Ureaplasma parvum infection induces inflammatory changes in vaginal epithelial cells independent of sialidase.

BACKGROUND: Ureaplasma, a genus of the order Mycoplasmatales and commonly grouped with Mycoplasma as genital mycoplasma is one of the most common microbes isolated from women with infection/inflammation-associated preterm labor (PTL). Mycoplasma spp. produce sialidase that cleaves sialic acid from glycans of vaginal mucous membranes and facilitates adherence and invasion of the epithelium by pathobionts, and dysregulated immune response. However, whether Ureaplasma species can induce the production of sialidase is yet to be demonstrated. We examined U. parvum-infected vaginal epithelial cells (VECs) for the production of sialidase and pro-inflammatory cytokines. METHODS: Immortalized VECs were cultured in appropriate media and treated with U. parvum in a concentration of 1 × 105 DNA copies/ml. After 24 h of treatment, cells and media were harvested. To confirm infection and cell uptake, immunocytochemistry for multi-banded antigen (MBA) was performed. Pro-inflammatory cytokine production and protein analysis for sialidase confirmed pro-labor pathways. RESULTS: Infection of VECs was confirmed by the presence of intracellular MBA. Western blot analysis showed no significant increase in sialidase expression from U. parvum-treated VECs compared to uninfected cells. However, U. parvum infection induced 2-3-fold increased production of GM-CSF (p = 0.03), IL-6 (p = 0.01), and IL-8 (p = 0.01) in VECs compared to controls. CONCLUSION: U. parvum infection of VECs induced inflammatory imbalance associated with vaginal dysbiosis but did not alter sialidase expression at the cellular level. These data suggest that U. parvum's pathogenic effect could be propagated by locally produced pro-inflammatory cytokines and, unlike other genital mycoplasmas, may be independent of sialidase.

Silencing P38 MAPK reduces cellular senescence in human fetal chorion trophoblast cells.

PROBLEM: Amniochorion senescence generates mechanistic signals to initiate parturition. Activation of p38 mitogen-activated kinase (MAPK) in fetal amnion cells is a key mediator of senescence as well as epithelial-mesenchymal transition (EMT) of amnion cells. However, the impact of p38 MAPK in chorion trophoblast cells (CTCs) is unclear. We tested if eliminating p38 will reduce oxidative stress (OS) induced cell fates like cellular senescence, EMT, and inflammation induced by these processes in CTCs. METHODS: p38MAPK in CTCs was silenced using CRISPR/Cas9. OS was evoked by cigarette smoke extract (CSE) exposure. EMT was evoked by transforming growth factor (TGF)-ß treatment. Cell cycle, senescence, EMT, and inflammation were analyzed. RESULTS: CSE-induced changes in the cell cycle were not seen in p38KO CTCs compared to WT cells. OS induced by CSE evoked senescence and senescence-associated secretory phenotype (SASP as indicated by IL-6 and IL-8 increase) in WT but not in p38MAPK KO CTCs. No changes were noted in HLA-G expression regardless of the status of p38MAPK. Neither CSE nor TGF-ß evoked EMT in either WT or p38 KO CTCs. CONCLUSION: Senescence and senescence-associated inflammation in human fetal CTCs are mediated by p38MAPK. Compared to amnion epithelial cells, CTCs are resistant to EMT. This refractoriness may help them to maintain the barrier functions at the choriodecidual interface.

Fetal Membranes Contribute to Drug Transport across the Feto-Maternal Interface Utilizing the Breast Cancer Resistance Protein (BCRP).

During pregnancy, the placenta is established as a primary organ for drug transport at the maternal-fetal interface. The fetal membranes (FM) also form an interface with maternal tissues; however, their role in drug transport has not been previously investigated. Knowledge of drug transport across this feto-maternal interface along with the placenta can improve new drug development and testing for use during pregnancy. We also hypothesize that extracellular vesicles (exosomes 30-160 nm) released from the FM and placental cells may also contain drug transport proteins and might impact drug trafficking across the feto-maternal interfaces. The objectives were to (1) localize the breast cancer resistance protein (BCRP) in human FM; (2) determine the drug transport function of BCRP in chorion trophoblast cells (CTCs) of the FM; and (3) investigate the presence of BCRP in FM cell-derived exosomes, as a paracrine modifier of the tissue environment for transport functions. The gene and protein expressions of ABCG2/BCRP in FMs were determined by quantitative real-time PCR (qRT-PCR) and western blotting (WB) and were localized by immunohistochemistry (IHC). The surface expression of BCRP in FM cells was determined by flow cytometry. The functional role of BCRP was assessed by an EFFLUX dye multidrug resistance assay. The presence of BCRP in exosomes derived from CTCs and BeWo cells was examined using ExoView®. Data derived from CTCs are compared with placental trophoblast cells (BeWo). BCRP is expressed and localized in the fetal membrane, primarily in the chorion trophoblast cell layer and scarcely in the amnion epithelial layer (AEC), and primarily localized on both AEC and CTC cell surfaces. Efflux assay data showed that FM cells have similar drug resistance activity as BeWo cells, suggesting that FM also have drug transportation capabilities. BeWo- and CTC-derived exosomes expressed limited BCRP protein on the surface, so it was predominantly contained in the exosomal lumen. As far as we are aware, this is the first study to report BCRP expression in fetal membrane cells and as cargo in fetal membrane-derived exosomes. We report that fetal membrane cells are capable of drug transportation. Based on these results, investigational drug trials should include the FM and its exosomes as possible drug transportation routes in pregnancy.

