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Problem: To determine whether altered concentrations of various inflammation/immune-, acute phase-, extracellular matrix-, adhesion-, and serine protease-related proteins in the amniotic fluid (AF) are independently associated with microbial invasion of the amniotic cavity and/or intra-amniotic inflammation (MIAC/IAI), imminent spontaneous preterm delivery (SPTD; ≤7 days), and major neonatal morbidity/mortality (NMM) in women with early preterm prelabor rupture of membranes (PPROM)., Method of Study: This was a retrospective cohort study involving 111 singleton pregnant women with PPROM (24-31 weeks) undergoing amniocentesis to diagnose MIAC/IAI. The following proteins were measured in stored AF samples by enzyme-linked immunosorbent assay (ELISA): APRIL, DKK-3, Gal-3BP, IGFBP-2, IL-8, VDBP, lumican, MMP-2, MMP-8, SPARC, TGFBI, TGF-β1, E-selectin, ICAM-5, P-selectin, haptoglobin, hepcidin, SAA1, kallistatin, and uPA., Results: Multivariate logistic regression analyses revealed that (i) elevated APRIL, IL-8, MMP-8, and TGFBI levels in the AF, reduced lumican and SPARC levels in the AF, and high percentages of samples above the lower limit of quantification for AF TGF-β1 and uPA were significantly associated with MIAC/IAI; (ii) elevated AF levels of IL-8 and MMP-8 were significantly associated with SPTD within 7 days; and (iii) elevated AF IL-6 levels were significantly associated with increased risk for major NMM, when adjusted for baseline covariates., Conclusion: ECM (lumican, SPRAC, TGFBI, and TGF-β1)- and serine protease (uPA)-associated proteins in the AF are involved in the regulation of the host response to infection/inflammation in the amniotic cavity, whereas AF inflammation (IL-8, MMP-8, and IL-6)-associated mediators are implicated in the development of preterm parturition and major NMM in early PPROM., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Šket, Ramuta, Starčič Erjavec and Kreft.)

Preterm birth is an important determinant of neonatal morbidity and mortality and intra-amniotic infection (IAI) and inflammation play a causative role. The constitutive proteasome and immunoproteasome are key players in maintenance of proteostasis and their alteration outside pregnancy has been linked to pathogenesis of numerous inflammatory diseases. Our goal was to evaluate the levels, activities, and potential origin of amniotic fluid (AF) proteasome in women with preterm birth induced by infection and/or inflammation. Total proteasome and immunoproteasome concentrations were measured in AF retrieved by trans-abdominal amniocentesis from 155 pregnant women. Proteasome activities were measured with fluorogenic substrates targeting caspase-like (CAS-L), trypsin-like (TRY-L), or chymotrypsin-like (CHE-L) lytic activities. We found that IAI significantly upregulated AF concentrations of total proteasome and of the immunoproteasome (P<0.001 for both) with no differences based on gestational age. Based on substrate preference and profile of pharmacologic inhibition, we identified the CHE-L activity of the immunoproteasome as the primary lytic activity upregulated in AF of pregnancies complicated by IAI. When compared with matched maternal blood and cord blood, proteasome activity was by far the highest in AF and this was further elevated in IAI. Western blot confirmed β5 (PSMB5) and β5i (PSMB8) subunits of the constitutive proteasome and immunoproteasome are present in AF and IHC staining of fetal membranes pointed to chorio-decidua as a potential source. In conclusion, IAI is associated with increased AF immunoproteasome activity that by analogy with other inflammatory diseases may generate antigenic oligopeptides and may play a role in triggering preterm birth., (© 2021. Society for Reproductive Investigation.)

Objectives: Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5-12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia., Methods: This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration ≥2.6 ng/mL., Results: (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis ; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40-58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases., Conclusions: Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood., (© 2020 Walter de Gruyter GmbH, Berlin/Boston.)

Intra-amniotic infection, the invasion of microbes into the amniotic cavity resulting in inflammation, is a clinical condition that can lead to adverse pregnancy outcomes for the mother and fetus as well as severe long-term neonatal morbidities. Despite much research focused on the consequences of intra-amniotic infection, there remains little knowledge about the innate immune cells that respond to invading microbes. We performed RNA-seq of sorted amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection to determine the transcriptomic differences between these innate immune cells. Further, we sought to identify specific transcriptomic pathways that were significantly altered by the maternal or fetal origin of amniotic fluid neutrophils and monocytes/macrophages, the presence of a severe fetal inflammatory response, and pregnancy outcome (i.e., preterm or term delivery). We show that significant transcriptomic differences exist between amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection, indicating the distinct roles these cells play. The transcriptome of amniotic fluid immune cells varies based on their maternal or fetal origin, and the significant transcriptomic differences between fetal and maternal monocytes/macrophages imply that those of fetal origin exhibit impaired functions. Notably, transcriptomic changes in amniotic fluid monocytes/macrophages suggest that these immune cells collaborate with neutrophils in the trafficking of fetal leukocytes throughout the umbilical cord (i.e., funisitis). Finally, amniotic fluid neutrophils and monocytes/macrophages from preterm deliveries display enhanced transcriptional activity compared to those from term deliveries, highlighting the protective role of these cells during this vulnerable period. Collectively, these findings demonstrate the underlying complexity of local innate immune responses in women with intra-amniotic infection and provide new insights into the functions of neutrophils and monocytes/macrophages in the amniotic cavity., (The Author(s). Published by S. Karger AG, Basel.)

Objective: Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Some preterm births are associated with clinical chorioamnionitis; yet, this condition has been poorly investigated. Herein, we characterized the amniotic fluid cellular immune responses in women with preterm clinical chorioamnionitis., Methods and Subjects: Amniotic fluid samples were obtained from women with preterm clinical chorioamnionitis and a positive or negative microbiological culture (n = 17). The cellular composition of amniotic fluid was evaluated using fluorescence microscopy, scanning and transmission electron microscopy, and flow cytometry. Women without preterm clinical chorioamnionitis were also examined (n = 10)., Results: Amniotic fluid from women with preterm clinical chorioamnionitis and a positive culture had: (1) abundant neutrophils associated with viable and non-viable bacteria, (2) neutrophils performing phagocytosis, (3) neutrophils forming NETs, (4) increased numbers of neutrophils, monocytes/macrophages, and CD4+ T cells, and (5) high expression of IL-1β by neutrophils and monocytes/macrophages. Amniotic fluid from women with preterm clinical chorioamnionitis and proven infection tended to have fewer monocytes/macrophages and CD4+ T cells compared to those without chorioamnionitis., Conclusion: We provide the first morphologic and phenotypic characterization of the cellular immune responses in the amniotic cavity of women with preterm clinical chorioamnionitis, a condition associated with adverse neonatal outcomes.

Objective: The objective of this study is to determine whether the amniotic fluid (AF) concentration of soluble CXCR3 and its ligands CXCL9 and CXCL10 changes in patients whose placentas show evidence of chronic chorioamnionitis or other placental lesions consistent with maternal anti-fetal rejection., Methods: This retrospective case-control study included 425 women with (1) preterm delivery (n=92); (2) term in labor (n=68); and (3) term not in labor (n=265). Amniotic fluid CXCR3, CXCL9 and CXCL10 concentrations were determined by ELISA., Results: (1) Amniotic fluid concentrations of CXCR3 and its ligands CXCL9 and CXCL10 are higher in patients with preterm labor and maternal anti-fetal rejection lesions than in those without these lesions [CXCR3: preterm labor and delivery with maternal anti-fetal rejection placental lesions (median, 17.24 ng/mL; IQR, 6.79-26.68) vs. preterm labor and delivery without these placental lesions (median 8.79 ng/mL; IQR, 4.98-14.7; P=0.028)]; (2) patients with preterm labor and chronic chorioamnionitis had higher AF concentrations of CXCL9 and CXCL10, but not CXCR3, than those without this lesion [CXCR3: preterm labor with chronic chorioamnionitis (median, 17.02 ng/mL; IQR, 5.57-26.68) vs. preterm labor without chronic chorioamnionitis (median, 10.37 ng/mL; IQR 5.01-17.81; P=0.283)]; (3) patients with preterm labor had a significantly higher AF concentration of CXCR3 than those in labor at term regardless of the presence or absence of placental lesions., Conclusion: Our findings support a role for maternal anti-fetal rejection in a subset of patients with preterm labor.

