Department of Biology, Faculty of Basic Sciences, Hamedan Branch, Islamic Azad University, Hamedan, Iran.
Chronic Respiratory Diseases Research Center, National Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University Of Medical Sciences, Tehran, Iran.
Cryobiology. 2020 Oct;96:166-174. doi: 10.1016/j.cryobiol.2020.07.001. Epub 2020 Jul 8.
The aim of this study was to examine the effects of alpha lipoic acid (ALA) supplementation during semen cryopreservation on the sperm quality, chromatin integrity, oxidative stress, and expression level of BAX, BCL2, HSP70 and iNOS genes in semen samples obtained from infertile men with asthenoteratozoospermia.
Twenty freshly ejaculated semen samples were cryopreserved with sperm freezing medium supplemented with 0.00, 0.02, 0.05, 0.1, 0.5, and 1 mmol/mL of ALA. The samples were analyzed according to the WHO guidelines before and after freezing. Sperm ROS production level, DNA fragmentation and cryo-capacitation were assessed using flow cytometry, TUNEL assay and chlortetracycline (CTC) test, respectively. Expression level of stress protein (HSP70), pro-apoptotic Bax, anti-apoptotic Bcl-2, and iNOS genes was assessed by real-time PCR assay.
The effective concentrations of ALA (0.02 and 0.5 mM) significantly improved the motility, viability and morphology of the frozen-thawed sperms compared to the control group treated with 0.00 mM of ALA. During cryopreservation, treatment of semen with 0.02 mM of ALA, as the optimal concentration, significantly decreased DNA fragmentation and oxidative stress level (P < 0.05), protected the acrosome integrity, and led to insignificant reduction in BAX gene expression level and significant increase in expression level of BCL2, HSP70, and iNOS genes compared with control group.
Our findings revealed that the adding ALA to semen samples obtained from infertile men with asthenoteratozoospermia plays a significant protective role against cryodamage by preserving the sperm functional parameters.
本研究旨在探讨在精子冷冻保存过程中添加α-硫辛酸（ALA）对弱精子症畸形精子症患者精液质量、染色质完整性、氧化应激以及 BAX、BCL2、HSP70 和 iNOS 基因表达水平的影响。
将 20 份新鲜射出的精液用含有 0.00、0.02、0.05、0.1、0.5 和 1 mmol/ml ALA 的精子冷冻培养基进行冷冻保存。根据世界卫生组织（WHO）的指南，在冷冻前后对样本进行分析。使用流式细胞术评估精子 ROS 产生水平、DNA 碎片化和冷冻诱导顶体反应，使用 TUNEL 测定法和氯四环素（CTC）试验分别评估 DNA 碎片化和冷冻诱导顶体反应。通过实时 PCR 测定法评估应激蛋白（HSP70）、促凋亡 Bax、抗凋亡 Bcl-2 和 iNOS 基因的表达水平。
与 0.00 mmol/ml ALA 处理的对照组相比，有效浓度为 0.02 和 0.5 mmol/ml 的 ALA 可显著提高冷冻-解冻精子的活力、存活率和形态。在冷冻过程中，用 0.02 mmol/ml 的 ALA（最佳浓度）处理精液可显著降低 DNA 碎片化和氧化应激水平（P < 0.05），保护顶体完整性，导致 BAX 基因表达水平显著降低，BCL2、HSP70 和 iNOS 基因表达水平显著升高，与对照组相比无显著差异。
我们的研究结果表明，在弱精子症畸形精子症患者的精液样本中添加 ALA 对冷冻损伤具有显著的保护作用，可保持精子功能参数。
