Haematopoietic innate interleukin 17A production drives immunopathology in female mouse genital Chlamydia muridarum infection.

Chlamydia trachomatis infection is the leading cause of bacterial urogenital infection and has been demonstrated to drive inflammation and scarring of the reproductive tract. Recent studies have identified key triggers of proinflammatory adaptive immune responses driven by innate leukocytes and epithelia driving immunopathology. Utilizing chimeric mouse models, we investigated the definitive source and role of IL17 and IL17 signalling receptors during early Chlamydia muridarum infection of the female urogenital tract. Bone marrow transplants from wild-type (WT) and IL17A-/- mice to recipients demonstrated equivocal infection kinetics in the reproductive tract, but interestingly, adoptive transfer of IL17A-/- immune cells to WT recipients resulted in no infertility, suggesting a haematopoietic (as opposed to tissue) source of IL17 driving immunopathology. To further delineate the role of IL17 in immunopathology, we infected WT and IL17 receptor A (IL17RA)-/- female mice and observed a significant reduction in immunopathology in IL17RA-/- mice. WT bone marrow transplants to IL17RA-/- recipient mice prevented hydrosalpinx, suggesting signalling through IL17RA drives immunopathology. Furthermore, early chemical inhibition of IL17 signalling significantly reduced hydrosalpinx, suggesting IL17 acts as an innate driver of disease. Early during the infection, IL17 was produced by γδ T cells in the cervico-vagina, but more importantly, by neutrophils at the site of infertility in the oviducts. Taken together, these data suggest innate production of IL17 by haematopoietic leukocytes drives immunopathology in the epithelia during early C. muridarum infection of the female reproductive tract.

IgG exacerbates genital chlamydial pathology in females by enhancing pathogenic CD8+ T cell responses.

Chlamydia trachomatis infections are an important sexually transmitted infection that can lead to inflammation, scarring and hydrosalpinx/infertility. However, infections are commonly clinically asymptomatic and do not receive treatment. The underlying cause of asymptomatic immunopathology remains unknown. Here, we demonstrate that IgG produced during male infection enhanced the incidence of immunopathology and infertility in females. Human endocervical cells expressing the neonatal Fc Receptor (FcRn) increased translocation of human IgG-opsonized C. trachomatis. Using total IgG purified from infected male mice, we opsonized C. muridarum and then infected female mice, mimicking sexual transmission. Following infection, IgG-opsonized Chlamydia was found to transcytose the epithelial barrier in the uterus, where it was phagocytosed by antigen-presenting cells (APCs) and trafficked to the draining lymph nodes. APCs then expanded both CD4+ and CD8+ T cell populations and caused significantly more infertility in female mice infected with non-opsonized Chlamydia. Enhanced phagocytosis of IgG-opsonized Chlamydia significantly increased pro-inflammatory signalling and T cell proliferation. As IgG is transcytosed by FcRn, we utilized FcRn-/- mice and observed that shedding kinetics of Chlamydia were only affected in FcRn-/- mice infected with IgG-opsonized Chlamydia. Depletion of CD8+ T cells in FcRn-/- mice lead to a significant reduction in the incidence of infertility. Taken together, these data demonstrate that IgG seroconversion during male infection can amplify female immunopathology, dependent on FcRn transcytosis, APC differentiation and enhanced CD8 T cell responses.

Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

AIMS: To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). METHODS AND RESULTS: The WD-NECs from healthy adult donors (N = 6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. CONCLUSIONS: These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

Enhanced clearance of C. muridarum infection using azithromycin-loaded liposomes.

