The cross-sectional study (Phase A) suggests that women who smoke cigarettes are significantly more likely to have a vaginal microbiota characterized by low proportions of Lactobacillus spp. In Phase B, with one of three women shifting from a vaginal Lactobacillus-deficient CST to a Lactobacillus-dominated CST during smoking cessation without the aid of antibiotics, we hypothesize that smoking cessation could benefit some women struggling with recurrent BV. In order to establish the causal association that smoking directly affects the vaginal microbiome and recurrence of BV, a larger smoking cessation study design is needed that also includes clinical evaluation for BV.
Several participants in Phase B fluctuated between a low-Lactobacillus state and a L. iners dominated CST with ID#1 appearing to transition fully to a L. iners-dominated CST. There has been recent species-specific attention to L. iners
L. iners is commonly found in the vagina  and has been associated with both BV and healthy states [53–56]. In addition, L. iners is often the first Lactobacillus species to recover after treatment for BV [46, 56]. Our group’s prior work suggest there are strains of L. iners which are highly stable over time while others are associated with a rapidly changing vaginal microbiota tending toward a BV state [46, 50]. Ongoing work is evaluating the genomic heterogeneity of L. iners and if different strains are associated with STI or BV outcomes .
This pilot study provides important preliminary data for future studies. Attrition was high among participants attempting smoking cessation in Phase B (55%), although those who remained in the study were very compliant with smoking cessation, nicotine patch use, attending weekly counseling sessions, and daily collection of vaginal swabs, vaginal pH, vaginal smears and diary entries. Women who were lost to follow-up were also compliant with smoking cessation as indicated by biomarkers. Loss to follow-up rates greater than 30% are not unusual in smoking cessation trials [58–61], and therefore a future study may need to recruit two-fold more women as our study indicates. Weekly visits confirmed that samples were collected in the week stipulated. We advocate that future studies include frequent self-collection of vaginal swabs (daily or 2–3 times weekly) with careful collection of the menstrual cycle, antibiotic use and behavioral data [50, 62, 63]. In addition, longer duration of follow-up (>12 weeks) is likely necessary to capture all women if their microbiome is to rebound to Lactobacillus-dominated CSTs.
There are a number of limitations to this study. The research was designed as a pilot, and therefore, sample size and funds were limited. We were unable to conduct broad testing for sexually transmitted infections, and larger studies may be able to detect other known CSTs, such as CSTs dominated by L. gasseri and L. jensenii. It should also be noted that there are known racial and ethnic differences in nicotine metabolism, which may affect biomarkers of smoking exposure [64–66]. Cotinine and carbon monoxide levels matched self-report of smoking in our study. Also due to sample size, we were unable to control for important confounders. For example, another important factor which may be driving the inverse association between Lactobacillus-dominated CSTs (and also Nugent scores) with smoking status is hormonal contraception (HC). Use of HC in most epidemiological studies has been associated with a reduced risk of bacterial vaginosis . Sixty percent of non-smokers versus 25% of smokers were using HC. The differences in HC use likely reflects clinicians’ prescribing patterns in which smokers are not prescribed HC due to potential cardiovascular side effects . Further, smokers tended to be older (over age 40 and possibly peri-menopausal) and therefore less likely to use HCs. It was not possible for this analysis to use HC in statistical modeling because eight of the 40 women in Phase A declined to answer the contraceptive questions and the sample size became prohibitive. In addition, HC use was not different between smokers and non-smokers in univariate analysis (P value =0.19). It remains important for future studies to collect HC formulation data and control for it in analysis.
It was also surprising to observe 5% of the non-smokers versus 40% of smokers had high Gram stain scores indicative of BV. This could have been the result of recruitment of “healthier” women than an average non-smoking cohort, and therefore, the non-smoking group was biased by women with Lactobacillus-dominated microbiota. However, non-smokers and smokers were recruited using the same methods of advertising and outreach, and more importantly, our data demonstrated that there were statistically significant dose-responses for increasing cotinine concentration and CO exhalation with increasing Nugent score. Numerous studies indicate BV is more common in smokers than non-smokers, smoking has a dose-dependent relationship with BV and the associations persist after controlling for confounders [1–12]. The current study also utilized saliva cotinine and carbon monoxide exhalation measures (which prior studies have not collected) coupled with extensive surveys to properly categorize women as smokers and non-smokers. A larger study with more representative sampling, coupled with biomarker detection of smoking, could resolve this issue.
A major strength of this study is that we are able to provide preliminary data for future studies and self-report of smoking status was confirmed by biomarkers. The study also utilized comprehensive questionnaires, weekly counseling in smoking cessation and high-throughput DNA sequencing technologies. Participants self-collected vaginal swabs daily so the dynamics of the microbiota could be intensively followed in smoking cessation.