Negative Amniotic Fluid Culture Research Articles

Evidence for participation of rage (receptor for advanced glycation end products), soluble rage (sRAGE) and S100A12 proteins, in control of the inflammatory response of amniotic cavity during pregnancy

END PRODUCTS), SOLUBLE RAGE (SRAGE) AND S100A12 PROTEINS, IN CONTROL OF THE INFLAMMATORY RESPONSE OF AMNIOTIC CAVITY DURING PREGNANCY IRINA A. BUHIMSCHI, GUOMAO ZHAO, CHRISTIAN PETTKER, MERT BAHTIYAR, LISSA MAGLOIRE, CATALIN S. BUHIMSCHI, Yale University, Obstetrics, Gynecology, and Reprod. Sciences, New Haven, Connecticut OBJECTIVE: RAGE (NF-B activator) is a novel membrane cell-surface receptor with role in inflammation. S100A12 (proteomic biomarker of inflammation) is a ligand for RAGE. sRAGE competitively inhibits RAGE ligands. Studies were conducted to understand the relationships among RAGE, sRAGE and S100A12 in the amniotic fluid (AF) and fetal tissues of women with PTL in the presence or absence of intra-AF infection/inflammation (IAI/I). STUDY DESIGN: AF was retrieved by amniocentesis in 93 consecutive women stratified as follows: i) CAFC: positive AF culture, n=20, GA=27.1G0.7wks; ii) AFC: negative AF culture, n=30, GA=28.7G0.9 wks; iii) 2nd trim. control, n=15, GA=18.5G0.4wks; iv) 3rd trim. control, n=28, GA=36.7G0.4wks. sRAGE and IL-6 levels were measured by highly specific immunoassay. Each sample was subjected to mass spectrometry (SELDI) for detection of S100A12. Fetal membranes, cord and placenta were stained for RAGE and S100A12 protein. RESULTS: The S100A12 [10.4kDa] biomarker was present in 70%(14/20) of women withCAFC but only in 10%(3/30) of women with –AFC(p!0.001). Its presence correlated with AF levels of IL-6 (r=0.63, p!0.001) and AF WBC count (r=0.71, p!0.001). No control had the S100A12 SELDI biomarker peak. Levels of the inhibitor sRAGE were not influenced by presence or absence of IAI/I but increased directly with increasing GA (r=0.67, p!0.001) [Figure]. Staining for RAGE was present in all fetal membranes, and did not vary with IAI/I. Staining for S100A12 was selectively and markedly increased in the amnion and chorio-decidua of patients with IAI/I.

Evaluation of amniotic fluid cytokines in preterm labor and intact membranes

Objective. To compare the amniotic fluid (AF) concentration of pro-inflammatory cytokines between women with preterm labor and intact membranes that delivered within 7 days, with those that delivered after 7 days of the amniocentesis according to the result of the AF culture.Methods. Fifty-two women with preterm labor and intact membranes between 21 and 35 weeks of gestation were included in the study. Transabdominal amniocentesis was performed to rule out intra-amniotic infection, and AF concentrations of interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor (TNF) were determined with sensitive and specific enzyme-linked immunosorbent assays. Amniotic fluid was cultured for aerobic and anaerobic bacteria, Ureaplasma urealyticum, and Mycoplasma hominis. Exclusion criteria included preterm premature rupture of membranes, vaginal bleeding, multiple gestations, uterine anomalies, fetal congenital anomalies, ominous fetal heart rate tracings and fetal deaths. Proportions were compared using χ2 or Fisher's exact test. Receiver operator characteristic (ROC) curve analysis was performed for each cytokine for the prediction of delivery within 7 days.Results. Sixty-two percent (32/52) of women delivered within 7 days and 38% (20/52) delivered after 7 days of amniocentesis. All women that delivered after 7 days of the procedure had negative AF cultures. In contrast, 28% (9/32) of women that delivered within 7 days had positive AF cultures and 72% (23/32) had negative AF cultures. Women that delivered within 7 days regardless of AF cultures had a lower birth weight and a shorter amniocentesis-to-delivery interval than those that delivered after 7 days of amniocentesis. Among women that delivered within 7 days, those with positive AF cultures had a lower gestational age at delivery and a higher frequency of histologic chorioamnionitis than those with negative AF cultures. The AF concentrations of all cytokines were significantly higher in women that delivered within 7 days with positive AF cultures than in those with negative AF cultures. Similarly, the AF concentrations of IL-1α, IL-6, and IL-8 were significantly higher in women that delivered within 7 days than those that delivered after 7 days of the amniocentesis, regardless of the AF culture results. Diagnostic indexes were calculated for all cytokines using critical values derived from ROC curve analysis for the prediction of delivery within 7 days.Conclusions. Women with preterm labor and intact membranes that delivered within 7 days had higher AF concentrations of pro-inflammatory cytokines than those who delivered after 7 days of the amniocentesis regardless of the AF culture results.

