## 2 Materials and Methods
### Clinical Study Protocol and Sample Collection  
Forty-eight female subjects (aged between 22 and 53) diagnosed with bacterial vagnosis were initially enrolled in this randomized, double-blind study. This study was approved by the Institutional Review Board of Kuo General Hospital, Tainan, Taiwan (IRB No. A-16 K002, NCT 03116789 in ClinicalTrials.gov) and performed in accordance with the relevant guidelines and regulations. The inclusion criteria were as follows--Nugent score 4-10, age ranging from 20-55 years, normal menstruation cycle (18-35 days), without sexual intercourse within 72 h before each visit. The exclusion criteria were as follows--pregnancy, lactation, allergy of the probiotic product in this study, abnormal genital tract bleeding, congenital or acquired immunodeficiency, diabetes mellitus, mental disorders, cancers, applications of NuvaRing hormonal contraceptive vaginal ring, mechanical contraceptives (e.g., diaphragms, intrauterine contraceptive insert, except condom), and hormonal preparations (e.g., Vagifem, Ovestin and vaginal estrogens in reproductive period), mycotic vagnitis, any antibiotic and corticosteroid use during the trial or four weeks before the first visit, and participation in another clinical study. Subjects were informed of the purpose of the trial and gave their signed informed consents before participation. Upon diagnosis, the eligible subjects were randomly allocated to 2 groups: VGA-1 (\(n\) = 21, a combination of \(L\). _Hammosus_ GMNL-74 and \(L\). _acidophilus_ GMNL-185 as 1 \(\times\) 10\({}^{10}\) CFU) and VGA-2 (\(n\) = 22, a combination of \(L\). _Hammosus_ GMNL-680 and \(L\). _plantarum_ GMNL-682 as 1 \(\times\) 10\({}^{10}\) CFU), which received two oral capsules, once daily for 28 days. For allocation of the participants, the randomization sequence created using the SAS version 9.4 statistical software by Bestat Pharmaservices Corp. (Taipei, Taiwan) was used. The primary outcome of this clinical trial is the Nugent score. The study protocol is provided in the Supplementary Materials (Supplementary protocol) and the VGA-1 or VGA-2 supplements were prepared by GenMont Biotech Inc. (Tainan, Taiwan). The subjects were asked to return 2 weeks and 1 month later, for a gynecological examination to evaluate the outcome. At the first visit (baseline), the information of demographic characteristics, gynecological history, and current genital symptoms were collected. The subjects received probiotic capsules for the first 14 days at the first visit (baseline), returned their unused capsules and received probiotic capsules again for the next 14 days at 2 weeks (W2), and eventually gave back the unused capsules to principal investigators at 4 weeks (W4). Subject with poor medicine adherence (defined as less than 75%) were excluded from the study. BV recurrence of subjects after probiotic treatment, over the course of 3 months, was followed by reviewing the medical record or over the telephone. During the trial, vaginal samples were collected at the first visit (baseline), 2 weeks (W2), and 1 month (W4) after oral consumptions of VGA-1 or VGA-2. At each visit, four dry swabs (COPAN Diagnosis Inc., Murrieta, CA, USA) from the vaginal wall were individually taken--the first sample was used for genomic DNA extraction to determine the vaginal microbiota, the second one was used for RNA extraction to evaluate vaginal inflammatory conditions, the third one was used for Gram staining to evaluate the vaginal flora, according to the Nugent score, and the last one was used for the BVBlue test to diagnose bacterial vaginosis. Samples of the first two vaginal swabs were immediately stored at \(-20\)\({}^{\circ}\)C until assayed, while samples of the last two vaginal swabs were examined by the experienced microscopists at the Kuo General Hospital, within 2 h of collection. The swab collection sequence and the corresponding examination performed was based on the pretest results, as described in the Supplementary Information (Supplementary Table S1).