Microbial-associated Intra-amniotic Inflammation Research Articles

Evidence for the participation of CHCHD2/MNRR1, a mitochondrial protein, in spontaneous labor at term and in preterm labor with intra-amniotic infection

Objective Intra-amniotic inflammation (IAI), associated with either microbe (infection) or danger signals (sterile), plays a major role in the pathophysiology of preterm labor and delivery. Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 2 (CHCHD2) [also known as Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1)], a mitochondrial protein involved in oxidative phosphorylation and cell survival, is capable of sensing tissue hypoxia and inflammatory signaling. The ability to maintain an appropriate energy balance at the cellular level while adapting to environmental stress is essential for the survival of an organism. Mitochondrial dysfunction has been observed in acute systemic inflammatory conditions, such as sepsis, and is proposed to be involved in sepsis-induced multi-organ failure. The purpose of this study was to determine the amniotic fluid concentrations of CHCHD2/MNRR1 in pregnant women, women at term in labor, and those in preterm labor (PTL) with and without IAI. Methods This cross-sectional study comprised patients allocated to the following groups: (1) mid-trimester (n = 16); (2) term in labor (n = 37); (3) term not in labor (n = 22); (4) PTL without IAI who delivered at term (n = 25); (5) PTL without IAI who delivered preterm (n = 47); and (6) PTL with IAI who delivered preterm (n = 53). Diagnosis of IAI (amniotic fluid interleukin-6 concentration ≥2.6 ng/mL) included cases associated with microbial invasion of the amniotic cavity and those of sterile nature (absence of detectable bacteria, using culture and molecular microbiology techniques). Amniotic fluid and maternal plasma CHCHD2/MNRR1 concentrations were determined with a validated and sensitive immunoassay. Results (1) CHCHD2/MNRR1 was detectable in all amniotic fluid samples and women at term without labor had a higher amniotic fluid CHCHD2/MNRR1 concentration than those in the mid-trimester (p = 0.003); (2) the amniotic fluid concentration of CHCHD2/MNRR1 in women at term in labor was higher than that in women at term without labor (p = 0.01); (3) women with PTL and IAI had a higher amniotic fluid CHCHD2/MNRR1 concentration than those without IAI, either with preterm (p < 0.001) or term delivery (p = 0.01); (4) women with microbial-associated IAI had a higher amniotic fluid CHCHD2/MNRR1 concentration than those with sterile IAI (p < 0.001); (5) among women with PTL and IAI, the amniotic fluid concentration of CHCHD2/MNRR1 correlated with that of interleukin-6 (Spearman’s Rho = 0.7; p < 0.001); and (6) no correlation was observed between amniotic fluid and maternal plasma CHCHD2/MNRR1 concentrations among women with PTL. Conclusion CHCHD2/MNRR1 is a physiological constituent of human amniotic fluid in normal pregnancy, and the amniotic concentration of this mitochondrial protein increases during pregnancy, labor at term, and preterm labor with intra-amniotic infection. Hence, CHCHD2/MNRR1 may be released into the amniotic cavity by dysfunctional mitochondria during microbial-associated IAI.

Maternal Plasma and Amniotic Fluid LBP, Pentraxin 3, Resistin, and IGFBP-3: Biomarkers of Microbial Invasion of Amniotic Cavity and/or Intra-amniotic Inflammation in Women with Preterm Premature Rupture of Membranes.

BackgroundWe sought to determine whether lipopolysaccharide binding protein (LBP), pentraxin 3, resistin, and insulin-like growth factor binding protein (IGFBP)-3 in plasma and amniotic fluid (AF) can predict microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation (IAI), and microbial-associated IAI in women with preterm premature rupture of membranes (PPROM).MethodsThis was a retrospective cohort study involving 168 singleton pregnant women with PPROM. AF obtained via amniocentesis was cultured and assayed for interleukin (IL)-6 to define IAI and for IL-8 to compare with AF biomarkers. Plasma samples were collected at the time of amniocentesis, and C-reactive protein (CRP) levels in serum were compared with plasma biomarkers. The stored plasma and AF samples were assayed for LBP, pentraxin 3 (PTX3), resistin, and IGFBP-3 by ELISA.ResultsMultivariate logistic regression analysis revealed that: 1) elevated plasma and AF levels of LBP were independently associated with increased risks of MIAC, IAI, and microbial-associated IAI; 2) elevated AF, but not plasma, PTX3, and resistin levels were independently associated with increased risks of MIAC, IAI, and microbial-associated IAI; 3) decreased IGFBP-3 levels in the plasma were independently associated with only IAI, whereas those in the AF were associated with only microbial-associated IAI. Among the tested biomarkers, AF PTX3 and resistin had the highest predictive performance for MIAC, IAI, and microbial-associated IAI (area under the curves [AUC] = 0.85–0.95), which is similar to the performance of AF IL-8. The AUCs of the plasma LBP and IGFBP-3 were similar to that of serum CRP with respect to IAI.ConclusionMaternal plasma LBP and IGFBP-3 are potential biomarkers for the non-invasive identification of IAI in women with PPROM, with a similar accuracy to the serum CRP level. AF LBP, PTX3, resistin, and IGFBP-3 may be involved in the intra-amniotic inflammatory responses in PPROM complicated by MIAC.

