背景:辅助生殖技术(ARTs)已在人类和动物中得到验证,以解决诸如不育之类的生殖问题,老化,遗传选择/扩增和疾病。ART生物医学应用的持续差距在于概述卵巢卵泡发生的早期阶段,因此提供了将大量未成熟卵泡推向促性腺激素依赖性阶段的方案。组织工程正在成为可能概括卵巢结构的具体解决方案,大多依靠使用自体早期卵泡上的天然或合成支架。基于这些前提,本研究旨在验证使用聚(ε-己内酯)(PCL)制造的卵巢生物启发图案化电纺纤维支架用于多个窦前(PA)卵泡发育。
方法:从羔羊卵巢中分离的PA卵泡在PCL支架上培养,采用经过验证的单卵泡方案(Ctrl)或通过复制移植有5或10PA(AO5PA和AO10PA)的人工卵巢来模拟多卵泡条件。在评估基于支架的微环境适用性之前,将孵育延长14和18天,以在形态和功能水平上协助体外卵泡发生(ivF)和卵子发生。
结果:ivF结果表明,PCL支架产生适当的仿生卵巢微环境,通过支持卵泡生长和类固醇生成激活,支持多个PA卵泡向早期胃窦(EA)阶段过渡。PCL-多重生物工程ivF(AO10PA)长期产生,此外,通过增强减数分裂能力,大染色质重塑与孤雌生殖发育能力。
结论:该研究展示了下一代ART使用PCL型支架的概念证明,该支架旨在生成移植有自体早期卵泡的可移植人工卵巢或推进IVF技术,该技术具有3D生物启发基质,可促进生理长期多PA卵泡方案。
BACKGROUND: Assisted Reproductive Technologies (ARTs) have been validated in human and animal to solve reproductive problems such as infertility, aging, genetic selection/amplification and diseases. The persistent gap in ART biomedical applications lies in recapitulating the early stage of ovarian folliculogenesis, thus providing protocols to drive the large reserve of immature follicles towards the gonadotropin-dependent phase. Tissue engineering is becoming a concrete solution to potentially recapitulate ovarian structure, mostly relying on the use of autologous early follicles on natural or synthetic scaffolds. Based on these premises, the present study has been designed to validate the use of the ovarian bioinspired patterned electrospun fibrous scaffolds fabricated with poly(ε-caprolactone) (PCL) for multiple preantral (PA) follicle development.
METHODS: PA follicles isolated from lamb ovaries were cultured on PCL scaffold adopting a validated single-follicle protocol (Ctrl) or simulating a multiple-follicle condition by reproducing an artificial ovary engrafted with 5 or 10 PA (AO5PA and AO10PA). The incubations were protracted for 14 and 18 days before assessing scaffold-based microenvironment suitability to assist in vitro folliculogenesis (ivF) and oogenesis at morphological and functional level.
RESULTS: The ivF outcomes demonstrated that PCL-scaffolds generate an appropriate biomimetic ovarian microenvironment supporting the transition of multiple PA follicles towards early antral (EA) stage by supporting follicle growth and steroidogenic activation. PCL-multiple
bioengineering ivF (AO10PA) performed in long term generated, in addition, the greatest percentage of highly specialized gametes by enhancing meiotic competence, large chromatin remodeling and parthenogenetic developmental competence.
CONCLUSIONS: The study showcased the proof of concept for a next-generation ART use of PCL-patterned scaffold aimed to generate transplantable artificial ovary engrafted with autologous early-stage follicles or to advance ivF technologies holding a 3D bioinspired matrix promoting a physiological long-term multiple PA follicle protocol.