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Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Šket, Ramuta, Starčič Erjavec and Kreft.)

Objective: Necrotizing funisitis (NF) is defined as the presence of an arc (i.e., crescent/band/ring/halos) of infiltrated neutrophils and/or associated debris in Wharton's jelly (WJ) of umbilical-cord (UC). However, no information exists about the comparison in intra-amniotic inflammatory-response (IAIR) and inflammation in extra-placental membranes between the presence and absence of NF in the context of inflammation in WJ among spontaneous preterm births (PTBs). The objective of current study is to examine this issue., Materials and Methods: We examined IAIR and the frequency of amnionitis according to the progression of inflammation in UC (i.e. stage-1, umbilical phlebitis [inflammation in umbilical-vein(UV)] only; stage-2, involvement of at least one umbilical-artery[UA] and either the other UA or UV without extension into WJ; stage-3, the extension of inflammation into WJ without NF; stage-4, the extension of inflammation into WJ with NF) in 120singleton spontaneous PTBs (<37weeks). IAIR was gauged by AF MMP-8 (ng/ml) within 3days before birth., Results: 1) Stage-1, stage-2, stage-3, and stage-4 were present in 20%(24/120), 6%(7/120), 61%(73/120), and 13%(16/120) of cases respectively; 2) AF MMP-8 continuously increased (stage-1 vs. stage-2 vs. stage-3 vs. stage-4; median[ng/ml], range[ng/ml]; 207.2[16.8-1196.5] vs. 444.1[8.5-2608.0] vs. 458.8[0.4-3116.7] vs. 1859.7[912.3-5304.8]; Spearman's rank correlation-test, α = 0.454, P = 0.006), and the frequency of increased AF MMP-8 (≥854.1 ng/ml) elevated (stage-1 vs. stage-2 vs. stage-3 vs. stage-4; 13%[1/8] vs. 33%[1/3] vs. 32%[6/19] vs. 100%[5/5]; Linear-by-linear-association, P = 0.012) with the progression of inflammation in UC; 3) Moreover, there was a stepwise increase in the frequency of amnionitis according to the progression of inflammation in UC (stage-1, 33%[8/24]; stage-2, 43%[3/7]; stage-3, 62%[45/73]; stage-4, 81%[13/16]; Linear-by-linear-association, P = 0.001)., Conclusion: NF is an indicator that IAIR is more severe and amnionitis is more frequent in the context of the extension of inflammation into WJ. Therefore, current study confirms that NF is the most advanced stage in the progression of inflammation within UC., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2021. Published by Elsevier B.V.)

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.

Objective: Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process., Methods: Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining., Results: (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection., Conclusion: NETs are detectable in the amniotic fluid of women with intra-amniotic infection.

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.

Objective: To evaluate clinical outcomes of women with singleton pregnancies that underwent intra-amniotic dye instillation (amniodye test) following equivocal diagnosis of prelabor rupture of membranes (PROM)., Method: Records of 34 pregnant women who underwent amniodye test for equivocal PROM were reviewed. Comparisons of characteristics, amniotic fluid (AF) cultures, AF interleukin (IL)-6 concentrations, and placenta pathology results between women who tested positive and those who tested negative were performed. A sub-analysis of women who were amniodye test-negative was also performed., Results: (1) Commonest indication for amniodye test was a typical history of PROM with positive conventional tests and persistently normal AF volume, (2) amniodye test-positive women had a shorter procedure-to-delivery interval (p = 0.008), and a greater proportion of histologic acute chorioamnionitis (p = 0.04) and funisitis (p = 0.01) than amniodye-negative women, and (3) in addition to similarities to women with amniodye-positive test, amniodye test-negative women who delivered <34 weeks, had a greater proportion of women with risk for preterm birth (p = 0.04), than their counterparts who delivered between 34 0/7 and 36 6/7 weeks., Conclusion: Equivocal diagnosis of PPROM should warrant an amniodye test to avoid iatrogenic intervention in women with intact amniotic membranes. AF analysis should be performed in amniodye test-negative women.

Objective: The purpose of this study was to investigate whether amniotic fluid (AF) CA-125 in patients with preterm labor or preterm premature rupture of membranes can help predict intra-amniotic inflammation (IAI), microbial invasion of the amniotic cavity (MIAC) and imminent delivery., Methods: We recruited 36 women who admitted with impending preterm delivery and suspicious AF infection. AF matrix metalloproteinase-8 (MMP-8), white blood cell (WBC) count, glucose levels, and CA-125 levels were measured, and the MMP-8 bedside rapid test was also performed. AF culture and PCR were subsequently performed to confirm MIAC. We compared AF CA-125 levels according to the presence of IAI or MIAC and assessed its predictive value for delivery within 7 days of admission., Results: AF CA-125 levels were significantly higher in the IAI group than in the non-IAI group (mean ± standard deviation: 5608 ± 864 vs 904 ± 84 IU/ml; p = 0.001). AF CA-125 levels showed a negative correlation with gestational age and a positive correlation with AF WBC counts and MMP-8 levels. AF CA-125 levels were higher in the MIAC group, though this difference was not statistically significant (p = 0.064). Delivery within 7 days of admission was significantly more common in patients with higher AF CA-125 levels (cut-off: 1650 IU/ml, sensitivity: 71.4 %, specificity: 86.4 %, p = 0.005)., Conclusion: AF CA-125 levels are increased in patients with AF inflammation and can be a predictor of imminent preterm delivery.

Amniotic fluid sludge (AFS) is defined as the presence of particulate matter in the amniotic fluid in close proximity to the cervix. Although its prevalence is known to correlate with the risk of preterm delivery, initial reports describe a strong association between AFS and microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis. However, AFS is also present in uncomplicated pregnancies, and its prevalence appears to increase with gestational age. Recent evidence debates the usefulness of AFS as a marker of early preterm delivery risk. We present four cases with AFS diagnosed by transvaginal ultrasound at admission for cervical insufficiency between 20 and 24 weeks of gestation, with confirmed lower genital tract and intra-amniotic infections by amniocentesis and histological chorioamnionitis and funisitis. Our findings reinforce the presence of AFS as a useful marker of MIAC, chorioamnionitis and funisitis that increase the likelihood of preterm delivery at an extreme gestational age.

