Every year, 60000 babies are born prematurely in the UK (7% of all births) [1]. Complications arising from preterm birth (PTB) significantly contribute to neonatal death. Greater understanding of the mechanisms leading to spontaneous PTB are needed to improve management strategies and pregnancy outcomes. Focusing on the involvement of the cervico-vaginal environment and risk of inflammation and infection related to spontaneous PTB, we have studied cervico-vaginal fluid (CVF) components and their impact on vaginal epithelium integrity. Proteases are proteolytic enzymes involved in peptide bond hydrolysis that can disrupt tight junctions via the PAR2 receptor [2]. An association between reproductive tract cell tight junctions and PTB has also been reported, as a decrease in claudins is linked with preterm and term cervical ripening [3]. Twenty-two pregnant women, between 14-23+6 weeks gestation, were recruited to a prospective cohort study (INSIGHT REC No. 13/LO/0393; written informed consent) and provided CVF via low vaginal swabs (LVS). Proteins were quantified from CVF using a microBCA Protein Assay as per protocol (ThermoFisher). Protease activity was measured using a fluorescent assay as per protocol (ThermoFisher). To mimic the acidic vaginal environment protein and protease assays were repeated on n=6 samples using a reproductive buffer (pH 4) (60 mM potassium phosphate and 20 mM sodium chloride) [4] instead of PBS (pH 7). Cultured vaginal epithelial cells (cell line VK2, passage 53-56; density 15×104) were cultivated in 12 permeable Transwell inserts and monitored for eight days. Transepithelial electrical resistance (TEER) was measured using the EVOM2 voltohmmeter. Permeability assays were carried out 24 hours after the TEER measurement using a sodium fluoride dye. Protein was quantified and detected in twenty-two CVF samples taken from LVS. Proteases were undetectable in neat CVF samples and in samples normalised to contain 10 µg/µL total protein. In six samples, lowering the pH from pH 7 to pH 4 reduced the protein concentration; this did not enhance any detection of protease activity. However, the addition of CVF (from n=9 different samples) to VK2 cells reduced the TEER and increased cell permeability. We provide evidence that samples from LVS cannot be used to analyse CVF proteases. This may be due to the collection and/or processing technique, or because the low vagina does not contain pure CVF and is contaminated with other cells and debris. However, we do show that CVF from LVS impacts tight junction integrity as it lowers the TEER of cells and increases cell permeability. The presence of short chain fatty acids amongst other enzymes and proteins may be responsible for the reduction in tight junction integrity, but this requires further investigation.