Neutralization of Cytokine Activity at the Receptor Level Improves Sperm Motility in Men With Spinal Cord Injuries
The Miami Project to Cure Paralysis, University of Miami School of Medicine, PO Box 016960, Mail Code R-48, Miami, FL 33101 (e-mail: NBrackett@miami.edu).
ABSTRACT: Men with spinal cord injury (SCI) have a unique semen profile characterized by normal sperm concentrations but abnormally low sperm motility. Previous studies showed that elevated concentrations of cytokines in the seminal plasma of these men contribute to this condition. For example, when elevated concentrations of interlekin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were immunoneutralized in the semen of men with SCI, sperm motility improved. The present study investigated if these cytokines act on sperm cell receptors to inhibit sperm motility. Semen was collected from men with SCI and from healthy non-SCI men. Sperm were separated from the seminal plasma by centrifugation. Eight identical aliquots of 5000 sperm suspended in 50 μL of seminal plasma were prepared for each subject. Agents were added to the aliquots in order to neutralize IL-1β, IL-6, and TNF-α at the receptor level. In SCI subjects, sperm motility improved in each treatment group compared with the untreated group, but statistical significance was reached only when neutralizing agents to all 3 cytokines were added. Improvement was less pronounced in subjects with close to normal semen cytokine concentrations or close to normal pretreatment sperm motility. In control subjects, IL-1β, IL-6, and TNF-α were within normal values, and addition of receptor blockers to semen had no effect on sperm motility. These data support the hypothesis that cytokines act at the level of the sperm receptor to inhibit sperm motility. These data further support the notion that inactivating semen cytokines leads to improved sperm motility in SCI men. Our goal is to develop this finding into a treatment for low sperm motility in men with SCI.
Men with spinal cord injury (SCI) have a unique semen profile characterized by normal sperm concentration but abnormally low sperm motility (Brackett et al, 1996, 1997; DeForge et al, 2005; Brown et al, 2006). Factors in the seminal plasma contribute to this condition. For example, previous studies showed elevated concentrations of white blood cells and activated T-cell subpopulations in the semen of men with SCI (Basu et al, 2002). Additionally, elevated concentrations of cytotoxic cytokines were found in the seminal plasma of these men (Basu et al, 2004). When the elevated cytokine concentrations were immunoneutralized with monoclonal antibodies added to the semen, sperm motility improved (Cohen et al, 2004).
The present study was undertaken to determine if cytokine receptors are involved in mediating the effects of cytokines on sperm motility in men with SCI.
Materials and Methods
Subjects were men with traumatic SCI and healthy non-SCI subjects. All subjects were participants in the Male Fertility Research Program of the Miami Project to Cure Paralysis at the University of Miami Miller School of Medicine, Miami, Fla. The mean (± SEM) age of SCI subjects was 31.9 ± 2.3 years (range, 20–42). All SCI subjects were past the acute phase of injury, and their mean time after injury was 9 ± 2.0 years (range, 1–18). Levels of injury were C4 to T11. Each subject had undergone at least 4 ejaculations spaced 4 to 8 weeks apart prior to semen collection for this study. Control subjects were healthy non-SCI men with no history of infertility. Their mean age was 30.7 ± 3.6 years (range, 25–47). SCI subjects with a sperm motility less than or equal to 40% and control subjects with a sperm motility greater than or equal to 50% were selected for the study.
Semen was collected from SCI subjects by the standard method of penile vibratory stimulation (PVS) (Brackett, 1999). Only antegrade fractions were used in the study. Retrograde ejaculates and electroejaculates were not used in this study because these procedures have been shown to alter semen quality (Brackett and Lynne, 2000).
Non-SCI subjects collected their semen by masturbation following 2–3 days of abstinence from ejaculation.
Each specimen was allowed to liquefy (20–30 minutes) at room temperature. Semen specimens were analyzed by placing a volume of 5 μL of semen into a disposable semen analysis chamber (Cell-Vu; Fertility Technologies, Natick, Mass). Semen analysis was performed according to World Health Organization criteria (1999). All semen specimens were analyzed by the same technician. The technician was blind to the treatment groups.
Sperm motility was analyzed as follows. At least 200 sperm were analyzed. The motility of each spermatozoon was graded as “a,” “b,” “c,” or “d” according to whether it showed 1) rapid progressive motility, (greater than 20 μm/sec); 2) slow or sluggish motility; 3) nonprogressive motility (less than 5 μm/sec); or 4) immotility. Sperm motility was calculated as the number of sperm with grade a + b motility divided by the total number of sperm (×100). A repeat sperm motility assessment was performed on a separate aliquot of 5 μL from the same semen sample. If the 2 measures of sperm motility from the same specimen varied beyond the 95% confidence interval (World Health Organization, 1999: 11, Figure 2.3), 2 new slides were prepared and sperm motility reassessed. When the 2 measures were within the 95% confidence interval, the 2 measures were averaged and reported.
