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From * IVF and Andrology Laboratories, Toronto
Center for ART, Toronto, Ontario, Canada; the
Center for Advanced Research in Human
Reproduction, Infertility, and Sexual Function, Glickman Urological Institute,
The Cleveland Clinic Foundation, Cleveland, Ohio; and the
Department of Dermatology and Venereology,
Faculty of Medicine, South Valley University, Sohag, Egypt.
| Correspondence to: Dr Ashok Agarwal, HCLD, Director, Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Glickman Urological Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave, Desk A19.1, Cleveland, OH 44195 (e-mail: agarwaa{at}ccf.org). |
| Received for publication April 2, 2003; accepted for publication July 14, 2003. |
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Abstract |
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Key words: Male infertility, oxidative stress
Spermatozoa are particularly susceptible to OS-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids (Alvarez and Storey, 1995) and their cytoplasm contains low concentrations of scavenging enzymes (Aitken and Fisher, 1994; de Lamirande and Gagnon, 1995; Sharma and Agarwal, 1996). Oxidative stress attacks not only the fluidity of the sperm plasma membrane, but also the DNA integrity (Aitken, 1999; Saleh et al, 2002). Morphologically abnormal spermatozoa and seminal leukocytes are the main source of high ROS production in human ejaculates (Aitken and West, 1990; Kessopolou et al, 1992). It is important to determine the source of ROS in a given semen sample because the clinical implications of infiltrating leukocytes are quite different from those of pathological conditions in which spermatozoa themselves are the source of ROS (Aitken, 1995). Methods that are currently used for assessment of seminal OS, such as chemiluminescence assays, help measure the total amount of ROS in semen. However, such assays do not provide information on the differential contribution of spermatozoa and leukocytes to ROS production in semen or on the state of activation of individual cells.
The myeloperoxidase or the Endtz test is used to differentiate granulocytes such as neutrophils, polymorphonuclear leukocytes, and macrophages from germinal cells. Peroxidase-positive leukocytes (neutrophils and macrophages) are the main leukocytes present in semen and these are also the source of ROS formation by phagocytosis. Consequently, this test can be used an indicator of excessive ROS formation in semen (Shekkariz et al, 1995). A disadvantage of the peroxidase test is that it cannot be used to detect ROS generation by spermatozoa. On the other hand, nitroblue tetrazolium (NBT) is a yellow water-soluble nitro-substituted aromatic tetrazolium compound that reacts with cellular superoxide ions to form formazan derivative that can be monitored spectrophotometrically (Baehner et al, 1976; Armstrong et al, 2002). The cytoplasmic NADPH, which is produced by oxidation of glucose through the hexose monophosphate shunt, serves as an electron donor. The oxidase system available in the cytoplasm helps transfer electrons from NADPH to NBT and reduce NBT into formazan (Baehner et al, 1976). Thus, the NBT reaction indirectly reflects the ROS-generating activity in the cytoplasm of cells and therefore can help determine the cellular origin of ROS in a heterogeneous suspension such as the seminal ejaculate. This was the reason to justify development of the NBT test and use of NBT staining in individual cells, that is, spermatozoa and the leukocytes, and measurement of the formazan precipitation due to NBT reduction.
The objectives of our study were to determine the contribution of spermatozoa and leukocytes toward the amount of ROS production in semen, to examine the state of activation of leukocytes in semen of infertile men with various levels of leukocyte contamination, and to determine the correlation between levels of ROS and TAC in semen by chemiluminescence, ROS-TAC score and the NBT staining in spermatozoa and leukocytes.
