Skip to main content
NCBI home page
Reproductive Sciences logoLink to Reproductive Sciences
. 2018 Dec 20;26(11):1449–1459. doi: 10.1177/1933719118820468

Low-Dose Aspirin May Prevent Trophoblast Dysfunction in Women With Chlamydia Pneumoniae Infection

Luis M Gomez 1,✉, Lauren Anton 2, Shindu K Srinivas 2,3, Michal A Elovitz 2,3, Samuel Parry 2,3
PMCID: PMC6949957  PMID: 30572799

Abstract

Objective:

Previously, we demonstrated that live Chlamydia pneumoniae (Cp) impaired extravillous trophoblast (EVT) viability and invasion and that Cp DNA was detected in placentas from cases with preeclampsia. We sought to elucidate whether (1) inactive forms of Cp also affect EVT function; (2) potential therapeutic interventions protect against the effects of Cp; and (3) anti-Cp antibodies are associated with preeclampsia.

Methods:

Human first-trimester EVTs were infected with ultraviolet light-inactivated Cp. Subgroups of EVTs were pretreated with low-dose acetyl-salicylic acid (ASA), dexamethasone, heparin, and indomethacin. We conducted functional assays after infection with inactivated Cp and measured interleukin 8 (IL8), C-reactive protein (CRP), heat shock protein 60 (HSP60), and tumor necrosis factor-α (TNFα) in culture media. We measured anti-Cp IgG serum levels from women who developed preeclampsia (N = 105) and controls (N = 121).

Results:

Inactivated Cp reduced EVT invasion when compared to noninfected cells (P < .00001) without adversely affecting cell viability. Increased levels of IL8, CRP, HSP60, and TNFα were detected in EVTs infected with inactivated Cp compared to noninfected cells (P < .0001). Only pretreatment with low-dose ASA prevented reduced EVT invasion and decreased release of inflammatory mediators (P < .01). Elevated anti-Cp IgG antibodies were more prevalent in serum from cases with preeclampsia compared to controls (67/105 vs 53/121; adjusted P = .013); elevated IgG correlated significantly with elevated serum CRP and elevated soluble fms-like tyrosine kinase-1–placental growth factor ratio.

Conclusion:

Inactivated Cp induces decreased EVT invasion and a proinflammatory response; these effects were abrogated by pretreatment with low-dose ASA. Our results suggest an association between Cp infection, trophoblast dysfunction, and preeclampsia.

Keywords: extravillous trophoblast, Chlamydia pneumoniae, elementary body, MOMP, sFlt-1, placental growth factor, interleukin 8, tumor necrosis factor α, heat shock protein, cytokines

Introduction

Chlamydia pneumoniae (Cp) is an intracellular bacterium that causes human respiratory infections. Seroepidemiologic studies, direct pathologic analysis of plaques, and limited clinical intervention studies suggest that acute and chronic infection with Cp are also risk factors for atherosclerosis.1-9 Preeclampsia shares risk factors and pathophysiological features with atherosclerosis. Known hallmarks of preeclampsia include decreased trophoblast migration and atherosclerotic changes in the uterine vasculature.10-12

Chlamydia pneumoniae has a unique biphasic developmental cycle: In susceptible cells, live Cp replicates as in acute infection (elementary body) or enters a persistent state in a nonproductive form eliciting a chronic host response.13-15 Only the elementary body (live Cp) displays the major outer membrane protein (MOMP). Previously, our group demonstrated that (1) live Cp was able to infect human first-trimester primary extravillous trophoblast (EVT) cells by detecting the presence of MOMP-DNA in infected EVT cells; (2) this acute Cp infection led to decreased cell viability and impaired EVT cell invasion; and (3) Cp MOMP-DNA was detected more frequently in placentas from cases with preeclampsia than controls.16

Both live and nonproductive or inactivated forms of Cp (representing acute and chronic Cp infection, respectively) induce endothelial, smooth muscle, and macrophage cell changes leading to atherosclerosis.3,17-20 Epidemiologic studies showed that many young adults are IgG seropositive to Cp indicating chronic exposure.21,22 While we know the effects of live Cp on trophoblast cells related to acute infection,16 the effects of inactivated Cp (nonproductive bacterial form related to past infection) on EVT cells and the clinical impact in pregnancy have not been investigated. We hypothesize that, similar to what we observed with live Cp,16 EVT infection with inactivated Cp elicits cell dysfunction. Therefore, we sought to investigate: (1) whether inactivated Cp affects EVT cell invasion; (2) whether potential therapeutic interventions are protective against the effects of inactivated Cp on EVT cells; and (3) whether elevated maternal serum anti-Cp IgG levels are associated with adverse pregnancy outcomes as preeclampsia.

Materials and Methods

Preparation of EVT Cells for In Vitro Experiments

We conducted in vitro experiments utilizing primary human EVT cells because they display invasive properties through extracellular matrices (ECMs) that resemble in vivo invasion during placentation.23 Primary EVT cells were isolated and propagated from human first-trimester placental tissues (8-13 weeks’ gestation).24,25 Briefly, minced chorionic villi were cultured at 37°C with Dulbecco modified Eagle medium (Gibco BRL, Grand Island, New York) containing 10% fetal bovine serum, gentamicin (100 μg/mL), and amphotericin B (2.5 μg/mL). Extravillous trophoblast cells that outgrew from attached villous fragments were separated from villous trophoblast cells on days 10 to 12 of culture. The isolated EVT cells were seeded and propagated in the same culture medium.25,26

Primary EVT cells used in our experiments were characterized by immunostaining of human leukocyte antigen–G (HLA-G) and cytokeratin filaments 8 and 18 (Figure 1, A-C).27 In previous cell preparations in our laboratory, >95% of isolated EVT cells stained positive for cytokeratin-18 and HLA–G25 with low representation for HLA-A, -B, and -C and vimentin.25,28 In this study, further characterization of the purity of the EVT cells was accomplished by cytokeratin-7 and integrin α-I staining as shown by other investigators (Figure 1, D-E).26,29-31

Figure 1.

Figure 1.

Open in a new tab

A-F, Immunocytochemical staining of primary extravillous trophoblast cell markers.

