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From the * Department of Biophysics,
Medical Biology, and
Urology, Cerrahpa
a Medical Faculty,
Istanbul University, 
stanbul, Turkey.
| Correspondence to: Birsen Aydemir, PhD, Cobancesme Mah, Valide Sok, No: 23/3 Yenibosna, Bahcelievler 34197, Istanbul, Turkey (e-mail: birsenay2001{at}yahoo.com). |
| Received for publication April 18, 2007; accepted for publication July 30, 2007. |
 |
Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Key words: Male infertility, malondialdehyde, protein carbonyls
On the other hand, recent studies have shown that oxidative stress is associated with blood viscosity (Jain et al, 1990; Chung and Ho, 1999; Liu et al, 2004). It has been found that blood viscosity increases were correlated with certain diseases associated with oxidative damage, such as diabetes mellitus, coronary artery disease, hyperlipoproteinemia and chronic kidney disease (Vaya et al, 1993; Kesmarky et al, 2006; Marcinkowska-Gapinska and Kowal, 2006). Serum levels of malondialdehyde (MDA), one of the most studied unsaturated carbonyl products of oxidative stress, were also correlated with blood viscosity (Jain et al, 1990; Chung and Ho, 1999; Liu et al, 2004) and other oxidative stress parameters, such as serum protein carbonyl content (Turkoglu et al, 2000). Several research groups have studied the elevated blood viscosity of erythrocyte membrane proteins induced by oxidative stress and MDA and have suggested that MDA, by leading to protein cross-links, might be the cause of viscosity changes (Pfafferott et al, 1982; Pasini et al, 1991; Uyesaka et al, 1992).
If the increased oxidative damage in blood is correlated with increases in the viscosity of blood and plasma of certain diseases associated with oxidative damage, then it is possible that there is an association between seminal fluid viscosity and seminal oxidative damage in infertile males, although compositions of blood and seminal fluid are different.
Increased viscosity of ejaculate was reported to occur more frequently
among infertile couples than in fertile males
(Bunge, 1970;
Hubner et al, 1985;
Elzanaty et al, 2004). Several
conditions, such as concentrations of prostate-specific antigen, zinc and
calcium, and activity of neutral 
-glucosidase in seminal plasma, were
found to be correlated with changed semen viscosity
(Mendeluk et al, 2000;
Elzanaty et al, 2004;
Andrade-Rocha, 2005). Siciliano
et al (2001) has demonstrated
a severe impairment of both the high– and low–molecular weight
antioxidative systems in semen with hyperviscosity. However, there are no
reports studying the relationship of oxidative damage and semen viscosity in
the context of male infertility. Therefore, the general aim of this study was
to determine whether the viscosity of seminal fluid is associated with MDA and
protein carbonyl levels of sperm and seminal plasma in infertile patients.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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a Medical Faculty,
stanbul, Turkey. Semen specimens were obtained from 60 men aged
26–48 years with infertility between 2005 and 2006. Specimens were also
obtained from 42 male volunteers aged 24–49 years with normal semen
analysis according to World Health Organization
(WHO, 1999) guidelines to
serve as the fertile control. Institutional Ethical Committee approval was
taken in accordance with the principles of the Declaration of Helsinki.
Informed consent was obtained from each study subject. Individuals with a
significant medical history, signs suggestive of defective androgenization, or
abnormal testicular examinations were excluded from this study. Further
exclusion criteria for both groups included chromosomal disorders related to a
fertility disorder, cryptorchidism, vasectomy, abnormal liver function and
hormone tests, cigarette smoking, alcohol consumption, or the use of folic
acid, glutathione, vitamin C, or vitamin E supplements or medication within 3
months before recruitment. Criteria for study inclusion were infertility for
at least 12 months, with at least 1 semen parameter abnormality, semen
leukocyte count less than 1 x 106/mL, and negative semen
anti-sperm antibody on a mixed agglutination reaction test. Semen specimens
were collected by masturbation into a sterile wide-mouth metal-free plastic
container after at least 3 days (3–5 days) of abstinence and liquefied
at 25°C for 30 minutes.
