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The human plasma proteome is underexplored despite its potential value for monitoring health and disease. Herein, using a recently developed aptamer-based platform, we profiled 7288 proteins in 528 plasma samples from 91 normal pregnancies (Gene Expression Omnibus identifier GSE206454). The coefficient of variation was20% for 93% of analytes (median 7%), and a cross-platform correlation for selected key angiogenic and anti-angiogenic proteins was significant. Gestational age was associated with changes in 953 proteins, including highly modulated placenta- and decidua-specific proteins, and they were enriched in biological processes including regulation of growth, angiogenesis, immunity, and inflammation. The abundance of proteins corresponding to RNAs specific to populations of cells previously described by single-cell RNA-Seq analysis of the placenta was highly modulated throughout gestation. Furthermore, machine learning-based prediction of gestational age and of time from sampling to term delivery compared favorably with transcriptomic models (mean absolute error of 2 weeks). These results suggested that the plasma proteome may provide a non-invasive readout of placental cellular dynamics and serve as a blueprint for investigating obstetrical disease.

To comprehensively characterize monocyte and neutrophil responses to E. coli and its product [lipopolysaccharide (LPS) or endotoxin] in vitro during pregnancy.Peripheral blood was collected from pregnant women during the third trimester (n = 20) and from non-pregnant women (n = 20).The number, phagocytic activity, and reactive oxygen species (ROS) production of peripheral monocytes and neutrophils were investigated using flow cytometry. The phenotypes of peripheral monocytes and neutrophils after acute or chronic LPS stimulation were also determined using flow cytometry. Cytokine profiles were quantified for LPS-stimulated peripheral blood mononuclear cells (PBMCs) and a whole blood TruCultureIncreased number, phagocytic activity, and ROS production capacity of monocytes and neutrophils were found in pregnant compared to non-pregnant women. Additionally, specific subsets of pro-inflammatory monocytes (IL-6Pregnancy tailors the immune responses of circulating monocytes and neutrophils to endotoxin, a Gram-negative bacterial product.

Pregnant women are at increased risk of adverse outcomes, including preeclampsia and preterm birth, that may result from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Pregnancy imprints specific maternal immune responses that can modulate host susceptibility to microbial infection; therefore, recent studies have focused on the humoral response against SARS-CoV-2 in pregnant women. However, the pregnancy-specific cellular immune responses triggered by SARS-CoV-2 infection are poorly understood. In this study, we undertook an extensive in vitro investigation to determine the cellular immune responses to SARS-CoV-2 particles and proteins/peptides in pregnant women. First, we show that SARS-CoV-2 particles do not alter the pregnancy-specific oxidative burst of neutrophils and monocytes. Yet, SARS-CoV-2 particles/proteins shift monocyte activation from the classical to intermediate states in pregnant, but not in nonpregnant, women. Furthermore, SARS-CoV-2 proteins, but not particles or peptide pools, mildly enhance T cell activation during pregnancy. As expected, B cell phenotypes are heavily modulated by SARS-CoV-2 particles in all women; yet, pregnancy itself further modified such responses in these adaptive immune cells. Lastly, we report that pregnancy itself governs cytokine responses in the maternal circulation, of which IFN-β and IL-8 were diminished upon SARS-CoV-2 challenge. Collectively, these findings highlight the differential in vitro responses to SARS-CoV-2 in pregnant and nonpregnant women and shed light on the immune mechanisms implicated in coronavirus disease 2019 during pregnancy.

Pregnant women represent a high-risk population for severe/critical COVID-19 and mortality. However, the maternal-fetal immune responses initiated by SARS-CoV-2 infection, and whether this virus is detectable in the placenta, are still under investigation. Here we show that SARS-CoV-2 infection during pregnancy primarily induces unique inflammatory responses at the maternal-fetal interface, which are largely governed by maternal T cells and fetal stromal cells. SARS-CoV-2 infection during pregnancy is also associated with humoral and cellular immune responses in the maternal blood, as well as with a mild cytokine response in the neonatal circulation (i.e., umbilical cord blood), without compromising the T-cell repertoire or initiating IgM responses. Importantly, SARS-CoV-2 is not detected in the placental tissues, nor is the sterility of the placenta compromised by maternal viral infection. This study provides insight into the maternal-fetal immune responses triggered by SARS-CoV-2 and emphasizes the rarity of placental infection., As pregnant women are considered vulnerable to SARSCoV-2 infection, it is important to investigate the actual risks involved. The authors show here that, while a T cell-dominant inflammatory response is observed at the maternal-foetal interface, the virus remains undetectable in the placenta but triggers specific immune responses in the neonatal (umbilical cord blood) circulation.

Pregnancy represents a state of systemic immune activation that is primarily driven by alterations in circulating innate immune cells. Recent studies have suggested that cellular adaptive immune components, T cells and B cells, also undergo changes throughout gestation. However, the phenotypes and functions of such adaptive immune cells are poorly understood. Herein, we utilized high-dimensional flow cytometry and functional assays to characterize T-cell and B-cell responses in pregnant and non-pregnant women.Peripheral blood mononuclear cells from pregnant (n = 20) and non-pregnant (n = 25) women were used for phenotyping of T-cell and B-cell subsets. T-cell proliferation and B-cell activation were assessed by flow cytometry after in vitro stimulation, and lymphocyte cytotoxicity was evaluated by using a cell-based assay. Statistical comparisons were performed with linear mixed-effects models.Pregnancy was associated with modestly enhanced basal activation of peripheral CD4Maternal circulating T cells and B cells display distinct responses during pregnancy. The former may reflect the unique capacity of T cells to respond to potential threats without undergoing aberrant activation, thereby preventing systemic inflammatory responses that can lead to adverse perinatal consequences.

Preterm birth, the leading cause of perinatal morbidity and mortality, is associated with increased risk of short- and long-term adverse outcomes. For women identified as at risk for preterm birth attributable to a sonographic short cervix, the determination of imminent delivery is crucial for patient management. The current study aimed to identify amniotic fluid (AF) proteins that could predict imminent delivery in asymptomatic patients with a short cervix. This retrospective cohort study included women enrolled between May 2002 and September 2015 who were diagnosed with a sonographic short cervix ( 1.5 for each). The sensitivity at a 10% false-positive rate for the prediction of imminent delivery by a quantitative cervical length alone was 38%, yet it increased to 79% when combined with the abundance of four AF proteins (CXCL8, SNAP25, PTPN11, and MMP8). Neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity were biological processes impacted by protein dysregulation in women destined to deliver within two weeks of diagnosis. The combination of AF protein abundance and quantitative cervical length improves prediction of the timing of delivery compared to cervical length alone, among women with a sonographic short cervix.

The amniotic fluid (AF) cell-free RNA was shown to reflect physiological and pathological processes in pregnancy, but its value in the prediction of spontaneous preterm delivery is unknown. Herein we profiled cell-free RNA in AF samples collected from women who underwent transabdominal amniocentesis after an episode of spontaneous preterm labor and subsequently delivered within 24 h (n = 10) or later (n = 28) in gestation. Expression of known placental single-cell RNA-Seq signatures was quantified in AF cell-free RNA and compared between the groups. Random forest models were applied to predict time-to-delivery after amniocentesis. There were 2385 genes differentially expressed in AF samples of women who delivered within 24 h of amniocentesis compared to gestational age-matched samples from women who delivered after 24 h of amniocentesis. Genes with cell-free RNA changes were associated with immune and inflammatory processes related to the onset of labor, and the expression of placental single-cell RNA-Seq signatures of immune cells was increased with imminent delivery. AF transcriptomic prediction models captured these effects and predicted delivery within 24 h of amniocentesis (AUROC = 0.81). These results may inform the development of biomarkers for spontaneous preterm birth.

Objectives Spontaneous preterm labor is an obstetrical syndrome accounting for approximately 65–70% of preterm births, the latter being the most frequent cause of neonatal death and the second most frequent cause of death in children less than five years of age worldwide. The purpose of this study was to determine and compare to uncomplicated pregnancies (1) the frequency of placental disorders of villous maturation in spontaneous preterm labor; (2) the frequency of other placental morphologic characteristics associated with the preterm labor syndrome; and (3) the distribution of these lesions according to gestational age at delivery and their severity. Methods A case-control study of singleton pregnant women was conducted that included (1) uncomplicated pregnancies (controls, n=944) and (2) pregnancies with spontaneous preterm labor (cases, n=438). All placentas underwent histopathologic examination. Patients with chronic maternal diseases (e.g., chronic hypertension, diabetes mellitus, renal disease, thyroid disease, asthma, autoimmune disease, and coagulopathies), fetal malformations, chromosomal abnormalities, multifetal gestation, preeclampsia, eclampsia, preterm prelabor rupture of the fetal membranes, gestational hypertension, gestational diabetes mellitus, and HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome were excluded from the study. Results Compared to the controls, the most prevalent placental lesions among the cases were the disorders of villous maturation (31.8% [106/333] including delayed villous maturation 18.6% [62/333] vs. 1.4% [6/442], q Conclusions Disorders of villous maturation are present in nearly one-third of the cases of spontaneous preterm labor.

