Center for Advanced Research in Human Reproduction and Infertility, Departments of Urology, Gynecology-Obstetrics, Biostatistics and Epidemiology, The Cleveland Clinic Foundation, Cleveland, OH, USA
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Abstract |
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Key words: infertility/oxidative stress/reactive oxygen species/ROSTAC score/spermatozoa
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Introduction |
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There is growing evidence that oxidative stress significantly impairs sperm function, and plays a major role in the aetiology of defective sperm function. This may lead to the onset of male infertility via mechanisms involving the induction of peroxidative damage to the plasma membrane (Jones et al., 1979; Aitken and Clarkson, 1987
; Iwasaki and Gagnon, 1992
; Aitken and Fischel, 1994; Sharma and Agarwal, 1996
; Griveau and Le Lannou, 1997
). Both spermatozoa and seminal plasma possess antioxidant systems capable of counteracting the harmful affects of ROS. Studies have demonstrated that infertile men are more likely than fertile ones to have depressed total antioxidant capacity (TAC) and lower levels of individual antioxidants (Smith et al., 1996
; Lewis et al., 1995
, 1997
). The production of abnormal levels of ROS is thought to be involved in many aspects of male infertility, where spermatozoa are rendered dysfunctional by lipid peroxidation and altered membrane function, together with impaired metabolism, morphology, motility, and fertility (Cummins et al., 1994
). Men whose spermatozoa produce excessive ROS are less likely to impregnate their wives through natural intercourse. The incidence of spontaneous pregnancy is negatively correlated with the generation of ROS in approximately 50% of oligozoospermic patients who exhibit increased ROS production (Aitken et al., 1991
).
Various clinical diagnoses are unable to determine the underlying cause of sperm dysfunction and pathophysiology of infertility. Since oxidative stress has been demonstrated to play a key role in male infertility, it is imperative to identify measures that would help predict, with accuracy, if oxidative stress is a significant contributor of infertility in any given clinical setting (Aitken et al., 1991; Lewis et al., 1995
). Given that oxidative stress is associated with infertility, we postulated that it might be possible to validate the infertility status based on the initial clinical diagnoses. After measuring sperm ROS and seminal TAC we derived a composite ROSTAC score, a novel indicator of oxidative stress. The aim of this prospective study was to investigate the significance of ROS and TAC in different subsets of clinical diagnoses of male infertility. We also examined if the ROSTAC score could better identify infertile patients than either variable alone. Finally, we investigated whether the ROSTAC score could identify the probability of patients who were likely to initiate a pregnancy.
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Materials and methods |
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Reactive oxygen species measurement
Aliquots of liquefied semen were centrifuged at 300 g for 7 min. Seminal plasma was aliquoted and frozen at 20°C for later measurement of total antioxidant levels. The sperm pellet was washed with phosphate-buffered saline (PBS, pH 7.4) and resuspended in the same medium at a concentration of 2x106 sperm/ml. Levels of ROS were determined by chemiluminescence assay using luminol (5-amino-2,3 dihydro-1,4 phthalazinedione; Sigma Chemical Co., St. Louis, MO, USA) as the probe (Shekarriz et al., 1995). Measurements were made using a Berthold luminometer (Autolumat LB 953, Wallac Inc., Gaithersburg, MD, USA). Five-millimolar luminol prepared in dimethyl sulphoxide (Sigma Chemical Co.) was added to 400 µl of the washed sperm suspension. Levels of ROS were determined by measuring chemiluminescence for 15 min and results were expressed as x104 counted photons per minute (cpm).
Total antioxidant capacity measurement
Total antioxidant capacity was measured in the seminal plasma using an enhanced chemiluminescence assay (Kolettis et al., 1999). Aliquots of the seminal plasma were thawed at room temperature and assessed for their antioxidant capacity. Seminal plasma was diluted 1:10 with deionized water and filtered through a 20 µm Millipore filter (Allegiance Health Care Corporation, McGaw Park, IL, USA). Signal reagent was prepared using a chemiluminescence kit (Amersham Life Science, Buckingham, UK). A constant source of ROS was produced by horseradish peroxidase-linked immunoglobulin (HRP-linked Ig; Amersham Life Science, Buckingham, UK). Twenty microlitres of HRP-Ig was added to 4.98 ml dH2O and further diluted 1:1 to give a desired chemiluminescence output (3x107 cpm).
