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Enzymatic breakdown of the collagen-rich extracellular matrix (ECM) that connects the amnion and chorion layers of the fetal membranes is one of the key events leading to rupture of membranes. Oxidant stress caused by increased formation of reactive oxygen species and/or reduced antioxidant capacity may predispose to membrane rupture, a major cause of preterm birth. The aim of this study was to determine the effect of human labour and supracervical (SC) apposition on antioxidant enzymes and 8-isoprostane (a marker of lipid peroxidation). To determine the effect of human labour on oxidative stress status, fetal membranes from the SC site (SCS) were collected from women at term Caesarean section (no labour), and from the site of membrane rupture (SOR) after spontaneous labour onset and delivery (post labour). To determine the effect of SC apposition on oxidative stress status, amnion was collected from the SCS and a distal site (DS) in women at term Caesarean section in the absence of labour. The release of 8-isoprostane was significantly higher in amnion from the SCS compared to DS, and in fetal membranes from the SOR compared to the SCS. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were lower in amnion from the SC compared to DS. SOD gene expression and enzyme activity were lower in fetal membranes after labour. There was no difference in expression or activity in catalase, GPx and glutathione reductase (GSR) between no labour and post labour fetal membranes. In primary amnion cells, SOD supplementation significantly augmented IL-1β induced MMP-9 expression and activity. In summary, non-labouring SC fetal membranes are characterised by reduced antioxidant enzyme activity when compared to distal membranes, and, as such, may be more susceptible to oxidative damage and thus membrane rupture.

Autism spectrum disorders (ASDs) are highly heritable, yet relatively few associated genetic loci have been replicated. Copy number variations (CNVs) have been implicated in autism; however, the majority of loci contribute to

The Perinatal ResearchCenter, Centennial Women’s Hospital, Nashville, Tennessee, USACorrespondence: Dr R Menon, Department of Obstetrics & Gynecology, Division of Maternal–Fetal Medicine, University of Texas MedicalBranch, 301 University Blvd MRB, Room 11.138C, Galveston, TX 77555-1062, USA. Email ram.menon@utmb.eduAccepted 28 November 2011.

Spontaneous preterm birth (PTB; birth prior to 37 weeks of gestation) is a complex phenotype with multiple risk factors that complicate our understanding of its etiology. A number of recent studies have supported the hypothesis that epigenetic modifications such as DNA methylation induced by pregnancy-related risk factors may influence the risk of PTB or result in changes that predispose a neonate to adult-onset diseases. The critical role of timing of gene expression in the etiology of PTB makes it a highly relevant disorder in which to examine the potential role of epigenetic changes. Because changes in DNA methylation patterns can result in long-term consequences, it is of critical interest to identify the epigenetic patterns associated with adverse pregnancy outcomes. This review examines the potential role of DNA methylation as a risk factor for PTB and discusses several issues and limitations that should be considered when planning DNA methylation studies.

Objective To give an overview of the literature for evidence of nutrient deficiencies as contributors to the disparity in preterm birth (PTB) between African-American and Caucasian women. Design Structured literature survey. Methods We searched MEDLINE to identify observational and experimental studies that evaluated the relation between nutrient intake and/or supplementation and PTB. For nutrients for which studies supported an association, we searched MEDLINE for studies of the prevalence of deficiency in the USA by race. Main outcome measures Summarized findings on nutrients for which there is both evidence of a role in PTB and variability in the prevalence of deficiency by race. Results Nutrient deficiencies for which there are varying levels of evidence for an association with PTB and a greater burden among African-American compared with Caucasian women include deficiencies of iron, folic acid, zinc, vitamin D, calcium and magnesium, and imbalance of ω-3 and ω-6 polyunsaturated fatty acids. There are inadequate high-quality studies that investigate the role of nutrient deficiencies in PTB, their potential interaction with other risks, the proportion of excess risk for which they account, and whether supplementation can reduce the risk of, and racial disparities in, PTB in US populations. Conclusion Deficiencies of several nutrients have varying levels of evidence of association with PTB and are of greater burden among African-American compared with Caucasian women. Although further research is needed, strategies that improve the nutritional status of African-American women may be a means of addressing a portion of the racial disparity in PTB.

Objective. We seek to expand on a biopsychosocial framework underlying the etiology of excess preterm birth experienced by African-American women by exploring short inter-pregnancy intervals as a partial explanatory factor. Design. We conducted a qualitative analyses of published studies that met specified criteria for assessing the association of inter-pregnancy interval and preterm birth. Methods. We determine whether inter-pregnancy interval is associated with preterm birth, what the underlying causal mechanism may be, whether African-American women are more likely than Caucasian women to have short intervals, and whether achieving an optimal interval will result in reduced African-American–Caucasian gap in preterm births. Main Outcome Measures. Crude and adjusted odds ratios for preterm birth, with the referent group being the interval closest to the ‘ideal’ of 18–23 months and the exposed group having intervals

To evaluate amniotic fluid arachidonic acid metabolites using enzymatic and nonenzymatic (lipid peroxidation) pathways in spontaneous preterm birth and term births, and to estimate whether prostanoid concentrations correlate with risk factors (race, cigarette smoking, and microbial invasion of amniotic cavity) associated with preterm birth.In a case-control study, amniotic fluid was collected at the time of labor or during cesarean delivery. Amniotic fluid samples were subjected to gas chromatography, negative ion chemical ionization, and mass spectrometry for prostaglandin (PG) E2, PGF2α, and PGD2 and for 6-keto-PGF1α (thromboxane 2 and F2-isoprostane). Primary analysis examined differences between prostanoid concentrations in preterm birth (n=133) compared with term births (n=189). Secondary stratified analyses (by race, cigarette smoking, and microbial invasion of amniotic cavity) compared eicosanoid concentrations in three epidemiological risk factors.Amniotic fluid F2-isoprostane, PGE2, and PGD2 were significantly higher at term than in preterm birth, whereas PGF2α was higher in preterm birth 6-keto-PGF1α and thromboxane 2 concentrations were not different. Data stratified by race (African American or white) showed no significant disparity among prostanoid concentrations. Regardless of gestational age status, F2-isoprostane was threefold higher in smokers, and other eicosanoids were also higher in smokers compared with nonsmokers. Preterm birth with microbial invasion of amniotic cavity had significantly higher F2-isoprostane compared with preterm birth without microbial invasion of amniotic cavity.Most amniotic fluid eicosanoid concentrations (F2-isoprostane, PGE2, and PGD2), are higher at term than in preterm births. The only amniotic fluid eicosanoid that is not higher at term is PGF2α.