Amniotic fluid CD11b levels in pregnancies complicated by preterm prelabor rupture of membranes

Objective CD11b is an integrin molecule located on the surface of leukocytes. CD11b is involved in the processes of cell adhesion and migration. Expression of CD11b increases during inflammation. Therefore, this study was aimed at the evaluation of concentrations of CD11b in the amniotic fluid from pregnancies complicated by preterm prelabor rupture of the membranes (PPROM), with respect to the presence of microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation (IAI), and microbial-associated IAI (the presence of both MIAC and IAI). Methods Eighty women with singleton pregnancies complicated by PPROM were included. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid CD11b concentrations were determined by enzyme-linked immunosorbent assay. MIAC was determined by a non-cultivation approach. IAI was defined by a bedside amniotic fluid interleukin-6 concentration ≥745 pg/mL. Result Women with MIAC or microbial-associated IAI had higher CD11b concentrations in the amniotic fluid than women without these complications (with MIAC: median 0.31 ng/mL versus without MIAC: median 0.17 ng/mL, p = .001; with microbial associated-IAI: median 0.35 ng/mL versus without microbial-associated IAI: median 0.16 ng/mL; p =.02). The presence of IAI was not associated with elevated CD11b concentrations. A weak negative correlation was found between amniotic fluid CD11b concentrations and interleukin-6 concentrations (rho = 0.26; p = .02). Conclusions MIAC and microbial-associated IAI are characterized by higher amniotic fluid CD11b concentrations in pregnancies complicated by PPROM.

Amniotic fluid cell-free DNA in preterm prelabor rupture of membranes.

We evaluated the levels of cell-free nuclear DNA (nDNA) and cell-free mitochondrial DNA (mtDNA) in the amniotic fluid supernatant from pregnancies complicated by preterm prelabor rupture of membranes (PPROM) based on evidence of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI). A total of 155 women with PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. The levels of cell-free nDNA and mtDNA in the amniotic fluid supernatant were assessed and quantified by real-time polymerase chain reaction. The levels of cell-free nDNA and mtDNA were higher in women with MIAC and IAI than in women without these conditions (nDNA: with MIAC: median 3.9×104 genome equivalent [GE]/mL vs without MIAC: median 1.2×104 GE/mL, with IAI: median: 5.3×104 GE/mL vs without IAI: median 1.2×104 GE/mL; mtDNA: with MIAC: median 9.2×105 GE/mL vs without MIAC: median 2.5×105 GE/mL, with IAI: median 1.1×106 GE/mL vs without IAI: median 2.5×105 ; all P values≤0.01). Women with the microbial-associated IAI showed the highest levels of cell-free nDNA and mtDNA. Cell-free nDNA and mtDNA are constituents of the amniotic fluid supernatant from PPROM pregnancies. Both cell-free nDNA and mtDNA are involved in the intra-amniotic inflammatory response in women with PPROM.

Amniotic fluid pentraxins: Potential early markers for identifying intra-amniotic inflammatory complications in preterm pre-labor rupture of membranes.

In this study, pentraxin 3 (PTX3), C-reactive protein (CRP), and serum amyloid P component (SAP) concentrations in the amniotic fluid of women with preterm pre-labor rupture of membranes (PPROM) were evaluated based on evidence of microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation (IAI), and microbial-associated IAI. A total of 149 women with PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid PTX3, SAP, and CRP concentrations were assessed using enzyme-linked immunosorbent assay. PTX3 and CRP concentrations were higher in women with MIAC, IAI, and microbial-associated IAI than in women without these conditions. SAP concentrations were only higher in the presence of IAI and microbial-associated IAI. Amniotic fluid PTX3 concentrations of 11ng/mL were found to be the best value for identifying the presence of microbial-associated IAI and IAI in women with PPROM. To conclude, amniotic fluid pentraxins are involved in intra-amniotic inflammatory responses in pregnancies complicated by PPROM.