Chlamydia trachomatis is an intracellular bacterium which infects around 129 million people annually. Despite similar infection rates between sexes, most research investigating the effects of chlamydial infection on fertility has focused on females. There is now emerging evidence of a potential link between Chlamydia and impaired male fertility. The only treatments for chlamydial infection are antibiotics, with azithromycin (AZI) being one of the commonly used drugs. However, recent studies have suggested that optimizing the treatment regime is necessary, as higher concentrations of AZI may be required to effectively clear the infection in certain cell types, particularly testicular macrophages. To address this challenge, we have prepared liposomes consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) loaded with AZI for clearing Chlamydia. These liposomes exhibited stability over time and were readily taken up by both macrophages and epithelial cells. Moreover, they demonstrated significant enhancement of chlamydial clearance in both cell types. In a mouse model, the drug-loaded liposomes cleared Chlamydia within the penile urethra more efficiently than the same dose of unencapsulated drug. Furthermore, the liposome-drug treatment showed significant protective effects on sperm motility and morphology, suggesting potential benefits in reducing sperm damage caused by the infection.

Intolerance of Uncertainty, Negative Reinforcement Alcohol Use Motives, and Hazardous Drinking in College Students with Clinically Elevated Worry.

Background: Hazardous drinking has remained high for college students in recent years, and individuals who endorse drinking to cope with emotional distress or to conform socially report higher levels of alcohol use. Intolerance of uncertainty, a core process underlying generalized anxiety disorder, has been linked to negative reinforcement drinking motives; however, no research, to date, has examined the role of intolerance of uncertainty in terms of alcohol use motives and hazardous drinking among individuals with generalized anxiety disorder. Objective: The current study was designed to examine the relationships between intolerance of uncertainty, coping and conformity alcohol use motives, and hazardous drinking in an analogue generalized anxiety disorder sample. Methods: Participants were 323 college students (Mage = 19.25, SD = 2.23, Range = 18-40) who endorsed past-year alcohol use and clinically elevated levels of worry. Self-report measures were completed online for course credit. Results: Partially consistent with our hypotheses, uncertainty paralysis predicted greater levels of coping motives, but not conformity motives. Desire for predictability did not predict either drinking motive. Mediation analyses revealed that there was a significant indirect effect of uncertainty paralysis on more hazardous drinking through greater coping motives. Conclusion: Overall, these findings highlight the potential utility of targeting behavioral inhibition due to uncertainty to reduce unhealthy coping via alcohol use and subsequent hazardous alcohol use.

Characterisation of the koala (Phascolarctos cinereus) pouch microbiota in a captive population reveals a dysbiotic compositional profile associated with neonatal mortality.

BACKGROUND: Captive koala breeding programmes are essential for long-term species management. However, breeding efficacy is frequently impacted by high neonatal mortality rates in otherwise healthy females. Loss of pouch young typically occurs during early lactation without prior complications during parturition and is often attributed to bacterial infection. While these infections are thought to originate from the maternal pouch, little is known about the microbial composition of koala pouches. As such, we characterised the koala pouch microbiome across the reproductive cycle and identified bacteria associated with mortality in a cohort of 39 captive animals housed at two facilities. RESULTS: Using 16S rRNA gene amplicon sequencing, we observed significant changes in pouch bacterial composition and diversity between reproductive time points, with the lowest diversity observed following parturition (Shannon entropy - 2.46). Of the 39 koalas initially sampled, 17 were successfully bred, after which seven animals lost pouch young (overall mortality rate - 41.18%). Compared to successful breeder pouches, which were largely dominated by Muribaculaceae (phylum - Bacteroidetes), unsuccessful breeder pouches exhibited persistent Enterobacteriaceae (phylum - Proteobacteria) dominance from early lactation until mortality occurred. We identified two species, Pluralibacter gergoviae and Klebsiella pneumoniae, which were associated with poor reproductive outcomes. In vitro antibiotic susceptibility testing identified resistance in both isolates to several antibiotics commonly used in koalas, with the former being multidrug resistant. CONCLUSIONS: This study represents the first cultivation-independent characterisation of the koala pouch microbiota, and the first such investigation in marsupials associated with reproductive outcomes. Overall, our findings provide evidence that overgrowth of pathogenic organisms in the pouch during early development is associated with neonatal mortality in captive koalas. Our identification of previously unreported, multidrug resistant P. gergoviae strains linked to mortality also underscores the need for improved screening and monitoring procedures aimed at minimising neonatal mortality in future. Video Abstract.

