1
Human and pathogen factors associated with Chlamydia
2
trachomatis-related infertility in women
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4
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Menon S. 1, Timms P. 1,2, Allan J. A.3,4, Alexander K.1, Rombauts L.5, Horner P.67, Keltz
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M.8,9, Hocking J.10, Huston W. M.1
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1. Institute of Health and Biomedical Innovation, 60 Musk Ave, Q Block, Queensland
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University of Technology, Kelvin Grove, QLD, 4059, Australia.
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2. Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast,
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Maroochydore, QLD, 4558, Australia.
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3. Wesley and St Andrews Research Institute, The Wesley Hospital, Auchenflower, QLD,
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4066, Australia.
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4. UC Health Clinical School, The Wesley Hospital, Auchenflower, QLD, 4066.
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5. MIMR-PH Institute of Medical Research, Monash, VIC, Australia, Australia
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6. School of Social and Community Medicine, University of Bristol, Bristol, UK
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7. Bristol Sexual Health Centre, University Hospital Bristol NHS Foundation Trust, Bristol,
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UK
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8. WESTMED Reproductive Services, New York, USA
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9. Icahn School of Medicine at Mt Sinai Hospital, New York, USA
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10. Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global
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Health, University of Melbourne, Carlton South, VIC, Australia
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23
Correspondence: Wilhelmina (Willa) Huston, Institute of Health and Biomedical
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Innovation, School of Biomedical Sciences, Queensland University of Technology, 60 Musk
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Ave, Kelvin Grove, QLD, 4059, Brisbane, Australia. Tel: +61 7 31386258; email:
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[email protected]
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Table of contents
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Introduction
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The evidence for the association of Chlamydia with infertility
31
Proposed models for the development of infertility following …..
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C. trachomatis infection in women
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33
Ascension
12
34
Persistence
13
35
Cellular paradigm
15
36
cHSP60 induced delayed hypersensitivity
16
37
Host/human factors associated with chlamydial infertility in women
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38
Repeat infection and infertility
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39
Do co-infections or the microbiome play a role in chlamydial pathology?
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40
Sexual behaviour and age of sexual contact
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41
Hormonal status at time of infection
21
42
Human genotypic factors associated with C. trachomatis infertility in women
22
43
Immune responses associated with Chlamydia trachomatis in women
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Immune responses from reproductive sites and tissues
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Immune responses in peripheral blood mononuclear cells (PBMC) in women
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3
6
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Immune pathway elucidated by in vitro culture models
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47
Conclusions
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48
Chlamydial factors that may influence the development of pathology
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49
Chlamydial serovar can associate with repeat infection and symptoms that….
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may link to a propensity to cause pathology and infertility
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Chlamydial genotypes can mediate tissue tropism and may be a factor …..
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in pathology development
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53
Conclusions
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54
References
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Authors Bios
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56
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4
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Introduction
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Chlamydia (C.) trachomatis is one of the most common bacterial sexually transmitted
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infections (STI) worldwide, with WHO’s most recent estimates indicating approximately 100
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million new infections annually (1), and in the United States of America alone,
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approximately 2.86 million Chlamydia infections occurred in 2008 (2). The infection can
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result in serious reproductive consequences, including pelvic inflammatory diseases (PID),
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ectopic pregnancy, and infertility in women. Infertilty is defined as the inability to have a
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healthly live baby after 12 months of unprotected timed intercourse. The majority of studies
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reviewed for this document state their definition of infertility as inability to achieve
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successful pregnancy as greater than 12 months. C. trachomatis is a Gram negative, obligate
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intracellular bacterial pathogen with a unique biphasic developmental cycle (3). The
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organism has been characterised using biology and genomics as a highly evolved or ancient
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pathogen with evidence of a reduced genome that is tailored for the obligate intracellular
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human niche (4). This unique intracellular niche has meant that until recent times, the
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organism has not been investigated using molecular microbial methods, however, animal
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models have been used to demonstrate Koch’s postulates in relation to infection and
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reproductive pathology (with all the hallmarks of pathology including presence of Th1-like
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cytokines, fibrosis, mononuclear infiltrates and lymphoid follicle formation), with more
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extensive pathology reported after repeat infections (recently reviewed, (5)). Apari et al., (6)
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recently proposed the appealing but somewhat controversial and untested hypothesis that the
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ability to cause infertility may actually be a specialised and evolved trait of STI pathogens
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(such as Chlamydia and Neisseria). This infertility was proposed to result in increased sexual
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promiscuity of the host in the seeking of fecundity, thus enabling enhanced transmission and
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positive selection of those strains of the pathogen that cause infertility (6).
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5
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In spite of the considerable worldwide burden of chlamydial disease it is still not known what
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the host and pathogen determinants are that result in infertility. We have chosen to
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specifically focus on infertility in women. WHY do review now wh insert We have used
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PUBMED searches and reading of the relevant literature to find and review the evidence
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(focussing on direct human evidence) and to identify the gaps in our knowledge that may be
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addressed in future to further advance our understanding of the risk factors that could predict
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or determine infertility outcomes in women.
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The evidence for the association of Chlamydia with infertility
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Genital Chlamydia infection is asymptomatic in over 70% of cases and as a result there are
95
few population-based prevalence or incidence estimates available (7). Available population-
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based data from the USA, Australia, and UK suggest that between 3 and 5% of under 30
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years of age will have Chlamydia at any point in time (8-12). Estimates of the incidence of
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disease sequelae (specifically pelvic inflammatory disease, ectopic pregnancy and infertility
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in women) are lacking, largely because there are very few natural history studies of
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Chlamydia infections in humans. Although a continuous-time Markov model analysing
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existing evidence from randomised control trials and observational studies estimated the
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probability of PID from a chlamydial infection was 0.16 (95% credible interval: 0.06-0.25)
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(13). These studies that have been conducted are typically limited to the relatively short time
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frame between diagnosis and treatment, which may not capture the true occurrence of
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pathology and reproductive consequences (14, 15). Generally, there are two main ways in
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which clinicians and scientists characterise or associate chlamydial infection with infertility
107
or other infection sequelae. The first is based on longitudinal information that includes
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infection (cervical), upper reproductive tract symptoms resulting in pelvic inflammatory
6
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disease (typically symptomatic and detected) and subsequent infertility (specifically tubal
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infertility) (14, 15). The second is a retrospective approach based on women presenting with
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tubal damage or unexplained infertility combined with evidence of chlamydial infection
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history either by medical history review for previous diagnosed infection, history of PID, or
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serological assessment to indicate previous infection history (16). Current infection with
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Chlamydia also prevents conception, although this is also the case in males which may
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confound this data (17). However, this infertility may be a consequence of local response to
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the active infection and not necessarily indicate permanent reproductive damage; therefore
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this review will not cover this form of infertility.
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Infertility is defined by the world health organisation as ‘a disease of the reproductive system
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that is defined by the failure to achieve a clinical pregnancy after 12 months or more of
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regular unprotected sexual intercourse’, and this is the definition we have used for the
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purpose of this review. Some studies refer to sub-fertility which is defined as reduced fertility
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with a prolonged time of unwanted non-conception and we consider this compatible with the
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definition of infertility for the purpose of this review. Pelvic inflammatory disease (PID) and
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ectopic pregnancy are also reviewed here, with ectopic pregnancy defined as tubal
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development of the embryo. Chlamydial infertility typically is detected some years after the
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infection and so the gold standard NAATs used for active infection diagnosis are not useful
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in these cases.
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A systematic review attempting to establish the attributable risk of infertility among women
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following a genital Chlamydia infection concluded that there is currently not sufficient
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longitudinal evidence to accurately determine this risk (18). However, there is robust
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evidence that women who have experienced PID are more likely to develop infertility, and
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robust evidence for Chlamydia causing PID. One large longitudinal cohort study was
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conducted by Westrom et al. (19) to evaluate PID and fertility outcomes. The study followed
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1844 women with abnormal laparoscopic findings upon suspicion of acute PID (cases) (not
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limited to Chlamydia) and 657 women who had normal findings (controls). Follow up
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identified that upon attempting to fall pregnant 16.5% of the cases and 2.7% of the controls
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failing to conceive (19). Further investigations demonstrated that 141 (10.8%) of the PID
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cases and none of the controls had developed tubal factor infertility (19). In addition, the
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ectopic pregnancy rate was also higher in the PID cases, with 9.1% ectopic pregnancy in
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cases and 1.4% in the controls (19). However, this study did not detail the infectious agent
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underling the initial PID.