Cervical fluid interleukin 6 and intra-amniotic complications of preterm prelabor rupture of membranes

Objective: To determine if cervical fluid interleukin (IL)-6 concentrations in women with preterm prelabor rupture of membranes (PPROM) allows identification of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI).Methods: One hundred forty-four women with singleton pregnancies complicated by PPROM were included in this prospective cohort study. Cervical and amniotic fluids were collected at the time of admission and concentrations of IL-6 were measured using an ELISA and point-of-care test, respectively. Cervical fluid was obtained using a Dacron polyester swab and amniotic fluid was obtained by transabdominal amniocentesis. MIAC was diagnosed based on a positive PCR result for Ureaplasma species, M. hominis, and/or C. trachomatis and/or by positivity for the 16 S rRNA gene. IAI was defined as amniotic fluid point-of-care IL-6 concentrations ≥745 pg/mL. The women were assigned to four subgroups based on the presence of MIAC and/or IAI: microbial-associated IAI (both MIAC and IAI), sterile IAI (IAI alone), MIAC alone, and without either MIAC or IAI.Results: (1) Women with microbial-associated IAI had higher cervical fluid IL-6 concentrations (median 560 pg/mL) than did women with sterile IAI (median 303 pg/mL; p = .001), women with MIAC alone (median 135 pg/mL; p = .0004), and women without MIAC and IAI (median 180 pg/mL; p = .0001). (2) No differences were found in cervical fluid IL-6 concentrations among women with sterile IAI, with MIAC alone, and without MIAC and IAI. (3) A positive correlation was observed between cervical fluid IL-6 concentrations and the amount of Ureaplasma species in amniotic fluid (copies DNA/mL; rho = 0.57, p < .0001). (4) A weak positive correlation was detected between cervical and amniotic fluid IL-6 concentrations (rho = 0.33, p < .0001).Conclusions: The presence of microbial-associated IAI is associated with the highest cervical fluid IL-6 concentrations. Cervical IL-6 can be helpful in the identification of microbial-associated IAI.

305: Impact of microbial associated intra-amniotic inflammation and sterile inflammation on short-term neonatal outcome in women with preterm labor and intact membranes

305: Impact of microbial associated intra-amniotic inflammation and sterile inflammation on short-term neonatal outcome in women with preterm labor and intact membranes

Amniotic fluid clusterin in pregnancies complicated by the preterm prelabor rupture of membranes

Objective: The aim of this study was to evaluate clusterin concentrations in amniotic fluid in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) with respect to the presence of the microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation (IAI) and microbial-associated IAI.Methods: One hundred thirty-six women with singleton pregnancies complicated by PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid clusterin concentrations were assessed by enzyme-linked immunosorbent assay. MIAC was determined by a non-cultivation approach. IAI was defined as an amniotic fluid bedside interleukin-6 concentration ≥745 pg/mL. Microbial-associated IAI was characterized as the presence of both MIAC and IAI.Result: Women with MIAC, IAI and microbial-associated IAI had lower amniotic fluid clusterin concentrations than women without these complications (with MIAC: median 1314 ng/mL versus without MIAC: median 1633 ng/mL, p = 0.003; with IAI: median 1281 ng/mL versus without IAI: median 1575 ng/mL, p = 0.04; with microbial associated-IAI: median 1220 ng/mL versus without microbial-associated IAI: median 1575 pg/mL; p = 0.008). A week negative correlation between amniotic fluid clusterin concentrations and gestational age at sampling was revealed (rho= −0.30; p = 0.0005).Conclusions: The presence of MIAC, IAI and microbial-associated IAI was characterized by lower amniotic fluid clusterin concentrations in pregnancies complicated by PPROM.

Amniotic fluid cathepsin-G in pregnancies complicated by the preterm prelabor rupture of membranes

Objective: The aim of this study was to evaluate the amniotic fluid cathepsin-G concentrations in women with preterm prelabor rupture of membranes (PPROM) based on the presence of the microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI). Methods: A total of 154 women with singleton pregnancies complicated by PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid cathepsin-G concentrations were assessed by ELISA. MIAC was determined using a non-cultivation approach. IAI was defined as an amniotic fluid bedside interleukin-6 concentration ≥ 745 pg/mL. Results: Women with MIAC had higher amniotic fluid cathepsin-G concentrations than women without MIAC (with MIAC: median 82.7 ng/mL, versus without MIAC: median 64.7 ng/mL; p = 0.0003). Women with IAI had higher amniotic fluid cathepsin-G concentrations than women without this complication (with IAI: median 103.0 ng/mL, versus without IAI: median 66.2 ng/mL; p < 0.0001). Women with microbial-associated (with both MIAC and IAI) IAI and sterile (IAI without MIAC) IAI had higher amniotic fluid cathepsin-G concentrations than women with colonization (MIAC without IAI) and women without both MIAC and IAI (p < 0.0001). Conclusions: The presence of either microbial-associated or sterile IAI was associated with increased amniotic fluid cathepsin-G concentrations in pregnancies complicated by PPROM. Amniotic fluid cathepsin-G appears to be a potential marker of IAI.

