Xenobiotic metabolites modify immune responses of the cervicovaginal epithelium: potential mechanisms underlying barrier disruption.

OBJECTIVE: Xenobiotic metabolites are exogenous biochemicals that can adversely impact reproductive health. We previously identified xenobiotics in cervicovaginal fluid during pregnancy in association with short cervix. In other organ systems, xenobiotics can modify epithelial barrier function. We hypothesise that xenobiotics dysregulate epithelial cell and macrophage immune responses as a mechanism to disrupt the cervicovaginal barrier. DESIGN: In vitro cell culture system. SETTING: Laboratory within academic institution. SAMPLE: Vaginal, ectocervical and endocervical epithelial cell lines and primary macrophages. METHODS: Cells were treated with diethanolamine (2.5 mM), ethyl glucoside (5 mM) or tartrate (2.5 mM) for 24 h. MAIN OUTCOME MEASURES: Cytokines and matrix metalloproteinases were measured in cell supernatants (n = 3 per condition). One-way analysis of variance (ANOVA) with Dunnett's test for multiple comparisons was performed. RESULTS: Diethanolamine induces inflammatory cytokines, whereas ethyl glucoside and tartrate generally exert anti-inflammatory effects across all cells. Diethanolamine increases interleukin 6 (IL-6), IL-8, interferon γ-induced protein 10 kDa (IP-10), growth-regulated oncogene (GRO), fractalkine, matrix metalloproteinase 1 (MMP-1), MMP-9 and MMP-10 (p < 0.05 for all), factors involved in acute inflammation and recruitment of monocytes, neutrophils and lymphocytes. Ethyl glucoside and tartrate decrease multiple cytokines, including RANTES and MCP-1 (p < 0.05 for all), which serve as chemotactic factors. Vaginal cells exhibit heightened inflammatory tone compared with cervical cells and macrophages, with a greater number of differentially expressed analytes after xenobiotic exposure. CONCLUSIONS: Xenobiotic metabolites present in the cervicovaginal space during pregnancy modify immune responses, unveiling potential pathways through which environmental exposures may contribute to the pathogenesis of cervical remodelling preceding preterm birth. Future work identifying xenobiotic sources and routes of exposure offers the potential to modify environmental risks to improve pregnancy outcomes.

Second trimester short cervix is associated with decreased abundance of cervicovaginal lipid metabolites.

BACKGROUND: A short cervix is a risk factor for preterm birth. The molecular drivers of a short cervix remain elusive. Metabolites may function as mediators of pathologic processes. OBJECTIVE: We sought to determine if a distinct cervicovaginal metabolomic profile is associated with a short cervix (<25 mm) to unveil the potential mechanisms by which premature cervical remodeling leads to a short cervix. STUDY DESIGN: This was a secondary analysis of a completed prospective pregnancy cohort. Cervicovaginal fluid was obtained between 20 and 24 weeks' gestation. The participants selected for metabolomic profiling were frequency-matched by birth outcome and cervicovaginal microbiota profile. This analysis included 222 participants with cervical length measured. A short cervix was defined as one having length <25 mm, as measured by transvaginal ultrasound. Unpaired t-tests were performed with a Bonferroni correction for multiple comparisons. RESULTS: There were 27 participants with a short cervix, and 195 with normal cervical length. Of the 637 metabolites detected, 26 differed between those with a short cervix and those with normal cervical lengths; 22 were decreased, of which 21 belonged to the lipid metabolism pathway (all P<.000079). Diethanolamine, erythritol, progesterone, and mannitol or sorbitol were increased in the cases of short cervix. Among participants with Lactobacillus-deficient microbiota, only diethanolamine and mannitol or sorbitol differed between short cervix (n=17) and normal cervical length (n=75), both increased. CONCLUSION: A short cervix is associated with decreased cervicovaginal lipid metabolites, particularly sphingolipids. This class of lipids stabilizes cell membranes and protects against environmental exposures. Increased diethanolamine-an immunostimulatory xenobiotic-is associated with a short cervix. These observations begin to identify the potential mechanisms by which modifiable environmental factors may invoke cell damage in the setting of biological vulnerability, thus promoting premature cervical remodeling in spontaneous preterm birth.