Objective: Microbial invasion of the amniotic cavity is associated with spontaneous preterm labor and adverse pregnancy outcome, and Mycoplasma hominis often is present. However, the pathogenic process by which M hominis invades the amniotic cavity and gestational tissues, often resulting in chorioamnionitis and preterm birth, remains unknown. We hypothesized that strains of M hominis vary genetically with regards to their potential to invade and colonize the amniotic cavity and placenta., Study Design: We sequenced the entire genomes of 2 amniotic fluid isolates and a placental isolate of M hominis from pregnancies that resulted in preterm births and compared them with the previously sequenced genome of the type strain PG21. We identified genes that were specific to the amniotic fluid/placental isolates. We then determined the microbial burden and the presence of these genes in another set of subjects from whom samples of amniotic fluid had been collected and were positive for M hominis., Results: We identified 2 genes that encode surface-located membrane proteins (Lmp1 and Lmp-like) in the sequenced amniotic fluid/placental isolates that were truncated severely in PG21. We also identified, for the first time, a microbial gene of unknown function that is referred to in this study as gene of interest C that was associated significantly with bacterial burden in amniotic fluid and the risk of preterm delivery in patients with preterm labor., Conclusion: A gene in M hominis was identified that is associated significantly with colonization and/or infection of the upper reproductive tract during pregnancy and with preterm birth., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Intra-amniotic infection/inflammation are major causes of spontaneous preterm labor and delivery. However, diagnosis of intra-amniotic infection is challenging because most are subclinical and amniotic fluid (AF) cultures take several days before results are available. Several tests have been proposed for the rapid diagnosis of microbial invasion of the amniotic cavity (MIAC) or intra-amniotic inflammation. The aim of this study was to examine the diagnostic performance of the AF Mass Restricted (MR) score in comparison with interleukin-6 (IL-6) and matrix metalloproteinase-8 (MMP-8) for the identification of MIAC or inflammation., Methods: AF samples were collected from patients with singleton gestations and symptoms of preterm labor (n = 100). Intra-amniotic inflammation was defined as >100 white blood cells/mm(3) (WBCs) in AF; MIAC was defined as a positive AF culture. AF IL-6 and MMP-8 were determined using ELISA. The MR score was obtained using the Surface-Enhanced Laser Desorption Ionization Time of Flight (SELDI-TOF) mass spectrometry. Sensitivity and specificity were calculated and logistic regression models were fit to construct receiver-operating characteristic (ROC) curves for the identification of each outcome. The McNemar's test and paired sample non-parametric statistical techniques were used to test for differences in diagnostic performance metrics., Results: (1) The prevalence of MIAC and intra-amniotic inflammation was 34% (34/100) and 40% (40/100), respectively; (2) there were no significant differences in sensitivity of the three tests under study (MR score, IL-6 or MMP-8) in the identification of either MIAC or intra-amniotic inflammation (using the following cutoffs: MR score >2, IL-6 >11.4 ng/mL, and MMP-8 >23 ng/mL); (3) there was no significant difference in the sensitivity among the three tests for the same outcomes when the false positive rate was fixed at 15%; (4) the specificity for IL-6 was not significantly different from that of the MR score in identifying either MIAC or intra-amniotic inflammation when using previously reported thresholds; and (5) there were no significant differences in the area under the ROC curve when comparing the MR score, IL-6 or MMP-8 in the identification of these outcomes., Conclusions: IL-6 and the MR score have equivalent diagnostic performance in the identification of MIAC or intra-amniotic inflammation. Selection from among these three tests (MR score, IL-6 and MMP-8) for diagnostic purposes should be based on factors such as availability, reproducibility, and cost. The MR score requires a protein chip and a SELDI-TOF instrument which are not widely available or considered "state of the art". In contrast, immunoassays for IL-6 can be performed in the majority of clinical laboratories.

Problem: The diagnosis of microbial invasion of the amniotic cavity (MIAC) has been traditionally performed using traditional cultivation techniques, which require growth of microorganisms in the laboratory. Shortcomings of culture methods include the time required (days) for identification of microorganisms, and that many microbes involved in the genesis of human diseases are difficult to culture. A novel technique combines broad-range real-time polymerase chain reaction with electrospray ionization time-of-flight mass spectrometry (PCR/ESI-MS) to identify and quantify genomic material from bacteria and viruses., Method of Study: AF samples obtained by transabdominal amniocentesis from 142 women with preterm labor and intact membranes (PTL) were analyzed using cultivation techniques (aerobic, anaerobic, and genital mycoplasmas) as well as PCR/ESI-MS. The prevalence and relative magnitude of intra-amniotic inflammation [AF interleukin 6 (IL-6) concentration ≥ 2.6 ng/mL], acute histologic chorioamnionitis, spontaneous preterm delivery, and perinatal mortality were examined., Results: (i) The prevalence of MIAC in patients with PTL was 7% using standard cultivation techniques and 12% using PCR/ESI-MS; (ii) seven of ten patients with positive AF culture also had positive PCR/ESI-MS [≥17 genome equivalents per PCR reaction well (GE/well)]; (iii) patients with positive PCR/ESI-MS (≥17 GE/well) and negative AF cultures had significantly higher rates of intra-amniotic inflammation and acute histologic chorioamnionitis, a shorter interval to delivery [median (interquartile range-IQR)], and offspring at higher risk of perinatal mortality, than women with both tests negative [90% (9/10) versus 32% (39/122) OR: 5.6; 95% CI: 1.4-22; (P < 0.001); 70% (7/10) versus 35% (39/112); (P = 0.04); 1 (IQR: <1-2) days versus 25 (IQR: 5-51) days; (P = 0.002), respectively]; (iv) there were no significant differences in these outcomes between patients with positive PCR/ESI-MS (≥17 GE/well) who had negative AF cultures and those with positive AF cultures; and (v) PCR/ESI-MS detected genomic material from viruses in two patients (1.4%)., Conclusion: (i) Rapid diagnosis of intra-amniotic infection is possible using PCR/ESI-MS; (ii) the combined use of biomarkers of inflammation and PCR/ESI-MS allows for the identification of specific bacteria and viruses in women with preterm labor and intra-amniotic infection; and (iii) this approach may allow for administration of timely and specific interventions to reduce morbidity attributed to infection-induced preterm birth., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Objective: This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology., Methods: A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology., Results: The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1., Conclusions: The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.

Introduction: Intrauterine infection is a global problem and a significant contributor to morbidity and perinatal death. The host response to infection causes an inflammatory state that acts synergistically with microbial insult to induce preterm birth and fetal damage. Prompt and accurate diagnosis of intra-amniotic infection in the asymptomatic stage of the disease is critical for improved maternal and neonatal outcomes., Areas Covered: This article provides an overview of the most recent progress, challenges, and opportunities for discovery and clinical implementation of various maternal serum, cervicovaginal, and amniotic fluid biomarkers in pregnancies complicated by intra-amniotic infection., Expert Opinion: Clinically relevant biomarkers are critical to the accurate diagnostic of intrauterine infection. Front-end implementation of such biomarkers will also translate in lower incidence of early-onset neonatal sepsis (EONS) which is an important determinant of neonatal morbidity and mortality associated with prematurity. However, of the hundreds of differentially expressed proteins, only few may have clinical utility and thus function as biomarkers. The small number of validation studies along with barriers to implementation of technological innovations in the clinical setting are current limitations.

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×10⁴ CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32-170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.

