Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on medication adherence

LIVE: Thursday 27th June 2024, 18:00-19:30 (CEST)
Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.
ADVANCED SEARCH
Log in
Top
Journal of Assisted Reproduction and Genetics
Published in: Journal of Assisted Reproduction and Genetics 5/2015

01-05-2015 | Reproductive Physiology and Disease

Reactive Oxygen Species (ROS) in human semen: determination of a reference range

Authors: Sheryl T. Homa, Wayne Vessey, Ana Perez-Miranda, Tripat Riyait, Ashok Agarwal

Published in: Journal of Assisted Reproduction and Genetics | Issue 5/2015

Login to get access

Abstract

Purpose

High levels of reactive oxygen species (ROS) are a leading cause of male factor infertility. Measurement of ROS has been hampered by a lack of standardisation and confounding variables including choice of controls and sample selection. This study aimed to determine a reference range for ROS in human semen.

Methods

Semen samples were obtained from men attending for routine semen analysis who gave informed consent for the study. Samples were assigned groups: Group 1 (N = 94) normal semen parameters, no leucocytospermia; Group 2 (N = 100) abnormal semen parameters, no leucocytospermia; Group 3 (N = 41) any semen parameters with leucocytospermia. ROS levels were assayed in fresh neat semen using a chemiluminescence assay measured in a single tube luminometer. Data are reported in relative light units (RLU)/sec/106 sperm

Results

ROS levels were significantly different between Groups 1, 2 and 3 (19.75 ± 8.12, 95.03 ± 33.63, 890.17 ± 310.23 RLU/sec/106 sperm respectively; p < 0.001). Group 3 gave the highest value confirming this group as the optimum choice for positive controls. The reference range < 24.1 RLU/sec/106 sperm was determined by ROC analysis that differentiates a reference population (Group 1) from a positive control group (Group 3), optimising the sensitivity and specificity (80.5 and 87.2 % respectively) of the test.

