Spatial Activation of Autophagy in Human Placenta-Related Tissue During Labor: A Possible Mechanism for Labor Onset.

INTRODUCTION: To explore the mechanisms of labor by investigating the autophagy of placental and fetal membranes tissue in normal pregnant women. METHODS: Placenta and fetal membranes were collected from women with singleton pregnancies without any medical complications and from women who delivered vaginally (labor-initiated group; L group) or by caesarean section (labor-noninitiated group; NL group). Autophagosomes were observed by transmission electron microscopy (TEM). Immunofluorescence and western blotting (WB) were used to detect protein levels of the autophagy markers LC3A and LC3B. TEM, immunohistochemistry (IHC), and WB were used to compare autophagy in different parts of the placenta and fetal membranes in the L and NL groups. The expression of LC3B/LC3A, ROCK1, and ROCK2 in the placenta of nonpregnant and pregnant rats was detected by WB and IHC. RESULTS: TEM and IHC results showed an increase in the number of autophagosomes and autophagic lysosomes in the L group, and WB results indicated an increase in the LC3B/A ratio between the placenta and fetal membranes in the L group. Autophagy was significantly increased on the maternal side of the placenta in the L group, and the level of autophagy became higher near rupture in the fetal membranes and near the point where the umbilical cord joins the placenta in the L group. The LC3B/A ratio increased and ROCK1 and ROCK2 levels decreased in postnatal rats. DISCUSSION: Autophagy can occur in the placenta and fetal membranes and its activity is higher at the onset of labor, suggesting a role in labor.

A dynamic flow fetal membrane organ-on-a-chip system for modeling the effects of amniotic fluid motion.

Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.

A multi-organ, feto-maternal interface organ-on-chip, models pregnancy pathology and is a useful preclinical extracellular vesicle drug trial platform.

Pregnant women and their fetuses are often excluded from clinical trials due to missing drug-related pre-clinical trial information at the human feto-maternal interface (FMi). The two interfaces-placenta/decidua and fetal membranes/decidua are gatekeepers of drug transport; however, testing their functions is impractical during pregnancy. Limitations of current in-vivo/in-vitro models have hampered drug development and testing during pregnancy. Hence, major complications like preterm births and maternal and neonatal mortalities remain high. Advancements in organ-on-chip (OOC) platforms to test drug kinetics and efficacy and novel extracellular vesicle-based fetal drug delivery are expected to accelerate preclinical trials related to pregnancy complications. Here we report the development and testing of a humanized multi-organ fetal membrane/placenta (fetal)-decidua (maternal) interface OOC (FMi-PLA-OOC) that contains seven cell types interconnected through microchannels to maintain intercellular interactions as seen in-utero. Cytotoxicity, propagation, mechanism of action, and efficacy of engineered extracellular vesicles containing anti-inflammatory interleukin (IL)-10 (eIL-10) were evaluated to reduce FMi inflammation associated with preterm birth. A healthy and disease model (lipopolysaccharide-infectious inflammation) of the FMi-PLA-OOC was created and co-treated with eIL-10. eIL-10 propagated from the maternal to fetal side within 72-hours, localized in all cell types, showed no cytotoxicity, activated IL-10 signaling pathways, and reduced lipopolysaccharide-induced inflammation (minimized NF-kB activation and proinflammatory cytokine production). These data recapitulated eIL-10s' ability to reduce inflammation and delay infection-associated preterm birth in mouse models, suggesting FMi-PLA-OOC as an alternative approach to using animal models. Additionally, we report the utility of eIL-10 that can traverse through FMis to reduce inflammation-associated pregnancy complications.

Pregnancy state before the onset of labor: a holistic mechanical perspective.

