微量滴注快速冷冻法：一种简单新颖的方法，可用于冷冻严重少精症样本。
Tip-microVapour Fast Freezing: A novel easy method for cryopreserving severe oligozoospermic samples.
机构信息
Unit of Andrology, Women's Endocrinology and Gender Incongruence, Centre for the Prevention, Diagnosis and Treatment of Infertility, Azienda Ospedaliera Universitaria Careggi Hospital, Florence, Italy.
Department of Experimental and Clinical Biomedical Sciences 'Mario Serio', University of Florence, Florence, Italy.
出版信息
Andrology. 2024 May;12(4):862-869. doi: 10.1111/andr.13531. Epub 2023 Sep 14.
BACKGROUND
Sperm cryopreservation is an important procedure for oligozoospermic subjects at risk of azoospermia and after surgical recovery of spermatozoa in non-obstructive azoospermic men. Conventional procedures for sperm cryopreservation might be, however, not suitable for samples with a very low sperm number.
OBJECTIVES
In this pilot study, we investigated the recoveries of sperm motility and viability in severe oligozoospermic subjects (n = 39) after cryopreservation with a tip-microVapour Fast Freezing, a procedure previously developed by our group for men with good semen quality. Sperm DNA fragmentation was also evaluated in a second group of oligozoospermic samples (n = 16).
MATERIALS AND METHODS
We used a Vapour Fast Freezing procedure using 10 μL tips as carrier, and Test Yolk Buffer as freezing medium (tip-microVapour Fast Freezing). In a subset of samples (n = 22), we compared recovery of motility and viability as obtained with tip-microVapour Fast Freezing and with a Vapour Fast Freezing procedure using 500 μL straws. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test.
RESULTS
We found a recovery rate (median [interquartile range]) of 0.29 (0.13-0.41) for progressive motility, 0.30 (0.21-0.52) for total motility and 0.48 (0.29-0.60) for viability. Interestingly, we observed that samples with the poorest motility were apparently less damaged by freezing/thawing. In a subset of samples (n = 22), we directly compared values of viability, progressive motility and total motility by freezing/thawing with tip-microVapour Fast Freezing and Vapour Fast Freezing conducted with 500 μL straws. We found much better values of all sperm parameters in samples after freezing/thawing with tip-microVapour Fast Freezing than with Vapour Fast Freezing in 500 μL straws: that is, progressive motility: 7.00 (3.00-8.50)% versus 2.00 (0.00-4.25)%, p < 0.001; total motility: 12.00 (8.00-16.25)% versus 6.50 (1.00-9.25)%, p < 0.001; viability: 29.75 (23.75-45.25) versus 22.50 (13.75-28.13), p < 0.001, respectively. In the second group of oligozoospermic samples, we found that tip-microVapour Fast Freezing produced lower levels of sperm DNA fragmentation than straws (33.00 [19.75-36.00]% vs. 36.00 [22.75-41.87]%, p < 0.001).
DISCUSSION AND CONCLUSION
Tip-microVapour Fast Freezing appears to be a very promising method to cryopreserve semen samples from severe oligozoospermic patients.
背景
精子冷冻保存对于有发生无精子症风险的少精子症患者以及非梗阻性无精子症患者的精子在手术后恢复是一项重要的程序。然而，常规的精子冷冻保存程序可能不适合精子数量极低的样本。
目的
在这项初步研究中，我们调查了 39 例严重少精子症患者（n=39）的精子活力和活力在经过我们小组先前为精液质量良好的男性开发的尖端微蒸气快速冷冻冷冻保存后的恢复情况。我们还在第二组少精子症样本（n=16）中评估了精子 DNA 碎片化。
材料和方法
我们使用 10 μL 尖端作为载体的蒸气快速冷冻程序，以及测试蛋黄缓冲液作为冷冻介质（尖端微蒸气快速冷冻）。在亚组样本（n=22）中，我们比较了使用尖端微蒸气快速冷冻和使用 500 μL 吸管的蒸气快速冷冻程序获得的运动和活力恢复情况。精子 DNA 碎片化通过精子染色质弥散试验进行评估。
结果
我们发现渐进性运动的恢复率（中位数[四分位数范围]）为 0.29（0.13-0.41），总运动为 0.30（0.21-0.52），活力为 0.48（0.29-0.60）。有趣的是，我们观察到运动能力最差的样本显然受到冷冻/解冻的损伤较小。在亚组样本（n=22）中，我们直接比较了用尖端微蒸气快速冷冻和用 500 μL 吸管进行的蒸气快速冷冻冷冻/解冻的活力、渐进性运动和总运动的值。我们发现，在使用尖端微蒸气快速冷冻冷冻/解冻的样本中，所有精子参数的值都明显好于使用 500 μL 吸管进行的蒸气快速冷冻：即，渐进性运动：7.00（3.00-8.50）%比 2.00（0.00-4.25）%，p<0.001；总运动：12.00（8.00-16.25）%比 6.50（1.00-9.25）%，p<0.001；活力：29.75（23.75-45.25）%比 22.50（13.75-28.13）%，p<0.001。在第二组少精子症样本中，我们发现尖端微蒸气快速冷冻产生的精子 DNA 碎片化水平低于吸管（33.00[19.75-36.00]%比 36.00[22.75-41.87]%，p<0.001）。
讨论和结论
尖端微蒸气快速冷冻似乎是一种很有前途的方法，可以冷冻保存严重少精子症患者的精液样本。
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