Intra-amniotic Inflammation Research Articles

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.

Expression of antimicrobial peptides in the amniotic fluid of women with cervical insufficiency.

Cervical insufficiency (CI) is associated with intra-amniotic infection or inflammation. Antimicrobial peptides (AMPs) in amniotic fluid may protect the fetus against microbial invasion, giving their broad-spectrum microbiocidal properties. We evaluated changes in amniotic fluid AMP expression in women with CI and assessed whether these changes are related to their pregnancy outcomes. We evaluated amniotic fluid human neutrophil peptide 3 (HNP-3), human β-defensin-2 (hBD-2), and LL-37 levels in 66 women with CI and 25 normal controls at 16-24 weeks of gestation. The CI group was divided into short cervix and cervical dilation groups, and the cervical dilation group was further divided into preterm and full-term delivery groups according to the pregnancy outcomes, and AMP expression was analyzed in each group. HNP-3 and hBD-2 levels were higher in women with CI than in normal controls and in the cervical dilation as compared to the short cervix group. Among women with cervical dilation, 22 delivered at full-term, and 23 had spontaneous preterm births. The hBD-2 level in amniotic fluid mid-pregnancy was higher in the full-term delivery than in the preterm delivery groups. However, LL-37 levels in amniotic fluid were low in women with CI and normal controls. Amniotic fluid HNP-3 and hBD-2 levels increased in women with CI compared with normal controls. Moreover, increased amniotic fluid hBD-2 levels mid-pregnancy were associated with favorable pregnancy outcomes in women with CI. AMPs in the amniotic fluid may participate in host defense against ascending infection in women with CI.

Lamellar body count: Marker for foetal lung maturation promoted by intra-amniotic infection and/or inflammation

ObjectiveThere is evidence indicating that the risk of respiratory distress syndrome is reduced in preterm neonates exposed to intra-amniotic infection and/or inflammation. We hypothesised that foetal lung maturation promoted by intra-amniotic infection and/or inflammation results in elevated lamellar body count (LBC) in amniotic fluid (AF). This study aimed to determine the relationship between LBC in AF and intra-amniotic infection and/or inflammation in patients with threatened preterm birth. Study designThis was a retrospective cohort study of patients with threatened preterm birth. A total of 104 consecutive pregnant women underwent amniocentesis in the early preterm period [gestational age < 34 weeks] to evaluate intra-amniotic infection and/or inflammation and foetal lung maturity. Intra-amniotic infection was confirmed by positive AF culture results for aerobic/anaerobic bacteria, fungi, and genital mycoplasma. Intra-amniotic inflammation was defined as a positive AF matrix metalloproteinase-8 rapid test. Outcomes of the study population were compared according to LBC in AF using a cut-off of 15,000/mm3. ResultsThe rates of elevated LBC and intra-amniotic infection and/or inflammation were 23% (24/104) and 52% (54/104), respectively. The median LBC was significantly higher in patients with intra-amniotic infection and/or inflammation than in those without [median LBC, 9,000/mm3 (interquartile range, IQR: 3,000–39,000) vs. 3,000/mm3 (IQR: 2,750–5,000), p < 0.001]. Intra-amniotic infection and/or inflammation was observed in 96% (23/24) of patients with elevated LBC and 39% (31/80) of patients without elevated LBC (p < 0.001). On multivariable analysis, the presence of intra-amniotic infection and/or inflammation was significantly associated with elevated LBC with an odds ratio (OR) of 66.0 [95% confidence interval (CI) 6.6–664.4, p < 0.001], even after accounting for gestational age at amniocentesis being a significantly related factor for predicting elevated LBC with an OR of 1.5 (95% CI 1.1–2.0, p = 0.004). ConclusionLBC elevation was independently associated with the presence of intra-amniotic infection and/or inflammation in women with early threatened preterm birth (gestational age < 34 weeks). This finding may support the view that an intra-amniotic inflammatory response promotes foetal lung maturation that can be detected by elevated LBC in AF.

Development of a Rat Model of Intra-Amniotic Inflammation via Ultrasound-Guided Administration of a Triggering Agent in the Gestational Sac to Enable Analysis of Individual Amniotic Fluid Samples.