Intra-amniotic infection, the invasion of microbes into the amniotic cavity resulting in inflammation, is a clinical condition that can lead to adverse pregnancy outcomes for the mother and fetus as well as severe long-term neonatal morbidities. Despite much research focused on the consequences of intra-amniotic infection, there remains little knowledge about the innate immune cells that respond to invading microbes. We performed RNA-seq of sorted amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection to determine the transcriptomic differences between these innate immune cells. Further, we sought to identify specific transcriptomic pathways that were significantly altered by the maternal or fetal origin of amniotic fluid neutrophils and monocytes/macrophages, the presence of a severe fetal inflammatory response, and pregnancy outcome (i.e., preterm or term delivery). We show that significant transcriptomic differences exist between amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection, indicating the distinct roles these cells play. The transcriptome of amniotic fluid immune cells varies based on their maternal or fetal origin, and the significant transcriptomic differences between fetal and maternal monocytes/macrophages imply that those of fetal origin exhibit impaired functions. Notably, transcriptomic changes in amniotic fluid monocytes/macrophages suggest that these immune cells collaborate with neutrophils in the trafficking of fetal leukocytes throughout the umbilical cord (i.e., funisitis). Finally, amniotic fluid neutrophils and monocytes/macrophages from preterm deliveries display enhanced transcriptional activity compared to those from term deliveries, highlighting the protective role of these cells during this vulnerable period. Collectively, these findings demonstrate the underlying complexity of local innate immune responses in women with intra-amniotic infection and provide new insights into the functions of neutrophils and monocytes/macrophages in the amniotic cavity.

Parturition is a well-orchestrated process characterized by increased uterine contractility, cervical ripening, and activation of the chorioamniotic membranes; yet, the transition from a quiescent to a contractile myometrium heralds the onset of labor. However, the cellular underpinnings of human parturition in the uterine tissues are still poorly understood. Herein, we performed a comprehensive study of the human myometrium during spontaneous term labor using single-cell RNA sequencing (scRNA-Seq). First, we established a single-cell atlas of the human myometrium and unraveled the cell type-specific transcriptomic activity modulated during labor. Major cell types included distinct subsets of smooth muscle cells, monocytes/macrophages, stromal cells, and endothelial cells, all of which communicated and participated in immune (e.g., inflammation) and nonimmune (e.g., contraction) processes associated with labor. Furthermore, integrating scRNA-Seq and microarray data with deconvolution of bulk gene expression highlighted the contribution of smooth muscle cells to labor-associated contractility and inflammatory processes. Last, myometrium-derived single-cell signatures can be quantified in the maternal whole-blood transcriptome throughout pregnancy and are enriched in women in labor, providing a potential means of noninvasively monitoring pregnancy and its complications. Together, our findings provide insights into the contributions of specific myometrial cell types to the biological processes that take place during term parturition.

Objectives To determine whether placental vascular pathology and impaired placental exchange due to maturational defects are involved in the etiology of spontaneous preterm labor and delivery in cases without histologic acute chorioamnionitis. Methods This was a retrospective, observational study. Cases included pregnancies that resulted in spontaneous preterm labor and delivery ( Results There were 184 cases and 2471 controls, of which 95 and 1178 had Doppler studies, respectively. The frequency of lesions of maternal vascular malperfusion was greater in the placentas of patients with preterm labor than in the control group [14.1% (26/184) vs. 8.8% (217/2471) (p=0.023)]. Disorders of villous maturation were more frequent in the group with preterm labor than in the control group: 41.1% (39/95) [delayed villous maturation in 31.6% (30/95) vs. 2.5% (13/519) in controls and accelerated villous maturation in 9.5% (9/95) vs. none in controls]. Conclusions Maturational defects of placental villi were associated with approximately 41% of cases of unexplained spontaneous preterm labor and delivery without acute inflammatory lesions of the placenta and with delivery of appropriate-for-gestational-age fetuses.

The cell-free transcriptome in amniotic fluid (AF) has been shown to be informative of physiologic and pathologic processes in pregnancy; however, the change in AF proteome with gestational age has mostly been studied by targeted approaches. The objective of this study was to describe the gestational age-dependent changes in the AF proteome during normal pregnancy by using an omics platform. The abundance of 1310 proteins was measured on a high-throughput aptamer-based proteomics platform in AF samples collected from women during midtrimester (16–24 weeks of gestation, n = 15) and at term without labor (37–42 weeks of gestation, n = 13). Only pregnancies without obstetrical complications were included in the study. Almost 25% (320) of AF proteins significantly changed in abundance between the midtrimester and term gestation. Of these, 154 (48.1%) proteins increased, and 166 (51.9%) decreased in abundance at term compared to midtrimester. Tissue-specific signatures of the trachea, salivary glands, brain regions, and immune system were increased while those of the gestational tissues (uterus, placenta, and ovary), cardiac myocytes, and fetal liver were decreased at term compared to midtrimester. The changes in AF protein abundance were correlated with those previously reported in the cell-free AF transcriptome. Intersecting gestational age-modulated AF proteins and their corresponding mRNAs previously reported in the maternal blood identified neutrophil-related protein/mRNA pairs that were modulated in the same direction. The first study to utilize an aptamer-based assay to profile the AF proteome modulation with gestational age, it reveals that almost one-quarter of the proteins are modulated as gestation advances, which is more than twice the fraction of altered plasma proteins (~ 10%). The results reported herein have implications for future studies focused on discovering biomarkers to predict, monitor, and diagnose obstetrical diseases.

The complex physiologic process of parturition includes the onset of labor, which requires the orchestrated stimulation of a common pathway involving uterine contractility, cervical ripening, and chorioamniotic membrane activation. However, the labor-specific processes taking place in these tissues have limited use as predictive biomarkers unless they can be probed in non-invasive samples, such as the peripheral blood. Herein, we utilized a transcriptomic dataset to assess labor-specific changes in the peripheral blood of women who delivered at term. We identified a set of genes that were differentially expressed with labor and enriched for immunological processes, and these gene expression changes were strongly correlated with results from prior studies, providing in silico validation of our findings. We then identified significant correlations between labor-specific transcriptomic changes in the maternal circulation and those detected in the chorioamniotic membranes, myometrium, and cervix of women at term, demonstrating that tissue-specific labor signatures are partly mirrored in the peripheral blood. Finally, we demonstrated a significant overlap between the peripheral blood transcriptomic changes in term parturition and those observed in asymptomatic women, prior to the diagnosis of preterm prelabor rupture of the membranes, who ultimately delivered preterm. Collectively, we provide evidence that the normal process of labor at term is characterized by a unique immunological expression signature, which may serve as a useful tool for assessing labor status and for potentially identifying women at risk for preterm birth.

Summary Identification of pregnancies at risk of preterm birth (PTB), the leading cause of newborn deaths, remains challenging given the syndromic nature of the disease. We report a longitudinal multi-omics study coupled with a DREAM challenge to develop predictive models of PTB. The findings indicate that whole-blood gene expression predicts ultrasound-based gestational ages in normal and complicated pregnancies (r = 0.83) and, using data collected before 37 weeks of gestation, also predicts the delivery date in both normal pregnancies (r = 0.86) and those with spontaneous preterm birth (r = 0.75). Based on samples collected before 33 weeks in asymptomatic women, our analysis suggests that expression changes preceding preterm prelabor rupture of the membranes are consistent across time points and cohorts and involve leukocyte-mediated immunity. Models built from plasma proteomic data predict spontaneous preterm delivery with intact membranes with higher accuracy and earlier in pregnancy than transcriptomic models (AUROC = 0.76 versus AUROC = 0.6 at 27–33 weeks of gestation)., Graphical abstract, Highlights Blood gene expression predicts gestational age in normal and complicated pregnancies RNA changes preceding preterm prelabor rupture of the membranes are shared between cohorts Plasma proteomic profiles from asymptomatic women predict spontaneous preterm birth, Harnessing the wisdom of crowds in a DREAM Challenge, Tarca et al. developed methods to predict gestational age and preterm birth from longitudinal multi-omics data. The authors show that blood RNAs predict ultrasound-based gestational age, and they identify molecular changes preceding a diagnosis of spontaneous preterm birth in asymptomatic women.

Proteoglycan macromolecules play key roles in several physiological processes (e.g., adhesion, proliferation, migration, invasion, angiogenesis, and apoptosis), all of which are important for placentation and healthy pregnancy. However, their precise roles in human reproduction have not been clarified. To fill this gap, herein, we provide an overview of the proteoglycans’ expression and role in the placenta, in trophoblast development, and in pregnancy complications (pre-eclampsia, fetal growth restriction), highlighting one of the most important members of this family, syndecan-1 (SDC1). Microarray data analysis showed that of 34 placentally expressed proteoglycans, SDC1 production is markedly the highest in the placenta and that SDC1 is the most upregulated gene during trophoblast differentiation into the syncytiotrophoblast. Furthermore, placental transcriptomic data identified dysregulated proteoglycan genes in pre-eclampsia and in fetal growth restriction, including SDC1, which is supported by the lower concentration of syndecan-1 in maternal blood in these syndromes. Overall, our clinical and in vitro studies, data analyses, and literature search pointed out that proteoglycans, as important components of the placenta, may regulate various stages of placental development and participate in the maintenance of a healthy pregnancy. Moreover, syndecan-1 may serve as a useful marker of syncytialization and a prognostic marker of adverse pregnancy outcomes. Further studies are warranted to explore the role of proteoglycans in healthy and complicated pregnancies, which may help in diagnostic or therapeutic developments.

