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Background: Human Amniotic Membrane (hAM) is endowed with several biological activities and might be considered an optimal tool in surgical treatment for different ophthalmic pathologies. We pioneered the surgical use of hAM to treat retinal pathologies such as macular holes, tears, and retinal detachments, and to overcome photoreceptor damage in age-related macular degeneration. Although hAM contributed to improved outcomes, the mechanisms of its effects are not yet fully understood. The characterization and explanation of the effects of hAM would allow the adoption of this new natural product in different retinal pathologies, operative contexts, and hAM formulations. At this end, we studied the properties of a hAM extract (hAME) on the ARPE-19 cells., Methods and Results: A non-denaturing sonication-based technique was developed to obtain a suitable hAME. Viability, proliferation, apoptosis, oxidative stress, and epithelial-mesenchymal transition (EMT) were studied in hAME-treated ARPE-19 cells. The hAME was able to increase ARPE-19 cell viability even in the presence of oxidative stress (H 2 O 2 , TBHP). Moreover, hAME prevented the expression of EMT features, such as EMT-related proteins, fibrotic foci formation, and migration induced by different cytokines., Conclusions: Our results demonstrate that the hAME retains most of the properties observed in the whole tissue by others. The hAME, other than providing a manageable research tool, could represent a cost-effective and abundant drug to treat retinal pathologies in the future., (© 2024. The Author(s).)

The role and mechanisms of progesterone in preterm premature rupture of membranes (PPROM) remains unclear. This study aims to investigate the molecular mechanisms of action of progesterone in pre-labor full-term fetal amniotic membrane cells with and without stimulation by microbial, pro-inflammatory, or thrombogenic agents. Fetal amniotic membranes were collected from 30 women with a normal singleton pregnancy undergoing elective cesarean section at term prior to the onset of labor. The human amniotic epithelial cells isolated were pretreated with and without medroxyprogesterone acetate for 24 h. Then, cells were treated with and without TLR/NLR agonists, pro-inflammatory cytokines, or thrombin for 48 h. Semi-quantitative RT-PCR, Western blot, and caspase-3 activity measurement were performed. Progesterone stimulation decreased the expression of TLR2, TLR5, and Nod2 genes (alone and/or in combination with TLR/NLR agonists) and decreased the expression of IL-1β and IL-8 genes increased by stimulation with specific agonists for TLR2, TLR4, TLR5, Nod1, and Nod2. Moreover, progesterone decreased thrombin-induced IL-8 gene expression. Progesterone also decreased expression of Bax and Bid proteins (pro-apoptotic factors) increased by stimulation with pro-inflammatory cytokines (TNF-α, NGAL, IL-18, and IL-1β) and thrombin. Progesterone stimulation alone as well as co-stimulation with TNF-α, NGAL, IL-18, IL-1β, or thrombin with progesterone either increased, decreased, or did not change the expression of Bcl-2, Bcl-XL, or XIAP genes (anti-apoptotic factors). These data suggest progesterone plays protective roles against PPROM through anti-microbial, anti-inflammatory, and anti-thrombogenic actions on human-term fetal amniotic membrane cells. Progesterone alters pro-inflammatory cytokine- and thrombin-induced apoptosis by controlling the expression of pro-apoptotic and anti-apoptotic factors., (© 2021. Society for Reproductive Investigation.)

Introduction: Uterine distension with pressurised carbon dioxide (CO 2 ) (amniotic insufflation) is used clinically to improve visibility during keyhole fetal surgery. However, there are concerns that amniotic insufflation with unconditioned (cold, dry) CO 2 damages the fetal membranes which leads to post-operative preterm prelabour rupture of membranes (iatrogenic PPROM). We assessed whether heating and humidifying the insufflated CO 2 could reduce fetal membrane damage in sheep., Methods: Thirteen pregnant ewes at 103-106 days gestation underwent amniotic insufflation with cold, dry (22 °C, 0-5% humidity, n = 6) or heated, humidified (40 °C, 95-100% humidity, n = 7) CO 2 at 15 mmHg for 180 min. Twelve non-insufflated amniotic sacs acted as controls. Fetal membrane sections were collected after insufflation and analysed for molecular and histological markers of cell damage (caspase 3 and high mobility group box 1 [HMGB1]), inflammation (interleukin 1-alpha [IL1-alpha], IL8 and vascular cell adhesion molecule [VCAM]) and collagen weakening (matrix metalloprotease 9 [MMP9])., Results: Exposure to cold, dry CO 2 increased mRNA levels of caspase 3, HMGB1, IL1-alpha, IL8, VCAM and MMP9 and increased amniotic epithelial caspase 3 and HMGB1 cell counts relative to controls. Exposure to heated, humidified CO 2 also increased IL8 levels relative to controls however, HMGB1, IL1-alpha and VCAM mRNA levels and amniotic epithelial HMGB1 cell counts were significantly lower than the cold, dry group., Discussion: Amniotic insufflation with cold, dry CO 2 damaged the amniotic epithelium and induced fetal membrane inflammation. Heated, humidified insufflation partially mitigated this damage and inflammation in sheep and may prove an important step in reducing the risk of iatrogenic PPROM following keyhole fetal surgery., Competing Interests: Declaration of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This efficacy trial evaluated the effects of two polyphenolic stilbenes, resveratrol and pterostilbene, mostly found in grapes, on the brush border membrane functionality, morphology and gut microbiome. This study applied the validated Gallus gallus intra-amniotic approach to investigate the effects of stilbene administration versus the controls. Three treatment groups (5% resveratrol; 5% pterostilbene; and synergistic: 4.75% resveratrol and 0.25% pterostilbene) and three controls (18 MΩ H 2 O; no injection; 5% inulin) were employed. We observed beneficial morphological changes, specifically an increase in the villus length, diameter, depth of crypts and goblet cell diameter in the pterostilbene and synergistic groups, with concomitant increases in the serum iron and zinc concentrations. Further, the alterations in gene expression of the mineral metabolism proteins and pro-inflammatory cytokines indicate a potential improvement in gut health and mineral bioavailability. The cecal microbiota was analyzed using 16S rRNA sequencing. A lower α-diversity was observed in the synergistic group compared with the other treatment groups. However, beneficial compositional and functional alterations in the gut microbiome were detected. Several key microbial metabolic pathways were differentially enriched in the pterostilbene treatment group. These observations demonstrate a significant bacterial-host interaction that contributed to enhancements in intestinal functionality, morphology and physiological status. Our data demonstrate a novel understanding of the nutritional benefits of dietary stilbenes and their effects on intestinal functionality, morphology and gut microbiota in vivo.

Maternal smoking is a risk factor of preterm prelabor rupture of the fetal membranes (pPROM), which is responsible for 30% of preterm births worldwide. Cigarettes induce oxidative stress and inflammation, mechanisms both implicated in fetal membranes (FM) weakening. We hypothesized that the receptor for advanced glycation end-products (RAGE) and its ligands can result in cigarette-dependent inflammation. FM explants and amniotic epithelial cells (AECs) were treated with cigarette smoke condensate (CSC), combined or not with RAGE antagonist peptide (RAP), an inhibitor of RAGE. Cell suffering was evaluated by measuring lactate dehydrogenase (LDH) medium-release. Extracellular HMGB1 (a RAGE ligand) release by amnion and choriodecidua explants were checked by western blot. NF-κB pathway induction was determined by a luciferase gene reporter assay, and inflammation was evaluated by cytokine RT-qPCR and protein quantification. Gelatinase activity was assessed using a specific assay. CSC induced cell suffering and HMGB1 secretion only in the amnion, which is directly associated with a RAGE-dependent response. CSC also affected AECs by inducing inflammation (cytokine release and NFκB activation) and gelatinase activity through RAGE engagement, which was linked to an increase in extracellular matrix degradation. This RAGE dependent CSC-induced inflammation associated with an increase of gelatinase activity could explain a pathological FM weakening directly linked to pPROM.

Corneal transplantation, by which the damaged cornea is replaced by a new one, suffers from limited access to HLA-compatible-donors and high maintenance and surgical costs. Therefore, artificial corneas are considered as alternative tools with promising prospects. In our previous study, a two-part-polymeric artificial cornea was composed of enhanced hydrophilic surface electrospun poly(ε-caprolactone) nanofibrous scaffold that is thermally connected to a polyvinyl alcohol-based hydrogel disk was prepared. Characterization tests revealed the prepared artificial cornea had similar biocompatible and structural characteristics regarding the natural cornea. In current study, human amniotic membrane extract containing growth factors, cytokines, anti-inflammatory factors, and anti-angiogenic factors was prepared, nano-encapsulated in chitosan-dextran nanoparticles, and physically decorated on the poly(ε-caprolactone)-polyvinyl-alcohol artificial cornea. Physicochemical and biological characterizations revealed the nano-decorated artificial cornea has more biocompatibility than the unmodified one. Our study demonstrated the bioactive macromolecules loaded on chitosan-dextran nanoparticles enhanced the anti-angiogenic property of artificial cornea through the sustained release of anti-angiogenic factors such as thrombospondin-1, endostatin, and heparin sulfate proteoglycan. Real-time-PCR and flow-cytometry assessments elucidated the vascularization was inhibited through a decrease in the expression of cluster of differentiation 31 and von-Willebrand-Factor. Our study proposed the use of biocompatible artificial cornea could be a promising strategy in corneal transplantation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Collagen has been widely shown to promote osteogenesis of bone marrow mesenchymal stromal cells (BM-MSCs). Due to the invasive procedure of obtaining BM-MSCs, MSCs from other tissues have emerged as a promising alternative for regenerative therapy. MSCs originated from different sources, exhibiting different differentiation potentials. Therefore, the applicability of collagen type I (COL), combining with amniotic membrane (AM)-MSCs was examined through proliferation and differentiation assays together with the expression of surface markers and genes associated with stemness and differentiation under basal or induction conditions. No increase in cell growth was observed because AM-MSCs might be directed toward spontaneous osteogenesis. This was evidenced by the calcium deposition and elevated expression of osteogenic genes when AM-MSCs were cultured in collagen plate with basal media. Under the osteogenic condition, reciprocal expression of OCN and CEBPA suggested a shift toward adipogenesis. Surprisingly, adipogenic genes were not elevated upon adipogenic induction, although oil droplets deposition was observed. In conclusion, our findings demonstrated that collagen causes spontaneous osteogenesis in AM-MSCs. However, the presence of exogenous inductors could shift the direction of adipo-osteogenic gene regulatory network modulated by collagen., (© 2020 The Author(s).)

