Blockade of IL-6R prevents preterm birth and adverse neonatal outcomes.

BACKGROUND: Preterm birth preceded by spontaneous preterm labour often occurs in the clinical setting of sterile intra-amniotic inflammation (SIAI), a condition that currently lacks treatment. METHODS: Proteomic and scRNA-seq human data were analysed to evaluate the role of IL-6 and IL-1α in SIAI. A C57BL/6 murine model of SIAI-induced preterm birth was developed by the ultrasound-guided intra-amniotic injection of IL-1α. The blockade of IL-6R by using an aIL-6R was tested as prenatal treatment for preterm birth and adverse neonatal outcomes. QUEST-MRI evaluated brain oxidative stress in utero. Targeted transcriptomic profiling assessed maternal, foetal, and neonatal inflammation. Neonatal biometrics and neurodevelopment were tested. The neonatal gut immune-microbiome was evaluated using metagenomic sequencing and immunophenotyping. FINDINGS: IL-6 plays a critical role in the human intra-amniotic inflammatory response, which is associated with elevated concentrations of the alarmin IL-1α. Intra-amniotic injection of IL-1α resembles SIAI, inducing preterm birth (7% vs. 50%, p = 0.03, Fisher's exact test) and neonatal mortality (18% vs. 56%, p = 0.02, Mann-Whitney U-test). QUEST-MRI revealed no foetal brain oxidative stress upon in utero IL-1α exposure (p > 0.05, mixed linear model). Prenatal treatment with aIL-6R abrogated IL-1α-induced preterm birth (50% vs. 7%, p = 0.03, Fisher's exact test) by dampening inflammatory processes associated with the common pathway of labour. Importantly, aIL-6R reduces neonatal mortality (56% vs. 22%, p = 0.03, Mann-Whitney U-test) by crossing from the mother to the amniotic cavity, dampening foetal organ inflammation and improving growth. Beneficial effects of prenatal IL-6R blockade carried over to neonatal life, improving survival, growth, neurodevelopment, and gut immune homeostasis. INTERPRETATION: IL-6R blockade can serve as a strategy to treat SIAI, preventing preterm birth and adverse neonatal outcomes. FUNDING: NICHD/NIH/DHHS, Contract HHSN275201300006C. WSU Perinatal Initiative in Maternal, Perinatal and Child Health.

A single-cell atlas of murine reproductive tissues during preterm labor.

Preterm birth, the leading cause of perinatal morbidity and mortality worldwide, frequently results from the syndrome of preterm labor. The best-established causal link to preterm labor is intra-amniotic infection, which involves premature activation of the parturition cascade in the reproductive tissues. Herein, we utilize single-cell RNA sequencing (scRNA-seq) to generate a single-cell atlas of the murine uterus, decidua, and cervix in a model of infection-induced preterm labor. We show that preterm labor affects the transcriptomic profiles of specific immune and non-immune cell subsets. Shared and tissue-specific gene expression signatures are identified among affected cells. Determination of intercellular communications implicates specific cell types in preterm labor-associated signaling pathways across tissues. In silico comparison of murine and human uterine cell-cell interactions reveals conserved signaling pathways implicated in labor. Thus, our scRNA-seq data provide insights into the preterm labor-driven cellular landscape and communications in reproductive tissues.

Clarithromycin prevents preterm birth and neonatal mortality by dampening alarmin-induced maternal-fetal inflammation in mice.

BACKGROUND: One of every four preterm neonates is born to a woman with sterile intra-amniotic inflammation (inflammatory process induced by alarmins); yet, this clinical condition still lacks treatment. Herein, we utilized an established murine model of sterile intra-amniotic inflammation induced by the alarmin high-mobility group box-1 (HMGB1) to evaluate whether treatment with clarithromycin prevents preterm birth and adverse neonatal outcomes by dampening maternal and fetal inflammatory responses. METHODS: Pregnant mice were intra-amniotically injected with HMGB1 under ultrasound guidance and treated with clarithromycin or vehicle control, and pregnancy and neonatal outcomes were recorded (n = 15 dams each). Additionally, amniotic fluid, placenta, uterine decidua, cervix, and fetal tissues were collected prior to preterm birth for determination of the inflammatory status (n = 7-8 dams each). RESULTS: Clarithromycin extended the gestational length, reduced the rate of preterm birth, and improved neonatal mortality induced by HMGB1. Clarithromycin prevented preterm birth by interfering with the common cascade of parturition as evidenced by dysregulated expression of contractility-associated proteins and inflammatory mediators in the intra-uterine tissues. Notably, clarithromycin improved neonatal survival by dampening inflammation in the placenta as well as in the fetal lung, intestine, liver, and spleen. CONCLUSIONS: Clarithromycin prevents preterm birth and improves neonatal survival in an animal model of sterile intra-amniotic inflammation, demonstrating the potential utility of this macrolide for treating women with this clinical condition, which currently lacks a therapeutic intervention.

