Seminal leukocyte concentration and related specific reactive oxygen species production in patients with male accessory gland infections

E. Vicari

Andrology Centre, Department of Internal Medicine, University of Catania, Catania, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to determine whether differences occur in seminal concentrations of white blood corpuscles (sWBC) and whether WBC production of reactive oxygen species (ROS) is related to the infiltration of one or more male sexual glands. We studied 70 infertile patients affected by bacterial male accessory gland infections (MAGI) who were divided clinically and, by ultrasound (US), into four well-characterized, aged-matched groups. Three of the groups presented an abnormal US scan (MAGI US+ groups): group P with prostatitis alone (n = 15), group PV with prostato-vesiculitis (n = 19), and group PVE with prostato-vesiculo-epididymitis (n = 22). The fourth group presented with a normal US scan (MAGI US– group) and was diagnosed with presumptive MAGI according to laboratory criteria (n = 14). In addition, 20 fertile males acted as controls. All patients underwent seminal and microbiological analyses as well as US scans. In addition, the WBC concentrations of whole semen and the WBC-rich 45% Percoll fraction (Pf45) as well as WBC-specific ROS production in the same sperm fraction were analysed. Semen samples from the PVE patient group exhibited significantly (P < 0.01) lower values of sperm parameters than those obtained from P, PV, MAGI US– and the control groups. The sWBC and Pf45 WBC concentration as well as baseline and fMLP-stimulated ROS counts in each MAGI US+ group were significantly (P < 0.01) higher than those found in the MAGI US– group and controls.

Key words: male accessory gland infections/reactive oxygen species/seminal white blood cells/sperm parameters/ultrasound


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although the current diagnosis of leukocytospermia is based upon a conventional normal threshold of 1x106 white blood cells (WBC)/ml semen [World Health Organization (WHO), 1992], several studies have indicated that WBC concentrations above this limit may be associated with normal fertility. Furthermore, the degree of WBC infiltration observed in infertile patients is still the subject of controversy, because their seminal WBC concentrations have been found to be in the normal to high range when compared with normal fertile controls (Wolff and Anderson, 1988Go; Tomlinson et al., 1993Go). It is unlikely that such differences, based on immunocytochemical techniques, could be explained on technical grounds (Wolff and Andersson, 1988; WHO, 1992; Wolff, 1995Go). Indeed, it is more likely that leukocytospermia depends, both quantitatively and qualitatively in terms of `functionally negative equivalents' (Kovalski et al., 1991Go) (i.e. hyperproduction of reactive oxygen species, ROS) (Aitken, 1989Go; Aitken et al., 1992Go; Kessopoulou et al., 1992Go), on certain WBC-related factors. One of these may be the specific infiltrated sexual gland site, which is the cause of the WBC infiltration process (Aitken and Baker, 1995Go; Wolff, 1995Go). Therefore it is doubtful whether ROS production by sperm suspensions in-vitro is related to the presence of oxidative stress in the male reproductive tract. This is a key question in deciding whether ROS production can be used as a criterion to select men for antioxidant therapy (Ford and Whittington, 1998Go). Apart from that, antioxidant therapy is a promising strategy to improve human reproductive function by achieving a re-equilibrium between leukocyte and/or sperm ROS production and anti/pro-oxidant scavenger efficacy in seminal plasma (Ford and Whittington, 1998Go; Geva et al., 1998Go; Lenzi et al., 1998Go; for review, see Tarin et al., 1998Go). To this end, the study of male accessory gland infections (MAGI) represents a valid clinical model, both to analyse the effects on semen quality of leukocyte infiltration into the epididymis and accessory sexual glands and to seek a rationale for the use of antioxidant scavengers, antiphlogistic drugs or broad-spectrum antibiotic treatment in these clinical categories of infertile patients in whom leukocytes rather than a primary sperm defect are responsible for oxidative stress (Geva et al., 1998Go).

Therefore, the study of MAGI and its consequences on male infertility requires comprehensive clinical investigation, including a characterization of the different MAGI infertile patient subgroups according to ultrasound findings, microbiological investigation, immunocytochemical WBC determination, and biochemical measurement of specific ROS production (Purvis and Christiansen, 1993Go).