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A review of studies characterising the risk of sequelae direct from chlamydial infection by
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Haggerty et al. (2010) reported that PID rates after untreated chlamydial infection (typically
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in the short time between diagnosis and treatment) found that in ‘high-risk’ settings 2-5% of
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women developed PID, but this may be lower in an asymptomatic population (20). However,
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in a randomised controlled trial to evaluate if chlamydial screening can prevent pelvic
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inflammatory disease (POPI trial), it was identified that the risk of PID after chlamydial
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infection in a one year time frame was 9.5% (95% CI 4.7%-18.3%) compared to 1.6%
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women who had not tested positive at baseline during the trial (21). In a population wide
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retrospective medical chart review approach in Sweden, it was identified that women who
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had a positive test for Chlamydia (n=2965) had an adjusted odds ratio of 1.27 (95% CI 1.04 -
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1.55, P<0.0001) of having had treatment for PID compared to women who tested Chlamydia
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negative (n=2853) (16). Mathematical modelling suggested that PID can occur at any time
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point during the natural history of infection, therefore annual chlamydial screening programs
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may reduce the incidence of PID (22), although a separate model concluded that it is unclear
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if there is a different risk of PID earlier or later in the infection, but that annual screening
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could prevent 61% (95% credible interval 55-67) of chlamydial associated PID (13).
8
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The use of medical record review to retrospectively identify previous diagnosed chlamydial
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genital infection in women who present with infertility is rare. However, in the same
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previously mentioned study in Sweden using a retrospective hospital record review process, it
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was identified that women who had a positive Chlamydia test had an adjusted hazards ratio of
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1.31 (95% confidence 1.09-1.57, P<0.0001) of having infertility compared to women who
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tested negative for Chlamydia, although there was no significant difference in ectopic
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pregnancy outcomes (16). Therefore, at least based on these limited studies, a relatively small
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proportion of women, who are diagnosed with the infection develop infertility.
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Much of the evidence of the association between Chlamydia and infertility comes from
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studies that have used Chlamydia serology to determine previous history of chlamydial
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infection. The studies typically find a significant association with either the presence of or
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higher titres of chlamydial antibodies in women who have tubal pathology compared to
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women who have other forms of infertility. Serology prediction of a past history of
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chlamydial infection relies on the accuracy of the test. The serological tests are
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microimmunoflourescence or ELISA (enzyme linked immunosorbant based assay) based
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tests.
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There have been attempts to evaluate the performance of these tests by comparing them on
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the same participants. Using this approach one study found that the pELISA MEDAC was the
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best of three ELISAs (compared with Savyon and Labsystems) for detection of tubal
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pathology in infertile women at a fertility treatment clinic with 55% sensitivity and 87%
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specificity (23). The same study compared two MIF tests (n=315, with 51 tubal pathology
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cases) reported that with a cut-off titre of 1/64 the Biomerieux test had 71% sensitivity and
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74% specificity, compared to 47% sensitivity and 95% specificity for the Labsystems test
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(23). A meta-analysis conducted in 2011 compared MIF and several ELISA and found that
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MIF had the highest accuracy (area under the curve) for detection of tubal pathology
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P<0.001, and P=0.01 (for bilateral occlusion) (24). However, in the absence of a longitudinal
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cohort of participant’s directly linking diagnosed active chlamydial infection with subsequent
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tubal infertility and detectable serological responses it is difficult to assess the accuracy of
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these tests.
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Regardless of test performance, numerous studies have identified a correlation of Chlamydia
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serology positivity (Chlamydia antibody testing: CAT) with diagnosed tubal factor infertility
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(typically using a case:control design of tubal infertility compared to other causes of
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infertility). These studies include the following: a significantly higher percentage of women
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with tubal pathology were CAT positive (50.2%, n=141) compared to infertile for other
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reasons (29.6%, n= 326, p<0.001) using microimmunoflourescence (MIF) (25). In a separate
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study high CAT titres using MIF correlated with presence of tubal damage n=434 compared
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to all women with infertility n=1006 (p<0.001), as did pelvic adhesions (26). Another study
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reported CAT (MIF) was 74% sensitive and 93% specific for detection of tubal disease (total
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210 patients) p<0.001 (27). In contrast, in Rwanda infertile participants that had a high
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proportion of tubal pathology (185 of 303 infertile participants), however, when CAT was
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conducted using several ELISA kits compared to fertile post-natal controls (n=312) the
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sensitivity and specificity of three different chlamydial ELISA were poor for tubal pathology,
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suggesting that Chlamydia is not a major cause of tubal pathology in this population (28).The
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MEDAC pELISA (MOMP peptides) in the Netherlands was able to predict tubal disease in
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infertile women (n=40 CAT positive, compared to n=167 CAT negative) with a sensitivity of
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0.37 (95% CI: 0.26-0.49) and specificity of 0.88 (95% CI 0.82-0.92) (29). Chlamydia heat
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shock protein 60 (cHSP60) ELISA detection of CAT was reported to detect women with
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Chlamydia-associated tubal infertility (n= 72) with a sensitivity and specificity of 81.3%, and
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97.5% respectively. However, these women were first tested by laparoscopy for tubal damage
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and for a positive result in the MIF serological assay to assign case group before testing the
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ELISA so this study has likely over-reported the test performance (30). In a subsequent study
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of a further 77 participants this group further reported that cHSP60 antibodies were 92%
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specific and 44% sensitive for tubal factor infertility (31). cHSP60 ELISA positivity was also
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found to be significantly more frequent in women with tubal factor (n=88) compared to
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controls of infertile women with other know factors for infertility or healthy blood donors
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(n=163) (43.2% vs 13.5%) in a study conducted in Helsinki (32).
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A prospective observational study at a reproductive health centre in New York used MIF
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serological prediction of previous C. trachomatis infection to compare with infertility
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diagnosis and treatment outcomes. A total of 1279 participants were screened and 70 (5.5%)
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were positive for C. trachomatis (33). Of those selected for investigation of tubal disease
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sero-positive women were significantly more likely to have hysterosalpingography detected
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tubal blockage (37.5% compared to 10.1%; P= 0.001) or laparoscopically diagnosed tubal
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damage (85.7% compared to 48.9%, P= 0.002) (33). Sero-positive women also had 57% less
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pregnancy rates than sero-negative women prior to IVF but once IVF treatment commenced
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they were equally likely to conceive by IVF as women with other causes of infertility (33). A
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mathematical model, including a number of assumptions, and adjusting for serology test
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sensitivity and specificity estimated that 45% of tubal factor infertility episodes are likely to
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be attributable to C. trachomatis (CI: 28-62%) (34). Combined, these studies provide
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substantial evidence that a past history of chlamydial infection is significantly associated with
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tubal pathology and infertility in women, regardless of serological assay performance.
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However, they do not allow us to determine how frequently this tubal infertility is an
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outcome of primary infection, and therefore the attributable risk of Chlamydia for infertility
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is not known.
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Proposed models for the development of infertility following C. trachomatis
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infection in women
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We describe what we consider to be the four models that are most commonly referred to
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within the field to explain the process that underlies the development of infertility following
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Chlamydia infection. The models are not exclusive of each other and there is evidence for
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and against each model. We will briefly summarise these models so that the host and
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pathogen data that we present later can be considered in the context of each model (Fig. 1.).
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Ascension
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This model suggests that the infection ascends to the upper reproductive tract in only some
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women, and this ascension is usually typified by the presence of symptoms, development of
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PID and if unchecked consequent development of tubal pathology. There is considerable
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direct evidence that the chlamydial infection can ascend beyond the cervix, however, the
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proportion of cases in which this ascension occurs is unknown. Evidence for ascension
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includes the following studies; Chlamydia DNA has been detected in salpingectomy
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specimens from one woman with ectopic pregnancy, and from several endometrial and cervix
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specimens from women with subsequent ectopic pregnancy although not in their ectopic
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salpingectomy specimens (35). C. trachomatis DNA was detected in tubal specimens from
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women with ectopic pregnancy in Myamar (36). C. trachomatis DNA was detected in
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endometrium, fallopian tube and ovaries of as many as 56% of women with ectopic
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pregnancy or tubal factor infertility (37). C. trachomatis organism presence was associated
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with endometritis (plasma cell influx in the endometrium) (38, 39). A small study in the UK
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identified the presence of C. trachomatis in the upper genital tract from 4 out of 10 women all
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of whom had no symptoms suggesting that is it possible to have asymptomatic ascension and
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that ascension may not occur in all women (40). This ascension model is also supported by
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substantial evidence of chlamydial inducing a pathological immune response in human cell
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and tissues (discussed later) and is mutually inclusive with the Persistence and/or Cellular
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Paradigm models. However, there is only limited evidence to support that; 1. Ascension of
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the infection occurs in some but not all women or that; 2. that lower genital tract symptoms
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and/or signs typify upper reproductive infection because mucopurulent cervicitis and other
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cervical/genital symptoms/signs have been reported in both the presence and absence of
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upper reproductive tract symptoms or with laparoscopic evidence of salpingitis (ascending
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infections) (41, 42). However, it is important to note that in the field it is generally agreed
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that upper genital tract infection does not happen in the absence of cervicitis/lower genital
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tract signs of inflammation, and these were included in the diagnostic criteria from both the
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Westrom and PEACH PID studies (19, 43).
