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Acute cervical insufficiency is frequently associated with subclinical intra-amniotic inflammation and intra-amniotic infection. Amniotic fluid analysis has been recommended prior to the placement of a cervical cerclage given that preexisting infection is associated with adverse pregnancy outcome. We report a case for which commonly available laboratory tests—amniotic fluid Gram stain, white blood cell count, and glucose concentration—did not detect either intra-amniotic inflammation, diagnosed by elevated amniotic fluid interleukin-6, or intra-amniotic infection, diagnosed by cultivation. Following cerclage placement, the patient developed clinical chorioamnionitis and bacteremia and experienced a spontaneous mid-trimester pregnancy loss. This case illustrates the need for a rapid and sensitive point-of-care test capable of detecting infection or inflammation, given recent evidence in support of treatment of intra-amniotic infection and intra-amniotic inflammation with antimicrobial agents. [ABSTRACT FROM AUTHOR]

The cell-free transcriptome in amniotic fluid (AF) has been shown to be informative of physiologic and pathologic processes in pregnancy; however, the change in AF proteome with gestational age has mostly been studied by targeted approaches. The objective of this study was to describe the gestational age-dependent changes in the AF proteome during normal pregnancy by using an omics platform. The abundance of 1310 proteins was measured on a high-throughput aptamer-based proteomics platform in AF samples collected from women during midtrimester (16–24 weeks of gestation, n = 15) and at term without labor (37–42 weeks of gestation, n = 13). Only pregnancies without obstetrical complications were included in the study. Almost 25% (320) of AF proteins significantly changed in abundance between the midtrimester and term gestation. Of these, 154 (48.1%) proteins increased, and 166 (51.9%) decreased in abundance at term compared to midtrimester. Tissue-specific signatures of the trachea, salivary glands, brain regions, and immune system were increased while those of the gestational tissues (uterus, placenta, and ovary), cardiac myocytes, and fetal liver were decreased at term compared to midtrimester. The changes in AF protein abundance were correlated with those previously reported in the cell-free AF transcriptome. Intersecting gestational age-modulated AF proteins and their corresponding mRNAs previously reported in the maternal blood identified neutrophil-related protein/mRNA pairs that were modulated in the same direction. The first study to utilize an aptamer-based assay to profile the AF proteome modulation with gestational age, it reveals that almost one-quarter of the proteins are modulated as gestation advances, which is more than twice the fraction of altered plasma proteins (~ 10%). The results reported herein have implications for future studies focused on discovering biomarkers to predict, monitor, and diagnose obstetrical diseases. [ABSTRACT FROM AUTHOR]

Objective: Preterm labor and birth are strongly associated with sterile intra-amniotic inflammation, a clinical condition that is proposed to be initiated by danger signals, or alarmins. The aim of this study was to investigate whether the intra-amniotic administration of the alarmin heat shock protein 70 (HSP70) induces preterm labor/birth and adverse neonatal outcomes.Methods: Pregnant mice received an intra-amniotic injection of 200 ng (n = 8), 400 ng (n = 6), 500 ng (n = 10), or 1 µg of HSP70 (n = 6). Control mice were injected with saline (n = 10). Following injection, the rates of preterm labor/birth and neonatal mortality were recorded. Neonatal weights at weeks 1, 2, and 3 were also recorded.Results: The intra-amniotic injection of 400 ng [late preterm birth 16.7 ± 16.7% (1/6)], 500 ng [early and late preterm birth 10 ± 10% (1/10) each], or 1 µg [early preterm birth 16.7 ± 16.7% (1/6)] of HSP70 induced low rates of preterm/birth. However, the intra-amniotic injection of 500 ng or 1 µg of HSP70 induced significantly higher rates of neonatal mortality compared to controls [saline 14.2% (10/74), 200 ng 9.8% (6/61), 400 ng 17.9% (9/45), 500 ng 28.8% (23/78), and 1 µg 21.4% (13/49)]. Neonates born to dams injected with 200, 500 ng, or 1 µg HSP70 were leaner than controls (p ≤ .05).Conclusion: Intra-amniotic administration of the alarmin HSP70 did not induce high rates of preterm labor/birth; yet, it did indeed result in adverse neonatal outcomes. [ABSTRACT FROM AUTHOR]

