Wang A W, Zhang H, Ikemoto I, Anderson D J, Loughlin K R
Department of Surgery and Fearing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Urology. 1997 Jun;49(6):921-5. doi: 10.1016/s0090-4295(97)00070-8.
To test the hypothesis that conventionally used procedures for semen cryopreservation may cause an increase in the production of reactive oxygen species (ROS) by sperm or by seminal leukocytes, which may contribute to poor sperm function following cryopreservation.
Eighteen semen specimens with normal parameters from healthy male donors 22 to 40 years of age were each divided into two portions. The first portion was combined 1:1 with Test Yolk Buffer-Glycerol Freezing Medium and was frozen by gradual cooling into liquid nitrogen (-196 degrees C). The second portion was washed and the cells were resuspended in Sperm Washing Medium (SWM) and incubated at room temperature to serve as controls. After a period of treatment, frozen samples were thawed and semen cells were washed and resuspended in SWM. ROS generation by semen cells from each treatment group was measured on a luminometer. Sperm motility, sperm viability, and sperm membrane integrity were also measured in both control and freeze-thaw samples. To further assess ROS generation by semen cells during the cooling process, aliquots of washed semen cells and purified polymorphonuclear leukocytes (PMNs) were incubated separately at different temperature conditions (37 degrees C, 22 degrees C, 4 degrees C, and -20 degrees C). ROS activity in each treatment group was measured and compared with each other.
In both semen cells and PMNs, ROS activity increased significantly during the cooling process. The highest ROS levels were recorded in both groups when cooled to 4 degrees C. The ROS levels were extremely low in samples cooled to -20 degrees C and in freeze-thaw samples, probably due to marked loss of cell viability.
Gradual reduction of temperature during the process of semen cryopreservation can cause a significant ROS generation by semen cells. ROS is particularly elevated during cooling if the semen sample is contaminated by more than 0.5 x 10(6) leukocytes. Removal of leukocytes from semen samples or treatment with antioxidants prior to cryopreservation may improve sperm viability and function.
检验如下假设，即传统的精液冷冻保存程序可能会导致精子或精浆白细胞产生的活性氧（ROS）增加，这可能是冷冻保存后精子功能不佳的原因之一。
选取18份来自22至40岁健康男性供者、参数正常的精液标本，每份标本均分为两部分。第一部分与测试卵黄缓冲液 - 无甘油冷冻培养基按1:1混合，通过逐步冷却至液氮（-196℃）进行冷冻。第二部分进行洗涤，细胞重悬于精子洗涤培养基（SWM）中，在室温下孵育作为对照。经过一段时间的处理后，将冷冻样本解冻，精液细胞洗涤并重悬于SWM中。用发光计测量各处理组精液细胞产生的ROS。同时在对照样本和冻融样本中测量精子活力、精子存活率和精子膜完整性。为了进一步评估冷却过程中精液细胞产生的ROS，将洗涤后的精液细胞和纯化的多形核白细胞（PMN）等分试样分别在不同温度条件（37℃、22℃、4℃和 -20℃）下孵育。测量各处理组的ROS活性并相互比较。
在精液细胞和PMN中，冷却过程中ROS活性均显著增加。两组在冷却至4℃时记录到最高的ROS水平。冷却至 -20℃的样本和冻融样本中的ROS水平极低，可能是由于细胞活力明显丧失。
精液冷冻保存过程中温度的逐渐降低可导致精液细胞产生大量ROS。如果精液样本被超过0.5×10⁶白细胞污染，冷却过程中ROS会特别升高。在冷冻保存前从精液样本中去除白细胞或用抗氧化剂处理可能会提高精子活力和功能。
