Sterile Intra-amniotic Inflammation Research Articles

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

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. 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. 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. IL-6R blockade can serve as a strategy to treat SIAI, preventing preterm birth and adverse neonatal outcomes. NICHD/NIH/DHHS, Contract HHSN275201300006C. WSU Perinatal Initiative in Maternal, Perinatal and Child Health.

Thymic stromal lymphopoietin participates in the host response to intra-amniotic inflammation leading to preterm labor and birth

The aim of this study was to establish the role of thymic stromal lymphopoietin (TSLP) in the intra-amniotic host response of women with spontaneous preterm labor (sPTL) and birth. Amniotic fluid and chorioamniotic membranes (CAM) were collected from women with sPTL who delivered at term (n = 30) or preterm without intra-amniotic inflammation (n = 34), with sterile intra-amniotic inflammation (SIAI, n = 27), or with intra-amniotic infection (IAI, n = 17). Amnion epithelial cells (AEC), Ureaplasma parvum, and Sneathia spp. were also utilized. The expression of TSLP, TSLPR, and IL-7Rα was evaluated in amniotic fluid or CAM by RT-qPCR and/or immunoassays. AEC co-cultured with Ureaplasma parvum or Sneathia spp. were evaluated for TSLP expression by immunofluorescence and/or RT-qPCR. Our data show that TSLP was elevated in amniotic fluid of women with SIAI or IAI and expressed by the CAM. TSLPR and IL-7Rα had detectable gene and protein expression in the CAM; yet, CRLF2 was specifically elevated with IAI. While TSLP localized to all layers of the CAM and increased with SIAI or IAI, TSLPR and IL-7Rα were minimal and became most apparent with IAI. Co-culture experiments indicated that Ureaplasma parvum and Sneathia spp. differentially upregulated TSLP expression in AEC. Together, these findings indicate that TSLP is a central component of the intra-amniotic host response during sPTL.

Amniotic fluid CD36 in pregnancies complicated by spontaneous preterm delivery: a retrospective cohort study

Objective The aim of this study was to evaluate CD36 concentrations in amniotic fluid in pregnancies complicated by spontaneous delivery with intact fetal membranes (preterm labor, PTL) and preterm prelabor rupture of membranes (PPROM) with respect to the presence of the intra-amniotic infection. Methods A total of 80 women with PPROM and 71 with PTL were included in the study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid CD36 concentrations were assessed by enzyme-linked immunosorbent assay. Microbial colonization of the amniotic cavity (MIAC) was determined by the cultivation and non-cultivation approach. Intra-amniotic inflammation (IAI) was defined as an amniotic fluid bedside interleukin-6 concentration ≥3000 pg/mL. Intra-amniotic infection was characterized by the presence of both MIAC and IAI. Results Women with PPROM with intra-amniotic infection had higher amniotic fluid CD36 concentrations than women without infection (with infection: median 346 pg/mL, IQR 262–384 vs. without infection: median 242 pg/mL, IQR 199–304; p = .006) A positive correlation between amniotic fluid CD36 concentrations and interleukin-6 concentrations was found (rho = 0.48; p < .0001). In PTL pregnancies, no statistically significant difference was found in the amniotic fluid level of CD36 between intra-amniotic infection, sterile IAI, and negative amniotic fluid. Conclusions The presence of intra-amniotic infection is characterized by higher amniotic fluid CD36 concentrations in pregnancies complicated by PPROM. An amniotic fluid CD36 cutoff value of 252.5 pg/mL was found to be optimal for the prediction of intra-amniotic infection. In PTL pregnancies, no statistically significant change in CD36 concentration was found with respect to the presence of intra-amniotic infection.