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It seems possible or even likely that ascension may be independently moderated by numerous
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other factors that could explain the low frequency of pathology development, supporting that
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it only occurs in some women. These moderating factors could include host factors such as
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cycle stage or hormone status at time of infection, as chlamydial or gonococcal endometritis
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was found to be more likely to be detected in women in the proliferative phase (44). Equally
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important moderating factors could include immune status, genotypic factors, other co-
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infections, or reproductive tract microbiome composition. Unfortunately, there is no
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published evidence on these factors and direct evidence of ascension or not. Furthermore,
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pathogen factors could also moderate the likelihood of ascension such as infectious burden
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(although chlamydial serovar alone has not been able to be significantly associated with
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pathogenic potential or clinical manifestations (41)).
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Persistence
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Chlamydia persistence, or the presence of viable but non-culturable chlamydial organisms,
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develops in response to adverse conditions, and can remain dormant but present for
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considerable lengths of time (reviewed (45)). Persistence in vivo has been proposed as a
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model where this on-going presence of the organism may drive a long-term pathological
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immune response resulting in the tissue damage to the fallopian tube (certainly the high
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expression of the dominant antigen cHSP60 has been detected in laboratory persistence
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models) (reviewed, (46)). This is a difficult model to test in vivo, although it was recently
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attempted by Arsovic et al., 2014 who defined patients with a persistent infection as those
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who had IgG and/or IgA MOMP (MEDAC pELISA) and IgG cHSP60 seropositivity, patients
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who had IgG or IgA with no cHSP60 IgG were defined as recent or past chlamydial infection
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and sero-negative for all three were considered negatives (47). However, this study included
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a spontaneous miscarriage group for comparison which is arguably likely highly distinct from
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tubal infertility and did not include groups with other causes of infertility nor fertile controls,
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nor recent diagnosed chlamydial infected participants. There are studies that have presented
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evidence of C. trachomatis DNA or antigens being present in the tubal material of women
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with tubal infertility or ectopic pregnancy or salpingitis, supporting that organism persistence
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in some form occurs in vivo (37, 48-50). Recently, morphologies consistent with laboratory
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models of persistence were observed in endocervix samples associated with IFN-, further
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supporting the likely in vivo relevance of laboratory models of persistence (51). It is difficult
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to determine if chlamydial persistence has always occurred leading to pathology. Persistent
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infection may resolve at any time after pathology develops and prior to detection, given the
14
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difficulties in specimen collection from the upper genital tract the evidence is reasonably
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compelling that chlamydial persistence (either consistent with the lab models or a distinct in
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vivo form) could be a relevant factor in pathology. It is important to highlight a recent review
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that suggested that some forms of persistence may be ‘colonisation’ and in fact not associated
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with ‘pathogenesis’ and disease (52). Our suggestion is that perhaps the two are distinct and
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there is pathology associated persistence (given the evidence reviewed here) and separate
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cases of non-damaging colonisation. Specifically, the evidence that 1. chlamydial persistence
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is induced by a range of conditions in vitro (reviewed, (45, 46)), and 2. persistence like
313
morphology and/or chlamydial antigen/DNA detection in the absence of active infection has
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been observed in clinical samples, leads us to feel confident to suggest that persistence may
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be a relevant physiological status in vivo particularly in the case of ascension to the upper
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reproductive tract tissues, and therefore likely a determinant of pathological outcome from
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infection.
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Cellular paradigm
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The cellular paradigm model proposes that the chlamydial infected epithelium or
321
endothelium immediately responds to the presence of the Chlamydia or specific antigens
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from Chlamydia in a pro-inflammatory manner, typified by pro-inflammatory cytokines and
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growth factors which in turn induces a pro-inflammatory secondary immune cell activation
324
and migration to form a local lymphoid follicle resulting in cell damage and fibrosis or
325
scarring (comprehensively presented and reviewed elsewhere (53)). This model is widely
326
supported by the published evidence of human epithelia and immune cell responses to
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Chlamydia and a primary tissue ex vivo model that is discussed in more detail later in this
328
review. This model also presumes that chlamydial ascension is associated with pathology and
15
329
also mutually allows for persistence of Chlamydia in the upper reproductive tract (53). A
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pathological adaptive memory response proposed as the secondary component of this model
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is also supported by the existing data that repeat infections increase the likelihood of
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pathological outcomes in women (54), and by numerous immunological results (discussed
333
later in this review).
334
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cHSP60 induced delayed hypersensitivity
336
There has been considerable focus on a pathology model that involves delayed
337
hypersensitivity and/or molecular mimicry in response to specific chlamydial antigens
338
(reviewed (55)). This model has largely resulted from a body of literature demonstrating high
339
titre human antibody response to cHSP60 in participants with tubal pathology and
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additionally that in several animal models tissue pathology developed after repeated
341
inoculations with cHSP60 protein (reviewed (53)). However, this model is subject to
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considerable controversy due to; inconsistencies between the different published findings;
343
problems with protein preparations that involved a hypersensitive detergent being used in
344
some animal models; and cross reactivity or poor specificity for some of the antibody tests
345
(56-62). On the other hand there is no doubt that cHSP60 is a predominant chlamydial
346
antigen that does frequently elicit an immune response, and is likely a major antigenic
347
contributor to the pathology development. Furthermore, in human trachoma cases it is
348
frequently possible to observe ongoing follicles present and disease pathology in the absence
349
of PCR detectable Chlamydia suggesting that there is continuing immune activity in the
350
absence of organism during pathology development (63).
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Interestingly, one group proposed that cHSP60 antibodies detected in the follicular fluid of
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female IVF patients may induce early destruction of the embryo by cross reacting against
16
353
embryo human HSP60 and result in lower transfer success (64). They observed a 74.1%
354
cHSP60 sero-positivity in women without embryo implantation (n=47) and 47.9% sero-
355
positivity in women with successful embryo implantation (n=91) (p=0.0004). However, this
356
observation directly contradicts a finding in separate and much larger study (n=1279) that
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cumulative IVF cycle pregnancy rates are the same for women who are chlamydial
358
seropositive compared to those who are seronegative (33). A study in France also
359
contradicted the possible role of adverse embryo implantation impact of anti-chlamydial
360
immunity as it that for found for both men and women with PCR or serological evidence of
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Chlamydia (n=52) the IVF pregnancy rate and semen characteristics were not different
362
compared to controls that were negative for chlamydial testing (n=119) (65).
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Host/human factors associated with chlamydial infertility in women
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Repeat infection and infertility
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Repeat infection represents a substantial proportion of chlamydial infections detected
367
annually (re-infection review; (66)). It is likely that these repeat infections are made up of
368
reinfection, treatment failure, and persistent infections (14). Batteiger et al., (2010) conducted
369
a longitudinal cohort of 210 adolescent women (14-17 years old) participants (USA) and
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identified that 121 experienced a repeat infection (14). In a longitudinal cohort in Australia,
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1116 women (16-25 year old) were followed, and 14 re-infections were observed from a total
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of 81 women who were at risk of repeat infection (three of these had two episodes of re-
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infection) (cumulative risk over 12 months of 20.3%, 95% CI 13.2-37.6%) (67). Interestingly,
374
in this study in Australia organism load was lower in re-infections compared to prevalent
375
infections detected at the study baseline (67), but there were no associations between
376
participant characteristics and re-infection (67) (although this was possibly a statistical power
17
377
issue). However, careful consideration into study design is needed when evaluating repeat
378
infection, the Batteiger and Walker studies tested quarterly for Chlamydia, and PCR may
379
detect residual DNA from dead organisms causing a false positive. A study conducted in
380
Vancouver, showed that the repeat infection occurred in 8 of 42 participants in a longitudinal
381
study (both genders) indicating a cumulative incidence of 29% (95% CI 12-46%), although
382
the study was confounded by re-testing immediately conducted in some participants (68). A
383
study in the UK showed re-infection was 29.9 (19.7-45.4) per 100 person years from a GP
384
clinic setting (69). Infection and repeat infection significantly associates with sexual
385
behaviour risk factors as we would expect, such as new partners or failure to use condoms
386
(67). These repeat infection rates appear to be higher than the reported infertility outcome
387
rates in women who have had a positive Chlamydia test in the Low et al., (2006)
388
retrospective cohort study (16), suggesting that repeat infection alone is not the sole
389
determinant of infertility or pathology. However, there is some evidence to suggest that
390
repeat infection increases the risk of developing infertility. A retrospective cohort study by
391
Hillis and co-workers in Wisconsin examined the risks of hospitalisation for ectopic
392
pregnancy or PID in 11000 Wisconsin women who had one or more chlamydial infections.