Exact morphometry and echopattern of testicular and epididymal volume by sonography, as well as of prostate and seminal vesicles by transrectal sonography, has been shown to be useful to detect functional abnormalities of the seminal vesicles of infertile patients or those with chronic urogenital infections (Behre et al., 1995Go). This is particularly important when the palpation of these swollen sexual organs is difficult, i.e. in the presence of large hydrocele, post-phlogistic didymo-epididymal adhesions, and/or of dishomogeneous prostatic consistency and/or enlargment vesicles overlapping the superior prostatic area. Moreover, extensive pathological changes in the sex glands may often be present without clear symptoms, and the prostate gland and seminal vesicles may show simultaneous evidence of inflammatory changes (Purvis and Christiansen, 1993Go).

The aim of the study was, therefore, to determine if leukocytospermia and WBC-specific ROS production are related to the WBC infiltration of one or more sexual glands.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General study population features
The study population included 70 consecutive and well-characterized male patients with MAGI, aged 28–42 years, who were under investigation for infertility, and a control group of 20 males with recently proven fertility. The infertile patients presented with primary infertility (mean 6.2 years, range 2–10), with presumptive diagnosis of MAGI ultimately confirmed, according to clinical and laboratory criteria recommended by (Rowe et al., 1993Go). Moreover, the results of the post-coital test and of the sperm–bovine cervical mucus penetration test (Alexander's test) were repeatedly negative in 42 (60%) and 45 (64.3%) of the cases respectively. Respective female partners were considered to be ovulatory on the basis of biphasic basal body temperature and/or ultrasound (US) follicular scans, luteal phase progesterone concentrations, or endometrial biopsy study. Tubal patency was assessed by hysterosalpingogram or laparoscopy in all but five of the women. A complete reproductive and sexual history was obtained and a physical examination performed on each male patient.

Clinical characterization of infected categories
Selection criteria
All infertile patients were affected by presumptive MAGI, suspected upon the basis of oligo- or astheno- or teratozoospermia and fulfilled the following criteria: two or more combined factors: the presence of one or two conventional ejaculate signs (Rowe et al., 1993Go), one or two cultures with significant bacteriospermia [>=105 colony-forming units (CFU)/ml] or Chlamydia trachomatis or Ureaplasma urealyticum detected in cultures of urethral swab obtained after prostatic massage, signs of accessory sexual gland inflammation at physical examination. All patients underwent US scans (both rectal US prostate-vesicular and scrotal didymo-epididymal examination).

In order to accurately distinguish the levels of expressed MAGI, we used certain US findings considered indicative of chronic inflammation and to some extent reported by the literature (Di Trapani et al., 1988Go; Doble and Carter, 1989Go; Christiansen and Purvis, 1991Go; Purvis and Christiansen, 1993Go; Holden and List, 1994Go; Vicari and Mongioì, 1995) (Table IGo).


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Table I. Ultrasound findings considered indicative of male accessory gland infection
 
Exclusion criteria (to eliminate potential confounding)
Patients affected by MAGI (P, PV or PVE) were excluded from the present study if they also had the following characteristics: (i) semen analyses: azoospermia; (ii) semen cultures: no significant bacteriospermia (0–104 CFU/ml); (iii) clinical: history of clear primary testicular disease (cryptorchidism, varicocele) or testicular size <12 ml according to US scans; (iv) other determinants: history of smoking, alcoholic consumption, occupational chemical exposure, fever or drugs taken within 3 months of the start of the study, significantly abnormal renal, hepatic or connective tissue disorders.

Control group
The control group included 20 males, aged 25–42 years, who had fathered children within the previous 6 months and had attended our Andrology Centre for previous fertility counselling. The controls underwent semen analysis, semen culture, and sexual accessory gland US examinations.