Objectives: To develop a rat model of intra-amniotic inflammation, characterized by the concentration of interleukin-6 in the amniotic fluid, induced by an ultrasound-guided transabdominal administration of lipopolysaccharide into individual gestational sacs. Methods: An ultrasound-guided transabdominal intra-amniotic administration of lipopolysaccharide or phosphate-buffered saline (PBS) as control was performed in rats on embryonic day 18. Only accessible gestational sacs with precise recording of their positions were injected. Twenty-four hours later, individual amniotic fluid samples were collected from the gestational sacs of laparotomized animals. The gestational sacs were divided into four subgroups: (i) with lipopolysaccharide: injected gestational sacs from rats undergoing lipopolysaccharide administration; (ii) without lipopolysaccharide: non-injected gestational sacs from rats undergoing lipopolysaccharide administration; (iii) with PBS: injected gestational sacs from rats undergoing PBS administration; and (iv) without PBS: non-injected gestational sacs from rats undergoing PBS administration. The concentration of interleukin-6 in individual amniotic fluid samples was assessed using ELISA. Results: In the group of five animals receiving lipopolysaccharide, 24 (33%) and 48 (77%) gestational sacs were and were not injected, respectively. The amniotic fluid was obtained from 21 (88%) injected and 46 (95%) non-injected sacs. In the control group of five animals receiving phosphate-buffered saline, 28 (35%) and 52 (75%) gestational sacs were and were not injected, respectively. The amniotic fluid was obtained from 18 (64%) injected and 50 (96%) non-injected sacs. No labor occurred, and only one fetal death was observed in a gestational sac injected with lipopolysaccharide. Differences in concentrations of interleukin-6 in the amniotic fluid were found among the subgroups of the gestational sacs (with lipopolysaccharide: median 762 pg/ml; without lipopolysaccharide: median 35.6 pg/ml; with PBS: median 35.6 pg/ml; and without PBS: median 35.6 pg/ml; p < 0.0001). Concentrations of interleukin-6 in the amniotic fluid from the gestational sacs with lipopolysaccharide were significantly higher than those in the three remaining subgroups (p < 0.0001). No differences in concentrations of interleukin-6 in the amniotic fluid were identified between the three remaining subgroups. Conclusion: The ultrasound-guided transabdominal intra-amniotic administration of lipopolysaccharide with a subsequent collection and analysis of amniotic fluid samples is feasible in rats. The intra-amniotic administration of lipopolysaccharide led to the development of intra-amniotic inflammation without leading to fetal mortality or induction of labor.

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.

Effects of maternal ritodrine hydrochloride administration on the heart rate of preterm fetal sheep with intraamniotic inflammation.

Ritodrine hydrochloride is used for pregnancy prolongation and intrauterine fetal resuscitation. However, its clinical significance in intraamniotic inflammation during preterm labor and intrauterine fetal distress is unclear. We investigated the effects of maternal ritodrine hydrochloride administration (MRA; 200 μg/min for 2 h, followed by 800 μg/min for 2 h after 24 h) on fetal physiological parameters. For this purpose, we used chronically instrumented pregnant sheep at 113–119 d (term = 145 d) of gestation without (Group 1, n = 5) and with (Group 2, n = 5) intraamniotic inflammation induced by lipopolysaccharide injection into the amniotic cavity. The changes in fetal heart rate (FHR) and short-term variability (STV) and long-term variability (LTV) in FHR, fetal blood pressure, and fetal arterial blood gas (FABG) values were measured before and at 1 and 2 h after initiating MRA. Before MRA, all parameters were similar between Groups 1 and 2; however, there was significantly higher STV in Group 2 than in Group 1 before MRA at 800 μg/min, significantly higher partial arterial pressure of carbon dioxide in FABG in Group 2 than in Group 1 before MRA at 200 μg/min, and significantly lower blood glucose (BG) in Group 2 than in Group 1 before MRA at 800 μg/min. One hour after MRA, the FHR, STV, and LTV were significantly higher at 800 μg/min than those at the baseline in Group 1, as determined by the Friedman test; however, no significant difference was observed in Group 2. Additionally, the FABG pH significantly decreased 1 h after MRA at 800 μg/min in Group 2, whereas FABG lactate and BG significantly increased 2 h after MRA at 800 μg/min in Groups 1 and 2. Thus, short-term MRA at 800 μg/min increased the FHR, STV, and LTV significantly; these values were further modified under intraamniotic inflammation.

