Observational cohort study of the effect of a single lubricant exposure during transvaginal ultrasound on cell-shedding from the vaginal epithelium.

The outer layers of the vaginal epithelium (VE) are important because they accumulate glycogen which, under optimal conditions, Lactobacillus spp. consume to grow and acidify the vaginal microenvironment with lactic acid. We hypothesized that exposure to lubricant, for example in the conduct of a transvaginal ultrasound (TVUS), may contribute to the shedding of mature epithelial cells, exposing immature cells. Cervicovaginal fluid (CVF) was sampled at four time points by menstrual cup (Softdisc™) from 50 women referred for TVUS, during which a controlled volume of lubricant was applied to the TVUS wand. Samples were collected (1) immediately before TVUS and (2) 6-12 hours, (3) within one week, and (4) two weeks after TVUS. Clinical vaginal lubricants are similar to commercial lubricants, and often have a high osmolality or pH, and contain bactericides such as methylparaben and propylparaben. The number and maturity of epithelial cells in each CVF sample were measured by quantitative and differential fluorimetry (maturity index, MI). Comparisons of cell-counts and maturity were made by paired Wilcoxon signed-rank tests. Among women with a high pre-TVUS MI (> 3), there was a decrease in median cell-count and mean MI in the sample collected 6-12 hours after TVUS (p<0.001, n = 26 and p < 0.001, n = 26, respectively). For these women, cell-count and MI remained lower in the sample collected within the subsequent week (p<0.001, n = 29 and p<0.01, n = 29, respectively), and MI remained lower in the sample collected within two weeks of TVUS (p<0.01, n = 25), compared to the pre-TVUS sample. Among participants with a low pre-TVUS MI (< 3), cell-count was higher in the sample collected within two weeks of TVUS compared to the pre-TVUS sample (p = 0.03, n = 15), but no significant changes in MI were observed. Results were similar when restricted to reproductive-age women. This preliminary data indicates hypertonic vaginal lubricants may increase vaginal epithelial cell shedding.

Introduction

The vaginal epithelium (VE) is the site of immunological [1], microbial [2] and biophysical [3] defenses against reproductive tract infections. The VE consists of approximately 30 layers of cells [4]. Cell proliferation in the basal zone pushes parabasal cells outwards []. In the intermediate zone, maturing cells become flattened, heavily glycogenated, and organized into continuous sheets connected by desmosomes. In the superficial zone, dying cells detach from underlying layers and are shed singly or in aggregates. Glycogenated superficial cells and free glycogen in vaginal fluid may promote colonization by vaginal Lactobacillus spp. [5, 6], which produce lactic acid and acidify the vagina (pH <4) [7]. Lactic acid is a broad-spectrum bactericide and viricide [8-11], and also has a significant anti-inflammatory effect on the VE [12, 13]. Detachment/shedding of the outer layer of mature cells in excess of proliferation/maturation rates will expose the underlying living cells [], and it has been hypothesized that this may influence susceptibility or resistence to reproductive tract infections [14, 15]. The spermicide N9 causes an increase in detachment/shedding of epithelial cells that is associated with increased risk of HIV infection [16].

A. The vaginal epithelium undergoes morphological changes during maturation. As cells mature, they migrate from the basal layer, to the parabasal and intermediate layers to the superficial layer from which they are shed. B. Morphological changes of the vaginal epithelium during maturation include decrease in nuclear and increase in cytoplasmic sizes, which provides the basis of the Maturity Index value (values shown are representative of each layer). C. Shedding of immature cells indicates depletion of superficial cells, and loss of structural integrity of the vaginal epithelial barrier.

Personal lubricants are used to enhance sexual enjoyment [17, 18] and increase acceptability of condom-use [19, 20]. Lubricants also reduce the discomfort of clinical speculum examinations [21, 22] and are often recommended for symptomatic relief of some vulvovaginal symptoms associated with the genitourinary syndrome of menopause [23, 24]. Some lubricants have shown toxicity to epithelial cells in vitro [25-27], in a three-dimensional vaginal epithelium tissue-model [27-29] and in mice [30]. In most cases, the level of toxicity correlates with the hyperosmolality of the lubricant [31, 32] (i.e., the higher concentration of solutes in the lubricant compared with the tissues to which it is applied), as well as the presence of bacteriostatic preservatives (e.g. chlorhexidine, methylparaben and propylparaben).

Evaluating the effects of lubricant use with an observational study design (as compared to randomized trials) is challenging, as women use a variety of commercial lubricants with different composition, at varying doses and duration. We sought to study women presenting for transvaginal ultrasound because it provided the opportunity to observationally study lubricant use incidental to transvaginal ultrasound (TVUS), during which each woman is exposed to the same amount and type of lubricant that is consistently applied, and for approximately the same length of time. We hypothesized that exposure in vivo to a hyperosmolal vaginal lubricant would transiently increase the shedding of mature cells. Here, we report a longitudinal observational study which assessed cell shedding from the VE following a single exposure to lubricant gel during a TVUS exam.

Materials and methods

Participants

Eighty-six participants were recruited between May 2017 and May 2019 to take part in the Gynecology and Lubricant Effects (GALE) Study, a 10-week observational cohort study in Baltimore, MD which sought to investigate changes in the vaginal microenvironment among women exposed to a single use of approximately 3g of vaginal lubricant (E-Z™, Chester laboratory, 2,400 mOsm/kg and pH 5.5) during transvaginal ultrasound (TVUS). An a priori sample size calculation was not conducted for this secondary exploratory analysis. The GALE study was powered to detect an increase in molecular-BV [33] following TVUS.

Women over 18 years of age referred for TVUS at the Diagnostic Radiology and Nuclear Medicine Department at the University of Maryland Medical Center were eligible for the study. Reasons for referral included pelvic pain, pelvic mass and abnormal uterine bleeding []. Exclusion criteria included cancer, diabetes, or other immunosuppressive conditions, diagnosis of laboratory-confirmed genital Chlamydia trachomatis, Trichomonas vaginalis, or Neisseria gonorrhea (BD MAX CT/GC/TV), syphilis (BD Macro-Vue), or HIV (Abbot ARCHITECT HIV Ag/Ab Combo), pregnancy, antibiotic or antifungal use in the month prior to TVUS, and self-reported lubricant use in the week prior to TVUS. Participants self-collected vaginal samples during the week prior to TVUS and for nine weeks following TVUS, and attended three clinical visits at enrollment, within one week of TVUS, and at the completion of the study. No lubricant was used during the three clinical visits, and sample collection occurred prior to the pelvic exam. Participants also completed daily sexual health and behavior diaries and met with study coordinators weekly to drop off samples, which they stored in their home freezers and transported to appointments in a cooler with an ice-pack. Participants gave written informed consent under a protocol approved by the Human Research Protection Office at the University of Maryland Baltimore.

