Stepped vitrification technique for human ovarian tissue cryopreservation

Author(s)

Rivas Leonel, Ellen Cristina ^{[1, 2]} ; Corral, Ariadna ^[3] ; Risco, Ramon ^{[3, 4]} ; Camboni, Alessandra ^{[2, 5]} ; Taboga, Sebastiao Roberto ^[1] ; Kilbride, Peter ^[6] ; Vazquez, Marina ^{[6, 4]} ; Morris, John ^[6] ; Dolmans, Marie-Madeleine ^{[2, 7]} ; Amorim, Christiani A. ^[2]

Affiliation

^[1] Sao Paulo State Univ UNESP, Inst Biosci Humanities & Exact Sci, Dept Biol, Rua Cristovao Colombo 2265, BR-15054000 Jardim Nazareth, Sao Jose Do Rio - Brazil

^[2] Catholic Univ Louvain, Inst Rech Expt & Clin, Pole Rech Gynecol, Ave Mounier 52, Bte B1-52-02, B-1200 Brussels - Belgium

^[3] Univ Seville, CNA, Calle Thomas Alva Edison 7, Seville 41092 - Spain

^[4] Univ Seville, Engn Sch Sevilla, Camino Descubrimientos S-N, Seville 41092 - Spain

^[5] Clin Univ St Luc, Serv Anat Pathol, Ave Hippocrate 10, B-1200 Brussels - Belgium

^[6] Gen Elect Healthcare, Sovereign House, Vis Pk, Cambridge CB24 9BY - England

^[7] Clin Univ St Luc, Gynecol & Androl Dept, Ave Hippocrate 10, B-1200 Brussels - Belgium

• Document type: Journal article
• Source: SCIENTIFIC REPORTS; v. 9, DEC 27 2019.
• Web of Science Citations: 0

Abstract

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. We aimed to test this method for human ovarian tissue cryopreservation. Ovarian cortex was taken from 7 fertile adult women. Samples were subjected to an SV protocol performed in an automatic freezer, which allowed sample transfer to ever higher concentrations of dimethyl sulfoxide (DMSO) as the temperature was reduced. Histological evaluation of the vitrified-warmed tissue showed large numbers of degenerated follicles after 24 hours of in vitro culture. We therefore evaluated DMSO perfusion rates by X-ray computed tomography, ice crystal formation by freeze-substitution, and cell toxicity by transmission electron microscopy, seeking possible reasons why follicles degenerated. Although cryoprotectant perfusion was considered normal and no ice crystals were formed in the tissue, ultrastructural analysis detected typical signs of DMSO toxicity, such as mitochondria degeneration, alterations in chromatin condensation, cell vacuolization and extracellular matrix swelling in both stromal and follicular cells. The findings indicated that the method failed to preserve follicles due to the high concentrations of DMSO used. However, adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants. (AU)

• FAPESP's process: 16/22947-8 - Techniques of female reproductive tissue morphology and viability assessment as tools for enhancing cancer research success
• Grantee: Ellen Cristina Rivas Leonel
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate