Releasing, activating, and maturing follicles from cortical tissue utilizing dynamic synthetic microenvironment

项目总结/摘要 卵巢组织冷冻保存（OTC）是癌症患者和其他增加生育能力的患者的生育能力保存选择。 理想情况下，在性腺毒性治疗之前进行不孕症的风险，已使>140人的生活 出生然而，生育力恢复目前仅限于OTC组织移植，一些患者可能 不能使用这个选项，因为有重新引入疾病的风险。尚未开发的替代品 对于临床来说，将使用冷冻保存的原始卵泡，卵母细胞和支持细胞单位， OTC，并进行体外生长和成熟（IVGM）以产生卵子。当前辅助生殖 技术需要20 - 30个卵子才能提供生育孩子的良好机会。然而，目前的IVGM方案 在研究实验室进行的卵子移植效率不高，每10个或更多的病人只能得到几个卵子。 重要的是，目前从卵巢组织中分离原始卵泡的方法的成功与否 在患者之间，最好的情况下，只有一小部分是完整的。次级卵泡更容易 保持完整，但在OTC期间很少冷冻保存。因此，原始卵泡必须被激活才能生长， 次级卵泡，如果OTC组织用于产卵。推进IVGM的限速步骤为：（1） 有效且可靠地分离被激活生长的健康原始卵泡，和（2）有效且 次级卵泡可靠地生长和成熟为优质卵子。3D打印的生物支架 结构可以支持次级卵泡的活力和生长，通过卵子体外成熟，以及 如原始卵泡的生长和成熟，通过卵子成熟、排卵和小鼠的活产。这些 有希望的结果奠定了基础，以进一步探索使用专门设计的生物支架，以解决 IVGM的局限性。这个应用程序测试了一个假设，即一个动态的合成微环境将 提供必要的机械和结构线索以诱导基质细胞迁移出卵巢组织， 释放嵌入的卵泡，并支持卵泡生长和卵母细胞成熟为卵子。3D打印 使用可调的高度多孔的生物材料， 用于诱导毛囊从皮质组织迁移，并提供动态环境， 随着卵泡的生长。牛卵巢在大小、皮质密度、卵泡生长等方面模拟人类卵巢 （1）确定支持基质细胞迁移的生物支架， 随后的原始卵泡的释放、激活和生长;以及（2）确定诱导原始卵泡生长的生物支架。 体外分离的次级卵泡的生长和成熟。这些研究将确定的具体性质， 合成微环境，可以增强卵泡分离卵巢组织，以及那些支持 卵泡发育成熟为卵子。如果成功，这项工作将使研究人员能够研究 在受控环境中，从原始卵泡激活到卵子的卵泡发生，将揭示 这是IVGM的一种全新方法，并支持人类IVGM未来技术的发展。

PROJECT SUMMARY/ABSTRACT Ovarian tissue cryopreservation (OTC), a fertility preservation option for cancer patients and others at increased risk of developing infertility that is ideally performed prior to the gonadotoxic treatment, has enabled >140 live births. However, fertility restoration is currently limited to transplantation of OTC tissue, and some patients may not be able to use this option due to the risk of reintroducing their disease. An alternative that is not yet developed for the clinic, would be to use the primordial follicles, the oocyte and support cell units that are cryopreserved in OTC, and perform in vitro growth and maturation (IVGM) to produce eggs. Current assisted reproductive technologies require 20 – 30 eggs to offer a good chance of producing a child. However, current IVGM protocols performed in the research lab are not efficient and yield only a few eggs for every 10 or more patients. Importantly, the success of current methods for isolating primordial follicles from ovarian tissue drastically differs between patients and, at best, only a small percentage are obtained intact. Secondary follicles more easily remain intact, but few are cryopreserved during OTC. Therefore, primordial follicles must be activated to grow to secondary follicles if OTC tissue is used to make eggs. The rate limiting steps for advancing IVGM are: (1) efficiently and reliably isolating healthy primordial follicles that are activated to grow, and (2) efficiently and reliably growing and maturing secondary follicles into good quality eggs. 3D-printed bioscaffolds of specific architectures can support the viability and growth of secondary follicles through egg maturation in vitro, as well as primordial follicle growth and maturation through egg maturation, ovulation, and live birth in mice. These promising results lay the foundation to further explore the use of specifically designed bioscaffolds to address current limitations of IVGM. This application tests the hypothesis that a dynamic synthetic microenvironment will provide the necessary mechanical and architectural cues to induce stromal cell migration out of ovarian tissue, release of the embedded follicles, and support follicle growth and oocyte maturation into an egg. 3D-printed bioscaffolds of varying mechanical and remodeling properties using tunable highly porous biomaterials will be utilized to induce follicle migration from cortical tissue and provide a dynamic environment that remodels over time as the follicles grow. Bovine ovaries as used to mimic human ovaries in size, cortical density, follicle growth and maturation for the following aims: (1) to define the bioscaffolds that support stromal cell migration and subsequent release, activation, and growth of primordial follicles; and (2) to define the bioscaffolds that induce growth and maturation of isolated secondary follicles in vitro. These studies will identify specific properties of synthetic microenvironments that can enhance follicle isolation from ovarian tissue, as well as those that support the growth and maturation of follicles into eggs. If successful, this work would enable researchers to study folliculogenesis from primordial follicle activation to an egg in a controlled environment, would uncover a completely novel approach to IVGM and support the development of future technologies for IVGM in humans.