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.

Betamethasone as a potential treatment for preterm birth associated with sterile intra-amniotic inflammation: a murine study.

OBJECTIVES: Preterm birth remains the leading cause of perinatal morbidity and mortality worldwide. Preterm birth is preceded by spontaneous preterm labor, which is commonly associated with sterile intra-amniotic inflammation; yet, no approved treatment exists for this clinical condition. Corticosteroids are the standard of care to improve neonatal outcomes in women at risk of preterm birth. Herein, we first validated our model of alarmin-induced preterm birth. Next, we investigated whether treatment with betamethasone could prevent preterm birth resulting from sterile intra-amniotic inflammation in mice. METHODS: Under ultrasound guidance, the first cohort of dams received an intra-amniotic injection of the alarmin high-mobility group box-1 (HMGB1, n=10) or phosphate-buffered saline (PBS, n=9) as controls. A second cohort of dams received HMGB1 intra-amniotically and were subcutaneously treated with betamethasone (n=15) or vehicle (n=15). Dams were observed until delivery, and perinatal outcomes were observed. RESULTS: Intra-amniotic HMGB1 reduced gestational length (p=0.04), inducing preterm birth in 40% (4/10) of cases, of which 100% (4/4) were categorized as late preterm births. Importantly, treatment with betamethasone extended the gestational length (p=0.02), thereby reducing the rate of preterm birth by 26.6% (from 33.3% [5/15] to 6.7% [1/15]). Treatment with betamethasone did not worsen the rate of neonatal mortality induced by HMGB1 or alter weight gain in the first three weeks of life. CONCLUSIONS: Treatment with betamethasone prevents preterm birth induced by the alarmin HMGB1. This study supports the potential utility of betamethasone for treating women with sterile intra-amniotic inflammation.

Clinical chorioamnionitis at term X: microbiology, clinical signs, placental pathology, and neonatal bacteremia - implications for clinical care.

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.

Gasdermin D: in vivo evidence of pyroptosis in spontaneous labor at term.

Objective: Pyroptosis is an inflammatory form of programmed cell death that is mediated by the activation of the inflammasome and depends on the pore-forming function of gasdermin D. Therefore, the detection of gasdermin D represents in vivo evidence of pyroptosis. We recently showed that there is intra-amniotic inflammasome activation in spontaneous labor at term; however, evidence of pyroptosis is lacking. The objectives of this study were to investigate (1) whether gasdermin D is detectable in the amniotic fluid of women who delivered at term; (2) whether amniotic fluid gasdermin D concentrations are associated with the process of spontaneous labor at term; and (3) whether gasdermin D is expressed in the chorioamniotic membranes from these patients.Methods: This retrospective cross-sectional study included amniotic fluid samples from 41 women who underwent spontaneous labor at term (n = 17) or delivered at term without labor (n = 24). As a readout of pyroptosis, gasdermin D was determined in amniotic fluid samples using a specific and sensitive ELISA kit. The 90th percentile of amniotic fluid gasdermin D concentrations was calculated among women without spontaneous labor at term (reference group). The association between high amniotic fluid gasdermin D concentrations (≥90th percentile in the reference group) and spontaneous labor at term was tested using the Fisher's exact test. A p value <.05 was considered significant. Multiplex immunofluorescence staining and phenoptics (multispectral imaging) were performed to determine gasdermin D expression in the chorioamniotic membranes and to colocalize this protein with the inflammasome-related molecules caspase-1 and interleukin-1ß.Results: (1) Gasdermin D is present in the amniotic fluid of women who delivered at term; (2) the 90th percentile of amniotic fluid gasdermin D concentrations in women who delivered at term without spontaneous labor was 3.4 ng/mL; (3) the proportion of women with amniotic fluid gasdermin D concentrations above the threshold was higher in those who underwent term labor than in those who delivered at term without labor; (4) amniotic fluid concentrations of gasdermin D > 3.4 ng/mL were significantly associated with the presence of spontaneous labor in women who delivered at term (odds ratio 6.0, p-value .048); and (5) the protein expression of gasdermin D is increased in the chorioamniotic membranes of women who underwent spontaneous labor at term and is colocalized with caspase-1 and IL-1ß.Conclusions: Gasdermin D is increased in the amniotic fluid and chorioamniotic membranes of women who underwent spontaneous labor at term compared to those without labor. These data provide evidence implicating pyroptosis in the mechanisms that lead to the sterile inflammatory process of term parturition.

