Stepped vitrification technique for human ovarian tissue cryopreservation

Ellen Cristina Rivas Leonel,Marina Vazquez,John Morris,Peter Kilbride,Ramon Risco,Christiani A Amorim,Alessandra Camboni,Sebastião Roberto Taboga,Ariadna Corral,Marie-Madeleine Dolmans

doi:10.1038/s41598-019-56585-7

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.

Highlights

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations
The main challenge faced during freezing is formation of ice within the tissue, as reducing the temperature leads to ice crystal formation due to ice nucleation
An increase in viscosity requires high concentrations of cryoprotective agents (CPAs) in order to extract water from the tissue, which may create other problems, namely toxic effects suffered by cells[11]

Summary

Introduction

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. Adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants These days, human fertility preservation should be considered a complement of cancer treatments[1]. When the temperature falls very fast, there is no time or energy for molecular rearrangement, so there is no ice crystal formation; the natural disorder of liquid molecules in tissue is maintained, mitigating any disturbance to the system. This forms the basis of the vitrification concept, as the absence of crystals prevents cell membrane damage normally caused by their mechanical. An increase in viscosity requires high concentrations of cryoprotective agents (CPAs) in order to extract water from the tissue, which may create other problems, namely toxic effects suffered by cells[11]

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Conclusion