Intrauterine Inflammation Leads to Select Sex- and Age-Specific Behavior and Molecular Differences in Mice.

Sex-specific differences in behavior have been observed in anxiety and learning in children exposed to prenatal inflammation; however, whether these behaviors manifest differently by age is unknown. This study assesses possible behavioral changes due to in utero inflammation as a function of age in neonatal, juvenile, and adult animals and presents potential molecular targets for observed differences. CD-1 timed pregnant dams were injected in utero with lipopolysaccharide (LPS, 50 µg/animal) or saline at embryonic day 15. No differences in stress responses were measured by neonatal ultrasonic vocalizations between LPS- and saline-exposed groups of either sex. By contrast, prenatal inflammation caused a male-specific increase in anxiety in mature but not juvenile animals. Juvenile LPS-exposed females had decreased movement in open field testing that was not present in adult animals. We additionally observed improved memory retrieval after in utero LPS in the juvenile animals of both sexes, which in males may be related to a perseverative phenotype. However, there was an impairment of long-term memory in only adult LPS-exposed females. Finally, gene expression analyses revealed that LPS induced sex-specific changes in genes involved in hippocampal neurogenesis. In conclusion, intrauterine inflammation has age- and sex-specific effects on anxiety and learning that may correlate to sex-specific disruption of gene expression associated with neurogenesis in the hippocampus.

Microbial supernatants from Mobiluncus mulieris, a bacteria strongly associated with spontaneous preterm birth, disrupts the cervical epithelial barrier through inflammatory and miRNA mediated mechanisms.

Recent human clinical studies have identified Mobiluncus mulieris, a fastidious strict anaerobic bacterium present in the cervicovaginal (CV) space, as being strongly associated with spontaneous preterm birth (sPTB). However, the molecular mechanisms that underlie this association remain unknown. As disruption of the cervical epithelial barrier has been shown to contribute to the premature cervical remodeling that precedes sPTB, we hypothesize that M. mulieris, a microbe strongly associated with sPTB in humans, has the ability to alter cervical epithelial function. We investigated if bacteria-free supernatants of M. mulieris were able to disrupt the cervical epithelial barrier through immunological and epigenetic based mechanisms in an in vitro model system. Ectocervical cells were treated with supernatant from cultured M. mulieris and epithelial cell permeability, immune cytokines and microRNAs (miRNAs) were investigated. M. mulieris supernatant significantly increased cell permeability and the expression of two inflammatory mediators associated with cervical epithelial breakdown, IL-6 and IL-8. Moreover, treatment of the ectocervical cells with the M. mulieris supernatant also increased the expression of miRNAs that have been associated with either sPTB or a shorter gestational length in humans. Collectively, these results suggest that M. mulieris induces molecular and functional changes in the cervical epithelial barrier thought to contribute to the pathogenesis of sPTB, which allows us to hypothesize that targeting CV bacteria such as M. mulieris could provide a therapeutic opportunity to reduce sPTB rates.

Downregulation of apelin in the human placental chorionic villi from preeclamptic pregnancies.