Cigarette smoking and bacterial infections are two major risk factors associated with preterm prelabor rupture of membranes (pPROM). We hypothesized that exposure of fetal membranes to cigarette smoke extracts might induce oxidative stress (OS) and fetal membrane apoptosis, culminating in an alternate pathway to that commonly activated by infection. To test this, we characterized the production of prostanoids and biomarkers of apoptosis in normal term human fetal membrane explant cultures. Fetal membrane explants collected at term (from cesarean deliveries, not in labor) were stimulated with cigarette smoke extract (CSE) for 24 h. Two classes of prostanoids, F2-Isoprostane (F2-IsoP), a marker of OS and PGF2α, a classical uterotonin, were measured by gas chromatography/mass spectrometry. Western blot analyses of tissue lysates were performed to quantify the anti-apoptotic protein Bcl2 and actin (as a control). Fetal membrane apoptosis was detected by immunohistochemistry for active caspase 3 and confirmed by TUNEL staining for nuclear fragmentation. CSE exposure resulted in significantly more F2-IsoP production from fetal membranes (242.8 ± 79.3 pg/ml/mg of total membrane protein) compared to unstimulated controls (131.5 ± 53.1 pg/ml/mg; p < 0.0001). By contrast, PGF2α was not different in CSE vs. controls (1083 ± 527 vs. 1136 ± 835 pg/ml/mg of protein; p = 0.80). CSE-exposed tissues demonstrated a dose-dependent decrease in Bcl2 expression and increases in active caspase 3 and nuclear fragmentation in both amnion and chorion cells compared to controls. In summary, fetal membranes exposed to CSE manifest evidence of OS and apoptosis. The differential pattern of prostanoid production observed in this study supports the hypothesis that an alternate non-inflammatory pathway mediated by OS and apoptosis in pPROM may promote proteolysis resulting in membrane weakening and rupture.

Fetal membranes, a vital component that helps maintain pregnancy and contribute to parturition signaling, are often studied in segments due to its structural complexity. Transwells are traditionally used to study cell interactions; however, their usefulness is limited. To overcome these difficulties, a fetal membrane-organ-on-chip (FM-OO-C) was created to study interactive properties of amnion epithelial cells (AECs) and decidual cells compared to transwell systems.Primary AECs and decidual cells from term, nonlaboring fetal membranes were cultured in a 2-chamber (AEC/decidual cell) FM-OO-C device and sandwiched between a semipermeable membrane. Cells were treated with cigarette smoke extract (CSE) or dioxin, and membrane permeability and cellular senescence were measured after 48 hours. The same experiments were conducted in transwells for comparisons.Compared to transwell cultures, FM-OO-C model produced better membrane permeability readings regardless of the side of treatment or time point. Membrane permeabilization was higher in AECs directly treated with CSE (1.6 fold) compared to similar treatment on the decidual side (1.2 fold). In FM-OO-C, treatments forced changes between cellular layers. This was evident when CSE and dioxin-induced senescence on one side of the chamber produced similar changes on the opposite side. This effect was minimal in the transwell system.The controlled environment of an FM-OO-C allows for improved signal propagation between cells by minimizing noise and highlighting the small changes between treatments that cannot be seen in conventional transwell devices. Fetal membrane-organ-on-chip provides a better interaction between cell types that can be used to study fetal-maternal signaling during pregnancy in future studies.

Background Fetal endocrine signals are generally considered to contribute to the timing of birth and the initiation of labor. Fetal tissues under oxidative stress release inflammatory mediators that lead to sterile inflammation within the maternal-fetal interface. Importantly, these inflammatory mediators are packaged into exosomes, bioactive cell-derived extra cellular vesicles that function as vectors and transport them from the fetal side to the uterine tissues where they deposit their cargo into target cells enhancing uterine inflammatory load. This exosome-mediated signaling is a novel mechanism for fetal-maternal communication. Objective This report tested the hypothesis that oxidative stress can induce fetal amnion cells to produce exosomes, which function as a paracrine intermediary between the fetus and mother and biochemically signal readiness for parturition. Study Design Primary amnion epithelial cells were grown in normal cell culture (control) or exposed to oxidative stress conditions (induced by cigarette smoke extract). Exosomes were isolated from cell supernatant by sequential ultracentrifugation. Exosomes were quantified and characterized based on size, shape, and biochemical markers. Myometrial, decidual, and placental cells (BeWo) were treated with 2 × 105, 2 × 107, and 2 × 109 control or oxidative stress–derived amnion epithelial cell exosomes for 24 hours. Entry of amnion epithelial cell exosomes into cells was confirmed by confocal microscopy of fluorescent-labeled exosomes. The effect of amnion epithelial cell exosomes on target cell inflammatory status was determined by measuring production of interleukin-6, interleukin-8, interleukin-1β, tumor necrosis factor-α, and prostaglandin E2 by enzyme-linked immunosorbent assay and inflammatory gene transcription factor (nuclear factor-κβ) activation status by immunoblotting for phosphorylated RelA/p65. Localization of NANOG in term human myometrium and decidua obtained from women before labor and during labor was performed using immunohistochemistry. Data were analyzed by Wilcoxon-Mann-Whitney test to compare effects of exosomes from control and oxidative stress-treated amnion epithelial cells on inflammatory status of target cells. Results Amnion epithelial cells released ∼125 nm, cup-shaped exosomes with ∼899 and 1211 exosomes released per cell from control and oxidative stress–induced cells, respectively. Amnion epithelial cell exosomes were detected in each target cell type after treatment using confocal microscopy. Treatment with amnion epithelial cell exosomes increased secretion of interleukin-6, interleukin-8, and PGE2 and activation of NF-κβ (each P Conclusion In vitro, amnion epithelial cell exosomes lead to an increased inflammatory response in maternal uterine cells whereas placental cells showed refractoriness. Fetal cell exosomes may function to signal parturition by increasing maternal gestational cell inflammation.

Problem Oxidative stress (OS)-induced, p38 mitogen-activated protein kinase (p38MAPK)-mediated chorioamniotic senescence and inflammation (senescence-associated secretory phenotype [SASP]) are associated with parturition. In response to OS-inducing risk factors, premature senescence contributes to preterm premature rupture of the membranes (pPROM) and spontaneous preterm birth (PTB). We determined the effect of simvastatin, rosuvastatin, and progesterone in downregulating p38MAPK-mediated senescence and SASP. Method of study Normal term, not-in-labor fetal membranes (n = 8) were exposed to cigarette smoke extract (CSE: OS inducer) alone or combined with simvastatin (100 and 200 ng/mL), rosuvastatin (100 and 200 ng/mL), and progesterone (10-6 mol/L). p38MAPK expression changes were studied by Western blot, senescence was determined by senescence-associated β-Galactosidase (SA-β-Gal) staining, and multiplex analysis determined changes associated with 4 SASP markers (IL-8, IL-10, TNF-α, and GM-CSF). A pairwise comparison between groups was conducted by ANOVA. Results Compared to untreated controls, CSE induced p38MAPK-mediated senescence and SASP. CSE cotreatment with simvastatin and rosuvastatin significantly reduced p38MAPK activation, senescence (decrease in SA-β-Gal) and SASP markers, GM-CSF, and TNF, but not IL-8, while increasing anti-inflammatory IL-10 in a dose-dependent manner. Cotreatment of CSE and progesterone had no effect on reducing p38MAPK activation, senescence, or SASP. Conclusion Both simvastatin and rosuvastatin downregulated OS-induced p38MAPK activation, senescence, and SASP, while rosuvastatin showed a pronounced effect. Progesterone did not reduce OS-induced fetal membrane senescence and SASP. Simvastatin or rosuvastatin may reduce the incidences of OS-associated PTB and pPROM by preventing premature senescence and SASP.