Successful pregnancy highly depends on the complex interaction between the uterine body, cervix, and fetal membrane. This interaction is synchronized, usually following a specific sequence in normal vaginal deliveries: (1) cervical ripening, (2) uterine contractions, and (3) rupture of fetal membrane. The complex interaction between the cervix, fetal membrane, and uterine contractions before the onset of labor is investigated using a complete third-trimester gravid model of the uterus, cervix, fetal membrane, and abdomen. Through a series of numerical simulations, we investigate the mechanical impact of (i) initial cervical shape, (ii) cervical stiffness, (iii) cervical contractions, and (iv) intrauterine pressure. The findings of this work reveal several key observations: (i) maximum principal stress values in the cervix decrease in more dilated, shorter, and softer cervices; (ii) reduced cervical stiffness produces increased cervical dilation, larger cervical opening, and decreased cervical length; (iii) the initial cervical shape impacts final cervical dimensions; (iv) cervical contractions increase the maximum principal stress values and change the stress distributions; (v) cervical contractions potentiate cervical shortening and dilation; (vi) larger intrauterine pressure (IUP) causes considerably larger stress values and cervical opening, larger dilation, and smaller cervical length; and (vii) the biaxial strength of the fetal membrane is only surpassed in the cases of the (1) shortest and most dilated initial cervical geometry and (2) larger IUP.

A Dynamic Flow Fetal Membrane Organ-on-a-Chip System for Modeling the Effects of Amniotic Fluid Motion.

Fetal membrane(amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. An amnion membrane (AM) organ-on-chip (OOC) was utilized to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 hours flow culture to mimic fluid motion. Static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control for pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to CK-18 ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a flow culture environment is not necessary to mimic any in utero physiologic cellular conditions of fetal membrane-derived cells.

TLR8-activating miR-146a-3p is an intermediate signal contributing to fetal membrane inflammation in response to bacterial LPS.

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)-8 and the proinflammatory cytokine, IL-1ß. While FMs can respond to infections through innate immune sensors, such as toll-like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non-classical microRNAs (miR-21a, miR-29a, miR-146a-3p, Let-7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro-inflammatory roles of TLR7/TLR8-activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR-146a-3p was significantly increased in both human FM explants and wild-type mouse FMs. Expression of miR-146a-3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL-8 and inflammasome-mediated IL-1ß production in response to LPS was dependent on miR-146a-3p and TLR8 downstream of TLR4 activation. In wild-type mice, LPS exposure increased FM IL-8 and IL-1ß production and induced preterm birth. In TLR7-/-/TLR8-/- mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal-fetal interface in which TLR8-activating miR-146a-3p acts as an intermediate danger signal to drive FM inflammasome-dependent and -independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth.

Group B streptococcus induces cellular senescence in human amnion epithelial cells through a partial interleukin-1-mediated mechanism.

Group B streptococcus (GBS) infection is a significant public health concern associated with adverse pregnancy complications and increased neonatal mortality and morbidity. However, the mechanisms underlying the impact of GBS on the fetal membrane, the first line of defense against pathogens, are not fully understood. Here, we propose that GBS induces senescence and inflammatory factors (IL-6 and IL-8) in the fetal membrane through interleukin-1 (IL-1). Utilizing the existing transcriptomic data on GBS-exposed human fetal membrane, we showed that GBS affects senescence-related pathways and genes. Next, we treated primary amnion epithelial cells with conditioned medium from the choriodecidual layer of human fetal membrane exposed to GBS (GBS collected choriodecidual [CD] conditioned medium) in the absence or presence of an IL-1 receptor antagonist (IL-1Ra). GBS CD conditioned medium significantly increased ß-galactosidase activity, IL-6 and IL-8 release from the amnion epithelial cells. Cotreatment with IL1Ra reduced GBS-induced ß-galactosidase activity and IL-6 and IL-8 secretion. Direct treatment with IL-1α or IL-1ß confirmed the role of IL-1 signaling in the regulation of senescence in the fetal membrane. We further showed that GBS CD conditioned medium and IL-1 decreased cell proliferation in amnion epithelial cells. In summary, for the first time, we demonstrate GBS-induced senescence in the fetal membrane and present evidence of IL-1 pathway signaling between the choriodecidua and amnion layer of fetal membrane in a paracrine manner. Further studies will be warranted to understand the pathogenesis of adverse pregnancy outcomes associated with GBS infection and develop therapeutic interventions to mitigate these complications.

