Detection of oxidative stress in seminal plasma and fractionated sperm from subfertile male patients

Abstract

Objectives: Oxidative stress in the reproductive system is thought to affect the fertilizing ability of sperm. Since 8-hydroxydeoxyguanosine (8-OHdG) and lipid peroxides are widely used as markers to quantify oxidative stress, we compared 8-OHdG and lipid peroxide concentrations in seminal plasma and spermatozoa from subfertile and fertile men. Study design: Semen obtained from 37 men of subfertile couples (21 men with normozoospermia and 16 with asthenozoospermia) and from eight fertile volunteers were examined. Seminal plasma and spermatozoa were fractionated by four-step discontinuous Percoll gradient centrifugation. 8-OHdG in seminal plasma was measured by ELISA, and lipid peroxides in seminal plasma and spermatozoa were determined using a thiobarbituric acid (TBA) assay. Results: The concentrations of 8-OHdG and lipid peroxides in the seminal plasma of the subfertile group were significantly higher than those of the fertile group. There were no significant differences in these values between patients with normozoospermia and asthenozoospermia. In all four fractions obtained by Percoll gradient fractionation, the lipid peroxide levels in spermatozoa recovered from subfertile males were significantly higher than those of fertile controls. Conclusions: Seminal plasma and spermatozoa from subfertile males showed elevated levels of oxidative stress that were detectable in ejaculated semen specimens by ELISA or TBA assay. Even the spermatozoa fraction considered to be mature and normal showed elevated oxidative stress in the subfertile group. Our results confirm the importance of oxidative stress in male reproductive function, and could be applied for the selection of patients for antioxidant therapy.