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.

Diagnosis of intraamniotic inflammation by measuring vaginal interleukin-6 in patients with cervical insufficiency: could amniocentesis be avoided?

Background Cervical insufficiency is a recurrent, passive, and painless dilation of the cervix in the second trimester. The etiology is unclear, but there may be an association with subclinical intraamniotic infection. Interleukin-6 (IL-6) production in the amniotic cavity is induced by bacterial invasion, it is the major proinflammatory cytokine released in response to infection. Although the gold standard method to measure it is through an amniocentesis, the procedure constitutes an invasive technique with several associated risks. The objective of this study is to determine if there is a correlation between intraamniotic and vaginal IL-6 in patients with cervical insufficiency and bulging membranes during the second trimester of pregnancy, in order to avoid an amniocentesis before the rescue cerclage. Methods A cohort study was performed in which all patients with cervical insufficiency and bulging membranes admitted into our tertiary hospital between 2019 and 2020 were included, and a control group of asymptomatic women in the second trimester of gestation where studied at the same time. Patients with bulging membranes underwent an amniocentesis to quantify amniotic IL-6, and a sample of vaginal fluid for vaginal IL-6 determination was obtained from both the study and the control group. Results A total of 20 women were included in each group. Median gestational age at diagnosis was 22 weeks in patients with bulging membranes, and 21 weeks in the control group. Vaginal IL-6 in control group (10.875 pg/mL) is much lower than the study group one (1308.77 pg/ml). In patients with bulging membranes, vaginal IL-6 expression was lower in the vagina than in the amniotic cavity [average IL-6 in the amniotic cavity 26890.07 pg/mL, vs 1308.77 pg/mL in the vagina (p < .01)]. Through a Spearman coefficient correlation rank [rho = 0.709 (p < .001)], there is a positive correlation between amniotic and vaginal IL-6 values. The best value of this correlation was calculated with the ROC curve, being the area under the curve 0.929 (CI 95% 0.721–0.995), and the cutoff of point less than 61.4 pg/ml (sensitivity 83.33%; specificity 92.86%). Patients with vaginal IL-6 < 61.4 pg/ml associated a longer latency time between diagnosis and delivery, a higher neonatal weight and a lower perinatal mortality. Rescue cerclage in vaginal IL-6 < 61.4 pg/ml was the best predictor of good pregnancy outcome. Conclusion There is a correlation between intraamniotic and vaginal IL-6 in patients with cervical insufficiency and bulging membranes during the second trimester of pregnancy. However, further studies are needed in order to considerate the avoidance of an amniocentesis before an emergency cerclage.

Preterm prelabor rupture of membranes without microbial invasion of the amniotic cavity and intra-amniotic inflammation: a heterogeneous group with differences in adverse outcomes