Ultrasound examination
All patients and controls underwent US examinations. The didimo-epididymal regions were carefully assessed with scrotal ultrasonography by means of an Aloke US scanner with a 7.5 MHz linear transducer. Scrotal US was performed systematically in various longitudinal, transverse and oblique scans with patients lying in a supine position. The examination always included measurement of testicular volume, documentation of testicular homogeneity and echogenicity; epididymal morphometry, including cranio-caudal diameter of the caput and cauda epididymis, echogenicity evaluation of multiple cysts and/or of a large hydrocele. The prostate-vesicular region was assessed at rectal ultrasonography by means of a 7 MHz monoplan and linear transducer through transverse and longitudinal scans. Prostate volume was measured using the planimetric method by scanning the organ at 5 mm intervals in transverse sections (Behre et al., 1994). Pathological seminal vesicles were either increased in diameter (with thickness >14 mm), asymmetric, hypoplastic (with thickness <7 mm) or atrophic.

US criteria considered indicative of chronic inflammation are summarized in the Table IGo.

Semen cultures and analysis, and sperm preparation
All semen specimens were collected by masturbation into sterile containers after 2–4 days of sexual abstinence. An aliquot from all samples was cultured aerobically and anaerobically after a 1:2 dilution in saline solution. Standard bacteriological methods were used to quantify and identify all organisms according to previously published methods (Vicari et al., 1986Go; Vicari and Mongioì, 1995Go); the overall population (patients and controls) also underwent tests for Chlamydia and Mycoplasma in cultures of urethral swabs obtained by prostate massage.

Another aliquot from the same sample was processed for semen analysis, assessed in terms of sperm concentration (using the Makler chamber), total sperm count, motility, morphology (percentage of oval forms) and WBC concentration, according to WHO (1992) guidelines. In whole semen and in a 45% Percoll-generated fraction, WBC concentrations were determined after morphological identification by conventional immunocytochemical staining (WHO, 1992). In addition, to evaluate and quantify a specific biochemical WBC activity, we measured the ROS generated by WBC in the 45% Percoll fractions, which is the WBC-rich sperm fraction.

All semen samples were prepared by a two-step Percoll 45/90% discontinuous gradient separation technique. Briefly, 1 ml aliquots were layered on the top of discontinuous 45–90% Percoll gradients consisting of 1 ml fractions, in 15 ml conical-based centrifuge tubes. Isotonic 90% Percoll was generated by supplementing 10x concentrated medium 199 (Flow Laboratories, Irvine, UK) with bovine serum albumin (BSA), sodium pyruvate and sodium lactate, and adding 90 ml of Percoll (Pharmacia, Uppsala, Sweden). 45% Percoll was prepared by dilution with an equal volume of Biggers–Whitten–Whittingham (BWW) medium (Biggers et al., 1971Go). The gradients were centrifuged at 500 g for 20 min, the seminal plasma was discarded and the cells were collected from the interface (45% fraction). These cells were resuspended in 7 ml of BWW, centrifuged at 500 g for 5 min and finally resuspended at a sperm concentration of 2.5x106 spermatozoa/ml.

Morphological identification of WBC
In whole semen and in the 45% Percoll-generated fraction white blood cell (WBC) concentrations were determined by morphological identification using conventional immunocytochemical staining (WHO, 1992). Briefly, the samples were centrifuged at 500 g x5 min and the cells resuspended in Dulbecco's phosphate-buffered saline (PBS; Flow Laboratories, Irvine, UK) at a concentration of 5x106 cells/ml. Then, 5 µl of each cell suspension was air-dried on separate wells of Hendley slides (C.A.Hendley, Essex, UK) wrapped in aluminium foil and stored at –70°C. WBC were identified using an anti-alkaline phosphatase monoclonal antibody CD45 (Dako Italia, Milan, Italy). Alkaline phosphatase activity was detected by incubating the slides for 18 min with a substrate containing naphthol As-MX phosphate (0.5 mM), 2% dimethylformamide, 0.01 M levamisole and 3.9 mM Fast Red TR in 0.1 M Tris buffer, pH 8.2. The slides were finally counterstained with haematoxylin and mounted in Apathy's aqueous mounting medium. The concentration of WBC was evaluated by counting the number of red-stained round cells in 20–30 microscopic high-power fields (hpf; x40 objective) under light microscopy. The analysis was carried out in duplicate. The total number of positive cells in duplicate spots was recorded, averaged and multiplied by the dilution factor (x200) to give the number of WBC per ml of semen (Cohen et al., 1985Go). The concentrations of WBC in Percoll fractions were expressed relative to the concentration of spermatozoa. The minimal number of WBC that could be detected with this method was 0.01x104 WBC/107 spermatozoa. Nucleated round cells that did not stain with the monoclonal antibody were considered to be germ cell precursors (Wolff and Anderson, 1988Go).