We examined the association of 166 single nucleotide polymorphisms (SNPs) in cytokines and cytokine related genes with cytokine concentrations (IL-1beta, IL-8, and IL-10) in the amniotic fluid (AF). These cytokines have been associated with spontaneous preterm birth (PTB) and their genetic regulation may play a role in disease risk. These associations were studied in both PTB and term births in African Americans and Caucasians; maternal and fetal genotypes were studied separately. Analyses modeled genotype, pregnancy status, and marker by pregnancy status (case/control) interaction with cytokine concentration as outcome. Our results indicate that AF cytokines (IL-1beta and IL-10) were associated with interactions between pregnancy status and both maternal and fetal SNPs, with the most significant interactions being observed for African Americans with IL-1beta concentration (maternal at IL1RAP rs1024941 p < 10(-3), fetal IL1RAP rs3773953 p < 10(-3)). AF IL-10 concentrations also showed evidence for association with SNPs in both ethnicities with the most significant interaction in Caucasian maternal samples (IL10 rs1800896 p < 10(-3)). Our data indicate that the genetic regulation of cytokine concentrations in PTB likely differs by ethnicity. AF cytokine concentrations were associated with interactions between genotype and PTB in African Americans, but less so in Caucasians.

Chorioamnionitis, inflammation of the amniochorionic membrane (fetal membranes) is a very common disease but a complex syndrome associated with pregnancy. It presents a clinical impasse due to lack of knowledge of specific etiologies associated with this condition making confident clinical interventions difficult. Recent reports provide insight into genetic, epigenetic, behavioral, psychosocial, molecular and pathophysiological factors that are associated with chorioamnionitis. However, a coordinated approach in understanding causality and lack of early indicators (clinical and biomarkers) has hampered gaining knowledge about the disease status preventing proper intervention. Several reviews have provided in-depth analysis of the histologic and clinical evidence associated with chorioamnionitis. In this review, we provide a novel perspective on chorioamnionitis based on recent evidences from scientific literature on inflammation, apoptosis and genetics.

Resumen Incidencia mundial de parto prematuro: revision sistematica de la morbilidad y mortalidad maternas Objetivo Analizar las tasas de prematuridad a nivel mundial para evaluar la incidencia de este problema de salud publica, determinar la distribucion regional de los partos prematuros y profundizar en el conocimiento de las actuales estrategias de evaluacion.Metodos Los datos utilizados sobre las tasas de prematuridad a nivel mundial se extrajeron a lo largo de una revision sistematica anterior de datos publicados e ineditos sobre la mortalidad y morbilidad maternas notificados entre 1997 y 2002. Esos datos se complementaron mediante una busqueda que abarco el periodo 2003–2007. Las tasas de prematuridad de los paises sin datos se estimaron mediante modelos de regresion multiple especificos para cada region.Resultados Estimamos que en 2005 se registraron 12,9 millones de partos prematuros, lo que representa el 9,6% de todos los nacimientos a nivel mundial. Aproximadamente 11 millones (85%) de ellos se concentraron en Africa y Asia, mientras que en Europa y America del Norte (excluido Mexico) se registraron 0,5 millones en cada caso, y en America Latina y el Caribe, 0,9 millones. Las tasas mas elevadas de prematuridad se dieron en Africa y America del Norte (11,9% y 10,6% de todos los nacimientos, respectivamente), y las mas bajas en Europa (6,2%).Conclusion El parto prematuro es un problema de salud perinatal importante en todo el mundo. Los paises en desarrollo, especialmente de Africa y Asia meridional, son los que sufren la carga mas alta en terminos absolutos, pero en America del Norte tambien se observa una tasa elevada. Es necesario comprender mejor las causas de la prematuridad y obtener estimaciones mas precisas de la incidencia de ese problema en cada pais si se desea mejorar el acceso a una atencion obstetrica y neonatal eficaz.

Preterm birth (PTB

BACKGROUND: Preterm birth (PTB) is hypothesized to be an inflammatory response disease. However, no single factor alone is likely to explain PTB risk. It is more probable that coordinated networks of cytokines affect risk. METHODS: Therefore, we examined the relationships between amniotic fluid (AF) cytokines/chemokines and related biomarkers in PTB and normal term deliveries in African Americans and Caucasians. Data were obtained from African American (41 preterm labor and 91 term labor) and Caucasian (105 preterm labor and 100 term labor) pregnant mothers. Pro-inflammatory cytokines and related molecules interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor- (TNF)-α, TNF soluble receptors (sTNFR1 and sTNFR2), and anti-inflammatory cytokine IL-10 that were all previously associated with PTB were studied. Correlations between biomarkers were calculated; differences of correlation coefficients between AF from African American and Caucasian samples in preterm labor and term labor were measured. RESULTS: Multiple differences were observed between African American and Caucasian preterm and term birth groups. In term birth the strongest differences were between pro- and anti-inflammatory correlations, whereas in PTB differences were equally distributed between pro-inflammatory/anti-inflammatory and pro-inflammatory/pro-inflammatory correlations. Three correlation patterns differed significantly between AF from PTB African Americans with and without microbial invasion of the intra-amniotic cavity (MIAC); no differences were observed in Caucasians with MIAC. CONCLUSION: Correlation analyses of cytokine measurements suggest coordinated interplay during pregnancy; significant differences exist between African Americans and Caucasians. Such analyses can serve as a means of understanding risk factors in these populations.