Problem: To investigate whether amniotic fluid (AF) "sludge" in patients with preterm labor (PTL) with intact membranes is related to intra-amniotic infection or inflammation., Method of Study: 105 PTL patients before 29 weeks' gestation were enrolled. AF "sludge" was evaluated by transvaginal sonography. Microorganisms were identified in AF by our newly established PCR method using a eukaryote-made thermostable DNA polymerase., Results: AF "sludge" was present in 18.1% (19/105) of patients. The results obtained in the AF "sludge" group vs the no "sludge" group were as follows: (i) a similar positive rate of microorganisms in AF by PCR, 31.6% (6/19) vs 38.4% (33/86); (ii) a higher level of AF interleukin-8, 15.2 (0.2-381.5) ng/mL vs 5.8 (0.1-413.7) ng/mL; P = .005); and (3) a higher frequency of histological chorioamnionitis, 52.6% (10/19) vs 23.3% (20/86); P = .010., Conclusion: The presence of AF "sludge" is related to intra-amniotic inflammation with or without microorganisms., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Neutrophils are the most abundant white blood cells found in the amniotic cavity of women with intraamniotic infection and/or inflammation. The current belief is that these neutrophils are of fetal origin. However, abundant neutrophils have been found in the amniotic fluid of women with a severe acute maternal inflammatory response but without a severe fetal inflammatory response in the placenta, suggesting that these innate immune cells can also be of maternal origin or a mixture of both fetal and maternal neutrophils., Objective: We sought to investigate the origin of amniotic fluid neutrophils from women with intraamniotic infection and/or inflammation and to correlate these findings with acute histologic maternal and fetal inflammatory responses in the placenta., Study Design: Amniotic fluid was collected from 15 women with suspected intraamniotic infection and/or inflammation (positive microbiological cultures and/or interleukin-6 concentrations ≥2.6 ng/mL). Amniotic fluid neutrophils were purified by fluorescence-activated cell sorting, DNA was extracted, and DNA fingerprinting was performed. DNA fingerprinting was also performed in the umbilical cord and maternal blood DNA. Fluorescence in situ hybridization was assayed in women with male neonates. Blinded placental histopathological evaluations were conducted., Results: First, DNA fingerprinting revealed that 43% (6/14) of women who underwent a single amniocentesis had mostly fetal neutrophils in the amniotic fluid. Second, DNA fingerprinting showed that 36% (5/14) of the women who underwent a single amniocentesis had predominantly maternal neutrophils in the amniotic fluid. Third, DNA fingerprinting indicated that 21% (3/14) of the women who underwent a single amniocentesis had an evident mixture of fetal and maternal neutrophils in the amniotic fluid. Fourth, DNA fingerprinting revealed that a woman who underwent 2 amniocenteses (patient 15) had fetal neutrophils first, and as infection progressed, abundant maternal neutrophils invaded the amniotic cavity. Fifth, fluorescence in situ hybridization confirmed DNA fingerprinting results by showing that both fetal and maternal neutrophils were present in the amniotic fluid. Sixth, most of the women who had predominantly amniotic fluid neutrophils of fetal origin at the time of collection delivered extremely preterm neonates (71% [5/7]). Seventh, all of the women who had predominantly amniotic fluid neutrophils of maternal origin at the time of collection delivered term or late preterm neonates (100% [6/6]). Eighth, 2 of the women who had an evident mixture of fetal and maternal neutrophils in the amniotic fluid at the time of collection delivered extremely preterm neonates (67% [2/3]), and the third woman delivered a term neonate (33% [1/3]). Finally, most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (87% [13/15])., Conclusion: Amniotic fluid neutrophils can be either predominantly of fetal or maternal origin, or a mixture of both fetal and maternal origin, in women with intraamniotic infection and/or inflammation. The findings herein provide evidence that both fetal and maternal neutrophils can invade the amniotic cavity, suggesting that both the fetus and the mother participate in the host defense mechanisms against intraamniotic infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Problem: Neutrophils are capable of performing phagocytosis, a primary mechanism for microbial killing. Intra-amniotic infection is characterized by an influx of neutrophils into the amniotic cavity. Herein, we investigated whether amniotic fluid neutrophils could phagocytize bacteria found in the amniotic cavity of women with intra-amniotic infection., Methods: Amniotic fluid neutrophils from women with intra-amniotic infection were visualized by transmission electron microscopy (n=6). The phagocytic activity of amniotic fluid neutrophils from women with intra-amniotic infection and/or inflammation (n=10) or peripheral neutrophils from healthy individuals (controls, n=3) was tested using ex vivo phagocytosis assays coupled with live imaging. Phagocytosis by amniotic fluid neutrophils was also visualized by confocal microscopy (n=10) as well as scanning and transmission electron microscopy (n=5)., Results: (i) Intra-amniotic infection-related bacteria including cocci (eg Streptococcus agalactiae), bacilli (eg Bacteriodes fragilis and Prevotella spp.), and small bacteria without a cell wall (eg Ureaplasma urealyticum) were found inside of amniotic fluid neutrophils; (ii) peripheral neutrophils (controls) rapidly phagocytized S. agalactiae, U. urealyticum, Gardnerella vaginalis, and Escherichia coli; (iii) amniotic fluid neutrophils rapidly phagocytized S. agalactiae and G. vaginalis; and (iv) amniotic fluid neutrophils slowly phagocytized U. urealyticum and E. coli; yet, the process of phagocytosis of the genital mycoplasma was lengthier., Conclusion: Amniotic fluid neutrophils can phagocytize bacteria found in the amniotic cavity of women with intra-amniotic infection, namely S. agalactiae, U. urealyticum, G. vaginalis, and E. coli. Yet, differences in the rapidity of phagocytosis were observed among the studied microorganisms. These findings provide a host defense mechanism whereby amniotic fluid neutrophils can kill microbes invading the amniotic cavity., (Published 2017. This articles is a U.S. Government work and is in the public domain in the USA.)

Objectives: 1) To characterize the cellular composition of the amniotic fluid of patients diagnosed with clinical chorioamnionitis at term, as a function of the presence or absence of microorganisms determined by cultivation techniques, and 2) to characterize the cytokine production by white blood cells present in the amniotic fluid using flow cytometry-based techniques., Materials and Methods: Amniotic fluid samples from 20 women who had the diagnosis of clinical chorioamnionitis at term were analyzed using cultivation techniques (for aerobic and anaerobic bacteria as well as genital Mycoplasmas). Amniotic fluid IL-6 concentrations were determined by an enzyme-linked immunosorbent assay. Amniotic fluid leukocytes were visualized by using hematoxylin and eosin staining and immunofluorescence. Immunophenotyping of surface markers and cytokines was performed in amniotic fluid leukocytes using flow cytometry., Results: 1) Neutrophils (CD45+CD15+ cells) were the most common leukocyte subset found in the amniotic fluid, followed by monocytes (CD45+CD14+ cells); other white blood cells (such as lymphocytes and natural killer cells) were scarce in the amniotic fluid; 2) the absolute counts of neutrophils and monocytes were significantly higher in patients with microorganisms found in the amniotic fluid than in those without detectable microorganisms, using cultivation techniques; 3) there was a significant correlation between the absolute counts of neutrophils and monocytes determined by flow cytometry (Spearman's correlation=0.97; P<0.001); 4) there was a significant correlation between the absolute white blood cell count determined with a hemocytometer chamber and by flow cytometric analysis (Spearman's correlation=0.88; P<0.001); and 5) the profile of cytokine expression differed between monocytes and neutrophils; while neutrophils predominantly produced TNF-α and MIP-1β, monocytes expressed higher levels of IL-1β and IL-1α., Conclusion: Flow cytometry analysis of the amniotic fluid of patients with intra-amniotic infection and clinical chorioamnionitis at term demonstrated that neutrophils and monocytes are the most common cells participating in the inflammatory process. We have characterized, for the first time, the differential cytokine expression by these cells in this important complication of pregnancy.