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

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

Cervical excisional treatment increases the risk of intraamniotic infection in subsequent pregnancy complicated by preterm prelabor rupture of membranes

Excisional treatment of cervical intraepithelial neoplasia or very early stages of cervical cancer increases the risk of preterm prelabor rupture of membranes in subsequent pregnancies. The risk increases with the length of the excised cone. The subset of cases with preterm prelabor rupture of membranes and a history of cervical excisional treatment could also be at higher risk of intraamniotic infection/inflammation. However, there is a paucity of relevant information on this subject. This study aimed to assess the differences in the rates of intraamniotic infection/inflammation and early-onset neonatal sepsis between singleton preterm prelabor rupture of membranes pregnancies without and with a history of cervical excisional treatment, and to investigate the association between these complications of preterm prelabor rupture of membranes and the excised cone length. This retrospective cohort study included 770 preterm prelabor rupture of membranes pregnancies in which transabdominal amniocentesis was performed as part of standard clinical management to assess the intraamniotic environment. The maternal and perinatal medical records of all included women were reviewed to obtain information on the absence or presence of history of cervical excisional treatment and neonatal outcomes. Women whose records contained any information on history of cervical excisional treatment were contacted by phone and in writing to inform them of the study and request permission to collect relevant information from their medical records. Women were divided into 4 subgroups according to the presence of microorganisms and/or their nucleic acids (through culturing and molecular biology methods) in amniotic fluid and/or intraamniotic inflammation (through amniotic fluid interleukin-6 concentration evaluation): intraamniotic infection (presence of both), sterile intraamniotic inflammation (intraamniotic inflammation alone), microbial invasion of the amniotic cavity without inflammation (presence of microorganisms and/or their nucleic acids in amniotic fluid alone), and negative amniotic fluid for infection/inflammation (absence of both). A history of cervical excisional treatment was found in 10% (76/765) of the women. Of these, 82% (62/76) had a history of only 1 treatment, and information on cone length was available for 97% (60/62) of them. Women with a history of cervical excisional treatment had higher rates of intraamniotic infection (with, 25% [19/76] vs without, 12% [85/689]; adjusted odds ratio, 2.5; adjusted P=.004), microbial invasion of the amniotic cavity without inflammation (with, 25% [19/76] vs without, 11% [74/689]; adjusted odds ratio, 3.1; adjusted P<.0001), and early-onset neonatal sepsis (with, 8% [11/76] vs without, 3% [23/689]; adjusted odds ratio, 2.9; adjusted P=.02) compared with those without such history. Quartiles of cone length (range: 3-32 mm) were used to categorize the women into 4 quartile subgroups (first: 3-8 mm; second: 9-12 mm; third: 13-17 mm; and fourth: 18-32 mm). Cone length of ≥18 mm was associated with higher rates of intraamniotic infection (with, 29% [5/15] vs without, 12% [85/689]; adjusted odds ratio, 3.0; adjusted P=.05), microbial invasion of the amniotic cavity without inflammation (with, 40% [6/15] vs without, 11% [74/689]; adjusted odds ratio, 6.1; adjusted P=.003), and early-onset neonatal sepsis (with, 20% [3/15] vs without, 3% [23/689]; adjusted odds ratio, 5.7; adjusted P=.02). History of cervical excisional treatment increases risks of intraamniotic infection, microbial invasion of the amniotic cavity without inflammation, and development of early-onset neonatal sepsis in a subsequent pregnancy complicated by preterm prelabor rupture of membranes.

Amniotic fluid soluble CD93 is elevated in the presence of intra-amniotic inflammation in preterm prelabor rupture of the fetal membranes.

To determine the soluble form of CD93 (sCD93) concentration in amniotic fluid from pregnancies complicated by preterm prelabor rupture of membranes (PPROM) based on the presence of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation. A total of 144 women with a singleton pregnancy complicated by PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. MIAC was determined by the combination of cultivation and non-cultivation techniques. Intra-amniotic inflammation was characterized as a concentration of interleukin-6 3,000 pg/mL in amniotic fluid. Women were categorized in the following groups: i) intra-amniotic infection (both MIAC and intra-amniotic inflammation), ii) sterile intra-amniotic inflammation (intra-amniotic inflammation per se), iii) colonization of the amniotic cavity (MIAC per se), and iv) negative amniotic fluid (without both MIAC and intra-amniotic inflammation). Levels of sCD93 in amniotic fluid were assessed by ELISA. A difference in the levels of sCD93 in amniotic fluid was found among the groups of women with intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid (intra-amniotic infection: median 22.3 ng/mL, sterile intra-amniotic inflammation: median 21.0 ng/mL, colonization of the amniotic cavity: 8.7 ng/mL, negative: median 8.7 ng/mL; P < 0.0001). Intra-amniotic inflammation in PPROM, irrespectively of the presence or absence of MIAC, is associated with the elevation of the level of sCD93 in amniotic fluid.