Spontaneous preterm birth (PTB

The objective of this study is to determine if BPA exposure, as measured by maternal plasma (MP) and amniotic fluid (AF) BPA concentrations is associated with an increased risk of spontaneous preterm birth (PTB) and preterm premature rupture of membranes (pPROM).In this nested case-control study, MP samples from women in term labor (n = 30), preterm labor that ended with preterm delivery (n = 25), or who had pPROM (n = 30) and amniotic fluid samples from term labor (n= 45), preterm labor (n = 60), and pPROM (n = 35) were assayed for BPA by enzyme immunoassay.BPA was detectible in 100% of MP and AF samples. Women with MP BPA concentrations in the fourth quartile were at increased risk of PTB (cOR = 4.12, 95% CI = 1.32-12.87; aOR = 4.78, 95% CI = 1.14-20) but not pPROM. High (fourth quartile) AF BPA values also tended to increase the risk of pPROM (cOR = 2.47, 95% CI = 0.96-6.37) but results were not statistically significant.Increased BPA concentration is associated with an increased risk for PTB or pPROM depending on the maternal-fetal compartment(s) affected. High MP plasma BPA concentrations are associated with PTB with intact membranes but high AF BPA concentrations may weakly be associated with pPROM.

At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readiness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs) that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that i) exosomes act as carriers of signals in utero-placental compartments and ii) exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to determine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC). We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor placentae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exosomes isolated by differential centrifugations. Both growth conditions (normal and oxidative stress induced) produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H) 3, heat shock protein (HSP) 70 and activated form of pro-senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK) (P-p38 MAPK) co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress conditions than standard conditions (p

Summary Preterm birth (PTB) is a significant neonatal health problem that is more common in African-Americans (AA) than in European-Americans (EA). Part of this disparity is likely to result from the differing genetic architectures of EA and AA. To begin assessing the role of these differences, patterns of genetic variation in two previously proposed candidate genes, encoding interleukin 6 (IL6) and its receptor (IL6R), were analyzed in mothers and fetuses from 496 EA birth-events (149 cases and 347 controls) and 397 birth-events in AA (76 cases and 321 controls). IL-6 levels in amniotic fluid (AF) samples were determined in a subset of these pregnancies. Case-control comparisons revealed a single SNP in IL6R associated with PTB (p=0.04 for allelic and p=0.05 for genotype association). In addition, all of the SNPs studied showed significant frequency differences between AA and EA in at least one comparison, significantly in excess of that expected from general population databases. Higher IL-6 concentrations were associated with the IL6 SNP -661 in EA preterm samples (p=0.0056), and this result seems to be driven by microbial invasion of the amniotic cavity, indicating a gene by infection interaction. These findings indicate that, as a function of IL6 genotype, EA and AA women respond differently to infection with respect to their expression of IL-6. Our data support differential genetic control of levels of IL-6 in amniotic fluid between EA and AA.

Objective The objective of this study is to examine TNF-α and its soluble and membrane bound receptors in fetal membranes derived from blacks and whites in response to in vitro infectious stimulus, and the balance between TNF-α and the receptors. Fetal membranes collected from black and white women at term were maintained in an organ explant system and stimulated with lipopolysaccharide (LPS). TNF-α, soluble TNF receptors (sTNFR1 and sTNFR2) in culture media and membrane bound TNF receptors (TNFR1 and TNFR2) in tissue homogenates were measured. Molar ratio (TNF/sTNFR) was calculated between LPS stimulated and unstimulated (controls) cultures in both races. TNF-α was increased in both races after LPS stimulation and showed no difference between races ( p = 0.7). LPS decreased sTNFR1 in blacks, but increased in whites, showing a significant difference between races ( p = 0.001). In blacks sTNFR2 also decreased and increased in whites, but the results were not significant between races ( p = 0.4). Both TNFR1 and TNFR2 were increased in blacks after LPS stimulation whereas no such changes were seen in whites compared to controls that were also significant between races. After LPS stimulation TNF-α bioavailability was increased in blacks with a drop in soluble receptors and with an increase in membrane receptors. This was not evident in whites because in whites soluble receptors were increased with no change in membrane receptors. Our data demonstrated that LPS stimulation results in a molar ratio switch favoring TNF-α biofunction in blacks, but not in whites.

Spontaneous preterm birth, caused by preterm labor (contractions before 37 weeks' gestation) or preterm premature rupture of the membranes (pPROM) (membrane rupture before the onset of labor) or both account for approximately 80% of preterm deliveries. pPROM is associated with 30-40% of preterm deliveries and the incidence of pPROM has increased in the past decade. The question we address here is why some women experience pPROM and some experience preterm labor with no rupture of membranes (ROM) when the etiologic factors associated with both these pathologic complications are the same. To date, studies had evaluated the markers that are commonly elevated in both preterm labor and pPROM. A better understanding of the similarities and differences between the biomolecular pathways leading to each of these conditions may open new avenues for research and intervention. In this chapter we review the role of inflammatory mediators (cytokines and matrix metalloproteinases), and programmed cell death (apoptosis) in preterm labor with no ROM and preterm labor with pPROM to delineate the differences in pathways between the two conditions.

Inflammation is a physiologic component of pregnancy and parturition. Overwhelming intrauterine inflammatory load promotes quiescent feto-maternal tissues into a contractile phenotype. Like inflammation, oxidative stress is an inevitable component of both pregnancy and parturition. Pathologic activation of host innate immune response to adverse pregnancy conditions can lead to premature activation of inflammatory and oxidative stress. Inflammation and oxidative stress markers seen with both sterile and infectious inflammation are often similar; therefore, it is difficult to understand causality of conditions like spontaneous preterm birth. This review demonstrates potential mechanistic pathways of activation of sterile and infectious inflammation. We demonstrate the activation of two unique pathways of inflammation by factors that are well-documented proxies for oxidative stress (cigarette smoke extract) and infection (lipopolysaccharide). Sterile inflammation seen after exposure to an oxidative stress inducer is due to cellular elemental damage resulting in p38 mitogen-activated protein kinase (MAPK) induced cellular senescence. Infectious inflammation is through activation of transcription factor NF-κB and independent of oxidative stress-associated damages and p38 MAPK-induced senescence. Understanding the differences in the inflammatory pathway activation by various risk factors is important to design better screening, diagnostic and intervention strategies to reduce the risks of adverse pregnancy outcomes.