The Quantose Insulin Resistance Test for Maternal Insulin Resistance: A Pilot Study.

OBJECTIVE: Insulin resistance (IR) increases during pregnancy which can lead to hyperinsulinemia, gestational diabetes mellitus (GDM), and neonatal hypoglycemia (NH), especially in obese women. Glucose tolerance testing (GTT) is used clinically to evaluate IR in pregnancy. Quantose IR score index is a novel blood screen of IR validated in nonpregnant individuals. The score is generated using an algorithm that combines insulin and three biomarkers of fatty acid pathways (α-hydroxybutyrate, oleic acid, linoleoyl-glycerophospocholine). Our objective was to determine the validity of Quantose IR test (Metabolan Inc. Morrisville, NC) in assessing IR in pregnant obese women, as compared with the homeostatic model assessment of insulin resistance (HOMA-IR), and its ability to predict GDM and NH. STUDY DESIGN: Women between 100/7 and 136/7 weeks of gestation with a pre-pregnancy or early pregnancy body mass index more than 30 kg/m2, and no pregestational diabetes, were included. Fasting blood samples were collected at 100/7 to 136/7 (T1) and 240/7 to 280/7 (T2) weeks. Quantose IR and HOMA-IR were calculated. All women underwent an early (T1; indicated for women with obesity) and a T2 glucose tolerance tests. GDM was diagnosed using the two-step approach, and NH was defined as a neonatal glucose less than 40 mg/dL in the first 24 hours of life. Linear regression and receiver operating characteristic curves were used for analysis. RESULTS: The trial enrolled 100 patients. Ten subjects (10%) were diagnosed with GDM in the second trimester and none in the first trimester. At T1, Quantose IR (R2 = 0.48), but not 1-hour glucose tolerance test (R2 = 0.07), correlated with HOMA-IR. Similar correlations were observed at T2. The 1-hour glucose tolerance test followed by HOMA-IR and Quantose IR (area under the curve [AUC]: 0.82, 0.68, and 0.62, respectively) were predictors of GDM. Quantose IR (AUC: 0.74) and 1-hour glucose tolerance test (AUC: 0.72) at T1 and T2 (AUC: 0.75; AUC: 0.93; respectively) were best predictors of NH. The best cut offs, sensitivities, and specificities for prediction of NH were determined. CONCLUSION: Similar to nonpregnant individuals, Quantose IR appears to be a valid measure of IR in obese pregnant women. First trimester Quantose IR is a predictor of GDM diagnosed in the second trimester and NH. Given that it requires a single blood draw and no glucose challenge, it may be a useful test to evaluate and monitor IR in pregnancy. Our findings may be used as pilot data to explore the potential use of Quantose IR in pregnancy further. KEY POINTS: · Traditional testing methods for insulin resistance in pregnancy are often performed late, are time consuming, and unpleasant to patients.. · The first trimester one-step Quantose IR test reflects insulin resistance in pregnancy and predicts GDM and neonatal hypoglycemia.. · This is the first known prospective clinical study validating Quantose IR score index in an obstetrical population at risk for developing GDM..

Development of a mouse model of ascending infection and preterm birth.

