Figure 1.
RF-EMR exposure decreases motility and vitality of human sperm while also inducing intracellular ROS.
Percoll-purified spermatozoa (5×106 cells) were suspended in 1 ml BWW in a 35 mm Petri dish and placed within the waveguide while control cells placed outside the waveguide. A frequency of 1.8 GHz at a SAR of 27.5 W/kg was used and all samples were incubated for 16 h at 21°C. A, Sperm vitality was significantly reduced from the control value of 82%±4% to 29%±4% for the exposed cells (***p<0.001). B, Sperm motility was also significantly reduced from the control value of 82%±4% to 28%±1% (**p<0.01). C, ROS production was increased after RF-EMR exposure such that 28%±1% of the cells were producing ROS, while only 7%±0.4% of the controls contributed to ROS production (***p<0.001). D, 24%±1% of the exposed cells generated mitochondrial ROS, while the only 12%±1% of the control cells produced ROS from this source (***p<0.001). All results are based on 4 independent samples.
Figure 2.
RF-EMR exposure reduces motility and vitality of human spermatozoa, in an SAR dependent manner.
Percoll-purified spermatozoa (5×106 cells) were suspended in 1 ml BWW in a 35 mm Petri dish and placed within the waveguide while control cells (closed circles) were placed outside the waveguide. Cells in the waveguide were exposed to 1.8 GHz RF-EMR at SAR levels of 0.4, 1.0 2.8 4.3 10.1 and 27.5 W/kg (open circles) for 16 h at 21°C. Both vitality and motility were reduced in a dose dependent manner. A, Vitality was significantly reduced at a SAR of 1.0 W/kg from 89%±3% to 65%±1% (**p<0.01). B, Motility was also significantly reduced at a SAR of 1.0 W/kg from 86%±2% to 68%±2% (*p<0.05). All results are based on 4 independent samples.
Figure 3.
RF-EMR induces ROS generation in human spermatozoa, in an SAR-dependent manner unrelated to thermal effects.
Percoll-purified spermatozoa (5×106 cells) were suspended in 1 ml BWW in a 35 mm Petri dish and placed within the waveguide while control cells placed outside the waveguide (closed circles). Cells in the waveguide were exposed to 1.8 GHz RF-EMR at SAR levels between 0.4 and 27.5 W/kg (open circles) for 16 h at 21°C. Also, purified sperm cells were subjected to incubation temperatures ranging from 21°C–50°C for 2 h. As the power levels were increased, the cellular generation of ROS increased in a dose-dependent manner. ROS levels were also observed to increase as a result of incubation temperature, but such results were not significant until the temperature exceeded 40°C. A, ROS generation (DHE response) was significantly increased from control levels after exposure to 1.0 W/kg (*p<0.05) and above (***p<0.001). B, RF-EMR induces ROS generation by the sperm mitochondria as monitored by MSR; significant increases were observed at SAR values of 2.8 W/kg (***p<0.001) and above. All results are based on 4 independent samples. C, In order to control for thermal effects, the impact of temperature of cellular ROS generation was monitored; a significant increase in ROS generation was observed as temperatures rose above 40°C (p<0.001). D, Across the entire data set, the total level of ROS generation by human spermatozoa (DHE positive cells) was highly correlated with the level of ROS generation by the mitochondria (MSR positive cells: R2 = 0.823).
Figure 4.
RF-EMR induces oxidative DNA damage in human spermatozoa.
Following Percoll fractionation, 5×106 high density, spermatozoa were suspended in 1 ml BWW. The cells were placed in 35 mm Petri dishes and placed inside a waveguide. 5×106 cells in 1 ml BWW were placed outside the waveguide as a control (closed circle). The cells in the waveguide were exposed to 1.8 GHz RF-EMR at SAR levels between 0.4 and 27.5 W/kg (open circles) and all samples were incubated for 16 h at 21°C. Following incubation, Fe2+ and H2O2 was added to cells to act as a positive control, incubated for 1 h, then 100 µl 2 mM DTT/BWW solution was added and incubated for 45 min at 37°C. Cells were fixed and labeled with 100 µl charcoal purified anti-8-OH-dG, FITC tagged antibody at a dilution of 1∶50, incubated at 21°C for 1 h, washed and then assessed by flow cytometry. A, As the power levels were increased, the amount of oxidative DNA damage expressed also increased. A significant amount of oxidative DNA damage was observed in cells exposed to 2.8 W/kg (*p<0.05) RF-EMR and above (**p<0.01; ***p<0.001). Results are based on 4 independent samples. B, The levels of 8-OH-dG expression were positively correlated with the levels of ROS generation by the mitochondria (R2 = 0.727).
Figure 5.
RF-EMR induces DNA fragmentation in human spermatozoa.
Following Percoll fractionation, 5×106 high density spermatozoa were resuspended in 1 ml BWW, pipetted into 35 mm Petri dishes and placed inside a waveguide. 5×106 cells in 1 ml BWW were placed outside the waveguide as a control (closed circle). The cells in the waveguide were exposed to 1.8 GHz RF-EMR at SAR levels between 0.4 and 27.5 W/kg (open circles) and all samples were incubated for 16 h at 21°C. Following incubation, cells were fixed; DNase-I was used as a positive control. After 1 h incubation at 37°C, 50 µl of label and enzyme master mixes were added to the cells and incubated for 1 h at 37°C. Cells were then washed and assessed by flow cytometry. A, Significant levels of DNA fragmentation was observed in exposed spermatozoa at 2.8 W/kg (*p<0.05) and above (***p<0.001). B, DNA fragmentation was positively correlated with ROS production by the mitochondria as monitored by MSR (R2 = 0.861). C, 8-OH-dG was also positively correlated with DNA fragmentation (R2 = 0.725). Results are based on 4 independent samples.