Comparative analysis of the histological architecture of ovarian tissue following slow-freezing cryopreservation

Czech version

Authors: Veronika Hončová ¹ ; Jan Vodička ¹ ; Zuzana Slobodová ² ; Radovan Pilka ¹
Authors‘ workplace: Porodnicko-gynekologická klinika LF UP a FN Olomouc ¹; Ústav klinické a molekulární patologie, LF UP a FN Olomouc ²
Published in: Ceska Gynekol 2025; 90(5): 349-359
Category: Original Article
doi: https://doi.org/10.48095/cccg2025349

Overview

Objective: The aim of this study was to validate the methodology of ovarian tissue cryopreservation using the slow freezing technique as a fertility preservation approach and to assess its potential implementation in clinical practice at the Assisted Reproduction Centre of the University Hospital Olomouc. In parallel, the technical procedure of cryopreservation was optimized and standardized. Materials and methods: The study was conducted between April 2022 and December 2024 at the Department of Obstetrics and Gynecology, University Hospital Olomouc, and included six transgender patients aged 19–25 years who underwent laparoscopic hysterectomy with bilateral adnexectomy as part of gender-affirming surgery. Ovarian tissue obtained during the procedure was immediately processed and cryopreserved using the slow freezing method at the Assisted Reproduction Centre of the University Hospital Olomouc. Histopathological evaluation of the ovarian tissue was performed both prior to cryopreservation and after thawing at the Department of Clinical and Molecular Pathology. Results: Morphological assessment of the ovarian tissue after cryopreservation confirmed preservation of structural characteristics of follicles and stromal components, without signs of significant degeneration. Conclusion: The results confirm that the applied slow freezing protocol for ovarian tissue cryopreservation is appropriate and sufficiently gentle for clinical use. This method represents a reliable option for fertility preservation in patients undergoing gonadectomy, with potential applications in subsequent autologous transplantation or in vitro follicle culture.

Keywords:

assisted reproduction – In vitro fertilization – oncofertility – fertility preservation – ovarian tissue cryopreservation – slow-freezing – transgender

Sources

1. Spears N, Lopes F, Stefansdottir A et al. Ovarian damage from chemotherapy and current approaches to its protection. Hum Reprod Update 2019; 25 (6): 673–693. doi: 10.1093/humupd/dmz027.

2. Sheshpari S, Shahnazi M, Mobarak H et al. Ovarian function and reproductive outcome after ovarian tissue transplantation: a systematic review. J Transl Med 2019; 17 (1): 396. doi: 10.1186/s12967-019-02149-2.

3. Clark RA, Mostoufi-Moab S, Yasui Y et al. Predicting acute ovarian failure in female survivors of childhood cancer: a cohort study in the Childhood Cancer Survivor Study (CCSS) and the St Jude Lifetime Cohort (SJLIFE). Lancet Oncol 2020; 21 (3): 436–445. doi: 10.1016/S1470-2045 (19) 30818-6.

4. Anderson RA, Wallace WH, Telfer EE. Ovarian tissue cryopreservation for fertility preservation: clinical and research perspectives. Hum Reprod Open 2017; 2017 (1): hox001. doi: 10.1093/hropen/hox001.

5. Andersen CY, Bollerup AC, Kristensen SG. Defining quality assurance and quality control in ovarian tissue cryopreservation and transplantation: a call to action. Hum Reprod 2018; 33 (7): 1201–1204. doi: 10.1093/humrep/ dey105.

6. Diaz-Garcia C, Domingo J, Garcia-Velasco JA 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 (3): 478.e2–485.e2. doi: 10.1016/j.fertnstert.2017.11.018.

7. Gellert SE, Pors SE, Kristensen SG et al. Transplantation of frozen-thawed ovarian tissue: update on worldwide activity published in peer-reviewed papers and on the Danish cohort. J Assist Reprod Genet 2018; 35 (4): 561–570. doi: 10.1007/s10815-018-1144-2.

