Optimising the collection of female genital tract fluid for cytokine analysis in pregnant women

Abstract

IntroductionTo better understand the immunology of pregnancy, study of female genital tract fluid (FGF) is desirable. However the optimum method of collection of FGF in pregnant women for immunological methods, specifically cytokine measurement, is unknown.MethodsA prospective study of HIV-uninfected pregnant women comparing two methods of FGF collection: polyvinyl acetal sponge collection of cervical fluid (CF) and menstrual cup collection of cervicovaginal fluid (CVF). Samples were collected at 3 time points across the second and third trimesters: 14–21, 22–25 and 26–31 weeks. Multiplex chemi-luminescent assays were used to measure: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13 and TNF-α. Optimal methodology for cytokine normalisation (sample weight, volume and total protein) was explored.ResultsAll cytokines were measurable in both fluid types. IL-1β, IL-8 and IL-6 were detected at the highest concentrations (ranking order CF > CVF > plasma). CVF collection was simpler, provided the largest volume of sample (median 0.5 g) with the potential for undiluted usage, and allowed for self-insertion. CF cytokine concentrations were intrinsically associated with sample weight and protein concentration however CVF cytokines were independent of these.ConclusionBoth methods of collection are robust for measurement of FGF cytokines during pregnancy. We recommend CVF collection using a menstrual cup as a viable option in pregnant women for high dimensional biological techniques.

Inflammatory Changes and Composition of Collagen during Cervical Ripening in Cows

“…In the present study, IL-8 levels increased two weeks before parturition, and PMN% peaked one week before parturition. Thus, as in previous reports in humans, IL-8 levels were increased by suppressing the cervical progesterone receptor, suggesting that progesterone and its receptor may be involved in cervical ripening [ 25 , 26 ]. In the future, the relationship between IL-8 variation and progesterone should be clarified by immunostaining of the receptor and measurement of the blood progesterone levels to elucidate the mechanism of cervical ripening further.…”

Lactobacillus-Depleted Vaginal Microbiota in Pregnant Women Living With HIV-1 Infection Are Associated With Increased Local Inflammation and Preterm Birth

“…CVF was extracted from the soft cup as previously published by this group ( Short et al., 2018 ). Multiplex chemiluminescent assays (V-plex Human Pro-inflammatory cytokine panel, Meso Scale Discovery ® (MSD)) were used to measure concentrations of ten cytokines: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, and TNF-α according to the manufacturer’s instructions on duplicate samples at a four-fold dilution in extraction buffer containing Protease-inhibitor cocktail, previously described ( Short et al., 2018 ).…”

Vaginal Microbiota, Genital Inflammation and Extracellular Matrix Remodelling Collagenase: MMP-9 in Pregnant Women With HIV, a Potential Preterm Birth Mechanism Warranting Further Exploration

“…CVF was thawed and extracted from the soft cup as previously described ( Short et al., 2018 ) and diluted in an extraction buffer containing a protease inhibitor cocktail ( Castle et al., 2004 ) in a dynamic range to fall within the standard curve of the intended assay. ELISA assays (Human Quantikine ® , R and D Systems™) were used to measure concentrations of MMP-9 and TIMP-1 according to manufacturer’s instructions (dilution range 40-800-fold).…”