Idiopathic vaginal bleeding during pregnancy as the only clinical manifestation of intrauterine infection

Objective. To determine the frequency and clinical significance of microbial invasion of the amniotic cavity (MIAC) in patients with vaginal bleeding in the absence of placenta previa, preterm labor or preterm premature rupture of membranes (PROM).Study design. This retrospective cohort study included patients who presented with vaginal bleeding between 18 and 35 weeks, and underwent an amniocentesis shortly after admission for the assessment of the microbiologic status of the amniotic cavity and/or fetal lung maturity. Amniotic fluid was cultured for aerobic and anaerobic bacteria, as well as genital mycoplasmas. Patients presenting with preterm labor, preterm PROM, placenta previa, overt placental abruption, and an intrauterine device in situ were excluded, as well as those with local cervical bleeding. MIAC was defined as a positive amniotic fluid culture. Analysis was conducted with non-parametric statistics.Results. One hundred and fourteen patients met the entry criteria. MIAC was detected in 14% of cases (16/114). Patients with vaginal bleeding and a gestational age < 28 weeks at the time of amniocentesis had a significantly higher frequency of MIAC than those with a gestational age ⩾ 28 weeks [25% (13/52) vs. 4.8% (3/62), respectively; p < 0.01]. Ureaplasma urealyticum was the microorganism most frequently isolated from the amniotic fluid. Except for one case admitted at 33 weeks, all patients with MIAC had an early preterm delivery ⩽ 32 weeks. Patients with vaginal bleeding and MIAC had a shorter procedure-to-delivery interval than those without MIAC [MIAC, median survival 19 days (95% CI 10–27 days) vs. no MIAC, median survival 50 days (95% CI 37–62 days); p < 0.0001]. Patients with vaginal bleeding and MIAC had a significantly lower gestational age at delivery and lower birth weight than those with vaginal bleeding and negative amniotic fluid cultures (for gestational age, median 25 weeks, range 21–33 weeks vs. median 37 weeks, range 19–42 weeks, respectively; p < 0.01, and for birth weight, median 750 grams, range 520–1820 grams vs. 2800 grams, range 520–4880 grams, respectively; p < 0.01), as well as a higher frequency of subsequent preterm PROM [81.3% (13/16) vs. 9.2% (9/98); p < 0.01].Conclusions. MIAC was detected in 14% of patients with ‘idiopathic’ vaginal bleeding and was associated with subsequent preterm PROM and early preterm delivery. Vaginal bleeding may be the only clinical manifestation of MIAC, and it predisposes to adverse outcome.

Clinical significance of intra-amniotic inflammation in patients with preterm premature rupture of membranes

This study was conducted to determine the frequency and clinical significance of intra-amniotic inflammation in patients with preterm premature rupture of the membranes. Amniotic fluid was retrieved from 219 patients with preterm premature rupture of the membranes; the fluid was cultured for aerobic and anaerobic bacteria and mycoplasmas and assayed for neutrophil collagenase, which is also known as matrix metalloproteinase-8. Matrix metalloproteinase-8 was used because previous studies indicated that this was a sensitive and specific index of inflammation and that is correlated with the amniotic fluid white blood cell count. Intra-amniotic inflammation was defined as an elevated amniotic fluid matrix metalloproteinase-8 concentration (>23 ng/mL). Nonparametric and survival techniques were used for statistical analysis. The overall rate of intra-amniotic inflammation was 42% (93/219 samples); proven intra-amniotic infection was detected only in 23% (50/219 samples). Intra-amniotic inflammation with a negative amniotic fluid culture for micro-organisms was found in 23% (51/219 samples) and was as common as proven intra-amniotic infection. Pregnancy outcome was worse in patients with intra-amniotic inflammation and a negative culture than in those patients with a negative culture and without inflammation. There were no differences in the interval-to-delivery or rate of complications between patients with intra-amniotic inflammation and a negative culture and patients with proven amniotic fluid infection. We conclude that intra-amniotic inflammation, regardless of culture result, is present in 42% of patients with preterm premature rupture of the membranes and that it is a risk factor for impending preterm delivery and adverse outcome. We propose that intra-amniotic inflammation, rather than infection, be used to classify and treat patients with preterm premature rupture of the membranes.