Determination of WBC-specific ROS production
ROS production was measured as previously reported (Aitken and Clarkson, 1987Go; Aitken and West, 1990Go; D'Agata et al., 1990Go). The chemiluminescent probe Luminol (Sigma Chemical Co., St. Louis, MO, USA) (4 µl of a 25 mM stock solution in dimethylsulphoxide, DMSO) containing 8.5 U horseradish peroxidase (Type VI, 310 U/mg; Sigma, St Louis, MO, USA) to sensitize the assay for the generation of extracellular hydrogen peroxide (Vilim and Wilhelm, 1989Go; Aitken and West, 1990Go) was added to 400 µl cell suspension. The resultant chemiluminescent signal was measured on a Berthold LB 9505 luminometer at a chamber temperature of 37°C. After 15 min, once the steady-state signal was established, 2 µl (final concentration 50 µM) of the WBC chemoattractant peptide fMLP (formyl-leucyl-phenylalanine) (Sigma) dissolved in DMSO was added and the WBC-dependent chemiluminescence was monitored for a further 15 min.

Statistical analysis
Results are reported as mean and median with the 10th and 90th percentile in parenthesis. Statistical analysis was performed employing the non-parametric Mann–Whitney (U-test). P < 0.05 was accepted as statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the 70 patients with MAGI, 14 were found to have a normal US scan or only a single abnormality (MAGI US–) and the remaining 56 had abnormal US findings (MAGI US+). All patients in the MAGI US+ groups presented abnormal US signs at each infected glandular site as outlined in Table IGo: patients in the P group presented two or more abnormalities, those in the PV group presented four or more abnormalities, while those in the PVE group presented six or more abnormalities.

The microbiological results of the four patient groups and the fertile control group are reported in the Table IIGo.


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Table II. Microbiological results [colony-forming units (CFU)/ml >=105] for the different categories of infertile male accessory sperm gland infection (MAGI) list patients affected by prostatitis (group P), prostato-vesiculitis (group PV), prostato-vesiculo-epididymitis (group PVE), MAGI ultrasound (US) negative (exhibiting 0–1 abnormal ultrasound signs) patients and fertile controls
 
Results of conventional semen analyses in the patient groups and in the controls are summarized in Table IIIGo. Semen samples from the PVE patient group exhibited significantly (P < 0.01) lower values than those values in the other patient groups or in the control group. The values were not significantly different between P and PV patient groups, the MAGI US– group versus the fertile control group, or between P and PV patients groups with respect to the MAGI US– group and the fertile control group.


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Table III. Semen analysis parameters from patients and controls
 
Seminal WBC concentrations in all MAGI US+ subgroups were significantly (P < 0.01) higher than those found in the MAGI US– group and the controls. Among MAGI US+ categories, the PVE group had significantly (P < 0.01) higher values than those in the P patient group, but not higher than those of the PV patient group (Table IVGo).


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Table IV. White blood cell (WBC) concentrations in semen and 45% Percoll fraction; basal and fMLP-stimulated WBC-specific reactive oxygen species (ROS) generation in the 45%-Percoll fraction from patient and control groups
 
WBC concentrations obtained from the 45% Percoll fraction were significantly (P < 0.01) higher in all MAGI US+ patient subgroups than in the MAGI US– group and the control group: no significant difference in WBC concentration was noted among the various MAGI US+ groups (Table IVGo), or between the MAGI US– group and controls.

