P-728: Vitrification may be a promising approach for cryopreservation of human ovarian tissue for auto- and xenotransplantation

Abstract

ObjectiveVitrification is a potential alternative for cryopreservation of human oocytes and ovarian tissues, where traditional methods fail to produce acceptable results. Ovarian tissue cryopreservation is required not only for autotransplantation to preserve fertility in cancer patients but also in ovarian tissue banking as an alternative to egg donation. In this study, various vitrification protocols were examined to develop an effective method for bovine and human ovarian tissue cryopreservation.DesignIce crystal formation during cooling, and post thaw survival rate of oocytes in bovine and human ovaries were examined by using varying sample sizes, concentrations of cryoprotectants, and equilibration times.Materials and methodsOvaries were obtained at local abattoirs from 3-years-old heifers, or from 26 to 29 years old patients with ovarian cancers with informed consent. Ovarian cortex was cut into either 0.5x0.5x0.5 mm (small sections), 10x10x1 mm or 20x10x1 mm (large sections) tissues (similar to that used in successful fresh ovarian transplantation cases; Silber, ASRM 2005). Vitrification was performed with the Cryotop method (Kuwayama, ASRM 2004). Tissues were incubated at room temperature, first in the equilibration solution (7.5% ethylene glycol; EG+7.5% dimethylsulphoxide; DMSO), then transferred to 5 different vitrification solutions (15+15%; 17.5+17.5%; 20+20%; 22.5+22.5% and 25+25% EG and DMSO, respectively), all supplemented with 0.5M sucrose for 5, 10, 15, 20, 25, 30, 35 and 40 min, respectively. Ovarian tissue samples were then cooled by submerging into liquid nitrogen. Vitrification of the ovarian tissue was evaluated by direct visual inspection: successfully vitrified samples remained transparent, while a milky transformation was regarded as sign of ice formation. Ovaries where appropriate vitrification was achieved were warmed by immersing them in 37°C thawing solution containing 1 M sucrose for 3 min. Subsequently, cryoprotectants were diluted in 0.5 M sucrose solution for 5 min, followed by 30 min incubation in isotonic solutions. Oocytes in the preantral follicles recovered from the vitrified ovarian tissue were first observed under visible light, then stained by Hoechst 33342 and propidium iodide and exposed to UV light to judge their survival and membrane integrity.ResultsVitrification of large sections of ovarian tissue was successfully obtained in the groups with > 15 min incubation and in 17.5+17.5% or higher concentration of cryoprotectants in vitrification solution. Post-warming survival rates of oocytes varied between 0 to 91%, depending on the size of the ovarian tissue, incubation time and cryoprotectant concentration in the vitrification solution. With small tissue samples, the highest (91%) survival rate was obtained after 10 min incubation in the 15+15% group.ConclusionThe excellent post-warming survival results indicate that the tested vitrification method may be a promising approach to cryopreserve human ovaries both for autotransplantation to maintain fertility and for ovarian tissue donation through ovarian tissue banking. ObjectiveVitrification is a potential alternative for cryopreservation of human oocytes and ovarian tissues, where traditional methods fail to produce acceptable results. Ovarian tissue cryopreservation is required not only for autotransplantation to preserve fertility in cancer patients but also in ovarian tissue banking as an alternative to egg donation. In this study, various vitrification protocols were examined to develop an effective method for bovine and human ovarian tissue cryopreservation. Vitrification is a potential alternative for cryopreservation of human oocytes and ovarian tissues, where traditional methods fail to produce acceptable results. Ovarian tissue cryopreservation is required not only for autotransplantation to preserve fertility in cancer patients but also in ovarian tissue banking as an alternative to egg donation. In this study, various vitrification protocols were examined to develop an effective method for bovine and human ovarian tissue cryopreservation. DesignIce crystal formation during cooling, and post thaw survival rate of oocytes in bovine and human ovaries were examined by using varying sample sizes, concentrations of cryoprotectants, and equilibration times. Ice crystal formation during cooling, and post thaw survival rate of oocytes in bovine and human ovaries were examined by using varying sample sizes, concentrations of cryoprotectants, and equilibration times. Materials and methodsOvaries were obtained