Objectives: Excessive formation of reactive oxygen species (ROS) in human semen has been associated with impaired sperm function and fertility potential. The presence of ROS in semen specimens from normal fertile men emphasizes the importance of defining a normal range of ROS formation. The purpose of this study was to establish a normal range of ROS generation and to investigate the effect of sperm concentration on the ROS level.
Methods: ROS was determined in 15 healthy donors and 20 men with suspected infertility. After the sperm concentration in normal donors was adjusted to 20 x 10(6)/mL, ROS was measured by chemiluminescence using luminol in a Berthold luminometer. A specimen was regarded as positive (abnormal) when the value was at least 10 x 10(4) counts per minute (cpm). ROS was also evaluated at 4 sperm concentrations (60, 30, 15, and 7.5 x 10(6)/mL) from samples obtained from the patients with suspected infertility. In addition, ROS was measured in 7 ROS-positive specimens at a sperm concentration of 15 x 10(6)/mL and 60 x 10(6)/mL.
Results: Results showed that ROS formation was negative in all 15 healthy donors (median, 0.9 x 10(4) cpm; interquartile range, 0 to 1.48 x 10(4) cpm). The ROS formation value among all the donors was less than 5.5 x 10(4) cpm. ROS formation was positive in 8 (40%) of the suspected infertile patients. ROS levels were significantly lower at sperm concentrations of 15 x 10(6)/mL or 7.5 x 10(6)/mL compared with 30 x 10(6)/mL or 60 x 10(6)/mL (P = 0.05). The ROS level increased after centrifugation for 10 minutes at 500 g in all 7 specimens at both 15 and 60 x 10(6)/mL. However, the increase in ROS formation at 60 x 10(6)/mL was significantly greater than that at 15 x 10(6)/mL (P < 0.001).
Conclusions: A range of ROS formation of 0 to 5.5 x 10(4) cpm at a sperm concentration of 20 x 10(6)/mL may be considered as normal for healthy donor semen. The positive relationship between ROS formation and sperm concentration at the time of measurement emphasizes the importance of concentration adjustment before analysis when comparing ROS levels between different specimens.