Semen Analysis
A semen analysis was carried out according to WHO guidelines to obtain
volume, pH, sperm concentration, motility, and morphology. Sperm concentration
was determined with a Makler Counting Chamber (Seti-Medical Instruments,
Haifa, Israel). Motility was expressed as a percentage of motile spermatozoa
and their mean velocity. Morphology was determined according to the WHO
criteria after incubation of the sample with trypsin for 10 minutes at
25°C according to the methylene blue eosin staining procedure, feathering
and fixation by flame. At least 100 cells were examined at a final
magnification of 1000x. Semen visco-elasticity was assessed using glass
pipettes as recommended in the WHO guidelines and semen samples showing a
thread of more than 2 cm long were considered highly visco-elastic, whereas
the viscoelasticity was considered normal when the thread length was 2 cm or
less.
Measurement of Seminal Plasma Viscosity
After liquefaction, at least 1 mL of semen was centrifuged at 400 x
g for 15 minutes at 25°C. Seminal plasma was collected at the
top. Manual viscosity measurements were made according to the recommendations
of the International Committee for Standardization in Haematology
(1984) with the use of a
Harkness capillary viscometer (Coulter Electronics Ltd, serial No: 6083,
Luton, United Kingdom) and were evaluated in relation to distilled water
(relative viscosity), the water bath of which was maintained at 35°C
(Harkness, 1963). This system
comprises a glass capillary tube 0.30 mm in internal diameter and 200 mm long
through which a sample of 0.5 mL is forced at a constant positive pressure of
17.2 kPa. Seminal fluid viscosity is proportional to its flow time through the
capillary. The seminal fluid viscosities were expressed in millipascal seconds
(mPa·s). The intra-assay coefficients of variation for seminal plasma
viscosity was 6.7%. With this device, we calculated the viscosity of seminal
fluid according to the formula of
Poiseuille (1840),
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is viscosity (mPa·s), 
P is the pressure
gradient (pascals), r is the radius of the capillary (m), L
is length (m), Q is flow rate (m3/s), and t is
time (s).
Spermatozoa Preparation
After liquefaction, spermatozoa were fractionated on Percoll gradients
(40%–95%) according to WHO guidelines. Semen was layered on top of the
gradient and centrifuged at 400 x g for 20 minutes at 25°C.
Spermatozoa in the 95% Percoll layer were collected and washed twice at 400
x g for 6 minutes at 25°C with added Tris, sodium, and EDTA
(TNE) buffer (0.15 mol/L NaCl, 0.01 mol/L Tris-HCl, 1 mmol/L
Na2-EDTA, pH 7.4) (Alkan et al,
1997). The remaining spermatozoa were frozen without preservatives
and stored for up to 1 month at -70°C before being assayed for
malondialdehyde and protein carbonyls.
Measurement of Lipid Peroxidation
The lipid peroxide levels in the seminal plasma and spermatozoa were
measured with a thiobarbituric acid (TBA)–reactive substance assay,
which monitors MDA production on the basis of the method of Buege and Aust
(1978). Briefly, to a 100-µL
sample of seminal plasma (or 1 x 106 spermatozoa/mL), 200
µL of cold 1.15% (wt/vol) KCl was sonicated for 30 seconds on ice and added
to 1.8 mL of 3% phosphoric acid and 0.6 mL of 0.6% TBA. These mixtures were
heated in boiling water for 45 minutes. After cooling, the MDA was extracted
by centrifugation at 1500 x g for 10 minutes at 25°C, and
the intensity was measured at 535 nm by ultraviolet-visible spectrophotometry
(Shimadzu UV-1601, Tokyo, Japan). The MDA level was determined from the molar
absorption coefficient of the MDA at 535 nm, 1.56 x 105 mol/L
cm-1.
Measurement of Protein Carbonyls
Because carbonyl groups (aldehydes and ketones) might be introduced into
proteins by ROS and free radicals, quantitation of protein carbonyls was
carried out by incubating equal volumes of the sample (seminal plasma or 1
x 106 spermatozoa/mL) and 2,4-dinitrophenylhydrazine (3.4
mg/10 mL of 1 mol/L HCl) at 50°C for 1 hour. After the reaction, proteins
were precipitated with 20% trichloroacetic acid, and the unreacted dye was
removed by centrifugation. The pellet was dissolved in 1 mol/L NaOH, and the
absorbance at 450 nm was recorded. The molar absorbance coefficient (
=
25 500 mol/L cm-1) was used to calculate the carbonyl content
(Levine et al, 1990). Protein
concentrations were determined by the Lowry method, with bovine serum albumin
as the standard (Lowry et al,
1951).