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-β) 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-β 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.

Background: Corneal neovascularization can result from many pathological processes affecting the ocular surface leading to disturbances and opacifications that reduce corneal clarity and may impact vision. In veterinary medicine, the use of topical corticosteroid is contraindicated in the presence of ulcerative keratitis, and there is sparse research regarding safe medical alternatives to inhibit corneal neovascularization in dogs to improve visual outcome., Aim: To investigate the pigment epithelium-derived factor (PEDF) concentration in equine amniotic membrane homogenate (EAMH) and its in-vitro vascular endothelial growth factor (VEGF) inhibition in tears of dogs with vascularized ulcerative keratitis., Methods: Homogenates from 10 equine amniotic membranes (AM) were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of equine PEDF and VEGF. Forty tear samples were collected from both eyes of dogs diagnosed with vascularized ulcerative keratitis, and 50 samples from healthy dogs. Samples from affected eyes were allocated to G1 - affected undiluted tears; G2 - affected tears diluted with phosphate-buffer solution; G3 - affected tears treated with low-concentrated EAMH; and G4 - affected tears treated with high-concentrated EAMH. Tears from the unaffected contralateral eyes were composed in G5, while G6 was composed by tears from healthy dogs (control). The presence and levels of VEGF were evaluated in all groups by Western blot and ELISA., Results: The PEDF:VEGF ratio in EAMH was 110:1. An increase in VEGF levels was observed in tears from eyes with vascularized corneal ulcers (G1) as well as in contralateral tears (G5), compared to normal dogs (G6). High-concentrated EAMH provided a greater decrease in VEGF levels in-vitro compared to low-concentrated EAMH., Conclusion: EAMHs exhibited high concentrations of PEDF in comparison to VEGF and were able to partially decrease VEGF levels in tears of dogs with vascularized ulcers, in-vitro . Our results suggest that VEGF concentration is elevated in tears of dogs with active vascularized ulcerative keratitis in both affected and contralateral eyes compared to that of healthy dogs., Competing Interests: The authors declare that there is no conflict of interest.

Introduction: Inflammation and infection, including dental infectious diseases, are factors that can induce preterm birth. We previously reported that mice with dental Porphyromonas gingivalis infection could be used as a model of preterm birth. In this model, cyclooxygenase (COX)-2 and interleukin (IL)-1β levels are increased, and P. gingivalis colonies are observed in the fetal membrane. However, the mechanism underlying fetal membrane inflammation remains unknown. Therefore, we investigated the immune responses of human amnion to P. gingivalis in vitro., Methods: Epithelial and mesenchymal cells were isolated from human amnion using trypsin and collagenase, and primary cell cultures were obtained. Confluent cells were stimulated with P. gingivalis lipopolysaccharide (P.g-LPS) or P. gingivalis. mRNA expressions of IL-1β, IL-8, IL-6 and COX-2, protein expressions of nuclear factor (NF)-κB pathway components and culture medium levels of prostaglandin E 2 were evaluated., Results: Following stimulation with 1 μg/mL P.g-LPS, the mRNA expression levels of IL-1β, IL-8, IL-6 and COX-2 in mesenchymal cells were increased 5.9-, 3.3-, 4.2- and 3.1-fold, respectively. Similarly, the expression levels of IL-1β, IL-8, IL-6 and COX-2 in mesenchymal cells were increased by 7.6-, 8.2-, 13.4- and 9.3-fold, respectively, after coculture with P. gingivalis. Additionally, stimulation with P.g-LPS or P. gingivalis resulted in the activation of NF-κB signaling and increased production of IL-1β and prostaglandin E 2 . In contrast, no significant changes were observed in epithelial cells., Discussion: Our findings suggest that mesenchymal cells might mediate the inflammatory responses to P. gingivalis and P.g-LPS, thereby producing inflammation that contributes to the induction of preterm birth., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: To maintain the normal pregnancy, suppression of inflammatory signaling pathway is a crucial physiologic response. Dexmedetomidine has been used for labor analgesia or supplement of inadequate regional analgesia during delivery. And it has been reported that dexmedetomidine has an anti-inflammatory effect. In this study, we examined the influence of dexmedetomidine on the expression of cyclooxygenase-2 (COX-2), prostaglandin E 2 (PGE 2 ) and inflammatory cytokines in lipopolysaccharide (LPS)-stimulated human amnion-derived WISH cells. In addition, we evaluated the association of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathway in anti-inflammatory effect of dexmedetomidine. Methods: Human amnion-derived WISH cells were pretreated with various concentrations of dexmedetomidine (0.001-1 µg/ml) for 1 h and after then treated with LPS (1 µg/ml) for 24 h. MTT assay was conducted to evaluate the cytotoxicity. Nitric oxide (NO) production was analyzed using Griess-reaction microassay. RT-PCR was performed for analysis of mRNA expressions of COX-2, PGE 2 , tumor necrosis factor (TNF)-α and interlukin (IL)-1β. Protein expressions of COX-2, PGE 2 , p38 and NF-κB were analyzed by western blotting. Results: LPS and dexmedetomidine had no cytotoxic effect on WISH cells. There was no difference in NO production after dexmedetomidine pretreatment. The mRNA and protein expressions of COX-2 and PGE 2 were decreased by dexmedetomidine pretreatment in LPS-treated WISH cells. Dexmedetomidine also attenuated the LPS-induced mRNA expression of TNF-α and IL-1β. The activation of p38 and NF-κB was suppressed by dexmedetomidine pretreatment in LPS-treated WISH cells. Conclusion: We demonstrated that dexmedetomidine pretreatment suppressed the expressions of inflammatory mediators increased by LPS. In addition, this study suggests that anti-inflammatory effect of dexmedetomidine on WISH cells was mediated by the inhibitions of p38 and NF-κB activation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Infection/inflammation is an important causal factor in spontaneous preterm birth (sPTB). Most mechanistic studies have concentrated on the role of bacteria, with limited focus on the role of viruses in sPTB. Murine studies support a potential multi-pathogen aetiology in which a double or sequential hit of both viral and bacterial pathogens leads to a higher risk preterm labour. This study aimed to determine the effect of viral priming on bacterial induced inflammation in human in vitro models of ascending and haematogenous infection. Methods: Vaginal epithelial cells, and primary amnion epithelial cells and myocytes were used to represent cell targets of ascending infection while interactions between peripheral blood mononuclear cells (PBMCs) and placental explants were used to model systemic infection. To model the effect of viral priming upon the subsequent response to bacterial stimuli, each cell type was stimulated first with a TLR3 viral agonist, and then with either a TLR2 or TLR2/6 agonist, and responses compared to those of each agonist alone. Immunoblotting was used to detect cellular NF-κB, AP-1, and IRF-3 activation. Cellular TLR3, TLR2, and TLR6 mRNA was quantified by RT-qPCR. Immunoassays were used to measure supernatant cytokine, chemokine and PGE2 concentrations. Results: TLR3 ("viral") priming prior to TLR2/6 agonist ("bacterial") exposure augmented the pro-inflammatory, pro-labour response in VECs, AECs, myocytes and PBMCs when compared to the effects of agonists alone. In contrast, enhanced anti-inflammatory cytokine production (IL-10) was observed in placental explants. Culturing placental explants in conditioned media derived from PBMCs primed with a TLR3 agonist enhanced TLR2/6 agonist stimulated production of IL-6 and IL-8, suggesting a differential response by the placenta to systemic inflammation compared to direct infection as a result of haematogenous spread. TLR3 agonism generally caused increased mRNA expression of TLR3 and TLR2 but not TLR6. Conclusion: This study provides human in vitro evidence that viral infection may increase the susceptibility of women to bacterial-induced sPTB. Improved understanding of interactions between viral and bacterial components of the maternal microbiome and host immune response may offer new therapeutic options, such as antivirals for the prevention of PTB., (Copyright © 2020 Rasheed, Lee, Kim, Rai, Ruano, Anucha, Sullivan, MacIntyre, Bennett and Sykes.)