Frontiers in the Etiology and Treatment of Preterm Premature Rupture of Membrane: From Molecular Mechanisms to Innovative Therapeutic Strategies.

Preterm premature rupture of membranes (pPROM) poses a significant threat to fetal viability and increases the risk for newborn morbidities. The perinatal period of preterm infants affected by pPROM is often characterized by higher rates of mortality and morbidity, with associated risks of cerebral palsy, developmental delays, compromised immune function, respiratory diseases, and sensory impairments. pPROM is believed to result from a variety of causes, including but not limited to microbially induced infections, stretching of fetal membranes, oxidative stress, inflammatory responses, and age-related changes in the fetal-placental interface. Maternal stress, nutritional deficiencies, and medically induced procedures such as fetoscopy are also considered potential contributing factors to pPROM. This comprehensive review explores the potential etiologies leading to pPROM, delves into the intricate molecular mechanisms through which these etiologies cause membrane ruptures, and provides a concise overview of diagnostic and treatment approaches for pPROM. Based on available therapeutic options, this review proposes and explores the possibilities of utilizing a novel composite hydrogel composed of amniotic membrane particles for repairing ruptured fetal membranes, thereby holding promise for its clinical application.

Anatomy of the fetal membranes: insights from spinning disk confocal microscopy.

PURPOSE: The fetal membranes are essential for the maintenance of pregnancy, and their integrity until parturition is critical for both fetal and maternal health. Preterm premature rupture of the membranes (pPROM) is known to be an indicator of preterm birth, but the underlying architectural and mechanical changes that lead to fetal membrane failure are not yet fully understood. The aim of this study was to gain new insights into the anatomy of the fetal membrane and to establish a tissue processing and staining protocol suitable for future prospective cohort studies. METHODS: In this proof of principle study, we collected fetal membranes from women undergoing vaginal delivery or cesarean section. Small membrane sections were then fixed, stained for nucleic acids, actin, and collagen using fluorescent probes, and subsequently imaged in three dimensions using a spinning disk confocal microscope. RESULTS: Four fetal membranes of different types were successfully processed and imaged after establishing a suitable protocol. Cellular and nuclear outlines are clearly visible in all cases, especially in the uppermost membrane layer. Focal membrane (micro) fractures could be identified in several samples. CONCLUSION: The presented method proves to be well suited to determine whether and how the occurrence of membrane (micro) fractures and cellular jamming correlate with the timing of membrane rupture and the mode of delivery. In future measurements, this method could be combined with mechanical probing techniques to compare optical and mechanical sample information.

Development of a multilayer fetal membrane material model calibrated using bulge inflation mechanical tests.

The fetal membranes are an essential mechanical structure for pregnancy, protecting the developing fetus in an amniotic fluid environment and rupturing before birth. In cooperation with the cervix and the uterus, the fetal membranes support the mechanical loads of pregnancy. Structurally, the fetal membranes comprise two main layers: the amnion and the chorion. The mechanical characterization of each layer is crucial to understanding how each layer contributes to the structural performance of the whole membrane. The in-vivo mechanical loading of the fetal membranes and the amount of tissue stress generated in each layer throughout gestation remains poorly understood, as it is difficult to perform direct measurements on pregnant patients. Finite element analysis of pregnancy offers a computational method to explore how anatomical and tissue remodeling factors influence the load-sharing of the uterus, cervix, and fetal membranes. To aid in the formulation of such computational models of pregnancy, this work develops a fiber-based multilayer fetal membrane model that captures its response to previously published bulge inflation loading data. First, material models for the amnion, chorion, and maternal decidua are formulated, informed, and validated by published data. Then, the behavior of the fetal membrane as a layered structure was analyzed, focusing on the respective stress distribution and thickness variation in each layer. The layered computational model captures the overall behavior of the fetal membranes, with the amnion being the mechanically dominant layer. The inclusion of fibers in the amnion material model is an important factor in obtaining reliable fetal membrane behavior according to the experimental dataset. These results highlight the potential of this layered model to be integrated into larger biomechanical models of the gravid uterus and cervix to study the mechanical mechanisms of preterm birth.

