Li Lingyu, Yang Jian, Jia Longzhong, Liang Jing, Cheng Kaixin, Yang Xuebing, Mu Lu, Wang Ge, Geng Kaiying, Hu Qiuxian, Xu Xueqiang, Li Zhen, Xia Guoliang, Guo Ting, Zhang Jiawei, Zhang Yan, Zhang Hua
State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
Institute of Women, Children, and Reproductive Health, Shandong University, Ji'nan, Shandong, 250012, China.
Reprod Biol Endocrinol. 2025 Jul 3;23(1):95. doi: 10.1186/s12958-025-01422-y.
Ovarian tissue cryopreservation and transplantation (OTCT) is a promising approach for fertility preservation. However, significant follicle loss after transplantation challenges long-term reproductive recovery. Although primordial follicle loss and ischemic damage are known contributors, the underlying mechanisms and effective strategies to mitigate these damages are still lacking.
Ovarian tissues from wild-type or Tek-CreER;mTmG female mice were cryopreserved using vitrification. Follicle dynamics after OTCT were analyzed through histology, proteomics, and high-resolution imaging. To assess protective effects, cryopreserved ovaries were treated with 500 nM rapamycin (Rapa), an inhibitor of primordial follicle activation; 5 ng/mL vascular endothelial growth factor A (VEGFA), which promotes angiogenesis; or both (Cryo + VRCS, VEGFA-Rapa Combination Strategy). DMSO or HO served as controls. Grafts were collected at 3, 7, 14, and 120 days post-transplantation, with measurements of vascular density, tip cell density, follicle activation, and remaining follicles. Oocyte quality was evaluated via in vitro fertilization, and graft lifespan was estimated through estrous cycle monitoring.
Our study observed a rapid decline in follicle numbers shortly after transplantation in a mouse model. Proteomic analysis and high-resolution 3D imaging revealed that this depletion was primarily due to damage to tip cells, which are crucial for angiogenesis, and the overactivation of dormant primordial follicles. Damage to tip cells compromised vascular reconstruction, leading to ischemic injury, while mechanical handling during tissue isolation and cryopreservation triggered excessive follicle activation. We implemented a combination therapy using rapamycin to inhibit follicle activation and VEGFA to promote angiogenesis prior to transplantation. This approach significantly improved follicle survival, extended reproductive function, and enhanced oocyte quality.
Our study provides a practical strategy for preserving the reproductive potential of cryopreserved ovarian tissues by simultaneously targeting vascular integrity and follicle stability through the dual drug strategy of VEGFA and rapamycin combine treatment.
卵巢组织冷冻保存与移植（OTCT）是一种很有前景的生育力保存方法。然而，移植后显著的卵泡丢失对长期生殖恢复构成挑战。虽然已知原始卵泡丢失和缺血性损伤是导致这一问题的因素，但减轻这些损伤的潜在机制和有效策略仍然缺乏。
使用玻璃化冷冻保存野生型或Tek-CreER;mTmG雌性小鼠的卵巢组织。通过组织学、蛋白质组学和高分辨率成像分析OTCT后的卵泡动态。为评估保护作用，将冷冻保存的卵巢用500 nM雷帕霉素（Rapa）（一种原始卵泡激活抑制剂）、5 ng/mL血管内皮生长因子A（VEGFA）（促进血管生成）或两者（Cryo + VRCS，VEGFA-雷帕霉素联合策略）进行处理。二甲基亚砜（DMSO）或透明质酸钠（HO）作为对照。在移植后3、7、14和120天收集移植物，测量血管密度、尖端细胞密度、卵泡激活情况和剩余卵泡数量。通过体外受精评估卵母细胞质量，并通过发情周期监测估计移植物寿命。
我们的研究在小鼠模型中观察到移植后不久卵泡数量迅速下降。蛋白质组学分析和高分辨率三维成像显示，这种卵泡减少主要是由于对血管生成至关重要的尖端细胞受损以及休眠原始卵泡的过度激活。尖端细胞受损损害了血管重建，导致缺血性损伤，而组织分离和冷冻保存过程中的机械操作引发了过度的卵泡激活。我们在移植前实施了联合治疗，使用雷帕霉素抑制卵泡激活，VEGFA促进血管生成。这种方法显著提高了卵泡存活率，延长了生殖功能，并提高了卵母细胞质量。
我们的研究提供了一种实用策略，通过VEGFA和雷帕霉素联合治疗的双重药物策略同时靶向血管完整性和卵泡稳定性，来保存冷冻保存的卵巢组织的生殖潜力。
