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To examine associations between the composition of the vaginal microbiota and genitourinary menopausal symptoms, serum estrogen, and vaginal glycogen.For this cross-sectional study, 88 women aged 40 to 62 years, enrolled in a hot flash treatment trial, provided vaginal swabs and a blood sample at enrollment. Bacterial communities were characterized using 16S rRNA PCR and deep sequencing targeting the V3-V4 region. Quantities of Lactobacillus crispatus and Lactobacillus iners were measured using qPCR. Self-reported genitourinary symptoms included: presence and severity of individual symptoms and identification of most bothersome symptom. Glycogen was measured fluorometrically in swab eluate. Serum estradiol (E2) and estrone (E1) were measured by liquid chromatography/mass spectrometry. Associations between bacteria, symptoms, glycogen, and serum estrogens were tested by linear regression or Wilcoxon signed-rank test, adjusted for multiple comparisons. Comparisons between groups used Kruskall-Wallis or Fisher's exact test.Of the 88 women, 33 (38%) had a majority of Lactobacillus species, whereas 58 (66%) had any Lactobacillus detected. Over half (53%) reported at least one vulvovaginal symptom (most commonly dryness), but symptoms were not associated with the presence of Lactobacillus species. Women with Lactobacillus-dominant communities had higher unconjugated serum estrone, but no difference in vaginal glycogen levels, compared with those with non-Lactobacillus-dominant communities. Higher serum E2 and E1 were not associated with either higher vaginal glycogen or detection of individual genera.Presence of Lactobacillus-dominant vaginal microbiota was not associated with fewer vulvovaginal symptoms. Serum estrone was higher in women with Lactobacillus dominance, but vaginal-free glycogen was not associated with composition of the vaginal microbiota.