Objective: A previous study has demonstrated that term labor is associated with an increased risk of microbial invasion of the amniotic cavity, intra-amniotic inflammation and histologic chorioamnionitis. This study was performed to determine when the risk of intra-amniotic infection, inflammation and histologic chorioamnionitis begins to increase during the course of labor., Study Design: Amniotic fluid (AF) was obtained from 926 term singleton pregnant women with intact membranes during cesarean section. AF was cultured for aerobic, anaerobic bacteria and genital mycoplasmas. An AF white blood cell (WBC) count was determined. Patients were divided into five groups according to the absence or presence of regular uterine contractions and the degree of cervical dilatation. Intra-amniotic inflammation was defined as an AF WBC ≥ 19/mm(3). Histologic chorioamnionitis was defined as the presence of acute inflammatory changes in the extra-placental membranes or the chorionic plate of the placenta, and funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton's jelly., Results: (1) The more advanced the cervical dilatation, the greater the risk of intra-amniotic infection and/or inflammation and histologic chorioamnionitis; (2) The risk of intra-amniotic infection and/or inflammation increased after the cervix began to dilate (7.8% in group 1 [without regular uterine contractions] vs. 18.3% in group 2 [regular uterine contractions and cervical dilation ≤1 cm], p < 0.01); (3) however, histologic chorioamnionitis was significantly more common in women in group 3 than in group 2 (4.2% in group 2 [regular uterine contractions and cervical dilatation ≤1 cm] vs. 23.1% in group 3 [regular uterine contractions and a cervical dilatation of 2-3 cm], p < 0.05)., Conclusions: The risk of intra-amniotic infection and/or inflammation increases after the cervix begins to dilate and that of histologic chorioamnionitis increases after the cervix dilates 2 cm in term pregnant women with regular uterine contractions with intact membranes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: Microbial invasion of the amniotic cavity (MIAC) has been detected in women with preterm labor, preterm prelabor rupture of membranes (PROM), and in patients at term with PROM or in spontaneous labor. Intrauterine infection is recognized as a potential cause of fetal growth restriction; yet, the frequency of MIAC in pregnancies with small-for-gestational-age (SGA) fetuses is unknown. The aim of this study was to determine the frequency, diversity and relative abundance of microbes in amniotic fluid (AF) of women with an SGA neonate using a combination of culture and molecular methods., Method: AF from 52 subjects with an SGA neonate was analyzed with both cultivation and molecular methods in a retrospective cohort study. Broad-range and group-specific PCR assays targeted small subunit rDNA, or other gene sequences, from bacteria, fungi and archaea. Results of microbiologic studies were correlated with indices of the host inflammatory response., Results: 1) All AF samples (n=52) were negative for microorganisms based on cultivation techniques, whereas 6% (3/52) were positive based on PCR; and 2) intra-amniotic inflammation was detected in one of the three patients with a positive PCR result, as compared with three patients (6.1%) of the 49 with both a negative culture and a negative PCR (P=0.2)., Conclusion: MIAC is detected by PCR in some patients with an SGA fetus who were not in labor at the time of AF collection.

Objective: Infection has been implicated in the pathogenesis of preeclampsia, yet the association between microbial invasion of the amniotic cavity (MIAC) and preeclampsia has not been determined. The aim of this study was to determine the prevalence, and microbial diversity associated with MIAC, as well as the nature of the host response to MIAC in patients with preeclampsia., Method of Study: Amniotic fluid (AF) from 62 subjects with preeclampsia, not in labor, was analyzed with both cultivation and molecular methods. Broad-range and group-specific PCR assays targeting small subunit ribosomal DNA, or other gene sequences, from bacteria, fungi and archaea were used. Results were correlated with measurements of host inflammatory response, including AF white blood cell count and AF concentrations of glucose, interleukin-6 (IL-6) and MMP-8., Results: 1) The rate of MIAC in preeclampsia was 1.6% (1/62) based on cultivation techniques, 8% (5/62) based on PCR, and 9.6% (6/62) based on the combined results of both methods; 2) among the six patients diagnosed with MIAC, three had a positive PCR for Sneathia/Leptotrichia spp.; and 3) patients with MIAC were more likely to have evidence of an inflammatory response in the amniotic cavity than those without MIAC, as determined by a higher median AF IL-6 [1.65 ng/mL interquartile range (IQR): 0.35-4.62 vs. 0.22 ng/mL IQR: 0.12-0.51; P=0.002)., Conclusion: The prevalence of MIAC in preeclampsia is low, suggesting that intra-amniotic infection plays only a limited role in preeclampsia. However, the unexpectedly high number of positive AF specimens for Sneathia/Leptotrichia warrants further investigation.

Acute chorioamnionitis is a response to amniotic fluid (AF) infection. However, it remains unclear whether substantial bacterial propagation in the chorioamniotic membranes (CAMs) precedes microbial invasion of the amniotic cavity (MIAC), which is inconsistent with characteristic 'amniotropic neutrophil migration' in acute chorioamnionitis. This study was performed to determine whether CAMs have widespread bacterial infection during MIAC and whether bacteria normally colonize CAMs. AF pellets and CAMs from the following groups were studied: group 1, patients with positive (n=18) or negative (n=22) AF cultures; group 2, patients with or without acute chorioamnionitis in which the amnion and chorion were studied separately (n=60); and group 3, patients at term who underwent a cesarean delivery (n=30). SYTO 9/propidium iodide fluorescent staining and fluorescent in situ hybridization for 16S rRNA were performed. Real-time quantitative PCR for 16S rDNA and PCR for genital mycoplasmas were also conducted. Bacteria were more frequently detected in AF than in CAMs of patients with positive AF culture (100 vs. 33%; P<0.0001). Bacteria were detected more frequently in CAMs as the severity of chorioamnionitis increased (P<0.01). The median 16S rRNA gene copy number in the amnion was significantly greater than in the chorion (group 2; P<0.0001). Bacteria were not detected in CAMs or AF in women at term before labor (group 3). A fraction of patients with chorioamnionitis or MIAC did not have bacteria in CAMs. Collectively, the findings herein indicate that MIAC does not follow widespread infection of CAMs, but precedes it. We propose a model of MIAC: the initial stage is intra-amniotic bacterial invasion through a discrete region of the CAMs, followed by intra-amniotic proliferation, and bacterial invasion of CAMs primarily extends from the amniotic fluid. This study emphasizes the importance of assessing the intra-amniotic compartment for diagnosis and treatment of preterm birth.

The paper aims at analyzing the histopathological modifications induced by the amniotic infection on present fetal membranes and in the absence of clinical signs of chorioamnionitis. Such an evaluation is important in the context of postpartum fetomaternal complications. The objective was to determine the relation between the microbial invasion in the amniotic cavity and the severity of inflammatory lesions of the placenta, of the membranes and the umbilical chord.

Objective: Amnionitis (inflammation of the amnion) is the final stage of extra-placental chorioamniotic inflammation. We propose that patients with "amnionitis", rather than "chorionitis" have a more advanced form of intra-uterine inflammation/infection and, thus, would have a more intense fetal and intra-amniotic inflammatory response than those without "amnionitis"., Study Design: The relationship between the presence of amnionitis, and a fetal and an intra-amniotic inflammatory response was examined in 290 singleton preterm births (

Objective: The amniotic cavity is normally sterile for bacteria. However, experimental evidence indicates that regular uterine contractions exert a suction-like effect whereby vaginal fluid ascends into the uterine cavity with contractions (demonstrated by sonohysterography contrast media). Consequently, this study was conducted to determine whether the presence and progress of labor are associated with an increased risk of microbial invasion of the amniotic cavity (MIAC), intraamniotic inflammation, and histologic chorioamnionitis in women with term pregnancies with intact membranes., Study Design: Amniotic fluid (AF) was obtained from term singleton pregnant women with intact membranes at the time of cesarean delivery. AF was cultured for aerobic and anaerobic bacteria and genital mycoplasmas, and white blood cell (WBC) count was determined. Patients were divided into 3 groups according to the presence or absence of labor and the progress of labor. Nonparametric statistics were used for analysis., Results: Results included: (1) a total of 884 pregnant women were enrolled and divided into 3 groups: group 1, not in labor (n = 775); group 2, in early labor (cervical dilatation less than 4 cm) (n = 86); and group 3, in active labor (cervical dilatation 4 cm or greater) (n = 23); (2) the frequency of MIAC was 1% (6 of 775) in women not in labor, 3.5% (3 of 86) in patients with early labor, and 13% (3 of 23) in patients with active labor; and (3) the median AF WBC count and the frequency of histologic chorioamnionitis were also higher in the presence of labor than in the absence of labor., Conclusion: We came to the following conclusions: (1) labor is associated with an increased risk of MIAC, a higher median AF WBC count, and histologic chorioamnionitis in term pregnancy with intact membranes; (2) the more advanced the cervical dilatation, the greater the risk of MIAC, a higher median AF WBC count, and histologic chorioamnionitis; and (3) in contrast, fetal inflammation (funisitis) did not increase with the presence of labor or as a function of cervical dilatation. We propose that labor predisposes to MIAC, a higher median AF WBC count, and histologic chorioamnionitis.