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Materials and Methods |
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1 x
106 PPL/mL; n = 13). Donors were selected on the basis of normal semen parameters according to World Health Organization (WHO) guidelines (WHO, 1999). All specimens were collected by masturbation at the clinical andrology laboratory after an abstinence period of 48-72 hours. After liquefaction, manual semen analysis was performed to measure sperm concentration and percentage of motility by using the WHO classification. Smears of neat semen were prepared for sperm morphology assessment. The smears were fixed and stained by using the Diff-Quik kit (Baxter Healthcare Corporation, Inc, McGaw Park, Ill). Immediately after staining, the smears were rinsed in distilled water and air-dried. Smears were scored for sperm morphology by using WHO criteria.
Quantification of Seminal Leukocytes
We assessed the presence of granular leukocytes (neutrophils and
macrophages) in semen by myeloperoxidase staining or the Endtz test (Shekkariz
et al, 1995). Briefly, a 20-µL volume of liquefied semen specimen was
placed in a Corning 2.0-mL cryogenic vial (Corning Costar Corp, Cambridge,
Mass) with 20 µL of phosphate-buffered saline (PBS) (pH 7.2) and 40 µL
of benzidine solution. The solutions were mixed and allowed to sit at room
temperature for 5 minutes. PPL staining brown were counted in all 100 squares
in a Makler's chamber under the bright-field objective (magnification,
20x) in 5 different fields and an average count was determined.
Leukocytospermia was defined as counts higher than 1 x 106
PPL/mL of semen.
Density Gradient Centrifugation
Aliquots of 1 mL of the liquefied semen were loaded onto a 47% and 90%
discontinuous ISolate gradient (Irvine Scientific, Santa Ana, Calif) and
centrifuged at 500 x g for 20 minutes at room temperature. The
resulting interfaces between seminal plasma and 47% (containing leukocytes)
and 47% and 90% (containing abnormal spermatozoa) were aspirated and
transferred to separate test tubes. Both fractions (leukocytes and abnormal
spermatozoa) were diluted in 1 volume of human tubal fluid (HTF) medium and
centrifuged at 500 x g for 7 minutes. The pellet was then
resuspended in 1 mL of HTF. An aliquot from whole ejaculate and each fraction
(leukocytes and abnormal spermatozoa) was examined for sperm and leukocyte
concentration, ROS production, and NBT staining.
Measurement of Reactive Oxygen Species
Levels of ROS were measured in the whole ejaculate and immature and mature
spermatozoa by chemiluminescence assay by using luminol
(5-amino-2,3,-dihydro-1,4-phthalazinedione; Sigma Chemical Co, St Louis, Mo)
as a probe (Kobayashi et al,
2001). Measurements were made with a Berthold luminometer
(Autolumat: LKB 953, Wallace Inc, Gaithersburg, Md). Eight microliters of
horseradish peroxidase (HRP) (12.4 U of HRP type VI, 310 U/mg; Sigma) were
added to 400 µL of the whole ejaculate, leukocytes, and abnormal
spermatozoa to allow sensitization of the assay for measurement of
extracellular hydrogen peroxide. Ten microliters of luminol prepared as 5-mM
stock in dimethylsulfoxide was added to the mixture. Ten microliters of 5-mM
luminol was added to 400 µL of PBS as a negative control. Levels of ROS
were determined by measuring chemiluminescence in the integrated mode for 15
minutes. Results were expressed as x 106 counted photons per
minute (cpm) per 20 x 106 cells/mL.
Measurement of Total Antioxidant Capacity
Total nonenzymatic antioxidant capacity was measured in the seminal plasma
by using the enhanced chemiluminescence assay
(Saleh et al, 2002). The
liquefied semen samples were centrifuged at 300 x g for 7
minutes, and the seminal plasma was removed and placed into a test tube. The
seminal plasma was centrifuged at 300 x g for 10 minutes to
remove all cellular contaminants. Aliquots of the seminal plasma were stored
at -80°C until use. The frozen seminal plasma was thawed at room
temperature and immediately assessed for nonenzymatic antioxidant capacity.