Made available in DSpace on 2015-12-07T15:31:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2015. Added 1 bitstream(s) on 2015-12-07T15:52:45Z : No. of bitstreams: 1 PMC4580414.pdf: 8286022 bytes, checksum: 53177f4f8c45fc0ba7b10a9f5c72cf41 (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Oxidative stress (OS)-induced senescence of the amniochorion has been associated with parturition at term. We investigated whether telomere fragments shed into the amniotic fluid (AF) correlated with labor status and tested if exogenous telomere fragments (T-oligos) could induce human and murine amnion cell senescence. In a cross-sectional clinical study, AF telomere fragment concentrations quantitated by a validated real-time PCR assay were higher in women in labor at term compared to those not in labor. In vitro treatment of primary human amnion epithelial cells with 40 μM T-oligos ([TTAGGG]2) that mimic telomere fragments, activated p38MAPK, produced senescence-associated (SA) β-gal staining and increased interleukin (IL)-6 and IL-8 production compared to cells treated with complementary DNA sequences (Cont-oligos, [AATCCC]2). T-oligos injected into the uteri of pregnant CD1 mice on day 14 of gestation, led to increased p38MAPK, SA-β-gal (SA β-gal) staining in murine amniotic sacs and higher AF IL-8 levels on day 18, compared to saline treated controls. In summary, term labor AF samples had higher telomere fragments than term not in labor AF. In vitro and in situ telomere fragments increased human and murine amnion p38MAPK, senescence and inflammatory cytokines. We propose that telomere fragments released from senescent fetal cells are indicative of fetal cell aging. Based on our data, these telomere fragments cause oxidative stress associated damages to the term amniotic sac and force them to release other DAMPS, which, in turn, provide a sterile immune response that may be one of the many inflammatory signals required to initiate parturition at term. Division of Maternal-Fetal Medicine and Perinatal Research, Department of Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America; Department of Pathology, Botucatu Medical School, UNESP-Univ. Estadual Paulista, Botucatu, Sao Paulo, Brazil. Division of Maternal-Fetal Medicine and Perinatal Research, Department of Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America. Department of Epidemiology & Biostatistics, Texas A&M University System Health Science Center, College Station, Texas, United States of America. Department of Obstetrics and Gynecology, Wake Forest University, Winston Salem, North Carolina, United States of America. Division of Maternal-Fetal Medicine and Perinatal Research, Department of Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America; Department of Pathology, Botucatu Medical School, UNESP-Univ. Estadual Paulista, Botucatu, Sao Paulo, Brazil.

Study hypothesis In women with preterm premature rupture of the membranes (PPROM), increased oxidative stress may accelerate premature cellular senescence, senescence-associated inflammation and proteolysis, which may predispose them to rupture. Study finding We demonstrate mechanistic differences between preterm birth (PTB) and PPROM by revealing differences in fetal membrane redox status, oxidative stress-induced damage, distinct signaling pathways and senescence activation. What is known already Oxidative stress-associated fetal membrane damage and cell cycle arrest determine adverse pregnancy outcomes, such as spontaneous PTB and PPROM. Study design, samples/materials, methods Fetal membranes and amniotic fluid samples were collected from women with PTB and PPROM. Molecular, biochemical and histologic markers were used to document differences in oxidative stress and antioxidant enzyme status, DNA damage, secondary signaling activation by Ras-GTPase and mitogen-activated protein kinases, and activation of senescence between membranes from the two groups. Main results and the role of chance Oxidative stress was higher and antioxidant enzymes were lower in PPROM compared with PTB. PTB membranes had minimal DNA damage and showed activation of Ras-GTPase and ERK/JNK signaling pathway with minimal signs of senescence. PPROM had higher numbers of cells with DNA damage, prosenescence stress kinase (p38 MAPK) activation and signs of senescence. Limitations, reasons for caution Samples were obtained retrospectively after delivery. The markers of senescence that we tested are specific but are not sufficient to confirm senescence as the pathology in PPROM. Wider implications of the findings Oxidative stress-induced DNA damage and senescence are characteristics of fetal membranes from PPROM, compared with PTB with intact membranes. PTB and PPROM arise from distinct pathophysiologic pathways. Oxidative stress and oxidative stress-induced cellular damages are likely determinants of the mechanistic signaling pathways and phenotypic outcome. Study funding and competing interests This study is supported by developmental funds to Dr R. Menon from the Department of Obstetrics and Gynecology at The University of Texas Medical Branch at Galveston and funds to Dr M. Kacerovský from the Ministry of Health Czech Republic (UHHK, 001799906). The authors report no conflict of interest.

African Americans are at increased risk for spontaneous preterm birth (PTB). Though PTB is heritable, genetic studies have not identified variants that account for its intergenerational risk, prompting the hypothesis that epigenetic factors may also contribute. The objective of this study was to evaluate DNA methylation from maternal leukocytes to identify patterns specific to PTB and its intergenerational risk. DNA from peripheral leukocytes from African American women that delivered preterm (24–34 weeks; N = 16) or at term (39–41 weeks; N = 24) was assessed for DNA methylation using the HumanMethylation450 BeadChip. In maternal samples, 17,829 CpG sites associated with PTB, but no CpG site remained associated after correction for multiple comparisons. Examination of paired maternal-fetal samples identified 5,171 CpG sites in which methylation of maternal samples correlated with methylation of her respective fetus (FDR < 0.05). These correlated sites were enriched for association with PTB in maternal leukocytes. The majority of correlated CpG sites could be attributed to one or more genetic variants. They were also significantly more likely to be in genes involved in metabolic, cardiovascular, and immune pathways, suggesting a role for genetic and environmental contributions to PTB risk and chronic disease. The results of this study may provide insight into the factors underlying intergenerational risk for PTB and its consequences.

Polybrominated diphenyl ethers (PBDEs) are documented to increase the risk for spontaneous preterm birth (PTB). We hypothesize that PBDEs cause oxidative stress (OS) that leads to fetal cell senescence and inflammation associated with PTB.Primary amnion epithelial cells (n = 5) isolated from term, not in labor pregnancies, were exposed to PBDE congeners 47 and 99 (each 5 μM). ROS kinetics was monitored. Morphologic changes, phospho-p38 MAPK (P-p38) activation, development of senescence, and induction of uterotonins (COX-2 expression) were quantified using light microscopy, Western blot, senescence-associated β-galactosidase (SA β-gal) staining, and qRT-PCR, respectively, after 48 and 72 hr of exposure.Both PBDE congeners induced ROS within 2 min compared to controls (P0.05). P-p38 activation was significant after PBDE-99 treatment than controls (P0.05). After 72 hr of treatment, both PBDE-treated cells showed cell death-associated morphologic changes with significantly higher SA β-gal-stained cells than control. COX-2 expression was higher after 72 hr of treatment with PBDE-99. Overall, the PBDE-99 response was more pronounced than PBDE-47.Congener-dependent OS response, p38 MAPK activation, senescence, and COX-2 expression were seen in human amnion cells by PBDEs. These findings demonstrate environment pollutant-induced senescence activation and inflammation can lead to pathways resulting in PTB.

Spontaneous preterm birth significantly contributes to the overall neonatal morbidity associated with preterm deliveries. Nearly 50% of cases are associated with microbial invasion of the amniotic cavity followed by an inflammatory response. Robust diagnostic tools for neonates jeopardized by infection and inflammation may thus decrease the overall neonatal morbidity substantially. Amniotic fluid retrieved during labor retains fetal and pregnancy-related protein fingerprint and its sampling does not place any unwanted stress on women. Using exploratory and targeted methods we analyzed proteomes of amniotic fluid sampled at the end of spontaneous preterm labor prior to delivery from women with and without infection and inflammation. Exploratory data indicated several amniotic fluid proteins to be associated with infectious-inflammatory complications in spontaneous preterm birth. LC-SRM analysis subsequently verified statistically significant changes in lipocalin-1 (P=0.047and AUC = 0.67,P=0.046), glycodelin (P=0.013and AUC = 0.73,P=0.013), and nicotinamide phosphoribosyltransferase (P=0.018and AUC = 0.71,P=0.01).