A Protocol for Evaluating Vital Signs and Maternal-Fetal Parameters Using High-Resolution Ultrasound in Pregnant Mice.

Pregnancy is a unique physiological state in which two individuals coexist: the mother and the fetus. Disruption of maternal-fetal crosstalk leads to pregnancy and neonatal pathologies. Therefore, assessing maternal-fetal well-being is essential for understanding the physiological and pathological processes occurring during pregnancy. Herein, we provide a protocol that allows for the determination of body temperature, blood pressure, and the evaluation of uterine and umbilical arteries as well as maternal and fetal heart rate using high-resolution ultrasound in pregnant mice. For complete details on the use and execution of this protocol, please refer to Gomez-Lopez et al. (2020).

Cellular immune responses in amniotic fluid of women with preterm prelabor rupture of membranes.

Background Preterm birth is the leading cause of perinatal morbidity and mortality. Preterm prelabor rupture of membranes (pPROM) occurs in 30% of preterm births; thus, this complication is a major contributor to maternal and neonatal morbidity. However, the cellular immune responses in amniotic fluid of women with pPROM have not been investigated. Methods Amniotic fluid samples were obtained from women with pPROM and a positive (n = 7) or negative (n = 10) microbiological culture. Flow cytometry was performed to evaluate the phenotype and number of amniotic fluid leukocytes. The correlation between amniotic fluid immune cells and an interleukin-6 (IL-6) concentration or a white blood cell (WBC) count in amniotic fluid was calculated. Results Women with pPROM and a positive amniotic fluid culture had (1) a greater number of total leukocytes in amniotic fluid, including neutrophils and monocytes/macrophages and (2) an increased number of total T cells in amniotic fluid, namely CD4+ T cells and CD8+ T cells, but not B cells. The numbers of neutrophils and monocytes/macrophages were positively correlated with IL-6 concentrations and WBC counts in amniotic fluid of women with pPROM. Conclusion Women with pPROM and a positive amniotic fluid culture exhibit a more severe cellular immune response than those with a negative culture, which is associated with well-known markers of intra-amniotic inflammation.

Cell-Free Fetal DNA Increases Prior to Labor at Term and in a Subset of Preterm Births.

Cell-free fetal DNA in the maternal circulation has been associated with the onset of labor at term. Moreover, clinical studies have suggested that cell-free fetal DNA has value to predict pregnancy complications such as spontaneous preterm labor leading to preterm birth. However, a mechanistic link between cell-free fetal DNA and preterm labor and birth has not been established. Herein, using an allogeneic mouse model in which a paternal green fluorescent protein (GFP) can be tracked in the fetuses, we established that cell-free fetal DNA (Egfp) concentrations were higher in late gestation compared to mid-pregnancy and were maintained at increased levels during the onset of labor at term, followed by a rapid decrease after birth. A positive correlation between cell-free fetal DNA concentrations and the number of GFP-positive pups was also observed. The increase in cell-free fetal DNA concentrations prior to labor at term was not linked to a surge in any specific cytokine/chemokine; yet, specific chemokines (i.e., CCL2, CCL7, and CXCL2) increased as gestation progressed and maintained elevated levels in the postpartum period. In addition, cell-free fetal DNA concentrations increased prior to systemic inflammation-induced preterm birth, which was associated with a strong cytokine response in the maternal circulation. However, cell-free fetal DNA concentrations were not increased prior to intra-amniotic inflammation-induced preterm birth, but in this model, a mild inflammatory response was observed in the maternal circulation. Collectively, these findings suggest that an elevation in cell-free fetal DNA concentrations in the maternal circulation precedes the physiological process of labor at term and the pathological process of preterm labor linked with systemic inflammation, but not that associated with intra-amniotic inflammation.

Human ß-defensin-3 participates in intra-amniotic host defense in women with labor at term, spontaneous preterm labor and intact membranes, and preterm prelabor rupture of membranes.

