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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.

Objectives The heterogeneous nature of preeclampsia is a major obstacle to early screening and prevention, and a molecular taxonomy of disease is needed. We have previously identified four subclasses of preeclampsia based on first-trimester plasma proteomic profiles. Herein, we expanded this approach by using a more comprehensive panel of proteins profiled in longitudinal samples. Methods Proteomic data collected longitudinally from plasma samples of women who developed preeclampsia (n=109) and of controls (n=90) were available from our previous report on 1,125 proteins. Consensus clustering was performed to identify subgroups of patients with preeclampsia based on data from five gestational-age intervals by using select interval-specific features. Demographic, clinical, and proteomic differences among clusters were determined. Differentially abundant proteins were used to identify cluster-specific perturbed KEGG pathways. Results Four molecular clusters with different clinical phenotypes were discovered by longitudinal proteomic profiling. Cluster 1 involves metabolic and prothrombotic changes with high rates of early-onset preeclampsia and small-for-gestational-age neonates; Cluster 2 includes maternal anti-fetal rejection mechanisms and recurrent preeclampsia cases; Cluster 3 is associated with extracellular matrix regulation and comprises cases of mostly mild, late-onset preeclampsia; and Cluster 4 is characterized by angiogenic imbalance and a high prevalence of early-onset disease. Conclusions This study is an independent validation and further refining of molecular subclasses of preeclampsia identified by a different proteomic platform and study population. The results lay the groundwork for novel diagnostic and personalized tools of prevention.

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.

Pregnant women are at greater risk of adverse outcomes, including mortality, as well as obstetrical complications resulting from COVID-19. However, pregnancy-specific changes that underlie such worsened outcomes remain unclear. Herein, we profiled the plasma proteome of pregnant and non-pregnant COVID-19 patients and controls and showed alterations that display a dose-response relationship with disease severity; yet, such proteomic perturbations are dampened during pregnancy. In both pregnant and non-pregnant state, the proteome response induced by COVID-19 showed enrichment of mediators implicated in cytokine storm, endothelial dysfunction, and angiogenesis. Shared and pregnancy-specific proteomic changes were identified: pregnant women display a tailored response that may protect the conceptus from heightened inflammation, while non-pregnant individuals display a stronger response to repel infection. Furthermore, the plasma proteome can accurately identify COVID-19 patients, even when asymptomatic or with mild symptoms. This study represents the most comprehensive characterization of the plasma proteome of pregnant and non-pregnant COVID-19 patients.

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.

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.

ObjectivesPregnant women are more susceptible to certain infections; however, this increased susceptibility is not fully understood. Herein, systems biology approaches were utilized to elucidate how pregnancy modulates tissue-specific host responses to a bacterial product, endotoxin.MethodsPregnant and non-pregnant mice were injected with endotoxin or saline on 16.5 dayspost coitum(n=8–11 per group). The uterus, cervix, liver, adrenal gland, kidney, lung, and brain were collected 12 h after injection and transcriptomes were measured using microarrays. Heatmaps and principal component analysis were used for visualization. Differentially expressed genes between groups were assessed using linear models that included interaction terms to determine whether the effect of infection differed with pregnancy status. Pathway analysis was conducted to interpret gene expression changes.ResultsWe report herein a multi-organ atlas of the transcript perturbations in pregnant and non-pregnant mice in response to endotoxin. Pregnancy strongly modified the host responses to endotoxin in the uterus, cervix, and liver. In contrast, pregnancy had a milder effect on the host response to endotoxin in the adrenal gland, lung, and kidney. However, pregnancy did not drastically affect the host response to endotoxin in the brain.ConclusionsPregnancy imprints organ-specific host immune responses upon endotoxin exposure. These findings provide insight into the host-response against microbes during pregnancy.

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.

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.

We describe and provide an array of diverse methods to predict cellular positions in tissue from RNA-seq data selecting mapping genes according to their spatial/statistical properties and their effect on improving the cell positioning., Single-cell RNA-sequencing (scRNAseq) technologies are rapidly evolving. Although very informative, in standard scRNAseq experiments, the spatial organization of the cells in the tissue of origin is lost. Conversely, spatial RNA-seq technologies designed to maintain cell localization have limited throughput and gene coverage. Mapping scRNAseq to genes with spatial information increases coverage while providing spatial location. However, methods to perform such mapping have not yet been benchmarked. To fill this gap, we organized the DREAM Single-Cell Transcriptomics challenge focused on the spatial reconstruction of cells from the Drosophila embryo from scRNAseq data, leveraging as silver standard, genes with in situ hybridization data from the Berkeley Drosophila Transcription Network Project reference atlas. The 34 participating teams used diverse algorithms for gene selection and location prediction, while being able to correctly localize clusters of cells. Selection of predictor genes was essential for this task. Predictor genes showed a relatively high expression entropy, high spatial clustering and included prominent developmental genes such as gap and pair-rule genes and tissue markers. Application of the top 10 methods to a zebra fish embryo dataset yielded similar performance and statistical properties of the selected genes than in the Drosophila data. This suggests that methods developed in this challenge are able to extract generalizable properties of genes that are useful to accurately reconstruct the spatial arrangement of cells in tissues.