Vaginal fluid interleukin-6 concentrations as a point-of-care test is of value in women with preterm prelabor rupture of membranes

Preterm prelabor rupture of membranes is frequently complicated/accompanied by infection and inflammation in the amniotic cavity. A point-of-care determination of amniotic fluid interleukin-6 has been shown to be a potentially clinically useful approach to assess inflammatory status of the amniotic cavity. Amniocentesis in preterm prelabor rupture of membranes is not broadly used in clinical practice, and therefore, a shift toward a noninvasive amniotic fluid sampling method is needed. The first aim of this study was to evaluate the association between the point-of-care vaginal and amniotic fluid interleukin-6 concentrations in fresh unprocessed samples obtained simultaneously. The second goal was to determine the diagnostic indices and predictive value of the point-of-care assessment of vaginal fluid interleukin-6 concentration in the identification of microbial invasion of the amniotic cavity, intraamniotic inflammation, and microbial-associated intraamniotic inflammation in patients with preterm prelabor rupture of membranes. A prospective cohort study was conducted in women with singleton gestation complicated by preterm prelabor rupture of membranes at between 24+0 and 36+6 weeks. A total of 153 women with singleton pregnancies were included in this study. Vaginal fluid was obtained from the posterior vaginal fornix by aspiration with a sterile urine sample tube with a suction tip. Amniotic fluid was obtained by transabdominal amniocentesis. Interleukin-6 concentrations were assessed with a lateral flow immunoassay in both fluids immediately after sampling. Microbial invasion of the amniotic cavity was determined based on a positive polymerase chain reaction analysis. Intraamniotic inflammation was defined as an amniotic fluid point-of-care interleukin-6 concentration ≥745 pg/mL. Several results were obtained in this study. First, it was possible to perform the point-of-care assessment of interleukin-6 in vaginal fluid in 92% of the women (141 of 153), and only those women were included in the analyses. Second, the rate of microbial invasion of the amniotic cavity and intraamniotic inflammation was 26% (36 of 141) and 19% (27 of 141), respectively. Microbial-associated intraamniotic inflammation was identified in 12% of the women (17 of 141). Third, a strong positive correlation was found between the interleukin-6 concentrations in vaginal and amniotic fluids (Spearman rho 0.68; P < .0001). Fourth, the presence of microbial invasion of the amniotic cavity, intraamniotic inflammation, or microbial-associated intraamniotic inflammation was associated with higher vaginal fluid interleukin-6 concentrations in both crude and adjusted analyses. Fifth, a vaginal fluid interleukin-6 concentration of 2500 pg/mL was determined to be the best cutoff value for the identification of microbial invasion of the amniotic cavity (sensitivity of 53% [19 of 36], specificity of 89% [93 of 104], positive predictive value of 63% [19 of 30], negative predictive value of 85% [93 of 110], positive likelihood ratio of 5.0 [95% confidence interval, 2.5-9.5], and negative likelihood ratio of 0.5 [95% confidence interval, 0.4-0.8]); intraamniotic inflammation (sensitivity of 74% [20/27], specificity of 91% [104/114], positive predictive value of 67% [20 of 30], negative predictive value of 94% [104 of 111], positive likelihood ratio of 8.4 [95% confidence interval, 4.5-15.9], and negative likelihood ratio of 0.3 [95% confidence interval, 0.2-0.5]); and microbial-associated intraamniotic inflammation (sensitivity of 100% [17 of 17], specificity of 90% [111 of 124), positive predictive value of 57% [17 of 30], negative predictive value of 100% [111 of 111], positive likelihood ratio of 9.5 [95% confidence interval, 5.7-16.0], and negative likelihood ratio of 0). The point-of-care assessment of interleukin-6 in vaginal fluid is an easy, rapid, noninvasive, and inexpensive method for the identification of intraamniotic inflammation and microbial-associated intraamniotic inflammation in preterm prelabor rupture of membranes pregnancies, showing good specificity and negative predictive value.

