Improving Primordial Follicle Survival After Transplantation of Cryopreserved Hum

描述（由申请人提供）：卵巢组织冷冻保存是保存生育能力的关键方法之一。它不需要卵巢刺激，可以在相对较短的时间内完成，甚至在未成年人中。虽然以前的研究表明，原始卵泡损失相对较小的冷冻和解冻后的卵巢组织，大部分的卵巢储备是在卵巢移植后的初始缺血损失。因此，任何能够促进卵巢移植血管重建的方法都有可能改善手术结果。因此，该项目的长期目标是开发新的策略来提高人类卵巢组织移植后的组织存活率。我们的建议的首要假设是，原始卵泡的生存和卵巢移植功能可以通过增强血管生成的药物治疗来改善。这将在以下具体目标下进行研究：1。在人卵巢异种移植模型中，确定神经酰胺诱导的细胞死亡途径抑制剂1磷酸鞘氨醇（S1 P）是否可保护卵巢原始卵泡池免受移植后缺血诱导的损伤。最近的研究表明，S1 P，神经酰胺诱导的细胞凋亡的抑制剂，可以增强生理血管化过程。因此，我们假设S1 P通过促进移植卵巢的新生血管形成，在卵巢移植后的初始缺血期提高原始卵泡的存活率。为了测试S1 P在增强卵巢移植新血管形成中的有效性，我们将采用基于严重联合免疫缺陷（SCID）小鼠的人卵巢异种移植模型。我们将通过原始卵泡存活、新生血管形成的组织学和分子学评估、HIF-1a缺氧的证据以及利用活体染料注射和创新的活体成像技术来确定S1 P是否改善移植物存活和新生血管形成。2.确定S1 P治疗是否改善异种移植模型中冻融卵巢组织移植物的功能。虽然S1 P可以增强卵巢移植物的血管化并增加原始卵泡的存活，但这并不一定意味着这些移植物将具有扩展的功能。因此，我们假设不仅S1 P将改善原始卵泡在卵巢异种移植物中的存活，而且这些卵泡将具有功能。一个更大的存活的功能性原始卵泡池应该转化为卵巢移植物的扩展功能。为了证明S1 P治疗可以增强卵巢移植的功能，我们将进行长期的卵巢异种移植实验。异种移植16周后，通过分析卵泡和卵母细胞形态、卵母细胞成熟和对hCG给药的排卵率、类固醇激素产生以及孤雌激活，评估并比较S1 P处理和对照移植物的功能。

DESCRIPTION (provided by applicant): Ovarian tissue cryopreservation is one of the key approaches to fertility preservation. It does not require ovarian stimulation and can be done in a relatively short time and even in minors. While previous research indicated that primordial follicle losses are relatively small after freezing and thawing of ovarian tissue, a large fraction of the ovarian reserve is lost during the initial ischemia after ovarian transplantation. Thus any approach that can enhance and facilitate revascularization of ovarian transplants is likely to improve the outcome of the procedure. Hence, the long-term objective of this project is to develop novel strategies to improve tissue survival after transplantation of ovarian tissue in humans. The overarching hypothesis of our proposal is that the primordial follicle survival and ovarian graft function can be improved by pharmacological treatments enhancing vasculogenesis. This will be studied under the following specific aims: 1. To determine whether a ceramide-induced cell death pathway inhibitor, sphingosine-1 phosphate (S1P), protects against post-transplant ischemia-induced damage to ovarian primordial follicle pool in a human ovarian xenograft model. Recent studies have shown that S1P, an inhibitor of ceramide-induced apoptotic cell death, can enhance physiological vascularization processes. Thus we hypothesize that S1P will enhance primordial follicle survival during the initial ischemic phase after ovarian transplantation by promoting neovascularization of ovarian grafts. To test the effectiveness of S1P in augmenting ovarian transplant neovascularization, we will employ a human ovarian xenograft model based on Severe-Combined- Immunodeficiency (SCID) mice. We will determine whether S1P improves graft survival and neovascularization by primordial follicle survival, histological and molecular assessment of neovascularization, evidence of hypoxia by HIF-1a, as well as by utilization of intravital dye injections and innovative live imaging techniques. 2. To determine whether S1P treatment improves the functionality of frozen-thawed ovarian tissue transplants in a xenograft model. While S1P may enhance vascularization of ovarian transplants and increase survival of primordial follicles, this does not necessarily mean that these grafts will have extended function. Thus, we hypothesize that not only S1P will improve primordial follicle survival in ovarian xenografts but these follicles will be functional. A larger surviving functional primordial follicle pool should translate into extended functionality of ovarian grafts. To show that S1P treatment can thus enhance functionality of ovarian transplants, we will conduct long-term ovarian xenografting experiments. After 16 weeks of xenografting, functionality will be assessed and compared between S1P treated and control grafts by analysis of follicle and oocyte morphology, oocyte maturation and ovulation rates in response to hCG administration, steroid hormone production, as well as parthenogenic activation.