Objective: Human ß-defensin-3 (HBD-3) has a broad spectrum of antimicrobial activity, and activity and, therefore, plays a central role in host defense mechanisms against infection. Herein, we determined whether HBD-3 was a physiological constituent of amniotic fluid during midtrimester and at term and whether the concentration of this defensin was increased in amniotic fluid of women with spontaneous preterm labor and intact membranes and those with preterm prelabor rupture of membranes (pPROM) with intra-amniotic inflammation or intra-amniotic infection.Methods: Amniotic fluid was collected from 219 women in the following groups: (1) midtrimester who delivered at term (n = 35); (2) with or without spontaneous labor at term (n = 50); (3) spontaneous preterm labor with intact membranes who delivered at term (n = 29); (4) spontaneous preterm labor with intact membranes who delivered preterm with or without intra-amniotic inflammation or intra-amniotic infection (n = 69); and (5) pPROM with or without intra-amniotic infection (n = 36). Amniotic fluid HBD-3 concentrations were determined using a sensitive and specific ELISA kit.Results: (1) HBD-3 is a physiological constituent of amniotic fluid; (2) the amniotic fluid concentration of HBD-3 did not change with gestational age (midtrimester versus term not in labor); (3) amniotic fluid concentrations of HBD-3 were higher in women with spontaneous labor at term than in those without labor; (4) in the absence of intra-amniotic inflammation, amniotic fluid concentrations of HBD-3 were similar between women with spontaneous preterm labor who delivered preterm and those who delivered at term; (5) among patients with spontaneous preterm labor who delivered preterm, amniotic fluid concentrations of HBD-3 were greater in women with intra-amniotic infection than in those without this clinical condition; (6) among patients with spontaneous preterm labor, amniotic fluid concentrations of HBD-3 were higher in women with intra-amniotic inflammation or intra-amniotic infection who delivered preterm than in those without these clinical conditions who delivered at term; and (7) women with pPROM and intra-amniotic infection had higher median amniotic fluid concentrations of HBD-3 than those without this clinical condition.Conclusion: Human ß-defensin-3 is a physiological constituent of amniotic fluid and increases during the process of labor at term. Amniotic fluid concentrations of HBD-3 were increased in women with spontaneous preterm labor with intact membranes or pPROM with intra-amniotic inflammation or intra-amniotic infection, indicating that this defensin participates in the host defense mechanisms in the amniotic cavity against microorganisms or danger signals. These findings provide insight into the soluble host defense mechanisms against intra-amniotic inflammation and intra-amniotic infection.

ELABELA plasma concentrations are increased in women with late-onset preeclampsia.

Objective: ELABELA is a newly discovered peptide hormone that appears to be implicated in the mechanisms leading to preeclampsia, independently of angiogenic factors. The aim of the current study was to investigate whether women with early- or late-onset preeclampsia have altered ELABELA plasma concentrations compared to gestational-age-matched normal pregnant women.Methods: This retrospective cross-sectional study focused on the maternal plasma samples collected from 232 women with a singleton pregnancy who were allocated into the following groups: (1) early-onset preeclampsia (<34 weeks of gestation, N = 56); (2) late-onset preeclampsia (≥34 weeks of gestation, N = 57); and (3) gestational-age-matched controls with a normal pregnancy [(<34 weeks of gestation, N = 59); (≥34 weeks of gestation, N = 60)]. ELABELA plasma concentrations were determined using a validated enzyme immunoassay.Results: (1) ELABELA plasma concentrations are higher in patients with late-onset preeclampsia compared with those from gestational-age-matched controls with a normal pregnancy [median: 7.99 ng/mL (IQR, 5.3-13.95 ng/mL) versus median: 4.17 ng/mL (IQR, 3-11.19 ng/mL), p =.001]; (2) ELABELA plasma concentrations in patients with early-onset preeclampsia do not differ from those of normal pregnant women [median: 6.09 ng/mL (IQR, 2.8-10.66 ng/mL) versus median: 4.02 ng/mL (IQR, 3.26-7.49), p = .32]; and (3) ELABELA plasma concentrations are higher in patients with late-onset preeclampsia compared to those with early-onset preeclampsia [median: 7.99 ng/mL (IQR, 5.3-13.95 ng/mL) versus median: 6.09 ng/mL (IQR, 2.8-10.66 ng/mL), p = .01].Conclusion: ELABELA plasma concentrations are higher in patients with late-onset preeclampsia than in those with a normal pregnancy. However, women with early-onset preeclampsia have similar ELABELA plasma concentrations to those with a normal pregnancy. These findings provide insight into the ELABELA axis during the human syndrome of preeclampsia. In addition, these data support the concept that different pathophysiologic mechanisms are implicated in early- and late-onset preeclampsia.