Intra-amniotic inflammation and birth weight in pregnancies with preterm labor with intact membranes: A retrospective cohort study.

To assess the association between newborn birth weight and the presence of intra-amniotic infection, presence of sterile intra-amniotic inflammation, and absence of intra-amniotic inflammation in pregnancies with preterm labor with intact membranes. A total of 69 pregnancies with preterm labor with intact membranes between gestational ages 22 + 0 and 34 + 6 weeks who delivered within seven days of admission were included in this retrospective cohort study. Transabdominal amniocentesis to determine the presence of microorganisms and/or their nucleic acids in amniotic fluid (through culturing and molecular biology methods) and intra-amniotic inflammation (according to amniotic fluid interleukin-6 concentrations) were performed as part of standard clinical management. The participants were further divided into three subgroups: intra-amniotic infection (presence of microorganisms and/or nucleic acids along with intra-amniotic inflammation), sterile intra-amniotic inflammation (intra-amniotic inflammation alone), and without intra-amniotic inflammation. Birth weights of newborns were expressed as percentiles derived from the INTERGROWTH-21st standards for (i) estimated fetal weight and (ii) newborn birth weight. No difference in birth weights, expressed as percentiles derived from the standard for estimated fetal weight, was found among the women with intra-amniotic infection, with sterile intra-amniotic inflammation, and without intra-amniotic inflammation (with infection, median 29; with sterile inflammation, median 54; without inflammation, median 53; p = 0.06). Differences among the subgroups were identified in the birth weight rates, expressed as percentiles derived from the standard for estimated fetal weight, which were less than the 10th percentile (with infection: 20%, with inflammation: 13%, without inflammation: 0%; p = 0.04) and 25th percentile (with infection: 47%, with inflammation: 31%, without inflammation: 9%; p = 0.01). No differences among the subgroups were observed when percentiles of birth weight were derived from the birth weight standard. The presence of intra-amniotic inflammatory complications in pregnancies with preterm labor with intact membranes prior to the gestational age of 35 weeks was associated with a higher rate of newborns with birth weight less than the 10th and 25th percentile, when percentiles of birth weight were derived from the standard for estimated fetal weight.

Intra-amniotic inflammation in the mid-trimester of pregnancy is a risk factor for neuropsychological disorders in childhood.

Intra-amniotic inflammation is a subclinical condition frequently caused by either microbial invasion of the amniotic cavity or sterile inflammatory stimuli, e.g., alarmins. An accumulating body of evidence supports a role for maternal immune activation in the genesis of fetal neuroinflammation and the occurrence of neurodevelopmental disorders such as cerebral palsy, schizophrenia, and autism. The objective of this study was to determine whether fetal exposure to mid-trimester intra-amniotic inflammation is associated with neurodevelopmental disorders in children eight to 12years of age. This is a retrospective case-control study comprising 20 children with evidence of prenatal exposure to intra-amniotic inflammation in the mid-trimester and 20 controls matched for gestational age at amniocentesis and at delivery. Amniotic fluid samples were tested for concentrations of interleukin-6 and C-X-C motif chemokine ligand 10, for bacteria by culture and molecular microbiologic methods as well as by polymerase chain reaction for eight viruses. Neuropsychological testing of children, performed by two experienced psychologists, assessed cognitive and behavioral domains. Neuropsychological dysfunction was defined as the presence of an abnormal score (<2 standard deviations) on at least two cognitive tasks. Neuropsychological dysfunction was present in 45% (9/20) of children exposed to intra-amniotic inflammation but in only 10% (2/20) of those in the control group (p=0.03). The relative risk (RR) of neuropsychological dysfunction conferred by amniotic fluid inflammation remained significant after adjusting for gestational age at delivery [aRR=4.5 (1.07-16.7)]. Of the 11 children diagnosed with neuropsychological dysfunction, nine were delivered at term and eight of them had mothers with intra-amniotic inflammation. Children exposed to intra-amniotic inflammation were found to have abnormalities in neuropsychological tasks evaluating complex skills, e.g., auditory attention, executive functions, and social skills, whereas the domains of reasoning, language, and memory were not affected in the cases and controls. Asymptomatic sterile intra-amniotic inflammation in the mid-trimester of pregnancy, followed by a term birth, can still confer to the offspring a substantial risk for neurodevelopmental disorders in childhood. Early recognition and treatment of maternal immune activation in pregnancy may be a strategy for the prevention of subsequent neurodevelopmental disorders in offspring.

