Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 94976, Nitra, Slovakia.
Unit of Genetics, Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain.
Cell Tissue Bank. 2021 Sep;22(3):379-387. doi: 10.1007/s10561-020-09883-8. Epub 2020 Nov 17.
Among the currently available strategies for sperm freezing, vitrification may be considered as the leading alternative to conventional cryopreservation. Nevertheless, a direct comparison of both techniques with respect to the iatrogenic sperm DNA damage has not been performed yet. As such, this study was focused to assess the static and dynamic behavior of human sperm DNA damage following thawing of cryopreserved or vitrified spermatozoa. Semen samples were obtained from fifty donors with a normal spermiogram, and divided into four aliquots. The first aliquot represented the neat sample. In the second aliquot the seminal plasma was discarded, and the resulting sperm pellet was resuspended in PBS. The third fraction was used for slow freezing and the fourth fraction was subjected to vitrification. Each set of samples was incubated at 37 °C for 24 h and sperm DNA damage (SDF) was assessed using the chromatin-dispersion test following 0 h, 2 h, 4 h and 24 h of incubation. When comparing the rate of DNA fragmentation (r-SDF) at 2 h, significant differences were observed between the PBS group, cryopreserved (p .000) or vitrified semen (p .015). Furthermore, the sperm longevity comparison using Kaplan-Meier survival curves revealed significant differences between cryopreservation and vitrification (p .000). Our data suggest that exposure of spermatozoa to low temperatures, independently of the chosen freezing protocol, leads to a higher susceptibility of sperm DNA towards damage. This damage is nevertheless lower following vitrification in comparison to traditional cryopreservation. As vitrification leads to a smaller proportion of spermatozoa with DNA damage, we may recommend its use in reproductive techniques which rely on a longer sperm survival, such as artificial insemination.
在目前可用的精子冷冻策略中，玻璃化可能被认为是传统冷冻保存的首选替代方法。然而，这两种技术在医源性精子 DNA 损伤方面的直接比较尚未进行。因此，本研究旨在评估冷冻或玻璃化解冻后人类精子 DNA 损伤的静态和动态行为。从 50 名具有正常精子图谱的供体中获得精液样本，并将其分为四等份。第一份代表原始样本。在第二份中，丢弃精液中的精浆，将得到的精子沉淀重新悬浮在 PBS 中。第三部分用于缓慢冷冻，第四部分进行玻璃化处理。每组样本在 37°C 下孵育 24 小时，并在孵育 0 小时、2 小时、4 小时和 24 小时后使用染色质分散试验评估精子 DNA 损伤（SDF）。当比较 2 小时时的 DNA 碎片化率（r-SDF）时，PBS 组、冷冻（p.000）或玻璃化精液（p.015）之间观察到显著差异。此外，使用 Kaplan-Meier 生存曲线比较精子寿命显示，冷冻保存和玻璃化处理之间存在显著差异（p.000）。我们的数据表明，无论选择哪种冷冻方案，精子暴露于低温都会导致精子 DNA 更容易受到损伤。然而，与传统冷冻保存相比，玻璃化处理后 DNA 损伤的精子比例较低。由于玻璃化处理导致具有 DNA 损伤的精子比例较小，因此我们可能建议在依赖精子更长生存时间的生殖技术中使用玻璃化处理，例如人工授精。
