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 (LN2) surface for 60 s during freezing and 2 cm above the LN2 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.

中文翻译：

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封闭半吸管载体系统中的连续间隔微滴加载：一种方便有效的极端少精子症超快速冷冻方法

以低浓度冷冻保存人类精子，同时保持足够的解冻后活力仍然是男性生育力保存的主要挑战。开发了一种方便、高效的超快速冷冻方法，用于在封闭的半吸管载体系统 (CHS) 中对少量人类精子进行冷冻。来自 60 名健康男性的精子参与了参数精炼测试，另外 15 名极端少精子症标本被分配进行验证测试。研究中使用了商业化的精子冷冻培养基，Quinn's Advantage® 精子冷冻培养基（甘油和蔗糖作为冷冻保护剂）。结果表明，通过 2 μl 单液滴顺序间隔加载的方法可以获得最高的回收率，在冷冻过程中将吸管置于液氮 (LN2) 表面上方 1 厘米处 60 秒，在解冻过程中将吸管置于液氮 (LN2) 表面上方 2 厘米处 2 分钟。该方法应用于正常精子标本的冷冻保存，并与传统的慢速冷冻方法进行了比较。结果在总运动恢复率（77.8±11.2% vs 56.6±11.9%，P < 0.01）、进行性运动恢复率（77.6±13.2% vs 47.7±14.6%，P < 0.01）方面优于对照组。 )、24 小时存活指数（60.9 ± 13.4% 对 42.1 ± 14.1%，P < 0.01）和精子 DNA 片段指数（4.2 ± 3.7% 对 5.8 ± 3.7%，P = 0.126）。该方法适用于少精子症标本。超速冻组15例中有12例可见活动精子，而对照组仅7例。