Objective This study was undertaken to investigate the association of tumor necrosis factor-α (TNF-α) single nucleotide polymorphism (G-308>A) and risk of preterm birth by performing a systematic review and a meta-analysis of available studies. In addition, association between this variant and TNF-α concentration in amniotic fluid (AF) in preterm birth was also investigated. Study design Articles were chosen based on a Medline and EMBASE searches (1990-2005) with no language restrictions. An ongoing case-control study conducted in Nashville, TN, was also included. Articles evaluating the association between G-308>A and preterm birth were screened according to specific inclusion criteria. Meta-analysis was performed by using a random effect model. Association between maternal –308 genotype and AF-TNF-α concentration was determined by sandwich immunoassays. Results Titles and abstracts of 6851 citations identified through the search were screened. Including our own study, a total of 7 studies were included for meta-analysis. Only 2 reported a statistically significant increased risk based on –308 genotype. Meta-analysis of the case-control studies on a pooled dataset (a total of 1846 subjects, 638 cases, and 1208 controls) showed no significant association between the –308 genotype and the risk of preterm birth (odds ratio [OR] 1.41; CI 0.90-2.19). A nonsignificant increase of AF TNF-α concentration was demonstrated with the GG genotype in cases compared with the presence of allele A. Conclusion Meta-analysis of available evidence documented no statistically significant association between a single nucleotide polymorphism in the TNF-α gene (G-308>A) and preterm birth. Analyses of AF-TNF-α concentration demonstrated no increase in TNF-α in the presence of the minor allele (A).These results suggest that this single nucleotide polymorphism does not independently associate strongly with preterm birth.

Problem The prematurity rate is higher in African-Americans (AA) compared with Caucasians (C). As spontaneous preterm labor has been hypothesized to be a host inflammatory response disease racial differences in human placental membrane inflammatory cytokine and prostaglandin pathway gene expression patterns between AA and C were examined in this report. Method of study Placental membranes (amniochorion) collected from AA and C women from cesareans at term were maintained in an organ explant system and stimulated with endotoxin (lipopolysaccharide, LPS). Microarray analysis and enzyme-linked immunosorbent assay was performed on mRNAs and culture media from AA- and C-derived membranes to document any differences in mRNA expression and protein production of IL-1, IL-6, IL-8, IL-10 and expression of cyclooxygenase 1 (COX-1), COX-2 and 15-hydroxyprostaglandin dehydrogenase (PGDH). Results Increased mRNA expression of IL-1, IL-8 and COX-2 in AA and IL-6, IL-10, COX-1 and PGDH in C were documented after LPS stimulation. Concentration of IL-1 was significantly higher in media derived from AA whereas IL-6 and IL-10 concentrations were higher in C with no differences observed in IL-8 after LPS stimulation compared with respective unstimulated controls. Conclusion These data document ethnic diversity in placental membrane immune response.

Objectives: Spontaneous preterm birth (PTB) has a significant ethnic disparity with people of African descent having an almost 2-fold higher incidence than those of European descent in the United States. This disparity may be caused by differences in the distribution of genetic risk factors. The objective of this study is to examine genetic differences between African-Americans and European Americans for single nucleotide polymorphisms (SNPs) in candidate genes for PTB. Methods: We examined patterns of variation in 19 SNPs in 3 candidate genes for preterm birth: TNF-α, TNF-receptor 1 and TNF-receptor 2. Allele, genotype and haplotype frequencies were compared between African-Americans (AA) and European-Americans (EA) in cases and controls separately. Both maternal and fetal genotypes were studied, as it is unclear whether one or both of these are important in the etiology of PTB. Results: The vast majority of the SNPs differed significantly between ethnic groups, although there are only a few suggestive results comparing cases and controls within an ethnic group. For TNF-α, four of six SNPs; for TNF-R1, 5/6; and for TNF-R2, 6/7 showed significant differences between ethnic groups in either allele and/or genotype frequency. Conclusions: Our data demonstrate highly significant genetic differences between ethnic groups in genes that may play a role in the risk of PTB.

Objective. The objectives of this study were to document the expression of IL-19 and IL-20, localize their expression in human fetal membranes and to examine their influence on the production of other inflammatory cytokines (IL-1, IL-6, IL-8, and TNF-alpha) from placental membranes.Methods. Human fetal membranes collected at term from normal pregnancies were stimulated with either recombinant human IL-19, IL-20, bacterial endotoxin (LPS) alone or the cytokine + LPS. The expression of IL-19 and IL-20 was studied by reverse transcriptase polymerase chain reaction (RT-PCR) and localized using immunohistochemistry. Concentrations of IL-1, IL-6, IL-8, and TNF-alpha were measured with multiplex sandwich immunoassay using microsphere technology.Results. RT-PCR documented IL-19 and IL-20 gene expression in fetal membranes. Immunohistochemistry localized both peptides to amnion and chorion layers. LPS stimulated the production of all four cytokines (IL-1, IL-6, IL-8, and TNF-alpha) from fetal membranes compared to unstimulated controls. No change in IL-1 and IL-8 concentration was seen after IL-19 or IL-20 stimulation, whereas IL-6 concentration was three- and two-fold higher after IL-19 and IL-20 treatment, respectively. TNF levels were unchanged after IL-19 and IL-20 treatment; however, TNF levels were significantly decreased in membranes treated with IL-19 or IL-20 + LPS compared to LPS alone.Conclusion. Fetal membranes are a source of IL-19 and IL-20. These cytokines act as an inhibitory agent to LPS-induced TNF production whereas they stimulate IL-6 production and have no effect on IL-1 and IL-8 production from human fetal membranes. The effect of IL-19 and IL-20 in pregnancy will be dependent on their concentrations and other environmental factors such as infection.