Objectives Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5–12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia. Methods This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration ≥2.6 ng/mL. Results (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40–58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases. Conclusions Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood.

BACKGROUND: The current approach to predict preeclampsia combines maternal risk factors and evidence from biophysical markers [mean arterial pressure (MAP), Doppler velocimetry of the uterine arteries (UtA-PI)], and maternal blood proteins (PlGF, sVEGFR-1, PAPP-A). Such models require the transformation of biomarker data into Multiples of the Mean (MoM) values by using population and site-specific models. Previous studies have focused on a narrow window in gestation, and have not included the maternal blood concentration of soluble endoglin (sEng), an important anti-angiogenic factor upregulated in preeclampsia. OBJECTIVES: 1) To develop models for calculation of MoM values for MAP and biochemical markers; 2) to build and assess the predictive models for preeclampsia based on maternal risk factors, the biophysical (MAP) and biochemical (PlGF, sVEGFR-1, and sEng) markers collected throughout pregnancy; and 3) to evaluate how prediction accuracy is affected by the presence of chronic hypertension and gestational age. STUDY DESIGN: This longitudinal case-cohort study included 1,150 pregnant women: women without preeclampsia with (n=49) and without chronic hypertension (n=871), and those who developed preeclampsia (n=166), or superimposed preeclampsia (n=64). MAP and immunoassay-based maternal plasma PlGF, sVEGFR-1 and sEng concentrations were available throughout pregnancy (median of 5 observations per patient). A prior-risk model for preeclampsia was established by using Poisson regression based on maternal characteristics and obstetrical history. Next, multiple regression was used to fit biophysical and biochemical marker data as a function of maternal characteristics by using data collected at 8–15(+6), 16–19(+6), 20–23(+6), 24–27(+6), 28–31(+6) and 32–36(+6) week intervals, and observed values were converted into MoM values. Then, multivariable prediction models for preeclampsia were fit based on the biomarker MoM data and prior-risk estimates. Separate models were derived for overall, preterm, and term preeclampsia, which were evaluated by receiver operating characteristic curves and sensitivity at fixed false-positive rates (FPR). RESULTS: 1) Inclusion of sEng in prediction models for all preeclampsia, together with the prior-risk estimates, MAP, PlGF, and sVEGFR1, increased the sensitivity (at a fixed FPR of 10%) for early prediction of superimposed preeclampsia, with the largest increase (from 44% to 54%) being noted at 20–23(+6) weeks (McNemar test, p

Background The amniotic fluid (AF) cell-free transcriptome is modulated by physiologic and pathologic processes during pregnancy. AF gene expression changes with advancing gestation reflect fetal development and organ maturation; yet, defining normal expression and splicing patterns for biomarker discovery in obstetrics requires larger heterogeneous cohorts, evaluation of potential confounding factors, and novel analytical approaches. Methods Women with a normal pregnancy who had an AF sample collected during midtrimester (n = 30) or at term gestation (n = 68) were included. Expression profiling at exon level resolution was performed using Human Transcriptome Arrays. Differential expression was based on moderated t-test adjusted p 1.25; for differential splicing, a splicing index > 2 and adjusted p In-silico validation studies were performed using publicly available datasets. Results 1) 64,071 genes were detected in AF, with 11% of the coding and 6% of the non-coding genes being differentially expressed between midtrimester and term gestation. Expression changes were highly correlated with those previously reported (R > 0.79, p p Conclusions This represents the largest AF transcriptomics study in normal pregnancy, reporting for the first time that single-cell genomic signatures can be tracked in the AF and display complex patterns of expression during gestation. We also demonstrate a role for alternative splicing in tissue-identity acquisition, organ development, and immune processes. The results herein may have implications for the development of fetal testing to assess placental function and fetal organ maturity.

Background Normal development of the human placenta, referred to as villous tree maturation, entails formation of the vasculosyncytial membranes. These structures develop by the approximation of syncytiotrophoblasts with the villous capillary endothelium and constitute the most efficient sites of gaseous exchange in the placenta. Defective maturation of the villous tree can lead to deficient vasculosyncytial membranes, implicated in the high incidence of hypoxic complications. Hypoxia, in turn, can stimulate production of erythropoietin, whereby increased fetal plasma or amniotic fluid concentrations of this hormone reflect fetal hypoxemia. The current study was undertaken to determine whether delayed villous maturation is associated with changes in amniotic fluid erythropoietin concentrations. Methods Placental histologic examination was performed using hematoxylin and eosin. Subsequent to histologic assessment of delayed villous maturation, the diagnosis was confirmed with CD-15 immunohistochemistry. The controls (n = 61) were pregnancies without villous maturation abnormalities, and cases (n = 5) were pregnancies with delayed villous maturation. Amniotic fluid erythropoietin concentrations were measured using a specific immunoassay. Results Concentrations of erythropoietin in the amniotic fluid (1) of controls were less than the limit of detection and (2) of cases with delayed villous maturation were significantly higher than those of controls (P-value = 0.048). Conclusion Delayed villous maturation is associated with higher concentrations of amniotic fluid erythropoietin.

ObjectiveAmniotic fluid cytokines have been implicated in the mechanisms of preterm labor and birth. Cytokines can be packaged within or on the surface of extracellular vesicles. The main aim of this study was to test whether the protein abundance internal to and on the surface of extracellular vesicles changes in the presence of sterile intra-amniotic inflammation and proven intra-amniotic infection in women with preterm labor as compared to the women with preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection.Study designWomen who had an episode of preterm labor and underwent an amniocentesis for the diagnosis of intra-amniotic infection or intra-amniotic inflammation were classified into three groups: 1) preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection, 2) preterm labor with sterile intra-amniotic inflammation, and 3) preterm labor with intra-amniotic infection. The concentrations of 38 proteins were determined on the extracellular vesicle surface, within the vesicles, and in the soluble fraction of amniotic fluid.Results1) Intra-amniotic inflammation, regardless of detected microbes, was associated with an increased abundance of amniotic fluid cytokines on the extracellular vesicle surface, within vesicles, and in the soluble fraction. These changes were most prominent in women with proven intra-amniotic infection. 2) Cytokine changes on the surface of extracellular vesicles were correlated with those determined in the soluble fraction; yet the magnitude of the increase was significantly different between these compartments. 3) The performance of prediction models of early preterm delivery based on measurements on the extracellular vesicle surface was equivalent to those based on the soluble fraction.ConclusionsDifferential packaging of amniotic fluid cytokines in extracellular vesicles during preterm labor with sterile intra-amniotic inflammation or proven intra-amniotic infection is reported herein for the first time. The current study provides insights into the biology of the intra-amniotic fluid ad may aid in the development of biomarkers for obstetrical disease.

Pregnancy represents a unique immunological state in which the mother adapts to tolerate the semi-allogenic conceptus; yet, the cellular dynamics in the maternal circulation are poorly understood. Using exon-level expression profiling of up to six longitudinal whole blood samples from 49 pregnant women, we undertook a systems biology analysis of the cellular transcriptome dynamics and its correlation with the plasma proteome. We found that: (1) chromosome 14 was the most enriched in transcripts differentially expressed throughout normal pregnancy; (2) the strongest expression changes followed three distinct longitudinal patterns, with genes related to host immune response (e.g., MMP8, DEFA1B, DEFA4, and LTF) showing a steady increase in expression from 10 to 40 weeks of gestation; (3) multiple biological processes and pathways related to immunity and inflammation were modulated during gestation; (4) genes changing with gestation were among those specific to T cells, B cells, CD71+ erythroid cells, natural killer cells, and endothelial cells, as defined based on the GNF Gene Expression Atlas; (5) the average expression of mRNA signatures of T cells, B cells, and erythroid cells followed unique patterns during gestation; (6) the correlation between mRNA and protein abundance was higher for mRNAs that were differentially expressed throughout gestation than for those that were not, and significant mRNA-protein correlations were observed for genes part of the T-cell signature. In summary, unique changes in immune-related genes were discovered by longitudinally assessing the cellular transcriptome in the maternal circulation throughout normal pregnancy, and positive correlations were noted between the cellular transcriptome and plasma proteome for specific genes/proteins. These findings provide insights into the immunobiology of normal pregnancy.