Infection of EVT Cells With Inactivated Cp

For our experiments, we chose the Cp TWAR 183 strain (ATCC VR2282).16,32 The infectivity of Cp was assessed in HEp2 cells (ATCC CCL-23, a known human cell line host for Cp). Aliquots of supernatant containing Cp at 0.5 to 1.0 infecting forming units/cell were used in duplicate cultures of EVT cells overnight, after which the culture medium was replaced with fresh DMEM not containing Cp. To obtain inactivated Cp before infecting EVT cells, the bacteria contained in the supernatant were subjected to ultraviolet (UV) irradiation for 30 minutes at 15 cm from the UV source (1200×100 mJ/cm2; Hoefer UVC500, Hoefer, Inc, San Francisco, California).33,34

Assessment of Inactivated Cp Infection in EVT Cells

To assess the success of Cp inactivation and infection, a subset of EVT cells were infected with live bacteria for comparison purposes. Subsequently, primary EVT cells infected with live and inactivated forms of Cp were harvested for DNA extraction using Qiagen DNA mini kits (Qiagen, Valencia, California) as previously described.16 Extracted DNA from infected EVT cells were used for nested touchdown polymerase chain reaction (PCR) to assess whether MOMP sequences (key component of live Cp)35 were detected in infected cells. Each set of PCR amplifications included positive controls (DNA extracted from HEp2 cells infected with live Cp) and negative controls (noninfected EVT cells).

Functional Assays

For our experiments, we used the following EVT cell groups: (1) cells infected with inactivated Cp, (2) noninfected cells (negative controls), and (3) cells exposed to dilution buffer (second set of negative controls). All experiments were conducted in triplicate in 2 separate sets.

We assessed cell viability based on lactate dehydrogenase (LDH) release using Cyto Tox 96 Non-Radioactive Cytotoxicity Assay kit (Promega Corp, Madison, Wisconsin).36 After collecting cell culture medium, EVT cells infected with inactivated Cp and EVT cells that served as negative controls were lysed; equal volumes (50 μL) of medium and lysis buffer (containing LDH released from lysed cells) were transferred to separate 96-well plates. After adding a reconstituted substrate mix followed by a stop solution, absorbance based on the reaction between LDH and substrate was recorded using a microplate reader at 490 nm. Results were obtained after subtracting background values. We calculated the absorbance ratio of (lysis buffer) / (absorbance of lysis buffer + cell culture medium) to determine the percentage of cells that remained viable at 48 hours from Cp infection.37 For comparison purposes, we also assessed viability in EVT cells infected with live Cp (positive controls).

A separate set of primary EVT cells and culture media were collected to perform invasion assays 48 hours after Cp infection. We used 1 × 104 cells per well in 96-well plates (1 × 106 cells/mL). Invasiveness of EVT cells through an extracellular matrix (ECM) Matrigel (BD Biosciences, San Jose, California) was measured using Cell Invasion Assay Kits (Chemicon International, Temecula, California). Briefly, 1 × 106 cells/mL were placed in invasion chambers. After 48 hours, cells that invaded through the ECM and across the semipermeable membrane were stained with Cell Stain provided by the manufacturer and treated with 10% acetic acid. A volume of 150 μL of the dye/solute mixture was transferred to 96-well plates, and invasion was measured by colorimetric absorbance at optical density (OD) of 560 nm. Levels of invasion were determined by comparison of average OD values of Cp-infected primary EVT cells to those of noninfected cells.16,30 Invasion rates were normalized to 1.0 in noninfected EVT cells.

Enzyme–Linked Immunosorbent Assay to Assess Inflammation in EVT Cells Infected With Inactivated Cp

We studied the expression of interleukin 8 (IL8), C-reactive protein (CRP), heat shock protein 60 (HSP60), and tumor necrosis factor-α (TNFα) in culture media of EVT cells infected with Cp. Supernatants of EVT cell cultures were collected at 48 hours and stored at −30°C until assayed. Levels of IL8, CRP, HSP60, and TNFα were measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Inc., Minneapolis, Minnesota). Briefly, 96-well ELISA microplates were coated with a capture antibody; after blocking with 5% BSA, supernatant samples were added to the coated plates. This was followed with a biotin-conjugated detection antibody. Antibody binding was measured with horseradish peroxidase-conjugated avidin along with a soluble colorimetric substrate. The absorbance (OD) was read at 450 nm with an ELISA photometer (VWR, International, Bridgeport, New Jersey). Each assay was performed in triplicate for all studied EVT cell subgroups, and the average OD was calculated.

Assessment of Invasion and Inflammation in EVT Cells Pretreated With Individual Pharmacologic Agents Prior to Infection With Cp

Physiological concentrations in culture media of low-dose acetyl-salicylic acid—aspirin—(ASA) 1 and 2.5 mmol/L, dexamethasone 1.2 µmol/L, heparin IU/mL, and indomethacin 10µmol/L were used to pretreat a subset of EVT cells for 24 hours before infection with inactivated Cp. After pretreatment, the culture media were replaced, and EVT cells were maintained in fresh media for 24 hours before infection. Invasion assays and ELISA were performed in triplicate as described earlier. The concentrations of the pharmacologic agents (SIGMA-ALDRICH, St Louis, Missouri) used in these experiments were based on prior reports and equivalent to low dose (ASA and heparin) or routine dose (dexamethasone and indomethacin) medications used in clinical settings.38-52

Case–Control Study Design and ELISA Experiments to Correlate Maternal Cp Infection With Pregnancy Outcomes

We conducted a secondary analysis of an already existing case–control study, “Preeclampsia: Mechanisms and Consequences” (PMC study).53 Institutional review board approval was obtained. Cases consisted of women diagnosed with preeclampsia;54 controls included women admitted in labor who delivered at term with no obstetric complications. Serum samples were collected prospectively in the third trimester from participants at or within 24 hours of enrollment.53

Samples were centrifuged for 10 minutes at 10 000g; the supernatant serum was aspirated and stored at –80°C. Serum samples were available for 226 participants. The ELISA kits (Sigma-Aldrich) containing human anti-Cp IgG (positive control) and dilution buffer (negative control) were used in 96-well plates. The concentrations of anti-Cp IgG were measured, and absorbance was read at 450 nm with an ELISA photometer (VWR International, Bridgeport, New Jersey). Each assay was performed in duplicate, and the average OD was calculated.

The results were expressed as index value calculated by dividing the patient’s OD by the mean OD absorbance value of the cutoff point. An index value >1.1 was considered positive.55-57 In our PMC case-control study,53 biomarkers of inflammation and angiogenesis had been obtained by ELISA: CRP (median values, mg/L), and soluble fms-like tyrosine kinase-1/placental growth factor (a validated sFlt-1/PlGF ratio >38 is associated with preeclampsia).58

Statistical Analysis

We used SPSS statistical software (SPSS Inc, Chicago, Illinois) for our analyses. Mean OD values and standard errors (SEs) were calculated and compared between Cp-exposed and control EVTs using analysis of variance (ANOVA). Mean, median values, and standard deviations were used for comparison of demographics and outcomes between cases and controls. χ2 or Fisher’s exact tests were used for dichotomous variables, and unpaired t tests for continuous variables. For our case–control study categorical outcomes, we used cross-tabulation to estimate odds ratio (OR) and 95% confidence intervals (CIs). Logistic regression analyses were used where applicable. A 2-sided P value of <.05 was indicative of significance.