Problem: To determine whether amniotic fluid (AF) CXCL10 concentration is associated with histologic chronic chorioamnionitis in patients with preterm labor (PTL) and preterm prelabor rupture of the membranes (PROM)., Method of Study: This study included 168 women who had an episode of PTL or preterm PROM. AF interleukin (IL)-6 and CXCL10 concentrations were determined by immunoassay., Results: (i) Increased AF CXCL10 concentration was associated with chronic (OR: 4.8; 95% CI: 1.7-14), but not acute chorioamnionitis; (ii) increased AF IL-6 concentration was associated with acute (OR: 4.2; 95% CI: 1.3-13.7) but not chronic chorioamnionitis; and (iii) an increase in AF CXCL10 concentration was associated with placental lesions consistent with maternal anti-fetal rejection (OR: 3.7; 95% CI: 1.3-10.4). (iv) All patients with elevated AF CXCL10 and IL-6 delivered preterm., Conclusion: Increased AF CXCL10 concentration is associated with chronic chorioamnionitis or maternal anti-fetal rejection, whereas increased AF IL-6 concentration is associated with acute histologic chorioamnionitis., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA. American Journal of Reproductive Immunology published by John Wiley & Sons Ltd.)

Background: Although Ureaplasma species are the most common organisms associated with prematurity, their effects on the maternal and fetal immune system remain poorly characterized., Methods: Rhesus macaque dams at approximately 80% gestation were injected intra-amniotically with 10 7 colony-forming units of Ureaplasma parvum or saline (control). Fetuses were delivered surgically 3 or 7 days later. We performed comprehensive assessments of inflammation and immune effects in multiple fetal and maternal tissues., Results: Although U. parvum grew well in amniotic fluid, there was minimal chorioamnionitis. U. parvum colonized the fetal lung, but fetal systemic microbial invasion was limited. Fetal lung inflammation was mild, with elevations in CXCL8, tumor necrosis factor (TNF) α, and CCL2 levels in alveolar washes at day 7. Inflammation was not detected in the fetal brain. Significantly, U. parvum decreased regulatory T cells (Tregs) and activated interferon γ production in these Tregs in the fetus. It was detected in uterine tissue by day 7 and induced mild inflammation and increased expression of connexin 43, a gap junction protein involved with labor., Conclusions: U. parvum colonized the amniotic fluid and caused uterine inflammation, but without overt chorioamnionitis. It caused mild fetal lung inflammation but had a more profound effect on the fetal immune system, decreasing Tregs and polarizing them toward a T-helper 1 phenotype., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: In patients with preterm premature rupture of membranes, intrauterine inflammation and/or infection is frequently present, can lead to fetal inflammatory response syndrome, and is associated with adverse neonatal outcome. Clinical decision making requires balancing the potential benefits of pregnancy prolongation against the risk of intrauterine infection. Diagnostic tests in maternal serum are of moderate prediction value and amniocentesis is an invasive procedure. Therefore, markers obtained noninvasively would be helpful in patients with expectant management., Objectives: To determine the predictive values of amniotic fluid interleukin-6 and tumor necrosis factor-α in vaginal secretions for fetal inflammatory response syndrome and/or histologic funisitis and for adverse neonatal outcome in patients with preterm premature rupture of membranes., Study Design: In this prospective multicenter case-control study, vaginal secretions were sampled daily with a noninvasive method from 99 women with preterm premature rupture of membranes and expectant management. Amniotic fluid interleukin-6 and tumor necrosis factor-α were measured by 2 different immunoassays (an automated chemiluminescent enzyme immunoassay and a lateral flow immunoassay). After delivery, patients were divided into a control or a fetal inflammatory response syndrome group according to neonatal interleukin-6 in cord plasma and/or the presence of funisitis. Univariate and multivariate regression analyses were performed and prediction models were developed by calculating receiver operating characteristic curves., Results: Gestational age at delivery was lower and latency period was longer in the fetal inflammatory response syndrome group compared to the control group. The strongest risk factor for composite adverse neonatal outcome was fetal inflammatory response syndrome (odds ratio, 2.48; confidence interval, 1.40-4.38). The median concentrations of amniotic fluid interleukin-6 and tumor necrosis factor-α in vaginal secretions were significantly higher in the fetal inflammatory response group compared to the control group in both immunoassays (P < .001). The area under the curve of the clinical reference model (including common clinical parameters) was 0.66. Adding interleukin-6 and tumor necrosis factor-α into the model improved the area under the curve to 0.92 (in both assays, interleukin-6 IMMULITE and QuickLine); 0.87 (tumor necrosis factor-α IMMULITE) and 0.94 (tumor necrosis factor-α QuickLine), respectively., Conclusion: The strongest risk factor for worse neonatal outcome (composite neonatal outcome) was fetal inflammatory response syndrome. Amniotic fluid interleukin-6 and tumor necrosis factor-α seem to be good predictors for fetal inflammatory response syndrome and for histologic funisitis and may improve the clinical management of patients with preterm premature rupture of membranes. The noninvasive technique of sampling amniotic fluid from vaginal secretions facilitates daily measurements and bedside assessment of cytokines and is in this respect preferable to invasive amniocentesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Recent studies indicate that clinical chorioamnionitis is a heterogeneous condition and only approximately one-half of the patients have bacteria in the amniotic cavity, which is often associated with intra-amniotic inflammation. The objective of this study is to characterize the nature of the inflammatory response within the amniotic cavity in patients with clinical chorioamnionitis at term according to the presence or absence of 1) bacteria in the amniotic cavity and 2) intra-amniotic inflammation., Materials and Methods: A retrospective cross-sectional case-control study was conducted to examine cytokine and chemokine concentrations in the amniotic fluid (AF). Cases consisted of women with clinical chorioamnionitis at term (n=45). Controls were women with uncomplicated pregnancies at term who did not have intra-amniotic inflammation and were in labor (n=24). Women with clinical chorioamnionitis were classified according to the results of AF cultures, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry, and AF concentration of interleukin-6 (IL-6) into those: 1) without intra-amniotic inflammation, 2) with microbial-associated intra-amniotic inflammation, and 3) with intra-amniotic inflammation without detectable bacteria. The AF concentrations of 29 cytokines/chemokines were determined using sensitive and specific V-PLEX immunoassays., Results: 1) The AF concentrations of pro- and anti-inflammatory cytokines/chemokines such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-4 (IL-4), macrophage inflammatory protein-1 beta (MIP-1β), and interleukin-8 (IL-8) (except Eotaxin-3) were significantly higher in women with clinical chorioamnionitis at term than in controls (term labor without intra-amniotic inflammation); 2) patients with microbial-associated intra-amniotic inflammation, and those with intra-amniotic inflammation without detectable bacteria, had a dramatic differential expression of cytokines and chemokines in AF compared to patients with spontaneous labor without intra-amniotic inflammation. However, no difference could be detected in the pattern of the intra-amniotic inflammatory response between patients with intra-amniotic inflammation with and without detectable bacteria; and 3) in patients with clinical chorioamnionitis at term but without intra-amniotic inflammation, the behavior of cytokines and chemokines in the AF was similar to those in spontaneous labor at term., Conclusions: Patients with clinical chorioamnionitis who had microbial-associated intra-amniotic inflammation or intra-amniotic inflammation without detectable bacteria had a dramatic upregulation of the intra-amniotic inflammatory response assessed by amniotic fluid concentrations of cytokines. A subset of patients with term clinical chorioamnionitis does not have intra-amniotic infection/inflammation, as demonstrated by elevated AF concentrations of inflammation-related proteins, when compared to women in term labor with uncomplicated pregnancies, suggesting over-diagnosis. These observations constitute the first characterization of the cytokine/chemokine network in the amniotic cavity of patients with clinical chorioamnionitis at term.

