Distinct Cellular Immune Responses to SARS-CoV-2 in Pregnant Women.

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

IL-22 Plays a Dual Role in the Amniotic Cavity: Tissue Injury and Host Defense against Microbes in Preterm Labor.

IL-22 is a multifaceted cytokine with both pro- and anti-inflammatory functions that is implicated in multiple pathologies. However, the role of IL-22 in maternal-fetal immunity in late gestation is poorly understood. In this study, we first showed that IL-22+ T cells coexpressing retinoic acid-related orphan receptor γt (ROR-γt) are enriched at the human maternal-fetal interface of women with preterm labor and birth, which was confirmed by in silico analysis of single-cell RNA sequencing data. T cell activation leading to preterm birth in mice was preceded by a surge in IL-22 in the maternal circulation and amniotic cavity; however, systemic administration of IL-22 in mice did not induce adverse perinatal outcomes. Next, using an ex vivo human system, we showed that IL-22 can cross from the choriodecidua to the intra-amniotic space, where its receptors (Il22ra1, Il10rb, and Il22ra2) are highly expressed by murine gestational and fetal tissues in late pregnancy. Importantly, amniotic fluid concentrations of IL-22 were elevated in women with sterile or microbial intra-amniotic inflammation, suggesting a dual role for this cytokine. The intra-amniotic administration of IL-22 alone shortened gestation and caused neonatal death in mice, with the latter outcome involving lung maturation and inflammation. IL-22 plays a role in host response by participating in the intra-amniotic inflammatory milieu preceding Ureaplasma parvum-induced preterm birth in mice, which was rescued by the deficiency of IL-22. Collectively, these data show that IL-22 alone is capable of causing fetal injury leading to neonatal death and can participate in host defense against microbial invasion of the amniotic cavity leading to preterm labor and birth.

A single-cell atlas of the myometrium in human parturition.

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

Maternal-fetal immune responses in pregnant women infected with SARS-CoV-2.

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

Resolution of acute cervical insufficiency after antibiotics in a case with amniotic fluid sludge.

Cervical insufficiency generally refers to a condition in which there is mid-trimester cervical dilatation or protruding chorioamniotic membranes in the absence of uterine contractions. Such condition is a risk factor for spontaneous mid-trimester abortion or early preterm birth, and is associated with adverse neonatal outcomes. Both intra-amniotic infection and inflammation ascertained by amniocentesis have been identified in patients with cervical insufficiency, and are poor prognostic factors. A subset of patients with intra-amniotic inflammation will have no demonstrable microorganisms detected via cultivation or molecular methods, and therefore represent cases of sterile intra-amniotic inflammation. Amniotic fluid sludge (free-floating hyperechogenic material within the amniotic fluid in close proximity to the uterine cervix) identified on sonography is a biomarker for intra-amniotic infection and inflammation. Recent evidence suggests that intra-amniotic infection, as well as sterile intra-amniotic inflammation can be treated successfully using antimicrobial agents. We report a unique case in which administration of antibiotics in the presence of mid-trimester cervical insufficiency, sterile intra-amniotic inflammation, and amniotic fluid sludge was associated with resolution of the cervical findings, as demonstrated on both sonographic and speculum examination. The patient successfully underwent elective cesarean delivery at 36-2/7 weeks of gestation. This case illustrates that antibiotic therapy may be effective despite the presence of several high-risk pregnancy conditions, and that successful outcome is possible.

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.

Specific innate immune cells uptake fetal antigen and display homeostatic phenotypes in the maternal circulation.

Pregnancy represents a period when the mother undergoes significant immunological changes to promote tolerance of the fetal semi-allograft. Such tolerance results from the exposure of the maternal immune system to fetal antigens (Ags), a process that has been widely investigated at the maternal-fetal interface and in the adjacent draining lymph nodes. However, the peripheral mechanisms of maternal-fetal crosstalk are poorly understood. Herein, we hypothesized that specific innate immune cells interact with fetal Ags in the maternal circulation. To test this hypothesis, a mouse model was utilized in which transgenic male mice expressing the chicken ovalbumin (OVA) Ag under the beta-actin promoter were allogeneically mated with wild-type females to allow for tracking of the fetal Ag. Fetal Ag-carrying Ly6G+ and F4/80+ cells were identified in the maternal circulation, where they were more abundant in the second half of pregnancy. Such innate immune cells displayed unique phenotypes: while Ly6G+ cells expressed high levels of MHC-II and CD80 together with low levels of pro-inflammatory cytokines, F4/80+ cells up-regulated the expression of CD86 as well as the anti-inflammatory cytokines IL-10 and TGF-ß. In vitro studies using allogeneic GFP+ placental particles revealed that maternal peripheral Ly6G+ and F4/80+ cells phagocytose fetal Ags in mid and late murine pregnancy. Importantly, cytotrophoblast-derived particles were also engulfed in vitro by CD15+ and CD14+ cells from women in the second and third trimester, providing translational evidence that this process also occurs in humans. Collectively, this study demonstrates novel interactions between specific maternal circulating innate immune cells and fetal Ags, thereby shedding light on the systemic mechanisms of maternal-fetal crosstalk.