Results

Primary EVT cells infected with live Cp demonstrated MOMP-coding gene sequences similar to positive controls indicating successful infection. Meanwhile, MOMP-DNA was not detected by PCR in EVT cells infected with inactivated Cp (comparable to negative controls: non-infected EVT cells and cells exposed to dilution buffer) indicating successful UV inactivation (Figure 2).

Figure 2.

Figure 2.

Open in a new tab

Detection of Chlamydia pneumoniae major outer membrane protein DNA sequences in extravillous trophoblast cells by polymerase chain reaction.

Viability Assay

At 48 hours postinfection, there was no significant difference in the viability of primary EVT cells infected with inactivated Cp when compared to noninfected EVT cells and EVT cells exposed to dilution buffer (84.5%, 93.9%, and 91.3% viable, respectively; P = .108; Figure 3). Conversely, the viability of EVT cells infected with live Cp was decreased significantly (71.8% viable) when compared to the other cell groups (P = .00015).

Figure 3.

Figure 3.

Open in a new tab

Viability of extravillous trophoblast cells infected with Chlamydia pneumoniae.

Invasive Properties in EVT Cells Infected With Inactivated Cp: Effect of Pretreatment With Pharmacologic Agents

Because we were interested in studying the effects of inactivated Cp on trophoblast cell function, we performed invasion assays on cells infected with Cp after UV-light inactivation. Compared to negative controls, EVT cells infected with inactivated Cp displayed impaired invasiveness. After normalizing invasion rates to 1.0 in noninfected EVT cells, invasion through an ECM was reduced significantly after infection with inactivated Cp, 0.73 invasion (range 0.72-0.74; P < .00001; Figure 4).

Figure 4.

Figure 4.

Open in a new tab

Extravillous trophoblast cell invasion through extracellular matrices.

Trophoblast invasion through an ECM was restored in EVT cells pretreated with low-dose ASA 1 mmol/L prior to infection with inactivated Cp. Invasion rates were normalized to 1.0 in noninfected EVT cells, and invasion rate in infected cells pretreated with ASA 1 mmol/L was 0.97 (range 0.95-0.98; P = .093) comparable to negative controls. Similar restored invasion rates were observed when EVT cells were pretreated with ASA 2.5 mmol/L (invasion 0.96, range 0.94-0.98; P = .064). Pretreatment with dexamethasone, heparin, and indomethacin did not have a significant effect on preventing reduced EVT invasion induced by inactivated Cp; degree of invasion in these cells remained impaired when compared to noninfected cells (P < .0001) and was similar to that observed in nonpretreated infected EVT cells (Figure 5).

Figure 5.

Figure 5.

Open in a new tab

Extravillous trophoblast cell invasion after pretreatment with pharmacologic agents.

Inflammation in EVT Cells Infected With Inactivated Cp: Effect of Pretreatment With Pharmacologic Agents

The expression of inflammatory markers was significantly elevated in culture media of EVT cells infected with inactivated Cp. Compared to noninfected cells, mean secretion of IL8, CRP, HSP60 and TNFα was 4.7-, 2.5-, 8.8- and 3.4-fold greater in EVT cells infected with UV-inactivated Cp (all P < .0001; Figure 6, A-D).

Figure 6.

Figure 6.

Open in a new tab

A-D, Release of inflammatory mediators by extravillous trophoblast cells infected with ultraviolet-inactivated Chlamydia pneumoniae.

Pretreatment of EVT cells with low-dose ASA 1 mmol/L and 2.5 mmol/L before infection with inactivated Cp significantly lowered levels of IL8, CRP, HSP60, and TNFα in culture media compared to nonpretreated EVT cells (P < .01; Figure 7). Pretreatment with dexamethasone, heparin, or indomethacin had no major impact in the release of IL8, CRP, HSP60, and TNFα in infected EVT cells; levels of these inflammatory mediators were comparable to those in nonpretreated infected cells (Figure 7, A-D).

Figure 7.

Figure 7.

Open in a new tab

A-D, Release of inflammatory mediators by extravillous trophoblasat cells infected with ultraviolet-inactivated Chlamydia pneumoniae after cell pretreatment with pharmacologic agents.

Case–Control Study Design: ELISA Experiments to Detect Anti-Cp IgG Antibodies in Maternal Serum

Demographic characteristics are shown in Table 1. Age, parity, and body mass index were similar among groups. Women were predominantly African American. Rates of preexisting hypertension and diabetes were comparable among cases and controls. Based on our study design, the mean gestational age at delivery was lower for cases with preeclampsia than for controls (36.4 ± 3.5 weeks vs 38.9 ± 1.2 weeks, respectively; P < .00001).

Table 1.

Demographics and Outcomes Data for Case–Control Study.

Variable Cases With Preeclampsia, n = 105 Controls, n = 121 P Value
Age, yearsa 27.3 ± 7.3 26.1 ± 7.1 .1947
African American race, n (%) 84 (80%) 91 (75%) .4838
Body mass index, kg/m2a 30.5 ± 16.1 28.6 ± 7.1 .2584
Obesity, n (%) 41 (39%) 43 (35.5%) .6843
Chronic hypertension, n (%) 9 (8.6%) 6 (4.9%) .4121
Diabetes mellitus, n (%) 6 (5.7%) 3 (2.5%) .369
Gestational age at delivery, weeksa 36.4 ± 3.5 38.9 ± 1.2 < .00001
Small for gestational age, n (%)b 25 (23.8%) 19 (15.7%) .1719
Open in a new tab

a Data are given as mean ± standard deviation.

b Birthweight <10th percentile for gestational age.

We used 105 serum samples from cases with preeclampsia and 121 from controls. Women who developed preeclampsia had higher titers of anti-Cp IgG compared to controls (median, 1.64 vs 0.84 μg/mL; P < .00001). Among our participants, 63.8% of women with preeclampsia (67/105) had positive IgG titers >1.1 compared to 43.8% of control women (53/121); P = .004; OR 2.26, 95% CI 1.32-3.87; Table 2). After controlling for confounders (obesity, diabetes mellitus, gestational diabetes, hypertension, history of preeclampsia, and fetal growth restriction), positive anti-Cp IgG antibodies remained significantly associated with preeclampsia (adjusted P = .013; OR 1.65, 95% CI 1.13-2.11; Table 2). Women with anti-Cp IgG >1.1 had a greater prevalence of sFlt-1–PlGF ratio >38 (90/120 vs 40/106, respectively; P = .0054) and higher serum CRP levels (median 37.5 vs 18.4 mg/L, respectively; P = .00001) when compared to those with IgG levels <1.1 (data not shown). Furthermore, positive anti-Cp IgG antibodies still showed a significant association with preeclampsia after accounting for CRP and sFlt-1–PlGF levels too (P = .028; OR 1.46, 95% CI 1.12-2.01); data not shown.

