Association between cigarette smoking and the vaginal microbiota: a pilot study

Phase A: cross-sectional study

In 2010–2011, 20 smokers and 20 non-smokers were recruited to a cross-sectional study (single visit) at the Center for Health Behavior Research (CHBR) at the University of Maryland School of Public Health (UMSPH). Inclusion criteria were non-pregnant, non-lactating women, aged 18 to 45 years. Women had to be healthy as determined by medical history, with absence of acute or chronic illnesses, including serious psychiatric disorders or current depression. In addition, participants were excluded if they had used an antibiotic or antimycotic in the prior 30 days or reported a known history of other drug or alcohol dependence in the prior 12 months. Current smoking status was determined by self-report and confirmed by carbon monoxide (CO) exhalation and saliva cotinine measures (described below). Non-smokers also denied smoking in the prior year. Participants completed questionnaires on demographics, nicotine withdrawal, smoking urges, smoking history and tobacco use behavior, depression, nicotine dependence, and reproductive health history.

In a private clinic room, participants self-collected two mid-vaginal swabs (Copan flocked nylon elution-swab and Starplex double headed rayon swab), measured their vaginal pH using the VpH glove (Inverness Medical) and prepared a vaginal smear on a slide for Nugent Gram-stain analysis [29]. The Nugent score reflects the relative abundance of large Gram-positive rods (lactobacilli), Gram-negative and Gram-variable rods and cocci (i.e., G. vaginalis, Prevotella, Porphyromonas, and peptostreptococci), and curved Gram-negative rods (i.e., Mobiluncus). This technique allows assessment of relative numbers of bacteria, allowing for classification of bacterial load. It is based on a linear scale ranging from 0–10. A score of 0–3 is considered normal, 4–6 is an intermediate state, and 7–10 is indicative of BV.

Self-collection of mid-vaginal swabs for microbiota analysis has been validated in several studies comparing self-collected to clinician-collected samples [30–32]. We have also validated the use of a home freezer (−20°C) in a vaginal microbiota study which compared cold chain protocols [33].

Phase B: a pilot longitudinal study of smoking cessation

Phase B participants (n = 9) were recruited using the same criteria as Phase A. Additional inclusion criteria for the Phase B smoking cessation trial were being a current smoker who smoked at least 10 cigarettes/day, no reported period of smoking abstinence greater than three months in the prior year, no history of hypersensitivity to nicotine or adhesives, and motivation to quit smoking. Participants completed questionnaires on demographics, nicotine withdrawal, smoking urges, smoking history and current behavior, depression, nicotine dependence, and reproductive health history at their baseline visit.

Daily procedures at the participant’s home

Each day for the 12-week study, participants self-collected two mid-vaginal swabs (Copan flocked nylon elution-swab and Starplex double headed rayon swab), measured their vaginal pH (VpH glove, Inverness Medical) and prepared a vaginal smear on a slide for Nugent Gram stain analysis [29]. The two vaginal swabs were stored at the participants’ home in the freezer and the smear was stored in a slide container at room temperature. Participants transported samples to the CHBR weekly in coolers with cold packs to maintain frozen samples. The participants also completed daily diaries which documented time-varying factors including sexual activities, menstrual cycle, feminine hygiene, smoking and nicotine patch use.

Weekly procedures at the study site

All participants received one-on-one behavioral counseling each week [34] that consisted of an initial 20-minute session focused on setting the target quit date and preparing to quit (baseline visit) and approximately 10-minute weekly sessions thereafter, focused on quitting or preventing relapse. Participants were given a modified copy of the National Cancer Institute’s booklet entitled “Clearing the Air”, a take-home aid for individuals in smoking cessation programs [35].

During the first ten weeks of the study, participants were asked to use Nicoderm CQ® patches daily as an aid to quitting smoking and were individually trained on proper patch application by the study staff. Participants stepped down the nicotine patch levels according to a known efficacious dosing schedule: a 21 mg patch in weeks 1 to 6, 14 mg patch in weeks 7 to 8, and 7 mg patch in weeks 9 to 10 [36]. There was no prescribed patch use in weeks 11–12 which represented a two-week nicotine-free observation period. At each weekly visit, smoking status and patch use was verified using saliva cotinine and CO measurements.

All participants in Phase A and Phase B provided written informed consent. Ethical approval was obtained from the Institutional Review Boards of the University of Maryland Baltimore (UMB) and the UMSPH.

Biomarkers of smoking

Smoking status was evaluated using both the semi-quantitative NicAlert® saliva cotinine test and Bedfont Micro Smokerlyzer® to measure CO exhalation [37–40]. Breath CO has a 5-6- hour half-life so it is accurate in detecting a 24-hour time frame [41]. The half-life for cotinine is approximately 20 hours and therefore can identify longer term abstinence [42]. The cotinine test can detect six levels of concentrations from 0 to 2000+ ng/ml. In this study, a CO of greater than 8 parts per million (ppm) and a cotinine measurement of greater than 200 ng/ml defined a smoker. When a participant has quit smoking and is actively using the nicotine patch, they are expected to have slightly elevated cotinine levels [43].