The role of the endogenous apelin system in pregnancy is not well understood. Apelin's actions in pregnancy are further complicated by the expression of multiple forms of the peptide. Using radioimmunoassay (RIA) alone, we established the expression of apelin content in the chorionic villi of preeclamptic (PRE) and normal pregnant women (NORM) at 36-38 wk of gestation. Total apelin content was lower in PRE compared with NORM chorionic villi (49.7±3.4 vs. 72.3±9.8 fmol/mg protein; n=20-22) and was associated with a trend for lower preproapelin mRNA in the PRE. Further characterization of apelin isoforms by HPLC-RIA was conducted in pooled samples from each group. The expression patterns of apelin peptides in NORM and PRE villi revealed little or no apelin-36 or apelin-17. Pyroglutamate apelin-13 [(Pyr1)-apelin-13] was the predominant form of the peptide in NORM and PRE villi. Angiotensin-converting enzyme 2 (ACE2) activity was higher in PRE villi (572.0±23.0 vs. 485.3±24.8 pmol·mg(-1)·min(-1); n=18-22). A low dose of ANG II (1 nM; 2 h) decreased apelin release in NORM villous explants that was blocked by the ANG II receptor 1 (AT1) antagonist losartan. Moreover, losartan enhanced apelin release above the 2-h baseline levels in both NORM and PRE villi (P<0.05). In summary, these studies are the first to demonstrate the lower apelin content in human placental chorionic villi of PRE subjects using quantitative RIA. (Pyr1)-apelin-13 is the predominant form of endogenous apelin in the chorionic villi of NORM and PRE. The potential mechanism of lower apelin expression in the PRE villi may involve a negative regulation of apelin by ANG II.

Can microRNA profiling in maternal blood identify women at risk for preterm birth?

OBJECTIVE: MicroRNAs (miRNAs), which are highly conserved single-stranded noncoding RNAs that play a crucial role in gene regulation, have now been identified as important players in many diseases states. MiRNAs have also been demonstrated to be reliable and useful biomarkers to identify those women who are at risk for specific adverse outcomes. The objective of this study was to determine whether miRNA profiles in maternal blood are different in women who are destined to have a preterm, compared with a term, birth. STUDY DESIGN: A nested case-control study was performed with maternal serum that was collected as part of a larger prospective cohort. MiRNA expression profiles in maternal serum were compared between women who ultimately had a preterm birth (n = 40) compared with term birth (n = 40). MiRNA expression profiles were created with the use of the Affymetrix GeneChip miRNA Array. The data were analyzed with the significance of analysis of microarrays and principle components analyses. A false discovery rate of 20% was used to determine the most differentially expressed miRNAs. RESULTS: Of the 5640 miRNAs that were analyzed on the array, 4 miRNAs were significantly different between cases and control subjects. Two of the 4 miRNAs were mature miRNAs. The fold difference in expression was <2 for all 4 miRNAs. CONCLUSION: MiRNA profiles in maternal blood were not significantly different in women who were destined to have a preterm, compared with a term, birth. MiRNAs in maternal blood are unlikely to become clinically useful biomarkers for the prediction of preterm birth.

miR-210 inhibits trophoblast invasion and is a serum biomarker for preeclampsia.

Preeclampsia is characterized by hypertension and proteinuria in pregnant women. Its exact cause is unknown. Preeclampsia increases the risk of maternal and fetal morbidity and mortality. Although delivery, often premature, is the only known cure, early targeted interventions may improve maternal and fetal outcomes. Successful intervention requires a better understanding of the molecular etiology of preeclampsia and the development of accurate methods to predict women at risk. To this end, we tested the role of miR-210, a miRNA up-regulated in preeclamptic placentas, in first-trimester extravillous trophoblasts. miR-210 overexpression reduced trophoblast invasion, a process necessary for uteroplacental perfusion, in an extracellular signal-regulated kinase/mitogen-activated protein kinase-dependent manner. Conversely, miR-210 inhibition promoted invasion. Furthermore, given that the placenta secretes miRNAs into the maternal circulation, we tested if serum expression of miR-210 was associated with the disease. We measured miR-210 expression in two clinical studies: a case-control study and a prospective cohort study. Serum miR-210 expression was significantly associated with a diagnosis of preeclampsia (P = 0.007, area under the receiver operator curves = 0.81) and was predictive of the disease, even months before clinical diagnosis (P < 0.0001, area under the receiver operator curve = 0.89). Hence, we conclude that aberrant expression of miR-210 may contribute to trophoblast function and that miR-210 is a novel predictive serum biomarker for preeclampsia that can help in identifying at-risk women for monitoring and treatment.

