Deciphering maternal-fetal cross-talk in the human placenta during parturition using single-cell RNA sequencing.

Labor is a complex physiological process requiring a well-orchestrated dialogue between the mother and fetus. However, the cellular contributions and communications that facilitate maternal-fetal cross-talk in labor have not been fully elucidated. Here, single-cell RNA sequencing (scRNA-seq) was applied to decipher maternal-fetal signaling in the human placenta during term labor. First, a single-cell atlas of the human placenta was established, demonstrating that maternal and fetal cell types underwent changes in transcriptomic activity during labor. Cell types most affected by labor were fetal stromal and maternal decidual cells in the chorioamniotic membranes (CAMs) and maternal and fetal myeloid cells in the placenta. Cell-cell interaction analyses showed that CAM and placental cell types participated in labor-driven maternal and fetal signaling, including the collagen, C-X-C motif ligand (CXCL), tumor necrosis factor (TNF), galectin, and interleukin-6 (IL-6) pathways. Integration of scRNA-seq data with publicly available bulk transcriptomic data showed that placenta-derived scRNA-seq signatures could be monitored in the maternal circulation throughout gestation and in labor. Moreover, comparative analysis revealed that placenta-derived signatures in term labor were mirrored by those in spontaneous preterm labor and birth. Furthermore, we demonstrated that early in gestation, labor-specific, placenta-derived signatures could be detected in the circulation of women destined to undergo spontaneous preterm birth, with either intact or prelabor ruptured membranes. Collectively, our findings provide insight into the maternal-fetal cross-talk of human parturition and suggest that placenta-derived single-cell signatures can aid in the development of noninvasive biomarkers for the prediction of preterm birth.

A mitochondrial regulator protein, MNRR1, is elevated in the maternal blood of women with preeclampsia.

OBJECTIVE: Preeclampsia, one of the most serious obstetric complications, is a heterogenous disorder resulting from different pathologic processes. However, placental oxidative stress and an anti-angiogenic state play a crucial role. Mitochondria are a major source of cellular reactive oxygen species. Abnormalities in mitochondrial structures, proteins, and functions have been observed in the placentae of patients with preeclampsia, thus mitochondrial dysfunction has been implicated in the mechanism of the disease. Mitochondrial nuclear retrograde regulator 1 (MNRR1) is a newly characterized bi-organellar protein with pleiotropic functions. In the mitochondria, this protein regulates cytochrome c oxidase activity and reactive oxygen species production, whereas in the nucleus, it regulates the transcription of a number of genes including response to tissue hypoxia and inflammatory signals. Since MNRR1 expression changes in response to hypoxia and to an inflammatory signal, MNRR1 could be a part of mitochondrial dysfunction and involved in the pathologic process of preeclampsia. This study aimed to determine whether the plasma MNRR1 concentration of women with preeclampsia differed from that of normal pregnant women. METHODS: This retrospective case-control study included 97 women with preeclampsia, stratified by gestational age at delivery into early (<34 weeks, n = 40) and late (≥34 weeks, n = 57) preeclampsia and by the presence or absence of placental lesions consistent with maternal vascular malperfusion (MVM), the histologic counterpart of an anti-angiogenic state. Women with an uncomplicated pregnancy at various gestational ages who delivered at term served as controls (n = 80) and were further stratified into early (n = 25) and late (n = 55) controls according to gestational age at venipuncture. Maternal plasma MNRR1 concentrations were determined by an enzyme-linked immunosorbent assay. RESULTS: 1) Women with preeclampsia at the time of diagnosis (either early or late disease) had a significantly higher median (interquartile range, IQR) plasma MNRR1 concentration than the controls [early preeclampsia: 1632 (924-2926) pg/mL vs. 630 (448-4002) pg/mL, p = .026, and late preeclampsia: 1833 (1441-5534) pg/mL vs. 910 (526-6178) pg/mL, p = .021]. Among women with early preeclampsia, those with MVM lesions in the placenta had the highest median (IQR) plasma MNRR1 concentration among the three groups [with MVM: 2066 (1070-3188) pg/mL vs. without MVM: 888 (812-1781) pg/mL, p = .03; and with MVM vs. control: 630 (448-4002) pg/mL, p = .04]. There was no significant difference in the median plasma MNRR1 concentration between women with early preeclampsia without MVM lesions and those with an uncomplicated pregnancy (p = .3). By contrast, women with late preeclampsia, regardless of MVM lesions, had a significantly higher median (IQR) plasma MNRR1 concentration than women in the control group [with MVM: 1609 (1392-3135) pg/mL vs. control: 910 (526-6178), p = .045; and without MVM: 2023 (1578-8936) pg/mL vs. control, p = .01]. CONCLUSIONS: MNRR1, a mitochondrial regulator protein, is elevated in the maternal plasma of women with preeclampsia (both early and late) at the time of diagnosis. These findings may reflect some degree of mitochondrial dysfunction, intravascular inflammation, or other unknown pathologic processes that characterize this obstetrical syndrome.

Preeclampsia at term: evidence of disease heterogeneity based on the profile of circulating cytokines and angiogenic factors.