WITHDRAWN: Vaginal fluid IL-6 concentrations as a point-of-care test is of value in women with preterm PROM

Preterm prelabor rupture of membranes (PROM) is frequently complicated/accompanied by infection and inflammation in the amniotic cavity. A point-of-care determination of amniotic fluid interleukin-6 has been shown to be a potentially clinically useful approach to assess inflammatory status of the amniotic cavity. Amniocentesis in preterm PROM is not broadly used in clinical practice, and therefore a shift towards a non-invasive amniotic fluid sampling method is needed. The first aim of this study was to evaluate the association between the point-of-care vaginal and amniotic fluid interleukin-6 concentrations in fresh unprocessed samples obtained simultaneously. The second goal was to determine the diagnostic indices and predictive value of the point-of-care assessment of vaginal fluid interleukin-6 concentration in the identification of microbial invasion of the amniotic cavity, intra-amniotic inflammation, and microbial-associated intra-amniotic inflammation in patients with preterm PROM. A prospective cohort study was conducted in women with singleton gestation complicated by preterm PROM at between 24+0 and 36+6 weeks. A total of 153 women with singleton pregnancies were included in this study. Vaginal fluid was obtained from the posterior vaginal fornix by aspiration with a sterile urine sample tube with a suction tip. Amniotic fluid was obtained by transabdominal amniocentesis. Interleukin-6 concentrations were assessed with a lateral flow immunoassay in both fluids immediately after sampling. Microbial invasion of the amniotic cavity was determined based on a positive PCR analysis. Intra-amniotic inflammation was defined as an amniotic fluid point-of-care interleukin-6 concentration ≥745 pg/mL. 1) It was possible to perform the point-of-care assessment of interleukin-6 in vaginal fluid in 92% (141/153) of the women, and only those women were included in the analyses. 2) The rate of microbial invasion of the amniotic cavity and intra-amniotic inflammation was 26% (36/141) and 19% (27/141), respectively. Microbial-associated intra-amniotic inflammation was identified in 12% (17/141) of the women. 3) A strong positive correlation was found between the interleukin-6 concentrations in vaginal and amniotic fluids (Spearman rho 0.68; p<0.0001). 4) The presence of microbial invasion of the amniotic cavity, intra-amniotic inflammation or microbial-associated intra-amniotic inflammation was associated with higher vaginal fluid interleukin-6 concentrations in both crude and adjusted analyses. 5) A vaginal fluid interleukin-6 concentration of 2500 pg/mL was determined to be the best cutoff value for the identification of microbial invasion of the amniotic cavity [sensitivity of 53% (19/36), specificity of 89% (93/104), positive predictive value of 63% (19/30), negative predictive value of 85% (93/110), positive likelihood ratio of 5.0 (95% CI 2.5-9.5), and negative likelihood ratio of 0.5 (95% CI 0.4-0.8)], intra-amniotic inflammation [sensitivity of 74% (20/27), specificity of 91% (104/114), positive predictive value of 67% (20/30), negative predictive value of 94% (104/111), positive likelihood ratio of 8.4 (95% CI 4.5-15.9), and negative likelihood ratio of 0.3 (95%CI 0.2-0.5)], and microbial-associated intra-amniotic inflammation (sensitivity of 100% (17/17), specificity of 90% (111/124), positive predictive value of 57% (17/30), negative predictive value of 100% (111/111), positive likelihood ratio of 9.5 (95% CI 5.7-16.0), and negative likelihood ratio of 0]. The point-of-care assessment of interleukin-6 in vaginal fluid is an easy, rapid, non-invasive, and inexpensive method for the identification of intra-amniotic inflammation and microbial-associated intra-amniotic inflammation in preterm PROM pregnancies, showing good specificity and negative predictive value.

Amniotic fluid calreticulin in pregnancies complicated by the preterm prelabor rupture of membranes

Objective: This study aimed to determine the amniotic fluid calreticulin concentrations in women with the preterm prelabor rupture of membranes (PPROM) based on the microbial invasion of the amniotic cavity (MIAC), intraamniotic inflammation (IAI) and microbial-associated IAI. Methods: One hundred sixty-eight women with singleton pregnancies were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis and were assayed for calreticulin concentrations by ELISA. IAI was defined as an amniotic fluid interleukin-6 concentration > 745 pg/ml. Microbial-associated IAI was defined as the presence of both MIAC and IAI. Result: Women with MIAC (with MIAC: median 54.4 ng/ml, versus without MIAC: median 32.6 ng/ml; p < 0.0001), IAI (with IAI: median 66.8 ng/ml, versus without IAI: median 33.0 ng/ml; p < 0.0001) and microbial-associated IAI (with microbial-associated IAI: median 82.5 ng/ml, versus without microbial-associated IAI: median 33.7 ng/ml; p < 0.0001) had higher concentrations of calreticulin than women without these complications. An amniotic fluid calreticulin concentration of 81.4 ng/ml was found to be the best cutoff point for identifying women with microbial-associated IAI. Conclusions: The presence of microbial-associated IAI is associated with increased amniotic fluid calreticulin concentrations. Calreticulin seems to be a promising marker for the early identification of PPROM complicated by microbial-associated IAI.

