicular growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice. Eur J Obstet Gynecol Reprod Biol. 2001;97:193–201.PubMedCrossRefGoogle Scholar
  43. 43.
    Hovatta O, Silye R, Abir R, Krausz T, Winston RM. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture. Hum Reprod. 1997;12:1032–6.PubMedCrossRefGoogle Scholar
  44. 44.
    Isachenko V, Montag M, Isachenko E, van der Ven K, Dorn C, Roesing B, et al. Effective method for in-vitro culture of cryopreserved human ovarian tissue. Reprod Biomed Online. 2006;13:228–34.PubMedCrossRefGoogle Scholar
  45. 45.
    Picton HM, Gosden RG. In vitro growth of human primordial follicles from frozen-banked ovarian tissue. Mol Cell Endocrinol. 2000;166:27–35.PubMedCrossRefGoogle Scholar
  46. 46.
    Telfer EE, McLaughlin M, Ding C, Thong KJ. A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin. Hum Reprod. 2008;23:1151–8.PubMedCrossRefGoogle Scholar
  47. 47.
    Chambers EL, Gosden RG, Yap C, Picton HM. In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility. Hum Reprod. 2010;25:2559–68.PubMedCrossRefGoogle Scholar
  48. 48.
    Kristensen SG, Rasmussen A, Byskov AG, Andersen CY. Isolation of pre-antral follicles from human ovarian medulla tissue. Hum Reprod. 2011;26:157–66.PubMedCrossRefGoogle Scholar
  49. 49.
    Allison AC, Young MR. Uptake of dyes and drugs by living cells in culture. Life Sci. 1964;3:1407–14.PubMedCrossRefGoogle Scholar
  50. 50.
    Brankin V, Mitchell MR, Webb B, Hunter MG. Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro. Reprod Biol Endocrinol. 2003;1:55.PubMedCrossRefGoogle Scholar
  51. 51.
    Shores EM, Picton HM, Hunter MG. Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture. J Reprod Fertil. 2000;118:211–9.PubMedGoogle Scholar
  52. 52.
    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
  53. 53.
    Hansen KR, Craig LB, Zavy MT, Klein NA, Soules MR. Ovarian primordial and nongrowing follicle counts according to the Stages of Reproductive Aging Workshop (STRAW) staging system. Menopause. 2012;19:164–71.PubMedCrossRefGoogle Scholar
  54. 54.
    Rodgers RJ, Irving-Rodgers HF. Morphological classification of bovine ovarian follicles. Reproduction. 2010;139:309–18.PubMedCrossRefGoogle Scholar
  55. 55.
    Amorim CA, Dolmans MM, David A, Jaeger J, Vanacker J, Camboni A, et al. Vitrification and xenografting of human ovarian tissue. Fertil Steril. 2012;98:1291–8.PubMedCrossRefGoogle Scholar
  56. 56.
    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;12:519–35.PubMedCrossRefGoogle Scholar
  57. 57.
    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;21:413–20.PubMedCrossRefGoogle Scholar
  58. 58.
    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;96:379–83.PubMedCrossRefGoogle Scholar
  59. 59.
    Pegg DE. The role of vitrification techniques of cryopreservation in reproductive medicine. Hum Fertil (Camb). 2005;8:231–9.CrossRefGoogle Scholar
  60. 60.
    Chang HJ, Moon JH, Lee JR, Jee BC, Suh CS, Kim SH. Optimal condition of vitrification method for cryopreservation of human ovarian cortical tissues. J Obstet Gynaecol Res. 2011;37:1092–101.PubMedCrossRefGoogle Scholar
  61. 61.
    Choi J, Lee JY, Lee E, Yoon BK, Bae D, Choi D. Cryopreservation of the mouse ovary inhibits the onset of primordial follicle development. Cryobiology. 2007;54:55–62.PubMedCrossRefGoogle Scholar
  62. 62.
    Mazoochi T, Salehnia M, Pourbeiranvand S, Forouzandeh M, Mowla SJ, Hajizadeh E. Analysis of apoptosis and expression of genes related to apoptosis in cultures of follicles derived from vitrified and non-vitrified ovaries. Mol Hum Reprod. 2009;15:155–64.PubMedCrossRefGoogle Scholar
  63. 63.
    Salehnia M, Sheikhi M, Pourbeiranvand S, Lundqvist M. Apoptosis of human ovarian tissue is not increased by either vitrification or rapid cooling. Reprod Biomed Online. 2012;25:492–9.Google Scholar
  64. 64.
    Depalo R, Lorusso F, Bettocchi S, Selvaggi L, Cavallini A, Valentini AM, et al. Assessment of estrogen receptors and apoptotic factors in cryopreserved human ovarian cortex. Syst Biol Reprod Med. 2009;55:236–43.PubMedCrossRefGoogle Scholar
  65. 65.
    Paynter SJ, Cooper A, Fuller BJ, Shaw RW. Cryopreservation of bovine ovarian tissue: structural normality of follicles after thawing and culture in vitro. Cryobiology. 1999;38:301–9.PubMedCrossRefGoogle Scholar
  66. 66.
    Tsuribe PM, Gobbo CA, Landim-Alvarenga FC. Viability of primordial follicles derived from cryopreserved ovine ovarian cortex tissue. Fertil Steril. 2009;91:1976–83.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Fariba Khosravi
    • 1
    • 3
  • Robert L. Reid
    • 2
  • Ashraf Moini
    • 4
  • Farid Abolhassani
    • 3
  • Mojtaba R. Valojerdi
    • 5
  • Frederick W. K. Kan
    • 1
    Email author
  1. 1.Department of Biomedical and Molecular Sciences, Faculty of Health SciencesQueen’s UniversityKingstonCanada
  2. 2.Department of Obstetrics and Gynecology, Faculty of Health SciencesQueen’s UniversityKingstonCanada
  3. 3.Department of Anatomy, Medical SchoolTehran University of Medical SciencesTehranIran
  4. 4.Department of Obstetrics and Gynecology, Medical SchoolTehran University of Medical SciencesTehranIran
  5. 5.Department of Anatomy, Faculty of Medical SciencesTarbiat Modares UniversityTehranIran

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