Journal of Assisted Reproduction and Genetics

, Volume 36, Issue 9, pp 1823–1835 | Cite as

In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification

  • Yann LocatelliEmail author
  • L. Calais
  • N. Duffard
  • L. Lardic
  • D. Monniaux
  • P. Piver
  • P. Mermillod
  • M. J. Bertoldo
Fertility Preservation



Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation.


Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9 days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media.


No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments.


Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.


Fertility preservation Ovarian cortex Cryopreservation Vitrification In vitro folliculogenesis 



The authors wish to thank Thierry Delpuech for the collection of ovaries at slaughterhouse, Dr. Charlène Rico for the help with setup of culture conditions, and Corinne Laclie and Anne-Lyse Lainé from Laboratoire Phénotypage-Endocrinologie for the P4 assay.

Author contributions

YL designed and performed the experiments and analysis, wrote the manuscript, and secured the funding. LC performed the experiments and analysis and contributed to first draft of the manuscript. ND performed the experiments and LL performed the assay for estradiol. DM, PP, and PM contributed to experimental design and revised the manuscript. MJB performed the experiments and analysis, wrote, and revised the manuscript.


Dr. Michael J. Bertoldo, Laure Calais, and the laboratories involved in the present study were supported by a grant from “Région Centre” (CRYOVAIRE, Grant number no. 320000268).


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.DMJZ, Muséum National d’Histoire Naturelle, Laboratoire de la RéserveZoologique de la Haute ToucheObterreFrance
  2. 2.INRA UMR Physiologie de la Reproduction et des Comportements, INRANouzillyFrance
  3. 3.Service de gynécologie-obstétrique, hôpital Mère-EnfantCHU de LimogesLimogesFrance
  4. 4.Fertility and Research Centre, School of Women’s and Children’s HealthUniversity of New South Wales SydneySydneyAustralia
  5. 5.School of Medical SciencesUniversity of New South Wales SydneySydneyAustralia

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