Objectives To investigate mechanisms of in utero death in normally formed fetuses by measuring amniotic fluid (AF) biomarkers for hypoxia (erythropoietin [EPO]), myocardial damage (cardiac troponin I [cTnI]) and brain injury (glial fibrillary acidic protein [GFAP]), correlated with risk factors for fetal death and placental histopathology. Methods This retrospective, observational cohort study included intrauterine deaths with transabdominal amniocentesis prior to induction of labor. Women with a normal pregnancy and an indicated amniocentesis at term were randomly selected as controls. AF was assayed for EPO, cTnI and GFAP using commercial immunoassays. Placental histopathology was reviewed, and CD15-immunohistochemistry was used. Analyte concentrations >90th centile for controls were considered “raised”. Raised AF EPO, AF cTnI and AF GFAP concentrations were considered evidence of hypoxia, myocardial and brain injury, respectively. Results There were 60 cases and 60 controls. Hypoxia was present in 88% (53/60), myocardial damage in 70% (42/60) and brain injury in 45% (27/60) of fetal deaths. Hypoxic fetuses had evidence of myocardial injury, brain injury or both in 77% (41/53), 49% (26/53) and 13% (7/53) of cases, respectively. Histopathological evidence for placental dysfunction was found in 74% (43/58) of these cases. Conclusion Hypoxia, secondary to placental dysfunction, was found to be the mechanism of death in the majority of fetal deaths among structurally normal fetuses. Ninety-one percent of hypoxic fetal deaths sustained brain, myocardial or both brain and myocardial injuries in utero. Hypoxic myocardial injury was an attributable mechanism of death in 70% of the cases. Non-hypoxic cases may be caused by cardiac arrhythmia secondary to a cardiac conduction defect.

Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.

Objective: To determine whether a soft cervix identified by shear-wave elastography between 18 and 24 weeks of gestation is associated with increased frequency of spontaneous preterm delivery (sPTD). Materials and methods: This prospective cohort study included 628 consecutive women with a singleton pregnancy. Cervical length (mm) and softness [shear-wave speed: (SWS) meters per second (m/s)] of the internal cervical os were measured at 18–24 weeks of gestation. Frequency of sPTD th percentile). Results: There were 31/628 (4.9%) sPTD Conclusions: A soft cervix at 18–24 weeks of gestation increases the risk of sPTD

ObjectiveTo determine the frequency and type of histopathologic lesions in placentas delivered by women with a normal pregnancy outcome.MethodsThis retrospective cohort study included placental samples from 944 women with a singleton gestation who delivered at term without obstetrical complications. Placental lesions were classified into the following four categories as defined by the Society for Pediatric Pathology and by our unit: (1) acute placental inflammation, (2) chronic placental inflammation, (3) maternal vascular malperfusion and (4) fetal vascular malperfusion.Results(1) Seventy-eight percent of the placentas had lesions consistent with inflammatory or vascular lesions; (2) acute inflammatory lesions were the most prevalent, observed in 42.3% of the placentas, but only 1.0% of the lesions were severe; (3) acute inflammatory lesions were more common in the placentas of women with labor than in those without labor; (4) chronic inflammatory lesions of the placenta were present in 29.9%; and (5) maternal and fetal vascular lesions of malperfusion were detected in 35.7% and 19.7%, respectively. Two or more lesions with maternal or fetal vascular features consistent with malperfusion (high-burden lesions) were present in 7.4% and 0.7%, respectively.ConclusionMost placentas had lesions consistent with inflammatory or vascular lesions, but severe and/or high-burden lesions were infrequent. Mild placental lesions may be interpreted either as acute changes associated with parturition or as representative of a subclinical pathological process (intra-amniotic infection or sterile intra-amniotic inflammation) that did not affect the clinical course of pregnancy.

OBJECTIVE: The objective of this study was to determine the profiles of maternal plasma soluble adhesion molecules in patients with preeclampsia, small-for-gestational-age (SGA) fetuses, acute pyelonephritis, preterm labor with intact membranes (PTL), preterm prelabor rupture of the membranes (preterm PROM), and fetal death. MATERIALS AND METHODS: A cross-sectional study was conducted to determine maternal plasma concentrations of sE-selectin, sL-selectin, and sP-selectin as well as sICAM-1, sVCAM-1, and sPECAM-1 in patients with 1) an uncomplicated pregnancy (control, n=100); 2) preeclampsia (n=94); 3) SGA fetuses (in women without preeclampsia/hypertension, n=45); 4) acute pyelonephritis (n=25); 5) PTL (n=53); 6) preterm PROM (n=24); and 7) fetal death (n=34). Concentrations of soluble adhesion molecules and inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-8) were determined with sensitive and specific enzyme-linked immunoassays. RESULTS: In comparison to women with a normal pregnancy, 1) women with preeclampsia had higher median concentrations of sE-selectin, sP-selectin, and sVCAM-1, and a lower concentration of sL-selectin (all p-values

Preterm preeclampsia is associated with the failure of trophoblast invasion, placental hypoxic/ischemic injury and the release of toxic substances, which promote the terminal pathway of preeclampsia. In term preeclampsia, factors yet unknown trigger the placenta to induce the terminal pathway. The contribution of the villous trophoblast to these pathologic events has not been fully elucidated. Here we aimed to study how stress and signaling pathways influence trophoblastic functions in various subforms of preeclampsia. Tissue microarrays (TMAs) were constructed from placentas obtained from pregnant women in the following groups: 1-2) preterm preeclampsia with (n = 8) or without (n = 7) HELLP syndrome; 3) late-onset preeclampsia (n = 8); 4-5) preterm (n = 5) and term (n = 9) controls. TMA slides were stained for phosphorylated Akt-1, ERK1/2, JNK, and p38 kinases, and trophoblastic immunostainings were semi-quantitatively evaluated. BeWo cells were kept in various stress conditions, and the expression of FLT1, GCM1, LEP, and PGF was profiled by qRT-PCR, while Akt-1, ERK1/2, JNK, and p38 kinase activities were measured with phospho-kinase immunoassays. We found that: 1) Placental LEP and FLT1 expression was up-regulated in preterm preeclampsia with or without HELLP syndrome compared to controls; 2) Mean pp38 immunoscore was higher in preterm preeclampsia, especially in cases with HELLP syndrome, than in controls. 3) Mean pERK1/2 immunoscore was higher in preterm preeclampsia with HELLP syndrome than in controls. 4) In BeWo cells, ischemia up-regulated LEP expression, and it increased JNK and decreased ERK1/2 activity. 5) Hypoxia up-regulated FLT1 and down-regulated PGF expression, and it increased ERK1/2, JNK and p38 activity. 6) IL-1β treatment down-regulated PGF expression, and it increased JNK and p38 activity. 7) The p38 signaling pathway had the most impact on LEP, FLT1 and PGF expression. In conclusion, hypoxic and ischemic stress, along with unknown factors, activates trophoblastic p38 signaling, which has a key role in villous trophoblastic functional changes in preterm preeclampsia. The activation of ERK1/2 signaling may induce additional trophoblastic functional changes in HELLP syndrome, while distinct mechanisms may promote late-onset preeclampsia.

The dysregulation of maternal-fetal immune tolerance is one of the proposed mechanisms leading to preeclampsia. Galectins are key regulator proteins of the immune response in vertebrates and maternal-fetal immune tolerance in eutherian mammals. Previously we found that three genes in a Chr19 cluster encoding for human placental galectin-13 (PP13), galectin-14 and galectin-16 emerged during primate evolution and may confer immune tolerance to the semi-allogeneic fetus.This study involved various methodologies for gene and protein expression profiling, genomic DNA methylation analyses, functional assays on differentiating trophoblasts including gene silencing, luciferase reporter and methylation assays. These methods were applied on placental specimens, umbilical cord blood cells, primary trophoblasts and BeWo cells. Genomic DNA sequences were analyzed for transposable elements, transcription factor binding sites and evolutionary conservation.The villous trophoblastic expression of Chr19 cluster galectin genes is developmentally regulated by DNA methylation and induced by key transcription factors of villous placental development during trophoblast fusion and differentiation. This latter mechanism arose via the co-option of binding sites for these transcription factors through promoter evolution and the insertion of an anthropoid-specific L1PREC2 transposable element into the 5' untranslated region of an ancestral gene followed by gene duplication events. Among placental Chr19 cluster galectin genes, the expression of LGALS13 and LGALS14 is down-regulated in preterm severe preeclampsia associated with SGA. We reveal that this phenomenon is partly originated from the dysregulated expression of key transcription factors controlling trophoblastic functions and galectin gene expression. In addition, the differential DNA methylation of these genes was also observed in preterm preeclampsia irrespective of SGA.These findings reveal the evolutionary origins of the placental expression of Chr19 cluster galectins. The complex dysregulation of these genes in preeclampsia may alter immune tolerance mechanisms at the maternal-fetal interface.

