Cryopreservation and Transplantation of Ovarian Cortical Tissue for Fertility Preservation

 描述(由申请人提供)：这项建议的长期目标是开发强大和可重复的卵巢组织冷冻保存和移植方法，作为癌症或其他卵巢功能受损的良性疾病幸存者保留生育能力的选择。虽然目前80%的年轻患者在癌症中存活下来，但化疗或放射治疗的破坏性副作用使女性幸存者面临卵巢衰竭导致的不孕不育。对于青春期前、青少年和年轻的癌症女性以及需要立即进行癌症治疗的成年患者，冷冻保存卵巢组织仍然是唯一的选择。但是，人类卵巢组织的冷冻保存被认为是实验性的，并受到多种因素的阻碍。美国的胚胎学诊所倾向于胚胎和卵母细胞玻璃化冷冻，而不是慢速冷冻，但卵巢组织的玻璃化冷冻没有统一的方案。尽管有报道称有30名婴儿是通过慢速冷冻和移植到原位出生的，但只有4人可以声称移植的组织是已知的卵母细胞来源。大多数年轻的幸存者将不会有一个健康的卵巢血管床作为癌症治疗后的移植部位。冷冻保存也是卵巢移植禁忌患者的唯一选择，因为有再次引入恶性细胞的风险。虽然体外卵泡培养正在进行中，但大多数研究使用的是从未冷冻保存的组织中分离出来的卵泡。我们成功地设计了一种封闭系统中玻璃化冷冻恒河猴卵巢组织的方法，并在体内和体外验证了融化后的功能。当移植到异位部位时，玻璃化组织恢复卵巢功能，并产生能够植入前胚胎发育的成熟卵母细胞。从玻璃化组织中分离出的次级卵泡被包裹进行三维(3D)培养，在体外存活并生长到胃窦期并产生类固醇激素，但卵母细胞质量较差。由于卵巢皮质由复杂的细胞类型组成，这使得冷冻保存变得复杂，单个卵泡的玻璃化冷冻和随后的3D培养是保存配子的一种新方法，适用于患有高侵袭性癌症的年轻患者。一个由合作研究人员和合作者组成的精英小组带来了最先进的冷冻生物学方面的专业知识以及临床卵巢移植方面的卓越经验，将帮助我们推进当前的技术，为患者更快地将其转化为临床实践。利用恒河猴，我们建议确定青春期前和青春期卵巢皮质组织最佳生育功能的移植部位，通过局部应用对血运重建和卵泡存活至关重要的因素来改善长期移植功能，并优化分离的腔前卵泡的玻璃化冷冻及其在体外培养后产生合格卵母细胞的能力。在这些对照研究之后，在非人类灵长类动物中出生LIV将在临床翻译之前确定实验疗法的安全性和可行性，这样女孩和年轻女性就可以在癌症幸存下来后获得最好的成为母亲的机会。

 DESCRIPTION (provided by applicant): The long-range goals of this proposal are to develop robust and reproducible methods for ovarian tissue cryopreservation and transplantation as options for fertility preservation in survivors of cancer or other benign conditions wherein ovaria function is compromised. While 80% of young patients currently survive their cancers, devastating side effects of chemo- or radiotherapies leave female survivors facing infertility due to ovarian failure. Cryopreservation of ovarian tissue remains the only option for prepubertal, adolescent and young women with cancer as well as adult patients for whom immediate cancer therapy is required. But, human ovarian tissue cryopreservation is considered experimental and hampered by several factors. Embryology clinics in the U.S. favor embryo and oocyte vitrification over slow-rate freezing, but there is no uniform protocol for vitrification of ovaria tissue. Although 30 human births have been reported from slow-rate freezing and transplantation to orthotopic sites, only 4 could claim that the transplanted tissue was known to be the source of the oocytes. Most young survivors will not have a healthy ovarian vascular bed as a transplantation site following cancer therapies. Cryopreservation is also the only option for patients contraindicated for ovarian transplantation due to the risk of reintroducing malignant cells. While in vitro follicle culture is under development, most studies use follicles isolated frm non-cryopreserved tissues. We successfully devised a method for vitrification of rhesus monkey ovarian tissue in a closed system, and demonstrated post-thaw function in vivo and in vitro. When transplanted to heterotopic sites, vitrified tissue resumes ovarian function, and yields mature oocytes capable of preimplantation embryonic development. Secondary follicles isolated from vitrified tissue and encapsulated for 3-dimensional (3D) culture survive, grow to antral stages and produce steroid hormones in vitro, but oocyte quality is poor. Since the ovarian cortex is composed of complex cell types that complicate cryopreservation, vitrification of individual follicles and subsequent 3D culture is a novel approach toward preserving gametes for young patients with highly invasive cancers. An elite panel of co-investigators and collaborators that bring expertise in both state-of-the-art cryobiology as well as pre-eminent experience in clinical ovarian transplantation will help us advance the current technology for more rapid translation to clinical practice for patients. Using the rhesus monkey, we propose to identify the transplantation site for optimal fertile function of both prepubertal and young adult ovarian cortical tissue, improve long-term transplant function via local administration of factors important for revascularization as well as follicular survival, and optimize vitrification of isolaed preantral follicles and their ability to yield competent oocytes following 3D culture in vitro. Liv births in nonhuman primates after these controlled studies will establish the safety and feasibilit of experimental therapies prior to clinical translation so girls and young women can be offered the best chances of becoming mothers after surviving cancer.