Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa
Open accessAbstract
To investigate the role of increased seminal leukocytes in enhancing reactive oxygen species (ROS) production by human spermatozoa.A prospective study. Male infertility clinic.Forty-eight infertile men. Standard semen analysis. Assessment of sperm nuclear DNA damage by sperm chromatin structure assay. Incubation of spermatozoa from nonleukocytospermic samples with blood neutrophils. Spontaneous and phorbol 12-myristate 13-acetate (PMA)-induced ROS production in pure-sperm suspensions (after removal of leukocytes) as measured by a chemiluminescence assay. Levels of spontaneous and PMA-induced ROS production in pure-sperm suspensions from the infertile men with a diagnosis of leukocytospermia (n = 16) were significantly higher compared with the case of infertile men without leukocytospermia (n = 32) and with the case of a control group of healthy volunteers (n = 13). A similar pattern of increased ROS was observed when spermatozoa were incubated with blood neutrophils. Leukocytospermia was associated with a significant decrease in sperm motility and increase in DNA damage. Increased seminal leukocytes may play a role in stimulating ROS production by human spermatozoa. Such stimulation may be mediated via direct cell-cell contact or by soluble products released by leukocytes. Poor sperm quality in leukocytospermic samples may be due to leukocyte-mediated oxidative stress.
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Seminal OS develops due to production of excessive amounts of ROS in numerous medical conditions, including varicocele, leukocytospermia, diabetes mellitus, and obesity.
“Due to the production of excessive amounts of ROS in numerous medical conditions, including varicocele [43], leukocytospermia [44], diabetes mellitus [45], and obesity [46], seminal OS develops.”
From The Sixth Edition of the WHO Manual for Human Semen Analysis: A Critical Review and SWOT Analysis (2021) · cites this paper as [44]
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“damaging sperm have been described, including lipopolysaccharides (LPS) exposure [22,23], leukocyte 120 activation [24], anti-sperm antibodies [25], and cytolysin production [26].”
The Role of Genetics and Oxidative Stress in the Etiology of Male Infertility—A Unifying Hypothesis?
Aitken, R., & Baker, M. (2020) · Frontiers in Endocrinology · doi:10.3389/fendo.2020.581838 · Cited by 92 · see citing works
“However, if the leukocyte numbers exceed this leukocytospermic threshold, or are just below it, then a state of oxidative stress prevails, and sperm function is compromised (95).”
BackgroundRole of Antioxidants in Assisted Reproductive Techniques
Agarwal, A., & Majzoub, A. (2017) · The World Journal of Men's Health · doi:10.5534/wjmh.2017.35.2.77 · Cited by 92 · see citing works
“Finally, seminal leukocytes may in fact induce ROS production by human spermatozoa through a mechanism that is not clearly understood [26], but possibly due to disturbing the mitochondrial membrane potential and activation of caspase 3/7 which eventually leads to an increased sperm superoxide production [27].”
BackgroundSelf-citationSperm DNA Integrity Assessment: A New Tool in Diagnosis and Treatment of Fertility
Bungum, M. (2011) · Obstetrics and Gynecology International · doi:10.1155/2012/531042 · Cited by 92
Ribas-Maynou, J., & Yeste, M. (2020) · Biology · doi:10.3390/biology9040077 · Cited by 88 · see citing works
Reference values of reactive oxygen species in seminal ejaculates using chemiluminescence assay
Agarwal, A., Ahmad, G., & Sharma, R. (2015) · Journal of Assisted Reproduction and Genetics · doi:10.1007/s10815-015-0584-1 · Cited by 87 · see citing works
Self-citationOxidation-reduction potential as a new marker for oxidative stress: Correlation to male infertility
Agarwal, A., & Bui, A. (2017) · Investigative and Clinical Urology · doi:10.4111/icu.2017.58.6.385 · Cited by 86 · see citing works
“Accumulated leukocytes in the seminal plasma (leukocytospermia) increase ROS production and lead to abnormal semen parameters [93,94].”