Objective The absence of microbial invasion of the amniotic cavity and intra-amniotic inflammation at the time of hospital admission is the most common condition associated with preterm prelabor rupture of membranes (PPROM). Although the intensity of intra-amniotic inflammatory response does not exceed the threshold for the diagnosis of intra-amniotic inflammation in this subgroup of PPROM, whether there could be differences in outcomes concerning the intensity of intra-amniotic inflammatory response remains unclear. Therefore, the main aims of this study on PPROM without microbial invasion of the amniotic cavity and intra-amniotic inflammation were (i) to characterize the association between the intensity of intra-amniotic inflammatory response, measured according to amniotic fluid interleukin (IL)-6 concentrations, and the presence of acute histological chorioamnionitis and acute inflammation in the amnion; (ii) to characterize the association between the intensity of intra-amniotic inflammatory response and fetal inflammatory response, and (iii) to describe the short-term morbidity of infants based on the intensity of intra-amniotic inflammatory response. Methods This retrospective study included 131 women with singleton pregnancies with PPROM without microbial invasion of the amniotic cavity and intra-amniotic inflammation between gestational ages of 24 + 0 and 36 + 6 weeks and who had delivered within 72 h of membrane rupture. Microbial invasion of the amniotic cavity was assessed based on a combination of cultivation and non-cultivation methods. Intra-amniotic inflammation was characterized based on the amniotic fluid IL-6 concentration. In addition, a histopathological assessment of the placenta was performed. Fetal inflammatory response syndrome was characterized according to IL-6 concentration in the umbilical cord blood of >11 pg/mL. Based on the quartiles of IL-6 concentrations in the amniotic fluid, these women were divided into four subgroups (from the lowest to the highest IL-6 concentrations). Results IL-6 concentrations in amniotic fluid were higher in women with acute histological chorioamnionitis (median: 819 pg/mL vs. 520 pg/mL; p = .003) and with acute inflammation of the amnion (median: 1116 pg/mL vs. 533 pg/mL; p = .0002) than in women without these complications. The rates of acute histological chorioamnionitis and acute inflammation of the amnion were the highest in the subgroup with IL-6 concentrations above the 75th percentile in amniotic fluid (chorioamnionitis, p = .02; amnion, p = .0002). No differences in IL-6 concentrations in amniotic fluid were identified between women with and without a fetal inflammatory response syndrome (p = .40). The rate of fetal inflammatory response syndrome did not vary among the amniotic fluid IL-6 quartile subgroups of women. Moreover, no differences were noted in short-term neonatal outcomes among the amniotic fluid IL-6 quartile subgroups. Conclusion A higher intensity of the intra-amniotic inflammatory response, measured by amniotic fluid IL-6 concentrations, is associated with a higher rate of acute inflammatory lesions in the placenta in the subset of PPROM pregnancies without microbial invasion of the amniotic cavity and intra-amniotic inflammation.

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.

Development of a mouse model of ascending infection and preterm birth.

Microbial invasion of the intraamniotic cavity and intraamniotic inflammation are factors associated with spontaneous preterm birth. Understanding the route and kinetics of infection, sites of colonization, and mechanisms of host inflammatory response is critical to reducing preterm birth risk. This study developed an animal model of ascending infection and preterm birth with live bacteria (E. coli) in pregnant CD-1 mice with the goal of better understanding the process of microbial invasion of the intraamniotic cavity and intraamniotic inflammation. Multiple experiments were conducted in this study. To determine the dose of E. coli required to induce preterm birth, CD-1 mice were injected vaginally with four different doses of E. coli (103, 106, 1010, or 1011 colony forming units [CFU]) in 40 μL of nutrient broth or broth alone (control) on an embryonic day (E)15. Preterm birth (defined as delivery before E18.5) was monitored using live video. E. coli ascent kinetics were measured by staining the E. coli with lipophilic tracer DiD for visualization through intact tissue with an in vivo imaging system (IVIS) after inoculation. The E. coli were also directly visualized in reproductive tissues by staining the bacteria with carboxyfluorescein succinimidyl ester (CFSE) prior to administration and via immunohistochemistry (IHC) by staining tissues with anti-E. coli antibody. Each pup's amniotic fluid was cultured separately to determine the extent of microbial invasion of the intraamniotic cavity at different time points. Intraamniotic inflammation resulting from E. coli invasion was assessed with IHC for inflammatory markers (TLR-4, P-NF-κB) and neutrophil marker (Ly-6G) for chorioamnionitis at 6- and 24-h post-inoculation. Vaginally administered E. coli resulted in preterm birth in a dose-dependent manner with higher doses causing earlier births. In ex vivo imaging and IHC detected uterine horns proximal to the cervix had increased E. coli compared to the distal uterine horns. E. coli were detected in the uterus, fetal membranes (FM), and placenta in a time-dependent manner with 6 hr having increased intensity of E. coli positive signals in pups near the cervix and in all pups at 24 hr. Similarly, E. coli grew from the cultures of amniotic fluid collected nearest to the cervix, but not from the more distal samples at 6 hr post-inoculation. At 24 hr, all amniotic fluid cultures regardless of distance from the cervix, were positive for E. coli. TLR-4 and P-NF-κB signals were more intense in the tissues where E. coli was present (placenta, FM and uterus), displaying a similar trend toward increased signal in proximal gestational sacs compared to distal at 6 hr. Ly-6G+ cells, used to confirm chorioamnionitis, were increased at 24 hr compared to 6 hr post-inoculation and control. We report the development of mouse model of ascending infection and the associated inflammation of preterm birth. Clinically, these models can help to understand mechanisms of infection associated preterm birth, determine targets for intervention, or identify potential biomarkers that can predict a high-risk pregnancy status early in pregnancy.