Pathogenic NKT cells attenuate urogenital chlamydial clearance and enhance infertility.

Urogenital chlamydial infections continue to increase with over 127 million people affected annually, causing significant economic and public health pressures. While the role of traditional MHCI and II peptide presentation is well defined in chlamydial infections, the role of lipid antigens in immunity remains unclear. Natural killer (NK) T cells are important effector cells that recognize and respond to lipid antigens during infections. Chlamydial infection of antigen-presenting cells facilitates presentation of lipid on the MHCI-like protein, CD1d, which stimulates NKT cells to respond. During urogenital chlamydial infection, wild-type (WT) female mice had significantly greater chlamydial burden than CD1d-/- (NKT-deficient) mice, and had significantly greater incidence and severity of immunopathology in both primary and secondary infections. WT mice had similar vaginal lymphocytic infiltrate, but 59% more oviduct occlusion compared to CD1d-/- mice. Transcriptional array analysis of oviducts day 6 post-infection revealed WT mice had elevated levels of Ifnγ (6-fold), Tnfα (38-fold), Il6 (2.5-fold), Il1ß (3-fold) and Il17a (6-fold) mRNA compared to CD1d-/- mice. In infected females, oviduct tissues had an elevated infiltration of CD4+ -invariant NKT (iNKT) cells, however, iNKT-deficient Jα18-/- mice had no significant differences in hydrosalpinx severity or incidence compared to WT controls. Lipid mass spectrometry of surface-cleaved CD1d in infected macrophages revealed an enhancement of presented lipids and cellular sequestration of sphingomyelin. Taken together, these data suggest an immunopathogenic role for non-invariant NKT cells in urogenital chlamydial infections, facilitated by lipid presentation via CD1d via infected antigen-presenting cells.

Prophylactic and therapeutic vaccination protects sperm health from Chlamydia muridarum-induced abnormalities.

Chlamydia is the most common bacterial sexually transmitted infection worldwide and it is widely acknowledged that controlling the rampant community transmission of this infection requires vaccine development. In this study, for the first time, we elucidate the long-term response to male mouse chlamydial vaccination with chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) both prophylactically and in a novel therapeutic setting. Vaccination significantly reduced and, in some cases, cleared chlamydial burden from the prostates, epididymides, and testes, which correlates with high IgG and IgA tires in tissues and serum. Important markers of sperm health and fertility were protected including sperm motility and proteins associated with fertility in men. Within splenocytes, expression of IFNγ, TNFα, IL17, IL13, IL10, and TGFß were changed by both infection and vaccination within CD4 and CD8 T cells and regulatory T cells. Within the testicular tissue, phenotypic and concentration changes were observed in macrophages and T cells (resident and transitory). This revealed some pathogenic phenotypes associated with infection and critically that vaccination allows maintenance of testicular homeostasis, likely by preventing significant influx of CD4 T cells and promoting IL10 production. Finally, we demonstrated the testes contained immature (B220+) B cells and mature (CD138+) Chlamydia-specific plasma cells. Thus, through vaccination, we can maintain the healthy function of the testes, which is vital to protection of male fertility.

Central and peripheral nervous system responses to chronic and paced hyperventilation in anxious and healthy subjects.

