Skip to main content

Advertisement

SpringerLink
Log in

Quality and functionality of human ovarian tissue after cryopreservation using an original slow freezing procedure

  • Fertility Preservation
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

To evaluate the efficiency of an original slow freezing protocol on the quality and function of human ovarian cortex.

Methods

Human ovarian tissues were cryopreserved using a freezing medium supplemented with propanediol and raffinose as cryoprotectants and antioxidants (L-glutamine, taurine). Samples were then frozen using a faster cooling rate than the usual one. Viability and morphology of follicles, DNA fragmentation in follicles and stroma as well as histology of the vascular endothelium were analyzed before and after freezing/thawing. Moreover, a functional analysis was performed based on the evaluation of follicular growth and development in thawed ovarian tissues that were cultured in vitro.

Results

Our freezing/thawing protocol allows preservation of a high proportion of viable follicles and the preservation of the different follicle developmental stages (p > 0.05 versus fresh control). 70.5 ± 5.2 % of follicles retained an intact morphology after cryopreservation (p = 0.04). Stroma cells but not follicles exhibited a slight increase of DNA fragmentation after thawing (p < 0.05). Microvessel endothelium within thawed tissues appeared to be preserved. Granulosa cells showed signs of proliferation in follicles cultured for 12 days. Secretion of 17β-oestradiol significantly increased during in vitro culture.

Conclusions

This protocol leads to good preservation of ovarian integrity and functionality post-thawing and thus appears as a suitable technique of ovarian tissue cryopreservation in clinical settings. Further research could be extended to optimize conditions of in vitro culture.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Abir R, Orvieto R, Raanani H, Feldberg D, Nitke S, Fisch B. Parameters affecting successful transplantation of frozen-thawed human fetal ovaries into immunodeficient mice. Fertil Steril. 2003;80(2):421–8.

    Article  PubMed  Google Scholar 

  2. Boland NI, Humpherson PG, Leese HJ, Gosden RG. Pattern of lactate production and steroidogenesis during growth and maturation of mouse ovarian follicles in vitro. Biol Reprod. 1993;48(4):798–806.

    Article  PubMed  CAS  Google Scholar 

  3. Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C. Pregnancies and births after oocyte cryopreservation. Fertil Steril. 2004;82(3):601–5. doi:10.1016/j.fertnstert.2004.04.025.

    Article  PubMed  Google Scholar 

  4. Cabrita E, Ma S, Diogo P, Martinez-Paramo S, Sarasquete C, Dinis MT. The influence of certain aminoacids and vitamins on post-thaw fish sperm motility, viability and DNA fragmentation. Anim Reprod Sci. 2011;125(1–4):189–95. doi:10.1016/j.anireprosci.2011.03.003.

    Article  PubMed  CAS  Google Scholar 

  5. 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. doi:10.1016/j.fertnstert.2005.03.079.

    Article  PubMed  Google Scholar 

  6. Demirci B, Lornage J, Salle B, Frappart L, Franck M, Guerin JF. Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols. Fertil Steril. 2001;75(4):754–62.

    Article  PubMed  CAS  Google Scholar 

  7. Demirci B, Salle B, Frappart L, Franck M, Guerin JF, Lornage J. Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep. Fertil Steril. 2002;77(3):595–600.

    Article  PubMed  Google Scholar 

  8. Ding CC, Thong KJ, Krishna A, Telfer EE. Activin A inhibits activation of human primordial follicles in vitro. J Assist Reprod Genet. 2010;27(4):141–7. doi:10.1007/s10815-010-9395-6.

    Article  PubMed  Google Scholar 

  9. Donnez J, Silber S, Andersen CY, Demeestere I, Piver P, Meirow D, et al. Children born after autotransplantation of cryopreserved ovarian tissue. a review of 13 live births. Ann Med. 2011;43(6):437–50. doi:10.3109/07853890.2010.546807.

    Article  PubMed  Google Scholar 

  10. Eroglu A. Cryopreservation of mammalian oocytes by using sugars: intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates. Cryobiology. 2010;60(3 Suppl):S54–9. doi:10.1016/j.cryobiol.2009.07.001.

    Article  PubMed  CAS  Google Scholar 

  11. Fabbri R, Pasquinelli G, Keane D, Magnani V, Paradisi R, Venturoli S. Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum. Reprod BioMed Online. 2010;21(6):819–28. doi:10.1016/j.rbmo.2010.07.008.