Neutralization of Cytokines at the Receptor Level
The following agents were used to interfere with cytokine receptors: monoclonal anti—interleukin-6 receptor antibody from mouse (MAB to IL-6 R; Sigma-Aldrich; St Louis, Mo; Catalog No. I0649), recombinant human soluble tumor necrosis factor receptor type I (sTNF RI; R&D Systems, Minneapolis, Minn; Catalog No. 636-R1–025), and recombinant human soluble IL-1 receptor type II, (sIL-1 RII; R&D Systems; Catalog No. 263–2R-050). Doses used in the experiment were adjusted based on ED50 (median effective dose [ie, dose which produces the desired effect in 50% of the population])information provided by the manufacturer.
Sperm were separated from the seminal plasma by centrifugation at 500 × g for 10 minutes. For each subject, 8 aliquots were prepared, each containing 5000 sperm suspended in 50 μL of seminal plasma. The aliquots were treated as follows to provide all individual and combined anticytokine-receptor complex preparations: treatment group 1: sperm aliquot with nothing added (untreated control); treatment group 2: sperm aliquot treated with 1.8 ng of recombinant human sTNF RI per microliter of semen; treatment group 3: sperm aliquot treated with 50 ng of recombinant human sIL-1 RII per microliter of semen; treatment group 4: sperm aliquot treated with 2 ng of monoclonal anti—IL-6 receptor antibody per microliter of semen; treatment group 5: sperm aliquot treated with 1.8 ng of sTNF RI plus 50 ng of sIL-1 RII per microliter of semen; treatment group 6: sperm aliquot treated with 1.8 ng of sTNF RI plus 2 ng of MAB to IL-6 R per microliter of semen; treatment group 7: sperm aliquot treated with 50 ng of sIL-1 RII plus 2 ng of MAB to IL-6 R per microliter of semen; and treatment group 8, sperm aliquot treated with 1.8 ng of sTNF RI plus 50 ng of sIL-1 RII plus 2 ng MAB to IL-6 R per microliter of semen.
The treatment group preparations were incubated at room temperature for 1 hour. Because protein-ligand binding is a reversible phenomenon, we evaluated sperm motility each 20 minutes during the incubation period. The highest sperm motility obtained during the 1-hour incubation was designated as the “treated” motility.
The protease inhibitor phenylmethylsulfonylfluoride (0.5 mM) was added to the remaining seminal plasma, which was then stored at −80°C until used for cytokine determination by enzyme-linked immunosorbent assay (ELISA). IL-1β, IL-6, and TNF-α were measured in the seminal plasma of SCI and control subjects using ELISA kits (R&D Systems). Seminal plasma samples were added to the wells of microtitration plates precoated with a specific anticytokine monoclonal antibody.
After incubation at room temperature for 2 hours, the unbound components were removed by washing. The second anti-human cytokine biotin-conjugated antibodies were added and incubated for 2 hours at room temperature. After washing the wells, streptavidin—horseradish peroxidase (polyconjugated) was added and incubated for 20 minutes at room temperature. Finally, substrate was added, color was developed for 15 minutes, and the reaction was stopped with 2N sulfuric acid. Absorbance was measured at 450 nm with an ELISA reader. Each sample was assayed in duplicate. The minimum detectable dose (ie, sensitivity) for IL-6, IL-1β, and TNF-α was 0.70 pg/mL, 1 pg/mL, and 1.6 pg/mL, respectively.
The sperm motility of each semen specimen was analyzed in duplicate (see semen analysis). The duplicates were averaged and reported as percent sperm motility for each semen specimen. These values were used in the statistical analysis, and the mean sperm motility for each group (ie, groups 1 through 8) was calculated. Group means were compared by analysis of variance using the 2003 version of Microsoft Excel.
In SCI subjects, sperm motility improved in each treatment group when compared with the untreated control group, but statistical significance was reached only when there was interference with receptors to all 3 cytokines (Table 1).
Table 1. . Sperm motility of spinal cord injury (SCI) subjects following treatment of semen with various combinations of neutralization agents*
|% Motility||17.4 ± 3.4||19.7 ± 3.3||24.9 ± 4.2||21.0 ± 3||23.6 ± 3.7||22.8 ± 3.5||24.9 ± 3.8||33.7 ± 4.2|
|Significance||NA||NS||NS||NS||NS||NS||NS||P < .03|
The degree of improvement in sperm motility seemed to be related to the cytokine concentrations or starting sperm motility in the semen of SCI subjects (Table 2). For example, semen treated with all 3 neutralizing agents had higher sperm motility than untreated semen in all SCI patients, but improvement was less pronounced in cases 2, 5, and 6 (Table 2). In case 2, cytokine concentrations were nearly normal. In case 5, TNF-α was undetectable, and IL-6 and IL-1β concentrations were not greatly elevated. In case 6, pretreatment sperm motility was approaching normal (normal is greater than 50%).
