- Email: [email protected]
Th17 cytokines in serum of Chlamydia trachomatis-infected women undergoing incomplete spontaneous abortion
Accepted Manuscript Differential expression of circulating Th1/ Th2/ Th17 cytokines in serum of Chlamydia trachomatis-infected women undergoing incomplete spontaneous abortion Priya Prasad, Namita Singh, Banashree Das, Sheikh Raisuddin, Mridu Dudeja, Sangita Rastogi PII:
S0882-4010(16)30921-4
DOI:
10.1016/j.micpath.2017.06.031
Reference:
YMPAT 2323
To appear in:
Microbial Pathogenesis
Received Date: 23 December 2016 Revised Date:
16 June 2017
Accepted Date: 21 June 2017
Please cite this article as: Prasad P, Singh N, Das B, Raisuddin S, Dudeja M, Rastogi S, Differential expression of circulating Th1/ Th2/ Th17 cytokines in serum of Chlamydia trachomatis-infected women undergoing incomplete spontaneous abortion, Microbial Pathogenesis (2017), doi: 10.1016/ j.micpath.2017.06.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title Page
Manuscript title: Differential expression of circulating Th1/ Th2/ Th17 cytokines in serum of
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Chlamydia trachomatis-infected women undergoing incomplete spontaneous abortion
Authors’ names:
Authors’ affiliations: Mrs. Priya Prasad Senior Research Fellow, Microbiology laboratory,
Sriramachari Bhawan,
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National Institute of Pathology (ICMR),
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Priya Prasad1, Namita Singh1, Banashree Das2, Sheikh Raisuddin3, Mridu Dudeja4, Sangita Rastogi1,*
Safdarjung hospital campus, Post Box No. 4909,
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New Delhi-110 029, India
e-mail: [email protected]
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Mrs. Namita Singh
Senior Research Fellow,
Microbiology laboratory,
National Institute of Pathology (ICMR), Sriramachari Bhawan, Safdarjung hospital campus, Post Box No. 4909, New Delhi-110 029, India
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e-mail: [email protected] Dr. Banashree Das Consultant & Professor,
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Department of Obstetrics & Gynecology,
New Delhi-110 029, India e-mail: [email protected]
Dr. Sheikh Raisuddin Professor & Advisor (Research)
Jamia Hamdard (Hamdard University)
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Department of Medical Elementology & Toxicology,
Hamdard Nagar, New Delhi-110 062, India e-mail: [email protected]
Dr. Mridu Dudeja
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Professor & HOD
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Vardhaman Mahavir Medical College (VMMC) & Safdarjung hospital,
Department of Microbiology,
Hamdard Institute of Medical Sciences and Research, Hamdard Nagar, New Delhi-110 044, India
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e-mail: [email protected]
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Dr. Sangita Rastogi
Scientist ‘F’ & Senior Deputy Director, Microbiology laboratory,
National Institute of Pathology (ICMR), Sriramachari Bhawan,
Safdarjung hospital campus, Post Box No. 4909, New Delhi-110 029, India e-mail: [email protected] 2
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Corresponding author: Name and address:
*Dr. Sangita Rastogi Microbiology laboratory,
Sriramachari Bhawan, Microbiology laboratory, Safdarjung hospital campus,
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Post Box No. 4909,
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National Institute of Pathology (ICMR),
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New Delhi-110 029, India
+ 91 - 011-26198 402 - 406 (Ext. 314)
Mobile no.:
+ 91 - 098102 17260
e-mail:
[email protected]
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Tel. no.:
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ABSTRACT The study aimed to elucidate role of Th1/ Th2/ Th17 cytokines in the immunopathogenesis of spontaneous abortion in Chlamydia trachomatis (Ct)-positive first-trimester aborters. Endometrial
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curettage tissue and serum were collected from 145 aborters (spontaneous abortion (SA) group, n = 85; recurrent miscarriage (RM) group, n = 60) and 120 controls attending Department of Obstetrics & Gynecology at Safdarjung hospital, New Delhi (India). Polymerase chain reaction was used to detect
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Ct plasmid/ MOMP, while commercial cytometric bead array kit was utilized to estimate circulating serum cytokines. 13.7% aborters were Ct-positive, however, none was found to be infected among
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controls. IFN-γ, TNF-α, IL-2, IL-6 and IL-17A cytokines were significantly increased in SA group/ RM group (Ct-infected) versus controls. IL-4 showed no difference between groups, while IL-10 was significantly elevated in controls versus Ct-infected subjects in SA group/ RM group. Furthermore, IFN-γ, TNF-α, IL-6, IL-17A cytokines were significantly elevated in Ct-positive RM group versus
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Chlamydia-infected SA group. However, IL-2, IL-4 and IL-10 cytokines showed no significant difference between Ct-positive SA group versus infected RM group. Positive correlation was found between few cytokines (TNF-α and IFN-γ/ IL-17A; IL-17A and IFN-γ/ IL-6) in Ct-positive aborters.
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Our study clearly established the role of Th1/ Th2/ Th17 cytokines in the pathogenesis of spontaneous abortion in Ct-infected subjects and found that Chlamydia-positive recurrent aborters
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had a predominant Th1/ Th17 bias.
Key words: Chlamydia trachomatis; Cytokine bead array; Recurrent miscarriage; Spontaneous abortion; Pathogenesis; Th1/ Th2/ Th17 cytokines
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1.
Introduction Chlamydia trachomatis (Ct) is the leading cause of sexually transmitted infection with the
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annual detection of 92 million new cases worldwide, including 43 million from south-east Asia [1]. A high prevalence of genital Ct infection has also been reported in our country, particularly in females [2-7]. In pregnant women, the presence of Ct is even more critical as it may affect normal intra- and
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extra-uterine development. Intra-uterine infection can threaten pregnancy by gaining access to the placenta, decidua and foetus since pregnancy increases the risk of Ct colonization and alters immune
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response. Pregnant women with chlamydial infection are tenfold more likely to suffer an adverse obstetric outcome and various studies have reported an increased risk of spontaneous abortion in Ctinfected pregnant women [8-11]. It is likely that spontaneous abortion is induced by persistent asymptomatic Ct infection spreading to the foetal tissue or the endometrium. The suggested mechanisms by which Ct can trigger abortion are invasion of the microorganism into the
chorioamnionitis.
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choriodecidual space and subsequent immune responses and placental inflammation, especially
Immune effector cell dysfunction has been implicated in the pathogenesis of early pregnancy
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loss [12]. This dysfunction may involve defects in cytokines, growth factors and immunosuppressive
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factors at the materno-foetal interface. Cytokines are important mediators in the bi-directional interaction between the maternal immune system and the reproductive system during pregnancy. This complex interplay between maternal and fetal immune mechanisms changes temporally as pregnancy progresses [13]. If this delicate balance is adversely affected, immunoregulatory mechanisms may be insufficient to restore homeostasis and this may lead to pregnancy failure; also, majority of cytokines are part of a cascade of cytokines released in succession and often act in synergy, and in most cases, are counter-regulated by inhibitory cytokines or soluble receptors [14]. In this regard, T-helper (Th)
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cells play a central role in modulating immune responses [15]. As a rule of thumb is that Th1 cytokines including (TNF-α, IFN-γ, IL-2) are abortive factors [15] and Th2 cytokines (IL-4, IL-5, IL6 and IL-10) are associated with normal pregnancy [16] while Th17 cytokines (IL-17A) are
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associated with foetal loss [17].
