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From the * Henry Wellcome Laboratories for Medical
Research, School of Medicine and Biomedical Sciences, The University of
Sheffield Medical School, Sheffield, United Kingdom; the
Avesina Research Institute, Shahidi Beheshti
University, Evin, Tehran, Iran; and the
Department of Medical Genetics, Tehran
University of Medical Sciences, Tehran, Iran.
| Correspondence to: Adrian Eley, Henry Wellcome Laboratories for Medical Research, Department of Infection and Immunity, School of Medicine and Biomedical Sciences, The University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, United Kingdom (e-mail: a.r.eley{at}sheffield.ac.uk). |
| Received for publication May 12, 2009; accepted for publication September 16, 2009. |
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Abstract |
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Key words: Chlamydia trachomatis, interleukin-8, interleukin-6, semen quality
Higher numbers of leukocytes (>1 x 106 per mL) are thought to be a sign or marker of microbiologically-induced inflammation (WHO, 1999). Nevertheless, there is much controversy as to whether leukocytospermia is closely related to the presence of pathogenic microorganisms (Trum et al, 1998) and whether markers such as leukocytospermia can be interpreted as an aid for the diagnosis of infections in asymptomatic patients (Barratt et al, 1990; Eggert-Kruse et al, 1992; Tomlinson et al, 1992; Aitken and Baker, 1995; Kiessling et al, 1995; Yanushpolsky et al, 1996). A number of studies have specifically excluded a correlation between leukocytospermia and semen quality (Eggert-Kruse et al, 2001; Ludwig et al, 2003). However, because the commonly used method for diagnosis of leukocytospermia only detects granulocytes that are intact, degranulated granulocytes could be missed (Kopa et al, 2005), suggesting that additional markers of inflammation such as cytokines in the seminal plasma might be of value in diagnosing infections in semen.
Cytokines are regulatory proteins produced by leukocytes and other cells that control inflammation. Certain proinflammatory cytokines, such as interleukin (IL)-6 and IL-8, are involved in inflammatory processes. Previous studies have investigated levels of seminal IL-6, IL-8, or both in infertile patients, and controversy exists as to whether elevated cytokine levels are related to semen quality (Comhaire et al, 1994; Dousset et al, 1997; Eggert-Kruse et al, 2001; Kopa et al, 2005). To our knowledge, there has been no study in which levels of IL-6 and IL-8 have been measured in semen from males infected with C trachomatis and comparisons made with a group shown not to be infected with chlamydia. This focus on C trachomatis–infected patients was suggested by Eggert-Kruse et al (2001), who found 1 subfertile male who was positive for C trachomatis and had high IL-6 and IL-8 concentrations in seminal plasma. Therefore, the purpose of the present study was to determine concentrations of IL-6 and IL-8 in seminal plasma, as well as numbers of seminal leukocytes in male partners of infertile couples, some of whom were C trachomatis–positive, and relate the findings to semen quality.
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Materials and Methods |
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Before semen analysis, the men were provided with written information on the study and requested to abstain from sexual intercourse for at least 48 hours, but no longer than 5 days, before attending the clinic. A record was made of the number of days of abstinence at the time of sample production. All semen samples were produced on site and collected into standard containers previously shown to have no cytotoxic effects on human spermatozoa according to the methods outlined in WHO (1999). In addition, all men were asked to bring 20 mL of first void urine (FVU) in a sterile pot to the clinic for detection of C trachomatis.
Semen Quality
Immediately after semen production, the sample was placed in an incubator
and allowed to liquefy at 37°C for up to 30 minutes before analysis. Semen
analysis was performed according to WHO
(1999) guidelines, with all
measures of semen quality being completed within 1 hour, apart from sperm
morphology, which was completed later after slides had been stained. Sperm
morphology was observed on Papanicolaou-stained smears by an experienced
technician according to WHO
(1999) criteria. The presence
of leukocytes in semen was determined within 1 hour by the peroxidase test as
recommended by WHO (1999).
