Conceived and designed the experiments: DBD RR EMB DAR. Performed the experiments: DBD HPA. Analyzed the data: DBD RR HPA JPK EMB FG CJK OE SE DAR. Contributed reagents/materials/analysis tools: RR DAR. Wrote the paper: DBD RR JPK EMB FG OE SE DAR. Placental pathology diagnosis: CJK. Determination of IL-6 concentrations in amniotic fluid: SE. Collection of data (demographic and clinical): OE FG JPK RR.
The authors have declared that no competing interests exist.
Preterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking.
In parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of
The amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship.
Preterm birth is the leading cause of neonatal mortality worldwide
A strong body of evidence suggests that occult intra-uterine infection plays a major role in preterm labor and delivery
Molecular methods such as polymerase chain reaction (PCR) can detect microbes independently of culture. Moreover, broad-range PCR assays that amplify highly-conserved but phylogenetically-informative gene sequences can identify microbes across broad taxonomic levels, including previously uncharacterized species. By overcoming investigator biases inherent to more specific detection methods, sequencing of broad-range PCR products has emerged as a powerful approach for revealing previously unsuspected microbial diversity in various anatomic niches in human health
The application of broad-range PCR to amniotic fluid has been limited to date. In particular, molecular investigations that characterize the microbial diversity of the amniotic cavity in a systematic manner, and evaluate findings within a coherent causal framework, are lacking. As an early step in defining the potential role of diverse microbial sequence types, including uncultivated taxa, in preterm delivery, we conducted a broad-range molecular investigation. In parallel with traditional amniotic fluid cultures, we used qualitative and quantitative PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of patients with spontaneous preterm labor and intact membranes. We examined sequence diversity in samples with detectable rDNA, and correlated findings with pre-specified measures of host inflammation, as well as pregnancy and neonatal outcome. We sought evidence for the types of associations that have been proposed as alternatives to Koch's postulates for inferring causality from molecular data (e.g., associations of space, time and dose)
A retrospective cohort study was conducted by searching our clinical database to identify patients with the diagnosis of spontaneous preterm labor with intact membranes, enrolled at Hutzel Women's Hospital (Detroit, MI) between October 1998 and December 2002, who provided written informed consent and for whom an adequate volume of amniotic fluid was available with which to conduct these research assays after clinically-indicated conventional analyses were completed. Patients were included if they met the following criteria: 1) singleton gestation; 2) gestational age between 18 and 35 weeks; and 3) had an amniocentesis with microbiological studies of amniotic fluid. Patients were excluded from the study if: 1) rupture of the chorioamniotic membranes occurred before amniotic fluid collection; 2) the amniotic fluid was collected transvaginally; 3) delivery occurred elsewhere and/or clinical data were unavailable; and 4) a major fetal congenital anomaly was present.
A total of 166 subjects were included in this study. The pre-specified control group consisted of study subjects who delivered at term. All women provided written informed consent prior to the collection of biological samples. The utilization of samples for research purposes was approved by the Institutional Review Boards of Wayne State University, the National Institute of Child Health and Human Development (NICHD/NIH/DHHS), and Stanford University.
