Immunosequencing and Profiling of T Cells at the Maternal-Fetal Interface of Women with Preterm Labor and Chronic Chorioamnionitis.

T cells are implicated in the pathophysiology of preterm labor and birth, the leading cause of neonatal morbidity and mortality worldwide. Specifically, maternal decidual T cells infiltrate the chorioamniotic membranes in chronic chorioamnionitis (CCA), a placental lesion considered to reflect maternal anti-fetal rejection, leading to preterm labor and birth. However, the phenotype and TCR repertoire of decidual T cells in women with preterm labor and CCA have not been investigated. In this study, we used phenotyping, TCR sequencing, and functional assays to elucidate the molecular characteristics and Ag specificity of T cells infiltrating the chorioamniotic membranes in women with CCA who underwent term or preterm labor. Phenotyping indicated distinct enrichment of human decidual effector memory T cell subsets in cases of preterm labor with CCA without altered regulatory T cell proportions. TCR sequencing revealed that the T cell repertoire of CCA is characterized by increased TCR richness and decreased clonal expansion in women with preterm labor. We identified 15 clones associated with CCA and compared these against established TCR databases, reporting that infiltrating T cells may possess specificity for maternal and fetal Ags, but not common viral Ags. Functional assays demonstrated that choriodecidual T cells can respond to maternal and fetal Ags. Collectively, our findings provide, to our knowledge, novel insight into the complex processes underlying chronic placental inflammation and further support a role for effector T cells in the mechanisms of disease for preterm labor and birth. Moreover, this work further strengthens the contribution of adaptive immunity to the syndromic nature of preterm labor and birth.

A key role for NLRP3 signaling in preterm labor and birth driven by the alarmin S100B.

Preterm birth remains the leading cause of neonatal morbidity and mortality worldwide. A substantial number of spontaneous preterm births occur in the context of sterile intra-amniotic inflammation, a condition that has been mechanistically proven to be triggered by alarmins. However, sterile intra-amniotic inflammation still lacks treatment. The NLRP3 inflammasome has been implicated in sterile intra-amniotic inflammation; yet, its underlying mechanisms, as well as the maternal and fetal contributions to this signaling pathway, are unclear. Herein, by utilizing a translational and clinically relevant model of alarmin-induced preterm labor and birth in Nlrp3-/- mice, we investigated the role of NLRP3 signaling by using imaging and molecular biology approaches. Nlrp3 deficiency abrogated preterm birth and the resulting neonatal mortality induced by the alarmin S100B by impeding the premature activation of the common pathway of labor as well as by dampening intra-amniotic and fetal inflammation. Moreover, Nlrp3 deficiency altered leukocyte infiltration and functionality in the uterus and decidua. Last, embryo transfer revealed that maternal and fetal Nlrp3 signaling contribute to alarmin-induced preterm birth and neonatal mortality, further strengthening the concept that both individuals participate in the complex process of preterm parturition. These findings provide novel insights into sterile intra-amniotic inflammation, a common etiology of preterm labor and birth, suggesting that the adverse perinatal outcomes resulting from prematurity can be prevented by targeting NLRP3 signaling.

Pregnancy-specific responses to COVID-19 revealed by high-throughput proteomics of human plasma.

BACKGROUND: 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. METHODS: Plasma samples were collected from pregnant women and non-pregnant individuals (male and female) with (n = 72 pregnant, 52 non-pregnant) and without (n = 29 pregnant, 41 non-pregnant) COVID-19. COVID-19 patients were grouped as asymptomatic, mild, moderate, severe, or critically ill according to NIH classifications. Proteomic profiling of 7,288 analytes corresponding to 6,596 unique protein targets was performed using the SOMAmer platform. RESULTS: Herein, we profile the plasma proteome of pregnant and non-pregnant COVID-19 patients and controls and show 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 shows enrichment of mediators implicated in cytokine storm, endothelial dysfunction, and angiogenesis. Shared and pregnancy-specific proteomic changes are 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. CONCLUSION: This study represents the most comprehensive characterization of the plasma proteome of pregnant and non-pregnant COVID-19 patients. Our findings emphasize the distinct immune modulation between the non-pregnant and pregnant states, providing insight into the pathogenesis of COVID-19 as well as a potential explanation for the more severe outcomes observed in pregnant women.