Production of chemokines and cytokines by the trophoblast is believed to be important for the maintenance of a normal pregnancy by recruiting immune cells to the maternal-fetal interface and
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influencing their activation status. Tom Wegmann was the first investigator to propose that the Th1/ Th2 paradigm of pregnancy is critical for foetal survival [18]. Various earlier studies in chlamydial
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immunobiology demonstrated a close link between cytokine production patterns and the type of immune responses to this pathogen, with emphasis on the production of Th1/ Th2 cells with different functions and pattern of cytokine release [19-21]. Presently, the Th1/ Th2 paradigm of cytokine expression in chlamydial pathogenesis has been expanded into the Th1/ Th2/ Th17 cells paradigm
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[17,22]. The latter cells (Th17) have been identified as a new lineage of helper T-cells and have been shown to be important in host defense against extracellular infectious agents/ chronic inflammatory diseases. Recently, Th17 cells have also been shown to participate in successful pregnancy, as well as
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in the pathogenesis of diseases of pregnancy, such as recurrent spontaneous abortion [23]. Altered phenotypes and functions of dendritic cells promote the abnormal balance of Th1 and Th17 in the
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development of preeclampsia [24]. Further, in recurrent aborters, increased levels of inflammatory cytokines, including IL-6 and IL-1β have also been observed, and these cytokines have a positive correlation with the proportion of Th-17 cells [25]. It is thus apparent that a greater understanding of the exact mechanism of T-cell/ cytokine
involvement in the pathogenesis of spontaneous abortion in Ct-infected women will hopefully identify more targets for the development of new and effective therapies. To the best of our
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knowledge, till date, there are limited studies on the functional insights of the immunopathology of events ending in early spontaneous abortion in Ct-infected women. The present study aimed to understand the expression pattern/ involvement of circulating Th1/ Th2/ Th17 cytokines in the serum
Materials and methods
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Study population
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of Ct-positive women in the first trimester of pregnancy undergoing spontaneous abortion.
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After obtaining hospital Ethical Committee permission, a total of 145 women (mean age = 25 ± 5.0 years) presenting at Department of Obstetrics & Gynecology, Safdarjung hospital, New Delhi (India) with bleeding per vagina/ passage of clots, etc. and undergoing incomplete spontaneous abortion in the first trimester of pregnancy, were enrolled. All these women underwent dilatation and evacuation and were divided into SA (spontaneous abortion) group; n = 85) and RM (recurrent
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miscarriage group; n = 60). Controls constituted 120 age-matched asymptomatic pregnant undergoing medical termination of pregnancy (mean age = 28 ± 4.0 years) by dilatation and curettage at the hospital. Criteria for enrollment of subjects were as described earlier [7]. Informed written consent
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was obtained from each patient. At the time of recruitment, a detailed clinical questionnaire was
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administered to each patient.
Collection of clinical samples
A viable intrauterine pregnancy was confirmed via ultrasound in each patient. The passed
Endometrial Curettage Tissue (ECT) viz.: the products of conception were collected in Phosphate Buffered Saline (PBS) from all patients in SA group/ RM group/ controls and transported on ice to
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the laboratory. The ECT was used for deoxyribonucleic acid (DNA) extraction while 3.0 ml nonheparinized blood was collected for separation of serum.
Detection of Chlamydia trachomatis in endometrial curettage tissue
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2.3
Polymerase Chain Reaction (PCR) assay was performed for the detection of Ct infection by targeting the Ct plasmid (200 bp) [26] and Major Outer Membrane Protein (MOMP) gene (537 bp)
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[27] in SA group, RM group and controls. DNA was extracted by using a commercial kit (Wizard Genomic DNA Isolation kit, Promega, USA) as per the manufacturer’s instructions and the
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amplification was performed as described earlier [28].
Estimation of cytokines by cytometric bead array (CBA)
A commercial BD CBA Human Th1/ Th2/ Th17 cytokines kit (Becton Dickinson, USA) was
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used to quantitatively measure IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-17A protein levels in serum using the bead array technology as described by the manufacturer. Compared with the traditional ELISA method, this new technology allows for evaluation of multiple cytokines in a single
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sample and utilization of minimal sample volumes to obtain data. During the assay, a mixture of 07 capture beads (with distinct fluorescent intensities) coated with capture antibodies specific for each
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cytokine were utilized. Briefly, the 07 bead populations were mixed together to form the bead array which was resolved in a red channel (i.e., FL3/ FL4) of a flow cytometer. Subsequently, mixed captured beads, unknown serum sample/ recombinant standard, and phycoerythrin (PE) detection reagent were added consecutively to each assay tube and incubated to form sandwich complexes. The intensity of PE fluorescence of each sandwich complex revealed the concentration of the particular cytokine. Subsequently, samples were washed, centrifuged and the bead pellet was resuspended in
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buffer. Thereafter, samples were acquired and measured on the BD FACS ARIA flow cytometer and analyzed by FCAP Array software to generate results in graphical/ tabular format. For calculation, data obtained from a set of diluted standards was used to generate a standard curve for each cytokine
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and the final calculations of cytokine concentrations were made accordingly. Individual cytokine concentrations were indicated by their fluorescent intensities. Cytokine standards were serially diluted to facilitate the construction of calibration curves, which were necessary for determining the
Statistical analysis
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2.5
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protein concentrations of test samples.
Statistical analysis was performed using the Graph Pad prism software (version 5.0, San Diego, CA). Data resulting from cytokine measurement was expressed as median and inter-quartile range while differences between study and control groups were assessed by Mann- Whitney test and
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correlations were measured between variables by Spearman's rank correlation test. ‘p’ value < 0.05 was considered to be significant.
Results
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Detection of Chlamydia trachomatis infection in endometrial curettage tissue of aborters
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Overall 13.7% (20/ 145) patients were Ct-positive in ECT for the cryptic plasmid (n=18/ 20)
and MOMP gene (n=14/ 20) by PCR assays in SA group and RM group in the first trimester. Among these, 12 out of 85 patients (14.1%) in SA group and 8 out of 60 patients (13.3%) in RM group were found to be infected with Ct. All control patients were Ct-negative. The clinical characteristics of enrolled women have been summarized in Table 1.
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Table 1 Baseline characteristics of aborters (N = 265)
Clinical
Range
SA group + RM group
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____________________________________________________________________________________ Controls
‘p’
value
18 – 38
(years)
Gestational age
5- 12
(weeks)
1-6
1-7
0.7401*
7.15 ± 2.15
0.364*
1.55 ± 0.11
2.56 ± 0.084
0.121*
3.74 ± 0.107
0.332*
2.81 ± 0.137
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Gravida
25.52 ± 0.41
7.80 ± 2.30
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Parity
24.08 ± 5.1
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Maternal age
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Characteristics (n =145) (n = 120) ____________________________________________________________________________________
* statistically non-significant
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Abbreviations: SA- spontaneous abortion; RM- recurrent miscarriage
Estimation of circulating Th1/ Th2/ Th17 cytokines in serum of aborters IFN-γ level in serum of Ct-infected SA group (median = 52.2 pg/ ml; IQR = 36.7 - 88.3, ‘p’ <
0.001) and Ct-infected RM group (median = 157.6 pg/ ml; IQR = 122.9 - 191.8, ‘p’ < 0.0001) was significantly high as compared to control patients (median = 30.9 pg/ ml; IQR = 22.9 - 41.8). IFN-γ
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was also found to be significantly upregulated in infected patients (RM group versus SA group; ‘p’ = 0.0008) (Fig. 1A). Significantly high TNF-α production was observed in Ct-positive SA group (median = 51.6 pg/ ml; IQR = 31.2 - 86.1, ‘p’ = 0.0021) and Ct-positive RM group (median = 189 pg/
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ml; IQR = 97.2 - 196, ‘p’ < 0.0001) as compared to controls (median = 30.4 pg/ ml; IQR = 22.4 37.2). Also, TNF-α level between infected SA (i.e. SA group vs RM group) was found to be statistically significant (‘p’ = 0.0023) (Fig. 1B). Further, IL-2 level in serum of Ct-infected SA group
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(median = 38.5 pg/ ml; IQR = 32.0 - 59.0, ‘p’ = 0.0018) and Ct-infected RM group (median = 47.2 pg/ ml; IQR = 30.3 - 114.8, ‘p’= 0.0032) was significantly high as compared to controls (median =
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23.1 pg/ ml; IQR = 15.0 - 32.5). However, IL-2 in Ct-positive SA group vs RM group was not statistically significant (‘p’= 0.512) (Fig. 1C).