The seminal plasma was diluted 1:10 with deionized water (dH2O) and
filtered through a 0.20-µm Millipore filter (Allegiance Healthcare
Corporation, McGaw Park, Ill). Signal reagent was prepared by adding 30 µL
of H2O2 (8.8 molar/L), 10 µL of para-iodophenol stock
solution (41.72 µM), and 110 µL of luminol stock solution (3.1 mM) to 10
mL of Tris buffer (0.1 M, pH 8.0). HRP working solution was prepared from an
HRP stock solution by making a dilution of 1:1 of dH2O to give a
luminescence output of 3 x 107 cpm.
Trolox (6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble tocopherol analogue, was added as a standard solution at concentration of 25, 50, and 75 µM for TAC calibration. The antioxidant capacity of the seminal plasma was then expressed in molar Trolox equivalents. With the luminometer in the kinetic mode, 100 µL of signal reagent and 100 µL of HRP working solution were added to 700 µL of dH2O and mixed. The mixture was equilibrated to the desired level of chemiluminescent output (between 2.8 and 3.2 x 107 cpm) for 100 seconds. One hundred microliters of the prepared seminal plasma was immediately added to the mixture, and the chemiluminescence was measured. Suppression of luminescence and the time from the addition of seminal plasma to 10% recovery of the initial chemiluminescence was recorded. Antioxidant capacity was expressed as molar Trolox equivalents.
Calculating the ROS-TAC Score
Reactive oxygen species values were log-transformed (log(ROS + 1)) to
normalize the data distribution. The ROS and TAC values from the controls were
used to create a scale of these 2 variables that used the control values as
reference points as discussed in our earlier work
(Sharma et al, 1999). In
brief, both log(ROS + 1) and TAC were standardized to their z-scores
and then analyzed with principal component analysis, which provided linear
combinations (or weighted sums) that accounted for the most variability among
correlated variables. The first principal component provided the following
linear equation:
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To ensure that the distribution of the standardized ROS-TAC score would
have a mean of 50 and standard deviation of 10 in the normal donors, the
ROS-TAC score was transformed as:
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Nitroblue Tetrazolium Test
Nitroblue tetrazolium (NBT) (0.1%) was prepared in PBS by adding 10 mg of
NBT powder (Sigma) to 100 mL of PBS (pH 7.2) and stirred at room temperature
for 1 hour. NBT solution was filtered with a 0.2-µm filter (Allegiance
Health Care). NBT staining was done for whole ejaculate, leukocytes, and
abnormal spermatozoa by adding equal volumes of 0.1% of NBT solution and
incubated for 30 minutes at 37°C. The tubes were centrifuged at 250
x g for 5 minutes and smears were prepared from the pellet and
air-dried. The air-dried smears were stained with Wright stain and a total of
100 spermatozoa and 100 leukocytes were scored under 100x magnification.
Three skilled observers (N.E., R.A.S., and R.S.) scored the NBT-stained slides
in a blinded manner. When the scoring differences were comparable between
these observers, 1 person (N.E.) scored the actual test samples in a blinded
fashion.
Leukocytes in the smears were scored as follows: no detectable formazan (-), scattered or few formazan granules (+), intermediate density (++), and cells filled with formazan (+++) (Figure 1). Spermatozoa in the smears were scored as follows: formazan occupying 50% or less of the cytoplasm (+) and more than 50% of the cytoplasm (++) (Figure 2).
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Statistical Analysis
Comparisons between groups and comparison of immature and mature
spermatozoa within the same individuals were performed by Wilcoxon rank-sum
test. The Spearman method was used to calculate the correlation between
peroxidase test and the NBT test and Dunn's pairwise comparison comparing the
3 groups for peroxidase and NBT test. The ROS-TAC score was compared with NBT
staining in the 3 groups. Receiver operating characteristic (ROC) curves were
used to predict a peroxidase level greater than 1 by NBT test. ROC curves
illustrate the sensitivity and specificity over the entire range. The area
under the curve (AUC) also was calculated. This can range from 50% to 100%
with diagnostic tests that approach 100% indicating a perfect predictor and
50% indicating random chance, or no predictive ability. The AUC is interpreted
as the probability that a randomly drawn leukocytospermic sample (peroxidase
values > 1 x 106 PPL/mL) has a higher value of NBT than a
randomly drawn nonleukocytospermic sample (peroxidase value < 1 x
106 PPL/mL). We also examined the various cutoff values for NBT
test to identify the best NBT cutoff points associated with sensitivity and
specificity.