Pregnant COVID-19 patients are at increased risk of experiencing complications and severe outcomes compared to the general population. However, the reasons for this heightened risk are still unclear. We measured the proteins present in the blood of pregnant and non-pregnant patients with COVID-19 and compared these to healthy individuals. We found that some COVID-19-associated proteins were present at lower levels in pregnant women, which could help to protect the fetus from harmful inflammation, the body's natural response to infection. While some proteins affected by COVID-19 are shared between pregnant and non-pregnant patients, others were distinctly affected only in pregnant women, providing a potential explanation for the more severe outcomes in this group.

Molecular subclasses of preeclampsia characterized by a longitudinal maternal proteomics study: distinct biomarkers, disease pathways and options for prevention.

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.

Human Plasma Proteome During Normal Pregnancy.

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 was <20% 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.

Pregnancy-specific responses to COVID-19 are revealed by high-throughput proteomics of human plasma.

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.

Fetal and maternal NLRP3 signaling is required for preterm labor and birth.

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. One of every 4 preterm neonates is born to a mother with intra-amniotic inflammation driven by invading bacteria. However, the molecular mechanisms underlying this hostile immune response remain unclear. Here, we used a translationally relevant model of preterm birth in Nlrp3-deficient and -sufficient pregnant mice to identify what we believe is a previously unknown dual role for the NLRP3 pathway in the fetal and maternal signaling required for the premature onset of the labor cascade leading to fetal injury and neonatal death. Specifically, the NLRP3 sensor molecule and/or inflammasome is essential for triggering intra-amniotic and decidual inflammation, fetal membrane activation, uterine contractility, and cervical dilation. NLRP3 also regulates the functional status of neutrophils and macrophages in the uterus and decidua, without altering their influx, as well as maternal systemic inflammation. Finally, both embryo transfer experimentation and heterozygous mating systems provided mechanistic evidence showing that NLRP3 signaling in both the fetus and the mother is required for the premature activation of the labor cascade. These data provide insights into the mechanisms of fetal-maternal dialog in the syndrome of preterm labor and indicate that targeting the NLRP3 pathway could prevent adverse perinatal outcomes.

The amniotic fluid proteome predicts imminent preterm delivery in asymptomatic women with a short cervix.

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 (< 25 mm) at 16-32 weeks of gestation. Amniocenteses were performed to exclude intra-amniotic infection; none of the women included had clinical signs of infection or labor at the time of amniocentesis. An aptamer-based multiplex platform was used to profile 1310 AF proteins, and the differential protein abundance between women who delivered within two weeks from amniocentesis, and those who did not, was determined. The analysis included adjustment for quantitative cervical length and control of the false-positive rate at 10%. The area under the receiver operating characteristic curve was calculated to determine whether protein abundance in combination with cervical length improved the prediction of imminent preterm delivery as compared to cervical length alone. Of the 1,310 proteins profiled in AF, 17 were differentially abundant in women destined to deliver within two weeks of amniocentesis independently of the cervical length (adjusted p-value < 0.10). The decreased abundance of SNAP25 and the increased abundance of GPI, PTPN11, OLR1, ENO1, GAPDH, CHI3L1, RETN, CSF3, LCN2, CXCL1, CXCL8, PGLYRP1, LDHB, IL6, MMP8, and PRTN3 were associated with an increased risk of imminent delivery (odds ratio > 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.

Maternal circulating concentrations of soluble Fas and Elabela in early- and late-onset preeclampsia.

OBJECTIVE: The Fas/Fas ligand (FASL) system and Elabela-apelin receptor signaling pathways are implicated in the pathophysiology of preeclampsia. The aim of the current study was to investigate whether a model combining the measurement of sFas and Elabela in the maternal circulation may serve as a clinical biomarker for early- and/or late-onset preeclampsia more effectively than measures of each biomarker individually. METHODS: Blood samples were collected from 214 women in the following groups: (1) normal pregnancy sampled <34 weeks of gestation (n = 56); (2) patients who developed early-onset preeclampsia (n = 54); (3) normal pregnancy sampled ≥34 weeks of gestation (n = 52); (4) patients who developed late-onset preeclampsia (n = 52). Maternal circulating soluble Fas and Elabela concentrations were determined using sensitive and validated immunoassays. Two sample t-tests, multivariate logistic regression, and receiver operating characteristic curves were used for analyses. RESULTS: (1) Women with early-onset preeclampsia, and those with late-onset preeclampsia with placental lesions of maternal vascular malperfusion, had increased concentrations of sFas compared to their gestational age-matched normal controls; (2) women with late-onset preeclampsia, but not those with early-onset preeclampsia, had increased concentrations of Elabela compared to their gestational age-matched counterparts; and (3) an increase in both Elabela and sFas concentrations was more strongly associated with late-onset preeclampsia than early-onset preeclampsia relative to models including either of the markers alone. CONCLUSIONS: A combined model of maternal sFas and Elabela concentrations provides a stronger association with late-onset preeclampsia than either protein alone. This finding demonstrates the possibility to improve the classification of late-onset preeclampsia by combining the results of both molecular biomarkers.

