Effect of Oral Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the Vaginal Microbiota, Cytokines and Chemokines in Pregnant Women

Abstract

Spontaneous preterm birth is associated with vaginal microbial dysbiosis. As certain strains of lactobacilli help restore homeostasis in non-pregnant women, the goal was to determine the effect of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 administered orally, twice daily for 12 weeks on the vaginal microbiota, cytokines and chemokines of low-risk pregnant women. A double-blind, placebo-controlled, randomized trial comparing probiotic lactobacilli to placebo daily was performed in 86 asymptomatic pregnant women who had an Intermediate or Bacterial Vaginosis Nugent score at 13 weeks. After drop outs, 32 women receiving probiotics and 34 receiving placebo completed the study. The Nugent score returned to normal in 30% of the women in both groups at 28 weeks and was maintained until 35 weeks. The majority of subjects had normal pregnancy outcomes. Ninety-three bacterial species were detected at 13 weeks, with Lactobacillus iners, Lactobacillus crispatus, Gardnerella vaginalis and Atopobium vaginae being the most abundant across pregnancy. There was no difference in the Shannon diversity index between the probiotic and placebo groups at 13, 28 or 35 weeks. Almost all subjects consumed fermented foods and many of the organisms in the vagina are also known to be present in fermented foods. Interleukin-4 in the placebo group and Interleukin-10 in both probiotic and placebo groups increased slightly at 28 weeks but were not different at 35 weeks when compared to 13 weeks. In conclusion, this study showed no adverse issues resulting from 12 week use of probiotic Lactobacillus strains GR-1 and RC-14 during pregnancy in women at low risk for premature birth. The vaginal microbiota demonstrated flux irrespective of this oral probiotic administration.

Keywords: bacterial vaginosis; chemokines; cytokines; microbiota; pregnancy; probiotics.

Figure 1

Consort flow chart of pregnant women enrolled in the study.

Figure 2

Vaginal microbiota clustered by bacterial similarity in pregnant women prior to treatment, at 13 weeks of gestation (n = 66). Each bar represents the vaginal microbiota of a single woman and corresponds to the participant identification (ID) number labeled in the dendogram, clustered using average linkage cluster analysis. Species found in >1% abundance are represented by a unique color. Species with <1% abundance in the sample are pooled into a single fraction at the top of the bar in grey color. Women who have a single bacterial species which dominated more than 40% of their vaginal microbiota are identified with a color dot below their identification number that corresponds to the dominant species (Dark green, Atopobium vaginae, n = 4; Very light blue, Lactobacillus (L.) jensenii, n = 1; blue, L. iners, n = 12; light blue, L. crispatus, n = 9 and red, Gardnerella vaginalis, n = 4). Black rectangles are used to denote women with a bacterial vaginosis (BV) Nugent score, and white rectangles are used to identify women with an Intermediate Nugent score.

Figure 3

Vaginal microbiota clustered by bacteria similarity in pregnant women with a BV (n = 24) or an Intermediate (n = 42) Nugent score prior to treatment, at 13 weeks of gestation. Each bar represents the vaginal microbiota of one woman and corresponds to the identification number labeled in the dendogram, clustered using average linkage cluster analysis. A unique color is used to represent species found in >1% abundance. Species with <1% abundance are pooled into a fraction at the top in grey color.

Figure 4

Vaginal microbiota across pregnancy clustered by bacteria similarity in pregnant women who received either placebo (n = 34) or probiotic (n = 32) treatment. Y Axis = Microbiota Fraction. Each bar represents the vaginal microbiota of a single woman and corresponds to the identification number labeled in the dendogram, clustered using average linkage cluster analysis. Species found in >1% abundance are represented by a unique color and species that have <1% abundance are pooled into a single fraction at the top of the bar in grey color. Women were aligned in the same vertical column at 13, 28 and 35 weeks of gestation. Women who have undergone preterm birth (PTB) (n = 2) in the probiotic group are denoted with white squares. There was no effect due to BV or Intermediate status.

Figure 5

Shannon diversity index (SDI) across gestation in pregnant women who received either placebo or probiotic treatment. Results are mean values ± standard deviation (SD) and are expressed in ratios. Comparisons between the probiotic (n = 32) and placebo (n = 34) groups at 13, 28 and 35 weeks of gestation were assessed with Two-Way Repeated Measure Analysis of Variance (ANOVA) followed by the Holm–Sidak post hoc test (p > 0.05).

Figure 6

Concentrations of cervico-vaginal cytokines IL-4, IL-10 and CSF3 across gestation in pregnant women who received either placebo or probiotic treatment. Results are mean values ± SD and are expressed in picogram per milliliter. Comparison between the placebo group (n = 33) and the probiotic group (n = 31) was assessed with the Generalized Estimation Equation model in R. * = p < 0.05.