Table 2.

Prevalence of Elevated Anti-Chlamydia Pneumoniae Immunoglobulin G Class Antibodies in Serum Among Cases With Preeclampsia and Controls.

Variable Cases With Preeclampsia, N = 105 Control, N = 121 P Value Odds Ratio (95% CI) Adjusted P Value Adjusted Odds Ratio (95% CI)
Anti-Cp IgG >1.1 67 53 .004 2.26 (1.32-3.87) .013 1.65 (1.13-2.11)
Anti-Cp IgG <1.1 38 68
Open in a new tab

Abbreviations: CI, confidence interval; Cp, Chlamydia pneumoniae; Ig, immunoglobulin.

Discussion

Our results show that inactivated Cp (a model of the nonproductive bacterial state that elicits chronic infection) negatively affects human EVT invasive properties and that these effects occur in the setting of increased release of proinflammatory mediators in infected trophoblast cells. We also demonstrated an association between elevated maternal serum anti-Cp IgG antibody titers, representing chronic Cp infection, and preeclampsia, the histopathologic hallmark of which is decreased trophoblast invasion. Finally, we demonstrated that low-dose ASA pretreatment of EVT cells prior to infection with inactivated Cp prevents the negative effects of Cp on trophoblast invasion and attenuates the release of proinflammatory mediators from infected trophoblast cells. The protective in vitro effects of ASA on EVT cells were not observed with dexamethasone, heparin, or indomethacin. For this research, we focused on the effects of inactivated Cp on primary EVT cells, since we previously reported the effects on trophoblast cells infected with live forms of Cp bacteria.

A strength of our research is that we conducted translational experiments that supported our case–control study findings. We decided to use human first-trimester EVT cells in our experiments rather than transformed trophoblasts (HTR-8/SVneo or choriocarcinoma BeWo cells) because the former are pure primary EVT cells, exhibit well-characterized invasive properties that mimic what occurs in vivo during human placentation, and represent a validated model for trophoblast migration used in prior research publications.16,23,30,31 The purity of the primary EVT cells used in our experiments was characterized not only by immunostaining of HLA-G and cytokeratins -8 and -18 but25,27 also by specific EVT cell markers such as cytokeratin-7 and integrin α-I.26,29-31 The Cp strain used in our experiments, TWAR 183, has been used successfully to conduct cell function assays in prior studies.16,32,59 We demonstrated adequate Cp inactivation by UV light after EVT cells infected with inactivated Cp failed to express MOMP-DNA (conversely, MOMP-DNA was detected by PCR in EVT cells infected with live Cp). We demonstrated that reduced cell viability occurred predominantly in EVT cells infected with live Cp compared to those infected with inactivated Cp. The effects of inactivated Cp on cell invasion and inflammation were not observed on both sets of negative controls used in our translational experiments (noninfected EVT cells and EVT cells exposed only to dilution buffer). All of the above increased our confidence that changes in cell invasion and production of proinflammatory mediators were due predominantly to the action of inactivated Cp. Another strength of our study is that the use of ASA and other pharmacologic agents for cell pretreatment before Cp infection was conducted at doses that resemble those used in clinical scenarios.38-52 Finally, the observation from our case–control study that demonstrated an association between elevated levels of anti-Cp IgG in serum of women with preeclampsia remained significant after we controlled for confounders.

Our study has limitations. The results of our in vitro assays should be interpreted with caution when applied to in vivo conditions. Although we did not demonstrate Cp MOMP-DNA by PCR in cells infected with UV-inactivated Cp, we did not assess other markers of the nonproductive form of Cp. The pathological effects observed in EVTs infected with inactivated Cp could be attributed partially to cell detachment from the culture plates; however, the comparable viability rates of noninfected cells with EVT cells infected with inactivated Cp along with the use of appropriate negative controls allowed us to conclude that impaired invasion and increased inflammation occurred as a consequence of the effects of inactivated Cp on primary EVT cells. Although we noted a correlation of decreased trophoblast invasion with increased release of inflammatory mediators in EVT cells infected with inactivated Cp, the mechanism(s) by which these actions occur need(s) to be elucidated by future work. We acknowledge that during in vivo scenarios, chronic Cp infection precedes pregnancy and potential use of ASA. In our experiments, we did not study the effects of ASA after Cp infection of EVT cells; this is a potential area of research. Finally, our case–control study was a secondary analysis, and the external validity of our findings are limited by the demographic characteristics of our participants.

Our interest in Cp as a potential risk factor for preeclampsia emerged from the association of Cp with atherosclerosis. Atherosclerosis shares many risk factors and pathophysiological features with preeclampsia.1,2,4 Our group demonstrated previously that live Cp was able to infect EVT cells leading to decreased trophoblast invasion.16 For the current work, we were interested in assessing whether similar effects on trophoblast invasion could be elicited by inactivated Cp. Since Cp may enter a persistent nonproductive state in target cells that are chronically infected,14,15 we inactivated live Cp with UV-light as a surrogate bacterial model of the nonproductive Cp form representing chronic infection. Previous reports have demonstrated that inactivated forms of Cp also are able to induce cellular pathologic effects leading to atherogenicity.18,20,60,61 Our experiments showed negative effects of inactivated Cp on EVT cell invasion, suggesting that live forms of Cp are not always necessary to impair trophoblast function.

Chlamydia pneumoniae induces atherosclerosis by eliciting inflammation and release of interleukins, HSP, and TNFα among other mediators.19,33,62-68 Similarly, preeclampsia is associated with endothelial dysfunction and intravascular inflammation. Levels of TNFα, interleukins, CRP, and HSP circulate at elevated concentrations in maternal serum of women who develop preeclampsia.69-76 High levels of CRP correlate with increased titers of anti-Cp IgG antibodies in women with preeclampsia requiring delivery before term.77 We do not consider that the early gestational age at delivery seen in cases with preeclampsia influenced the elevated levels of anti-Cp IgG antibodies, CRP, or increased sFlt-1/PlGF ratio when compared to healthy controls. Furthermore, the greater prevalence of positive anti-Cp IgG antibodies in cases with preeclampsia was not influenced by levels of CRP or sFlt-1/PlGF as the association with preeclampsia continued to be significant after controlling for these markers. Our in vitro experiments showed a proinflammatory response (elevated IL8, CRP, HSP60, and TNFα) observed in culture media of EVT cells infected with inactivated Cp. Other investigators have found that exogenous IL8 actually enhances trophoblast invasion in HTR-8/SVneo and BeWo trophoblast cells.78,79 Our different results may be explained because we used a different cell model: primary human EVT rather than transformed trophoblast cells. Using primary EVT cells, our group also observed that elevated IL8 induced by lipopolysaccharide is associated with decreased trophoblast invasion.80 We hypothesize that in the setting of inflammation the effects of IL8 on trophoblast cells differ and lead to decreased invasiveness.