Intra-amniotic infection, defined by the presence of microorganisms in the amniotic cavity, is often accompanied by intra-amniotic inflammation. Occasionally, laboratories report the growth of bacteria or the presence of microbial nucleic acids in amniotic fluid in the absence of intra-amniotic inflammation. This study was conducted to determine the clinical significance of the presence of bacteria in amniotic fluid samples in the absence of intra-amniotic inflammation. A retrospective cross-sectional study included 360 patients with preterm labor and intact membranes who underwent transabdominal amniocentesis for evaluation of the microbial state of the amniotic cavity as well as intra-amniotic inflammation. Cultivation techniques were used to isolate microorganisms, and broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was utilized to detect the nucleic acids of bacteria, viruses, and fungi. Patients whose amniotic fluid samples evinced microorganisms but did not indicate inflammation had a similar perinatal outcome to those without microorganisms or inflammation [amniocentesis-to-delivery interval (p=0.31), spontaneous preterm birth before 34 weeks (p=0.83), acute placental inflammatory lesions (p=1), and composite neonatal morbidity (p=0.8)]. The isolation of microorganisms from a sample of amniotic fluid in the absence of intra-amniotic inflammation is indicative of a benign condition, which most likely represents contamination of the specimen during the collection procedure or laboratory processing rather than early colonization or infection. [ABSTRACT FROM AUTHOR]

The amniotic fluid (AF) cell-free RNA was shown to reflect physiological and pathological processes in pregnancy, but its value in the prediction of spontaneous preterm delivery is unknown. Herein we profiled cell-free RNA in AF samples collected from women who underwent transabdominal amniocentesis after an episode of spontaneous preterm labor and subsequently delivered within 24 h (n = 10) or later (n = 28) in gestation. Expression of known placental single-cell RNA-Seq signatures was quantified in AF cell-free RNA and compared between the groups. Random forest models were applied to predict time-to-delivery after amniocentesis. There were 2385 genes differentially expressed in AF samples of women who delivered within 24 h of amniocentesis compared to gestational age-matched samples from women who delivered after 24 h of amniocentesis. Genes with cell-free RNA changes were associated with immune and inflammatory processes related to the onset of labor, and the expression of placental single-cell RNA-Seq signatures of immune cells was increased with imminent delivery. AF transcriptomic prediction models captured these effects and predicted delivery within 24 h of amniocentesis (AUROC = 0.81). These results may inform the development of biomarkers for spontaneous preterm birth. [ABSTRACT FROM AUTHOR]

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

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

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

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

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

Background: The amniotic fluid (AF) cell-free transcriptome is modulated by physiologic and pathologic processes during pregnancy. AF gene expression changes with advancing gestation reflect fetal development and organ maturation; yet, defining normal expression and splicing patterns for biomarker discovery in obstetrics requires larger heterogeneous cohorts, evaluation of potential confounding factors, and novel analytical approaches. Methods: Women with a normal pregnancy who had an AF sample collected during midtrimester (n = 30) or at term gestation (n = 68) were included. Expression profiling at exon level resolution was performed using Human Transcriptome Arrays. Differential expression was based on moderated t-test adjusted p < 0.05 and fold change > 1.25; for differential splicing, a splicing index > 2 and adjusted p < 0.05 were required. Functional profiling was used to interpret differentially expressed or spliced genes. The expression of tissue-specific and cell-type specific signatures defined by single-cell genomics was quantified and correlated with covariates. In-silico validation studies were performed using publicly available datasets. Results: 1) 64,071 genes were detected in AF, with 11% of the coding and 6% of the non-coding genes being differentially expressed between midtrimester and term gestation. Expression changes were highly correlated with those previously reported (R > 0.79, p < 0.001) and featured increased expression of genes specific to the trachea, salivary glands, and lung and decreased expression of genes specific to the cardiac myocytes, uterus, and fetal liver, among others. 2) Single-cell RNA-seq signatures of the cytotrophoblast, Hofbauer cells, erythrocytes, monocytes, T and B cells, among others, showed complex patterns of modulation with gestation (adjusted p < 0.05). 3) In 17% of the genes detected, we found differential splicing with advancing gestation in genes related to brain development processes and immunity pathways, including some that were missed based on differential expression analysis alone. Conclusions: This represents the largest AF transcriptomics study in normal pregnancy, reporting for the first time that single-cell genomic signatures can be tracked in the AF and display complex patterns of expression during gestation. We also demonstrate a role for alternative splicing in tissue-identity acquisition, organ development, and immune processes. The results herein may have implications for the development of fetal testing to assess placental function and fetal organ maturity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