Conclusions

We have determined a reference range for ROS in human semen and identified a patient population that falls outside the normal range. This simple, cost effective assay can be incorporated into routine diagnostic testing to aid in the diagnosis of male infertility, especially with regard to unexplained infertility.
Literature
1.
2.
McLachlan R, de Kretser D. Male infertility: the case for continued research. MJA. 2001;174:116–7.PubMed
3.
WHO. WHO laboratory manual for the examination and processing of human semen. 5th ed. Cambridge: Cambridge University Press; 2010.
4.
Moghissi KS, Wallach EE. Unexplained infertility. Fertil Steril. 1983;39:5–21.PubMed
5.
Pizzol D, Ferlin A, Garolla A, Lenzi A, Bertoldo A, Foresta C. Genetic and molecular diagnostics of male infertility in the clinical practice. Front Biosci (Landmark Ed). 2014;19:291–303.CrossRef
6.
Agarwal A, Makker K, Sharma R. Clinical relevance of oxidative stress in male factor infertility: an update. Am J Reprod Immunol. 2008;59:2–11.CrossRefPubMed
7.
Tremellen K. Oxidative stress and male infertility – a clinical perspective. Hum Reprod Update. 2008;14:243–53.CrossRefPubMed
8.
Mahfouz RZ, Sharma R, Thiyagarajan A, Kale V, Gupta S, Sabanegh E, et al. Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil Steril. 2010;94:2141–6.CrossRefPubMed
9.
10.
11.
Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. J Androl. 1987;8:338–48.PubMed
12.
Plante M, de Lamirande E, Gagnon C. Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility. Fertil Steril. 1994;62:387–93.PubMed
13.
De Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod. 1995;10:15–21.CrossRefPubMed
14.
Aitken RJ, Curry BJ. Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid Redox Signal. 2011;14:367–81.CrossRefPubMed
15.
Shi T-Y, Chen G, Huang X, Yuan Y, Wu X, Wu B, et al. Effects of reactive oxygen species from activated leucocytes on human sperm motility, viability and morphology. Andrologia. 2011;44:696–703.CrossRefPubMed
16.
Chen SJ, Allam JP, Duan YG, Haidi G. Influence of reactive oxygen species on human sperm functions and fertilizing capacity including therapeutical approaches. Arch Gynecol Obstet. 2013;288:191–9.CrossRefPubMed
17.
Morielli T, O’Flaherty C. Oxidative stress impairs function and increases redox protein modifications in human spermatozoa. Reproduction. 2015;149:113–23.CrossRefPubMed
18.
Ichikawa T, Oeda T, Ohmori H, Schill WB. Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa. Int J Androl. 1999;22:37–42.CrossRefPubMed
19.
Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod. 1998;59:1037–46.CrossRefPubMed
20.
Zorn B, Vidmar G, Meden-Vrtovec H. 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. 2003;26:279–85.CrossRefPubMed
21.
Agarwal A, Allamaneni SS, Nallella KP, George AT, Mascha E. Correlation of reactive oxygen species levels with the fertilization rate after in vitro fertilization: a qualified meta-analysis. Fertil Steril. 2005;84:228–31.CrossRefPubMed
22.
Ghaleno LR, Valojerdi MR, Hassani F, Chehrazi M, Janzamin E. High level of intracellular sperm oxidative stress negatively influences embryo pronuclear formation after intracytoplasmic sperm injection treatment. Andrologia. 2014;46:1118–27.CrossRefPubMed
23.
Imam SN, Shamsi MV, Kumar K, Deka D, Dada R. Idiopathic recurrent pregnancy loss: role of paternal factors; a pilot study. J Reprod Infertil. 2011;12:267–76.PubMedCentralPubMed
24.
Aitken RJ, Baker MA. Oxidative stress, sperm survival and fertility control. Mol Cell Endocrinol. 2006;250:66–9.CrossRefPubMed
25.
Athayde KS, Cocuzza M, Agarwal A, Krajcir N, Lucon AM, Srough M, et al. Development of normal reference values for seminal reactive oxygen species and their correlation with leukocytes and semen parameters in a fertile population. J Androl. 2007;28:613–20.CrossRefPubMed
26.
Moein MR, Dehghani VO, Tabibnejad N, Vahidi S. Reactive oxygen species (ROS) level in seminal plasma of infertile men and healthy donors. Iran J Reprod Med. 2007;5:51–5.
27.
Desai N, Sharma R, Makker K, Sabanegh E, Agarwal A. Physiologic and pathologic levels of reactive oxygen species in neat semen of infertile men. Fertil Steril. 2009;92:1626–31.CrossRefPubMed
28.
Guz J, Gackowski D, Foksinski M, Rozalski R, Zarakowska E, Siomek A, et al. Comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. PLoS One. 2013;8:e68490.CrossRefPubMedCentralPubMed
29.
Kullisaar T, Türk S, Kilk K, Ausmees K, Punab M, Mändar R. Increased levels of hydrogen peroxide and nitric oxide in male partners of infertile couples. Andrology. 2013;1:850–8.CrossRefPubMed
30.
Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl. 1988;9:367–76.PubMed
31.
Agarwal A, Ikemoto I, Loughlin KR. Effect of sperm washing on levels of reactive oxygen species in semen. Arch Androl. 1994;33:157–62.CrossRefPubMed
32.
Shekarriz M, DeWire DM, Thomas Jr AJ, Agarwal A. A method of human semen centrifugation to minimise iatrogenic sperm injuries caused by reactive oxygen species. Eur Urol. 1995;28:31–5.PubMed
33.
Venkatesh S, Shamsi MB, Dudeja S, Kumar R, Dada R. Reactive oxygen species measurement in neat and washed semen: comparative analysis and its significance in male infertility assessment. Arch Gynaecol Obstet. 2011;283:121–6.CrossRef
34.
Li Z, Zhou Y, Liu R, Lin H, Liu W, Xiao W, et al. Effects of semen processing on the generation of reactive oxygen species and mitochondrial membrane potential of human spermatozoa. Andrologia. 2012;44:157–63.CrossRefPubMed
35.