BACKGROUND: Microbial invasion of the intraamniotic cavity and intraamniotic inflammation are factors associated with spontaneous preterm birth. Understanding the route and kinetics of infection, sites of colonization, and mechanisms of host inflammatory response is critical to reducing preterm birth risk. OBJECTIVES: This study developed an animal model of ascending infection and preterm birth with live bacteria (E. coli) in pregnant CD-1 mice with the goal of better understanding the process of microbial invasion of the intraamniotic cavity and intraamniotic inflammation. STUDY DESIGN: Multiple experiments were conducted in this study. To determine the dose of E. coli required to induce preterm birth, CD-1 mice were injected vaginally with four different doses of E. coli (103, 106, 1010, or 1011 colony forming units [CFU]) in 40 µL of nutrient broth or broth alone (control) on an embryonic day (E)15. Preterm birth (defined as delivery before E18.5) was monitored using live video. E. coli ascent kinetics were measured by staining the E. coli with lipophilic tracer DiD for visualization through intact tissue with an in vivo imaging system (IVIS) after inoculation. The E. coli were also directly visualized in reproductive tissues by staining the bacteria with carboxyfluorescein succinimidyl ester (CFSE) prior to administration and via immunohistochemistry (IHC) by staining tissues with anti-E. coli antibody. Each pup's amniotic fluid was cultured separately to determine the extent of microbial invasion of the intraamniotic cavity at different time points. Intraamniotic inflammation resulting from E. coli invasion was assessed with IHC for inflammatory markers (TLR-4, P-NF-κB) and neutrophil marker (Ly-6G) for chorioamnionitis at 6- and 24-h post-inoculation. RESULTS: Vaginally administered E. coli resulted in preterm birth in a dose-dependent manner with higher doses causing earlier births. In ex vivo imaging and IHC detected uterine horns proximal to the cervix had increased E. coli compared to the distal uterine horns. E. coli were detected in the uterus, fetal membranes (FM), and placenta in a time-dependent manner with 6 hr having increased intensity of E. coli positive signals in pups near the cervix and in all pups at 24 hr. Similarly, E. coli grew from the cultures of amniotic fluid collected nearest to the cervix, but not from the more distal samples at 6 hr post-inoculation. At 24 hr, all amniotic fluid cultures regardless of distance from the cervix, were positive for E. coli. TLR-4 and P-NF-κB signals were more intense in the tissues where E. coli was present (placenta, FM and uterus), displaying a similar trend toward increased signal in proximal gestational sacs compared to distal at 6 hr. Ly-6G+ cells, used to confirm chorioamnionitis, were increased at 24 hr compared to 6 hr post-inoculation and control. CONCLUSION: We report the development of mouse model of ascending infection and the associated inflammation of preterm birth. Clinically, these models can help to understand mechanisms of infection associated preterm birth, determine targets for intervention, or identify potential biomarkers that can predict a high-risk pregnancy status early in pregnancy.

Inflammatory response elicited by Ureaplasma parvum colonization in human cervical epithelial, stromal, and immune cells.

Ureaplasma parvum is a commensal bacterium in the female reproductive tract but has been associated with pregnancy complications such as preterm prelabor rupture of membranes and preterm birth (PTB). However, the pathologic effects of U. parvum in the cervix, which prevents ascending infections during pregnancy, are still poorly understood. To determine the impact of U. parvum on the cervix, ectocervical (ecto) and endocervical (endo) epithelial and stromal cells were incubated with U. parvum. Macrophages were also tested as a proxy for cervical macrophages to determine the antigenicity of U. parvum. The effects of U. parvum, including influence on cell cycle and cell death, antimicrobial peptide (AMP) production, epithelial-to-mesenchymal transition (EMT), and inflammatory cytokine levels, were assessed. U. parvum colonized cervical epithelial and stromal cells 4 h post-infection. Like uninfected control, U. parvum neither inhibited cell cycle progression and nor caused cell death in cervical epithelial and stromal cells. U. parvum increased the production of the AMPs cathelicidin and human ß-defensin 3 and exhibited weak signs of EMT evidenced by decreased cytokeratin 18 and increased vimentin expression in cervical epithelial cells. U. parvum induced a proinflammatory environment (cytokines) and increased MMP-9 in cervical epithelial cells but promoted pro- and anti-inflammatory response in cervical stromal cells and macrophages. U. parvum may colonize the cervical epithelial layer, but induction of AMPs and anti-inflammatory response may protect the cervix and may prevent ascending infections that can cause PTB. These findings suggest that U. parvum is a weak inducer of inflammation in the cervix.

Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response.

BACKGROUND: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. METHODS: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. RESULTS: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. CONCLUSION: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract.

Cross talk: trafficking and functional impact of maternal exosomes at the feto-maternal interface under normal and pathologic states†.

Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (ecto), endocervical epithelial cells (endo), and cervical stromal cells (stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual cells, chorion trophoblast cells (CTC), chorion mesenchymal cells (CMC), amnion mesenchymal cells (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. Enzyme-linked immunosorbent assay for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi.

