Supplement Andrea Jones

项目总结 这项拟议研究的长期目标是为妇女建立一个广泛的生育保护选择 接受促性腺激素抗癌治疗并面临不孕不育。这项提案的总体目标是 朝着提出的目标努力并降低与自体移植相关的风险是创建一个 从体外原始阶段促进人类卵泡发育的仿生环境。低谷 小卵泡培养的成功率在很大程度上归因于复杂而鲜为人知的旁分泌， 滤泡细胞、邻近滤泡和基质细胞之间的自分泌和内分泌信号。中环 假说是，人类卵泡的转录图谱将揭示推动发育和 通过设计一种保留细胞分泌的仿生水凝胶来重建卵巢微环境 细胞外基质(ECM)将在体外支持人卵泡的发育。拟议工作的理由 是通过破译驱动卵泡激活和早期发育的机制，并概括 自然卵巢微环境在细胞外基质隔离水凝胶中，未来的培养系统可以转化为 临床用于冷冻卵巢卵泡成熟及随后的受精和妊娠。在第一个 目的：利用单细胞RNA测序技术对人卵巢卵泡和支持基质细胞进行测序。在……里面 第二个目标是将细胞外基质隔离多肽引入仿生聚乙二醇(PEG)中。 迈克尔型加成化学促进ECM组分沉积的水凝胶体系及其模拟 天然的卵巢组织。卵泡的基底膜由ECM蛋白组成，它的功能是 对毛囊细胞的结构支持，对进入毛囊的分子的选择性屏障，以及 保留可溶的生长因子和细胞因子。它在卵泡发育过程中不断重塑，但细胞- 分泌的ECM分子不能附着在未经修饰的聚乙二醇上进行自组装。通过集成ECM- 在聚乙二醇水凝胶中隔离多肽，细胞外基质的结构和生物学作用可以在 体外卵泡发育。这项工作的贡献将是生殖年龄卵巢的单细胞图谱。 强调卵泡发育的机制和基质细胞在卵泡发生和发育中的支持作用 一种支持人类卵泡发育的新型体外卵泡培养系统。这项工作的贡献将是 具有重要意义，因为它将指导人类成熟标准化体外培养的发展 卵泡和安全的生殖力保存选项，用于因以下原因无法产生成熟卵子的患者 促性腺激素治疗。拟议的工作具有创新性，因为它将是来自Healthy的第一个单细胞数据集 育龄妇女和人类卵泡在人工ECM隔离基质中培养的第一例。

PROJECT SUMMARY The long-term goal of the proposed research is to establish a broad fertility preservation option for women undergoing gonadotoxic anticancer treatments and facing infertility. The overall objective of this proposal in working towards the presented goal and mitigating the risks associated with autotransplantation is to create a biomimetic environment that promotes human follicle development from the primordial stage in vitro. The low success rates of small follicle culture are largely attributed to the complex and poorly understood paracrine, autocrine and endocrine signaling between follicular cells, neighboring follicles, and stromal cells. The central hypothesis is that transcriptional profiling of human follicles will reveal mechanisms driving development and recreating the ovarian microenvironment through design of a biomimetic hydrogel which retains cell-secreted extracellular matrix (ECM) will support human follicle development in vitro. The rationale for the proposed work is that by deciphering the mechanisms driving follicle activation and early development, and recapitulating the natural ovarian microenvironment in an ECM-sequestering hydrogel, future culture systems can be translated to the clinic for maturation of cryopreserved ovarian follicles and subsequent fertilization and pregnancy. In the first aim, single cell RNA sequencing will be used to profile human ovarian follicles and supportive stromal cells. In the second aim, ECM-sequestering peptides will be incorporated in a biomimetic poly(ethylene glycol) (PEG) hydrogel system using Michael-type addition chemistry to promote deposition of ECM components and mimic the native ovarian tissue. The follicle’s basement membrane is composed of ECM proteins and it functions as structural support for follicular cells, a selective barrier for molecules entering the follicle, and a scaffold for retaining soluble growth factors and cytokines. It is continuously remodeled during follicle development, but cell- secreted ECM molecules are unable to adhere to unmodified PEG for self-assembly. By integrating ECM- sequestering peptides in the PEG hydrogels, the structural and biological roles of ECM can be restored for in vitro follicle development. The contribution of this work will be a single cell atlas of the reproductive-age ovary highlighting mechanisms driving follicle development and stromal cells’ supportive roles in folliculogenesis and a novel in vitro follicle culture system that supports human follicle development. The contribution of this work will be significant because it will guide the development of a standardized in vitro culture for maturation of human follicles and a safe fertility preservation option for patients unable to produce mature eggs as a result of gonadotoxic treatments. The proposed work is innovative in that it will be the first single cell dataset from healthy reproductive aged women and the first instance of human follicle culture in a synthetic ECM-sequestering matrix.