Chlamydiae in the ejaculate: their influence on the quality and morphology of sperm

Acta Obstet Gynecol Scand 2004: 83: 656--660 Copyright # Acta Obstet Gynecol Scand 2004 Printed in Denmark. All rights reserved Acta Obstetricia et Gynecologica Scandinavica ORIGINAL ARTICLE Chlamydiae in the ejaculate: their influence on the quality and morphology of sperm ZDENEK VEZNIK 1,LEOPOLD POSPISIL 1,DRAHOMIRA SVECOVA 1,ATANASKA ZAJICOVA 1 AND VIT UNZEITIG 2 From the 1 Veterinary Research Institute, Brno and the 2 Department of Gynecology and Obstetric, Medical Faculty of Masaryk University, Brno, Czech Republic Acta Obstet Gynecol Scand 2004; 83: 656 660. # Acta Obstet Gynecol Scand 83 2004 Background. Given the lack of information concerning the role of Chlamydia trachomatis in male fertility, the aim of this study was to ascertain and analyze the quality of Chlamydiae-positive and -negative semen. Material and methods. Sperm count was performed according to the 1999 World Health Organization (WHO) laboratory manual for examination of human semen and sperm cervical mucus interaction, and sperm survival was assessed by a 120-min test. The evaluation of the morphological examination of ejaculates was carried out using the SASMO (strict morphological analysis of ejaculates) computer program. Chlamydiae were detected by immunofluorescent reaction using the Progen Biotechnik GmbH diagnostic set. Fisher s exact test and the -quadrate test were used for statistical analysis. Results. Of the total of 627 sperm samples examined, Chlamydiae were detected in 136 cases (21.7%). Sperm analysis showed significant differences between Chlamydiae-positive and -negative samples. The Chlamydiae-contaminated group showed normal sperm morphology 14.4% lower, volume 6.4% lower, concentration 8.3% lower, motility 7.8% and velocity 9.3% lower than in Chlamydiae-negative samples. The average values for normal spermatozoa and motility in the Chlamydiae-positive group were also significantly reduced. Conclusion. Chlamydia trachomatis was found to be a possible factor in sperm pathology. These results could help to elucidate the role of Chlamydia trachomatis in male infertility. Key words: male infertility; Chlamydia trachomatis; semen analysis; sperm morphology Submitted 28 July, 2003 Accepted 10 February, 2004 Introduction Given the number of studies on genital chlamydiosis, there are surprisingly few dealing with the presence of Chlamydiae in the ejaculate and their potential influence on its quality. At five recent symposia on chlamydial infections held between 1999 and 2002 (1 5), only a few of the papers dealt with this issue (6,7). Nevertheless, there is sufficient information available on this topic. Abbreviations: WHO: World Health Organization; SASMO: strict morphological sperm analysis; SD: standard deviation; SV: starting value; 2V: values after 120 min; IgA: immunoglobulin A; IgG: immunoglobulin G; LCR: ligase chain reaction. The questions of to what extend chlamydial infections can bring about pathological changes in male gonads and, consequently, fertility disorders have not yet been addressed in sufficient detail. In their 2001 study, Bollman et al. attempted to show a connection between the quality of sperm, the function of male gonads and Chlamydia trachomatis infections detected by both direct and indirect detection methods. Only in rare cases did the authors succeed in demonstrating a connection between the poorer quality of sperm and the Chlamydia trachomatis infection (8). Similarly, a study by Videau et al. (9) did not demonstrate any changes in sperm relating to the immunoglobulin A (IgA) Chlamydia trachomatis

Detection of Chlamydiae in human ejaculate 657 antibodies. However, the authors did suggest that the presence of these antibodies in the semen of asymptomatic, infertile men might function as an indication of a Chlamydia trachomatis infection in their female partners (9). The presence of Chlamydia trachomatis in the semen of males in infertile couples was detected in 35.9% of the 92 examined men in a 2001 study by Gdoura et al. However, they do not consider the presence of Chlamydia trachomatis in the semen to be inevitable proof of male infertility (10). In contrast to these authors, Rezacova et al. (7) state that 62% of males examined with IgA Chlamydia trachomatis antibodies detected in their seminal plasma were also diagnosed with spermiogram abnormalities. Earlier studies by Veznik et al. (11,12) showed connections between qualitative changes in spermiograms of males with Chlamydiae detected in their semen. According to Vigil et al. (13), the influence of Chlamydia trachomatis infections on male infertility is highly controversial. The authors believe that spermatozoa are active carriers involved in the dissemination of Chlamydia trachomatis, which does not directly affect the quality of semen, but their transmission to the female partner results in infectious processes and the production antispermatic antibodies. The fact that Chlamydiae can negatively influence spermatozoa motility was shown in an experimental study by Hosseinzadeh et al. (14). In a simultaneous incubation of spermatozoa and Chlamydiae, spermatozoa motility dropped significantly (14). The authors claim that this is a consequence of the presence of a live microorganism rather than the presence of soluble components or membrane elements. Pospisil et al. (15) demonstrated in an experiment involving Chlamydial orchitis in rabbits that both antichlamydial and antispermatic antibodies can develop. As for the diagnosis of chlamydial