Objective This study compares the immune responsiveness of amniochorionic membranes (AC) derived from African American (AA) and white (C) women to an infectious stimulus ex vivo. Study design AC derived from AA and C women were placed in an organ explant culture for 48 hours and then stimulated with endotoxin. Enzyme-linked immunosorbent assay measured the concentration of matrix metalloproteinase 9 (MMP9), tumor necrosis factor-α (TNF-α), and soluble TNF receptors (sTNFR1and sTNFR2) in culture media from stimulated and unstimulated AC. Results The C group produced 8-fold more TNF-α after stimulation than did the AA group. Both soluble receptor (R1 and R2) production increased in the C group and decreased in the AA group after stimulation. Although the C group–derived membranes produced more MMP9 at rest, a 6-fold increase in MMP9 concentration was seen in the AA group–derived membranes after stimulation. No change in MMP9 concentration was seen after stimulation of the C group–derived membranes. Conclusion Although the C group produced more TNF, they also produce higher sTNFRs, which may serve a protective role. The increased MMP9 release by the AA group may be suggestive of the greater risk of premature rupture of membranes in the AA group.

OBJECTIVE: To examine the expression, site of production and a role of collagenase-3 in human fetal membranes and amniotic fluid (AF). METHODS: Amniochorion collected at the time of elective repeat cesareans prior to labor from normal term gestation, were placed in an organ explant system for 72 hr. The AF was collected from the following groups of women: women at term; women at third trimester pregnancy: women at mid-trimester pregnancy. Women with premature rupture of the membranes (PROM) with or without pre-term labor and infection delivered by cesareans before term; women with pre-term labor and no rupture of membranes and delivered by cesareans before term; The mRNA expression of collagenase-3 was studied by reverse transcriptase–polymerase chain reaction (RT–PCR) and protein concentrations in the AF were assayed by enzyme-linked immunosorbent assay (ELISA). Site of collagenase-3 production was documented by immunohistochemistry. Statistical comparisons were made using ANOVA. All statistical hypotheses were adjusted for multiple comparisons using the Scheffe method of adjustment. RESULTS: Amniochorion at term expressed collagenase-3 mRNA. Immunohistochemistry localized collagenase-3 in both amnion and chorion. Collagenase-3 was seen in AF from all gestational age groups with lowest levels at mid-trimester [167.8 pg/mL] and highest during term labor (323.92 pg/mL) with no statistically significant difference between term and third trimester (310.11 pg/mL). AF levels of collagenase-3 were significantly higher in women with pre-term labor (370.02 pg/mL; Padj = 0.046) or pre-term labor and infection (628.73 pg/mL; Padj = 0.002) compared with PROM with pre-term labor and infection (87.19 pg/mL). CONCLUSION: Collagenase-3 mRNA is constitutively expressed and produced in amniochorion. It is a normal physiologic constituent of AF. PROM is not associated with an increase in the AF collagenase-3.

Objective Lysyl oxidase (LOX) and LOX like enzymes (LOXL1-4) physiologically remodel extracellular matrix and pathologically contribute to cellular senescence under oxidative stress (OS). We characterized LOX and LOXL expressions and activity in human fetal membranes. Methods Human fetal membranes from women with uncomplicated pregnancies at term, preterm birth with intact membranes (PTB) or preterm prelabor rupture of membranes (pPROM), and in vitro fetal membranes stimulated with water-soluble cigarette smoke extract (CSE), an OS inducer, were analyzed by real-time PCR and immunohistochemistry for LOX and LOXL (1-4) expression and localization. LOX activity was measured by fluorometric assay. Results LOX gene expression was ∼2.5-fold higher in fetal membranes from pPROM compared to PTB and term (P=0.02). LOX and LOXL1, 2 and 4 were localized to both amniotic and chorionic cells, whereas LOXL3 was limited to chorion. LOX and LOXL isoform expressions were not different between CSE treated and untreated groups, while LOX activity was increased in the presence of an antioxidant (P=0.02). Conclusions Increase of LOX expression in pPROM, an OS-related disease, and the apparent inhibition of LOX activity by CSE restored by antioxidant treatment suggest that reactive oxygen species might influence LOX-mediated tissue remodeling in fetal membranes. Balanced antioxidant supplementation during pregnancy may reduce the risk of pPROM by increasing LOX activity.

Objective To characterize subgroups of preterm prelabor rupture of membranes (PPROM) and short-term neonatal outcomes based on the presence and absence of intraamniotic inflammation (IAI) and/or microbial invasion of the amniotic cavity (MIAC). Methods One hundred and sixty-six Caucasian women with singleton pregnancies were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis (n=166) and were assayed for interleukin-6 levels by a lateral flow immunoassay. The presence of Ureaplasma species, Mycoplasma hominis, Chlamydia trachomatis, and 16S rRNA was evaluated in the amniotic fluid. IAI was defined as amniotic fluid IL-6 values, measured by a point of care test, higher than 745 pg/mL. Results Microbial-associated IAI (IAI with MIAC) and sterile intraamniotic inflammation (IAI alone) were found in 21% and 4%, respectively, of women with PPROM. Women with microbial-associated IAI had higher microbial loads of Ureaplasma species in the amniotic fluid than women with MIAC alone. No differences in the short-term neonatal morbidity with respect to the presence of microbial-associated IAI, sterile IAI and MIAC alone were found after adjusting for the gestational age at delivery in women with PPROM. Conclusions Microbial-associated but not sterile intraamniotic inflammation is common in Caucasian women with PPROM. The gestational age at delivery but not the presence of inflammation affects the short-term neonatal morbidity of newborns from PPROM pregnancies.

PROBLEM We investigated p38MAPK activation-induced fetal membrane cell senescence in response to inflammation (tumour necrosis factor-alpha [TNF-α]) and infection (lipopolysaccharide [LPS]), factors associated with spontaneous preterm birth. METHOD OF STUDY Primary amnion epithelial cells (AECs) were exposed to TNF-α, 50 ng/mL and LPS, 100 ng/mL. Cigarette smoke extract (CSE), a known OS inducer, was used as positive control. AECs were cotreated with the antioxidant N-acetyl cysteine (NAC) and p38MAPK inhibitor SB203580 to determine the effect of OS and p38MAPK. Western blot analysis was performed for active (Phospho-p38MAPK) and total p38MAPK. Senescence was determined by flow cytometry, and culture supernatants were tested for IL-6 using ELISA. RESULTS TNF-α, but not LPS, increased p38MAPK activation compared to untreated cells (P = .01). The number of senescent cells and senescence-associated IL-6 was higher in both TNF-α and LPS-treated cells compared to control (P = .001, P = .01, respectively). Antioxidant NAC inhibited p38MAPK activation by TNF-α. p38MAPK inhibitor SB203580 reduced the development of senescence and IL-6 by TNF-α and LPS. CSE treatment validated our current data. CONCLUSION TNF-α caused OS-mediated p38MAPK induction, senescence, and IL-6 increase from AECs. LPS also induced senescence and IL-6 increase. Inflammatory and infectious factors may cause premature fetal cell senescence contributing to preterm birth pathophysiology.