We found more commonly positive anti-Cp IgG antibodies in serum from cases with preeclampsia (63.8%) than controls (43.8%), suggesting chronic Cp exposure. Circulating maternal IgG antibodies against Cp have been reported previously in patients with preeclampsia.81-83 Although the frequency of anti-Cp IgG in healthy controls may appear high, this is not markedly different from what has been reported before.81,84 At least 50% of the general population over 20 years old are seropositive for Cp.21,22 Following the model seen in atherosclerosis induced by Cp,7-9 we hypothesize that being seropositive to Cp is not enough for the development of chronic diseases related to the bacterium; susceptible individuals who have positive anti-Cp IgG antibodies may require not only repetitive chronic active infection but also being subject to other risk factors in order to develop adverse outcomes such as preeclampsia.Importantly, the results of our case–control study still showed a significant higher prevalence of anti-Cp IgG in cases with preeclampsia after adjusting for confounders, and the presence of positive anti-Cp antibodies was associated with elevated CRP and sFlt-1–PlGF ratio. Our translational experiments and clinical observations suggest that, in a subset of susceptible pregnant women, chronic Cp infection may be associated with an abnormal profile of angiogenesis-related biomarkers and may have a role in the pathogenesis of preeclampsia.

Low-dose ASA has been shown to reduce the risk of adverse pregnancy outcomes including preeclampsia and medically-indicated preterm births, and it is used widely in clinical settings.85-87 Aspirin has been shown to inhibit Cp growth and its associated inflammation in endothelial cells.88,89 Thus, we decided to test the effects of low-dose ASA and also other pharmacologic agents on EVT cells exposed to inactivated Cp. Our results suggest a potential protective effect of low-dose ASA against the deleterious effects of Cp in human trophoblast cells.

Collectively, our data indicate that inactivated Cp adversely reduced human first-trimester EVT cell invasion and induced an inflammatory response. We observed that low-dose ASA prevented these adverse effects more efficiently than other potential prophylactic drugs. Our case–control study establishes a relationship between chronic Cp infection, abnormal angiogenesis biomarkers, and preeclampsia, possibly secondary to the effects of Cp on trophoblast invasion. Importantly, our clinical findings remained significant after controlling for conditions known to be associated with preeclampsia and correlate with other markers of inflammation (elevated CRP) and abnormal angiogenesis (elevated sFlt-1/PlGF ratio). The findings of our research provide a scientific basis for the continued investigation of the effects of Cp on trophoblast cells that could lead to pregnancy complications related to placental dysfunction and potential therapeutic strategies that can reverse the effects of chronic Cp infection.

Footnotes

Authors’ Note: This research was conducted at the Center for Research on Reproduction on Women’s Health at the University of Pennsylvania School of Medicine. The funding sources of this research had no involvement in the study design, collection, analysis and interpretation of the data.

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article supported by the following institutional grants: Maternal-Fetal Medicine Fellowship Training Program grant; Child Health Research Fund, University of Pennsylvania; National Institute of Health Grant 42100.

ORCID iD: Luis M. Gomez, MD, MScE Inline graphic https://orcid.org/0000-0002-8924-6386