Sample collection and processing

Participants’ cervicovaginal fluid (CVF) was sampled (1) immediately before TVUS (pre-TVUS), (2) at bedtime (approximately 6–12 hours after TVUS) on the day of the ultrasound, (post-TVUS), (3) at a follow-up visit within one week of TVUS (W1-post-TVUS) and (4) within two weeks of TVUS (W2-post-TVUS). CVF was sampled using a disposable menstrual device, the Softdisc™ (The Flex Company, Venice CA). Samples were self-collected by participants except for the W1-post-TVUS sample, which was collected by the clinician at the post-TVUS follow-up visit. As previously described [34], this sampling method permits collection and recovery of relatively large volumes of undiluted CVF. At each sampling, a Softdisc™ was inserted into the vagina and immediately removed, then placed in a 50 mL plastic conical tube. Tubes were immediately frozen at -20°C in the participants’ home freezer and transported frozen to the clinical visits, where they were stored at -20˚C until processing (typically less than three weeks). Previous control experiments suggested that freezing undiluted vaginal fluid samples did not affect cell count or maturity index scores compared to fresh condition (data not shown).

At processing, each tube was thawed at room temperature for ten minutes, then centrifuged at 1250 rpm for three minutes to recover the collected CVF from the Softdisc™. The Softdisc™ was discarded. Approximately 50 μL of the collected CVF was transferred, using a wide bore pipette tip, to a tared 1.5 mL Flex-Tube® (Eppendorf North America, Hauppauge NY). Each tube was reweighed and the exact mass of the CVF sample was calculated. Each sample was diluted with 1 mL of PBS and centrifuged at 10,000 rpm for ten minutes. The supernatant was drawn off and each pellet was resuspended in 1 mL trypsin solution (0.5% v/v trypsin-EDTA in PBS). Tubes were incubated at 37˚C for 40 minutes and vortexed for a few seconds every ten minutes. After incubation each tube was vortexed again, and 3 μL of each trypsinized suspension was added to 3 μL of trypan blue solution (0.4% w/v trypan blue in PBS) in a well of a 12-well microscope slide (Tekdon Inc., Myakka City FL). Complete trypsinization was confirmed by examination at 100x total magnification (i.e., the preparation was a single-cell suspension without cell clumps). The tubes were then centrifuged at 10,000 rpm for five minutes. The supernatant was drawn off as completely as possible without disturbing the pellet. Using a 1 μL to 1 mg equivalence, and the exact mass of each CVF sample processed, ten volumes of PBS were added to the pellet and the pellet thoroughly resuspended by gently pipetting. Of the approximately 500 μL of trypsinized cells in PBS produced, a 100 μL aliquot was stored at -20˚C for later measurement of cell maturity (typically less than three months).

Cell count measurements

The number of cells was measured using bulk fluorimetry and fluorescence microscopy. A 100 μL aliquot of the freshly trypsinized cells in PBS was transferred to a fresh 1.5 mL Flex-Tube®; 100 μL of SYTO™ 13 (Molecular Probes, Eugene OR) staining solution (0.5% v/v SYTO™ 13 in TAE) was added to each tube. The tubes were incubated for 40 minutes, at room temperature protected from light. An 80 μL aliquot of stained cell suspension was loaded into each of two replicate wells of a 96-well spectrophotometry plate. Fluorescence was measured at 488 nm510nm. The measured fluorescence emissions of the two replicates were averaged. To convert fluorescence emission values to cell counts, the remaining 40 μL of stained cells were diluted with TAE to bring the measured fluorescence to approximately 100 (at which the concentration of cells becomes almost countable by microscopy) and the factor of dilution was noted. A further serial three-fold dilution with TAE was made, to give a total of six descending concentrations. A 10 μL aliquot of each concentration was spotted into two replicate wells of a 12-well microscope slide; the slides were partially air-dried, at room temperature and protected from light. Each well was surveyed at 100x total magnification, and individual cells were counted if the well’s cell counts were not too numerous (< approximately 250 cells per well). Every sample yielded at least two wells with countable numbers of cells: the cell count of each well was corrected for the three-fold dilution and the counts averaged. This value was further corrected for the variable dilution factor (necessary to reach 488 nm510nm of approximately 100), to give the estimated cell count in the samples measured by bulk fluorimetry and finally corrected ten-fold to give the cell count in 10 μL native CVF sample.

Cell maturity measurements

As VE cells mature, the ratio of cytoplasm to nucleus increases [35] []. Cell maturity in CVF was measured using differential fluorescence staining of cytoplasm and nuclei [36]. Frozen 100 μL aliquots of trypsinized cells in PBS were thawed on ice and 500 μL of chilled standard saline containing 20% v/v ethanol and 0.025% w/v thymol was added prior to incubation at 4°C for approximately 24 hours, after which the liquid was removed. One mL of acridine orange solution (0.010% w/v in water) was added. The mixture was incubated for ten minutes at room temperature and transferred to a 15 mL plastic conical tube containing 12 mL of 5M salt solution. After centrifugation at 5,000 rpm for five minutes, the liquid was poured off. The cell pellet was resuspended in 400 μL of standard saline. A 180 μL aliquot of this suspension was loaded into two replicate wells of a 96-well spectrophotometry plate. Fluorescence was measured at 488nm650nm (cytoplasm) and at 488nm550nm (nuclei). The ratios of emission650nm to emission550nm for the two replicates were averaged and reported as Maturity Index (MI).

Statistical analyses

The exposure was lubricant use during TVUS. Cell-count and MI of each pre-TVUS sample were compared, pairwise, to each participant’s cell-count and MI of her corresponding post-TVUS, within one week (W1-post-TVUS) and within two weeks (W2-post-TVUS) samples. Comparisons of cell-count and MI are presented as median difference (interquartile range Q1-Q3) and p value from nonparametric Wilcoxon signed rank tests. Not all women provided samples with a useable volume of CVF at all four time points (22% at post-TVUS, 16% at W1-post-TVUS, and 18% at W2-post-TVUS did not have usable volume), therefore n is reported separately for each comparison.