Statistical Methods
Values reported are 
± SD.
All data were normally distributed and underwent equal variance testing.
Statistical significance of differences was determined by SPSS program version
11.5 for Windows (SPSS Inc, Chicago; Ill). Average comparison between 2
subgroups was made by Student's t test. Correlation between seminal
fluid viscosity, and seminal malondialdehyde and carbonyl levels was tested
with a Pearson correlation model. Data were considered statistically
significant at P < .05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Considering the oxidative stress biomarkers, we compared the levels of MDA and protein carbonyls in spermatozoa and seminal plasma between infertile patients and controls. As shown in Table 2, protein carbonyls and MDA levels in the seminal plasma and spermatozoa were significantly higher in the infertile group.
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When viscoelasticity of semen samples was assessed according to the WHO guidelines, all of the control and patient subjects had normal viscoelasticity. However, when the seminal viscosity of patients and controls were compared after the capillary viscometer was used to study seminal plasma viscosity changes, the mean seminal plasma viscosity value of the patient group was significantly higher then the control group (P < .05) (Table 2). Additionally, oxidative damage was associated with seminal plasma viscosity in subfertile and control subjects. Correlation analysis (Pearson test) revealed a significant positive relationship of seminal fluid viscosity with seminal plasma MDA and sperm MDA (r = .676, P < .01; r = .482, P < .01, respectively) in the subfertile group (Table 3; Figure). In addition, seminal plasma viscosity was significantly but weakly correlated with sperm and seminal plasma protein carbonyl concentrations (r = .276, P < .05; r = .308, P < .05, respectively). However, these observed correlations were not found in the control groups (Table 3).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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On the basis of semen samples from a group of 60 patients, seminal plasma MDA and protein carbonyl levels were found to be increased in infertile subjects. Our findings are in accordance with other investigators' reports (Chen et al, 1997; Aydemir et al, 2007). When we applied the WHO standard method for assessment of semen viscoelasticity, all subjects were normoviscous for semen. However, analysis of the viscosity values measured with a capillary viscometer (Table 2) showed a statistically significant difference in levels of seminal plasma viscosity between the group of healthy patients and control subjects with normal viscoelasticity. The simplest explanation for lack of difference in semen viscosity between our infertile and control subjects, when viscoelasticity was assessed with glass pipettes, might be because this method is qualitative and might be inefficient for estimating the differences in semen viscosity. Moreover, sperm concentration might be an important determinant of semen viscosity because the mean sperm concentration values were significantly higher in control subjects than in infertile subjects. However, an exact explanation for this situation could not be made by Hubner et al (1985) and Lin et al (1992), who found increased viscosity in the semen samples of infertile subjects by rotational viscometer. Viscometry by capillary viscometers is a well-accepted method frequently used for analysis of non-Newtonian liquids in the laboratory and is a good indicator of samples containing macromolecules. To our knowledge, our results are the first to report seminal fluid viscosity measured by capillary viscometer.
Furthermore, our study demonstrated significant associations between
viscosity of seminal fluid and MDA levels in seminal plasma and sperm of
infertile subjects. Again, seminal fluid viscosity was also correlated
positively with protein carbonyl levels of sperm and seminal plasma in
infertile patients, although it was weakly correlated. Some investigators have
found that some conditions, including concentrations of prostate-specific
antigens zinc and calcium and activity of neutral -glucosidase in
seminal fluid, were correlated with changed semen viscosity
(Bunge, 1970;
Mendeluk et al, 2000;
Elzanaty et al, 2004;
Andrade-Rocha, 2005). Siciliano
et al (2001) have reported
that the lowering of catalase activity and total antioxidant status values
appeared to be associated with semen hyperviscosity. The present experimental
data suggest that MDA and protein oxidation might contribute to viscosity
increase in seminal fluid. The possible explanation for high seminal viscosity
could be a change in protein-protein interactions in the seminal plasma. MDA,
which is an end product of lipid peroxidation, is known to cross-bind or
induce secondary oxidative damage in the plasma proteins
(Traverso et al, 2004). Small
molecular aldehydes can react with membrane proteins and modify their
structure. Malondialdehyde also causes decreased protein solubility, which is
related to changes in viscosity
(Ingemansson et al 1995). In
addition, it has suggested that proteins modified directly by ROSs with the
eventual formation of oxidized amino acids and proteins modified indirectly
with reactive carbonyl compounds formed by the autoxidation of carbohydrates
and lipids might be cause of the viscosity changes
(Esterbauer et al, 1991). Such
a biological side-reaction inevitably might cause alterations in the
physicochemical properties of biological materials, resulting in changes in
the membrane fluidity of sperm, leading to viscosity alteration. However, the
high seminal plasma viscosity observed in our patients could be the result of
a rise in many other products of oxidative damage (eg, unsaturated aldehydes)
and other factors. On the other hand, Mendeluk et al
(2000) reported that protein
disulfide bonds in the gel network of seminal plasma are responsible for the
differential rheological properties observed in the hyperviscous group. It is
known that oxidative stress leads to disulfide bond formation of sulfoxidation
in sulfhydryl residues in proteins.