8. Marin L, Bedoschi G, Kawahara T et al. History and current state of ovarian tissue auto-transplantation with cryopreserved tissue: a successful translational research journey from 1999 to 2020. Reprod Sci 2020; 27 (4): 955–962. doi: 10.1007/s43032-019-00066-9.

9. Donnez J, Dolmans MM, Demylle D et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004; 364 (9443): 1405–1410. doi: 10.1016/S0140-6736 (04) 1 7222-X.

10. Practice Committee of the American Society for Reproductive Medicine. Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion. Fertil Steril 2019; 112 (6): 1022–1033. doi: 10.1016/ j.fertnstert.2019.09.013.

11. Haering C, Coyne K, Daunov K et al. Ovarian tissue cryopreservation for fertility preservation in patients with hemoglobin disorders: a comprehensive review. J Clin Med 2024; 13 (13): 3631. doi: 10.3390/jcm13133631.

12. Dolmans MM, von Wolff M, Poirot C et al. Transplantation of cryopreserved ovarian tissue in a series of 285 women: a review of five leading European centers. Fertil Steril 2021; 115 (5): 1102–1115. doi: 10.1016/j.fertnstert. 2021.03.008.

13. Meirow D, Hardan I, Dor J et al. Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients. Hum Reprod 2008; 23 (5): 1007–1013. doi: 10.1093/humrep/ den055.

14. Soares M, Saussoy P, Maskens M et al. Elimination of malignant cells from cryopreserved ovarian tissue is possible in leukaemia patients. Br J Heamatol 2017; 178 (2): 231–239. doi: 10.1111/bjh.14657.

15. Kim SS. Assessment of long term endocrine function after transplantation of frozen--thawed human ovarian tissue to the heterotopic site: 10 year longitudinal follow-up study. J Assist Reprod Genet 2012; 29 (6): 489–493. doi: 10.1007/s10815-012-9757-3.

16. Marco A, Gargallo M, Ciriza J et al. Current fertility preservation steps in young women suffering from cancer and future perspectives. Int J Mol Sci 2024; 25 (8): 4360. doi: 10.3390/ijms25084360.

17. Donnez J, Dolmans MM. Fertility preservation in women. N Engl J Med 2017; 377 (17): 1657–1665. doi: 10.1056/NEJMra16 14676.

18. Martínek J, Vacek Z. Histologický atlas. 1. vyd. Praha: Grada Publishing 2009.

19. Gartner LP, Hiatt JL. Color textbook of histology. 4th ed. Philadelphia: Elsevier 2017.

20. Richards JS, Pangas SA. The ovary: basic biology and clinical implications. J Clin Invest 2010; 120 (4): 963–972. doi: 10.1172/JCI4 1350.

21. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1986; 1 (2): 81–87. doi: 10.1093/oxfordjournals.humrep.a136365.

22. Sadler TW. Langman’s medical embryology. 14th ed. Philadelphia: Wolters Kluwer 2019.

23. Zhang T, He M, Zhang J et al. Mechanisms of primordial follicle activation and new pregnancy opportunity for premature ovarian failure patients. Front Physiol 2023; 14 : 1113684. doi: 10.3389/fphys.2023.1113684.

24. Williams CJ, Erickson GF. Morphology and physiology of the ovary. In: Endotext. 2005 [online]. Available from: https: //www.ncbi.nlm.nih.gov/books/NBK278951/.

25. Baerwald AR, Adams GP, Pierson RA. A new model for ovarian follicular development during the human menstrual cycle. Fertil Steril 2003; 80 (1): 116–122. doi: 10.1016/s0015 -⁠ 0282 (03) 00544-2.

26. Tilly JL. Apoptosis and ovarian function. Rev Reprod 1996; 1 (3): 162–172. doi: 10.1530/ror. 0.0010162.

27. Knight PG, Glister C. TGF-b superfamily members and ovarian follicle development. Reproduction 2006; 132 (2): 191–206. doi: 10.1530/rep.1.01074.