Context and Objectives: Inflammation is the leading mechanism involved in both physiological and pathological rupture of fetal membranes. Our aim was to obtain a better characterization of the inflammasome-dependent inflammation processes in these tissues, with a particular focus on the nucleotide-binding oligomerization domain (NOD)-like receptor, pyrin domain containing protein 7 (NLRP7) inflammasome. Methods: The presence of NLRP7 inflammasome actors [NLRP7, apoptosis-associated speck-like protein containing a CARD domain (ASC), and caspase-1] was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) in human amnion and choriodecidua at the three trimesters and at term. The protein concentrations were then determined by enzyme-linked immunosorbent assay in term tissues, with or without labor. The presence of Mycoplasma salivarium and Mycoplasma fermentans in human fetal membranes was investigated using a PCR approach. Human amnion epithelial cells (AECs) were treated for 4 or 20 h with fibroblast-stimulating lipopeptide-1 (FSL-1), a M. salivarium -derived ligand. Transcripts and proteins quantity was then measured by RT-quantitative PCR and Western blotting, respectively. NLRP7 and ASC colocalization was confirmed by immunofluorescence. Western blots allowed analysis of pro-caspase-1 and gasdermin D cleavage. Results: NLRP7, ASC, and caspase-1 transcripts were expressed in both sheets of human fetal membranes during all pregnancy stages, but only ASC protein expression was increased with labor. In addition, M. salivarium and M. fermentans were detected for the first time in human fetal membranes. NLRP7 and caspase-1 transcripts, as well as NLRP7, ASC, and pro-caspase-1 protein levels, were increased in FSL-1-treated AECs. The NLRP7 inflammasome assembled around the nucleus, and pro-caspase-1 and gasdermin D were cleaved into their mature forms after FSL-1 stimulation. Conclusion: Two new mycoplasmas, M. salivarium and M. fermentans , were identified in human fetal membranes, and a lipopeptide derived from M. salivarium was found to induce NLRP7 inflammasome formation in AECs., (Copyright © 2020 Lavergne, Belville, Choltus, Gross, Minet-Quinard, Gallot, Sapin and Blanchon.)

This study aims to evaluate the changes of fragility and ultrastructure of amniotic membrane after cross-linking by UVA/riboflavin.Forty-nine fresh amniotic membranes were randomly divided into 3 groups. Eighteen were in group A (CX group) and immersed in 0.1% riboflavin solution for 10 min for UVA/riboflavin cross-linking. Sixteen were in group B (B2 group), soaked for 10 min with 0.1% riboflavin. After soaking, membranes in group A and B were transferred into corneal preservation solution. Fifteen pieces were in group C, directly into corneal preservation solution. The biomechanical and ultrastructural changes of the amniotic tissue before and after cross-linking were examined (CX group = 13, B2 group = 11, C group = 15). The amniotic membrane tissue of group A (n = 5) and B (n = 5) was transplanted into 16 eyes of the rabbits, respectively, and the dissolution time of the amniotic membrane tissue was investigated.After cross-linking, compared with the control group, the elastic modulus of the low-stress area of the amniotic membrane (Elow) was higher, while the elastic modulus of the high-stress area of the amniotic membrane (Ehigh) was lower, with no significant difference in the tensile strength. Also, the collagen fibers showed coarse and bamboo-like changes. In group A, amniotic membranes began to dissolve 4 weeks after conjunctiva transplantation, and all amniotic membranes were dissolved and absorbed 6 weeks after conjunctiva transplantation. In group B, some amniotic membrane tissues were still visible 6 weeks after conjunctiva transplantation.This study suggested that after amniotic membrane cross-linking, the brittleness was increased, the hardness was enhanced, and the morphology of the collagen fiber was changed. The cross-linked amniotic membrane showed resistance to tissue dissolution.

Introduction: Prostaglandins are critical for the onset and progression of labor in mammals, and are formed by the metabolism of arachidonic acid. The products of arachidonic acid, 2-arachidonoylglycerol (2-AG), and anandamide (AEA) have a similar lipid back bone but differing polar head groups, meaning that identification of these products by immunoassay can be difficult., Materials and Methods: In the current study, we present the use of mass spectrometry as multiplex method of identifying the specific end products of arachidonic and anandamide metabolism by human derived amnion explants treated with either an infectious agent (LPS) or inflammatory mediator (IL-1β or TNF-α)., Results: Human amnion tissue explants treated with LPS, IL-1β, or TNF-α increased production of prostaglandin E 2 (PGE 2 ; p < 0.05) but decreased PGFM. Overall, PGE 2 production was greater compared to the other prostaglandins and prostamides irrespective of treatment., Conclusions: The findings of the current study are in keeping with the literature which describes amnion tissues as predominantly producing PGE 2 . The use of mass spectrometry for the differential identification of prostaglandins, prostamides, and other eicosanoids may help better elucidate mechanisms of preterm labor, and lead to new targets for the prediction of risk for preterm labor and/or birth., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Context: Prenatal exposure to bisphenol A (BPA) is linked to obesity and diabetes but the molecular mechanisms driving these phenomena are not known. Alterations in deoxyribonucleic acid (DNA) methylation in amniocytes exposed to BPA in utero represent a potential mechanism leading to metabolic dysfunction later in life., Objective: To profile changes in genome-wide DNA methylation and expression in second trimester human amniocytes exposed to BPA in utero., Design: A nested case-control study was performed in amniocytes matched for offspring sex, maternal race/ethnicity, maternal age, gestational age at amniocentesis, and gestational age at birth. Cases had amniotic fluid BPA measuring 0.251 to 23.74 ng/mL. Sex-specific genome-wide DNA methylation analysis and RNA-sequencing (RNA-seq) were performed to determine differentially methylated regions (DMRs) and gene expression changes associated with BPA exposure. Ingenuity pathway analysis was performed to identify biologically relevant pathways enriched after BPA exposure. In silico Hi-C analysis identified potential chromatin interactions with DMRs., Results: There were 101 genes with altered expression in male amniocytes exposed to BPA (q < 0.05) in utero, with enrichment of pathways critical to hepatic dysfunction, collagen signaling and adipogenesis. Thirty-six DMRs were identified in male BPA-exposed amniocytes and 14 in female amniocyte analysis (q < 0.05). Hi-C analysis identified interactions between DMRs and 24 genes with expression changes in male amniocytes and 12 in female amniocytes (P < 0.05)., Conclusion: In a unique repository of human amniocytes exposed to BPA in utero, sex-specific analyses identified gene expression changes in pathways associated with metabolic disease and novel DMRs with potential distal regulatory functions., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The amnion is considered to be the load-bearing part of the fetal membranes. We investigated the influence of osmolarity of the testing medium and hydration on its fracture toughness. Mode I fracture tests revealed that physiological variations in the bath osmolarity do not influence the tear resistance of amnion, while larger changes, i.e. from physiological saline solution to distilled water, lead to a significant reduction of the fracture toughness. Uniaxial tensile tests on collagen hydrogels confirmed the reduction in toughness, suggesting that lower bath osmolarity triggers changes in the failure properties of single collagen fibers. Prenatal surgeries, in particular fetoscopic procedures with partial amniotic carbon dioxide insufflation, might result in dehydration of the amnion. Dehydration induced a brittle behavior; however, subsequent rehydration for 15 min resulted in a similar tear resistance as for the fresh tissue., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: We have previously demonstrated two associations of PPROM, (1) inflammation/infection (modeled by tumor necrosis factor (TNF)) and (2) decidual bleeding (modeled by thrombin), both decrease fetal membrane (FM) rupture strength in-vitro. Furthermore, Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) induced by both TNF and thrombin is a critical intermediate, necessary and sufficient for weakening by either agent. The amnion is the strength component of FM and must weaken for FM to rupture. It is unclear whether GM-CSF weakens amnion (AM) directly, or initially targets choriodecidua (CD) which secondarily releases agents to act on amnion., Methods: Full thickness FM fragments were treated with/without GM-CSF. Some were preincubated with alpha-lipoic acid (LA), a known inhibitor of FM weakening. The FM fragments were then strength-tested. Separately, FM fragments were initially separated to AM and CD. AM fragments were cultured with Medium ± GM-CSF and then strength-tested. In other experiments, CD fragments were cultured with Medium, GM-CSF, LA, or LA + GM-CSF. Conditioned medium from each group was then incubated with AM. AM was then strength-tested. Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) were analyzed by Mutiplex Elisa., Results: GM-CSF weakened intact FM which was blocked by LA. GM-CSF did not weaken isolated AM. However, GM-CSF conditioned CD media weakened AM and this weakening was inhibited by LA. GM-CSF treatment of CD increased MMPs 2, 9, and 10, and decreased TIMPs 1-3. LA reversed these effects., Conclusions: GM-CSF does not weaken amnion directly; GM-CSF acts on CD to increase proteases and decrease anti-proteases which secondarily weaken the amnion., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2019. Published by Elsevier Ltd.)

