Takeshima Teppei, Yumura Yasushi, Kuroda Shinnosuke, Kawahara Takashi, Uemura Hiroji, Iwasaki Akira
a Department of Urology, Reproductive Center , Yokohama City University Medical Center , Yokohama City , Kanagawa , Japan.
b Department of Urology and Renal Transplantation , Yokohama City University Medical Center , Yokohama City , Kanagawa , Japan.
Syst Biol Reprod Med. 2017 Jun;63(3):192-198. doi: 10.1080/19396368.2017.1294214. Epub 2017 Mar 23.
Density gradient centrifugation can separate motile sperm from immotile sperm and other cells for assisted reproduction, but may also remove antioxidants from seminal plasma, resulting in oxidative stress. Therefore, we investigated reactive oxygen species (ROS) concentrations and distribution in semen before and after density gradient centrifugation. We assessed semen volume, sperm concentration, sperm motility, and ROS levels before and after density gradient centrifugation (300 x g for 20 minutes) in 143 semen samples from 118 patients. The ROS removal rate was evaluated in ROS-positive samples and ROS formation rate in ROS-negative samples. Thirty-eight of 143 untreated samples (26.6%) were ROS-positive; sperm motility was significantly lower in these samples than in ROS-negative samples (p < 0.05). After density gradient centrifugation, only seven of the 38 ROS-positive samples (18.42%) exhibited a ROS-positive lower layer (containing motile sperm) with a ROS removal rate of 81.58%, whereas the upper layer was ROS-positive in 24 samples (63.16%). In the ROS-negative group (n = 105), ROS was detected in 19 samples after centrifugation (18.10%, ROS generation rate), of which 18 were ROS-positive only in the upper layer or interface and the other was ROS-positive in both layers. Density gradient centrifugation can separate motile sperm from immotile sperm as well as remove ROS (including newly generated ROS). This data supports the view that density gradient centrifugation can select motile spermatozoa without enhancing oxidative stress.
ROS: reactive oxygen species; SOD: superoxide dismutase; GPx: glutathione peroxidase; DNA: deoxyribonucleic acid; DGC: density gradient centrifugation; IUI: intrauterine insemination; IVF: in vitro fertilization; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA: ethylenediaminetetraacetic acid; HTF: HEPES-buffered human tubal fluid; IMSI: intracytoplasmic morphologically selected sperm injection; SMAS: sperm motility analyzing system; CASA: computer-assisted semen analyzer; WHO: World Health Organization.
密度梯度离心可将活动精子与不活动精子及其他细胞分离，用于辅助生殖，但也可能从精浆中去除抗氧化剂，导致氧化应激。因此，我们研究了密度梯度离心前后精液中活性氧（ROS）的浓度及分布情况。我们评估了118例患者的143份精液样本在密度梯度离心（300×g，20分钟）前后的精液体积、精子浓度、精子活力和ROS水平。对ROS阳性样本评估ROS去除率，对ROS阴性样本评估ROS生成率。143份未处理样本中有38份（26.6%）为ROS阳性；这些样本中的精子活力显著低于ROS阴性样本（p<0.05）。密度梯度离心后，38份ROS阳性样本中只有7份（18.42%）下层（含活动精子）为ROS阳性，ROS去除率为81.58%，而24份样本（63.16%）上层为ROS阳性。在ROS阴性组（n = 105）中，离心后19份样本（18.10%，ROS生成率）检测到ROS，其中18份仅在上层或界面为ROS阳性，另1份两层均为ROS阳性。密度梯度离心可将活动精子与不活动精子分离，同时去除ROS（包括新生成的ROS）。这些数据支持这样一种观点，即密度梯度离心可选择活动精子而不增强氧化应激。
ROS：活性氧；SOD：超氧化物歧化酶；GPx：谷胱甘肽过氧化物酶；DNA：脱氧核糖核酸；DGC：密度梯度离心；IUI：宫腔内人工授精；IVF：体外受精；HEPES：4-（2-羟乙基）-1-哌嗪乙磺酸；EDTA：乙二胺四乙酸；HTF：HEPES缓冲的人输卵管液；IMSI：胞浆内形态学选择精子注射；SMAS：精子活力分析系统；CASA：计算机辅助精液分析仪；WHO：世界卫生组织。
