In general it is believed that apoptosis does not occur in C. trachomatis-infected host cells. However, using three different methods, our findings clearly indicate that co-incubation of sperm with C. trachomatis LPS results in cellular death which is in part due to apoptosis and is caspase-mediated. These findings provide an explanation as to how C. trachomatis can mediate premature death in human sperm.

We have shown previously that the in vitro exposure of spermatozoa to elementary bodies (EBs) of Chlamydia trachomatis can lead to sperm death over a number of hours of incubation. As such, we have hypothesized that the ejaculates of men with a chlamydial infection could contain increased numbers of nonmotile (dead) spermatozoa if they are exposed to EBs prior to ejaculation. To test this hypothesis, the ejaculates of 642 men undergoing diagnostic semen analysis as part of ongoing infertility investigations with their partner were examined. All men were without symptoms of genitourinary infections and semen analysis was performed according to World Health Organisation (WHO) 1999 methods after a 3-5 day abstinence period. In addition to semen analysis, nested plasmid polymerase chain reaction (PCR) was undertaken on the ejaculate to detect the presence of C trachomatis DNA. A total of 31 semen specimens (4.9%) were found to be positive, and in 28 of these, the diagnosis was confirmed using the ligase chain reaction (LCR). Men whose ejaculates were PCR positive for chlamydial DNA had a significantly (P <.05) higher mean concentration of leukocytes (1.71 +/- 2.20 x 10(6) per mL) and a higher mean ejaculate volume (3.45 +/- 1.52 mL) than in those whose ejaculates were PCR negative (leukocyte concentration: 0.67 +/- 2.59 x 10(6) per mL; volume 2.93 +/- 1.38 mL). Leukocytospermia was twice as common in men that were PCR positive for chlamydial DNA (P <.05) but it was not always associated with the presence of chlamydial DNA in semen. However, there was no difference in the mean percent motility between the 2 groups and the proportion of asthenozoospermia also did not differ. Because these results do not confirm the hypothesis proposed from our in vitro experiments, further work needs to be undertaken to understand whether human spermatozoa are actually exposed to elementary bodies of C trachomatis in an infected individual prior to ejaculation.

The aim of this work was to investigate the effect of elementary bodies (EB) of Chlamydia trachomatis serovars E and LGV on sperm motility, viability and acrosomal status. Highly motile preparations of spermatozoa from normozoospermic patients were co-incubated for 6 h with 0.54x10(6) EB per ml. At 1, 3 and 6 h of incubation, sperm motility was determined by computer-assisted semen analysis (CASA) and the proportion of dead cells determined by the hypo-osmotic swelling (HOS) test. Acrosomal status was also examined using a standard monoclonal antibody assay. In the absence of EB, the percentage of motile spermatozoa remained >69% over the 6h incubation and the proportion of dead spermatozoa at <12%. However, during the incubation with EB of serovar E there was a significant decline in the percentage of motile spermatozoa (P < 0.05), and a corresponding increase in the proportion of dead spermatozoa (P < 0.05) at all time-points. However, following incubation with serovar LGV, only the percentage of dead spermatozoa after 6 h incubation was significantly different from the control (P < 0.05). The amount of acrosome-reacted spermatozoa remained unchanged (<16%) in all incubations at all time-points. Dose-response experiments indicated that increasing the concentration of EB to 2.5x10(6) per ml did not significantly alter the results. Furthermore, co-incubation of spermatozoa with dead EB (killed by heat treatment) abolished the chlamydia-mediated response, indicating that the effect is a result of the live organism and not soluble components or membrane elements. These data suggest that a detrimental effect on sperm function by some serovars may be an as yet unrecognized component of infertility problems.

Elementary bodies (EBs) of Chlamydia trachomatis serovar E are more toxic to sperm than those from serovar LGV. In this study, lipopolysaccharide (LPS) was prepared from the EBs of both serovars and incubated with human spermatozoa at concentrations that matched the LPS concentration of EBs. The effects of EBs and LPS on sperm motility, viability and acrosomal status were then determined. Sperm motility was measured by computer-assisted sperm analysis and the hypo-osmotic swelling test was used to determine the proportion of dead cells. Acrosomal status was examined using a standard mAb assay. Over a 6 h incubation, LPS from both serovars resulted in a marked reduction in sperm motility (and a concomitant increase in the proportion of dead spermatozoa) in a manner similar to that seen in response to EBs of serovar E. In addition, when sperm were incubated with a range of doses of EBs and LPS, probit analysis revealed that the greater spermicidal effects of EBs from serovar E (when compared with serovar LGV) were not observed when sperm were incubated with LPS from the two serovars. This suggests that the more potent effect of EBs of serovar E cannot be explained entirely by differences in the composition of LPS. Interestingly, Escherichia coli LPS was required in doses 500 times more concentrated than chlamydial LPS in order to kill a similar proportion of sperm, suggesting that bacterial LPSs may differ in their spermicidal properties. However, that chlamydial LPS was spermicidal was demonstrated by the use of polymyxin B (a polycationic antibiotic known to neutralize LPS effects), confirming that the effects observed were primarily a result of LPS activity.