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From the * Department of Urology, University of
Sao Paulo, Sao Paulo, Brazil; and Reproductive
Research Center, Glickman Urological Institute and Department of
Obstetrics-Gynecology, Cleveland Clinic, Cleveland, Ohio.
| Correspondence to: Dr Jorge Hallak, Rua Dr Eneas de Carvalho Aguiar, 255 7o andar - Sala 7123, ICHC - Pós-Graduação Divisão de Urologia, CEP: 05422-970 Brazil – Sao Paulo (e-mail: hallakj{at}androscience.com.br). |
| Received for publication October 11, 2006; accepted for publication March 22, 2007. |
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Abstract |
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Key words: Oxidative stress, male infertility, Endtz test, spermatozoa, standardization
The World Health Organization (WHO) considers leukocytospermia to be a condition in which leukocyte levels are equal to or exceed 1 x 106/mL. Studies have reported oxidative stress in sperm cells from subfertile men even when the leukocyte concentration is low. However, the minimum leukocyte level that can cause oxidative stress in semen has yet to be determined (Sharma et al, 2001).
Other possible sources of ROSs include xenobiotics, pollutants, and abnormal spermatozoa (Aitken and Krausz, 2001). High levels of ROSs generated by abnormal spermatozoa negatively affect fertilization as well as pregnancy rates after in vitro fertilization and are associated with increased sperm DNA damage (Aitken et al, 1991; Zorn et al, 2003). In addition, clinical studies have found that urogenital inflammation significantly influences acrosomal functionality by the presence of inflammatory mediators such as ROSs (Henkel et al, 2006).
Many methods are available to measure ROS levels in semen, but chemiluminescence is the most widely used in both neat and washed semen samples (Agarwal et al, 2004). Sperm-washing techniques include many steps such as multiple centrifugations, resuspension, and vortexing. These steps may artificially increase ROS levels and produce inaccurate test results (Agarwal et al, 1994; Twigg et al, 1998). When seminal plasma is extracted during the washing procedure, so too are protective antioxidants. As a result, levels of ROSs increase. Determining ROS levels in washed semen may reflect an inaccurate measure of the in vivo oxidative stress status (Allamaneni et al, 2005).
The aims of this study were to 1) determine the normal reference levels of ROSs in neat and washed semen of a proven fertile population and 2) evaluate the correlation between ROSs, seminal parameters, and low leukocyte levels (Endtz test, less than 1 x 106/mL).
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Materials and Methods |
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Study Population
In this cross-sectional study, the population consisted of 114 fertile and
47 subfertile males. The fertile group was composed of men seeking voluntary
sterilization by vasectomy at the Division of Urology, Medical School,
University of São Paulo between May 2004 and January 2006. The fertile
group included healthy males with no history of fertility problems and whose
partners were pregnant or had delivered a child in the past. These patients
constituted the proven fertile population. The fertile population was divided
into 2 groups based on the presence of leukocytes in semen: 39 fertile men
with leukocytes (Endtz, more than 0 and less than 1 x 106/mL)
and 75 fertile men without leukocytes (Endtz, 0). A group of 47 subfertile
patients from couples who had failed to achieve pregnancy after more than 1
year of regular unprotected intercourse served as the positive control. All
female partners of these men had undergone gynecologic evaluation and had
normal results.
Both groups were evaluated by 2 male infertility specialists. Patients were excluded from the study when there was history of illicit drug use; exposure to any environmental or occupational toxicants; use of medication with proven toxicity on fertility; exposure to radiation or heat; mumps with orchitis; sexually transmitted or systemic diseases; cryptorchidism regardless of treatment; testicular torsion; genitourinary anomalies; epydidimal or vas deferens alterations; and/or scrotal or inguinal surgery. The same exclusion criteria were applied to the fertile and infertile groups.
All samples were subjected to semen analysis, leukocytospermia testing (Endtz), and chemiluminescence to determinate ROS levels in neat and washed semen.
Semen Analysis
Semen was collected by masturbation after 48 to 72 hours of sexual
abstinence. After liquefaction, all semen analyses were performed manually.