Analysis was conducted in aggregate for all participants, as well as stratified by baseline MI, as baseline MI may restrict the scope of further MI changes. Baseline MI was categorized as low (MI < 3) or high (MI > 3); MI = 3 approximates an equal prevalence of mature and immature cells in a sample. No samples had a MI = 3. Baseline demographic and clinical characteristics were compared between women with low and high pre-TVUS MI using chi-square or Fisher’s exact tests for categorical variables and Wilcoxon rank sum tests for continuous variables. At W2-post-TVUS, cell-count and MI were also compared between women with low versus high pre-TVUS MI using Wilcoxon rank sum tests. A sensitivity analysis was performed by repeating the analysis only with samples from reproductive-age participants. For this analysis, pre-, peri- and post-menopause status was defined by a combination of variables including age, STRAW +10 menstrual cycle criteria [37], and use of hormonal contraception.

Results

Study participant characteristics

Eighty-six women were enrolled in the GALE study, of whom 56 provided samples at all four time points, and 50 contributed a useable sample to determine baseline MI and cell-count for pairwise comparisons. The most common indication for TVUS referral was pelvic pain, followed by assessment of pelvic mass. The most common diagnosis from TVUS was fibroids (13/50, 27%), but a plurality of participants (19/50, 36%) had no significant finding from TVUS. The median number of days between collection of the pre-TVUS sample and the W1-post-TVUS and W2-post-TVUS samples were 4 days (IQR: 3–5 days) and 7 days (IQR: 6–9 days), respectively.

There were a few differences in baseline factors between women with high MI versus low MI at the pre-TVUS sampling (). The median age was not statistically different between the high pre-TVUS MI (31 years) vs low pre-TVUS MI (35 years), although women in the low pre-TVUS MI group were more likely to be peri/post-menopausal. Abnormal uterine bleeding as the indication for referral for TVUS was more common in women with high pre-TVUS MI, although women with a low MI were significantly more likely to have no findings on TVUS. There was no association between hormonal contraception use or menses reported on TVUS day and pre-TVUS MI. Both groups reported similar patterns of sexual activity and menses based on analysis of behavioral diaries on sampling days; however, receptive oral sex was more commonly reported by women with low pre-TVUS MI than by women with high pre-TVUS MI (12% versus 3% of sampling days, respectively, p = 0.03).

Within-woman comparisons

Cell counts

The pre-TVUS median cell count of samples was 17,421/10 μL (IQR 3,616/10 μL to 24,225/10 μL), and the median pre-TVUS MI was 3.29 (IQR 2.01 to 4.11). We found that post-TVUS samples taken 6–12 hours after TVUS contained significantly fewer VE cells than pre-TVUS samples, with a median change in cell count of -6,693/10 μL (IQR -19,768/10 μL to 63/10 μL), p = 0.02, n = 39 []. There was no significant difference in cell count between pre-TVUS samples and W1-post-TVUS samples taken within one week of TVUS, which had a median change in cell count of -1,968/10 μL (IQR -13,112/10 μL to 3,171/10 μL), p = 0.1, n = 42. Similarly, there was not a significant difference in cell count between pre-TVUS samples and W2-post-TVUS samples taken within two weeks of TVUS, which had a median change in cell count of 879/10 μL (IQR -5,383/10 μL to 11,273/10 μL), p = 0.2, n = 40.

Dashed lines represent change = 0.

Stars represent significant pairwise differences from pre-TVUS samples. A. For all participants, the change in VE cell count per 10 μL CVF in samples taken approximately 12 hours later (post-TVUS), within one week (W1-post-TVUS), and within two weeks (W2-post-TVUS), compared to immediately before TVUS. B. For all participants, the change in maturity index (MI) of samples taken approximately 12 hours later (post-TVUS), within one week (W1-post-TVUS), and within two weeks (W2-post-TVUS), compared to immediately before TVUS. C. For only participants with a pre-TVUS MI > 3, the change in VE cell count per 10 μL CVF in samples approximately 12 hours later (post-TVUS), within one week (W1-post-TVUS), and within two weeks (W2-post-TVUS), compared to immediately before TVUS. D. For only participants with a pre-TVUS MI > 3, the change in maturity index (MI) of samples taken approximately 12 hours later (post-TVUS), within one week (W1-post-TVUS), and within two weeks (W2-post-TVUS), compared to immediately before TVUS. E. For only participants with a pre-TVUS MI < 3, the change in VE cell count per 10 μL CVF in samples taken approximately 12 hours later (post-TVUS), within one week (W1-post-TVUS), and within two weeks (W2-post-TVUS), compared to immediately before TVUS. F. For only participants with a pre-TVUS MI < 3, the change in maturity index (MI) of samples taken approximately 12 hours later (post-TVUS), within one week (W1-post-TVUS), and within two weeks (W2-post-TVUS), compared to immediately before TVUS.

Maturity Index (MI)

Considering all participants together, the MI of post-TVUS samples was significantly lower than that of pre-TVUS samples, with a median change of -0.93 (IQR -2.15 to -0.02), p < 0.01, n = 39 []. The difference in MI between pre-TVUS samples and W1-post-TVUS samples was significant, with a median change of -1.01 (IQR -2.29 to 0.45), p = 0.03, n = 42. The difference in MI between pre-TVUS samples and W2-post-TVUS samples was not significant, with a median change of -0.54 (IQR -1.76 to 0.58), p = 0.1, n = 40.

Because the state of the epithelium before exposure to lubricant might affect findings, we stratified analysis into two groups: those whose pre-TVUS MI was less than 3 (n = 18), indicating a paucity of mature superficial cells and predominance of immature intermediate and parabasal cells, and those whose pre-TVUS MI was greater than 3 (n = 32), indicating predominance of mature superficial cells.

Analysis of women with high pre-TVUS Maturity Index

Among participants with high pre-TVUS MI (> 3), post-TVUS samples contained significantly fewer VE cells than pre-TVUS samples, with a median change in cell count of -16,690/10 μL (IQR -20,130/10 μL to -4,997/10 μL), p < 0.001, n = 26 []. Among these participants, W1-post-TVUS samples also contained significantly fewer VE cells than pre-TVUS samples, with a median change in cell count of -6,491/10 μL (IQR -14,439/10 μL to 1,268/10 μL), p = 0.01, n = 29. However, there was no significant difference between the cell count of pre-TVUS samples and W2-post-TVUS samples, with a median change of 731/10 μL (IQR -13,058/10 μL to 8,317/10 μL), p = 0.9, n = 25.

Among participants with high pre-TVUS MIs (> 3), the MI of post-TVUS samples was significantly lower than that of pre-TVUS samples, with a median change of -1.37 (IQR -2.70 to -0.62), p < 0.001, n = 26 []. The MI of W1-post-TVUS samples was significantly lower than that of pre-TVUS samples, with a median change of -1.71 (IQR -2.78 to -0.45), p < 0.01, n = 29; the MI of W2-post-TVUS samples was also significantly lower than that of pre-TVUS samples, with a median change of -0.80 (IQR -2.21 to 0.31), p < 0.01, n = 25.