References

  • 1. Gayston JT, Kuo CC, Wang SP, Altman J. A new Chlamydia psitacci strain, TWAR, isolated in acute respiratory tract infections. N Engl J Med. 1986;315(3):161–168. [DOI] [PubMed] [Google Scholar]
  • 2. Saikku P, Leinonen M, Matilla K, et al. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet. 1988;2(8618):983–986. [DOI] [PubMed] [Google Scholar]
  • 3. Gaydos CA, Summergill JT, Sahney NN, Ramírez JA, Quinn TC. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells. Infect Immun. 1996;64(5):1614–1620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Sander D, Winbeck K, Klingelhöfer J, Etgen T, Conrad B. Enhanced progression of early carotid atherosclerosis is related to Chlamydia pneumoniae (Taiwan acute respiratory) seropositivity. Circulation. 2001;103(10):1390–1395. [DOI] [PubMed] [Google Scholar]
  • 5. Sander D, Winbeck K, Klingelhofer J, Etgen T, Conrad B. Reduced progression of early carotid atherosclerosis after antibiotic treatment and Chlamydia pneumoniae seropositivity. Circulation. 2002;106(19):2428–2433. [DOI] [PubMed] [Google Scholar]
  • 6. Saikku P. Seroepidemiology in Chlamydia pneumoniae-atherosclerosis association. Eur Heart J. 2002;23(4):263–264. [DOI] [PubMed] [Google Scholar]
  • 7. Belland RJ, Oulellette SP, Gieffers J, Byrne GI. Chlamydia pneumoniae and atherosclerosis. Cell Microbiol. 2004;6(2):117–127. [DOI] [PubMed] [Google Scholar]
  • 8. Romano Carratelli C, Nuzzo I, Cozzolino D, Bentivoglio C, Paolillo R, Rizzo A. Relationship between Chlamydia pneumoniae infection, inflammatory markers, and coronary heart diseases. Int Immunopharmacol. 2006;6(5):848–853. [DOI] [PubMed] [Google Scholar]
  • 9. Watson C, Alp NJ. Role of Chlamydia pneumoniae in atherosclerosis. Clin Sci. 2008;114(8):509–531. [DOI] [PubMed] [Google Scholar]
  • 10. Kong TY, DeWolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by preeclampsia and by small-for-gestational age infants. BJOG. 1986;93(10):1049–1059. [DOI] [PubMed] [Google Scholar]
  • 11. Frusca T, Morassi L, Pecorelli S, Grigolato P, Gastaldi A. Histological features of uteroplacental vessels in normal and hypertensive patients in relation to birthweight. BJOG. 1989;96(7):835–839. [DOI] [PubMed] [Google Scholar]
  • 12. Meekins JW, Pijnenborg R, Hanssens M, McFadyen IR, van Asshe A. A study of placental bed spiral arteries and trophoblast invasion in normal and severe preeclamptic pregnancies. BJOG. 1994;101(8):669–674. [DOI] [PubMed] [Google Scholar]
  • 13. Wolf K, Fischer E, Mead D, et al. Chlamydia pneumoniae major outer membrane protein is a surface-exposed antigen that elicits antibodies primarily directed against conformation-dependent determinants. Infect Immun. 2001;69(5):3082–3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. JAMA. 2002;288(21):2274–2231. [DOI] [PubMed] [Google Scholar]
  • 15. Kern JM, Maass V, Maass M. Chlamydia pneumoniae-induced pathological signaling in the vasculature. FEMS Immunol Med Microbiol. 2009;55(2):131–139. [DOI] [PubMed] [Google Scholar]
  • 16. Gomez LM, Parry S. Trophoblast infection with Chlamydia pneumoniae and adverse pregnancy outcomes associated with placental dysfunction. Am J Obstet Gynecol. 2009;200(5):526.e1-e7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Godzik KL, O’Brien ER, Wang SK, Kuo CC. In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae. J Clin Microbiol. 1995;33(9):2411–2414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Baer JT, Du Laney TV, Wyrick PB, et al. Nuclear factor-kappaB activation in endothelium by Chlamydia pneumoniae without active infection. J Infect Dis. 2003;188(8):1094–1097. [DOI] [PubMed] [Google Scholar]
  • 19. Yang X, Coriolan D, Schultz K, Golenbock DT, Beasley D. Toll-like receptor 2 mediates persistent chemokine release by Chlamydia pneumoniae-infected vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2005;25(11):2308–2314. [DOI] [PubMed] [Google Scholar]
  • 20. Liu W, He P, Cheng B, Mei CL, Wang YF, Wan JJ. Chlamydia pneumoniae disturbs cholesterol homeostasis in human THP-1 macrophages via JNK-PPARγ dependent signal transduction pathways. Microbes Infect. 2010;12(14-15):1226–1235. [DOI] [PubMed] [Google Scholar]
  • 21. Grayston JT, Campbell LA, Kuo CC, et al. A new respiratory tract pathogen: Chlamydia pneumoniae strain TWAR. J Infect Dis. 1990;161(4):618–625. [DOI] [PubMed] [Google Scholar]
  • 22. Kanamoto Y, Ouchi K, Mizui M, Usui T. Prevalence of antibody to Chlamydia pneumoniae TWAR in Japan. J Clin Microbiol. 1991;29(4):816–818 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Appleton SD, Lash GE, Marks GS, et al. Effect of glucose and oxygen deprivation on hem oxygenase expression in human chorionic villi explants and immortalized trophoblast cells. Am J Physiol Regul Integr Com Physiol. 2003;285(6):R1453–R1460. [DOI] [PubMed] [Google Scholar]
  • 24. Graham CH, Hawley TS, Hawley RG, et al. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res. 1993;206(2):204–211. [DOI] [PubMed] [Google Scholar]
  • 25. Koi H, Zhang J, Makrigiannakis A., et al. Syncytiotrophoblast is a barrier to maternal-fetal transmission of herpes simplex virus. Biol Reprod. 2002;67(5):1572–1579. [DOI] [PubMed] [Google Scholar]
  • 26. Graham CH, Lysiak JJ, McCrae KR, Lala PK. Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation. Biol Reprod. 1992;46(4):561–572. [DOI] [PubMed] [Google Scholar]
  • 27. Neudeck H, Oei SL, Stiemer B, Hopp H, Graf R. Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes. Histochem J. 1997;29(5):419–430. [DOI] [PubMed] [Google Scholar]
  • 28. O’Brien PJ, Koi H, Parry S, et al. Thrombin receptors and protease-activated receptor-2 in human placentation: receptor activation mediates extravillous trophoblast invasion in vitro. Am J Pathol. 2003;163(4):1245–1254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Getsios S, Chen GT, Huang DT, MacCalman CD. Regulated expression of cadherin-11 in human extravillous cytotrophoblasts undergoing aggregation and fusion in response to transforming growth factor beta. J Reprod Fertil. 1998;114(2):357–363. [DOI] [PubMed] [Google Scholar]
  • 30. Anton L, Brown AG, Parry S, Elovitz MA. Lipopolysaccharide induces cytokine production and decreases extravillous trophoblast invasion through a mitogen-activated protein kinase-mediated pathway: possible mechanisms of first trimester placental dysfunction. Hum Reprod. 2011;27(1):61–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Gomez LM, Anton L, Srinivas SK, Elovitz MA, Parry S. Effects of increased human fetuin-A in human trophoblast cells and associated pregnancy outcomes. Am J Obstet Gynecol. 2012;207(6):484, e1–e8. [DOI] [PubMed] [Google Scholar]
