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 30-50% of cases that remain unexplained.

 

ROS image

Reactive Oxygen Species (ROS)

There is now growing evidence to support a link between oxidative stress and male infertility1-3. 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 metabolism4. 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 antioxidant 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 antioxidant pathways to maintain appropriate low levels, then oxidative stress occurs, leading to pathological effects1-4. ROS initiates peroxidation of membrane lipids, proteins and DNA, which leads to the formation of potentially genotoxic and mutagenic adducts, damaging membrane function, ion gradients and receptor-mediated signal transduction as well as interfering with DNA methylation. This affects the fertilisation processas well as causing DNA fragmentation and gene mutations2,3. Manifestations of oxidative stress include semen parameter impairment, particularly a reduction in motility and vitality1,2.

 

Effects of ROS-induced Oxidative Stress on Sperm

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

What are the Causes of Oxidative Stress?

While cytoplasmic droplets retained on immature sperm are a source of excess ROS production, leukocytes in semen are up to a thousand fold more effective at generating ROS7. The major causes of oxidative stress include infections8 and varicocoele9 which is the most common known cause of infertility in men. In addition, there are many lifestyle factors that are responsible for oxidative stress10. A list of the major external sources of ROS are given below.

 

Medical Causes of Oxidative Stress 

  • Genito-urinary tract infection
  • Prostatitis
  • Vasectomy reversal
  • Varicocoele
  • Cryptorchidism
  • Chronic disease 
  • Advanced age
  • Obesity

External Causes of Oxidative Stress

  • Xenobiotics
  • Chemical pollutants and occupational hazards
  • Heavy metal exposure
  • Drugs – cyclophosphamide, aspirin, paracetamol, recreational
  • Smoking 
  • Alcohol
  • Excessive exercise 
  • Heat exposure

 

Oxidative Stress and Testicular Dysfunction

Approximately 45% of male fertility patients are diagnosed with varicocoele, cryptorchidism, testicular torsion or endocrine imbalance. All of these conditions are associated with oxidative stress and impaired blood flow which in turn can increase germ cell apoptosis resulting in oligozoospermia1,9,11. Sperm are vulnerable to ROS attack in the testicular environment as they are not in contact with the protective antioxidants of the semen.

 

Genitourinary Tract or Male Accessory Gland Infection (MAGI)

Large numbers of leukocytes are found in semen as a result of genitourinary tract infection or MAGI8,12. Sperm are particularly susceptible to ROS attack once they leave the testes especially during epididymal transit – especially if there is inflammation resulting from infection.

 

Seminal Oxidative Stress and Infertility

Several 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 their semen parameters1-4. Indeed, high levels of ROS have been observed in 30 – 80% of infertile men. High ROS levels impair semen parameters particularly motility and vitality2 as well as affecting fertilisation3,4 and blastocyst development. Oxidative stress is associated with reduced pregnancy rates after IVF5and is correlated with an increased time to natural conception3. There is also an association between oxidative stress levels and recurrent miscarriage6.

 

Indications for Male Patients Who May Benefit From ROS Measurement:

  • Unexplained infertility
  • Poor sperm motility or vitality
  • 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
  • Multiple failed IVF / ICSI treatment
  • Advanced age
  • Exposure to harmful substances
  • Men with prostatitis
  • Men with varicocoele

How Is Oxidative Stress Measured? 

There are two tests we use to measure oxidative stress:


1. Reactive Oxygen Species (ROS) Assay 13,14
The ROS test is a relatively simple chemiluminescence test. 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 luminometer. The test has been fully validated and is CE marked.


2. MiOXSYS assay 15
This is an electrochemical assay that takes into consideration the balance between the total oxidants and antioxidants in the semen. The result gives the oxidation reduction potential of the sample. This test is also CE marked.

 

Both tests require a fresh semen sample produced by masturbation after 2 – 3 days sexual abstinence. Measurement is performed in semen within 15 - 30 minutes of ejaculation. The result can be reported within 24 hours. The tests 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, increasing the chances of natural conception and reducing the need for assisted conception treatment. Varicocoele is a major cause of male infertility and is associated with oxidative stress. Current evidence shows that repair of clinical varicocoele not only significantly improves semen parameters and reduces oxidative stress and DNA damage, but also increases pregnancy rates, both naturally and with assisted conception16. Thus information about oxidative stress levels in men with varicocoele may aid urologists in the decision to perform varicocoele repair. Treatment of infections would also be expected to reduce ROS levels1,17. A large randomised study compared men with Chlamydia or Ureaplasma infection with and without antibiotics for 3 months17. Those treated showed a significant fall in ROS levels, improved sperm motility and a significant increase in pregnancy rates. If there is no known cause for the oxidative stress, ROS levels may be reduced with a change in lifestyle and a diet rich in anti-oxidants, designed to protect against oxidative stress1,10,18. Randomised placebo controlled studies have shown that oral antioxidant treatment can decrease seminal ROS levels and sperm DNA damage, and improve pregnancy rates1,18. Initiatives to reduce the levels of ROS can be assessed by undertaking a second test three months after the first1. If levels remain high, a sperm DNA fragmentation test may be considered.