Analysis of women with low pre-TVUS Maturity Index

Among participants with low pre-TVUS MIs (< 3), there was no significant difference in cell count between pre-TVUS and post-TVUS samples, with a median change of 25/10 μL (IQR -85/10 μL to 20,560/10 μL), p = 0.5, n = 13 []. There was no significant difference in cell count between pre-TVUS sample and W1-post-TVUS samples, with a median change of 1,379/10 μL (IQR 56/10 μL to 8,608), p = 0.2, n = 13. However, there was a significant increase in cell count from pre-TVUS samples to W2-post-TVUS samples, with a median change of 2,586/10 μL (IQR -5/10 μL to 14,031/10 μL), p = 0.03, n = 15.

Among participants with low pre-TVUS MI (< 3), there was no significant difference in MI between pre-TVUS sample and post-TVUS samples, with a median change of -0.28 (IQR -0.81 to 0.70), p = 0.9, n = 13 []. Similarly, there was no significant difference in MI between pre-TVUS samples and W1-post-TVUS samples, with a median change of 0.78 (IQR 0.18 to 0.99), p = 0.09, n = 13; or between pre-TVUS samples and W2-post-TVUS samples, with a median change of 0.30 (IQR -0.69 to 1.56), p = 0.3, n = 15.

Analysis of reproductive-age women

Among 12 reproductive-age participants with low pre-TVUS MI (< 3), there was no significant difference in cell count between pre-TVUS samples and either the post-TVUS, W1-post-TVUS, or W2-post-TVUS samples []. Among 30 reproductive-age participants with high pre-TVUS MI (> 3), cell counts were significantly decreased in the post-TVUS and W1-post-TVUS samples, with a median decrease in cell count of 16,452/10 μL and 6444/10 μL, respectively.

Among 12 reproductive-age participants with low pre-TVUS MI (< 3), there was no significant difference in MI between pre-TVUS samples and either the post-TVUS, W1-post-TVUS, or W2-post-TVUS samples. Among 30 reproductive-age participants with high pre-TVUS MI (> 3), MI was significantly decreased in the post-TVUS, W1-post-TVUS, and W2-post-TVUS samples, with a median decrease of 1.51, 1.79, and 0.72, respectively.

Comparison of women with high and low pre-TVUS Maturity Index at W2-post-TVUS

To assess whether there was any association between the initial condition of the epithelium and the condition of the epithelium two weeks following lubricant exposure, we also compared W2-post-TVUS samples from women with high pre-TVUS MI to samples from women with low pre-TVUS MI.

Cell count

The median cell-count of W2-post-TVUS samples from women with low pre-TVUS MIs was not significantly different than that of women with pre-TVUS MIs > 3, p = 0.2, n = 40 [].

Stars represent significant difference between low MI and high MI samples.

A. Cell-count per 10 μL CVF in samples taken within two weeks of TVUS (W2-post-TVUS) with lubricant exposure, comparing women with a pre-TVUS maturity index < 3 and women with a pre-TVUS maturity index > 3. B. Maturity index of samples taken within two weeks of TVUS (W2-post-TVUS) with lubricant exposure, comparing women with a pre-TVUS maturity index < 3 and women with a pre-TVUS maturity index > 3.

The MIs of W2-post-TVUS samples from women with low pre-TVUS MIs were significantly lower than that from women with high pre-TVUS MIs, with a median W2-post-TVUS MI of 2.09 (IQR 0.68 to 3.27) in women with low pre-TVUS MI and median W2-post-TVUS MI of 3.21 (IQR 2.29 to 4.15) in women with high pre-TVUS MIs, p = 0.02, n = 40 [].

Plots of individual trajectories for MI and cell counts are included in .

Discussion

We hypothesized that a single vaginal lubricant exposure, as part of a TVUS protocol, would promote shedding of VE cells, resulting in loss of fully mature upper layers of VE cells and increased exposure of immature intermediate and parabasal cells.

Our original expectation was that CVF samples taken approximately 12 hours after TVUS would contain more VE cells than samples taken immediately before TVUS, indicating increased detachment/shedding. In fact, participant’s post-TVUS samples contained significantly fewer cells than their pre-TVUS samples, and post-TVUS samples also had a significantly lower MI than paired pre-TVUS samples. One possible explanation for these findings is that rapid detachment/shedding and subsequent clearance of VE cells may have occurred very soon after lubricant exposure, resulting in the observed decreases in cell maturity. This rapid loss hypothesis is supported by published in vivo research showing that rectal epithelial denudation caused by hyperosmolal lubricants increases within 1.5 hours of lubricant application [32]. Future studies could include additional sampling time-points immediately following exposure to lubricant to investigate the rapidity of the shedding response.

The osmolality of the lubricant used in this study (approximately 2,400 mOsm/kg [27]) is in the middle of the range for personal lubricants [32] and the length of exposure (all TVUS procedures were completed in approximately 30 minutes) is reasonably consistent with use of lubricants for sexual activities. The effects observed in this study are, therefore, likely to pertain to non-clinical use of many other personal lubricants as well. Additionally, while it is not possible with this observational study design to separate the effects of lubricant from other possible factors, including effects of the TVUS probe or SoftDiscTM collection device, in vitro studies support that hyperosmolal lubricants alone are capable of reducing epithelial cell barrier integrity and causing tissue morphological damage [29, 38].

The epithelial cell changes observed in this study occurred almost entirely in participants whose pre-TVUS sample had high MI (> 3). It has been hypothesized that exfoliation of mature superficial cells is one mechanism by which the vaginal epithelium provides protection against infection [14]. However, shedding of immature cells reflects a depletion of superficial cells and a loss of vaginal epithelial barrier integrity. Following the single use of vaginal lubricant during TVUS, we observed the shedding of fewer, and more immature, VE cells in both the W1-post-TVUS and W2-post-TVUS samples, suggesting this proposed protective mechanism may be disrupted.

MI did not change significantly in samples from participants with a low pre-TVUS MI (< 3). VE cell-count was significantly increased in the W2-post-TVUS sample, but not in the post-TVUS or the W1-post-TVUS samples. In other words, in women whose VE is already depleted of mature cells, lubricant exposure does not appear to produce much discernable change in the amount or maturity of cells shed as measured by our methodology. Indeed, W2-post-TVUS samples from these women had a significantly lower MI than W2-post-TVUS samples from women with pre-TVUS MI > 3, suggesting that there may be a prior cause for low MI in these women that persisted. Although there was no association between hormonal contraception use or menstrual bleeding and pre-TVUS MI, the low pre-TVUS MI group did have a significantly higher percentage of post-menopausal women, consistent with the expected morphology of the post-menopausal vaginal epithelium [39]. However, when restricting the analysis to reproductive-age women, there were still no significant changes in MI or cell-count among women with a low pre-TVUS MI. Overall, this finding argues against the change in women with high pre-TVUS MI being due to regression to the mean, in which case one would expect to see an equally prevalent increase in MI among women with a low pre-TVUS MI.