Microvesicles and exosomes released by amnion epithelial cells under oxidative stress cause inflammatory changes in uterine cells†.

Extracellular vesicles play a crucial role in feto-maternal communication and provide an important paracrine signaling mechanism in pregnancy. We hypothesized that fetal cells-derived exosomes and microvesicles (MVs) under oxidative stress (OS) carry unique cargo and traffic through feto-maternal interface, which cause inflammation in uterine cells associated with parturition. Exosomes and MVs, from primary amnion epithelial cell (AEC) culture media under normal or OS-induced conditions, were isolated by optimized differential centrifugation method followed by characterization for size (nanoparticle tracking analyzer), shape (transmission electron microscopy), and protein markers (western blot and immunofluorescence). Cargo and canonical pathways were identified by mass spectroscopy and ingenuity pathway analysis. Myometrial, decidual, and cervical cells were treated with 1 × 107 control/OS-derived exosomes/MVs. Pro-inflammatory cytokines were measured using a Luminex assay. Statistical significance was determined by paired T-test (P < 0.05). AEC produced cup-shaped exosomes of 90-150 nm and circular MVs of 160-400 nm. CD9, heat shock protein 70, and Nanog were detected in exosomes, whereas OCT-4, human leukocyte antigen G, and calnexin were found in MVs. MVs, but not exosomes, were stained for phosphatidylserine. The protein profiles for control versus OS-derived exosomes and MVs were significantly different. Several inflammatory pathways related to OS were upregulated that were distinct between exosomes and MVs. Both OS-derived exosomes and MVs significantly increased pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating factor, interleukin 6 (IL-6), and IL-8) in maternal cells compared with control (P < 0.05). Our findings suggest that fetal-derived exosomes and MVs under OS exhibited distinct characteristics and a synergistic inflammatory role in uterine cells associated with the initiation of parturition.

Extracellular vesicle mediated feto-maternal HMGB1 signaling induces preterm birth.

Preterm birth (PTB; <37 weeks of gestation) impacts ∼11% of all pregnancies and contributes to 1 million neonatal deaths worldwide annually. An understanding of the feto-maternal (F-M) signals that initiate birthing (parturition) at term is critical to design strategies to prevent their premature activation, resulting in PTB. Although endocrine and immune cell signaling are well-reported, fetal-derived paracrine signals capable of transitioning quiescent uterus to an active state of labor are poorly studied. Recent reports have suggested that senescence of the fetal amnion membrane coinciding with fetal growth and maturation generates inflammatory signals capable of triggering parturition. This is by increasing the inflammatory load at the feto-maternal interface (FMi) tissues (i.e., amniochorion-decidua). High mobility group box 1 protein (HMGB1), an alarmin, is one of the inflammatory signals released by senescent amnion cells via extracellular vesicles (exosomes; 40-160 nm). Increased levels of HMGB1 in the amniotic fluid, cord and maternal blood are associated with term and PTB. This study tested the hypothesis that senescent amnion cells release HMGB1, which is fetal signaling capable of increasing FMi inflammation, predisposing them to parturition. To test this hypothesis, exosomes from amnion epithelial cells (AECs) grown under normal conditions were engineered to contain HMGB1 by electroporation (eHMGB1). eHMGB1 was characterized (quantity, size, shape, markers and loading efficiency), and its propagation through FMi was tested using a four-chamber microfluidic organ-on-a-chip device (FMi-OOC) that contained four distinct cell types (amnion and chorion mesenchymal, chorion trophoblast and decidual cells) connected through microchannels. eHMGB1 propagated through the fetal cells and matrix to the maternal decidua and increased inflammation (receptor expression [RAGE and TLR4] and cytokines). Furthermore, intra-amniotic injection of eHMGB1 (containing 10 ng) into pregnant CD-1 mice on embryonic day 17 led to PTB. Injecting carboxyfluorescein succinimidyl ester (CFSE)-labeled eHMGB1, we determined in vivo kinetics and report that eHMGB1 trafficking resulting in PTB was associated with increased FMi inflammation. This study determined that fetal exosome mediated paracrine signaling can generate inflammation and induce parturition. Besides, in vivo functional validation of FMi-OOC experiments strengthens the reliability of such devices to test physiologic and pathologic systems.

Progesterone alters human cervical epithelial and stromal cell transition and migration: Implications in cervical remodeling during pregnancy and parturition.