The aim of the present study was to examine self-report, peripheral nervous system, and central nervous system correlates of naturally-occurring, chronic hyperventilation (HV, assessed by hypocapnia or low resting state low end-tidal CO2), and to examine the additional effect of acute, experimentally-induced HV in anxious and healthy participants. By identifying the biomarkers of anxiety-related chronic HV and examining responses to acute HV, we hope to identify meaningful, mechanistic targets for further treatment development. Seventy anxious patients and 34 healthy control participants completed electroencephalogram (EEG) and peripheral nervous system recording at baseline and following a paced breathing task. Diagnosis x baseline hypnocapnia group analyses indicated that anxious/hypocapnic patients exhibited greater nonspecific skin conductance response amplitude than did anxious/normocapnic patients, and the anxious group reported greater HV-related symptoms and anxiety sensitivity than did the control group. However, no EEG abnormalities were noted as a function of anxiety group or baseline hypocapnia status. Following paced HV, anxious patients (but not controls) exhibited an increase in left-frontal alpha 1 power. Hypocapnic, but not normocapnic, participants exhibited an increase in skin conductance levels. Anxious patients reported an increase in negative cognitive appraisals of HV symptoms, and anxious/hypocapnic participants reported an increase in affective responses to HV. Thus, chronic HV is associated with greater arousal, and increased self-reported and physiological sensitivity to paced HV. Patients who chronically hyperventilate appear to be more sensitive to respiratory distress, responding with higher levels of anxiety and poorer tolerance of the physiological sensations accompanying acute HV.

Primary oral vaccination followed by a vaginal pull protects mice against genital HSV-2 infection.

PROBLEM: HSV-2 infected more than 491 million people aged 15-49 world-wide in 2016. The morbidity associated with recurrent infections and the increased risk of HIV infection make this a major health problem. To date there is no effective vaccine. Because HSV-2 ascends to the dorsal route ganglion within 12-18 h of infection, an effective vaccine will need to elicit a strong local resident CD8+ T cell response to prevent the infection from becoming life-long. METHOD OF STUDY: Using a mouse model we investigated the potential of oral immunization with a novel lipid adjuvant (LiporaleTM ) followed by local vaginal application of an inflammatory agents to protect against primary HSV-2 infections. RESULTS: Oral vaccination of mice with live-attenuated HSV-2 in Liporale followed by vaginal application of DNFB or CXCL9/10 led to recruitment of tissue-resident CD8+ memory cells into the genital epithelia. This prime and pull vaccination strategy provided complete protection against wild-type HSV-2 challenge and prevented viral dissemination to the spinal cords. CONCLUSIONS: Activation of mucosal immunity by oral immunization, combined with induction of transient local genital inflammation can recruit long-lived tissue resident CD8+ T cells into the genital epithelium, providing significant protection against primary HSV-2 infection.

Brief adjunctive mindfulness-based cognitive therapy via Telehealth for anxiety during the COVID-19 pandemic.

BACKGROUND AND OBJECTIVES: While cognitive-behavioral therapy is a highly efficacious treatment for anxiety, additional research is needed to identify adjunctive interventions that may augment treatment outcome. DESIGN: In response to the COVID-19 pandemic, we conducted an open feasibility trial of brief (i.e., four 75- to 90-minute sessions) mindfulness-based cognitive therapy (MBCT) for anxiety via telehealth for patients (N = 23) receiving individual CBT at an outpatient specialty clinic. METHODS: Self-report measures of home practice compliance (weekly), intervention acceptability (post-intervention), mindfulness and self-compassion, anxiety and depressive symptoms, and transdiagnostic processes (pre- and post-intervention) were administered as part of routine clinical practice. RESULTS: Results indicated good retention and attendance rates, few technical difficulties, good home practice compliance, and high levels of perceived importance. Participants indicated that they would highly recommend the group to others and also recommended extending the group beyond four sessions. There were significant improvements in mindfulness and self-compassion and reductions in intolerance of uncertainty, anxiety sensitivity, distress intolerance, emotion dysregulation, and anxiety symptoms from pre- to post-intervention. CONCLUSIONS: Brief MBCT as an adjunctive treatment for anxiety via telehealth is feasible and acceptable, and shows promise in terms of engaging treatment targets and transdiagnostic processes and reducing anxiety symptoms.