Preterm labor was diagnosed by the presence of at least two regular uterine contractions every 10 minutes associated with cervical changes that required hospital admission before 37 weeks of gestation. Preterm delivery was defined as delivery before 37 weeks of gestation. Clinical chorioamnionitis was diagnosed according to criteria previously proposed
Enrolled subjects underwent transabdominal, ultrasound-guided amniocentesis, which is within the standard of care at Hutzel Women's Hospital for evaluating possible microbial invasion of the amniotic cavity of patients with spontaneous preterm labor. Amniotic fluid was immediately transported in a capped sterile syringe to the clinical laboratory where it was cultured for aerobic and anaerobic bacteria, including genital mycoplasmas. White blood cell (WBC) count and Gram stain of amniotic fluid were also performed shortly after collection. Amniotic fluid not required for clinical assessment was centrifuged for 10 minutes at 4°C shortly after the amniocentesis, and the supernatant was aliquoted and stored at −70°C until analysis. Amniotic fluid IL-6 concentrations were determined after delivery for research purposes, and these results were not used in patient management. A flowchart of our overall approach to amniotic fluid analysis is illustrated in Supporting
Microbial genomic DNA was extracted from 200 µl of each amniotic fluid sample. Qualitative analysis was achieved by means of three separate broad-range end-point PCR assays targeting rDNA of either bacteria, fungi, or archaea. Cloned amplicons (up to 24 clones per positive PCR reaction) were bidirectionally sequenced, the sequences aligned, and then subjected to phylogenetic analysis (621 nucleotide positions). Quantitative rDNA analysis was achieved by means of three real-time PCR assays corresponding to each of the three microbial groups targeted by end-point PCR. Methodologic details of the key aspects of our molecular approach (including nucleic acid extraction, PCR assays, phylogenetic analysis, and contamination prevention) appear under Supporting
In order to assess the clinical significance of the microbes detected with molecular methods, pre-specified outcome variables from four broad categories were measured: 1) markers of intra-amniotic inflammation (including amniotic fluid WBC count
Statistical analyses were performed using ‘R’ (open source,
Of 166 subjects, 53 (32%) delivered at term and 113 (68%) delivered preterm (
Panel A shows outcomes of enrolled subjects. Panel B summarizes results of culture and PCR analysis of amniotic fluid. PCR refers to end-point PCR for bacteria, fungi and archaea. Culture refers to the use of routine cultivation methods for bacteria (aerobic, anaerobic and genital mycoplasmas) and fungi. Circle areas are not to scale.
Microbial invasion of the amniotic cavity – as defined by either a positive end-point PCR or culture of amniotic fluid – was found in 15% (25/166) of patients. Of these, bacteria were cultivated from 15 and a fungus from 1, whereas bacterial rDNA was amplified from 17 and fungal rDNA from 2. Six culture-positive samples were negative by PCR, and nine PCR-positive samples were negative by culture (
Among the 25 patients whose amniotic fluid tested positive by culture or end-point PCR, 17 bacterial species (belonging to 5 phyla) and 1 fungal species were identified. Of the 17 bacterial taxa identified, 6 were detected by both culture and PCR (
Phylogeny of the 17 bacterial taxa identified in this study, based on a maximum likelihood algorithm. Colored boxes indicate the number of subjects who were positive for a given taxon by culture (gray) or PCR (blue) (some samples were polymicrobial). For most individual taxa, the larger of the two numbers in the corresponding gray or blue box represents the total number of positive subjects; for taxa where neither method detected all positive subjects, the total number is shown in the white box. A 99% sequence similarity cutoff threshold was used for phylotype assignment, which was based on 621 unambiguous nucleotide positions.
A host inflammatory response has been implicated in the pathogenesis of preterm delivery
Amniotic fluid concentrations of white blood cells (WBCs) (Panel A) and interleukin-6 (IL-6) (Panel B) as a function of PCR and culture results. Each circle represents one subject; each horizontal bar and adjacent number indicates the median value for that group. P values were calculated by means of the Mann-Whitney U test. PCR refers to end-point PCR for bacteria, fungi and archaea. Culture refers to the use of routine cultivation methods for bacteria (aerobic, anaerobic and genital mycoplasmas) and fungi.
To evaluate associations of microbial DNA with inflammation of maternal and fetal tissues, results from end-point PCR and culture were correlated with histologic chorioamnionitis, which has been previously associated with preterm delivery
Variable | Histologic Chorioamnionitis |
Funisitis |
Composite Neonatal Morbidity and Mortality |
|||
Univariate | Multivariate | Univariate | Multivariate | Univariate | Multivariate | |
Positive amniotic fluid PCR | 37 (4.8–288) | 20 (2.4–172) | 24 (5.2–109) | 18 (3.1–99) | 16.2 (3.6–73) | 5.2 (0.84–32) |
Positive amniotic fluid culture | 31 (4.0–243) | 8.6 (1.0–76) | 20 (4.2–91) | 5.8 (1.1–32) | 23 (2.9–179) | 7.1 (0.66–76) |
Maternal age | 0.99 (0.93–1.0) | 0.97 (0.90–1.0) | 0.95 (0.89–1.0) | 0.9 (0.84–0.98) | 1.0 (0.97–1.1) | NA |
Gestational age at amniocentesis | 0.87 (0.80–0.94) | 0.89 (0.81–0.97) | 0.9 (0.81–0.96) | 0.89 (0.83–0.98) | 0.84 (0.78–92) | 0.80 (0.72–0.88) |
Cervical dilatation | 1.1 (0.91–1.4) | NA | 1.1 (0.90–1.4) | NA | 1.4 (1.1–1.7) | 1.6 (1.2–2.1) |
Results are reported as odds ratios (95% confidence interval).