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

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

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

Objective: Upon inflammasome activation, the adaptor protein of the inflammasome ASC (apoptosis-associated speck-like protein containing a CARD) forms intracellular specks, which can be released into the extracellular space. The objectives of this study were to investigate whether (1) extracellular ASC is present in the amniotic fluid of women who delivered at term; (2) amniotic fluid ASC concentrations are greater in women who underwent spontaneous labor at term than in those who delivered at term in the absence of labor; and (3) amniotic epithelial and mesenchymal cells can form intracellular ASC specks in vitro. Methods: This retrospective cross-sectional study included amniotic fluid samples from 41 women who delivered at term in the absence of labor (n = 24) or underwent spontaneous labor at term (n = 17). Amniotic epithelial and mesenchymal cells were also isolated from the chorioamniotic membranes obtained from a separate group of women who delivered at term (n = 3), in which ASC speck formation was assessed by confocal microscopy. Monocytes from healthy individuals were used as positive controls for ASC speck formation (n = 3). Results: (1) The adaptor protein of the inflammasome ASC is detectable in the amniotic fluid of women who delivered at term; (2) amniotic fluid ASC concentration was higher in women who underwent spontaneous labor at term than in those who delivered at term without labor; and (3) amniotic epithelial and mesenchymal cells are capable of forming ASC specks and/or filaments in vitro. Conclusion: Amniotic fluid ASC concentrations are increased in women who undergo spontaneous labor at term. Amniotic epithelial and mesenchymal cells are capable of forming ASC specks, suggesting that these cells are a source of extracellular ASC in the amniotic fluid. These findings provide in vivo evidence that there is inflammasome activation in the amniotic cavity during the physiological process of labor at term. [ABSTRACT FROM AUTHOR]

Introduction: A bioassay based on the detection of beta-glucan, a constituent of the cell wall of fungi, has been successfully used to diagnose fungal infections in a variety of biological fluids but not yet in the amniotic fluid.Objective: To determine the diagnostic performance of a beta-glucan bioassay in the detection of Candida species in the amniotic fluid of women who either did or did not have an intrauterine contraceptive device (IUD) in place during an episode of spontaneous preterm parturition.Methods: The study population comprised women who had a singleton pregnancy without congenital or chromosomal abnormalities, who experienced preterm labor or preterm prelabor rupture of the fetal membranes, and who underwent a transabdominal amniocentesis for clinical indications. Samples of amniotic fluid were cultured for aerobic and anaerobic bacteria, genital mycoplasmas, and Candida species, and assayed for beta-glucan, using the (1→3)-beta-d-glucan-specific Limulus amebocyte lysate test (beta-glucan assay) in all cases. Amniotic fluid interleukin (IL)-6 assay results were also available for all cases. The beta-glucan assay takes about 1 hour to run: a concentration >80 pg/mL was considered positive for fungi. Sterile intra-amniotic inflammation of the amniotic cavity was defined by the presence of an amniotic fluid IL-6 concentration ≥2.6 ng/mL and a negative amniotic fluid culture.Results: (1) One hundred ninety-seven (197) women met the study criteria, of whom 58 (29.4%) had an IUD in place; (2) 20 (10.2%) women had a culture of proven intra-amniotic Candida species-related infection, 19 of whom had a positive beta-glucan assay [sensitivity, 95% (19/20; 95% confidence interval (CI): 75.1-99.9%)]; and (3) the specificity of the beta-glucan assay was 75.1% [133/177; 95% CI: 68.1-99.9%]. It was affected by the presence of nonfungal intra-amniotic infections and an IUD, but not by the presence of sterile intra-amniotic inflammation, and there was a significant interaction between the presence of an IUD and nonfungal intra-amniotic infections (estimated for the interaction effect = 2.1923, p value =.026). The assay's specificity was reduced when nonfungal intra-amniotic infections were diagnosed but only in women who did not have an IUD. Among women without an IUD, the assay's specificity was 91.4% (117/128); it was 93% (106/114) for those without intra-amniotic infection, and 78.6% (11/14) for those with a nonfungal intra-amniotic infection; the difference was not significant (p = .09). Among women with an IUD, the assay's specificity was 32.7% (16/49); 42.9% (9/21) for those with a nonfungal intra-amniotic infection; and 25% (7/28) for those without intra-amniotic infection; and the difference was significant (p = .03).Conclusions: The beta-glucan assay is a sensitive, rapid, point-of-care test used to diagnose intra-amniotic Candida species-related infection, and it has a high specificity in pregnant women who did not have an IUD in place. [ABSTRACT FROM AUTHOR]