The clinical significance of detecting Ureaplasma urealyticum by the polymerase chain reaction in the amniotic fluid of patients with preterm labor

Objective This study was undertaken to determine the clinical significance of a detection of Ureaplasma urealyticum by using the polymerase chain reaction (PCR) in the amniotic fluid of patients with preterm labor and intact membranes. Study design Amniocentesis was performed in 257 patients with preterm labor and intact membranes. Amniotic fluid was cultured for aerobic and anaerobic bacteria as well as genital mycoplasmas. U urealyticum was detected by PCR using specific primers. Patients were divided into 3 groups according to the results of amniotic fluid culture and PCR for U urealyticum: those with a negative culture and negative PCR (n = 228), those with a negative culture but positive PCR (n = 6), and those with a positive culture regardless of the results of PCR (n = 23). Results The prevalence of positive amniotic fluid culture was 9% (23 of 257). U urealyticum was detected by PCR in 6% (15 of 254) of cases. Of the 15 cases with positive PCR for U urealyticum, amniotic fluid culture was negative in 40% (6 of 15). Patients with a negative culture but positive PCR for U urealyticum had significantly shorter median amniocentesis-to-delivery interval and higher amniotic fluid interleukin-6 and white blood cell count than those with a negative amniotic fluid culture and negative PCR ( P<.01 for each). Patients with a positive PCR for U urealyticum but a negative amniotic fluid culture had a higher rate of significant neonatal morbidity than those with a negative culture and negative PCR ( P<.05). However, no significant differences in perinatal outcome were observed between patients with a negative culture but positive PCR and those with a positive amniotic fluid culture. Conclusion Patients with preterm labor and a positive PCR for U urealyticum but negative amniotic fluid culture are at risk for impending preterm delivery and adverse perinatal outcome.

Differences in the fetal interleukin-6 response to microbial invasion of the amniotic cavity between term and preterm gestation

Background/objective: Fetal inflammatory response has been implicated as a mechanism of multi-system organ injury in preterm and term neonates. Microbial invasion of the amniotic cavity (MIAC) isfrequently associated with a fetal inflammatory response. However, there are no studies comparing the fetal response to MIAC in term and preterm gestations. The purpose of this study was to compare theumbilical cord plasma interleukin-6 (IL-6) concentrations in term and preterm neonates in the presence or absence of MIAC.Study design: Umbilical cord blood was obtained at birth from 252 neonateswhose mothers had an amniocentesis within 48 h of delivery (preterm delivery, n = 62; term delivery, n = 190). MIAC was defined as a positive amniotic fluid culture for bacteria or genital mycoplasmas.IL-6 was measured by a sensitive and specific immunoassay.Results: The median IL-6 concentration in umbilical cord plasma was significantly higher in preterm neonates than in term neonates (median13.4 pg/ml, range 0.1-676 pg/ml vs. median 3.2 pg/ml, range 0.1-408 pg/ml; p < 0.0001). In the context of MIAC, the median umbilical cord plasma IL-6 concentration was significantly higher inpreterm than in term neonates (median 31.6 pg/ml, range 1.4-676 pg/ml vs. median 11.7 pg/ml, range 1.3-82 pg/ml, respectively; p < 0.05). Neonates born to mothers with a positive amniotic fluidculture had a significantly higher median IL-6 concentration than neonates born to mothers with a negative amniotic fluid culture (preterm: median 31.6, range 1.4-676 pg/ml vs. median 8.0, range 0.1-656pg/ml; p < 0.05 and term: median 11.7, range 1.3-82 pg/ml vs. median 3.1, range 0.1-408 pg/ml; p < 0.01, respectively).Conclusions: The preterm fetus is capable of mountinga systemic cytokine response as measured by IL-6 in its peripheral blood. In the setting of MIAC, a fetal IL-6 response is higher in preterm than in term gestation.

Chronic intrauterine infection and inflammation in the preterm rabbit, despite antibiotic therapy

Objective: In a pregnant rabbit model using intracervical inoculation of Escherichia coli with delayed antibiotic therapy, we investigated the rate of positive cultures and histologic inflammation of maternal and fetal compartments and the concentration of tumor necrosis factor-α in the amniotic fluid for up to 5 days. Study Design: New Zealand White rabbits at 70% gestation were inoculated intracervically with 103,4 colony-forming units of E coli per uterine horn. At varying intervals after inoculation (0.5-4.0 hours), antibiotic therapy was initiated with ampicillin-sulbactam. Primary outcomes were positive cultures and histologic inflammation score. Tumor necrosis factor–α levels in the amniotic fluid were determined by bioassay. Results: A total of 60 animals were inoculated with E coli. At the endpoint, uterine cultures were positive more commonly than in the fetus or amniotic fluid (41.8% vs 27.5% vs 17.3%, respectively), which was consistent with an ascending pathway of infection. Inflammation scores were similar in uterus and placenta but lower in fetal lung and absent in fetal brain (2.8 vs 3.1 vs 0.84 vs 0.0, respectively). Comparing the durations of delay in antibiotic administration, we found a significant increase in positive uterine cultures and a significant increase in histologic inflammation score with increasing delay. The proportion of dead pups within a litter was significantly associated with the log of the tumor necrosis factor–α concentration in amniotic fluid and the degree of histologic inflammation in the uterus, but not with amniotic fluid or other culture positivity. Conclusion: The administration of therapeutic doses of antibiotic does not consistently eradicate bacteria from the rabbit uterus nor, more importantly, from the fetus and the amniotic fluid. Obtaining a negative amniotic fluid culture does not exclude either infection in the decidua or the fetus or histologic inflammation with tumor necrosis factor–α elaboration. (Am J Obstet Gynecol 2002;186:234-9.)