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

ObjectiveThe aims of this study were to ascertain the frequency of disorders of villous maturation in fetal death and to also delineate other placental histopathologic lesions in fetal death.MethodsThis was a retrospective observational cohort study of fetal deaths occurring among women between January 2004 and January 2016 at Hutzel Women’s Hospital, Detroit, MI, USA. Cases comprised fetuses with death beyond 20 weeks’ gestation. Fetal deaths with congenital anomalies and multiple gestations were excluded. Controls included pregnant women without medical/obstetrical complications and delivered singleton, term (37–42 weeks) neonate with 5-min Apgar score ≥7 and birthweight between the 10thand 90thpercentiles.ResultsNinety-two percent (132/143) of placentas with fetal death showed placental histologic lesions. Fetal deaths were associated with (1) higher frequency of disorders of villous maturation [44.0% (64/143) vs. 1.0% (4/405), P ConclusionThis study demonstrates that placentas of womem with fetal death were 44 times more likely to present disorders of villous maturation compared to placentas of those with normal pregnancy. This suggests that the burden of placental disorders of villous maturation lesions is substantial.

Background The prevalence of allergic diseases such as asthma and allergic rhinitis is high in the general population, and aeroallergens are the most common allergens that cause airway inflammation. Skin prick testing (SPT) is a validated method to diagnose IgE-mediated allergic diseases. The knowledge of allergen sensitivity pattern in this part of India is limited. The present study was undertaken to identify common aeroallergens prevalent in this area using a standardized SPT. Methods We did a cross-sectional hospital-based study. A total of 330 patients of proven allergic airway disease underwent SPT. We used a validated skin prick test which included 22 allergen extracts, 1 positive control, and 1 negative control. Results A total of 327 patients were included in the study. Two hundred seventy-one (82.27%, n = 327) patients had a positive SPT to one or more aeroallergen extract. The sensitivity of Dermatophagoides pteronyssinus (49.85%) was seen in the maximum number of people followed by Dermatophagoides farinae (47.70%). Conclusions In our study, dust mites were the most common aeroallergen seen in maximum study subjects.

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.

Additional file 1: Figure S1. Representation of chromosome-specific differential expression between midtrimester and term gestation groups. The outer circle shows the chromosomes, with significant enrichment being marked with * (q

Additional file 6: Figure S6. Correlation of expression changes with maternal obesity between studies. Each dot represents a unique annotated gene. The y axis represents the log2 fold change (obese/lean) obtained in the current study. The x-axis represents the log2 expression of 182 genes detected as present in the current study among those reported as differentially expressed with obesity by Edlow et al. [99]. R: Spearman’s correlation coefficient

Additional file 4: Figure S4. Representation of chromosome-specific differential expression between pregnancies with a male those with a female fetus. The outer circle shows the chromosomes with significant enrichment being marked with * (q

Additional file 2: Figure S2. Changes in the expression of tissue-specific signatures with gestational age superposed to patient-specific values. For each tissue, the expression of the top 20 most-specific genes (based on the Gene Atlas dataset) was transformed into a Z-score and averaged in each AF sample (dots). A Robust Locally Weighted Regression and Smoothing Scatterplots (LOESS) model fit through the Z-scores as a function of gestational age is shown using lines. All differentially expressed tissue signatures (term versus midtrimester or linear correlation within the midtrimester group) are shown. AF, amniotic fluid

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.

Additional file 5: Figure S5. Example of differential splicing with fetal sex for TXLNGY gene. Details as shown in Fig. 5

Additional file 3: Figure S3. Changes in the expression of RNA Seq single-cell signatures with gestational age. For each single-cell signature, the expression the all specific genes (based on Tsang et al. [105]) was transformed into a Z-score and averaged in each AF sample (dots). A Robust Locally Weighted Regression and Smoothing Scatterplots (LOESS) model fit through the Z-scores as a function of gestational age is shown using lines. AF, amniotic fluid

Single-cell RNA-seq (scRNAseq) technologies are rapidly evolving and a growing number of datasets are now available. While very informative, in standard scRNAseq experiments the spatial organization of the cells in the organism or tissue of origin is lost. Conversely, spatial RNA-seq technologies designed to keep the localization of the cells have limited throughput and gene coverage. Mapping scRNAseq to data of genes with spatial information can thus increase coverage while providing spatial location. However, methods to perform such a mapping are still in their infancy and have not been benchmarked in an unbiased manner. To bridge the gap, we organized the DREAM Single-Cell Transcriptomics challenge to evaluate methods for reconstructing the spatial arrangement of single cells from single-cell RNA sequencing data. The challenge focused on the spatial reconstruction of cells from the Drosophila embryo from single-cell transcriptomic and, leveraging as gold standard, in situ hybridization data of a set of selected driver genes from the Berkeley Drosophila Transcription Network Project reference atlas. The 34 participating teams used an array of different algorithms for gene selection and location prediction. We devised a novel scoring and cross-validation scheme to evaluate the robustness of the best performing algorithms. Participants were able to correctly and robustly localize rare subpopulations of cells, accurately mapping both spatially co-localized and scattered groups of cells. The selection of predictor genes was essential for accurately locating the cells in the embryo. Among the most frequently selected set of genes we measured a relatively high expression entropy, high spatial clustering and the presence of prominent developmental genes such as gap and pair-ruled genes and tissue defining markers.

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.

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