Reactive oxygen species in human semen Validation and qualification of a chemiluminescence assay

Reactive oxygen species can be measured accurately and reliably with the use of a simple luminol- based chemiluminescence assay. This test can be incorporated into routine testing of human semen for male infertility.

Wayne Vessey, B.Sc., Ana Perez-Miranda, Ph.D., Rachel Macfarquhar, B.M.L.S., Ashok Agarwal, Ph.D., Sheryl Homa, Ph.D.

Volume 102, Issue 6, Pages 1576-1583


To standardize and validate an assay for reactive oxygen species (ROS) in human semen.

ROS levels assayed in blanks, negative and positive control samples (30% H2O2), and human semen, with the use of a luminol-based chemiluminescence assay measured in a single tube luminometer.

Andrology laboratory.

Semen samples from 19 men attending for routine semen analysis.


Main Outcome Measure(s):
ROS levels reported in relative light units (RLU) per second, adjusted for sperm concentration.

The ROS assay equipment performed according to expectations, generating a chemiluminescence signal for positive control samples and semen samples that decayed rapidly and was captured within 10 minutes. Blanks and negative control samples gave negligible readings. There was no significant intra- or interassay variation. Interference from extraneous factors was negligible. The assay distinguished changes in ROS over a wide range of concentrations and provided consistent results between reagent batches. Working reagents remained stable for 3 months. Acceptable levels for negative and positive control samples were established to set criteria for the test passing or failing on any given day. The assay was sensitive to ambient temperature >25°C. ROS declined significantly with time after ejaculation. Mechanical agitation doubled ROS production in semen.

These results validate the ROS assay and demonstrate that it is a highly reliable and accurate diagnostic test.

  • msamplaski

    It is useful to have an alternative test for ROS assessment. However, what this begs is to determine the role of ROS testing in the assessment of the infertile male. At present it is unclear if assessment of ROS will impact therapy, aside from treatment of causal factors and the initiation of oral antioxidant therapy. While compelling, the authors need to demonstrate how this test is superior to currently used tests, and how it will change clinical management.

    • Sheryl Homa

      ROS testing is a very useful adjunct to standard semen
      analysis for male infertility, since it has been shown to have a negative
      effect on sperm parameters, molecular events involved in fertilisation and
      fertility outcome. It is important to identify the causal factors leading to oxidative
      stress in each patient with high ROS levels, in order to manage it effectively.
      For example, infections and varicocoele as well as certain lifestyle factors, are
      associated with oxidative stress and patients should be investigated
      accordingly. Both antibiotic treatment and varicocoele repair, in addition to lifestyle
      modification and antioxidant supplementation, may improve semen parameters and sperm
      genetic integrity to the point where assisted conception treatment is no longer

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