Clinical significance of intra-amniotic inflammation in patients with preterm labor and intact membranes

Objective: The purpose of this study was to determine the frequency and clinical significance of intraamniotic inflammation in patients with preterm labor and intact membranes. Study Design: Amniocentesis was performed in 206 patients with preterm labor and intact membranes. Amniotic fluid was cultured for aerobic and anaerobic bacteria and mycoplasmas. The diagnosis of intraamniotic inflammation was made in patients with a negative amniotic fluid culture on the basis of amniotic fluid concentrations of interleukin-6 (>2.6 ng/mL, derived from receiver operating characteristic curve analysis). Statistical analysis was conducted with contingency tables and survival techniques. Results: Intra-amniotic inflammation (negative amniotic fluid culture but elevated amniotic fluid interleukin-6) was more common than intra-amniotic infection (positive amniotic fluid culture regardless of amniotic fluid interleukin-6 concentration; 21% [44/206 women] vs 10% [21/206 women]; P <.001). The amniocentesisto-delivery interval was significantly shorter in patients with intra-amniotic inflammation than in patients with a negative culture and without an inflammation (median, 20 hours [range, 0.1-2328 hours] vs median, 701 hours [range, 0.1-3252 hours], respectively; P <.0001). Spontaneous preterm delivery of <37 weeks was more frequent in patients with intra-amniotic inflammation than in those with a negative culture and without inflammation (98% vs 35%; P <.001). Patients with intra-amniotic inflammation had a significantly higher rate of adverse outcome than patients with a negative culture and without intra-amniotic inflammation. Adverse outcomes included clinical and histologic chorioamnionitis, funisitis, early preterm birth, and significant neonatal morbidity. There were no significant differences in the rate of adverse outcomes between patients with a negative culture but with intra-amniotic inflammation and patients with intra-amniotic infection (positive culture regardless of amniotic fluid interleukin-6 concentration). Conclusion: Intra-amniotic inflammation/infection complicates one third of the patients with preterm labor (32%; 65/206 women), and its presence is a risk factor for adverse outcome. The outcome of patients with microbiologically proven intra-amniotic infection is similar to that of patients with intra-amniotic inflammation and a negative amniotic fluid culture. We propose that the treatment of patients in preterm labor be based on the operational diagnosis of intra-amniotic inflammation rather than the diagnosis of intra-amniotic infection because the latter diagnosis cannot be undertaken rapidly. (Am J Obstet Gynecol 2001;185:1130-6.)

Amniotic fluid lipopolysaccharide-binding protein and soluble CD14 as mediators of the inflammatory response in preterm labor

Objective: Our purpose was to determine the association of lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14) with the proinflammatory response among women in preterm labor. The binding of lipopolysaccharide (LPS) with LBP and sCD14 activates macrophages at LPS concentrations up to 1000 times lower than required with LPS alone. LBP and sCD14 in amniotic fluid could explain the high concentrations of cytokines present in the amniotic fluid of culture-positive women and the presence of cytokines in the amniotic fluid of culture-negative women. Study Design: A cohort of 169 afebrile women in preterm labor with intact membranes had amniotic fluid collected by transabdominal amniocentesis for culture and for LBP, sCD14, and interleukin 6 (IL-6) determinations by enzyme-linked immunosorbent assay. IL-6 levels of >2 ng/mL were considered elevated. Statistical analyses included analysis of variance, multiple comparisons with Bonferroni correction, and linear regression. Results: All 169 amniotic fluid samples had measurable LBP and sCD14. Subjects were categorized by amniotic fluid culture results and IL-6 concentration into 3 groups: (1) positive amniotic fluid culture, (2) negative amniotic fluid culture, elevated IL-6 concentration, and (3) negative amniotic fluid culture, low IL-6 level. Geometric mean LBP and sCD14 levels differed significantly among groups such that levels were approximately twice as high among pregnancies with positive amniotic fluid culture or elevated IL-6 compared with those without evidence of inflammation (both P <.001). sCD14 was inversely associated with enrollment gestational age independent of amniotic fluid culture results and IL-6 concentrations. Among culture negative, low IL-6 pregnancies, sCD14 decreased 3.5% for each additional week of gestation (95% confidence interval [CI], 0.01%-6.4%; P =.02). LBP levels showed a similar trend in this group (P =.09). One hundred eleven subjects had detectable IL-6 levels. Among these subjects, IL-6 increased by 2.1-fold for every 10-fold increase in LBP (95% CI, 1.1-4.0; P =.02) and by 28.4-fold for every 10-fold increase in sCD14 (95% CI, 10.4-77.4; P <.001) with adjustment for gestational age by linear regression. Conclusions: LBP and sCD14 are present in amniotic fluid of preterm pregnancies and are linearly associated with amniotic fluid IL-6 concentrations. These molecules may amplify the cytokine response and thereby help explain the presence of cytokines in amniotic fluid when culturable quantities of microbes are absent. (Am J Obstet Gynecol 2001;184:1241-8.)