    Article  PubMed  CAS  Google Scholar 

  12. Fauque P, Ben Amor A, Joanne C, Agnani G, Bresson JL, Roux C. Use of trypan blue staining to assess the quality of ovarian cryopreservation. Fertil Steril. 2007;87(5):1200–7. doi:10.1016/j.fertnstert.2006.08.115.

    Article  PubMed  Google Scholar 

  13. Gidoni Y, Holzer H, Tulandi T, Tan SL. Fertility preservation in patients with non-oncological conditions. Reprod Biomed Online. 2008;16(6):792–800.

    Article  PubMed  Google Scholar 

  14. Gook DA, Edgar DH, Stern C. Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2-propanediol. Hum Reprod. 1999;14(8):2061–8.

    Article  PubMed  CAS  Google Scholar 

  15. 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(4):597–603.

    PubMed  CAS  Google Scholar 

  16. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod. 1986;1(2):81–7.

    PubMed  CAS  Google Scholar 

  17. Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev. 1996;17(2):121–55.

    PubMed  CAS  Google Scholar 

  18. Hardikar AA, Risbud MV, Remacle C, Reusens B, Hoet JJ, Bhonde RR. Islet cryopreservation: improved recovery following taurine pretreatment. Cell Transplant. 2001;10(3):247–53.

    PubMed  CAS  Google Scholar 

  19. Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, et al. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod. 1996;11(6):1268–72.

    Article  PubMed  CAS  Google Scholar 

  20. Hreinsson JG, Scott JE, Rasmussen C, Swahn ML, Hsueh AJ, Hovatta O. Growth differentiation factor-9 promotes the growth, development, and survival of human ovarian follicles in organ culture. J Clin Endocrinol Metab. 2002;87(1):316–21.

    Article  PubMed  CAS  Google Scholar 

  21. Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, et al. Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation. Reproduction. 2009;138(2):319–27. doi:10.1530/REP-09-0039.

    Article  PubMed  CAS  Google Scholar 

  22. Jain A, Mohanka R, Orloff M, Abt P, Kashyap R, Cullen J, et al. University of Wisconsin versus histidine-tryptophan-ketoglutarate for tissue preservation in live-donor liver transplantation. Exp Clin Transplant. 2006;4(1):451–7.

    PubMed  Google Scholar 

  23. Kedem A, Fisch B, Garor R, Ben-Zaken A, Gizunterman T, Felz C, et al. Growth differentiating factor 9 (GDF9) and bone morphogenetic protein 15 both activate development of human primordial follicles in vitro, with seemingly more beneficial effects of GDF9. J Clin Endocrinol Metab. 2011;96(8):E1246–54. doi:10.1210/jc.2011-0410.

    Article  PubMed  CAS  Google Scholar 

  24. Kedem A, Hourvitz A, Fisch B, Shachar M, Cohen S, Ben-Haroush A, et al. Alginate scaffold for organ culture of cryopreserved-thawed human ovarian cortical follicles. J Assist Reprod Genet. 2011;28(9):761–9. doi:10.1007/s10815-011-9605-x.

    Article  PubMed  Google Scholar 

  25. 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(7):1670–83. doi:10.1093/humrep/dep079.

    Article  PubMed  CAS  Google Scholar 

  26. Konc J, Kanyo K, Varga E, Kriston R, Cseh S. Births resulting from oocyte cryopreservation using a slow freezing protocol with propanediol and sucrose. Syst Biol Reprod Med. 2008;54(4–5):205–10. doi:10.1080/19396360802415778.

    Article  PubMed  CAS  Google Scholar 

  27. Louhio H, Hovatta O, Sjoberg J, Tuuri T. The effects of insulin, and insulin-like growth factors I and II on human ovarian follicles in long-term culture. Mol Hum Reprod. 2000;6(8):694–8.

    Article  PubMed  CAS  Google Scholar 

  28. Marsella T, Sena P, Xella S, La Marca A, Giulini S, De Pol A, et al. Human ovarian tissue cryopreservation: effect of sucrose concentration on morphological features after thawing. Reprod Biomed Online. 2008;16(2):257–67.

    Article  PubMed  Google Scholar 

  29. Muhlbacher F, Langer F, Mittermayer C. Preservation solutions for transplantation. Transplant Proc. 1999;31(5):2069–70.

    Article  PubMed  CAS  Google Scholar 

  30. 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(1):122–9.

