CASE REPORT Journal of Andrological Sciences 2009;16:130-132 Chlamydia trachomatis attacks young male spermatozoon T. Cai, S. Mazzoli*, D. Bani**, T. Sacchi Bani**, R. Bartoletti Department of Urology, University of Florence, Italy; * STDs Centre, Santa Maria Annunziata Hospital, Florence, Italy; ** Department of Anatomy, Histology & Forensic Medicine, University of Florence, Italy Summary Chlamydia trachomatis infection, even if has a potential role in chronic prostatitis pathogenesis, has a controversial impact on male fertility. The role of Chlamydia trachomatis infections of the upper male genital tract as probable aetiological factors for male infertility has been fully argued, without any convincing demonstration of a clear correlation. In this case report, an electron transmission microscopic evaluation of spermatozoa obtained from a patients with chronic prostatitis due to Chlamydia trachomatis infection has been showed. In this case, we stress the role of Chlamydia trachomatis infection in young males, highlighting the role of Chlamydia trachomatis in spermatozoa damaging and fertility decreasing. Key words Chlamydia trachomatis • Spermatozoa • Fertility • Prostatitis Introduction Chlamydia trachomatis (C. trachomatis) is the most prevalent bacterial cause of sexually transmitted infections1. Moreover, World Health Organization estimates that more than 92 million of C. trachomatis infections occurred worldwide in the last years 1. This high diffusion is probably due also to the fact that approximately 75% of C. trachomatis infections in women and up to 50% of those in men are asymptomatic and this is why, due to the low reported incidence rates of genital chlamydial infections in the population 2. Recent reports have established that C. trachomatis causes symptomatic infection in the lower male genital tract and that should have a potential role in chronic prostatitis pathogenesis 3. Furthermore, we recently showed a clear correlation between poor semen quality and C. trachomatis infections in young male patients affected by chronic prostatitis due to C. trachomatis infection4. Even if the pathophysiology of C. trachomatis infection damage on human fertility is also unclear for the moment, few hypotheses have been, recently, purposed 5-6. Some Authors state that C. trachomatis could directly damage male sperm5 while others suggested a probable immuno-mediated damage. In fact, the presence of anti-chlamydial immunoglobulin (Ig) A in semen from males with a previous contact with C. trachomatis is associated with elevated levels of T lymphocytes, that have been also correlated with the presence of antisperm antibodies 6. In our previous study, we have found a statistically significant differences between patients affected by C. trachomatis infection and those with uropathogens Corresponding author: Tommaso Cai, Department of Urology, University of Florence, via dell’Antella 58, 50011 Florence, Italy – Tel. +39 0552496347 – Fax +39 0552496452 – E-mail: [email protected] 130 Chlamydia trachomatis attacks young male spermatozoon infections in terms of sperm concentration, percentage of motile sperm and that of normal morphological forms, highlithing the probable direct damage of C. trachomatis against spermatozoa 4. In addition, we have found a strong correlation between positivity to Heat Shock Protein 60, 70 and sperm concentration and normal morphological forms, highlighting the immuno-mediated damage too 4. Recently, the use of electron microscopy in the study of male affected by C. trachomatis infections has been improved, due to the extreme small size of this microrganisms. We present, here, a case of a 30-year-old patient affected by chronic prostatitis, positive to C. trachomatis infection markers, in which an electron transmission microscopic evaluation has been performed. Case report A 30-year-old patient affected by clinically demonstrated chronic prostatitis underwent Meares-Stamey test, performed according to European Association of Urology (EAU) guidelines by using a 4-glass test 7. All genital samples were collected, in accordance with indications described in our previous report 8. All biological samples was analysed for the detection of Chlamydia trachomatis (CT), urogenital Mycoplasmata, Neisseria gonorrhoeae, Human Papilloma virus, Herpes virus 1 and 2 by PCR (Polymerase Chain Reaction) and aerobic, anaerobic and microaerophylic common bacteria and yeasts by culture 8. The patient showed positivity both for CT plasmidic DNA and mucosal IgA in total ejaculate, while was negative for all the other tests. In order to perform the electron microscopy analysis, sperm sample has been fixed in Karnovsky’s reagent, rinsed overnight in 0.1 mol/L cacodylate buffer (pH 7.2), postfixed in 1% buffered OsO4, dehydrated, and embedded in Epon-Araldite (Fluka, Milan, Italy). Ultrathin sections were cut with an LKB ultramicrotome (Vienna, Austria) and stained with uranyl acetate and lead citrate. Observations were made with a TEM CM 10 (Philips, Eindhoven, the Netherlands), at magnifications of