Bolu Abant Izzet Baysal University Medical Faculty, Histology and Embryology Department, Bolu, Turkey.
Acta Histochem. 2019 Oct;121(7):798-803. doi: 10.1016/j.acthis.2019.07.006. Epub 2019 Jul 22.
The study consisted of semen samples of 20 male individuals who applied to Abant Izzet Baysal University Faculty of Medicine and participated in a spermiogram. The aim of this study was to determine how to obtain the healthiest spermatozoa by employing a variety of swim-up methods over differing time periods and without the use of centrifuge. Ejaculate samples were taken from the 20 patients and each patient's homogenized semen sample was divided into 4 groups without centrifugation. Group 1 was taken as the sample of untreated semen. For the other 3 groups, 250 μl of medium was added in the semen samples. Afterwards, the samples were kept at 37 °C for different time periods, 30 min for Group 2, 60 min for Group 3 and 90 min for Group 4 in order for the spermatozoa to swim to the media in the upper layer. At the end of the periods, 10 μl of propagation preparations were prepared from the swim-up fluid. Using Aniline Blue for chromatin condensation analysis, two hundred cells were immunostained by Caspase 3 for apoptotic analysis. Subsequently, the result of the four groups were compared for each test. The spermatozoa obtained at the end of the 30 min. of swim-up was compared to the spermatozoa obtained from the swim-up of 60 min., the swim-up of 90 min. It was found that the control group had statistically significant lower rates of apoptosis and was healthier in terms of chromatin integrity. The swim-up method without centrifugation is the best suited sperm preparation, based on sperm DNA integrity and sperm chromatin condensation.
本研究纳入了 20 名申请阿布山伊泽特·贝萨尔大学医学院并接受精液分析的男性个体的精液样本。本研究旨在确定如何通过采用多种不同时间的泳动方法并避免使用离心来获得最健康的精子。从 20 名患者中采集精液样本，每个患者的匀浆精液样本未经离心分为 4 组。第 1 组为未经处理的精液样本。对于其他 3 组，在精液样本中加入 250μl 培养基。然后，将样本在 37°C 下保持不同时间：第 2 组为 30 分钟，第 3 组为 60 分钟，第 4 组为 90 分钟，以使精子游向上层培养基。在各时间段结束时，从泳动液中制备 10μl 繁殖准备液。使用苯胺蓝进行染色质凝聚分析，用 Caspase 3 对 200 个细胞进行免疫染色以进行凋亡分析。随后，对每个测试的四个组的结果进行比较。与 60 分钟和 90 分钟泳动获得的精子相比，在 30 分钟泳动结束时获得的精子。结果发现，对照组的凋亡率具有统计学显著差异，并且在染色质完整性方面更健康。基于精子 DNA 完整性和精子染色质凝聚，无需离心的泳动方法是最适合的精子制备方法。