Objective: To estimate the stage of histological chorioamnionitis (h-CAM) antenatally using clinical data., Materials and Methods: Four hundred and twenty-eight singleton mothers were recruited. Clinical data including the levels of white blood cell count (WBC), C-reactive protein (CRP), amniotic fluid interleukin-8 (AF-IL-8) at Cesarean section, and maternal body temperature (MBT) were collected., Results: Histological chorioamnionitis was present in 45.3% of the cases. Poor neonatal prognosis was highest (59.1%) in cases with h-CAM stage III. AF-IL-8 (odds ratio: 8.5, 95% CI: 5.1-14.8, P < 0.0001) and MBT (odds ratio: 2.3, 95% CI: 1.13-4.1, P = 0.0192) were independent risk factors for h-CAM. The cutoff value of AF-IL-8 for predicting each stage of h-CAM (stage I or higher, stage II or higher, and stage III) were ≥9.9 ng/mL, ≥17.3 ng/mL, and ≥55.9 ng/mL, respectively., Conclusion: The stage of h-CAM was able to be predicted accurately by the level of AF-IL-8 before delivery., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: The objectives of this study were: 1) to determine the amniotic fluid (AF) microbiology of patients with the diagnosis of clinical chorioamnionitis at term using both cultivation and molecular techniques; and 2) to examine the relationship between intra-amniotic inflammation with and without microorganisms and placental lesions consistent with acute AF infection., Methods: The AF samples obtained by transabdominal amniocentesis from 46 women with clinical signs of chorioamnionitis at term were analyzed using cultivation techniques (for aerobic and anerobic bacteria as well as genital mycoplasmas) and broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). The frequency of microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation [defined as an AF interleukin 6 (IL-6) concentration ≥2.6 ng/mL], and placental lesions consistent with acute AF infection (acute histologic chorioamnionitis and/or acute funisitis) were examined according to the results of AF cultivation and PCR/ESI-MS as well as AF IL-6 concentrations., Results: 1) Culture identified bacteria in AF from 46% (21/46) of the participants, whereas PCR/ESI-MS was positive for microorganisms in 59% (27/46) – combining these two tests, microorganisms were detected in 61% (28/46) of patients with clinical chorioamnionitis at term. Eight patients had discordant test results; one had a positive culture and negative PCR/ESI-MS result, whereas seven patients had positive PCR/ESI-MS results and negative cultures. 2) Ureaplasma urealyticum (n=8) and Gardnerella vaginalis (n=10) were the microorganisms most frequently identified by cultivation and PCR/ESI-MS, respectively. 3) When combining the results of AF culture, PCR/ESI-MS and AF IL-6 concentrations, 15% (7/46) of patients did not have intra-amniotic inflammation or infection, 6.5% (3/46) had only MIAC, 54% (25/46) had microbial-associated intra-amniotic inflammation, and 24% (11/46) had intra-amniotic inflammation without detectable microorganisms. 4) Placental lesions consistent with acute AF infection were significantly more frequent in patients with microbial-associated intra-amniotic inflammation than in those without intra-amniotic inflammation [70.8% (17/24) vs. 28.6% (2/7); P=0.04]., Conclusion: Microorganisms in the AF were identified in 61% of patients with clinical chorioamnionitis at term; 54% had microbial-associated intra-amniotic inflammation, whereas 24% had intra-amniotic inflammation without detectable microorganisms.

Intra-amniotic infection (IAI) is a common cause of pre-term labour. Manual WBC count on amniotic fluid (AF) has been suggested as a diagnostic test for IAI using a threshold of 50 cells/mm(3). However, no validation studies assessing the accuracy of this method have been performed. AF samples were selected for cell count analysis. WBCs were introduced to 47 AF samples. The results from two technologists' counts were compared with the calculated expected value for WBCs in these samples. Results showed that a comparison between the technologists' WBC count to the expected WBC count yielded R(2) coefficients of 0.62 and 0.78, indicating moderate accuracy. Percentage agreement between the technologists was 67%, indicating low reproducibility. It was concluded that there was moderate correlation between the manual and the expected WBC in the spiked AF samples. Clinicians should be aware of the inaccuracy and imprecision associated with this test when evaluating a patient for IAI.

Aims: Chronic chorioamnionitis is a histological manifestation of maternal anti-fetal cellular rejection. As failure of graft survival is the most catastrophic event in organ transplantation, we hypothesized that fetal death could be a consequence of maternal rejection. The aim of this study was to assess whether there is evidence of cellular and antibody-mediated rejection in fetal death., Methods and Results: Placental histology was reviewed for the presence of chronic chorioamnionitis in unexplained preterm fetal death (n=30) and preterm live birth (n=103). Amniotic fluid CXCL10 concentrations were measured with a specific immunoassay. Chronic chorioamnionitis was more frequent in fetal death than in live birth (60.0% versus 37.9%; P<0.05) and fetal death had a higher median amniotic fluid CXCL10 concentration than live birth (2.0 versus 1.8 ng/ml, P<0.05), after adjusting for gestational age at amniocentesis. Maternal anti-human leucocyte antigen class II panel-reactive seropositivity determined by flow cytometry was higher in fetal death compared to live birth (35.7% versus 10.9%; P<0.05)., Conclusions: Chronic chorioamnionitis is a common pathologic feature in unexplained preterm fetal death. This novel finding suggests that cellular and antibody-mediated anti-fetal rejection of the mother is associated with fetal death (graft failure) in human pregnancy., (© 2011 Blackwell Publishing Limited.)

Dendrimers have emerged as topical microbicides to treat vaginal infections. This study explores the in vitro, in vivo antimicrobial activity of PAMAM dendrimers, and the associated mechanism. Interestingly, topical cervical application of 500 microg of generation-4 neutral dendrimer (G(4)-PAMAM-OH) showed potential to treat the Escherichia coli induced ascending uterine infection in guinea pig model of chorioamnionitis. Amniotic fluid collected from different gestational sacs of infected guinea pigs posttreatment showed absence of E. coli growth in the cultures plated with it. The cytokine level [tumor necrosis factor (TNFalpha) and interleukin (IL-6 and IL-1beta)] in placenta of the G(4)-PAMAM-OH treated animals were comparable to those in healthy animals while these were notably high in infected animals. Since, antibacterial activity of amine-terminated PAMAM dendrimers is known, the activity of hydroxyl and carboxylic acid terminated PAMAM dendrimers was compared with it. Though the G(4)-PAMAM-NH(2) shows superior antibacterial activity, it was found to be cytotoxic to human cervical epithelial cell line above 10 microg/mL, while the G(4)-PAMAM-OH was non-cytotoxic up to 1mg/mL concentration. Cell integrity, outer (OM) and inner (IM) membrane permeabilization assays showed that G(4)-PAMAM-OH dendrimer efficiently changed the OM permeability, while G(4)-PAMAM-NH(2) and G(3.5)-PAMAM-COOH damaged both OM and IM causing the bacterial lysis. The possible antibacterial mechanism are G(4)-PAMAM-NH(2) acts as polycation binding to the polyanionic lipopolysaccharide in E. coli, the G(4)-PAMAM-OH forms hydrogen bonds with the hydrophilic O-antigens in E. coli membrane and the G(3.5)-PAMAM-COOH acts as a polyanion, chelating the divalent ions in outer cell membrane of E. coli. This is the first study which shows that G(4)-PAMAM-OH dendrimer acts as an antibacterial agent., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Purpose of Review: There is overwhelming evidence that intrauterine infection and inflammation play an important role in the pathogenesis of spontaneous preterm labor, preterm prelabor rupture of the membranes and fetal injury resulting in long-term sequelae. Early diagnosis of subclinical infection and inflammation may therefore aid clinicians institute interventions focusing on such adverse outcomes., Recent Findings: Biomarkers of intrauterine inflammation such as interleukin-6, although sensitive, are not specific. Thus, decision to deliver remote from term because of intrauterine infection and/or inflammation should be based on clinical signs and/or bacterial culture or Gram stain of amniotic fluid. In patients with preterm contractions and intact membranes, the risk of delivery is 1% within the week following a negative fetal fibronectin in cervicovaginal secretions. This aids to decide whether antenatal steroids should be administered to patients presenting with preterm contractions between 24 and 34 weeks' gestation. Biomarkers in cervical secretions and amniotic fluid identify those who may benefit from cerclage when the cervix is shortened (<25 mm) and dilated in the second trimester., Summary: So far, few interventions utilizing inflammatory biomarkers have shown clinical benefit. Future efforts should focus on the quest for accurate biomarkers that can be obtained noninvasively and allow early prediction of subclinical disease to initiate appropriate risk-specific intervention.

