Study design

This retrospective case-control study was performed at the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology (the Ott Institute), St. Petersburg, Russia. Repository cervicovaginal samples from outpatients attending for routine gynecological care, which had been submitted to the Laboratory of Microbiology of the Ott Institute from January 2014 to February 2019 for diagnostic PCR testing for at least one of the non-viral STIs mentioned above, were used in the study. The routine diagnostic testing for C. trachomatis, M. genitalium, T. vaginalis, and N. gonorrhoeae had been performed using PCR assays developed by InterLabService (Moscow, Russia) and DNA Technology (Moscow, Russia), and all the used PCR assays have been previously validated in comparison with PCR assays commercially available internationally [35–38]. The cervicovaginal swabs had been collected, after removing cervical mucus, from the cervix and vagina with either two separate swabs into the same tube of transport media provided by the manufacturers of the used PCR assays (see above) or, most frequently, the same swab from both sites. The samples after routine testing had been stored at − 70 °C. The samples (STI positive cases and STI negative controls) were selected consecutively in reverse chronological order. Patients’ data (age, pregnancy status, and results of STI testing) were obtained from the database of the Laboratory of Microbiology. The inclusion criteria were reproductive age (18–50 years), not being pregnant at the time of sample collection, and not being tested positive for BV and/or any of the four STIs within 2 months prior to enrollment. Sample size was estimated with the use of Fleiss’s criterion with continuity correction at the Open Source Epidemiologic Statistics for Public Health (OpenEpi version 3.01) [39] based on assumed proportions of the main exposure (BV-associated vaginal microbiota) of 15% in controls, 40% in C. trachomatis and M. genitalium cases, and 90% in T. vaginalis cases. These proportions were estimated via testing the first 30 STI-negative and 30 STI-positive samples (C. trachomatis (n = 13), M. genitalium (n = 12), and T. vaginalis (n = 5)) included in the present study. None of the few N. gonorrhoeae samples tested positive during this period was available or eligible. The estimated minimum numbers of positive samples for overall STIs were 57, for C. trachomatis and M. genitalium 41 each, and for T. vaginalis five samples. The study was approved by the Ethical Committee at the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology (approval number 95/2019), and waiver of informed consent was obtained.