Intestinal microbiota associate with menstrual cycle-dependent complaints and plasma female hormones in young Japanese women

To evaluate the hypothesis that intestinal microbiota affects menstrual cycle-dependent complaints and plasma levels of female hormones, we conducted an observational pilot study in healthy young females for 12 weeks (2 or 3 menstrual cycles). We recruited 10 subjects (mean age: 20.9 ? 1.3 years, body mass index: 20.67 ? 1.71) from the college student. Fecal samples were collected on a weekly basis. From the samples, we got the DNA extraction by using commercially available kit (QIAamp DNA stool mini kit, Qiagen) and RNase A (Qiagen). Pyrosequencing of 16S rRNA gene was performed with the samples to amplify the V1-V2 region of 16S rRNA gene by using 454 GS JUNIOR sequencing (Roche Applied Science, Penzberg, Germany). Menstrual cycle was classified into 4 phases, an ovulation phase (12 to 16 days before the beginning of menstruation), a luteal phase (1 to 11 days before the beginning of menstruation), a menstruation phase (during menstruation) and a follicular phase (between the menstruation phase and the ovulation phase) based on the Ogino-Knaus theory. Fecal samples also classified the 4 phases.