  • 32. Grayston JT, Wang SP, Kuo CC, Campbell LA. Current knowledge on Chlamydia pneumoniae strain TWAR, an important cause of pneumonia and other acute respiratory diseases. Eur J Clin Microbiol Infec Dis. 1989;8(3):191–202. [DOI] [PubMed] [Google Scholar]
  • 33. Sasu S, LaVerda D, Qureshi N, Golenbock DT, Beasley D. Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation. Circ Res. 2001;89(3):244–250. [DOI] [PubMed] [Google Scholar]
  • 34. Vielma SA, Krings G, Lopes-Virella MF. Chlamydophila pneumoniae induces ICAM-1 expression in human aortic endothelial cells via protein kinase C-dependent activation of nuclear factor-kappaB. Circ Res. 2003;92(10):1130–1137. [DOI] [PubMed] [Google Scholar]
  • 35. Tong CY, Sillis M. Detection of Chlamydia pneumoniae and Chlamydia psitacci in sputum samples by PCR. J Clin Pathol. 1993;46(4):313–317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Decker T, Lohmann-Matthes ML. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods. 1988;115(1):61–69. [DOI] [PubMed] [Google Scholar]
  • 37. Gómez LM, Ma Y, Ho C, McGrath, Nelson DB, Parry S. Placental infection with human papillomavirus is associated with spontaneous preterm delivery. Hum Reprod. 2008;23(3):709–715. [DOI] [PubMed] [Google Scholar]
  • 38. Dekker GA, Sibai BM. Low-dose aspirin in the prevention of pre-eclampsia and fetal growth retardation: rationale, mechanisms and clinical trials. Am J Obstet Gynecol. 1993;168(1 pt 1):214–227. [DOI] [PubMed] [Google Scholar]
  • 39. Quenby S, Mountfield S, Cartwright JE, Whitley GS, Vince G. Effects of low-molecular-weight and unfractionated heparin on trophoblast function. Obstet Gynecol. 2004;104(2):354–361. [DOI] [PubMed] [Google Scholar]
  • 40. Higuchi A, Uchiyama S, Demura M, et al. Enhanced CEA production associated with aspirin in a culture of CW-2 cells on some polymeric films. Cytotechnology. 1999;31(3):233–242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Orendi K, Gauster M, Moser G, Meiri H, Huppertz B. Effects of vitamins C and E, acetylsalicylic acid and heparin on fusion, beta-hCG and PP13 expression in BeWo cells. Placenta. 2010;31(5):431–438. [DOI] [PubMed] [Google Scholar]
  • 42. Han CS, Mulla MJ, Brosens JJ, et al. Aspirin and heparin effect on basal and antiphospholipid antibody modulation of trophoblast function. Obstet Gynecol. 2011;118(5):1021–1028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Kleppa MJ, Erlenwein SV, Darashchonak N, von Kaisenberg CS, von Versen-Höynck F. Hypoxia and the anticoagulants dalteparin and acetylsalicylic acid affect human placental amino acid transport. PLoS One. 2014;9(6):e99217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Huang Y, Lichtenberger LM, Taylor M, et al. Antitumor and Antiangiogenic Effects of Aspirin-PC in Ovarian Cancer. Mol Cancer Ther. 2016;15(12):2894–2904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Zhou Y, Ling EA, Dheen ST. Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia. J Neurochem 2007;102(3):667–678. [DOI] [PubMed] [Google Scholar]
  • 46. Audette MC, Greenwood SL, Sibley CP, et al. Dexamethasone stimulates placental system A transport and trophoblast differentiation in term villous explants. Placenta. 2010;31(2):97–105. [DOI] [PubMed] [Google Scholar]
  • 47. Bohlmann MK, Hoerster JC, Bundschuh D, Boemicke P, Luedders DW, Köster F. Growth-modulatory effects of heparin and VEGF165 on the choriocarcinoma cell-line JEG-3 and its expression of heparanase. Anticancer Res. 2014;34(12):7071–7076. [PubMed] [Google Scholar]
  • 48. Faioni EM, Fontana G, Razzari C, et al. Activation of Protein C in Human Trophoblasts in Culture and Downregulation of Trophoblast Endothelial Protein C Receptor by TNF-α. Reprod Sci. 2015;22(8):1042–1048. [DOI] [PubMed] [Google Scholar]
  • 49. Lee JH, Oh SH, Kim TH, Go YY, Song JJ. Anti-apoptotic effect of dexamethasone in an ototoxicity model. Biomaterials Research. 2017;21:4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Ding ZQ, Rowe J, Ng B, Sinosich MJ, Gallery ED. Modulation of prostacyclin and thromboxane secretion by cytotrophoblasts from normal and pre-eclamptic human pregnancies. Placenta. 2002;23(8-9):594–599. [DOI] [PubMed] [Google Scholar]
  • 51. Julan L, Guan H, van Beek JP, Yang K. Peroxisome proliferator-activated receptor delta suppresses 11beta-hydroxysteroid dehydrogenase type 2 gene expression in human placental trophoblast cells. Endocrinology. 2005;146(3):1482–1490. [DOI] [PubMed] [Google Scholar]
  • 52. Park HR, Loch-Caruso R. Protective effect of (±)α-tocopherol on brominated diphenyl ether-47-stimulated prostaglandin pathways in human extravillous trophoblasts in vitro. Toxicol In Vitro. 2015;29(7):1309–1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53. Srinivas SK, Sammel MD, Bastek J, et al. Evaluating the association between all components of the metabolic syndrome and pre-eclampsia. J Matern Fetal Neonatal Med. 2009;22(6):501–509. [DOI] [PubMed] [Google Scholar]
  • 54. Hypertension in Pregnancy. Report of the American College of Obstetricians and Gynecologists’ Task Force on Hypertension in Pregnancy. Obstet Gynecol. 2013;122(5):1122–1231. [DOI] [PubMed] [Google Scholar]
  • 55. Piechowski-Jóźwiak B, Mickielewicz A, Gaciong Z, Berent H, Kwieciński H. Elevated levels of anti-Chlamydia pneumoniae IgA and IgG antibodies in young adults with ischemic stroke. Acta Neurol Scand. 2007;116:144–149. [DOI] [PubMed] [Google Scholar]
  • 56. Rai NK, Choudhary R, Bhatia R, et al. Chlamydia pneumoniae seropositivity in adults with acute ischemic stroke: A case–control study. Ann Indian Acad Neurol. 2011;14(2):93–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57. Jaworowska A, Bazylak G. Chlamydophila pneumoniae antibodies may be independently associated with increased BMI and percentage of body fat among women. Int J Obes. 2011;35(9):1225–1232. [DOI] [PubMed] [Google Scholar]
  • 58. Zeisler H, Llurba E, Chantraine F, et al. Predictive value of the sFlt-1: PlGF ratio in women with suspected preeclampsia. NEJM. 2016;374(1):13–22. [DOI] [PubMed] [Google Scholar]
  • 59. Rajalingam K, Al-Younes H, Müller A, Meyer TF, Szczepek A, Rudel T. Epithelial cells infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) are resistant to apoptosis. Infect Immun. 2001;69(12):7880–7888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60. Kaukoranta-Tolvanen SS, Ronni T, Leinonen M, Saikku P, Laitinen K. Expression of adhesion molecules on endothelial cells stimulated by Chlamydia pneumoniae. Microb Pathog. 1996;21(5):407–411. [DOI] [PubMed] [Google Scholar]
  • 61. Rizzo A, Paolillo R, Lanza AG, Guida L, Annunziata M, Carratelli CR. Chlamydia pneumoniae induces interleukin-6 and interleukin-10 in human gingival fibroblasts. Microbiol Immunol. 2008;52(9):447–454. [DOI] [PubMed] [Google Scholar]