28. Eppig JJ. Oocyte control of ovarian follicular development and function in mammals. Reproduction 2001; 122 (6): 829–838. doi: 10.1530/rep.0.1220829.

29. Richards JS, Jahnsen T, Hedin L et al. Ovarian follicular development: from physiology to genes. Recent Prog Horm Res 1987; 43 : 231–276. doi: 10.1016/b978-0-12-571143-2. 50012-5.

30. Jensen AK, Kristensen SG, Macklon KT et al. Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark. Hum Reprod 2015; 30 (12): 2838–2845. doi: 10.1093/humrep/dev230.

31. Anderson RA, Wallace WH, Telfer EE. Ovarian tissue cryopreservation for fertility preservation: clinical and research perspectives. Hum Reprod Open 2017; 2017 (1): hox001. doi: 10.1093/hropen/hox001.

32. Amorim CA, Shikanov A. The artificial ovary: current status and future perspectives. Future Oncol 2016; 12 (20): 2323–2332. doi: 10.2217/fon-2016-0202.

33. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984; 247 (3 Pt 1): C125–C142. doi: 10.1152/ajpcell.1984.247.3. C125.

34. Newton H, Aubard Y, Rutherford A et al. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996; 11 (7): 1487–1491. doi: 10.1093/oxfordjournals.humrep.a019423.

35. Perelli F, Fusi G, Lonati L et al. Laparoscopic ovarian tissue collection for fertility preservation in children with malignancies: a multicentric experience. Front Surg 2024; 11 : 1352698. doi: 10.3389/fsurg.2024.1352 698.

36. Beckmann MW, Dittrich R, Lotz L et al. Fertility protection: complications of surgery and results of removal and transplantation of ovarian tissue. Reprod Biomed Online 2018; 36 (2): 188–196. doi: 10.1016/j.rbmo.2017. 10.109.

37. Duncan FE, Zelinski M, Gunn AH et al. Ovarian tissue transport to expand access to fertility preservation: from animals to clinical practice. Reproduction 2016; 152 (6): R201–R210. doi: 10.1530/REP-15-0598.

38. Hončová V. Kryokonzervace ovariální tkáně jako metoda pro zachování fertility u žen. Ceska Gynekol 2023; 88 (1): 44–51. doi: 10.48095/cccg202344.

39. Bastings L, Liebenthron J, Westphal JR et al. Efficacy of ovarian tissue cryopreservation in a major European center. J Assist Reprod Genet 2014; 31 (8): 1003–1012. doi: 10.1007/s10815-014-0239-7.

40. Dittrich R, Hackl J, Lotz L et al. Pregnancies and live births after 20 transplantations of cryopreserved ovarian tissue in a single center. Fertil Steril 2015; 103 (2): 462–468. doi: 10.1016/ j.fertnstert.2014.10.045.

41. HARVEY MAIN. Planer Kryo 370. 2025 [online]. Available from: https: //www.harveymain.com/products/Planer/Kryo370.php.

42. Donnez J, Jadoul P, Squifflet J et al. Ovarian tissue cryopreservation and transplantation in cancer patients. Best Pract Res Clin Obstet Gynaecol 2010; 24 (1): 87–100. doi: 10.1016/ j.bpobgyn.2009.09.003.

43. Rosendahl M, Greve T, Andersen CY. The safety of transplanting cryopreserved ovarian tissue in cancer patients: a review of the literature. J Assist Reprod Genet 2013; 30 (1): 11–24. doi: 10.1007/s10815-012-9912-x.

ORCID autorů

V. Hončová 0000-0001-7066-7853

J. Vodička 0000-0003-0446-4454

Z. Slobodová 0000-0001-7755-048X

R. Pilka 0000-0001-8797-1894

Doručeno/Submitted: 26. 7. 2025

Přijato/Accepted: 30. 7. 2025

Mgr. Veronika Hončová

Porodnicko-gynekologická klinika

LF UP a FN Olomouc

Zdravotníků 248/7

779 00 Olomouc