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From the Miami Project to Cure Paralysis and the Department of Urology, University of Miami School of Medicine, Miami, Florida.
| Correspondence to: Dr 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 January 6, 2004; accepted for publication June 20, 2004. |
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Abstract |
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), all 3 cytokines having been previously detected at
high concentrations in the seminal plasma of patients with SCI. In a group of
17 SCI men with low sperm motility (mean ± SE, 20.1% ± 3.1%),
treatment with the 3 monoclonal antibodies at the median neutralization dose
concentrations for 1.0 to 1.5 hours improved sperm motility in all cases.
Effectiveness was higher in those specimens with a pretreatment sperm motility
between 11% and 30% (from 19.3% ± 1.4% to 41.9% ± 4.9%, P <
.0002), suggesting that pretreatment sperm motility might represent an
indicator of cell damage and, therefore, a factor that influences monoclonal
antibody effectiveness. To the best of our knowledge, these results represent
the first rational treatment for improving low sperm motility in these
severely affected patients.
Key words: Infertility, ejaculation, semen, IL1-ß, IL6, TNF-
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Materials and Methods |
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Semen Collection and Analysis
Only antegrade semen (ie, no retrograde semen) was collected from subjects
by the standard method of penile vibratory stimulation
(Brackett, 1999) because semen
quality can be altered in retrograde ejaculates or if semen is collected by
electroejaculation (Brackett and Lynne,
2000). Semen analysis was performed according to World Health
Organization criteria (1999).
Each semen specimen was first allowed to liquefy at room temperature. Sperm
parameters were assessed by placing 6 µL of the semen specimen on a
disposable semen analysis chamber (Cell-Vu; Fertility Technologies, Natick,
Mass). Sperm motility was evaluated in semen specimens before exposure to
monoclonal antibodies. The study was evaluator blind, in which the operator
did not know the treatment conditions of the specimen being evaluated. Sperm
motility was calculated by adding the percentage of rapid and sluggish sperm
with forward movement. The same operator evaluated all specimens.
Monoclonal Antibodies
Specific monoclonal antibodies to human cytokine interleukin 1 beta
(IL1-ß), interleukin 6 (IL6), and tumor necrosis factor alpha
(TNF-) were used to neutralize cytokine activity in the seminal plasma.
These agents were selected according to our previous finding that
concentrations of these specific cytokines are elevated in the seminal plasma
of patients affected by SCI (Basu et al,
2004).
Monoclonal antibodies to human IL1-ß, IL6, and TNF- (catalog
numbers MAB 601, MAB 206, and MAB 610, respectively; R&D Systems,
Minneapolis, Minn) were reconstituted in sterile phosphate-buffered saline
(PBS, pH 7.2), and a stock solution of 500 µg/mL of each cytokine was
prepared. Aliquots (200 µL) of the stock solution were transferred to
sterile microfuge tubes and frozen at -20°C. The stock solution was
further diluted to 10 µL/mL and 1 µL/mL with PBS and frozen at -20°C
until used.
Experimental Design
Each semen specimen was separated into eight 50-µL aliquots, with each
aliquot placed in a 1.5-mL microfuge tube. Specific monoclonal antibodies to
IL1-ß, IL6, and TNF- were added directly to the semen aliquots in
the tubes. Monoclonal antibodies were added singly and in all possible
combinations as described below. Doses were adjusted according to the median
neutralization dose information provided by the manufacturers. There were 8
different treatment groups for each specimen: 1) 50 µL semen + 10 µL
buffer (untreated control), 2) 50 µL semen + 10 µL monoclonal antibody
(mAb) to TNF- (1 µg/mL), 3) 50 µL semen + 10 µL mAb to
IL1-ß (1 µg/mL), 4) 50 µL semen + 10 µL mAb to IL6 (1 µg/mL),
5) 50 µL semen + 10 µL mAb to TNF- (1 µg/mL) + 10 µL mAb to
IL1-ß (1 µg/mL), 6) 50 µL semen + 10 µL mAb to TNF- (1
µg/mL) + 10 µL mAb to IL6 (1 µg/mL), 7) 50 µL semen + 10 µL mAb
to IL1-ß (1 µg/mL) + 10 µL mAb to IL6 (1 µg/mL), 8) 50 µL
semen + 10 µL mAb to TNF- (1 µg/mL) + 10 µL mAb to IL1-ß
(1 µg/mL) + 10 µL mAb to IL6 (1 µg/mL). Concentrations of all
cytokines were 1 µg/mL.
After 1.0 to 1.5 hours incubation at room temperature, sperm motility was analyzed in each preparation. Mean sperm motility in untreated vs treated preparations was compared by analysis of variance.
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Results |
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Discussion |
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, IL1-ß, and IL6 (Basu et
al, 2004). The actual source of these cytokines in the semen is
unknown, but apparently the cytokines are restricted to the urogenital system
because they are not detected in patient blood samples. Additional studies suggest that seminal plasma contributes to low sperm motility in men with SCI. For example, seminal plasma from men with SCI rapidly reduces sperm motility of normal men (Brackett et al, 1996a). Another study showed that sperm collected from the vas deferens of men with SCI (ie, sperm that had not been in contact with the seminal plasma) had higher motility than sperm from their ejaculates, whereas control subjects had similar sperm motility in their vas deferens vs their ejaculates (Brackett et al, 2000).