The immunobiology of preterm labor and birth: intra-amniotic inflammation or breakdown of maternal-fetal homeostasis.

The syndrome of preterm labor comprises multiple established and novel etiologies. This review summarizes the distinct immune mechanisms implicated in preterm labor and birth and highlights potential strategies for its prevention. Preterm birth, the leading cause of neonatal morbidity and mortality worldwide, results from preterm labor, a syndrome that includes multiple etiologies. In this review, we have summarized the immune mechanisms implicated in intra-amniotic inflammation, the best-characterized cause of preterm labor and birth, as well as novel etiologies non-associated with intra-amniotic inflammation (i.e. formally known as idiopathic). While the intra-amniotic inflammatory responses driven by microbes (infection) or alarmins (sterile) have some overlap in the participating cellular and molecular processes, the distinct natures of these two conditions necessitate the implementation of specific approaches to prevent adverse pregnancy and neonatal outcomes. Intra-amniotic infection can be treated with the correct antibiotics, whereas sterile intra-amniotic inflammation could potentially be treated by administering a combination of anti-inflammatory drugs (e.g. betamethasone, inflammasome inhibitors, etc.). Recent evidence also supports the role of fetal T-cell activation as a newly described trigger for preterm labor and birth in a subset of cases diagnosed as idiopathic. Moreover, herein we also provide evidence of two maternally-driven immune mechanisms responsible for preterm births formerly considered to be idiopathic. First, the impairment of maternal Tregs can lead to preterm birth, likely due to the loss of immunosuppressive activity resulting in unleashed effector T-cell responses. Secondly, homeostatic macrophages were shown to be essential for maintaining pregnancy and promoting fetal development, and the adoptive transfer of homeostatic M2-polarized macrophages shows great promise for preventing inflammation-induced preterm birth. Collectively, in this review, we discuss the established and novel immune mechanisms responsible for preterm birth and highlight the potential targets for novel strategies aimed at preventing the multi-etiological syndrome of preterm labor leading to preterm birth.

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

BackgroundOne 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.MethodsPregnant 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).ResultsClarithromycin 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.ConclusionsClarithromycin 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.

Prevalence and Load of Cervical Ureaplasma Species With Respect to Intra-amniotic Complications in Women With Preterm Prelabor Rupture of Membranes Before 34weeks.

Objectives: To determine the prevalence and load of Ureaplasma spp. DNA in the cervical fluid of women with singleton pregnancies complicated by preterm prelabor rupture of membranes (PPROM) with respect to intra-amniotic infection, sterile intra-amniotic inflammation, and colonization of the amniotic fluid. Methods: A total of 217 women with PPROM between gestational ages 24 + 0 and 33 + 6 weeks were included in this study. Paired amniotic and cervical fluid samples were collected at the time of admission via transabdominal amniocentesis and using a Dacron polyester swab, respectively. Microbial invasion of the amniotic cavity was diagnosed using a combination of culture and molecular biology methods. Intra-amniotic inflammation was determined based on the concentration of interleukin-6 in the amniotic fluid. Based on the presence or absence of these conditions, the women were stratified into the following subgroups: intra-amniotic infection (with both), sterile intra-amniotic inflammation (with inflammation only), colonization (with microorganisms only), and negative amniotic fluid (without either). The Ureaplasma spp. DNA load in the cervical fluid was assessed using PCR. Results: Ureaplasma spp. DNA in the cervical fluid was found in 61% (133/217) of the women. Women with negative amniotic had similar prevalence of Ureaplasma spp. DNA in cervical fluid (55%) to those with sterile intra-amniotic inflammation (54%) but lower than those with intra-amniotic infection (73%) and colonization (86%; p < 0.0001). Women with negative amniotic fluid had a lower load of Ureaplasma spp. DNA in their cervical fluid (median: 4.7 × 103 copies of DNA/ml) than those with intra-amniotic infection (median: 2.8 × 105 copies DNA/ml), sterile intra-amniotic inflammation (median: 5.3 × 104 copies DNA/ml), and colonization (median: 1.2 × 105 copies DNA/mL; p < 0.0001). Conclusion: In conclusion, in PPROM at <34 weeks, the presence of intra-amniotic infection, sterile intra-amniotic inflammation, or colonization of the amniotic fluid was associated with a higher prevalence and/or load of Ureaplasma spp. DNA in the cervical fluid than the absence of intra-amniotic complications.