393
They identified elevated risks of ectopic pregnancy in women who had two (OR 2.1 95% CI
394
1.3-3.4), and three or more infections (OR 4.5 95% CI 1.8-5.3) (54). Pelvic inflammatory
395
disease risk was also increased for women who had two (OR 4.0 95% CI 1.6 - 9.9) and three
396
or more chlamydial infections (OR 6.4 95% CI 2.2-18.4) (54). Therefore, repeat infection is
397
likely to be a significant contributing factor in at least some cases of chlamydial infertility.
398
Treatment failures or persistence induced by antibiotics have also been proposed to be
399
contributing to repeat infection rates (reviewed (70)). Batteiger et al., (14) showed that repeat
400
infections in 13.7% of the patients were due to possible or probable treatment failures.
401
Additionally, in vitro studies have shown that azithromycin induces chlamydial persistence
18
402
(71). Therefore, treatment failure or persistence (presenting as treatment failure) could be a
403
contributing factor to development of infertility in some women via all four of the proposed
404
pathology models.
405
The rise of repeat infections detected over time has resulted in the proposal that increased
406
public health investment into chlamydial screening and treatment is preventing the
407
development of natural immunity to the infection (arrested immunity hypothesis) (72, 73).
408
Furthermore, the authors propose that these repeat infections may be the source of
409
pathological sequelae that may not occur in women who have a protective immunity from a
410
naturally resolved infection (72, 73).
411
412
Do co-infections or the microbiome play a role in chlamydial pathology?
413
Co-infections with other STIs are not uncommon, for example 46% of female patients
414
positive for N. gonorrhoeae were also be positive for Chlamydia in a family planning clinic
415
in the USA (74). However, a serology study in Zimbabwe, demonstrated that women with
416
antibodies to either C. trachomatis of N. gonorrhoeae were significantly more at risk of
417
having PID, ectopic pregnancy, or abnormal fallopian tubes than those women with
418
antibodies to both pathogens (75), suggesting that it is not necessary to have had infection
419
with both pathogens to result in reproductive pathology. Patients with bacterial vaginosis who
420
were a recent contact of a male with chlamydial urethritis had an odds ratio of 3.4 (95 % CI
421
1.5-7.8) to test positive for chlamydial infection. The presence of H2O2 producing lactobacilli
422
in the vagina were protective against acquisition of infection (odds ratio 0.4, 95% CI 0.2-0.8)
423
(76). Recently an in vitro model was used to demonstrate that it is actually the acid or pH
424
lowering impact of the lactobacilli that confers the anti-chlamydial activity (77). It has been
425
hypothesized that indole producing organisms that increase in the microbiome during or
19
426
subsequent to bacterial vaginosis enable C. trachomatis to synthesize tryptophan from indole
427
(78), consequently evading the activity of interferon gamma at the infected site and thus may
428
facilitate the infection (78, 79). Bacterial vaginosis during C. trachomatis or N. gonorrhoeae
429
infection was recently found to be associated with risk of PID, although the authors conclude
430
that difficult to determine if the bacteria underlying the vaginosis facilitated ascension and
431
PID caused by the STIs or alternatively if the bacteria responsible for vaginosis cause the PID
432
(80). Clearly, there is increased chlamydial infection risk associated with co-infection or
433
bacterial vaginosis, and also evidence for an association with PID, supporting that co-
434
infections could therefore result in an increased likelihood of development of infertility.
435
436
Sexual behaviour and age of sexual contact
437
Age is a risk factor for pelvic inflammatory disease, and in most studies generally PID is
438
typically significantly associated with increasing age, whereas younger women tend to have
439
the highest chlamydial infection incidence. Age is also a risk factor in likelihood of
440
contracting chlamydial infection (12, 81). Cross national studies from 1999-2008 in
441
Denmark, Australia and Sweden showed that increasing PID rates were associated with
442
increasing age, with the exception of New Zealand, where the rates were higher in women of
443
ages 15-19 (82). The highest incidence of C. trachomatis infection is in women of ages 16-24
444
(83). Consistent with these studies, being under 20, having cervical symptoms were all risk
445
factors for endocervical chlamydial infection in women, and the organism burden was also
446
higher in younger women when measured by viable culture counts (84). Potentially the
447
organism burden may influence the ability to ascend to the upper reproductive tract and
448
ability to maintain a longer or persistent infection, therefore even though there is no direct
20
449
evidence, there seems to be the potential for younger age of acquisition of chlamydial
450
infection to increase the risk of pathological outcomes.
451
452
Sexual behaviour (new partners or higher number of partners) is often significantly associated
453
with increased risk of C. trachomatis infection (9, 85). As shown in the study by Skjeldestad
454
et al., 2009 (86) , where there was a 37% probability of a woman acquiring C. trachomatis
455
infection if she has had 3 more partners within 42 months. A younger age at first coitus,
456
higher number of sexual partners, and self-reported history of medically diagnosed sexually
457
transmitted infection were all significantly associated with tubal infertility compared to fertile
458
controls (p<0.01) (87). However, as these are also known to be significant factors for
459
increased likelihood of acquisition of chlamydial infection it is not certain if they directly
460
influence the likelihood of pathology outcomes from the infection.
461
462
Hormonal status at time of infection
463
Oral contraceptive use was found to be a risk factor for C. trachomatis infection (p=0.006),
464
and the organism burden was found to be higher in oral contraceptive users (p<0.001) (84).
465
Prospective recruitment of young female participants (>17) attending genitourinary medicine
466
clinics in the UK identified that those who had a Chlamydia infection were also more likely
467
to have elevated progesterone concentrations (p=0.05) (88). In contrast, oral contraceptive
468
usage has been linked to protection against subsequent infertility or conception problems by
469
two different studies (19, 89). However, it would be extremely difficult to link hormone
470
status at time of infection with subsequent pathological outcomes such as infertility, even in a
21
471
longitudinal study, given the high numbers of oral contraceptive users, and the independent
472
importance of these factors in the risk of acquiring an infection.
473
474
475
Human genotypic factors associated with C. trachomatis infertility in women
476
The development of C. trachomatis related infertility in only some women may be a
477
consequence of a genotypic pre-disposition (reviewed comprehensively (90)). These studies
478
are described below; however, for the sake of brevity we have documented the sample sizes,
479
statistics and details of the alleles in table format (Table 1). Note the above mentioned
480
comprehensive review of this topic has recently been published therefore we have been brief
481
in this section (90).
482
Toll-like receptors (TLRs) recognize and bind to antigens of pathogen, and signal to
483
upregulate an immune response. There are 10 Toll like receptors (TLR) identified in the
484
human body, and TLR 1-4 expressed in the female genital tract (91). TLR2 and TLR4 are
485
also expressed on macrophages, dendritic cells and epithelial cells and they bind chlamydial
486
lipopolysaccharide (92). In a prospective genotypic study with female participants attending a
487
sexual health clinic and a fertility clinic in Amsterdam, Karimi et al. (2009) (93)
488
demonstrated a possible protective role of a TLR2 haplotype. The haplotype consisting of
489
two distinct single nucleotide polymorphisms (SNPS) was found to be present in a
490
significantly higher proportion of women with a history of Chlamydia who did not have tubal
491
pathology and in sexual health clinic attendees was present in a higher proportion of women
492
who were asymptomatic (93). In the PEACH PID study TLR1 and TLR4 polymorphisms
493
were found to significantly associate with chlamydial PID (94), whilst SNPS from TLR2
22
494
(different SNPs from those included in the Karimi study), TLR6, Myd88, and TIRAP did not
495
significantly associate with chlamydial PID (94).