We investigated whether low sperm motility in men with SCI could be
improved by inactivating cytokines in seminal plasma with the use of specific
monoclonal antibodies to TNF-, IL1-ß, and IL6. In vitro treatments
were based on the 3 monoclonal antibodies mentioned above, and 8 different
treatment groups were designed to compare all possible treatment combinations.
The results showed that the low sperm motility of the untreated control group
could be improved by treatment with monoclonal antibodies, singly and in all
possible combinations; however, statistical significance was reached only in
the group treated with all 3 monoclonal antibodies (group 8).
Although the number of subjects studied is small, treatment success appears
to be related to PSM (Table).
The groups at either extreme (ie, the group with PSM 
10% or the group
with PSM 
31%) showed small but nonsignificant improvement in sperm
motility in all treatment combinations. The groups with PSMs of 11% to 19% and
20% to 30% showed significant improvement when treated with the combination of
all 3 monoclonal antibodies (ie, from 17.3% to 37.2% and from 21.4% to 47.6%,
respectively).
The reason for these differences is currently not known. Possible causes for these observations are topics for further investigation. For example, in the group with the highest mean PSM (41.6% ± 2.9%), with motility close to normal, other mechanisms at play might be higher concentrations of stimulatory cytokines, lower concentrations of reactive oxygen species, or different cytokine receptor expression (Fierro et al, 2002). For the group with the lowest mean PSM (1.6% ± 1.6%), these low sperm motilities might represent overwhelming irreparable damage related to any of the above-mentioned factors or simply to higher concentrations of toxic cytokines in the seminal plasma.
Cytokines rarely act alone and generally are expressed in groups or patterns in response to a stimulus. Their effects on other cells and cytokine pathways could be stimulatory or inhibitory (Tanaka, 2000; Petty, 2003). How cytokines negatively affect sperm motility is not fully understood. Cytokines are thought to modulate pro-oxidant and antioxidant activities in the seminal plasma, with resultant negative effects on sperm motility and fertilization (Sanocka et al, 2003). Sperm membrane phospholipid composition might be affected by oxidative damage with a resultant negative effect on fertilization potential (Zalata et al, 1998). There have been strong associations in other systems between elevations of certain seminal plasma cytokines and poor sperm motility (Sikka et al, 2001).
A relationship between reactive oxygen species (ROS) and sperm parameters has been well reported. ROS might act as intracellular signals to mediate the effects of cytokines (Sanocka et al, 2003). Our study looked only at a group of cytokines noted to be greatly elevated in the seminal plasma of SCI men. It did not account for other cytokine populations less profoundly elevated or even those found markedly lower in this group of subjects. Finally, the testing method used did not allow for analysis of each subject's seminal plasma cytokine concentrations. This information would have allowed the individualization of the amount of monoclonal antibodies for specific cytokines in each subject. Given these constraints, it is remarkable that simple addition of a combination of monoclonal antibodies resulted in significant improvement in sperm motility.
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Conclusion |
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Footnotes |
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References |
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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, Davi RC, Padron O, Lynne C. Seminal plasma of spinal cord injured men inhibits sperm motility of normal men. J Urol. 1996a;155: 1632 -1635.[Medline]
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, Lynne C, Aballa T, Ferrell S. Sperm motility from the vas deferens of spinal cord injured men is higher than from the ejaculate. J Urol. 2000;164: 712 -715.[Medline]
Brackett NL, Nash MS, Lynne CM. Male fertility following spinal
cord injury: facts and fiction. Phys Ther. 1996b; 76: 1221
-1231.
Brackett NL, Padron OF, Lynne CM. Semen quality of spinal cord injury men is better when obtained by vibratory stimulation versus electroejaculation. J Urol. 1997; 157: 151 -157.[Medline]
de Lamirande E, Leduc BE, Iwasaki A, Hassouna M, Gagnon C. Increased reactive oxygen species formation in semen of patients with spinal cord injury. Fertil Steril. 1995; 63: 637 -642.[Medline]
Fierro R, Schwed P, Foliguet B, Grignon G, Bene MC, Faure G. Expression of IL-2 alpha and IL-2 beta receptors on the membrane surface of human sperm. Arch Androl. 2002; 48: 397 -404.[Medline]
Petty HR. Detection of cytokine signal transduction "Cross-Talk" in leukocyte activation. Methods Mol Biol. 2003;249: 219 -228.
Sanocka D, Jedrzejczak P, Szumala-Kakol 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.
Sikka SC, Champion HC, Bivalacqua TJ, Estrada LS, Wang R, Rajasekaran M, Aggarwal BB, Hellstrom WJ. Role of genitourinary inflammation in infertility: synergistic effect of lipopolysaccharide and interferon-gamma on human spermatozoa. Int J Androl. 2001; 24: 136 -141.[Medline]
Tanaka Y. Integrin activation by chemokines: relevance to inflammatory adhesion cascade during T cell migration. Histol Histopathol. 2000; 15: 1169 -1176 [review].[Medline]
World Health Organization. WHO Laboratory Manual for Examination of Human Semen and Sperm-Cervical Mucous Interactions. Cambridge, United Kingdom: Cambridge University Press; 1999.
Zalata AA, Christophe AB, Depuydt CE. White blood cells cause oxidative damage to the fatty acid composition of phospholipids of human spermatozoa. Int J Androl. 1998; 21: 154 -162.[Medline]
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