Objective: Pro-inflammatory chemokines have been associated with preterm parturition. Monocyte chemotactic protein-1 (MCP-1) is a chemokine capable of recruiting monocytes/macrophages into sites of inflammation, as well as stimulating the respiratory burst required for macrophage activation. MCP-1 transcripts and immuno-reactivity are expressed by uterine tissues (i.e., decidual cells and myometrium) and, thus, may participate in the process of labor. This study was conducted to determine if preterm labor leading to preterm delivery and intra-amniotic infection (IAI) are associated with changes in the amniotic fluid concentrations of MCP-1., Study Design: A cross-sectional study was designed to examine the amniotic fluid concentrations of MCP-1 in women in two groups: 1) those presenting with preterm labor with intact membranes; and 2) those with preterm prelabor rupture of membranes (PROM). Amniotic fluid was obtained by transabdominal amniocentesis from 131 women in preterm labor with intact membranes and 105 women with preterm PROM. IAI was defined by a positive amniotic fluid culture for microorganisms. Group 1 included women with preterm labor and intact membranes (n=131), and was subdivided into the following groups: a) delivery at term in the absence of IAI (n=42); b) preterm delivery (< 37 weeks) in the absence of IAI (n=58); and c) preterm delivery in the presence of IAI (n=31). Group 2 consisted of women with PROM (n=105), and was subdivided into women with (n=51) and without (n=54) IAI. Non-parametric statistics were used for data analysis., Results: 1) Immuno-reactive MCP-1 was detected in all amniotic fluid samples; 2) IAI, regardless of the membrane status, was associated with a significantly higher median amniotic fluid concentration of immuno-reactive MCP-1 than those without IAI (p<0.001 for both comparisons); 3) women in preterm labor who delivered preterm without IAI had a significantly higher median amniotic fluid concentration of immuno-reactive MCP-1 than those who delivered at term (p<0.001); 4) histological chorioamnionitis was associated with increased amniotic fluid concentrations of immuno-reactive MCP-1; and 5) a significant relationship existed between the amniotic fluid concentrations of immuno-reactive MCP-1 and the interval from amniocentesis to delivery., Conclusions: Amniotic fluid concentrations of immuno-reactive MCP-1 were increased in women in preterm labor with IAI, those without IAI who delivered preterm, and those who displayed acute inflammatory lesions in the extra-placental membranes. These findings suggest that MCP-1 may play a role in preterm labor regardless of the presence of IAI.

Objective: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are expressed in many inflammatory conditions and contribute to connective tissue breakdown. Stromelysin 1 [matrix metalloproteinase 3 (MMP-3)], a novel member of this family, is produced in the context of infection and is able to activate the latent forms of other MMPs. The purpose of this study was to determine if parturition (either term or preterm), premature rupture of the membranes (PROM), and microbial invasion of the amniotic cavity are associated with changes in amniotic fluid concentrations of MMP-3., Study Design: A cross-sectional study was conducted, which included women who underwent transabdominal amniocentesis (n = 365) in the following categories: (1) mid-trimester with a subsequent normal pregnancy outcome (n = 84) and a subsequent fetal loss (n = 10); (2) preterm labor with intact membranes without microbial invasion of the amniotic cavity who delivered at term (n = 36), or prematurely (n = 50), and preterm labor with microbial invasion of the amniotic cavity (n = 25); (3) preterm PROM with (n = 25) and without (n = 26) microbial invasion of the amniotic cavity; (4) term with intact membranes in the absence of microbial invasion of the amniotic cavity, in labor (n = 52) and not in labor (n = 31); and (5) term with PROM in the absence of microbial invasion of the amniotic cavity and not in labor (n = 26). MMP-3 concentrations in amniotic fluid were measured by a sensitive and specific immunoassay that was validated for amniotic fluid. MMP-3 concentrations were normalized using logarithmic transformation for statistical analysis. Parametric statistics were used and a p value < 0.05 was considered statistically significant., Results: (1) MMP-3 was detected in 99.5% (363/365) of amniotic fluid samples, and its concentration did not change with advancing gestational age. (2) Spontaneous parturition at term and preterm was associated with a significant increase in amniotic fluid MMP-3 concentrations (p = 0.04 and p = 0.002, respectively). (3) Spontaneous rupture of membranes in term and preterm gestations was not associated with significant changes in amniotic fluid MMP-3 concentrations. (4) Intra-amniotic infection was associated with a significant increase in amniotic fluid MMP-3 concentrations in both women with preterm labor and intact membranes (p = 0.03), and women with preterm PROM (p = 0.02). (5) Subsequent fetal loss after genetic amniocentesis was not associated with significant changes in mid-trimester concentrations of amniotic fluid MMP-3., Conclusions: (1) MMP-3 is a physiologic constituent of amniotic fluid. (2) MMP-3 may play a role in the mechanisms of human parturition and in the regulation of the host response to intrauterine infection.

Intraamniotic infections caused by viruses, bacteria or mycoplasmas are frequently followed by damage of fetus or increased perinatal morbidity and mortality. Cytokines are key substances regulating a number of biological processes including reproductive and inflammatory processes. An association between intraamniotic infections, rising concentrations of inflammatory cytokines in amniotic fluid and preterm labor is suggested. A great effort is made to find reliable markers typical for intraamniotic infections with high predictive value that make possible prompt identification of patients with intraamniotic infection. This review concerns inflammatory mediators, especially IL-1, IL-6, IL-8, TNF-alpha, and other important biologically active substances as prostaglandins and NO metabolites and their roles in intraamniotic infections. Finally, we discuss their relevance for diagnosis of intraamniotic infections.

Objective: To evaluate the correlation of amniotic fluid (AF) markers (AFMs) of intra-amniotic infection with histopathologic findings in cases of preterm labor with intact membranes, between 22 and 36 weeks' gestation., Study Design: We reviewed the charts of patients admitted in preterm labor with intact membranes between January 1993 and December 1996. Those having amniocentesis were identified, and AFMs were compared with histopathology in patients who delivered within 48 hours of the amniocentesis. The AFMs evaluated were glucose, polymorphonuclear leukocytes, Gram stain, and culture. All placentae were reviewed by a single pathologist blinded to the AF findings. Histologic evidence of acute inflammation was defined by findings of both subchorial intervillositis and marginating choriodeciduitis. The sensitivities, specificities, and positive and negative predictive values of the various AFMs were calculated., Results: Of 556 women with intact membranes presenting in preterm labor, 181 (32.6%) had amniocentesis and 88 delivered within 48 hours of the amniocentesis. Histopathologic chorioamnionitis was seen in 53 patients (60.2%). The findings (with their sensitivity, specificity, and positive and negative predictive values) were: polymorphonuclear leukocytes at > 10/high-power field (22.6%, 97.2%, 92.3%, and 46.1%), positive Gram stain (26.4%, 94.6%, 87.5%, and 47.3%), culture (28.3%, 92.1%, 83.3%, and 47.9%), and glucose of < 15 mg/dl (28.3%, 94.6%, 88.2%, and 47.9%), respectively. Using a receiver-operator characteristic curve for different level of AF glucose, a glucose level of < 20 mg/dl was the most sensitive AF predictor of histologic chorioamnionitis., Conclusion: Histopathologic evidence of chorioamnionitis was present in 60.2% of cases of preterm births due to preterm labor in women who at our institution were offered and accepted amniocentesis and subsequently delivered within 48 hours. AFMs may be useful predictors of histologic chorioamnionitis. The most efficient AFM for chorioamnionitis in this group of patients was glucose at < 20 mg/dl.