Peroxidase-positive cells (leukocytes) that were brown and round in shape were
counted with a hemocytometer. Throughout the study, the laboratory was a
member of a national external quality assessment scheme (NEQAS) for andrology
(UK NEQAS, St. Mary's Hospital, Manchester, United Kingdom).
Sample Transportation From Tehran to Sheffield
After semen analysis, both semen and FVU samples were lyophilized in
microcentrifuge tubes, and transportation of the lyophilized samples to
Sheffield was carried out in carrier vessels (Airsea Container Ltd,
Birkenhead, United Kingdom) at room temperature. Lyophilization with the use
of an Alpha 1–2 ld plus (Martin Christ, Osterode am Herz, Germany) was
carried out according to the manufacturer's instructions. In Sheffield, both
semen and FVU samples were reconstituted with sterile, endotoxin-free water on
the basis of their initial volume and tested immediately as described
below.
Strand Displacement Amplification for C trachomatis in Semen and Urine
Four milliliters of reconstituted urine or 200 µL of reconstituted semen
were tested by strand displacement amplification (SDA; Becton Dickinson,
Cowley, United Kingdom) at the Northern General Hospital, Sheffield, United
Kingdom. The laboratory is a member of the NEQAS scheme for microbiology and
where this test is routinely performed on clinical samples for the Sheffield
Teaching Hospitals NHS Foundation Trust. Positive results were confirmed by
retesting the sample with the same SDA test. SDA is a DNA amplification
system, the BDProbeTec ET, based on simultaneous strand displacement
amplification and real-time fluorescence detection. The system uses sealed
microwells to minimize the release of amplicons into the environment. Although
commercial nucleic acid amplification tests (NAATs) such as SDA have now
become the method of choice for routine C trachomatis detection
(Hamdad and Orfila, 2005;
Gaydos et al, 2008), these
methods have rarely been applied to testing of semen, and the following nested
polymerase chain reaction (PCR) confirmatory test was therefore performed.
Nested Plasmid PCR Confirmatory Testing for C trachomatis in Semen and Urine
DNA extraction was carried out on semen and urine samples that tested
positive for C trachomatis by SDA with the use of a QIAamp DNA Mini
Kit (Qiagen, Hamburg, Germany) according to the manufacturer's instructions.
The extracted DNA was stored at –20°C until nested PCR analysis.
Initially the extracted DNA was tested for β-globulin according to the
method of Saiki et al (1985)
to check that no PCR inhibitors were in the samples. When samples were shown
to be β-globulin positive, they were tested by the nested PCR method
using primers directed against the cryptic plasmid, as described previously
(Hosseinzadeh et al, 2004).
Products were analyzed by gel electrophoresis in 1.5% agarose with ethidium
bromide staining. Each PCR run included C trachomatis DNA as a
positive control. The amplicon was sequenced and compared with GenBank
144462.
Human Interleukin-8 Immunoassay on Semen Samples
Each reconstituted sample of seminal plasma was diluted 1:4 before testing.
Each diluted sample (100 µL) was tested with a commercial quantitative
sandwich enzyme immunoassay (R+D Systems, Abingdon, United Kingdom) according
to the manufacturer's instructions. A monoclonal antibody specific to IL-8 had
been precoated onto a microplate. Standards and samples were pipetted into the
wells, and any IL-8 present was bound by the immobilized antibody. After
washing to remove any unbound substances, an enzyme-linked polyclonal antibody
specific for IL-8 was added to the wells. After a further washing step to
remove unbound antibody-enzyme reagent, a substrate solution was added to the
wells for 30 minutes, and the color was developed in proportion to the amount
of IL-8 bound in the initial step. Color development was stopped by adding 50
µL of stop solution to each well, and the intensity of color was measured.
A standard curve was constructed for each 96-well plate by plotting the mean
absorbance for each standard on the y axis against the concentration
on the x axis: a best fit curve was drawn through the points on the
graph. The minimum detectable level was 1.5 pg/mL.
Human Interleukin-6 Immunoassay on Semen Samples
Each sample of reconstituted seminal fluid was diluted 1:2 before testing.
