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。以往的研究表明，卵巢组织在冷冻和解冻后，原始卵泡的损失相对较小，但在卵巢移植后的初始缺血期间，卵巢储备的很大一部分会损失。因此，任何能够增强和促进卵巢移植血运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。我们的建议的总体假设是，原始卵泡的生存和卵巢移植功能可以通过药物治疗增强血管生成改善。这将在以下具体目标下进行研究：探讨神经酰胺诱导的细胞死亡途径抑制剂鞘氨醇-1磷酸（S1P）在人卵巢异种移植模型中是否能防止移植后缺血诱导的卵巢原始卵泡池损伤。最近的研究表明，S1P是神经酰胺诱导的凋亡细胞死亡的抑制剂，可以增强生理性血管化过程。因此，我们假设S1P通过促进卵巢移植物新生血管的形成来提高卵巢移植后初始缺血期的原始卵泡存活率。为了测试S1P在增强卵巢移植新生血管方面的有效性，我们将采用基于严重联合免疫缺陷（SCID）小鼠的人类卵巢异种移植模型。我们将通过原始卵泡存活、新生血管形成的组织学和分子评估、HIF-1a缺氧证据以及活体染料注射和创新的实时成像技术来确定S1P是否能改善移植物存活和新生血管形成。2. 在异种移植模型中，确定S1P治疗是否能改善冷冻解冻卵巢组织移植的功能。虽然S1P可以增强卵巢移植的血管化，提高原始卵泡的存活率，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S1P不仅可以改善卵巢异种移植的原始卵泡存活率，而且这些卵泡将具有功能。一个更大的存活功能原始卵泡池应该转化为卵巢移植物的扩展功能。为了证明S1P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、卵母细胞成熟和排卵率对hCG的反应、类固醇激素的产生以及单性生殖激活，评估和比较S1P处理和对照移植的功能。

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.