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Detailed Morphological Analysis of Cryoinjury in Human Ovarian Tissue Following Vitrification or Slow Freezing

  • Reproductive Biology: Original Article
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Abstract

Cryopreservation of human ovarian tissue represents a key procedure for fertility preservation. The two most widely used cryopreservation methods for human ovarian cortex samples are slow freezing\thawing (SF\T) and vitrification\warming (V\W). The aim of the present study was to analyze the effects of SF\T and V\W using a metal chamber, on specific follicle and oocyte structures and on the stromal organization post-cryopreservation. We did histology analysis of SF\T and V\W ovarian fragments from nine healthy subjects. Overall results showed that cryopreserved tissues presented significant rates of damage in primordial and primary follicles. Altered nuclear structure of primordial follicles and cell detachment from primordial and primary follicles were the main injuries observed after V/W and SF/T. The stromal components were similarly well preserved after cryopreservation. We conclude that both cryopreservation methods may be used for fertility preservation purposes with similar outcomes in terms of follicular and stromal integrity. Detachment of follicle cells from basal membrane represents an important cryoinjury that deserves further investigation.

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Data Availability

All authors include information of no funding, non-financial interests, study-specific approval by the appropriate ethics committee for research involving humans (Conceição Hospital Group Ethics Committee approval #17,135) and informed consent as the research involved human participants (Informed consent was given and signed by all participants).

Code Availability

All data and materials as well as software application or custom code support our published claims and comply with field standards.

References

  1. Abramson JH. WINPEPI updated: computer programs for epidemiologists, and their teaching potential. Epidemiol Perspect Innov. 2011; 8. https://doi.org/10.1186/1742-5573-8-1

  2. Amorim CA, Curaba M, Van LA, Dolmans MM, Donnez J. Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod Biomed Online. 2011;23:160–86. https://doi.org/10.1016/j.rbmo.2011.04.005.

    Article  PubMed  Google Scholar 

  3. Aquino D, Danielli L, Rigon P, Lothhammer N, Frantz N, Bos-Mikich A. Ovarian tissue vitrification: the use of a novel metal closed system for clinical grade cryopreservation. JBRA Assist Reprod. 2014;18:12–5. https://doi.org/10.5935/1518-0557.20140086.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bos-Mikich A, Marques L, Rodrigues JL, Lothhammer N, Frantz N. The use of a metal containner for vitrification of mouse ovaries as a clinical grade model for human ovarian tissue cryopreservation after different times and temperatures of transport. J Assist Reprod Genet. 2012;29:1267–71. https://doi.org/10.1007/s10815-012-9867-y.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B, Van Langendonckt A. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet. 2004;364:1405–10. https://doi.org/10.1016/S0140-6736(04)17222-X.

    Article  CAS  PubMed  Google Scholar 

  6. Fabbri R, Vicenti R, Macciocca M, Pasquinelli G, Paradisi R, Battaglia C, Martino NA, Venturoli S. Good preservation of stromal cells and no apoptosis in human ovarian tissue after vitrification. Biomed Res Int. 2014;2014:673537. https://doi.org/10.1155/2014/673537.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Fabbri R, Vicenti R, Macciocca M, Martino NA, Dell’Aquila ME, Pasquinelli G, Morselli-Labate AM, Seracchioli R, Paradisi R. Morphological, ultrastructural and functional imaging of frozen/thawed and vitrified/warmed human ovarian tissue retrieved from oncological patients. Hum Reprod. 2016;31:1838–49. https://doi.org/10.1093/humrep/dew134.

    Article  CAS  PubMed  Google Scholar 

  8. Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, Lass A, Winston RM. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod. 1996;11:1268–72. https://doi.org/10.1093/oxfordjournals.humrep.a019370.

    Article  CAS  PubMed  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. https://doi.org/10.1016/j.ejogrb.2008.03.002.

    Article  CAS  PubMed  Google Scholar 

  10. Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, Bader M, Weiss JM. Human ovarian tissue vitrification versus conventional freezing: morphological and molecular biological evaluation. Reproduction. 2009;138:319–27. https://doi.org/10.1530/REP-09-0039.

    Article  CAS  PubMed  Google Scholar 

  11. Kawamura K, Cheng Y, Suzuki N, Deguchi M, Sato Y, Takae S, et al. Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment. Proc Natl Acad Sci USA. 2013;110:17474–9. https://doi.org/10.1073/2Fpnas.1312830110.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Keros V, Xella S, Hultenby K, Pettersson K, Sheikhi M, Volpe A, Hreinsson J, Hovatta O. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod. 2009;24:1670–83. https://doi.org/10.1093/humrep/dep079.

    Article  CAS  PubMed  Google Scholar 

  13. Klocke S, Bundgen N, Koster F, Eichenlaub-Ritter U, Griesinger G. Slow-freezing versus vitrification for human ovarian tissue cryopreservation. Arch Gynecol Obstet. 2015;291:419–26. https://doi.org/10.1007/s00404-014-3390-6.

