Is oxygen availability a limiting factor for in vitro folliculogenesis?

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles’ health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes.为保留肿瘤患者生育能力而开展的卵巢组织移植，现已成为被广泛认可的临床实践手段。然而，移植过程中可能回输肿瘤细胞的风险，使得学界对完全体外卵泡发生（in vitro folliculogenesis）的研究热情日益高涨。目前人类尚未实现完全体外卵泡发生，究其原因可能是对体内调控卵泡发生的微环境认知不足。本研究的核心目标为探究卵巢组织条带体外培养（in vitro culture）过程中，氧供水平对卵泡健康状态与生长发育的影响。为此，本研究构建了可预测不同培养条件下组织内溶解氧浓度的模型。本研究以牛卵巢皮质条带作为动物模型，并辅以人类卵巢组织样本，分别在常规培养皿（conventional dish, CD）与透气培养皿（gas permeable dish, PD）中进行培养，设置不同的培养基柱高与气相氧分压条件，培养时长分别为3、6、9天。通过组织学分析，对卵泡质量、原始卵泡向初级阶段的激活情况，以及向次级阶段的发育进展进行评估。利用共聚焦扫描激光显微镜（confocal scanning laser microscope）开展死活染色检测，以评估卵泡活力。实验结果显示，在合适的培养基柱高与氧分压条件下，于PD培养皿中培养的牛卵巢条带，其卵泡质量与活力均更优异。本研究将牛样本中筛选出的最优培养条件应用于人类卵巢条带培养，同样实现了卵泡质量、活力与发育进展的提升。综上，本研究数据表明，在卵巢组织体外培养过程中，调控组织内氧供水平，对维持卵泡健康、保障其存活并向次级阶段发育起到关键作用。该培养条件可提升健康次级卵泡的获取量，便于后续进行卵泡分离与单个体培养，以获得具备发育潜能的卵母细胞。