All summary statistics are presented as median and interquartile values (25th and 75th percentiles). All statistical tests were 2-tailed with statistical significance considered at P < .05, and computed by using SAS version 8.1 (SAS Institute Inc, Cary, NC).
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Results |
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Reactive Oxygen Species
Production of ROS in whole ejaculate, leukocytes, and abnormal spermatozoa
among donors and patients is shown in Table
2. The differences for amount of ROS in whole ejaculate were
statistically significant across all 3 groups. The highest levels of ROS were
seen in whole ejaculate of leukocytospermic patients compared to donors
(P = .04) and nonleukocytospermic group (P = .06). In
leukocytospermic group, ROS levels showed significant positive correlation
with the concentration of round cells in leukocytospermic group (r =
0.88, P = .006).
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Seminal Total Antioxidant Capacity and ROS-TAC Score
The median (25th, 75th percentiles) seminal plasma TAC and ROS-TAC scores
for the whole ejaculate are shown in Table
2. TAC levels were comparable among the 3 groups. The lowest
median (25th, 75th percentiles) ROS-TAC score was observed in the
leukocytospermic patients compared to the donors (P = .04). The
ROS-TAC score for the nonleukocytospermic group did not differ significantly
from the donors. A significant negative correlation was found between the
ROS-TAC score and the leukocyte concentrations (r = -0.46, P
= .01), levels of ROS in the fraction containing leukocytes (r =
-0.39, P = .04), and levels of ROS in fraction containing abnormal
spermatozoa (r = -0.57, P = .002).
NBT Test Results
Median interquartile range (25th and 75th percentiles) of NBT results for
leukocytes and spermatozoa with cytoplasmic retention are shown in the
Table 3. In leukocytospermic
samples, 51% (41%, 67%) of leukocytes stained positive for NBT, of these 2.5%
(1%, 7%) were classified as (+++), 10.5 % (8%, 19%) as (++), and 36.5% (23%,
44.5%) as (+). In nonleukocytospermic samples, 6% (2%, 7%) were classified as
(+++), 1% (0, 3%) as (++), and 4% (1%, 6%) as (+). A strong positive
correlation was seen between ROS levels in whole ejaculate and an NBT-positive
response in leukocytes in whole ejaculate (r = 0.59; P =
.0006) and leukocytes (r = 0.7; P < .0001;
Figure 3). Similarly, positive
correlation was seen between spermatozoa with cytoplasmic retention in neat
semen (r = 0.5; P = .008) and abnormal spermatozoa
(r = 0.72; P < .001;
Figure 4). A strong negative
correlation was seen between ROS-TAC scores and NBT staining in leukocytes in
whole ejaculate (r = -0.60; P = .0007) and leukocytes
(r = -0.39, P = .04); and in sperm with cytoplasmic
retention in abnormal spermatozoa in abnormal spermatozoa fraction (r
= -0.38; P = .04).
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Comparison Between Peroxidase and NBT Test
Overall comparison of the 3 groups (donors and patients with and without
leukocytes) showed significant correlation between peroxidase and the NBT test
(P < .001; Figure
5A). The AUC was 0.96 with 95% confidence interval (CI) of 0.89,
1.0 (P < .001). An NBT test with a cutoff of 19% had a sensitivity
of 1.000 (0.631, 1.000) and specificity of 0.864 (0.651, 0.971). Similarly, a
cutoff of 23% and 27% had a sensitivity of 1.000 (0.631, 1.000) and 0.875
(0.473, 0.997) and specificity of 0.909 (0.708, 0.989), respectively.