BACKGROUND: Intravascular inflammation and an antiangiogenic state have been implicated in the pathophysiology of preeclampsia. On the basis of the profiles of their angiogenic/antiangiogenic factors, women with preeclampsia at term may be classified into 2 subgroups with different characteristics and prevalence of adverse outcomes. This study was undertaken to examine whether these 2 subgroups of preeclampsia at term also show differences in their profiles of intravascular inflammation. OBJECTIVE: This study aimed to determine the plasma profiles of cytokines and chemokines in women with preeclampsia at term who had a normal or an abnormal angiogenic profile. STUDY DESIGN: A nested case-control study was conducted to include women classified into 3 groups: women with an uncomplicated pregnancy (n=213) and women with preeclampsia at term with a normal (n=55) or an abnormal (n=41) angiogenic profile. An abnormal angiogenic profile was defined as a plasma ratio of placental growth factor and soluble fms-like tyrosine kinase-1 multiple of the median <10th percentile for gestational age. Concentrations of cytokines were measured by multiplex immunoassays. RESULTS: Women with preeclampsia at term and an abnormal angiogenic profile showed evidence of the greatest intravascular inflammation among the study groups. These women had higher plasma concentrations of 5 cytokines (interleukin-6, interleukin-8, interleukin-12/interleukin-23p40, interleukin-15, and interleukin-16) and 7 chemokines (eotaxin, eotaxin-3, interferon-γ inducible protein-10, monocyte chemotactic protein-4, macrophage inflammatory protein-1ß, macrophage-derived chemokine, and thymus and activation-regulated chemokine compared to women with an uncomplicated pregnancy. By contrast, women with preeclampsia at term and a normal angiogenic profile, compared to women with an uncomplicated pregnancy, had only a higher plasma concentration of monocyte chemotactic protein-4. A correlation between severity of the antiangiogenic state, blood pressure, and plasma concentrations of a subset of cytokines was observed. CONCLUSION: Term preeclampsia can be classified into 2 clusters. One is characterized by an antiangiogenic state coupled with an excessive inflammatory process, whereas the other has neither of these features. These findings further support the heterogeneity of preeclampsia at term and may explain the distinct clinical outcomes.

The Vaginal Microbiota of Pregnant Women Varies with Gestational Age, Maternal Age, and Parity.

The composition of the vaginal microbiota is heavily influenced by pregnancy and may factor into pregnancy complications, including spontaneous preterm birth. However, results among studies have been inconsistent due, in part, to variation in sample sizes and ethnicity. Thus, an association between the vaginal microbiota and preterm labor continues to be debated. Yet, before assessing associations between the composition of the vaginal microbiota and preterm labor, a robust and in-depth characterization of the vaginal microbiota throughout pregnancy in the specific study population under investigation is required. Here, we report a large longitudinal study (n = 474 women, 1,862 vaginal samples) of a predominantly African-American cohort-a population that experiences a relatively high rate of pregnancy complications-evaluating associations between individual identity, gestational age, and other maternal characteristics with the composition of the vaginal microbiota throughout gestation resulting in term delivery. The principal factors influencing the composition of the vaginal microbiota in pregnancy are individual identity and gestational age at sampling. Other factors are maternal age, parity, obesity, and self-reported Cannabis use. The general pattern across gestation is for the vaginal microbiota to remain or transition to a state of Lactobacillus dominance. This pattern can be modified by maternal parity and obesity. Regardless, network analyses reveal dynamic associations among specific bacterial taxa within the vaginal ecosystem, which shift throughout the course of pregnancy. This study provides a robust foundational understanding of the vaginal microbiota in pregnancy and sets the stage for further investigation of this microbiota in obstetrical disease. IMPORTANCE There is debate regarding links between the vaginal microbiota and pregnancy complications, especially spontaneous preterm birth. Inconsistencies in results among studies are likely due to differences in sample sizes and cohort ethnicity. Ethnicity is a complicating factor because, although all bacterial taxa commonly inhabiting the vagina are present among all ethnicities, the frequencies of these taxa vary among ethnicities. Therefore, an in-depth characterization of the vaginal microbiota throughout pregnancy in the specific study population under investigation is required prior to evaluating associations between the vaginal microbiota and obstetrical disease. This initial investigation is a large longitudinal study of the vaginal microbiota throughout gestation resulting in a term delivery in a predominantly African-American cohort, a population that experiences disproportionally negative maternal-fetal health outcomes. It establishes the magnitude of associations between maternal characteristics, such as age, parity, body mass index, and self-reported Cannabis use, on the vaginal microbiota in pregnancy.

Clinical chorioamnionitis at term is characterized by changes in the plasma concentration of CHCHD2/MNRR1, a mitochondrial protein.