Objective: The aim of the study was to compare the diagnostic utility of the Gram stain, the amniotic fluid glucose level, and the ratio of amniotic fluid glucose level to serum glucose level in detecting intra-amniotic infection., Study Design: We conducted a prospective study of 127 patients with preterm labor and 26 patients with preterm premature rupture of the membranes (153 total). All patients underwent amniocentesis to diagnose intra-amniotic infection. The diagnostic criterion for intra-amniotic infection was a positive amniotic fluid culture result., Results: The Gram stain is 80% sensitive and 91% specific when a positive is considered the presence of white blood cells or bacteria. Amniotic fluid glucose level and the ratio of amniotic fluid glucose level to serum glucose level are significantly lower when amniotic fluid culture results are positive, but as diagnostic tests they are inferior to the Gram stain. Logistic regression models that combine predictors yield superior accuracy with respect to individual tests. The most accurate combination was amniotic fluid glucose level and Gram stain with white blood cells or bacteria. Although the number of patients with preterm premature rupture of the membranes was small in this study (n = 26), analysis of our data suggests that the diagnostic performance levels of these tests were similar when used in patients with preterm labor and intact membranes and in patients with premature rupture of the membranes., Conclusions: The amniotic fluid glucose level and the ratio of amniotic fluid to serum glucose level have equivalent diagnostic utility and are inferior to the Gram stain. The combination of Gram stain with amniotic fluid glucose level is superior to any individual test.

Bacterial vaginosis is the most common lower genital tract infection encountered among women of reproductive age. This condition can best be considered as a vaginal syndrome associated with an alteration of the normal vaginal flora rather than an infection specific to any one microorganism. Bacterial vaginosis is a clinical condition with a complex microbiology that is characterized by a reduced concentration of a normally abundant Lactobacillus species along with high concentrations of gram-negative and anaerobic bacteria, particularly, Gardnerella vaginalis and Mobiluncus, Bacteroides, Prevotella, and Mycoplasma species. The exact make up of the microorganisms and their relative concentration vary among women who have this condition. Although it was previously regarded as a harmless condition, recent work has linked bacterial vaginosis to numerous upper genital tract complications such as preterm labor and preterm delivery, preterm premature rupture of the membranes, chorioamnionitis, and postpartum endometritis. The findings from recent prospective randomized trials suggest that treatment of bacterial vaginosis in certain women who are at high risk for preterm delivery decreases the rate of preterm birth.

Lactoferrin (Lf) has been found in most biological fluids including amniotic fluid and cervical mucoids in pregnant women, and released from neutrophils in response to the inflammation. As Lf possesses antimicrobial properties, it is widely considered to be an important component of the host defence against microbial infections. It is known that premature labor is caused by amniotic infection with the increase of prostaglandin production. High concentration of the inflammatory cytokines: interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in the amniotic fluid has been known. However, changes of Lf in amniotic fluid with infection has not been reported. In the present study, Lf concentrations in amniotic fluid were measured under the intra-uterine infections state and the biological significance of Lf was investigated. The effects of Lf on the IL-6 and IL-6mRNA production in cultured amnion cells were also investigated. The concentrations of Lf and IL-6 in amniotic fluid with CAM were 8.76 +/- 0.65 micrograms/ml and 6.92 +/- 4.88 ng/ml (n = 28) respectively and both were significantly higher (p < 0.01) than those without CAM [0.86 +/- 0.81 microgram/ml and 0.34 +/- 0.25 ng/ml (n = 31)]. Significant positive correlation (r = 0.91, p < 0.01) between Lf and IL-6 levels in amniotic fluid was found. IL-6 production induced by lipopolysaccharide (LPS) (100 ng/ml) in cultured amnion cells was significantly inhibited (p < 0.05) under the physiological concentration of Lf in amnion. Total RNA was extracted from the amniotic cells by guianizine solution. RT-PCR procedure and product analysis were performed from one microgram aliquote of total RNA. beta-actin was used as an international standard and c-DNA samples were followed by 30 cycles of PCR. RT-PCR product of IL-6 mRNA was detected by Southern hybridization. Expression of IL-6 mRNA was inhibited by the addition of Lf. From the results, the possibility that Lf might suppress amniotic IL-6 production under the condition of amniotic infection is suggested. It is also suggested that Lf might act as self defence mechanism from intra-uterine infection.

Objective: Our purpose was to determine whether amniotic fluid interleukin-6 is increased and inversely proportional to gestational age in women with chorioamnion colonization and spontaneous labor versus women delivered for medical or obstetric indications., Study Design: The chorioamnion and amniotic fluid were cultured at cesarean delivery for aerobic and anaerobic bacteria, fungi, mycoplasmas, Chlamydia trachomatis and Trichomonas vaginalis in 269 women with singleton gestations with intact membranes. The amniotic fluid interleukin-6 concentration was also determined., Results: Amniotic fluid interleukin-6 levels were (1) higher in women with spontaneous labor versus those with indicated deliveries (15.8 +/- 5.0 vs 2.2 +/- 0.2 ng/ml, p = 0.01), (2) inversely proportional to gestational age in women with spontaneous labor (< 34 weeks: 47.4 +/- 18.0 ng/ml vs > or = 34 weeks: 8.7 +/- 4.1 ng/ml, p = 0.001) but not in women with indicated deliveries (1.5 +/- 0.4 vs 2.4 +/- 0.3 ng/ml), (3) higher in women with a positive versus a negative chorioamnion (15.1 +/- 4.8 vs 3.0 +/- 0.8 ng/ml, p < 0.001) or amniotic fluid (17.4 +/- 7.7 vs 3.8 +/- 0.9 ng/ml, p < 0.001) culture, and (4) higher in women with a negative amniotic fluid but positive chorioamnion culture compared with women in whom both cultures were negative (10.0 +/- 4.4 vs 3.0 +/- 0.9 ng/ml, p = 0.002)., Conclusions: Amniotic fluid interleukin-6 levels are (1) higher and inversely proportional to gestational age in women with intact membranes and spontaneous labor versus indicated deliveries, (2) higher in women with one or more microorganisms in the chorioamnion or amniotic fluid, and (3) reflective of chorioamnion microbial colonization, even when the amniotic fluid culture is negative, and may be a useful clinical marker for infection-mediated preterm labor.