Therefore, a further study is desired to assess how oxidative damage might contribute to seminal viscosity changes.
The data from our study by capillary viscometer demonstrated positive relationships between seminal fluid viscosity and seminal malondialdehyde and carbonyl levels in infertile males. These results suggest that seminal fluid viscosity is influenced by the products of oxidative damage in the seminal plasma.
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Acknowledgments |
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enocak for statistical
advice and Dr Emre Basatemur (Barnet General Hospital, London, United Kingdom)
for critical revision of the manuscript. |
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aitken RJ, Buckingham D, Harkiss D. Use of a xanthine oxidase free
radical generating system to investigate the cytotoxic effects of reactive
oxygen species on human spermatozoa. J Reprod Fertil. 1993; 97: 441
–450.
Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod. 1989;40: 183 –197.
Alkan I, Simsek F, Haklar G, Kervancioglu E, Ozveri H, Yalcin S, Akdas A. Reactive oxygen species production by the spermatozoa of patients with idiopathic infertility: relationship to seminal plasma antioxidants. J Urol. 1997;157: 140 –143.[CrossRef][Medline]
Andrade-Rocha FT. Physical analysis of ejaculate to evaluate the secretory activity of the seminal vesicles and prostate. Clin Chem Lab Med. 2005; 43(11): 1203 –1210.[CrossRef][Medline]
Aydemir B, Onaran I, Kiziler AR, Alici B, Akyolcu MC. Increased oxidative damage of sperm and seminal plasma in men with idiopathic infertility is higher in patients with glutathione S-transferase Mu-1 null genotype. Asian J Androl. 2007; 9(1): 108 –115.[CrossRef][Medline]
Buege JA, Aust ST. Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302 –310.[Medline]
Bunge RG. Some observations on the male ejaculate. Fertil Steril. 1970; 21(9): 638 –644.
Chen CS, Chao H-T, Pan R-L, Wei Y-H. Hydroxyl radical–induced decline in motility and increase in lipid peroxidation and DNA modification in human sperm. Biochem Mol Biol Int. 1997; 43: 291 –303.[Medline]
Chung TW, Ho CP. Changes in viscosity of low shear rates and viscoelastic properties of oxidative erythrocyte suspensions. Clin. Hemorheol. Microcirc. 1999; 21: 99 –103.[Medline]
Elzanaty S, Malm J, Giwercman A. Visco-elasticity of seminal fluid in relation to the epididymal and accessory sex gland function and its impact on sperm motility. Int J Androl. 2004; 27(2): 94 –100.[CrossRef][Medline]
Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991; 11(1): 81 –128.[CrossRef][Medline]
Fraczek M, Kurpisz M. Inflammatory mediators exert human
spermatozoa. J Androl. 2007; 28: 325
–333.
Halliwell B, & Gutteridge JM, eds. Free Radicals in Biology and Medicine. 3rd ed. New York: Oxford University Press, 1999; 617–783.
Harkness J. A new method for the measurement of plasma viscosity. Lancet. 1963;2: 280 .[CrossRef][Medline]
Hubner HM, Heidl R, Krause W. Investigation of flow behaviour (viscosity) from human seminal fluid with a rotational viscosimeter. Andrologia. 1985; 17(6): 592 –597.[Medline]
Ingemansson T, Kaufmann P, Ekstrand B. Multivariate evaluation of lipid hydrolysis and oxidation data from light and dark muscle of frozen stored rainbow trout (Oncorhynchus mykiss). J Agric Food Chem. 1995;43: 2046 –2052.[CrossRef]
International Committee for Standardization in Heamatology,
Recommendation for a selected method for the measurement of plasma viscosity.
