Differing prevalence and diversity of bacterial species in fetal membranes from very preterm and term labor

Abstract

Background: Intrauterine infection may play a role in preterm delivery due to spontaneous preterm labor (PTL) and preterm prolonged rupture of membranes (PPROM). Because bacteria previously associated with preterm delivery are often difficult to culture, a molecular biology approach was used to identify bacterial DNA in placenta and fetal membranes.

Methodology/principal findings: We used broad-range 16S rDNA PCR and species-specific, real-time assays to amplify bacterial DNA from fetal membranes and placenta. 74 women were recruited to the following groups: PPROM <32 weeks (n = 26; 11 caesarean); PTL with intact membranes <32 weeks (n = 19; all vaginal birth); indicated preterm delivery <32 weeks (n = 8; all caesarean); term (n = 21; 11 caesarean). 50% (5/10) of term vaginal deliveries were positive for bacterial DNA. However, little spread was observed through tissues and species diversity was restricted. Minimal bacteria were detected in term elective section or indicated preterm deliveries. Bacterial prevalence was significantly increased in samples from PTL with intact membranes [89% (17/19) versus 50% (5/10) in term vaginal delivery p = 0.03] and PPROM (CS) [55% (6/11) versus 0% (0/11) in term elective CS, p = 0.01]. In addition, bacterial spread and diversity was greater in the preterm groups with 68% (13/19) PTL group having 3 or more positive samples and over 60% (12/19) showing two or more bacterial species (versus 20% (2/10) in term vaginal deliveries). Blood monocytes from women with PTL with intact membranes and PPROM who were 16S bacterial positive showed greater level of immune paresis (p = 0.03). A positive PCR result was associated with histological chorioamnionitis in preterm deliveries.

Conclusion/significance: Bacteria are found in both preterm and term fetal membranes. A greater spread and diversity of bacterial species were found in tissues of women who had very preterm births. It is unclear to what extent the greater bacterial prevalence observed in all vaginal delivery groups reflects bacterial contamination or colonization of membranes during labor. Bacteria positive preterm tissues are associated with histological chorioamnionitis and a pronounced maternal immune paresis.

Figure 1. The microbial prevalence in placental tissue and fetal membranes from term and very preterm deliveries.

The microbial prevalence was determined in the following groups term with vaginal delivery (V) or caesarean section (CS), indicated preterm delivery, preterm delivery with PPROM, and preterm delivery with intact membranes (PTL). An individual was deemed positive if any of the 5 tissue samples taken were positive for the amplification of bacterial DNA by either method described. The frequency of bacterial prevalence in each group is expressed as a percentage and the number in each group. Groups were compared and significance was determined by a Fisher's exact test.

Figure 2. The spread of bacteria in placental tissue and fetal membranes from term and very preterm deliveries.

A total of 5 samples were taken from each women. DNA was extracted and then subject to broad-range 16S rDNA endpoint PCR and species-specific real time PCR. The percentage (and number) of individuals that had 3–5 samples, 1–2 samples, and 0 samples positive for bacteria are shown for each group. (CS) caesarean section, (V) spontaneous vaginal delivery, (PTL) preterm labour with intact membranes, (PPROM) preterm prolonged rupture of membranes.

Figure 3. The diversity of bacterial species found in tissues from women with very preterm and term deliveries.

The percentage (and number) of women with placental tissue and fetal membranes positive for 2 or more, 1 or 0 bacterial species is shown for each group. (CS) caesarean section, (V) spontaneous vaginal delivery, (PTL) preterm labour with intact membranes, (PPROM) preterm prolonged rupture of membranes.

Figure 4. The presence of histological chorioamnionitis in fetal membranes from very preterm deliveries.

Fetal membranes of all deliveries before 32 weeks gestation were assessed routinely for histological chorioamnionitis. Here we show the association between the presence of bacteria in fetal membranes and placental tissue and histological chorioamnionitis in preterm labour with intact membranes (PTL), preterm prolonged rupture of membranes (PPROM), and indicated preterm delivery.

Figure 5. Monocyte MHC Class II expression in women with very preterm labour and preterm premature rupture of membranes with and without the presence of bacteria.

Maternal monocytes were analysed for the expression (median fluorescence intensity- MFI) of MHC Class II by flow cytometry. The differential expression of MHC Class II expression in very preterm deliveries (PTL with intact membranes and PPROM CS and V; n = 33 as only 33 blood samples were processed out of the possible 45 total of PPROM and PTL groups) with or without the presence of bacteria (as defined by positive bacterial DNA amplification by broad-range 16S rDNA PCR only) was analysed by Mann-Whitney U test.