Chlamydia trachomatis and sperm lipid peroxidation in infertile men

A. Segnini¹, M.I. Camejo², F. Proverbio¹

Asian J Androl 2003 Mar; 5: 47-49             

Keywords: Chlamydia trachomatis; lipid peroxidation; spermatozoa; male infertility

Abstract

Aim: To relate the presence of anti-Chlamydial trachomatis IgA in semen with sperm lipid membrane peroxidation and changes in seminal parameters. Methods: Semen samples of the male partners of 52 couples assessed for undiagnosed infertility were examined for the presence of IgA antibody against C. trachomatis. The level of sperm membrane lipid peroxidation was estimated by determining the malondialdehyde (MDA) formation. Results: Sperm membrane of infertile males with positive IgA antibodies against C. trachomatis showed a higher level of lipid peroxidation than that of infertile males with negative IgA antibody (P<0.05). There was a positive correlation (P< 0.01) between the level of C. trachomatis antibody and the magnitude of sperm membrane lipid peroxidation. All the other tested semen parameters were found to be similar in the two groups. Conclusion: The activation of immune system by C. trachomatis may promote lipid peroxidation of the sperm membrane. This could be the way by which C. trachomatis affects fertility.

1 Introduction

Chlamydia trachomatis infection is considered as one of the most common sexually transmitted diseases in industrial countries. Whereas the clinical importance of C. trachomatis induced pelvic inflammatory disease (PID) has been well established as an important cause of female infertility, the impact of chlamydial infection on semen parameters and male fertility is controversial. There are studies correlating sperm quality and genital chlamydial infection [1], but other studies have contradictory findings [2]. Male genital tract infection may lead to several consequences, including the release of pro-inflammatory cytokines and the production of reactive oxygen species (ROS) [3]. ROS generation has been indicated to impair sperm function and has been proposed as a potential cause of male infertility. ROS could result from peroxidation of unsaturated fatty acids of the sperm plasma membrane [4]. The purpose of this study was to relate the presence of anti-Chlamydial IgA in semen with sperm lipid membrane peroxidation and changes in sperm parameters.

2 Materials and methods

Fifty-two couples assessed for undiagnosed infertility were selected in a consecutive way. All patients with known infertility cause or with seminal WBC of more than 10⁶/mL (to avoid important contributions to the level of lipid peroxidation) were discarded. Informed consent was obtained from every patient.

Semen samples were examined in the laboratory of the Universidad Simn Bolivar. Semen was obtained by masturbation after 2~5 days of abstinence and was assessed using the guidelines of the World Health Organization [5]. The semen was centrifuged and the supernatant used for the determination of IgA antibodies to C. trachomatis (SeroELISA, Savyon Diagnostic, Beer-Sheva, Israel). This assay excludes cross-species reactive with C. pneumonia. To avoid further important peroxidation, all the samples were treated exactly the same way and all the pellets were frozen at -20 and utilized next day for the determination. The level of sperm membrane lipid peroxidation was estimated by measuring the thiobarbituric acid-reactive substances (TBARS) formation according to the method of Feix et al. [6], using a malondialdehyde (MDA) standard curve prepared by acid hydrolysis of 1, 1, 3, 3-tetramethoxypropane. The protein concentration of the samples was standardized to 0.6 to 0.8 mg/mL. The MDA determination was carried out as follows: 250 µL of sample, 50 µL of Butilate Hidroxitoluene (BHT, 50 mmol/L) and 750 µL of trichloric acetic acid (TCA 10 %) were incubated for 10 min at 4 and then centrifuged for 10 min at 600 rpm. Seven hundred and fifty µL of the supernatant were mixed with 750 µL of thiobar-bituric acid 5 %. This mixture was heated for 15 min at 100 , cooled and read at 532 nm. Values were expressed as nmols of MDA per mg protein.

Statistical analysis was performed by Student t test and non parametric Kedall Tau correlations test. All results are expressed as meanSE. P<0.05 was considered significant.