Self-citationNi, K., Steger, K., Yang, H., Wang, H., Hu, K., Zhang, T., & Chen, B. (2016) · Andrology · doi:10.1111/andr.12210 · Cited by 86
“, 2006), (iv) no other factors affecting seminal ROS level such as leukocytospermia (Saleh et al., 2002), age>40 years (Cocuzza et al.”
BackgroundMuriel, L., Goyanes, V., Segrelles, E., Gosálvez, J., Alvarez, J., & Fernández, J. (2006) · Journal of Andrology · doi:10.2164/jandrol.106.000067 · Cited by 84
Self-citationAssessment of Oxidative Stress in Sperm and Semen
Kashou, A. H., Sharma, R. K., & Agarwal, A. K. (2012) · Methods in molecular biology · doi:10.1007/978-1-62703-038-0_30 · Cited by 81 · see citing works
Self-citationBarranco, I., Tvarijonaviciute, A., Pérez-Patiño, C., Parrilla, I., Cerón, J. J., Martinez, E., Rodríguez-Martínez, H., & Roca, J. (2015) · Scientific Reports · doi:10.1038/srep18538 · Cited by 80
Lackner, J., Schatzl, G., Waldhör, T., Resch, K., Kratzik, C., & Marberger, M. (2005) · Fertility and Sterility · doi:10.1016/j.fertnstert.2005.05.049 · Cited by 76
The role of infections and leukocytes in male infertility
Henkel, R., Offor, U., & Fisher, D. (2020) · Andrologia · doi:10.1111/and.13743 · Cited by 74 · see citing works
“Leukocytospermia causes an increase in ROS and pro-inflammatory cytokines, especially IL-8 and IL-6, for which higher seminal concentrations have been shown in men with genital tract inflammation (Aghazarian, Stancik, Pflüger, & Lackner, 2013; Lotti & Maggi, 2013; Saleh et al., 2002).”
InfluentialSperm chromatin structure is associated with the quality of spermatogenesis in infertile patients.
Smit, M., Romijn, J., Wildhagen, M., Weber, R., & Dohle, G. (2010) · Fertility and Sterility · doi:10.1016/j.fertnstert.2009.10.030 · Cited by 73 · see citing works
“Studies that evaluated the associations between sperm DNA fragmentation and leukocytospermia (12, 13) or smoking (14, 15) in infertile men showed inconsistent results.”
ResultContemporary concepts in the evaluation and management of male infertility
Hwang, K., Walters, R. C., & Lipshultz, L. I. (2011) · Nature Reviews Urology · doi:10.1038/nrurol.2010.230 · Cited by 84 · see citing works
“Extended semen analysis White blood cell assay In the past, leukocytospermia has been associated with decreased sperm counts, reduced motility, abnormal morphology, and defective fertilization; however, these studies have demonstrated inconsistent results regarding the relationship between leukocytospermia and inferti…”
ResultFraczek, M., Hryhorowicz, M., Gill, K., Zarzycka, M., Gączarzewicz, D., Jędrzejczak, P., Bilińska, B., Piasecka, M., & Kurpisz, M. (2016) · Journal of Reproductive Immunology · doi:10.1016/j.jri.2016.08.006 · Cited by 68
Effects of Hepatitis B Virus S Protein Exposure on Sperm Membrane Integrity and Functions
Kang, X.-J., Xie, Q.-D., Zhou, X.-L., Li, F., Huang, J.-H., Liu, D.-L., & Huang, T.-H. (2012) · PLoS ONE · doi:10.1371/journal.pone.0033471 · Cited by 68
Effects of microwave at 2.45 GHz radiations on reproductive system of male rats
Kesari, K., & Behari, J. (2010) · Toxicological & Environmental Chemistry Reviews · doi:10.1080/02772240903233637 · Cited by 68 · see citing works
“It was suggested that sperm damage (DNA fragmentation) by ROS may occur due to microwave field exposure (Erenpreiss et al. 2002; Marchetti et al. 2002; Saleh et al. 2002).”
BackgroundMarkers of oxidative stress and sperm chromatin integrity.