Distinct cervical microRNA profiles are present in women destined to have a preterm birth.

OBJECTIVE: Although premature cervical remodeling is involved in preterm birth (PTB), the molecular pathways that are involved have not been elucidated fully. MicroRNAs (miRNAs) that are highly conserved single-stranded noncoding RNAs that play a crucial role in gene regulation have now been identified as important players in disease states. The objective of this study was to determine whether miRNA profiles in cervical cells are different in women who are destined to have a PTB compared with a term birth. STUDY DESIGN: A nested case-control study was performed. With the use of a noninvasive method, cervical cells were obtained at 2 time points in pregnancy. The cervical cell miRNA expression profiles were compared between women who ultimately had a PTB (n = 10) compared with a term birth (n = 10). MiRNA expression profiles were created with the Affymetrix GeneChip miRNA Array. The data were analyzed with the Significance of Analysis of Microarrays and Principle Components Analyses. A false-discovery rate of 20% was used to determine the most differentially expressed miRNAs. Validation was performed with quantitative polymerase chain reaction. In vitro studies were performed to confirm expression and regulation of select miRNAs. RESULTS: With a false-discovery rate of 20% of the 5640 miRNAs that were analyzed on the array, 99 miRNAs differed between those with a PTB vs a term birth. Qualitative polymerase chain reaction validated the array findings. In vitro studies confirmed expression of select miRNAs in cervical cells. CONCLUSION: MiRNA profiles in cervical cells may distinguish women who are at risk for PTB months before the outcome. With the large downstream effects of miRNAs on gene expression, these studies provide a new understanding of the processes that are involved in premature cervical remodeling and allow for the discovery of new therapeutic targets.

A functional genomics screen for microRNA regulators of NF-kappaB signaling.

BACKGROUND: The nuclear factor-KappaB (NF-κB) pathway is conserved from fruit flies to humans and is a key mediator of inflammatory signaling. Aberrant regulation of NF-κB is associated with several disorders including autoimmune disease, chronic inflammation, and cancer, making the NF-κB pathway an attractive therapeutic target. Many regulatory components of the NF-κB pathway have been identified, including microRNAs (miRNAs). miRNAs are small non-coding RNAs and are common components of signal transduction pathways. Here we present a cell-based functional genomics screen to systematically identify miRNAs that regulate NF-κB signaling. RESULTS: We screened a library of miRNA mimics using a NF-κB reporter cell line in the presence and absence of tumor necrosis factor (+/- TNF). There were 9 and 15 hits in the -TNF and +TNF screens, respectively. We identified putative functional targets of these hits by integrating computational predictions with NF-κB modulators identified in a previous genome-wide cDNA screen. miR-517a and miR-517c were the top hits, activating the reporter 86- and 126-fold, respectively. Consistent with these results, miR-517a/c induced the expression of endogenous NF-κB targets and promoted the nuclear localization of p65 and the degradation of IκB. We identified TNFAIP3 interacting protein1 (TNIP1) as a target and characterized a functional SNP in the miR-517a/c binding site. Lastly, miR-517a/c induced apoptosis in vitro, which was phenocopied by knockdown of TNIP1. CONCLUSIONS: Our study suggests that miRNAs are common components of NF-κB signaling and miR-517a/c may play an important role in linking NF-κB signaling with cell survival through TNIP1.

Vaginal microbes alter epithelial transcriptome and induce epigenomic modifications providing insight into mechanisms for susceptibility to adverse reproductive outcomes.