Chorioamnionitis (CAM) is primarily a polymicrobial bacterial infection involving chorionic and amniotic membranes that is associated with increased risk of preterm delivery. Epoxyeicosatrienoic acids (EETs) are eicosanoids generated from arachidonic acid by cytochrome P450 enzymes and further metabolized mainly by soluble epoxide hydrolase (sEH) to produce dihydroxyeicosatrienoic acids (DHETs). As a consequence of this metabolism of EETs, sEH reportedly exacerbates several disease states; however, its role in CAM remains unclear. The objectives of this study were to (1) determine the localization of sEH and compare the changes it undergoes in the gestational tissues (placentas and fetal membranes) of women with normal-term pregnancies and those with pregnancies complicated by acute CAM; (2) study the effects of lipopolysaccharide (LPS) on the expression of sEH in the human gestational tissues; and (3) investigate the effect of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a specific sEH inhibitor, on LPS-induced changes in 14,15-DHET and cytokines such as interleukin- (IL-) 1 β and IL-6 in human gestational tissues in vitro and in pregnant mice. We found that women with pregnancies complicated by acute CAM had higher levels of sEH mRNA and protein in fetal membranes and villous tissues compared to those in women with normal-term pregnancies without CAM. Furthermore, fetal membrane and villous explants treated with LPS had higher tissue levels of sEH mRNA and protein and 14,15-DHET than those present in the vehicle controls, while the administration of AUDA in the media attenuated the LPS-induced production of 14,15-DHET in tissue homogenates and IL-1 β and IL-6 in the media of explant cultures. Administration of AUDA also reduced the LPS-induced changes of 14,15-DHET, IL-1 β , and IL-6 in the placentas of pregnant mice. Together, these results suggest that sEH participates in the inflammatory changes in human gestational tissues in pregnancies complicated by acute CAM., Competing Interests: All the authors declare that they have no conflicts of interest., (Copyright © 2019 Tai-Ho Hung et al.)

Our hypothesis is that hyaluronic acid may regulate the differentiation of human amniotic epithelial cells (hAECs) into insulin-producing cells and help the treatment of type 1 diabetes. Herein, a protocol for the stepwise in vitro differentiation of hAECs into functional insulin-producing cells was developed by mimicking the process of pancreas development. Treatment of hAECs with hyaluronic acid enhanced their differentiation of definitive endoderm and pancreatic progenitors. Endodermal markers Sox17 and Foxa2 and pancreatic progenitor markers Pax6, Nkx6.1, and Ngn3 were upregulated an enhanced gene expression in hAECs, but hAECs did not express the β cell-specific transcription factor Pdx1. Interestingly, hyaluronic acid promoted the expression of major pancreatic development-related genes and proteins after combining with commonly used inducers of stem cells differentiation into insulin-producing cells. This indicated the potent synergistic effects of the combination on hAECs differentiation in vitro. By establishing a multiple injection transplantation strategy via tail vein injections, hAECs transplantation significantly reduced hyperglycemia symptoms, increased the plasma insulin content, and partially repaired the islet structure in type 1 diabetic mice. In particular, the combination of hAECs with hyaluronic acid exhibited a remarkable therapeutic effect compared to both the insulin group and the hAECs alone group. The hAECs' paracrine action and hyaluronic acid co-regulated the local immune response, improved the inflammatory microenvironment in the damaged pancreas of type 1 diabetic mice, and promoted the trans-differentiation of pancreatic α cells into β cells. These findings suggest that hyaluronic acid is an efficient co-inducer of the differentiation of hAECs into functional insulin-producing cells, and hAECs treatment with hyaluronic acid may be a promising cell-replacement therapeutic approach for the treatment of type 1 diabetes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human amniotic membrane (hAM) is considered as an attractive biological scaffold for tissue engineering. For this application, hAM has been mainly processed using cryopreservation, lyophilization and/or decellularization. However, no study has formally compared the influence of these treatments on hAM properties. The aim of this study was to develop a new decellularization-preservation process of hAM, and to compare it with other conventional treatments (fresh, cryopreserved and lyophilized). The hAM was decellularized (D-hAM) using an enzymatic method followed by a detergent decellularization method, and was then lyophilized and gamma-sterilized. Decellularization was assessed using DNA staining and quantification. D-hAM was compared to fresh (F-hAM), cryopreserved (C-hAM) and lyophilized/gamma-sterilized (L-hAM) hAM. Their cytotoxicity on human bone marrow mesenchymal stem cells (hBMSCs) and their biocompatibility in a rat subcutaneous model were also evaluated. The protocol was effective as judged by the absence of nuclei staining and the residual DNA lower than 50 ng/mg. Histological staining showed a disruption of the D-hAM architecture, and its thickness was 84% lower than fresh hAM (p < 0.001). Despite this, the labeling of type IV and type V collagen, elastin and laminin were preserved on D-hAM. Maximal force before rupture of D-hAM was 92% higher than C-hAM and L-hAM (p < 0.01), and D-hAM was 37% more stretchable than F-hAM (p < 0.05). None of the four hAM were cytotoxic, and D-hAM was the most suitable scaffold for hBMSCs proliferation. Finally, D-hAM was well integrated in vivo. In conclusion, this new hAM decellularization process appears promising for tissue engineering applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and -negative bacteria.

Progestins have been recommended for preterm birth prevention in high-risk women; however, their mechanism of action still remains an area of debate. Medroxyprogesterone acetate (MPA) has previously been shown to significantly inhibit tumor necrosis factor α (TNFα)-induced matrix metalloproteinase 9 (MMP9) messenger RNA (mRNA) expression and activity in primary amnion epithelial cells, a process that may lead to preterm premature rupture of membranes. A mechanism that explains MPA's inhibition of TNFα-induced MMP9 mRNA expression and activity in primary amnion epithelial cells is unclear since these cells lack the classic nuclear progesterone receptor but express a membrane-associated progesterone receptor-progesterone receptor membrane component 1 (PGRMC1) along with the glucocorticoid receptor (GR). Primary amnion epithelial cells harvested from healthy term pregnant women at cesarean section were treated with PGRMC1 (to knockdown PGRMC1 expression), GR (to knockdown GR expression), or control small interfering RNA (siRNA; 10 nm) for 72 hours, pretreated with ethanol or MPA (10 -6 M) for 6 hours, and then stimulated with or without TNFα 10 ng/mL for 24 hours. Real-time quantitative polymerase chain reaction and gelatin zymography were used to quantify MMP9 mRNA expression and activity, respectively. Experimental groups were compared using 1-way analysis of variance. Both TNFα-induced MMP9 mRNA expression and activity were significantly inhibited by pretreatment with MPA; however, only the inhibition of TNFα-induced MMP9 activity was partially reversed with PGRMC1 siRNA. However, GR siRNA reversed both the inhibition of TNFα-induced MMP9 mRNA expression and activity by MPA. This study demonstrates that MPA mediates its anti-inflammatory effects primarily through GR and partially through PGRMC1 in primary amnion epithelial cells.

During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Problem: Cortisol, which is regenerated from biologically inactive cortisone by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in human fetal membranes, may play an important role in human parturition. Recently, we have demonstrated that human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute-phase protein of inflammation, and SAA1 may be engaged in multiple actions associated with human parturition. It remains to be determined whether SAA1 can interact with cortisol in the regulation of 11β-HSD1 in the fetal membranes., Method of Study: In the current study, we examined the regulation of 11β-HSD1 expression by SAA1, and the interaction between SAA1 and cortisol in the regulation of 11β-HSD1 expression in primary human amnion fibroblasts and amnion tissue., Results: Either SAA1 or cortisol induced 11β-HSD1 expression in a concentration-dependent manner. Combination of SAA1 and cortisol synergistically enhanced 11β-HSD1 expression. Mechanism studies revealed that SAA1 and cortisol induced the phosphorylation of the transcription factor STAT3 in a sequential order with the induction by SAA1 preceding the induction by cortisol. Furthermore, the induction of 11β-HSD1 expression by either SAA1 or cortisol or combination of SAA1 and cortisol was blocked by STAT3 inhibition with its antagonist S3I-201 or siRNA-mediated knockdown., Conclusion: This study has demonstrated that SAA1 and cortisol can reinforce each other in the induction of 11β-HSD1 expression through sequential phosphorylation of STAT3. The synergistic enhancement of 11β-HSD1 expression by SAA1 and cortisol may lead to excessive cortisol accumulation in the fetal membranes at parturition., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Inflammation plays a pivotal role in the terminal process of human labor and delivery, including myometrial contractions and membrane rupture. TNF-alpha-induced protein 8-like-2 (TIPE2) is a novel inflammation regulator; however, there are no studies on the role of TIPE2 in human labor. We report that in myometrium, there is decreased TIPE2 mRNA expression during late gestation which was further decreased in labor. In fetal membranes, TIPE2 mRNA expression was decreased with both term and preterm labor compared to no labor samples. Knockdown of TIPE2 by siRNA in primary myometrium and amnion cells was associated with an augmentation of IL1B and TNF-induced expression of pro-inflammatory cytokines and chemokines; expression of contraction-associated proteins and secretion of the uterotonic prostaglandin PGF2α and expression of extracellular matrix degrading enzymes. In TIPE2-deficient myometrial cells treated with inhibitors of NF-κB or ERK1/2, the secretion of pro-labor mediators was reduced back to control levels. In conclusion, these in vitro experiments indicate that loss of TIPE2 exacerbates the inflammatory response.