Complement Activation Fragments in Cervicovaginal Fluid Are Associated with Intra-Amniotic Infection/Inflammation and Spontaneous Preterm Birth in Women with Preterm Premature Rupture of Membranes.

We sought to determine whether the levels of complement and other inflammatory and angiogenic mediators in cervicovaginal fluid (CVF) are independently associated with intra-amniotic infection and/or inflammation (IAI) and imminent spontaneous preterm birth (SPTB, £48 hours of sampling) in women with preterm premature rupture of membranes (PPROM). This was a retrospective study consisting of 85 singleton pregnant women with PPROM at 200/7 to 336/7 weeks. Amniotic fluid (AF) obtained via amniocentesis was cultured and assayed for interleukin-6. CVF samples collected at the time of amniocentesis were assayed for complement C3a, C4a, and C5a, HSP70 (heat shock protein 70), M-CSF (macrophage colony-stimulating factor), M-CSF-R (macrophage colony-stimulating factor-receptor), S100 A8, S100 A9, thrombospondin-2, VEGF (vascular endothelial growth factor-receptor), and VEGFR-1 (vascular endothelial growth factor-receptor 1) by enzyme-linked immunosorbent assay. Multivariate logistic regression analyses revealed that elevated CVF concentrations of complement C3a, 4a, and 5a were significantly associated with an increased risk of IAI and imminent SPTB, whereas those of M-CSF were associated with IAI, but not imminent SPTB (p = 0.063), after adjustment for baseline covariates (e.g., gestational age at sampling). However, univariate, and multivariate analyses showed that the CVF concentrations of angiogenic (thrombospondin-2, VEGF, and VEGFR-1) and inflammatory (HSP70, M-CSF-R, S100 A8, and S100 A9) proteins were not associated with either IAI or imminent SPTB. In women with PPROM, elevated CVF concentrations of complement C3a, C4a, and C5a are independently related to an increased risk of IAI and imminent SPTB. These findings suggest that complement activation in CVF is significantly involved in mechanisms underlying preterm birth and in the host response to IAI in the context of PPROM. · Elevated CVF levels of C3a, 4a and 5a are associated with IAI and SPTB.. · CVF C3a, 4a and 5a have better predictability for SPTB, compared to AF WBC.. · Elevated CVF levels of M-CSF were associated with IAI, but not SPTB..

84 Looking back to move forward; can fetal placental inflammatory response play a role in risk of sepsis and clinical decision making in preterm infant