OBJECTIVE: Mitochondrial dysfunction was observed in acute systemic inflammatory conditions such as sepsis and might be involved in sepsis-induced multi-organ failure. Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 2 (CHCHD2), also known as Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1), a bi-organellar protein located in the mitochondria and the nucleus, is implicated in cell respiration, survival, and response to tissue hypoxia. Recently, the reduction of the cellular CHCHD2/MNRR1 protein, as part of mitochondrial dysfunction, has been shown to play a role in the amplification of inflammatory cytokines in a murine model of lipopolysaccharide-induced systemic inflammation. The aim of this study was to determine whether the plasma concentration of CHCHD2/MNRR1 changed during human normal pregnancy, spontaneous labor at term, and clinical chorioamnionitis at term. METHODS: We conducted a cross-sectional study that included the following groups: 1) non-pregnant women (n = 17); 2) normal pregnant women at various gestational ages from the first trimester until term (n = 110); 3) women at term with spontaneous labor (n = 50); and 4) women with clinical chorioamnionitis at term in labor (n = 25). Plasma concentrations of CHCHD2/MNRR1 were assessed by an enzyme-linked immunosorbent assay. RESULTS: 1) Pregnant women at term in labor with clinical chorioamnionitis had a significantly higher plasma CHCHD2/MNRR1 concentration than those in labor without chorioamnionitis (p = .003); 2) CHCHD2/MNRR1 is present in the plasma of healthy non-pregnant and normal pregnant women without significant differences in its plasma concentrations between the two groups; 3) there was no correlation between maternal plasma CHCHD2/MNRR1 concentration and gestational age at venipuncture; and 4) plasma CHCHD2/MNRR1 concentration was not significantly different in women at term in spontaneous labor compared to those not in labor. CONCLUSIONS: CHCHD2/MNRR1 is physiologically present in the plasma of healthy non-pregnant and normal pregnant women, and its concentration does not change with gestational age and parturition at term. However, plasma CHCHD2/MNRR1 is elevated in women at term with clinical chorioamnionitis. CHCHD2/MNRR1, a novel bi-organellar protein located in the mitochondria and the nucleus, is released into maternal plasma during systemic inflammation.

Further Evidence that an Episode of Premature Labor Is a Pathologic State: Involvement of the Insulin-Like Growth Factor System.

INTRODUCTION: Approximately 47% of women with an episode of preterm labor deliver at term; however, their infants are at greater risk of being small for gestational age and for neurodevelopmental disorders. In these cases, a pathologic insult may disrupt the homeostatic responses sustaining pregnancy. We tested the hypothesis of an involvement of components of the insulin-like growth factor (IGF) system. METHODS: This is a cross-sectional study in which maternal plasma concentrations of pregnancy-associated plasma protease (PAPP)-A, PAPP-A2, insulin-like growth factor-binding protein 1 (IGFBP-1), and IGFBP-4 were determined in the following groups of women: (1) no episodes of preterm labor, term delivery (controls, n = 100); (2) episode of preterm labor, term delivery (n = 50); (3) episode of preterm labor, preterm delivery (n = 100); (4) pregnant women at term not in labor (n = 61); and (5) pregnant women at term in labor (n = 61). Pairwise differences in maternal plasma concentrations of PAPP-A, PAPP-A2, IGFBP-1, and IGFBP-4 among study groups were assessed by fitting linear models on log-transformed data and included adjustment for relevant covariates. Significance of the group coefficient in the linear models was assessed via t-scores, with p < 0.05 deemed a significant result. RESULTS: Compared to controls, (1) women with an episode of premature labor, regardless of a preterm or a term delivery, had higher mean plasma concentrations of PAPP-A2 and IGFBP-1 (each p < 0.05); (2) women with an episode of premature labor who delivered at term also had a higher mean concentration of PAPP-A (p < 0.05); and (3) acute histologic chorioamnionitis and spontaneous labor at term were not associated with significant changes in these analytes. CONCLUSION: An episode of preterm labor involves the IGF system, supporting the view that the premature activation of parturition is a pathologic state, even in those women who delivered at term.

Meconium-stained amniotic fluid.

Green-stained amniotic fluid, often referred to as meconium-stained amniotic fluid, is present in 5% to 20% of patients in labor and is considered an obstetric hazard. The condition has been attributed to the passage of fetal colonic content (meconium), intraamniotic bleeding with the presence of heme catabolic products, or both. The frequency of green-stained amniotic fluid increases as a function of gestational age, reaching approximately 27% in post-term gestation. Green-stained amniotic fluid during labor has been associated with fetal acidemia (umbilical artery pH <7.00), neonatal respiratory distress, and seizures as well as cerebral palsy. Hypoxia is widely considered a mechanism responsible for fetal defecation and meconium-stained amniotic fluid; however, most fetuses with meconium-stained amniotic fluid do not have fetal acidemia. Intraamniotic infection/inflammation has emerged as an important factor in meconium-stained amniotic fluid in term and preterm gestations, as patients with these conditions have a higher rate of clinical chorioamnionitis and neonatal sepsis. The precise mechanisms linking intraamniotic inflammation to green-stained amniotic fluid have not been determined, but the effects of oxidative stress in heme catabolism have been implicated. Two randomized clinical trials suggest that antibiotic administration decreases the rate of clinical chorioamnionitis in patients with meconium-stained amniotic fluid. A serious complication of meconium-stained amniotic fluid is meconium aspiration syndrome. This condition develops in 5% of cases presenting with meconium-stained amniotic fluid and is a severe complication typical of term newborns. Meconium aspiration syndrome is attributed to the mechanical and chemical effects of aspirated meconium coupled with local and systemic fetal inflammation. Routine naso/oropharyngeal suctioning and tracheal intubation in cases of meconium-stained amniotic fluid have not been shown to be beneficial and are no longer recommended in obstetrical practice. A systematic review of randomized controlled trials suggested that amnioinfusion may decrease the rate of meconium aspiration syndrome. Histologic examination of the fetal membranes for meconium has been invoked in medical legal litigation to time the occurrence of fetal injury. However, inferences have been largely based on the results of in vitro experiments, and extrapolation of such findings to the clinical setting warrants caution. Fetal defecation throughout gestation appears to be a physiologic phenomenon based on ultrasound as well as in observations in animals.

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.

A biomarker for bacteremia in pregnant women with acute pyelonephritis: soluble suppressor of tumorigenicity 2 or sST2.