The Effects of Combined Exposure to Bisphenols and Perfluoroalkyls on Human Perinatal Stem Cells and the Potential Implications for Health Outcomes.

The present study investigates the impact of two endocrine disruptors, namely Bisphenols (BPs) and Perfluoroalkyls (PFs), on human stem cells. These chemicals leach from plastic, and when ingested through contaminated food and water, they interfere with endogenous hormone signaling, causing various diseases. While the ability of BPs and PFs to cross the placental barrier and accumulate in fetal serum has been documented, the exact consequences for human development require further elucidation. The present research work explored the effects of combined exposure to BPs (BPA or BPS) and PFs (PFOS and PFOA) on human placenta (fetal membrane mesenchymal stromal cells, hFM-MSCs) and amniotic fluid (hAFSCs)-derived stem cells. The effects of the xenobiotics were assessed by analyzing cell proliferation, mitochondrial functionality, and the expression of genes involved in pluripotency and epigenetic regulation, which are crucial for early human development. Our findings demonstrate that antenatal exposure to BPs and/or PFs may alter the biological characteristics of perinatal stem cells and fetal epigenome, with potential implications for health outcomes at birth and in adulthood. Further research is necessary to comprehend the full extent of these effects and their long-term consequences.

Modeling an ascending infection by Ureaplasma parvum and its cell signaling and inflammatory response at the feto-maternal interface.

PROBLEM: Ascending bacterial infection is associated with ∼ 40% of spontaneous preterm birth (PTB), and Ureaplasma spp. is one of the most common bacteria isolated from the amniotic fluid. Developing novel in vitro models that mimic in vivo uterine physiology is essential to study microbial pathogenesis. We utilized the feto-maternal interface organ-on-chip (FMi-OOC) device and determined the propagation of Ureaplasma parvum, and its impact on cell signaling and inflammation. METHOD OF STUDY: FMi-OOC is a microphysiologic device mimicking fetal membrane/decidua interconnected through microchannels. The impact of resident decidual CD45+ leukocytes was also determined by incorporating them into the decidual chamber in different combinations with U. parvum. We tested the propagation of live U. parvum from the decidual to the amniochorion membranes (immunocytochemistry and quantitative PCR), determined its impact on cytotoxicity (LDH assay), cell signaling (JESSTM Western Blot), cellular transition (immunostaining for vimentin and cytokeratin), and inflammation (cytokine bead array). RESULTS: U. parvum transversed the chorion and reached the amnion epithelium after 72 hours but did not induce cell signaling kinases (p38MAPK and JNK) activation, or cellular transition (epithelial-mesenchymal), regardless of the presence of immune cells. The inflammatory response was limited to the choriodecidual interface and did not promote inflammation in the amnion layer. CONCLUSIONS: Our data suggest that U. parvum is poorly immunogenic and does not produce massive inflammatory changes at the feto-maternal interface. We speculate that the presence of U. parvum may still compromise the feto-maternal interface making it susceptible to other pathogenic infection.

New Insights on the Biomechanics of the Fetal Membrane.

During pregnancy, the Fetal Membrane (FM) is subjected to mechanical stretching that may result in preterm labor. The structural integrity of the FM is maintained by its collagenous layer. Disconnection and reconnection of molecular bonds between collagen fibrils is the fundamental process that governs the irreversible mechanical and supramolecular changes in the FM. At a critical threshold strain, bundling and alignment of collagen fibrils alter the super-molecular structure of the collagenous layer. Recent studies indicate that these changes are associated with inflammation and/or expression of specific proteins that are known to be related to uterine contractions and labor. The potential healing of stretching-induced damages in the FM by mediators involved in mechano-transduction is discussed.

The Trace Element Concentrations and Oxidative Stress Parameters in Afterbirths from Women with Multiple Pregnancies.