at local abattoirs from 3-years-old heifers, or from 26 to 29 years old patients with ovarian cancers with informed consent. Ovarian cortex was cut into either 0.5x0.5x0.5 mm (small sections), 10x10x1 mm or 20x10x1 mm (large sections) tissues (similar to that used in successful fresh ovarian transplantation cases; Silber, ASRM 2005). Vitrification was performed with the Cryotop method (Kuwayama, ASRM 2004). Tissues were incubated at room temperature, first in the equilibration solution (7.5% ethylene glycol; EG+7.5% dimethylsulphoxide; DMSO), then transferred to 5 different vitrification solutions (15+15%; 17.5+17.5%; 20+20%; 22.5+22.5% and 25+25% EG and DMSO, respectively), all supplemented with 0.5M sucrose for 5, 10, 15, 20, 25, 30, 35 and 40 min, respectively. Ovarian tissue samples were then cooled by submerging into liquid nitrogen. Vitrification of the ovarian tissue was evaluated by direct visual inspection: successfully vitrified samples remained transparent, while a milky transformation was regarded as sign of ice formation. Ovaries where appropriate vitrification was achieved were warmed by immersing them in 37°C thawing solution containing 1 M sucrose for 3 min. Subsequently, cryoprotectants were diluted in 0.5 M sucrose solution for 5 min, followed by 30 min incubation in isotonic solutions. Oocytes in the preantral follicles recovered from the vitrified ovarian tissue were first observed under visible light, then stained by Hoechst 33342 and propidium iodide and exposed to UV light to judge their survival and membrane integrity. Ovaries were obtained at local abattoirs from 3-years-old heifers, or from 26 to 29 years old patients with ovarian cancers with informed consent. Ovarian cortex was cut into either 0.5x0.5x0.5 mm (small sections), 10x10x1 mm or 20x10x1 mm (large sections) tissues (similar to that used in successful fresh ovarian transplantation cases; Silber, ASRM 2005). Vitrification was performed with the Cryotop method (Kuwayama, ASRM 2004). Tissues were incubated at room temperature, first in the equilibration solution (7.5% ethylene glycol; EG+7.5% dimethylsulphoxide; DMSO), then transferred to 5 different vitrification solutions (15+15%; 17.5+17.5%; 20+20%; 22.5+22.5% and 25+25% EG and DMSO, respectively), all supplemented with 0.5M sucrose for 5, 10, 15, 20, 25, 30, 35 and 40 min, respectively. Ovarian tissue samples were then cooled by submerging into liquid nitrogen. Vitrification of the ovarian tissue was evaluated by direct visual inspection: successfully vitrified samples remained transparent, while a milky transformation was regarded as sign of ice formation. Ovaries where appropriate vitrification was achieved were warmed by immersing them in 37°C thawing solution containing 1 M sucrose for 3 min. Subsequently, cryoprotectants were diluted in 0.5 M sucrose solution for 5 min, followed by 30 min incubation in isotonic solutions. Oocytes in the preantral follicles recovered from the vitrified ovarian tissue were first observed under visible light, then stained by Hoechst 33342 and propidium iodide and exposed to UV light to judge their survival and membrane integrity. ResultsVitrification of large sections of ovarian tissue was successfully obtained in the groups with > 15 min incubation and in 17.5+17.5% or higher concentration of cryoprotectants in vitrification solution. Post-warming survival rates of oocytes varied between 0 to 91%, depending on the size of the ovarian tissue, incubation time and cryoprotectant concentration in the vitrification solution. With small tissue samples, the highest (91%) survival rate was obtained after 10 min incubation in the 15+15% group. Vitrification of large sections of ovarian tissue was successfully obtained in the groups with > 15 min incubation and in 17.5+17.5% or higher concentration of cryoprotectants in vitrification solution. Post-warming survival rates of oocytes varied between 0 to 91%, depending on the size of the ovarian tissue, incubation time and cryoprotectant concentration in the vitrification solution. With small tissue samples, the highest (91%) survival rate was obtained after 10 min incubation in the 15+15% group. ConclusionThe excellent post-warming survival results indicate that the tested vitrification method may be a promising approach to cryopreserve human ovaries both for autotransplantation to maintain fertility and for ovarian tissue donation through ovarian tissue banking. The excellent post-warming survival results indicate that the tested vitrification method may be a promising approach to cryopreserve human ovaries both for autotransplantation to maintain fertility and for ovarian tissue donation through ovarian tissue banking.