Agarwal, A., Varghese, A., & Sharma, R. (2009) · Methods in molecular biology · doi:10.1007/978-1-60327-378-7_24 · Cited by 66
Self-citationOkazaki, T., Mihara, T., Fujita, Y., Yoshida, S., Teshima, H., & Shimada, M. (2010) · Theriogenology · doi:10.1016/j.theriogenology.2010.05.019 · Cited by 65 · see citing works
Ji, G.-X., Gu, A., Wang, Y.-B., Huang, C., Hu, F., Zhou, Y., Song, L., & Wang, X.-R. (2012) · Free Radical Biology & Medicine · doi:10.1016/j.freeradbiomed.2011.11.032 · Cited by 64
“Although the origin of DNA damage in spermatozoa is still controversial, several studies have now reported a link between oxidative stress and sperm DNA damage using various techniques such as TUNEL, sperm chromatin structure assay, and measurement of the by-product of DNA oxidation, 8-OHdG [32–38].”
MethodGavriliouk, D., & Aitken, R. (2015) · Advances in Experimental Medicine and Biology · doi:10.1007/978-3-319-18881-2_2 · Cited by 64
Gambera, L., Serafini, F., Morgante, G., Focarelli, R., de Leo, V., & Piomboni, P. (2007) · Human Reproduction · doi:10.1093/humrep/del490 · Cited by 63
“Different studies suggest a negative effect of leukocytes and reactive-oxygen species, primarily produced by leukocytes, on semen quality (Sharma et al., 2001; Erenpreiss et al., 2002; Saleh et al., 2002).”
BackgroundThe Effects of Oxidative Stress on Outcomes of Assisted Reproductive Techniques
Oral, O., Kutlu, T., Aksoy, E., Ficicioglu, C., Uslu, H., & Tuğrul, S. (2006) · Journal of Assisted Reproduction and Genetics · doi:10.1007/s10815-005-9010-4 · Cited by 63 · see citing works
What should it take to describe a substance or product as 'sperm-safe'
Mortimer, D., Barratt, C. L. R., Björndahl, L., de Jager, C., Jequier, A. M., & MULLER, C. H. (2013) · Human Reproduction Update · doi:10.1093/humupd/dmt008 · Cited by 64 · see citing works
“However, immature spermatozoa may produce comparable levels of ROS in the presence of pro-inflammatory factors (Saleh et al., 2002).”
BackgroundAgarwal, A., Bragais, F. M., & Sabanegh, E. (2008) · Urologic clinics of North America · doi:10.1016/j.ucl.2008.01.012 · Cited by 62
“Leukocytospermia is associated with increased ROS levels and can serve as indirect measurement of ROS [23].”
BackgroundSelf-citationShahin, S., Mishra, V., Singh, S. P., & Chaturvedi, C. (2014) · Free radical research · doi:10.3109/10715762.2014.888717 · Cited by 59 · see citing works
Brugnon, F., Ouchchane, L., Pons-Rejraji, H., Artonne, C., Farigoule, M., & Janny, L. (2013) · Human Reproduction · doi:10.1093/humrep/det253 · Cited by 59
“and apoptotic-like damage during the freezing/thawing process, with lethal and sublethal consequences (Saleh et al., 2002; Zorn et al., 2010).”
BackgroundValue of counting white blood cells (WBC) in semen samples to predict the presence of bacteria.
Lackner, J., Schatzl, G., Horvath, S., Kratzik, C., & Marberger, M. (2006) · European Urology · doi:10.1016/j.eururo.2005.08.017 · Cited by 59
Das, J., Choi, Y., Song, H., & Kim, J. (2016) · Human Reproduction Update · doi:10.1093/humupd/dmw020 · Cited by 66 · see citing works
“Saleh et al. (2002) also demonstrated that immature spermatozoa could produce excess ROS in the presence of proinflammatory factors.”
BackgroundEmpirical treatment of low-level leukocytospermia with doxycycline in male infertility patients.