The cervicovaginal microbiome is highly associated with women's health, with microbial communities dominated by Lactobacillus species considered optimal. Conversely, a lack of lactobacilli and a high abundance of strict and facultative anaerobes, including Gardnerella vaginalis, have been associated with adverse reproductive outcomes. However, how host-microbial interactions alter specific molecular pathways and impact cervical and vaginal epithelial function remains unclear. Using RNA-sequencing, we characterized the in vitro cervicovaginal epithelial transcriptional response to different vaginal bacteria and their culture supernatants. We showed that G. vaginalis upregulates genes associated with an activated innate immune response. Unexpectedly, G. vaginalis specifically induced inflammasome pathways through activation of NLRP3-mediated increases in caspase-1, IL-1ß and cell death, while live L. crispatus had minimal transcriptomic changes on epithelial cells. L. crispatus culture supernatants resulted in a shift in the epigenomic landscape of cervical epithelial cells that was confirmed by ATAC-sequencing showing reduced chromatin accessibility. This study reveals new insights into host-microbe interactions in the lower reproductive tract and suggests potential therapeutic strategies leveraging the vaginal microbiome to improve reproductive health.

Extracellular vesicles from vaginal Gardnerella vaginalis and Mobiluncus mulieris contain distinct proteomic cargo and induce inflammatory pathways.

Colonization of the vaginal space with bacteria such as Gardnerella vaginalis and Mobiluncus mulieris is associated with increased risk for STIs, bacterial vaginosis, and preterm birth, while Lactobacillus crispatus is associated with optimal reproductive health. Although host-microbe interactions are hypothesized to contribute to reproductive health and disease, the bacterial mediators that are critical to this response remain unclear. Bacterial extracellular vesicles (bEVs) are proposed to participate in host-microbe communication by providing protection of bacterial cargo, delivery to intracellular targets, and ultimately induction of immune responses from the host. We evaluated the proteome of bEVs produced in vitro from G. vaginalis, M. mulieris, and L. crispatus, identifying specific proteins of immunologic interest. We found that bEVs from each bacterial species internalize within cervical and vaginal epithelial cells, and that epithelial and immune cells express a multi-cytokine response when exposed to bEVs from G. vaginalis and M. mulieris but not L. crispatus. Further, we demonstrate that the inflammatory response induced by G. vaginalis and M. mulieris bEVs is TLR2-specific. Our results provide evidence that vaginal bacteria communicate with host cells through secreted bEVs, revealing a mechanism by which bacteria lead to adverse reproductive outcomes associated with inflammation. Elucidating host-microbe interactions in the cervicovaginal space will provide further insight into the mechanisms contributing to microbiome-mediated adverse outcomes and may reveal new therapeutic targets.

Extracellular vesicles alter trophoblast function in pregnancies complicated by COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and resulting coronavirus disease (COVID-19) causes placental dysfunction, which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests that pathophysiology associated with maternal COVID-19, rather than direct placental infection, is responsible for placental dysfunction and alteration of the placental transcriptome. We hypothesized that maternal circulating extracellular vesicles (EVs), altered by COVID-19 during pregnancy, contribute to placental dysfunction. To examine this hypothesis, we characterized maternal circulating EVs from pregnancies complicated by COVID-19 and tested their effects on trophoblast cell physiology in vitro . We found that the gestational timing of COVID-19 is a major determinant of circulating EV function and cargo. In vitro trophoblast exposure to EVs isolated from patients with an active infection at the time of delivery, but not EVs isolated from Controls, altered key trophoblast functions including hormone production and invasion. Thus, circulating EVs from participants with an active infection, both symptomatic and asymptomatic cases, can disrupt vital trophoblast functions. EV cargo differed between participants with COVID-19 and Controls, which may contribute to the disruption of the placental transcriptome and morphology. Our findings show that COVID-19 can have effects throughout pregnancy on circulating EVs and circulating EVs are likely to participate in placental dysfunction induced by COVID-19.

Prevention of preterm birth by progestational agents: what are the molecular mechanisms?