  • 62. Heinemann M, Susa M, Simnacher U, Marre R, Essig A. Growth of Chlamydia pneumoniae induces cytokine production and expression of CD14 in a human monocytic cell line. Infect Immun. 1996; 64(11):4872–4875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63. Kalayoglu MV, Byrne GI. A Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccharide. Infect Immun. 1998;66(11):5067–5072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64. Kol A, Sukhova G, Lichtman AH, Libby P. Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-a and matrix metalloproteinase expression. Circulation. 1998;98(4):300–307. [DOI] [PubMed] [Google Scholar]
  • 65. Kol A, Bourcier T, Lichtman AH, Libby P. Chlamydial and human heat shock protein 60 s activate human vascular endothelium, smooth muscle cells, and macrophages. J Clin Invest. 1999;103(4):571–577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66. Molestina RE, Dean D, Miller RD, Ramirez JA, Summersgill JT. Characterization of a strain of Chlamydia pneumoniae isolated from a coronary atheroma by analysis of the omp1 gene and biological activity in human endothelial cells. Infect Immun. 1998;66(4):1370–1376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67. Dechend R, Maass M, Gieffers J, et al. Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-κB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circulation. 1999;100(13):1369–1373. [DOI] [PubMed] [Google Scholar]
  • 68. Netea MG, Selzman CH, Kullberg BJ, et al. Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells. Eur J Immunol. 2000;30(2):541–549. [DOI] [PubMed] [Google Scholar]
  • 69. Redman CWG, Sacks GP, Sargent IL. Pre-eclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol. 1999;180(2 pt 1):499-506. [DOI] [PubMed] [Google Scholar]
  • 70. LaMarca BD, Ryan MJ, Gilbert JS, Murphy SR, Granger JP. Inflammatory cytokines in the pathophysiology of hypertension during preeclampsia. Curr Hypertens Rep. 2007;9(6):480–485. [DOI] [PubMed] [Google Scholar]
  • 71. Tosun M, Celik H, Avci B, Yavuz E, Alper T, Malatyalioğlu E. Maternal and umbilical serum levels of interleukin-6, interleukin-8, and tumor necrosis factor-alpha in normal pregnancies and in pregnancies complicated by preeclampsia. J Matern Fetal Neonatal Med. 2010;23(8):880–886. [DOI] [PubMed] [Google Scholar]
  • 72. Xie F, Hu Y, Speert DP, et al. ; Toxaemia Study Group. Toll-like receptor gene polymorphisms and preeclampsia risk: A case–control study and data synthesis. Hypertens Pregnancy 2010;29(4);390–398. [DOI] [PubMed] [Google Scholar]
  • 73. Kashanian M, Aghbali F, Mahali N. Evaluation of the diagnostic value of the first-trimester maternal serum high-sensitivity C-reactive protein level for prediction of pre-eclampsia. J Obstet Gynaecol Res. 2013;39(12):1549–1554. [DOI] [PubMed] [Google Scholar]
  • 74. Lau SY, Guild SJ, Barrett CJ, et al. Tumor necrosis factor-alpha, interleukin-6, and interleukin-10 levels are altered in preeclampsia: A systematic review and meta-analysis. Am J Reprod Immunnol. 2013;70(5):412–427. [DOI] [PubMed] [Google Scholar]
  • 75. Álvarez-Cabrera MC, Barrientos-Galeana E, Barrera-García A, et al. Secretion of heat shock -60, -70 kD protein, IL-1β and TNFα levels in serum of a term normal pregnancy and patients with pre-eclampsia development. J Cell Mol Med. 2018;22(11):1–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76. Black KD, Horowitz JA. Inflammatory Markers and Preeclampsia: A Systematic Review. Nurs Res. 2018;67(3):242–251. [DOI] [PubMed] [Google Scholar]
  • 77. Karinen L, Leinonen M, Bloigu A, et al. Maternal serum Chlamydia pneumoniae antibodies and CRP levels in women with preeclampsia and gestational hypertension. Hypertens Pregnancy. 2008;27(2):143–158. [DOI] [PubMed] [Google Scholar]
  • 78. Jovanović M, Stefanoska I, Radojcić L, Vićovac L. Interleukin-8 (CXCL8) stimulates trophoblast cell migration and invasion by increasing levels of matrix metalloproteinase (MMP)2 and MMP9 and integrins alpha5 and beta1. Reproduction. 2010;139(4):789–798. [DOI] [PubMed] [Google Scholar]
  • 79. Ryu BJ, Han JW, Kim RH, et al. Activation of NOD-1/JNK/IL-8 signal axis in decidual stromal cells facilitates trophoblast invasion. Am J Reprod Immunol. 2017;78(2):e12672. [DOI] [PubMed] [Google Scholar]
  • 80. Anton L, Brown AG, Parry S, Elovitz MA. Lipopolysaccharide induces cytokine production and decreases extravillous trophoblast invasionthrough a mitogen-activated protein kinase-mediated pathway: possible mechanisms of first trimester placental dysfunction. Hum Reprod. 2012;27(1):61–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 81. Heine RP, Ness RB, Roberts JM. Seroprevalence of antibodies to Chlamydia pneumoniae in women with preeclampsia. Obstet Gynecol. 2003;101(2):221–226. [DOI] [PubMed] [Google Scholar]
  • 82. von Dadelszen P, Magee LA, Krajden M, et al. Levels of antibodies against cytomegalovirus and Chlamydophila pneumoniae are increased in early onset preeclampsia. BJOG 2003;110(8):725–730. [PubMed] [Google Scholar]
  • 83. Goulis DG, Chappell L, Gibbs RG, Williams D, Dave JR, Taylor P. Association of raised titres of antibodies to Chlamydia pneumoniae with a history of preeclampsia. BJOG. 2005;112(3):299–305. [DOI] [PubMed] [Google Scholar]
  • 84. Teran E, Escudero C, Calle A. Seroprevalence of antibodies to Chlamydia pneumoniae in women with preeclampsia [Letter to the Editor]. Obstet Gynecol. 2003;102(1):198–199. [DOI] [PubMed] [Google Scholar]
  • 85. LeFevre ML. on behalf of the U.S. Preventive Services Task Force. Low-Dose Aspirin Use for the Prevention of Morbidity and Mortality From Preeclampsia: U.S. Preventive Services Task Force Recommendation Statement. Ann Intern Med. 2014;161(11):819–826. [DOI] [PubMed] [Google Scholar]
  • 86. Roberge S, Odibo A, Bujold E. Aspirin for the prevention of preeclampsia and intrauterine growth restriction. Clin Lab Med. 2016;36(2):319–329. [DOI] [PubMed] [Google Scholar]
  • 87. Roberge S, Nicolaides K, Demmers S, Hyett J, Chaillet N, Bujold E. The role of aspirin dose on the prevention of preeclampsia and fetal growth restriction: systematic review and meta-analysis. Am J Obstet Gynecol. 2017;216(2):110–120.e6. [DOI] [PubMed] [Google Scholar]
  • 88. Tiran A, Gruber HJ, Graier WF, Wagner AH, van Leeuwen EB, Tiran B. Aspirin Inhibits Chlamydia pneumoniae–Induced Nuclear Factor-κB Activation, Cytokine Expression, and Bacterial Development in Human Endothelial Cells. Arterioscler Thromb Vasc Biol. 2002;22(7):1075–1080. [DOI] [PubMed] [Google Scholar]
  • 89. Yoneda H, Miura K, Matsushima H, et al. Aspirin inhibits Chlamydia pneumoniae-induced NF-kB activation, cyclo-oxygenase 2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth. J Med. Microbiol. 2003;52(pt 5):409–415. [DOI] [PubMed] [Google Scholar]

Articles from Reproductive Sciences are provided here courtesy of Society for Reproductive Investigation

RESOURCES