496
Den Hartog et al (95) identified that the risk of tubal pathology was higher in C. trachomatis
497
IgG positive women with multiple TLR polymorphisms. The study also showed a trend
498
towards association of various individual polymorphisms in TLR9 and TLR4 with increased
499
risk of tubal pathology in C. trachomatis IgG positive women (95). However, the small
500
sample size in the study makes it difficult to draw a correlation between the polymorphisms
501
and level of risk for female infertility.
502
NLRP3, a component of the inflammasome that activates a pro-inflammatory response has
503
been identified to have polymorphisms reported to result in altered IL-1secretion (a pro-
504
inflammatory cytokine) (96). Several NLRP3 polymorphisms were tested in women
505
prospectively recruited at a sexual health clinic and fertility clinics in Amsterdam, women
506
heterozygous or homozygous with the one of the NLRP3 polymorphisms were at significant
507
risk of developing abdominal pain during C. trachomatis infection (96).
508
Mannose-binding lectin (MBL) is a pro-inflammatory protein that activates complement and
509
is locally synthesised in the vagina (97). MBL and MBL promoter polymorphisms have been
510
previously demonstrated to alter the levels of MBL produced (97). It was identified that
511
women with tubal factor infertility who were chlamydial sero-positive more frequently had
512
the low MBL producing genotypes compared to healthy controls (97). However, this was not
513
the case in an ectopic pregnancy group who were less likely to have the MBL-deficient
514
genotypes (97). This later finding sheds some doubt on the role of MBL genotypes, given that
515
it seems unlikely that the pathology associate with ectopic pregnancy is distinct from tubal
516
infertility.
23
517
Human leukocyte antigen (HLA) class II molecules (DR, DQ and DP) present peptides to
518
CD4 T cells and induce adaptive immunity. DQ polymorphisms were more frequent in
519
women with tubal infertility who were also C. trachomatis sero-positive (98).Whilst another
520
polymorphism was negatively associated with C. trachomatis positive tubal infertility (98).
521
Wang et al., (99) showed that in adolescent females with recurrent chlamydial infections,
522
certain HLA polymorphisms were significantly associated with recurrent infections. A recent
523
study attempted to link different immune genotypes to immunological outcome by testing a
524
functional role for HLA DQ alleles combined with an IL-10 allele for both frequency in a
525
tubal factor infertility (Chlamydia positive) cohort compared to controls and also for
526
lymphocyte proliferative response to cHSP60 (100). The HLA alleles and IL-10 allele were
527
more frequently identified in the chlamydial tubal factor cohort compared to the control,
528
however the lymphocyte proliferation in response to cHSP60 was not significantly different
529
in relation to the alleles (100). However, in a different study, distinct IL-10 and IFN-
530
polymorphisms were found to significantly impact on lymphocyte proliferation in response to
531
chlamydial antigens (101).
532
533
A range of cytokine polymorphisms have been shown to significantly associate with women
534
with C. trachomatis tubal infertility, including: IL-10 (102), TNF- (102) and IL-12 (103).
535
Polymorphism in IL-1 and receptor genes were not associated with C. trachomatis related
536
tubal pathology (104). Adolescent females with recurrent chlamydial infections had a lower
537
frequency of three IL-10 promoter polymorphisms (99). Eng et al. (105) demonstrated that a
538
CD14 allele (TLR-4 co-receptor) was associated with increased TNF-α production when
539
whole blood was stimulated with C. trachomatis and C. pneumoniae (106).
24
540
Whilst none of these studies were able to account for all chlamydial tubal infertility cases, it
541
is a convincing body of evidence that human genotype may be a factor in pathology
542
development. It is interesting that to date the vast majority of studies have focussed on
543
immune factors, yet as an obligate intracellular pathogen Chlamydia has key host nutritional
544
requirements, inclusion vacuole requirements, and therefore the ability to ascend and cause
545
pathology may in fact be independently determined by a as yet unknown non-immune host
546
genotype.
547
548
Immune responses associated with Chlamydia trachomatis in women
549
Insight into the immunological factors that may be involved in C. trachomatis infertility in
550
women has been obtained through various human studies, including; local secretions
551
(cervical and vaginal lavages), human tissues, peripheral blood mononuclear cells (PBMC),
552
as well as through in vitro cell culture models. The innate response to infection is clearly
553
critical for the infection outcome, and as outlined in the cellular paradigm model, could be
554
the major determinant of either a pathological or resolution outcome from the infection. A
555
commonly used framework to categorize the adaptive immune response is the Th1:Th2
556
paradigm (reviewed (107)). This paradigm suggests that the immune response can polarize
557
towards a cytotoxic pathological response (Th1) or a humoral antibody mediated response
558
(Th2). The profiles of these responses include Th1 subset of T helper lymphocytes (Th) and
559
cytokine production such as: IL-2, IL-6, tumor necrosis factor (TNF-α), and interferon-
560
gamma (IFN-γ)(108, 109); and Th2 responses involve IL-4, IL-5, IL-6, IL-10 and IL-13
561
(108). In addition to Th1:Th2 regulatory immune profiles typified by IL-17 or IL-23 have
562
also recently been described (reviewed (110)). Here we summarize the existing evidence for
25
563
the human immune response against C. trachomatis and how this evidence fits the four
564
different models of infertility development using these immune categories.
565
566
Immune responses from reproductive sites and tissues
567
Genital secretions have been used to reveal the immunological responses that occur at the site
568
of infection, although it is not possible to link these with subsequent pathology. IFN-γ levels
569
were found to be five times higher in the endocervical secretions of women with C.
570
trachomatis infection detected by culture (n=47) compared to uninfected women (n=52)
571
(111). Analysis of immune factors using multiplex immunoassay in cervical-vaginal lavages
572
of women attending a STD clinic with acute C. trachomatis infection (n=5) compared to
573
controls with no infections (n=13), revealed significantly higher levels of IL-1β, lactoferrin,
574
TNF-α, IL-8, VEGF, G-CSF, IL-10, IL-3, IL-7, IL-12 and IL-6 (112). However, in a separate
575
study in India IFN- levels were only significantly higher in women with recurrent C.
576
trachomatis infections compared to controls but not higher than those with primary infection
577
(113). IL-17 and IL-22 were 5 times and 3 times higher in the cervical secretion of C.
578
trachomatis positive women (n= 27) compared to negative controls (n=17) (114). IgG and
579
IgA antibodies to C. trachomatis EBs were higher in the cervical washes of C. trachomatis
580
positive fertile women during primary infection compared to recurrent infection (113).
581
However, cervical IgG to specific chlamydial antigens (cHSP60 and cHSP10) was higher in
582
recurrent infection than compared to primary infection, which is what would be expected
583
(113). Flow cytometric analysis of lymphocytes present at the cervix of C. trachomatis
584
infected women identified the increased presence of a unique cell type; 47+CLA+memory
585
T cells (115). Stimulation of cervical monocytes with chlamydial EBs showed an increased
586
expression of Toll-like receptors (TLRs) TLR2 and 4, and both receptors were also found to
26
587
be expressed at higher levels in cervical cells from women with infection compared to
588
uninfected women (116). The levels of IL-1, IL-4, IL-5, IL-6 and IL-10 were reported to be
589
significantly higher from enriched cervical T cells stimulated with chlamydial inclusion
590
membrane (Inc) proteins from C. trachomatis related infertile women (n=18) as compared to
591
C. trachomatis positive fertile women (n=14) (117). Whilst not always a Chlamydia related
592
pathology, a study by Balasubramaniam et al., (2012) that found that level of cytokines IL-8
593
and IL-6 were significantly upregulated in the fallopian tube immediately in the location of
594
implantation of ectopic pregnancy, but not in the fallopian tubes of women undergoing
595
benign hysterectomy (118). A very recent study identified that differences in IFN- levels
596
correlated with differences in the chlamydial cellular morphologies at the cervix when
597
comparing two patients (51). These local responses all support that immune mediated
598
pathology and key chlamydial antigens are likely involved in the development of infertility,
599
although they are not directly correlated to the fallopian tube.
600
601
The effects of chlamydial infection in human tissue ex vivo studies provided a controlled
602
insight into the cellular responses that likely occur in vivo during the immediate primary
603
infection. Though ex vivo fallopian tube studies, Hvid et al. (119) showed that addition of IL-
604
1RA receptor antagonists, blocked IL-1 and IL-8 production preventing the pathology from
605
chlamydial infection (119). This confirms that IL-1 along with IL-8 are likely major factors
606
for tubal pathology (119), supporting the cellular paradigm of pathology development and not
607
supporting the hypersensitivity model. Furthermore, implicating the innate immune response
608
as a possible determinant of infection outcome. Fallopian tube explants from ectopic
609
pregnancy cases that were C. trachomatis sero-positive were used to demonstrate that C.