Among patients presenting with preterm labor and intact membranes, those with intra-amniotic inflammation have adverse obstetrical and neonatal outcomes. The diagnosis of intra-amniotic inflammation can easily be made by detecting an elevated concentration of the cytokine interleukin (IL)-6 or the enzyme neutrophil collagenase, also known as matrix metalloproteinase (MMP)-8. The diagnostic performances of MMP-8 and IL-6 enzyme-linked immunosorbent assay tests are similar. Recently, a rapid test has become available for point-of-care determination of either MMP-8 or IL-6. The objectives of this study were to compare the diagnostic indices and predictive values between the rapid MMP-8 and IL-6 tests for the identification of intra-amniotic inflammation in patients with preterm labor and intact membranes.We performed a retrospective cohort study including 124 women with singleton pregnancies who presented with symptoms of preterm labor and underwent transabdominal amniocentesis for the evaluation of microbial invasion of the amniotic cavity (MIAC). MIAC was defined according to amniotic fluid culture results (aerobic and anaerobic bacteria as well as genital Mycoplasmas). Amniotic fluid white blood cell (WBC) counts were determined using a hemocytometer chamber. An elevated amniotic fluid MMP-8 concentration was assessed using Yoon's MMP-8 Check(1) The rapid tests had the same sensitivity for the detection of intra-amniotic inflammation [85.7% (18/21) for all]; (2) the specificity of the rapid MMP-8 test was higher than that of the rapid IL-6 test (cutoff: 745 pg/mL) for the identification of intra-amniotic inflammation [72.8% (75/103) vs. 64.1% (66/103); p 0.05]; and (3) there were no differences in the sensitivity and specificity between the rapid MMP-8 test and the rapid IL-6 test (cutoff:1000 pg/mL) in the identification of intra-amniotic inflammation. Of 13 patients with discrepant results between the rapid MMP-8 and rapid IL-6 tests, two had a positive MMP-8 but a negative rapid IL-6 test, and both delivered preterm - one within 24 h, and the other within 10 days - and both had acute histologic chorioamnionitis. On the other hand, there were 11 patients with a positive rapid IL-6 but a negative rapid MMP-8 result: 10 delivered preterm, 3 had acute histologic chorioamnionitis and 1 had subacute chorionitis.We conclude that the rapid MMP-8 test has a better specificity than the rapid IL-6 (cutoff: 745 pg/mL) assay for the detection of intra-amniotic infection. Moreover, we observed that among patients who were not identified as having intra-amniotic infection or inflammation by the standard cultivation technique and amniotic fluid WBC count, those who had a positive MMP-8 rapid test delivered preterm and had acute histologic chorioamnionitis.

Objective Clinical chorioamnionitis is the most common infection/inflammatory process diagnosed in labor and delivery units worldwide. The condition is a syndrome that can be caused by (1) intra-amniotic infection, (2) intra-amniotic inflammation without demonstrable microorganisms (i.e. sterile intra-amniotic inflammation), and (3) maternal systemic inflammation that is not associated with intra-amniotic inflammation. The presence of intra-amniotic inflammation is a risk factor for adverse maternal and neonatal outcomes in a broad range of obstetrical syndromes that includes clinical chorioamnionitis at term. Although the diagnosis of intra-amniotic infection has relied on culture results, such information is not immediately available for patient management. Therefore, the diagnosis of intra-amniotic inflammation could be helpful as a proxy for intra-amniotic infection, while results of microbiologic studies are pending. A rapid test is now available for the diagnosis of intra-amniotic inflammation, based on the determination of neutrophil collagenase or matrix metalloproteinase-8 (MMP-8). The objectives of this study were (1) to evaluate the diagnostic indices of a rapid MMP-8 test for the identification of intra-amniotic inflammation/infection in patients with the diagnosis of clinical chorioamnionitis at term, and (2) to compare the diagnostic performance of a rapid MMP-8 test to that of a conventional enzyme-linked immunosorbent assay (ELISA) interleukin (IL)-6 test for patients with clinical chorioamnionitis at term. Materials and methods A retrospective cohort study was conducted. A transabdominal amniocentesis was performed in patients with clinical chorioamnionitis at term (n=44). Amniotic fluid was analyzed using cultivation techniques (for aerobic and anaerobic bacteria as well as genital Mycoplasmas) and broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Amniotic fluid IL-6 concentrations were determined by ELISA, and rapid MMP-8 results were determined by Yoon's MMP-8 Check®. Intra-amniotic inflammation was defined as an elevated amniotic fluid IL-6 concentration ≥2.6 ng/mL, and intra-amniotic infection was diagnosed by the presence of microorganisms in the amniotic fluid accompanied by intra-amniotic inflammation. The diagnostic indices of Yoon's MMP-8 Check® for the identification of intra-amniotic inflammation were calculated. In order to objectively compare Yoon's MMP-8 Check® with the ELISA IL-6 test for the identification of intra-amniotic inflammation, we used an amniotic fluid white blood cell (WBC) count ≥50 cells/mm3 to define intra-amniotic inflammation. Results (1) A positive rapid MMP-8 test had a sensitivity of 82.4% (28/34), specificity of 90% (9/10), positive predictive value of 96.6% (28/29), negative predictive value of 60% (9/15), positive likelihood ratio 8.2 (95% CI 1.3-53.2), and negative likelihood ratio 0.2 (95% CI 0.1-0.4) for the identification of intra-amniotic inflammation (prevalence 77.3%); (2) a positive rapid MMP-8 test had a sensitivity of 91.7% (22/24), specificity of 65% (13/20), positive predictive value of 75.9% (22/29), negative predictive value of 86.7% (13/15), positive likelihood ratio of 2.6 (95% CI 1.4-4.8), and negative likelihood ratio of 0.1 (95% CI 0.03-0.5) for the identification of intra-amniotic infection; (3) the rapid MMP-8 test had a significantly higher specificity than the ELISA IL-6 test in the identification of intra-amniotic inflammation as determined by an amniotic fluid WBC count ≥50 cells/mm3. The sensitivity and accuracy of the rapid MMP-8 test were comparable to those of the ELISA IL-6 test; and (4) importantly, the rapid MMP-8 test had 100% sensitivity and 100% negative predictive value in the identification of neonates affected with fetal inflammatory response syndrome (FIRS). Conclusion The rapid diagnosis of intra-amniotic inflammation is possible by analysis of amniotic fluid using a point-of-care test for MMP-8. Patients with a positive test are at risk of delivering a neonate affected with systemic inflammation, a risk factor for adverse neonatal outcome.

Objective: Human pregnancy is characterized by activation of the innate immune response and suppression of adaptive immunity. The former is thought to provide protection against infection for the mother, and the latter, tolerance against paternal antigens expressed in fetal cells. Acute pyelonephritis is associated with an increased risk of acute respiratory distress syndrome and sepsis in pregnant (vs. nonpregnant) women. The objective of this study was to describe the gene expression profile (transcriptome) of maternal whole blood in acute pyelonephritis. Method: A case-control study was conducted to include pregnant women with acute pyelonephritis (n=15) and women with a normal pregnancy (n=34). Affymetrix HG-U133 Plus 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were used for gene expression profiling. A linear model was used to test the association between the presence of pyelonephritis and gene expression levels while controlling for white blood cell count and gestational age. A fold change of 1.5 was considered significant at a false discovery rate of 0.1. A subset of differentially expressed genes (n=56) was tested with real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (cases, n=19; controls, n=59). Gene ontology and pathway analyses were applied. Results: A total of 983 genes were differentially expressed in acute pyelonephritis: 457 were upregulated and 526 were downregulated. Significant enrichment of 300 biological processes and 63 molecular functions was found in pyelonephritis. Significantly impacted pathways in pyelonephritis included (a) cytokine-cytokine receptor interaction, (b) T-cell receptor signaling, (c) Jak-STAT signaling, and (d) complement and coagulation cascades. Of 56 genes tested by qRT-PCR, 48 (85.7%) had confirmation of differential expression. Conclusion: This is the first study of the transcriptomic signature of whole blood in pregnant women with acute pyelonephritis. Acute infection during pregnancy is associated with the increased expression of genes involved in innate immunity and the decreased expression of genes involved in lymphocyte function.

Intra-amniotic infection/inflammation (IAI) is causally linked with spontaneous preterm labor and delivery. The ST2L receptor and its soluble form (sST2) are capable of binding to interleukin (IL)-33, a member of the IL-1 superfamily. Members of this cytokine family have been implicated in the onset of spontaneous preterm labor in the context of infection. Soluble ST2 has anti-inflammatory properties, and plasma concentrations are elevated in systemic inflammation, such as sepsis, acute pyelonephritis in pregnancy and the fetal inflammatory response syndrome. The aims of this study were to examine: (1) whether amniotic fluid concentrations of sST2 change with IAI, preterm, and term parturition; and (2) if mRNA expression of ST2 in the chorioamniotic membranes changes with acute histologic chorioamnionitis in women who deliver preterm.A cross-sectional study was conducted to determine amniotic fluid concentrations of sST2 in: (1) women with preterm labor (PTL) who delivered at term (n=49); (2) women with PTL who delivered preterm without IAI (n=21); (3) women with PTL who delivered preterm with IAI (n=31); (4) term pregnancies not in labor (n=13); and (5) term pregnancies in labor (n=43). The amniotic fluid concentration of sST2 was determined by ELISA. The mRNA expression of ST2 in the chorioamniotic membranes of women who delivered preterm with (n=24), and without acute histologic chorioamnionitis (n=19) was determined by qRT-PCR.(1) Patients with PTL who delivered preterm with IAI had a lower median amniotic fluid concentration of sST2 compared to those with PTL who delivered preterm without IAI [median 410 ng/mL, inter-quartile range (IQR) 152-699 ng/mL versus median 825 ng/mL, IQR 493-1216 ng/mL; p=0.0003] and those with PTL who delivered at term [median 410 ng/mL, IQR 152-699 ng/mL versus median 673 ng/mL, IQR 468-1045 ng/mL; p=0.0003]; (2) no significant differences in the median amniotic fluid concentration of sST2 were observed between patients with PTL who delivered at term and those who delivered preterm without IAI (p=0.4), and between women at term in labor and those at term not in labor (p=0.9); (3) the mean mRNA expression of ST2 was 4-fold lower in women who delivered preterm with acute histologic chorioamnionitis than in those without this lesion (p=0.008).The median sST2 amniotic fluid concentration and mRNA expression of ST2 by chorioamniotic membranes is lower in PTL associated with IAI and acute histologic chorioamnionitis than in PTL without these conditions. Changes in the median amniotic fluid sST2 concentration are not observed in preterm and term parturition without IAI. Thus, amniotic fluid sST2 in the presence of IAI behaves differently when compared to sST2 in the plasma of individuals affected by fetal inflammatory response syndrome, acute pyelonephritis in pregnancy, and adult sepsis. Decreased concentrations of sST2 in IAI are likely to promote a pro-inflammatory response, which is important for parturition in the context of infection.