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) a water-soluble tocopherol analogue was added as the standard at concentrations between 50 to 150 µmol/l. The antioxidant capacity of the seminal plasma was expressed in molar Trolox equivalents. With the luminometer in the kinetic mode, 100 µl of signal reagent and 100 µl of HRP were added to 700 µl of dH2O and mixed. This solution was equilibrated to the desired level of chemiluminescence output (between 2 and 3x107 cpm) for 10 s. One hundred microlitres of the prepared seminal plasma was immediately added to the signal reagent and HRP, and the chemiluminescence was measured. Suppression of chemiluminescence and the time from the addition of seminal plasma to 10% recovery of the initial chemiluminescence were recorded. Antioxidant capacity was expressed as molar Trolox equivalents.
In this study, the samples used as the reference point in the scaling of the ROSTAC score were obtained from donors who were healthy, normal men. Other control groups should be within 1 standard deviation (SD) (4060) of the ROSTAC score approximately two-thirds of the time; any group falling outside this range cannot be employed for calculating the reference value.
Statistical analyses
Comparisons among diagnosis groups were performed with a one-way ANOVA and post-hoc t-tests for pairwise comparisons. A multiple comparison procedure (Dunnett, 1995) was used to adjust the P values. The ROS and TAC values from controls were used to create a scale of these two variables that uses the control values as reference points. The log of (ROS + 1) was used in calculations so that both values were normalized to the same distribution. First, both log (ROS + 1) and TAC were standardized to the z-scores (mean = 0, SD = 1) so that both would have the same variability. These standardized scores were calculated by subtracting the mean value of the controls from each individual's observed value and dividing by the SD of the control population.
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These two standardized variables were then analysed with principal component analysis which provided linear combinations (or weighted sums) that account 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 ROSTAC score would have a mean of 50 and SD of 10 in normal donors, the ROSTAC score was transformed as:
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For example, if a donor specimen gives an ROS value of 10.3 and a TAC of 2499.0, initially the ROS would be converted to log (10.3 + 1), or 1.05. Next, the values would be standardized as follows:
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This will then be converted to the ROSTAC score as follows:
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By comparison, a patient with a high ROS value of 92436.36 and low TAC of 1106.85 would have an ROSTAC score of 5.33.
Receiver operating characteristics (ROC) curves were used to examine the diagnostic ability of ROSTAC score to predict fertility. ROC curves illustrate the sensitivity and specificity over the entire range of the ROSTAC score. The area under the curve 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. ROS, TAC, and ROSTAC score variables were compared using DeLong's non-parametric comparison (DeLong et al., 1988).
Statistical significance was assessed at P < 0.05, and summary statistics presented as mean ± SD. Data were analysed by the SAS statistical software package (version 6.12, SAS Institute Inc., Cary, NC, USA).
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Results |
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Total antioxidant capacity
The total antioxidant capacity was significantly higher in the control group (1650.93 ± 532.22) than in any other infertile groups (P < 0.006), with the exception of the infertile vasectomy reversal group. Levels of TAC were lowest in the idiopathic infertility group (1051.98 ± 380.88), and were significantly lower than levels in the infertile vasectomy reversal group (P = 0.02). The TAC in fertile vasectomy reversal patients did not differ significantly from the controls.
ROSTAC score
ROSTAC score was significantly higher in the control than in the infertile patient groups (P 0.0002; Table I
). The lowest ROSTAC score was observed in the varicocele with prostatitis group (22.39 ± 13.48). This score was significantly lower than the varicocele (P = 0.01), and infertile vasectomy reversal (P = 0.01) scores. The average ROSTAC score for the fertile vasectomy reversal group was 49.35 ± 14.4, which did not differ significantly from the control group.
Receiver operating characteristic curves
The effectiveness of oxidative stress in discriminating fertile from infertile men with different clinical diagnoses was studied by generating receiver operating characteristic (ROC) curves. In addition, the percentage of area under the curve, and their 95% confidence intervals (95% CI) were also calculated (Table II; Figure 1AD
). Areas under the curve correspond to the percentage of men correctly classified to the proper fertility status. The area under the curve for ROS was 94.8% (95% CI: 86.8100%) in discriminating controls from patients with varicocele with prostatitis. However, the area under the curve for ROS was <80% in both the infertile varicocele group (68.9%: 95% CI, 57.080.7%) and idiopathic infertility group (74.3%: 95% CI, 60.488.1%).
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The ROSTAC score was significantly better at discriminating infertility among all diagnostic groups versus controls than either ROS or TAC alone. For all the infertile diagnoses, the area under the curve was >80%. The ROSTAC score was better than ROS alone in identifying patients with varicocele (P = 0.002); and patients with idiopathic infertility (P = 0.005) (Table II, Figure 1AD
). The ROS and ROSTAC scores were comparable in the varicocele with prostatitis and vasectomy reversal groups. Also, the ROSTAC score was better than TAC alone in identifying the vasectomy reversal group (P = 0.04). The only group in which the ROSTAC score did not perform significantly better than either ROS or TAC was the varicocele with prostatitis group, and this may be because of the small sample size (n = 8).
