Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital of Bonn;
Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital of Bonn.
J Vis Exp. 2024 Aug 9(210). doi: 10.3791/66801.
Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or for pre-pubertal girls, it is often the only option for fertility protection. Cryopreservation can be performed either by vitrification or by slow freezing. Slow freezing is currently the standard approach. An increasing number of studies indicate that vitrification can replace slow freezing in the state-of-the-art in vitro fertilization (IVF) laboratories, significantly improving thawing survival rates and simplifying the technical aspects of cryopreservation. A metal grid-based, high-throughput protocol for rapid vitrification of ovarian cortex tissue, suitable for clinical routine, is described. The sterilization of metal grids and liquid nitrogen ensures high quality, meeting good manufacturing practice (GMP) standards. Vitrification was conducted to ensure ultra-rapid cooling rates. Instead of slowly thawing, samples were rapidly warmed. To assess follicular viability, calcein staining was performed both prior to cryopreservation and after rapid warming. The successful application of vitrification and rapid warming using metal grids is reported. No significant differences in follicular viability were observed prior to vitrification and after rapid warming. These results substantiate the high capacity of tissue vitrification for clinical routine applications as a potential substitute for the widely used slow-freezing method.
卵巢组织冷冻保存（OTC）是生育力保存的重要选择。对于那些性腺毒性治疗不能推迟的患者，或者对于青春期前的女孩来说，这通常是生育保护的唯一选择。冷冻保存可以通过玻璃化或慢速冷冻来进行。慢速冷冻目前是标准方法。越来越多的研究表明，玻璃化可以在最先进的体外受精（IVF）实验室中取代慢速冷冻，显著提高解冻存活率并简化冷冻保存的技术方面。本文描述了一种基于金属网格的高通量快速卵巢皮质组织玻璃化方案，适用于临床常规。金属网格和液氮的消毒确保了高质量，符合良好生产规范（GMP）标准。玻璃化是为了确保超快速冷却速率。样本不是缓慢解冻，而是快速升温。为了评估卵泡活力，在冷冻保存前和快速升温后都进行了钙黄绿素染色。报告了使用金属网格进行玻璃化和快速升温的成功应用。在玻璃化前和快速升温后，卵泡活力没有显著差异。这些结果证实了组织玻璃化在临床常规应用中的高能力，可以作为广泛使用的慢速冷冻方法的潜在替代品。