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Donors (median and 25th and 75th percentiles; 0.66 [0, 1.0]) could be identified correctly from patients with leukocytospermia (2.25 [1.3, 11.7]; P < .001), both by peroxidase and NBT test (0 [0, 27] versus 51 [23, 77]; P < .001). Similarly, patients with and without leukocytospermia (0.06 [0, 0.4] versus 2.25 [1.3, 11.7]; P < .001) could be identified both by peroxidase and by NBT test (6.0 [0, 71] versus 51 [23, 77]; P < .004). Thus, both peroxidase and NBT test are sensitive to identify leukocytes in a given specimen. The high sensitivity (1.0; CI of 0.63, 1.0) and specificity (0.91; CI of 0.71, 0.99) of the NBT test provides the ability of predicting specimens with peroxidase values >1 x 106 PPL/mL of the seminal ejaculate (Figure 5B).
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Discussion |
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Activated leukocytes are capable of producing 100-fold higher amounts of ROS than nonactivated leukocytes (Plante et al, 1994). Leukocytes may be activated in response to a variety of stimuli including inflammation and infection (Pasqualotto et al, 2000). Activated leukocytes increase NADPH production via the hexose monophosphate shunt. The myeloperoxidase system of both PMN leukocytes and macrophages also is activated, which leads to respiratory burst and production of high levels of ROS (Blake et al, 1987). Such an oxidative burst is an early and effective defense mechanism in cases of infection for killing the microbes (Saran et al, 1999).
Sperm damage from ROS that is produced by leukocytes occurs if seminal leukocyte concentrations are abnormally high, that is, leukocytospermia (Shekarriz et al, 1995) or if seminal plasma is removed during sperm preparation for assisted reproduction (Ochsendorf, 1999). Excessive ROS production that exceeds critical levels can overwhelm all antioxidant defense strategies of spermatozoa and seminal plasma, causing OS (Sikka et al, 1995; Sharma and Agarwal, 1996; de Lamirande et al, 1997; Sikka, 2001). Despite the fact that OS has been established as a major factor in the pathogenesis of male infertility, consensus is lacking as to the clinical utility of seminal OS testing in an infertility clinic. One important reason for the inability to utilize an OS test in clinical practice may be the lack of a simple method that can reliably measure ROS in semen.
One of the limitations of the myeloperoxidase test is its inability to stain spermatozoa unlike the leukocytes, and hence its inability to indicate ROS levels produced by the spermatozoa. Earlier studies showed that NBT reduction and formazan deposition in blood neutrophils are related to their phagocytic activity (Park et al, 1968; Segal and Levi, 1973). In the same studies, the state of activation of neutrophils was determined by scoring the blue-black formazan granules deposited in the cytoplasm. Therefore, we attempted to evaluate the NBT assay as an indicator of ROS production in both leukocytes and spermatozoa.
To the best of our knowledge, this is the first study examining ROS-generating activity in individual cells (sperm and leukocytes) in semen based on their morphological characteristics acquired by deposition of formazan granules in ROS positive cells. Myeloperoxidase or other cytochemistry tests such as determination of granulocyte-elastase level and immunocytochemistry with monoclonal antibodies currently are used for identifying the leukocytes in semen (Wolff, 1995). These tests yield a static value for leukocyte concentration, but they do not provide any clear indication of leukocyte viability or activity. However, the NBT reduction test can be used for 2 purposes: to determine the ROS-generating activity and to detect and identify the neutrophils. We believe that the NBT has this advantage over the peroxidase test, in that the NBT test, as a histochemical method, can assess the ROS-generating activity in morphologically abnormal and immature spermatozoa with cytoplasmic retention. The NBT test can detect neutrophils at a concentration of 0.5 x 106/mL or higher (Kovalski et al, 1991). This level of sensitivity is appropriate for detecting the cutoff value of 1.0 x 106/mL established by the WHO for leukocytospermia. The NBT assay described is an indirect reflection of ROS generation. The mechanism of ROS generation in human sperm recently was found to depend upon a novel NADPH-oxidase (NOX5) resembling the multicomponent NADPH-oxidase of white blood cells (WBC). A recent study with electron spin resonance analysis, chemiluminescence, and NBT reduction indicated that the ROS-producing activity of spermatozoa may be different and significantly lower than the WBC-NADPH-oxidase (Armstrong et al, 2002).