Objective: To compare aztreonam in a standard dose with two gentamicin doses in the early treatment of experimental intra-amniotic infection in rabbits induced by intracervical inoculation with Escherichia coli., Methods: Timed pregnant rabbits on day 21 (70% of gestation) were inoculated intracervically with 10(4)-10(5) colony-forming units of E coli. After inoculation, the animals were treated with one of three regimens: 1) aztreonam at 90 mg/kg/day ("standard" dose in humans), 2) gentamicin at 4.5 mg/kg/day ("standard" dose in humans), or 3) higher-dose gentamicin at 6.0 mg/kg/day, each given in three divided doses daily. Outcomes included fever, delivery, and presence of a live fetus. At necropsy, cultures were taken from endometrium, amniotic fluid, and blood. Data were analyzed by Fisher exact test because the expected cell size was fewer than five., Results: Compared with rabbits treated with aztreonam, those treated with gentamicin 4.5 mg/kg/day delivered significantly more often (P = .002), had more positive cultures (P < .001), and had significantly fewer live fetuses (P < .001). Compared with rabbits treated with gentamicin 6.0 mg/kg/day, those treated with gentamicin 4.5 mg/kg/day delivered more often (P = .003), had fewer live fetuses (P = .02), and had more positive cultures (P = .02). There were no significant differences between the aztreonam and gentamicin 6.0 mg/kg/day groups., Conclusions: This study demonstrates in an animal model that aztreonam and gentamicin at 6.0 mg/kg/day are more effective than gentamicin at 4.5 mg/kg/day (a dose that is widely used empirically in humans) in the early treatment of experimental intra-amniotic infection in rabbits. Aztreonam was as effective as gentamicin at 6.0 mg/kg/day. In this rabbit model, in which intra-amniotic infection is accompanied by maternal sepsis, 4.5 mg/kg/day of gentamicin was not adequate for the treatment of severe maternal infection.

Objectives: Our purpose was to (1) determine the value of amniotic fluid interleukin-6 determination in the detection of microbial invasion of the amniotic cavity and (2) compare interleukin-6 to other rapid tests in the evaluation of preterm labor., Study Design: Amniotic fluid interleukin-6 was determined quantitatively by enzyme-linked immunosorbent assay in 91 amniotic fluid specimens obtained by amniocentesis in 89 patients with preterm labor. Amniotic fluid cultures for aerobes, anaerobes, and mycoplasma species were performed. Receiver-operator characteristic curve analysis, logistic regression analysis, and Cox's proportional-hazards model were used to explore the relationship between several explanatory and outcome variables. Diagnostic index values of interleukin-6, glucose level, Gram stain, leukocyte esterase, and limulus amebocyte lysate assay for prediction of a positive amniotic fluid culture, preterm delivery, clinical infection, and neonatal sepsis were calculated., Results: The prevalence of positive amniotic fluid cultures was 13% (12/89). The median interleukin-6 concentration in patients with positive cultures was 241.8 ng/ml, in contrast to 0.291 ng/ml in patients with negative cultures (p. < 0.005). Sensitivity and specificity of an interleukin-6 level > or = 6.17 ng/ml was 75% and 79%, in contrast to that of glucose, < or = 12 mg/dl (83% and 86%) for a positive amniotic fluid culture and sensitivity (p = 0.26, z test). All patients with an interleukin-6 level > 6.17 ng/ml were delivered preterm, in contrast to 85.2% of patients with a glucose level < or = 12 mg/dl. When all rapid tests and clinical parameters were considered simultaneously in the logistic model, only interleukin-6 maintained a significant relationship to preterm birth (odds ratio 35, p = 0.003). Cox's proportional analysis demonstrated a strong relationship between interleukin-6 and the amniocentesis-to-delivery interval after clinical variables were controlled for (hazard ratio 3.01, p < 0.00001)., Conclusion: Amniotic fluid interleukin-6 determination may be a useful adjunct to our armamentarium of rapid tests to exclude infection and predict delivery in patients with preterm labor and intact membranes.

Problem: This study was conducted to determine whether: (1) gestational age, parturition, and microbial invasion of the amniotic cavity (MIAC) are associated with changes in amniotic fluid concentrations of immunoreactive macrophage inflammatory protein-1 alpha; (2) amniotic fluid concentrations of macrophage inflammatory protein-1 alpha are correlated with the white blood cell count and the concentrations of interleukin-8 in amniotic fluid., Method: Amniotic fluid was retrieved by amniocentesis from 126 patients; 54 women with preterm labor and intact membranes (no MIAC-delivery at term, N = 21; no MIAC-preterm delivery, N = 16; MIAC-preterm delivery, N = 17); 62 patients at term (no labor, N = 19; labor-no MIAC, N = 20; labor-MIAC, N = 23); and 10 patients in the midtrimester of pregnancy. Amniotic fluid was cultured for aerobic, anaerobic and Mycoplasma species. Determinations of amniotic fluid macrophage inflammatory protein-1 alpha and interleukin-8 were performed with immunoassays validated for amniotic fluid (sensitivity: 14.2 pg/ml and 0.3 ng/ml, respectively). Kruskal-Wallis analysis of variance (ANOVA) for censored data, Mann-Whitney U test and Spearman's rank correlation were performed for analysis., Results: 1) Amniotic fluid macrophage inflammatory protein-1 alpha was present in only 31.0% (9/29) of patients not in labor (midtrimester and term). 2) Patients with preterm labor and MIAC had higher amniotic fluid concentrations of macrophage inflammatory protein-1 alpha than those without MIAC (no MIAC-delivery at term: median 0.0 pg/ml, range 0.0-221.2; no MIAC-preterm delivery: median 37.4 pg/ml, range 0.0-494.6; MIAC-preterm delivery: median 7171.0 pg/ml, range 402.5-37994.0; P < 0.00001). 3) Among patients at term, MIAC was associated with higher concentrations of amniotic fluid macrophage inflammatory protein-1 alpha than patients without MIAC (no labor: median 0.0 pg/ml, range 0.0-25.6; labor-no MIAC: median 16.7 pg/ml, range 0.0-161.6; labor-MIAC: median 103.8 pg/ml, range 0.0-4349.0, P < 0.001). 4) Among patients in preterm labor, a strong correlation was found between amniotic fluid concentrations of macrophage inflammatory protein-1 alpha and interleukin-8 (r = 0.9, P < 0.00001) and between amniotic fluid macrophage inflammatory protein-1 alpha concentrations and amniotic fluid white blood cell count (r = 0.6, P < 0.0001)., Conclusions: (1) Macrophage inflammatory protein-1 alpha is undetectable in most amniotic fluid samples from patients in the midtrimester of pregnancy and at term not in labor. (2) Microbial invasion of the amniotic cavity is associated with increased concentrations of immunoreactive amniotic fluid macrophage inflammatory protein-1 alpha in both term and preterm gestations. (3) Amniotic fluid macrophage inflammatory protein-1 alpha concentrations significantly correlate with interleukin-8 levels and white blood cell count in amniotic fluid. Our data strongly suggest a role for macrophage inflammatory protein-1 alpha in the mechanisms responsible for the recruitment of leukocytes into the amniotic cavity during the course of intrauterine infection.

