Clinical grade vitrification of human ovarian tissue: an ultrastructural analysis of follicles and stroma in vitrified tissue

Abstract

BACKGROUNDCancer therapy is one of many conditions which may diminish the ovarian reserve. Banking of human ovarian tissue has become an option for the preservation of female fertility. We have shown that vitrification is an excellent method to cryopreserve ovarian tissue. To carry out vitrification in a clinical setting, we have developed a clinical grade closed system to avoid direct contact of ovarian tissue with liquid nitrogen.METHODSOvarian tissue was obtained by biopsy from 12 consenting women undergoing Caesarean section. Tissues were vitrified in cryotubes, using dimethyl sulphoxide, 1,2-propanediol, ethylene glycol and polyvinylpyrrolidon as cryoprotectants. Non-vitrified and warmed-vitrified tissue was compared by light and electron microscopic morphology of the follicles within the tissues.RESULTSWe did not see any differences in the light or electron microscopic ultrastructure of oocytes between non-vitrified and vitrified tissues. No irreversible subcellular alterations in vitrified tissues were seen.CONCLUSIONSThe ultrastructure of follicles within the vitrified human ovarian tissue was well preserved using cryotube in a closed vitrification system to avoid direct contact of liquid nitrogen. The system is compatible with the European tissue directive.

The use of a metal container for vitrification of mouse ovaries, as a clinical grade model for human ovarian tissue cryopreservation, after different times and temperatures of transport

“…Vitrification, as a choice of cryopreservation, has shown promising outcomes in terms of follicle and stromal preservation in rodents and other species including humans [1,2,4,10,13,16,17,19,21]. Despite these promising results, little attention has been paid to how the tissue should be handled before cryopreservation, to guarantee the best possible post-storage outcome.…”

“…This issue has impelled scientists to develop systems, in which the biological material is never directly exposed to the LNi during cryopreservation and subsequent cryostorage. In this regard, closed systems were developed for the vitrification of macaque and the human ovarian tissue using cryovials [17,20]. However, both studies employed plastic devices to enclose the ovarian fragments, and attention should be given to alternative materials that present faster heat-conduction, a key factor for successful vitrification.…”

Good manufacturing practice requirements for the production of tissue vitrification and warming and recovery kits for clinical research

“…[16] Xenograft and recovery of primordial or secondary follicle-containing bovine tissue into mice [17] X X PrOH, PVP Histology and ultrastructure in human [11,12] X X PVP Histology, follicle count, and ultrastructure in cynomolgus macaques [18] Autotransplant, histology and ultrastructure, vascularization of graft, serum E2 and P4, ova stimulation, and embryo development through ICSI in cynomolgus macaques [19] Autotransplant, serum E2 and P4, ova stimulation, and embryo development through ICSI, 2 successful deliveries in human [20] X X X PVP K-10 Apoptosis, proliferation, and fibrosis of human xenografted in mice [21] Autotransplant, histology, follicle count, proliferation, apoptosis, graft vascularization and fibrosis, AMH immunohistochemistry in Papio anubis [22] Autotransplant, histology, follicle count, proliferation, apoptosis, graft vascularization and fibrosis, AMH immunohistochemistry in cynomolgus macaques…”

“…1. Vitrification of ovarian tissue has already been tested as a potential way to preserve and restore quality ovarian tissue in rhesus monkeys and humans [7][8][9][10][11][12]. The goal for developing the Human Ovarian Tissue Vitrification Kit and Human Ovarian Tissue Warming & Recovery Kit were to maintain consistent media formulations and sets of instructions for handling the human ovarian tissue for establishing ovarian tissue vitrification as a way to preserve and restore quality ovarian tissue in future clinical trials.…”

Advances in ovarian tissue cryopreservation in goats and sheep

“…Comumente, as amostras são congeladas e mantidas em nitrogênio líquido, cuja temperatura de -196 °C é conhecida como temperatura criogênica (Rubinsky, 2003). Nessa temperatura todas as reações químicas, processos biológicos, bem como as atividades intra e extracelulares estão suspensas, portanto, teoricamente, uma célula ou tecido podem ser mantidos criopreservados indefinidamente (Sheikhi et al, 2011). Porém, as células e tecidos são extremamente sensíveis a baixas temperaturas.…”