Evidence that intra-amniotic infections are often the result of an ascending invasion - a molecular microbiological study.

Background Microbial invasion of the amniotic cavity resulting in intra-amniotic infection is associated with obstetrical complications such as preterm labor with intact or ruptured membranes, cervical insufficiency, as well as clinical and histological chorioamnionitis. The most widely accepted pathway for intra-amniotic infection is the ascension of microorganisms from the lower genital tract. However, hematogenous dissemination of microorganisms from the oral cavity or intestine, retrograde seeding from the peritoneal cavity through the fallopian tubes, and introduction through invasive medical procedures have also been suggested as potential pathways for intra-amniotic infection. The primary reason that an ascending pathway is viewed as most common is that the microorganisms most often detected in the amniotic fluid are those that are typical inhabitants of the vagina. However, thus far, no studies have shown that microorganisms in the amniotic cavity are simultaneously present in the vagina of the woman from which they were isolated. The objective of the study was to determine the frequency with which microorganisms isolated from women with intra-amniotic infection are also present in the lower genital tract. Methods This was a cross-sectional study of women with intra-amniotic infection with intact membranes. Intra-amniotic infection was defined as a positive culture and elevated concentrations of interleukin-6 (IL-6) (>2.6 ng/mL) in amniotic fluid and/or acute histologic chorioamnionitis and funisitis. Microorganisms isolated from bacterial cultures of amniotic fluid were taxonomically identified through matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and 16S ribosomal RNA (rRNA) gene sequencing. Vaginal swabs were obtained at the time of amniocentesis for the identification of microorganisms in the lower genital tract. The overall bacterial profiles of amniotic fluids and vaginal swabs were characterized through 16S rRNA gene sequencing. The bacterial profiles of vaginal swabs were interrogated for the presence of bacteria cultured from amniotic fluid and for the presence of prominent (>1% average relative abundance) operational taxonomic units (OTUs) within the overall 16S rRNA gene bacterial profiles of amniotic fluid. Results (1) A total of 75% (6/8) of women had bacteria cultured from their amniotic fluid that are typical residents of the vaginal ecosystem. (2) A total of 62.5% (5/8) of women with bacteria cultured from their amniotic fluid also had these bacteria present in their vagina. (3) The microorganisms cultured from amniotic fluid and also detected in the vagina were Ureaplasma urealyticum, Escherichia coli, and Streptococcus agalactiae. (4) 16S rRNA gene sequencing revealed that the amniotic fluid of women with intra-amniotic infection had bacterial profiles dominated by Sneathia, Ureaplasma, Prevotella, Lactobacillus, Escherichia, Gardnerella, Peptostreptococcus, Peptoniphilus, and Streptococcus, many of which had not been cultured from the amniotic fluid samples. (5) Seventy percent (7/10) of the prominent (>1% average relative abundance) OTUs found in amniotic fluid were also prominent in the vagina. Conclusion The majority of women with intra-amniotic infection had bacteria cultured from their amniotic fluid that were typical vaginal commensals, and these bacteria were detected within the vagina at the time of amniocentesis. Molecular microbiological interrogation of amniotic fluid from women with intra-amniotic infection revealed that the bacterial profiles of amniotic fluid were largely consistent with those of the vagina. These findings indicate that ascension from the lower genital tract is the primary pathway for intra-amniotic infection.

The origin of amniotic fluid monocytes/macrophages in women with intra-amniotic inflammation or infection.