Objective: Intra-amniotic infection/inflammation (IAI) is one of the most important mechanisms of disease in preterm birth. Triggering receptor expressed on myeloid cells (TREM)- 1 is a transmembrane glycoprotein expressed by neutrophils, macrophages and mature monocytes. TREM-1 is upregulated in biological fluids and tissues infected by Gram (+) and Gram (-) bacteria and fungi, amplifies the production of pro-inflammatory cytokines and chemokines, and its soluble form (sTREM-1) is released in the presence of infection. The aim of this study was to determine the effect of gestational age, parturition (term and preterm) and IAI in the amniotic fluid (AF) concentrations of sTREM-1., Study Design: This cross-sectional study included 434 patients in the following groups: (1) mid-trimester of pregnancy (14-18 weeks, n = 38); (2) normal pregnant women at term with (n = 39) and without (n = 39) labor; (3) patients with spontaneous preterm labor (PTL) and intact membranes classified into: (a) PTL who delivered at term (n = 99); (b) PTL who delivered preterm (<37 weeks gestation) without IAI (n = 80); and (c) PTL with IAI (n = 59); and (4) women with preterm prelabor rupture of membranes (PROM) with (n = 40) and without (n = 40) IAI. The AF concentration of sTREM-1 was determined by enzyme-linked immunoassay. Non-parametric statistics were used for analyses., Results: (1) sTREM-1 was detected in all the AF samples; (2) the median AF sTREM-1 concentration at term was higher than in the mid-trimester (4277.6 pg/ml vs. 1140.4 pg/ml; p < 0.001); (3) among patients with PTL, the median AF sTREM-1 concentration was higher in patients with IAI than in those without IAI (6154.4 pg/ml vs. 3282.8 pg/ml; p < 0.001) and those with PTL who delivered at term (6154.4 pg/ml vs. 2794 pg/ml; p < 0.001); (4) patients with preterm PROM with IAI had a higher median AF sTREM-1 concentration than those without IAI (7893.1 pg/ml vs. 3386.6 pg/ml; p < 0.001); (5) no differences were observed in the median AF sTREM-1 concentration between patients with spontaneous labor at term and those at term not in labor (4712.4 pg/ml vs. 4277.6 pg/ml; respectively p = 0.4); and 6) an AF sTREM-1 concentration >or=6416 pg/ml (derived from a ROC curve) had a sensitivity of 72% and a specificity of 89% for the diagnosis of intra-amniotic infection., Conclusions: sTREM-1 is a physiologic constituent of the AF, and its concentration: (1) is significantly elevated in the presence of IAI; (2) increases with advancing gestation; and (3) does not change in the presence of spontaneous labor at term. We propose that sTREM-1 play a role in the innate immune response against intra-amniotic infection.

Objective: Fragment Bb is an activator of the alternative pathway of the complement system. Recently, increased first trimester maternal plasma concentrations of this fragment were reported in patients destined to have a spontaneous preterm delivery before 34 weeks of gestation. The aim of this study was to determine whether the amniotic fluid (AF) concentrations of fragment Bb change with gestational age, spontaneous labor (term and preterm) and in the presence of intra-amniotic infection/inflammation (IAI)., Study Design: This cross-sectional study included patients in the following groups: (1) mid-trimester (n = 64); (2) term in spontaneous labor (n = 70); (3) term not in labor (n = 43); (4) spontaneous preterm labor (PTL) who delivered at term (n = 76); (5) PTL without IAI who delivered preterm (n = 73); (6) PTL with IAI (n = 76); (7) preterm prelabor rupture of membranes (PROM) without IAI (n = 71); and (8) preterm PROM with IAI (n = 71). Fragment Bb concentration in AF was determined by an enzyme-linked immunoassay. Non-parametric statistics were used for analyses., Results: (1) Fragment Bb was detected in all AF samples (n = 544); (2) The median AF concentration of fragment Bb in patients at term not in labor was significantly higher than that of those in the mid-trimester [2.42 microg/ml, interquartile range (IQR) 1.78-3.22 vs. 1.64 microg/ml, IQR 1.06-3.49; p < 0.001]; (3) Among patients with PTL, those with IAI had a higher median AF fragment Bb concentration than that of woman without IAI, who delivered preterm (4.82 microg/ml, IQR 3.32-6.08 vs. 3.67 microg/ml, IQR 2.35-4.57; p < 0.001) and than that of women with an episode of PTL, who delivered at term (3.21 microg/ml, IQR 2.39-4.16; p < 0.001); (4) Similarly, among patients with preterm PROM, the median AF fragment Bb concentration was higher in individuals with IAI than in those without IAI (4.24 microg/ml, IQR 2.58-5.79 vs. 2.79 microg/ml, IQR 2.09-3.89; p < 0.001). (5) Among patients at term, the median AF fragment Bb concentration did not differ between women with spontaneous labor and those without labor (term in labor: 2.47 microg/ml, IQR 1.86-3.22; p = 0.97)., Conclusions: (1) Fragment Bb, an activator of the alternative complement pathway, is a physiologic constituent of the AF, and its concentration increases with advancing gestational age; (2) AF concentrations of fragment Bb are higher in pregnancies complicated with IAI; and (3) labor at term is not associated with changes in the AF concentrations of fragment Bb. These findings suggest a role for fragment Bb in the host immune response against IAI.

Objective: The aim of this study was to characterize the pregnant guinea pig cytokine time course following a maternal inflammatory insult., Study Design: Pregnant guinea pigs (n = 34) were injected intraperitoneally with 100 microg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours, or 5 days. Control animals were euthanized at 70% gestation without LPS exposure. Interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) were quantified in the maternal serum and amniotic fluid by an enzyme-linked immunosorbent assay., Results: IL-6 and IL-1beta concentrations were elevated in the maternal serum at 24 hours and returned to control levels by 5 days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1beta at 24 hours. TNF-alpha levels were not significantly increased., Conclusion: Maternal intraperitoneal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid within 5 days, further implicating the cytokines as mediators of fetal white matter damage.

Objective: The purpose of this study was to determine the frequency and clinical significance of intraamniotic inflammation in patients with acute cervical insufficiency., Study Design: Amniocentesis was performed in 52 patients with acute cervical insufficiency (cervical dilation, > or =1.5 cm) and intact membranes and without regular uterine contractions (gestational age, 17-29 weeks). Amniotic fluid (AF) was cultured for aerobic and anaerobic bacteria and genital mycoplasmas and assayed for matrix metalloproteinase-8. Intraamniotic inflammation was defined as an elevated AF matrix metalloproteinase-8 concentration (>23 ng/mL). Nonparametric statistics and survival techniques were used for analysis., Results: The prevalence of intraamniotic inflammation was 81% (42/52); the prevalence of a positive AF culture was 8% (4/52). Intraamniotic inflammation was present in all cases with a positive AF culture. Preterm delivery within 7 days occurred in 50% of cases (19/38), and delivery before 34 weeks of gestation occurred in 84% of cases (32/38) with intraamniotic inflammation but without AF infection. Fifty-five percent of newborn infants (21/38) who were born to mothers with intraamniotic inflammation but without AF infection died immediately after birth (<1 day). The amniocentesis-to-delivery interval was shorter in patients with intraamniotic inflammation than in those without inflammation (P < .05). There were no differences in the interval-to-delivery or the rate of adverse outcome between patients with intraamniotic inflammation and a negative culture and patients with proven AF infection., Conclusion: Intraamniotic inflammation, regardless of AF culture result, is present in approximately 80% of patients with acute cervical insufficiency and is a risk factor for impending preterm delivery and adverse outcomes.

