A Comparison of Lower Genital Tract Glycogen and Lactic Acid Levels in Women and Macaques: Implications for HIV and SIV Susceptibility

Understanding factors that affect eterosexual transmission of HIV in women is of great importance. Lactobacilli in the lower genital tract of women utilize glycogen in vaginal epithelial cells as an energy source and produce lactic acid. The resultant vaginal acidity is believed to provide protection against HIV infection. Conversely, bacterial vaginosis (BV) is characterized by less lactic acid and a higher pH, and is associated with increased susceptibility to HIV infection. Because vaginal infection of macaques with simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) is used as a model to study HIV sexual transmission, and because previous studies have shown a paucity of lactobacilli in rhesus macaques’ lower genital tract, we compared lactic acid and glycogen levels in the genital fluid of rhesus and pigtail macaques with levels found in humans. The levels of lactic acid were lower in both rhesus (median = 1.2mol lactate/mg protein) and pigtail macaques (median = 0.7mol/mg) compared to women with healthy genital microbiota (median = 4.2mol/mg). Glycogen levels were significantly lower in both rhesus (median = 0.004 lg glycogen/lg protein) and pigtail macaques (median = 0 lg/lg) than in women (median = 0.2 lg/lg). No significant differences in glycogen or lactate levels were observed comparing longitudinally collected samples from cycling pigtail macaques. These data show that the previously reported scarcity of lactobacilli in macaques correlates with low glycogen and lactic acid levels. These findings have important implications for studies of vaginal infection of macaques with SIV or SHIV and further our understanding of how the bacterial microbiota influences HIV infection

Glycogen Levels in Undiluted Genital Fluid and Their Relationship to Vaginal pH, Estrogen, and Progesterone

<div><p>Background</p><p>Colonization of the female lower genital tract with <i>Lactobacillus</i> provides protection against STIs and adverse pregnancy outcomes. Growth of genital <i>Lactobacillus</i> is postulated to depend on epithelial cell-produced glycogen. However, the amount of cell-free glycogen in genital fluid available for utilization by <i>Lactobacillus</i> is not known.</p><p>Methods</p><p>Eighty-five genital fluid samples from 7 pre-menopausal women taken over 4–6 weeks were obtained using the Instead SoftCup^® (EvoFem, Inc., San Diego, CA, USA) by consented donors. Cell-free glycogen and glucose in genital fluids and estrogen and progesterone in blood were quantified.</p><p>Findings</p><p>Glycogen ranged from 0.1–32 μg/μl. There were significant differences between women in glycogen over the observation period. There was a strong negative correlation between glycogen and vaginal pH (r = -0.542, p<0.0001). In multivariable analysis, free glycogen levels were significantly negatively associated with both vaginal pH and progesterone (p < 0.001 and p = 0.004, respectively). Estrogen, glucose, age, sexual intercourse 24 hours prior to visit, and days after the initial visit were not significantly associated with free glycogen levels.</p><p>Conclusion</p><p>Cell-free glycogen concentrations can be very high, up to 3% of genital fluid, and are strongly associated with acidic vaginal pH. However, the fluctuations in glycogen levels in individuals and differences between individuals do not appear to be associated with estrogen.</p></div

Longitudinal vaginal pH, serum estrogen and progesterone.

<p>Vaginal pH (A) was determined utilizing a sterile pH probe as described in Methods. Blood was drawn from the seven subjects shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153553#pone.0153553.g001" target="_blank">Fig 1</a>. Serum levels of estrogen (B) and progesterone (C) were measured by ELISA.</p

Free Glycogen in Vaginal Fluids Is Associated with <i>Lactobacillus</i> Colonization and Low Vaginal pH

<div><p>Objective</p><p><i>Lactobacillus</i> dominates the lower genital tract microbiota of many women, producing a low vaginal pH, and is important for healthy pregnancy outcomes and protection against several sexually transmitted pathogens. Yet, factors that promote <i>Lactobacillus</i> remain poorly understood. We hypothesized that the amount of free glycogen in the lumen of the lower genital tract is an important determinant of <i>Lactobacillus</i> colonization and a low vaginal pH.</p><p>Methods</p><p>Free glycogen in lavage samples was quantified. Pyrosequencing of the 16S rRNA gene was used to identify microbiota from 21 African American women collected over 8–11 years.</p><p>Results</p><p>Free glycogen levels varied greatly between women and even in the same woman. Samples with the highest free glycogen had a corresponding median genital pH that was significantly lower (pH 4.4) than those with low glycogen (pH 5.8; p<0.001). The fraction of the microbiota consisting of <i>Lactobacillus</i> was highest in samples with high glycogen versus those with low glycogen (median = 0.97 vs. 0.05, p<0.001). In multivariable analysis, having 1 vs. 0 male sexual partner in the past 6 months was negatively associated, while BMI ≥30 was positively associated with glycogen. High concentrations of glycogen corresponded to higher levels of <i>L. crispatus</i> and <i>L. jensenii,</i> but not <i>L. iners</i>.</p><p>Conclusion</p><p>These findings show that free glycogen in genital fluid is associated with a genital microbiota dominated by <i>Lactobacillus,</i> suggesting glycogen is important for maintaining genital health. Treatments aimed at increasing genital free glycogen might impact <i>Lactobacillus</i> colonization.</p></div

Subjects’ characteristics at index visit, n = 7.

<p>Subjects’ characteristics at index visit, n = 7.</p

Heat map of bacterial relative abundance by Community State Type and HIV status.

<p>Heat map of relative bacterial abundance for 25 most commonly detected bacterial taxa by (A) community state type (CST) and (B) HIV status. (C) Shannon diversity index calculated for 581 observations.</p

Analysis of factors associated with glycogen concentration (n = 29).

<p>Analysis of factors associated with glycogen concentration (n = 29).</p

Results of mixed effects multinomial modeling^*: Multivariable^{^} adjusted odds of Community State Type.

<p>*Reference category is community type 1.</p><p>^The model is adjusted for all variables presented.</p><p>OR = Odds Ratio; CI = Confidence Interval.</p><p>^$ p<0.01;</p><p>⁺p<0.05.</p><p>Results of mixed effects multinomial modeling^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116894#t005fn001" target="_blank">*</a>}: Multivariable^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116894#t005fn002" target="_blank">^</a>} adjusted odds of Community State Type.</p

Relative Abundance of <i>Lactobacillus</i> Species by Category of Glycogen Concentration.

<p>Free glycogen was analyzed as a binary variable dichotomized about the median and categorically using cutpoints according quartiles of the distribution, as indicated. The distribution of <i>Lactobacillus</i> species relative abundance is summarized by glycogen as a continuous variable by glycogen quartiles. Shaded portions represent interquartile range, horizontal bars represent medians, and whiskers represent 95% confidence intervals. *p<0.05 using extension of the Kruskal-Wallis test with adjustment for clustering among repeated measurements.</p

Longitudinal cell-free glycogen and glucose in undiluted vaginal secretions.

<p>Vaginal fluid was collected from seven pre-menopausal subjects using the Instead SoftCup^® as described in Methods. Glycogen (A) and glucose (B) levels were measured in undiluted vaginal fluid.</p