Background: The inflammasome has been implicated in the mechanisms that lead to spontaneous labor at term. However, whether the inflammasome is activated in the amniotic cavity of women with clinical chorioamnionitis at term is unknown. Herein, by measuring extracellular ASC [apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (CARD)], we investigated whether there is in vivo inflammasome activation in amniotic fluid of patients with clinical chorioamnionitis at term with sterile intra-amniotic inflammation and in those with intra-amniotic infection. Methods: This was a retrospective cross-sectional study that included amniotic fluid samples collected from 76 women who delivered after spontaneous term labor with diagnosed clinical chorioamnionitis. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin (IL)-6 concentration ≥2.6 ng/mL, and intra-amniotic infection was diagnosed by the presence of microbial invasion of the amniotic cavity (MIAC) accompanied by intra-amniotic inflammation. Patients were classified into the following groups: (1) women without intra-amniotic inflammation or infection (n=16); (2) women with MIAC but without intra-amniotic inflammation (n=5); (3) women with sterile intra-amniotic inflammation (n=15); and (4) women with intra-amniotic infection (n=40). As a readout of in vivo inflammasome activation, extracellular ASC was measured in amniotic fluid by enzyme-linked immunosorbent assay. Acute inflammatory responses in the amniotic fluid and placenta were also evaluated. Results: In clinical chorioamnionitis at term: (1) amniotic fluid concentrations of ASC (extracellular ASC is indicative of in vivo inflammasome activation) and IL-6 were greater in women with intra-amniotic infection than in those without intra-amniotic inflammation, regardless of the presence of MIAC; (2) amniotic fluid concentrations of ASC and IL-6 were also higher in women with sterile intra-amniotic inflammation than in those without intra-amniotic inflammation, regardless of the presence of MIAC; (3) amniotic fluid concentrations of IL-6, but not ASC, were more elevated in women with intra-amniotic infection than in those with sterile intra-amniotic inflammation; (4) a positive and significant correlation was observed between amniotic fluid concentrations of ASC and IL-6; (5) no differences were observed in amniotic fluid ASC and IL-6 concentrations between women with and without MIAC in the absence of intra-amniotic inflammation; (6) women with intra-amniotic infection had elevated white blood cell counts and reduced glucose levels in amniotic fluid compared to the other three study groups; and (7) women with intra-amniotic infection presented higher frequencies of acute maternal and fetal inflammatory responses in the placenta than those with sterile intra-amniotic inflammation. Conclusion: The intra-amniotic inflammatory response, either induced by alarmins or microbes, is characterized by the activation of the inflammasome – as evidenced by elevated amniotic fluid concentrations of extracellular ASC – in women with clinical chorioamnionitis at term. These findings provide insight into the intra-amniotic inflammatory response in women with clinical chorioamnionitis at term. [ABSTRACT FROM AUTHOR]