All MAGI US+ patient groups showed significantly (P < 0.01) higher baseline and fMLP-stimulated ROS productions than those found in the MAGI US– group and controls (Table IVGo). Moreover, in the PV and PVE groups the median baseline ROS productions were significantly (P < 0.01) higher than in the P group; the highest fMLP-stimulated ROS production was observed in the PVE groups.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peroxidative damage to the sperm plasma membrane induced by overproduction of ROS (Aitken et al., 1992Go; Kessopoulou et al., 1992Go) is one of the major causes of defective sperm function (Aitken and Clarkson, 1987Go; Aitken et al., 1989Go; Iwasaki and Gagnon, 1992Go; Kessopoulou et al., 1992Go; Sikka et al., 1995Go). Clinical studies have shown increased production of ROS in semen of some infertile patients (D'Agata et al., 1990Go; Iwasaki and Gagnon, 1992Go) and those with oligozoospermia (Aitken et al., 1992Go). Although overproduction is known to be due to increased infiltration of the reproductive system by WBC or spermatozoa, the question remains as to which is the critical site: prostate, seminal vesicle or epididymis. In cases with MAGI a deleterious effect on sperm quality may be exerted through WBC production of ROS and/or particular cytokines [interleukin (IL)-1{alpha} and IL-8] and their soluble receptor (IL-1 RA) (Depuydt et al., 1996Go). Data in this area are limited because most clinical laboratories do not accurately screen semen samples for leukocytospermia, but merely equate round cells in semen with WBC. Patients with complaints of MAGI are often also inadequately assessed clinically (physical examination and rectal ultrasound-confirmed abnormalities). Thus, a heterogeneous population, which includes patients with temporary inflammatory episodes, chronic uncomplicated (urethro-prostatitis) or complicated (prostato-vesiculitis or prostato-vesiculo-epididymitis), has often been enrolled into an antibiotic trial, resulting in confusing and conflicting clinical data.

The source and the role of WBC in the human ejaculate remains unclear and the subject of controversy (Aitken and Baker, 1995Go). Certainly the conservative threshold of 1x106 WBC/ml of semen needs to be critically revised, because the overall WBC population (predominantly granulocytes) is comprised of several morphologically and biochemically distinct cell subpopulations. The total WBC population and its subpopulations may be associated with infiltration of a specific sexual gland (epididymis, prostate, vesicles), a specific infectious aetiology (i.e. bacteria, viruses, Chlamydia, Ureaplasma), the presence of non-infectious inflammatory conditions due to the presence of chemical irritants (cigarettes, alcohol, marijuana) (Close et al., 1990Go) or to the presence of scavenging abnormal spermatozoa. Moreover, the potential contact times between spermatozoa and WBC differ widely in the various regions of the male genital tract. Indeed, spermatozoa may be exposed only very transiently to WBC of an intermediate or large number which originate from the prostate or seminal vesicles respectively, while the epididymis is a site of intense contact between spermatozoa and macrophages and lymphocytes (Wolff, 1995Go).

As a result of this, it is probable that a single `functional equivalent' of WBC (i.e. specific ROS production), rather than strict and `static numerical concentration equivalents' (Kovalski et al., 1991Go) (even detected by immunocytochemical technique), may correlate best with the clinical findings.

Given that WBC are mainly responsible for ROS bursts even when present at very low concentration (Aitken et al., 1992Go; Kessopoulou et al., 1992Go; Vicari et al., 1995Go), the methods used to identify and quantify WBC must be specific and sensitive enough to accurately determine the cell source of ROS in fractionated sperm samples.

In this study the origin of WBC was well documented in three categories of patients who presented ultrasound abnormalities (MAGI US+ groups: P, PV and PVE), and in one category with normal US scans but with semen and microbial abnormalities presumptive of MAGI (MAGI US– group) (Rowe et al., 1993Go). In the MAGI US+ groups, patients always presented two or more abnormal US signs at each infected site (i.e. the P group had two or more US abnormalities, the PV group had four or more abnormalities, and the PVE group had six or more abnormalities). In this study, we chose to focus on the evaluation of the cell components from the low-density region of the Percoll gradient because this fraction contains the highest WBC concentration (Aitken and Clarkson, 1988Go). The WBC concentration was evaluated by specific immunostaining and confirmed by response to fMLP, a chemotactic peptide capable of stimulating oxidative stress selectively from WBC (Krausz et al., 1992Go). Using accurate and reliable methods to measure WBC concentrations and WBC-specific ROS production, our data suggest that all MAGI US+ (P, PV, PVE) patient groups have different sperm parameters, WBC concentrations and WBC-specific ROS productions which are significantly different from the MAGI US– patients and the fertile control group. Among the different MAGI US+ categories, the sperm parameters of the PVE patient group were significantly lower than those in the P and PV patient groups, in the MAGI US– group and in the fertile control group, although these parameters were not significantly different between the P and PV patient groups and the controls. The PVE group also had seminal WBC concentration significantly higher than in the P group, but not higher than those in the PV group. Thirdly, whereas WBC concentrations in the low density 45% Percoll fractions were not significantly different among the patient groups, baseline median ROS production was significantly higher in the PV and PVE groups than in the P group; furthermore, the highest fMLP-stimulated ROS production was in PVE groups.