Implementation of a structured practical activity to analyse student healthcare worker perceptions and compliance with prescribed infection control procedures.

BACKGROUND: Non-compliance with infection control guidelines has been reported within healthcare settings. Infection control education in undergraduate healthcare education programs forms a critical component in preparing student healthcare workers for vocational roles. METHODS: Clinical sciences students (nutrition science, paramedicine, pharmacy, podiatry, optometry studying for qualifications recognised by the Australian Health Practitioner Regulation Agency) self-reported hygiene perceptions and practices and collected microbiological swabs from personal or medical equipment items before and after recommended disinfection procedures. RESULTS: Cultivable microorganisms were isolated from 95% of student medical equipment items. Disinfection significantly reduced microbial growth on student medical equipment items (P < 0.05). CONCLUSIONS: Student perceptions of infection control procedures do not always correlate with infection control practice. Infection control education of undergraduate healthcare students requires ongoing assessment to ensure successful translation into clinical practice.

A randomized controlled trial evaluating the efficacy of a brief computerized anxiety sensitivity reduction intervention for health anxiety.

It is estimated that individuals with severe health anxiety (HA) utilize 41 %-78 % more healthcare resources than individuals with identified medical diagnoses. Thus, identifying targets for intervention and prevention efforts for HA that are appropriate for primary care or specialty clinic settings is imperative. The aim of the present investigation was to evaluate the effect of a single-session, computerized anxiety sensitivity (AS) intervention on AS and HA. Participants were 68 university students (79.4 % female; Mage = 19.68) with elevated levels of AS and HA. Participants were randomized to either the AS intervention condition or an active control condition and completed self-report and behavioral follow-up assessments at post-intervention, 1-week follow-up, and 1-month follow-up. Results indicated a significant Time x Condition interaction for ASI-3 at each follow-up assessment (all ps < .001), such that individuals in the active condition exhibited greater reductions in AS compared to the control condition. There was no significant Time x Condition interaction for HA at any follow-up. Mediation analyses revealed a significant indirect effect of Condition on changes in HA through changes in AS. No significant effects were observed for behavioral outcomes. Findings suggest that this intervention successfully reduces AS among those who are high in HA and AS and may indirectly contribute to reductions in HA over time through reductions in AS.

Assessment of intolerance of uncertainty: Validation of a modified anagram task.

BACKGROUND AND OBJECTIVES: Intolerance of uncertainty (IU) is an individual difference factor that reflects difficulty tolerating emotional distress in the context of uncertainty and is a robust transdiagnostic risk factor for emotional disorders. A limitation of prior research on IU is the heavy reliance on self-report measures to assess this construct. The aim of the present study was to examine the validity of a brief, computerized anagram task modified to assess IU (the PACT Anagram Task; PAT). METHODS: Participants were 221 unselected undergraduate students (71% female; Mage = 19; 89% Caucasian) who completed the PAT and a series of self-report measures of convergent, discriminant, and incremental validity, and measures of anxiety and depression symptoms online for course credit. RESULTS: Greater PAT-Distress was positively correlated measures of convergent validity and demonstrated incremental validity in relation to self-reported IU above and beyond attentional control. None of the PAT indicators were associated with compassion or empathic concern, evidencing discriminant validity. PAT-Distress and PAT-Answers were each associated with greater worry above and beyond self-reported IU. LIMITATIONS: Study limitations include the cross-sectional design and lack of diversity in terms of sample demographics. Furthermore, we did not examine convergence between this task and other behavioral measures of IU. CONCLUSION: Subjective distress following completion of the PAT may serve as one indicator of IU. Further research is needed in order to replicate these results and validate the use of the PAT in clinical samples.

A meta-analysis of relapse rates in cognitive-behavioral therapy for anxiety disorders.