Objective: Sepsis is a leading cause of maternal death, and its diagnosis during the golden hour is critical to improve survival. Acute pyelonephritis in pregnancy is a risk factor for obstetrical and medical complications, and it is a major cause of sepsis, as bacteremia complicates 15-20% of pyelonephritis episodes in pregnancy. The diagnosis of bacteremia currently relies on blood cultures, whereas a rapid test could allow timely management and improved outcomes. Soluble suppression of tumorigenicity 2 (sST2) was previously proposed as a biomarker for sepsis in non-pregnant adults and children. This study was designed to determine whether maternal plasma concentrations of sST2 in pregnant patients with pyelonephritis can help to identify those at risk for bacteremia.Study design: This cross-sectional study included women with normal pregnancy (n = 131) and pregnant women with acute pyelonephritis (n = 36). Acute pyelonephritis was diagnosed based on a combination of clinical findings and a positive urine culture. Patients were further classified according to the results of blood cultures into those with and without bacteremia. Plasma concentrations of sST2 were determined by a sensitive immunoassay. Non-parametric statistics were used for analysis.Results: The maternal plasma sST2 concentration increased with gestational age in normal pregnancies. Pregnant patients with acute pyelonephritis had a higher median (interquartile range) plasma sST2 concentration than those with a normal pregnancy [85 (47-239) ng/mL vs. 31 (14-52) ng/mL, p < .001]. Among patients with pyelonephritis, those with a positive blood culture had a median plasma concentration of sST2 higher than that of patients with a negative blood culture [258 (IQR: 75-305) ng/mL vs. 83 (IQR: 46-153) ng/mL; p = .03]. An elevated plasma concentration of sST2 ≥ 215 ng/mL had a sensitivity of 73% and a specificity of 95% (area under the receiver operating characteristic curve, 0.74; p = .003) with a positive likelihood ratio of 13.8 and a negative likelihood ratio of 0.3 for the identification of patients who had a positive blood culture.Conclusion: sST2 is a candidate biomarker to identify bacteremia in pregnant women with pyelonephritis. Rapid identification of these patients may optimize patient care.

Evidence for the participation of CHCHD2/MNRR1, a mitochondrial protein, in spontaneous labor at term and in preterm labor with intra-amniotic infection.

OBJECTIVE: Intra-amniotic inflammation (IAI), associated with either microbe (infection) or danger signals (sterile), plays a major role in the pathophysiology of preterm labor and delivery. Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 2 (CHCHD2) [also known as Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1)], a mitochondrial protein involved in oxidative phosphorylation and cell survival, is capable of sensing tissue hypoxia and inflammatory signaling. The ability to maintain an appropriate energy balance at the cellular level while adapting to environmental stress is essential for the survival of an organism. Mitochondrial dysfunction has been observed in acute systemic inflammatory conditions, such as sepsis, and is proposed to be involved in sepsis-induced multi-organ failure. The purpose of this study was to determine the amniotic fluid concentrations of CHCHD2/MNRR1 in pregnant women, women at term in labor, and those in preterm labor (PTL) with and without IAI. METHODS: This cross-sectional study comprised patients allocated to the following groups: (1) mid-trimester (n = 16); (2) term in labor (n = 37); (3) term not in labor (n = 22); (4) PTL without IAI who delivered at term (n = 25); (5) PTL without IAI who delivered preterm (n = 47); and (6) PTL with IAI who delivered preterm (n = 53). Diagnosis of IAI (amniotic fluid interleukin-6 concentration ≥2.6 ng/mL) included cases associated with microbial invasion of the amniotic cavity and those of sterile nature (absence of detectable bacteria, using culture and molecular microbiology techniques). Amniotic fluid and maternal plasma CHCHD2/MNRR1 concentrations were determined with a validated and sensitive immunoassay. RESULTS: (1) CHCHD2/MNRR1 was detectable in all amniotic fluid samples and women at term without labor had a higher amniotic fluid CHCHD2/MNRR1 concentration than those in the mid-trimester (p = 0.003); (2) the amniotic fluid concentration of CHCHD2/MNRR1 in women at term in labor was higher than that in women at term without labor (p = 0.01); (3) women with PTL and IAI had a higher amniotic fluid CHCHD2/MNRR1 concentration than those without IAI, either with preterm (p < 0.001) or term delivery (p = 0.01); (4) women with microbial-associated IAI had a higher amniotic fluid CHCHD2/MNRR1 concentration than those with sterile IAI (p < 0.001); (5) among women with PTL and IAI, the amniotic fluid concentration of CHCHD2/MNRR1 correlated with that of interleukin-6 (Spearman's Rho = 0.7; p < 0.001); and (6) no correlation was observed between amniotic fluid and maternal plasma CHCHD2/MNRR1 concentrations among women with PTL. CONCLUSION: CHCHD2/MNRR1 is a physiological constituent of human amniotic fluid in normal pregnancy, and the amniotic concentration of this mitochondrial protein increases during pregnancy, labor at term, and preterm labor with intra-amniotic infection. Hence, CHCHD2/MNRR1 may be released into the amniotic cavity by dysfunctional mitochondria during microbial-associated IAI.

Proteomic profile of extracellular vesicles in maternal plasma of women with fetal death.