 

 

REFERENCES

 

1. Agarwal A, Parekh N, Panner Selvam MK, Henkel R, Shah R, Homa ST et al, (2019) Male Oxidative Stress Infertility (MOSI): Proposed Terminology and Clinical Practice Guidelines for Management of Idiopathic Male Infertility. The World Journal of Men's Health May 8. doi: 10.5534/wjmh.190055. [Epub ahead of print] Review.

 

2. Aitken RJ, Smith TB, Jobling MS, Baker MA, De Iuliis G.N. (2014) Oxidative stress and male reproductive health. Asian J. Androl. 16,31–38

 

3. Tremellen, K (2008) Oxidative stress and male infertility: a clinical perspective. Hum Reprod Update 14, 243–258.

 

4. Aitken RJ (1995) Free radicals, lipid peroxidation and sperm function. Reprod fertile Dev 7(4):659-68.

 

5. Zorn B, Vidmar G and Meden-Vrtovec H (2003) Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection. Int. J Androl. 26 (5): 279-85.

 

6. Jayasena CN, Radia UK, Figueiredo M, Revill LF, Dimakopoulou A, Osagie M, Vessey W, Regan L, Rai R, Dhillo WS(2019) Reduced Testicular Steroidogenesis andIncreased Semen Oxidative Stress in Male Partners as Novel Markers of Recurrent Miscarriage. Clin Chem. 2019 Jan;65(1):161-169.

 

7. Henkel RR (2011) Leukocytes and oxidative stress: dilemma for sperm function and male fertility. Asian J Androl 13: 43–52

 

8. Agarwal A, Rana M, Qiu E, AlBunni H, Bui AD, Henkel R (2018) Role of oxidative stress, infection and inflammation in male infertility. Andrologia. 2018 Dec;50(11):e13126.

 

9.Hamada A, Esteves SC, Agarwal A (2013) Insight into oxidative stress in varococele-associated male infertility: part 2. Nat Rev Urol 10 :26-37

 

10. Wright C, Milne S, Leeson H (2014) Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod Biomed Online 28: 684-703

 

11.Turner TT and Lysiak JJ (2008) Oxidative stress: a common factor in testicular dysfunction. J Androl. 2008 Sep-Oct;29(5):488-98.

 

12. Ihsan AU, Khan FU, Khongorzul P, Ahmad KA, Naveed M, Yasmeen S, Cao Y, Taleb A, Maiti R, Akhter F, Liao X, Li X, Cheng Y, Khan HU, Alam K, Zhou X (2018) Role of oxidative stress in pathology of chronic prostatitis/chronic pelvic pain syndrome and male infertility and antioxidants function in ameliorating oxidative stress. Biomed Pharmacother. 2018 Oct;106:714-723.

 

13. Vessey W, Perez-Miranda A, Macfarquhar R, Agarwal A, Homa S. (2014) Reactive oxygen species (ROS) in human semen: validation and qualification of a chemiluminescence assay. Fertil Steril. 102:1576-1583

 

14. Homa ST, Vessey W, Perez-Miranda A, Riyait T, Agarwal A (2015) Reactive oxygen species (ROS) in human semen: determination of a reference range. J Assist Reprod Genet 32(5):757-64

 

15.Agarwal A, Panner Selvam MK, Arafa M, Okada H, Homa S, Killeen A, Balaban B, Saleh R, Armagan A, Roychoudhury S, Sikka S. (2019) Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen Asian J Androl. 29: 1-5

 

16. Yan S, Shabbir M, Yap T, Homa S, Ramsay J, McEleny K and Minhas S (2019) Should the current guidelines for the treatment of varicoceles in infertile men be re-evaluated? Human Fertil. 23: 1-15

 

17. Vicari E (2000) Effectiveness and limits of antimicrobial treatment on seminal leukocyte concentration and related reactive oxygen species production in patients with male accessory gland infection. Hum Reprod. 15(12): 2536-44.

 

18. Showell MG, Mackenzie-Proctor R, Brown J, Yazdani A, Stankiewicz MT and Hart RJ (2014) Antioxidants for male subfertility. Cochrane Database Syst Rev Dec 15;(12):CD007411.