The aim of this study was to evaluate the intensity of oxidative stress by measuring the concentrations of lipid peroxidation products (LPO) in fetal membrane, umbilical cord, and placenta samples obtained from women with multiple pregnancies. Additionally, the effectiveness of protection against oxidative stress was assessed by measuring the activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR). Due to the role of iron (Fe), copper (Cu), and zinc (Zn) as cofactors for antioxidant enzymes, the concentrations of these elements were also analyzed in the studied afterbirths. The obtained data were compared with newborn parameters, selected environmental factors, and the health status of women during pregnancy to determine the relationship between oxidative stress and the health of women and their offspring during pregnancy. The study involved women (n = 22) with multiple pregnancies and their newborns (n = 45). The Fe, Zn, and Cu levels in the placenta, umbilical cord, and fetal membrane were determined using inductively coupled plasma atomic emission spectroscopy (ICP-OES) using an ICAP 7400 Duo system. Commercial assays were used to determine SOD, GPx, GR, CAT, and LPO activity levels. The determinations were made spectrophotometrically. The present study also investigated the relationships between trace element concentrations in fetal membrane, placenta, and umbilical cord samples and various maternal and infant parameters in women. Notably, a strong positive correlation was observed between Cu and Zn concentrations in the fetal membrane (p = 0.66) and between Zn and Fe concentrations in the placenta (p = 0.61). The fetal membrane Zn concentration exhibited a negative correlation with shoulder width (p = -0.35), while the placenta Cu concentration was positively correlated with placenta weight (p = 0.46) and shoulder width (p = 0.36). The umbilical cord Cu level was positively correlated with head circumference (p = 0.36) and birth weight (p = 0.35), while the placenta Fe concentration was positively correlated with placenta weight (p = 0.33). Furthermore, correlations were determined between the parameters of antioxidative stress (GPx, GR, CAT, SOD) and oxidative stress (LPO) and the parameters of infants and maternal characteristics. A negative correlation was observed between Fe and LPO product concentrations in the fetal membrane (p = -0.50) and placenta (p = -0.58), while the Cu concentration positively correlated with SOD activity in the umbilical cord (p = 0.55). Given that multiple pregnancies are associated with various complications, such as preterm birth, gestational hypertension, gestational diabetes, and placental and umbilical cord abnormalities, research in this area is crucial for preventing obstetric failures. Our results could serve as comparative data for future studies. However, we advise caution when interpreting our results, despite achieving statistical significance.

Fetal membranes exhibit similar nutrient transporter expression profiles to the placenta.

INTRODUCTION: During pregnancy, the growth of the fetus is supported by the exchange of nutrients, waste, and other molecules between maternal and fetal circulations in the utero-placental unit. Nutrient transfer, in particular, is mediated by solute transporters such as solute carrier (SLC) and adenosine triphosphate-binding cassette (ABC) proteins. While nutrient transport has been extensively studied in the placenta, the role of human fetal membranes (FM), which was recently reported to have a role in drug transport, in nutrient uptake remains unknown. OBJECTIVES: This study determined nutrient transport expression in human FM and FM cells and compared expression with placental tissues and BeWo cells. METHODS: RNA sequencing (RNA-Seq) of placental and FM tissues and cells was done. Genes of major solute transporter groups, such as SLC and ABC, were identified. Proteomic analysis of cell lysates was performed via nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to confirm expression at a protein level. RESULTS: We determined that FM tissues and cells derived from the fetal membrane tissues express nutrient transporter genes, and their expression is similar to that seen in the placenta or BeWo cells. In particular, transporters involved in macronutrient and micronutrient transfer were identified in both placental and FM cells. Consistent with RNA-Seq findings, carbohydrate transporters (3), vitamin transport-related proteins (8), amino acid transporters (21), fatty acid transport-related proteins (9), cholesterol transport-related proteins (6) and nucleoside transporters (3) were identified in BeWo and FM cells, with both groups sharing similar nutrient transporter expression. CONCLUSION: This study determined the expression of nutrient transporters in human FMs. This knowledge is the first step in improving our understanding of nutrient uptake kinetics during pregnancy. Functional studies are required to determine the properties of nutrient transporters in human FMs.

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.

Acute and chronic maternal exposure to fine particulate matter and prelabor rupture of the fetal membranes: A nation-wide survey in China.