Exosomes (30- to 150-nm particles), originating from multivesicular bodies by the invagination of the endosomal membrane, are communication channels between cells. Exosomes are released by various cell types and cargo proteins, lipids, and nucleic acids reflecting the physiologic status of their cells of origin and cause functional changes in recipient cells, which are likely dependent on their quantity and/or cargo contents. Recently, placental exosomes, produced by various placental cell types, have been isolated from maternal blood using the placental protein-specific marker, placental alkaline phosphatase (PLAP). PLAP-positive exosomes are seen in maternal blood as early as the first trimester of pregnancy and increase as gestation progresses, with maximum numbers seen at term. Although the functional relevance of placental exosomes is still under investigation, several studies have linked placental exosomes changes (quantity and cargo) reflecting placental dysfunctions associated with adverse pregnancy events. As placental exosomes can be isolated from maternal blood, they are liquid biopsies reflecting placental functions. Hence, they are useful as biomarkers of placental functions and dysfunctions obtainable through non-invasive approaches. This review summarizes the biogenesis, release, and functions of exosomes and specifically expounds the role of placental-specific exosomes and their significance associated with pregnancy complications.

Objective : Endogenous activation of matrix metalloproteinase (MMP) in human fetal membranes is hypothesized to contribute to membrane weakening leading to early rupture and is also involved in the initiation of labor. Our laboratory and several others have studied the source and action of some of these MMPs. The objective of this study is to document the expression pattern of most of the MMPs cloned and sequenced so far in amniochorion during preterm premature rupture of membranes (pPROM), at term not in labor and during term labor. Materials and Methods : Placentas were collected from women with PROM, term not in labor after C-sections and from women after term vaginal delivery. Membranes were separated from the placenta and a section away from the rupture site was selected. Amniochorion were separated from the placenta. RT-PCR was performed to study the expression pattern of MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP18, MMP20, MMP23, MMP24 (MT5-MMP), MMP25 (MT6-MMP), and MMP 26 using specific primers. Results : A differential pattern of expression was noted for some of the novel MMPs screened in this study in human fetal membranes. mRNA for most of the MMPs were expressed by amniochorion. MMP16 [membrane type metalloproteinase 3], MMP20 [enamelysin], and MMP26 [matrilysin] were not expressed. Conclusion : Amniochorion expresses several of the MMP genes at the time of pPROM, term not in labor and during active labor. We have previously reported the expression pattern of other MMPs and their inhibitors and their potential role in PROM. These findings support our hypothesis that amniochorion has a fully functional MMP system.

Purpose: Cervical dilatation during parturition is associated with a significant increase of interleukin (IL)-6 and IL-8. The purpose of this study was to localize the respective messenger RNA and to quantitate their protein levels during various stages of dilatation. Methods: Protein extracts from 34 biopsy specimens of the lower uterine segment at various stages of cervical dilatation ( 6 cm) were subjected to enzyme linked immunosorbent assay for IL-6 and IL-8 to document their concentration. The respective mRNA expression was studied using reverse transcriptase PCR using IL-6 and IL-8 specific primers. Localization of IL-6 and IL-8 was done by in situ hybridization using biotinylated probes. Results: The IL-6 and IL-8 concentrations increased significantly in the lower uterine segment tissues at 4–6 cm of dilatation. Expression of IL-8 was seen in all the tissues at or more than 4 cm dilatation, whereas IL-6 expression was inconsistent. In situ hybridization localized IL-6 and IL-8 mRNA to the glandular epithelium. Conclusions: The increase in the inflammatory cytokines (IL-6 and IL-8) during parturition is associated with cervical effacement.

Purpose: Increased amniotic fluid tumor necrosis factor (TNF) is a marker of infection when associated with preterm labor and preterm premature rupture of the amniochorionic membranes (PROM). We have noted increased apoptosis in membranes derived from women with PROM. This study examines the role of TNF in promoting fetal membrane apoptosis. Methods: Amniochorion (n = 8), collected at the time of elective repeat cesarean section prior to labor from normal term gestation, were placed in an organ explant system. After 48 h in culture, the membranes were stimulated with recombinant TNF-α (20 ng/mL) for 24 h. Tissue frozen after stimulation was subjected to RT-PCR to study the expression of TNF-induced caspase genes. ELISA assayed the levels of proapoptotic p53 in tissues and cell death related nuclear matrix protein (NMP) in tissue culture supernatants. The activity of caspases in tissue homogenates was measured using substrates specific for caspase 2, 3, 6, 8, and 9. Results were analyzed by using the Wilcoxon nonparametric test for paired samples. A p < 0.05 was considered significant. Results: RT-PCR showed induction of caspases 2, 8, and 9 (caspase cascade initiators) in human fetal membranes after TNF stimulation. Caspases 3 and 6 (effector caspases) expression was constitutive in both TNF stimulated- and control membranes. Caspases, 2, 3, 8, and 9 activity was significantly higher in TNF-stimulated tissues compared with control, whereas, no significant change in caspase 6 activity was noticed. TNF-stimulated tissues released increased levels of NMP (24.03 U/mL) compared with control (13.5U/mL) (p = 0.03). TNF also increased p53 levels in the tissues (0.05 ng/mL) compared with control cultures (0.03 ng/mL; p = 0.02). Conclusions: TNF increases proapoptotic p53 levels and caspase activities in fetal membranes. Increased NMP reflects cell death.

NICHD Parturition at term is characterized by inflammatory overload in both feto-maternal tissues. Despite the large number of individual studies on changes in inflammatory biomarkers linked to labor, a comprehensive profile of them in each of the uterine compartments is not available to better understand their mechanistic contributions to labor. This systematic review investigated the pro- and anti-inflammatory biomarkers reported in intra-uterine tissues (amnion, chorion, decidua, placenta, and myometrium) at term labor. We conducted a systematic review of studies on pro- and anti-inflammatory biomarkers (mRNA and/or protein) reported in feto-maternal tissues during normal human term labor, published in English (1980-2016), in 3 electronic data bases. From a total of 3712 citations, 172 were included for final review. Each tissue expresses a unique set of biomarkers at the time of term labor, but there is significant overlap between tissues. All tissues had IL-6, IL-8, IL-1, COX-2, PGE-2, TNF-, and hCAP18 in common at term labor. Common and unique inflammatory biomarkers are expressed in various feto-maternal compartments at term labor. Increase in pro-inflammatory markers in all gestational tissue signifies their harmonious functional role in promoting labor. Anti-inflammatory markers at term labor are hardly reported. Univ Texas Med Branch, Dept Obstet & Gynecol, Div Maternal Fetal Med & Perinatal Res, Galveston, TX 77555 USA Univ Fed Sao Paulo, Dept Obstet & Gynecol, Sao Paulo, Brazil Univ Fed Sao Paulo, Dept Obstet & Gynecol, Sao Paulo, Brazil NICHD: 1R03HD086354-01 Web of Science