Abstract Primary Subject area Neonatal-Perinatal Medicine Background Sepsis adversely impacts the survival of very low birth weight infants (VLBW), with a mortality risk up to 50%. Its diagnosis in premature infants is challenging. Conversely, prolonged antibiotic use is associated with perilous potential consequences, propelling the need to better identify those at risk of sepsis. The presence of a histological intra-amniotic inflammation (IAI) response may be associated with an increased risk of early onset sepsis (EOS). Currently, it is unknown how the diagnosis of histological IAI impacts the risk of sepsis. Objectives Our research goal is to explore histological fetal and maternal inflammation in the placenta in VLBW infants and to evaluate if specific recommendations about antibiotic management of VLBW infants with histological fetal response can be proposed. Design/Methods Retrospective cohort study of all infants &amp;lt; 1500 g born to a mother with histologically confirmed IAI. Demographic information about the pregnancy, delivery and postnatal course up to 28 days of life was extracted. Descriptive statistical analysis was conducted to compare the characteristics of infants with histological fetal response using χ2 test or Fisher’s exact test and Wilcoxon rank sum test or ANOVA. Results Seventy-three mother-baby pairs were reviewed. EOS prevalence (19%) in our IAI group of VLBW infants is much higher than EOS observed in all VLBW infants from the Canadian Neonatal Network database (below 3%). In our cohort, the majority had fetal inflammatory stage 1 (31.6%) and fetal inflammatory grade 0 (50%). There was no statistically significant distribution amongst the fetal stages or grades. Time to sepsis event analysis showed that the earlier fetal inflammatory grade was associated with positive cultures occurring earlier, while in the later grades there demonstrated longer latency to positive cultures. This trend was also true when looking at the maternal inflammatory stages. Nine infants who had antibiotics discontinued from day of life (DOL) 2-5 developed a positive blood culture ≤ DOL8. Conclusion Our results suggest that the presence of mild fetal inflammatory changes is associated with earlier positive cultures. We hypothesize that later grades may be associated with longer infection exposure leading to prolonged maternal antibiotics, resulting in less EOS. Majority of positive cultures were within the 3-8 day window, suggesting a role of knowing the fetal inflammatory response when deciding duration of antibiotic treatment.

The Role of Innate Immune System in the Human Amniotic Membrane and Human Amniotic Fluid in Protection Against Intra-Amniotic Infections and Inflammation

Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI.

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.

Direct administration of the non-competitive interleukin-1 receptor antagonist rytvela transiently reduced intrauterine inflammation in an extremely preterm sheep model of chorioamnionitis.

Intraamniotic inflammation is associated with up to 40% of preterm births, most notably in deliveries occurring prior to 32 weeks' gestation. Despite this, there are few treatment options allowing the prevention of preterm birth and associated fetal injury. Recent studies have shown that the small, non-competitive allosteric interleukin (IL)-1 receptor inhibitor, rytvela, may be of use in resolving inflammation associated with preterm birth (PTB) and fetal injury. We aimed to use an extremely preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela in response to established intra-amniotic (IA) lipopolysaccharide (LPS) exposure. We hypothesized that rytvela would reduce LPS-induced IA inflammation in amniotic fluid (AF) and fetal tissues. Sheep with a single fetus at 95 days gestation (estimated fetal weight 1.0 kg) had surgery to place fetal jugular and IA catheters. Animals were recovered for 48 hours before being randomized to either: i) IA administration of 2 ml saline 24 hours before 2 ml IA and 2 ml fetal intravenous (IV) administration of saline (Saline Group, n = 7); ii) IA administration of 10 mg LPS in 2 ml saline 24 hours before 2 ml IA and 2 ml fetal IV saline (LPS Group, n = 10); 3) IA administration of 10 mg LPS in 2 ml saline 24 hours before 0.3 mg/fetal kg IA and 1 mg/fetal kg fetal IV rytvela in 2 ml saline, respectively (LPS + rytvela Group, n = 7). Serial AF samples were collected for 120 h. Inflammatory responses were characterized by quantitative polymerase chain reaction (qPCR), histology, fluorescent immunohistochemistry, enzyme-linked inmmunosorbent assay (ELISA), fluorescent western blotting and blood chemistry analysis. LPS-treated animals had endotoxin and AF monocyte chemoattractant protein (MCP)-1 concentrations that were significantly higher at 24 hours (immediately prior to rytvela administration) relative to values from Saline Group animals. Following rytvela administration, the average MCP-1 concentrations in the AF were significantly lower in the LPS + rytvela Group relative to in the LPS Group. In delivery samples, the expression of IL-1β in fetal skin was significantly lower in the LPS + rytvela Group compared to the LPS Group. A single dose of rytvela was associated with partial, modest inhibition in the expression of a panel of cytokines/chemokines in fetal tissues undergoing an active inflammatory response.