Composition of vaginal bacterial communities

DNA extraction from vaginal swabs, PCR amplification and 454 pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA genes were performed as previously described [44] using primers 27 F* [45] and 534R [46].

The QIIME split_libraries.py script (version 1.7.0) [44] was used to demultiplex and quality filter sequence reads that had a perfect match to the unique sample-specific barcode sequence by: 1) removing primer and barcode sequences; 2) truncating reads to the first ambiguous base; 3) truncating reads to the first base of a 25 bp sliding window where the average quality within the window dropped below Q25; and 4) including only reads with lengths between 300–600 bp. Quality filtered reads were de-replicated (99% similarity) using the UCLUST software package [47] and potential chimeric sequences were removed using the UCHIME component of UCLUST [48] prior to taxonomic assignment. Genus level taxonomic assignments were performed by using the RDP Naïve Bayesian Classifier [49], and further species level assignments of Lactobacillus sp. were performed using higher order Markov Chain Lactobacillus species models using the software speciateIT (speciateIT.sourceforge.net) [44]. For each sample, vectors of phylotype proportions were clustered into community state types (CSTs) as previously described by Gajer et al. [50].

Statistical analysis

Fisher’s exact test and logistic regression were used to determine the association between vaginal bacterial CST and smoking status. Factors collected from questionnaires that had been identified on the basis of previous literature and biologic plausibility were evaluated in analyses. To assess differences in the bacterial community structure between smokers and non-smokers, we also utilized the classification and regression tree (CART) analysis. Data were analyzed using STATA/SE 10.0 for Windows (Stata Corporation, College Station, Texas) and the CART was performed in R (R Foundation for Statistical Computing, Vienna, Austria).

Phase A: cross-sectional study

Vaginal microbiota clustered into three community state types (CSTs) [44]; two dominated by Lactobacillus spp. (L. iners (termed CST III), L. crispatus (CST I)), and one lacking significant numbers of Lactobacillus spp. and characterized by anaerobic and strictly anaerobic bacteria (CST-IV) (Figure 1). In prior work we split CST IV into CST IV-A and IV-B, [50] however, all but two of the samples were assigned to CST IV-B, and therefore CST IV-A samples were combined to one CST IV group as in Ravel et al. [44].

CST was significantly associated with smoking status (P value =0.04). Fifty percent of smokers versus 15 % of non-smokers were classified to the low-Lactobacillus CST IV (Table 1). Similarly, fewer smokers had a L. crispatus-dominated CST I (30% in smokers versus 65% in non-smokers). Smokers tended to have higher Nugent Gram-stain scores (P value = 0.02) and higher vaginal pH (P value = 0.13) indicative of BV diagnosis compared to non-smokers. Among women observed to have high cotinine concentration (>1000 ng/ml) and CO exhalation (>11 ppm), 56% were categorized to the low-Lactobacillus CST IV (P value < 0.04). There were also statistically significant trends for both increasing cotinine concentration and CO exhalation with increasing Nugent score (P value = 0.004 and P value = 0.005, respectively). Smokers may also have had more sexual exposures with 83% of smokers versus 15% of non-smokers reporting a history of eight or more sexual partners (p < 0.01).

In the CART analysis, bacteria from the genera Peptostreptococcus and Veillonella were identified as the most important bacterial predictors for smoking status among the 133 bacteria observed.

Phase B: a pilot longitudinal study of smoking cessation

Of the nine women who enrolled in Phase B, four completed the 12-week study, three discontinued at 5–8 weeks, and two dropped out immediately. None of the women in Phase B reported a clinician’s diagnosis of symptomatic BV or antibiotic use during the study. Cotinine and CO levels of enrolled women suggest they maintained nicotine patch use (high cotinine levels) and quit smoking or reduced smoking (low CO levels) (Figure 2). Even among those women who were lost to follow-up at weeks 5–8, cotinine and CO levels suggest good compliance with smoking cessation (data not shown).

Of the four women who completed the 12-week study, three entered the study with high Nugent scores and vaginal microbiota belonging to the low-Lactobacillus CST IV (Figure 2). Of these three women, one woman (ID#1) demonstrated a possible response to smoking cessation. At week 10, coinciding with completion of the nicotine patch and smoking cessation program, her CSTs switched from CST IV to CST III (a L. iners-dominated) and CST I (L. crispatus-dominated) with a concomitant drop in Nugent scores. She did not report a vaginitis diagnosis during the study or in the two months prior to study entry, nor any antibiotic use during the study or any change in behaviors which she documented on the daily diaries. She reported intermittent vaginal odor and discharge on daily diaries but had no such complaints in the final two weeks of observation when she had ceased smoking, finished the nicotine patch protocol and switched to the Lactobacillus-dominated CSTs. The other 2 women (ID#s 2 and 3) fluctuated between CST IV and CST III. The fourth woman (ID#6) had low Nugent scores with L. crispatus-dominated CST I throughout (Figure 2). Among the three women that did not complete follow-up, Nugent scores fluctuated between low and intermediate categories. Longitudinal multivariate modeling was not possible in Phase B due to the small sample size (n = 4) of this pilot study, and therefore, we are limited to descriptive findings.