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Diverse Approaches to Ovarian Tissue Cryopreservation Have Equivalent Outcomes in Markers of Tissue Viability

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Abstract

Ovarian tissue cryopreservation (OTC) is an accepted method of fertility preservation. However, OTC is not standardized and many variations exist in the freezing strategy, tissue processing, and surgical approach. In this pilot study, we used a sheep model to compare slow freezing versus vitrification techniques, as well as the feasibility of processing ovarian tissue into a hyaluronan suspension of small ovarian units. Twelve ovaries were harvested from six female ewes. Paired tissues from each animal were assigned to different treatments and underwent freezing, thawing, autotransplantation, and second-look surgery, for a total of 18 surgical procedures and 3 measured time points. Treatments included whole tissue strips versus gel suspension and slow freezing versus vitrification. At each of the time points, tissue viability was measured by immunohistochemical analysis of CD31 and cleaved caspase-3 (CCASP3). CD31 and CCASP3 expression levels were equivalent between slow freezing and vitrification, and between whole ovarian tissue strips and gel suspension of fragmented ovarian tissue, at all time points. These preliminary data using a sheep model suggest that ovarian tissue is robust and likely to be minimally affected by aggressive fragmentation using a hyaluronan suspension. Furthermore, we provide evidence in support of vitrification as a viable option in OTC. Hyaluronan suspension of ovarian cortical fragments is novel and may represent a desirable method for reimplantation of frozen-thawed ovarian tissue in patients where occult malignant cells are a concern.

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References

  1. Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Options to preserve fertility before oncological treatment: cryopreservation of ovarian tissue and its clinical application. Acta Clin Belg. 2006;61:259–63.

    Article  CAS  Google Scholar 

  2. Meirow D, Roness H, Kristensen SG, Andersen CY. Optimizing outcomes from ovarian tissue cryopreservation and transplantation; activation versus preservation. Hum Reprod. 2015;30:2453–6.

    Article  Google Scholar 

  3. Donnez J, Dolmans M-M. Fertility preservation in women. Nat Rev Endocrinol. 2013;9:735–49.

    Article  CAS  Google Scholar 

  4. Kim S, Lee Y, Lee S, Kim T. Ovarian tissue cryopreservation and transplantation in patients with cancer. Obstet Gynecol Sci. 2018;61:431–42.

    Article  Google Scholar 

  5. Fisch B, Abir R. Female fertility preservation: past, present and future. Reproduction. 2018;156:F11–27.

    Article  CAS  Google Scholar 

  6. Donnez J, Dolmans M-M. Fertility preservation in women. N Engl J Med. 2017;377:1657–65.

    Article  Google Scholar 

  7. Ladanyi C, Mor A, Christianson MS, Dhillon N, Segars JH. Recent advances in the field of ovarian tissue cryopreservation and opportunities for research. J Assist Reprod Genet. 2017;34:709–22.

    Article  Google Scholar 

  8. Kim SK, Lee JR, Samuel KS. Chapter 2 Utility of animal models for human ovarian tissue cryopreservation. Methods Mol Biol. 2017;1568:23–31.

    Article  CAS  Google Scholar 

  9. 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.

    Article  CAS  Google Scholar 

  10. Chi H-J, Koo J-J, Kim M-Y, Joo J-Y, Chang S-S, Chung K-S. Cryopreservation of human embryos using ethylene glycol in controlled slow freezing. Hum Reprod. 2002;17:2146–51.

    Article  CAS  Google Scholar 

  11. Desai N, Abdelhafez F, Calabro A, Falcone T. Three dimensional culture of fresh and vitrified mouse pre-antral follicles in a hyaluronan-based hydrogel: a preliminary investigation of a novel biomaterial for in vitro follicle maturation. Reprod Biol Endocrinol. 2012;10:29.

    Article  CAS  Google Scholar 

  12. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82.

    Article  CAS  Google Scholar 

  13. Carnevale G, Pisciotta A, Riccio M, De Biasi S, Gibellini L, Ferrari A, et al. Optimized cryopreservation and banking of human bone-marrow fragments and stem cells. Biopreserv Biobank. Mary Ann Liebert, Inc., publishers. 2016;14:138–48.

    Article  CAS  Google Scholar 

  14. Chatdarong K, Thuwanut P, Morrell JM. The development of cat testicular sperm cryopreservation protocols: effects of tissue fragments or sperm cell suspension. Theriogenology. 2016;85:200–6.

    Article  CAS  Google Scholar 

  15. Ferreira M, Bos-Mikich A, Frantz N, Rodrigues JL, Brunetto AL, Schwartsmann G. The effects of sample size on the outcome of ovarian tissue cryopreservation. Reprod Domest Anim. 2010;45:99–102.

    Article  CAS  Google Scholar 

  16. Gastal GDA, Alves BG, Alves KA, Souza MEM, Vieira AD, Varela AS, et al. Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C. Reproduction. 2017;153:577–87.

    Article  CAS  Google Scholar 

  17. Friedler S, Schachter M, Strassburger D, Esther K, Ron El R, Raziel A. A randomized clinical trial comparing recombinant hyaluronan/recombinant albumin versus human tubal fluid for cleavage stage embryo transfer in patients with multiple IVF-embryo transfer failure. Hum Reprod. 2007;22:2444–8.

    Article  CAS  Google Scholar 

  18. Saito H, Kaneko T, Takahashi T, Kawachiya S, Saito T, Hiroi M. Hyaluronan in follicular fluids and fertilization of oocytes. Fertil Steril. 2000;74:1148–52.

