Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark.
Department of Gynecological Minimal Invasive Center, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China.
Hum Reprod. 2018 Dec 1;33(12):2276-2284. doi: 10.1093/humrep/dey318.
Can follicle survival in frozen-thawed human ovarian tissue be quantified in situ using the dye Neutral Red (NR) to stain viable follicles specifically?
A follicle survival rate within ovarian tissue can be calculated using NR followed by histological evaluation and evidence for a consistently high follicle survival in a series of ovarian tissue from 25 Danish girls and women undergoing ovarian tissue cryopreservation (OTC) was obtained.
Securing follicle survival in cryopreserved ovarian tissue is crucial for proper quality control when centers wish to implement OTC. The only established technique for validation of follicle survival is xenografting of thawed ovarian tissue to immunodeficient mice. However, this functional test is expensive, time consuming, requires animal facilities and only provides a qualitative-not quantitative-measure for follicle survival.
STUDY DESIGN SIZE, DURATION: Quantification of follicle survival in human ovarian tissue donated from 30 girls and women having tissue cryopreserved for fertility preservation from 2000 to 2015 at the Laboratory of Reproductive Biology in Copenhagen, Denmark.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Cryopreserved ovarian cortex was donated from 25 girls and young women aged 10-36 years (mean age: 25 years) and the average storage time in liquid nitrogen was 9.1 ± 5.6 years, ranging from 1.6 to 17.9 years. In 12 of the cases, the ovarian tissue was collected from the local hospital and in the other 13 cases the ovarian tissue was transported on ice up to 6 h prior to freezing. Donated fresh ovarian surplus tissue was obtained from five women aged 23-34 years (mean age: 27 years). Ovarian tissues were chopped into small fragments and incubated in culture medium containing 50 mg/ml NR for 3-4 h. Fragments of ovarian tissue containing clearly NR-stained follicles were selected for counting, encapsulated in 4% agar and were processed for histology to calculate a follicular survival rate.
The mean follicle survival rate in the 25 patients after freezing and thawing was 84% ± 11 (mean ±SD), ranging from 50% to 98%. The high follicle survival rate in this clinical series of patients reflects a constant high-quality service performed in our center and confirms the robustness of the slow freezing protocol. No significant association between follicle survival rates and storage time was found using linear regression analysis, suggesting that storage in liquid nitrogen does not affect viability of the tissue. No significant association in follicle survival rates was found between ovarian tissues collected at the local hospital compared to tissues transported on ice prior to freezing, supporting that prolonged cooling does not seem to greatly affect the follicle survival. For the fresh ovarian tissue, the average follicle survival rate was 91% ± 5 (mean ± SD) in five patients, ranging from 81% to 95%.
LIMITATIONS, REASONS FOR CAUTION: Even though the NR staining requires active incorporation of the dye, the test is merely a short in situ test that cannot completely replace the functional value of xenografting studies in which the integrity and developmental potential of the ovarian follicles are assessed.
OTC is now being employed around the world but to date it has been difficult for centers to evaluate the effectiveness of their program and perform proper quality control. NR staining combined with histological evaluation is the first quantitative method to provide a survival rate for follicles in frozen-thawed human ovarian tissue and offer a valuable and easily applicable tool to validate the cryopreservation procedure when implementing OTC or as routine quality control for the overall freezing performance within tissue banking facilities.
STUDY FUNDING/COMPETING INTEREST(S): The Research Pools of Rigshospitalet, the Danish Cancer Foundation, Dagmar Marshalls Foundation, and the Novo Nordic Foundation are thanked for having funded this study. The authors have no conflicts of interest.
使用中性红（NR）特异性染色存活卵泡，能否定量评估冷冻-解冻人类卵巢组织中的卵泡存活？
通过 NR 染色后进行组织学评估，可以计算卵巢组织中的卵泡存活率，并且在对来自 25 名丹麦女孩和妇女的一系列卵巢组织进行的研究中获得了卵巢组织冷冻保存（OTC）中卵泡存活率始终较高的证据。
确保冷冻保存的卵巢组织中的卵泡存活对于中心希望实施 OTC 时的适当质量控制至关重要。验证卵泡存活的唯一既定技术是将解冻的卵巢组织异种移植到免疫缺陷小鼠中。然而，这种功能测试昂贵、耗时、需要动物设施，并且只能提供卵泡存活的定性而不是定量测量。
研究设计、规模、持续时间：在丹麦哥本哈根生殖生物学实验室，对 2000 年至 2015 年期间因生育保护而冷冻保存卵巢组织的 30 名女孩和妇女捐赠的卵巢组织进行卵泡存活的定量分析。
参与者/材料、设置、方法：从 25 名年龄 10-36 岁（平均年龄 25 岁）的女孩和年轻女性中捐赠冷冻卵巢皮质，在液氮中的平均储存时间为 9.1±5.6 年，范围从 1.6 到 17.9 年。在 12 例病例中，卵巢组织从当地医院采集，在其他 13 例病例中，卵巢组织在冷冻前用冰运输长达 6 小时。从 5 名年龄 23-34 岁（平均年龄 27 岁）的女性中获得新鲜卵巢剩余组织。卵巢组织切成小块，在含有 50mg/ml NR 的培养基中孵育 3-4 小时。选择含有明显 NR 染色卵泡的卵巢组织片段进行计数，包裹在 4%琼脂中，并进行组织学处理以计算卵泡存活率。
25 名患者冷冻和解冻后的平均卵泡存活率为 84%±11（平均值±SD），范围为 50%至 98%。该患者临床系列中高卵泡存活率反映了我们中心始终如一的高质量服务，并证实了缓慢冷冻方案的稳健性。线性回归分析未发现卵泡存活率与储存时间之间存在显著相关性，表明储存在液氮中不会影响组织的活力。未发现卵巢组织在当地医院采集与冷冻前在冰上运输之间的卵泡存活率存在显著相关性，支持长时间冷却似乎不会对卵泡存活产生重大影响。对于新鲜卵巢组织，5 名患者的平均卵泡存活率为 91%±5（平均值±SD），范围为 81%至 95%。
局限性、谨慎的原因：尽管 NR 染色需要染料的主动掺入，但该测试仅是一种短期的原位测试，不能完全替代异种移植研究中的功能价值，异种移植研究中评估了卵巢卵泡的完整性和发育潜力。
OTC 现在在全球范围内使用，但迄今为止，中心很难评估其计划的有效性并进行适当的质量控制。NR 染色结合组织学评估是第一个提供冷冻-解冻人类卵巢组织中卵泡存活率的定量方法，并提供了有价值且易于应用的工具，可在实施 OTC 或作为组织库设施整体冷冻性能的常规质量控制时验证冷冻程序。
研究资金/利益冲突：感谢 Rigshospitalet 研究池、丹麦癌症基金会、Dagmar Marshalls 基金会和 Novo Nordic 基金会为这项研究提供资金。作者没有利益冲突。