Transcriptome changes in maternal peripheral blood during term parturition mimic perturbations preceding spontaneous preterm birth†.

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.

Gestational Age Dependence of the Maternal Circulating Long Non-Coding RNA Transcriptome During Normal Pregnancy Highlights Antisense and Pseudogene Transcripts.

In the post-genomic era, our understanding of the molecular regulators of physiologic and pathologic processes in pregnancy is expanding at the whole-genome level. Longitudinal changes in the known protein-coding transcriptome during normal pregnancy, which we recently reported (Gomez-Lopez et al., 2019), have improved our definition of the major operant networks, yet pregnancy-related functions of the non-coding RNA transcriptome remain poorly understood. A key finding of the ENCODE (Encyclopedia of DNA Elements) Consortium, the successor of the Human Genome Project, was that the human genome contains approximately 60,000 genes, the majority of which do not encode proteins. The total transcriptional output of non-protein-coding RNA genes, collectively referred to as the non-coding transcriptome, is comprised mainly of long non-coding RNA (lncRNA) transcripts (Derrien et al., 2012). Although the ncRNA transcriptome eclipses its protein-coding counterpart in abundance, it has until recently lacked a comprehensive, unbiased, genome-scale characterization over the timecourse of normal human pregnancy. Here, we annotated, characterized, and selectively validated the longitudinal changes in the non-coding transcriptome of maternal whole blood during normal pregnancy to term. We identified nine long non-coding RNAs (lncRNAs), including long intergenic non-coding RNAs (lincRNAs) as well as lncRNAs antisense to or otherwise in the immediate vicinity of protein-coding genes, that were differentially expressed with advancing gestation in normal pregnancy: AL355711, BC039551 (expressed mainly in the placenta), JHDM1D-AS1, A2M-AS1, MANEA-AS1, NR_034004, LINC00649, LINC00861, and LINC01094. By cross-referencing our dataset against major public pseudogene catalogs, we also identified six transcribed pseudogenes that were differentially expressed over time during normal pregnancy in maternal blood: UBBP4, FOXO3B, two Makorin (MKRN) pseudogenes (MKRN9P and LOC441455), PSME2P2, and YBX3P1. We also identified three non-coding RNAs belonging to other classes that were modulated during gestation: the microRNA MIR4439, the small nucleolar RNA (snoRNA) SNORD41, and the small Cajal-body specific ncRNA SCARNA2. The expression profiles of most hits were broadly suggestive of functions in pregnancy. These time-dependent changes of the non-coding transcriptome during normal pregnancy, which may confer specific regulatory impacts on their protein-coding gene targets, will facilitate a deeper molecular understanding of pregnancy and lncRNA-mediated molecular pathways at the maternal-fetal interface and of how these pathways impact maternal and fetal health.

The Distinct Immune Nature of the Fetal Inflammatory Response Syndrome Type I and Type II.