3 Results

In the 52 patients, 25 semen samples were found to be negative (48 %) and 27 positive (52 %) to C. trachomatis. As shown in Table 1, the two groups, C trachomatis positive and negative, showed values statistically similar for the routine semen parameters (sperm concentration, motility and morphology), while the level of lipid peroxidation was significantly higher in the C. trachomatis positive than in the negative group (P< 0.01). There was a certain degree of overlap in the lipid peroxidation values of the two groups, thus this parameter cannot be taken as a predictive criterion for the presence of chlamydial antibody. However, the distribution of the patients, as shown in Figure 1, shows a positive correlation between the level of IgA against C. trachomatis and the extent of lipid peroxidation (P<0.01).

Table 1. Relationship between seminal parameters and anti-C. trachomatis IgA in seminal plasma. ^cP<0.01, compared with IgA anti-C. trachomatis negative.

Figure 1. Correlation study (non parametric Kedall Tau correlations test) between levels of specific IgA anti-C. trachomatis in seminal plasma and lipid peroxidation (nmol of malondialdehyde, MDA, per milligram of protein) of undiagnosed infertile men (n=51) (P<0.01).

4 Discussion

The routine semen parameters were found to be similar between the C. trachomatis negative and positive groups. However, the level of sperm membrane lipid peroxidation was found to be strongly elevated for the C. trachomatis positive group. Moreover, for the studied samples, the magnitude of lipid peroxidation was directly proportional to the magnitude of anti-C. trachomatis IgA antibodies (Figure 1).

Variations in the sensitivity and specificity of the tests employed and the use of specimens from different anatomical locations may produce inconsistent results in the detection of C. trachomatis [7], which is the reason that this technique can not be utilized as a diagnostic test. The presence of IgA antibodies in the semen does not mean chlamydial infection [8], but has been associated with an inflammatory response in the male genital tract[9, 10], either due to an elevation of the inflammatory marker polymorphonuclear granulocyte-elastase (PMN-elastase) or to a significant diminution of the citric acid level in the semen. Besides, cytokine liberation has been observed during C. trachomatis infection [11] and in a previous work we have found a clear direct correlation between the levels of IL-6 in the seminal plasma and the levels of sperm membrane lipid peroxidation [12].

Accordingly, the presence of IgA-chlamydial antibodies in semen can be associated with an activated immune system and consequently with an increased ROS formation. It was suggested that ROS produced during C. trachomatis infection might cause membrane lipid peroxidation [13]. If this increased ROS formation overcomes the antioxidant protection mechanisms of the semen, it could promote sperm membrane lipid peroxidation [14]. Granulocytes and macrophages are known to produce ROS, which could increase the level of lipid peroxidation. To avoid a significant interference by this factor, we utilized only semen samples with WBC concentrations under 106/mL.

Experiments in vitro have shown that low levels of lipid peroxidation are important for normal sperm processes, such as sperm activation, capacitation, acrosome reaction and sperm binding to the zona pellucida [15]. However, in in vivo condition the spermatozoa should travel a long distance to reach the oviduct and any extemporaneous increase in the level of lipid peroxidation could accelerate spontaneous sperm hyperactivation, which, in turn, could impair sperm transport along the female reproductive tract or lead to a premature capacitation and acrosome reaction [16]. Increased lipid peroxidation has been typically associated with decreased sperm motility [17]. In the present study, the positive anti C. trachomatic IgA group showed a 16 % decrease in sperm motility, which is, however, statistically insignificant; probably a larger sample study may be necessary.

Increased membrane lipid peroxidation disturbs membrane fluidity, membrane associated enzymes such as Ca-ATPase and Na,K-ATPase activities [17] and other membrane functions, including spermatozoa capacitation, acrosome reaction and ovocite junction. This could be the way by which C. trachomatis infection, by increasing sperm membrane lipid peroxidation, affects fertility.

References

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Correspondence to: Dr. Mara Isabel Camejo, Departamento de Biologa de Organismos, Universidad Simn Bolvar, Baruta, Edo. Miranda, Venezuela.
Tel/fax: +58-2-906 3077
E-mail: mcamejo@usb.ve
Received 2002-05-27      Accepted 2003-01-14