Necrotizing funisitis is an indicator that intra-amniotic inflammatory response is more severe and amnionitis is more frequent in the context of the extension of inflammation into Wharton's jelly

ObjectiveNecrotizing funisitis (NF) is defined as the presence of an arc (i.e., crescent/band/ring/halos) of infiltrated neutrophils and/or associated debris in Wharton's jelly (WJ) of umbilical-cord (UC). However, no information exists about the comparison in intra-amniotic inflammatory-response (IAIR) and inflammation in extra-placental membranes between the presence and absence of NF in the context of inflammation in WJ among spontaneous preterm births (PTBs). The objective of current study is to examine this issue. Materials and methodsWe examined IAIR and the frequency of amnionitis according to the progression of inflammation in UC (i.e. stage-1, umbilical phlebitis [inflammation in umbilical-vein(UV)] only; stage-2, involvement of at least one umbilical-artery[UA] and either the other UA or UV without extension into WJ; stage-3, the extension of inflammation into WJ without NF; stage-4, the extension of inflammation into WJ with NF) in 120singleton spontaneous PTBs (<37weeks). IAIR was gauged by AF MMP-8 (ng/ml) within 3days before birth. Results1) Stage-1, stage-2, stage-3, and stage-4 were present in 20%(24/120), 6%(7/120), 61%(73/120), and 13%(16/120) of cases respectively; 2) AF MMP-8 continuously increased (stage-1 vs. stage-2 vs. stage-3 vs. stage-4; median[ng/ml], range[ng/ml]; 207.2[16.8–1196.5] vs. 444.1[8.5–2608.0] vs. 458.8[0.4–3116.7] vs. 1859.7[912.3–5304.8]; Spearman's rank correlation-test, α = 0.454, P = 0.006), and the frequency of increased AF MMP-8 (≥854.1 ng/ml) elevated (stage-1 vs. stage-2 vs. stage-3 vs. stage-4; 13%[1/8] vs. 33%[1/3] vs. 32%[6/19] vs. 100%[5/5]; Linear-by-linear-association, P = 0.012) with the progression of inflammation in UC; 3) Moreover, there was a stepwise increase in the frequency of amnionitis according to the progression of inflammation in UC (stage-1, 33%[8/24]; stage-2, 43%[3/7]; stage-3, 62%[45/73]; stage-4, 81%[13/16]; Linear-by-linear-association, P = 0.001). ConclusionNF is an indicator that IAIR is more severe and amnionitis is more frequent in the context of the extension of inflammation into WJ. Therefore, current study confirms that NF is the most advanced stage in the progression of inflammation within UC.

Birth weight and intra-amniotic inflammatory and infection-related complications in pregnancies with preterm prelabor rupture of membranes: a retrospective cohort study

Objective To assess the association between the birth weight of newborns and microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation in pregnancies with preterm prelabor rupture of membranes. Methods A total of 528 pregnancies with preterm prelabor rupture of membranes were included in this retrospective cohort study. Transabdominal amniocentesis to determine the presence of MIAC (through culturing and molecular biology methods) and intra-amniotic inflammation (according to amniotic fluid interleukin-6 level) was performed as part of standard clinical management. Based on the presence of MIAC and/or intra-amniotic inflammation, the participants were divided into four subgroups: with intra-amniotic infection (presence of both), with sterile IAI (intra-amniotic inflammation alone), with colonization (MIAC alone), and with negative amniotic fluid (absence of both). Birth weights of newborns are expressed as percentiles derived from INTERGROWTH-21st standards for (i) newborn birth weight and (ii) estimated fetal weight. Results No differences in birth weights, expressed as percentiles derived from newborn weight standards (infection: median 52; sterile: median 54; colonization: median 50; negative amniotic fluid: median 51; p = .93) and estimated fetal weight standards (infection: median 47; sterile: median 51; colonization: median 47; negative amniotic fluid: median 53; p = .48) were found among the four subgroups. No differences in percentiles (derived from both standards) were found in the subset of participants who delivered within 72 h after rupture of membranes (newborn weight standard, p = .99; estimated fetal weight standard, p = .81). Conclusions No association was identified between the birth weight of newborns and the presence of intra-amniotic inflammatory and infection-related complications in pregnancies with preterm prelabor rupture of membranes.