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

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

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.

Maternal-Fetal Immune Responses in Pregnant Women Infected with SARS-CoV-2.

Pregnant women are a high-risk population for severe/critical COVID-19 and mortality. However, the maternal-fetal immune responses initiated by SARS-CoV-2 infection, and whether this virus is detectable in the placenta, are still under investigation. Herein, we report that SARS-CoV-2 infection during pregnancy primarily induced specific maternal inflammatory responses in the circulation and at the maternal-fetal interface, the latter being governed by T cells and macrophages. SARS-CoV-2 infection during pregnancy was also associated with a cytokine response in the fetal circulation (i.e. umbilical cord blood) without compromising the cellular immune repertoire. Moreover, SARS-CoV-2 infection neither altered fetal cellular immune responses in the placenta nor induced elevated cord blood levels of IgM. Importantly, SARS-CoV-2 was not detected in the placental tissues, nor was the sterility of the placenta compromised by maternal viral infection. This study provides insight into the maternal-fetal immune responses triggered by SARS-CoV-2 and further emphasizes the rarity of placental infection.

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.

The fetal inflammatory response syndrome: the origins of a concept, pathophysiology, diagnosis, and obstetrical implications.

The fetus can deploy a local or systemic inflammatory response when exposed to microorganisms or, alternatively, to non-infection-related stimuli (e.g., danger signals or alarmins). The term "Fetal Inflammatory Response Syndrome" (FIRS) was coined to describe a condition characterized by evidence of a systemic inflammatory response, frequently a result of the activation of the innate limb of the immune response. FIRS can be diagnosed by an increased concentration of umbilical cord plasma or serum acute phase reactants such as C-reactive protein or cytokines (e.g., interleukin-6). Pathologic evidence of a systemic fetal inflammatory response indicates the presence of funisitis or chorionic vasculitis. FIRS was first described in patients at risk for intraamniotic infection who presented preterm labor with intact membranes or preterm prelabor rupture of the membranes. However, FIRS can also be observed in patients with sterile intra-amniotic inflammation, alloimmunization (e.g., Rh disease), and active autoimmune disorders. Neonates born with FIRS have a higher rate of complications, such as early-onset neonatal sepsis, intraventricular hemorrhage, periventricular leukomalacia, and death, than those born without FIRS. Survivors are at risk for long-term sequelae that may include bronchopulmonary dysplasia, neurodevelopmental disorders, such as cerebral palsy, retinopathy of prematurity, and sensorineuronal hearing loss. Experimental FIRS can be induced by intra-amniotic administration of bacteria, microbial products (such as endotoxin), or inflammatory cytokines (such as interleukin-1), and animal models have provided important insights about the mechanisms responsible for multiple organ involvement and dysfunction. A systemic fetal inflammatory response is thought to be adaptive, but, on occasion, may become dysregulated whereby a fetal cytokine storm ensues and can lead to multiple organ dysfunction and even fetal death if delivery does not occur ("rescued by birth"). Thus, the onset of preterm labor in this context can be considered to have survival value. The evidence so far suggests that FIRS may compound the effects of immaturity and neonatal inflammation, thus increasing the risk of neonatal complications and long-term morbidity. Modulation of a dysregulated fetal inflammatory response by the administration of antimicrobial agents, anti-inflammatory agents, or cell-based therapy holds promise to reduce infant morbidity and mortality.

Cellular immune responses in amniotic fluid of women with a sonographic short cervix.

Objectives A sonographic short cervix is one of the strongest predictors of preterm delivery. However, the cellular immune composition of amniotic fluid in women with a short cervix has not yet been described. Herein, we determined cellular and soluble immune responses in amniotic fluid from pregnant women with a mid-trimester asymptomatic short cervix. Methods Amniotic fluid samples (n=77) were collected from asymptomatic women with a cervical length between 15 and 25 mm (n=36, short cervix) or ≤15 mm (n=41, severely short cervix) diagnosed by ultrasound. Flow cytometry and multiplex measurement of cytokines/chemokines were performed. Results (1) The cellular immune composition of amniotic fluid did not differ between women with a severely short cervix (≤15 mm) and those with a short cervix 15-25 mm; (2) amniotic fluid concentrations of multiple cytokines/chemokines were higher in women with a severely short cervix (≤15 mm) than in those with a short cervix 15-25 mm; (3) the cellular immune composition of amniotic fluid did not differ between women with a severely short cervix (≤15 mm) who ultimately underwent preterm delivery and those who delivered at term; and (4) amniotic fluid concentrations of IL-2, but not other immune mediators, were increased in women with a severely short cervix (≤15 mm) who ultimately delivered preterm compared to those who delivered at term. Conclusions Women with a severely short cervix (≤15 mm) have increased concentrations of pro-inflammatory mediators in the amniotic cavity; yet, these do not translate to changes in the cellular immune response.