    Article  CAS  Google Scholar 

  19. Babayan A, Neuer A, Dieterle S, Bongiovanni AM, Witkin SS. Hyaluronan in follicular fluid and embryo implantation following in vitro fertilization and embryo transfer. J Assist Reprod Genet. 2008;25:473–6.

    Article  Google Scholar 

  20. Friedman O, Orvieto R, Fisch B, Felz C, Freud E, Ben-Haroush A, et al. Possible improvements in human ovarian grafting by various host and graft treatments. Hum Reprod. 2012;27:474–82.

    Article  CAS  Google Scholar 

  21. 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;43:437–50.

    Article  Google Scholar 

  22. Donnez J, Dolmans M-M, 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.

    Article  Google Scholar 

  23. Donnez J, Dolmans M-M. Ovarian cortex transplantation: 60 reported live births brings the success and worldwide expansion of the technique towards routine clinical practice. J Assist Reprod Genet. 2015;32:1167–70.

    Article  Google Scholar 

  24. Amorim CA, Curaba M, Van Langendonckt A, Dolmans M-M, Donnez J. Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod BioMed Online. 2011;23:160–86.

    Article  Google Scholar 

  25. Campos ALM, Guedes J d S, Rodrigues JK, Pace WAP, Fontoura RR, Caetano JPJ, et al. Comparison between slow freezing and vitrification in terms of ovarian tissue viability in a bovine model. Rev Bras Ginecol Obstet. 2016;38:333–9.

    Article  Google Scholar 

  26. Dalman A, Deheshkar Gooneh Farahani NS, Totonchi M, Pirjani R, Ebrahimi B, Rezazadeh Valojerdi M. Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: an evaluation of histological changes, WNT signaling pathway and apoptotic genes expression. Cryobiology. 2017;79:29–36.

    Article  CAS  Google Scholar 

  27. Diaz-Garcia C, Domingo J, Garcia-Velasco JA, Herraiz S, Mirabet V, Iniesta I, et al. Oocyte vitrification versus ovarian cortex transplantation in fertility preservation for adult women undergoing gonadotoxic treatments: a prospective cohort study. Fertil Steril. 2018;109:478–485.e2.

    Article  Google Scholar 

  28. Suzuki N, Yoshioka N, Takae S, Sugishita Y, Tamura M, Hashimoto S, et al. Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency. Hum Reprod. 2015;30:608–15.

    Article  Google Scholar 

  29. Shi Q, Xie Y, Wang Y, Li S. Vitrification versus slow freezing for human ovarian tissue cryopreservation: a systematic review and meta-anlaysis. Sci Rep. 2017;7:8538.

    Article  Google Scholar 

  30. Klocke S, Bündgen N, Köster F, Eichenlaub-Ritter U, Griesinger G. Slow-freezing versus vitrification for human ovarian tissue cryopreservation. Arch Gynecol Obstet. 2015;291:419–26.

    Article  CAS  Google Scholar 

  31. Sanfilippo S, Canis M, Smitz J, Sion B, Darcha C, Janny L, et al. Vitrification of human ovarian tissue: a practical and relevant alternative to slow freezing. Reprod Biol Endocrinol. 2015;13:67.

  32. Locatelli Y, Calais L, Duffard N, Lardic L, Monniaux D, Piver P, et al. In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification. J Assist Reprod Genet. 2019;36:1823–35.

    Article  Google Scholar 

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Funding

This research was made possible by internal funds from the Research Program Committee at Cleveland Clinic Lerner Research Institute.

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Author notes

  1. Rebecca Flyckt

    Present address: Reproductive Endocrinology and Infertility, University Hospitals, 1000 Auburn Dr., Suite 310, Beachwood, OH, 44122, USA

Authors and Affiliations

  1. Women’s Health Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH, 44915, USA

    Anne Cashmore Davis, Elliott G. Richards, Tommaso Falcone, Nina Desai & Rebecca Flyckt

  2. Division of Reproductive Endocrinology and Infertility, Baylor College of Medicine, Houston, TX, 77030, USA

    Sara E. Arian

  3. Department of Qualitative Health Sciences, Cleveland Clinic, Cleveland, OH, 44195, USA

    Meng Yao

  4. Pathology and Laboratory Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH, USA

    Andres Chiesa-Vottero

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  1. Anne Cashmore Davis
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Contributions

Anne Cashmore Davis, MD: study design, conduct of the experiments, data collection, data interpretation, and manuscript writing.

Elliott G. Richards, MD: conduct of the experiments, data collection, data interpretation, and manuscript writing.

Sara E. Arian, MD: study design and manuscript review.

Tommaso Falcone, MD: study design, conduct of the experiments, data collection, data interpretation, and manuscript review.

Nina Desai, HCLD, PhD: study design, data interpretation, and manuscript review.

Meng Yao, MS: data interpretation and manuscript review.

Andres Chiesa-Vottero, MD: data collection, data interpretation, and manuscript review.

Rebecca Flyckt, MD: study design, conduct of the experiments, data collection, data interpretation, and manuscript writing.

Corresponding author

Correspondence to Elliott G. Richards.

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The authors declare no competing interests.

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The authors consider that Anne Cashmore Davis and Elliott G. Richards should be regarded as the joint first authors.

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Davis, A.C., Richards, E.G., Arian, S.E. et al. Diverse Approaches to Ovarian Tissue Cryopreservation Have Equivalent Outcomes in Markers of Tissue Viability. Reprod. Sci. 28, 2129–2135 (2021). https://doi.org/10.1007/s43032-021-00486-6

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  • DOI: https://doi.org/10.1007/s43032-021-00486-6

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