Untimely activation of the inflammatory response by sterile or infective insults in uterine tissues can result in preterm birth. Pro-inflammatory cytokines and pathogenic activation of toll-like receptors (TLRs) initiate a biochemical cascade of events leading to myometrial activation and contractility, cervical dilatation, and rupture of the chorioamniotic membranes. GIT2 is a signaling protein known to play a role in innate and adaptive immunity; however, its role in the inflammatory pathways of human labor is not known. In this article, we report that GIT2 expression is lower in human myometrium and fetal membranes with term labor, and in preterm amnion with histological chorioamnionitis. GIT2 knockdown by siRNA in primary myometrial and amnion cells exhibited reduced expression of pro-inflammatory cytokines and chemokines in response to inflammatory challenge by cytokines or TLR ligands. In addition, the pro-inflammatory cytokines IL1B and TNF could not induce the expression of extracellular matrix degrading enzymes in GIT2-deficient amnion cells. Myometrial activation in response to pro-inflammatory cytokines was also significantly suppressed in GIT2-deficient cells as evidenced by decreased prostaglandin release and expression of contraction-associated proteins. Further to this, collagen gel assays demonstrated that TNF had a reduced ability to induce myometrial contractility in situ in GIT2-deficient myometrial cells compared to control-transfected cells. In summary, the loss of GIT2 diminishes the effects inflammatory mediators have in promoting myometrial contraction and fetal membrane rupture in vitro, suggesting that GIT2 could be a possible target for preterm birth therapies., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Dexamethasone (Dex), a corticosteroid hormone, is used during the perinatal period to help fetal lung and other organ development. Conversely, Dex-induced cell proliferation has been associated with accelerated aging. Using primary amnion epithelial cells (AECs) from term, not in labor, fetal membranes, we tested the effects of Dex on cell proliferation, senescence, and inflammation. Primary AECs treated with Dex (100 and 200 nM) for 48 h were tested for cell viability (crystal violet dye exclusion), cell cycle progression and/or type of cell death (flow cytometry), expression patterns of steroid receptors (glucocorticoid receptor, progesterone receptor membrane component 1&2), inflammatory mediators (IL-6 and IL-8), and telomere length (quantitative RT-PCR). Mechanistic mediators of senescence (p38MAPK and p21) were determined by western blot analysis. Dex treatment did not induce AEC proliferation, cell cycle, influence viability, or morphology. However, Dex caused dependent telomere length reduction and p38MAPK-independent but p21-dependent (confirmed by treatment with p21 inhibitor UC2288). Senescence was not associated with an increase in inflammatory mediators, which is often associated with senescence. Co-treatment with RU486 produced DNA damage, cell cycle arrest, and cellular necrosis with an increase in inflammatory mediators. The effect of Dex was devoid of changes to steroid receptors, whereas RU486 increased GR expression. Dex treatment of AECs produced nonreplicative and noninflammatory senescence. Extensive use of Dex during the perinatal period may lead to cellular senescence, contributing to cellular aging associated pathologies during the perinatal and neonatal periods., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Background: Preterm labor is a leading risk factor for neonatal death and long-term impairment and linked closely with inflammation. Non-obstetric surgery is occasionally needed during pregnancy and the anesthetic drugs or surgery itself can give rise to inflammation. Here, we examined the influence of propofol pretreatment on the expression of cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) after lipopolysaccharide (LPS) stimulation. In addition, we evaluated the expression of pro-inflammatory cytokines and nuclear factor kappa B (NF-κB)., Methods: Human amnion-derived WISH cells were used to investigate the effect of propofol on the LPS-induced expression of inflammatory substances involved in preterm labor. For the experiment, WISH cells were pretreated with various concentrations propofol (0.01-10 μg/ml) for 1 h and then treated with LPS (1 μg/ml) for 24 h. Cytotoxicity was evaluated using MTT assay. PGE 2 concentration was assessed by ELISA. Protein expressions of COX-2, PGE 2 and NF-κB were analyzed by western blotting analysis. RT-PCR was used for analysis of mRNA expression of COX-2, PGE 2 , interlukin (IL)-1β and tumor necrosis factor (TNF)-α., Results: Propofol showed no cytotoxicity on the WISH cells. LPS-induced PGE 2 production and COX-2 and PGE 2 expression were decreased after propofol pretreatment. Propofol also attenuated the LPS-induced mRNA expression of IL-1β and TNF-α. Moreover, the activation of NF-κB was inhibited by propofol pretreatment on LPS-stimulated WISH cells., Conclusion: We demonstrated that propofol suppresses the expression of inflammatory substances enhanced by LPS stimulation. Furthermore, this inhibitory effect of propofol on the inflammatory substance expression is mediated by suppression of NF-κB activation., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest.

Glucocorticoid use during pregnancy is known to increase the risk of preterm birth and preterm premature rupture of membranes (pPROM). Here, we investigated the mechanism of how glucocorticoids weaken the fetal membranes. The amnion mesenchymal layer was significantly thinner in pregnant women treated with prednisolone and in corticosterone-injected mice than in control groups. Matrix metalloproteinase (MMP)-9 mRNA and its activity, COX2 mRNA levels, and prostaglandin E2 synthesis were increased, whereas type 1 collagen (COL1A1) mRNA levels were decreased in the fetal membranes of corticosterone-injected mice. Unexpectedly, the proliferation and migration of macrophages were observed around the corticosterone-injected amnion, and IL-1β was released from these macrophages. In human amnion mesenchymal cells, cortisol did not change MMP mRNA expression, whereas IL-1β treatment robustly increased MMP and COX2 mRNA expression. COL1A1 mRNA level was decreased by both cortisol and IL-1β. These data suggest that the recruitment of amniotic macrophages by glucocorticoids plays a pivotal role in weakening of the fetal membranes, leading to the pathogenesis of pPROM., (Copyright © 2019 Endocrine Society.)

Preterm premature rupture of fetal membranes precedes 30-40% of preterm births. Activation of matrix metalloproteases (MMPs) is the one of the major causes of extracellular matrix (ECM) degradation in membrane rupture. Increased cortisol, regenerated by 11β-hydroxysteroid dehydrogenase 1 in the amnion at parturition, is known to participate in a number of parturition-pertinent events. However, whether cortisol has a role in the regulation of MMPs in the membranes is not known. Here, we addressed this issue using human amnion tissue, the most tensile layer of the membranes. RNA-sequencing revealed that cortisol induced MMP7 expression dramatically in amnion fibroblasts, which was confirmed by real-time quantitative RT-PCR and Western blotting analysis in cortisol-treated amnion explants and fibroblasts. Measurement of collagen IV α5 chain (COL4A5), a substrate for MMP-7, showed that cortisol reduced its extracellular abundance, which was blocked by an antibody against MMP-7. Moreover, increased MMP-7 but decreased COL4A5 abundance was observed in the amnion tissue following labor-initiated spontaneous rupture of membranes. Mechanistic studies showed that cortisol increased the phosphorylation of c-Jun and the expression of c-Fos, the 2 major components of activated protein 1 (AP-1), respectively. The knocking down of c-Fos or c-Jun significantly attenuated the induction of MMP7 expression by cortisol. Chromatin immunoprecipitation assays showed that cortisol stimulated the enrichment of c-Fos and c-Jun at the AP-1 binding site in the MMP7 promoter. The data suggest that induction of MMP7 by cortisol via AP-1 may be a contributing factor to ECM degradation in membrane rupture at parturition.-Wang, L.-Y., Wang, W.-S., Wang, Y.-W., Lu, J.-W., Lu, Y., Zhang, C.-Y., Li, W.-J., Sun, K., Ying, H. Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.

Background: Chemotherapy can induce premature ovarian insufficiency (POI) and reduce fertility in young female patients. Currently, there is no effective therapy for POI. Human amnion-derived mesenchymal stem cells (hAD-MSCs) may be a promising seed cell for regenerative medicine. This study investigated the effects and mechanisms of hAD-MSC transplantation on chemotherapy-induced POI in rats., Methods: Chemotherapy-induced POI rat models were established by intraperitoneal injection of cyclophosphamide. Seventy-two female SD rats were randomly divided into control, POI, and hAD-MSC-treated groups. hAD-MSCs were labeled with PKH26 and injected into the tail veins of POI rats. To examine the underlying mechanisms, the differentiation of transplanted hAD-MSCs in the POI ovaries was analyzed by immunofluorescent staining. The in vitro expression of growth factors secreted by hAD-MSCs in hAD-MSC-conditioned media (hAD-MSC-CM) was analyzed by ELISA. Sixty female SD rats were divided into control, POI, and hAD-MSC-CM-treated groups, and hAD-MSC-CM was injected into the bilateral ovaries of POI rats. After hAD-MSC transplantation or hAD-MSC-CM injection, serum sex hormone levels, estrous cycles, ovarian pathological changes, follicle counts, granulosa cell (GC) apoptosis, and Bcl-2, Bax, and VEGF expression in ovaries were examined., Results: PKH26-labeled hAD-MSCs mainly homed to ovaries after transplantation. hAD-MSC transplantation reduced ovarian injury and improved ovarian function in rats with POI. Transplanted hAD-MSCs were only located in the interstitium of ovaries, rather than in follicles, and did not express the typical markers of oocytes and GCs, which are ZP3 and FSHR, respectively. hAD-MSCs secreted FGF2, IGF-1, HGF, and VEGF, and those growth factors were detected in the hAD-MSC-CM. hAD-MSC-CM injection improved the local microenvironment of POI ovaries, leading to a decrease in Bax expression and an increase in Bcl-2 and endogenous VEGF expression in ovarian cells, which inhibited chemotherapy-induced GC apoptosis, promoted angiogenesis and regulated follicular development, thus partly reducing ovarian injury and improving ovarian function in rats with POI., Conclusions: hAD-MSC transplantation can improve ovarian function in rats with chemotherapy-induced POI at least partly through a paracrine mechanism. The presence of a paracrine mechanism accounting for hAD-MSC-mediated recovery of ovarian function might be attributed to the growth factors secreted by hAD-MSCs.