The cervix undergoes extensive remodeling throughout pregnancy and parturition. This process involves both ECM collagen degradation and cellular remodeling, which includes cell proliferation, transition and migration. Progesterone (P4) has been used clinically to delay cervical ripening and prevent preterm birth (PTB). However, the mechanisms by which progesterone affects cell transition and the migration of cervical epithelial and stromal cells are not yet fully known. In this study, we documented the role of a gestational level of P4 in the cellular transition (epithelial-mesenchymal transition [EMT] and mesenchymal-epithelial transition [MET]), cell migration, and inflammatory responses of endocervical epithelial cells (EEC) and cervical stromal cells (CSC). EEC and CSC were treated with LPS and P4 for 6 days. The epithelial:mesenchymal ratio (regular microscopy and cell shape index analysis), shift in intermediate filaments (immunofluorescence microscopy and western blot analyses for cytokeratin [CK]-18 and vimentin), adhesion molecules and transcription factors (western blot analyses for E-cadherin, N-cadherin and SNAIL), were used to determine growth characteristics and EMT and MET changes in EEC and CSC under the indicated conditions. To test cell remodeling, scratch assays followed by cellular analyses as mentioned above were performed. Inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor α [TNFα]) and matrix metallopeptidase 9 (MMP9) were measured by ELISA. LPS promoted EMT (decreased cell shape index, decreased CK-18 and E-cadherin, increased vimentin, N-cadherin, and SNAIL), and increased IL-6 and MMP9 production by EEC. A gestational level of P4 prevented LPS-induced EMT in EEC and exhibited anti-inflammatory effect in both EEC and CSC. LPS slowed down wound healing in CSC but P4 treatment prevented the negative impact of LPS in CSC wound healing. These results may explain the cellular mechanisms by which P4 helps to stabilize the cervical epithelial barrier and preserve the mechanical and tensile strength of the cervical stromal layer, which are important in normal cervical remodeling processes during pregnancy.

Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation.

The fetal inflammatory response, a key contributor of infection-associated preterm birth (PTB), is mediated by nuclear factor kappa B (NF-kB) activation. Na+/H+ exchanger regulatory factor-1 (NHERF1) is an adapter protein that can regulate intracellular signal transduction and thus influence NF-kB activation. Accordingly, NHERF1 has been reported to enhance proinflammatory cytokine release and amplify inflammation in a NF-kB-dependent fashion in different cell types. The objective of this study was to examine the role of NHERF1 in regulating fetal membrane inflammation during PTB. We evaluated the levels of NHERF1 in human fetal membranes from term labor (TL), term not in labor (TNIL), and PTB and in a CD1 mouse model of PTB induced by lipopolysaccharide (LPS). Additionally, primary cultures of fetal membrane cells were treated with LPS, and NHERF1 expression and cytokine production were evaluated. Gene silencing methods using small interfering RNA targeting NHERF1 were used to determine the functional relevance of NHERF1 in primary cultures. NHERF1 expression was significantly (p < 0.001) higher in TL and PTB membranes compared to TNIL membranes, and this coincided with enhanced (p < 0.01) interleukin (IL)-6 and IL-8 expression levels. LPS-treated animals delivering PTB had increased levels of NHERF1, IL-6, and IL-8 compared to phosphate-buffered saline (PBS; control) animals. Silencing of NHERF1 expression resulted in a significant reduction in NF-kB activation and IL-6 and IL-8 production as well as increased IL-10 production. In conclusion, downregulation of NHERF1 increased anti-inflammatory IL-10, and reducing NHERF1 expression could be a potential therapeutic strategy to reduce the risk of infection/inflammation associated with PTB.

Interleukin (IL)-6: A Friend or Foe of Pregnancy and Parturition? Evidence From Functional Studies in Fetal Membrane Cells.