Allamaneni SSR, Agarwal A, Nallella KP, Sharma RK, Thomas Jr AJ, Sikka SC. Characterization of oxidative stress status by evaluation of reactive oxygen species levels in whole semen and isolated spermatozoa. Fertil Steril. 2005;83:800–3.CrossRefPubMed
36.
Fingerova H, Oborna I, Novotny J, Svobodova M, Brezinova J, Radova L. The measurement of reactive oxygen species in human neat semen and in suspended spermatozoa: a comparison. Reprod Biol Endocrinol. 2009;7:118–23.CrossRefPubMedCentralPubMed
37.
Henkel R, Kierspel E, Stalf T, Mehnert C, Menkveld R, Tinneberg HR, et al. Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non- leukocytospermic patients. Fertil Steril. 2005;83:635–42.CrossRefPubMed
38.
Mupfiga C, Fisher D, Kruger T, Henkel R. The relationship between seminal leukocytes, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa. Syst Biol Reprod Med. 2013;59:304–11.CrossRefPubMed
39.
Ochsendorf FR. Infections in the male genital tract and reactive oxygen species. Hum Reprod Update. 1999;5:399–420.CrossRefPubMed
40.
Sakamoto Y, Ishikawa T, Kondo Y, Yamaguchi K, Fujisawa M. The assessment of oxidative stress in infertile patients with varicocele. BJU Int. 2008;101:1547–52.CrossRefPubMed
41.
Kothari S, Thompson A, Agarwal A, du Plessis SS. Free radicals: their beneficial and detrimental effects on sperm function. Indian J Exp Biol. 2010;48:425–35.PubMed
42.
Lavranos G, Balla M, Tzortzopoulou A, Syriou V, Angelopoulou R. Investigating ROS sources in male infertility: a common end for numerous pathways. Reprod Toxicol. 2012;34:298–307.CrossRefPubMed
43.
Kobayashi H, Gil-Guzman E, Mahran AM, Sharma RK, Nelson DR, Thomas Jr AJ, et al. Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay. J Androl. 2001;22:568–74.PubMed
44.
Vessey W, Perez-Miranda A, Macfarquhar R, Agarwal A, Homa S. Reactive oxygen species (ROS) in human semen: validation and qualification of a chemiluminescence assay. Fertil Steril. 2014;102:1576–83.CrossRefPubMed
45.
Agarwal A, Allamaneni SS, Said TM. Chemiluminsecence technique for measuring reactive oxygen species. Reprod Biomed Online. 2004;9:466–8.CrossRefPubMed
46.
Saleh RA, Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice. J Androl. 2002;23:737–52.PubMed
47.
48.
Kashou AH, Sharma R, Agarwal A. Assessment of oxidative stress in sperm and semen. Methods Mol Biol. 2013;927:351–61.CrossRefPubMed
49.
WHO. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press; 1999.
50.
Agarwal A, Sharma RK, Nallella KP, Thomas Jr AJ, Alvarez JG, Sikka SC. Reactive oxygen species as an independent marker of male factor infertility. Fertil Steril. 2006;86:878–85.CrossRefPubMed
51.
Desai NR, Mahfouz R, Sharma R, Gupta S, Agarwal A. Reactive oxygen species levels are independent of sperm concentration, motility and abstinence in a normal, healthy, proven fertile man: a longitudinal study. Fertil Steril. 2010;94:1541–3.CrossRefPubMed
52.
Pasqualotto FF, Sharma RK, Kobayashi H, Nelson DR, Thomas Jr AJ. Oxidative stress in normospermic men undergoing infertility evaluation. J Androl. 2001;22:316–22.PubMed
53.
Aitken RJ, West K. Relationship between reactive oxygen species generation and leukocyte infiltration in fractions isolated from the human ejaculate on Percoll gradients. Int J Androl. 1990;13:433–51.CrossRefPubMed
54.
Deepinder F, Cocuzza M, Agarwal A. Should seminal oxidative stress measurement be offered routinely to men presenting for infertility evaluation? Endocr Pract. 2008;14:484–91.CrossRefPubMed
55.
Wright C, Milne S, Leeson H. Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod Biomed Online. 2014;28:684–703.CrossRefPubMed
56.
Showell MG, Brown J, Yazdani A, Stankiewicz MT, Hart RJ. Antioxidants for male subfertility. Cochrane Database Syst Rev. 2011;19(1), CD007411.
57.
Gharagozloo P, Aitken RJ. The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod. 2011;26:1628–40.CrossRefPubMed
58.
Vicari E. 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. 2000;15:2536–44.CrossRefPubMed
59.
Hamada A, Esteves SC, Agarwal A. Insight into oxidative stress in varococele-associated male infertility: part 2. Nat Rev Urol. 2013;10:26–37.CrossRefPubMed
60.
Ramya T, Misro MM, Sinha D, Nandan D. Sperm function and seminal oxidative stress as tools to identify sperm pathologies in infertile men. Fertil Steril. 2010;93:297–300.CrossRefPubMed
Metadata
Title
Reactive Oxygen Species (ROS) in human semen: determination of a reference range
Authors
Sheryl T. Homa
Wayne Vessey
Ana Perez-Miranda
Tripat Riyait
Ashok Agarwal
Publication date
01-05-2015
Publisher
Springer US
Published in
Journal of Assisted Reproduction and Genetics / Issue 5/2015
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI
https://doi.org/10.1007/s10815-015-0454-x

Other articles of this Issue 5/2015

Journal of Assisted Reproduction and Genetics 5/2015 Go to the issue

Assisted Reproduction Technologies

Laparoscopic excision of ovarian endometrioma does not exert a qualitative effect on ovarian function: insights from in vitro fertilization and single embryo transfer cycles

Gamete Biology

Age-associated alterations in the micromechanical properties of chromosomes in the mammalian egg

Genetics

Genetic association between FXIII and β-fibrinogen genes and women with recurrent spontaneous abortion: a meta- analysis

Assisted Reproduction Technologies

Systemic oxidative stress could predict assisted reproductive technique outcome

Epigenetics

Dysregulation of methylation and expression of imprinted genes in oocytes and reproductive tissues in mice of advanced maternal age

Assisted Reproduction Technologies

Is the presence of a non-cleaved embryo on day 3 associated with poorer quality of the remaining embryos in the cohort?