Reference values of reactive oxygen species in seminal ejaculates using chemiluminescence assay

A physiological balance exists between seminal reactive oxygen species (ROS) and antioxidant capacity. An overproduction of ROS which exceeds the seminal plasma antioxidant capacity results in oxidative stress (OS). The aim of the present study was to describe a detailed protocol to measure ROS in a diagnostic laboratory and revise our previous cutoff value of ROS in seminal ejaculates in a larger cohort of infertile men and controls with proven and unproven fertility.

A total of 258 infertile men and 92 controls were enrolled in the study. Following initial semen analysis, ROS measurement in whole ejaculates was carried out using luminol-based chemiluminescence assay. Chemiluminescence was measured for 15 min with a Berthold luminometer. Results were expressed as relative light units (RLU/s/10(6) sperm). The test's specificity, sensitivity, and cutoff values were calculated using the receiver operating characteristic (ROC) curve.

Significantly higher ROS levels were seen in infertile men compared to controls (p < 0.001). The optimal cutoff value to differentiate between controls and infertile men was 102.2 RLU/s/10(6) sperm. At this cutoff value, the test was 76.4% sensitive and 53.3% specific. The positive and negative predictive values of the test were 82.1% and 44.5%, respectively. A total of 76.4% infertile population was above this cutoff value compared to 46.7% of controls.

The luminol-based chemiluminescence assay can be used in routine diagnostic screening to test for male infertility diagnosis in a clinical setting. The current ROS cutoff value substantially distinguishes infertile from normal controls. Patients with elevated ROS must be evaluated for the underlying cause of ROS production.