REACTIVE OXYGEN SPECIES GENERATION BY SEMINAL CELLS DURING CRYOPRESERVATION

Objectives. 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, whic h may contribute to poor sperm function following cryopreservation. Me thods. Eighteen semen specimens with normal parameters from healthy ma le donors 22 to 40 years of age were each divided into two portions. T he first portion was combined 1:1 with Test Yolk Buffer-Glycerol Freez ing Medium and was frozen by gradual cooling into liquid nitrogen (-19 6 degrees C). The second portion was washed and the cells were resuspe nded in Sperm Washing Medium (SWM) and incubated at room temperature t o serve as controls. After a period of treatment, frozen samples were thawed and semen cells were washed and resuspended in SWM. ROS generat ion by semen cells from each treatment group was measured on a luminom eter. Sperm motility, sperm viability, and sperm membrane integrity we re also measured in both control and freeze-thaw samples. To further a ssess ROS generation by semen cells during the cooling process, aliquo ts of washed semen cells and purified polymorphonuclear leukocytes (PM Ns) 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. Re sults. In both semen cells and PMNs, ROS activity increased significan tly during the cooling process. The highest ROS levels were recorded i n both groups when cooled to 4 degrees C. The ROS levels were extremel y low in samples cooled to -20 degrees C and in freeze-thaw samples, p robably due to marked loss of cell viability. Conclusions. Gradual red uction of temperature during the process of semen cryopreservation can cause a significant ROS generation by semen cells. ROS is particularl y 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 s perm viability and function. (C) 1997, Elsevier Science Inc.