Reactive oxygen species measurement in neat and washed semen: comparative analysis and its significance in male infertility assessment

Abstract

Oxidative stress (OS) is a major concern in idiopathic male infertility as elevated levels of reactive oxygen species (ROS) in the semen is believed to adversely affect sperm functional competence and damage both nuclear and mitochondrial DNA. Therefore, identifying infertile men with seminal OS may be used as a valuable tool in the assessment of male infertility. Semen is a complex mixture of spermatozoa and seminal plasma which is rich in enzymatic and non-enzymatic antioxidants. However, the measurement of ROS in the semen and its application in male infertility assessment is poorly understood. The aim of the present study was to compare the significance of ROS measurement in washed and neat semen. The study included 65 infertile men with abnormal semen (SA) parameters, 17 infertile men with normal semen (NS) parameters and 43 fertile controls (FC). ROS levels in both washed and neat semen were measured by luminol-dependent chemiluminescence assay and the values were expressed as 10(4)RLU per minute per 20 million spermatozoa. The levels of ROS in both washed and neat semen were found to be significantly higher (P<0.0001) in infertile men with abnormal and normal semen parameters as compared with FC (P<0.0001). The lowest median level of ROS was found in FC (neat semen 0.03, washed semen 0.24), whereas infertile men with SA group had the highest median ROS level (neat semen 3.44, washed semen 27.42). In all subjects, ROS levels were found to be higher in washed semen than in neat semen. A strong positive correlation (r=0.847, P<0.0001) of ROS levels between neat and washed semen was observed. Measurement of ROS in neat semen reflects the original oxidative status in the actual sperm microenvironment and is an easy, simple, inexpensive and rapid method compared with ROS measurement from washed semen. ROS measurement in washed semen is done to assess ROS levels following sperm processing as in cases opting for assisted conception. As both ROS values in neat and washed show a positive correlation, neat semen may be used as a valuable tool for identifying infertile men with seminal OS. ROS levels are elevated in nearly 70% men with idiopathic infertility. Hence, ROS analysis in neat semen has both good diagnostic and prognostic value in male infertility assessment.