Preeclampsia is characterized by maternal systemic anti-angiogenic and pro-inflammatory states. Syndecan-1 is a cell surface proteoglycan expressed by the syncytiotrophoblast, which plays an important role in angiogenesis and resolution of inflammation. Our aim was to examine placental syndecan-1 expression in preeclampsia with or without hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome. Placentas were obtained from women in the following groups: (1) late-onset preeclampsia (n = 8); (2) early-onset preeclampsia without (n = 7) and (3) with HELLP syndrome (n = 8); (4) preterm controls (n = 5); and (5) term controls (n = 9). Tissue microarrays (TMAs) were constructed from paraffin-embedded placentas. TMA slides were immunostained for syndecan-1 and evaluated using microscopy, virtual microscopy, and semi-automated image analysis. Maternal sera from patients with preeclampsia (n = 49) and controls (n = 32) were immunoassayed for syndecan-1. BeWo cells were treated with Forskolin or Latrunculin B or kept in ischemic conditions. SDC1 expression and syndecan-1 production were investigated with qRT-PCR, confocal microscopy, and immunoassays. Syndecan-1 was localized to the syncytiotrophoblast apical membrane in normal placentas. Syndecan-1 immunoscores were higher in late-onset preeclampsia (p = 0.0001) and early-onset preeclampsia with or without HELLP syndrome (p = 0.02 for both) than in controls. Maternal serum syndecan-1 concentration was lower in preeclampsia (median, 673 ng/ml; interquartile range, 459-1,161 ng/ml) than in controls (1,158 ng/ml; 622-1,480 ng/ml). SDC1 expression and syndecan-1 immunostainings in BeWo cells and syndecan-1 concentrations in supernatants increased during cell differentiation. Disruption of the actin cytoskeleton with Latrunculin B decreased syndecan-1 release, while ischemic conditions increased it. Syncytiotrophoblastic syndecan-1 expression depends on the differentiation of villous trophoblasts, and trophoblastic syndecan-1 release is decreased in preeclampsia and HELLP syndrome. This phenomenon may be related to the disturbed syncytiotrophoblastic cortical actin cytoskeleton and associated with maternal anti-angiogenic and pro-inflammatory states in these syndromes.

Decidual macrophages (dMφ) of the mother and placental macrophages (Hofbauer cells, HC) of the fetus are deployed at a critical location: the feto-maternal interface. This study was conducted to compare the DNA methylome of maternal and fetal monocytes, dMφ, and HC and thereby to determine the immunobiological importance of DNA methylation in pregnancy.Paired samples were obtained from normal pregnant women at term not in labor and their neonates. Maternal monocytes (MMo) and fetal monocytes (FMo) were isolated from the peripheral blood of mothers and fetal cord blood, respectively. dMφ and HC were obtained from the decidua of fetal membranes and placentas, respectively. DNA methylation profiling was performed using the Illumina Infinium Human Methylation27 BeadChip. Quantitative real-time PCR and Western Blot were performed for validation experiments.(i) Significant differences in DNA methylation were found in each comparison (MMo versus FMo, 65 loci; dMφ versus HC, 266 loci; MMo versus dMφ, 199 loci; FMo versus HC, 1030 loci). (ii) Many of the immune response-related genes were hypermethylated in fetal cells (FMo and HC) compared to maternal cells (MMo and dMφ). (iii) Genes encoding markers of classical macrophage activation were hypermethylated, and genes encoding alternative macrophage activation were hypomethylated in dMφ and HC compared to MMo and FMo, respectively. (iv) mRNA expressions of DNMT1, DNMT3A, and DNMT3B were significantly lower in dMφ than in HC. (v) 5-azacytidine treatment increased expression of INCA1 in dMφ.The findings herein indicate that DNA methylation patterns change during monocyte-macrophage differentiation at the feto-maternal interface. It is also suggested that DNA methylation is an important component of the biological machinery conferring an anti-inflammatory phenotype to macrophages at the feto-maternal interface.

The cervical mucus plug (CMP) differs from the cervical secretions of non-pregnant women, and is the ultimate sealant of the uterine cavity during pregnancy. Although several studies have analyzed biochemical properties of large glycoproteins in the CMP, comprehensive information about its protein composition is yet unavailable. We hypothesized that protein profiling of the CMP could provide key clues to its physiological functions in pregnancy. For this purpose, five CMPs obtained from women in labor at term were analyzed by LC–MS/MS. Out of 291 total proteins identified, 137 were detected in two or more samples, which included S100A8, S100A9, and complement proteins (C3, C4a, C4b, C6, and C8g). Several proteins, which have not been described in the cervical mucus of non-pregnant women or in cervicovaginal fluids, such as CD81 antigen and pregnancy zone protein, were also identified. Gene ontology analysis of identified proteins showed significant enrichment of 28 biological processes such as ‘activation of plasma proteins involved in acute inflammatory response’ and ‘positive regulation of cholesterol esterification’. We report the proteome of CMPs from pregnant women at term for the first time, and the overall findings strongly suggest an important role for the CMP in the maintenance of pregnancy and parturition.

Background The assessment of fetal growth disorders requires a standard. Current nomograms for the assessment of fetal growth in African American women have been derived either from neonatal (rather than fetal) biometry data or have not been customized for maternal ethnicity, weight, height, and parity and fetal sex. Objective We sought to (1) develop a new customized fetal growth standard for African American mothers; and (2) compare such a standard to 3 existing standards for the classification of fetuses as small (SGA) or large (LGA) for gestational age. Study Design A retrospective cohort study included 4183 women (4001 African American and 182 Caucasian) from the Detroit metropolitan area who underwent ultrasound examinations between 14-40 weeks of gestation (the median number of scans per pregnancy was 5, interquartile range 3-7) and for whom relevant covariate data were available. Longitudinal quantile regression was used to build models defining the “normal” estimated fetal weight (EFW) centiles for gestational age in African American women, adjusted for maternal height, weight, and parity and fetal sex, and excluding pathologic factors with a significant effect on fetal weight. The resulting Perinatology Research Branch/Eunice Kennedy Shriver National Institute of Child Health and Human Development (hereinafter, PRB/NICHD) growth standard was compared to 3 other existing standards--the customized gestation-related optimal weight (GROW) standard; the Eunice Kennedy Shriver National Institute of Child Health and Human Development (hereinafter, NICHD) African American standard; and the multinational World Health Organization (WHO) standard--utilized to screen fetuses for SGA ( 90th centile) based on the last available ultrasound examination for each pregnancy. Results First, the mean birthweight at 40 weeks was 133 g higher for neonates born to Caucasian than to African American mothers and 150 g higher for male than female neonates; maternal weight, height, and parity had a positive effect on birthweight. Second, analysis of longitudinal EFW revealed the following features of fetal growth: (1) all weight centiles were about 2% higher for male than for female fetuses; (2) maternal height had a positive effect on EFW, with larger fetuses being affected more (2% increase in the 95th centile of weight for each 10-cm increase in height); and (3) maternal weight and parity had a positive effect on EFW that increased with gestation and varied among the weight centiles. Third, the screen-positive rate for SGA was 7.2% for the NICHD African American standard, 12.3% for the GROW standard, 13% for the WHO standard customized by fetal sex, and 14.4% for the PRB/NICHD customized standard. For all standards, the screen-positive rate for SGA was at least 2-fold higher among fetuses delivered preterm than at term. Fourth, the screen-positive rate for LGA was 8.7% for the GROW standard, 9.2% for the PRB/NICHD customized standard, 10.8% for the WHO standard customized by fetal sex, and 12.3% for the NICHD African American standard. Finally, the highest overall agreement among standards was between the GROW and PRB/NICHD customized standards (Cohen's interrater agreement, kappa = 0.85). Conclusion We developed a novel customized PRB/NICHD fetal growth standard from fetal data in an African American population without assuming proportionality of the effects of covariates, and without assuming that these effects are equal on all centiles of weight; we also provide an easy-to-use centile calculator. This standard classified more fetuses as being at risk for SGA compared to existing standards, especially among fetuses delivered preterm, but classified about the same number of LGA. The comparison among the 4 growth standards also revealed that the most important factor determining agreement among standards is whether they account for the same factors known to affect fetal growth.