Clinical characteristics of colonization of the amniotic cavity in women with preterm prelabor rupture of membranes, a retrospective study

To determine the main clinical characteristics of preterm prelabor rupture of membranes (PPROM) complicated by colonization of the amniotic cavity (microbial invasion of the amniotic cavity without intra-amniotic inflammation). A total of 302 women with PPROM were included. Transabdominal amniocentesis was performed and amniotic fluid was assessed. Based of microbial invasion of the amniotic cavity and intra-amniotic inflammation (interleukin-6 ≥ 3000 pg/mL), the women were divided into following groups: intra-amniotic infection, sterile intra-amniotic inflammation, colonization of the amniotic cavity, and negative amniotic fluid. Colonization was found in 11% (32/302) of the women. The most common bacteria identified in the amniotic fluid were Ureaplasma spp. with a lower burden than those with intra-amniotic infection (p = 0.03). The intensity of intra-amniotic inflammatory response measured by interleukin-6 was higher in women with colonization than in those with negative amniotic fluid (medians: 961 pg/mL vs. 616 pg/mL; p = 0.04). Women with colonization had higher rates of acute inflammatory placental lesions than those with negative amniotic fluid. In PPROM, colonization, caused mainly by microorganisms from the lower genital tract, might represent an early stage of microbial invasion of the amniotic cavity with a weak intra-amniotic inflammatory response.

Calprotectin levels in amniotic fluid in relation to intra-amniotic inflammation and infection in women with preterm labor with intact membranes: A retrospective cohort study

ObjectiveTo evaluate the concentrations of calprotectin in amniotic fluid with respect to intra-amniotic inflammation and infection and to assess the presence or absence of bacteria in the amnio-chorionic niche with respect to presence or absence of intra-amniotic inflammation. Study designSeventy-nine women with singleton pregnancies and preterm labor with intact membranes (PTL) were included in the study. Amniotic fluid was collected at the time of admission by amniocentesis and calprotectin levels were analyzed from frozen/thawed samples using ELISA. Interleukin (IL)-6 concentration was measured by point-of-care test. Samples from amniotic fluid and the amnio-chorionic niche (space between amniotic and chorionic membranes) were microbiologically analyzed. Microbial invasion of the amniotic cavity (MIAC) was diagnosed based on a positive PCR result for Ureaplasma species, Mycoplasma hominis, 16S rRNA or positive culture. Intra-amniotic inflammation (IAI) was defined as amniotic fluid point-of-care IL-6 concentration ≥ 745 pg/mL. The cohort of included women was divided into 4 subgroups based on the presence or absence of IAI/MIAC; i) intra-amniotic infection, ii) sterile IAI, iii) intra-amniotic colonization and iv) neither MIAC nor IAI. ResultsWomen with intra-amniotic infection had a significantly higher intra-amniotic calprotectin concentration (median; 101.6 µg/mL) compared with women with sterile IAI (median; 9.2 µg/mL), women with intra-amniotic colonization (median; 2.6 µg/mL) and women with neither MIAC nor IAI (median 4.6 µg/mL) (p = 0.001). Moreover, significantly higher amniotic fluid calprotectin concentration was seen in women who delivered within 7 days (p = 0.003). A significant negative correlation was found between amniotic fluid calprotectin and gestational age at delivery (rho = 0.32, p = 0.003). Relatively more bacteria in the amnio-chorionic niche were found in the sterile IAI group compared with the other groups. ConclusionsCalprotectin concentrations in amniotic fluid were significantly higher in the intra-amniotic infection group compared with the other groups. Moreover, the bacterial presence in the amnio-chorionic niche was higher in IAI group.

