Section of Andrology, Endocrinology and Internal Medicine, Department of Biomedical Sciences, University of Catania, Italy
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Abstract |
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Key words: carnitines/leucocytospermia/prostato-vesiculo-epididymitis/reactive oxygen species/sperm parameters
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Introduction |
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Some antioxidants (vitamins E, A and C, glutathione) are able to chain-break ROS-induced lipid peroxidation within the sperm membrane or in the seminal plasma (Sikka, 1995); others (i.e. carnitine/acetyl-carnitine complex) seem to exert a repairing effect through the removal of elevated intracellular toxic acetyl-coenzyme A (acetyl-CoA) and/or the replacement of fatty acid in membrane phospholipids (Arduini, 1992). Furthermore, high concentrations of carnitine, a 3 hydroxy-4 trimethylaminobutyric acid, are present in both seminal plasma and spermatozoa, where carnitine is efficient in the transport of fatty acids through mitochondrial membranes and in the intracellular storage of acetate moieties derived from acetyl-CoA. Acetyl-carnitine, generated by the carnitine acetyltransferase reaction, is a useful compound in the energetic-metabolic cellular economy through the transport of acetyl-groups into mitochondria, where acetyl-CoA serves as fuel for the Krebs cycle to supply ATP for sperm motility. The concentration of this biochemical complex increases continuously during the epididymal transit, that is when sperm motility and fertilizing ability develop, thus suggesting that the carnitine-acetylcarnitine complex may play an important role in these processes (Brooks, 1980
). Since the important antioxidant role of the epididymal microenvironment has been pointed out (Potts et al., 1999
), we thought that a combined treatment with carnitines (carnitine and acetyl-carnitine complex) might be rational in conditions of acetyl-CoA accumulation. Indeed, carnitine and acetyl-carnitine act as the first and second scavenger agent respectively to remove acetyl-CoA from the cell, and possibly replace unsaturated fatty acids in the plasma membrane phospholipids (Arduini, 1992
). In addition, a combined treatment reduces the dosage of each antioxidant and reproduces more closely the biochemical conditions present in the epididymal milieu. For these reasons, we administered carnitine plus acetyl-carnitine to 54 asymptomatic infertile patients with ultrasound evidence of PVE. The patients became abacterial after antimicrobial treatment but proved to have a persistent seminal ROS over-production and ultrasound evidence of PVE. On the basis of seminal WBC concentration, they were divided into two groups: group A with normal WBC (<1x106/ml) and group B with elevated WBC (>1x106/ml) concentration. Sperm parameters, ROS production and spontaneous pregnancy rates were evaluated before, during and following treatment with carnitines.
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Materials and methods |
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Inclusion criteria
All infertile patients were affected by chronic abacterial PVE, suspected on the basis of the following eligibility criteria: (i) oligo- (sperm concentration <20x106/ml), astheno- (<50% spermatozoa with forward progression, a and b categories) or terato- (<30% spermatozoa with normal oval form) -zoospermia; (ii) clinical signs (at the physical examination) and ultrasound findings considered indicative of chronic PVE, as previously described (Vicari, 1999, 2000
); (iii) achievement of a bacteriological cure (<1x103 colony-forming units, CFU/ml), following antimicrobial treatment with ofloxacin (200 mg orally every 12 h; Flobacin®, SigmaTau, Italy) or doxycycline (100 mg orally once daily; Bassado®, Poli, Italy) for 14 days/month over a 3 month period, in patients with one or two consecutive cultures with significant bacteriospermia (
105 CFU/ml), or eradication of Chlamydia trachomatis or Ureaplasma urealyticum from cultures of urethral swabs obtained after prostatic massage, following the same antimicrobial treatment; and (iv) seminal ROS over-production after antimicrobial treatment.
Exclusion criteria
(i) PVE patients with a significant microbial reinfection (with growth of 105 Streptococcus faecalis or Staphylococcus aureus species) resistant (minimal inhibitory concentration, MIC, 816 mg/l) to both drugs (ofloxacin or doxycyclin). The cut off value of
105 CFU/ml, much higher than the value mentioned in the WHO semen manual (WHO, 1992) was chosen because of its recently reported strong association with ultrasound abnormalities (Vicari, 1999
); (ii) azoospermia, severe oligozoospermia (<5x106/ml), terato-zoospermia (normal forms <14%) according to the WHO criteria (World Health Organization, 1992
), and/or necrozoospermia (sperm viability <10%); (iii) elevated (>10 mIU/ml) serum FSH concentrations; (iv) history or presence of primary testicular disease (cryptorchidism, orchitis, varicocele) or testicular volume
12 ml; (v) smoking habit, alcohol consumption, occupational chemical exposure; history of major renal and hepatic disorders and myopathy; and (vi) treatment with other drugs within the 3 months before enrolment in this study.
