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.

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.

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.

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.

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.

Gardnerella vaginalis alters cervicovaginal epithelial cell function through microbe-specific immune responses.

BACKGROUND: The cervicovaginal (CV) microbiome is highly associated with vaginal health and disease in both pregnant and nonpregnant individuals. An overabundance of Gardnerella vaginalis (G. vaginalis) in the CV space is commonly associated with adverse reproductive outcomes including bacterial vaginosis (BV), sexually transmitted diseases, and preterm birth, while the presence of Lactobacillus spp. is often associated with reproductive health. While host-microbial interactions are hypothesized to contribute to CV health and disease, the mechanisms by which these interactions regulate CV epithelial function remain largely unknown. RESULTS: Using an in vitro co-culture model, we assessed the effects of Lactobacillus crispatus (L. crispatus) and G. vaginalis on the CV epithelial barrier, the immune mediators that could be contributing to decreased barrier integrity and the immune signaling pathways regulating the immune response. G. vaginalis, but not L. crispatus, significantly increased epithelial cell death and decreased epithelial barrier integrity in an epithelial cell-specific manner. A G. vaginalis-mediated epithelial immune response including NF-κB activation and proinflammatory cytokine release was initiated partially through TLR2-dependent signaling pathways. Additionally, investigation of the cytokine immune profile in human CV fluid showed distinctive clustering of cytokines by Gardnerella spp. abundance and birth outcome. CONCLUSIONS: The results of this study show microbe-specific effects on CV epithelial function. Altered epithelial barrier function through cell death and immune-mediated mechanisms by G. vaginalis, but not L. crispatus, indicates that host epithelial cells respond to bacteria-associated signals, resulting in altered epithelial function and ultimately CV disease. Additionally, distinct immune signatures associated with Gardnerella spp. or birth outcome provide further evidence that host-microbial interactions may contribute significantly to the biological mechanisms regulating reproductive outcomes. Video Abstract.

Gardnerella vaginalis induces matrix metalloproteinases in the cervicovaginal epithelium through TLR-2 activation.

Lactobacillus-deficient cervicovaginal microbiota, including Gardnerella vaginalis, are implicated in cervical remodeling and preterm birth. Mechanisms by which microbes drives outcomes are not fully elucidated. We hypothesize that Gardnerella vaginalis induces matrix metalloproteinases through TLR-2, leading to epithelial barrier dysfunction and premature cervical remodeling. Cervicovaginal cells were treated with live Gardnerella vaginalis or Lactobacillus crispatus or their bacteria-free supernatants for 24 h. For TLR-2 experiments, cells were pretreated with TLR-2 blocking antibody. A Luminex panel was run on cell media. For human data, we conducted a case-control study from a prospective pregnancy cohort of Black individuals with spontaneous preterm (sPTB) (n = 40) or term (n = 40) births whose vaginal microbiota had already been characterized. Cervicovaginal fluid was obtained between 20 and 24 weeks' gestation. Short cervix was defined as < 25 mm by second trimester transvaginal ultrasound. MMP-9 was quantified by ELISA. Standard analytical approaches were used to determine differences across in vitro conditions, as well as MMP-9 and associations with clinical outcomes. Gardnerella vaginalis induced MMP-1 in cervical cells (p = 0.01) and MMP-9 in cervical and vaginal (VK2) cells (p ≤ 0.001 for all). TLR-2 blockade mitigated MMP-9 induction by Gardnerella vaginalis. MMP-9 in cervicovaginal fluid is higher among pregnant individuals with preterm birth, short cervix, and Lactobacillus-deficient microbiota (p < 0.05 for all). MMP-9 is increased in the cervicovaginal fluid of pregnant individuals with subsequent sPTB. Our in vitro work ascribes a potential mechanism by which a cervicovaginal microbe, commonly associated with adverse pregnancy outcomes, may disrupt the cervicovaginal epithelial barrier and promote premature cervical remodeling in spontaneous preterm birth.

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.

IFNγ-Producing γ/δ T Cells Accumulate in the Fetal Brain Following Intrauterine Inflammation.