A limitation of this study is that we were unable to assess the effect of any pre-study lubrication products. If pre-TVUS samples were affected by prior lubricant use, this could lead to an underestimation of the effect of lubricant used during TVUS on the vaginal epithelium. However, the risk of bias is minimized due to study exclusion criteria that prohibited the use of lubricant in the week prior to TVUS, and this non-use period was longer for most participants. Eighty-four percent reported they had not used a lubricant in the two months prior to enrollment. In addition, the use of a cohort of women seeking clinical care may affect the generalizability of these results to all women, although 37% of participants had no significant findings from TVUS. Fibroids were a common finding on TVUS; however, there are no data relating fibroids to the vaginal epithelium. The vaginal microbiota was not evaluated, and we have previously shown that asymptomatic bacterial vaginosis (BV) is associated with the shedding of fewer, lower MI epithelial cells [15]. While menses did not appear to significantly influence our analysis and there was no association between hormonal contraception and baseline MI, we were unable to model menstrual cycle phase or specific types of hormonal contraception due to sample size limitations. Patton et al found evidence for only small changes in the mean number of epithelial cell layers throughout the menstrual cycle (27.8 on days 1–5, 28.1 on days 7–12, and 26.0 on days 19–24) [4]. Future studies could explore the association between lubricant use and the vaginal epithelium controlling for a wide variety of baseline and time-varying factors. Lastly, we were unable to make comparisons with pre-TVUS MI and cell-count for 22%, 16%, and 18% of samples at the post-TVUS, W1-post-TVUS, and W2-post-TVUS timepoints because the SoftDiscTM did not recover a large enough sample volume for experiments. It is possible that removal of VE cells by the SoftDisc at the pre-TVUS sample may have contributed to the higher proportion of unusable samples at the post-TVUS timepoint. 45% of samples without a usable volume occurred among women with a low pre-TVUS MI, and 55% occurred among women with a high pre-TVUS MI.

Strengths of this study include the longitudinal within-woman analysis, collection of confounding time-varying behavioral data that might affect results and uniform exposure of each woman to the same volume of lubricant product, used in the same way for approximately the same duration of exposure.

Conclusions

These preliminary data raise potentially important questions about the effect of lubricant on the vaginal epithelium. If, as our findings suggest, even a single lubricant exposure during transvaginal ultrasound can cause significant rapid loss of mature VE cells and exposure of immature ones, then lubricant exposure may meaningfully decrease VE barrier integrity. To our knowledge, no direct measurements of the VE have been made before and after lubricant exposure, perhaps one can envision using optical coherence tomography (OCT) which can accurately measure the thickness of the VE in vivo without disrupting its integrity [40]. Future studies should explore whether changes in VE barrier integrity might be associated with an increased inflammation and susceptibility to reproductive tract infections. Certainly, our findings support the importance and urgency of finding less cytotoxic formulations for lubricants and other related feminine products.

Wilcoxon signed-rank test to evaluate changes in maturity index and cell count from pre-TVUS to post-TVUS, W1-post-TVUS, and W2-post-TVUS among reproductive age women, stratified by pre-TVUS maturity index.

(DOCX)

Click here for additional data file.

A and B. Plots of individual trajectories for cell-counts and MI, respectively, in women with a pre-TVUS maturity index > 3. C and D. Plots of individual trajectories for MI and cell counts, respectively, in women with a pre-TVUS maturity index < 3.

(TIF)

Click here for additional data file.

1 Oct 2020

PONE-D-20-18637

Observational cohort study of the effect of a single lubricant exposure on cell-shedding from the vaginal epithelium

PLOS ONE

Dear Dr. Brotman,

Thank you for submitting your manuscript to PLOS ONE, and thank you for your patience in what has been a long review process. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

You will see that Reviewer #1 has raised major concerns around the potential effect of confounders on the validity of the conclusions drawn, in particular the ability without a control group to attribute changes to the lubricant use rather than prior application of the softcup or the ultrasound probe. They also felt that the data were not sufficient to conclude that lubricant use "may increase infection susceptibility", and raised concerns around the effect of hormonal milieu and the microbiome. Any resubmission will need to deal with these concerns around the validity of conclusions very carefully.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

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Rupert Kaul

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review for PLOS ONE (PONE-D-20-18637)

Title: Observational cohort study of the effect of a single lubricant exposure on cell-shedding from the vaginal epithelium

This study is a non-randomized, single group cohort study of non-pregnant women 18+ years undergoing transvaginal ultrasound for a variety of reasons and willing to collect vaginal fluid using a softcup immediately prior to and at times following the transvaginal ultrasound. The authors evaluated the collected vaginal fluid for number maturity index of epithelial cells. Overall, I have several major concerns about premise as outlined by the authors and the study design, which suffers from exposure confounders that impede interpretation of the results.

1. The opening premise is that “the other layers of the vaginal epithelium form a physical barrier of dead and dying cells that protect the inner maturing cell layers.” It is unclear what this premise is based on as there are no references provided to this specific point. This actually seems to be a hypothesis for the function of the outer, dead layers of the vaginal epithelium. Other hypotheses also exist, such as that these layers provide a food source for commensal bacteria and/or that they don’t have a specific function and rather simply are a byproduct of the epithelial cell maturation and sloughing process.

2. The authors hypothesize that a single vaginal exposure to commonly used lubricating jelly “accelerates” shedding of the vaginal epithelium. However, in order to test “acceleration” then there must be a comparison to a non-exposed group. The study was not designed this way, rather the women all had multiple variables that may have impacted the measured outcomes (epithelial cell number and maturation index): 1. softcup application #1, 2. TVUS probe, 3. Lubricant. Further, the vaginal epithelium is clearly hormonally responsive (drastic changes from pre-pubertal-->reproductive age-->menopausal and there are more subtle though still quite perceptible changes in reproductive aged-women through the menstrual cycle as well as with initiation and use/changes in exogenous hormone exposure (hormonal contraceptives)), yet there was no characterization of participants based on age/menopausal status, phase of menses, type of contraception use, etc—all of which are critically important to understand. Thus, the study consisted of an uncharacterized, likely quite heterogeneous in terms of hormonal status, women who all had 3 exposures, and any observed results may have been attributed to any of these exposures, yet the authors conclude that the observed results are based only on the lubricant exposure. This is very problematic.

