Novel in vitro methods aiming at improving fertility preservation

Abstract: The well-established fertility preservative methods for female cancer patients include the cryopreservation of embryos, mature oocytes and ovarian tissue before the initiation of gonadotoxic cancer treatment. Due to a diversity of clinical and practical situations, there are limitations of these methods, creating the necessity to develop further solutions, and there is also a lack of knowledge on the effect of recent gonadotoxic cancer treatment in the ovarian potential. Importantly, the mechanisms of chemotherapy-induced ovarian damage have not been fully explained. In Study I and II of this thesis, the ovarian effect of cyclophosphamide (CPA) at three different doses was investigated in juvenile and prepubertal mice. The feasibility of isolating appropriate secondary follicles from CPA-exposed ovaries, in vitro follicle culture and oocyte maturation (Study II) were evaluated. Both studies supported the feasibility of these methods. The final outcomes regarding follicle yield and follicle survival rates were shown to be dose-dependent and age-dependent. Additionally, Study II showed that mature oocytes with normal spindle configuration and chromosomes structures could be obtained through in vitro maturation. In Study III, the feasibility of retrieving secondary follicles for in vitro culture from superovulated adult mouse ovaries was evaluated. The study showed that albeit a reduced secondary follicle yield in comparison to control unstimulated ovaries (46% less in superovulated ovaries), several parameters including follicle survival rates, oocyte maturation rates and ratios of follicles with normal-looking spindle configuration and chromosomes structures were similar between the two groups. Mouse models have previously proved that follicle culture could achieve fully competent oocytes for fertilization and further development, resulting in the birth of pups. In my studies, the method of follicle culture was central for the investigation of viability, survival and follicle competence following a given gonadotoxic treatment, aiming at mimicking specific clinical situations, such as when patients need to start cancer treatment without delay and would request to undergo fertility preservation few days after the initiation of chemotherapy. Additionally, I also investigated if the superovulated ovary could be a source of follicles to be cultured in vitro up to mature oocytes using the mouse model, thus mimicking the clinical case when gonadotropin stimulation aiming at fertility preservation has been unsuccessful in oocyte recovery. For such patients, there are no further options to offer, as the ovarian tissue is considered unsuitable for cryopreservation after ovarian stimulation. In my studies, isolation of secondary follicles, in vitro follicle growth and oocyte maturation were found efficient. The potential benefit of these methods was supported by the observation of normal-looking spindle configuration and chromosomes structures in the mature oocytes obtained after culture. In Study IV, the mechanisms involved in CPA-induced primordial follicle depletion were investigated, this question was also approached through Study I and Study II. In Study IV new-born mouse ovaries were exposed to 4-hydroperoxycyclophosphamide (4-HC) in culture. Histological characteristics and gene expression analysis revealed the involvement of apoptosis during primordial follicle depletion. Significantly reduced primordial follicle density was observed 24 h after 4-HC exposure comparing to a control group and the strongest apoptotic signals on histological assessment were also found at 24 h, and there was transcriptional up-regulation of apoptosis related genes (Noxa, Bax, Casp 6 and 8) while Tap63 was down-regulated. There was no evidence of significantly increased density of growing follicles following 4-HC exposure, nor significant changes in follicle development related gene expression (Mki67, Pcna) or primordial follicle dormancy maintenance related gene (Pten) in the experiments. Thus follicle over-activation was not supported by Study IV, where a single dose of 4-HC was used, thus might have caused a direct and acute primordial follicle depletion.