| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From * The Miami Project to Cure Paralysis and the
Department of Urology, University of Miami
Miller School of Medicine, Miami, Florida.
| Correspondence to: Nancy L. Brackett, 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{at}miami.edu). |
| Received for publication October 27, 2006; accepted for publication May 3, 2007. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsSemen AnalysisResultsDiscussionReferences |
|---|
(TNF-) 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-1ß, IL-6, and
TNF- 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-1ß, IL-6, and TNF- 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.
Key words: Infertility, ejaculation, semen, TNF-, IL-1ß, IL-6
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 |
|---|
|
TopAbstractMaterials and MethodsSemen AnalysisResultsDiscussionReferences |
|---|
Semen Collection
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.
|
Semen Analysis |
|---|
|
TopAbstractMaterials and MethodsSemen AnalysisResultsDiscussionReferences |
|---|
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 (x100). 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.
Experimental Design
Sperm were separated from the seminal plasma by centrifugation at 500
x 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.
Cytokine Determination
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-1ß, IL-6, and TNF- 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-1ß, and TNF- was 0.70 pg/mL, 1 pg/mL,
and 1.6 pg/mL, respectively.
Statistical Analysis
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.
|
Results |
|---|
|
TopAbstractMaterials and MethodsSemen AnalysisResultsDiscussionReferences |
|---|
|
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- was undetectable, and IL-6 and IL-1ß concentrations were
not greatly elevated. In case 6, pretreatment sperm motility was approaching
normal (normal is greater than 50%).
|
In healthy non-SCI subjects, the neutralizing agents had no effect on sperm
motility (Table 2). TNF-
was undetectable by ELISA in all cases (100%), and IL-1ß and IL-6
concentrations were within normal values
(Table 2).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsSemen AnalysisResultsDiscussionReferences |
|---|
Our previous study showed 3 cytokines to be elevated in the seminal plasma
of men with SCI: IL-6, IL-1ß and TNF-
(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-1ß, and TNF- 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-; 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-1ß, and
TNF- 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.
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsSemen AnalysisResultsDiscussionReferences |
|---|
Basu S, Aballa TC, Ferrell SM, Lynne CM, Brackett NL. Inflammatory
cytokine concentrations are elevated in seminal plasma of men with spinal cord
injuries. J Androl. 2004; 25: 250
-254.
Basu S, Lynne CM, Ruiz P, Aballa TC, Ferrell SM, Brackett NL.
Cytofluorographic identification of activated T-cell subpopulations in the
semen of men with spinal cord injuries. J Androl. 2002; 23: 551
-556.
Brackett NL. Semen retrieval by penile vibratory stimulation in men
with spinal cord injury. Hum Reprod Update. 1999; 5: 216
-222.
Brackett NL, Lynne CM. The method of assisted ejaculation affects the outcome of semen quality studies in men with spinal cord injury: a review. NeuroRehabilitation. 2000; 15: 89 -100.[Medline]
Brackett NL, Nash MS, Lynne CM. Male fertility following spinal
cord injury: facts and fiction. Phys Ther. 1996; 76: 1221
-1231.
Brown DJ, Hill ST, Baker HW. Male fertility and sexual function after spinal cord injury. Prog Brain Res. 2006; 152: 427 -439.[Medline]
Cohen DR, Basu S, Randall JM, Aballa TC, Lynne CM, Brackett NL.
Sperm motility in men with spinal cord injuries is enhanced by inactivating
cytokines in the seminal plasma. J Androl. 2004; 25: 922
-925.
DeForge D, Blackmer J, Garritty C, et al. Fertility following spinal cord injury: a systematic review. Spinal Cord. 2005; 43: 693 -703.[CrossRef][Medline]
Fierro R, Schwed P, Foliguet B, Grignon G, Bene MC, Faure G. Expression of IL-2alpha and IL-2beta receptors on the membrane surface of human sperm. Arch Androl. 2002; 48: 397 -404.[CrossRef][Medline]
Gruschwitz MS, Brezinschek R, Brezinschek HP. Cytokine levels in
the seminal plasma of infertile males. J Androl. 1996; 17: 158
-163.
Huang YW, Vitetta ES. A monoclonal anti-human IL-6 receptor antibody inhibits the proliferation of human myeloma cells. Hybridoma. 1993; 12: 621 -630.[Medline]
Huleihel M, Levy A, Lunenfeld E, Horowitz S, Potashnik G, Glezerman M. Distinct expression of cytokines and mitogenic inhibitory factors in semen of fertile and infertile men. Am J Reprod Immunol. 1997; 37: 304 -309.[Medline]
National Spinal Cord Injury Statistical Center. Spinal cord injury. Facts and figures at a glance. J Spinal Cord Med. 2005; 28: 379 -380.[Medline]
Sanocka D, Jedrzejczak P, Szumala-Kaekol A, Fraczek M, Kurpisz M.
Male genital tract inflammation: the role of selected interleukins in
regulation of pro-oxidant and antioxidant enzymatic substances in seminal
plasma. J Androl. 2003; 24: 448
-455.
Symons JA, Young PR, Duff GW. Soluble type II interleukin 1 (IL-1)
receptor binds and blocks processing of IL-1 beta precursor and loses affinity
for IL-1 receptor antagonist. Proc Natl Acad Sci U S
A. 1995;92: 1714
-1718.
Van Zee KJ, Kohno T, Fischer E, Rock CS, Moldawer LL, Lowry SF.
Tumor necrosis factor soluble receptors circulate during experimental and
clinical inflammation and can protect against excessive tumor necrosis factor
alpha in vitro and in vivo. Proc Natl Acad Sci U S A. 1992; 89: 4845
-4849.
World Health Organization. Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucous Interaction. Cambridge, United Kingdom: Cambridge University Press, 1999 .
Yoshida S, Harada T, Iwabe T, et al. A combination of interleukin-6
and its soluble receptor impairs sperm motility: implications in infertility
associated with endometriosis. Hum Reprod. 2004; 19: 1821
-1825.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