Hamada, A., Agarwal, A., Sharma, R., French, D., Ragheb, A., & Sabanegh, E. (2011) · Urology · doi:10.1016/j.urology.2011.08.062 · Cited by 58 · see citing works
Self-citationMale infertility : contemporary clinical approaches, andrology, ART & antioxidants
Parekattil, S., & Agarwal, A. (2012) · Elsevier eBooks · doi:10.1016/b978-1-4160-6911-9.00021-9 · Cited by 58 · see citing works
Self-citationNot every sperm is sacred; a perspective on male infertility.
Aitken, R. (2018) · Molecular human reproduction · doi:10.1093/molehr/gay010 · Cited by 57 · see citing works
The photon emission, ATP level and motility of boar spermatozoa during liquid storage.
Gogol, P., Szczesniak-fabianczyk, B., & Wierzchoś-Hilczer, A. (2009) · Reproductive Biology · doi:10.1016/s1642-431x(12)60093-x · Cited by 57
“During this process they face new offensive peroxidation environments especially in the uterus, where an invasion of leukocytes adds massive amounts of radicals into the uterine fluid [1, 23].”
BackgroundSperm DNA Fragmentation: Consequences for Reproduction.
Simon, L., Emery, B., & Carrell, D. (2019) · Advances in Experimental Medicine and Biology · doi:10.1007/978-3-030-21664-1_6 · Cited by 57 · see citing works
Sperm function and assisted reproduction technology
Henkel, R., Maa, G., Bödeker, R., Scheibelhut, C., Stalf, T., Mehnert, C., Schuppe, H., Jung, A., & Schill, W. (2005) · Reproductive Medicine and Biology · doi:10.1111/j.1447-0578.2005.00087.x · Cited by 55
Yavas, Y., & Selub, M. (2003) · Fertility and Sterility · doi:10.1016/j.fertnstert.2004.04.061 · Cited by 55
Laboratory tests for oxidative stress
Agarwal, A. K., & Majzoub, A. A. (2017) · Indian Journal of Urology · doi:10.4103/iju.iju_9_17 · Cited by 67 · see citing works
“[24] Leukocytospermia, a condition characterized by abnormally high levels of peroxidase-positive leukocytes in seminal fluid, was detected in up to 20% of infertile men and associated with significant elevations in seminal ROS levels and sperm DNA damage.[24] A recent study by Lobascio et al.”
BackgroundSelf-citationRecent scenario of obesity and male fertility
Shukla, K., Chambial, S., Dwivedi, S., Misra, S., & Sharma, P. (2014) · Andrology · doi:10.1111/andr.270 · Cited by 54 · see citing works
“Hence, oxidative stress induced by excess amount of ROS may lead to changes in the tertiary structure and expression of proteins, protein membrane receptors, and membrane transport proteins, eventually resulting in disturbed ionic balance (Saleh et al., 2002; Chambial et al., 2013).”
BackgroundSeminal leukocytes are Good Samaritans for spermatozoa.
Barraud-Lange, V., Pont, J., Ziyyat, A., Pocate, K., Sifer, C., Cédrin-Durnerin, I., Fechtali, B., Ducot, B., & Wolf, J. (2011) · Fertility and Sterility · doi:10.1016/j.fertnstert.2011.09.035 · Cited by 54
One abstinence day decreases sperm DNA fragmentation in 90 % of selected patients
Pons, I., Cercas, R., Villas, C., Braña, C., & Fernández-Shaw, S. (2013) · Journal of Assisted Reproduction and Genetics · doi:10.1007/s10815-013-0089-8 · Cited by 48 · see citing works
Reactive oxygen species in seminal plasma as a cause of male infertility.
Kumar, N., & Singh, A. (2018) · Journal of Gynecology Obstetrics and Human Reproduction · doi:10.1016/j.jogoh.2018.06.008 · Cited by 46 · see citing works
Sharma, R., Gupta, S., Agarwal, A., Henkel, R., Finelli, R., Parekh, N., Saleh, R., Arafa, M., Ko, E. Y., Zini, A., et al. (2021) · The World Journal of Men's Health · doi:10.5534/wjmh.210063 · Cited by 44 · see citing works
“There are numerous reports indicating that leukocytospermia has a negative impact on male fertility [7,12,13,29].”