BACKGROUND: Prelabor rupture of the fetal membranes (PROM) is a major contributor to adverse perinatal outcomes. Some epidemiologic studies explored the association between maternal PM2.5 exposure and PROM but failed to treat the labor induction and prelabor cesarean section as censored observations. OBJECTIVE: We aimed to evaluated whether acute and chronic maternal ambient PM2.5 exposure may increase the risk of PROM in China. METHODS: This study was based on the China Labor and Delivery Survey, a nationwide multicenter investigation. Included in the current analysis were 45,879 singleton spontaneous births in 96 hospitals in mainland China from 2015 to 2017. Outcomes were PROM, preterm PROM (<37 weeks' gestation) and term PROM (≥37 weeks' gestation). Daily concentration of PM2.5 at 1 km spatial resolution was estimated by gap-filling model. Generalized linear mixed model and mixed effects Cox model were applied to assess the associations of acute (from 0 to 4 days before delivery) and chronic (average gestational and trimester-specific) ambient PM2.5 exposure with outcomes, respectively. RESULTS: Significant associations were found between acute PM2.5 exposures (per interquartile range increase) and the risk of preterm PROM (OR = 1.11; 95 % CI: 1.03, 1.19 for PM2.5 on delivery day; OR = 1.10; 95 % CI: 1.02, 1.18 for PM2.5 1 day before delivery) but not for term PROM. An interquartile range increase in PM2.5 during the second trimester was associated with elevated risks of PROM (HR = 1.14; 95 % CI: 1.07, 1.22), preterm PROM (HR = 1.22; 95 % CI: 1.02, 1.45) and term PROM (HR = 1.13; 95 % CI: 1.06, 1.22), respectively. Women who were less educated, obese, or gave birth in a cold season appeared to be more sensitive to ambient PM2.5 exposure. CONCLUSION: Our findings suggest that both acute and chronic maternal exposures to ambient PM2.5 during pregnancy are risk factors for PROM.

Magnetic resonance imaging of the supra-cervical fetal membrane detects an increased risk of prelabor rupture of membranes.

Objective: In 10% of term deliveries and 40% of preterm deliveries, the fetal membrane (FM) ruptures before labor. However, the ability to predict these cases of premature rupture of membranes (PROM) and preterm premature rupture of membranes (PPROM) is very limited. In this paper, our objective was to determine whether a prediction method based on T2 weighted magnetic resonance imaging (MRI) of the supra-cervical FM could predict PROM and PPROM. Methods: This prospective cohort study enrolled 77 women between the 28th and 37th weeks of gestation. Two indicators of fetal membrane defects, including prolapsed depth >5 mm and signal abnormalities, are investigated for our prediction. Fisher's exact test was used to determine whether prolapsed depth >5 mm and/or signal abnormalities were associated with PROM and PPROM. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated for prolapsed depth >5 mm, signal abnormalities, and the combination of prolapsed depth >5 mm and signal abnormalities. Result: Among 12 women with PROM (5 preterm and 7 term, prior to labor onset), 9 had membrane prolapse >5 mm and 5 had FM signal abnormalities. Among 65 women with rupture of membranes at term, 2 had membrane prolapse >5 mm and 1 had signal abnormalities. By Fisher's exact test both indicators, membrane prolapse >5 mm and signal abnormalities, were associated with PROM (P<0.001, P<0.001) and PPROM (P=0.001, P<0.001). Additionally, membrane prolapse >5 mm, signal abnormalities, and the combination of the two indicators all demonstrated high specificity for predicting PROM (96.9%, 98.5%, and 100%, respectively) and PPROM (90.3%, 97.2%, and 100%, respectively). Conclusion: MRI can distinguish the supra-cervical fetal membrane in vivo and may be able to identify women at high risk of PPROM.

Exosomes from Ureaplasma parvum-infected ectocervical epithelial cells promote feto-maternal interface inflammation but are insufficient to cause preterm delivery.