Within male factor diagnoses of varicocele, varicocele with prostatitis, and vasectomy reversal, 52 patients had a follow-up for an average of 17.4 ± 12.3 months (median, 12.6 months) to determine if subsequent pregnancies were achieved. Out of these 52, 13 (25%; 12 vasectomy reversal and 1 varicocele) patients eventually initiated normal pregnancies. Patients with partner pregnancies had significantly higher ROSTAC scores (47.7 ± 13.2) compared to those who did not (35.8 ± 15.0; P = 0.01). The expected probability of the successful pregnancy during the average time frame of 1218 months was calculated over a range of values of the ROSTAC score based on logistic regression results (Figure 2). The probability of successful pregnancy was estimated at <10% for values of ROSTAC score <30, but increased as the ROSTAC score increased. Using logistic regression estimates, the expected pregnancy rates for a patient with a ROSTAC score of 30 would be 13.9, 21.0, and 31.6% for 12, 24, and 36 months respectively; whereas a score of 50 would have expected pregnancy rates of 35.1, 48.9, and 54.3% over the same intervals. Based on the expected probabilities, the estimated number of successful pregnancies among the 28 idiopathic infertility patients (assuming no female factor diagnoses) over a 12-month interval is 5.6 pregnancies (95% CI: 3.212.8).
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Discussion |
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The clinical significance of seminal oxidative stress is suggested by several independent studies indicating a link between peroxidative damage to human spermatozoa and the incidence of male infertility (Jones et al., 1979; Aitken and Clarkson, 1987
; Iwasaki and Gagnon, 1992
; Weese et al., 1993
; Mazzilli et al., 1994
). Oxidative stress at the testicular level has also been implicated in the disruption of spermatogenesis observed during cryptorchidism, vitamin E depletion, and exposure to xenobiotics (Peltola et al., 1994
, 1995
). Furthermore, it has been proposed that oxidative damage is a possible cause of idiopathic male infertility involving disruption of spermatogenesis (Lenzi et al., 1993
). Significantly high levels of free radicals (superoxide anion) were reported in the seminal fluid of infertile (87%) and fertile (55%) normozoospermic patients (Mazzilli et al., 1994
). Evidence of high levels of lipid peroxidation was also reported in 12% of normozoospermic men (Huszar and Vigue, 1994
). This information suggests that a high incidence of ROS formation may be associated with reduced sperm fertilizing potential in both infertile and fertile males. In our study, we observed significantly higher levels of ROS in all infertile groups. The highest ROS levels were seen in the varicocele with prostatitis group followed by the infertile vasectomy reversal group and the idiopathic infertility group. The spontaneous formation of ROS has been associated with decreased spermegg interaction and reduced in-vivo fertility (Aitken and Clarkson, 1987
; Aitken et al., 1989
). The same group (Aitken and Clarkson, 1987
) showed that the production of ROS by human spermatozoa is inversely related to their ability to exhibit spermoocyte fusion. High lipid peroxidation may reduce the capacity of the sperm to fertilize (Aitken et al., 1989
).
In our study, we found depressed TAC levels in all infertile patients. The idiopathic infertility group had the lowest levels of TAC, followed by the varicocele with inflammation, and varicocele groups. These levels were significantly different (>2 SD) compared to controls.