Intra-Amniotic Infection with Ureaplasma parvum Causes Preterm Birth and Neonatal Mortality That Are Prevented by Treatment with Clarithromycin.

Intra-amniotic infection is strongly associated with adverse pregnancy and neonatal outcomes. Most intra-amniotic infections are due to Ureaplasma species; however, the pathogenic potency of these genital mycoplasmas to induce preterm birth is still controversial. Here, we first laid out a taxonomic characterization of Ureaplasma isolates from women with intra-amniotic infection, which revealed that Ureaplasma parvum is the most common bacterium found in this clinical condition. Next, using animal models, we provided a causal link between intra-amniotic inoculation with Ureaplasma species and preterm birth. Importantly, the intra-amniotic inoculation of Ureaplasma species induced high rates of mortality in both preterm and term neonates. The in vivo potency of U. parvum to induce preterm birth was not associated with known virulence factors. However, term-derived and preterm-derived U. parvum isolates were capable of inducing an intra-amniotic inflammatory response. Both U. parvum isolates invaded several fetal tissues, primarily the fetal lung, and caused fetal inflammatory response syndrome. This bacterium was also detected in the placenta, reproductive tissues, and most severely in the fetal membranes, inducing a local inflammatory response that was replicated in an in vitro model. Importantly, treatment with clarithromycin, a recently recommended yet not widely utilized antibiotic, prevented the adverse pregnancy and neonatal outcomes induced by U. parvum These findings shed light on the maternal-fetal immunobiology of intra-amniotic infection.IMPORTANCE Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Multiple etiologies are associated with preterm birth; however, 25% of preterm infants are born to a mother with intra-amniotic infection, most commonly due to invasion of the amniotic cavity by Ureaplasma species. Much research has focused on establishing a link between Ureaplasma species and adverse pregnancy/neonatal outcomes; however, little is known about the taxonomy of and host response against Ureaplasma species. Here, we applied a multifaceted approach, including human samples, in vivo models, and in vitro manipulations, to study the maternal-fetal immunobiology of Ureaplasma infection during pregnancy. Furthermore, we investigated the use of clarithromycin as a treatment for this infection. Our research provides translational knowledge that bolsters scientific understanding of Ureaplasma species as a cause of adverse pregnancy/neonatal outcomes and gives strong evidence for the use of clarithromycin as the recommended treatment for women intra-amniotically infected with Ureaplasma species.

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.

Microbial burden and inflammasome activation in amniotic fluid of patients with preterm prelabor rupture of membranes.

Background Intra-amniotic inflammation, which is associated with adverse pregnancy outcomes, can occur in the presence or absence of detectable microorganisms, and involves activation of the inflammasome. Intra-amniotic inflammasome activation has been reported in clinical chorioamnionitis at term and preterm labor with intact membranes, but it has not yet been investigated in women with preterm prelabor rupture of membranes (preterm PROM) in the presence/absence of detectable microorganisms. The aim of this study was to determine whether, among women with preterm PROM, there is an association between detectable microorganisms in amniotic fluid and intra-amniotic inflammation, and whether intra-amniotic inflammasome activation correlates with microbial burden. Methods Amniotic fluids from 59 cases of preterm PROM were examined for the presence/absence of microorganisms through culture and 16S ribosomal RNA (rRNA) gene quantitative real-time polymerase chain reaction (qPCR), and concentrations of interleukin-6 (IL-6) and ASC [apoptosis-associated spec-like protein containing a caspase recruitment domain (CARD)], an indicator of inflammasome activation, were determined. Results qPCR identified more microbe-positive amniotic fluids than culture. Greater than 50% of patients with a negative culture and high IL-6 concentration in amniotic fluid yielded a positive qPCR signal. ASC concentrations were greatest in patients with high qPCR signals and elevated IL-6 concentrations in amniotic fluid (i.e. intra-amniotic infection). ASC concentrations tended to increase in patients without detectable microorganisms but yet with elevated IL-6 concentrations (i.e. sterile intra-amniotic inflammation) compared to those without intra-amniotic inflammation. Conclusion qPCR is a valuable complement to microbiological culture for the detection of microorganisms in the amniotic cavity in women with preterm PROM, and microbial burden is associated with the severity of intra-amniotic inflammatory response, including inflammasome activation.

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.

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.

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.