This study aimed to evaluate the use of Insulin-Transferrin-Selenium (ITS) medium in place of fetal bovine serum (FBS) to culture human amnion mesenchymal stem cells (hAMSCs). Cell morphology, ultrastructure, proliferation, migration and MSC related markers were assessed accordingly. The hAMSCs were induced to osteocyte, chondrocyte, adipocyte and keratinocyte by culturing in appropriate induction medium. hAMSCs mRNA expression was detected for the matrix metalloproteinases 2 (MMP2), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I), Platelet-derived Growth Factor (PDGF), and transforming growth factor beta 1 (TGF-β) by real-time quantitative RT-PCR. Our results showed that hAMSCs cultured in ITS medium exhibited similar proliferation rates, demonstrated a statistically significant increased migration and expressed similar levels of MSC markers(CD73+, CD90+, CD105+, CD45-, CD34-) compared with those cultured in FBS. Osteoblasts, chondrocytes, adipocytes and keratinocytes were differentiated. Results of transmission electron microscope (TEM) revealed that hAMSCs cultured in ITS medium underwent active metabolism. The mRNA expression of MMP2, VEGF, KGF, TGF-β, IGF-I and PDGF upregulated in ITS medium. In conclusion, ITS medium has the potential to be used for the expansion of hAMSCs before clinical application., (© 2019 by the Association of Clinical Scientists, Inc.)

Intrauterine infection is a major cause of immune imbalance at the maternal-fetal interface, which leads to spontaneous abortion, premature rupture of the fetal membranes, and preterm birth. Human amniotic epithelial cells (hAECs) play a fundamental role in the maintenance of pregnancy. We hypothesize that bacteria influence the immunomodulatory effects of hAECs through stimulation of Toll-like receptors (TLRs). Here, we investigated how lipopolysaccharide (LPS) as a bacterial component affects anti-inflammatory and pro-inflammatory cytokines production of hAECs. Human placentas were obtained from six healthy pregnant women and hAECs were isolated. The phenotypic characteristics of hAECs were determined by flow cytometry. The hAECs (4 × 10 5 cells/ml) were cultured in the presence or absence of LPS (5 μg/ml). The viability of the cells was assessed and culture supernatants of hAECs were collected after 24, 48 and 72 h of incubation. The levels of transforming growth factor-beta1 (TGF-β1), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α), interleukin-17 A (IL-17A), and interferon-gamma (IFN-γ) were measured by ELISA. Our data showed that LPS treatment did not affect the viability of hAECs, while had a stimulatory effect on TGF-β1 production of hAECs (p < 0.001). A significant reduction in IL-4 production of LPS-stimulated hAECs was observed (p < 0.05). LPS enhanced the production of TNF-α and IL-17 A of hAECs (p < 0.05-0.0001). The IFN-γ level was only detectable in two culture supernatants of hAECs, and the level was unchanged after stimulation with LPS. Based on these findings, LPS may play a pivotal role in immune imbalance at the feto-maternal interface through affecting anti-inflammatory and pro-inflammatory cytokines production of hAECs., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Epidemiological evidence suggests that diabetes mellitus (DM) is an important factor in promoting periodontitis. It not only affects the attachment of connective tissue but also causes loss of alveolar bone. Hence, there is an urgent need to find an effective treatment for DM-induced bone deficiency. This study aimed to investigate the effects of human amniotic mesenchymal stem cells (HAMSCs) on the proliferation and osteogenic differentiation of DM-induced human bone marrow mesenchymal stem cells (HBMSCs). High glucose and palmitic acid (GP) were used to mimic DM-induced glucolipotoxicity. The proliferation levels were measured using flow cytometry. Alkaline phosphatase activity substrate assays, Alizarin red S staining, and western blotting were used to investigate osteogenic differentiation. Oxidative stress was measured by assaying the levels of reactive oxygen species. This study found that glucolipotoxicity caused by GP remarkably inhibited cell proliferation and osteogenesis, and upregulated the oxidative stress level in HBMSCs. However, HAMSCs attenuated HBMSC dysfunction through antioxidant activity by influencing p38 mitogen-activated protein kinase and vascular endothelial growth factor secretion. In conclusion, our findings indicate that HAMSCs might be suitable for treating DM-mediated bone deficiency.

Background: The aim of this study is to evaluate the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2), human amnion mesenchymal stem cells (hAMSCs), and nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA) in tissue engineering to provide potential approaches for periodontal bone regeneration., Methods: hAMSCs were isolated from discarded amniotic membrane samples and cultured in vitro. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to evaluate the osteoblast (OB) differentiation ability of hAMSCs. Three groups were divided: the experimental group (cells transfected with pcDNA3.1-rhBMP-2), the blank group (cells without gene transfection), and the control group (cells transfected with empty plasmid). RT-PCR and western blot were used to examine whether rhBMP-2 has been successfully expressed. 3-(4,5)-dimethylthiahiazol(-z-y1)-3,5-di-phenytetrazo-liumromide assay (MTT) was done to detect the effect of rhBMP-2 on hAMSCs seeded on nHAC/PLA. ALP activity, mineral formation assay, calcium, phosphate and osteocalcin (OCN) content, and OCN and RUNX2 expression of hAMSCs were detected to evaluate osteogenic differentiation capability of rhBMP-2 on hAMSCs seeded on nHAC/PLA., Results: hAMSCs exhibited intense ALP staining, obvious calcium deposition, and mineralization nodules, and rhBMP-2 were highly expressed in the experimental group. The proliferation of the hAMSCs with rhBMP-2 on nHAC/PLA was significantly higher than the cells without rhBMP-2, and the cells all increased in a time-dependent manner. rhBMP-2 significantly increased the OCN and phosphate content, mineral formation, ALP activity, osteogenic biomarkers OCN, and Runx2, and decreased calcium content in hAMSCs seeded on the nHAC/PLA scaffold., Conclusions: This finding demonstrated that hAMSCs has an ideal OB differentiation ability. rhBMP-2 facilitates the proliferation and osteogenesis of hAMSCs. The nHAC/PLA could act as a good scaffold for hAMSCs seeding, proliferation, and osteogenic differentiation. The application of rhBMP-2, nHAC/PLA, and hAMSCs in tissue engineering may offer promising possibilities for periodontal bone regeneration.

Purpose: The amniotic membrane (AM) is the transparent innermost layer of the placenta and it facilitates rapid wound healing in a diversity of ocular surface disorders. However, extended periods of cryopreservation before use induce significant impairment of cell viability due to oxidative stresses and inflammatory responses. We investigated the effect of supplementing ulinastatin (ULI), a known serine protease inhibitor, and relevant mechanisms of action in AM preservation solution through the hypothermic continuum on inflammatory and apoptotic signals and viability of AM tissue., Materials and Methods: The expression of inflammatory signal factors, including high mobility group box 1 (HMGB1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and anti-TNF-inducible gene 6 (TSG-6) which is a TNF-α-inducible anti-inflammatory protein, and the expression of apoptotic signal factors, including caspase (Cas)-9 and Cas-8, the initiators, and Cas-3, the executioner caspase and Bax were analyzed with or without ULI during hypothermic preservation of human AM. Subsequently, the actual viability of human AM tissue was verified with or without ULI supplementation throughout hypothermic continuum (both hypothermic- and cryopreservation)., Results: Hypothermic AM preservation with ULI for 48 h resulted in downregulated expression of cold-inducible inflammatory factors, including HMGB1 and NF-κB, as well as RIPK3. In addition, ULI suppressed apoptotic signals related with Cas-9, Cas-8, and Cas-3 under hypothermic conditions. Furthermore, ULI supplementation during hypothermic- and cryopreservation of AM significantly enhanced viability of AM tissue and amniotic epithelial cells., Conclusions: Supplementation of ULI during human AM preservation through the hypothermic continuum may be a feasible dual anti-inflammatory and anti-apoptotic strategy that enhances the viability of AM tissue.