Each diluted sample (100 µL) was tested with the use of a commercial
quantitative sandwich enzyme immunoassay (R+D Systems) according to the
manufacturer's instructions. Essentially, the assay method for IL-6 was as
described above for IL-8, but a luminol, rather than a colorimetric substrate
was used. This necessitated the use of a microplate luminometer to measure the
intensity of the light emitted. The minimum detectable level was 0.7
pg/mL.
Statistical Analysis
Data were processed by SPSS 12.0 for Windows (SPSS Inc, Chicago, Illinois).
Independent Student's t tests or Mann-Whitney tests were employed,
depending on whether data were normally distributed or not. P <
.05 was accepted as significantly different. Pearson or Spearman correlation
coefficients were appropriately used to investigate the correlation between
variables.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The median seminal plasma concentration of IL-8 was 400 (range 70–12 000) pg/mL. Concentrations of greater than 788 pg/mL, defined as high levels of IL-8 (based on 75% percentile) were found in 62 samples. When the median IL-8 concentrations in C trachomatis–infected and –noninfected groups were compared, a statistically significant difference (P < .05) was found (Table 2), with IL-8 concentrations significantly greater in C trachomatis–infected compared with -uninfected individuals. In 6 samples, insufficient semen was available for testing.
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The median seminal plasma concentration of IL-6 was 6.1 (range, 1–150) pg/mL. Concentrations of greater than 13.5 pg/mL, defined as high levels of IL-6 (based on 75% percentile), were found in 62 samples. When median IL-6 concentrations in C trachomatis–infected and –noninfected groups were compared, there was no statistical significance between them (Table 2). In 6 samples, insufficient semen was available for testing.
A degree of correlation was seen between IL-8 and IL-6 concentrations (r = .376, P < .001) with respect to all samples tested. The presence of C trachomatis had no effect on this correlation.
A bivariate analysis of the independent correlation of different semen parameters with levels of seminal plasma IL-6 and IL-8 found that semen volume was correlated with IL-8 (r = .18; P < .01) and leukocyte count was correlated with IL-6 (r = .51; P < .01) and IL-8 (r = .28; P < .01), respectively. These results are presented in Table 3.
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At semen analysis, it was found that the percentage of progressively motile sperm was lower in subjects infected with C trachomatis but that there were no significant differences in the percentage of immotile or viable sperm (P < .05; Table 4). The age of the subjects, the duration of infertility, and the days of sexual abstinence were not significantly different between the infected and uninfected groups (Table 4). Interestingly, C trachomatis–infected men had a significantly raised pH in semen (P < .05; Table 4), although this difference was small and probably clinically insignificant.
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Table 4 shows that the mean seminal leukocyte counts were significantly (P < .05) greater in subjects showing evidence of infection with C trachomatis (1.0 ± 0.6 x 106 leukocytes/mL) compared with those who did not (0.2 ± 0.6 x 106 leukocytes/mL). Moreover, 5 of 16 (31.2%) patients with C trachomatis infection, in contrast to only 12 of 239 (5.0%) patients without C trachomatis infection, showed leukocytospermia according to the WHO (1999) definition.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Controversy exists as to whether elevated cytokine levels are related to semen quality. Previous studies suggested that elevated cytokine levels were not related to semen quality (Comhaire et al, 1994; Dousset et al, 1997; Matalliotakis et al, 1998; Eggert-Kruse et al, 2001; Matalliotakis et al, 2002), although more recent studies have supported such a relationship (Furuya et al, 2003; Kopa et al, 2005). The advantage of the present study is that it is the first to specifically investigate levels of both IL-6 and IL-8 in semen from C trachomatis–infected and –noninfected patients. Of course, the realization that other microorganisms could be present in semen, and that they could be responsible for altered cytokine levels, might be a complicating factor in interpreting the results. However, Eggert-Kruse et al (2001) found no relationship between a wide range of bacteria present in semen samples and interleukin concentrations, and Bezold et al (2007) showed that pathogen DNA in semen was not associated with inflammatory markers. Nevertheless, the fact that a relationship between raised IL-8 levels and semen volume was found would lean toward our previous findings of increased semen volume levels in C trachomatis–infected patients (Hosseinzadeh et al, 2004), which was not observed in our present study. Not surprisingly, raised IL-6 and IL-8 levels were associated with an increased leukocyte count in semen because both these cytokines have neutrophil chemotactic and activating factors (Eggert-Kruse et al, 2001). No other semen parameters were associated with raised levels of either IL-6 or IL-8.