Vaginal indicators of amniotic fluid infection in preterm labor

Objective: To determine whether vaginal interleukin-6, interleukin-8, neutrophils, bacterial vaginosis, and selected vaginal bacteria are predictors of amniotic fluid (AF) infection among women in preterm labor. Methods: One hundred ninety-seven afebrile women in preterm labor with intact membranes had vaginal and AF samples collected for Gram stain, culture, and interleukin-8 and interleukin-6 determinations. Vaginal interleukin-6, interleukin-8, neutrophils, and vaginal flora were compared in women with positive and negative AF cultures. The negative AF culture group was subdivided according to AF interleukin-6 concentration. Logistic regression was used to examine the associations between vaginal cytokines and flora and AF infection or elevated AF interleukin-6. Results: The vaginal interleukin-8 concentration and neutrophil count were significantly higher with both AF infection and elevated concentrations of AF interleukin-6 and interleukin-8. The vaginal interleukin-6 concentration was not associated with AF infection or high concentration of AF cytokines. Amniotic fluid infection was associated with bacterial vaginosis or intermediate vaginal flora by Gram stain, absence of hydrogen peroxide–producing Lactobacillus, and presence of vaginal Bacteroides ureolyticus and Fusobacterium. Vaginal interleukin-8 levels greater than 30 ng/mL had 80% sensitivity and a positive predictive value of 35%, and an abnormal vaginal Gram stain (more than five neutrophils per 400× field, bacterial vaginosis species, or intermediate flora) had 90% sensitivity and a positive predictive value of 27% to detect AF infection or elevated AF interleukin-6. Conclusion: A high vaginal interleukin-8 concentration, abnormal vaginal Gram stain, absent hydrogen peroxide–producing Lactobacillus, and anaerobic vaginal flora were strongly associated with AF infection among women in preterm labor.

Clinical implications of detection of Ureaplasma urealyticum in the amniotic cavity with the polymerase chain reaction

Objective: The objective of this study was to determine the frequency and clinical significance of the detection of Ureaplasma urealyticum by means of the polymerase chain reaction with specific primers in the amniotic fluid of patients with preterm premature rupture of membranes. Study Design: Amniocentesis was performed in 154 patients with preterm premature rupture of membranes. Amniotic fluid was cultured for aerobic and anaerobic bacteria and for mycoplasmas. Ureaplasma urealyticum was detected by means of the polymerase chain reaction with specific primers. Patients were divided into the following 3 groups according to the results of amniotic fluid culture and polymerase chain reaction for U urealyticum: those with a negative amniotic fluid culture and a negative polymerase chain reaction (n = 99), those with a negative amniotic fluid culture but a positive polymerase chain reaction (n = 18), and those with a positive amniotic fluid culture regardless of the results of the polymerase chain reaction (n = 37). Contingency table and survival techniques were used for analysis. Results: (1) U urealyticum was detected by polymerase chain reaction in 28% (43/154) of patients and by culture in 16% (25/154). (2) Among the 43 patients with a positive polymerase chain reaction for U urealyticum, amniotic fluid culture was negative in 42% (18/43). (3) Patients with a negative amniotic fluid culture for U urealyticum but a positive polymerase chain reaction had a significantly shorter median interval from amniocentesis to delivery and a higher amniotic fluid interleukin 6 and white blood cell count than did those with a negative amniotic fluid culture and a negative polymerase chain reaction (interval to delivery; median, 53 hours; range, 0.3-335 hours; vs median, 141 hours; range, 0.1-3552 hours; P <.05; amniotic fluid white blood cell count: median, 513 cells/mm3; range, 1-2295 cells/mm3; vs median, 1 cell/mm3; range, 0-7956 cells/mm3; amniotic fluid interleukin 6: median, 16.6 ng/mL; range, 0.3-53.0 ng/mL; vs median 0.4 ng/mL; range, 0-69.8 ng/mL; P <.0001 for all). (4) Patients with a positive polymerase chain reaction for U urealyticum but a negative amniotic fluid culture had a higher rate of significant neonatal morbidity than did those with both a negative culture and a negative polymerase chain reaction (P <.05). (5) No significant differences in perinatal outcome were observed between patients with a negative culture but a positive polymerase chain reaction and those with a positive amniotic fluid culture. Conclusion: (1) Culture techniques for mycoplasmas missed 40% of cases of microbial invasion of the amniotic cavity with U urealyticum. (2) Patients with a positive polymerase chain reaction but a negative amniotic fluid culture are at risk for adverse outcomes. (3) The use of molecular microbiologic techniques is likely to increase the detection of infection among patients with obstetric complications. (Am J Obstet Gynecol 2000;183:1130-7.)