Study design and treatments
Before initiating antioxidative treatment, patients were subdivided into two groups according to their seminal WBC concentration: group A (n = 34) had normal seminal WBC (<1x106/ml); group B (n = 20) had elevated seminal WBC (>1x106/ml). The patients of both groups received carnitine (1 g, Carnitene®, Sigma-Tau, Pomezia-Rome, Italy) and acetyl-carnitine (500 mg, Nicetile®, Sigma-Tau) orally every 12 h for 3 months, followed by a wash-out period of 3 months.
This study was approved by the Committee for Ethics of the Medical Faculty, University of Catania. All patients provided their written informed consent. They completed the entire trial, including treatment and follow-up examinations.
Sampling management
All the patients underwent semen analyses before (T0) and during treatment (T3), and 90 days (range 90101 days) after completion of therapy (T6). After liquefaction, semen samples were analysed for sperm concentration, total sperm number, forward motility percentage (% grade a + b, after 1 h), sperm morphology (percentages of normal oval forms), sperm viability (percentages of viable spermatozoa) and seminal WBC concentration according to the WHO guidelines (World Health Organization, 1992). In particular, conventional immunocytochemical staining (World Health Organization, 1992
) was used to assess seminal WBC concentration, according to previously published methods (Vicari, 1999
). All semen analyses were performed by the same investigator in a blinded fashion, to minimize methodological errors. Spermatozoa were separated by means of a two-step 45/90% discontinuous Percoll gradient; basal and formyl-methionyl-leucyl-phenylalanine (Sigma Chemicals Co., St Louis, MO, USA) (fMLP)-stimulated ROS measurements were then carried out on 400 µl aliquots of cell suspension both from the 90% Percoll layer and the 45/90% Percoll interface (= 45% Percoll fraction), as previously reported (Vicari, 1999
). ROS production was measured in aliquots containing a maximum of 2.5x106 spermatozoa/ml to reduce the number of WBC in each aliquot and consequently the number of `overflow' samples. The counts obtained in aliquots containing <2.5x106 spermatozoa/ml were adjusted to this number.
Safety assessment
Safety assessment included medical history, physical examinations, haematological and serum chemistry profiles at all visits, and the monitoring of drug-related adverse events by means of indirect questioning through patients' diaries.
Statistical analysis
In every phase of the trial, sperm results are the mean of two consecutive semen specimens, collected 35 days apart. Throughout the text, results are shown as median and the 10th and 90th percentiles in parentheses. One-way analysis of variance (ANOVA) followed by Duncan's multiple range test was applied to evaluate the effects of treatment within each group. The effects of carnitine treatment between the two groups were evaluated by means of the MannWhitney U-test for unpaired data. Fisher's exact test was used to compare the rates of spontaneous pregnancy (and pregnancy rate per cycle) between the groups. P value < 0.05 was considered statistically significant.
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Results |
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Safety assessment
The carnitines administered throughout the trial were generally well tolerated. No laboratory abnormalities were observed.
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Discussion |
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Previous studies have demonstrated an oxidative sperm stress secondary to an unbalanced pro-oxidant/antioxidant ratio in patients with MAGI. They have shown a direct relationship between seminal WBC concentrations and the seminal concentration of some cytokines, and an inverse relationship between ROS production and the seminal concentrations of gamma-glutamyltransferase (-GT) and
-glucosidase, which are known biochemical markers of epididymal origin (Depuydt et al., 1996
). Furthermore, antimicrobial treatment produced only a limited antioxidant effect on these patients, since it improved some sperm parameters associated with ameliorated antioxidant proprieties of the seminal plasma through increased interleukin (IL)-4 levels, whilst the levels of IL-2 and IL-8 remained low (Omu et al., 1998). In other words, this limited antioxidant effect may be explained by an increased seminal IL-4 concentration, which has marked inhibitory effects on the expression and release of the pro-inflammatory cytokines produced by T helper 1-type lymphocyte (IL-2), monocytes and macrophages (IL-8). Moreover, peroxidative damage could be caused by the growth of bacterial mycotoxins, whose effects have been counteracted by in-vitro addition of antioxidants (co-enzyme Q-10 plus carnitine) (Atroshi et al., 1998
). Recently, we have shown that, in comparison with patients affected by prostatitis or prostato-vesiculitis, patients with PVE have the highest leukocytic/juxta-spermatozoa ROS over-production (Vicari, 1999
) associated with the lowest antimicrobial efficacy, in terms of bacteriological cure rate, pregnancy rate, sperm outcome and a persistent ROS over-production (Vicari, 2000
). This suggests that PVE patients may represent a well-defined clinical model of oxidative stress for evaluating the antioxidant effects of carnitines.