This study determined if exosomes from ectocervical epithelial (ECTO) cells infected with Ureaplasma parvum (U. parvum) can carry bacterial antigens and cause inflammation at the feto-maternal interface using two organ-on-chip devices, one representing the vagina-cervix-decidua and another one mimicking the feto-maternal interface, and whether such inflammation can lead to preterm birth (PTB). Exosomes from U. parvum-infected ECTO cells were characterized using cryo-electron microscopy, nanoparticle tracking analysis, Western blot, and Exoview analysis. The antigenicity of the exosomes from U. parvum-infected ECTO cells was also tested using THP-1 cells and our newly developed vagina-cervix-decidua organ-on-a-chip (VCD-OOC) having six microchannel-interconnected cell culture chambers containing cells from the vagina, ectocervical, endocervical, transformation zone epithelia, cervical stroma, and decidua. The VCD-OOC was linked to the maternal side of our previously developed feto-maternal interface organ-on-a-chip (FMi-OOC). Cell culture media were collected after 48 h to determine the cytokine levels from each cell line via ELISA. For physiological validation of our in vitro data, high-dose exosomes from U. parvum-infected ECTO cells were delivered to the vagina of pregnant CD-1 mice on E15. Mice were monitored for preterm birth (PTB, < E18.5 days). Exosomes from ECTO cells infected with U. parvum (UP ECTO) showed significant downregulation of exosome markers CD9, CD63, and CD81, but contained multiple banded antigen (MBA), a U. parvum virulence factor. Monoculture experiments showed that exosomes from UP ECTO cells delivered MBA from the host cell to uninfected endocervical epithelial cells (ENDO). Moreover, exposure of THP-1 cells to exosomes from UP ECTO cells resulted in increased IL-8 and TNFα and reduced IL-10. The OOC experiments showed that low and high doses of exosomes from UP ECTO cells produced a cell type-specific inflammatory response in the VCD-OOC and FMi-OOC. Specifically, exosomes from UP ECTO cells increased pro-inflammatory cytokines such as GM-CSF, IL-6, and IL-8 in cervical, decidual, chorion trophoblast, and amnion mesenchymal cells. The results from our OOC models were validated in our in vivo mice model. The inflammatory response was insufficient to promote PTB. These results showed the potential use of the VCD-OOC and FMi-OOC in simulating the pathophysiological processes in vivo.

Expression of CYP450 enzymes in human fetal membranes and its implications in xenobiotic metabolism during pregnancy.

BACKGROUND: Environmental exposure to toxicants is a major risk factor for spontaneous preterm birth (PTB, <37 weeks). Toxicants and drugs administered to patients are metabolized primarily by the cytochrome P450 (CYP450) system. Along with the adult and fetal liver, the placenta, a critical feto-maternal interface organ, expresses CYP450 enzymes that metabolize these xenobiotics. However, the contribution of the fetal membranes, another tissue of the feto-maternal interface, to the expression of CYP450 enzymes and the detoxification of xenobiotics remains unknown. AIMS: This study characterized CYP450 expression and determined the functional activity of CYP450 enzymes in fetal membranes. MAIN METHODS: RNA sequencing (RNA-Seq) of placental and fetal membrane tissues and cells was done. Differential expressions of CYP450 genes were compared and validated via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) between the two tissues. The functional activity of major CYP450 enzymes was determined using a fluorophore-based enzymatic assay in the presence and absence of their corresponding inhibitors. KEY FINDINGS: With the exception of genes that regulate cholesterol metabolism, the expression profile of CYP450 genes was similar between placental and fetal membranes tissues/cells. RT-qPCR analysis confirmed these findings with significant levels of mRNA for major CYP450 genes being detectable in amnion epithelial cells (AECs) and chorion trophoblasts cells (CTCs). Biochemical analyses revealed significant CYP450 enzymatic activities that were sensitive to specific inhibitors for both AECs and CTCs, suggesting that the genes were expressed as functional enzymes. SIGNIFICANCE: This is the first study to determine global expression of CYP450 enzymes in fetal membranes which may play a role in xenobiotic metabolism during pregnancy. Given that many women are exposed to environmental toxins or require medications during pregnancy, a better understanding of their role in metabolism is required to develop safer therapeutics and prevent adverse outcomes.