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ABSTRACT Despite the increase in the incidence of cancer, the number of women who survive cancer treatment is growing. However, one of the principal results of chemotherapy is premature ovarian failure (POF). The aim of this study was to use the in situ culture preantral follicles as an in vitro model to evaluate the toxicity of two anticancer drugs, doxorubicin (DXR) and paclitaxel (PTX), on the integrity and development of ovarian follicles. Fragments of the ovarian cortex of goats were cultured in vitro for 1 or 7 days in α-MEM+ supplemented with different concentrations of DXR (0.003, 0.03, or 0.3 µg/mL) and PTX (0.001, 0.01, or 0.1 µg/mL). Analyses were performed before and after culture to evaluate tissue integrity by classical histology, apoptosis by TUNEL assay, DNA laddering kit and the detection of activated caspase 3, and DNA damage by the immune detection of phosphorylated histone H2A.x (H2AXph139). Both DXR and PTX reduced the number of morphologically normal primordial and developing follicles. Positive staining for TUNEL and active caspase 3 was detected in all the samples ( P < 0.05). Therefore, we propose the in situ culture of caprine preantral follicles as a useful experimental model for assessing the toxic effects of the chemotherapeutic agents on ovarian folliculogenesis. Microsc. Res. Tech. 79:773-781, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Tissue transplantation and in vitro ovarian follicle culture have been investigated as alternative techniques to restore fertility in young women who are facing fertility-threatening diseases or treatments following ovarian tissue cryopreservation. Although transplants of fresh or frozen ovarian tissue have successfully yielded healthy live births in different species including humans, the risks of reintroducing cancer cells back into the patient, post treatment, have limited its clinical purpose. The in vitro ovarian follicle culture minimizes these risks and provides a way to harvest more mature oocytes, however its clinical translation has yet to be determined. Not only is it possible for tissue cryopreservation to safeguard fertility in cancer patients, this technique also allows the maintenance of germplasm banks for animals of high commercial value or for those animals that are at risk of extinction. Given the importance of managing female genetic material, this paper reviews the progress of the methods used to preserve and restore female fertility in different species to demonstrate the results obtained in the past 50 years of research, the current achievements and the future directions on this field. [ABSTRACT FROM PUBLISHER]

Abstract: The main objective was to compare the efficiency of vitrification techniques and solutions on the preservation of morphology, ultrastructure and viability of sheep preantral follicles enclosed in ovarian tissue fragments. The fragments were cryopreserved by using macrotube vitrification (MTV), solid-surface vitrification (SSV) or conventional vitrification (CV). These techniques were combined with one of the six solutions containing 6M ethylene glycol (EG) and with or without sucrose (SUC) (0.25 or 0.50M) and with or without fetal calf serum (FCS) (10%). After one week, samples were warmed and histological analysis was performed, showing that the percentage of normal follicles after CV (66.20±8.87%) using a solution containing 6M EG, 0.25M SUC and 10% FCS (vitrification solution 4 – VS4) was similar to fresh control (79.40±7.83%), MTV (53.40±10.60%) and SSV (56.75±15.33%), all of them with the same vitrification solution (P <0.05). For follicular viability evaluation, ovarian fragments were vitrified as described above. After warming, follicles were assessed by trypan blue dye. Controversially, the highest percentage of viable follicles was observed in MTV (97.06%) and was similar to fresh control (92.62%) (P <0.05), but was significantly different from SSV (81.08%) and CV (83.81%) (P <0.05). These results were validated by transmission electron microscopy that showed normal follicles observed in MTV and in fresh control. In addition, to verify the MTV with VS4 (a combination of the best technique plus the best solution), follicle viability was evaluated after 48h in vitro culture. The viability assay was performed by fluorescence microscopy (calcein-AM and ethidium homodimer-1) analysis as follows: follicles isolated from fresh tissue were forthwith analyzed or underwent 48h in vitro culture before analysis, whereas others fragments were vitrified/warmed and immediately analyzed or underwent 48h in vitro culture before analysis. These results showed that, although follicular viability after MTV/VS4 (65%) was reduced when compared to the non-vitrified follicles at day 0 (100%), follicular viability after MTV/VS4 at day 2 (36.5%) was similar to follicles vitrified at day 0 (65%) and similar to non-vitrified follicles at day 2 (62.5%) (P >0.05). As the decrease of viability in non-vitrified follicles at day 2 was similar to the decrease of MTV/VS4 in the same time, follicle viability at day 2 is not affected by MTV/VS4. In conclusion, using the experimental conditions of the present study, an efficient solution (VS4: 6M EG, 0.25M SUC and 10% FCS) and technique (MTV) were successfully used to vitrify ovine ovarian tissue. [Copyright &y& Elsevier]