Interleukin-6 Concentrations in Cervical Secretions in the Prediction of Intrauterine Infection in Preterm Premature Rupture of the Membranes

The objective of this study was to determine the value of interleukin-6 (IL-6) in cervical secretion to diagnose microbial invasion of the amniotic cavity in patients with premature rupture of the membranes. Cervical secretions were sampled immediately before amniocentesis in 124 patients with singleton pregnancies and preterm premature rupture of the membranes. Gestational age ranged between 24 and 32 weeks. Amniotic fluid was cultured and IL-6 measured in amniotic fluid and cervical secretions. A total of 33.8% (21/124) of the amniotic fluid cultures had positive results. In cervical secretions the median concentration of IL-6 was 672 pg/ml (range 5–1,250) in the presence of intra-amniotic infection in contrast to 95.5 pg/ml (range 12–640) in women with negative amniotic fluid culture (p ≤0.001). There were no differences between IL-6 concentrations in the cervical secretions of patients with or without obvious leakage of amniotic fluid. A significant relationship was found between IL-6 levels in amniotic fluid and in cervical secretions (ρ = 0.74, p ≤0.001). An IL-6 level in cervical secretions >200 pg/ml had a sensitivity of 78.5%, a specificity of 73.1% and a relative risk of 4.6 for intra-amniotic infection. Receiver-operator characteristics curve analysis showed that IL-6 assay in cervical secretions is comparable to IL-6 assay in amniotic fluid in predicting intra-amniotic infection (p = 0.468).In conclusion, intra-amniotic infection is associated with increased levels of IL-6 and concentrations in cervical secretions are related to amniotic levels. The measurement of IL-6 in cervical secretions may help to noninvasively identify intra-amniotic infection among pregnancies with preterm premature rupture of membranes.

Isolation of Ureaplasma urealyticum From the Amniotic Cavity and Adverse Outcome in Preterm Labor

Objective: To determine the relationship between the presence of Ureaplasma urealyticum in the amniotic cavity and adverse maternal and perinatal outcome in women with preterm labor. Methods: Amniocentesis was performed in 181 patients with preterm labor and intact membranes. Amniotic fluid (AF) was cultured for aerobic and anaerobic bacteria and mycoplasmas. Patients were divided into three groups according to the results of AF culture: those with negative AF cultures ( n = 160), those with positive AF cultures and in whom the only microbial isolate was U urealyticum ( n = 11), and those with positive cultures for non-ureaplasmas or mixed microorganisms ( n = 10). Survival techniques were used for analysis. Results: The prevalence of positive AF cultures in which the only microbial isolate was U urealyticum was 6.1% (11 of 181), and of positive cultures with non-ureaplasmas or mixed microorganisms was 5.5% (10 of 181). The amniocentesis-to-delivery interval was significantly shorter in patients with positive cultures limited to U urealyticum than in those with negative cultures (median 7 [range 0.1–149] hours versus median 264 [0.1–2659] hours, P < .001). Preterm delivery within 48 hours, 72 hours, and 7 days was more frequent in patients with U urealyticum in the AF than in those with sterile AF (48 hour: 91% versus 33%; 72 hour: 91% versus 36%; 7 days: 100% versus 45%, P < .001 for each). Patients with positive AF cultures limited to U urealyticum had a significantly higher rate of adverse perinatal outcome than those with negative culture. Adverse outcomes included low gestational age at birth, low birth weight, histologic chorioamnionitis, significant neonatal morbidity, and perinatal death. Conclusion: Microbial invasion of the amniotic cavity with U urealyticum is a risk factor for impending preterm delivery and adverse perinatal outcome.

