title: Reactive oxygen species in human semen: validation and qualification of a chemiluminescence assay.
abstract: OBJECTIVE
  To standardize and validate an assay for reactive oxygen species (ROS) in human semen.
  
  
  METHODS
  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.
  
  
  METHODS
  Andrology laboratory.
  
  
  METHODS
  Semen samples from 19 men attending for routine semen analysis.
  
  
  METHODS
  None.
  
  
  METHODS
  ROS levels reported in relative light units (RLU) per second, adjusted for sperm concentration.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  These results validate the ROS assay and demonstrate that it is a highly reliable and accurate diagnostic test.
authors.affiliation: Andrology Department, The Doctors Laboratory, London, United Kingdom.
authors.forename: Wayne
authors.initials: W
authors.lastname: Vessey
authors.affiliation: Andrology Department, The Doctors Laboratory, London, United Kingdom.
authors.forename: Ana
authors.initials: A
authors.lastname: Perez-Miranda
authors.affiliation: Andrology Department, The Doctors Laboratory, London, United Kingdom.
authors.forename: Rachel
authors.initials: R
authors.lastname: Macfarquhar
authors.affiliation: Andrology Center, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Ashok
authors.initials: A
authors.lastname: Agarwal
authors.affiliation: Andrology Department, The Doctors Laboratory, London, United Kingdom; Andrology Solutions, London, United Kingdom. Electronic address: s.homa@andrologysolutions.co.uk.
authors.forename: Sheryl
authors.initials: S
authors.lastname: Homa
mesh_terms: D006801:Humans; D006861:Hydrogen Peroxide; D007248:Infertility, Male / Q000175:diagnosis*; D049449:Luminescence; D008163:Luminescent Measurements; D008297:Male; D017382:Reactive Oxygen Species / Q000032:analysis*; D015203:Reproducibility of Results; D012661:Semen / Q000737:chemistry*; D055101:Semen Analysis
chemical_list: D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide
keywords: Reactive oxygen species; chemiluminescence; human semen; male infertility; oxidative stress