Background Monocytes, after neutrophils, are the most abundant white blood cells found in the amniotic cavity of women with intra-amniotic inflammation/infection. However, the origin of such cells has not been fully investigated. Herein, we determined (1) the origin of amniotic fluid monocytes/macrophages from women with intra-amniotic inflammation/infection, (2) the relationship between the origin of amniotic fluid monocytes/macrophages and preterm or term delivery and (3) the localization of monocytes/macrophages in the placental tissues. Methods Amniotic fluid samples (n = 16) were collected from women with suspected intra-amniotic inflammation or infection. Amniotic fluid monocytes/macrophages were purified by fluorescence-activated cell sorting, and DNA fingerprinting was performed. Blinded placental histopathological evaluations were conducted. Immunohistochemistry was performed to detect CD14+ monocytes/macrophages in the placental tissues. Results DNA fingerprinting revealed that (1) 56.25% (9/16) of amniotic fluid samples had mostly fetal monocytes/macrophages, (2) 37.5% (6/16) had predominantly maternal monocytes/macrophages and (3) one sample (6.25% [1/16]) had a mixture of fetal and maternal monocytes/macrophages. (4) Most samples with predominantly fetal monocytes/macrophages were from women who delivered early preterm neonates (77.8% [7/9]), whereas all samples with mostly maternal monocytes/macrophages or a mixture of both were from women who delivered term or late preterm neonates (100% [7/7]). (5) Most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (85.7% [12/14]). (6) Women who had mostly fetal monocytes/macrophages in amniotic fluid had abundant CD14+ cells in the umbilical cord and chorionic plate, whereas women with mostly maternal amniotic fluid monocytes/macrophages had abundant CD14+ cells in the chorioamniotic membranes. Conclusion Amniotic fluid monocytes/macrophages can be of either fetal or maternal origin, or a mixture of both, in women with intra-amniotic inflammation or infection. These immune cells could be derived from the fetal and maternal vasculature of the placenta.

Fetal T Cell Activation in the Amniotic Cavity during Preterm Labor: A Potential Mechanism for a Subset of Idiopathic Preterm Birth.

Prematurity is the leading cause of perinatal morbidity and mortality worldwide. In most cases, preterm birth is preceded by spontaneous preterm labor, a syndrome that is associated with intra-amniotic inflammation, the most studied etiology. However, the remaining etiologies of preterm labor are poorly understood; therefore, most preterm births are categorized as idiopathic. In this study, we provide evidence showing that the fetal immune system undergoes premature activation in women with preterm labor without intra-amniotic inflammation, providing a potential new mechanism of disease for some cases of idiopathic preterm birth. First, we showed that fetal T cells are a predominant leukocyte population in amniotic fluid during preterm gestations. Interestingly, only fetal CD4+ T cells were increased in amniotic fluid of women who underwent idiopathic preterm labor and birth. This increase in fetal CD4+ T cells was accompanied by elevated amniotic fluid concentrations of T cell cytokines such as IL-2, IL-4, and IL-13, which are produced by these cells upon in vitro stimulation, but was not associated with the prototypical cytokine profile observed in women with intra-amniotic inflammation. Also, we found that cord blood T cells, mainly CD4+ T cells, obtained from women with idiopathic preterm labor and birth displayed enhanced ex vivo activation, which is similar to that observed in women with intra-amniotic inflammation. Finally, we showed that the intra-amniotic administration of activated neonatal CD4+ T cells induces preterm birth in mice. Collectively, these findings provide evidence suggesting that fetal T cell activation is implicated in the pathogenesis of idiopathic preterm labor and birth.

Gasdermin D: Evidence of pyroptosis in spontaneous preterm labor with sterile intra-amniotic inflammation or intra-amniotic infection.

PROBLEM: Pyroptosis, inflammatory programmed cell death, is initiated through the inflammasome and relies on the pore-forming actions of the effector molecule gasdermin D. Herein, we investigated whether gasdermin D is detectable in women with spontaneous preterm labor and sterile intra-amniotic inflammation or intra-amniotic infection. METHOD OF STUDY: Amniotic fluid samples (n = 124) from women with spontaneous preterm labor were subdivided into the following groups: (a) those who delivered at term (n = 32); and those who delivered preterm (b) without intra-amniotic inflammation (n = 41), (c) with sterile intra-amniotic inflammation (n = 32), or (d) with intra-amniotic infection (n = 19), based on amniotic fluid IL-6 concentrations and the microbiological status of amniotic fluid (culture and PCR/ESI-MS). Gasdermin D concentrations were measured using an ELISA kit. Multiplex immunofluorescence staining was also performed to determine the expression of gasdermin D, caspase-1, and interleukin-1ß in the chorioamniotic membranes. Flow cytometry was used to detect pyroptosis (active caspase-1) in decidual cells from women with preterm labor and birth. RESULTS: (a) Gasdermin D was detected in the amniotic fluid and chorioamniotic membranes from women who underwent spontaneous preterm labor/birth with either sterile intra-amniotic inflammation or intra-amniotic infection, but was rarely detected in those without intra-amniotic inflammation. (b) Amniotic fluid concentrations of gasdermin D were higher in women with intra-amniotic infection than in those with sterile intra-amniotic inflammation, and its expression in the chorioamniotic membranes was associated with caspase-1 and IL-1ß (inflammasome mediators). (c) Decidual stromal cells and leukocytes isolated from women with preterm labor and birth are capable of undergoing pyroptosis given their expression of active caspase-1. CONCLUSION: Pyroptosis can occur in the context of sterile intra-amniotic inflammation and intra-amniotic infection in patients with spontaneous preterm labor and birth.