Amniotic fluid prostaglandin E2 in pregnancies complicated by preterm prelabor rupture of the membranes

Objective: To determine amniotic fluid prostaglandin E2 concentrations in women preterm prelabor rupture of the membranes (PPROM) with respect to microbial invasion of the amniotic cavity (MIAC), intraamniotic inflammation (IAI), microbial-associated IAI, histological chorioamnionitis, and short-term neonatal morbidity.Methods: One hundred forty-five women with singleton pregnancies were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis and were assayed for prostaglandin E2 concentrations by ELISA. IAI was defined as amniotic fluid interleukin-6 >745 pg/mL. Microbial-associated IAI was defined as the presence of both MIAC and IAI.Result: No differences in prostaglandin E2 concentrations were found between women with and without MIAC (p = 0.27). Women with IAI (p = 0.0008) and microbial-associated IAI (p = 0.01) had higher prostaglandin E2 concentrations than women without these complications. Women with histological chorioamnionitis had higher prostaglandin E2 concentrations only in crude analysis (p = 0.02), but not after adjustment for gestational age at sampling (p = 0.10). No associations between amniotic fluid prostaglandin E2 concentrations and the selected conditions of severe neonatal morbidity were found.Conclusions: The intraamniotic inflammatory response either to infectious or to non-infectious stimulus, but not MIAC per se, seems to be a main factor associated with the elevation of the amniotic fluid PGE2 concentrations in women with PPROM.

Evidence of perturbations of the cytokine network in preterm labor

Intraamniotic inflammation/infection is the only mechanism of disease with persuasive evidence of causality for spontaneous preterm labor/delivery. Previous studies about the behavior of cytokines in preterm labor have been largely based on the analysis of the behavior of each protein independently. Emerging evidence indicates that the study of biologic networks can provide insight into the pathobiology of disease and improve biomarker discovery. The goal of this study was to characterize the inflammatory-related protein network in the amniotic fluid of patients with preterm labor. A retrospective cohort study was conducted that included women with singleton pregnancies who had spontaneous preterm labor and intact membranes (n = 135). These patients were classified according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry, and amniotic fluid concentration of interleukin (IL)-6 into the following groups: (1) those without intraamniotic inflammation (n = 85), (2) those with microbial-associated intraamniotic inflammation (n = 15), and (3) those with intraamniotic inflammation without detectable bacteria (n = 35). Amniotic fluid concentrations of 33 inflammatory-related proteins were determined with the use of a multiplex bead array assay. Patients with preterm labor and intact membranes who had microbial-associated intraamniotic inflammation had a higher amniotic fluid inflammatory-related protein concentration correlation than those without intraamniotic inflammation (113 perturbed correlations). IL-1β, IL-6, macrophage inflammatory protein (MIP)-1α, and IL-1α were the most connected nodes (highest degree) in this differential correlation network (degrees of 20, 16, 12, and 12, respectively). Patients with sterile intraamniotic inflammation had correlation patterns of inflammatory-related proteins, both increased and decreased, when compared to those without intraamniotic inflammation (50 perturbed correlations). IL-1α, MIP-1α, and IL-1β were the most connected nodes in this differential correlation network (degrees of 12, 10, and 7, respectively). There were more coordinated inflammatory-related protein concentrations in the amniotic fluid of women with microbial-associated intraamniotic inflammation than in those with sterile intraamniotic inflammation (60 perturbed correlations), with IL-4 and IL-33 having the largest number of perturbed correlations (degrees of 15 and 13, respectively). We report for the first time an analysis of the inflammatory-related protein network in spontaneous preterm labor. Patients with preterm labor and microbial-associated intraamniotic inflammation had more coordinated amniotic fluid inflammatory-related proteins than either those with sterile intraamniotic inflammation or those without intraamniotic inflammation. The correlations were also stronger in patients with sterile intraamniotic inflammation than in those without intraamniotic inflammation. The findings herein could be of value in the development of biomarkers of preterm labor.

Intraamniotic Inflammation in Women with Preterm Prelabor Rupture of Membranes.