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality worldwide. Although intra-amniotic infection is a recognized cause of spontaneous preterm labor, the noninfection-related etiologies are poorly understood. In this article, we demonstrated that the expansion of activated CD1d-restricted invariant NKT (iNKT) cells in the third trimester by administration of α-galactosylceramide (α-GalCer) induced late PTB and neonatal mortality. In vivo imaging revealed that fetuses from mice that underwent α-GalCer–induced late PTB had bradycardia and died shortly after delivery. Yet, administration of α-GalCer in the second trimester did not cause pregnancy loss. Peroxisome proliferator–activated receptor (PPAR)γ activation, through rosiglitazone treatment, reduced the rate of α-GalCer–induced late PTB and improved neonatal survival. Administration of α-GalCer in the third trimester suppressed PPARγ activation, as shown by the downregulation of Fabp4 and Fatp4 in myometrial and decidual tissues, respectively; this suppression was rescued by rosiglitazone treatment. Administration of α-GalCer in the third trimester induced an increase in the activation of conventional CD4+ T cells in myometrial tissues and the infiltration of activated macrophages, neutrophils, and mature dendritic cells to myometrial and/or decidual tissues. All of these effects were blunted after rosiglitazone treatment. Administration of α-GalCer also upregulated the expression of inflammatory genes at the maternal–fetal interface and systemically, and rosiglitazone treatment partially attenuated these responses. Finally, an increased infiltration of activated iNKT-like cells in human decidual tissues is associated with noninfection-related preterm labor/birth. Collectively, these results demonstrate that iNKT cell activation in vivo leads to late PTB by initiating innate and adaptive immune responses and suggest that the PPARγ pathway has potential as a target for prevention of this syndrome.

Microbial invasion of the fetus due to intra-amniotic infection can lead to a systemic inflammatory response characterized by elevated concentrations of cytokines in the umbilical cord plasma/serum. Clinical chorioamnionitis represents the maternal syndrome often associated with intra-amniotic infection, although other causes of this syndrome have been recently described. The objective of this study was to characterize the umbilical cord plasma cytokine profile in neonates born to mothers with clinical chorioamnionitis at term, according to the presence or absence of bacteria and/or intra-amniotic inflammation.A cross-sectional study was conducted, including patients with clinical chorioamnionitis at term (n=38; cases) and those with spontaneous term labor without clinical chorioamnionitis (n=77; controls). Women with clinical chorioamnionitis were classified according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) and amniotic fluid interleukin (IL)-6 concentration into three groups: 1) no intra-amniotic inflammation; 2) intra-amniotic inflammation without detectable microorganisms; or 3) microbial-associated intra-amniotic inflammation. A fetal inflammatory response syndrome (FIRS) was defined as an umbilical cord plasma IL-6 concentration11 pg/mL. The umbilical cord plasma concentrations of 29 cytokines were determined with sensitive and specific V-PLEX immunoassays. Nonparametric statistical methods were used for analysis, adjusting for a false discovery rate of 5%.1) Neonates born to mothers with clinical chorioamnionitis at term (considered in toto) had significantly higher median umbilical cord plasma concentrations of IL-6, IL-12p70, IL-16, IL-13, IL-4, IL-10 and IL-8, but significantly lower interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF)-α concentrations than neonates born to mothers with spontaneous term labor without clinical chorioamnionitis; 2) neonates born to mothers with clinical chorioamnionitis at term but without intra-amniotic inflammation had higher concentrations of IL-6, IL-12p70, IL-13, IL-4, IL-5, and IL-8, but lower IFN-γ, than neonates not exposed to clinical chorioamnionitis, suggesting that maternal fever in the absence of intra-amniotic inflammation leads to a change in the fetal cytokine network; 3) there were significant, positive correlations between maternal and umbilical cord plasma IL-6 and IL-8 concentrations (IL-6: Spearman correlation=0.53; P0.001; IL-8: Spearman correlation=0.42; P0.001), consistent with placental transfer of cytokines; 4) an elevated fetal plasma IL-6 (11 pg/mL), the diagnostic criterion for FIRS, was present in 21% of cases (8/38), and all these neonates were born to mothers with proven intra-amniotic infection; and 5) FIRS was associated with a high concentration of umbilical cord plasma IL-8, IL-10 and monocyte chemoattractant protein (MCP)-1.Neonates born to mothers with clinical chorioamnionitis at term had higher concentrations of umbilical cord plasma cytokines than those born to mothers without clinical chorioamnionitis. Even neonates exposed to clinical chorioamnionitis but not to intra-amniotic inflammation had elevated concentrations of multiple cytokines, suggesting that intrapartum fever alters the fetal immune response.

Fever is a major criterion for clinical chorioamnionitis; yet, many patients with intrapartum fever do not have demonstrable intra-amniotic infection. Some cytokines, such as interleukin (IL)-1, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), can induce a fever. The objective of this study was to determine whether maternal plasma concentrations of cytokines could be of value in the identification of patients with the diagnosis of clinical chorioamnionitis at term who have microbial-associated intra-amniotic inflammation.A retrospective cross-sectional study was conducted, including patients with clinical chorioamnionitis at term (n=41; cases) and women in spontaneous labor at term without clinical chorioamnionitis (n=77; controls). Women with clinical chorioamnionitis were classified into three groups according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), and amniotic fluid IL-6 concentration: 1) no intra-amniotic inflammation; 2) intra-amniotic inflammation without detectable microorganisms; or 3) microbial-associated intra-amniotic inflammation. The maternal plasma concentrations of 29 cytokines were determined with sensitive and specific V-PLEX immunoassays. Nonparametric statistical methods were used for analysis, adjusting for a false discovery rate of 5%.1) The maternal plasma concentrations of pyrogenic cytokines (IL-1β, IL-2, IL-6, IFN-γ, and TNF-α) were significantly higher in patients with clinical chorioamnionitis at term than in those with spontaneous term labor without clinical chorioamnionitis; 2) the maternal plasma concentrations of cytokines were not significantly different among the three subgroups of patients with clinical chorioamnionitis (intra-amniotic inflammation with and without detectable bacteria and those without intra-amniotic inflammation); and 3) among women with the diagnosis of clinical chorioamnionitis, but without evidence of intra-amniotic inflammation, the maternal plasma concentrations of pyrogenic cytokines were significantly higher than in patients with spontaneous labor at term. These observations suggest that a fever can be mediated by increased circulating concentrations of these cytokines, despite the absence of a local intra-amniotic inflammatory response.1) The maternal plasma concentrations of pyrogenic cytokines (e.g. IL-1β, IL-2, IL-6, IFN-γ, and TNF-α) are higher in patients with intra-partum fever and the diagnosis of clinical chorioamnionitis at term than in those in spontaneous labor at term without a fever; and 2) maternal plasma cytokine concentrations have limited value in the identification of patients with bacteria in the amniotic cavity. Accurate assessment of the presence of intra-amniotic infection requires amniotic fluid analysis.

Background Neurocognitive impairment among children born preterm may arise from complex interactions between genes and the intra-uterine environment. Objectives (1) To characterize the transcriptomic profiles of chorioamniotic membranes in preterm neonates with and without neurocognitive impairment via microarrays and (2) to determine if neonates with neurocognitive impairment can be identified at birth. Materials/Methods A retrospective case-control study was conducted to examine the chorioamniotic transcriptome of gestational-age matched very preterm neonates with and without neurocognitive impairment at 18–24 months’ corrected-age defined by a Bayley-III Cognitive Composite Score 1.5); 2) Gene ontology analysis indicated enrichment of 19 biological processes and 3 molecular functions; 3)PADOG identified 4 significantly perturbed KEGG pathways: oxidative phosphorylation, Parkinson’s disease, Alzheimer’s disease and Huntington’s disease (q-value

Recent studies indicate that clinical chorioamnionitis is a heterogeneous condition and only approximately one-half of the patients have bacteria in the amniotic cavity, which is often associated with intra-amniotic inflammation. The objective of this study is to characterize the nature of the inflammatory response within the amniotic cavity in patients with clinical chorioamnionitis at term according to the presence or absence of 1) bacteria in the amniotic cavity and 2) intra-amniotic inflammation.A retrospective cross-sectional case-control study was conducted to examine cytokine and chemokine concentrations in the amniotic fluid (AF). Cases consisted of women with clinical chorioamnionitis at term (n=45). Controls were women with uncomplicated pregnancies at term who did not have intra-amniotic inflammation and were in labor (n=24). Women with clinical chorioamnionitis were classified according to the results of AF cultures, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry, and AF concentration of interleukin-6 (IL-6) into those: 1) without intra-amniotic inflammation, 2) with microbial-associated intra-amniotic inflammation, and 3) with intra-amniotic inflammation without detectable bacteria. The AF concentrations of 29 cytokines/chemokines were determined using sensitive and specific V-PLEX immunoassays.1) The AF concentrations of pro- and anti-inflammatory cytokines/chemokines such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-4 (IL-4), macrophage inflammatory protein-1 beta (MIP-1β), and interleukin-8 (IL-8) (except Eotaxin-3) were significantly higher in women with clinical chorioamnionitis at term than in controls (term labor without intra-amniotic inflammation); 2) patients with microbial-associated intra-amniotic inflammation, and those with intra-amniotic inflammation without detectable bacteria, had a dramatic differential expression of cytokines and chemokines in AF compared to patients with spontaneous labor without intra-amniotic inflammation. However, no difference could be detected in the pattern of the intra-amniotic inflammatory response between patients with intra-amniotic inflammation with and without detectable bacteria; and 3) in patients with clinical chorioamnionitis at term but without intra-amniotic inflammation, the behavior of cytokines and chemokines in the AF was similar to those in spontaneous labor at term.Patients with clinical chorioamnionitis who had microbial-associated intra-amniotic inflammation or intra-amniotic inflammation without detectable bacteria had a dramatic upregulation of the intra-amniotic inflammatory response assessed by amniotic fluid concentrations of cytokines. A subset of patients with term clinical chorioamnionitis does not have intra-amniotic infection/inflammation, as demonstrated by elevated AF concentrations of inflammation-related proteins, when compared to women in term labor with uncomplicated pregnancies, suggesting over-diagnosis. These observations constitute the first characterization of the cytokine/chemokine network in the amniotic cavity of patients with clinical chorioamnionitis at term.

