Presented at the Neonatal Society 2003 Autumn Meeting.
Cahill R1, Sullivan MHF4, Steel J4, Kennea N3, Feldman R2, Dougan G1, Edwards AD3
1 Centre of Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, SW7 2AZ, UK
2 Vmax Limited, 545 Eskdale Road, Winnersh Triangle, Wokingham, Berks, RG41 5TU, UK
3 Department of Paediatrics, 4Department of Obstetrics and Gynaecology, Imperial College, London, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK
Purpose: An association between intrauterine infection, inflammation in fetal membranes or placenta, fetal inflammatory responses and early delivery has been found in many studies. The identification of different organisms found in pregnancy tissues after preterm labour, led to the theory that pre-term labour is a generic response to organisms, rather than being caused by some particular pathogens. These studies however are limited by traditional technologies as a full screen of organisms requires many different culture methods, and some organisms are notoriously difficult to culture reproducibly. In this study we have use molecular approaches to (a) localise any organisms in fetal membranes and (b) identify these organisms.
Methods: Fetal membranes were collected from a cohort (1 calender year) of preterm deliveries (=32 weeks gestation) (n=50). Fetal membranes were also obtained after delivery at term by elective Caesarean section (n=26) or after normal vaginal delivery (n=26). Organisms in fetal membranes were visualised by fluorescent in situ hybridisation (FISH) with a probe generic for 16S ribosomal RNA (rRNA) of many organisms. To identify the organisms present in fetal membranes DNA was extracted from frozen tissue samples, and the integrity confirmed by PCR for b-actin. Universal degenerate primers were designed and used to amplify bacterial 16S ribosomal DNA (rDNA). These PCR products were sub-cloned, amplified and sequenced. This data was compared with published 16S rDNA sequences by BLAST search. A probe for generic conserved Fusobacterium spp. 16S rDNA was designed and used for FISH.
Results: Fetal membranes contained organisms after term (19/26) and preterm (10/12) elective delivery, as well as after term labour (12/26), preterm labour (13/14) or prolonged premature rupture of the fetal membranes (PPROM) (18/22). There were no statistically significant differences between these patient groups. PCR was performed on a sub-group of 42 samples (6 term, 36 preterm). Organisms could be identified in a minority of tissues (15/42); this applied to 10/12 PPROM tissues, 4/14 preterm labour tissues, 1/11 preterm no labour tissues, and 0/6 term tissues. The most common organism was Fusobacterium nucleatum (9 samples). 7 of these were in PPROM tissues, giving an incidence of 70% in this patient group. FISH with a probe to a generic Fusobacterium 16S rDNA sequence visualised these organisms in PCR+, but not PCR- tissues.
Conclusions: These preliminary data suggest that the presence of organisms in fetal membranes is not sufficient to cause preterm labour. However, Fusobacterium nucleatum within the pregnant uterus may be an important cause of PPROM.