In our previous study in the United Kingdom that investigated the presence of C trachomatis in semen of men with asymptomatic chlamydial infection who were undergoing infertility investigations, a prevalence of 4.9% according to NAATs was observed (Hosseinzadeh et al, 2004). The prevalence rate of 6.2% in the current study involving Iranian men was therefore similar. An earlier study that investigated the presence of C trachomatis with the use of cell culture in male patients attending a genitourinary medicine clinic in Tehran showed a prevalence rate of 8.8% (Darougar et al, 1982), whereas a more recent study conducted on women attending obstetrics and gynecology clinics in Tehran gave an overall prevalence rate of at least 6.4%, using both SDA and PCR testing (Chamani-Tabriz, 2007). This suggests that the level of C trachomatis infection in adult males and females in Iran is relatively high and comparable to that in the United Kingdom.
With regard to the relationship between semen parameters and chlamydial infection, the current study found that men infected with C trachomatis had a lower percent progressive sperm motility, a higher leukocyte count, and a raised concentration of IL-8 compared with men without infection. In a previous study (Hosseinzadeh et al, 2004), a raised leukocyte count was also observed in semen from patients with a chlamydial infection, but no difference was observed in percent motile sperm. However, the present study is different in that urine was also examined for C trachomatis, and a newer molecular method was used to test for C trachomatis. Therefore, direct comparisons with our previous study cannot be made. In the current study, a much higher percentage of patients with leukoctyospermia had C trachomatis infection than in those without C trachomatis infection (31.2% vs 5.0%, respectively), and these findings are again similar to Hosseinzadeh et al (2004).
Rather unexpectedly in the current study, using SDA, twice as many semen samples (n = 18) as FVU samples (n = 9) were found to be positive for C trachomatis. This is in spite of there being insufficient semen sample left for 2 patients to run confirmatory tests (and therefore defined as negative). It is acknowledged that there can be a discrepancy between detection of C trachomatis in FVU and semen from the same patient (Pannekoek et al, 2003; Gdoura et al, 2008). As suggested by Gdoura et al (2008), the presence of C trachomatis DNA in FVU and its absence in semen might indicate a urethral infection, whereas its presence only in semen could indicate an infection of the epididymis or seminal vesicles. It is also generally believed that a higher number of positive results are found in urine (Pannekoek et al, 2003; Hamdad-Daoudi et al, 2004). However, a recent study by Gdoura et al (2008) showed a good correlation between PCR detection of C trachomatis in FVU and in semen, with a marginally higher proportion of C trachomatis positives detected in semen (42.3%) compared with FVU (39.4%). Similarly, in a study by Bornman et al (1998), more semen samples (35 of 131) were positive for C trachomatis than FVU samples (33 of 131). These reports, along with the data presented here, therefore raise an important question as to which is the best test specimen in males to look for C trachomatis infection in the genital tract. This is compounded by the fact that unlike FVU, there is no approved method of testing for C trachomatis in semen. It is difficult to fully understand why in our study more patients were positive for C trachomatis in semen than in FVU samples. One suggestion is that despite clear guidelines, there could have been a misunderstanding of the patients on the strict requirement to collect a urine sample immediately on waking and not at a later time in the morning. However, in the light of the above findings, it is therefore suggested that further comparative studies of C trachomatis testing in semen and FVU be undertaken and efforts made to determine and recommend the best test to detect the presence of C trachomatis in the male.
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Acknowledgments |
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Footnotes |
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± SD) of men with and without C trachomatis infection (n =
255)