OBJECTIVE: Clinically, vaginal progesterone (VP) and 17 alpha-hydroxyprogesterone caproate (17P) have been shown to prevent preterm birth (PTB) in high-risk populations. We hypothesize that treatment with these agents may prevent PTB by altering molecular pathways involved in uterine contractility or cervical remodeling. STUDY DESIGN: Using a mouse model, on embryonic day (E)14-E17 CD-1 pregnant mice were treated with: (1) 0.1 mL of 25 mg/mL of 17P subcutaneously; (2) 0.1 mL of castor oil subcutaneously; (3) 0.1 mL of 10 mg/mL of progesterone in a long-lasting Replens (Lil' Drug Store Products, Inc., Cedar Rapids, IA); or (4) 0.1 mL of the same Replens, with 4 dams per treatment group. Mice were sacrificed 6 hours after treatment on E17.5. Cervices and uteri were collected for molecular analysis. RESULTS: Exposure to VP significantly increased the expression of defensin 1 compared to Replens (P < .01) on E17.5. Neither VP nor 17P altered the expression of uterine contraction-associated proteins, progesterone-mediated regulators of uterine quiescence, microRNA involved in uterine contractility, or pathways involved in cervical remodeling. In addition, neither agent had an effect on immune cell trafficking or collagen content in the cervix. CONCLUSION: Neither VP nor 17P had any effect on the studied pathways known to be involved in uterine contractility or quiescence. In the cervix, neither VP nor 17P altered pathways demonstrated to be involved in cervical remodeling. Administration of VP was noted to increase the expression of the antimicrobial protein defensin 1. Whether this molecular change from VP results in a functional effect and is a key mechanism by which VP prevents PTB requires further study.

Vaginal microbes alter epithelial transcriptomic and epigenomic modifications providing insight into the molecular mechanisms for susceptibility to adverse reproductive outcomes.

The cervicovaginal microbiome is highly associated with women's health with microbial communities dominated by Lactobacillus spp. being considered optimal. Conversely, a lack of lactobacilli and a high abundance of strict and facultative anaerobes including Gardnerella vaginalis, have been associated with adverse reproductive outcomes. However, the molecular pathways modulated by microbe interactions with the cervicovaginal epithelia remain unclear. Using RNA-sequencing, we characterize the in vitro cervicovaginal epithelial transcriptional response to different vaginal bacteria and their culture supernatants. We showed that G. vaginalis upregulated genes were associated with an activated innate immune response including anti-microbial peptides and inflammasome pathways, represented by NLRP3-mediated increases in caspase-1, IL-1ß and cell death. Cervicovaginal epithelial cells exposed to L. crispatus showed limited transcriptomic changes, while exposure to L. crispatus culture supernatants resulted in a shift in the epigenomic landscape of cervical epithelial cells. ATAC-sequencing confirmed epigenetic changes with reduced chromatin accessibility. This study reveals new insight into host-microbe interactions in the lower reproductive tract and suggest potential therapeutic strategies leveraging the vaginal microbiome to improve reproductive health.

Intrauterine colonization with Gardnerella vaginalis and Mobiluncus mulieris induces maternal inflammation but not preterm birth in a mouse model.

PROBLEM: Preterm birth (PTB) remains a leading cause of childhood mortality. Recent studies demonstrate that the risk of spontaneous PTB (sPTB) is increased in individuals with Lactobacillus-deficient vaginal microbial communities. One proposed mechanism is that vaginal microbes ascend through the cervix, colonize the uterus, and activate inflammatory pathways leading to sPTB. This study assessed whether intrauterine colonization with either Gardnerella vaginalis and Mobiluncus mulieris alone is sufficient to induce maternal-fetal inflammation and induce sPTB. METHOD OF STUDY: C56/B6J mice, on embryonic day 15, received intrauterine inoculation of saline or 108 colony-forming units of G. vaginalis (n = 30), M. mulieris (n = 17), or Lactobacillus crispatus (n = 16). Dams were either monitored for maternal morbidity and sPTB or sacrificed 6 h post-infusion for analysis of bacterial growth and cytokine/chemokine expression in maternal and fetal tissues. RESULTS: Six hours following intrauterine inoculation with G. vaginalis, M. mulieris, or L. crispatus, live bacteria were observed in both blood and amniotic fluid, and a potent immune response was identified in the uterus and maternal serum. In contrast, only a limited immune response was identified in the amniotic fluid and the fetus after intrauterine inoculation. High bacterial load (108 CFU/animal) of G. vaginalis was associated with maternal morbidity and mortality but not sPTB. Intrauterine infusion with L. crispatus or M. mulieris at 108 CFU/animal did not induce sPTB, alter pup viability, litter size, or maternal mortality. CONCLUSIONS: Despite inducing an immune response, intrauterine infusion of live G. vaginalis or M. mulieris is not sufficient to induce sPTB in our mouse model. These results suggest that ascension of common vaginal microbes into the uterine cavity alone is not causative for sPTB.