The table includes variables that exhibited a statistically significant association with one or more of the three measured outcomes. For each outcome, the multivariate model was comprised of those variables exhibiting a significant association in the univariate model. Other variables that were subject to univariate analysis but did not exhibit a significant association with any of these outcomes were: African-American race, nulliparity, previous preterm delivery, cigarette smoking, and recreational drug use.
Diagnosed based on the presence of inflammatory cells in the chorionic plate and/or chorioamniotic membranes.
Defined as the presence of neutrophils in the wall of the umbilical vessels and/or Wharton's jelly.
Defined as the presence of any one or more of the following: bronchopulmonary dysplasia, respiratory distress syndrome, necrotizing enterocolitis, intraventricular hemorrhage of grade≥III, sepsis, respiratory failure requiring mechanical ventilation, and neonatal death.
NA Not applicable.
To evaluate the association of microbial DNA with gestational outcomes, results from end-point PCR and culture were correlated with gestational age at delivery. All subjects testing positive by either PCR or culture delivered preterm; this complete separation with respect to test result precluded logistic regression analysis. However, when the study population was stratified according to clinically-relevant gestational-age cut offs, the frequency distribution of positive PCR assays exhibited a clear inverse relationship with gestational age at delivery (
Panel A shows the proportion of subjects from each gestational age cohort who were positive by culture or PCR. The total of number of subjects in each cohort appears across the top of the graph, and the number of positive subjects appears above each bar. Panel B is a Kaplan-Meier analysis of amniocentesis-to-delivery interval according to culture and PCR results. Subjects in whom labor was augmented were censored and are represented by crosses. P values were calculated by means of the Mantel-Haenszel log-rank test. The inset table shows the median interval for each group. For both panels, PCR results refer to end-point PCR for bacteria, fungi and archaea, and culture refers to the use of routine cultivation methods for bacteria (aerobic, anaerobic and genital mycoplasmas) and fungi.
Outcome | Preterm delivery (<37 weeks) | Very preterm delivery (<32 weeks) | Extremely preterm delivery (<25 weeks) | Delivery within one day of amniocentesis |
Prevalence | 68% (113/166) | 49% (58/118) | 40% (17/43) | 17% (28/166) |
PPV of PCR | 100% (19/19) | 100% (16/16) | 100% (8/8) | 68% (13/19) |
PPV of culture | 100% (16/16) | 100% (14/14) | 89% (8/9) | 69% (11/16) |
PPV of PCR and culture combined | 100% (10/10) | 100% (8/8) | 100% (5/5) | 100% (10/10) |
To evaluate the association of microbial DNA with neonatal outcomes, results from end-point PCR and culture were correlated with a composite of neonatal morbidity and mortality by means of simple and multiple logistic regression. Similar to the outcome of preterm delivery, both PCR and culture exhibited near-complete positive correlation: all but two PCR-positive cases, and all but one culture-positive case were associated with neonatal morbidity and mortality (Supporting
To evaluate the association of microbial DNA with timing of delivery, results from end-point PCR and culture were correlated with the interval from amniocentesis to delivery. Kaplan-Meier survival estimates, censored for subjects in whom labor was augmented, and evaluated for differences by means of the Mantel-Haenszel log-rank test, revealed an overall association between a positive test result (PCR and/or culture) and a shortened amniocentesis-to-delivery interval (P<0.001) (
To evaluate dose-response associations, bacterial rDNA concentration was estimated by broad-range real-time PCR, and correlated with gestational age at delivery. Regression modeling revealed a significant correlation (r2 = 0.42; P<0.002) (
Data are for samples yielding detectable results within the sensitivity and dynamic range of the real-time PCR assay (∼250–1×108 genes/µl amniotic fluid).