J Clin Pathol. 1984; 37: 1147
–1152.
Jain SK, Ross JD, Levy GJ, Duett J. The effect of malonyldialdehyde on viscosity of normal and sickle red blood cells. Biochem Med Metab Biol. 1990;44: 37 –41.[CrossRef][Medline]
Kesmarky G, Feher G, Koltai K, Horvath B, Toth K. Viscosity, hemostasis and inflammation in atherosclerotic heart diseases. Clin Hemorheol Microcirc. 2006; 35(1–2): 67 –73.[Medline]
Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Shaltiel S, Stadtman ER. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990; 186: 464 –478.[Medline]
Lin MC, Tsai TC, Yang YS. Measurement of viscosity of human semen with a rotational viscometer. J Formos Med Assoc. 1992; 91(4): 419 –423.[Medline]
Liu X, Qin W, Yin D. Biochemical relevance between oxidative/carbonyl stress and elevated viscosity of erythrocyte suspensions. Clin Hemorheol Microcirc. 2004; 31(2): 149 –156.[Medline]
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement
with the Folin phenol reagent. J Biol Chem. 1951; 193: 265
–275.
Marcinkowska-Gapinska A, Kowal P. Blood fluidity and thermography in patients with diabetes mellitus and coronary artery disease in comparison to healthy subjects. Clin Hemorheol Microcirc. 2006; 35(4): 473 –479.[Medline]
Mendeluk G, Flecha FLG, Castello PR, Bregni C. Factors involved in the biochemical etiology of human seminal plasma hyperviscosity. J Androl. 2000; 21(2): 262 –267.[Abstract]
Pasini FL, Pasqui AL, Pieragalli D, Acciavatti A, Saletti M, Guideri F, Galigani C, Perri TD. Soluble haemoglobin–induced rheological impairment: a role for oxygen free radical formation. Clin Hemorheol. 1991;11: 361 –367.
Pfafferott C, Meiselman HJ, Hochstein P. The effect of
malonyldialdehyde on erythrocyte deformability. Blood. 1982; 59: 12
–15.
Poiseuille JLM. Recherches experimentales sur le mouvement des liquides dans les tubes de tres petits diameters. Compt Rend Acad Sci. 1840;11: 961 –1041.
Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 1994;233: 359 .
Siciliano L, Tarantino P, Longobardi F, Rago V, de Stefano C, Carpino A. Impaired seminal antioxidant capacity in human semen with hyperviscosity oligoasthenozoospermia. J Androl. 2001; 22(5): 798 –803.[Abstract]
Singhal SS, Saxena M, Ahmad H, Awasthi S, Haque AK, Awasthi YC. Glutathione S-transferases of human lung: characterization and evaluation of the protective role of the alpha-class isozymes against lipid peroxidation. Arch Biochem Biophys. 1992; 299: 232 –241.[CrossRef][Medline]
Traverso N, Menini S, Maineri EP, Patriarca S, Odetti P, Cottalasso
D, Marinari UM, Pronzato MA. Malondialdeyde, a lipoperoxidation-derived
aldehyde, can bring about secondary oxidative damage to proteins. J
Gerontol A Biol Sci Med Sci. 2004; 59(9): B890
–B895.
Turkoglu UM, Dogru-Abbasoglu S, Aykac-Toker G, Mirsal H, Beyazyürek M, Uysal M. Increased lipid and protein oxidation and DNA damage in patients with chronic alcoholism. J Lab Clin Med. 2000;136: 287 .[CrossRef][Medline]
Uyesaka N, Hasegawa S, Ishioka N, Ishioka R, Shio H, Schechter AN. Effect of superoxide anions on red cell deformability and membrane proteins. Biorheology. 1992; 29: 217 –229.[Medline]
Vaya A, Martinez M, Carmena R, Aznar J. The lipid composition of red blood cells and their hemorheological behavior in patients with primary hyperlipoproteinemia. Clin Hemorheol. 1993; 13: 447 –457.
World Health Organization. Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 3rd ed. New York: Cambridge University Press, 1999.
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