Cellular immune responses in amniotic fluid of women with preterm labor and intra-amniotic infection or intra-amniotic inflammation.

PROBLEM: Preterm birth is commonly preceded by preterm labor, a syndrome that is causally linked to both intra-amniotic infection and intra-amniotic inflammation. However, the stereotypical cellular immune responses in these two clinical conditions are poorly understood. METHOD OF STUDY: Amniotic fluid samples (n = 26) were collected from women diagnosed with preterm labor and intra-amniotic infection (amniotic fluid IL-6 concentrations ≥2.6 ng/mL and culturable microorganisms, n = 10) or intra-amniotic inflammation (amniotic fluid IL-6 concentrations ≥2.6 ng/mL without culturable microorganisms, n = 16). Flow cytometry was performed to evaluate the phenotype and number of amniotic fluid leukocytes. Amniotic fluid concentrations of classical pro-inflammatory cytokines, type 1 and type 2 cytokines, and T-cell chemokines were determined using immunoassays. RESULTS: Women with spontaneous preterm labor and intra-amniotic infection had (a) a greater number of total leukocytes, including neutrophils and monocytes/macrophages, in amniotic fluid; (b) a higher number of total T cells and CD4+ T cells, but not CD8+ T cells or B cells, in amniotic fluid; and (c) increased amniotic fluid concentrations of IL-6, IL-1ß, and IL-10, compared to those with intra-amniotic inflammation. However, no differences in amniotic fluid concentrations of T-cell cytokines and chemokines were observed between these two clinical conditions. CONCLUSION: The cellular immune responses observed in women with preterm labor and intra-amniotic infection are more severe than in those with intra-amniotic inflammation, and neutrophils, monocytes/macrophages, and CD4+ T cells are the main immune cells responding to microorganisms that invade the amniotic cavity. These findings provide insights into the intra-amniotic immune mechanisms underlying the human syndrome of preterm labor.

QUEST MRI assessment of fetal brain oxidative stress in utero.

PURPOSE: To achieve sufficient precision of R1 (=1/T1) maps of the fetal brain in utero to perform QUEnch-assiSTed (QUEST) MRI in which a significant anti-oxidant-induced reduction in R1 indicates oxidative stress. METHODS: C57BL/6 mouse fetuses in utero were gently and non-surgically isolated and secured using a homemade 3D printed clip. Using a commercial receive-only surface coil, brain maps of R1, an index sensitive to excessive and continuous free radical production, were collected using either a conventional Cartesian or a non-Cartesian (periodically rotated overlapping parallel lines with enhanced reconstruction) progressive saturation sequence. Data were normalized to the shortest TR time to remove bias. To assess oxidative stress, brain R1 maps were acquired on the lipopolysaccharide (LPS) model of preterm birth ±â€¯rosiglitazone (ROSI, which has anti-oxidant properties); phosphate buffered saline (PBS) controls ±â€¯ROSI were similarly studied. RESULTS: Sufficient quality R1 maps were generated by a combination of the 3D printed clip, surface coil detection, non-Cartesian sequence, and normalization scheme ensuring minimal fetal movement, good detection sensitivity, reduced motion artifacts, and minimal baseline variations, respectively. In the LPS group, the combined caudate-putamen and thalamus region R1 was reduced (p < 0.05) with ROSI treatment consistent with brain oxidative stress; no evidence for oxidative stress was found in the pons region. In the PBS control group, brain R1's did not change with ROSI treatment. CONCLUSION: The sensitivity and reproducibility of the combined approaches described herein enabled first-time demonstration of regional oxidative stress measurements of the fetal brain in utero using QUEST MRI.

The prediction of early preeclampsia: Results from a longitudinal proteomics study.

