Omes C, Savio M, Mazzini G, Citterio C, Casasco A, Nappi R E, Riva F
Center for Reproductive Medicine, Obstetrics and Gynecology Unit 2, Woman and Child Health Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.
Department of Molecular Medicine, Immunology and General Pathology Unit, University of Pavia, Italy.
Cryo Letters. 2025 Jul-Aug;46(4):261-273.
Human semen and epididymal spermatozoa cryopreservation are crucial for men's fertility preservation, particularly for those patients facing neoplastic, autoimmune, urological, and neurological conditions where medical or surgical treatments may pose a risk to fertility or where obstructive or secretory azoospermia is documented. However, there are currently no standardized methods to assure optimal cryosurvival rates.
To determine the best freezing protocol out of five selected methods based on routine sperm analysis and additional assays including cytofluorimetric analysis, comet assay, and transmission electron microscopy.
The study is a cross-sectional analysis of 26 fresh semen samples frozen using five different freezing protocols (or methods, M), varying in cooling phase time and temperatures, and utilizing TEST-Yolk Buffer (TYB) as a cryoprotectant. Data on sperm motility, viability, membrane integrity, DNA fragmentation, and ultrastructural shape post-thawing were collected.
Our findings showed that the method 1 (M1) and method 3 (M3) (involving a three-phase cooling process with a phase at +4 degree C, followed by 10 min of exposure to the gas phase of liquid nitrogen before immersion in liquid nitrogen) yielded the best protocols, resulting in minimal deterioration of semen quality.
These results highlight the importance of a pre-freezing phase at +4 degree C when using TYB cryoprotectant on untreated semen, regardless of the duration, despite the less-than-optimal survival rate achieved. It is crucial to use a range of assays to study the effects of cryopreservation procedures, not only assessing sperm motility and viability, but also evaluating membrane integrity, DNA fragmentation, and ultrastructural shape. https://doi.org/10.54680/fr25410110212.
人类精液和附睾精子冷冻保存对于男性生育力保存至关重要，特别是对于那些面临肿瘤、自身免疫、泌尿和神经疾病的患者，在这些疾病中，药物或手术治疗可能会对生育力构成风险，或者已记录存在梗阻性或分泌性无精子症的情况。然而，目前尚无标准化方法来确保最佳冷冻存活率。
基于常规精子分析以及包括细胞荧光分析、彗星试验和透射电子显微镜在内的其他检测方法，从五种选定方法中确定最佳冷冻方案。
本研究是对26份新鲜精液样本进行的横断面分析，这些样本使用五种不同的冷冻方案（或方法，M）进行冷冻，冷却阶段的时间和温度各不相同，并使用测试蛋黄缓冲液（TYB）作为冷冻保护剂。收集解冻后精子活力、存活率、膜完整性、DNA片段化和超微结构形状的数据。
我们的研究结果表明，方法1（M1）和方法3（M3）（涉及三相冷却过程，其中一个阶段为+4℃，随后在浸入液氮之前在液氮气相中暴露10分钟）产生了最佳方案，导致精液质量的恶化最小。
这些结果突出了在使用TYB冷冻保护剂处理未处理精液时，+4℃预冷冻阶段的重要性，无论持续时间如何，尽管实现的存活率并非最佳。使用一系列检测方法来研究冷冻保存程序的效果至关重要，不仅要评估精子活力和存活率，还要评估膜完整性、DNA片段化和超微结构形状。https://doi.org/10.54680/fr25410110212