Table 2. . Cytokine concentrations and sperm motility in spinal cord injury (SCI) and control subjects*
|SCI case no.|| || || || || |
| SCI 1||59.2||132.0||43.0||33||52|
| SCI 2||3.0||13.0||6.0||18||20|
| SCI 3||244.4||11.0||36.0||19||49|
| SCI 4||3.6||428.0||44.0||19||34|
| SCI 5||Undetectable||56.5||33.0||15||21|
| SCI 6||14.4||103.2||Undetectable||41||49|
| SCI 7||245.0||101.4||11.3||11||34|
| SCI 8||58.3||623.0||50.2||0||7|
| SCI 9||104.2||320.5||Undetectable||17||42|
| SCI 10||3.0||428.0||28.3||15||34|
| SCI 11||247.0||58.0||3.8||4||29|
|Control case no.|| || || || || |
| Control 1||Undetectable||3.6||5.1||52||52|
| Control 2||Undetectable||8.2||4.0||56||54|
| Control 3||Undetectable||5.7||3.6||51||54|
| Control 4||Undetectable||12.1||5.0||52||55|
| Control 5||Undetectable||9.0||2.1||58||54|
In healthy non-SCI subjects, the neutralizing agents had no effect on sperm motility (Table 2). TNF-α was undetectable by ELISA in all cases (100%), and IL-1β and IL-6 concentrations were within normal values (Table 2).
SCI occurs most often to young men at the peak of their reproductive health. In the United States, more than 80% of new injuries occur to men between the ages of 16–45 (National Spinal Cord Injury Statistical Center, 2005). Men with SCI have impaired semen quality characterized by low sperm motility, increased concentrations of activated T cells, and high levels of inflammatory cytokines in their seminal plasma (Gruschwitz et al, 1996; Basu et al, 2002; Sanocka et al, 2003).
Our previous study showed 3 cytokines to be elevated in the seminal plasma of men with SCI: IL-6, IL-1β and TNF-α (Basu et al, 2004). When these cytokines were inactivated in vitro using monoclonal antibodies, sperm motility improved in men with SCI (Cohen et al, 2004). The present study investigated if inhibiting receptors to these cytokines would improve sperm motility in men with SCI. Agents that inhibit or neutralize IL-6, IL-1β, and TNF-α activity at the receptor level were used in the study (Aderka et al, 1992; Van Zee et al, 1992; Huang and Vitetta, 1993; Symons et al, 1995).
Semen was collected from SCI or control subjects. Each semen specimen was divided into 8 aliquots. Each aliquot received 0, 1, 2, or 3 neutralizing agents. Sperm motility was evaluated every 20 minutes in each preparation. A machine to perform computer-assisted semen analysis was not available for this study; therefore, care was taken to assure sperm motility was analyzed by the highest possible standards for manual methods (see “Materials and Methods”).
The results showed higher sperm motility in the treated compared with untreated samples in all SCI subjects. Improvement was less pronounced in 3 subjects: 1 had normal cytokine levels; another had minimal elevated levels of IL-6 and TNF-α; and the last subject had nearly normal pretreatment sperm motility. Our previous experiment using monoclonal antibodies in vitro (Cohen et al, 2004) showed that the best response was obtained when the pretreatment sperm motility was between 11% and 30%. In the present study, 7 of the 9 subjects (77.8%) that lie in this group showed significant improvement in their sperm motility. In addition, the overall improvement in sperm motility was statistically significant in the group treated with all 3 neutralizing agents (Table 1, group 8).
The results of the present study are consistent with our previous results using monoclonal antibodies to the same 3 cytokines (Cohen et al, 2004). Collectively, these results indicate that inactivating semen cytokines leads to improved sperm motility in SCI males and the improvement is more profound in those specimens with very low sperm motility and high semen cytokine concentrations. These data further emphasize the concept of developing treatments that individualize regimens according to predetermined seminal plasma cytokine concentrations and sperm motilities.
Our data support the notion that cytokines interact with sperm receptors to inhibit sperm motility. Previous studies also support this hypothesis. For example, adding a combination of IL-6 and sIL-6 R reduced the percentage of motile and rapidly moving sperm in 20 normospermic fertile volunteers. In the same experiment, adding anti IL-6 R antibody abolished these adverse effects (Yoshida et al, 2004). Cytokine receptors were identified and localized on human sperm and correlated with poor sperm concentration in 20 oligospermic infertile men attending an in vitro fertilization program (Fierro et al, 2002). During genital infection, cytokines and various soluble receptors of immunoregulatory cytokines were expressed distinctly in seminal plasma (Huleihel et al, 1997). For example, sTNF-1 receptor was significantly lower in patients with oligoasthenoteratospermia (OATS) who also had genital infection. Compared with fertile controls, IL-1 receptor antagonist was higher in patients with azoospermia and higher in patients with OATS, but the highest concentrations were found in OATS patients who also had genital infection (Huleihel et al, 1997).
In conclusion, interference with the activity of IL-6, IL-1β, and TNF-α at the receptor level improved sperm motility in men with SCI whose semen had elevated concentrations of these cytokines. Neutralizing the actions of elevated semen cytokines offers a promising approach for treating low sperm motility in this severely affected group of patients.
Supported by the Christopher Reeve Paralysis Foundation, State of Florida Specific Appropriations, and the Miami Project to Cure Paralysis.