    Article  CAS  PubMed  Google Scholar 

  14. 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. JARG. 2019;36:1823–35.

    Google Scholar 

  15. Mao M, Alavi MV, Labelle-Dumais C, Gould DB, in: J.H. Miner (Eds.) Current topics in membranes, Cambridge, 2015, pp. 61–116.

  16. Margarone JE, Natiella JR, Vaughan CD. Artifacts in oral biopsy specimens. J Oral Maxillofac Surg. 1985;43:163–72. https://doi.org/10.1016/0278-2391(85)90154-5.

    Article  CAS  PubMed  Google Scholar 

  17. Marques LS, Rodrigues JL, Lothhammer N, Frantz N, Bös-Mikich A. Follicle and stroma integrity after holding ovarian tissue for up to 8 hours before vitrification in a metal closed system. JBRA Assist Reprod. 2013;17:169–72. https://doi.org/10.5935/1518-0557.20130056.

    Article  Google Scholar 

  18. Martinez F. Update on fertility preservation from the Barcelona International Society for Fertility Preservation-ESHRE-ASRM 2015 expert meeting: indications, results and future perspectives. Fertil Steril. 2017;108:407–15. https://doi.org/10.1016/j.fertnstert.2017.05.024.

    Article  PubMed  Google Scholar 

  19. Massignam ET, Ferreira M, Sanguinet E, Dupont A, Klamt F, Frantz N, Bös-Mikich A. Antioxidant defence capacity of ovarian tissue after vitrification in a metal closed system. JBRA Assist Reprod. 2018;22:199–204. https://doi.org/10.5935/2F1518-0557.20180044.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Ovary and ovulation: Low temperature storage and grafting of human ovarian tissue. Hum Reprod. 1996;11:1487–91. https://doi.org/10.1093/oxfordjournals.humrep.a019423.

    Article  CAS  PubMed  Google Scholar 

  21. Rosendahl M, Schmidt KT, Ernst E, Rasmussen PE, Loft A, Byskov AG, Andersen AN, Andersen CY. Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique. Reprod Biomed Online. 2011;22:162–71. https://doi.org/10.1016/j.rbmo.2010.10.015.

    Article  PubMed  Google Scholar 

  22. Sanfilippo S, Canis M, Smitz J, Sion B, Darcha C, Janny L, Brugnon F. Vitrification of human ovarian tissue: a practical and relevant alternative to slow freezing. Reprod Biol Endocrinol. 2015;13:67. https://doi.org/10.1186/s12958-015-0065-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Santana LN, Van den Hurk R, Oskam IC, Brito AB, Brito DC, Domingues SFS, Santos RR. Vitrifcation of ovarian tissue from primates and domestic ruminants: an overview. Biopreserv Biobank. 2012;10:288–94. https://doi.org/10.1089/bio.2011.0048.

    Article  PubMed  Google Scholar 

  24. Schmidt KLT, Byskov AG, Nyboe Andersen A, Muller J, Yding AC. Density and distribution of primordial follicles in single pieces of cortex from 21 patients and in individual pieces of cortex from three entire human ovaries. Hum Reprod. 2003;18:1158–64. https://doi.org/10.1093/humrep/deg246.

    Article  CAS  PubMed  Google Scholar 

  25. Silber S, Fan Y, Goldsmith S. World wide update: results with cryopreserved ovarian tissue transplant. Fertil Steril. 2019;112:85. https://doi.org/10.1016/j.fertnstert.2019.07.342.

    Article  Google Scholar 

  26. 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. https://doi.org/10.1093/humrep/deu353.

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank Mr Maikel Oliveira (Department of Morphological Sciences, ICBS, Federal University of Rio Grande do Sul) for helping with the histology procedures.

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Authors and Affiliations

  1. Department of Morphological Sciences, ICBS, Federal University of Rio Grande Do Sul, Rua Sarmento Leite 500, Porto Alegre, RS, 90050-170, Brazil

    L. Ramos & A. Bos-Mikich

  2. FÊMINA Hospital, Porto Alegre, RS, Brazil

    S. Galbinski & A. Nacul

  3. Department of Gynecology and Obstetrics, Federal University of Health Sciences of Porto Alegre, Porto Alegre, RS, Brazil

    M. F. Jiménez

  4. Nilo Frantz Research and Human Reproduction Center, Porto Alegre, RS, Brazil

    N. Frantz

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  1. L. Ramos
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  2. S. Galbinski
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  5. N. Frantz
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Correspondence to A. Bos-Mikich.

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The questionnaire and methodology for this study was approved by the Human Research Ethics committee of the Conceição Hospital Group (Ethics approval number:17135).

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Written informed consent was obtained from the parents.

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Patients signed informed consent regarding publishing their data and photographs.

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Ramos, L., Galbinski, S., Nacul, A. et al. Detailed Morphological Analysis of Cryoinjury in Human Ovarian Tissue Following Vitrification or Slow Freezing. Reprod. Sci. 29, 2374–2381 (2022). https://doi.org/10.1007/s43032-021-00716-x

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