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A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4. Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-γ mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9. For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.

. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4 .
Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell-culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5 . Further limitations are given by the fact that central signaling cascades (e.g. IFN-γ mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7 . We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9 . For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.

Preparation of Human Fallopian Tube
1. Store human fallopian tubes (HFT) from women undergoing hysterectomy immediately after surgery in RPMI containing 5% FCS without antibiotics in 4 °C until preparation. The protocol was approved by the ethical committee of the University of Lübeck (09-153). Women included in the study (age 34 until 53) had no history of prior C. trachomatis infection. Tissue of the Fallopian tubes was collected at peripartum sterilisation on maternal request during caesarean sections or during hysterectomies due to symptomatic uterine fibroids in the luteal phase. All HFT were tested negative for C. trachomatis by PCR. 2. Dissect the HFT in a Petri dish containing RPMI with 5% FCS without antibiotics. During the whole processing the tissue should be submerged in media to prevent drying. 3. Cut off and discard connective tissue and tissue destroyed during surgery. 4. After dissection open the HFT carefully with a small scalpel. 5. For further experiments prepare pieces of tissue in the size of 0.5 cm x 0.5 cm up to 1 cm x 1 cm. 6. For infection store HFT specimen in 1 ml RPMI containing 5% FCS without antibiotics. HFT specimens were infected with 5.5x10 5 IFU

Discussion
Visualization of pathogen-induced harm to the infected tissue is arduous and often limited to experimental procedures in mice. We established an ex vivo infection model in human fallopian tubes to analyze C. trachomatis infections of the upper female genital tract. By the use of this method, we were able to visualize C. trachomatis-induced damage to human fallopian tube epithelium within one day post infection. Cellular swelling and lysis were typical morphological changes that were observed in C. trachomatis-infected fallopian tubes but not in non-infected controls. TEM analyses revealed that chlamydiae implement a typical intracellular developmental cycle within infected tissues, showing large inclusions with re-differentiated elementary bodies but also smaller inclusions with enlarged reticulate bodies that might indicate persistence. However, the morphological picture alone does not suffice to discriminate between replicating and persistent infections, which are characterized by reduced metabolism and increased resistance to antimicrobials 10 .
Destruction of the epithelium in infected fallopian tubes is either due to disruption of C. trachomatis-infected epithelial cells after completion of the intracellular developmental cycle and release of infectious elementary bodies or the release of pro-inflammatory cytokines 8 . Pathogeninduced tissue damage and host-induced immune reactions against C. trachomatis are supposed to be the major factors leading to loss of epithelial cell function, fibroblast remodeling and finally tubal infertility in vivo 8,11 . One of the major limitations of this model is the absence of inflammatory cells which hampers the analysis of secondary effects to the initial C. trachomatis infection of the fallopian tube epithelium. However, the model is appropriate to investigate different environmental conditions (e.g. oxygen content) and the initial host-immune response in acute C. trachomatis infections 8,9 . Further plans are to establish a model for testing of antimicrobials against C. trachomatis in whole human fallopian tubes and to characterize the metabolic states of the different developmental stages observed in the tubal epithelium by 2-photon laser scanning microscopy.

Disclosures
No conflicts of interest declared.