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 (R2 = 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. Published 2010. This article is a US Government work and is in the public domain in the USA.