M. STOJANOV¹, J. GYGER¹, V. CASTELLA², N. VULLIEMOZ³, D. BAUD<sup>1

1</sup>Materno-fetal and Obstetrics Research Unit, Department “Femme-Mère-enfant”, Maternity, University Hospital, Lausanne, Switzerland, ²University Center of Legal Medicine, University Hospital, Lausanne, Switzerland, ³Reproductive Medicine Unit, Department “Femme-Mère-enfant”, Maternity, University Hospital, Lausanne, Switzerland

Introduction: Intracellular bacteria, which fail to grow on media used routinely to isolate human pathogens, could represent yet unrecognized agents of infertility and miscarriages. Similarly to Chlamydia spp., Waddlia chondrophila is an emerging intracellular pathogen that has been associated to adverse pregnancy outcomes both in humans and animals. Our recent observations indicate that seroprevalence of anti-Waddlia antibodies is higher in male patients from couples with infertility problems, suggesting that this bacterium might play impact reproduction in humans.

Material and methods: We established an in vitro model of infection of human spermatozoa by Waddlia. Controls included filtrate of cell culture used to grow Waddlia (mock) and latex beads (0.6 μm diameter). Attachment to spermatozoa and internalization of Waddlia was monitored by confocal microscopy using a specific antibody. Viability of spermatozoa was assessed with flow cytometry using the LIVE/DEAD Sperm Viability Kit. The amount of bacteria was monitored with a specific quantitative RT-PCR.

Results: Using confocal microscopy we observed that, despite multiple washing steps, Waddlia was able to attach to spermatozoa. However, internalization and replication of bacteria were not observed. Presence of Waddlia decreased significantly viability of spermatozoa, with almost a 2-fold increase compared to control at 72 h post-infection (32% versus 18%). Decrease of viability was not observed for spermatozoa incubated with mock or latex beads.

Conclusions: We showed in this study that Waddlia had a negative impact on spermatozoa viability. Bacteria were observed in close contact with spermatozoa membrane, suggesting the presence of specific interactions. Bacterial replication was not observed. Future analysis will focus on the impact of Waddlia on spermatozoa motility, a key parameter for the evaluation of sperm quality. Moreover, specificity of the Waddlia-spermatozoa interaction will be analysed at the molecular level, with the goal to determine specific receptors involved in the attachment process. Taken together, our data suggest that Waddlia might be associated with fertility impairment in men.