    Article  PubMed  CAS  Google Scholar 

  31. Oktem O, Urman B. Options of fertility preservation in female cancer patients. Obstet Gynecol Surv. 2010;65(8):531–42. doi:10.1097/OGX.0b013e3181f8c0aa.

    Article  PubMed  Google Scholar 

  32. Poirot C, Schubert B. Fertility preservation in prepubertal children. Bull Cancer. 2011;98(5):489–99. doi:10.1684/bdc.2011.1362.

    PubMed  Google Scholar 

  33. Rimon E, Cohen T, Dantes A, Hirsh L, Amit A, Lessing JB, et al. Apoptosis in cryopreserved human ovarian tissue obtained from cancer patients: a tool for evaluating cryopreservation utility. Int J Oncol. 2005;27(2):345–53.

    PubMed  CAS  Google Scholar 

  34. Sadeu JC, Smitz J. Growth differentiation factor-9 and anti-Mullerian hormone expression in cultured human follicles from frozen-thawed ovarian tissue. Reprod Biomed Online. 2008;17(4):537–48.

    Article  PubMed  CAS  Google Scholar 

  35. Sanchez F, Romero S, Albuz FK, Smitz J. In vitro follicle growth under non-attachment conditions and decreased FSH levels reduces Lhcgr expression in cumulus cells and promotes oocyte developmental competence. J Assist Reprod Genet. 2012;29(2):141–52. doi:10.1007/s10815-011-9690-x.

    Article  PubMed  Google Scholar 

  36. Sanfilippo S, Canis M, Ouchchane L, Botchorishvili R, Artonne C, Janny L, et al. Viability assessment of fresh and frozen/thawed isolated human follicles: reliability of two methods (Trypan blue and Calcein AM/ethidium homodimer-1). J Assist Reprod Genet. 2011;28(12):1151–6. doi:10.1007/s10815-011-9649-y.

    Article  PubMed  Google Scholar 

  37. Schmidt KT, Larsen EC, Andersen CY, Andersen AN. Risk of ovarian failure and fertility preserving methods in girls and adolescents with a malignant disease. BJOG. 2010;117(2):163–74. doi:10.1111/j.1471-0528.2009.02408.x.

    Article  PubMed  CAS  Google Scholar 

  38. Schubert B, Canis M, Darcha C, Artonne C, Pouly JL, Dechelotte P, et al. Human ovarian tissue from cortex surrounding benign cysts: a model to study ovarian tissue cryopreservation. Hum Reprod. 2005;20(7):1786–92. doi:10.1093/humrep/dei002.

    Article  PubMed  Google Scholar 

  39. Schubert B, Canis M, Darcha C, Artonne C, Smitz J, Grizard G. Follicular growth and estradiol follow-up after subcutaneous xenografting of fresh and cryopreserved human ovarian tissue. Fertil Steril. 2008;89(6):1787–94. doi:10.1016/j.fertnstert.2007.03.101.

    Article  PubMed  Google Scholar 

  40. Shiva Shankar Reddy N, Jagan Mohanarao G, Atreja SK. Effects of adding taurine and trehalose to a tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm quality following cryopreservation. Anim Reprod Sci. 2010;119(3–4):183–90. doi:10.1016/j.anireprosci.2010.01.012.

    Article  PubMed  CAS  Google Scholar 

  41. Silber SJ. Ovary cryopreservation and transplantation for fertility preservation. Mol Hum Reprod. 2012;18(2):59–67. doi:10.1093/molehr/gar082.

    Article  PubMed  CAS  Google Scholar 

  42. Smitz J, Dolmans MM, Donnez J, Fortune JE, Hovatta O, Jewgenow K, et al. Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation. Hum Reprod Update. 2010;16(4):395–414. doi:10.1093/humupd/dmp056.

    Article  PubMed  CAS  Google Scholar 

  43. Sordet O, Khan QA, Pommier Y. Apoptotic topoisomerase I-DNA complexes induced by oxygen radicals and mitochondrial dysfunction. Cell Cycle. 2004;3(9):1095–7.

    Article  PubMed  CAS  Google Scholar 

  44. Tjer GC, Chiu TT, Cheung LP, Lok IH, Haines CJ. Birth of a healthy baby after transfer of blastocysts derived from cryopreserved human oocytes fertilized with frozen spermatozoa. Fertil Steril. 2005;83(5):1547–9. doi:10.1016/j.fertnstert.2005.01.007.

