Cryopreservation and Transplantation of Ovarian Cortical Tissue for Fertility Preservation

 描述（由申请人提供）：本提案的长期目标是开发用于卵巢组织冷冻保存和移植的稳健且可重复的方法，作为癌症或卵巢功能受损的其他良性疾病幸存者的生育力保存选择。虽然目前有80%的年轻患者在癌症中幸存下来，但化疗或放疗的毁灭性副作用使女性幸存者因卵巢功能衰竭而面临不孕症。冷冻保存卵巢组织仍然是青春期前、青少年和年轻女性癌症患者以及需要立即进行癌症治疗的成年患者的唯一选择。但是，人类卵巢组织冷冻保存被认为是实验性的，受到几个因素的阻碍。美国的胚胎学诊所倾向于胚胎和卵母细胞的玻璃化冷冻，而不是慢速冷冻，但是对于卵巢组织的玻璃化冷冻没有统一的方案。虽然有30例人类出生的报道是通过慢速冷冻和移植到原位，但只有4例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在癌症治疗后将没有健康的卵巢血管床作为移植部位。冷冻保存也是由于再次引入恶性细胞的风险而禁忌卵巢移植的患者的唯一选择。虽然体外卵泡培养正在开发中，但大多数研究使用从非冷冻保存组织中分离的卵泡。我们成功地设计了一种在封闭系统中玻璃化冷冻恒河猴卵巢组织的方法，并在体内和体外证明了解冻后的功能。当移植到异位部位时，玻璃化组织恢复卵巢功能，并产生能够进行植入前胚胎发育的成熟卵母细胞。从玻璃化冷冻组织中分离出的次级卵泡在体外可存活并生长至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，使冷冻保存复杂化，因此单个卵泡的玻璃化和随后的3D培养是一种为患有高度侵袭性癌症的年轻患者保存配子的新方法。一个由共同研究者和合作者组成的精英小组，将带来最先进的低温生物学专业知识以及临床卵巢移植方面的卓越经验，这将有助于我们推进当前的技术，以便更快地将其转化为患者的临床实践。使用恒河猴，我们建议，以确定最佳的生育功能的移植部位的青春期前和年轻的成年人卵巢皮质组织，改善长期移植功能，通过局部管理的因素，重要的血管重建以及卵泡存活，并优化玻璃化冷冻的孤立腔前卵泡和他们的能力，以产生主管卵母细胞后的三维体外培养。在这些对照研究之后，非人类灵长类动物的liv分娩将在临床转化之前建立实验疗法的安全性和可行性，以便为女孩和年轻女性提供在癌症幸存后成为母亲的最佳机会。

 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.