Identification of functional sets of genes associated with conditions of interest from omics data was first reported in 1999, and since, a plethora of enrichment methods were published for systematic analysis of gene sets collections including Gene Ontology and biological pathways. Despite their widespread usage in reducing the complexity of omics experiment results, their performance is poorly understood. Leveraging the existence of disease specific gene sets in KEGG and Metacore® databases, we compared the performance of sixteen methods under relaxed assumptions while using 42 real datasets (over 1,400 samples). Most of the methods ranked high the gene sets designed for specific diseases whenever samples from affected individuals were compared against controls via microarrays. The top methods for gene set prioritization were different from the top ones in terms of sensitivity, and four of the sixteen methods had large false positives rates assessed by permuting the phenotype of the samples. The best overall methods among those that generated reasonably low false positive rates, when permuting phenotypes, were PLAGE, GLOBALTEST, and PADOG. The best method in the category that generated higher than expected false positives was MRGSE.

Objective: The molecular basis of failure to progress in labor is poorly understood. This study was undertaken to characterize the myometrial transcriptome of patients with an arrest of dilatation (AODIL). Study design: Human myometrium was prospectively collected from women in the following groups: (1) spontaneous term labor (TL; n=29) and (2) arrest of dilatation (AODIL; n=14). Gene expression was characterized using Illumina® HumanHT-12 microarrays. A moderated Student’s t-test and false discovery rate adjustment were used for analysis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of selected genes was performed in an independent sample set. Pathway analysis was performed on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database using Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). The MetaCore knowledge base was also searched for pathway analysis. Results: (1) Forty-two differentially expressed genes were identified in women with an AODIL; (2) gene ontology analysis indicated enrichment of biological processes, which included regulation of angiogenesis, response to hypoxia, inflammatory response, and chemokine-mediated signaling pathway. Enriched molecular functions included transcription repressor activity, heat shock protein (Hsp) 90 binding, and nitric oxide synthase (NOS) activity; (3) MetaCore analysis identified immune response chemokine (C-C motif) ligand 2 (CCL2) signaling, muscle contraction regulation of endothelial nitric oxide synthase (eNOS) activity in endothelial cells, and triiodothyronine and thyroxine signaling as significantly overrepresented (false discovery rate NOS3); hypoxic ischemic factor 1A (HIF1A); Chemokine (C-C motif) ligand 2 (CCL2); angiopoietin-like 4 (ANGPTL4); ADAM metallopeptidase with thrombospondin type 1, motif 9 (ADAMTS9); G protein-coupled receptor 4 (GPR4); metallothionein 1A (MT1A); MT2A; and selectin E (SELE) in an AODIL. Conclusion: The myometrium of women with AODIL has a stereotypic transcriptome profile. This disorder has been associated with a pattern of gene expression involved in muscle contraction, an inflammatory response, and hypoxia. This is the first comprehensive and unbiased examination of the molecular basis of an AODIL.

Excess reactive oxygen species (ROS) generation and oxidative stress in vascular tissue is associated with many diseases. Glutathione (GSH), one of the most abundant low molecular weight non-protein thiols, modulates physiological levels of ROS and is involved in the cell's oxidative stress response. The GSH/GSSG redox couple is commonly used in measuring oxidative stress status. The imbalance of GSH is reported in many disease states including atherosclerosis, cancer, neurodegenerative disease, and aging. The importance of GSH in modulation of intracellular ROS involves both its protective defense against the damaging effects of oxidative stress and its role in facilitating ROS cell signaling. In this paper, we review significant results obtained from mass balance and kinetic reactions based computational and mathematical models of GSH participation in oxidative stress. The focus is on the mediation of ROS and oxidative stress with respect to the antioxidant capacity of the cell. We discuss the role of GSH in the redox state of the cell, maintaining homeostasis through GSH synthesis, scavenging of free radicals, modulating hydrogen peroxide level and interacting with nitric oxide pathways.

Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis.Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4.Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed.Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced.Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT.

The unique conformation of the active site in calpains along with the implication of their role in several diseases has prompted widespread research interest in the scientific community. Structural studies devoted to m- and μ-calpains have proposed a two-stage calcium-dependent activation mechanism for calpains. In this work, we performed conventional and targeted molecular dynamics simulations to investigate global and local changes in the structure of the protease core of m-calpain upon calcium binding. Simulations were performed on the protease core of calcium free (pdbid: 1kfu) and calcium bound (pdbid: 3df0) m-calpain with and without the presence of calcium ions. Our results indicate that the inactive, open conformation of the protease core does not transform into the active, closed conformation simply upon removal of constraints from the neighbor domains. The role of other factors, including calcium binding and the subsequent formation of an Arg94–Glu305 inter-domain salt bridge and the change in the orientation of Trp288 side chain, in the activation of the protease core is elicited.

CD300a is an immunomodulatory molecule of the immunoglobulin receptor superfamily expressed in the leukocytes of myeloid and lymphoid lineages. However, its biological function on CD8+ T lymphocytes remains largely unknown. This study was conducted to assess the biological significance of CD300a expression in T lymphocytes and to determine whether its expression in peripheral T lymphocytes changes in pregnant women presenting with antifetal rejection.Microarray analysis was performed using total RNA isolated from peripheral CD300a+ and CD300a- T lymphocytes. Flow cytometric analysis of the peripheral blood samples of pregnant women and pathologic examination of the placentas were conducted. A large number of genes (N = 1245) were differentially expressed between CD300a- and CD300a+ subsets of CD8+ T lymphocytes, which included CCR7, CD244, CX3CR1, GLNY, GZMB, GZMK, IL15, ITGB1, KLRG1, PRF1, and SLAMF7. Gene ontology analysis of differentially expressed genes demonstrated enrichment of biological processes such as immune response, cell death, and signal transduction. CD300a expression in CD8+ T lymphocytes was coupled to a more cytotoxic molecular signature. Of note, the proportion of CD300a+CD8+ T lymphocytes increased in pregnant women with chronic chorioamnionitis (antifetal rejection of the chorioamniotic membranes; P0.05). The findings of this study strongly suggest an increase in systemic T-lymphocyte-mediated cytotoxicity in pregnant women with chronic chorioamnionitis as a manifestation of maternal antifetal rejection.

Background The human amnion plays a pivotal role in parturition. Two of its compartments, the placental amnion and the reflected amnion, have distinct transcriptome and are functionally coordinated for parturition. This study was conducted to determine the microRNA (miRNA) expression pattern and its significance in the placental amnion and the reflected amnion in association with labor at term. Methodology/Principal Findings MicroRNA microarray, real-time quantitative RT-PCR (qRT-PCR), and miRNA in situ hybridization analyses of the placental amnion and the reflected amnion (n = 20) obtained at term were conducted. Luciferase assay, transfection, and qRT-PCR analyses of primary amnion epithelial cells (AECs) and amnion mesenchymal cells (AMCs) were performed. MicroRNA microarray analysis demonstrated differential expression of 32 miRNAs between the placental amnion and the reflected amnion after labor. Thirty-one (97%) miRNAs, which included miR-143 and miR-145, a cardiovascular-specific miRNA cluster, were down-regulated in the reflected amnion. Analyses of miR-143 and miR-145 by qRT-PCR confirmed microarray results, and further demonstrated their decreased expression in the reflected amnion with labor. Interestingly, expression of miR-143 and miR-145 was higher in AMCs than in AECs (p

Background The identification of gene sets that are significantly impacted in a given condition based on microarray data is a crucial step in current life science research. Most gene set analysis methods treat genes equally, regardless how specific they are to a given gene set. Results In this work we propose a new gene set analysis method that computes a gene set score as the mean of absolute values of weighted moderated gene t-scores. The gene weights are designed to emphasize the genes appearing in few gene sets, versus genes that appear in many gene sets. We demonstrate the usefulness of the method when analyzing gene sets that correspond to the KEGG pathways, and hence we called our method Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). Unlike most gene set analysis methods which are validated through the analysis of 2-3 data sets followed by a human interpretation of the results, the validation employed here uses 24 different data sets and a completely objective assessment scheme that makes minimal assumptions and eliminates the need for possibly biased human assessments of the analysis results. Conclusions PADOG significantly improves gene set ranking and boosts sensitivity of analysis using information already available in the gene expression profiles and the collection of gene sets to be analyzed. The advantages of PADOG over other existing approaches are shown to be stable to changes in the database of gene sets to be analyzed. PADOG was implemented as an R package available at: http://bioinformaticsprb.med.wayne.edu/PADOG/or http://www.bioconductor.org.