It would actually be quite interesting to have conducted a study evaluating the epithelial cell numbers and maturity index in cohorts of women separated out by hormonal status: pre-menopausal, naturally cycling in follicular phase; pre-menopausal, naturally cycling in luteal phase; pre-menopausal, on progestogen-only hormonal contraception; pre-menopausal, on combined progestogen/estrogen containing hormonal contraception; post-menopausal. This information could then inform understanding a major factor driving the maturity index, which the authors then used to ‘bin’ the participants. Indeed, the results demonstrate that the only women with significant changes after the ultrasound/lubricant exposure, were those with high MI pre-ultrasound. These women are likely to represent a more hormonally similar grouping, which would be important to know.

The authors state that a strength of the study is the longitudinal within-woman analysis—however the authors do not account for the fact that naturally cycling women and women on cyclic hormonal contraceptives are not in a static state over time with respect to the outcomes of interest—thus, the purported strength is actually a weakness for evaluation of these outcomes. This could have been addressed with a study design that considered hormonal status and timing of collections, however, this study design did not account for these confounding changes.

3. Omission of evaluation of the vaginal microbiota, another significant factor contributing to the vaginal microenvironment. In the discussion, the authors do allude to the microbiota having potential impacts on the MI, which is true—indeed it would be important to understand the contributions to the MI from the hormonal state and microbiological state independently.

4. The statement that a single exposure to vaginal lubricant ‘may increase infection susceptibility’ is overreaching and inappropriate given that the study did not assess this in any way.

Reviewer #2: In this study, the authors used 50 women being exposed to the lubricant EZ, being used for transvaginal ultrasound and took the opportunity to enroll them for pre and several post u/s time points, to see how the lubricant, with a relatively high osmolality, affected cell shedding and maturity. The women self collected cervico vaginal fluid using a Softdisc and froze the samples at home at -20oC until they were brought to the clinic. Clinicians also collected a sample using the same device at 1 visit (Visit 3). Samples were thawed and tested for cell count and maturity index and analyzed by maturity index > or less than 3.

The authors had expected that lubricant exposure would increase shedding of vaginal epithelial (VE) cells. However, at the 6-12 hr time point (1st time post u/s sampled) and several subsequent time points they found less cells, suggesting perhaps they had missed the peak of VE shedding, perhaps within minutes or a few hours, and by the time they sampled what was being shed were deeper layers of less mature cells. Their data do support early increased shedding, that has been observed by others in rectal studies, but they would need to confirm this. Nevertheless, certainly the lubricants are doing something to alter VE balance, finding cells with lower maturity index post u/s in women with higher maturity index (>3) at baseline. The maturity index cut off of 3 was based on an equal prevalence of mature and immature cells in a sample. Having many immature cells (which have low cytoplasm: nuclear ratios) indicates that upper layers (more mature) of VE are lost, potentially exposing deeper layers of VE, reducing protection of the mucosa against abrasion and infections. Hence the interest as to whether the lubricant in this study increased shedding and of which cells.

I found some parts of the manuscript hard to follow and had some questions/clarifications.

Methods

Clarify if exclusion criteria and STI results were self-report, or clinician/lab confirmed.

It is stated that the study continued for 9 weeks, but the paragraph on sample collection and the results only show data for 4 time points (pre, 6-12 h, 1 week, 2 weeks). Was data from post 9 weeks part of the study, and if so clarify where?

What did participants do to carry their -20 oC samples from home to the clinic to keep them frozen?

Cell counts: These were performed on cells thawed from the Softdisc collections. But no DMSO or other cell membrane preservative appears to have been included in the collections/freezing. Could the authors explain why cell counts could be performed using the methods they describe as usually freezing of cells lyses membranes and reduces/alters cell counts? Or perhaps epithelial cells freeze well and can be counted following freezing in absence of DMSO? Similarly for maturity index, is this something that is stable to freeze thaw? If it’s based on cytoplasm/nuclear ratio, if cell membranes rupture during freeze/thaw, is cytoplasm volume altered?

Results.

The text of Fig 2 and 3 legends is a bit confusing. Although the Y axis labels and title of the Fig legend indicate the graphs refer to “change” the text reports “cell count” or “maturity index” not “change in cell count” or “change in maturity index”, so I spent a while looking in the Figs for the actual #s.

Discussion.

The authors could add (end of para 2) that future studies with earlier time points (within a few hours) would confirm the theoretical increased VE shedding that the authors think they missed.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Janet M McNicholl

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Dec 2020

Please see response to reviewer .doc file.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

14 Jan 2021

PONE-D-20-18637R1

Observational cohort study of the effect of a single lubricant exposure on cell-shedding from the vaginal epithelium

PLOS ONE

Dear Dr. Brotman,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

While the revised version has resolved the minor concerns raised by Reviewer #1, our second peer reviewer continues to have substantial concerns regarding the validity of the conclusions drawn. Most importantly, they do not feel that the study setup permits the research team to definitely attribute the genital changes observed to the gel (rather than to the probe). Therefore, they require rewording of the manuscript to reflect this uncertainty, as outlined in their comments below.

Please submit your revised manuscript by Feb 28 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Rupert Kaul

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review for PLOS ONE (PONE-D-20-18637R1)

Title: Observational cohort study of the effect of a single lubricant exposure on cell-shedding from the vaginal epithelium

This study is a non-randomized, single group cohort study of non-pregnant women 18+ years undergoing transvaginal ultrasound for a variety of reasons and willing to collect vaginal fluid using a softcup immediately prior to and at times following the transvaginal ultrasound. The authors evaluated the collected vaginal fluid for number and maturity index of epithelial cells. Overall, the manuscript is greatly improved compared to the original submission.