IL-4 level in serum of Ct-infected SA group (median = 63.6 pg/ ml; IQR = 28.2 - 90.1, ‘p’ = 0.728) and Ct-infected RM group (median = 67 pg/ ml; IQR = 35.6 – 96.3, ‘p’ = 0.161) was not
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significant high as compared to control patients (median = 32 pg/ ml; IQR = 31.1 – 55.3). IL-4 was also not found to be significant in infected patients (RM group versus SA group; ‘p’ = 0.088) (Fig. 1D). Significantly upregulated IL-6 level was observed in Ct-positive SA group (median = 1045 pg/
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ml; IQR = 610 – 1245, ‘p’ = 0.0037) and Ct-positive RM group (median = 3229 pg/ ml; IQR = 1295 – 3229.0, ‘p’ = 0.0001) as compared to controls (median = 403.5 pg/ ml; IQR = 272.3 - 605.6). The
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IL-6 level in Ct-positive recurrent aborters (RM group) was also found to be significantly upregulated in comparison to the Chlamydia-infected SA group (‘p’ = 0.0049) (Fig. 1E). The level of IL-10 in Ctpositive SA group (median = 31.1 pg/ ml; IQR = 23.2 - 33.8, ‘p’ = 0.045) and Ct-positive RM group (median = 26.6 pg/ ml; IQR = 21.9 - 33.9, ‘p’ = 0.0394) was significantly low as compared to controls (median = 38.6 pg/ ml; IQR = 29.5 - 59.0). Also, IL-10 level in Ct-infected SA group and RM group was not found to be statistically significant (‘p’ = 0.847) (Fig. 1F).
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IL-17A was significantly high in Ct-positive SA group (median = 68.8 pg/ ml; IQR =34.1 90.3, ‘p’= 0.0015) and Ct-positive RM group (median = 160.3 pg/ ml; IQR = 112.3 – 210.1, ‘p’< 0.0001) as compared to controls (median = 27.1 pg/ ml; IQR = 21.1 - 38.4). Also, IL-17A was found
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to be significantly upregulated in the infected RM group aborters in comparison to the infected SA
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group (‘p’ = 0.0018) (Fig. 1G).
Fig. 1 (A-G): Estimation of serum Th1 (IFN-γ, TNF-α, IL-2), Th2 (IL-4, IL-6, IL-10) and Th17 (IL17A) cytokines in Chlamydia trachomatis-positive aborters (SA group, n = 12; RM group, n = 8) and asymptomatic controls (n = 20) by cytometric bead array (non-parametric, Mann-Whitney test). 12
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‘p’ value: * ‘p’ < 0.05, ** ‘p’ < 0.001, *** ‘p’ < 0.0001 Abbreviations: SA- spontaneous abortion group; RM- recurrent abortion group; NS- non-significant
Correlation among circulating Th1/ Th2/ Th17 cytokines in serum of Ct-positive spontaneous
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3.3
aborters
Positive significant correlation was found between TNF-α and IFN-γ (r = 0.809; ‘p’ < 0.0001)
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(Fig. 2A). A statistically significant correlation was also observed between TNF-α and IL-17A (r = 0.783; ‘p’ < 0.0001) (Fig. 2B). IL-17A showed significant positive correlation with IFN-γ (r =
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0.8319; ‘p’ < 0.0001) (Fig. 2C) and IL-6 (r = 0.7665; ‘p’ < 0.0001) (Fig. 2D). However, no statistically significant correlation was found among the remaining cytokines, viz.: IL-2, IL-4 and IL-
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Fig. 2: Correlation among serum IFN-γ, TNF-α, IL-6 and IL-17 cytokines in Chlamydia trachomatispositive aborters: TNF-α showed a significant correlation with (A) IFN-γ (r = 0.809; ‘p’ < 0.0001)
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and (B) IL-17 (r = 0.783.; ‘p’ < 0.0001); (C) IL-17A showed a significant positive correlation with
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IFN-γ (r = 0.8319; ‘p’ < 0.0001) and (D) IL-6 (r = 0.7665; ‘p’ < 0.0001).
Discussion
The exact pathologic mechanism of Chlamydia-induced tissue damage during spontaneous
abortion has not been elucidated, however, chronic inflammatory processes have been implicated and an association between cytokine profile and genital Ct infection has been reported in terms of either resolution or damage. Studies on human pregnancy failure support the concept that successful 14
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pregnancy occurs in a Th2-biased situation, while Th1 type immunity may lead to pregnancy failure. Recent studies showed an increased prevalence of Th17 cells in peripheral blood and decidua in unexplained recurrent spontaneous abortion patients [29]. However to the best of our knowledge,
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there are reportedly no published studies on the role of circulatory cytokines in aborters found infected with Ct. Th1 response, especially TNF-α, IFN-γ and IL-2, has been postulated to be detrimental to the survival of the conceptus [30,31]. When administered to pregnant mice, TNF-α,
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IFN-γ and IL-2 caused miscarriage [32] and it was further reported that miscarriage was reversed by administration of anti-TNF-α. In vitro, TNF-α and IL-2 inhibited outgrowth of human trophoblast
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cells [33] and synergistically stimulated apoptosis of human primary villous trophoblast cells [34]. IFN-γ, a proinflammatory cytokine produced in the uterus during early pregnancy, initiates endometrial vasculature remodelling and contributes to the normal health of the deciduas [35]. However, IFN-γ administration caused pregnancy failure in rabbits [36] and in mice [32]. A recent
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study suggested a new line of evidence correlating the deleterious effects of IFN-γ on pregnancy with the aberrant regulation of CX3CL1 and CD49b+ NK cells [37]. In the present study, Ct-infected aborters in SA group and RM group showed significantly upregulated serum IFN-γ level as compared
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to controls (‘p’ < 0.05); also, the level of IFN-γ was found to be significantly increased in RM group in comparison to SA group (‘p’ < 0.001). IFN-γ was also reported to be significantly higher (‘p’ <
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0.05) in non-pregnant women who had a history of at least three previous miscarriages compared to non-pregnant women with no history of recurrent miscarriage [38]. To the best of our knowledge, till date no studies have been reported showing elevated IFN-γ levels in Ct-infected aborters. Further, it was observed in our study that the level of TNF-α was significantly increased in Ct-infected SA group/ RM group as compared to controls; also, the level of TNF-α was found to be significantly increased in RM group in comparison to SA group (‘p’ < 0.05). Cells from Ct-infected sites release
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small amounts of TNF-α [39]. Likewise, another group observed decreased serum TNF-α concentration during early pregnancy to be associated with abortion in recurrent aborters [40]. However, a study from Iraq reported that aborted women positive for Toxoplasma gondii had
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significantly higher percentage of trophoblasts expressing TNF-α and IL-6 than both aborted women negative for T. gondii and women with induced abortion [41]. In ovine chlamydial abortion, the production of TNF-α by fetal macrophages expressing MHC II molecules was reported to be
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significant in the pathogenesis of abortion [42]. Significantly higher level of IFN-γ (‘p’ = 0.01) and a trend towards increased TNF-α production (‘p’ = 0.07) was found in women suffering recurrent
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pregnancy loss as compared to controls [43]. Furthermore, we observed that the level of serum IL-2 was also significantly elevated in Ct-infected aborters (SA group and RM group) as compared to controls; also IL-2 was high in RM group, however, no statistically significant difference was found in RM group in comparison to the SA group. High level of IL-2 was reported as a risk factor for
history of abortion [44].