OBJECTIVES: Fetal death is a complication of pregnancy caused by multiple etiologies rather than being the end-result of a single disease process. Many soluble analytes in the maternal circulation, such as hormones and cytokines, have been implicated in its pathophysiology. However, changes in the protein content of extracellular vesicles (EVs), which could provide additional insight into the disease pathways of this obstetrical syndrome, have not been examined. This study aimed to characterize the proteomic profile of EVs in the plasma of pregnant women who experienced fetal death and to evaluate whether such a profile reflected the pathophysiological mechanisms of this obstetrical complication. Moreover, the proteomic results were compared to and integrated with those obtained from the soluble fraction of maternal plasma. METHODS: This retrospective case-control study included 47 women who experienced fetal death and 94 matched, healthy, pregnant controls. Proteomic analysis of 82 proteins in the EVs and the soluble fractions of maternal plasma samples was conducted by using a bead-based, multiplexed immunoassay platform. Quantile regression analysis and random forest models were implemented to assess differences in the concentration of proteins in the EV and soluble fractions and to evaluate their combined discriminatory power between clinical groups. Hierarchical cluster analysis was applied to identify subgroups of fetal death cases with similar proteomic profiles. A p-value of <.05 was used to infer significance, unless multiple testing was involved, with the false discovery rate controlled at the 10% level (q < 0.1). All statistical analyses were performed by using the R statistical language and environment-and specialized packages. RESULTS: Nineteen proteins (placental growth factor, macrophage migration inhibitory factor, endoglin, regulated upon activation normal T cell expressed and presumably secreted (RANTES), interleukin (IL)-6, macrophage inflammatory protein 1-alpha, urokinase plasminogen activator surface receptor, tissue factor pathway inhibitor, IL-8, E-Selectin, vascular endothelial growth factor receptor 2, pentraxin 3, IL-16, galectin-1, monocyte chemotactic protein 1, disintegrin and metalloproteinase domain-containing protein 12, insulin-like growth factor-binding protein 1, matrix metalloproteinase-1(MMP1), and CD163) were found to have different plasma concentrations (of an EV or a soluble fraction) in women with fetal death compared to controls. There was a similar pattern of change for the dysregulated proteins in the EV and soluble fractions and a positive correlation between the log2-fold changes of proteins significant in either the EV or the soluble fraction (ρ = 0.89, p < .001). The combination of EV and soluble fraction proteins resulted in a good discriminatory model (area under the ROC curve, 82%; sensitivity, 57.5% at a 10% false-positive rate). Unsupervised clustering based on the proteins differentially expressed in either the EV or the soluble fraction of patients with fetal death relative to controls revealed three major clusters of patients. CONCLUSION: Pregnant women with fetal death have different concentrations of 19 proteins in the EV and soluble fractions compared to controls, and the direction of changes in concentration was similar between fractions. The combination of EV and soluble protein concentrations revealed three different clusters of fetal death cases with distinct clinical and placental histopathological characteristics.

The amniotic fluid proteome changes with term labor and informs biomarker discovery in maternal plasma.

The intra-uterine components of labor, namely, myometrial contractility, cervical ripening, and decidua/membrane activation, have been extensively characterized and involve a local pro-inflammatory milieu of cellular and soluble immune mediators. Targeted profiling has demonstrated that such processes extend to the intra-amniotic space, yet unbiased analyses of the proteome of human amniotic fluid during labor are lacking. Herein, we utilized an aptamer-based platform to characterize 1,310 amniotic fluid proteins and found that the proteome undergoes substantial changes with term labor (251 proteins with differential abundance, q < 0.1, and fold change > 1.25). Proteins with increased abundance in labor are enriched for immune and inflammatory processes, consistent with prior reports of labor-associated changes in the intra-uterine space. By integrating the amniotic fluid proteome with previously generated placental-derived single-cell RNA-seq data, we demonstrated the labor-driven upregulation of signatures corresponding to stromal-3 and decidual cells. We also determined that changes in amniotic fluid protein abundance are reflected in the maternal plasma proteome. Collectively, these findings provide novel insights into the amniotic fluid proteome in term labor and support its potential use as a source of biomarkers to distinguish between true and false labor by using maternal blood samples.

Acute pyelonephritis in pregnancy and plasma syndecan-1: evidence of glycocalyx involvement.