OBJECTIVE: Protection of the fetus within the amniotic sac is primarily attained by remodeling fetal membrane (amniochorion) cells through cyclic epithelial to mesenchymal and mesenchymal to epithelial (EMT and MET) transitions. Endocrine and paracrine factors regulate EMT and MET during pregnancy. At term, increased oxidative stress forces a terminal state of EMT and inflammation, predisposing to membrane weakening and rupture. IL-6 is a constitutively expressed cytokine during gestation, but it is elevated in term and preterm births. Therefore, we tested the hypothesis that IL-6 can determine the fate of amnion membrane cells and that pathologic levels of IL-6 can cause a terminal state of EMT and inflammation, leading to adverse pregnancy outcomes. METHODS: Primary amnion epithelial cells (AECs) were treated with recombinant IL-6 (330, 1,650, 3,330, and 16,000 pg/ml) for 48 h (N = 5). IL-6-induced cell senescence (aging), cell death (apoptosis and necrosis), and cell cycle changes were studied using flow cytometry. Cellular transitions were determined by immunocytochemistry and western blot analysis, while IL-6 signaling (activation of signaling kinases) was measured by immunoassay. Inflammatory marker matrix metalloproteinase (MMP9) and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations were measured using a Fluorokine E assay and ELISA, respectively. Amniotic membranes collected on gestational day (D) 12 and D18 from IL-6 knockout (KO) and control C57BL/6 mice (N = 3 each) were used to determine the impact of IL-6 on cell transitions. Fold changes were measured based on the mean of each group. RESULTS: IL-6 treatment of AECs at physiologic or pathologic doses increased JNK and p38MAPK activation; however, the activation of signals did not cause changes in AEC cell cycle, cellular senescence, apoptosis, necrosis, cellular transitions, or inflammation (MMP9 and GM-CSF) compared to control. EMT markers were higher on D18 compared to D12 regardless of IL-6 status in the mouse amniotic sac. CONCLUSION: Physiologic and pathologic concentrations of IL-6 did not cause amnion cell aging, cell death, cellular transitions, or inflammation. IL-6 may function to maintain cellular homeostasis throughout gestation in fetal membrane cells. Although IL-6 is a good biomarker for adverse pregnancies, it is not an indicator of an underlying pathological mechanism in membrane cells.

Inflammation, but not infection, induces EMT in human amnion epithelial cells.

A non-reversible state of epithelial to mesenchymal transition (EMT) at term accumulates proinflammatory mesenchymal cells and predisposes fetal membrane to weakening prior to delivery at term. We investigated the induction of EMT in amnion epithelial cells (AEC) in response to inflammation and infection associated with spontaneous preterm birth (SPTB). For this, membranes from SPTB were screened for EMT markers. Primary AEC in culture were treated with TNF-α (10 and 50 ng/mL) and LPS (50 and 100 ng/mL) for 72 h. Cell shape index (SI) was determined based on morphological shift (microscopy followed by ImageJ software analysis). Immunocytochemistry and Western blot assessed changes in epithelial markers (cytokeratin-18 and E-cadherin) and mesenchymal markers (vimentin and N-cadherin). Involvement of transforming growth factor beta (TGF-ß) in EMT induction and EMT associated inflammation was tested using specific markers (Western blot) and by measuring MMP9 (ELISA), respectively. We report that PTB is associated with fetal membrane EMT. TNF-α produced dose- and time-dependent induction of EMT; within 24 h by 50 ng/mL and after 72 h by 10 ng/mL. AEC showed mesenchymal morphology, lower E-cadherin, higher vimentin and N-cadherin and higher MMP9 compared to control. TNF-α-induced EMT was not associated with canonical TGF-ß pathway. LPS, regardless of dose or time, did not induce EMT in AEC. We conclude that PTB with intact membranes is associated with EMT. Our data suggest that inflammation, but not infection, is associated with non-canonical activation of EMT and inflammation that can predispose membrane to undergo weakening.

Lactation Leads to Modifications in Maternal Renin-Angiotensin System in Later Life.

The objective of this study was to evaluate whether the renin-angiotensin system (RAS) is associated with maternal cardioprotective phenotype observed in post-lactated mice later in life. Following the delivery, CD-1 female mice were randomized to one of the following groups: lactated (nursed pups for 3 weeks, n = 10) or non-lactated (pups were removed after birth, n = 10). The mice were sacrificed 6 months after the delivery, and tissues were collected. Protein levels of angiotensinogen, angiotensin type 1 and 2 receptors (AT1R, AT2R), angiotensin converting enzymes (ACE, ACE2), and MAS receptor were determined using Western blot. Results were analyzed using Student's t-test and Mann-Whitney test as appropriate (significance: P < 0.05). Angiotensinogen levels were significantly lower in the liver (P = 0.0002), and ACE was significantly decreased in the lungs (P = 0.04) and kidney (P = 0.001) from lactated mice as compared to non-lactated. The levels of AT2R in the kidney (P = 0.02) and visceral adipose tissue (VAT, P = 0.04), the ACE 2 in the VAT (P = 0.03) and heart (P = 0.04), and MAS receptor in VAT (P = 0.02) were significantly elevated in tissues from lactated mice. No other differences were found. Lactation led to the upregulation and downregulation of selected RAS components in lactated mice as compared to non-lactated group and may be a contributing factor to maternal cardioprotective phenotype later in life. Further studies are needed to dissect the mechanisms between lactation and the long-term maternal cardiometabolic benefits, which could lead to the therapies to prevent cardiovascular disease in women.

Changes in mediators of pro-cell growth, senescence, and inflammation during murine gestation.

