Reactive oxygen species measurement in neat and washed semen: comparative analysis and its significance in male infertility assessment
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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 104 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.
KeywordsReactive oxygen species (ROS) Neat semen Washed semen Oxidative stress Male infertility
The authors thank All India Institute of Medical Sciences (AIIMS) and Indian Council of Medical research (ICMR) for their financial support. S. Venkatesh is a senior research fellow awarded by ICMR.
Conflict of interest
We declare that we have no conflict of interest.
- 15.Kobayashi H, Gil-Guzman E, Mahran AM, Sharma RK, Nelson DR, Thomas AJ Jr, Agarwal A (2001) Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay. J Androl 22(4):568–574Google Scholar
- 17.Athayde KS, Cocuzza M, Agarwal A, Krajcir N, Lucon AM, Srougi M, Hallak J (2007) Development of normal reference values for seminal reactive oxygen species and their correlation with leukocytes and semen parameters in a fertile population. J Androl 28(4):613–620. doi: 10.2164/jandrol.106.001966 CrossRefPubMedGoogle Scholar
- 19.(1999) WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction, 4th edn. Cambridge University Press, CambridgeGoogle Scholar
- 21.Mahfouz R, Sharma R, Thiyagarajan A, Kale V, Gupta S, Sabanegh E, Agarwal A (2010) Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil Steril. doi: 10.1016/j.fertnstert.2009.12.030
- 23.Dada R (2010) Recurrent pregnancy loss: male factor. In: Deka D, Malhotra N (eds) An introduction to genetics and fetal medicine. Federation of obstetrics and gynecological society of India publication. Jaypee Brothers, New Delhi, pp 27–37Google Scholar
- 27.McLeod J (1943) The role of oxygen in the metabolism and motility of human spermatozoa. Am J Physiol 138:512–518Google Scholar
- 30.Hammadeh ME, Radwan M, Al-Hasani S, Micu R, Rosenbaum P, Lorenz M, Schmidt W (2006) Comparison of reactive oxygen species concentration in seminal plasma and semen parameters in partners of pregnant and non-pregnant patients after IVF/ICSI. Reprod Biomed Online 13(5):696–706CrossRefPubMedGoogle Scholar
- 34.Aziz N, Novotny J, Oborna I, Fingerova H, Brezinova J, Svobodova M (2010) Comparison of chemiluminescence and flow cytometry in the estimation of reactive oxygen and nitrogen species in human semen. Fertil Steril. doi: 10.1016/j.fertnstert.2010.03.022
- 35.Desai NR, Mahfouz R, Sharma R, Gupta S, Agarwal A (2010) Reactive oxygen species levels are independent of sperm concentration, motility, and abstinence in a normal, healthy, proven fertile man: a longitudinal study. Fertil Steril. doi:10.1016/j.fertnstert.2009.12.041
- 39.Venkatesh S, Singh G, Gupta NP, Kumar R, Deecaraman M, Dada R (2009) Correlation of sperm morphology and oxidative stress in infertile men. Iran J Reprod Med 7(1):29–34Google Scholar