Objectives: To investigate mechanisms of in utero death in normally formed fetuses by measuring amniotic fluid (AF) biomarkers for hypoxia (erythropoietin [EPO]), myocardial damage (cardiac troponin I [cTnI]) and brain injury (glial fibrillary acidic protein [GFAP]), correlated with risk factors for fetal death and placental histopathology. Methods: This retrospective, observational cohort study included intrauterine deaths with transabdominal amniocentesis prior to induction of labor. Women with a normal pregnancy and an indicated amniocentesis at term were randomly selected as controls. AF was assayed for EPO, cTnI and GFAP using commercial immunoassays. Placental histopathology was reviewed, and CD15-immunohistochemistry was used. Analyte concentrations >90th centile for controls were considered "raised". Raised AF EPO, AF cTnI and AF GFAP concentrations were considered evidence of hypoxia, myocardial and brain injury, respectively. Results: There were 60 cases and 60 controls. Hypoxia was present in 88% (53/60), myocardial damage in 70% (42/60) and brain injury in 45% (27/60) of fetal deaths. Hypoxic fetuses had evidence of myocardial injury, brain injury or both in 77% (41/53), 49% (26/53) and 13% (7/53) of cases, respectively. Histopathological evidence for placental dysfunction was found in 74% (43/58) of these cases. Conclusion: Hypoxia, secondary to placental dysfunction, was found to be the mechanism of death in the majority of fetal deaths among structurally normal fetuses. Ninety-one percent of hypoxic fetal deaths sustained brain, myocardial or both brain and myocardial injuries in utero. Hypoxic myocardial injury was an attributable mechanism of death in 70% of the cases. Non-hypoxic cases may be caused by cardiac arrhythmia secondary to a cardiac conduction defect. [ABSTRACT FROM AUTHOR]

Background: Normal development of the human placenta, referred to as villous tree maturation, entails formation of the vasculosyncytial membranes. These structures develop by the approximation of syncytiotrophoblasts with the villous capillary endothelium and constitute the most efficient sites of gaseous exchange in the placenta. Defective maturation of the villous tree can lead to deficient vasculosyncytial membranes, implicated in the high incidence of hypoxic complications. Hypoxia, in turn, can stimulate production of erythropoietin, whereby increased fetal plasma or amniotic fluid concentrations of this hormone reflect fetal hypoxemia. The current study was undertaken to determine whether delayed villous maturation is associated with changes in amniotic fluid erythropoietin concentrations. Methods: Placental histologic examination was performed using hematoxylin and eosin. Subsequent to histologic assessment of delayed villous maturation, the diagnosis was confirmed with CD-15 immunohistochemistry. The controls (n = 61) were pregnancies without villous maturation abnormalities, and cases (n = 5) were pregnancies with delayed villous maturation. Amniotic fluid erythropoietin concentrations were measured using a specific immunoassay. Results: Concentrations of erythropoietin in the amniotic fluid (1) of controls were less than the limit of detection and (2) of cases with delayed villous maturation were significantly higher than those of controls (P-value = 0.048). Conclusion: Delayed villous maturation is associated with higher concentrations of amniotic fluid erythropoietin. [ABSTRACT FROM AUTHOR]

Problem: The immune cellular composition of amniotic fluid is poorly understood. Herein, we determined: 1) the immunophenotype of amniotic fluid immune cells during the second and third trimester in the absence of intra‐amniotic infection/inflammation; 2) whether amniotic fluid T cells and ILCs display different phenotypical characteristics to that of peripheral cells; and 3) whether the amniotic fluid immune cells are altered in women with intra‐amniotic infection/inflammation. Method of Study: Amniotic fluid samples (n = 57) were collected from 15 to 40 weeks of gestation in women without intra‐amniotic infection/inflammation. Samples from women with intra‐amniotic infection/inflammation were also included (n = 9). Peripheral blood mononuclear cells from healthy adults were used as controls (n = 3). Immunophenotyping was performed using flow cytometry. Results: In the absence of intra‐amniotic infection/inflammation, the amniotic fluid contained several immune cell populations between 15 and 40 weeks. Among these immune cells: (i) T cells and ILCs were greater than B cells and natural killer (NK) cells between 15 and 30 weeks; (ii) T cells were most abundant between 15 and 30 weeks; (iii) ILCs were most abundant between 15 and 20 weeks; (iv) B cells were scarce between 15 and 20 weeks; yet, they increased and were constant after 20 weeks; (v) NK cells were greater between 15 and 30 weeks than at term; (vi) ILCs expressed high levels of RORγt, CD161, and CD103 (ie, group 3 ILCs); (vii) T cells expressed high levels of RORγt; (viii) neutrophils increased as gestation progressed; and (ix) monocytes/macrophages emerged after 20 weeks and remained constant until term. All of the amniotic fluid immune cells, except ILCs, were increased in the presence of intra‐amniotic infection/inflammation. Conclusion: The amniotic fluid harbors a diverse immune cellular composition during normal and complicated pregnancies. [ABSTRACT FROM AUTHOR]