infections in the male genital tract, some authors believe that direct detection of Chlamydiae gives reliable data; the evidence of antichlamydial antibodies in the serum is not, however, equally reliable (16). Despite the availability of modern molecular biological methods for the detection of Chlamydiae, an indisputable association between the isolation of the chlamydial agents and its origin in the ejaculate is limited by various factors. The main problem lies in the fact that the examined material the ejaculate only reflects contamination experienced in the urethra, through which it passes (17,18). This assumption is supported by Keck et al. (19), who believe that the presence of bacteria in the sperm may be the result of either a contamination or even an infection. So far, there is no ideal test for the detection of Chlamydiae in the ejaculate, even though the use of the ligase chain reaction (LCR) has resulted in further progress in this field (20). In this respect, the demonstration of IgA antibodies in seminal plasma seems to be a useful method (21). The whole issue of the role of Chlamydia trachomatis in the pathogenesis of male infertility is discussed in detail in the recent study by Eggert-Kruse et al. (6). In an effort to bring more light onto this issue, we are developing an analysis of the results of our own spermatological research. Material and methods Our study included 627 male ejaculates acquired from sperm donors. Each donor provided recorded information on alcohol abuse, smoking, sexual activity, profession, health, diseases, injuries and medication. Chlamydiae in the ejaculate were detected by a direct immunofluorescent reaction using the Progen Biotechnik GmbH diagnostic set. Each smear was examined for at least 15 min. The absence of Chlamydiae was marked as 0. Chlamydial elementary bodies detected were marked as follows: (þ) for the detection of between one and five elementary bodies in the smear; (þþ) for the detection of more than five elementary bodies; and (þþþ) for the detection of either elementary or reticular bodies in every viewing field. For the spermatological examination the following indicators of ejaculate quality were measured: the volume of semen, the density of the spermatozoa and the motility of the spermatozoa, the latter measured two times during microscopic examination to assess the short-term survivability of the spermatozoa, first at the starting point of the examination (SV) and then again after 120 min (2V). Similarly, the speed of the spermatozoa, measured by a propulsivity test, and the nature of the spermatozoa motion were measured at SV and at 2V. The percentage of live spermatozoa in the smear was also assessed at SV and at 2V using supravital staining tests. Morphological examination of the ejaculates and structural changes in the spermatozoa as well as the detection of Chlamydiae were carried out using a Nikon Labothop-2 microscope at a magnification of 1000. The classification of normal spermatozoa was in accordance with the definition of the 1999 WHO laboratory manual for the examination of human semen and sperm cervical mucus interaction (22). For the evaluation, we used the SASMO (strict morphological sperm analysis) computer program in a strict morphological analysis of the ejaculate (23). The evaluation of frequency of ejaculates contaminated by Chlamydiae was divided into two time periods, from 1995 to 1998 and from 1998 to 2001. Results A total of 627 men were examined with Chlamydiae detected in 136 (21.7%) of them. According to the intensity of the agents present in the 136 contaminated ejaculates, 48 smears (35.3%) were marked (þ) and 88 (64.7%) with (þþ)or(þþþ). In the spermatological examination of ejaculates with either positive or negative

658 Z. Veznik et al. Table I. Analysis of 627 male ejaculates (1995 99) Chlamydia negative Chlamydia positive Difference Ø SD Ø SD (%) Volume of ejaculate (ml) 3.01 1.53 2.82 1.53 6.4 Spermatozoa density (mm 3 ) 79.4 77.7 72.8 65.7 8.3 (in thousands) Spermatozoa motility (%) 52.0* 15.6 48.4* 14.3 7.8 Speed of spermatozoa (mm/s) 36.6 23.4 33.2 21.9 9.3 Normal spermatozoa (%) 43.2 16.6 37.0 16.7 14.4 Teratospermia index 1.29 0.12 1.38 0.14 6.9 *Statistically significant: p ¼ 0.05 High statistical significance: p ¼ 0.01 Chlamydiae detection, other differences were also found in the relative values of individual criteria (Table I). In the Chlamydiae-contaminated group the volume of semen was 6.4% lower, spermatozoa density 8.3% lower and spermatozoa motility at the starting point of the examination 7.8% lower than the values in the Chlamydiae-negative ejaculates. The Chlamydiae-contaminated ejaculates also showed a 9.3% lower speed of spermatozoa motility and the percentage of normal spermatozoa was lower by 14.4% compared to the Chlamydiaenegative ejaculates. The differences in the proportional representation of normal spermatozoa between ejaculates with and without Chlamydiae were statistically highly significant (43.2%, SD 16.7 and 37.0%, SD 16.7, respectively, at p ¼ 0.01). The differences in sperm motility were also statistically highly significant (p ¼ 0.05). The high frequency of pathological spermatozoa in Chlamydiae-contaminated ejaculates is shown in Fig. 1 by a significant shift of in the plot to the right. Teratospermatozoa, so designated based on the number of pathologically abnormal spermatozoa detections, showed a higher percentage of ejaculates with over 65% pathological spermatozoa (Table II), which was also represented by differences in the teratospermatozoa index (Table