1. The remaining major concern continues to relate to the study design and attribution of endpoints (epithelial cell number and MI change) solely to the exposure to lubricant, which was not the only exposure and thus attribution cannot be definitively declared as the authors have continued to do. These data were clearly generated from samples collected as a sub-study and as such, the study design is not optimal to definitively state that any observed changes were solely attributable to the gel exposure. This is a common situation for researchers to face and it does not mean that the data are not publishable. It does require clear description and acknowledgement of the multiple exposures, which the current manuscript continues to lack. Even the title of the manuscript directs the reader to believe the conclusion that all of the observed changes are attributable to the single gel exposure, however, there are no controls to demonstrate that the changes did not occur due to the softcup removal of vaginal fluid/cells and/or to the TVUS probe, which were both exposures that all participants in this cohort had. This needs to be clearly acknowledged in the manuscript. I would request a reframing of the title because although there was only a single exposure to gel, the gel was not the only exposure for the vagina. Within the body of the text, the authors did adjust the language of the TVUS probe slightly, to now read “…the effect of a single lubricant exposure incidental to TVUS on cell-shedding from the VE…” This needs to be made even more explicit. I recommend reframing to include the probe in the exposure as follows: “…the effect on cell-shedding from the VE of a single exposure to lubricant gel administered on a TVUS probe … This is truly the exposure. To address the issue with not having a control to assess the impact of the softcup ‘scraping/squeegee’ effect on the vaginal epithelium (for instance, best would be to have had a parallel cohort of women who performed all of the softcup collections at the same times and did not have the gel/TVUS exposure), this needs to be clearly stated in the discussion as a weakness of this study and an acknowledgement that removal of VE cells by softcup squeegee could have contributed to and/or caused the reduction in epithelial cell number and/or MI demonstrated by the data. Indeed, as noted by the authors, this collection method recovers “large volumes of CVF.” The data are what the data are—they simply need to be reported in a non-biased manner with all possibilities for the observed findings considered and discussed.

2. In the abstract, rather than stating, “We determined whether a typical clinical lubricant accelerates shedding of mature epithelial cells, exposing immature cells.”, consider reframing as “We hypothesized that exposure to a typical vaginal lubricant, used for clinical pelvic exams and during transvaginal ultrasonography (TVUS), may contribute to shedding of mature epithelial cells, exposing immature cells. To test this hypothesis, we enrolled women scheduled to undergo TVUS examination for a variety of indications and who were willing to undergo sampling of cervicovaginal fluid immediately before and at three timepoints after the single exposure to lubricant and TVUS (6-12 hours, within one week, and two weeks after).” Please make careful note that the word ‘accelerates’ is problematic as that implies a comparator, and in this study there are none. Same issue in the first sentence of the Discussion, which describes both ‘accelerated’ and ‘increased’ as findings to measures without comparators.

3. Please add references for the statement, “The balance between detachment/shedding and proliferation/maturation processes may influence susceptibility or resistance to reproductive tract infections.”

4. Figure 1. A—Please re-word “as they move up” and “through the parabasal…” as these phrases are non-scientific, vague, and confusing.

5. Figure 1. B—Please do not start with “These” as it is unclear to what this refers. Be specific in your written language.

6. Participants—please add that the exclusion criteria “lubricant use in the week prior to TVUS” was by self-report. Please confirm that at the enrollment visit there was nothing placed in the vagina prior to the softcup collection of vaginal fluid.

7. Was this study registered on ClinicalTrials.gov? If so, please add the registration number to the manuscript.

8. Do you have information about the length of time between the TVUS and the collection of CVF ‘at bedtime on the day of ultrasound’? What was the mean and median number of hours between these two timepoints?

9. Missing 22% of the post-TVUS samples is significant enough to deserve a comment in the weaknesses section of the discussion.

10. Beware that not all ‘hormonal contraception’ is comparable, particularly as it relates to impacting the genital milieu. Women using depot medroxyprogesterone acetate will have markedly thinned epithelia and endometria. Women using hormonal IUDs will also have markedly thinned endometria. Clearly, this study enrolled women using hormonal IUDs. Perhaps adding the various contraceptive methods to Table 1 under ‘Current use of any hormonal contraception’ would be useful.

11. Under ‘Comparison of women with high and low pre-TVUS Maturity Index at W2-post-TVUS’, paragraph starting ‘Maturity index (MI)…’, the verb ‘was’ should be ‘were’ in order to agree with the pleural ‘MIs’.

12. Discussion, paragraph starting, “The epithelial cell changes…” contains a sentence, “These participants began the study with optimal and balanced VE proliferation/maturation and detachment/shedding, the single use of vaginal lubricant incidental to TVUS seems to have caused a significant disruption of that balance.” I don’t see any evidence presented demonstrating “optima and balanced VE proliferation/maturation and detachment/shedding”—what is this based upon? What are the criteria for having an optimal balance? This statement is problematic. Also, the next sentence talks about ‘disruption’, which may or may not be accurate given the absence of data about what is normal at various points in the menstrual cycle. Overall, using language that is purely descriptive, such as “In W1- and W2-post-TVUS samples, we observed persistently low cell numbers and low MIs compared to baseline” and avoiding language that demonstrates bias, such as ‘disruption’, would be preferable. In the discussion it is fair to then hypothesize that the persistently low cell numbers and MI may represent significant and persistent disruption in the epithelium by using language such as this that clearly frames a hypothesis and not stated as fact.

13. Please re-word the vague and grammatically incorrect sentence, “This is not to say that these women’s VE is not affected by lubricant, only that these effects cannot be measured by our methodology.”

14. With respect to the following author responses: “1. Softcup: The softcup was used during all sampling time points and would not impact change in either MI or cell count from the pre-TVUS timepoint to the three post-TVUS time points. In addition, women who experienced a decrease in maturity index or cell count following TVUS had those metrics recover at later timepoints, suggesting that the softcup device is not responsible for the changes observed. Response to Reviewers 2. TVUS probe: It would not be possible to determine the effect of a TVUS probe alone on maturity index or cell count as a TVUS procedure will always include the use of a lubricant in conjunction with the wand. In this sense, the exposure of lubricant + TVUS wand reflects typical use in clinical practice.” 1. Softcup—if the softcup itself (which is an exposure) caused the removal of VE cells, prior to the gel/TVUS probe exposure, the change in MI and/or cell count caused by the softcup would be seen as a change from baseline to follow-up. The removed cells would be in the baseline cup—thus making that measurement ‘high’ relative to the follow-up measurement. 2. Agree that the exposure of the TVUS probe cannot be divorced from the gel, which is precisely why together, they need to be described as the ‘exposure’ and not just the gel.

Reviewer #2: Only request is in the methods section to indicate that the freeze/thaw method is assumed to not affect the assays, based on what the authors said in their response to my question.

Thanks,

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Janet McNicholl

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

16 Feb 2021

See response to reviewers in .doc attachments.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

8 Mar 2021

PONE-D-20-18637R2

OBSERVATIONAL COHORT STUDY OF THE EFFECT OF A SINGLE LUBRICANT EXPOSURE DURING TRANSVAGINAL ULTRASOUND ON CELL-SHEDDING FROM THE VAGINAL EPITHELIUM

PLOS ONE

Dear Dr. Brotman,

Thank you for submitting your manuscript to PLOS ONE. Based on review of the R1 revised manuscript, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor remaining concerns raised by the peer reviewer.