Macroscopic and microscopic parameters were performed according to WHO
guidelines (World Health Organization,
1999). For each fresh measurement, a 5-µL aliquot from patient
samples was loaded into a Makler chamber (Sefi Medical, Haifa, Israel). The
following variables were determined: sperm concentration; total motility;
percentage of spermatozoa grade A, grade B, grade C; total motile sperm; and
total sperm.
Sperm morphology was assessed using the methodology described by Kruger (Kruger et al, 1988) and by the WHO (World Health Organization, 1992). During morphology evaluation, seminal smears were stained with Giemsa stain (Diff-Quick; Baxter Healthcare, McGraw Park, Ill). Semen smears were performed in duplicate and stained at the clinical sites. Stained slides were air dried, and coverslips were applied with 2 drops of Cytoseal mounting medium (Richard-Allan Scientific, Kalamazoo, Mich). Manual morphologic assessment was performed at immersion magnification.
Leukocytospermia (Endtz) Test
Leukocyte concentrations in semen were quantified using a
myeloperoxidase-staining test (Shekarriz
et al, 1995a). A 20-µL volume of liquefied specimen was placed
in a 1.8-mL microtube. Twenty microliters of phosphate-buffered saline ([PBS]
pH 7.0) and 40 µL of benzidine solution were added. The mixture was
vortexed and allowed to sit for 5 minutes. Five microliters of this mixture
was placed in a Makler chamber and examined for brown stained cells,
indicating the presence of peroxidase. These brown stained cells were
therefore identified as granulocytes
(Shekarriz et al, 1995a).
Measurement of ROSs
ROSs in Washed Semen—
Aliquots of liquefied semen were centrifuged at 300 x g for
7 minutes. The sperm pellet was washed twice with human tubal fluid, HEPES
buffered (Irvine Scientific, Santa Ana, Calif), and resuspended in the same
medium at a concentration of 20 x 106 sperm per milliliter
(Shekarriz et al, 1995a). ROS
production was measured by the chemiluminescence assay method using luminol
(5-amino-2,3-dihydro 1,4-phthalazinedione; Sigma Chemical Co, St Louis, Mo) as
the probe. Ten microliters of 5-mM luminol prepared in dimethyl sulfoxide
(DMSO) (Sigma) was added to 400 µL of the washed sperm suspension.
ROSs in Neat Semen— Ten microliters of 5-mM luminol prepared in DMSO was added to 400 µL of liquefied semen.
For both neat and washed semen, ROS levels were determined by measuring chemiluminescence via a luminometer (MicroBeta TriLux, version 4.7; Perkin Elmer Life Sciences, Turku, Finland) for 15 minutes. The results were expressed as 104 counted photons per minute (cpm) per 20 x 106 sperm.
Statistical Analysis
The median and interquartile range (IQR) (25%, 75%) were used to describe
the populations. The Mann-Whitney U test, a nonparametric test, was
used to compare the groups. Correlation between variables was calculated using
the Spearman correlation coefficient for nonparametric data. The sensitivity,
specificity, and accuracy were calculated for different cutoffs to determine
reference values. Receiver operating characteristic (ROC) curves and the area
under the curve (AUC) were calculated to identify which cutoff value would be
superior in differentiating between the subfertile and fertile subjects.
Accordingly, different cutoffs were defined and evaluated. All analyses were
performed with MINITAB (version 14.2; State College, Pa), SAS (version 8.1;
Cary, NC), and SPSS (version 14.0; Chicago, Ill). A P value of less
than .05 was considered statistically significant.
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Results |
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Fertile men with leukocytes had higher sperm counts (106/mL) than the subfertile men with leukocytes (99.3 [59, 150] vs 41 [18, 81]) (P = .0001). In the same population, ROS levels (x 104 cpm /20 x 106 sperm) in the fertile men were lower in neat (0.75 [0.27, 1.71] vs 2.0 [0.97, 27.41]) (P = .001) and washed semen (15.85 [4.18, 62.16] vs 239.83 [33.4, 1193.75]) (P < .0001).
Similarly, the fertile men without leukocytes had a higher sperm count (106/mL) than the subfertile men without leukocytes (74 [50.5, 142] vs 23 [10.8, 41.5]) (P < .0001). In the same population, ROS levels (x 104 cpm/20 x 106 sperm) in the fertile men were lower in neat (0.29 [0.18, 0.54] vs 0.94 [0.38, 1.51]) (P = .001) and washed semen (5.73 [1.90, 14.71] vs 23.4 [9.46, 115.55]) (P = .001).