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patients with history of recurrent spontaneous abortion as compared to pregnant women with no
In the present study, the level of the Th2 cytokine, viz.: IL-4 showed no significant difference
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between Ct-positive SA group/ RM group as compared to the control group of uninfected aborters. Also, no significant difference was observed between Ct-positive SA group and RM group. Our
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findings were consistent with an Iranian study that also showed similar levels of IL-4 in women with recurrent miscarriages compared to pregnant women with no history of abortion [45]. The other Th2 cytokine viz.: IL-10 played a positive role in the prevention of spontaneous pregnancy failure in a mouse model as it was shown that the injection of IL-10 into abortion-prone mice resulted in the prevention of foetal wastage [46]. In our study, the level of serum IL-10 was significantly increased in controls as compared to Ct-infected SA group/ RM group. Also, there was no statistically
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significant difference between the Ct-positive SA group/ RM group. In other studies, IL-10 was also produced at higher concentrations by peripheral blood mononuclear cells (PBMCs) of women with normal pregnancy than those with a history of unexplained recurrent abortion [44]. Thus, IL-10 has
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emerged as an important Th2-type cytokine in the maintenance of normal pregnancy. Since it is directly involved in downregulating Th1 type activity by inhibiting IFN-γ production, IL-10 has been proposed to play an important immunoregulatory role in pregnancy by maintaining a bias away from
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the detrimental Th1-type of reactivity [46].
The pleiotropic cytokine, IL-6 is frequently evident in the altered cytokine profile
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characteristic of adverse obstetric outcome such as recurrent miscarriage, etc. There is compelling evidence indicating that an altered systemic IL-6 trans-signalling occurs in women prone to recurrent miscarriage, with excessive IL-6 bioavailability potentially inhibiting generation of CD4+ Tregulatory cells required for pregnancy tolerance. In our study, level of IL-6 was upregulated in Ct-
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infected SA group/ RM group as compared to controls; also, the level of IL-6 cytokine was found to be significantly increased in RM group in comparison to SA group (‘p’ < 0.05). Insufficient local IL6 may also contribute to fetal loss, since IL-6 expression is reduced in the endometrium of women
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with recurrent miscarriage and in the fetal-placental tissue of CBA×DBA/2 mice [47]. Other researchers have also shown the need of IL-6 for an early optimal host response against Chlamydia
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infection [48]. IL-6 has been reported to be significantly higher in the cervical mucus of patients who miscarried than in those who had a live birth [49]. Furthermore, our study showed that the Th17 cytokine, IL-17A was upregulated in Ct-
infected aborters (SA group and RM group) as compared to controls. Also, the level of IL-17A was found to be significantly increased in Ct-infected RM group in comparison to the infected SA group (‘p’ < 0.05). In a recent study, the percentage of Th17 cells in women with unexplained recurrent
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abortion in peripheral blood lymphocytes was higher than normal non-pregnant women [50]. Th17 cells have been shown to participate in successful pregnancy, as well as in the pathogenesis of diseases of pregnancy, such as recurrent spontaneous abortion. In recent years, it have been shown
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that Th17 cells produce an array of pro-inflammatory cytokines and host defense molecules and therefore, may play an important role in preventing pathological infection in pregnancy, although a defensive role for Th17 cells in reproductive organs has not yet been reported [23]. However, in
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human recurrent pregnancy loss, excessive Th17 cell numbers and high level of IL-1β, IL-6 and IL17 have been reported, indicating that uncontrolled Th17 cells may emerge as an important mediator
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of inflammation and tissue damage in the diseases of pregnancy [23]. Furthermore, it was reported in a recent study that Treg activation ensures pregnancy success and low Treg cell number increased the risk of pregnancy loss [17]. Our study also suggests the possibility that Th17 cells in implantation sites might be key regulators for maintenance of pregnancy in Ct-infected women. This is because a
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good correlation was observed between Th1/ Th17 cytokines as IFN-γ was found to be strongly correlated with IL-17A in Ct-infected patients belonging to SA group and RM group. It is well established that IFN-γ conditioning enhances the capacity of antigen-presenting cells to drive the
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expansion of memory Th17 cells, and the early Th17 response can induce an optimal Th1 response, facilitate the recruitment of Th1 cells to sites of infection and promote the development of Th1
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memory [51].
We performed two conventional PCR assays targeting MOMP-based and cryptic plasmid and
found that 2/ 20 aborters were positive with the MOMP PCR and negative with the PCR targeting the cryptic plasmid. Further, we found MOMP-based PCR to be negative in 6/ 20 aborters positive with the PCR targeting the plasmid DNA, possibly due to the fact that the plasmid is a high copy number target [52]. Detection of Swedish mutants was not the goal of this work and to the best of our
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knowledge, there are no published studies on the Swedish mutant in Indian population; hence, it is difficult to ascertain whether the Swedish mutant is not circulating or else, has a low prevalence in India. Several other members of the Chlamydiales order have been associated with miscarriage
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including Chlamydia abortus and Waddlia chondrophila and the mechanisms at play in miscarriage due to these chlamydiales encoding for the highly immunogenic MOMP and Polymorphic Membrane Protein (PMP) might also take place by similar pathogenetic mechanisms [53]. However, in order to
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obtain a clear association between cytokine profile and miscarriage, further studies are needed at the local uterine site before pregnancy loss is observed in Chlamydia-infected aborters. This shall firmly
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establish a link between cytokine pattern and abortion in Ct-infected women. Despite this limitation, our study clearly established the role of Th1/ Th2/ Th17 cytokines in spontaneous aborters found infected with Ct and concludes that Chlamydia-positive recurrent aborters had a predominant Th1/
Conflict of Interest
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Th17 bias.
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The authors declare no conflict of interest.
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Acknowledgments
PP is thankful to Council of Scientific and Industrial Research, New Delhi (India) for award
of Junior Research Fellowship (112831156). NS gratefully acknowledges the award of Senior Research Fellowship (SRF) (212830429) by University Grants Commission, New Delhi (India). PP and NS are also thankful to National Institute of Pathology, Indian Council of Medical Research, New Delhi (India) for SRF.
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Highlights Role of cytokines in pathogenesis of C. trachomatis-induced spontaneous abortion.
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Positive correlation between IL-6/ IL-17A cytokines in Chlamydia-positive aborters.
