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

项目摘要/摘要 卵巢组织冷冻保存(OTC)，癌症患者和其他正在增加的患者的生育能力保存选择 在性腺激素治疗之前，理想的不孕症风险使&gt；140得以存活 出生。然而，目前生育能力的恢复仅限于OTC组织的移植，一些患者可能 由于有再次感染他们的疾病的风险，无法使用此选项。一种尚未开发的替代方案 对于临床，将使用原始卵泡，卵母细胞和支持细胞单位被冷冻保存在 OTC，并进行体外生长和成熟(IVGM)以产生卵子。目前的辅助生殖 技术需要20-30个卵子才能提供很好的生育机会。然而，当前的IVGM协议 在研究实验室中进行的卵子移植效率不高，每10个或更多的患者只能产生几个卵子。 重要的是，目前从卵巢组织中分离原始卵泡的方法有很大的不同。 在患者之间，充其量只有一小部分是完好无损的。次级毛囊更容易 保持完好，但在非处方药期间很少被超低温保存。因此，原始卵泡必须被激活才能生长到 如果OTC组织用于产卵，则为次级卵泡。推进IVGM的限速步骤有：(1) 高效和可靠地分离被激活以生长的健康原始卵泡，以及(2)高效和 可靠地生长和成熟的次级卵泡形成高质量的卵子。3D打印的特定生物支架 建筑结构也可以通过卵子的体外成熟来支持次级卵泡的存活和生长。 作为原始卵泡在小鼠体内通过卵子成熟、排卵和活产而生长和成熟。这些 可喜的结果为进一步探索使用专门设计的生物支架来解决 目前IVGM的局限性。这个应用程序测试了一个动态的合成微环境将 提供必要的机械和建筑线索以诱导间质细胞迁移出卵巢组织， 释放嵌入的卵泡，支持卵泡生长和卵母细胞成熟为卵子。3D打印 使用可调高孔生物材料的不同机械和重塑性能的生物支架将被 用于诱导卵泡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、卵泡生长等方面与人类卵巢相似 和成熟以达到以下目的：(1)定义支持基质细胞迁移的生物支架和 随后原始卵泡的释放、激活和生长；以及(2)确定诱导 体外分离的次级卵泡的生长和成熟。这些研究将确定特定的属性 合成微环境可以加强卵泡与卵巢组织的隔离，以及那些支持 卵泡发育成熟为卵子的过程。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，将揭示一种 这是一种全新的IVGM方法，并支持未来人类IVGM技术的开发。

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.