Fertile Men With Lower Seminal Levels of Leukocytes (Endtz, More Than 0 and Less Than 1 x 106/mL)
Correlations between the 2 measurements of ROSs (neat and washed) and sperm
concentration; total motility; percentage of grade A, B, and C spermatozoa;
and morphology (strict criteria and WHO guidelines) were performed considering
ROSs as the sole predictor in each model
(Table 2). This analysis was
performed in all fertile men with leukocyte levels below 1 x
106/mL.
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In neat and washed semen, ROSs were negatively correlated with sperm concentration, total motility, and grade A and B motility. A negative correlation was found between ROSs, grade C motility, and morphology using both WHO and strict criteria. A significant positive correlation between ROSs in neat and washed semen (r = .62; P < .0001) and leukocyte levels was determined.
Fertile Men With Leukocytes (Endtz, Less Than 1 x 106/mL) and Fertile Men Without Leukocytes (Endtz, 0)
No statistically significant differences were found in the median (IQR)
value in age between fertile men with leukocytes (34.7 years [32, 40 years])
and fertile men without leukocytes (34.1 years [31, 37 years]) (P =
.29) (Table 3). Fertile men
with seminal leukocytes had significantly higher levels of ROSs in washed and
neat samples than the fertile men without seminal leukocytes. There were no
significant differences in sperm concentration, total motility, and grade A,
B, and C motility when the 2 populations of fertile men were compared.
Although sperm morphology according to the WHO criteria was higher in the men
with seminal leukocytes, no significant difference was found when using strict
criteria. ROS levels in neat and washed semen were significantly lower in the
fertile men without leukocytes than in the fertile men with leukocytes.
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ROC Curves
To determine whether the ROS test on neat and washed semen could better
differentiate fertile patients from subfertile patients, we examined various
cutoff values to determine one with a high sensitivity
(Table 4). This evaluation was
performed by generating ROC curves and the AUC, as shown in the Figure.
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ROSs in Neat and Washed Semen Without Leukocytes (Endtz, 0)
With a cutoff value of 0.2 (x 104 cpm/20 x
106 sperm) for ROSs in neat semen without leukocytes, the
sensitivity was high at 93.3% and the specificity was low at 34.7%. The
accuracy was 44.4% (defined as the positively diagnosed subfertile patients
and correctly excluded fertile patients). With a cutoff value of 0.4, the
sensitivity was 80%, but the specificity was 56%. With this cutoff value,
levels of ROSs could correctly identify 80% of the subfertile patients as
subfertile, but the accuracy to identify fertile patients from the subfertile
sample was only 60%.
An increase in specificity implies a corresponding decrease in sensitivity. One option to combat this was to give equal weight to both variables and evaluate accuracy. By giving equal weight to sensitivity and specificity, the cutoff value was 0.5 with 75.6% accuracy. The AUC for this test was 76.4% with a 95% confidence interval (CI) of 62.6% to 90.2%. Sixty-eight patients of 90 could be correctly classified using the ROS test in neat semen without leukocytes (true positive and true negative).
In the same group of patients, performing the test on washed samples with a cutoff of 3.0 yielded 93.3% sensitivity and 32% specificity and only 42.2% accuracy. Using 5.0 as a cutoff, the sensitivity was 86.7% and the specificity was 48%. The accuracy was 54.4%. By giving equal weight to sensitivity and specificity, the cutoff value was 10.0 with 68.9% accuracy. The AUC for this test was 77% with a 95% CI of 62.6% to 91.7%. Sixty-two patients of 90 could be correctly classified with the ROS test on washed semen without leukocytes (Table 4).
ROS in Neat and in Washed Semen with Leukocytes (Endtz, More than 0 and Less Than 1x 106/mL)
For patients with leukocytes in neat semen, a cutoff of 0.36 (x
104 cpm/20 x 106 sperm) yielded 90% sensitivity
but a very poor specificity of 33.3% and accuracy of 58.0%. Using the cutoff
of 0.6, the sensitivity was 80% and specificity was 61%. By giving equal
weight to sensitivity and specificity, the cutoff value was 1.25 with 69.6%
accuracy. The AUC for this test was 72.7% with a 95% CI of 60.8% to 84.7%.