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A predominant Th-1/ Th-17 bias observed in Chlamydia-infected recurrent abortion.
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S0882-4010(16)30921-4
DOI:
10.1016/j.micpath.2017.06.031
Reference:
YMPAT 2323
To appear in:
Microbial Pathogenesis
Received Date: 23 December 2016 Revised Date:
16 June 2017
Accepted Date: 21 June 2017
Please cite this article as: Prasad P, Singh N, Das B, Raisuddin S, Dudeja M, Rastogi S, Differential expression of circulating Th1/ Th2/ Th17 cytokines in serum of Chlamydia trachomatis-infected women undergoing incomplete spontaneous abortion, Microbial Pathogenesis (2017), doi: 10.1016/ j.micpath.2017.06.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title Page
Manuscript title: Differential expression of circulating Th1/ Th2/ Th17 cytokines in serum of
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Chlamydia trachomatis-infected women undergoing incomplete spontaneous abortion
Authors’ names:
Authors’ affiliations: Mrs. Priya Prasad Senior Research Fellow, Microbiology laboratory,
Sriramachari Bhawan,
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National Institute of Pathology (ICMR),
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Priya Prasad1, Namita Singh1, Banashree Das2, Sheikh Raisuddin3, Mridu Dudeja4, Sangita Rastogi1,*
Safdarjung hospital campus, Post Box No. 4909,
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New Delhi-110 029, India
e-mail: [email protected]
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Mrs. Namita Singh
Senior Research Fellow,
Microbiology laboratory,
National Institute of Pathology (ICMR), Sriramachari Bhawan, Safdarjung hospital campus, Post Box No. 4909, New Delhi-110 029, India
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e-mail: [email protected] Dr. Banashree Das Consultant & Professor,
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Department of Obstetrics & Gynecology,
New Delhi-110 029, India e-mail: [email protected]
Dr. Sheikh Raisuddin Professor & Advisor (Research)
Jamia Hamdard (Hamdard University)
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Department of Medical Elementology & Toxicology,
Hamdard Nagar, New Delhi-110 062, India e-mail: [email protected]
Dr. Mridu Dudeja
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Professor & HOD
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Vardhaman Mahavir Medical College (VMMC) & Safdarjung hospital,
Department of Microbiology,
Hamdard Institute of Medical Sciences and Research, Hamdard Nagar, New Delhi-110 044, India
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e-mail: [email protected]
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Dr. Sangita Rastogi
Scientist ‘F’ & Senior Deputy Director, Microbiology laboratory,
National Institute of Pathology (ICMR), Sriramachari Bhawan,
Safdarjung hospital campus, Post Box No. 4909, New Delhi-110 029, India e-mail: [email protected] 2
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Corresponding author: Name and address:
*Dr. Sangita Rastogi Microbiology laboratory,
Sriramachari Bhawan, Microbiology laboratory, Safdarjung hospital campus,
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Post Box No. 4909,
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National Institute of Pathology (ICMR),
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New Delhi-110 029, India
+ 91 - 011-26198 402 - 406 (Ext. 314)
Mobile no.:
+ 91 - 098102 17260
e-mail:
[email protected]
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Tel. no.:
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ABSTRACT The study aimed to elucidate role of Th1/ Th2/ Th17 cytokines in the immunopathogenesis of spontaneous abortion in Chlamydia trachomatis (Ct)-positive first-trimester aborters. Endometrial
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curettage tissue and serum were collected from 145 aborters (spontaneous abortion (SA) group, n = 85; recurrent miscarriage (RM) group, n = 60) and 120 controls attending Department of Obstetrics & Gynecology at Safdarjung hospital, New Delhi (India). Polymerase chain reaction was used to detect
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Ct plasmid/ MOMP, while commercial cytometric bead array kit was utilized to estimate circulating serum cytokines. 13.7% aborters were Ct-positive, however, none was found to be infected among
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controls. IFN-γ, TNF-α, IL-2, IL-6 and IL-17A cytokines were significantly increased in SA group/ RM group (Ct-infected) versus controls. IL-4 showed no difference between groups, while IL-10 was significantly elevated in controls versus Ct-infected subjects in SA group/ RM group. Furthermore, IFN-γ, TNF-α, IL-6, IL-17A cytokines were significantly elevated in Ct-positive RM group versus
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Chlamydia-infected SA group. However, IL-2, IL-4 and IL-10 cytokines showed no significant difference between Ct-positive SA group versus infected RM group. Positive correlation was found between few cytokines (TNF-α and IFN-γ/ IL-17A; IL-17A and IFN-γ/ IL-6) in Ct-positive aborters.
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Our study clearly established the role of Th1/ Th2/ Th17 cytokines in the pathogenesis of spontaneous abortion in Ct-infected subjects and found that Chlamydia-positive recurrent aborters
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had a predominant Th1/ Th17 bias.
Key words: Chlamydia trachomatis; Cytokine bead array; Recurrent miscarriage; Spontaneous abortion; Pathogenesis; Th1/ Th2/ Th17 cytokines
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1.
Introduction Chlamydia trachomatis (Ct) is the leading cause of sexually transmitted infection with the
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annual detection of 92 million new cases worldwide, including 43 million from south-east Asia [1]. A high prevalence of genital Ct infection has also been reported in our country, particularly in females [2-7]. In pregnant women, the presence of Ct is even more critical as it may affect normal intra- and
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extra-uterine development. Intra-uterine infection can threaten pregnancy by gaining access to the placenta, decidua and foetus since pregnancy increases the risk of Ct colonization and alters immune
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response. Pregnant women with chlamydial infection are tenfold more likely to suffer an adverse obstetric outcome and various studies have reported an increased risk of spontaneous abortion in Ctinfected pregnant women [8-11]. It is likely that spontaneous abortion is induced by persistent asymptomatic Ct infection spreading to the foetal tissue or the endometrium. The suggested mechanisms by which Ct can trigger abortion are invasion of the microorganism into the
chorioamnionitis.
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choriodecidual space and subsequent immune responses and placental inflammation, especially
Immune effector cell dysfunction has been implicated in the pathogenesis of early pregnancy
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loss [12]. This dysfunction may involve defects in cytokines, growth factors and immunosuppressive
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factors at the materno-foetal interface. Cytokines are important mediators in the bi-directional interaction between the maternal immune system and the reproductive system during pregnancy. This complex interplay between maternal and fetal immune mechanisms changes temporally as pregnancy progresses [13]. If this delicate balance is adversely affected, immunoregulatory mechanisms may be insufficient to restore homeostasis and this may lead to pregnancy failure; also, majority of cytokines are part of a cascade of cytokines released in succession and often act in synergy, and in most cases, are counter-regulated by inhibitory cytokines or soluble receptors [14]. In this regard, T-helper (Th)
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cells play a central role in modulating immune responses [15]. As a rule of thumb is that Th1 cytokines including (TNF-α, IFN-γ, IL-2) are abortive factors [15] and Th2 cytokines (IL-4, IL-5, IL6 and IL-10) are associated with normal pregnancy [16] while Th17 cytokines (IL-17A) are
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associated with foetal loss [17].