This study revealed significant associations between the presence and quantity of microbes or their DNA in amniotic fluid, and preterm delivery or other clinically-relevant outcomes. Microbial prevalence, as revealed by means of end-point PCR and culture combined (15%), was 56% higher than that found by culture alone (9.6%), which is the conventional diagnostic approach. PCR detected microbial DNA in culture-negative samples despite the limited sample volumes available for DNA extraction (200 µl per patient), and the fact that PCR was performed 2 to 6 years after sample collection and culturing, which may have limited the number of PCR-positive samples owing to DNA degradation
The microbial diversity revealed by end-point PCR and culture combined (18 taxa) was almost twice as rich as that found by culture alone (11 taxa). Among the taxa identified by PCR, but not culture, were phylotypes that would not have been predicted from prior studies. One taxon appeared to be a novel species, and possibly a novel genus. The sequence of this taxon (clone PL036-b24; GenBank accession no. EU932745) clustered nearest the genus
A single fungal phylotype,
Prior studies have applied species-specific
To overcome limitations of prior studies, we analyzed 166 subjects for microbes belonging to three broad taxonomic groups. Microbial diversity was assessed by sequencing up to 24 clones per subject. Microbial rDNA abundance was measured in parallel. Using this approach, we demonstrated greater microbial diversity than expected from prior studies. Compared to findings from culture, the molecular detection of these diverse microbes exhibited equivalent associations with indices of host inflammation, and with adverse gestational and neonatal outcomes.
Our study does not prove causation. To do so for multifactorial biological phenomena often entails successive investigations that expand knowledge within a coherent framework until sufficient evidence accumulates. Any single framework, however, may have limitations. The postulates of Robert Koch, for example, cannot be fulfilled for uncultivated pathogens
Despite this study's progress in defining the microbial diversity associated with preterm delivery, much likely awaits discovery. Many detected phylotypes, such as the uncultivated
This study has some limitations, some of which could affect results from molecular assays disproportionately, thus potentially contributing to a negative PCR in some culture-positive cases. First, DNA may have degraded during the interval from amniocentesis to molecular analysis (range, 2–6 years)
In conclusion, among patients with spontaneous preterm labor and intact membranes, the amniotic cavity harbors a greater diversity of microbes than previously suspected, including uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which detected microbes were associated with clinically relevant outcomes, including preterm delivery, suggest a causal relationship. Despite these insights, the microbial census of the amniotic cavity is unfinished. Taken together, our findings support a contributory role for occult intra-amniotic infection in preterm delivery and its neonatal sequelae, and argue for further large-scale prospective molecular investigations.
Supporting Materials and Methods
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Approach to Amniotic Fluid Analysis. Results reported in this study are shaded either grey (conventional analyses) or blue (molecular analyses). *PCR assays targeted the domain
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Baseline Subject Characteristics According to Results of PCR and Culture of Amniotic Fluid.
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Characteristics of Individual Subjects Who Tested Positive by PCR or Culture.
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Association of Demographic and Microbiologic Variables with Shortened Amniocentesis-to-Delivery Interval.
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Broad-range PCR Assays Used in this Study.
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We are grateful to study subjects. We thank Nicola Casali, University of California, Berkeley, for thoughtful comments on the manuscript. The authors wish to acknowledge essential contributions of staff of the Perinatology Research Branch and Detroit Medical Center, including Nancy Hauff, Sandy Field, Lorraine Nikita, Vicky Ineson, Evie Russell, Mahbubeh Mahmoudieh, Julie McKinley, Sue Rehel, Shannon Donegan, Linda Bouey, Carolyn Sudz, Sylvia Warren, Shelley Mullen, Gail Bartley, Denise Bayoneto, Judy Kerman, Barbara Steffy, Milagros Kitchen, and Leandra Ga-Pinlac, as well as Doctors Sonia Hassan, Pooja Mittal, Yoram Sorokin, Theodore Jones, and Susan Berman.