Differential expression of novel biomarkers (TLR-2, TLR-4, COX-2, and Nrf-2) of inflammation and oxidative stress in semen of leukocytospermia patients

Abstract

SUMMARYChronic genitourinary inflammation results in Leukocytospermia (LCS), an elevated number of white blood cells (WBCs) in semen, which, in association with oxidative stress, may suppress sperm function, and manifest as male factor infertility. The current clinical diagnosis of LCS employs manual enumeration of WBCs and requires complex staining and laboratory skills or measurement of inflammatory cytokines and chemokines levels. Many patients with idiopathic infertility are asymptomatic. In search of better inflammatory markers for LCS, we evaluated expression of toll-like receptors 2 and 4 (TLR-2/4), cyclooxygenase-2 (COX-2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) in semen samples of age-matched infertile patients with and without LCS. We employed the usage of specific Western blot evaluation, cytokine array; immunofluorescence microscopy (IFM) followed by computer-based analysis, and other molecular approaches. As compared with non-LCS patients (n = 38), semen samples from LCS patients (n = 47) displayed significantly lower total sperm count (p < 0.01), motility (p < 0.0001), normal head count (p < 0.0001), and a significantly higher white blood cell count (p < 0.0001). Differential cytokine profiling of seminal plasma by antibody array revealed up-regulation of several pro-inflammatory chemokines in LCS samples. Western blot analysis of LCS seminal plasma (n = 15) also showed a significant increase in expression of TLR-2 (p < 0.001) and 4 (p < 0.01), COX-2 (p < 0.001), and Nrf-2 (p < 0.001) as compared with semen samples from non-LCS patients (n = 15). Computer-based objective IFM analysis of spermatozoa from LCS patients showed increased expression of TLR-4 (p < 0.001), Cox-2 (p < 0.01), and (Nrf-2) (p < 0.01). Significant differences in the subcellular localization of these proteins were evident in the sperm head and tail segments of LCS samples. Altogether, these observations suggest that TLR-2/4, COX-2, and Nrf-2 can serve as novel biomarkers of inflammation and oxidative stress. Therefore, developing a rapid assay for these biomarkers may facilitate early diagnosis and management of LCS especially in idiopathic and asymptomatic male infertility patients.

Semen leukocytes and oxidative-dependent DNA damage of spermatozoa in male partners of subfertile couples with no symptoms of genital tract infection

“…the fertilization rate of metaphase II oocytes (Moilanen et al, 1998). Although the association between the presence of leukocytes and semen quality or sperm fertility potential is at best questionable, the proposed detrimental effect of leukocytes on spermatozoa is suggested to rely on the ability of leukocytes to release ROS and inflammatory mediators (Aitken & West, 1990;Plante et al, 1994;Whittington & Ford, 1999;Sharma et al, 2001;Henkel et al, 2005;Agarwal et al, 2014;Hagan et al, 2015). Accordingly, ROS are considered the main cause of sperm DNA damage as a consequence of leukocyte infiltration (Aitken et al, 2014).…”

“…Relevant methodological differences in the evaluation of semen leukocyte infiltration and sperm DNA fragmentation may account for the conflicting opinion between those who denied (Henkel et al, 2005;Moskovtsev et al, 2007;present study) or those who advocated (Agarwal et al, 2014;Lobascio et al, 2015) a positive association between sperm DNA fragmentation and the concentration of semen leukocyte. Furthermore, findings cannot be extended to ejaculates with a leukocyte infiltration associated to a symptomatic genital tract infection or to increased levels of inflammatory markers in the seminal plasma (Hagan et al, 2015). These limitations are balanced by the effort to apply an objective method that coupled cytofluorimetry and immunocytochemistry.…”

The change in serum Thiol/Disulphide homeostasis after transrectal ultrasound guided prostate biopsy

“…Recently, Ener et al evaluated the oxidative stress status and antioxidant capacity in patients with painful bladder syndrome, and used serum total antioxidant capacity, total oxidant status, binding capacity of exogenous cobalt to human albumin, serum advanced oxidation protein products, paraoxonase, arylesterase, IgE, and C-reactive protein as oxidative markers (17). Some investigators researched the levels of oxidative markers either in urine or semen (18, 19). When compared to these conventional oxidative stress markers, the novel assay developed by Drs.…”

Immunological tolerance, pregnancy and pre-eclampsia: the roles of semen microbes and the father1

“…Sperm cells are well endowed with TLRs [466; 710-712]. However, we can find only one study showing that increased semen expression of TLRs is indeed observed during inflammation and oxidative stress such as occurs during infection and infertility [713]. A more wide-ranging assessment of TLR expression in sperm cells as a function of fertility seems warranted.…”