Experimental engineering of ovarian grafting to promote angiogenesis for fertilit

描述(申请人提供)：随着癌症和其他疾病接受大剂量化疗和/或放射治疗的患者数量持续增加，更多的注意力集中在改善他们的生活质量上，生育是年轻人的首要任务之一。与男性相比，女性避免医源性不孕和更年期的选择更少，青春期前女孩几乎没有选择。胚胎的冷冻保存或玻璃化冷冻是一种护理标准，目前单身女性正在出现卵母细胞库，但这些辅助生殖技术都不是普遍适用或可接受的，也不能帮助儿童。低温储存卵巢组织切片，或许有一天可以储存整个卵巢，现在为这些人提供了一种选择，到目前为止，在移植解冻组织后，已经有6例足月妊娠，还有几例是在移植新鲜组织后，但三年后，没有移植物仍然起作用。这种保持生育能力的策略仍被认为是“试验性的”，尽管多年前已经在动物模型中建立了原理证明。这些研究表明，在移植物血运重建之前的最初2-5天内，大量不同比例的卵泡因缺血而丢失，而冷冻损伤的影响相对较小。根据我们的假设，缺血时间的缩短将有利于通过减少坏死组织的数量和废弃卵泡的比例来延长移植物的功能并增强生育能力。在目标1中，我们将描述卵巢移植物中血管生成的细胞和分子决定因素，以及它们与卵泡存活相关的时间进程。这一目标将通过研究候选因子，特别是血管内皮生长因子和PDGF的作用来填补知识空白，这些候选因子参与了将移植到骨骼肌中的小鼠卵巢和作为翻译模型的人卵巢移植到NOD-SCID小鼠中。这些研究将包括在移植后两周内通过显微超声无创监测再灌注的新方法，以及血管生长、血管生成因子、卵泡存活和细胞外基质中因子的分子和细胞定量。在目标2中，我们将测试两种通过组织工程改善血管灌流和卵泡存活的实验策略，无论是在未来的移植物部位还是移植物本身。首先，我们将藻酸盐水凝胶植入肌肉，用于局部、分阶段地释放血管生成因子，以刺激内皮细胞和周细胞，目的是加速小鼠移植物中稳定微循环的形成。在第二项研究中，我们将减少人卵巢组织中的胶原蛋白体积，以测试较高的纤维密度是否为血管入侵的屏障，以及是否可能导致较高比例的卵泡浪费。这两个实验中的任何一个的成功结果都可能导致计划将卵巢组织储存以保持生育能力的患者的临床实施。公共卫生相关性：迫切需要有效和可接受的生育保护技术，以服务于越来越多的在药物和放射治疗后幸存下来的癌症患者，这些药物和放射可能导致不孕不育和卵巢早衰。卵巢组织库已经被世界各地的数千名患者采用，因为它比胚胎/卵母细胞库有一些优势，特别是对儿童来说，但组织移植回来只产生了几个可存活的妊娠，而且只有几年的功能。这项研究项目将填补关于卵巢移植物血运重建生物学的知识空白，这限制了存活卵泡的比例，并将测试两种旨在促进血管生成的策略。

DESCRIPTION (provided by applicant): As the numbers of patients continue to rise after high-dose chemotherapy and/ or irradiation for cancer and other diseases, more attention is focused on improving their quality-of-life, with fertility one of the top priorities for young people. Women have had fewer options for avoiding iatrogenic infertility and menopause compared with men, and prepubertal girls have virtually none. Cryopreservation or vitrification of embryos is a standard of care, and oocyte banking is now emerging for single women, but neither of these assisted reproductive technologies is universally suitable or acceptable, nor can either help children. Low temperature banking of ovarian tissue slices, and perhaps whole ovaries one day, now offers an option for these people and, so far, six full-term pregnancies have been established after transplanting thawed tissue, and several others after fresh tissue, but no grafts were still functioning after three years. This strategy for preserving fertility is still considered 'experimental', though proof of principle was established many years ago in animal models. Those studies revealed a large and variable fraction of follicles is lost by ischemia during the first 2-5 days until the grafts are revascularized, whereas the impact of cryoinjury was comparatively minor. According to our hypothesis, a reduction in ischemia time will be beneficial for extending graft function and potentiating fertility by reducing the amount of necrotic tissue and the fraction of wasted follicles. In Aim 1, we will characterize the cellular and molecular determinants of angiogenesis in ovarian grafts and their temporal course in relation to follicular survival. This aim will fill a knowledge gap by investigating the role of candidate factors, notably VEGF and PDGF, involved in revascularizing mouse ovaries grafted into skeletal muscle and human ovarian xenografts in NOD-scid mice as a translational model. The studies will include novel methods to monitor reperfusion non-invasively by microultrasound during the first two weeks post-grafting, and molecular and cellular quantification of vascular growth, angiogenic factors, follicular survival and factors in the extracellular matrix. In Aim 2, we will test two experimental strategies for improving vascular perfusion and follicle survival by tissue engineering either at the future graft site or the graft itself. In the first, we will implant alginate hydrogels into muscle for local, phased release of angiogenic factors to stimulate endothelial cells and pericytes with the goal of accelerating the formation of a stable microcirculation in murine grafts. In the second, we will reduce collagen bulk in human ovarian tissue to test whether the higher fiber density is a barrier to vascular invasion and potentially responsible for the higher fraction of follicles wasted. A successful outcome of either of these experiments could lead to clinical implementation for patients who plan to bank their ovarian tissue to preserve fertility. PUBLIC HEALTH RELEVANCE: There is an urgent public health need for effective and acceptable fertility preservation technologies to serve rising numbers of patients surviving cancer treatment after drugs and irradiation that potentially cause infertility and premature ovarian failure. Ovarian tissue banking has been adopted by several thousand patients worldwide because it offers some advantages over embryo/ oocyte banking, especially for children, but tissue grafted back has only generated a few viable pregnancies so far and only functions for a few years. This research project will fill the knowledge gap about the biology of revascularization of ovarian grafts, which limits the fraction of surviving follicles, and will test two strategies aimed at enhancing angiogenesis.