BACKGROUND: Acute pyelonephritis, a risk factor for maternal sepsis, adult respiratory distress syndrome, and preterm labor, is a frequent cause of hospitalization. This condition is characterized by excessive intravascular inflammation and endothelial cell activation and dysfunction. Syndecan-1, a major component of the glycocalyx, is a gel-like layer that covers the luminal surface of healthy endothelial cells, preserving and mediating many endothelial functions. During pregnancy, there is an additional potential source of syndecan-1, the "syncytiotrophoblast glycocalyx," which lines the intervillous space. Insults that damage the glycocalyx lead to a shedding of syndecan-1 into the circulation. Hence, syndecan-1 has been proposed as a marker of endothelial injury in conditions such as sepsis, trauma, cardiovascular disease, and diabetes mellitus. OBJECTIVE: The objective of this study was to determine whether the plasma syndecan-1 concentration changes in women with acute pyelonephritis in the presence or absence of bacteremia. STUDY DESIGN: This cross-sectional study included three groups: (1) non-pregnant women (n = 25); (2) women with an uncomplicated pregnancy from whom samples were collected preterm (n = 61) or at term (n = 69); and (3) pregnant women diagnosed with acute pyelonephritis from whom samples were collected at the time of diagnosis during the second and third trimesters (n = 33). The diagnosis of acute pyelonephritis was based on clinical findings and a positive urine culture for bacteria. Blood culture results were available in 85% (28/33) of women with acute pyelonephritis. Plasma concentrations of syndecan-1 were determined by a validated immunoassay. RESULTS: (1) Women with an uncomplicated pregnancy had a higher plasma concentration of syndecan-1 than non-pregnant women. The geometric mean (95% confidence interval [CI]) of syndecan-1 concentration was 51.0 (12.1-216.1) ng/mL in non-pregnant controls; 1280 (365-4487) ng/mL in normal preterm gestations; and 1786 (546-5834) ng/mL in normal term gestations (adjusted p < .005 for all three between group comparisons); (2) plasma syndecan-1 concentrations increased with gestational age among women with a normal pregnancy (p < .001, R2 = 0.27); (3) syndecan-1 multiple of the mean (MoM) values in pregnant patients with acute pyelonephritis were higher than those in normal pregnant women based on second- and third-trimester samples (p = .048, 1.26-fold change). The increase was driven primarily by cases with a positive blood culture (p = .009, 1.74-fold change); (4) when data from third-trimester samples were compared, overall differences in syndecan-1 MoM values between cases and controls were slightly larger (p = .03, 1.36- fold change), which were especially contributed to by cases with a positive blood culture (p = .023, fold change 1.79-fold change). CONCLUSIONS: Plasma syndecan-1 concentration is higher in pregnant women and increases as a function of gestational age. Patients with acute pyelonephritis have a higher plasma concentration of syndecan-1, and this is particularly the case in the presence of bacteremia.

Maternal plasma syndecan-1: a biomarker for fetal growth restriction.

OBJECTIVE: The identification of fetal growth disorders is an important clinical priority given that they increase the risk of perinatal morbidity and mortality as well as long-term diseases. A subset of small-for-gestational-age (SGA) infants are growth-restricted, and this condition is often attributed to placental insufficiency. Syndecan-1, a product of the degradation of the endothelial glycocalyx, has been proposed as a biomarker of endothelial damage in different pathologies. During pregnancy, a "specialized" form of the glycocalyx-the "syncytiotrophoblast glycocalyx"-covers the placental villi. The purpose of this study was to determine whether the concentration of maternal plasma syndecan-1 can be proposed as a biomarker for fetal growth restriction. STUDY DESIGN: A cross-sectional study was designed to include women with normal pregnancy (n = 130) and pregnant women who delivered an SGA neonate (n = 50). Doppler velocimetry of the uterine and umbilical arteries was performed in women with an SGA fetus at the time of diagnosis. Venipuncture was performed within 48 h of Doppler velocimetry and plasma concentrations of syndecan-1 were determined by a specific and sensitive immunoassay. RESULTS: (1) Plasma syndecan-1 concentration followed a nonlinear increase with gestational age in uncomplicated pregnancies (R2 = 0.27, p < .001); (2) women with a pregnancy complicated with an SGA fetus had a significantly lower mean plasma concentration of syndecan-1 than those with an appropriate-for-gestational-age fetus (p = .0001); (3) this difference can be attributed to fetal growth restriction, as the mean plasma syndecan-1 concentration was significantly lower only in the group of women with an SGA fetus who had abnormal umbilical and uterine artery Doppler velocimetry compared to controls (p = .00071; adjusted p = .0028). A trend toward lower syndecan-1 concentrations was also noted for SGA with abnormal uterine but normal umbilical artery Doppler velocimetry (p = .0505; adjusted p = .067); 4) among women with an SGA fetus, those with abnormal umbilical and uterine artery Doppler findings had a lower mean plasma syndecan-1 concentration than women with normal Doppler velocimetry (p = .02; adjusted p = .04); 5) an inverse relationship was found between the maternal plasma syndecan-1 concentration and the umbilical artery pulsatility index (r = -0.5; p = .003); and 6) a plasma syndecan-1 concentration ≤ 850 ng/mL had a positive likelihood ratio of 4.4 and a negative likelihood ratio of 0.24 for the identification of a mother with an SGA fetus who had abnormal umbilical artery Doppler velocimetry (area under the ROC curve 0.83; p < .001). CONCLUSION: Low maternal plasma syndecan-1 may reflect placental diseases and this protein could be a biomarker for fetal growth restriction. However, as a sole biomarker for this condition, its accuracy is low.

Soluble suppression of tumorigenicity-2 in pregnancy with a small-for-gestational-age fetus and with preeclampsia.