The abnormal sperm morphology detected mainly in the PVE group does not have a clear explanation. However, the epididymal microenvironment can become hostile in the presence of leukocytic infiltration into the epididymis, and an improvement in sperm parameters has been reported following administration of acetyl carnitine in a group of patients with epididymitis who exhibited a iuxta-sperm ROS hyperproduction (Vicari, 1997Go). Hence, altered sperm morphology may reflect a disturbed lipid pattern during spermiogenesis, spermiation, sperm passage through the epididymis or after maturation (see Lenzi et al., 1998Go) in these patients. Another explanation may be that the abnormal sperm morphology found in the PVE group is secondary to coexisting aspecific vascular or inflammatory events which extend to the testicular level. A link between ROS production and the normal presence of abnormal or immature (with residual cytoplasm) spermatozoa that may in turn serve as a source of ROS provides a good rationale to treat patients with prostato-vesiculo-epididymitis with antioxidants during or following antimicrobials, thus emphasizing all the potential advantages of antioxidant therapy (Tarin et al., 1998Go), and integrating the conclusions of a recent debate (Geva et al., 1998Go; Ford and Whittington, 1998Go; Lenzi et al., 1998Go; Tarin et al., 1998Go).

This is the first comprehensive ultrasound, laboratory and clinical report suggesting a strong association between sexual gland post-inflammation damage with an abnormal US and severe abnormal WBC concentration and specific ROS production. This may be due to a different and/or reduced anti-oxidative protection in the seminal plasma of such patients. Indeed, it has been reported that the WBC concentration in semen is positively correlated with cytokine concentration and ROS production, and negatively correlated with {gamma}-glutamyltransferase and {alpha}-glucosidase activities, a measure of prostate and epididymal secretory function respectively (Depuydt et al., 1996Go). Recently, an unmodified dyspermia was reported in a group of patients with treated epididymitis. This was associated with iuxta-sperm ROS overproduction in the 90% Percoll fraction and in the seminal plasma, which persisted even after microbial eradication as well as 3 months after therapy withdrawal (Vicari et al., 1997Go).

These patients may benefit from treatment with antioxidants (Aitken and Clarkson, 1988Go; Lenzi et al., 1993Go; Vicari, 1997Go), possibly combined with antimicrobial treatment, since oxidative stress may play a causative role in their infertility (D'Agata et al., 1990Go; Aitken et al., 1991Go; Krausz et al., 1994Go; Vicari, 1997Go).


    Acknowledgments
 
The author would like to thank Dr Giuseppe Aranzulla, Division of Radiology, Garibaldi Hospital, Catania and Dr Francesco Caccamo, Institute of Microbiology, University of Catania, for their skilful technical assistance.


    Notes
 
Address for correspondence: Via A.Diaz 15, 95125 Catania, Italy


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aitken, R.J. (1989) The role of free oxygen radicals and sperm function. Int. J. Androl., 12, 95–97.[ISI][Medline]

Aitken, R.J. and Baker, G.H.W. (1995) Seminal leukocytes: passengers, terrorists or good Samaritans? Hum. Reprod., 10, 1736–1739.[ISI][Medline]

Aitken, R.J. and Clarkson, J.S. (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J. Reprod. Fertil., 81, 459–469.[Abstract]

Aitken, R.J. and Clarkson, J.S. (1988) Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J. Androl., 9, 367–376.[Abstract]

Aitken, R.J. and West, K.M. (1990) Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients. Int. J. Androl., 13, 433–451.[ISI][Medline]

