Study question How to improve in-vitro Folliculogenesis (ivF) protocols to address the enlarged demand of fertility preservation? Summary answer Tissue engineering-based approach opens new frontiers for ivF improving 3D-technologies addressed to support immature-ovarian-follicle-growth to obtain an increased number of competent oocytes enrolled in Assisted-Reproductive-Technology. What is known already ivF is a promising Assisted-Reproductive-Technology (ART) for preserving and restoring fertility. This technology potentially reproduces the early stages of folliculogenesis and oogenesis in-vitro allowing to move a large amount of oocyte on individual basis towards the validated protocol of in-vitro maturation/in-vitro fertilization (IVM/IVF). The current availability of biocompatible-supporting materials offers the challenging opportunity to mimic the native organ stroma in order to better reproduce the 3D environmental conditions leading to synergic follicles-oocyte development in-vitro with the aim to improve the performance of ivF in translational large sized mammal models. Study design, size, duration The present research aimed to compare preantral (PA) follicles culture on two different typologies of scaffolds fabricated using PCL(poly(epsilon caprolactone)), respectively made with patterned and randomly aligned fibers (PCL-Patterned/PCL-Randomic) with a standardized-single-follicle scaffold-free-method (3D-oil), widely validated on ovine model (Cecconi et al., 2004). The culture outcomes are compared analyzing follicle/oocyte growth, percentage of antrum differentiation and the incidence of meiotic competence, by exposing ivF growing oocytes to IVM protocol. Participants/materials, setting, methods PA follicles (mean size diameter: 250±4μm), mechanically isolated from slaughterhoused lamb ovaries, were individually cultured on electrospun PCL scaffolds (patterned vs randomic) or using the 3D-oil method. ivF were cultured alphaMEM-Fetal Bovine Serum free medium (5% Knockout Serum Replacement) supplemented with 4 IU/mL of equine Chorionic Gonadotropin (Di Berardino et al., 2021). At the end of ivF (14-days) the fully-grown oocytes isolated from early-antral follicles were tested on IVM. Main results and the role of chance PCL-Patterned electrospun scaffolds were able to strongly support a synergic oocyte and follicular growth. The 3D culture on Patterned electrospun scaffold supported the highest viability of follicles (87.5% vs 63% under 3D-oil conditions). On the contrary, the highest incidence of degenerated follicles was observed in cultures performed using PCL-Randomic materials (55 vs 37% vs 12.5% for PCL-Randomic vs 3D-oil vs PCL-Patterned, respectively; p <0.0004). The greatest follicle diameter increment (74.7±1 vs 70±0.4 vs 60.9±2%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, respectively p <0.0007) and rate of antrum differentiation (87.5% vs 45% and vs 63%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, for both p <0.0001) were observed in PA ovine follicles cultured on PCL-Patterned scaffolds. Furthermore, PCL-Patterned electrospun scaffolds supported a complete functional development of the oocyte compartment. More in detail, the majority of fully grown oocytes isolated from early- antral follicles grown on PCL-Patterned materials reached the metaphase-II stage (MII 80%) at the end of IVM in comparison to the significant lower percentage in 3D-oil (MII 68%, p =0.04) and PCL-Randomic (MII 18%, p <0.0001) protocols, respectively. Limitations, reasons for caution - Wider implications of the findings Tissue engineering scaffold-based approach represents a valid strategy generating a multi-organ in-vitro system, where different compartments may cooperate generating the complexity of paracrine-mechanism controlling early-follicles outcomes. Scaffold topology is essential to control early-follicles development. Indeed, exclusively PCL-Patterned can preserve long-term follicle 3D-microarchitecture supporting in-vitro oogenesis up to a complete meiotic-competence-acquisition. Trial registration number not applicable

中文翻译：

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P-440 静电纺丝支架拓扑结构对应用于前腔羊卵泡的体外卵泡发生性能的影响

研究问题如何改进体外卵泡生成 (ivF) 方案以解决生育力保存的扩大需求？摘要答案 基于组织工程的方法为 ivF 改进 3D 技术开辟了新的领域，这些技术旨在支持未成熟卵巢卵泡的生长，以获得更多参与辅助生殖技术的有能力的卵母细胞。众所周知，体外受精是一种很有前途的辅助生殖技术（ART），用于保存和恢复生育能力。该技术有可能在体外复制卵泡发生和卵子发生的早期阶段，允许在个体基础上将大量卵母细胞移向经过验证的体外成熟/体外受精 (IVM/IVF) 协议。目前生物相容性支持材料的可用性为模拟天然器官基质提供了具有挑战性的机会，以便更好地再现导致协同卵泡-卵母细胞体外发育的 3D 环境条件，旨在提高 ivF 在大尺寸转化中的性能哺乳动物模型。研究设计、规模、持续时间 本研究旨在比较使用 PCL（聚（ε-己内酯））制造的两种不同类型支架上的腔前 (PA) 卵泡培养，分别由图案化和随机排列的纤维 (PCL-Patterned/PCL-随机）采用标准化的单卵泡无支架方法（3D 油），在绵羊模型上得到广泛验证（Cecconi 等人，2004 年）。比较培养结果，分析卵泡/卵母细胞的生长，通过将 ivF 生长的卵母细胞暴露于 IVM 协议，胃窦分化的百分比和减数分裂能力的发生率。参与者/材料、设置、方法 PA 卵泡（平均尺寸直径：250±4μm），从屠宰的羔羊卵巢中机械分离，在静电纺丝 PCL 支架上单独培养（图案化 vs 随机）或使用 3D 油法。ivF 培养 alphaMEM-胎牛血清无培养基（5% 敲除血清替代物），辅以 4 IU/mL 马绒毛膜促性腺激素（Di Berardino 等人，2021）。在体外受精（14 天）结束时，从早期窦卵泡中分离出的完全生长的卵母细胞在体外受精上进行测试。主要结果和机会 PCL 图案静电纺丝支架的作用能够强烈支持协同卵母细胞和卵泡生长。图案化电纺支架上的 3D 培养支持最高的毛囊存活率（在 3D 油条件下为 87.5% 对 63%）。相反，在使用 PCL-Randomic 材料进行的培养中观察到退化卵泡的最高发生率（PCL-Randomic vs 3D-oil vs PCL-Patterned 分别为 55% vs 37% vs 12.5%；p <0.0004）。最大卵泡直径增量（74.7±1 vs 70±0.4 vs 60.9±2%，PCL-Patterned vs 3D-oil vs PCL-Randomic，分别为 p <0.0007）和窦腔分化率（87.5% vs 45% 和在 PCL 图案支架上培养的 PA 绵羊卵泡中观察到 PCL 图案与 3D 油与 PCL 随机对照的 63%（对于 p < 0.0001）。此外，PCL 图案的静电纺丝支架支持卵母细胞室的完整功能发展。更详细，从在 PCL 图案材料上生长的早期窦卵泡中分离出的大多数完全生长的卵母细胞在 IVM 结束时达到中期 II 阶段（MII 80%），而 3D 油中的百分比显着降低（MII 68%， p = 0.04) 和 PCL-随机 (MII 18%, p < 0.0001) 协议。限制，谨慎的原因 - 研究结果的更广泛意义基于组织工程支架的方法代表了一种有效的策略，可以产生多器官体外系统，其中不同的隔室可能会合作产生控制早期卵泡结果的旁分泌机制的复杂性。支架拓扑结构对于控制早期卵泡发育至关重要。的确，独家 PCL-Patterned 可以保留支持体外卵子发生的长期卵泡 3D 微结构，直至获得完整的减数分裂能力。试用注册号不适用