Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre-labor rupture of membranes

Abstract

Problem: The role played by microbial invasion of the amniotic cavity (MIAC) in preterm pre-labor rupture of membranes (pPROM) is inadequately characterized, in part because of reliance on cultivation-based methods.

Method of study: Amniotic fluid from 204 subjects with pPROM was analyzed with both cultivation and molecular methods in a retrospective cohort study. Broad-range and group-specific polymerase chain reaction (PCR) assays targeted small subunit ribosomal DNA (rDNA), or other gene sequences, from bacteria, fungi, and archaea. Results were correlated with measurements of host inflammation, as well as pregnancy and perinatal outcomes.

Results: The prevalence of MIAC was 34% (70/204) by culture, 45% (92/204) by PCR, and 50% (101/204) by both methods combined. The number of bacterial species revealed by PCR (44 species-level phylotypes) was greater than that by culture (14 species) and included as-yet uncultivated taxa. Some taxa detected by PCR have been previously associated with the gastrointestinal tract (e.g., Coprobacillus sp.), the mouth (e.g., Rothia dentocariosa), or the vagina in the setting of bacterial vaginosis (e.g., Atopobium vaginae). The relative risk for histologic chorioamnionitis was 2.1 for a positive PCR [95% confidence interval (CI), 1.4-3.0] and 2.0 for a positive culture (95% CI, 1.4-2.7). Bacterial rDNA abundance exhibited a dose relationship with gestational age at delivery (R(2) = 0.26; P < 0.01). A positive PCR was associated with lower mean birthweight, and with higher rates of respiratory distress syndrome and necrotizing enterocolitis (P < 0.05 for each outcome).

Conclusion: MIAC in pPROM is more common than previously recognized and is associated in some cases with uncultivated taxa, some of which are typically associated with the gastrointestinal tract. The detection of MIAC by molecular methods has clinical significance.

Figures

Figure 1. Distribution of subjects (n=204) according to results of PCR and culture of amniotic fluid

Data are from amniocentesis at presentation of 204 subjects for whom results from all culture assays were available. Culture refers to the aggregate results from routine cultivation methods for bacteria (aerobes, anaerobes and genital mycoplasmas) and for fungi. PCR refers to the aggregate results from end-point or real-time PCR targeting domain Bacteria, domain Archaea, genus Candida, and five specific bacterial groups (see Methods). Circle areas are not to scale.

Figure 2. Bacterial taxa detected by PCR or culture

Phylogeny of the bacterial taxa identified in this study, based on a neighbor-joining algorithm with Felsenstein correction and a 682-column filter. The scale bar represents evolutionary distance (10 substitutions per 100 nucleotides). The taxon in brackets and gray type (uncultured TM7-like bacterium) is a public database sequence included for reference and was not detected in this study. Colored boxes indicate the number of subjects who were positive for a given taxon by culture (gray) or PCR (blue) (some samples were polymicrobial). Because culture isolates were not sequenced, each is represented by a GenBank sequence that corresponds to the taxonomic resolution to which culture isolates were phenotypically identified. Two culture isolates are not represented because they were not characterized to a sufficiently narrow taxonomic resolution to allow tree placement (viridans group streptococcus, and gram positive bacillus, each detected in a separate subject). Candida was the lone fungal genus detected in the study population (not shown in this figure).

Figure 3. Bacterial taxa detected by PCR in those samples (n=26) for which culture data were incomplete

Phylogeny is based on a neighbor-joining algorithm with Felsenstein correction and a 682-column filter. The scale bar represents evolutionary distance (10 substitutions per 100 nucleotides).

Figure 4. Inflammatory markers in amniotic fluid at presentation, according to PCR and culture results

Panel A presents white blood cell counts. Panel B presents interleukin-6 concentrations. For both panels, P values were calculated using the Mann-Whitney U test. Data are from amniocentesis at presentation of 204 subjects for whom results from all culture assays were available.

Figure 5. Correlation of bacterial 16S rDNA concentration with pregnancy outcomes

Panel A presents results for gestational age at which subjects presented with preterm PROM. Panel B presents results for gestational age at delivery.

Figure 6. Kaplan-Meier survival plot of amniocentesis-to-delivery interval according to results of PCR and culture of amniotic fluid

Observations were right-censored for subjects who underwent cesarean section prior to labor onset, or who underwent labor induction. Prior to analysis, influential outliers, as defined by a dfbetas residual >3 standard deviations, were excluded (n=3). P values for differences in survival curves were calculated by means of the Mantel-Haenszel log-rank test.

Figure 7

Time interval from amniotic fluid collection to DNA extraction.