I). However, the differences in frequencies are statistically insignificant. Abnormalities in spermatozoa heads caused by Frequency 25 20 15 10 5 positive negative 0 0 10 20 30 40 50 60 70 80 90 100 Pathological sperm % Fig. 1. The frequency of pathological spermatozoa in Chlamydia-contaminated ejaculates. developmental deformations accounted for 44.27% (SD 15.3) in Chlamydiae-negative ejaculates and 50.28% (SD 16.6) in Chlamydiaepositive ejaculates; this is statistically a highly significant difference (p ¼ 0.01). The frequency of the ejaculates contaminated by Chlamydiae was ascertained to be 21.7%. The division of the study into two periods (1995 98 and 1998 2001) produced no statistically significant difference ( 2 0.728) in the frequency of the Chlamydiae-contaminated ejaculates. In the first phase, 360 ejaculates were examined with contamination detected in 81 cases (22.5%), and in the second phase 53 ejaculates (19.9%) were contaminated out of the total of 267 examined. Discussion Our frequency of Chlamydiae detection in male ejaculates (21.7%) corresponds with the findings of Hartung (21). By contrast, Rezacova et al. (7), using the LCR test, no positive Chlamydiae detection despite the presence of IgA and IgG antichlamydial antibodies in the ejaculates of 39% of the men examined. We cannot exclude the possibility that the negative results of the LCR test, which was originally designed as a noninvasive means of Chlamydiae detection in urine, could have been a consequence of the presence of seminal plasma inhibitors obstructing the process of the reaction (24). Men with a positive detection of antibodies in seminal plasma Table II. The occurrence of pathological spermatozoa in male ejaculates with negative and positive results for Chlamydia Pathological Chlamydia negative Chlamydia positive Statistical sperm (%) % SD % SD significance 40 13.2 6.20 12.4 4.37 40 65 54.4 6.69 45.8 6.98 p ¼ 0.01 65 32.4 7.84 41.8 7.85 p ¼ 0.01

Detection of Chlamydiae in human ejaculate 659 also showed qualitative changes in their spermiograms. Rezacova et al. (7) also focused on azithromycin therapy in 14 patients, 10 of whom showed improvement in the quality of semen (71%). This result is in accordance with the findings of our study with a limited number of patients. Thirteen patients were observed for the effect of therapy (their respective general practitioners were recommended to use azithromycin therapy). Eight of the 13 patients observed were Chlamydiae negative at their second or third examination (following therapy duration of at least 3 months). The therapy, then, helped to eliminate the ejaculate Chlamydiae contamination in 61.5% of the patients. In two patients the therapy resulted in a considerable reduction in the frequency of elementary bodies in the ejaculate. On the whole, a positive effect of the therapy was observed in 76.9% of the patients. In three patients the therapy resulted in no change in the frequency of ejaculate contamination (23%). The examination of the quality of the ejaculates after the therapy showed an overall improvement in individual criteria of spermatoanalysis. The volumes of ejaculates rose by 26.7% in comparison with the figures prior to therapy and the density of spermatozoa in the ejaculates rose by 58% on average. Spermatozoa motility improved by 11.6% at the initial value (SV) and 20% after the short-term survival test (2V). The speed of spermatozoa rose by 30% at SV, but results after 2V showed only a 3% improvement. The increase in the percentage of morphologically nonmutated spermatozoa was 15.4% on average. In the individual spermatoanalytical criteria, the following improvements were found: 78.5% in ejaculate volume, 71.4% in spermatozoa density, 57.2% in spermatozoa motility at SV and 64.3% at 2 V, approximately 50% in the speed of spermatozoa at both SV and 2V, and finally, 50% in the morphological analysis of the semen. The results of macrolide antibiotics therapy can therefore be regarded as very promising, and suitable for use in cases of genital chlamydiosis. The evidence of qualitative changes in ejaculates paralleling positive Chlamydiae detections cannot be considered as a generally valid parallel phenomenon (9). It is therefore of great significance that in a group of Chlamydiae-positivemaleejaculates, the percentage of morphologically abnormal spermatozoa was statistically much higher. Further evidence of the connection between morphologically abnormal spermatozoa and chlamydial contamination is their lower posttherapy frequency. Similarly positive results of the therapy were also noted by Wolff et al. (25), with the difference in the frequency of pathological changes in spermatozoa also of high statistical significance. Functional and morphological changes in spermatozoa in ejaculates reflect the locality of the agents relative activity. If the origin of the infection is in the adnexa and/or the excretory duct, the changes in ejaculates take the form of a lower level of functional indicators and a reduced membrane resistance leading to a decrease in survival abilities. The presence of agents in the parenchyma of the testis can affect two basic structural components, the interstitium and the spermogenetic epithelium. In both cases this results in morphological changes in spermatozoa, varying from teratospermia to oligospermia, of differing intensity. In such cases agents can be found adhering to spermatozoa or submembraneously incorporated in the acrosome locality, namely in the connecting portion. 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