Objective: To correlate cervical and amniotic fluid cytokines and macrophage-related chemokines to the development of histological chorioamnionitis (HCA) in patients with preterm labor (PTL) and preterm prelabor rupture of the membranes (PPROM)., Study Design: Cervical and amniotic fluid interleukin (IL)-6, IL-8, IL-18, monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 from pregnant women (at

Objective: Receptor for advanced glycation end products (RAGE) is a multiligand cell-surface receptor part of the immunoglobulin superfamily with crucial roles in inflammation. S100A12/ENRAGE, a biomarker of amniotic fluid (AF) inflammation, is a ligand for RAGE. sRAGE, a competitive soluble RAGE receptor, inhibits RAGE ligands. Here we aimed to investigate the presence and changes in components of the RAGE system in women with intra-amniotic infection and inflammation., Study Design: AF was retrieved by amniocentesis in 113 women stratified as follows: (1) positive AF culture (+AFC; GA = 27 [20-33] wk; n = 27); (2) negative AF culture (-AFC; GA = 30 [20-36] wk; n = 27); (3) second trimester control (2T-CRL; GA = 19 [15-25] wk; n = 31); (4) third trimester control (3T-CRL; GA = 36 [31-38] wk; n = 28). We used mass spectrometry (SELDI) to detect S100A12/ENRAGE in AF. sRAGE levels were measured using specific immunoassays. Placental pathology was interpreted in relationship to the presence or absence of histologic acute inflammation and immunoreactivity of S100A12/ENRAGE and RAGE. mRNA expression of S100A12/ENRAGE, sRAGE, and RAGE in amniochorion and placental villous tissue was investigated using quantitative real-time polymerase chain reaction (PCR)., Results: Presence of the S100A12/ENRAGE biomarker SELDI peak was confirmed in 70% of the +AFC but in only 10% of the -AFC samples (P < .001). The inflammatory biomarker was absent in all control samples. We further determined that the competitive inhibitor sRAGE is temporally regulated during gestation and that its AF levels are not influenced by the presence of either intra-amniotic infection or inflammation. Histologic choriamnionitis associated with intense staining for S100A12/ENRAGE, particularly in inflammatory cells. The immunoreactivity for extracellular domain of RAGE was localized exclusively to amnion epithelial, decidual, and extravillous trophoblast cells. Yet, acute histologic chorioamnionitis was related to increased gene expression of S100A12/ENRAGE in fetal membranes and decreased sRAGE and RAGE in the placenta., Conclusion: The S100A12/ENRAGE system is markedly upregulated in women with intra-amniotic infection and correlates with the degree of inflammation. Further studies remain to elucidate whether the gestational age dependence of the inhibitor molecule sRAGE may explain the higher incidence of infection-related preterm deliveries and especially rupture of the membranes at earlier gestational age.

Objective: Lipopolysaccharide-binding protein (LBP) is an acute-phase protein of predominantly hepatic origin, capable of binding the lipid A fraction of bacterial lipopolysaccharide (LPS). The complex LBP-LPS binds to CD14, and has been implicated in the host response to gram-negative infection. The purpose of this study was to determine whether microbial invasion of the amniotic cavity (MIAC) and parturition (term and preterm) are associated with changes in the amniotic fluid concentration of LBP., Study Design: Amniotic fluid was retrieved by amniocentesis from 343 patients in the following groups: (1) those in mid-trimester with a subsequent normal pregnancy outcome (n = 84); (2) those in mid-trimester with a fetal loss after the procedure (n = 10); (3) those with preterm labor and intact membranes without MIAC who delivered at term (n = 36) or prematurely (n = 52), and those with preterm labor with MIAC (n = 26); (4) those with preterm premature rupture of membranes (PROM) with (n = 26) and without (n = 26) MIAC; and (5) those delivering at term with intact membranes in the absence of MIAC, in labor (n = 52) and not in labor (n = 31). The concentration of LBP in amniotic fluid was determined with a specific and sensitive immunoassay. Non-parametric statistics were used. A p value of < 0.05 was considered significant., Results: LBP was detected in 98% (335/343) of the amniotic fluid samples. MIAC was associated with a significant increase in amniotic fluid concentration of LBP in women with preterm labor and intact membranes, but not in preterm PROM. Spontaneous preterm parturition was associated with a significant increase in amniotic fluid concentration of LBP. Patients who had a spontaneous fetal loss after a mid-trimester amniocentesis had a significantly higher median amniotic fluid LBP concentration than those who had a mid-trimester amniocentesis and a normal perinatal outcome., Conclusion: Preterm labor with MIAC and preterm parturition are associated with higher amniotic fluid concentrations of LBP than those with sterile amniotic fluid.

Objective: Endotoxin has been implicated in the mechanism responsible for the setting of infection in preterm labor. To exert its biological effects, endotoxin binds to a circulating protein known as lipopolysaccharide binding protein (LBP) and presents endotoxin monomers to CD14, which may be a membrane-bound receptor or a soluble molecule. The endotoxin-LBP-CD14 complex interacts with Toll-like receptor 4 and other regulatory proteins leading to cellular activation and an inflammatory response. The purpose of this study was to determine whether microbial invasion of the amniotic cavity (MIAC)/intra-amniotic inflammation (both preterm and term) and parturition at term are associated with changes in the amniotic fluid and umbilical plasma soluble concentrations of CD14 (sCD14)., Study Design: Amniotic fluid was retrieved by amniocentesis from 88 patients in the following groups: group 1, preterm labor with intact membranes with MIAC/intra-amniotic inflammation (n = 18) and without these conditions (n = 26); group 2, term gestations not in labor without MIAC/intra-amniotic inflammation (n = 11), in labor without MIAC/intra-amniotic inflammation (n = 12) and in labor with MIAC/intra-amniotic inflammation (n = 13); and group 3, patients who underwent genetic amniocentesis at mid-trimester (n = 8). A sample of cord blood was obtained after delivery in all patients except those in group 3. sCD14 was assayed with a sensitive and specific immunoassay. Non-parametric statistics were used for analysis. A p value of < 0.05 was considered significant., Results: sCD14 was detectable in 97% (85/88) of the amniotic fluid samples. Amniotic fluid sCD14 concentrations were lower in patients at term than in the mid-trimester of pregnancy (mid-trimester: median 482 ng/ml, range 258-838 ng/ml vs. term no labor: median 7 ng/ml, range 2-274 ng/ml, p = 0.01). Among patients with preterm labor with intact membranes, the median amniotic fluid sCD14 level of patients with MIAC/intra-amniotic inflammation was higher than in patients without these conditions (median 1568 ng/ml, range 98-5887 ng/ml vs. median 645 ng/ml, range 0-3961 ng/ml, respectively; p = 0.01). Among women at term in labor, those with MIAC/intra-amniotic inflammation had a higher median amniotic fluid sCD14 concentration than those without these conditions (median 85 ng/ml, range 2-1113 ng/ml vs. median 17 ng/ml, range 0-186 ng/ml; p = 0.01). MIAC/intra-amniotic inflammation in women with preterm labor with intact membranes was associated with a higher median umbilical venous plasma sCD14 concentration (median 744 ng/ml, range 0-3620 ng/ml vs. median 0 ng/ml, range 0-2060 ng/ml; p = 0.04). sCD14 was undetectable in plasma from umbilical cords of all neonates born to women at term. An increase in amniotic fluid concentration of sCD14 was observed in cases of intrauterine infection, not only by gram-negative bacteria, but also gram-positive bacteria and Ureaplasma spp., Conclusion: sCD14 is a physiological constituent of amniotic fluid, and its concentrations at term are lower than in the mid-trimester. Intrauterine infection/inflammation is associated with a higher median amniotic fluid sCD14 concentration in both preterm and term parturition. Neonates born from mothers with preterm labor with intact membranes and MIAC/intra-amniotic inflammation had a higher median concentration of sCD14 in umbilical cord plasma than those without these conditions. sCD14 concentrations are increased in the amniotic fluid and umbilical cord blood even in the absence of a microbiologically proven gram-negative infection. CD14 appears to participate in the host response to intrauterine infection even in cases involving genital mycoplasmas.