Cognitive-behavioral therapy (CBT) is a first-line treatment for anxiety and related disorders, with large pre- to post-treatment effect sizes. Rates of relapse, or the likelihood that a state of remission will be maintained once treatment is withdrawn, have been relatively neglected in CBT outcome studies. The present meta-analysis aimed to determine the overall rate of relapse in CBT for anxiety and related disorders. A secondary aim was to assess whether demographic, clinical, and methodological factors were associated with rates of relapse in CBT. Articles were identified from prior CBT meta-analyses and review papers and from literature searches using the PsycINFO and Medline electronic databases, with 17 full-length articles retained for meta-analysis (total N = 337 patients). Results showed an overall relapse rate of 14 %, which did not significantly differ between diagnoses. The way in which relapse was defined was significantly associated with relapse rates; when relapse was defined as meeting diagnostic criteria, estimates were lower than when alternative definitions were used. The findings indicate that relapse following symptom remission occurs in a minority of patients, suggesting that future treatment development and refinement efforts should focus on improving relapse prevention skills and interventions to minimize risk of relapse.

DNA damage contributes to transcriptional and immunological dysregulation of testicular cells during Chlamydia infection.

Chlamydia is the most commonly reported sexually transmitted bacterial infection, with 127 million notifications worldwide each year. Both males and females are susceptible to the pathological impacts on fertility that Chlamydia infections can induce. However, male chlamydial infections, particularly within the upper reproductive tract, including the testis, are not well characterized. In this study, using mouse testicular cell lines, we examined the impact of infection on testicular cell lineage transcriptomes and potential mechanisms for this impact. The somatic cell lineages exhibited significantly fragmented genomes during infection. Likely resulting from this, each of the Leydig, Sertoli and germ cell lineages experienced extensive transcriptional dysregulation, leading to significant changes in cellular biological pathways, including interferon and germ-Sertoli cell signalling. The cell lineages, as well as isolated spermatozoa from infected mice, also contained globally hypomethylated DNA. Cumulatively, the DNA damage and epigenetic-mediated transcriptional dysregulation observed within testicular cells during chlamydial infection could result in the production of spermatozoa with abnormal epigenomes, resulting in previously observed subfertility in infected animals and congenital defects in their offspring.

Testicular inflammation and infertility: Could chlamydial infections be contributing?

Despite the global incidence of both male infertility and sexually transmitted infections rising each year, the relationship between the two is relatively unstudied. Chlamydia is the most common bacterial sexually transmitted pathogen; however, the majority of research remains focussed on women, while the role of infection and resulting immunopathology in male factor infertility is largely unknown. Chlamydia was found in testicular biopsies from asymptomatic men with idiopathic infertility, which highlights this potential role. In animal models, testicular Chlamydia, and potentially other bacterial and viral infections, cause histopathology that is likely to adversely affect spermatogenesis and fertility. This likely occurs through infiltration of inflammatory cells, functional dysregulation of immunosuppressive testicular macrophages and Sertoli cells and destruction of key testicular cell types including sperm progenitors. Here, testicular damage due to infection and/or inflammation is reviewed, as it represents a probable underestimated and unrecognized factor leading to male infertility.

Chronic testicular Chlamydia muridarum infection impairs mouse fertility and offspring development†.

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality.

Detection of chlamydia infection within human testicular biopsies.

STUDY QUESTION: Can Chlamydia be found in the testes of infertile men? SUMMARY ANSWER: Chlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men. WHAT IS KNOWN ALREADY: Male chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility. STUDY DESIGN, SIZE, DURATION: We collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling. PARTICIPANTS/MATERIALS, SETTING, METHODS: The diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques. MAIN RESULTS AND THE ROLE OF CHANCE: Chlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia. LIMITATIONS, REASONS FOR CAUTION: No reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples. WIDER IMPLICATIONS FOR THE FINDINGS: Application of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure. STUDY FUNDING/COMPETING INTEREST(S): Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.