Regulatory T cell adoptive transfer alters uterine immune populations, increasing a novel MHC-IIlow macrophage associated with healthy pregnancy.

Intrauterine fetal demise (IUFD) - fetal loss after 20 weeks - affects 6 pregnancies per 1,000 live births in the United States, and the majority are of unknown etiology. Maternal systemic regulatory T cell (Treg) deficits have been implicated in fetal loss, but whether mucosal immune cells at the maternal-fetal interface contribute to fetal loss is under-explored. We hypothesized that the immune cell composition and function of the uterine mucosa would contribute to the pathogenesis of IUFD. To investigate local immune mechanisms of IUFD, we used the CBA mouse strain, which naturally has mid-late gestation fetal loss. We performed a Treg adoptive transfer and interrogated both pregnancy outcomes and the impact of systemic maternal Tregs on mucosal immune populations at the maternal-fetal interface. Treg transfer prevented fetal loss and increased an MHC-IIlow population of uterine macrophages. Single-cell RNA-sequencing was utilized to precisely evaluate the impact of systemic Tregs on uterine myeloid populations. A population of C1q+, Trem2+, MHC-IIlow uterine macrophages were increased in Treg-recipient mice. The transcriptional signature of this novel uterine macrophage subtype is enriched in multiple studies of human healthy decidual macrophages, suggesting a conserved role for these macrophages in preventing fetal loss.

Lipopolysaccharide induces cytokine production and decreases extravillous trophoblast invasion through a mitogen-activated protein kinase-mediated pathway: possible mechanisms of first trimester placental dysfunction.

BACKGROUND: Defects in extravillous trophoblast (EVT) function could contribute to placental dysfunction resulting in adverse obstetrical outcomes. Adverse obstetrical outcomes have been highly correlated with intrauterine infection; however, the mechanisms linking infection to placental dysfunction remain unclear. We investigated the effects of inflammation on EVT cytokine production and invasion early in pregnancy and determined the cell signaling pathways mediating this response. METHODS AND RESULTS: In our model of inflammation, EVT cells, isolated following first trimester pregnancy terminations (n= 6) were stimulated with lipopolysaccharide (LPS). LPS induced a dose-dependent increase in interleukin (IL)-8 and IL-6 protein production (P < 0.01) and decreased EVT invasion (P = 0.01) versus control. The LPS-mediated changes in cytokine production (P < 0.001) and invasion (P < 0.001) were reversed by dexamethasone (DEX). Exposure to LPS resulted in an increase in mitogen-activated protein kinase (MAPK) signaling pathway phosphorylation, including p44/42 MAPK (P < 0.01), p38 MAPK (P < 0.05), MAPK extracellular signal-regulated kinase 1/2 (MEK1/2) (P< 0.01) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK; P < 0.001), which was reversed by DEX (P < 0.05) for all MAPKs except p38. MAPK-specific inhibitors to MEK1/2 (U0126), p38 MAPK (SB 202190) and JNK (SP 600125) significantly reversed the LPS-mediated increase in IL-6 (P < 0.001) and IL-8 (P < 0.001) production. While U0126 reversed the LPS-induced decrease in EVT invasion (P < 0.001), SB 202190 (P < 0.001) and SP 600125 (P< 0.001) decreased EVT invasion, further indicating that MEK1/2 phosphorylation may be inflammation dependent while p38 MAPK and JNK phosphorylation occurs independently of an inflammatory stimulus. CONCLUSIONS: LPS increased IL-8 and IL-6 and decreased EVT invasion through activation of MAPK signaling. MEK1/2 activation may contribute to placental dysfunction, in the setting of inflammation-associated adverse obstetrical outcomes.