610
trachomatis induced higher expression of a prokineticin receptor via TLR2 binding (120).
27
611
The prokineticin pathway impacts on smooth muscle contraction and intrauterine
612
implantation as well as angiogenesis, which may imply a mechanism for C. trachomatis
613
damage leading to ectopic pregnancy (120). T lymphocytes from endometrial and salpingeal
614
tissues cultured ex vivo in the presence of Chlamydia or cHSP60 showed induction of
615
lymphocyte proliferation and IFN- secretion from PID or ectopic pregnancy cases (121). In
616
the same participants, higher levels of IFN-γ mRNA expression and lower levels of IL-5 in
617
fallopian tube and peritoneal cavity specimens of PID patients who had T cells proliferating
618
in response to the organism ex vivo further implicated Th1 cytokine production in C.
619
trachomatis related pathology (121). Primary endocervical cell cultures infected with C.
620
trachomatis also demonstrated a pro-inflammatory cytokine response with abundant
621
production of IL-8, IL-1 and TNF, all of which were maximally detected during live
622
infection with active bacterial protein synthesis (122). These primary endocervical cultures
623
indicate that the innate immune response to Chlamydia is typically pro-inflammatory, which
624
is what may be required to successfully resolve the infection. Combined these data
625
predominantly support the cellular paradigm and ascending infection models of pathology
626
and link to a Th1 profile of immunity. However, given that a cytotoxic response is needed to
627
resolve infection, differentiating between a successful Th1 profile resolving infection, and
628
one that results in tissue damage is not possible in the absence of longitudinal data..
629
630
Immune responses in peripheral blood mononuclear cells (PBMC) in women
631
Peripheral blood mononuclear cells (PBMC) are often used to provide insight into immune
632
cell responses to Chlamydia from different participant cohorts. PBMC proliferation induced
633
by purified C. trachomatis EBs showed a prominent secretion of IFN-γ in women with
634
genital infection (123). PBMC from women at high risk of acquiring an infection were
28
635
stimulated with cHSP60 and the production of IFN- strongly correlated with protection
636
against incident C. trachomatis infection (124). In a separate study PBMC from healthy
637
donors were incubated with C. trachomatis serovar K, leading to the finding that pro-
638
inflammatory gene expression (measured by microarray and validated by qRT-PCR) was
639
sustained for up to seven days, especially for IFN-, and IL-2receptor (125). However in a
640
separate study, cHSP60 PBMC production of IFN- was lower in women with PID or repeat
641
infections compared to women with current infections (126, 127). Human dendritic cells
642
prepared from human PBMC were infected with C. trachomatis serovar E or L2 that induced
643
production of IL-1, IL-6, IL-8, IL-12p70, IL-18 and TNF-α (128). A separate study also
644
detected production of IL-12 and TNF-α by human PBMC sourced dendritic cells infected
645
with C. trachomatis L2 (129). Hook et al. (130), showed that stimulation of PBMC with
646
chlamydial EBs elicited IFN-γ production by natural killer cells. Cohen et al., (124) showed
647
IFN-γ production from PBMC stimulated with cHSP60 correlated with participants who were
648
protected from acquisition of infection in a longitudinal study of 143 female participants
649
(124). This study also showed the PBMC production of IL-13 in response to chlamydial EBs
650
also correlated with a reduced risk of infection (124). Our own research has demonstrated IL-
651
6 production from endometrial and endocervical primary ex vivo cultures from live infections
652
with C. trachomatis (131).
653
654
Immune pathway elucidated by in vitro culture models
655
Epithelial cell culture are most commonly used for in vitro growth and characterisation for C.
656
trachomatis infection (132). C. trachomatis infection in HeLA, SiHa (human cervical
657
carcinoma epithelial cell) and HEp2 (human epithelial cell) were used to demonstrate that IL-
658
1α regulated IL-8 production from these cell lines during infection (133). Similarly, mRNA
29
659
measurement of cytokine expression after C. trachomatis infection of HeLa, SiHa cervix
660
squamous carcinoma cells and HT-29 colon adenocarcinoma cells showed increased levels of
661
IL-8, GROα, GM-CSF, IL-6 and IL-1α levels. These cytokines are potent chemoattractants,
662
activators of neutrophils, monocytes and T lymphocytes (134). A HeLa/THP-1 co-culture
663
model attempting to re-capitulate the in vivo cellular cross talk also identified sustained IL-6
664
and IL-8 cytokine secretion during C. trachomatis infection with IL-1transiently detected
665
(135). IL-6 and IL-8 were also detected in a separate study using infections of cervical and
666
epithelial cell models (122). A HeLa and THP-1 (mono-nuclear cell) co-cultures model
667
identified that there are distinct profiles of innate immune responses to C. trachomatis
668
serovar E compared to L2. The results also showed that the monocyte released innate
669
cytokines (such as TNF-) had different effectiveness in controlling the infection in infected
670
epithelia model cells between the two serovars (136). Using a similar co-culture model our
671
team has also demonstrated sustained IL-6 production in response to C. trachomatis L2
672
infection (122). A RNAseq methodology to examine host and chlamydial gene expression
673
enabled the investigators to identify that a fibrotic profile of gene expression being induced in
674
infected HEP-2 cells (137). The role of TNF-α in inhibiting chlamydial development was
675
evaluated by addition of recombinant TNF-α to Hep2 cells prior to infection which resulted
676
in smaller inclusion bodies (138). Furthermore, addition of IFN-γ to the cell line showed that
677
in combination with TNF-α, chlamydial replication was inhibited (138). Lu et al., (139)
678
showed that chlamydial infection of several epithelial cell lines resulted in the secretion of
679
mature IL-18. IL-18 is known to potentiate the Th1 immune response and enhance production
680
of IFN-γ (139).
681
682
Conclusion
30
683
Overwhelmingly, the studies of in vivo responses, ex vivo cultures, and laboratory cell culture
684
models all support a higher level or predominance of a Th1 or cytotoxic profile of immune
685
response to chlamydial infection, with IFN-, IL-8, IL-1, and IL-6 findings featuring almost
686
universally in the studies (although IFN- was also associated with protection against repeat
687
infection). However, it is not clear if the pathological response is moderated or different
688
between women who develop pathology or those who resolve the infection without
689
pathology. Longitudinal sampling and analysis of correlates of the local immune response,
690
PMBC response, infection clearance relative to initial infection burden with the development
691
of upper reproductive tract pathology is one possible study design that could help to clarify
692
the contribution of pro-inflammatory primary immunity.
693
694
695
696
697
Chlamydial factors that may influence the development of pathology
698
Chlamydial serovar can associate with repeat infection and symptoms that may link to a
699
propensity to cause pathology and infertility
700
Chlamydial serovars are assigned based on the outer membrane proteins, typically by
701
sequencing so studies that refer to serovars are actually frequently referring to genovars
702
(140). Serovar E and serovar F are generally found to be predominant in most countries
703
including: Australia, Netherlands, and Sweden (67, 141-144). Using omp1 PCR based RFLP
704
genotyping Lan et al., (145) reported that in women less than 30 years old, serovars D (4/21
31
705
asymptomatic women) and I (4/21 asymptomatic women) were associated with asymptomatic
706
infection, while serovar G was associated with symptomatic infections (4/30 symptomatic
707
cases). Similarly, Gao et al., (143) also reported the predominance of serovar G in
708
symptomatic infection, particularly lower abdominal pain (18/33 patients) as compared to
709
asymptomatic patients (P<0.001) amongst female sex workers and STD patients in China.
710
47.5% (28/59) asymptomatic patients had serovar E (143). Verweij et al., (2014) reported
711
that different serovars induced differential serological responses in 510 women with PCR
712
confirmed C. trachomatis infection (146). The study showed that serovar D (n=45) and E
713
(n=217) from serogroup B elicited the highest IgG response (Median IgG titre of 200)
714
followed by serovar H (n=20) (146). Women with persisting infections were found to have
715
twice as may serovar E infections (67%, 8/12) compared to women who naturally cleared
716
infections (33% 3/9) during a longitudinal study of women with asymptomatic C.
717
trachomatis infections, however this was not significantly different (147). However, several
718
study design flaws have been raised about the Morre et al., 2002 study and therefore it may
719
not be relevant to include these data to make conclusions (eg see (148, 149)). Whilst not
720
linked directly to infertility, the majority of evidence from these studies support that different
721
serovars do have different associations with symptoms, re-infection, or infection duration, all
722
of which may contribute to pathology development.