Production of chemokines and cytokines by the trophoblast is believed to be important for the maintenance of a normal pregnancy by recruiting immune cells to the maternal-fetal interface and
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influencing their activation status. Tom Wegmann was the first investigator to propose that the Th1/ Th2 paradigm of pregnancy is critical for foetal survival [18]. Various earlier studies in chlamydial
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immunobiology demonstrated a close link between cytokine production patterns and the type of immune responses to this pathogen, with emphasis on the production of Th1/ Th2 cells with different functions and pattern of cytokine release [19-21]. Presently, the Th1/ Th2 paradigm of cytokine expression in chlamydial pathogenesis has been expanded into the Th1/ Th2/ Th17 cells paradigm
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[17,22]. The latter cells (Th17) have been identified as a new lineage of helper T-cells and have been shown to be important in host defense against extracellular infectious agents/ chronic inflammatory diseases. Recently, Th17 cells have also been shown to participate in successful pregnancy, as well as
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in the pathogenesis of diseases of pregnancy, such as recurrent spontaneous abortion [23]. Altered phenotypes and functions of dendritic cells promote the abnormal balance of Th1 and Th17 in the
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development of preeclampsia [24]. Further, in recurrent aborters, increased levels of inflammatory cytokines, including IL-6 and IL-1β have also been observed, and these cytokines have a positive correlation with the proportion of Th-17 cells [25]. It is thus apparent that a greater understanding of the exact mechanism of T-cell/ cytokine
involvement in the pathogenesis of spontaneous abortion in Ct-infected women will hopefully identify more targets for the development of new and effective therapies. To the best of our
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knowledge, till date, there are limited studies on the functional insights of the immunopathology of events ending in early spontaneous abortion in Ct-infected women. The present study aimed to understand the expression pattern/ involvement of circulating Th1/ Th2/ Th17 cytokines in the serum
Materials and methods
2.1
Study population
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2.
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of Ct-positive women in the first trimester of pregnancy undergoing spontaneous abortion.
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After obtaining hospital Ethical Committee permission, a total of 145 women (mean age = 25 ± 5.0 years) presenting at Department of Obstetrics & Gynecology, Safdarjung hospital, New Delhi (India) with bleeding per vagina/ passage of clots, etc. and undergoing incomplete spontaneous abortion in the first trimester of pregnancy, were enrolled. All these women underwent dilatation and evacuation and were divided into SA (spontaneous abortion) group; n = 85) and RM (recurrent
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miscarriage group; n = 60). Controls constituted 120 age-matched asymptomatic pregnant undergoing medical termination of pregnancy (mean age = 28 ± 4.0 years) by dilatation and curettage at the hospital. Criteria for enrollment of subjects were as described earlier [7]. Informed written consent
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was obtained from each patient. At the time of recruitment, a detailed clinical questionnaire was
2.2
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administered to each patient.
Collection of clinical samples
A viable intrauterine pregnancy was confirmed via ultrasound in each patient. The passed
Endometrial Curettage Tissue (ECT) viz.: the products of conception were collected in Phosphate Buffered Saline (PBS) from all patients in SA group/ RM group/ controls and transported on ice to
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the laboratory. The ECT was used for deoxyribonucleic acid (DNA) extraction while 3.0 ml nonheparinized blood was collected for separation of serum.
Detection of Chlamydia trachomatis in endometrial curettage tissue
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2.3
Polymerase Chain Reaction (PCR) assay was performed for the detection of Ct infection by targeting the Ct plasmid (200 bp) [26] and Major Outer Membrane Protein (MOMP) gene (537 bp)
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[27] in SA group, RM group and controls. DNA was extracted by using a commercial kit (Wizard Genomic DNA Isolation kit, Promega, USA) as per the manufacturer’s instructions and the
2.4
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amplification was performed as described earlier [28].
Estimation of cytokines by cytometric bead array (CBA)
A commercial BD CBA Human Th1/ Th2/ Th17 cytokines kit (Becton Dickinson, USA) was
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used to quantitatively measure IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-17A protein levels in serum using the bead array technology as described by the manufacturer. Compared with the traditional ELISA method, this new technology allows for evaluation of multiple cytokines in a single
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sample and utilization of minimal sample volumes to obtain data. During the assay, a mixture of 07 capture beads (with distinct fluorescent intensities) coated with capture antibodies specific for each
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cytokine were utilized. Briefly, the 07 bead populations were mixed together to form the bead array which was resolved in a red channel (i.e., FL3/ FL4) of a flow cytometer. Subsequently, mixed captured beads, unknown serum sample/ recombinant standard, and phycoerythrin (PE) detection reagent were added consecutively to each assay tube and incubated to form sandwich complexes. The intensity of PE fluorescence of each sandwich complex revealed the concentration of the particular cytokine. Subsequently, samples were washed, centrifuged and the bead pellet was resuspended in
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buffer. Thereafter, samples were acquired and measured on the BD FACS ARIA flow cytometer and analyzed by FCAP Array software to generate results in graphical/ tabular format. For calculation, data obtained from a set of diluted standards was used to generate a standard curve for each cytokine
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and the final calculations of cytokine concentrations were made accordingly. Individual cytokine concentrations were indicated by their fluorescent intensities. Cytokine standards were serially diluted to facilitate the construction of calibration curves, which were necessary for determining the
Statistical analysis
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2.5
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protein concentrations of test samples.
Statistical analysis was performed using the Graph Pad prism software (version 5.0, San Diego, CA). Data resulting from cytokine measurement was expressed as median and inter-quartile range while differences between study and control groups were assessed by Mann- Whitney test and
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correlations were measured between variables by Spearman's rank correlation test. ‘p’ value < 0.05 was considered to be significant.
Results
3.1
Detection of Chlamydia trachomatis infection in endometrial curettage tissue of aborters
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3.
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Overall 13.7% (20/ 145) patients were Ct-positive in ECT for the cryptic plasmid (n=18/ 20)
and MOMP gene (n=14/ 20) by PCR assays in SA group and RM group in the first trimester. Among these, 12 out of 85 patients (14.1%) in SA group and 8 out of 60 patients (13.3%) in RM group were found to be infected with Ct. All control patients were Ct-negative. The clinical characteristics of enrolled women have been summarized in Table 1.
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Table 1 Baseline characteristics of aborters (N = 265)
Clinical
Range
SA group + RM group
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____________________________________________________________________________________ Controls
‘p’
value
18 – 38
(years)
Gestational age
5- 12
(weeks)
1-6
1-7
0.7401*
7.15 ± 2.15
0.364*
1.55 ± 0.11
2.56 ± 0.084
0.121*
3.74 ± 0.107
0.332*
2.81 ± 0.137
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Gravida
25.52 ± 0.41
7.80 ± 2.30
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Parity
24.08 ± 5.1
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Maternal age
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Characteristics (n =145) (n = 120) ____________________________________________________________________________________
* statistically non-significant
3.2
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Abbreviations: SA- spontaneous abortion; RM- recurrent miscarriage
Estimation of circulating Th1/ Th2/ Th17 cytokines in serum of aborters IFN-γ level in serum of Ct-infected SA group (median = 52.2 pg/ ml; IQR = 36.7 - 88.3, ‘p’ <
0.001) and Ct-infected RM group (median = 157.6 pg/ ml; IQR = 122.9 - 191.8, ‘p’ < 0.0001) was significantly high as compared to control patients (median = 30.9 pg/ ml; IQR = 22.9 - 41.8). IFN-γ
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was also found to be significantly upregulated in infected patients (RM group versus SA group; ‘p’ = 0.0008) (Fig. 1A). Significantly high TNF-α production was observed in Ct-positive SA group (median = 51.6 pg/ ml; IQR = 31.2 - 86.1, ‘p’ = 0.0021) and Ct-positive RM group (median = 189 pg/
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ml; IQR = 97.2 - 196, ‘p’ < 0.0001) as compared to controls (median = 30.4 pg/ ml; IQR = 22.4 37.2). Also, TNF-α level between infected SA (i.e. SA group vs RM group) was found to be statistically significant (‘p’ = 0.0023) (Fig. 1B). Further, IL-2 level in serum of Ct-infected SA group
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(median = 38.5 pg/ ml; IQR = 32.0 - 59.0, ‘p’ = 0.0018) and Ct-infected RM group (median = 47.2 pg/ ml; IQR = 30.3 - 114.8, ‘p’= 0.0032) was significantly high as compared to controls (median =
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23.1 pg/ ml; IQR = 15.0 - 32.5). However, IL-2 in Ct-positive SA group vs RM group was not statistically significant (‘p’= 0.512) (Fig. 1C).