OBJECTIVE: Preeclampsia and fetal growth disorders are pregnancy-specific conditions that share common pathophysiological mechanisms. Yet, why some patients develop preeclampsia while others experience fetal growth restriction, or a combination of both clinical presentations, is unknown. We propose that the difference in severity of the maternal inflammatory response can contribute to the clinical phenotypes of preeclampsia vs. small for gestational age (SGA). To assess this hypothesis, we measured maternal plasma concentrations of the soluble isoform of suppression of tumorigenicity-2 (sST2), a member of the interleukin-1 receptor family that buffers proinflammatory responses. Previous reports showed that serum sST2 concentrations rise in the presence of intravascular inflammation and Th1-type immune responses and are significantly higher in patients with preeclampsia compared to those with normal pregnancy. The behavior of sST2 in pregnancies complicated by SGA has not been reported. This study was conducted to compare sST2 plasma concentrations in normal pregnancies, in those with preeclampsia, and in those with an SGA fetus. METHODS: This retrospective cross-sectional study included women with an SGA fetus (n = 52), women with preeclampsia (n = 106), and those with normal pregnancy (n = 131). Maternal plasma concentrations of sST2 were determined by enzyme-linked immunosorbent assay. Doppler velocimetry of the uterine and umbilical arteries was available in a subset of patients with SGA (42 patients and 43 patients, respectively). RESULTS: (1) Women with an SGA fetus had a significantly higher median plasma concentration of sST2 than normal pregnant women (p = .008); (2) women with preeclampsia had a significantly higher median plasma concentration of sST2 than those with normal pregnancy (p < .001) and those with an SGA fetus (p < .001); (3) patients with SGA and abnormal uterine artery Doppler velocimetry had a higher median plasma concentration of sST2 than controls (p < .01) and those with SGA and normal uterine artery Doppler velocimetry (p = .02); (4) there was no significant difference in the median plasma sST2 concentration between patients with SGA who had normal uterine artery Doppler velocimetry and controls (p = .4); (5) among patients with SGA, those with abnormal and those with normal umbilical artery Doppler velocimetry had higher median plasma sST2 concentrations than controls (p = .001 and p = .02, respectively); and (6) there was no significant difference in the median plasma sST2 concentrations between patients with SGA who did and those who did not have abnormal umbilical artery Doppler velocimetry (p = .06). CONCLUSIONS: Preeclampsia and disorders of fetal growth are conditions characterized by intravascular inflammation, as reflected by maternal plasma concentrations of sST2. The severity of intravascular inflammation is highest in patients with preeclampsia.

Perturbations in kinetics of the thrombin generation assay identify women at risk of preeclampsia in the first trimester and provide the rationale for a preventive approach.

BACKGROUND: Activation of the coagulation system and increased thrombin generation have been implicated in the pathophysiology of preeclampsia, and this rationale supports the administration of low-molecular-weight heparin to prevent this syndrome in patients at risk. Yet, randomized trials of this prophylactic measure have yielded contradictory results. A possible explanation is that only a subset of patients with preeclampsia have excessive thrombin generation and would benefit from the administration of low-molecular-weight heparin. Therefore, the key questions are whether and when patients who subsequently develop preeclampsia present evidence of abnormal thrombin generation. OBJECTIVE: This study aimed to determine (1) the kinetics of thrombin generation throughout gestation in women with a normal pregnancy and in those with early and late preeclampsia, and (2) the diagnostic performance of in vivo thrombin generation parameters to predict the development of preeclampsia. STUDY DESIGN: This retrospective, nested case-control study was based on a prospective longitudinal cohort of singleton gestations. Cases comprised women who developed preeclampsia (n=49), and controls consisted of patients with a normal pregnancy (n=45). Preeclampsia was classified into early-onset (n=24) and late-onset (n=25). Longitudinal changes in the parameters of the thrombin generation assay (lag time, time to peak thrombin concentration, peak thrombin concentration, endogenous thrombin generation, and velocity index) throughout gestation were compared between the study groups, and normal pregnancy percentiles were derived from the control group. We tested whether a single parameter or a combination of parameters, derived from the kinetics of thrombin generation, could identify patients who subsequently developed preeclampsia. Time-related parameters <10th percentile were considered short, and concentration-related parameters >90th percentile were considered high. RESULTS: (1) Patients who developed preeclampsia (early- and late-onset) had abnormal thrombin generation kinetics as early as 8 to 16 weeks of pregnancy; (2) patients with a combination of a short lag time and high peak thrombin concentration at 8 to 16 weeks of pregnancy had an odds ratio of 43.87 for the subsequent development of preeclampsia (area under the curve, 0.79; sensitivity, 56.8%; specificity, 92.7%; positive likelihood ratio, 7.76); (3) at 16 to 22 weeks of gestation, patients with a combination of a short lag time and a high velocity index had an odds ratio of 16 for the subsequent development of preeclampsia (area under the curve, 0.78; sensitivity, 62.2%; specificity, 92.5%; positive likelihood ratio, 8.29). CONCLUSION: During early pregnancy, the thrombin generation assay can identify the subset of patients at a greater risk for the development of preeclampsia owing to accelerated and enhanced production of thrombin. This observation provides a rationale for testing the efficacy of low-molecular-weight heparin in this subset of patients. We propose that future research on the efficacy of low-molecular-weight heparin and other interventions targeting the coagulation system to prevent preeclampsia should be focused on patients with abnormal kinetics of thrombin generation.

One-third of patients with eclampsia at term do not have an abnormal angiogenic profile.

