Purpose Although oncological advances have improved survival rates of female cancer patients, they often suffer a reduced fertility due to treatment side effects. In the present study, we evaluated the potential fertoprotective effects of the specific inhibitor of SIRT1, EX-527, on the gonadotoxic action exerted by cyclophosphamide (CPM) on loss of primordial follicles (PFs). Methods The effects of the CPM metabolite phosphoramide mustard (PM) on follicle activation, growth and viability and the protective action of EX-527 against PM effects were evaluated on bovine ovarian cortical strips in vitro cultured for 1 or 6 days. To understand whether PFs exposed to PM plus EX-527 were able to activate and grow to the secondary stage after suspension of the treatment, strips cultured for 3 days in PM plus EX-527 for 3 days were transferred to plain medium until day 6. Follicle growth and health were evaluated through histology and viability assay at a confocal microscope. In order to investigate the molecular pathways underlying the ovarian response to PM in the presence of EX-527, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after 1 day of in vitro culture. Results We found that (1) PM, the main CPM active metabolite, promotes PF activation; (2) the ovarian stress response induced by PM includes a SIRT1-dependent pathway; and (3) EX-527 reduces PF activation and growth induced by PM. Conclusion SIRT1 can represent a candidate molecule to be targeted to protect ovarian follicles from alkylating agents and EX-527 could represent a potential fertoprotective agent for cancer patients.

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Study question Does enhancing oxygen availability during dynamic in vitro culture of bovine ovarian cortical tissue (BOCT) improve follicle growth and health? Summary answer Enhancing oxygen availability during dynamic in vitro culture of BOCT in perifusion bioreactors (PB) does improve follicle health and yield to secondary follicles What is known already Oxygen availability has been demonstrated to represent a key factor in follicle health and growth during in vitro culture of bovine and human ovarian cortical tissue (HOCT) under static culture conditions. Disruption of solutes gradients and application of physiological fluid mechanical stress, through in vitro dynamic culture of HOCT in a newly designed perifusion bioreactor have been shown to further enhance follicle growth and health. As it shows striking similarities with human, bovine folliculogenesis is considered a valuable model to study follicle growth in vitro Study design, size, duration Bovine ovaries from animals aged 8-24 months were collected at slaughterhouse. In each experiment (n = 3), BOCT strips from the same ovary were cultured for 6 days in perifusion bioreactors (PB, dynamic culture) and conventional dishes (CD, static culture). Culture outcome in static culture was analysed and compared to two bioreactor configurations in which medium oxygenation was kept low by using a standard tube reservoir (StPB) or was enhanced by using a gas-permeable dish reservoir (PB+O2). Participants/materials, setting, methods Slices of BOCT 0.5mm thick were cut with a tissue slicer and chopped into 1x1mm strips. In each experiment, fresh (D0) and cultured tissue (groups of ten strips) were analyzed. Follicle stages and health were assessed by histology (hematoxylin-eosin staining). Follicle viability was estimated by labelling with live-dead far-red and propidium iodide followed by clearing before analysis at the confocal laser scanning microscope. Main results and the role of chance Overall, 2417 follicles were analyzed (histology, 1476; viability, 941). At day 0 most follicles were primordial (primordial, 88.7%; primary, 10.6%; secondary, 0.7%), and had good quality (grade 1-2, 92.2%; grade 3, 7.8%), and high viability (91.8%). At day 6, follicle growth and health in StPB was superior than in CD (StPB vs CD - staging: primordial, 6.8 vs 16.3, P < 0.01; primary, 70.7 vs 74.1, NS; secondary, 22.5 vs 9.6%, P < 0.01; grading: grade 1 + 2, 71.4 vs 44.8, P < 0.01; grade 3, 28.6 vs 55.2%, P < 0.01). Dynamic culture in StPB better-preserved follicle viability compared to static culture in CD (StPB vs CD: 77.75 vs 64.9%, P < 0.01). Enhancing oxygen availability during dynamic culture increased follicle progression and viability (PB+O2 vs StPB - staging: primordial, 5.1 vs 6.8, NS; primary, 65.4 vs 70.7, NS; secondary, 29.5 vs 22.5%, P < 0.05; viability - 92 vs 77.75, P < 0.01). Overall, the obtained results demonstrate that i) disruption of stagnant layers of medium and application of shear stress to BOCT through dynamic culture improves follicle activation, growth and health; ii) enhancing oxygen availability by means of a gas-permeable medium reservoir further increases follicle progression and viability. Limitations, reasons for caution Although the bovine is considered a reliable model for human folliculogenesis, the study should be validated on human ovarian tissue. Wider implications of the findings A limiting step in the in vitro production of mature oocytes starting from primordial follicles is the low yield of secondary follicles after organ culture. The adoption of a newly designed dynamic bioreactor and modulation of oxygen availability could represent a valuable tool for multistep in vitro folliculogenesis. Trial registration number none

Study question Do tissue thickness and FSH supplementation affect follicle growth and health in the vitro culture of bovine ovarian cortical strips (BOCS) in gas-permeable dishes? Summary answer Culture of 0.5mm thin BOCS with 5ng/ml FSH does improve follicle health and yield to secondary follicles compared with 1mm thick BOCS What is known already Oxygen availability inside tissue has been demonstrated to represent a key factor in follicle health and growth during in vitro culture of bovine and human ovarian cortical strips (HOCS). Although, strip thickness can limit nutrients and gases diffusion in and out of the innermost tissue zone, the presence of the outer medulla in thick strips could positively affect follicle growth. The role of FSH on the progression of primordial to secondary follicles in ovarian organ culture is still debated. Study design, size, duration Bovine ovaries from animals aged 8-24 months were collected at a slaughterhouse. In each experiment (n = 3), BOCS of varying thickness collected from the same ovary were cultured with or without FSH in gas-permeable dishes for 10 and 15 days. Participants/materials, setting, methods Slices either 0.5mm or 1mm thick were cut with a tissue-slicer and were chopped into 1x1mm2 strips. BOCS were cultured for 10 or 15 days at the same tissue/medium volume ratio (groups of either five 1mm strips or ten 0.5mm strips in 5ml medium) with 0, 1, or 5ng/ml FSH. Follicle stages were assessed by histology. Follicle viability was estimated by labeling with live-dead far red and propidium iodide at the confocal laser scanning microscope. Main results and the role of chance Overall, 2314 follicles were analyzed (histology, 998; viability, 1316). At day 0 most follicles were primordial (primordial, 89.4%; primary, 8.7%; secondary, 1.9%), and had a high viability (94.69%). The best follicle growth and viability was observed in 0.5mm thin BOCS cultured with 5ng/ml FSH. In particular, when compared to 1mm thick BOCS cultured with 5ng/ml FSH, 0.5mm thin BOCS cultured with 5ng/ml FSH showed a higher and significant proportion of secondary follicles at day 10 (0.5 vs 1mm, % secondary follicles: 26.5 vs 10, P < 0.05) and a significantly higher proportion of viable follicles at day 15 (0.5 vs 1mm, % viable follicles: 89.4 vs 60.7, P < 0.01). These results demonstrate that smaller BOCS thickness and 5ng/ml FSH supplementation significantly improve the growth and health of secondary follicles. Limitations, reasons for caution Although the bovine is considered a reliable model for human folliculogenesis, the study should be validated on human ovarian tissue. Wider implications of the findings A limiting step in the production of mature oocytes starting from primordial follicles is the low yield to secondary follicles at the end of organ culture. Given the similarities between bovine and human folliculogenesis, the best culture conditions herein identified could contribute to the refinement of human in vitro folliculogenesis. Trial registration number Not applicable

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