Shekarriz M, DeWire D M, Thomas A J, Agarwal A
Department of Urology, Cleveland Clinic Foundation, OH 44195, USA.
Eur Urol. 1995;28(1):31-5. doi: 10.1159/000475016.
Current techniques of sperm preparation for in vitro fertilization or intrauterine insemination require centrifugation of human semen to separate spermatozoa from the seminal plasma. Centrifugation increases reactive oxygen species (ROS) formation in semen. Moreover, high levels of ROS are associated with sperm membrane injury through spontaneous lipid peroxidation, which may alter sperm function. We investigated the relationship between centrifugation variables (time and g-force) and ROS production to establish an optimal centrifugation protocol for sperm preparation techniques. Semen from 38 men (24 patients and 14 normal volunteers) was evaluated for the formation of ROS before centrifugation and after centrifugation at 200 g for 2 or 10 min and after 500 g for 2 or 10 min. The absence of white blood cells in semen which can also produce ROS was determined with the myeloperoxidase technique (Endtz test). All specimens were negative (< 1 x 10(6)/ml) by the Endtz test. The formation of ROS was measured by chemiluminescence. ROS formation was regarded as high (positive) when the chemiluminescence response was at least 10 x 10(4) counted photons/min (cpm). The sperm concentration in each sample was adjusted to 15-20 x 10(6) cells/ml before analysis. Eight specimens (7 patients and 1 donor) exhibited high levels of ROS before centrifugation. All 8 showed further, significant increases in ROS formation regardless of g-force or time. The increase in ROS was significantly less when semen was centrifuged for 2 as compared to 10 min (p < 0.001). Six specimens previously ROS-negative became ROS-positive after centrifugation for 10 min at 200 and 500g. We conclude that the time of centrifugation is more important than g-force for inducing ROS formation in semen. Based on these results, we recommend a shorter centrifugation period in the preparation of sperm for assisted reproductive techniques.
目前用于体外受精或宫内人工授精的精子制备技术需要对人类精液进行离心，以将精子与精浆分离。离心会增加精液中活性氧（ROS）的形成。此外，高水平的ROS通过自发脂质过氧化与精子膜损伤相关，这可能会改变精子功能。我们研究了离心变量（时间和离心力）与ROS产生之间的关系，以建立精子制备技术的最佳离心方案。对38名男性（24名患者和14名正常志愿者）的精液在离心前以及在200g离心2或10分钟后和500g离心2或10分钟后进行ROS形成评估。用髓过氧化物酶技术（恩茨试验）确定精液中不会产生ROS的白细胞是否存在。所有标本经恩茨试验均为阴性（<1×10⁶/ml）。通过化学发光测量ROS的形成。当化学发光反应至少为10×10⁴计数光子/分钟（cpm）时，ROS形成被视为高水平（阳性）。在分析前将每个样本中的精子浓度调整至15 - 20×10⁶细胞/ml。8个标本（7名患者和1名供体）在离心前表现出高水平的ROS。所有8个标本无论离心力或时间如何，ROS形成均进一步显著增加。与离心10分钟相比，精液离心2分钟时ROS的增加显著更少（p < 0.001）。6个先前ROS阴性的标本在200g和500g离心10分钟后变为ROS阳性。我们得出结论，在诱导精液中ROS形成方面，离心时间比离心力更重要。基于这些结果，我们建议在辅助生殖技术的精子制备中采用更短的离心时间。
