Longitudinal qPCR Study of 5 Key Vaginal Species Figure 1. The presence and average concentrations of the 5 species during the study in group N. Legend: The chart above represents the total presence (%) of the 5 species during the days that were analysed. In addition, the actual days present versus the total days analysed are also given. The chart below represents the average concentrations (cells/ml) of the 5 species during the days that were analysed. doi:10.1371/journal.pone.0045281.g001 Hydrogen-peroxide-production The hydrogen-peroxide-production was assessed by direct inoculation of 75 ml of ESwab medium onto TMBplus agar plates [41]. After a two day incubation period at 37uC in an anaerobic chamber (BugBox, LedTechno, Heusden-Zolder, Belgium), the plates were exposed to oxygen for 1.5 hours and photographed. Each plate was given a score according to the intensity of the blue color: no blue color = score 0 (no H2O2 production), score 1 (weak H2O2-production), score 2 (strong H2O2-production) and score 3 (very blue, very strong H2O2-production) [42]. Statistical analysis The statistical analysis of the results was performed with the Wilcoxon signed-rank test and the Mann–Whitney U test. Results Based on Gram stained vaginal smears, the 17 volunteers could be allocated into two groups, whereby group N(ormal) (n = 9) comprised the women with a predominantly stable normal VMF (grades Ia, Iab and Ib) and group D(isturbed) (n = 8) comprised the women with predominantly disturbed VMF (grades I-like, II, III and IV, i.e. non-grade I). Within group N, two women could be set further apart from the other 7 subjects, because they were the only two in group N with primarily grade Ib VMF, with L. crispatus only sporadically present. Jellyfish DNA qPCR was carried out for DNA extracts of vaginal swabs taken during 7 menses periods of 7 subjects. DNA- extracts (including a 100-fold dilution) taken from 4 days before the menses, during the menses and 4 days after the menses were tested for qPCR inhibition. No inhibition could be observed. File S1 depicts an overview of the qPCR results, representing the bacterial concentration of the five species tested and the concentration of the G. vaginalis sialidase gene, during the study period (two menstrual cycles) for each of the 17 women, with indication of the following parameters for each sampling moment: the VMF grade (according to Gram stain), the presence of gram positive cocci (GPC) and of yeast cells on Gram stain, the H2O2 production score, occurrence of menses and of sexual intercourse, usage of antibiotics and the hygienic practices. Bacterial concentrations during the menstruation cycle Lactobacillus crispatus. In group N, 7 of the 9 women had high concentrations of L. crispatus (average log7-log9 cells/ml). For two women of group N with primarily grade Ib VMF (#4, #20), L. crispatus was only sporadically present, but L. iners was present in high concentrations. For 6 of the 7 women with predominantly L. crispatus, the concentrations of this species declined significantly, i.e. 100-fold on average, during the menses . Within group D, different types of disturbances of the VMF and different prevalences of L. crispatus were observed. The two women PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45281