Objective: We investigated the role of interleukin (IL)-6 and IL-1 receptor antagonist (IL-1ra) in pre-term premature rupture of the membranes (PROM)., Method: Amniotic fluid samples were collected from 10 patients with pre-term PROM (group 1a), 13 patients undergoing genetic amniocentesis (group 2), seven patients with normal vaginal delivery (group 3a), and 11 patients with elective cesarean section (group 4a). Umbilical venous blood was collected from nine cases of pre-term PROM (group 1b), 19 cases of normal delivery (group 3b) and nine cases of elective cesarean section (group 4b)., Results: The concentration of IL-6 in the amniotic fluid in group 1a was significantly higher than in group 2 (P<0.001), and that in group 3a was significantly higher than in group 4a (P<0.001). The concentration of IL-1ra in amniotic fluid in group 1a was significantly higher than in group 2 (P<0.001). The concentrations of IL-6 in umbilical venous blood were not significant among the groups. The concentration of IL-1ra in umbilical venous blood in group 1b was significantly higher than in group 3b (P<0.05) and group 4b (P<0.05)., Conclusions: High concentrations of amniotic fluid IL-6 present in pre-term PROM, are indicative of intrauterine inflammation, probably due to sub-clinical infection. The high concentrations of IL-1ra in amniotic fluid and umbilical venous blood suggest the possibility that the presence of this anti-inflammatory cytokine serves to prevent the development of overt inflammation.

Background: Chorioamnionitis is considered to be one of the important causes of preterm labor. To investigate the relationship between inflammatory cytokines in amniotic fluid and the histologic evidence of chorioamnionitis, we studied amniotic fluid interleukin-6 (IL-6) levels in patients with preterm labor., Methods: Between 1993 and 1996, we obtained amniotic fluid by amniocentesis from 110 patients before 32 weeks of gestation who had preterm labor on admission. We measured IL-6 levels in the amniotic fluid with an ELISA method. Histologic examination of placentae and fetal membranes after delivery was evaluated. As controls, we measured IL-6 levels in the amniotic fluid of 37 patients without preterm labor. Seventy-eight patients delivered after 35 weeks of gestation, and 32 patients delivered before 34 weeks of gestation. Analysis was conducted using Mann-Whitney U test and Scheffe's multiple comparison test., Results: Amniotic fluid IL-6 levels in patients delivering before 34 weeks were significantly higher than those in patients delivering after 35 weeks (p<0.01). IL-6 levels in the amniotic fluid were significantly different among control patients, patients without chorioamnionitis and those with histologic stages I, II and III which means subchorionic intervillositis, chorionitis and amnionitis, respectively (controls vs patients with stage I, II, III: p<0.001, patients without chorioamnionitis vs those with stage II, III, stage I vs II, stage II vs III: p<0.01). The IL-6 concentration in the amniotic fluid associated with histologic stages II or III was above 3500 pg/ml (sensitivity: 87.5%, specificity: 89.5%)., Conclusions: Our findings indicate that amniotic fluid IL-6 may have a sensitive diagnostic and prognostic value in the management of preterm labor and is an index of the severity of chorioamnionitis during pregnancy.

Subclinical intrauterine infections have been proposed to be one of the leading causes of preterm labor. The determination of inflammatory cytokines IL-1 beta and IL-6 in the amniotic fluid may be useful to identify women who have infection-associated preterm labor. Amniotic fluid was collected from 99 women during amniocentesis, during cesarean section or at the time of amniotomy using sterile technique. IL-1 beta and IL-6 were determined by a specific ELISA. Fluid of each sample was cultured for aerobic and anaerobic bacteria and for Mycoplasma hominis and Ureaplasma urealyticum. Different populations were identified according to the criteria "gestational age", "active labor", "positive amniotic fluid cultures". Interleukin-6 was detectable in all samples of amniotic fluid. The second-trimester (weeks 14-27) amniotic fluid concentration of IL-6 (18-2270 pg/ml) was higher than in the third trimester (weeks 28-42, 4-329 pg/ml). The difference was significant. Women in active labor had higher levels of IL-6 in their amniotic fluid than women not in labor (p < 0.01). There is no significant difference between women with preterm labor and delivery (weeks 28-37, 597-8670 pg/ml) and with term labor and delivery (weeks 38-42, 24-8300 pg/ml). Only culture negative samples were included in this population. Interleukin-1 beta was not detectable in the majority (90%) of these samples. 30% of the women in labor had positive amniotic fluid cultures. The IL-6 concentration of this population was not elevated in comparison to women in labor with negative cultures. Interleukin-1 beta was present in high concentrations (5-1100 pg/ml) in all fluid samples with detectable bacteria. Our data suggest that IL-1 beta may indicate subclinical uterine infection. IL-6 is elevated in all fluid samples of women in active labor.

To determine if amniotic fluid interleukin-10 (IL-10) concentrations are elevated in women with labor, either at term or preterm, and in the setting of infection-associated preterm labor, amniotic fluid samples were collected from women: (1) at term, not in labor (n = 42); at term, in labor (n = 56), preterm contractions, undelivered within 1 week (n = 22), and preterm labor, delivered within 1 week (n = 31). IL-10 concentrations were assayed in each sample via ELISA (Pharmingen, San Diego, CA). In a subsequent analysis, 8 women with preterm labor associated with chorioamnionitis were matched for gestational age with women experiencing preterm contractions (undelivered within 7 days) and preterm labor (delivered within 7 days) and amniotic fluid IL-10 concentrations compared. Approximately 40-70% of amniotic fluid samples obtained from women in each group had detectable IL-10. However, there were no significant differences in amniotic fluid IL-10 concentrations among the patients. While 1 of 8 patients with chorioamnionitis had amniotic fluid IL-10 concentrations greater than 300 pg/ml, there were no statistically significant differences among the matched samples. Amniotic fluid IL-10 concentrations were not elevated in women with term labor, preterm labor, or chorioamnionitis. This finding contrasts with the elevated concentrations of pro-inflammatory cytokines and chemokines such as interleukin-1, tumor necrosis factor-alpha, IL-6, IL-8, MIP-1 alpha, and GRO alpha reported in previous studies. Because we did not detect elevations of the key anti-inflammatory cytokine IL-10 in amniotic fluid of women with infection-associated preterm labor, we suggest that anti-inflammatory processes in this setting may be attenuated.