IL-4 level in serum of Ct-infected SA group (median = 63.6 pg/ ml; IQR = 28.2 - 90.1, ‘p’ = 0.728) and Ct-infected RM group (median = 67 pg/ ml; IQR = 35.6 – 96.3, ‘p’ = 0.161) was not
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significant high as compared to control patients (median = 32 pg/ ml; IQR = 31.1 – 55.3). IL-4 was also not found to be significant in infected patients (RM group versus SA group; ‘p’ = 0.088) (Fig. 1D). Significantly upregulated IL-6 level was observed in Ct-positive SA group (median = 1045 pg/
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ml; IQR = 610 – 1245, ‘p’ = 0.0037) and Ct-positive RM group (median = 3229 pg/ ml; IQR = 1295 – 3229.0, ‘p’ = 0.0001) as compared to controls (median = 403.5 pg/ ml; IQR = 272.3 - 605.6). The
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IL-6 level in Ct-positive recurrent aborters (RM group) was also found to be significantly upregulated in comparison to the Chlamydia-infected SA group (‘p’ = 0.0049) (Fig. 1E). The level of IL-10 in Ctpositive SA group (median = 31.1 pg/ ml; IQR = 23.2 - 33.8, ‘p’ = 0.045) and Ct-positive RM group (median = 26.6 pg/ ml; IQR = 21.9 - 33.9, ‘p’ = 0.0394) was significantly low as compared to controls (median = 38.6 pg/ ml; IQR = 29.5 - 59.0). Also, IL-10 level in Ct-infected SA group and RM group was not found to be statistically significant (‘p’ = 0.847) (Fig. 1F).
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IL-17A was significantly high in Ct-positive SA group (median = 68.8 pg/ ml; IQR =34.1 90.3, ‘p’= 0.0015) and Ct-positive RM group (median = 160.3 pg/ ml; IQR = 112.3 – 210.1, ‘p’< 0.0001) as compared to controls (median = 27.1 pg/ ml; IQR = 21.1 - 38.4). Also, IL-17A was found
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to be significantly upregulated in the infected RM group aborters in comparison to the infected SA
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group (‘p’ = 0.0018) (Fig. 1G).
Fig. 1 (A-G): Estimation of serum Th1 (IFN-γ, TNF-α, IL-2), Th2 (IL-4, IL-6, IL-10) and Th17 (IL17A) cytokines in Chlamydia trachomatis-positive aborters (SA group, n = 12; RM group, n = 8) and asymptomatic controls (n = 20) by cytometric bead array (non-parametric, Mann-Whitney test). 12
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‘p’ value: * ‘p’ < 0.05, ** ‘p’ < 0.001, *** ‘p’ < 0.0001 Abbreviations: SA- spontaneous abortion group; RM- recurrent abortion group; NS- non-significant
Correlation among circulating Th1/ Th2/ Th17 cytokines in serum of Ct-positive spontaneous
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3.3
aborters
Positive significant correlation was found between TNF-α and IFN-γ (r = 0.809; ‘p’ < 0.0001)
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(Fig. 2A). A statistically significant correlation was also observed between TNF-α and IL-17A (r = 0.783; ‘p’ < 0.0001) (Fig. 2B). IL-17A showed significant positive correlation with IFN-γ (r =
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0.8319; ‘p’ < 0.0001) (Fig. 2C) and IL-6 (r = 0.7665; ‘p’ < 0.0001) (Fig. 2D). However, no statistically significant correlation was found among the remaining cytokines, viz.: IL-2, IL-4 and IL-
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10.
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Fig. 2: Correlation among serum IFN-γ, TNF-α, IL-6 and IL-17 cytokines in Chlamydia trachomatispositive aborters: TNF-α showed a significant correlation with (A) IFN-γ (r = 0.809; ‘p’ < 0.0001)
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and (B) IL-17 (r = 0.783.; ‘p’ < 0.0001); (C) IL-17A showed a significant positive correlation with
4.
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IFN-γ (r = 0.8319; ‘p’ < 0.0001) and (D) IL-6 (r = 0.7665; ‘p’ < 0.0001).
Discussion
The exact pathologic mechanism of Chlamydia-induced tissue damage during spontaneous
abortion has not been elucidated, however, chronic inflammatory processes have been implicated and an association between cytokine profile and genital Ct infection has been reported in terms of either resolution or damage. Studies on human pregnancy failure support the concept that successful 14
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pregnancy occurs in a Th2-biased situation, while Th1 type immunity may lead to pregnancy failure. Recent studies showed an increased prevalence of Th17 cells in peripheral blood and decidua in unexplained recurrent spontaneous abortion patients [29]. However to the best of our knowledge,
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there are reportedly no published studies on the role of circulatory cytokines in aborters found infected with Ct. Th1 response, especially TNF-α, IFN-γ and IL-2, has been postulated to be detrimental to the survival of the conceptus [30,31]. When administered to pregnant mice, TNF-α,
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IFN-γ and IL-2 caused miscarriage [32] and it was further reported that miscarriage was reversed by administration of anti-TNF-α. In vitro, TNF-α and IL-2 inhibited outgrowth of human trophoblast
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cells [33] and synergistically stimulated apoptosis of human primary villous trophoblast cells [34]. IFN-γ, a proinflammatory cytokine produced in the uterus during early pregnancy, initiates endometrial vasculature remodelling and contributes to the normal health of the deciduas [35]. However, IFN-γ administration caused pregnancy failure in rabbits [36] and in mice [32]. A recent
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study suggested a new line of evidence correlating the deleterious effects of IFN-γ on pregnancy with the aberrant regulation of CX3CL1 and CD49b+ NK cells [37]. In the present study, Ct-infected aborters in SA group and RM group showed significantly upregulated serum IFN-γ level as compared
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to controls (‘p’ < 0.05); also, the level of IFN-γ was found to be significantly increased in RM group in comparison to SA group (‘p’ < 0.001). IFN-γ was also reported to be significantly higher (‘p’ <
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0.05) in non-pregnant women who had a history of at least three previous miscarriages compared to non-pregnant women with no history of recurrent miscarriage [38]. To the best of our knowledge, till date no studies have been reported showing elevated IFN-γ levels in Ct-infected aborters. Further, it was observed in our study that the level of TNF-α was significantly increased in Ct-infected SA group/ RM group as compared to controls; also, the level of TNF-α was found to be significantly increased in RM group in comparison to SA group (‘p’ < 0.05). Cells from Ct-infected sites release
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small amounts of TNF-α [39]. Likewise, another group observed decreased serum TNF-α concentration during early pregnancy to be associated with abortion in recurrent aborters [40]. However, a study from Iraq reported that aborted women positive for Toxoplasma gondii had
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significantly higher percentage of trophoblasts expressing TNF-α and IL-6 than both aborted women negative for T. gondii and women with induced abortion [41]. In ovine chlamydial abortion, the production of TNF-α by fetal macrophages expressing MHC II molecules was reported to be
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significant in the pathogenesis of abortion [42]. Significantly higher level of IFN-γ (‘p’ = 0.01) and a trend towards increased TNF-α production (‘p’ = 0.07) was found in women suffering recurrent
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pregnancy loss as compared to controls [43]. Furthermore, we observed that the level of serum IL-2 was also significantly elevated in Ct-infected aborters (SA group and RM group) as compared to controls; also IL-2 was high in RM group, however, no statistically significant difference was found in RM group in comparison to the SA group. High level of IL-2 was reported as a risk factor for
history of abortion [44].
