| BackgroundIn recent years, advances in treatment, especially the progress of chemotherapy and radiotherapy have improved the survival rate of childhood and young cancer patients. However, these treatments were toxic to the gonads and could result in premature ovarian failure and infertility. Cryopreservation of ovarian tissue before cancer treatment made fertility preservation and maintenance of menstrual cycles in such women possible.Slow-freezing has widely used in the cryopreservation of embryo and oocyte. Slow-freezing of ovarian tissue has achieved pregnancies in mice, monkeys, goat and human. However, there are some problems in this protocol. The diversity of cell types in ovarian tissue demanded different parameters to avoid ice crystallization damage, and the programmed freezer is expensive and takes several hours. Recent studies showed that vitrification seems more effective. Vitrification provides a rapid cooling rate to induce glass-like solidification inside cells to protect them from injury by ice crystals. Comparing to slow-freezing, vitrification protocols are simpler, faster and cheaper. Further more, no special equipment is needed. The carried system is one of the critical parameters for efficient vitrification, which determines the cooling rate. Only when the critical cooling rate is reached, the vitrification solution can be transformd directly to a glassy state. Several special carried systems had been developed, such as cryovials, open pulled straw, cryoloop and so on. However, fewer studies of defining the optimal vitrification protocol and comparing it to slow-freezing method were reported.ObjectiveTo compare the protective effect on rabbit ovarian tissue of different cryopreservation methodsMethodsOvarian biopsies from15New Zealand doe rabbits were cut into slices of approximately2-3mm×1mm×1mm, and randomly distributed into fresh(Group F), DMSO slow-cooling (Group DMSO), PROH slow-freezing(Group PROH), tube vitrification(Group TV), solid-surface vitrification(Group SSV) groups. Morphologically normal follicles rates were compared between fresh and the post warming/thawing tissue. The levels of estradiol in culture medium were measured to compare the endocrinal functions of the post-cryopreservation tissue. Detect of apoptotic follicles in ovarian tissue by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assayResultCompared with Group F, the percentages of morphologically normal primordial follicles were reduced (P<0.05) in Group DMSO and TV. And there were no significant differences between Group F, PROH and SSV (P>0.05). The percentages of morphologically normal primary follicles did not show difference between5groups (P>0.05). After14days in-vitro culture, all cryopreservation groups show a significant reduction of the proportions of primordial follicles in cultured tissues (P<0.001). The proportions of primary follicles between5groups were equal (P=0.165). The levels of estradiol in culture medium increased during the14days in-vitro culture (P<0.001). The difference of estradiol levels between slow-cooling and vitrification was significant (P=0.021). We did not find a visible difference of the percentages of apoptotic follicles in all groups (P>0.05).ConclusionEither slow-cooling or solid-surface vitrification for rabbit ovarian tissue cryopreservation is effective. And the endocrinal functions of the post-cryopreservation tissue could be preserved satisfactorily.In slowing-freezing, the protecting effects are equal by using either DMSO or PROH as a cryoprotectant.Comparing to tube vitrification, solid-surface vitrification is a simple and effective method for rabbit ovarian tissue. |
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