Objective: We sought to determine and compare the value of several rapid diagnostic tests in the detection of intraamniotic infection., Study Design: Gram stain, intraamniotic glucose level determination, leukocyte esterase assay, and the Limulus amebocyte lysate assay were performed on 144 amniotic fluid specimens retrieved by transabdominal amniocentesis in 136 patients with preterm premature rupture of the membranes or preterm labor. Diagnostic indices for a positive amniotic fluid culture and the development of clinical infection were calculated for each rapid test. Receiver-operator characteristic curves were generated to help select the optimal glucose level and combination of tests to detect intraamniotic infection., Results: The greatest sensitivity for predicting either a positive culture or subsequent clinical infection in preterm labor patients and in predicting clinical infection in patients with preterm premature rupture of the membranes was demonstrated by a low glucose level. The Gram stain provided the greatest positive predictive value in patients with preterm labor. Combining the Gram stain and measurement of intraamniotic glucose levels did not improve sensitivity above glucose alone or positive predictive value above Gram stain alone., Conclusion: Leukocyte esterase determination and Limulus amebocyte lysate assay are insensitive indicators of intraamniotic infection. Selection of Gram stain or glucose level measurement alone or in combination as an appropriate screen for intraamniotic infection will depend on the clinicians' false-positive rate threshold.

Objective: The purpose of this study was to determine the prevalence and clinical significance of microbial invasion of the amniotic cavity in patients presenting with cervical dilatation in the midtrimester of pregnancy., Study Design: Amniocentesis for microbial studies was performed in women admitted with cervical dilatation > or = 2 cm, intact membranes, and without active labor between 14 and 24 weeks of gestation. Amniotic fluid was cultured for aerobic and anaerobic bacteria, as well as for mycoplasmas. Gram stain was performed on all samples., Results: The prevalence of microbial invasion of the amniotic cavity was 51.5% (17/33). The most common microbial isolates were Ureaplasma urealyticum, Gardnerella vaginalis, Candida albicans, and Fusobacterium sp. All patients with microbial invasion of the amniotic cavity had complications. Patients who underwent cervical cerclage in the presence of a positive amniotic fluid culture had rupture of membranes, clinical chorioamnionitis, or pregnancy loss. On the other hand, the prognosis of patients with a negative amniotic fluid culture was better than that of patients with a positive culture. Of 16 patients with a negative amniotic culture, nine were delivered at > 34 weeks., Conclusions: (1) Microbial invasion of the amniotic cavity occurs frequently in women presenting with cervical dilatation in the midtrimester; (2) the microbiologic state of the amniotic cavity is an important prognostic factor for pregnancy outcome; (3) amniocentesis to determine the microbiologic characteristics of the amniotic cavity should be considered before a cerclage is placed in women presenting with cervical dilatation in the midtrimester.

Objective: The purpose of this study was to determine the frequency, microbiologic characteristics, and clinical significance of microbial invasion of the amniotic cavity in women with premature rupture of membranes at term., Study Design: Amniocentesis was performed in 32 women with term premature rupture of membranes and amniotic fluid cultured for aerobic and anaerobic bacteria and Mycoplasmas., Results: The prevalence of positive amniotic fluid cultures was 34.3% (11/32). The most common isolates were Ureaplasma urealyticum, Peptostreptococcus sp., Lactobacillus sp., Bacteroides fragilis, and Fusobacterium sp. Clinical chorioamnionitis occurred only in one patient with a positive amniotic fluid culture. Her neonate had ophthalmitis. Three patients (9.4%) had endometritis. Among women who were delivered vaginally, those with a positive amniotic fluid culture had a significantly higher rate of endometritis than those with a negative culture (33% [3/9] vs 0% [0/20], respectively, p = 0.023)., Conclusions: These data indicate that microbial invasion of the amniotic cavity occurs in approximately one third of patients with preterm premature rupture of membranes. Microbial invasion of the amniotic cavity is a risk factor for endometritis in women with term premature rupture of membranes.

The purpose of this study was to determine the effect of gestational age, labor, and microbial invasion of the amniotic cavity on amniotic fluid concentrations of endothelin-1,2. Amniotic fluid was retrieved by amniocentesis from 148 women: patients at term with and without labor, patients with preterm labor with and without intraamniotic infection, and women in the second trimester of pregnancy. Endothelin-1,2 was measured by a sensitive and specific radioimmunoassay. Immunoreactive endothelin-1,2 was detectable in all samples of human amniotic fluid. Advancing gestational age and spontaneous term labor did not result in changes in amniotic fluid concentrations of endothelin-1,2. Women with preterm labor and positive amniotic fluid cultures for microorganisms had higher amniotic fluid concentrations of endothelin-1,2 than did those without microbial invasion of the amniotic cavity (p less than 0.05). These results support a role for endothelins in the mechanisms responsible for preterm delivery associated with intraamniotic infection.

Indirect evidence suggests that amnionitis, in the absence of maternal symptoms, contributes to neonatal morbidity. The incidence is unknown because diagnostic techniques are not available. A quantitative amniotic fluid analysis of bacteria, white blood cells, and lactic dehydrogenase levels was performed during labor among 28 patients considered to be a risk for infection. Thirteen of 16 patients with bacterial colony counts greater than 10(3)/ml subsequently developed maternal infection had a premature delivery, neonatal sepsis, or a combination of these factors. Maternal fever was a late sign in clinical infection and was frequently absent in cases of premature delivery associated with microbiologic evidence of amnionitis. Amniotic fluid analysis may be of value in diagnosing unrecognized amnionitis among patients delivering prematurely and those requiring nonelective cesarean section.

Thirty-seven consecutive patients with singleton pregnancies in "uncomplicated" preterm labor with intact membranes suitable for tocolysis were evaluated for evidence of silent chorioamnionitis by means of maternal serum C-reactive protein and amniotic fluid white blood cell count, Gram stain, and cultures. Abnormalities in these markers of infection were found to be significantly more common in cases that were refractory to tocolysis. These cases also showed both pathologic evidence of chorioamnionitis and a significantly greater neonatal early infectious morbidity. We conclude that silent chorioamnionitis is a significant cause of "uncomplicated" preterm labor refractory to conventional methods of tocolysis.

Our objective was to investigate the role of Gardnerella vaginalis in intra-amniotic infection by use of comparative, quantitative cultures on selective media and by detection of maternal antibody response. Amniotic fluid was collected from patients with intra-amniotic infection and from matched control women. In addition to media for aerobes, anaerobes, and mycoplasmas, we used V agar-selective (Remel, Lenexa, KS) to isolate G vaginalis. Acute and convalescent maternal sera were collected and assayed for antibodies by a microenzyme-linked immunosorbent assay (ELISA) prepared against whole cells of G vaginalis. Gardnerella vaginalis was isolated in the amniotic fluid of 24 (28%) of the 86 patients with intra-amniotic infection, but this was not significantly different from the isolation rate in amniotic fluid of 86 matched controls (21%). No patient exhibited G vaginalis bacteremia. The ELISA performed on paired sera of selected patients showed that 25 had intra-amniotic infection (eight G vaginalis-positive, 17 negative), and 18 were asymptomatic (seven G vaginalis-positive, 11 negative). The amount of G vaginalis antibodies detected by ELISA in acute sera was similar in all four groups. Mean changes during convalescence were small (.053-.084 optical density units) and not significantly different. Although G vaginalis is found commonly in amniotic fluid of patients with intra-amniotic infection, the data do not support a pathogenic role for this organism; however, a facilitating role in polymicrobial infection cannot be excluded.