OBJECTIVES: To identify maternal plasma protein markers for early preeclampsia (delivery <34 weeks of gestation) and to determine whether the prediction performance is affected by disease severity and presence of placental lesions consistent with maternal vascular malperfusion (MVM) among cases. STUDY DESIGN: This longitudinal case-control study included 90 patients with a normal pregnancy and 33 patients with early preeclampsia. Two to six maternal plasma samples were collected throughout gestation from each woman. The abundance of 1,125 proteins was measured using high-affinity aptamer-based proteomic assays, and data were modeled using linear mixed-effects models. After data transformation into multiples of the mean values for gestational age, parsimonious linear discriminant analysis risk models were fit for each gestational-age interval (8-16, 16.1-22, 22.1-28, 28.1-32 weeks). Proteomic profiles of early preeclampsia cases were also compared to those of a combined set of controls and late preeclampsia cases (n = 76) reported previously. Prediction performance was estimated via bootstrap. RESULTS: We found that 1) multi-protein models at 16.1-22 weeks of gestation predicted early preeclampsia with a sensitivity of 71% at a false-positive rate (FPR) of 10%. High abundance of matrix metalloproteinase-7 and glycoprotein IIbIIIa complex were the most reliable predictors at this gestational age; 2) at 22.1-28 weeks of gestation, lower abundance of placental growth factor (PlGF) and vascular endothelial growth factor A, isoform 121 (VEGF-121), as well as elevated sialic acid binding immunoglobulin-like lectin 6 (siglec-6) and activin-A, were the best predictors of the subsequent development of early preeclampsia (81% sensitivity, FPR = 10%); 3) at 28.1-32 weeks of gestation, the sensitivity of multi-protein models was 85% (FPR = 10%) with the best predictors being activated leukocyte cell adhesion molecule, siglec-6, and VEGF-121; 4) the increase in siglec-6, activin-A, and VEGF-121 at 22.1-28 weeks of gestation differentiated women who subsequently developed early preeclampsia from those who had a normal pregnancy or developed late preeclampsia (sensitivity 77%, FPR = 10%); 5) the sensitivity of risk models was higher for early preeclampsia with placental MVM lesions than for the entire early preeclampsia group (90% versus 71% at 16.1-22 weeks; 87% versus 81% at 22.1-28 weeks; and 90% versus 85% at 28.1-32 weeks, all FPR = 10%); and 6) the sensitivity of prediction models was higher for severe early preeclampsia than for the entire early preeclampsia group (84% versus 71% at 16.1-22 weeks). CONCLUSION: We have presented herein a catalogue of proteome changes in maternal plasma proteome that precede the diagnosis of preeclampsia and can distinguish among early and late phenotypes. The sensitivity of maternal plasma protein models for early preeclampsia is higher in women with underlying vascular placental disease and in those with a severe phenotype.

Effector and Activated T Cells Induce Preterm Labor and Birth That Is Prevented by Treatment with Progesterone.

Preterm labor commonly precedes preterm birth, the leading cause of perinatal morbidity and mortality worldwide. Most research has focused on establishing a causal link between innate immune activation and pathological inflammation leading to preterm labor and birth. However, the role of maternal effector/activated T cells in the pathogenesis of preterm labor/birth is poorly understood. In this study, we first demonstrated that effector memory and activated maternal T cells expressing granzyme B and perforin are enriched at the maternal-fetal interface (decidua) of women with spontaneous preterm labor. Next, using a murine model, we reported that prior to inducing preterm birth, in vivo T cell activation caused maternal hypothermia, bradycardia, systemic inflammation, cervical dilation, intra-amniotic inflammation, and fetal growth restriction, all of which are clinical signs associated with preterm labor. In vivo T cell activation also induced B cell cytokine responses, a proinflammatory macrophage polarization, and other inflammatory responses at the maternal-fetal interface and myometrium in the absence of an increased influx of neutrophils. Finally, we showed that treatment with progesterone can serve as a strategy to prevent preterm labor/birth and adverse neonatal outcomes by attenuating the proinflammatory responses at the maternal-fetal interface and cervix induced by T cell activation. Collectively, these findings provide mechanistic evidence showing that effector and activated T cells cause pathological inflammation at the maternal-fetal interface, in the mother, and in the fetus, inducing preterm labor and birth and adverse neonatal outcomes. Such adverse effects can be prevented by treatment with progesterone, a clinically approved strategy.