Biology of Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels 1090, Belgium.
Hum Reprod. 2013 Jul;28(7):1816-26. doi: 10.1093/humrep/det100. Epub 2013 Apr 7.
Is there a better alternative to the conventional cryopreservation protocols for human testicular tissue banking?
Uncontrolled slow freezing (USF) using 1.5 M dimethylsulphoxide (DMSO) and 0.15 M sucrose as cryoprotectants appears to be a user-friendly and efficient method for the cryopreservation of human testicular tissue.
Currently, time-consuming controlled slow freezing (CSF) protocols that need expensive equipment are commonly used for human testicular tissue banking. USF and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration with human tissue.
STUDY DESIGN, SIZE, DURATION: Fragments (n = 160) of testicular tissue from 14 patients undergoing vasectomy reversal were assigned to a fresh control group or one of the following cryopreservation procedures: CSF using DMSO at a concentration of 0.7 or 1.5 M in the presence (+S) or absence of sucrose (-S), USF using either 0.7 or 1.5 M DMSO combined with sucrose, solid-surface vitrification (SSV) or direct cover vitrification (DCV).
MATERIALS, SETTING, METHODS: Light microscopic evaluations were performed to study apoptosis, germ cell proliferation ability, spermatogonial survival, coherence of the seminiferous epithelium and integrity of the interstitial compartment after cryopreservation. Ultrastructural alterations were studied by scoring cryodamage to four relevant testicular cell types.
The USF 1.5 M DMSO + S protocol proved not solely to prevent cell death and to preserve seminiferous epithelial coherence, interstitial compartment integrity, SG and their potential to divide but also protected the testicular cell ultrastructure. A significant reduction in the number of SG per tubule from 21.4 ± 5.6 in control tissue to 4.9 ± 2.1, 8.2 ± 5.4, 11.6 ± 5.1, 8.8 ± 3.9, 12.6 ± 4.4 and 11.7 ± 5.7 was observed after cryopreservation combined with at least one other form of cryoinjury when using CSF 0.7 M DMSO -S, CSF 0.7 M DMSO + S, CSF 1.5 M DMSO + S, USF 0.7 M DMSO + S, SSV and direct cover vitrification (DCV), respectively (P < 0.001).
LIMITATIONS, REASONS FOR CAUTION: Supplementary research is required to investigate the effect on tissue functionality and to confirm this study's findings using prepubertal tissue.
An optimal cryopreservation protocol enhances the chances for successful fertility restoration. USF, being an easy and cost-effective alternative to CSF, would be preferable for laboratories in developing countries or whenever tissue is to be procured from a diseased child at a site distant from the banking facility.
是否有比传统的人类睾丸组织库冷冻保存方案更好的替代方案？
使用 1.5 M 二甲基亚砜（DMSO）和 0.15 M 蔗糖作为冷冻保护剂的非控制性慢速冷冻（USF）似乎是一种用户友好且高效的方法，可用于人类睾丸组织的冷冻保存。
目前，常采用耗时的控制性慢速冷冻（CSF）方案，该方案需要昂贵的设备，通常用于人类睾丸组织库。USF 和玻璃化是已成功应用于多种动物模型的冷冻保存技术，但需要进一步探索人类组织。
研究设计、大小和持续时间：从 14 名接受输精管复通术的患者中取出睾丸组织碎片（n = 160），分为新鲜对照组或以下冷冻保存程序之一：CSF 中使用浓度为 0.7 或 1.5 M 的 DMSO，存在（+S）或不存在蔗糖（-S），USF 使用 0.7 或 1.5 M DMSO 与蔗糖结合，固态表面玻璃化（SSV）或直接覆盖玻璃化（DCV）。
材料、设置和方法：进行光镜评估，以研究细胞凋亡、生殖细胞增殖能力、精原细胞存活、生精小管上皮的连贯性以及冷冻保存后间质腔的完整性。通过对四种相关睾丸细胞类型的冷冻损伤进行评分，研究超微结构改变。
USF 1.5 M DMSO + S 方案不仅被证明可以防止细胞死亡和保持生精小管上皮的连贯性、间质腔的完整性、SG 及其分裂能力，而且还可以保护睾丸细胞的超微结构。冷冻保存后，SG 数量从对照组组织的 21.4 ± 5.6 个/小管显著减少到以下各处理组的 4.9 ± 2.1、8.2 ± 5.4、11.6 ± 5.1、8.8 ± 3.9、12.6 ± 4.4 和 11.7 ± 5.7（P < 0.001）。CSF 0.7 M DMSO -S、CSF 0.7 M DMSO + S、CSF 1.5 M DMSO + S、USF 0.7 M DMSO + S、SSV 和直接覆盖玻璃化（DCV）分别组合冷冻保存时，观察到的SG 数量减少（P < 0.001）。
局限性、谨慎的原因：需要进行补充研究，以调查对组织功能的影响，并使用青春期前组织证实本研究的发现。
优化的冷冻保存方案增加了成功恢复生育能力的机会。与 CSF 相比，USF 作为一种简单且具有成本效益的替代方案，对于发展中国家的实验室或在远离银行设施的地方从患病儿童获取组织时，将是首选。