Objective: To better understand the role of the antiinflammatory cytokine interleukin-10 in preterm labor and infection, we evaluated the amniotic fluid interleukin-10 concentrations through pregnancy, in term, and in preterm labor., Study Design: Amniotic fluid interleukin-10 levels were measured in 147 women throughout pregnancy including patients in the second trimester, patients at term with and without labor, and in patients in preterm labor with and without an intrauterine infection. We compared the amniotic fluid interleukin-10 concentrations among these five groups using the Mann-Whitney U test., Results: Amniotic fluid interleukin-10 was detected in 70% to 91% of patients in each of the five study groups. Higher concentrations were found at term compared with the second trimester (p < 0.001) and concentrations were significantly greater in patients with preterm labor and intrauterine infection compared with those patients in preterm labor without infection (p < 0.001), patients at term in labor (p < 0.001), or patients at term not in labor (p < 0.001). When the patients in preterm labor with infection were analyzed by gestational age, those patients at < 30 weeks had significantly higher amniotic fluid concentrations of interleukin-10 (p = 0.014)., Conclusions: Interleukin-10 was present in the amniotic fluid of the majority of pregnancies, with higher concentrations found at term compared with the second trimester. Intrauterine infection was associated with significantly increased concentrations, with even higher concentrations found in the very premature pregnancies. Interleukin-10 has a prominent yet undefined role in pregnancy and preterm labor complicated by intrauterine infection.

Objective: Our purpose was to determine whether amniotic fluid concentrations of interleukin-6 are of value in the antenatal diagnosis of acute inflammatory lesions (histologic chorioamnionitis) of preterm placenta and in the prediction of perinatal morbidity and mortality., Study Design: The relation among placental histologic findings, perinatal outcome, and amniotic fluid interleukin-6 concentrations was examined in 50 consecutive patients who delivered preterm neonates within 72 hours after amniocentesis. Interleukin-6 was determined by enzyme-linked immunosorbent assays. Receiver-operator characteristic curve was used for analysis., Results: Patients with acute histologic chorioamnionitis had significantly higher median amniotic fluid interleukin-6 concentrations than patients without histologic chorioamnionitis (median 70.8 ng/ml, range 0.7 to 499.2 ng/ml vs median 2.9 ng/ml, range 0.8 to 16.0 ng/ml, respectively; p < 0.00001). An amniotic fluid interleukin-6 concentration > 17 ng/ml had a sensitivity of 79% (23/29) and a specificity of 100% (21/21) in the diagnosis of acute histologic chorioamnionitis and a sensitivity of 69% (18/26) and a specificity of 79% (19/24) in the prediction of significant neonatal morbidity (defined as neonatal sepsis, respiratory distress syndrome, pneumonia, intraventricular hemorrhage, bronchopulmonary dysplasia, or necrotizing enterocolitis) and mortality. These sensitivities were significantly higher than those of amniotic fluid culture (79% vs 38%, p < 0.005; 69% vs 27%, p < 0.01, respectively)., Conclusions: Amniotic fluid interleukin-6 is a sensitive test for the prospective diagnosis of acute histologic chorioamnionitis and the identification of neonates at risk for significant morbidity and mortality.

Accumulating evidence indicates an association between intraamniotic infection and raising concentrations of amniotic cytokines, resulting in preterm labor and preterm rupture of fetal membranes, because these cytokines are able to stimulate prostaglandin biosynthesis. Therefore the purpose of our study was to investigate if quantitative determination of Il-1 beta, Il-6, Il-8 and TNF-a in amniotic fluid may be a practicable method to diagnose intraamniotic infection. Since invasive amniocentesis doesn't allow repeated cytokine detection, in case of preterm rupture of fetal membranes, amniotic fluid also was obtained by placing a sterile gauze and cotton pad into the women's vagina, absorbing draining amniotic fluid for cytokine detection. Our results clearly indicate that Il-1 beta and TNF-a are not detectable in normal pregnancy, while Il-6 and Il-8 are produced in low, but constant levels. In contrast, in amniotic fluid of patients with intraamniotic infection high amounts of Il-6 and Il-8 were found, while Il-1 beta and TNF-a bioactivity became measurable, indicating that biosynthesis was activated. These results demonstrate, that infection associated cytokines detectable in amniotic fluid are highly sensitive markers for intraamniotic infection. In case of preterm rupture of fetal membranes recovery of amniotic fluid from a vaginal pad allows monitoring of cytokine bioactivity in daily intervals to control success of antibiotic treatment.

Problem: The purpose of this study was to determine if amniotic fluid concentrations of the interleukin-6 (IL-6) are of value in diagnosis of microbial invasion of the amniotic cavity and in the prediction of failure of tocolysis, preterm delivery and perinatal morbidity and mortality., Method: Amniotic fluid was obtained by transabdominal amniocentesis from 146 consecutive patients admitted with the diagnosis of preterm labor and intact membranes. Fluid was cultured for aerobic and anaerobic bacteria as well as for mycoplasmas. Amniotic fluid IL-6 levels were measured using a monoclonal antibody-based enzyme-linked immunosorbent assay with a sensitivity of 0.03 ng/ml. Logistic regression and Cox's proportional hazards model were used to examine the effect of several variables on dichotomous outcomes or interval to delivery., Results: Patients with a positive amniotic fluid culture had a significantly higher amniotic fluid IL-6 concentrations than patients with a negative culture (median 91.2 ng/ml, range 0.9 to 437 ng/ml versus median 0.4 ng/ml, range < 0.3 to 195 ng/ml, respectively; P < .0001). An amniotic fluid IL-6 concentration of greater than or equal to 11.3 ng/ml had a sensitivity of 93.3% (14 of 15) and a specificity of 91.6% (120 of 131). All patients with an amniotic fluid IL-6 concentration above 11.3 ng/ml and a negative amniotic fluid culture (N = 11) delivered preterm and all placenta available for examination (N = 7) had histologic evidence of chorioamnionitis. Amniotic fluid concentrations of IL-6 were an independent predictor of preterm delivery, amniocentesis-to-delivery interval and neonatal morbidity and mortality. Moreover, IL-6 concentrations added significant information to the prediction of these outcomes to that provided only by clinical information such as cervical dilatation, gestational age at admission or at delivery., Conclusion: IL-6 is a sensitive and rapid test for the detection of microbial invasion of the amniotic cavity and for identifying women at risk for spontaneous preterm delivery and neonates at risk for morbidity and mortality.

Objective: To assess the association between cytokines in the amniotic fluid (AF) and preterm delivery, the isolation of bacteria from the AF or chorioamnion, and histologic chorioamnionitis., Methods: Fifty afebrile women with intact membranes in preterm labor at or before 34 weeks' gestation underwent amniocentesis. Cytokine levels were measured in AF, and cultures were performed. Placentas were cultured and examined histologically., Results: Thirty-two (64%) of the 50 patients delivered at or before 34 weeks' gestation. Delivery at or before 34 weeks, compared with delivery after 34 weeks, was related to increased levels of interleukin-6 (IL-6) (88 versus 12%; P < .001), interleukin-1 (IL-1) alpha (50 versus 6%; P = .004), IL-1 beta (42 versus 0%; P = .002), and prostaglandin (PG) E2 (66 versus 22%; P = .008). Bacteria were recovered from the AF of nine (18%) of the 50 patients. All of the cytokines with increased levels, plus tumor necrosis factor (TNF)-alpha, were related to bacteria in the AF. Increased IL-6, IL-1 alpha, IL-1 beta, TNF-alpha, and PGE2 were also associated with histologic chorioamnionitis among women who delivered within 1 week of amniocentesis. Elevated cytokine levels were not related to chorioamnion infection., Conclusions: Elevated AF cytokines and PGE2 predicted delivery before 34 weeks' gestation and delivery within 7 days of the amniocentesis, as well as AF infection and histologic chorioamnionitis. These findings support the hypothesis that infection is one cause of preterm delivery, operating via a mechanism involving induction of cytokine production.

We measured the amniotic fluid Interleukin-8 (AF IL-8) levels of 80 women to see whether or not AF IL-8 levels were of value in the diagnosis of intraamniotic infection. Of twelve patients developing conventional signs of infection, 9 had an AF IL-8 concentration above 10,000 pg/ml serum. In two patients, whose baby had a serious neonatal infection, AF IL-8 concentration also exceeded 10,000 pg/ml. Only one out of 66 apparently uninfected patients had an AF IL-8 level above 10,000 pg/ml. We therefore suggest that measuring the AF IL-8 levels is of value in cases of suspected intraamniotic infection.