Intrauterine inflammation impacts prenatal neurodevelopment and is linked to adverse neurobehavioral outcomes ranging from cerebral palsy to autism spectrum disorder. However, the mechanism by which a prenatal exposure to intrauterine inflammation contributes to life-long neurobehavioral consequences is unknown. To address this gap in knowledge, this study investigates how inflammation transverses across multiple anatomic compartments from the maternal reproductive tract to the fetal brain and what specific cell types in the fetal brain may cause long-term neuronal injury. Utilizing a well-established mouse model, we found that mid-gestation intrauterine inflammation resulted in a lasting neutrophil influx to the decidua in the absence of maternal systemic inflammation. Fetal immunologic changes were observed at 72-hours post-intrauterine inflammation, including elevated neutrophils and macrophages in the fetal liver, and increased granulocytes and activated microglia in the fetal brain. Through unbiased clustering, a population of Gr-1+ γ/δ T cells was identified as the earliest immune cell shift in the fetal brain of fetuses exposed to intrauterine inflammation and determined to be producing high levels of IFNγ when compared to γ/δ T cells in other compartments. In a case-control study of term infants, IFNγ was found to be elevated in the cord blood of term infants exposed to intrauterine inflammation compared to those without this exposure. Collectively, these data identify a novel cellular immune mechanism for fetal brain injury in the setting of intrauterine inflammation.

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.

The tracking of lipopolysaccharide through the feto-maternal compartment and the involvement of maternal TLR4 in inflammation-induced fetal brain injury.

PROBLEM: Exposure to intrauterine inflammation (IUI) has been shown to induce fetal brain injury and increase the risk of acquiring a neurobehavioral disorder. The trafficking of the inflammatory mediator, lipopolysaccharide (LPS), in the pregnant female reproductive tract in the setting of IUI and the precise mechanisms by which inflammation induces fetal brain injury are not fully understood. METHOD OF STUDY: FITC-labeled LPS was utilized to induce IUI on E15, tissues were collected, and fluorescence was visualized via the Spectrum IVIS. Embryo transfer was utilized to create divergent maternal and fetal genotypes. Wild-type (WT) embryos were transferred into TLR4-/- pseudopregnant dams (TLR4-/-mat /WTfet ). On E15, TLR4-/-mat /WTfet dams or their WT controls (WTmat /WTfet ) received an intrauterine injection of LPS or phosphate-buffered saline (PBS). Endotoxin and IL-6 levels were assessed in amniotic fluid, and cytokine expression was measured via QPCR. RESULTS: Lipopolysaccharide trafficked to the uterus, fetal membranes, placenta, and the fetus and was undetectable in other tissues. Endotoxin was present in the amniotic fluid of all animals exposed to LPS. However, the immune response was blunted in TLR4-/-mat /WTfet compared with WT controls. CONCLUSION: Intrauterine administered LPS is capable of accessing the entire feto-placental unit with or without a functional maternal TLR4. Thus, bacteria or bacterial byproducts in the uterus may negatively impact fetal development regardless of the maternal genotype or endotoxin response. Despite the blunted immune response in the TLR4-deficient dams, an inflammatory response is still ignited in the amniotic cavity and may negatively impact the fetus.

HIF-1α Stabilization Increases miR-210 Eliciting First Trimester Extravillous Trophoblast Mitochondrial Dysfunction.

Preeclampsia is associated with first trimester placental dysfunction. miR-210, a small non-coding RNA, is increased in the preeclamptic placenta. The effects of elevated miR-210 on placental function remain unclear. The objectives of this study were to identify targets of miR-210 in first trimester primary extravillous trophoblasts (EVTs) and to investigate functional pathways altered by elevated placental miR-210 during early pregnancy. EVTs isolated from first trimester placentas were exposed to cobalt chloride (CoCl2), a HIF-1α stabilizer and hypoxia mimetic, and miR-210 expression by qPCR, HIF1α protein levels by western blot and cell invasion were assessed. A custom TruSeq RNA array, including all known/predicted miR-210 targets, was run using miR-210 and miR-negative control transfected EVTs. Mitochondrial function was assessed by high resolution respirometry in transfected EVTs. EVTs exposed to CoCl2 showed a dose and time-dependent increase in miR-210 and HIF1α and reductions in cell invasion. The TruSeq array identified 49 altered genes in miR-210 transfected EVTs with 27 genes repressed and 22 enhanced. Three of the top six significantly repressed genes, NDUFA4, SDHD, and ISCU, are associated with mitochondrial function. miR-210 transfected EVTs had decreased maximal, complex II and complex I+II mitochondrial respiration. This study suggests that miR-210 alters first trimester trophoblast function. miR-210 overexpression alters EVT mitochondrial function in early pregnancy. Mitochondrial dysfunction may lead to increased reactive oxygen species, trophoblast cell damage and likely contributes to the pathogenesis of preeclampsia.

Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction.

BACKGROUND: Genome-wide studies have begun to link subtle variations in both allelic DNA methylation and parent-of-origin genetic effects with early development. Numerous reports have highlighted that the placenta plays a critical role in coordinating fetal growth, with many key functions regulated by genomic imprinting. With the recent description of wide-spread polymorphic placenta-specific imprinting, the molecular mechanisms leading to this curious polymorphic epigenetic phenomenon is unknown, as is their involvement in pregnancies complications. RESULTS: Profiling of 35 ubiquitous and 112 placenta-specific imprinted differentially methylated regions (DMRs) using high-density methylation arrays and pyrosequencing revealed isolated aberrant methylation at ubiquitous DMRs as well as abundant hypomethylation at placenta-specific DMRs. Analysis of the underlying chromatin state revealed that the polymorphic nature is not only evident at the level of allelic methylation, but DMRs can also adopt an unusual epigenetic signature where the underlying histones are biallelically enrichment of H3K4 methylation, a modification normally mutually exclusive with DNA methylation. Quantitative expression analysis in placenta identified two genes, GPR1-AS1 and ZDBF2, that were differentially expressed between IUGRs and control samples after adjusting for clinical factors, revealing coordinated deregulation at the chromosome 2q33 imprinted locus. CONCLUSIONS: DNA methylation is less stable at placenta-specific imprinted DMRs compared to ubiquitous DMRs and contributes to privileged state of the placenta epigenome. IUGR-associated expression differences were identified for several imprinted transcripts independent of allelic methylation. Further work is required to determine if these differences are the cause IUGR or reflect unique adaption by the placenta to developmental stresses.

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

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.

Common Cervicovaginal Microbial Supernatants Alter Cervical Epithelial Function: Mechanisms by Which Lactobacillus crispatus Contributes to Cervical Health.

Cervicovaginal (CV) microbiota is associated with vaginal health and disease in non-pregnant women. Recent studies in pregnant women suggest that specific CV microbes are associated with preterm birth (PTB). While the associations between CV microbiota and adverse outcomes have been demonstrated, the mechanisms regulating the associations remain unclear. As the CV space contains an epithelial barrier, we postulate that CV microbiota can alter the epithelial barrier function. We investigated the biological, molecular, and epigenetic effects of Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis on the cervical epithelial barrier function and determined whether L. crispatus mitigates the effects of lipopolysaccharide (LPS) and G. vaginalis on the cervical epithelial barrier as a possible mechanism by which CV microbiota mitigates disease risk. Ectocervical and endocervical cells treated with L. crispatus, L. iners, and G. vaginalis bacteria-free supernatants alone or combined were used to measure cell permeability, adherens junction proteins, inflammatory mediators, and miRNAs. Ectocervical and endocervical permeability increased after L. iners and G. vaginalis exposure. Soluble epithelial cadherin increased after exposure to L. iners but not G. vaginalis or L. crispatus. A Luminex cytokine/chemokine panel revealed increased proinflammatory mediators in all three bacteria-free supernatants with L. iners and G. vaginalis having more diverse inflammatory effects. L. iners and G. vaginalis altered the expression of cervical-, microbial-, and inflammatory-associated miRNAs. L. crispatus mitigated the LPS or G. vaginalis-induced disruption of the cervical epithelial barrier and reversed the G. vaginalis-mediated increase in miRNA expression. G. vaginalis colonization of the CV space of a pregnant C57/B6 mouse resulted in 100% PTB. These findings demonstrate that L. iners and G. vaginalis alter the cervical epithelial barrier by regulating adherens junction proteins, cervical immune responses, and miRNA expressions. These results provide evidence that L. crispatus confers protection to the cervical epithelial barrier by mitigating LPS- or G. vaginalis-induced miRNAs associated with cervical remodeling, inflammation, and PTB. This study provides further evidence that the CV microbiota plays a role in cervical function by altering the cervical epithelial barrier and initiating PTB. Thus, targeting the CV microbiota and/or its effects on the cervical epithelium may be a potential therapeutic strategy to prevent PTB.