Fetal inflammatory response syndrome (FIRS) is strongly associated with neonatal morbidity and mortality and can be classified as type I or type II. Clinically, FIRS type I and type II are considered as distinct syndromes, yet the molecular underpinnings of these fetal inflammatory responses are not well understood because of their low prevalence and the difficulty of postdelivery diagnosis. In this study, we performed RNA sequencing of human cord blood samples from preterm neonates diagnosed with FIRS type I or FIRS type II. We found that FIRS type I was characterized by an upregulation of host immune responses, including neutrophil and monocyte functions, together with a proinflammatory cytokine storm and a downregulation of T cell processes. In contrast, FIRS type II comprised a mild chronic inflammatory response involving perturbation of HLA transcripts, suggestive of fetal semiallograft rejection. Integrating single-cell RNA sequencing-derived signatures with bulk transcriptomic data confirmed that FIRS type I immune responses were mainly driven by monocytes, macrophages, and neutrophils. Last, tissue- and cell-specific signatures derived from the BioGPS Gene Atlas further corroborated the role of myeloid cells originating from the bone marrow in FIRS type I. Collectively, these data provide evidence that FIRS type I and FIRS type II are driven by distinct immune mechanisms; whereas the former involves the innate limb of immunity consistent with host defense, the latter resembles a process of semiallograft rejection. These findings shed light on the fetal immune responses caused by infection or alloreactivity that can lead to deleterious consequences in neonatal life.

Crowdsourcing assessment of maternal blood multi-omics for predicting gestational age and preterm birth.

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).

Human Chorionic Gonadotropin Modulates the Transcriptome of the Myometrium and Cervix in Late Gestation.

Human chorionic gonadotropin (hCG) is a critical hormone for the establishment and maintenance of pregnancy. hCG administration prevents the onset of preterm labor in mice; yet, the transcriptomic changes associated with this tocolytic effect that take place in the myometrium and cervix have not been elucidated. Herein, we implemented both discovery and targeted approaches to investigate the transcriptome of the myometrium and cervix after hCG administration. Pregnant mice were intraperitoneally injected with 10 IU of hCG on 13.0, 15.0, and 17.0 days post coitum, and the myometrium and cervix were collected. RNA sequencing was performed to determine differentially expressed genes, enriched biological processes, and impacted KEGG pathways. Multiplex qRT-PCR was performed to investigate the expression of targeted contractility- and inflammation-associated transcripts. hCG administration caused the differential expression of 720 genes in the myometrium. Among the downregulated genes, enriched biological processes were primarily associated with regulation of transcription. hCG administration downregulated key contractility genes, Gja1 and Oxtr, but upregulated the prostaglandin-related genes Ptgfr and Ptgs2 and altered the expression of inflammation-related genes in the myometrium. In the cervix, hCG administration caused differential expression of 3348 genes that were related to inflammation and host defense, among others. The downregulation of key contractility genes and upregulation of prostaglandin-related genes were also observed in the cervix. Thus, hCG exerts tocolytic and immunomodulatory effects in late gestation by altering biological processes in the myometrium and cervix, which should be taken into account when considering hCG as a potential treatment to prevent the premature onset of labor.

RNA Sequencing Reveals Distinct Immune Responses in the Chorioamniotic Membranes of Women with Preterm Labor and Microbial or Sterile Intra-amniotic Inflammation.

Preterm labor precedes premature birth, the leading cause of neonatal morbidity and mortality worldwide. Preterm labor can occur in the context of either microbe-associated intra-amniotic inflammation (i.e., intra-amniotic infection) or intra-amniotic inflammation in the absence of detectable microorganisms (i.e., sterile intra-amniotic inflammation). Both intra-amniotic infection and sterile intra-amniotic inflammation trigger local immune responses that have deleterious effects on fetal life. Yet, the extent of such immune responses in the fetal tissues surrounding the amniotic cavity (i.e., the chorioamniotic membranes) is poorly understood. By using RNA sequencing (RNA seq) as a discovery approach, we found that there were significant transcriptomic differences involving host response to pathogens in the chorioamniotic membranes of women with intra-amniotic infection compared to those from women without inflammation. In addition, the sterile or microbial nature of intra-amniotic inflammation was associated with distinct transcriptomic profiles in the chorioamniotic membranes. Moreover, the immune response in the chorioamniotic membranes of women with sterile intra-amniotic inflammation was milder in nature than that induced by microbes and involved the upregulation of alarmins and inflammasome-related molecules. Lastly, the presence of maternal and fetal inflammatory responses in the placenta was associated with the upregulation of immune processes in the chorioamniotic membranes. Collectively, these findings provide insight into the immune responses against microbes or alarmins that take place in the fetal tissues surrounding the amniotic cavity, shedding light on the immunobiology of preterm labor and birth.

RNA Sequencing Reveals Diverse Functions of Amniotic Fluid Neutrophils and Monocytes/Macrophages in Intra-Amniotic Infection.

