Huang Wu-Jian, Zhang Duo, Hong Zhi-Wei, Chen Zhi-Biao, Dong Li-Hong, Zhang Yan, Chen Guo-Yong, Liu Yun, Yao Bing
Jinling Hospital Department Reproductive Medical Center, Southern Medical University, Nanjing, 210002, China; Center for Reproductive Medicine, The 900th Hospital of Joint Logistic Support Force, PLA, Fuzhou, 350025, China.
Research Center for Molecular Diagnosis of Genetic Diseases, The 900th Hospital of Joint Logistic Support Force, PLA, Fuzhou, 350025, China.
Cryobiology. 2020 Apr;93:75-83. doi: 10.1016/j.cryobiol.2020.02.005. Epub 2020 Feb 11.
Cryopreservation of human spermatozoa with low concentration while maintaining adequate post-thawing motility remains a major challenge for male fertility preservation. A convenient and efficient ultra-rapid freezing method for small amounts of human spermatozoa in a closed Hemi-Straw carrier system (CHS) was developed. Spermatozoa from 60 healthy men were involved in a parameter refining test and another 15 extreme oligozoospermic specimens were assigned to a verification test. A commercialized sperm freezing medium, Quinn's Advantage® Sperm Freeze medium (glycerol and sucrose as the cryoprotective agent) was used in the study. The results showed that the highest recovery rates would be obtained via the method of 2 μl single droplet sequential interval loading, by placing the straw at 1 cm above the liquid nitrogen (LN) surface for 60 s during freezing and 2 cm above the LN for 2 min during thawing. This method was applied in cryopreservation for the normozoospermic specimens and compared with a conventional slow freezing method. The results were better than those in the control group in the total motility recovery rate (77.8 ± 11.2% vs 56.6 ± 11.9%, P < 0.01), progressive motility recovery rate (77.6 ± 13.2% vs 47.7 ± 14.6%, P < 0.01), 24 h survival index (60.9 ± 13.4% vs 42.1 ± 14.1%, P < 0.01) and the sperm DNA fragment index (4.2 ± 3.7% vs 5.8 ± 3.7%, P = 0.126). This method was applied to the oligozoospermic specimens. Motile spermatozoa could be found in 12 of 15 cases in the ultra-rapid freezing group, while only in 7 cases in control group. The results indicated that this freezing method was simple, convenient and bio-safe for cryopreservation of severe oligozoospermic specimens.
在保持解冻后足够活力的同时，采用低浓度对人类精子进行冷冻保存，仍然是男性生育力保存面临的一项重大挑战。本研究开发了一种便捷高效的超快速冷冻方法，用于在封闭的半麦管载体系统（CHS）中对少量人类精子进行冷冻。选取60名健康男性的精子进行参数优化试验，并将另外15份严重少精子症样本用于验证试验。研究使用了一种商业化的精子冷冻培养基Quinn's Advantage® Sperm Freeze培养基（以甘油和蔗糖作为冷冻保护剂）。结果表明，采用2 μl单滴顺序间隔加载法，在冷冻时将麦管置于液氮（LN）表面上方1 cm处60 s，解冻时置于LN上方2 cm处2 min，可获得最高回收率。该方法应用于正常精子样本的冷冻保存，并与传统的慢速冷冻方法进行比较。在总活力恢复率（77.8 ± 11.2% vs 56.6 ± 11.9%，P < 0.01）、前向运动恢复率（77.6 ± 13.2% vs 47.7 ± 14.6%，P < 0.01）、24小时存活指数（60.9 ± 13.4% vs 42.1 ± 14.1%，P < 0.01）和精子DNA片段指数（4.2 ± 3.7% vs 5.8 ± 3.7%，P = 0.126）方面，该方法的结果均优于对照组。该方法还应用于少精子症样本。超快速冷冻组15例中有12例可发现活动精子，而对照组仅7例。结果表明，这种冷冻方法对严重少精子症样本的冷冻保存而言简单、便捷且生物安全性良好。