PROBLEM: Senescence of the fetal membranes and senescence-associated inflammation have been associated with parturition at term and pre-term in both mice and humans. Using a pregnant mouse model, we determined changes in multiple molecular signalers contributing to senescence and inflammation associated with parturition. METHOD OF STUDY: Fetal membranes were collected from timed-pregnant CD-1 mice on gestation days (E) 13, 15, 17, 18, and 19. Immunohistochemistry (IHC) localized pro-cell growth factors glycogen synthase kinase 3ß (GSK3ß) and ß-catenin. Gestational age-associated changes in pro-cell growth vs senescence mediators (p38 mitogen-activated protein kinase [p38MAPK]), prooxidants (heme oxygenase-1 [HO-1], peroxisome proliferator-activated receptor γ [PPARγ]), and pro- and anti-inflammatory cytokines (IL-6, IL-8, IL-10, and IL-1ß) were determined by Western blots and Luminex assays. RESULTS: Fetal membrane expressions of phosphorylated forms of GSK3ß (inactivation) and p38MAPK (activation) increased, while ß-catenin expression decreased, as gestation progressed. Antioxidant HO-1 expression decreased while PPARγ increased toward term gestation. IL-6 and IL-8 concentrations were highest on E19 (day of delivery), while IL-10 and IL-1ß concentrations were highest on E15. CONCLUSION: Mouse fetal membranes showed a progressive senescence marker increase coincided with downregulation of cell growth factors. Development of senescence is associated with inflammation. Senescence-associated changes are natural and physiologic and indicative of fetal membranes' readiness for parturition.

Oxidative stress-induced downregulation of glycogen synthase kinase 3 beta in fetal membranes promotes cellular senescence†.

OBJECTIVE: Oxidative stress (OS)-induced stress signaler p38 mitogen-activated protein kinase (p38MAPK) activation and fetal membrane senescence are associated with parturition. This study determined changes in glycogen synthase kinase 3 beta (GSK3ß) and its regulation by p38MAPK in effecting senescence to further delineate the molecular mechanism involved in senescence. METHODS: Primary human amnion epithelial cells and amnion mesenchymal cells were treated with cigarette smoke extract (CSE, OS inducer). Expression of total and phosphorylated GSK3ß and p38MAPK, and that of GSK3ß's downstream targets: beta-catenin (ß-Cat) and nuclear factor erythroid 2-related factor 2 (Nrf2) (western blot analysis), cell cycle regulation and senescence (flow cytometry) were determined. The specificity of GSK3ß and p38MAPK's mechanistic role was tested by co-treating cells with their respective inhibitors, CHIR99021 and SB203580. Exosomal secretion of ß-Cat from OS-induced cells was confirmed by immunofluorescence confocal microscopy and western blot. RESULTS: OS induced by CSE resulted in phosphorylation of GSK3ß (inactivation) and p38MAPK (activation) that was associated with cell cycle arrest and senescence. Inhibitors to GSK3ß and p38MAPK verified their roles. Glycogen synthase kinase 3 beta inactivation was associated with nuclear translocation of antioxidant Nrf2 and exosomal secretion of ß-Cat. CONCLUSIONS: OS-induced P-p38MAPK activation is associated with functional downregulation of GSK3ß and arrest of cell cycle progression and senescence of amnion cells. Lack of nuclear translocation of ß-Cat and its excretion via exosomes further supports the postulation that GSK3ß down-regulation by p38MAPK may stop cell proliferation preceding cell senescence. A better understanding of molecular mechanisms of senescence will help develop therapeutic strategies to prevent preterm birth.

Effect of Normal Pregnancy Followed by Lactation on Long-Term Maternal Health in a Mouse Model.

Although it has been widely accepted that pregnancies with complications are associated with increased maternal cardiovascular risk later in life, there is no consensus if noncomplicated pregnancy followed by lactation plays a protective role or is a risk factor. The objective of this study was to investigate the effects of normal pregnancy and lactation on long-term maternal health in a mouse model. CD-1 mice were allocated to breeding (primigravid [PG]) and nonbreeding (nulligravid [NG]) groups. The PG group proceeded through normal pregnancy and delivery. Using a telemetry system, blood pressure (BP) was analyzed in the PG group at 6 months postpartum and in age-matched NG mice. Serum analytes, gene expressions, and protein levels were determined using appropriate analysis methods. Primigravid mice had significantly lower systolic and diastolic BP and fasting glucose levels. Circulating oxytocin (OXT) levels were significantly higher in PG mice. Oxt gene expression was significantly higher in the heart and aorta and lower in visceral adipose tissue (VAT) from PG mice. The oxytocin receptor ( Oxtr) gene expression was significantly higher in the heart, aorta, and VAT from PG animals. The level of Oxtr DNA hypermethylation and the expression of mmu-miR-29a were significantly lower in the hearts of PG mice. In PG VAT, glucose transporter-4 expression was significantly higher. Our study demonstrates that a history of normal pregnancy followed by lactation was associated with lower maternal cardiovascular risk factors later in life in female mouse.

