Ebrahimi Bahareh, Keshtgar Sara
Department of Physiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Iran J Med Sci. 2020 May;45(3):188-198. doi: 10.30476/ijms.2019.45787.
Sperm cryopreservation-thawing process has damaging effects on the structure and function of sperm, namely cryoinjury. Calcium overload has been reported as a postulated mechanism for sperm damage during the first steps after thawing. This study was designed to assess the intracellular calcium (Ca ) after cryopreservation and to clarify the role of a calcium chelator ethylene glycol-bis (2-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) on human sperm quality.
Forty semen samples were obtained from fertile men (March 2017 to 2018). The samples were randomly divided into fresh (F) and cryopreserved-thawed (CT) groups. The F and CT samples were divided into control and 1 mM EGTA-treated groups. Sperm kinematics and membrane integrity were assessed. The reactive oxygen species (ROS) and adenosine triphosphate (ATP) were measured by luminescent methods. Ca , apoptotic rate, and mitochondrial membrane potential (MMP) were evaluated using flow cytometric methods. Data were compared using SPSS software, version 16.0 by ANOVA and Kruskal-Wallis test. P<0.05 was considered as significant.
Cryopreservation decreased sperm motility, viability, membrane integrity, Ca , MMP, and induced cell apoptosis and ROS production. EGTA could not protect the cryopreserved sperm from cryoinjury. It was found to have destructive effects on fresh sperm motility and viability (P=0.009) relative to cryopreserved sperm. ATP was reduced (P=0.02) and ROS production (P=0.0001) was increased in the EGTA-treated F and CT sperms.
Despite Ca reduction by EGTA, it had no protective effects on fresh or cryopreserved sperm. We concluded that sperm cryoinjury was not dependent on calcium overload, and it was suggested that cryoinjury was mainly related to cell membranes damage.
精子冷冻-解冻过程会对精子的结构和功能产生损害作用,即冷冻损伤。钙超载被报道为解冻后最初几步中精子损伤的一种假定机制。本研究旨在评估冷冻保存后的细胞内钙(Ca),并阐明钙螯合剂乙二醇双(2-氨基乙基醚)-N,N,N',N'-四乙酸(EGTA)对人类精子质量的作用。
从2017年3月至2018年的可育男性中获取40份精液样本。样本被随机分为新鲜(F)组和冷冻保存-解冻(CT)组。F组和CT组样本再分为对照组和1 mM EGTA处理组。评估精子运动学和膜完整性。通过发光法测量活性氧(ROS)和三磷酸腺苷(ATP)。使用流式细胞术方法评估Ca、凋亡率和线粒体膜电位(MMP)。数据使用SPSS软件16.0版通过方差分析和Kruskal-Wallis检验进行比较。P<0.05被认为具有显著性。
冷冻保存降低了精子活力、存活率、膜完整性、Ca、MMP,并诱导细胞凋亡和ROS产生。EGTA不能保护冷冻保存的精子免受冷冻损伤。相对于冷冻保存的精子,发现其对新鲜精子活力和存活率有破坏作用(P=0.009)。EGTA处理的F组和CT组精子中ATP降低(P=0.02),ROS产生增加(P=0.0001)。
尽管EGTA可降低Ca,但它对新鲜或冷冻保存的精子均无保护作用。我们得出结论,精子冷冻损伤不依赖于钙超载,提示冷冻损伤主要与细胞膜损伤有关。