Amniotic fluid sludge – a marker of intra-amniotic infection and histological chorioamnionitis in cervical insufficiency

Amniotic fluid sludge is an ultrasound demonstrable hyperechogenic mass close to the uterine cervix and is an independent risk factor for preterm birth and preterm premature rupture of membranes. Cervical insufficiency represents cervical dilatation in the mid-trimester of pregnancy regardless of uterine contractions. It appears to be associated with intraamniotic infection/inflammation. Chorioamnionitis is the inflammation of the fetal membranes (chorion and amnios) and is the consequence of the inflammatory response of the maternal organism to aggression that can be infectious or non-infectious (sterile intraamniotic inflammation). The paper illustrates the association between the amniotic fluid sludge and microbial invasion of the amniotic cavity and chorioamnionitis. Conclusion. Amniotic fluid sludge is a marker of intra-amniotic infection and histological chorioamnionitis in pregnant women with cervical insufficiency.

The alarmin S100A12 causes sterile inflammation of the human chorioamniotic membranes as well as preterm birth and neonatal mortality in mice†.

Sterile inflammation is triggered by danger signals, or alarmins, released upon cellular stress or necrosis. Sterile inflammation occurring in the amniotic cavity (i.e. sterile intra-amniotic inflammation) is frequently observed in women with spontaneous preterm labor resulting in preterm birth, the leading cause of neonatal morbidity and mortality worldwide; this condition is associated with increased amniotic fluid concentrations of alarmins. However, the mechanisms whereby alarmins induce sterile intra-amniotic inflammation are still under investigation. Herein, we investigated the mechanisms whereby the alarmin S100A12 induces inflammation of the human chorioamniotic membranes in vitro and used a mouse model to establish a causal link between this alarmin and adverse perinatal outcomes. We report that S100A12 initiates sterile inflammation in the chorioamniotic membranes by upregulating the expression of inflammatory mediators such as pro-inflammatory cytokines and pattern recognition receptors. Importantly, S100A12 induced the priming and activation of inflammasomes, resulting in caspase-1 cleavage and the subsequent release of mature IL-1β by the chorioamniotic membranes. This alarmin also caused the activation of the chorioamniotic membranes by promoting MMP-2 activity and collagen degradation. Lastly, the ultrasound-guided intra-amniotic injection of S100A12 at specific concentrations observed in the majority of women with sterile intra-amniotic inflammation induced preterm birth (rates: 17% at 200ng/sac; 25% at 300ng/sac; 25% at 400ng/sac) and neonatal mortality (rates: 22% at 200ng/sac; 44% at 300ng/sac; 31% at 400ng/sac), thus demonstrating a causal link between this alarmin and adverse perinatal outcomes. Collectively, our findings shed light on the inflammatory responses driven by alarmins in the chorioamniotic membranes, providing insight into the immune mechanisms leading to preterm birth in women with sterile intra-amniotic inflammation.

Intra-amniotic infection and sterile intra-amniotic inflammation in women with preterm labor with intact membranes are associated with a higher rate of Ureaplasma species DNA presence in the cervical fluid