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.

Pregnancy-specific transcriptional changes upon endotoxin exposure in mice.

Objectives Pregnant 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. Methods Pregnant and non-pregnant mice were injected with endotoxin or saline on 16.5 days post 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. Results We 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. Conclusions Pregnancy imprints organ-specific host immune responses upon endotoxin exposure. These findings provide insight into the host-response against microbes during pregnancy.

Regulatory T Cells Play a Role in a Subset of Idiopathic Preterm Labor/Birth and Adverse Neonatal Outcomes.

Regulatory T cells (Tregs) have been exhaustively investigated during early pregnancy; however, their role later in gestation is poorly understood. Herein, we report that functional Tregs are reduced at the maternal-fetal interface in a subset of women with idiopathic preterm labor/birth, which is accompanied by a concomitant increase in Tc17 cells. In mice, depletion of functional Tregs during late gestation induces preterm birth and adverse neonatal outcomes, which are rescued by the adoptive transfer of such cells. Treg depletion does not alter obstetrical parameters in the mother, yet it increases susceptibility to endotoxin-induced preterm birth. The mechanisms whereby depletion of Tregs induces adverse perinatal outcomes involve tissue-specific immune responses and mild systemic maternal inflammation, together with dysregulation of developmental and cellular processes in the placenta, in the absence of intra-amniotic inflammation. These findings provide mechanistic evidence supporting a role for Tregs in the pathophysiology of idiopathic preterm labor/birth and adverse neonatal outcomes.

Amniotic fluid cell-free transcriptome: a glimpse into fetal development and placental cellular dynamics during normal pregnancy.

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 < 0.05 and fold change > 1.25; for differential splicing, a splicing index > 2 and adjusted p < 0.05 were required. Functional profiling was used to interpret differentially expressed or spliced genes. The expression of tissue-specific and cell-type specific signatures defined by single-cell genomics was quantified and correlated with covariates. 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 < 0.001) and featured increased expression of genes specific to the trachea, salivary glands, and lung and decreased expression of genes specific to the cardiac myocytes, uterus, and fetal liver, among others. 2) Single-cell RNA-seq signatures of the cytotrophoblast, Hofbauer cells, erythrocytes, monocytes, T and B cells, among others, showed complex patterns of modulation with gestation (adjusted p < 0.05). 3) In 17% of the genes detected, we found differential splicing with advancing gestation in genes related to brain development processes and immunity pathways, including some that were missed based on differential expression analysis alone. 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.

Exhausted and Senescent T Cells at the Maternal-Fetal Interface in Preterm and Term Labor.

Successful pregnancy requires a tightly-regulated equilibrium of immune cell interactions at the maternal-fetal interface (i.e., the decidual tissues), which plays a central role in the inflammatory process of labor. Most of the innate immune cells in this compartment have been well characterized; however, adaptive immune cells are still under investigation. Herein, we performed immunophenotyping of the decidua basalis and decidua parietalis to determine whether exhausted and senescent T cells are present at the maternal-fetal interface and whether the presence of pathological (i.e., preterm) or physiological (i.e., term) labor and/or placental inflammation alter such adaptive immune cells. In addition, decidual exhausted T cells were sorted to test their functional status. We found that (1) exhausted and senescent T cells were present at the maternal-fetal interface and predominantly expressed an effector memory phenotype, (2) exhausted CD4+ T cells increased in the decidua parietalis as gestational age progressed, (3) exhausted CD4+ and CD8+ T cells decreased in the decidua basalis of women who underwent labor at term compared to those without labor, (4) exhausted CD4+ T cells declined with the presence of placental inflammation in the decidua basalis of women with preterm labor, (5) exhausted CD8+ T cells decreased with the presence of placental inflammation in the decidua basalis of women who underwent labor at term, (6) both senescent CD4+ and CD8+ T cells declined with the presence of placental inflammation in the decidua basalis of women who underwent preterm labor, and (7) decidual exhausted T cells produced IFNγ and TNFα upon in vitro stimulation. Collectively, these findings indicate that exhausted and senescent T cells are present at the human maternal-fetal interface and undergo alterations in a subset of women either with labor at term or preterm labor and placental inflammation. Importantly, decidual T cell function can be restored upon stimulation.