Following the removal of the microbial noxae, 20 out of 54 patients (37%) still exhibited an increased concentration of seminal WBC (group B). Activated leukocytes can enhance ROS production by mature and immature spermatozoa (Ochsendorf, 1999; Piquet-Pellorce et al., 2000
) thus having detrimental effects on sperm function. In contrast, the other PVE patients studied showed a normalization of seminal WBC concentration. Nevertheless, they had a persistent oxidative stress of sperm origin (basal but not fMLP-stimulated ROS production). This may relate to an absolute or relative increase in pro-inflammatory cytokines, such as tumour necrosis factor-alpha (TNF-
), IL-1ß or IL-8, etc., and/or their soluble receptors, not necessarily associated with an increase in seminal WBC concentration. It has been shown that male germ cells are able to produce TNF-
(De et al., 1993) and there is a mounting body of evidence that demonstrates the importance of cytokines and chemokines in testicular paracrine regulation (Dousset and Hussenet, 1997
; Hales et al., 1999
).
Treatment with carnitines improved sperm forward motility and viability in PVE patients with normal seminal WBC concentration, besides significantly increasing their otherwise poor (Vicari, 2000) reproductive performance. These effects may be explained by a re-equilibrium of the seminal oxidative balance resulting from an amelioration of the scavanger properties of the epididymal microenvironment. In addition, since exposure to spermiotoxic noxae present at the epididymal levels (`epididymal hostility') (Wilton et al., 1988
; Ochsendorf, 1999
) could be aggravated by the duration of epididymal transit, which seems to be slower in oligozoospermic patients (Johnson and Varver, 1988
), it cannot be excluded that the improvement of some sperm parameters may also be due to a more effective energy supply to spermatozoa. This, in turn, reduces transit time and/or protects the sperm pool of the `epididymal reserve'. The associated reduction in ROS over-production supports the hypothesis that these alterations may be caused by plasma membrane peroxidative damage and/or abnormal or unbalanced levels of pro-inflammatory cytokines. Accordingly, TNF-
and interferon-
have been shown to be capable of causing astheno-necrozoospermia in the absence of infection when co-incubated with normal spermatozoa (Depuydt et al., 1996
). Moreover, since several types of bacteria are capable of altering the host cell cytokine synthesis by degrading pro-inflammatory cytokines and/or using cytokine receptors as portals of entry for cellular invasion (Wilson et al., 1998
), it can be speculated that the administration of carnitines, as it acts on intraleukocytic metabolism, may use some components of the cytokine network to enhance sperm function. At the same time it may re-establish an equilibrium between pro-inflammatory and anti-inflammatory cytokines, reducing the former and/or increasing the latter. On the other hand, treatment with carnitines was only able to increase the percentage of viable spermatozoa without having any detectable effect on sperm forward motility and ROS over-production. None of the group B patients achieved a spontaneous pregnancy. Since carnitines have been shown to localize in polymorphonuclear leukocytes (Katrib et al., 1987
), it may be hypothesized that this mechanism contributes to annihilating their antioxidant and energetic effects at the spermatozoon level.
In conclusion, antioxidant treatment with carnitines is effective in patients with PVE, elevated ROS production and normal seminal WBC concentrations. In this group of patients, these compounds may represent a new rational, non-hormonal, therapeutic frontier. The lower efficacy of carnitine treatment in PVE patients with persistently elevated seminal WBC concentrations has suggested that a different anti-inflammatory/antioxidant strategy should be explored in this subgroup of patients.
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Acknowledgements |
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Notes |
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References |
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Submitted on April 25, 2001; accepted on July 19, 2001.