OBJECTIVES: An abnormal angiogenic profile is present in about one-half of women with preeclampsia at term. Few studies examined the roles of angiogenic biomarkers in eclampsia. The aims of this study were to determine (1) whether the degree of an anti-angiogenic state, reflected by a low placental growth factor (PlGF) to soluble fms-like tyrosine kinase-1 (sFlt-1) ratio, in women with eclampsia differed from that of women with severe preeclampsia; and (2) the prevalence of women who had an abnormal angiogenic profile at the diagnoses of preterm and term eclampsia. METHODS: A cross-sectional study was conducted to include women in the following groups: (1) uncomplicated pregnancy (n=40); (2) severe preeclampsia (n=50); and (3) eclampsia (n=35). Maternal serum concentrations of PlGF and sFlt-1 were determined by immunoassays. RESULTS: Women with preterm, but not term, eclampsia had a more severe anti-angiogenic state than those with severe preeclampsia (lower PlGF and PlGF/sFlt-1 ratio, each p<0.05). However, the difference diminished in magnitude with increasing gestational age (interaction, p=0.005). An abnormal angiogenic profile was present in 95% (19/20) of women with preterm eclampsia but in only 67% (10/15) of women with eclampsia at term. CONCLUSIONS: Angiogenic biomarkers can be used for risk assessment of preterm eclampsia. By contrast, a normal profile of angiogenic biomarkers cannot reliably exclude patients at risk for eclampsia at term. This observation has major clinical implications given that angiogenic biomarkers are frequently used in the triage area as a test to rule out preeclampsia.

Preeclampsia at term can be classified into 2 clusters with different clinical characteristics and outcomes based on angiogenic biomarkers in maternal blood.

BACKGROUND: An antiangiogenic state has emerged as a mechanism of disease in preeclampsia. Angiogenic biomarkers are used in the risk assessment of this syndrome, particularly of early disease. The role of an antiangiogenic state in late preeclampsia is unclear. OBJECTIVE: This study aimed to determine the prevalence, characteristics, and clinical significance of angiogenic/antiangiogenic factor abnormalities in women with preeclampsia stratified according to gestational age at delivery. STUDY DESIGN: Two studies were conducted: (1) a longitudinal nested case-control study comprising women with preeclampsia (n=151) and a control group (n=540); and (2) a case series of patients with preeclampsia (n=452). In patients with preeclampsia, blood was collected at the time of diagnosis. Plasma concentrations of placental growth factor and soluble fms-like tyrosine kinase-1 were determined by enzyme-linked immunosorbent assays. An abnormal angiogenic profile was defined as a plasma ratio of placental growth factor and soluble fms-like tyrosine kinase-1 expressed as a multiple of the median <10th percentile for gestational age based on values derived from the longitudinal study. The proportion of patients diagnosed with preeclampsia who had an abnormal angiogenic profile was determined in the case-series participants and stratified by gestational age at delivery into early (≤34 weeks), intermediate (34.1-36.9 weeks), and term (≥37 weeks) preeclampsia. The demographics, clinical characteristics, and pregnancy outcomes of women with preeclampsia with and without an abnormal angiogenic profile were compared. RESULTS: The prevalence of an abnormal angiogenic profile was higher in preterm than in term preeclampsia (for early, intermediate, and term in the case-control study: 90%, 100%, and 39%; for the case series: 98%, 80%, and 55%, respectively). Women with preeclampsia at term who had an abnormal angiogenic profile were more frequently nulliparous (57% vs 35%), less likely to smoke (14% vs 26%), at greater risk for maternal (14% vs 5%) or neonatal (7% vs 1%) complications, and more often had placental lesions consistent with maternal vascular malperfusion (42% vs 23%; all, P<.05) than those without an abnormal profile. Women with preeclampsia at term who had a normal angiogenic profile had a higher frequency of chronic hypertension (36% vs 21%) and were more likely to have class ≥2 obesity (41% vs 23%) than those with an abnormal profile (both, P<.05). CONCLUSION: Patients with early preeclampsia had an abnormal angiogenic profile in virtually all cases, whereas only 50% of women with preeclampsia at term had such abnormalities. The profile of angiogenic biomarkers can be used to classify patients with preeclampsia at term, on the basis of mechanisms of disease, into 2 clusters, which have different demographics, clinical characteristics, and risks of adverse maternal and neonatal outcomes. These findings provide a simple approach to classify preeclampsia at term and have implications for future clinical care and research.

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.

Maternal plasma cytokines and the subsequent risk of uterine atony and postpartum hemorrhage.

OBJECTIVES: To determine whether the maternal plasma concentrations of cytokines are higher in pregnant women with postpartum hemorrhage (PPH) compared to pregnant women without PPH. METHODS: A retrospective case-control study included 36 women with PPH and 72 matched controls. Cases and controls were matched for gestational age at delivery, labor status, delivery route, parity, and year of sample collection. Maternal plasma samples were collected up to 3 days prior to delivery. Comparison of the plasma concentrations of 29 cytokines was performed by using linear mixed-effects models and included adjustment for covariates and multiple testing. A false discovery rate adjusted p-value <0.1 was used to infer significance. Random forest models with evaluation by leave-one-out and 9-fold cross-validation were used to assess the combined value of the proteins in predicting PPH. RESULTS: Concentrations of interleukin (IL)-16, IL-6, IL-12/IL-23p40, monocyte chemotactic protein 1 (MCP-1), and IL-1ß were significantly higher in PPH than in the control group. This difference remained significant after adjustment for maternal age, clinical chorioamnionitis, and preeclampsia. Multi-protein random forest proteomics models had moderate cross-validated accuracy for prediction of PPH [area under the ROC curve, 0.69 (0.58-0.81) by leave-one-out cross validation and 0.73 (0.65-0.81) by 9-fold cross-validation], and the inclusion of clinical and demographic information did not increase the prediction performance. CONCLUSIONS: Pregnant women with severe PPH had higher median maternal plasma concentrations of IL-16, IL-6, IL-12/IL-23p40, MCP-1, and IL-1ß than patients without PPH. These cytokines could serve as biomarkers or their pathways may be therapeutic targets.