When we compared amniotic fluid and amniotic membranes, as a source of organisms at the time of cesarean section, amniotic membranes proved to be superior. During labor, intact membranes do not protect the amniotic cavity from contamination with pathogenic anaerobes and commensals. Ruptured membranes in the presence of prolonged labor predisposes the amniotic cavity to contamination with pathogenic anaerobes, commensals, and pathogenic aerobes. Cultures of the endometrial biopsy specimens taken from the site of incision at the time of cesarean section showed the presence of pathogens in 50% of the women with prolonged labor and ruptured membranes.

The purpose of this prospective investigation was to determine the incidence of subclinical intra-amniotic infection in asymptomatic patients who had intact membranes and refractory preterm labor. Refractory preterm labor was defined as persistent uterine contractions despite maximum recommended doses of parenteral tocolytics, or recurrent preterm labor within three days of successful transition to oral tocolytics. Amniotic fluid was cultured aerobically and anaerobically and prepared for Gram stain and group B streptococci latex fixation test. One of 24 women had a positive latex fixation test, but the culture was negative. One culture was positive for isolated colonies of Corynebacterium sp. None of the patients developed clinical evidence of intra-amniotic infection or postpartum endometritis. The mean prolongation of pregnancy was 31 days (range 1-63). None of the infants had evidence of sepsis in the immediate neonatal period. In this population, subclinical infection was an uncommon cause of refractory preterm labor.

A growing body of evidence supports a causal link between subclinical intrauterine infection and preterm labor. The mechanisms responsible for the onset of parturition in this setting have not been elucidated. The conventional view has been that bacterial products increase prostaglandin biosynthesis by intrauterine tissues and this, in turn, leads to the onset of labor. An alternative or complementary mechanism is that microbial products activate the host monocyte-macrophage system and that cytokines released during this process signal the initiation of parturition by stimulating prostaglandin biosynthesis by intrauterine tissues. This study was conducted to determine if cachectin-tumor necrosis factor is present in the amniotic fluid of women with intraamniotic infection and whether this cytokine can alter the rate of prostaglandin biosynthesis by intrauterine tissues. Amniotic fluid from 54 women was assayed for tumor necrosis factor. Tumor necrosis factor was not detectable in the amniotic fluid of women without intraamniotic infection regardless of the presence or absence of term or preterm labor. On the other hand, the amniotic fluid of 11 of 15 women with preterm labor and intraamniotic infection had measurable tumor necrosis factor. This cytokine stimulated prostaglandin E2 biosynthesis by amnion cells in monolayer culture in a dose-dependent fashion. These data support the concept that macrophage activation is involved in the onset of human parturition in the setting of infection. We propose that the host (fetus and/or mother) signals the onset of parturition through the secretion of inflammatory cytokines released in response to bacterial invasion.

Quantitative amniotic fluid cultures were performed on 12 patients delivered of premature infants (10-premature labor, 2-premature rupture of membranes). Specimens from patients in premature labor were obtained at the time of amniotomy via transcervical needle aspiration or intrauterine catheter. Of the seven patients with colony counts greater than 1,000 per ml, two mothers and four neonates had clinical evidence of infection. Blood cultures from two infants and one mother and cerebrospinal fluid cultures from one newborn infant were positive for the same organism isolated from amniotic fluid. Anearobic bacteria were the predominant isolates in four specimens and included Bacteriodes species in two. In contrast, the five mothers and infants associated with counts of less than 1,000 per ml exhibited no infectious morbidity. Three had no bacterial growth from amniotic fluid, and factors commonly associated with prematurity were recognized in four. These clinical and microbiologic observations suggest that current methods of evaluating patients with premature labor and delivery are incomplete, and the role of unrecognized amnionitis should be reevaluated.

In this review the mechanisms whereby amniotic fluid serves to protect the fetus from microbial disease have considered. It appears from the data reviewed that the principal mode of antibacterial action of amniotic fluid is bacteriostasis. Thus, the host is able to cope with a small number of organisms introduced into the amniotic cavity; however when the amniotic fluid is grossly contaminated the host resistance capacity of the amniotic fluid may be overwhelmed. This may be understood best by the quantitative description of disease theroized by Theobald Smith (94). Smith suggested that disease was a function of the number of organisms with which a host is initially infected multiplied by the virulence of the organism. The effects of the number and virulence of the organism in producing disease is lessened by the host's resistance capacity. This concept of disease is summarized by the equation: Disease equals (number) (virulence)/resistance. Although these parameters do not possess numeric values, it is possible to see at least conceptually their interplay with respect to intrauterine infection. For example, the number of organisms reaching the amniotic fluid may be increased by various modes, namely maternal viremia or bacteremia; premature rupture of the fetal membranes, antenatal vaginal examination and possibly intrauterine fetal monitoring. While these circumstances may result in increased rate of infection, some reports conversely indicate that minimal bacterial contamination in the amniotic fluid is not an extraordinary occurrence and may not result in any maternal or fetal complication (73,74). The intrinsic host resistance capacity of the amniotic fluid likewise represents an important part of the Smith equation for disease. We have found that amniotic fluids may vary in antibacterial efficacy from almost no inhibitory activity to profound bactericidal activity (90). Obviously, the likelihood of the production of disease by an equivalent inoculum of a particular organism would be quite different depending upon the intrinsic inhibitory capacity of the amniotic fluid. The measurement of the inhibitory capacity of amniotic fluid holds some promise for enabling a physician to determine which patients may be at special risk of intrauterine infection.

A prospective study of fetal behavior in cases complicated with preterm premature rupture of membranes was conducted in 41 patients. The length of time and the number of fetal breathing and gross body movements were correlated with the amniotic fluid culture results. An episode of fetal activity (body movements and breathing movements) of greater than or equal to 30 seconds during 30 minutes of observation was associated with the absence of intraamniotic infection in 100% of the cases. On the other hand, the absence of fetal breathing movements and gross body movements of less than a 50 second duration during 30 minutes of observation was associated with positive amniotic fluid cultures in all cases. If an episode of fetal breathing movements was present but lasted less than 30 seconds and/or the total time of gross body movements was greater than 50 seconds, 64% of patients had an intraamniotic infection.

A study was made of the outcome for mothers and their neonates with both clinical and bacteriologic evidence of intra-amniotic infection at term. Samples of amniotic fluid from patients with intra-amniotic infection showed greater than 10(2) colony-forming units per milliliter of a high-virulence isolate, whereas samples from control patients showed no growth or low-virulence isolates only. Control patients were uninfected during labor and were matched on the basis of gestational age, interval from rupture of membranes to delivery, and mode of delivery. There were 67 matched pairs. The mean interval from diagnosis of intra-amniotic infection to delivery was 3.1 +/- 2.2 hours (+/- SD). Mothers with intra-amniotic infection had a significantly longer hospital stay and greater fever index after delivery than did control patients. Intrapartum bacteremia was documented in six of 50 (12%) women with intra-amniotic infection. The cesarean birth rate was 36%. There was one case of probable septic shock and one of postpartum hemorrhage among women with intra-amniotic infection. Infants in the intra-amniotic infection group had a significantly longer hospital stay than did the control infants. Among 59 infants for whom blood culture results were available, bacteremia was documented in five (8%) with intra-amniotic infection. Definite radiographic evidence of pneumonia was present in 4%; there were no cases of meningitis. There was one perinatal death in the intra-amniotic infection group. Overall, the maternal and perinatal outcome after intra-amniotic infection at term was excellent.