Background: The deleterious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) is able to cross the placenta barrier and cause alterations in fetal growth, low birth weight and preterm labor. However, the effects of THC on the human placenta amnion are still unknown., Methods: The distributions of CB1R and CB2R in human amnion tissues were observed by immunohistochemistry (IHC). Human amniotic epithelial cell proliferation and migration in response to THC treatment were measured by MTS and transwell assays, respectively. The PCR array was performed to study the key regulators involved in the cell migration. The protein levels of CB1R, CB2R in amnion tissues and MMP2, MMP9 in cells were detected by western blotting. Small interfering RNAs (siRNAs) were used to knockdown MMP2 and MMP9 in WISH cells., Results: Our results indicated that both CB1R and CB2R primarily identified in the epithelial layer of human placental amnion tissue. The CB1R expression in the amnion tissue was higher in the preterm group than normal control. High-dose of THC (30uM, but not 20 and 10uM) significantly inhibited (p<0.01) human amniotic epithelial cell lines (WISH) proliferation. Meanwhile, THC at both 10uM and 20uM (p<0.05) significantly suppressed cells migration in both WISH and primary human amniotic epithelial cells. The PCR array data and siRNA experiments demonstrated that MMP2/9 were tightly involved in the regulation of THC-inhibited cell migration in WISH cells., Conclusion: These results suggested that THC inhibited the migration of human amniotic epithelial cell through the regulation of MMP2 and MMP9, which in turn altered the development of the amnion during the gestation and partially resulted in preterm labor and other adverse pregnancy outcomes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Preterm birth occurs in 9% to 13% of all human pregnancies and accounts for 80% of all neonatal morbidities and mortalities. Approximately 40% of all preterm births are idiopathic and about half are associated with infection and/or an activated inflammatory process. Further to studies showing anti-inflammatory effects of supernatant from the probiotic Lactobacillus rhamnosus GR-1 (GR-1), we tested its ability to modulate cytokine and chemokine production from amnion cells in response to stimulation by bacterial wall components, lipopolysaccharide (LPS), and lipoteichoic acid (LTA). Placentae were collected from women undergoing elective cesarean section at term. Amnion cells were cultured for 48 hours to confluence, serum starved for 12 hours, and then treated with GR-1 supernatant (1:20 dilution), followed after 12 hours by LPS (100 ng/mL) or LTA (10 ng/mL) for an additional 12 hours. Both LTA and LPS caused significant increases in the concentration of the pro-inflammatory cytokine, tumor necrosis factor α (TNF-α; 103.9 ± 67.5 pg/mL and 368.3 ± 65.7 pg/mL, respectively) in medium from cultured amnion cells compared to control (<4 pg/mL). There was no significant effect of GR-1 supernatant alone on TNF-α output, but there was significant reduction after LPS treatment. The basal output of the immunomodulatory cytokine, interleukin 6, was 613 ± 170 pg/mL and increased significantly after addition of GR-1 supernatant, LTA, LPS, and combinations of LTA/LPS with GR-1 supernatant. In conclusion, probiotic L rhamnosus GR-1 attenuates the effect of both LPS and LTA in stimulating the output of the pro-inflammatory cytokine TNF-α from mixed cultures of human amnion cells in keeping with previous findings in human trophoblast cells.

Phosphorylation of the transcription factors cyclic adenosine monophosphate response element-binding protein (CREB) and signal transducer and activator of transcription 3 (STAT3) by protein kinase A (PKA) is required for the cortisol-induced production of cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) in human amnion fibroblasts, which critically mediates human parturition (labor). We found that PKA was confined in the nucleus by A-kinase-anchoring protein 95 (AKAP95) in amnion fibroblasts and that this localization was key to the cortisol-induced expression of PTGS2 , the gene encoding COX-2. Cortisol increased the abundance of nuclear PKA by stimulating the expression of the gene encoding AKAP95. Knockdown of AKAP95 not only reduced the amounts of nuclear PKA and phosphorylated CREB but also attenuated the induction of PTGS2 expression in primary human amnion fibroblasts treated with cortisol, whereas the phosphorylation of STAT3 in response to cortisol was not affected. The abundances of AKAP95, phosphorylated CREB, and COX-2 were markedly increased in human amnion tissue after labor compared to those in amnion tissues from cesarean sections without labor. These results highlight an essential role for PKA that is anchored in the nucleus by AKAP95 in the phosphorylation of CREB and the consequent induction of COX-2 expression by cortisol in amnion fibroblasts, which may be important in human parturition., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Introduction: Nucleotide-binding oligomerization domain (NOD)-like receptors or NOD-like receptors (NLRs) have been implicated in several disease pathologies associated with inflammation. Since local and systemic inflammation is a hallmark of both term and preterm labour, a role for NLRs at the materno-fetal interface has been postulated., Methods: Gene expression and immunolocalisation of NLR family members in human placenta, choriodecidua, and amnion were examined. Tissue explants were used to examine the response to activators of NOD1 (Tri-DAP), NOD2 (MDP) and NLRP3 (nigericin). Cell/tissue-free supernatants were examined for the production of interleukin (IL)-1β, IL-6, IL-8 and IL-10 using specific ELISAs., Results: Expression of transcripts for NOD1, NOD2, NLRP3, NLRC4, NLRX1, NLRP1 and NAIP and protein expression of NOD1, NOD2 and NLRP3 were a broad feature of all term gestation-associated tissues. Production of cytokines was increased significantly in response to all ligands in placenta and choriodecidua, except for MDP-induced IL-10. Similarly, there was a significant in the amnion except for MDP induced IL-1β and IL-10 response to either agonist. IL-1β production was dependent on caspase-1 regardless of agonist used or tissue examined., Discussion: Term human gestation-associated tissues express functional NLRs which likely play a role in both sterile and pathogen-driven inflammatory responses at the materno-fetal interface., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: The preterm premature rupture of membranes (PPROM) is a frequent pathology responsible of more than 30% of preterm births. Tobacco smoking is one of the most frequently described risk factors identified and contributes to the pre term weakening of fetal membranes. As previously demonstrated, all-trans retinoic acid (atRA) regulates several genes involved in the extracellular matrix dynamics, an essential actor in fetal membrane ruptures. We hypothesized that cigarette smoke may affect this pathway in human amnion., Methods: Amnion was obtained from full-term fetal membranes collected from non-smoking women after cesarean births and used either as explants or for the isolation of derived epithelial cells. The pro-healing and transcriptomic effects of atRA were studied by a scratch assay experiment and quantitative RT-PCR, respectively, after treatment with dimethyl sulfoxyde (DMSO), atRA, DMSO + cigarette smoke condensate (CSC), or atRA + CSC., Results: Our results show a strong alteration of the retinoid pathway after CSC treatment on amnion-derived epithelial cells and explants. We first demonstrated that CSC inhibits the activity of the RARE reporter gene in amnion-derived epithelial cells. Then, atRA's effects on both the transcription of its target genes and wound healing were demonstrated to be inhibited or at least decreased by the CSC in human amnion epithelial cells., Discussion: Here, we demonstrated that CSC altered the retinoid signal, already known to have roles in fetal membrane physiopathology. These results highlight a potential negative action of maternal smoking on the retinoid pathway in human amnion and more generally on pregnancy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Study Question: Does A20 regulate mediators involved in the terminal processes of human labour in primary myometrial and amnion cells?, Summary Answer: A20 is a nuclear factor-kappa B (NF-κB) responsive gene that acts as a negative regulator of NF-κB-induced expression of pro-labour mediators., What Is Known Already: Inflammation is commonly implicated in spontaneous preterm birth and the processes involved in rupture of foetal membranes and uterine contractions. In myometrium and foetal membranes, the pro-inflammatory transcription factor NF-κB regulates the transcription of pro-labour mediators in response to inflammatory stimuli. In non-gestational tissues, A20 is widely recognised as an anti-inflammatory protein that inhibits inflammation-induced NF-κB signalling., Study Design, Size, Duration: Primary human amnion and myometrial cells were used to determine the effect of pro-inflammatory mediators on A20 expression and the effect of A20 siRNA on the expression and secretion of pro-labour mediators. The expression of A20 was assessed in myometrium and foetal membranes from non-labouring and labouring women at preterm and or term (n = 8 or nine samples per group)., Participants/materials, Setting, Methods: The effects of pro-inflammatory mediators and of A20 siRNA in cell cultures were determined by quantitative RT-PCR (qRT-PCR), western blots, immunoassays, gelatin zymography and luciferase assays. A20 expression in tissue samples was assessed by qRT-PCR. Statistical significance was ascribed to a P value < 0.05., Main Results and the Role of Chance: In primary cells isolated from myometrium and or amnion, the pro-inflammatory cytokines IL1B and TNF, the bacterial products flagellin and fsl-1, and the viral double stranded RNA analogue poly(I:C) significantly increased A20 mRNA expression via NF-κB. A20 siRNA studies in primary myometrial and amnion cells demonstrated an augmentation of inflammation-induced expression and or secretion of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL1, CXCL8, CCL2), adhesion molecules (ICAM1, VCAM1), contraction-associated proteins (PTGS2, PTGFR, PGF2α) and the extracellular matrix degrading enzyme MMP9, as well as NF-κB activation. Inhibition of NF-κB activity significant attenuated inflammation-induced expression of pro-labour mediators in A20 siRNA transfected cells. Finally, A20 mRNA expression was decreased in myometrium and foetal membranes with labour, and in foetal membranes with chorioamnionitis., Large Scale Data: Not applicable., Limitations, Reasons for Caution: The conclusions of this study are solely reliant on the data from in vitro experiments using cells isolated from myometrium and amnion., Wider Implications of the Findings: The results of this study raise the possibility that targeting A20 may be a therapeutic approach to reduce inflammation associated with spontaneous preterm birth., Study Funding and Competing Interest(s): Associate Professor Martha Lappas is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; grant no. 1047025). Funding for this study was provided by the NHMRC (grant no. 1058786), Norman Beischer Medical Research Foundation and the Mercy Research Foundation. There are no competing interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Chemical penetration enhancers (CPEs) have long been used for mass transport enhancement across membranes. Many CPEs are used in a solution or gel and could be a solvent. The use of CPEs is mainly limited due to their toxicity/irritation levels. This study presents the evaluation of encapsulated CPEs in nano-sized polymeric particles on the chorioamnion (CA) membrane mass transport. CPEs' mass encapsulated in nanoparticles was decreased by 10,000-fold. Interestingly, this approach resulted in a 6-fold increase in mass transport across the CA. This approach may also be used with other CPEs' base applications necessitating lower CPE concentration. Applying Ultrasound (US) has shown to increase the release rate of and also the mass transport across the CA membrane. It is proposed that encapsulated CPEs penetrate into the CA membrane thus prolonging their exposure, possibly extending their penetration into the CA membrane, while insonation also deepens their penetration into the CA membrane., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: Alkali burns are the most common, severe chemical ocular injuries, their functional prognosis depending on corneal wound healing efficiency. The purpose of our study was to compare the benefits of amniotic membrane (AM) grafts and homogenates for wound healing in the presence or absence of previous all-trans retinoic acid (atRA) treatment., Methods: Fifty male CD1 mice with reproducible corneal chemical burn were divided into five groups, as follows: group 1 was treated with saline solution; groups 2 and 3 received untreated AM grafts or grafts treated with atRA, respectively; and groups 4 and 5 received untreated AM homogenates or homogenates treated with atRA, respectively. After 7 days of treatment, ulcer area and depth were measured, and vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) were quantified., Results: AM induction by atRA was confirmed via quantification of retinoic acid receptor β (RARβ), a well-established retinoic acid-induced gene. Significant improvements of corneal wound healing in terms of ulcer area and depth were obtained with both strategies. No major differences were found between the efficiency of AM homogenates and grafts. This positive action was increased when AM was pretreated with atRA. Furthermore, AM induced a decrease in VEGF and MMP-9 levels during the wound healing process. The atRA treatment led to an even greater decrease in the expression of both proteins., Conclusions: Amnion homogenate is as effective as AM grafts in promoting corneal wound healing in a mouse model. A higher positive effect was obtained with atRA treatment.

