| With increasing incidences of early onset of cancer and the delay of reproductive age, the demand for fertility preservation in women have increased. Even though most results of oocyte cryopreservation have been obtained from ovarian stimulation protocols for in vitro fertilization (IVF), ovarian stimulation with gonadotropins may not be suitable for many women, especially those who are with hormone-dependent cancers, or those require immediate chemotherapy. However, it is agreed that the outcome of in vitro matured (IVM) oocytes are not as good as in vivo matured ones. In this thesis, we tested the interfering of oocytes during IVM, by the addition of antioxidant and the blocking of mitochondrial function, and observed their survival and the outcome of embryonic development after vitrification and IVF. For the first part, our study showed that the presence of the antioxidant, cysteamine during IVM significantly (P<0.05) reduced the fragmentation rate in cumulus-intact group from 61.9% to 40.8% following vitrification-thawing and IVF. However, the same trend wasn't shown in denuded groups. Nevertheless, there were no significant differences in terms of maturation and cleavage rate with the supplementation of cysteamine in either COC or denuded groups. We hypothesized that cysteamine facilitated effect during IVM might have occurred by affecting mtDNA accumulation during IVM. Fluorescent real-time quantitative analysis of mtDNA showed that the mtDNA copy number were similar in cysteamine treated and untreated group with a ratio close to 1, which implied that the effect of cysteamine during IVM may not be directly related to the mtDNA replication. In the second part, a mitochondria blocker, rotenone, was added to the IVM medium in order to test the vitrification outcome and the subsequent embryonic development. The testing of rotenone concentrations from 100nM to 50muM, showed a lethal dose of around 50muM where all the oocytes were lysed during IVM. With the increase of rotenone concentration from 2muM to 50muM, the oocyte maturation rate dropped significantly in a dose dependent manner and eventually reaching 0 when the concentration was at 50muM. Within the concentration window of 250nM to 2muM, where no difference has been found in terms of the oocyte death and maturation rates compared to the control, there is a significant drop of cryo-survival rate at a concentration of 2muM, from 93.3% (in the control) to 55.6%. The percentage of embryos that developed to 4-8 cell stage were significant lower in the 2muM group (17.5%), compared to the group with a lower concentration of 250nM (50%). This study confirmed the strong positive dependency of mitochondrial functionality during oocyte maturation on its later development as well as the cryopreservation outcomes. Our work suggested that as two individual ART applications, the understanding and improvement of IVM could improve the outcome of vitrification since the impact of IVM on the oocytes could significantly determine the oocytes ability to survival and to develop following vitrification and IVF. |