ObjectiveTo characterize subgroups of preterm prelabor rupture of membranes (PPROM) and short-term neonatal outcomes based on the presence and absence of intraamniotic inflammation (IAI) and/or microbial invasion of the amniotic cavity (MIAC).MethodsOne hundred and sixty-six Caucasian women with singleton pregnancies were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis (n=166) and were assayed for interleukin-6 levels by a lateral flow immunoassay. The presence of Ureaplasma species, Mycoplasma hominis, Chlamydia trachomatis, and 16S rRNA was evaluated in the amniotic fluid. IAI was defined as amniotic fluid IL-6 values, measured by a point of care test, higher than 745 pg/mL.ResultsMicrobial-associated IAI (IAI with MIAC) and sterile intraamniotic inflammation (IAI alone) were found in 21% and 4%, respectively, of women with PPROM. Women with microbial-associated IAI had higher microbial loads of Ureaplasma species in the amniotic fluid than women with MIAC alone. No differences in the short-term neonatal morbidity with respect to the presence of microbial-associated IAI, sterile IAI and MIAC alone were found after adjusting for the gestational age at delivery in women with PPROM.ConclusionsMicrobial-associated but not sterile intraamniotic inflammation is common in Caucasian women with PPROM. The gestational age at delivery but not the presence of inflammation affects the short-term neonatal morbidity of newborns from PPROM pregnancies.

Clinical chorioamnionitis at term III: how well do clinical criteria perform in the identification of proven intra-amniotic infection?

The diagnosis of clinical chorioamnionitis is based on a combination of signs [fever, maternal or fetal tachycardia, foul-smelling amniotic fluid (AF), uterine tenderness and maternal leukocytosis]. Bacterial infections within the amniotic cavity are considered the most frequent cause of clinical chorioamnionitis and an indication for antibiotic administration to reduce maternal and neonatal morbidity. Recent studies show that only 54% of patients with the diagnosis of clinical chorioamnionitis at term have bacteria in the AF and evidence of intra-amniotic inflammation. The objective of this study was to examine the performance of the clinical criteria for the diagnosis of chorioamnionitis to identify patients with microbial-associated intra-amniotic inflammation (also termed intra-amniotic infection). This retrospective cross-sectional study included 45 patients with the diagnosis of clinical chorioamnionitis at term, whose AF underwent analysis for: 1) the presence of microorganisms using both cultivation and molecular biologic techniques [polymerase chain reaction (PCR) with broad primers], and 2) interleukin (IL)-6 concentrations by enzyme-linked immunosorbent assay. The diagnostic performance (sensitivity, specificity, accuracy, and likelihood ratios) of each clinical sign and their combination to identify clinical chorioamnionitis were determined using microbial-associated intra-amniotic inflammation [presence of microorganisms in the AF using cultivation or molecular techniques and elevated AF IL-6 concentrations (≥2.6 ng/mL)] as the gold standard. The accuracy of each clinical sign for the identification of microbial-associated intra-amniotic inflammation (intra-amniotic infection) ranged between 46.7% and 57.8%. The combination of fever with three or more clinical criteria did not substantially improve diagnostic accuracy. In the presence of a fever during labor at term, signs used to diagnose clinical chorioamnionitis do not accurately identify the patient with proven intra-amniotic infection (i.e., those with microorganisms detected by culture or molecular microbiologic techniques and an associated intra-amniotic inflammatory response).

Clinical chorioamnionitis at term V: umbilical cord plasma cytokine profile in the context of a systemic maternal inflammatory response.