Inflammasomes are cytosolic signaling platforms that regulate the activation of caspase (CASP)-1, which induces the maturation of interleukin (IL)-1β and IL-18. Herein, we determined whether the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis express major inflammasome components and whether these changes are associated with the activation of CASP-1 and CASP-4 and the release of mature IL-1β and IL-18. When comparing the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the messenger RNA (mRNA) abundance of NLR family pyrin domain containing 3 ( NLRP3), NLR family CARD domain containing 4 ( NLRC4), absent in melanoma 2 ( AIM2), and nucleotide binding oligomerization domain 2 ( NOD2) was higher; (2) the NLRP3 and NLRC4 protein quantities were increased; (3) the mRNA and protein expressions of CASP-1 and its active forms were greater; (4) CASP-4 was increased at the mRNA level only; (5) the mRNA and protein expressions of IL-1β and its mature form were higher; and (6) a modest increase in the total protein concentration and abundance of the mature form of IL-18 was observed. In vitro incubation of the chorioamniotic membranes with the CASP-1 inhibitor, VX765, decreased the release of endotoxin-induced IL-1β and IL-18 (2-fold) but not IL-6 or tumor necrosis factor α. In conclusion, spontaneous labor at term with acute histologic chorioamnionitis is characterized by an upregulation of inflammasome components which, in turn, may participate in the activation of CASP-1 and lead to the release of mature IL-1β by the chorioamniotic membranes. These results support a role for the inflammasome in the mechanisms responsible for spontaneous labor at term with acute histologic chorioamnionitis.

Rupture of fetal membranes can initiate parturition at both term and preterm. Collagen is the crucial factor determining the tensile strength of the membranes. Toward the end of gestation, a feed-forward regeneration of cortisol via 11β-hydroxysteroid dehydrogenase 1 exists in fetal membranes. It remains undetermined whether cortisol contributes to collagen reduction in fetal membranes. In this study, we have examined whether cortisol accumulation is a causative factor for collagen reduction in human amnion fibroblasts, the major source of collagens in the membranes. Cortisol had no effect on collagen 1A1 (COL1A1) and 1A2 (COL1A2) messenger RNA (mRNA) abundance but decreased their protein abundance. The latter effect was affected by neither mRNA transcription inhibitor nor protein translation inhibitor. Mechanistic studies revealed that the reduction in COL1A1 but not COL1A2 protein by cortisol was blocked by lysosome inhibitor chloroquine or small interfering RNA (siRNA)-mediated knockdown of autophagy-related protein 7, an essential protein for autophagy, whereas the proteasome inhibitors MG132 and bortezomib were ineffective. Further analysis showed that cortisol dose dependently increased the ratio of LC3II/LC3I, a marker of lysosome activation, an effect blocked by the glucocorticoid receptor (GR) antagonist RU486 and siRNA-mediated knockdown of GR. Consistently, cortisol decreased COL1A1 and COL1A2 protein abundance in amnion tissue explants, and decreased COL1A1 and COL1A2 protein abundance was observed at parturition in the amnion tissue. Conclusively, cortisol regeneration in fetal membranes may contribute to rupture of fetal membranes at parturition by reducing collagen protein abundance. Lysosome-mediated autophagy accounts for the reduction in COL1A1 by cortisol, but the mechanism underlying the reduction in COL1A2 awaits further investigation., (Copyright © 2017 Endocrine Society.)

Context: Biochemical weakening of the amnion is a major factor preceding preterm premature rupture of membranes (PPROMs), leading to preterm birth. Activation of matrix metalloproteinases (MMPs) is known to play a key role in collagen degradation of the amnion; however, epithelial to mesenchymal transition (EMT) that is also induced by MMP activation has not been investigated as a mechanism for amnion weakening., Objective: To measure amniotic EMT associated with vaginal delivery (VD) compared with unlabored cesarean sections (CSs), and to assess changes in amniotic mechanical strength with pharmacologic inhibitors and inducers of EMT, thus testing the hypothesis that EMT is a key biochemical event that promotes amniotic rupture., Findings: (1) Amnions taken from VD contained a significantly increased number of mesenchymal cells relative to epithelial cells compared with unlabored CS by fluorescence-activated cell sorting analysis (60% vs 10%); (2) tumor necrosis factor (TNF)-α stimulation of amniotic epithelial cells increased expression of the mesenchymal marker vimentin after 2 days; (3) EMT inhibitor, etodolac, significantly increased the time and mechanical pressure required to rupture the amnion; and (4) TNF-α and another pharmacologic EMT inducer, ethacridine, decreased the time and mechanical pressure required for amnion rupture, further confirming that the mesenchymal phenotype significantly weakens the amnion., Conclusions: This work demonstrated amniotic cell EMT was associated with labor and EMT decreased the tensile strength of the amnion. These findings suggest a role for EMT in the pathophysiology of PPROM and may provide a basis for development of therapies to prevent preterm labor., (Copyright © 2017 Endocrine Society)

One of the most established roles of oxytocin (OT) is in inducing uterine contractions and labor. Apart from inducing contractions, our recent studies showed that OT can also activate proinflammatory pathways in both human myometrial and amnion cells, which suggests that the proinflammatory role of OT should be taken into account when developing tocolytics targeting the OT/oxytocin receptor (OTR) system. The OTR antagonist, atosiban, is currently used therapeutically for the treatment of preterm labor. We previously showed that atosiban fails to inhibit the proinflammatory effects of OT in human amnion; atosiban alone activates nuclear factor- κ B (NF- κ B) and mitogen activated protein kinases, thus upregulating downstream prolabor genes. In contrast with our findings with atosiban, the presence of the orally active OTR antagonist, nolasiban, reduced the effect of OT on NF- κ B and p38 kinase activation in both myometrial and amnion cells. Consistent with the activation of these inflammatory mediators, OT led to increases in the expression of cyclooxygenase-2 and phosphorylated cytosolic phospholipase A 2 , which was reflected in prostaglandin E 2 synthesis. Inhibition of NF- κ B activation by nolasiban also translated to suppression of downstream prolabor gene expression, such as cyclooxygenase-2, C-C motif chemokine ligand 2, interleukin-6, and interleukin-8. We also demonstrated that nolasiban treatment alone has no significant stimulatory effect on both the myometrium and amnion. In conclusion, our findings indicate that nolasiban possesses promising potential as a novel tocolytic agent for both acute and maintenance therapy, as it inhibits both myometrial contractions and the proinflammatory effects of OT without the biased agonist effects., (Copyright © 2017 by The Author(s).)

Despite the high incidence of trophoblast-related diseases, the molecular mechanism of inadequate early trophoblast development is still unclear due to the lack of an appropriate cellular model in vitro. In the present study, we reprogrammed the amniotic cells to be induced pluripotent stem cells (iPSCs) via a non-virus and non-integrated method and subsequently differentiated them into trophoblast-like cells by a modified BMP4 strategy in E6 medium. Compared with the previously studied trophoblast-like cells from ESCs, the iPSCs derived trophoblast-like cells behave similarly in terms of gene expression profiles and biofunctions. Also we confirmed the differentiating tendency from iPSCs to be syncytiotrophoblasts-like cells might be caused by inappropriate differentiating oxygen condition. Additionally, we preliminarily indicated in vitro "artificial" differentiation of iPSCs also undergoing a possible trophoblastic stem cell stage, as witnessed in vivo. In conclusion, we provided an in vitro cellular model to study early trophoblast development for specific individual, by using the feasible amnion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)