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patients with history of recurrent spontaneous abortion as compared to pregnant women with no
In the present study, the level of the Th2 cytokine, viz.: IL-4 showed no significant difference
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between Ct-positive SA group/ RM group as compared to the control group of uninfected aborters. Also, no significant difference was observed between Ct-positive SA group and RM group. Our
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findings were consistent with an Iranian study that also showed similar levels of IL-4 in women with recurrent miscarriages compared to pregnant women with no history of abortion [45]. The other Th2 cytokine viz.: IL-10 played a positive role in the prevention of spontaneous pregnancy failure in a mouse model as it was shown that the injection of IL-10 into abortion-prone mice resulted in the prevention of foetal wastage [46]. In our study, the level of serum IL-10 was significantly increased in controls as compared to Ct-infected SA group/ RM group. Also, there was no statistically
16
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significant difference between the Ct-positive SA group/ RM group. In other studies, IL-10 was also produced at higher concentrations by peripheral blood mononuclear cells (PBMCs) of women with normal pregnancy than those with a history of unexplained recurrent abortion [44]. Thus, IL-10 has
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emerged as an important Th2-type cytokine in the maintenance of normal pregnancy. Since it is directly involved in downregulating Th1 type activity by inhibiting IFN-γ production, IL-10 has been proposed to play an important immunoregulatory role in pregnancy by maintaining a bias away from
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the detrimental Th1-type of reactivity [46].
The pleiotropic cytokine, IL-6 is frequently evident in the altered cytokine profile
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characteristic of adverse obstetric outcome such as recurrent miscarriage, etc. There is compelling evidence indicating that an altered systemic IL-6 trans-signalling occurs in women prone to recurrent miscarriage, with excessive IL-6 bioavailability potentially inhibiting generation of CD4+ Tregulatory cells required for pregnancy tolerance. In our study, level of IL-6 was upregulated in Ct-
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infected SA group/ RM group as compared to controls; also, the level of IL-6 cytokine was found to be significantly increased in RM group in comparison to SA group (‘p’ < 0.05). Insufficient local IL6 may also contribute to fetal loss, since IL-6 expression is reduced in the endometrium of women
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with recurrent miscarriage and in the fetal-placental tissue of CBA×DBA/2 mice [47]. Other researchers have also shown the need of IL-6 for an early optimal host response against Chlamydia
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infection [48]. IL-6 has been reported to be significantly higher in the cervical mucus of patients who miscarried than in those who had a live birth [49]. Furthermore, our study showed that the Th17 cytokine, IL-17A was upregulated in Ct-
infected aborters (SA group and RM group) as compared to controls. Also, the level of IL-17A was found to be significantly increased in Ct-infected RM group in comparison to the infected SA group (‘p’ < 0.05). In a recent study, the percentage of Th17 cells in women with unexplained recurrent
17
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abortion in peripheral blood lymphocytes was higher than normal non-pregnant women [50]. Th17 cells have been shown to participate in successful pregnancy, as well as in the pathogenesis of diseases of pregnancy, such as recurrent spontaneous abortion. In recent years, it have been shown
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that Th17 cells produce an array of pro-inflammatory cytokines and host defense molecules and therefore, may play an important role in preventing pathological infection in pregnancy, although a defensive role for Th17 cells in reproductive organs has not yet been reported [23]. However, in
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human recurrent pregnancy loss, excessive Th17 cell numbers and high level of IL-1β, IL-6 and IL17 have been reported, indicating that uncontrolled Th17 cells may emerge as an important mediator
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of inflammation and tissue damage in the diseases of pregnancy [23]. Furthermore, it was reported in a recent study that Treg activation ensures pregnancy success and low Treg cell number increased the risk of pregnancy loss [17]. Our study also suggests the possibility that Th17 cells in implantation sites might be key regulators for maintenance of pregnancy in Ct-infected women. This is because a
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good correlation was observed between Th1/ Th17 cytokines as IFN-γ was found to be strongly correlated with IL-17A in Ct-infected patients belonging to SA group and RM group. It is well established that IFN-γ conditioning enhances the capacity of antigen-presenting cells to drive the
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expansion of memory Th17 cells, and the early Th17 response can induce an optimal Th1 response, facilitate the recruitment of Th1 cells to sites of infection and promote the development of Th1
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memory [51].
We performed two conventional PCR assays targeting MOMP-based and cryptic plasmid and
found that 2/ 20 aborters were positive with the MOMP PCR and negative with the PCR targeting the cryptic plasmid. Further, we found MOMP-based PCR to be negative in 6/ 20 aborters positive with the PCR targeting the plasmid DNA, possibly due to the fact that the plasmid is a high copy number target [52]. Detection of Swedish mutants was not the goal of this work and to the best of our
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knowledge, there are no published studies on the Swedish mutant in Indian population; hence, it is difficult to ascertain whether the Swedish mutant is not circulating or else, has a low prevalence in India. Several other members of the Chlamydiales order have been associated with miscarriage
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including Chlamydia abortus and Waddlia chondrophila and the mechanisms at play in miscarriage due to these chlamydiales encoding for the highly immunogenic MOMP and Polymorphic Membrane Protein (PMP) might also take place by similar pathogenetic mechanisms [53]. However, in order to
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obtain a clear association between cytokine profile and miscarriage, further studies are needed at the local uterine site before pregnancy loss is observed in Chlamydia-infected aborters. This shall firmly
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establish a link between cytokine pattern and abortion in Ct-infected women. Despite this limitation, our study clearly established the role of Th1/ Th2/ Th17 cytokines in spontaneous aborters found infected with Ct and concludes that Chlamydia-positive recurrent aborters had a predominant Th1/
Conflict of Interest
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Th17 bias.
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The authors declare no conflict of interest.
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Acknowledgments
PP is thankful to Council of Scientific and Industrial Research, New Delhi (India) for award
of Junior Research Fellowship (112831156). NS gratefully acknowledges the award of Senior Research Fellowship (SRF) (212830429) by University Grants Commission, New Delhi (India). PP and NS are also thankful to National Institute of Pathology, Indian Council of Medical Research, New Delhi (India) for SRF.
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chromosome-based polymerase chain reaction assays for detecting Chlamydia trachomatis
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Highlights Role of cytokines in pathogenesis of C. trachomatis-induced spontaneous abortion.
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Positive correlation between IL-6/ IL-17A cytokines in Chlamydia-positive aborters.
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A predominant Th-1/ Th-17 bias observed in Chlamydia-infected recurrent abortion.
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