x 7,500, by a dedicated evaluator. The Figure 1 shows a C. trachomatis elementary body (EB) (arrow) attached to the thin cytoplasmic layer of a spermatozoon (diameter 0.1 µm), while the Figure 2 shows Chlamydia forms free in the seminal fluid from the same patient, especially reticulary bodies (RB) and EB, that should be the infecting form of C. trachomatis. The patients underwent standard antibiotic therapy for C. trachomatis infections, in accordance with EAU guidelines (doxycycline 2 times daily 100 mg orally for 21 days) 7. At the follow-up visit, the patient showed improvement in symptoms relief and quality of life and, then, Figure 1. C. trachomatis elementary body (arrow) attached to the thin cytoplasmic layer of a spermatozoon (diameter 0.1 µm). Electron microscopy photo. Original magnification x 7,500. Figure 2. C. trachomatis forms free in the seminal fluid, elementary bodies and reticulary body. Electron microscopy photo. Original magnification x 7,500. underwent microbiological analyses that confirmed the absence of C. trachomatis infection. Discussion Chlamydia trachomatis, an obligate intracellular parasite, has a biphasic life cycle characterized by an EB with infective capacity and a reticular body RB that is able to replicate within eukaryotic cells 9. In details, the organism has a unique developmental cycle in which it exists in two alternating forms: an extracellular, metabolically inactive, infectious form, EB and an intracellular, metabolically active, reproducing form, RB. This developmental cycle is necessary because C. trachomatis need to utilize the intracellular machinery of a host cell in order to reproduce 10. An obvious consequence of this developmental cycle is that, at the site of infection, the reproductive tract will periodically contain significant numbers of highly infectious EB, and these may be encountered by any gametes that are present in the reproductive tract at that time 10. Hosseinzadeh 131 T. Cai, et al. and co-workers have, recently, demonstrated that EB can have a direct and negative effect on sperm physiology and this was seen primarily via a reduction in sperm motility, which was reflected in a corresponding increase in sperm death 11. The same group, have also demonstrated that Ct-induced death of human sperm is primarily caused by lipopolysaccharide (LPS) 10, by means of a caspase-mediated apoptosis 12. LPS is, then, a heat-stable complex and unique glycolipid that is present in all Gram-negative bacteria. It is the immunodominant antigen of most Gram-negative bacteria and is considered to be intimately associated with the virulence of the pathogen 13. C. trachomatis LPS is known to be particularly spermicidal 10 and is composed of a simple structure containing lipid A and Kdo 14. In particular, C. trachomatis LPS interacts with CD14 on the sperm surface (and, possibly, Toll-like receptors if present), leading to increased production of reactive oxygen species, resulting in caspase-mediated apoptosis 5. Excessive generation of reactive oxygen species is related to an increase in sperm defects both in vitro and in infertile men 15. Several studies showed that in vivo model, C. trachomatis were phagocytized and killed by polimorphonucler leukocytes and macrophages 16. However, C. trachomatis can resists in polimorphonucler leukocytes and macrophages inclusion as intact form, promoting the establishment of latent or chronic infections states and circumvents bactericidal immune mechanisms and selective drug therapy 16. This characteristic may act as a factor for dissemination of C. trachomatis infections to the female partners. The ability to cause persistent infection is, then, one of the major characteristics of C. trachomatis in its appropriate hosts. C. trachomatis has also been demonstrated to enter a persistent state after treatment with cytokines such as interferon-gamma (Interferon-gamma), treatment with antibiotics, or restriction of certain nutrients, or to enter this state spontaneously under certain culture conditions 17. While the organism is in the persistent state, metabolic activity is reduced, and the organism is often refractory to antibiotic treatment 17. In conclusion, the present case report underlines the role of C. trachomatis infection in young males and highlights the damage to spermatozoa by C. trachomatis. The role of C. trachomatis infection in male fertility decreasing should now be clearer, due to the many published studies and reported experiences. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 References World Health Organization. Global prevalence and incidence of selected curable sexually transmitted diseases: overview and estimates. Geneva: World Health Organization 2001. 2 Gonzales GF, Munoz G, Sanchez R, Henkel R, Gal1 132 legos-Avila G, Diaz-Gutierrez O, et al. Update on the impact of Chlamydia trachomatis infection on male fertility. Andrologia 2004;36:1-23. Ochsendorf FR. Sexually transmitted infections: impact on male fertility. Andrologia 2008;40:72-5. 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