    PubMed  Google Scholar 

  45. Touraine P, Beau I, Gougeon A, Meduri G, Desroches A, Pichard C, et al. New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype. Mol Endocrinol. 1999;13(11):1844–54.

    Article  PubMed  CAS  Google Scholar 

  46. Weissman A, Gotlieb L, Colgan T, Jurisicova A, Greenblatt EM, Casper RF. Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse. Biol Reprod. 1999;60(6):1462–7.

    Article  PubMed  CAS  Google Scholar 

  47. Woodruff TK, Shea LD. The role of the extracellular matrix in ovarian follicle development. Reprod Sci. 2007;14(8 Suppl):6–10. doi:10.1177/1933719107309818.

    Article  PubMed  Google Scholar 

  48. Xiao Z, Wang Y, Li L, Li SW. Cryopreservation of the human ovarian tissue induces the expression of Fas system in morphologically normal primordial follicles. Cryo-Letters. 2010;31(2):112–9.

    PubMed  Google Scholar 

  49. Xiao Z, Wang Y, Li L, Luo S, Li SW. Needle immersed vitrification can lower the concentration of cryoprotectant in human ovarian tissue cryopreservation. Fertil Steril. 2010;94(6):2323–8. doi:10.1016/j.fertnstert.2010.01.011.

    Article  PubMed  CAS  Google Scholar 

  50. Xiong Y, Connolly T, Futcher B, Beach D. Human D-type cyclin. Cell. 1991;65(4):691–9.

    Article  PubMed  CAS  Google Scholar 

  51. Zheng JH, Min ZL, Li YL, Zhu YH, Ye TJ, Li JQ, et al. A modified CZ-1 preserving solution for organ transplantation: comparative study with UW preserving solution. Chin Med J (Engl). 2008;121(10):904–9.

    CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank all the surgical team in the department of gynecology, CHU Estaing, Clermont-Ferrand (France) for their help to recruit patients. We are grateful to Dr Wassim Essamet for his expertise regarding the histology, Emmanuel Bourgeois and Christine Artonne for their precious technical assistance and Mrs Elizabeth Petit for language revision of the manuscript. Thanks are also due to the women who donated tissue for this research.

This work is supported by an industrial PhD fellowship (Convention Industrielle de Formation par la Recherche, CIFRE) with the Centre International de Chirurgie Endoscopique (CICE), France (Grant No: 176/2009).

Conflicts of interest

The authors declare that there are no conflicts of interest.

Author information

Authors and Affiliations

  1. Laboratoire de Biologie de la Reproduction, Université Clermont 1, UFR Médecine, E.A. 975, 28 Place Henri Dunant, 63001, Clermont-Ferrand Cedex 1, France

    Sandra Sanfilippo, Laurent Janny & Florence Brugnon

  2. Centre International de Chirurgie Endoscopique (CICE), 63001, Clermont-Ferrand Cedex 1, France

    Sandra Sanfilippo, Michel Canis & Jean-Luc Pouly

  3. Pôle Gynécologie obstétrique et Reproduction humaine, CHU Clermont-Ferrand, CHU Estaing, 63003, Clermont-Ferrand Cedex 1, France

    Michel Canis, Jean-Luc Pouly, Laurent Janny & Florence Brugnon

  4. Follicle Biology Laboratory, Vrije Universiteit Brussel, 1090, Jette, Belgium

    Sergio Romero & Johan Smitz

  5. Inserm U1107 NEURO-DOL, Laboratoire de Physiologie, Université Clermont 1, UFR Pharmacie, 63001, Clermont-Ferrand Cedex 1, France

    Benoît Sion

  6. Anatomie Pathologique, CHU Clermont-Ferrand, CHU Estaing, 63003, Clermont-Ferrand Cedex1, France

    Pierre Déchelotte

Authors
  1. Sandra Sanfilippo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michel Canis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sergio Romero
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Benoît Sion
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pierre Déchelotte
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jean-Luc Pouly
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Laurent Janny
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Johan Smitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Florence Brugnon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sandra Sanfilippo.

Additional information

Capsule An original slow freezing protocol able to protect both quality and functionality of human ovarian tissue, and easily applicable in a clinical setting.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sanfilippo, S., Canis, M., Romero, S. et al. Quality and functionality of human ovarian tissue after cryopreservation using an original slow freezing procedure. J Assist Reprod Genet 30, 25–34 (2013). https://doi.org/10.1007/s10815-012-9917-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-012-9917-5

Keywords

Search

Navigation