Aitken, R.J., Clarkson, J.S. and Fishel, S. (1989) Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biol. Reprod., 41, 183–197.[Abstract]

Aitken, R.J., Irvine, D.S. and Wu, F.C. (1991) Prospective analysis of sperm–oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am. J. Obstet. Gynecol., 164, 542–551.[ISI][Medline]

Aitken, R.J., Buckingham, D., West, K. et al. (1992) Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. J. Reprod. Fertil., 94, 451–462.[Abstract]

Behre, H.M., Kliesch, S., Schadel, F. and Nieschlag, E. (1995) Clinical relevance of scrotal and transrectal ultrasonography in andrological patients. Int. J. Androl., 18 (Suppl. 2), 27–31.[ISI][Medline]

Biggers, J.S., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In Daniel, J.C. Jr (ed.), Methods in Mammalian Embryology. Freeman, San Francisco, pp. 86–116.

Cohen, J., Edwards, R., Fehilly, C. et al. (1985) In vitro fertilization: a treatment for male infertility. Fertil. Steril., 43, 422–432.[ISI][Medline]

Christiansen, E. and Purvis, K. (1991) Diagnosis of chronic abacterial prostatovesiculitis by rectal ultrasonography in relation to symptoms and findings. Br. J. Urol., 67, 173–176.[ISI][Medline]

Close, C.E., Roberts, P.L. and Berger, R.E.. (1990) Cigarettes, alcohol and marijuana are related to pyospermia in infertile men. J. Urol., 144, 900–903.[ISI][Medline]

D'Agata, R., Vicari, E., Moncada, M.L. et al. (1990) Generation of reactive oxygen species in subgroups of infertile men. Int. J. Androl., 13, 344–351.[ISI][Medline]

Depuydt, C.E., Bosmans, E., Zalata, A. et al. (1996) The relation between reactive oxygen species and cytokines in andrological patients with or without male accessory gland infection. J. Androl., 17, 699–707.[Abstract/Free Full Text]

Di Trapani, D.D., Pavone, C., Serretta, V. et al. (1988) Chronic prostatitis and prostatodynia: ultrasonographic alteration of the prostate, bladder neck, seminal vesicles and periprostatic venous plexus. Eur. Urol., 15, 230–234.[ISI][Medline]

Doble, A. and Carter, S.S. (1989) Ultrasonographic findings in prostatitis. Urol. Clin. North. Am., 16, 763–772.[ISI][Medline]

Ford, W.C.L. and Whittington, K. (1998) Antioxidant treatment for male subfertility: a promise that remains unfulfilled. Debate: Is antioxidant therapy a promising strategy to improve human reproduction? Hum. Reprod., 13, 1415–1424.[Free Full Text]

Geva, E., Lessing, J.B., Lerner-Geva, L. and Amit, A. (1998) Free radicals, antioxidants and human spermatozoa: clinical implications. Debate: Is antioxidant therapy a promising strategy to improve human reproduction? Hum. Reprod., 13, 1415–1424.[Free Full Text]

Holden, A. and List, A. (1994) Extratesticular lesions: a radiological and pathological correlation. Austral. Radiol., 38, 98–105

Iwasaki, A. and Gagnon, C. (1992) Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil. Steril., 57, 409–416.[ISI][Medline]

Kessopoulou, E., Tomlinson, M.J., Barratt, C.L. et al. (1992) Origin of reactive oxygen species in human semen: spermatozoa or leucocytes? J. Reprod. Fertil., 94, 463–470.[Abstract]

Kovalski, N., deLamirande, E. and Gagnon, C. (1991) Determination of neutrophil concentration in semen by measurement of superoxide radical formation. Fertil. Steril., 56, 946–953.[ISI][Medline]

Krausz, C., West, K., Buckingham, D. et al. (1992) Development of a technique for monitoring the contamination of human semen samples with leukocytes. Fertil. Steril., 57, 1317–1325.[ISI][Medline]

Krausz, C., Mills, C., Rogers, S. et al. (1994) Stimulation of oxidant generation by human sperm suspensions using phorbol esters and formyl peptides: relationships with motility and fertilization in vitro. Fertil. Steril., 62, 599–605.[ISI][Medline]