723
724
Chlamydial genotypes can mediate tissue tropism and may be a factor in pathology
725
development
726
There are several published examples of C. trachomatis genotypic changes associating with
727
tissue tropism, however, no reports yet aligning chlamydial genotype with pathology
728
development and infertility. The most well know genotype that mediates tissue tropism is
32
729
actually polymorphisms in the trpAB operon and the ability to synthesis tryptophan from
730
indole between C. trachomatis ocular and genital strains (79, 150) which we will mention
731
here given the significance of this finding to the field even though this review is focussed on
732
infertility. There is also evidence of positively selected genotypes that align with tissue
733
trophism (ocular, genital, mononuclear/invasive), for example in a review of 59 C.
734
trachomatis genome sequences, it was reported that tarp, and the pmp genes have positively
735
selected polymorphisms that significantly cluster with tissue niche (151). Polymorphisms in
736
three distinct open reading frames have been found to associate with rectal but not cervical
737
serovar G isolates, and two further open reading frame polymorphisms were found to
738
associate with rectal and cervical tropism of serovars E, F and J (152). These studies do not
739
identify pathology associated polymorphisms, however they do support the theoretical
740
potential for chlamydial genotypes to exist that either improved ascension, or upper
741
reproductive tract survival or polymorphisms that alter the immune response. Any association
742
a chlamydial genotype with pathology development would only be possible by a longitudinal
743
study of a large group of women throughout their reproductive years with continual sampling
744
and analysis of infecting chlamydial strains.
745
746
Conclusions
747
A better understanding of the disease mechanism and the risk factors associated with C.
748
trachomatis infertility would enable specific screening for at risk women and inform vaccine
749
development targeted to prevent the progress of infection to infertility or pathology. Whilst
750
there are several convincing models of how the disease pathology potentially occurs, each
751
with some supporting evidence from the human data reviewed here, the actual process or host
752
and pathogen factors that result in infertility remain uncertain and require further
33
753
investigation. It is clear that a pro-inflammatory response is largely elicited in response to
754
chlamydial infection, whether this response or the extent of this response is the primary
755
determinant of pathology remains unclear. Furthermore, whilst there are tantalising
756
associations there are not yet definitive correlates of contributions of many host and pathogen
757
factors to pathology risk including; human genetic polymorphisms, C. trachomatis serovar
758
and genotype specific distinctions, co-infection or microbiome parameters, and risk
759
behaviours like repeat infections or age of sexual debut (summarised in Fig. 2.). A
760
comprehensive and extended longitudinal analysis of each of these factors using a very large
761
cohort study of women prior to any infection in early teen years until post-pregnancy age
762
(with adequate statistical power) would be the most robust way to monitor disease factors and
763
pathology outcomes in women. Identification of the major contributing factors to disease
764
pathology would better inform design of future vaccines and would also enable design of
765
molecular diagnostics for early detection of women at risk of the pathology development.
766
767
In the absence of such comprehensive knowledge and subsequent preventative interventions
768
(enhanced diagnosis or vaccine) there is a real opportunity to alleviate some of the risks and
769
costs associated with the pathological outcome from infection by improving guidelines for
770
screening, as recently proposed in the UK (153). Further to this we propose that improved use
771
of serological diagnosis of pathology risk expediting infertility diagnosis for women, and also
772
as a tool to predict the risk of ectopic pregnancy or PID in women (27, 33), who have had
773
chlamydial infections should also be rigorously evaluated and considered for routine
774
implementation in both fertility and general practice clinic settings.
775
34
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56
Author Biographies
Shruti Menon
Shruti Menon received her B.Sc. (Honours) in Biotechnology from Monash University,
Malaysia in 2011. She is currently doing her PhD in Microbiology at Queensland University
of Technology. Her project focuses on developing a novel peptide-based serological
diagnostic for women with Chlamydia trachomatis-related infertility. Her current interests
involve chlamydial diagnostics and mechanisms that lead to C.trachomatis –related infertility
in women.
Peter Timms
Peter Timms obtained his PhD from the University of Queensland, Australia in 1989 and was
made a Fellow of the Australian Society for Microbiology in 1991. He joined Queensland
University of Technology in 1991 and rose to the level of full professor in 2003. He moved
from QUT to University of Sunshine Coast, Australia in 2014 where he is Professor of
Microbiology. He has spent 25 years working on all aspects of Chlamydia, in both humans
and animals. His team has made major discoveries in chlamydial genomics, evolution,
vaccine development and basic mechanisms of pathogenesis.
John Allan
John A. Allan obtained his MB.BS from University of Queensland in 1973 and his fellowship
in Obstetrics and Gynaecology in 1984. Associate Professor John Allan was director of
Reproductive Medicine from 1988 till 2014 at the Wesley Hospital Brisbane and is currently
Head of UnitingCare Health Clinical School and Director of Gynaecology at The Wesley
Hospital. Dr Allan is a member of the Society of Pelvic Surgeons and his areas of research
have been in advanced laparoscopic surgery, investigation of the microbiome of
endometrium, fallopian tubes and follicular fluid. Dr Allan developed a technique for
freezing spermatozoa in the seminiferous tubule.
Kimberly Alexander
Kimberly E. Alexander is a Senior Lecturer within the School of Nursing at the Queensland
University of Technology, Brisbane, Australia. She is a registered nurse with a clinical
background in oncology. She has a Bachelor of Nursing, Bachelor of Health Science (Public
Health), a Graduate Certificate in Academic Practice, a Masters of Education (Higher
Education) and a PhD in epidemiology. Kim has cross-disciplinary research experience in
epidemiology, genetics, and nursing with a particular interest in patient-reported outcomes
such as symptoms and quality of life.
57
Luk Rombauts
Luk Rombauts was awarded a PhD in 1993 from the University of Leuven, Belgium, and
completed his general O&G training in 1996. He relocated to Australia in 1999 and
successfully completed his subspecialty training in Reproductive Endocrinology and
Infertility in 2001. He was appointed as Monash IVF Research Director in 2001, Clinical
Director of Monash IVF (2003-2008) and a Monash IVF Company Board Member between
2010-2012. He is an Adjunct Clinical Associate Professor in the Department of Obstetrics
and Gynaecology at Monash University. His main research interests include endometriosis
and embryo implantation. He is an associate editor for Human Reproduction. Associate Prof
Rombauts is a Board Member of the World Endometriosis Society since 2008 and is the
editor of the World Endometriosis Society's electronic Journal. In 2011 he was appointed to
the World Endometriosis Research Foundation Board of Trustees and to the Board of the
Fertility Society of Australia.
Paddy Horner
Paddy Horner has been a consultant at Bristol in Genitourinary Medicine since 1994 and
works at the Bristol Sexual Health Centre. In 2007 he was awarded a Walport Clinical Senior
Lectureship at the School of Social and Community Medicine, University of Bristol. He has
long standing research interests in the epidemiology diagnosis and treatment of Chlamydia
trachomatis, Mycoplasma genitalium, and non-gonococcal urethritis (NGU) on which he has
published widely in peer reviewed journals.
Martin Keltz
Martin Keltz, a board certified endocrinologist, established WESTMED Reproductive
Services in 2015. Dr Keltz graduated from Harvard College in 1985, the NYU School of
Medicine in 1989, and completed his OB/GYN residency at NYU/Bellevue in 1993. Since
completing his fellowship in reproductive endocrinology at Yale University in 1995, Dr Keltz
has been director of reproductive endocrinology at St Luke’s and Roosevelt Hospitals, where
is he now an associate professor at the Ican School of Medicine at Mt Sinai. He cares for
couples challenged by infertility and recurrent miscarriage as well as women suffering with
fibroids, endometriosis, or any reproductive disorder. His busy practise focuses on IVF, SHG,
IUI, and minimally invasive hysteroscopy and laparoscopy. Dr Keltz has been awarded the
“Top Reproductive Endocrinologist” in New York Magazine for the years 2012, 2013, and
2014. He has authored over 40 peer-reviewed articles and 50 abstracts in reproductive
medicine.
Jane S Hocking
58
Jane S Hocking is an epidemiologist with a particular interest in the epidemiology and control
of genital Chlamydia trachomatis. Dr Hocking is a Professor at the University of Melbourne,
Australia where she heads up the Sexual Health Unit in the Melbourne School of Population
and Global Health. She obtained a Master of Public Health in 1998 and a PhD in 2005, both
from the University of Melbourne. As part of her PhD, she generated Australia’s first
population-based estimates of genital chlamydia prevalence among young women aged 18 to
25 years. She has subsequently provided Australia’s first estimates of chlamydia incidence
and re-infection rates among young women. She currently leading a world first chlamydia
screening randomised controlled trial of a chlamydia testing intervention in general practice
that aims to determine whether chlamydia screening is cost-effective and should be
introduced in Australia. She is also interested in treatment efficacy for chlamydia.