Please submit your revised manuscript by Apr 22 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Rupert Kaul

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

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Reviewer #1: Review for PLOS ONE (PONE-D-20-18637R2)

Title: Observational cohort study of the effect of a single lubricant exposure during transvaginal ultrasound on cell-shedding from the vaginal epithelium

This study is a non-randomized, single group cohort study of non-pregnant women 18+ years undergoing transvaginal ultrasound for a variety of reasons and willing to collect vaginal fluid using a softcup immediately prior to and at times following the transvaginal ultrasound. The authors evaluated the collected vaginal fluid for number and maturity index of epithelial cells. Overall, the manuscript is greatly improved compared to the original submission and the R1 submission. The remainder of the comments are minor and intended to further strengthen this manuscript.

Abstract-

1. Please correct the punctuation in the 1st sentence.

2. The first sentence seems disconnected from the rest of the abstract and as currently written the connection of this sentence to the rest of the abstract may not be clear for all readers. This can be resolved by adding the concept that the outer layers are important because…

3. Please re-word the sentence that starts, “The clinical lubricant, EZTM lubricating jelly (Chester laboratory),…” As written, this sentence is awkward. You could say that vaginal lubricants used clinically commonly have high osmolality, pH~5.5, and contain bactericides.

Introduction-

1. Change ‘upwards’ in 3rd sentence—this directionality does not make physiologic sense. Perhaps ‘outwards’ or ‘more distal’ could substitute.

2. 2nd sentence of 2nd paragraph: recommend alternate word choice for ‘seem necessary’

Methods-

1. Participants, 1st sentence: change the tense of ‘seeks’ to match the rest of the sentence that is appropriately past tense.

2. Previous control experiments (RE: freezing of CVF)—please add ‘(data not shown)’

Table 1-contraception-

1. Were all of the oral contraceptive users on estrogen-containing pills? If so, please change to ‘combined oral contraceptives. If not or unknown, keep as oral contraceptives.

Within-woman comparisons-

1. Paragraph 3, 1st sentence: “…indicating depletion of mature…” Please consider changing ‘depletion’ to ‘paucity’ since depletion suggests a change and there is no baseline sampling and thus no comparator. Use of ‘paucity’ also would parallel your appropriate use of ‘predominance’ rather than suggesting that there is an increase.

Comparison of women with high and low pre-TVUS Maturity Index at W2-post-TVUS

1. Please remove the bias from sentence #1. Consider: ‘To assess correlation between the initial epithelial state (high or low pre-TVUS MI) and epithelial state at W2 post TVUS.

2. Maturity Index�it looks like women with low or high pre TVUS MI still had low or high W2 post TVUS MI. Did you assess the change in MI pre�post TVUS? That is, assessing change from baseline for each woman rather than the inter-participant evaluations that you are reporting here? Indeed, this is what is essentially shown in Supplementary Fig 1. Any significant changes? Based on just an eyeball look, it does not appear so, if true, this is important.

Discussion-

1. Second paragraph, sentence 3—please correct grammar. Also, please take great care to not over-state your findings. This study design is imperfect and the findings are not overly convincing, therefore to state as fact, such as “…resulting in depletion of fully mature cells, reduction of cells available for shedding, and increased exposure/loss of immature cells,…” is overstating the findings of this study.

**********

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Reviewer #1: No

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29 Mar 2021

Please see the response to reviewer .doc.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

1 Apr 2021

OBSERVATIONAL COHORT STUDY OF THE EFFECT OF A SINGLE LUBRICANT EXPOSURE DURING TRANSVAGINAL ULTRASOUND ON CELL-SHEDDING FROM THE VAGINAL EPITHELIUM

PONE-D-20-18637R3

Dear Dr. Brotman,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Rupert Kaul

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

22 Apr 2021

PONE-D-20-18637R3

OBSERVATIONAL COHORT STUDY OF THE EFFECT OF A SINGLE LUBRICANT EXPOSURE DURING TRANSVAGINAL ULTRASOUND ON CELL-SHEDDING FROM THE VAGINAL EPITHELIUM

Dear Dr. Brotman:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Rupert Kaul

Academic Editor

PLOS ONE

Table 1

Demographic and clinical description of study participants with a pre-TVUS epithelial cell maturity index (MI) less than or more than 3.

Characteristic	MI < 3	MI > 3	p-value
	N        Median	N        Median
	(%)        (Q1-Q3)	(%)        (Q1-Q3)
Age (years)	31 (27–51)	35 (31–40)	0.82c
Menopausal status			0.05a
Pre-menopausal	12 (67%)	29 (91%)
Peri/post-menopausal	6 (33%)	3 (9%)
Race			0.69a
Black or African American	12 (67%)	22 (69%)
White	4 (22%)	6 (19%)
Asian	2 (11%)	1 (3.1%)
Multiracial	0 (0.0%)	2 (6.3%)
Other	0 (0.0%)	1 (3.1%)
Current use of hormonal contraception	5 (29%)	13 (42%)	0.65b
Type of hormonal contraception
Oral contraceptive	2 (11%)	4 (13%)
IUD (progestin)	2 (11%)	4 (13%)
Implant	1 (6%)	5 (16%)
Menses reported on day of TVUS	3 (17%)	4 (13%)	0.70a
TVUS Indications
Assessment of pelvic mass	6 (33%)	11 (34%)	0.94b
Localization of intrauterine device	2 (11%)	3 (9.4%)	0.99a
Abnormal uterine bleeding	1 (5.6%)	10 (31%)	0.07a
Screening for malignancy	0 (0.0%)	1 (3.1%)	0.99a
Pelvic pain	10 (56%)	11 (34%)	0.14b
Other	3 (17%)	1 (3.1%)	0.13a
TVUS Findings
Adenomyosis	0 (0.0%)	3 (9.4%)	0.54a
Fibroids	4 (22%)	9 (28%)	0.75a
Cysts	1 (5.6%)	5 (16%)	0.40a
Other	4 (22%)	8 (25%)	0.99a
No significant findings	11 (61%)	8 (25%)	0.01b

The median weight of CVF recovered by clinician collection at W1-post-TVUS was 86 mg (IQR 28–172 mg); the median weight recovered by participant self-collection at W2-post-TVUS was 62 mg (IQR 9–176 mg). There was no significant difference in weight comparing paired clinician-collected and participant-collected samples at W1-post-TVUS and W2-post-TVUS, respectively, p = 0.81.

Differences across groups assessed by Fisher’s exact test (a), chi-square test (b), and Wilcoxon rank sum test (c).