Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice

Abstract

Ovarian tissue cryopreservation and transplantation can be used to preserve fertility for cancer patients. In this study, we assessed the viability and function of ovarian tissue from adult mice that was cryopreserved by solid surface vitrification or traditional slow-cooling using various in vitro and in vivo techniques, including allotransplantation, in vitro oocyte maturation, embryo culture in vitro, blastocyst cryopreservation, embryo transfer, and development. The importance of cumulus cells for oocyte maturation, fertilization, and embryo development was investigated. Graft recovery, follicle survival, and oocyte retrieval was similar in control, vitrified, and slow-cooled groups. High rates of oocyte maturation, cleavage, and blastocyst formation were achieved, with no significant differences between the control, vitrified or slow-cooled ovarian tissue grafts. The presence of cumulus cells was important for oocyte maturation, fertilization, and subsequent development. Cumulus-oocyte complexes with no surrounding cumulus cells (N-COCs) or with an incomplete layer (P-COCs) had significantly lower rates of oocyte maturation and blastocyst formation than cumulus-oocyte complexes with at least one complete layer of cumulus cells (F-COCs; maturation rate: 63, 78 vs 94%; blastocyst rate: 29, 49 vs 80%). Live births were achieved using vitrified blastocysts derived from oocytes taken from vitrified and slow-cooled ovarian tissue heterotypic allografts. Successful production of healthy offspring from these vitrified blastocysts suggests that this technique should be considered as a useful stage to pause in the assisted reproduction pathway. This provides an alternative protocol for restoring fertility and offering cancer patients a better indication of their chances of pregnancy and live birth.

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births

“…The follicle survival, oocyte maturation and fertilization rates, and subsequent developmental potential were similar to those reported previously using an immature mouse model (Liu et al 2001, O'Brien et al 2003, Hasegawa et al 2006. Moreover, the efficiency of in vitro follicle culture was similar to the tissue transplantation system reported by our group previously (Wang et al 2009), with 244 cultured pre-antral follicles resulting in 16 blastocysts, from which two pups were obtained (2/16 transferred, 12.5%). The transplantation study, using the same in vitro maturation (IVM), IVF, and embryo culture system, yielded 107 blastocysts from 26 vitrified quarterovary grafts (and several times more follicles than the number cultured in the follicle culture study) resulting in the same live birth rate of around 12.5%.…”

“…Histological examination confirmed that adult mouse ovarian tissue could be successfully cryopreserved using the solid surface vitrification method (Wang et al 2009). Though the percentage of morphologically normal follicles at different developmental stages in vitrified-warmed tissue was significantly lower than that in fresh ovarian tissue, a large proportion of pre-antral follicles (w80%) in the vitrified ovarian tissue retained normal morphology, and there was no difference in the follicle survival rate between fresh and vitrified-warmed follicles after 12 days in culture.…”

“…However, data on ovarian tissue vitrification are still limited, and the results are often conflicting and controversial (Choi et al 2008, Isachenko et al 2009, Kagawa et al 2009. We have previously shown that a solid surface vitrification protocol was successful in cryopreserving adult mouse ovarian tissue, and subsequent renal grafting of thawed vitrified tissue showed that this combination was successful for the preservation and restoration of fertility in mouse (Wang et al 2009). This study used a pre-antral follicle culture system to examine the viability of vitrified-warmed adult mouse ovarian tissue without the need for grafting or other surgical intervention to restore ovarian function and fertility.…”

“…Lower concentrations of cryoprotectant are required in freezing media for slow cooling, which reduces the risk of toxic and osmotic damage to cells, but this is insufficient to prevent ice crystal formation jeopardizing cell survival during the freezing procedures (Vajta 2006). Vitrification, though still regarded as a relatively novel freezing method, has recently been reported as an effective alternative method for the cryopreservation of ovarian tissue in various species, including mouse (Wang et al 2009), rat (Deng et al 2009), pig (Gandolfi et al 2006), goat (Santos et al 2007), sheep (Al-aghbari & Menino 2002, Courbiere et al 2006, monkey (Yeoman et al 2005), and human (Keros et al 2009, Zhou et al 2010. It combines a high cooling rate with a high concentration of cryoprotectant in the vitrification media, which rapidly dehydrates the cells and prevents water from precipitating as ice (Vajta 2006).…”

“…Ovarian tissue grafting techniques have been used to restore ovarian function after cryopreservation, and live births have been reported after transplantation of cryopreserved ovarian tissues in animals (Gosden et al 1994, Salle et al 2003, Wang et al 2009) and humans (Donnez et al 2004, Meirow et al 2005, Demeestere et al 2007, Andersen et al 2008, Silber et al 2008, Sánchez-Serrano et al 2010. However, extensive follicular damage and subsequent fibrosis are commonly observed after transplantation (Kim et al 2002), and autotransplantation procedures have the added risk of transferring malignant cells back to the patient (Kim et al 2001).…”