Lenzi, A., Culasso, F., Gandini, L. et al. (1993) Placebo-controlled, double-blind, cross-over trial of glutathione therapy in male infertility. Hum. Reprod., 8, 1657–1662.[Abstract]

Lenzi, A., Landini, G. and Picardo, M. (1998) A rationale for glutatione therapy. Debate: Is antioxidant therapy a promising strategy to improve human reproduction? Hum. Reprod., 13, 1415–1424.[Free Full Text]

Purvis, K and Christiansen, E. (1993) Infection in the male reproductive tract. Impact, diagnosis and treatment in relation to male infertility. Int. J. Androl., 16, 1–14.

Rowe, P.J., Comhaire, F.H., Hargreave, T.B. et al. (1993) WHO Manual for the Standardized Investigation and Diagnosis of the Infertile Couple. Cambridge University Press, Cambridge.

Sikka, S.C., Rajasekaran, M. and Hellstrom, W.J.G. (1995) Role of oxidative stress and antioxidants in male fertility. J. Androl., 16, 464–468.[Abstract/Free Full Text]

Tarin, J.J., Brines, J. and Cano, A. (1998) Antioxidants may protect against infertility. Debate: Is antioxidant therapy a promising strategy to improve human reproduction? Hum. Reprod., 13, 1415–1424.[Free Full Text]

Tomlinson, M.J., Barrat, C.L.R. and Cooke, I.D. (1993) Prospective study of leukocytes and leukocytes subpopulations in semen suggests they are not a cause of male infertility. Fertil. Steril., 60, 1069–1075.[ISI][Medline]

Vicari, E. (1997) Effectiveness of a short-term anti-oxidative high-dose therapy on IVF program outcome in infertile male patients with previous excessive sperm Radical Oxygen Species production, persistent even following antimicrobials administered for epididymitis: preliminary results. In Ambrosini, A., Melis, G.B., Dalla Pria, S. and Dessole, S. (eds), Int. Meeting on Infertility and Assisted Reproductive Technology (From Research to Therapy), Porto Cervo, June 11–14, 1997. Monduzzi, Ed, International Proceedings Division, Bologna, Italy, pp. 93–97.

Vicari, E. and Mongioì, A. (1995) Effectiveness of long-acting gonadotrophin-releasing hormone agonist treatment in combination with conventional therapy on testicular outcome in human orchitis/ epididymo-orchitis. Hum. Reprod., 10, 2072–2078.[Abstract]

Vicari, E., Mongioì, A., Speciale, A. et al. (1986) Enhancing detection of gonococcus in ejaculates of adult males using sperm dilution. Arch. Androl., 16, 19–23.[ISI][Medline]

Vicari, E., Sidoti, G. and Mongioì, A. (1995) Abnormal Radical Oxygen Species production by low leucocytospermia in high-density fractionated cell population from infertile patients. Arch. Ital. Urol. Androl., LXVII, 135–141.

Vicari, E., Barone, N. and D'Agata, R. (1997) Infertility or complete IVF failure in presence of persistent detection of high Reactive Oxygen Species (ROS) sperm production in a large number of patients affected by epididymitis previously treated with antimicrobials. In Ambrosini, A., Melis, G.B., Dalla Pria, S. and Dessole, S. (eds), Int. Meeting on Infertility and Assisted Reproductive Technology (From Research to Therapy), Porto Cervo, June 11–14, 1997. Monduzzi, Ed, International Proceedingsz Division, Bologna, Italy, pp. 99–103.

Vilim, V. and Wilhelm, J. (1989) What do we measure by a luminol-dependent chemiluminescence of phagocytes? Free Rad. Biol. Med., 6, 623–629.[ISI][Medline]

Wolff, H. (1995) The biologic significance of white blood cells in semen. Fertil. Steril., 63, 1143–1157.[ISI][Medline]

Wolff, H. and Anderson, D.J. (1988) Immunohistologic characterization and quantitation of leukocyte subpopulations in human semen. Fertil. Steril., 49, 497–504.[ISI][Medline]

World Health Organization (1992) Laboratory Manual for the Examination of the Human Semen and Sperm–Cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.

Submitted on January 7, 1999; accepted on April 9, 1999.