Objective To determine the prevalence of Ureaplasma spp. DNA and its load in the cervical fluid in women with preterm labor with intact membranes (PTL) complicated by intra-amniotic infection (the presence of both microbial invasion of the amniotic cavity and intra-amniotic inflammation) or sterile intra-amniotic inflammation (the presence of intra-amniotic inflammation alone). Methods Overall, 115 women with singleton pregnancies complicated by PTL between gestational ages of 22 + 0 and 34 + 6 weeks were included in this study. Paired amniotic and cervical fluid samples were collected at the time of admission via transabdominal amniocentesis using a Dacron polyester swab. Microbial invasion of the amniotic cavity was diagnosed based on a combination of culture and molecular biology methods. Intra-amniotic inflammation was determined based on the concentration of interleukin-6 in the amniotic fluid. Bacterial and Ureaplasma spp. DNA loads were assessed in the cervical fluid using PCR. Results Intra-amniotic infection and sterile inflammation were identified in 14% (16/115) and 25% (29/115) of the women, respectively. Ureaplasma spp. DNA in the cervical fluid was identified in 51% (59/115) of women. The presence of Ureaplasma spp. DNA in the cervical fluid was higher in women with intra-amniotic infection (75% (12/16)) and sterile intra-amniotic inflammation (76% (22/29)) than in women without intra-amniotic inflammation (36% (25/70); p = .0002). Concurrent presence of Ureaplasma spp. and Mycoplasma hominis DNA was higher in women with intra-amniotic infection (42% (5/12)) than women with sterile intra-amniotic inflammation (7% (2/29)) and women without intra-amniotic inflammation (7% (5/70); p = .001). There were no differences in the load of Ureaplasma spp. DNA in the cervical fluid among women with intra-amniotic infection, sterile intra-amniotic inflammation, and those without intra-amniotic inflammation (median values; infection: 1.2 × 104 copies DNA/mL; sterile: 5.0 × 105 copies DNA/mL; without: 8.4 × 104 copies DNA/mL; p = .18). Conclusions In PTL , both forms of intra-amniotic inflammation were associated with a higher prevalence of Ureaplasma spp. DNA in the cervical fluid. The presence of intra-amniotic infection was related to a higher rate of concurrent Ureaplasma spp. and M. hominis DNA in the cervical fluid.

Macrophage inflammatory protein-1α in amniotic and cervical fluids in spontaneous preterm labor with intact membranes with respect to intra-amniotic inflammation

Objective Macrophage inflammatory protein 1α is a chemokine produced by various immune, epithelial, mesothelial, and fibroblast cells after exposure to bacterial lipopolysaccharide or pro-inflammatory molecules. The primary aim of this study was to determine MIP-1α concentrations in amniotic and cervical fluids from pregnancy with spontaneous preterm labor with intact membranes (PTL) with respect to the presence of intra-amniotic infection (both microbial invasion of the amniotic cavity and intra-amniotic inflammation) and sterile intra-amniotic inflammation (intra-amniotic inflammation alone). The secondary aim was to assess the diagnostic indices of MIP-1α in predicting intra-amniotic infection. Materials and methods Seventy-four women with PTL were included in this study. Paired amniotic and cervical fluid samples were obtained using transabdominal amniocentesis and a Dacron polyester swab, respectively. Microbial invasion of the amniotic cavity was diagnosed based on a combination of culture and molecular biology methods. The concentration of IL-6 in the amniotic and cervical fluids was measured using an automated electrochemiluminescence immunoassay method. Intra-amniotic inflammation was defined as an amniotic fluid IL-6 concentration of ≥3000 pg/mL. The MIP-1α concentrations in the samples were assessed using an enzyme-linked immunosorbent assay. Results A difference in amniotic fluid MIP-1α was observed among women with intra-amniotic infection, sterile intra-amniotic inflammation, and negative amniotic fluid (infection: median 1779.0 pg/mL; sterile, median 102.7 pg/mL; negative, median 19.9 pg/mL; p < .0001). No difference in the concentrations of MIP-1α was identified in cervical fluid after adjustment for gestational age at sampling (infection: median 77.7 pg/mL, sterile: median 152.7 pg/mL, negative: median 18.0 pg/mL; p = .30). The presence of intra-amniotic infection was associated with elevated MIP-1α concentrations in amniotic fluid (presence: 1779.0 pg/mL vs. absence: 26.3 pg/mL, p < .0001, area under receiver operating characteristic curve = 0.87). Conclusions In PTL pregnancies with the presence of intra-amniotic infection, the concentration of MIP-1α is elevated in amniotic fluid but not in cervical fluid. Amniotic fluid MIP-1α may provide a useful marker for intra-amniotic infection in women with PTL.

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

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. 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. 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. 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.

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

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

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

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. 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≥2.6ng/mL. (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. 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.