Amniotic Fluid Complement C3 as a Marker of Intra-amniotic Infection

Objective: To determine the value of amniotic fluid (AF) complement C3 as a marker of intra-amniotic infection and to compare complement C3 with other rapid markers of intra-amniotic infection. Methods: One hundred four women with singleton gestations, in preterm labor with intact membranes, at 23–35 weeks’ gestation underwent transabdominal amniocentesis. Amniotic fluid was analyzed for white blood cell (WBC) count, lactate dehydrogenase (LDH), glucose, Gram stain, and complement C3. Cultures for aerobes, anaerobes, and mycoplasma species also were performed. The median values of complement C3, WBC, LDH, and glucose were compared between the culture-positive and -negative groups. Complement C3 was compared with WBC count, LDH, glucose, and Gram stain for sensitivity, specificity, positive and negative predictive values, and accuracy in the prediction of a positive AF culture. Descriptive statistics, receiver operating characteristic curve, Fisher exact test, and Wilcoxon rank-sum test were used for analysis. Results: The prevalence of positive cultures was 11.5% (12 of 104). The culture-positive group had a significantly higher median C3 (7.0 mg/dL) than the median C3 (3.0 mg/dL) of the culture-negative group ( P < .001). Also, the median values of WBC (1120.5 cells/mm 3) and LDH (2697 U/L) were significantly higher and the median glucose (6.5 mg/dL) was significantly lower among women with positive AF cultures than among women with negative AF cultures (WBC = 1 cell/mm 3; LDH = 165 U/L; glucose = 45 mg/dL; P < .001). Eleven of the 12 culture-positive cases had a C3 of 5 mg/dL or more, whereas four of the 92 culture-negative cases had a C3 of 5 mg/dL ( P < .001). Nine of the 12 culture-positive cases but none of the 92 culture-negative cases had a C3 of 6 mg/dL or more ( P < .001). The relative risks of a positive AF culture were 65.27 (95% confidence interval [CI] 9.08, 469.27) and 31.67 (95% CI 10.40, 96.43) times greater among women with AF complement C3 levels of 5 and 6 mg/dL or more, respectively. Depending on the cutoff point used, complement C3 had similar or higher sensitivity, specificity, positive predictive value, and negative predictive value for intra-amniotic infection when compared with WBC count, LDH, glucose and Gram stain. Conclusion: Amniotic fluid complement C3 has value in the diagnosis of intra-amniotic infection in preterm labor with intact membranes. Complement C3 is available readily and compares favorably with other rapid markers of AF infection. This study supports the general concept of fetal inflammatory response to microbial invasion of AF.

Prenatal diagnosis of fetal primary cytomegalovirus infection

To evaluate the validity of prenatal diagnosis work-up for congenital cytomegalovirus (CMV) in women with primary infection. Sixty-three pregnant women with primary cytomegalovirus disease (including two with twin pregnancies), referred to three tertiary perinatal centers over 4 years, underwent evaluation for congenital cytomegalovirus. Fetal diagnosis was made after 21 weeks' gestation by amniocentesis and fetal blood sampling (40 subjects), or amniocentesis only (23 subjects). Twenty-two (35%) pregnancies showed evidence of vertical transmission: 13 of them underwent funipuncture, but only ten (77%) of the 13 showed positive immunoglobulin (Ig)-M results in fetal blood. No cases of positive fetal serum Ig-M with negative amniotic fluid culture or polymerase chain reaction were observed. In nine (41%) of the 22 pregnancies with evidence of vertical transmission, abnormal ultrasonographic findings were recorded. Six (27%) women with evidence of vertical transmission continued their pregnancies and in only one (with prenatal ultrasonographic abnormalities) was an infant born with neurologic sequelae. In 41 (65%) pregnancies, no evidence of vertical transmission was found, and 37 continued to term. Only one newborn from this subgroup subsequently showed mild motor disability during a median of 23 months of follow-up. Among pregnant patients with primary CMV infection, analysis of amniotic fluid detected all of the infected fetuses. Thus, this is a reliable tool for counseling pregnant women with primary infection. This may guide the patient as to whether or not pregnancy can be continued with a high level of confidence.

The diagnostic value of interleukin-8 and fetal fibronectin concentrations in cervical secretions in patients with preterm labor and intact membranes.

The objectives of this study were 1) to evaluate interleukin-8 concentrations in cervical secretions in predicting preterm delivery, microbial invasion of the amniotic cavity and histologic chorioamnionitis in patients with preterm labor and intact membranes and 2) to compare the diagnostic value of interleukin-8 with fetal fibronectin determinations in predicting preterm delivery, microbial invasion of the amniotic cavity and histologic chorioamnionitis in patients with preterm labor and intact membranes. Interleukin-8 and fetal fibronectin were assayed in cervical secretions in 106 patients with singleton pregnancies and intact membranes admitted for preterm labor. Amniotic fluid obtained by amniocentesis was cultured and placentas (No = 43) analyzed for the presence of chorioamnionitis. The prevalence of pregnancies delivered preterm was 46.2% (49/106) and 15.09% (16/106) of amniotic fluid cultures were positive. Interleukin-8 levels in cervical secretions were significantly increased in patients who delivered preterm (p < or = 0.0001), in presence of positive amniotic fluid culture (p = 0.0016) and histological chorioamnionitis (p = 0.008) than in patients with negative findings. Receiver-operator characteristics curve analysis showed that an interleukin-8 value > 450 pg/ml is comparable to that of a fetal fibronectin value > 50 ng/ml in predicting preterm delivery (p = 0.247). Among patients who delivered preterm interleukin-8 concentrations > 860 pg/ml predicted a positive amniotic fluid culture with a sensitivity of 81.2% and a specificity 66.6%. Further, in patients who delivered preterm and had a negative amniotic fluid culture, IL-8 concentrations > 480 pg/ml predicted histological chorioamnionitis with a sensitivity 78.5% and specificity 61.5%. A positive fetal fibronectin > 50 ng/ml was not predictve of either a positive amniotic fluid culture or the presence of histological chorioamnionitis. In conclusion, increased concentrations of interleukin-8 and fetal fibronectin are associated with impending delivery and their diagnostic value seems comparable. However, interleukin-8 concentrations identify patients at risk of a positive amniotic fluid culture and the presence of histological chorioamnionitis. Measurement of interleukin-8 in cervical secretion is a non-invasive method to identify patients at risk for both preterm delivery and intrauterine infection.