Objective: Our purpose was to compare and correlate amniotic fluid GRO-alpha, interleukin-8, and L-selectin in patients with and without intraamniotic infection.Study Design: Amniocentesis was performed on 45 pregnant women with preterm contractions, labor, or rupture of membranes. Fourteen patients had intraamniotic infection, and 31 did not. Intraamniotic infection was defined as the presence of a positive amniotic fluid culture. Amniotic fluid tests for Gram stain, glucose, neutrophil counts, creatinine, pH, and specific gravity were performed. Amniotic fluid levels of soluble L-selectin, interleukin-8, and GRO-alpha were measured by an enzyme-linked immunoassay and normalized by amniotic fluid creatinine levels. The Mann-Whitney Utest and Spearman's rank correlation test were used for statistical analyses.Results: Amniotic fluid median levels of soluble L-selectin, interleukin-8, and GRO-alpha were significantly higher in pregnant women with intraamniotic infection than in those without intraamniotic infection (soluble L-selectin: median 3334.6 ng/mg creatinine, range 408.4 to 15,956.8 vs 717.2 ng/mg creatinine, range 129.4 to 4601.9, p = 0.009; GRO-alpha: median 841.6 ng/mg creatinine, range 28.1 to 8591.7 vs 56.8 ng/mg creatinine, range 0.0 to 440.2, p < 0.0001; interleukin-8: median 4932.7 ng/mg creatinine, range 0.0 to 55,058.7 vs 28.3 ng/mg creatinine, range 0.0 to 1161.6, p = 0.0004). Patients with intraamniotic infection had significantly higher amniotic fluid leukocyte counts and leukocyte esterase activities and significantly lower amniotic fluid glucose concentrations compared with those without intraamniotic infection. Amniotic fluid GRO-alpha, interleukin-8, and soluble L-selectin were positively correlated, and each was positively correlated with amniotic fluid leukocytes and negatively correlated with amniotic fluid levels of glucose.Conclusions: Our data indicate amniotic fluid GRO-alpha and interleukin-8 may be two potent leukocyte chemoattractants and activators, and L-selectin is rapidly shed from leukocytes in the amniotic fluid in patients with intraamniotic infection. [ABSTRACT FROM AUTHOR]

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

Problem: The inflammasome is implicated in the mechanisms that lead to spontaneous preterm labor (PTL). However, whether there is inflammasome activation in the amniotic cavity of women with PTL and intra‐amniotic infection (IAI) or sterile intra‐amniotic inflammation (SIAI) is unknown. Method of study: Amniotic fluid samples were collected from women with PTL who delivered at term (n = 31) or preterm without IAI or SIAI (n = 35), with SIAI (n = 27), or with IAI (n = 17). As a readout of inflammasome activation, extracellular ASC (apoptosis‐associated speck‐like protein containing a CARD) was measured in amniotic fluid by ELISA and the expression of ASC, caspase‐1, and interleukin (IL)‐1β was detected in the chorioamniotic membranes by multiplex immunofluorescence. Acute inflammatory responses in amniotic fluid and the placenta were also evaluated. Results: (a) Amniotic fluid concentrations of ASC and IL‐6 were higher in women with PTL and IAI or SIAI than in those who delivered preterm or at term without intra‐amniotic inflammation; (b) amniotic fluid concentrations of ASC and IL‐6 were lower in women with PTL and SIAI than in those with IAI; (c) there was a significant nonlinear correlation between ASC and IL‐6 amniotic fluid concentrations; (d) the expression of inflammasome‐related proteins (ASC, caspase‐1, and IL‐1β) in the chorioamniotic membranes was increased in women with PTL and IAI or SIAI than in those who delivered preterm or at term without intra‐amniotic inflammation; (e) inflammasome activation in the chorioamniotic membranes was weaker in women with PTL and SIAI than in those with IAI; (f) women with PTL and IAI had elevated amniotic fluid white blood cell counts compared to those without this clinical condition; and (g) severe acute placental inflammatory lesions were observed in women with PTL and IAI and in a subset of women with PTL and SIAI. Conclusion: Inflammasome activation occurs in the settings of intra‐amniotic infection and sterile intra‐amniotic inflammation during spontaneous preterm labor. [ABSTRACT FROM AUTHOR]