Forty-nine patients of 71 could be correctly classified with the ROS test on
neat semen with leukocytes.
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Using the ROS test on washed samples with leukocytes and a cutoff of 8.0, the sensitivity was 93.8% with 33.3% specificity and 60.6% accuracy. When this cutoff was increased to 12.0, the sensitivity was 87.5% but the specificity and accuracy were both 38.5%. By giving equal weight to sensitivity and specificity, the best cutoff value determined was 51.5 with 73.2% accuracy. The AUC for this test was 81% with a 95% CI of 70.3% to 91.6%. Fifty-two patients of 71 could be correctly classified with the ROS test on washed semen with leukocytes. There was no difference between the AUC of these tests, because their 95% CIs overlapped on at least 1 range/period.
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Discussion |
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Studies imply that oxidative sperm DNA damage is closely associated with impaired sperm function and male infertility. Strong evidence suggests that DNA fragmentation—commonly observed in the spermatozoa of subfertile men—is mediated by high levels of ROSs (Sun et al, 1997).
In approximately 25% of males, the cause of infertility cannot be determined (Alkan et al, 1997). Studies have shown that 40% to 88% of nonselected subfertile patients have high levels of ROSs (Padron et al, 1997). Developing a proper diagnostic and prognostic test to evaluate the oxidative stress status may contribute to a major understanding of idiopathic infertility. Therefore, defining the normal levels of a diagnostic test is crucial to allow future clinical practice interventions.
A test used as a diagnostic tool must identify a maximum number of patients during routine evaluation. It must be sensitive at a given cutoff to accurately differentiate fertile from subfertile patients. In this study, we have defined the levels of ROSs in neat and washed semen in a fertile population. We determined the sensitivity, specificity, and accuracy of different cutoff values. To determine reference values for normality, we considered different cutoffs statistically determined by ROC curves.
Shekarriz et al (1995b) described all ROS values of 10 x 104 cpm or more as abnormal. According to Sharma et al (1999), the mean level of ROSs was 1.39 ± 0.73 (Log ROS + 1) for fertile men, 2.1 ± 1.21 (Log ROS + 1) for subfertile men with varicocele, and 3.25 ± 0.89 (Log ROS + 1) for subfertile men with seminal infection. Allamaneni et al (2005) reported that the mean ROS level of washed semen was 11.1 x 104 cpm (0.075 x 104–0.41 x 104) (20 x 106 sperm) in donors, 16.8 x 104 cpm (0.022–0.546) in subfertile patients, and 29.0 x 104 cpm (0.058–1.43) in leukocytospermic patients. In the same study, the value for ROSs in neat semen for the fertile population was 2.9 x 104 cpm (0.008–0.13). Nallella et al (2005) found that in washed semen, a cutoff of 1.25 using Log ROS + 1 had 74.6% accuracy.
For washed semen, we found an optimal cutoff of 10.0 (accuracy, 69.0%) for samples without leukocytes and 51.5 (accuracy, 73.2%) for semen with leukocytes but without leukocytospermia. Although our results are different than those of Nallella's study, they are more similar to the reference values described by Shekarriz et al (abnormal values, 10.0 x 104 cpm or more) (Nallella et al, 2005; Shekarriz et al, 1995b). Nevertheless, in these studies, there are no considerations regarding the presence of low levels of leukocytes. In the present study, the established cutoff was much higher for samples with white cells (51.5). The AUC for the ROS test on washed semen was 77% (range, 62.2%–91.7%) for the samples without leukocytes and 80.9% (range, 70.3%–91.6%) for the samples with leukocytes. In the studies of Nallella et al (2005) and Sharma et al (2001), the AUC was 78.92% (range, 72.60%–85.23%) and 68.9% (range, 57.0%–80.7%), respectively. For washed samples, ROS test showed a sensitivity of 71.9% and 73.3% for samples with and without leukocytes, respectively, compared with 83.5% from Nallella's study (Nallella et al, 2005).