BackgroundSelf-citationA preliminary study of oscillating electromagnetic field effects on human spermatozoon motility
Iorio, R., Scrimaglio, R., Rantucci, E., Monache, S. D., Digaetano, A., Finetti, N., Francavilla, F., Santucci, R., Tettamanti, E., & Colonna, R. (2007) · Bioelectromagnetics · doi:10.1002/bem.20278 · Cited by 44
“For long culture experiments (24 h), the presence in the sperm preparation of other cells, particularly leucocytes, can further invalidate the life of spermatozoa [Saleh et al., 2002].”
BackgroundYamanaka, M., Tomita, K., Hashimoto, S., Matsumoto, H., Satoh, M., Kato, H., Hosoi, Y., Inoue, M., Nakaoka, Y., & Morimoto, Y. (2016) · Journal of reproduction and development · doi:10.1262/jrd.2016-112 · Cited by 43
Zhang, Q.-X., Xiao, Y.-X., Zhuang, W.-D., Cheng, B., Zheng, L., Cai, Y., Zhou, H., & Wang, Q. (2014) · Urology · doi:10.1016/j.urology.2014.04.014 · Cited by 43
Leukocytospermia: Overview of diagnosis, implications, and management of a controversial finding
Sandoval, J., Raburn, D., & Muasher, S. (2013) · doi:10.1016/j.mefs.2013.02.004 · Cited by 38
Oxidative stress in leucocytospermic prostatitis patients: preliminary results
Kullisaar, T., Türk, S., Punab, M., Korrovits, P., Kisand, K., Rehema, A., Zilmer, K., Zilmer, M., & Mändar, R. (2008) · Andrologia · doi:10.1111/j.1439-0272.2007.00816.x · Cited by 37
“Several studies have shown that peroxidase-positive leucocytes in semen (mostly polymorphonuclear leucocytes and macrophages) produce large amounts of reactive oxygen species (ROS) (reviewed by Saleh et al., 2002; Agarwal et al., 2003).”
BackgroundOxidation of glyceraldehyde-3-phosphate dehydrogenase decreases sperm motility
Elkina, Y., Atroshchenko, M., Bragina, E., Muronetz, V., & Schmalhausen, E. (2011) · Biochemistry (Moscow) · doi:10.1134/s0006297911020143 · Cited by 36
- Authors
- Saleh, R. A., Agarwal, A., Kandirali, E., Sharma, R. K., Thomas, A. J., Nada, E. A., Evenson, D. P., & Alvarez, J. G.
- Affiliations
- Cleveland Clinic (US)
- South Valley University (EG)
- South Dakota State University (US)
- Corresponding author
- Agarwal, Ashok
- Venue
- Fertility and Sterility
- Publisher
- Elsevier BV
- Published
- 2002-12
- Volume
- 78
- Issue
- 6
- Pages
- 1215-1224
- ISSN
- 0015-0282 (ISSN-L)
- OpenAlex
- W2129317574
- Cited by
- 259
- References
- 0
- Citations by year
- 2026: 6 · 2025: 5 · 2024: 6 · 2023: 11 · 2022: 9 · 2021: 13 · 2020: 11 · 2019: 5 · 2018: 11 · 2017: 14 · 2016: 10 · 2015: 13 · 2014: 21 · 2013: 11 · 2012: 16
- FWCI
- 7.45
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Fertility and Sterility (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Cleveland Clinic Foundation
- Topics
- Health Sciences › Medicine › Reproductive Medicine › Sperm and Testicular Function
- Health Sciences › Medicine › Rheumatology › Urologic and reproductive health conditions
- Health Sciences › Health Professions › General Health Professions › Male Reproductive Health Studies
- Keywords
- leukocytospermia, reactive oxygen species, spermatozoa, seminal leukocytes, neutrophils, oxidative stress, male infertility, sperm DNA damage, sperm motility, phorbol myristate acetate
- MeSH terms
- DNA Damage, Humans, Infertility, Male, Leukocytes, Male, Neutrophils, Prospective Studies, Reference Values, Sperm Motility, Spermatozoa, Tetradecanoylphorbol Acetate, Reactive Oxygen Species
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