Examination of our results indicated that the density of formazan deposition in spermatozoa and seminal leukocytes was directly correlated with the state of activation of these cells. In addition, a strong positive relationship was seen between results of the NBT test, expressed as percent NBT-positive cells, and the levels of ROS in the same cell suspensions as measured by chemiluminescence assay. Although luminol-dependent chemiluminescence assay helped measure the total amount of ROS in semen, the NBT test provided information on the differential contribution of spermatozoa and leukocytes to ROS production in semen and on the state of activation of these cells. It is true that NBT reacts with cellular superoxide ions to form formazan derivatives that can be monitored spectrophotometrically or as demonstrated in our study. However, many cellular reductases also can donate an electron to NBT, forming its radical, which under aerobic conditions reacts with environmental oxygen to form monoformazan. This formazan formation can be inhibited by superoxide dismutase (SOD), and the SOD-rich seminal plasma. In addition, changes in cellular content of various oxidoreductases also are responsible for alterations in rates of NBT reduction (Fridovich, 1997). However, we did not examine this aspect as this was not the focus of the present study.
In our study, overall comparison of the 3 groups (donors and patients with and without leukocytospermia) showed significant correlation between peroxidase and the NBT test. We examined the ROC curve predicting peroxidase level greater than 1 x 106 PPL/mL by the NBT test. The AUC was 0.96 with a 95% CI of 0.89, 1.0 (P < .001). We also examined the various cutoff values for the NBT test to identify the best NBT cutoff points associated with sensitivity and specificity. Our results indicate a strong positive relationship between the results of the NBT test, expressed as percent NBT-positive cells, and the level of ROS of the same cell suspension as measured by chemiluminescence.
Furthermore, results of the NBT test were inversely correlated with the ROS-TAC score, which has been recently introduced as an accurate measure of oxidative stress in infertile men (Sharma et al, 1999). The ROS-TAC score minimizes the variability present in the individual parameters of oxidative stress (ROS alone or TAC alone). In our earlier studies we stressed the importance of ROS-TAC scores as an index of oxidative stress that may explain previously unexplained cases of male infertility (Pasqualotto et al, 2001). In addition, both abnormal spermatozoa and leukocyte contamination influence ROS-TAC score, and lowest scores are seen in patients with leukocytospermia (Sharma et al, 2001). Examination of our results suggests that the NBT test can be used as a simple alternative to the ROS-TAC score for assessment of the levels of seminal oxidative stress. Therefore, the use of the NBT test in semen may have important implication both for the andrology research and clinical practice.
In conclusion, identification of particular cells in semen that produce ROS in excess may be the first step towards detection of the underlying inherent or acquired defect behind such abnormality. In addition, the NBT reduction test is readily available, easily performed, inexpensive, and has high sensitivity. This test can be used for assessment of seminal oxidative stress, and the differential contribution of cells to ROS generation, and to determine the state of activation of seminal leukocytes. Such a test can be added to the routine clinical andrology workup for assessment of seminal OS without the need for expensive equipment such as the luminometer.
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Footnotes |
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): whole ejaculate; (•): immature spermatozoa; (—):
regression line for correlation between ROS and NBT in whole ejaculate; and (-
- -): regression line for correlation between ROS and NBT in immature
spermatozoa.