Wilhelmina M Huston
Wilhelmina (Willa) Huston is a senior lecturer at Queensland University of Technology. Her
PhD was completed at The University of Queensland (conferred 2004). She held a
Postdoctoral Fellowship in the UK at The University of York (2003-2005), and returned to
Australia in 2005 as Postdoctoral Fellow at Queensland University of Technology in the field
of Chlamydia research (2005-2007). She held a NHMRC Peter Doherty Fellowship (20072010), Lecturer (2011), and Senior Lecturer (2012) positions at Queensland University of
Technology. Dr Huston is interested in the molecular microbiology of the human intracellular
pathogen Chlamydia, how we can better understand the pathogenesis mechanisms of this
organism leading to better treatment and diagnosis. Dr Huston has been in the Chlamydia
research field since 2005.
59
Fig. 1. The four major models of development of pathology associate with chlamydial
infertility in women. The figure shows the four models summarised here that are supported
within the field by evidence from human studies.
Fig. 2. Summary of host and pathogen factors that have some evidence of contribution
to the development of chlamydial infertility in women.
60
Summary
Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen
worldwide. The infection can result in serious reproductive pathologies including pelvic
inflammatory disease, ectopic pregnancy, and infertility in women. However, the processes
that result in these reproductive pathologies have not been well defined. Here we review the
evidence for the human disease burden of these chlamydial reproductive pathologies. We
present the four most widely accepted models of pathology development. We then review all
of human based evidence that links Chlamydia with reproductive pathologies in women and
how they support these models of disease. We present data supporting that host,
immunological, epidemiological, and pathogen factors may all contribute to the development
of infertility. Specifically, we review the existing evidence that host and pathogen genotypes,
host hormone status, age of sexual debut, sexual behaviour, co-infections and repeat
infections are all likely to be contributory factors to development of infertility. Pathogen
factors such as infectious burden, treatment failure and tissue tropism’s or ascension capacity
are also potential contributory factors. We highlight the limitations to the evidence and
propose future studies that could be beneficial to improve understanding of how chlamydial
infertility in women occurs and possible future interventions to reduce this disease burden.
Table 1: Summary of genotypes that have been significantly associated with chlamydial
infection or pathology
61
Locus
TLR1
(RS5743618) TT
Study parameters
Prospective recruitment
PEACH PID study:
PCR confirmed CT negative
women with PID n=111
PCR confirmed CT positive
women with PID n=94
Significance
TT increased risk of
developing PID with
chlamydial infection
(Adjusted Odds ratio: 2.8
95% CI 1.3-6.2; P=0.008)
TLR2
-16934 T>A
(rs4696480)
+2477 G>A
(rs5743708)
Fertility clinic:
CT IgG negative women with
Tubal pathology n=17)
CT positive women without
tubal pathology n=13
CT IgG positive women with
tubal pathology n=26 (CT
status MIF test titre >1:32)
Sexual Health Clinic:
CT IgG positive women n=147
CT IgG positive asymptomatic
women n=81
CT IgG positive symptomatic
women n=66
CT IgG negative women n=
321
Prospective recruitment
PEACH PID study: CT
negative (PCR) women with
PID n=111
CT positive (PCR) women
with PID (n=94)
Individual SNPs not
(93)
significantly associated
with infection, risk,
symptom or infertility
Haplotype: (-16934A with
+2477G) higher women
with CT but not tubal
pathology (P=0.015;
OR:0.28; 95% CI: 0.10.75), and decreased
proportion when women
ranked from asymptomatic
to symptomatic infections
to CT+ and tubal infertility
CC allele significantly
associated with being
chlamydial positive in PID
participants (P=0.002)
(94)
a
TT allele associated with
significantly higher TNF-
production when whole
blood stimulated with C.
trachomatis P<0.05
(relatively minor
differences in actual
concentration)
a
TLR4
Rs1927911 CC
allele
CD14 -260C>T
Whole blood from healthy
females n=44
Distribution of TNF-α allele
among male and female
participants in Taiwan (TT
n=26, CT, n=36, CC n=22)
b
Dutch women Caucasian
origin, CT positive test
determined by MIF with a titre
≥1:32.
62
No significant difference
in the distribution of CD14
Ref
(94)
(106)
(154)
b
STD clinic
CT IgG positive n= 135
CT IgG negative n=49
Subfertility clinic
CT IgG positive
women n= 41
CT IgG negative
women n =212
CT IgG positive with
tubal pathology n=28
CT IgG negative
women with tubal
pathology n=22
Healthy control group n=170
NLRP3
Sexual health clinic: CT
AG or AA at SNP positive n=99, CT positive
rs12065526
symptomatic n=44,
CT negative n=219,
CT negative symptomatic n=75
Healthy fertile control n = 93
MBL
High MBL2
producing
genotypes
(HYA/HYA)
(HYA/LYA)
CT IgG positive women with
TFI n=76 (n=157)
CT IgG positive women
without TFI n= 71(n=398)
HLA class II
allele
DRB1*1503
Infertility clinic, Kenya.
CT IgG positive women with
TFI (MIF titre ≥1:16) n=33
63
polymorphism in STD or
infertility cohort
AG or AA at SNP
(96)
rs12065526 were at
significant risk of
developing abdominal pain
when CT infected
(P=0.047, OR 2.9 95% CI
1.8-8.0) (not significantly
associated with infection
risk or tubal pathology)
High MBL2 producing
(92)
genotypes (HYA/HYA)
(P=0.054; OR 3.72; 95%
CI=1.07-12.78) and
(HYA/LYA) (P=0.019;
OR=0.28; 95% CI=0.110.75) higher in CT positive
women as compared to CT
positive TFI women.
MBL2 genotypes
(LXA/LXA) were higher
in CT positive TFI women
compared to CT positive
women without TFI
(P=0.025; OR 2.34;
95%CI 1.17-4.69)
DRB1*1503 detected in 7 (98)
TFI women who were CT
negative and absent in CT
DRB5*0101
CT IgG negative women with
TFI n=37
Cytokines
IL-10 1083AA
TNF- -308A
IL-12B
rs3212227 AC
Il-10 1082A
a
Infertility clinic at Helsinki
Infertile women n=114
Fertile control n=176
CT IgG positive women with
TFI (MEDAC ELISA kits) n=
96
b
TFI n=100
Pregnant fertile controls n=125
CT IgG positive with TFI n=94
CT IgG negative with TFI n=
69
CT cHSP60 IgG positive with
TFI n=90
CT cHSP60 IgG negative with
TFI n=73
c
TFI from IVF clinic, Helsinki
n=47
CT IgG antibodies n=23
Major
Infertility Clinic Chinahistocompatibility TFI women n=63
complex class I
Non TFI=151
chain related A
CT IgG positive women with
(MICA)
TFI (determined by
commercial immunoassay kits)
n= 42
CT IgG positive women
without TFI n=59
64
positive TFI women
(P=0.01; OR 0.05; 95%
CI=0-0.7).
DRB5*0101 was more
common in TFI CT
seronegative than TFI CT
seropositive (P=0.05;
OR=0.2; 95% CI=0.02,1)
a
IL-10 increased the risk of
tubal damage in CT
positive women (OR 7.3,
95% CI 1.3-4.2)
a
TNF- increased the risk
of tubal damage in CT
positive women (OR 4.0,
95% CI 1.0-16)
b
IL-12B AC heterozygote
increased risk of
chlamydial tubal factor
infertility (OR 2.44; 95%
CI 1.23-4.87)
c
Il-10 1082A allele
frequency higher in CT
positive TFI cases than CT
negative TFI cases (0.72
vs 0.46; P=0.01)
MICA *008 higher in
infertile patients without
CT infection than infertile
women with CT infection
(P=0.00367; OR=2.14;
95% CI=1.4-3.28)
MICA allele confers
protection against CT
tubal infertility.
a
(102)
(103)
c
(100)
b
(155)
Fig 1
65
Fig 2
66
Menon Bio pic
67
Timms Bio pic
68
Allan Bio pic
69
Alexander Bio pic
70
Luk Rombauts Bio pic
71
Horner Bio pic
72
Keltz bio pic
73
Hocking bio pic
74
Huston bio pic
75