Information about Reactive Oxygen Species and Male Infertility

Almost 50% of all cases of infertility may be associated with a male factor. Although a semen analysis has classically been used as the gold standard for determining a man’s fertility, this test may not detect abnormalities at the molecular level that may contribute to the 25% of cases that remain unexplained. 

Reactive Oxygen Species (ROS)

There is now growing evidence to support a link between oxidative stress and male infertility. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radicals (OH-) and superoxide anions (O2-) are generated by human sperm as part of their normal metabolism. At low levels, ROS enhance sperm capacitation and hyperactivation, as well as promoting the acrosome reaction and binding to the oocyte zona pellucida. ROS are maintained at low levels by effective anti-oxidant pathways, both within the sperm cytoplasm and more significantly in the seminal plasma, where high levels of ROS scavengers are found. However, if the production of ROS overwhelms the capacity of these anti-oxidant pathways to maintain appropriate low levels, then oxidative stress occurs, leading to pathological effects. Leukocytes in semen are a major source of ROS production, while cytoplasmic droplets retained on immature sperm are also a source of ROS production.



Effects of ROS-induced Oxidative Stress on Sperm

  • Lipid peroxidation which damages the sperm surface causing an abnormal morphology and impaired motility.
  • Damage to proteins on cell surface responsible for cell signalling and may affect enzyme function inside the cell.
  • Increased semen viscosity.
  • Peroxidation of DNA and subsequent unravelling or fragmentation.
  • Possible mutagenic effects.
  • Damage to seminiferous epithelium, damage to tubules, testicular atrophy, reduced spermatogenesis.
  • Decrease in sperm vitality.
  • Impaired fertilization by affecting sperm capacitation and the acrosome reaction.


Causes of Elevated ROS Levels

  • Genito-urinary tract infection
  • Prostatitis
  • Vasectomy reversal
  • Varicocoele
  • Cryptorchidism
  • Chronic disease 
  • Xenobiotics
  • Chemical pollutants and occupational hazards
  • Heavy metal exposure
  • Removal of seminal plasma during sperm preparation for assisted conception
  • Drugs – cyclophosphamide, aspirin, paracetamol
  • Smoking 
  • Alcohol
  • Excessive exercise 
  • Heat exposure
  • Obesity
  • Age


Seminal Oxidative Stress and Infertility

Recent studies show that there is a significant increase in ROS levels and a reduced antioxidant capacity in infertile men compared with fertile controls, irrespective of semen parameters. Indeed, 25 - 40% of infertile men have high levels of ROS. High ROS levels impair fertilisation, adversely affect blastocyst development and negatively affect pregnancy rates after IVF. Furthermore, elevated seminal ROS levels are correlated with an increased time to natural conception.


Oxidative Stress and Testicular Dysfunction

Approximately 45% of male fertility patients are diagnosed with either varicocoele, cryptorchidism, testicular torsion or endocrine imbalance. All of these conditions are associated with oxidative stress which can impair blood flow and increase germ cell apoptosis resulting in oligozoospermia. 


Genito-urinary Tract Infection

Large numbers of leukocytes are found in semen as a result of genito-urinary tract infection. Sperm are vulnerable to ROS attack in the testicular environment as they are not in contact with the protective antioxidants of the seminal plasma, and are particularly susceptible during epididymal transit – especially if there is inflammation from genital tract infection. A large randomised study compared men with Chlamydia or Ureaplasma infection with and without antibiotics for 3 months. Those treated showed a significant fall in ROS levels, improved sperm motility and a significant increase in pregnancy rates. 

Indications for Male Patients Who May Benefit From ROS Measurement:

  • Unexplained infertility
  • Poor sperm motility
  • Presence of high levels of immature sperm retaining cytoplasmic droplets
  • Increased semen viscosity
  • Poor fertilisation with IVF
  • Poor blastocyst development in IVF with no known female cause
  • Decreased pregnancy rate
  • Multiple failed IVF / ICSI treatment
  • Advanced age
  • Exposure to harmful substances
  • Men with prostatitis
  • Men with varicocoele

The ROS Test 

The Reactive Oxygen Species (ROS) test is a relatively simple chemiluminescence test requiring a fresh semen sample produced by masturbation after 2 – 3 days sexual abstinence. Measurement of ROS is performed in whole semen within 15 - 30 minutes of ejaculation. ROS are  measured indirectly using a probe such as luminol, which is oxidised in the presence of ROS, resulting in chemiluminescence.  The luminescence generated by this reaction is measured using a CE marked luminometer. The result can be reported within 24 hours.  The test can be requested alone or in conjunction with a semen analysis or any other test for male reproductive health. 


Management of High ROS Levels 

Identification of infertile patients who demonstrate oxidative stress in their semen may assist in the management of male infertility. Increased levels of free radicals may be reduced with a change in lifestyle and a diet rich in anti-oxidants, designed to protect against oxidative stress.  Randomised placebo controlled studies have shown that oral anti-oxidant treatment can decrease seminal ROS levels and sperm DNA damage, and improve pregnancy rates. Treatment of infection would also be expected to reduce ROS levels.  Initiatives to reduce the levels of ROS can be assessed by undertaking a second test three months after the first. If levels remain high, a sperm DNA fragmentation test may be considered. 




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