Whether TAC levels characterize the level of oxidative stress will depend on the source of ROS, i.e. whether it is produced by the abnormal spermatozoa and the neutrophils (extracellularly), or within the spermatozoa (intracellularly). Two ROS-generating systems have been proposed, an NADPH oxidase-like system at the sperm plasma membrane level, and a sperm diaphorase (an NADH-dependent oxido-reductase) located in the middle piece and integrated into the mitochondrial respiratory system of the spermatozoa (Aitken, 1997). ROS emanates in part from abnormal spermatozoa, which are characterized by the retention of excess residual cytoplasm as a result of defective spermiogenesis (Aitken et al., 1994
). Only one-third of the ROS produced by spermatozoa is released extracellularly (Plante et al., 1994
). The usual ROS scavengers such as catalase and superoxide dismutase, though very effective against ROS produced by an external source, are relatively ineffective when ROS production is intracellular. In a recent placebo-controlled study, patients with asthenozoospermia in association with peroxidative damage to the spermatozoa demonstrated a potential value of oral vitamin E supplementation (Suleiman et al., 1996
). In clinical situations where ROS production is extracellular, classical antioxidants would be very effective, and the TAC level may be indicative of the extent of oxidative stress. In the case of oligozoospermic males whose spermatozoa generate particularly high levels of ROS, the source of cytotoxic oxygen radicals is frequently intracellular (Aitken et al., 1992
; Gomez et al., 1996
) and TAC may not be able to differentiate the extent of oxidative stress. In such instances, albumin is reported to exhibit an excellent ability to sustain sperm motility (Twiggs et al., 1998
). A typical example of free-radical associated male pathology is oligozoospermia in which fertilization and pregnancy rates are low using in-vitro fertilization techniques. Indeed, half of these cases are associated with the generation of ROS above the normal fertile range (Aitken et al., 1989
, 1992
). Even though it is now clear that oxidative stress is harmful to the spermatozoa, the debate on the use of antioxidants in improving semen quality continues (Geva et al., 1998
; Lenzi et al., 1998
; Ford and Whittington, 1998
; Tarin et al., 1998
; Martin-Du Pan and Sakkas, 1998
; Saez et al., 1998
; Twiggs et al., 1998
). Antioxidants may be beneficial only in those infertile cases where infertility is due to oxidative stress. More controlled, randomized double-blind studies are needed to validate this issue.
The hypotheses that we wanted to test were whether ROS and TAC levels were abnormal in infertile men with various clinical diagnoses, and, more importantly, whether we could identify a variable that could discriminate fertile from infertile men better than ROS or TAC alone. The clinical diagnosis of infertility alone cannot identify the underlying pathophysiology, and routine semen analysis is inadequate to determine the underlying cause. Oxidative stress can be a major mediator of infertility, therefore any parameter that is able to quantify oxidative stress would also be able to predict whether the patients in a given clinical diagnosis are more likely to be infertile. It is important to recognize that there is a great deal of variability in the levels of oxidative stress even in infertile men (Alvarez et al., 1987). The ROSTAC score as proposed by us provides a measure derived both from the levels of ROS produced and the antioxidant levels present in a given set of patients. Furthermore, this score minimizes the variability present in the individual parameters of oxidative stress. The degree of oxidative stress can be effectively determined from the ROSTAC score which provides a measure derived from both the levels of ROS produced and the antioxidant capacity in a given set of patients.
In our study, the ROSTAC score was calculated from control comprising healthy normal men who had very low levels of ROS, had initiated pregnancies in the past 2 years, and were therefore fertile. The composite ROSTAC score calculated for these men was representative of the fertile group, and any scores significantly below these were indicative of the patients being infertile. Furthermore, using the area under the curve for the three variables (ROS, TAC, and ROSTAC score) indicated the probability of correctly identifying patients in any given clinical diagnosis as either fertile or infertile. As seen from our ROC curves, ROS levels effectively discriminate fertile and infertile populations in many clinical diagnoses. But across all clinical diagnoses, the ROSTAC score was a superior discriminator between fertile and infertile men than either ROS or TAC alone. It is quite likely that neither variable alone adequately quantifies seminal oxidative stress and the combination of these two variables is better at quantifying the overall oxidative stress affecting spermatozoa.
Furthermore, analyses of patients with male factor diagnoses indicated that those with subsequent successful pregnancies had an average ROSTAC score in the normal range compared to significantly lower ROCTAC scores in those without subsequent pregnancies. In addition, we found that the average ROSTAC score for the fertile vasectomy reversal group was nearly identical to the controls. It appears that individuals with ROSTAC scores <30, the lower limits of normal range (2 SD from 50), are at particular risk for prolonged inability to initiate pregnancies. Our observations are based on the assumption that there were no female factors present.
In conclusion, our study describes the ROSTAC score, a novel measure of oxidative stress as being superior to ROS or TAC alone in discriminating between fertile and infertile men. We found that infertile men with male factor or idiopathic diagnoses had significantly lower ROSTAC scores than controls, and that men with male factor diagnoses that eventually were able to initiate a successful pregnancy had significantly higher ROSTAC scores than those who failed. In addition, male partners of couples who achieved pregnancy did not have significantly different ROSTAC scores than controls. Therefore, the ROSTAC score may serve as an important measure in identifying those patients with a clinical diagnosis of male infertility who are likely to achieve a pregnancy over a period of time.
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Acknowledgments |
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Notes |
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* Presented at the Annual Meeting of the American Urological Association, Dallas, TX, May 16, 1999.
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References |
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Submitted on April 7, 1999; accepted on July 27, 1999.