Problem: Human β‐defensins (HBDs) are antimicrobial peptides that participate in the soluble innate immune mechanisms against infection. Herein, we determined whether HBD‐1 was present in amniotic fluid during normal pregnancy and whether its concentrations change with intra‐amniotic inflammation and/or infection. Method of Study: Amniotic fluid was collected from 219 women in the following groups: (a) midtrimester who delivered at term (n = 35); (b) term with (n = 33) or without (n = 17) labor; (c) preterm labor with intact membranes who delivered at term (n = 29) or who delivered preterm with (n = 19) and without (n = 29) intra‐amniotic inflammation and infection or with intra‐amniotic inflammation but without infection (n = 21); and (d) preterm prelabor rupture of membranes (pPROM) with (n = 19) and without (n = 17) intra‐amniotic inflammation/infection. Amniotic fluid HBD‐1 concentrations were determined using a sensitive and specific ELISA kit. Results: (a) HBD‐1 was detectable in all amniotic fluid samples; (b) amniotic fluid concentrations of HBD‐1 were changed with gestational age (midtrimester vs term no labor), being higher in midtrimester; (c) amniotic fluid concentrations of HBD‐1 were similar between women with and without spontaneous labor at term; (d) among patients with spontaneous preterm labor, amniotic fluid concentrations of HBD‐1 in women with intra‐amniotic inflammation/infection and in those with intra‐amniotic inflammation without infection were greater than in women without intra‐amniotic inflammation or infection who delivered preterm or at term; and (e) the presence of intra‐amniotic inflammation and infection in patients with pPROM did not change amniotic fluid concentrations of HBD‐1. Conclusion: HBD‐1 is a physiological constituent of amniotic fluid that is increased in midtrimester during normal pregnancy and in the presence of culturable microorganisms in the amniotic cavity. These findings provide insight into the soluble host defense mechanisms against intra‐amniotic infection. Amniotic fluid concentrations of human beta defensin‐1 (HBD‐1) in women with spontaneous preterm labor and intact membranes. Red lines indicate medians with interquartile ranges. [ABSTRACT FROM AUTHOR]

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OBJECTIVE: To assess the potential role of amniotic fluid (AF) matrix metalloproteinase-9 and interleukin-6 in predicting intra-amniotic infection.METHODS: Eighty-four women with singleton gestations with preterm contraction, preterm labor, preterm premature rupture of membranes, or clinical suspicion of intra-amniotic infection were studied. Amniotic fluid was obtained by transabdominal amniocentesis before starting any treatment. Intra-amniotic infection was defined as the presence of a positive AF culture. Amniotic fluid glucose concentration, leukocytes, matrix metalloproteinase-9, and interleukin-6 were determined.RESULTS: Amniotic fluid matrix metalloproteinase-9 and interleukin-6 levels were significantly higher in women with intra-amniotic infection than in those without. With intra-amniotic infection, levels of matrix metalloproteinase-9 significantly correlated with interleukin-6 (r = 0.813, P < .001). Each of matrix metalloproteinase-9 and interleukin-6 significantly correlated with AF leukocytes and inversely correlated with AF glucose. Using AF cutoff levels of 13.6 ng/mL for matrix metalloproteinase-9 and 11.4 ng/mL for interleukin-6, the sensitivity, specificity, and positive and negative predictive values for diagnosing intra-amniotic infection were 77% versus 73%, 100% versus 79%, 100% versus 61%, and 90% versus 86%, respectively. Combining AF matrix metalloproteinase-9 with interleukin-6 slightly improved the sensitivity and the negative predictive values in diagnosing intra-amniotic infection.CONCLUSIONS: Amniotic fluid matrix metalloproteinase-9 and interleukin-6 are significantly elevated in women with intra-amniotic infection. Amniotic fluid matrix metalloproteinase-9 is an accurate biochemical marker in predicting intra-amniotic infection with better sensitivity, specificity, and positive and negative predictive values than interleukin-6. [Copyright &y& Elsevier]