RESUMO
Modeling human pregnancy is challenging as two subjects, the mother and fetus, must be evaluated in tandem. To understand pregnancy, parturition, and adverse pregnancy outcomes, the two feto-maternal interfaces (FMi) that form during gestation (i.e., the placenta and fetal membrane) need to be investigated to understand their biological roles, and organ dysfunction can lead to adverse outcomes. Adverse pregnancy outcomes such as preterm rupture of the membranes, spontaneous preterm birth, preeclampsia, intra-uterine growth restriction, and gestational diabetes rates are on the rise worldwide, highlighting the need for future studies and a better understanding of molecular and cellular pathways that contribute to disease onset. Current in vivo animal models nor in vitro cell culture systems can answer these questions as they do not model the function or structure of human FMis. Utilizing microfabrication and soft-lithography techniques, microfluidic organ-on-chip (OOC) devices have been adapted by many fields to model the anatomy and biological function of complex organs and organ systems within small in vitro platforms.These techniques have been adapted to recreate the fetal membrane FMi (FMi-OOC) using immortalized cells and collagen derived from patient samples. The FMi-OOC is a four-cell culture chamber, concentric circle system, that contains both fetal (amniochorion) and maternal (decidua) cellular layers and has been validated to model physiological and pathological states of pregnancy (i.e., ascending infection, systemic oxidative stress, and maternal toxicant exposure). This platform is fully compatible with various analytical methods such as microscopy and biochemical analysis. This protocol will outline this device's fabrication, cell loading, and utility to model ascending infection-related adverse pregnancy outcomes.
Assuntos
Nascimento Prematuro , Recém-Nascido , Gravidez , Feminino , Animais , Humanos , Placenta/metabolismo , Membranas Extraembrionárias/metabolismo , Linhagem Celular , TecnologiaRESUMO
PROBLEM: Ascending bacterial infection is associated with â¼ 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.
Assuntos
Nascimento Prematuro , Ureaplasma , Recém-Nascido , Feminino , Humanos , Transdução de Sinais , Âmnio , InflamaçãoRESUMO
In indicated preterm births such a Gestational Diabetes Mellitus (GDM), little is known about the role of the amnion membranes. Investigating the role of amnion membrane inflammation in response GDM may suggest novel pathophysiologic mechanisms. We hypothesize that increased GDM inflammatory mediators may weaken the amnion membrane predisposing them to infection. Maternal and fetal serum and amnion membrane biopsies were collected from 20 GDM and 38 normoglycemic subjects (control) who underwent elective cesarean sections. Cytokines and adipokines were evaluated in serum and amnion culture supernatant samples. Amnion membrane biopsies from GDM and control subjects were studied: fresh frozen for RNA analysis for Toll-like receptor expression; cultured with LPS to test membrane permeability, and inflammation LPS + anti-TLR4 for testing mechanism. GDM was associated with higher fetal serum leptin (p = 0.004) and IL-10 (p = 0.04) compared to controls. Amnion membrane explants from GDM had higher levels of IL-6 (p = 0.019), and lower expression of Claudin-4 (p = 0.007) and increased permeability (p = 0.046) compared to controls. GDM membranes treated with LPS showed an increased expression of IL-10 (p = 0.013); IL-6 (p = 0.004) and TNF-α (p = 0.0005) but did not affect membrane permeability. LPS and anti-TLR4 antibody treatment reduced the production of TNF-α in controls (p = 0.03) and GDM (p = 0.007) compared to LPS alone. Fetal inflammatory response seems more balanced in GDM and does not impact membrane permeability function even with an infectious stimulus. Light fetal membrane inflammatory response may explain lack of preterm labor in GDM. Concluding, benign inflammation in the membranes may not be harmful for pregnancy maintenance.
Assuntos
Diabetes Gestacional/imunologia , Membranas Extraembrionárias/imunologia , Trabalho de Parto Prematuro/epidemiologia , Adulto , Biomarcadores/sangue , Glicemia/análise , Estudos de Casos e Controles , Diabetes Gestacional/sangue , Diabetes Gestacional/diagnóstico , Diabetes Gestacional/patologia , Membranas Extraembrionárias/patologia , Feminino , Humanos , Inflamação/sangue , Inflamação/complicações , Inflamação/diagnóstico , Inflamação/imunologia , Mediadores da Inflamação/sangue , Trabalho de Parto Prematuro/imunologia , Placenta/imunologia , Placenta/patologia , Gravidez , Índice de Gravidade de Doença , Adulto JovemRESUMO
OBJECTIVE: A physiologic increase of reactive oxygen species (ROS) is observed through pregnancy. ROS-induced damage to major cellular elements, specifically protein peroxidation, can lead to fetal and placental tissue senescence and inflammation often associated with normal parturition. The purpose of this study was to examine the effects of oxidative stress (OS) in inducing changes in proteins, senescence, and sterile inflammation in pregnant mice. METHODS: CD-1 mice (n = 5/group) on day 14 of gestation were subjected to minilaparotomy and the uterine horn between gestational sacs was injected with the following: saline (control), cigarette smoke extract (CSE) CSE diluted in saline and CSE + SB 203580 (SB) (a p38 mitogen-activated protein kinase (MAPK) inhibitor). Mice were sacrificed on day 18, and amniotic sacs, placentas and amniotic fluid (AF) were collected. Protein damage was evaluated by immunostaining for 3-Nitrotyrosine modified proteins (3-NT). Activation of prosenescence p38MAPK was evaluated by western blot. Senescence features, ß-galactosidase (SA-ß-Gal) and AF inflammatory cytokines were analyzed by immunostaining and multiplex luminex-based immunoassays, respectively. The data were analyzed by ANOVA and Tukey's test, p < .05 was used for significance. RESULTS: Amniotic sac from CSE-treated animals showed significant protein peroxidation compared to control as indicated by 3-NT staining. CSE activated p38MAPK phosphorylation in amniotic sac but not in placenta. Membrane p38MAPK activation was reduced after treatment with SB. CSE increased fetal membrane senescence (staining for SA-ß-Gal) and increased AF concentrations of all evaluated cytokines. High inflammation correlated with pup loss and a decrease in placental weight. Treatment with p38MAPK inhibitor (SB) minimized damages, senescence and sterile inflammation. CONCLUSION: OS induction by cigarette smoke extract cause fetal tissue protein damage, p38MAPK activation, senescence and sterile inflammation in the amniotic cavity of mouse. Prevention of p38MAPK activation can be a novel approach to prevention of adverse pregnancy outcomes related to OS induced premature senescence.