For neat semen, we identified an optimal cutoff of 0.56 (0.5575) for samples without leukocytes and 1.25 (1.2480) for semen with leukocytes (Endtz, more than 0 and less than 1 x 106/mL) whereas Allamaneni et al showed 18.5 as an optimum cutoff value independent of leukocyte presence (Allamaneni et al, 2005). When evaluating the AUC for the ROS tests on neat semen, our findings were 76.4% (range, 62.6%–90.2%) and 72.7% (range, 60.8%–84.7%) for samples without and with leukocytes, respectively, compared with 68% (range, 57%–80%) from Nallella et al (2005). In the same study, the authors showed a 50% sensitivity for the test. Our findings reported 73.3% for both samples with and without leukocytes in neat semen.
The identification of potential sources of ROSs is extremely important for guiding any clinical intervention. As reported previously, leukocytes are the major source of ROS production (Lemkecher et al, 2005). This strongly suggests the need for a more specific evaluation of their interference with ROS levels. When we compared the fertile groups with and without leukocytes (excluding samples with leukocytospermia), a significant difference was identified. In neat and washed semen, fertile men with the presence of leukocytes (less than 1 x 106/mL) had significantly higher levels of ROSs. Other parameters showed no statistical differences.
Like previous studies (Lemkecher et al, 2005; Saleh et al, 2002), we found that leukocytes interfere greatly with ROS levels. Novotny et al (2003) and Sharma et al (2001) reported that lower leukocyte levels were associated with significant ROS levels in semen. However, these 2 previous studies were based on subfertile patients whereas we showed that lower levels of leukocytes also affect ROS levels in a fertile population. Recently, Henkel et al reported that leukocyte counts of less than 1 x 106/mL were significantly correlated with ROS production as well as decreased DNA integrity (Henkel et al, 2003, 2005). These findings suggest that the established cutoff for leukocytospermia given by the WHO should be reevaluated.
The influence of ROSs on sperm motility can be explained by lipid peroxidation. This process results in a loss of plasmatic membrane solubility that decreases the phosphorylation of axonemal proteins and causes sperm immobilization (Agarwal et al, 2003). Decreased sperm count could be attributed to extended exposure of the seminiferous epithelium to high levels of ROSs produced by immature or abnormal spermatozoa. ROSs damage these tubules, leading to testicular atrophy, reduction of gamete production, motility loss, and DNA damage to mature sperm.
Previous studies have shown that ROS production is negatively correlated with the percentage of normal sperm, concentration, and motility and is positively correlated with increased percentages of abnormal spermatozoa (Aziz et al, 2004). Similarly, our study showed a negative correlation of ROS and sperm concentration and motility as well as positive correlation with leukocyte levels. In addition, we were able to show these results in a fertile population and in those with lower levels of leukocytes.
An important implication of our study is that the normal reference values were determined in a proven fertile population. There is a strong recommendation from the WHO to individualize the references for normal semen parameters in each laboratory. However, due to the difficulty of recruiting a fertile population, most studies have used normal healthy donors as controls. Because the ultimate evaluation of fertility is pregnancy, donors presenting with normal semen parameters cannot be considered fertile unless their fertility status is known. In addition, although most published studies have assessed ROSs in washed semen, our study measured ROS levels in both neat and washed semen. It is crucial to measure ROSs in washed semen because removing the seminal plasma is the first step in processing samples for assisted reproductive techniques. However, measuring ROSs in processed samples would be interesting because this is also an essential step during assisted reproductive procedures.
The most significant drawbacks of our study include the limited number of patients and the cross-sectional design. However, the data were collected prospectively, and the same exclusion criteria were applied to all patients. Patients selected for the subfertile group included men with idiopathic infertility, which may partially explain why the seminal parameter values in the subfertile patients were not severely abnormal. Although samples were adjusted for concentration (20 x 106 sperm per milliliter), increased seminal parameters, especially molitity, might have interfered in seminal ROS levels. Additional participants would improve our efficacy in identifying the cutoffs of normality in this population.
In conclusion, the present study determined normal reference values for ROSs in neat and washed semen in a population of men with proven fertility. These values can help to identify the pathologic levels of ROSs in infertile men and may guide in clinical therapeutic interventions. In addition, increased ROS levels were associated with leukocyte counts below 1 x 106/mL, and ROSs showed a significant negative correlation with sperm motility and count. Our findings suggest that patients with normal seminal parameters and lower seminal leukocyte levels may benefit from treatment to improve spontaneous pregnancy rates as well as assisted reproduction outcomes, especially in couples with unknown causes of infertility.
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