Differential senescence in feto-maternal tissues during mouse pregnancy.

BACKGROUND: Human studies show that fetal membranes have a limited lifespan and undergo telomere-dependent cellular senescence that is augmented by oxidative stress and mediated by p38 mitogen activated protein kinase (MAPK). Further, these studies suggest that fetal membranes are anatomically and physiologically positioned to transmit senescence signals that may initiate parturition at term. METHODS: Longitudinal evaluation of feto-maternal tissues from mouse pregnancies was undertaken to determine the molecular progression of senescence during normal pregnancy. On days 10-18 of gestation, C57BL/6 mice were euthanized. Fetal membranes, placenta, and decidua/uterus were collected. Tissues were examined for Telomere length (TL) and the presence of Phosphorylated (P) p38MAPK and p53, p21 and senescence associated ß-Galactosidase (SA- ß-Gal). FINDINGS: Linear regression modeling of observed telomere length as a function of gestational age revealed that beta (ß), the slope of the linear regression was negative and significantly different from zero for each tissue (fetal membranes, ß = -0.1901 ± 0.03125, p < 0.0001; placenta ß = -0.09000 ± 0.03474, p = 0.0135; decidua/uterus ß = -0.1317 ± 0.03264, p = 0.0003). Progressive activation p38MAPK was observed in all tissues from days 10 to day18, with the highest activation observed in fetal membranes. Activation of p53 was progressive in fetal membranes. In contrast, active p53 was constitutive in placenta and decidua/uterus throughout gestation. Detection of p21 indicated that pro-senescent change was higher in all compartments on day 18 as compared to other days. The number of SA-ß-Gal positive cells increased in fetal membranes as gestation progressed. However, in placenta and uterus and decidua/uterus SA-ß-Gal was seen only in days 15 and 18. CONCLUSIONS: Telomere dependent p38 and p53 mediated senescence progressed in mouse fetal membranes as gestation advanced. Although senescence is evident, telomere dependent events were not dominant in placenta or decidua/uterus. Fetal membrane senescence may significantly contribute to mechanisms of parturition at term.

Is There a Causal Relation between Maternal Acetaminophen Administration and ADHD?

OBJECTIVE: Recent epidemiological studies reported an association between maternal intake of acetaminophen (APAP) and attention deficit hyperactivity disorder (ADHD) in their children. However, none of these studies demonstrated causality. Our objective was to determine whether exposure to APAP during pregnancy result in hyperkinetic dysfunctions in offspring, using a murine model. MATERIAL AND METHODS: Pregnant CD1 mice (N = 8/group) were allocated to receive by gavage either APAP (150 mg/kg/day, equivalent to the FDA-approved maximum human clinical dose), or 0.5% carboxymethylcellulose (control group), starting on embryonic day 7 until delivery. Maternal serum APAP and alanine transaminase (ALT) concentrations were determined by ELISA and kinetic colorimetric assays, respectively. Open field locomotor activity (LMA) in the 30-day old mouse offspring was quantified using Photobeam Activity System. Mouse offspring were then sacrificed, whole brains processed for magnetic resonance imaging (MRI; 11.7 Tesla magnet) and for neuronal quantification using Nissl stain. The association between APAP exposure and LMA in mouse offspring was analyzed using a mixed effects Poisson regression model that accounted for mouse offspring weight, gender, random selection, and testing time and day. We corrected for multiple comparisons and considered P<0.008 as statistically significant. RESULTS: Maternal serum APAP concentration peaked 30 minutes after gavage, reaching the expected mean of 117 µg/ml. Serum ALT concentrations were not different between groups. There were no significant differences in vertical (rearing), horizontal, or total locomotor activity between the two rodent offspring groups at the P level fixed to adjust for multiple testing. In addition, no differences were found in volumes of 29 brain areas of interest on MRI or in neuronal quantifications between the two groups. CONCLUSION: This study refutes that hypothesis that prenatal exposure to APAP causes hyperkinetic dysfunction in mouse offspring. Due to lack of accurate assessment of ADHD in murine models, our results should be taken with caution when compared to the reported clinical data.