Interleukin-6 concentrations in cervical secretions identify microbial invasion of the amniotic cavity in patients with preterm labor and intact membranes

OBJECTIVE: The objectives of this study were to determine whether cytokine levels in cervical secretions were increased in the presence of microbial invasion of the amniotic cavity in patients with preterm labor and intact membranes and to relate concentrations to cytokine levels in amniotic fluid, cervicovaginal microflora, and the presence of chorioamnionitis. STUDY DESIGN: Cervical secretions were sampled immediately before amniocentesis in 92 patients admitted for preterm labor with singleton pregnancies and intact membranes. Amniotic fluid was cultured and the following cytokines were measured in amniotic fluid and cervical secretions: interleukin-1β, interleukin-1 receptor antagonist, tumor necrosis factor-α, and interleukin-6. The cervicovaginal microflora and placentas (n = 42) were also analyzed. RESULTS: A total of 19.56% (18/92) of the amniotic fluid cultures had positive results. All the cytokines tested showed significantly higher levels in cervical secretions in the presence of intraamniotic infection. There were significant relationships between the concentrations of interleukin-6 and interleukin-1 receptor antagonist in amniotic fluid and cervical secretions. A concentration of interleukin-6 in cervical secretions >410 pg/ml had a sensitivity of 66.8% and a specificity of 90.5% and a relative risk of 7.7 for intraamniotic infection, higher than the other cytokines tested. There were no relationships between the presence of bacterial vaginosis and cervicovaginal pathogens and cervical cytokine levels. In the presence of chorioamnionitis, cervical concentrations of interleukin-6 and interleukin-1 receptor antagonist were significantly increased in spite of negative amniotic fluid culture results. CONCLUSION: The measurement of interleukin-6 in cervical secretions may help to noninvasively identify intraamniotic infection among pregnancies with preterm labor and intact membranes. (Am J Obstet Gynecol 1996;175:812-7.)

Application of a new perchloric acid treatment method to measure endotoxin in both amniotic fluid and cord blood by an endotoxin-specific chromogenic Limulus test in intra-amniotic infection.

Endotoxin in both amniotic fluid and cord blood was measured to detect intra-amniotic fetal infection. Both amniotic fluid and cord blood plasma were pretreated by a perchloric acid treatment, and the endotoxin level was measured by Endospecy test. Cut off values for endotoxin in amniotic fluid and cord blood were 8.5 pg/mL and 7.6 pg/mL, respectively. Escherichia coli intra-amniotic infection caused respiratory distress syndrome (RDS)-mimicking pneumonia. Abnormally high values of endotoxin in both amniotic fluid and cord blood were detected. Intra-amniotic infection caused by Gram-positive bacteria (group B streptococci, Enterococcus fecalis) was shown to be endotoxin negative in both amniotic fluid and cord blood. In cases of negative amniotic fluid culture, measurement of the value of endotoxin in the amniotic fluid is useful in identifying intra-amniotic fetal infection.

Fetal haematological response to intra-uterine infection in preterm prelabour amniorrhexis.

The value of fetal haematological indices in the prediction of intra-uterine infection in 91 cases of preterm prelabour amniorrhexis was examined. Cordocentesis and amniocentesis were performed for the diagnosis of intra-uterine infection. The patients were subsequently divided into three groups, depending on the results of fetal blood and amniotic fluid cultures. In group 1 there were 53 patients with negative fetal blood and amniotic fluid cultures, group 2 consisted of 22 patients with negative fetal blood, but positive amniotic fluid cultures, and in group 3 there were 16 patients with positive fetal blood cultures. The mean leucocyte and neutrophil counts in all three groups were significantly higher than normal, and in group 3 the values were significantly higher than in group 1. The leucocyte and neutrophil counts were above the 95th centile of the normal range in 58% (22 cases) and 66% (25 cases), respectively, of the 38 cases with positive fetal blood and/or amniotic fluid cultures and in only 15% (8 cases) and 13% (7 cases), respectively, of the 53 patients with no infection. There were no significant differences between the groups, between the patients with amniorrhexis or for normal haemoglobin concentration, platelet count, or lymphocyte count. In the majority of the cases with positive fetal blood and/or amniotic fluid cultures, there is fetal leucocytosis. Since the results of the fetal leucocyte and neutrophil counts are available within a few minutes after cordocentesis, it would be reasonable to give antibiotics to all patients with a fetal leucocyte count above the 95th centile.

Amniotic fluid interleukin-6 determinations are of diagnostic and prognostic value in preterm labor.

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