Microbial invasion of the fetus due to intra-amniotic infection can lead to a systemic inflammatory response characterized by elevated concentrations of cytokines in the umbilical cord plasma/serum. Clinical chorioamnionitis represents the maternal syndrome often associated with intra-amniotic infection, although other causes of this syndrome have been recently described. The objective of this study was to characterize the umbilical cord plasma cytokine profile in neonates born to mothers with clinical chorioamnionitis at term, according to the presence or absence of bacteria and/or intra-amniotic inflammation. A cross-sectional study was conducted, including patients with clinical chorioamnionitis at term (n=38; cases) and those with spontaneous term labor without clinical chorioamnionitis (n=77; controls). Women with clinical chorioamnionitis were classified according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) and amniotic fluid interleukin (IL)-6 concentration into three groups: 1) no intra-amniotic inflammation; 2) intra-amniotic inflammation without detectable microorganisms; or 3) microbial-associated intra-amniotic inflammation. A fetal inflammatory response syndrome (FIRS) was defined as an umbilical cord plasma IL-6 concentration >11 pg/mL. The umbilical cord plasma concentrations of 29 cytokines were determined with sensitive and specific V-PLEX immunoassays. Nonparametric statistical methods were used for analysis, adjusting for a false discovery rate of 5%. 1) Neonates born to mothers with clinical chorioamnionitis at term (considered in toto) had significantly higher median umbilical cord plasma concentrations of IL-6, IL-12p70, IL-16, IL-13, IL-4, IL-10 and IL-8, but significantly lower interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF)-α concentrations than neonates born to mothers with spontaneous term labor without clinical chorioamnionitis; 2) neonates born to mothers with clinical chorioamnionitis at term but without intra-amniotic inflammation had higher concentrations of IL-6, IL-12p70, IL-13, IL-4, IL-5, and IL-8, but lower IFN-γ, than neonates not exposed to clinical chorioamnionitis, suggesting that maternal fever in the absence of intra-amniotic inflammation leads to a change in the fetal cytokine network; 3) there were significant, positive correlations between maternal and umbilical cord plasma IL-6 and IL-8 concentrations (IL-6: Spearman correlation=0.53; P<0.001; IL-8: Spearman correlation=0.42; P<0.001), consistent with placental transfer of cytokines; 4) an elevated fetal plasma IL-6 (>11 pg/mL), the diagnostic criterion for FIRS, was present in 21% of cases (8/38), and all these neonates were born to mothers with proven intra-amniotic infection; and 5) FIRS was associated with a high concentration of umbilical cord plasma IL-8, IL-10 and monocyte chemoattractant protein (MCP)-1. Neonates born to mothers with clinical chorioamnionitis at term had higher concentrations of umbilical cord plasma cytokines than those born to mothers without clinical chorioamnionitis. Even neonates exposed to clinical chorioamnionitis but not to intra-amniotic inflammation had elevated concentrations of multiple cytokines, suggesting that intrapartum fever alters the fetal immune response.

Clinical chorioamnionitis at term IV: the maternal plasma cytokine profile.

Fever is a major criterion for clinical chorioamnionitis; yet, many patients with intrapartum fever do not have demonstrable intra-amniotic infection. Some cytokines, such as interleukin (IL)-1, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), can induce a fever. The objective of this study was to determine whether maternal plasma concentrations of cytokines could be of value in the identification of patients with the diagnosis of clinical chorioamnionitis at term who have microbial-associated intra-amniotic inflammation. A retrospective cross-sectional study was conducted, including patients with clinical chorioamnionitis at term (n=41; cases) and women in spontaneous labor at term without clinical chorioamnionitis (n=77; controls). Women with clinical chorioamnionitis were classified into three groups according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), and amniotic fluid IL-6 concentration: 1) no intra-amniotic inflammation; 2) intra-amniotic inflammation without detectable microorganisms; or 3) microbial-associated intra-amniotic inflammation. The maternal plasma concentrations of 29 cytokines were determined with sensitive and specific V-PLEX immunoassays. Nonparametric statistical methods were used for analysis, adjusting for a false discovery rate of 5%. 1) The maternal plasma concentrations of pyrogenic cytokines (IL-1β, IL-2, IL-6, IFN-γ, and TNF-α) were significantly higher in patients with clinical chorioamnionitis at term than in those with spontaneous term labor without clinical chorioamnionitis; 2) the maternal plasma concentrations of cytokines were not significantly different among the three subgroups of patients with clinical chorioamnionitis (intra-amniotic inflammation with and without detectable bacteria and those without intra-amniotic inflammation); and 3) among women with the diagnosis of clinical chorioamnionitis, but without evidence of intra-amniotic inflammation, the maternal plasma concentrations of pyrogenic cytokines were significantly higher than in patients with spontaneous labor at term. These observations suggest that a fever can be mediated by increased circulating concentrations of these cytokines, despite the absence of a local intra-amniotic inflammatory response. 1) The maternal plasma concentrations of pyrogenic cytokines (e.g. IL-1β, IL-2, IL-6, IFN-γ, and TNF-α) are higher in patients with intra-partum fever and the diagnosis of clinical chorioamnionitis at term than in those in spontaneous labor at term without a fever; and 2) maternal plasma cytokine concentrations have limited value in the identification of patients with bacteria in the amniotic cavity. Accurate assessment of the presence of intra-amniotic infection requires amniotic fluid analysis.

Clinical chorioamnionitis at term II: the intra-amniotic inflammatory response.

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. 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. 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. 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.

Clinical chorioamnionitis at term I: microbiology of the amniotic cavity using cultivation and molecular techniques.

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. 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. 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]. 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.