Colonization of the cervicovaginal space with Gardnerella vaginalis leads to local inflammation and cervical remodeling in pregnant mice.

The role of the cervicovaginal (CV) microbiome in regulating cervical function during pregnancy is poorly understood. Gardnerella vaginalis (G. vaginalis) is the most common bacteria associated with the diagnosis of bacterial vaginosis (BV). While BV has been associated with preterm birth (PTB), clinical trials targeting BV do not decrease PTB rates. It remains unknown if G. vaginalis is capable of triggering molecular, biomechanical and cellular events that could lead to PTB. The objective of this study was to determine if cervicovaginal colonization with G. vaginalis, in pregnant mice, induced cervical remodeling and modified cervical function. CD-1 timed-pregnant mice received a 5X108 CFU/mL intravaginal inoculation of G. vaginalis or control on embryonic day 12 (E12) and E13. On E15, the mice were sacrificed and cervicovaginal fluid (CVF), amniotic fluid (AF), cervix, uterus, placentas and fetal membranes (FM) were collected. Genomic DNA was isolated from the CVF, placenta, uterus and FM and QPCR was performed to confirm colonization. IL-6 was measured in the CVF and AF and soluble e-cadherin (seCAD) was assessed in the CVF by ELISA. RNA was extracted from the cervices to evaluate IL-10, IL-8, IL-1ß, TNF-α, Tff-1, SPINK-5, HAS-1 and LOX expression via QPCR. Mucicarmine and trichrome staining was used to assess cervical mucin and collagen. Biomechanical properties of the cervix were studied using quasi-static tensile load-to-failure biomechanical tests. G. vaginalis successfully colonized the CV space. This colonization induced immune responses (increased IL-6 levels in CVF and AF, increased mRNA expression of cervical cytokines), altered the epithelial barrier (increased seCAD in the CVF), induced cervical remodeling (increased mucin production, altered collagen) and altered cervical biomechanical properties (a decrease in biomechanical modulus and an increase in maximum strain). The ability of G. vaginalis to induce these molecular, immune, cellular and biomechanical changes suggests that this bacterium may play a pathogenic role in premature cervical remodeling leading to PTB.

miR-143 and miR-145 disrupt the cervical epithelial barrier through dysregulation of cell adhesion, apoptosis and proliferation.

Molecular mechanisms regulating preterm birth (PTB)-associated cervical remodeling remain unclear. Prior work demonstrated an altered miRNA profile, with significant increases in miR-143 and miR-145, in cervical cells of women destined to have a PTB. The study objective was to determine the effect of miR-143 and miR-145 on the cervical epithelial barrier and to elucidate the mechanisms by which these miRNAs modify cervical epithelial cell function. Ectocervical and endocervical cells transfected with miR-negative control, miR-143 or miR-145 were used in cell permeability and flow cytometry assays for apoptosis and proliferation. miR-143 and miR-145 target genes associated with cell adhesion, apoptosis and proliferation were measured. Epithelial cell permeability was increased in miR-143 and miR-145 transfected cervical epithelial cells. Cell adhesion genes, JAM-A and FSCN1, were downregulated with overexpression of miR-143 and miR-145. miR-143 and miR-145 transfection decreased cervical cell number by increasing apoptosis and decreasing cell proliferation through initiation of cell cycle arrest. Apoptosis genes, BCL2 and BIRC5, and proliferation genes, CDK1 and CCND2, were repressed by miR-143 and miR-145. These findings suggest that miR-143 and miR-145 play a significant role in cervical epithelial barrier breakdown through diverse mechanisms and could contribute to premature cervical remodeling associated with PTB.