Acidform was found to be more irritating than HEC placebo gel, with a greater proportion of mild genital symptoms. A similar increased rate of mild AEs was observed in an earlier study when Acidform was compared to K-Y Jelly 
. Despite the findings of mild irritation associated with Acidform use, we observed no increase in pro-inflammatory cytokines or chemokines, although there was a significant decrease in the concentration of the anti-inflammatory protein, IL-1ra in CVL obtained from participants who applied Aciform compared to HEC gel. Whether the mild irritation and the decrease in IL-1ra observed in this study portend a risk for mucosal inflammation with more prolonged exposure to Acidform requires further study.
The median pH of the cervix and vagina 2 hours after a clinician administered Acidform gel was 3.5–3.7, which was associated with increased E. coli
bactericidal activity, and is low enough to completely immobilize spermatozoa if the pH is maintained following coitus. However, no significant differences in bactericidal activity or pH at the lateral vaginal wall, posterior fornix or cervix were observed at any other time-point, which suggests that the acid-buffering effects do not persist. The average time elapsed between gel dosing and CVL sampling for the Day 7 and 14 study visits was 13 and 16 hours, respectively. These findings suggest that Acidform may be effective as a topical contraceptive and may prevent vaginal E. coli
colonization, which has been observed after sexual intercourse with and without a condom 
. We recently demonstrated an inverse correlation between vaginal E. coli
colonization and bactericidal activity of CVL, indicating that the ex vivo
activity may translate to protection against colonization 
. However, the transient nature of the responses observed in the current study suggests that the product would have to be applied shortly prior to sex to be effective. The need to apply gel shortly prior to intercourse, the brief duration of activity, and the potential to develop genital irritation may limit adherence, acceptability and efficacy of Acidform gel as a topical contraceptive.
Despite potent antiviral activity in a mouse model 
, there was no significant increase in anti-HSV activity of CVL following Acidform gel use. These findings may reflect the pH of CVL (mean of 4 at 2 hours and >4.5 at all other visits), which was likely not sufficient to inhibit HSV. We previously demonstrated that HSV inactivation in vitro
is rapid and substantial at pH 3.5, but less effective at pH of 4.5 
. The anti-HSV activity of CVL correlates with concentrations of several immune mediators including lactoferrin, IL-8 and IgA; each of these was lower following Acidform use and this may have also contributed to the absence of any increase in anti-HSV activity. Notably, lactoferrin, an anti-bacterial glycoprotein produced by epithelial cells and neutrophils has been shown to inhibit HSV in vitro
. While we observed no loss in the anti-HSV activity of CVL in this study, the lower levels of lactoferrin and other immune mediators following Acidform gel application suggest that more prolonged or frequent exposure could potentially interfere with mucosal defense. Further studies are needed to determine the clinical significance of these findings.
The healthy human vaginal microbiome is dominated by Lactobacillus
species at high concentrations, including L. crispatus
and L. jensenii
, which maintain acidic vaginal pH by producing lactic acid. BV occurs when these beneficial vaginal lactobacilli are replaced by overgrowth of commensal vaginal anaerobes 
. In a previous Phase I study, there was no significant increase in hydrogen peroxide-producing lactobacilli following repeated vaginal application of Acidform gel, as measured by semiquantitative vaginal cultures 
. In the present study, qPCR was utilized to assess the impact of Acidform gel on absolute quantities of protective lactobacilli and BV-associated organisms. There was no increase in the concentrations of beneficial lactobacilli following use of Acidform gel. Notably, there was a decrease in the amount of G. vaginalis
recovered following Acidform, but not HEC gel application, on Day 14. Gardnerella vaginalis
is almost always present in the vaginas of women with BV, but is also found in 70% of women without BV 
. These findings suggest that the product may provide at least some colonization resistance against G. vaginalis
, although further studies with sexually active women are needed to determine the potential role Acidform may play in promoting a healthy microbiome. Future studies should include more comprehensive studies of the effects of Acidform on the vaginal microbiota.
This is the first study to apply the DSA to polyethylene applicators used to insert gel more than once daily. The DSA has been previously studied with methylcellulose, HEC, Carraguard, and PRO 2000 gels inserted once daily from low-density polyethylene (LDPE) applicators, with sensitivity and specificity of >90% 
. The sensitivity of the assay was decreased when studied with VivaGel gel applied twice daily from polypropylene applicators 
. The difference may have been due to gel remaining in the vagina from the previous insertion. Alternatively, the sensitivity and specificity of the DSA may vary with the type of applicator used (polyethylene vs. polypropylene). In a recent study of tenofovir gel applied once daily with polypropylene applicators 
, the sensitivity of the DSA was >90% but the specificity was <70%, suggesting that staining of polypropylene applicators may not be as effective as that of polyethylene. In the current study of twice daily dosing of Acidform or HEC from polyethylene applicators, the sensitivity of the DSA for two observers was 87% and 84%, respectively and the specificity was 69% and 85%, respectively. These results suggest that the DSA may not be an effective method to assess applicator use when gel is administered more than once daily, despite the type of applicator used. Important limitations of the DSA are the differences in technique used by different groups and the high degree of interobserver variability 
, indicating the need for standardization and training to reduce subjectivity. These findings also highlight the need for better markers of adherence. A recent study suggests that direct inspection of polypropylene applicators under ultraviolet light may provide a reliable assessment of adherence 
In summary, Acidform gel was associated with a decrease in pH and an increase in E. coli bactericidal activity 2 hours after gel application as well as a decrease in the concentration of G. vaginalis recovered on Day 14, suggesting that it may promote a healthier vaginal microbial environment. However, twice daily application of Acidform was associated with mild irritation and lower CVL levels of several immune mediators compared to HEC placebo gel. Determination of the clinical significance of these findings requires additional safety studies of this candidate non-hormonal contraceptive with sexually active populations and more prolonged product exposure.