Inflammation promotes a cytokine response and disrupts the cervical epithelial barrier: a possible mechanism of premature cervical remodeling and preterm birth.

OBJECTIVE: An inflammatory challenge disrupts the cervical epithelial barrier and promotes cervical remodeling. STUDY DESIGN: Immortalized ectocervical and endocervical cells were treated with lipopolysaccharide (LPS), and interleukin (IL)-6, IL-8, and soluble E-cadherin (SECAD) were assessed. Cells were then pretreated with dexamethasone prior to LPS exposure, and IL-6, IL-8, and SECAD levels were again assessed. The integrity of the epithelial cell barrier was determined using a permeability assay. RESULTS: LPS significantly increased IL-6 and IL-8 levels, and SECAD was significantly increased at 24 hours. LPS induced inflammation increased permeability for both cell lines. Dexamethasone pretreatment prior to LPS exposure significantly decreased IL-6 and IL-8 levels in both cell lines. There was no reduction in SECAD levels with dexamethasone pretreatment. Permeability decreased in the presence of dexamethasone for ectocervical cells only. CONCLUSION: These studies demonstrate an inflammatory challenge to cervical epithelial cells promotes a cytokine release and functionally alters the cervical epithelial barrier.

Effects of increased fetuin-A in human trophoblast cells and associated pregnancy outcomes.

OBJECTIVE: The purpose of this study was to determine whether fetuin-A affects trophoblast viability and invasion, whether growth factors that bind receptors that activate tyrosine kinase are impaired by fetuin-A, and whether elevated maternal serum fetuin-A levels are associated with adverse pregnancy outcomes. STUDY DESIGN: We studied viability and invasion in first-trimester extravillous trophoblast cells that were exposed to fetuin-A, insulin-like growth factor, and placental growth factor. Insulin receptor substrates expression was assessed. We compared serum fetuin-A levels in 111 preeclampsia cases and 95 controls. RESULTS: Fetuin-A reduced extravillous trophoblast cell viability and invasion in the presence or absence of growth factors. Fetuin-A reduced insulin receptor substrate-1 and tyrosine phosphorylated insulin receptor substrate-1 expression in extravillous trophoblast cells that had been treated with insulin-like growth factor. Elevated serum fetuin-A levels were more prevalent in preeclampsia cases than control subjects, even after we controlled for diabetes mellitus, hypertension, and obesity. CONCLUSION: Fetuin-A may decrease trophoblast viability and invasion caused by the inhibition of receptor tyrosine kinase activity. Elevated serum levels of fetuin-A may be associated with preeclampsia.

Examining the correlation between placental and serum placenta growth factor in preeclampsia.

OBJECTIVE: Decreased levels of serum placenta growth factor (PlGF) are associated with preeclampsia. We sought to determine whether serum and placental levels of PlGF (sPlGF and pPlGF) are associated with preeclampsia and whether there is a correlation between serum and placental PlGF levels. STUDY DESIGN: These analyses were part of a larger, prospective, case-control study. Cases were women with preeclampsia. Controls were women without preeclampsia who delivered at term. Analyses included nonparametric tests to compare medians, logistic regression to estimate odds, and calculation of correlation coefficients. RESULTS: Twenty-four cases (10 preterm, 14 term) were compared with 14 controls. Median levels of PlGF were significantly lower in cases than controls (pPlGF: 232.6 vs 363.4 pg/mL, P = .02; sPlGF: 85.5 vs 274.4 pg/mL, P < .001). Serum and placental PlGF were correlated (overall: 39%, P = .006; cases with preterm preeclampsia and growth restriction: 87%, P = .02). CONCLUSION: Serum and placental PlGF are independently associated with preeclampsia and correlated with each other.