For over a century it has been well documented that bacteria in the vagina maintain vaginal homeostasis, and that an imbalance or dysbiosis may be associated with poor reproductive and gynecologic health outcomes. Vaginal microbiota are of particular significance to postmenopausal women and may have a profound effect on vulvovaginal atrophy, vaginal dryness, sexual health and overall quality of life. As molecular-based techniques have evolved, our understanding of the diversity and complexity of this bacterial community has expanded. The objective of this review is to compare the changes that have been identified in the vaginal microbiota of menopausal women, outline alterations in the microbiome associated with specific menopausal symptoms, and define how hormone replacement therapy impacts the vaginal microbiome and menopausal symptoms; it concludes by considering the potential of probiotics to reinstate vaginal homeostasis following menopause. This review details the studies that support the role of Lactobacillus species in maintaining vaginal homeostasis and how the vaginal microbiome structure in postmenopausal women changes with decreasing levels of circulating estrogen. In addition, the associated transformations in the microanatomical features of the vaginal epithelium that can lead to vaginal symptoms associated with menopause are described. Furthermore, hormone replacement therapy directly influences the dominance of Lactobacillus in the microbiota and can resolve vaginal symptoms. Oral and vaginal probiotics hold great promise and initial studies complement the findings of previous research efforts concerning menopause and the vaginal microbiome; however, additional trials are required to determine the efficacy of bacterial therapeutics to modulate or restore vaginal homeostasis.
Colonization of the endometrium by pathogenic bacteria, ascending from the lower female reproductive tract (FRT), is associated with many gynecologic and obstetric health complications. To study these host-microbe interactions in vitro, we developed a human three-dimensional (3-D) endometrial epithelial cell (EEC) model using the HEC-1A cell line and rotating wall vessel (RWV) bioreactor technology. Our model, composed of 3-D EEC aggregates, recapitulates several functional/structural characteristics of human endometrial epithelial tissue including cell differentiation, junctional complexes/desmosomes, microvilli, membrane-associated mucins and Toll-like receptors (TLRs). TLR function was evaluated by exposing the EEC aggregates to viral and bacterial products. Treatment with polyinosinic-polycytidylic acid (poly(I:C)) and flagellin, but not with synthetic lipoprotein (FSL-1) or lipopolysaccharide (LPS), significantly induced proinflammatory mediators in a dose dependent manner. To simulate ascending infection, we infected EEC aggregates with commensal and pathogenic bacteria: Lactobacillus crispatus, Gardnerella vaginalis and Neisseria gonorrhoeae All vaginal microbiota and N. gonorrhoeae efficiently colonized the 3-D surface localizing to crevices of the EEC model and interacting with multiple adjacent cells simultaneously. However, only infection with pathogenic N. gonorrhoeae significantly induced proinflammatory mediators and significant ultrastructural changes to the host cells relative to other bacteria tested. This latter observation is consistent with clinical findings and illustrated the functional specificity of our system. Additionally, we highlighted the utility of 3-D EEC model to study N. gonorrhoeae pathogenesis using a well-characterized ΔpilT mutant. Overall, this study demonstrates that the human 3-D EEC model is a robust tool for studying host-microbe interactions and bacterial pathogenesis in the upper FRT.
We have developed an in vitro human vaginal epithelial cell (EC) model using the innovative rotating wall vessel (RWV) bioreactor technology that recapitulates in vivo structural and functional properties, including a stratified squamous epithelium with microvilli, tight junctions, microfolds, and mucus. This three-dimensional (3-D) vaginal model provides a platform for high-throughput toxicity testing of candidate microbicides targeted to combat sexually transmitted infections, effectively complementing and extending existing testing systems such as surgical explants or animal models. Vaginal ECs were grown on porous, collagen-coated microcarrier beads in a rotating, low fluid-shear environment; use of RWV bioreactor technology generated 3-D vaginal EC aggregates. Immunofluorescence and scanning and transmission electron microscopy confirmed differentiation and polarization of the 3-D EC aggregates among multiple cell layers and identified ultrastructural features important for nutrient absorption, cell-cell interactions, and pathogen defense. After treatment with a variety of toll-like receptor (TLR) agonists, cytokine production was quantified by cytometric bead array, confirming that TLRs 2, 3, 5, and 6 were expressed and functional. The 3-D vaginal aggregates were more resistant to nonoxynol-9 (N-9), a contraceptive and previous microbicide candidate, when compared to two-dimensional monolayers of the same cell line. A dose-dependent production of tumor necrosis factor-related apoptosis-inducing ligand and interleukin-1 receptor antagonist, biomarkers of cervicovaginal inflammation, correlated to microbicide toxicity in the 3-D model following N-9 treatment. These results indicate that this 3-D vaginal model could be used as a complementary tool for screening microbicide compounds for safety and efficacy, thus improving success in clinical trials.
Alternative strategies for controlling the growing herpes simplex virus type 2 (HSV-2) epidemic are needed. A novel class of immunomodulatory microbicides has shown promise as antiherpetics, including intravaginally applied CpG-containing oligodeoxynucleotides that stimulate toll-like receptor 9 (TLR9). In the current study, we quantified protection against experimental genital HSV-2 infection provided by an alternative nucleic acid-based TLR agonist, polyinosine-poly(C) (PIC) (TLR3 agonist). Using a protection quantification paradigm, groups of mice were PIC treated and then subdivided into groups challenged with escalating doses of HSV-2. Using this paradigm, a temporal window of PIC efficacy for single applications was defined as 1 day prior to (prophylactic) through 4 h after (therapeutic) viral challenge. PIC treatment within this window protected against 10-fold-higher HSV-2 challenges, as indicated by increased 50% infectious dose values relative to those for vehicle-treated controls. Disease resolution and survival were significantly enhanced by repetitive PIC doses. Using optimal PIC regimens, cytokine induction was evaluated in murine vaginal lavages and in human vaginal epithelial cells. Similar induction patterns were observed, with kinetics that explained the limited durability of PIC-afforded protection. Daily PIC delivery courses did not generate sustained cytokine levels in murine vaginal fluids that would be indicative of local immunotoxicity. No evidence of immunotoxicity was observed in selected organs that were analyzed following repetitive vaginal PIC doses. Animal and in vitro data indicate that PIC may prove to be a valuable preventative microbicide and/or therapeutic agent against genital herpes by increasing resistance to HSV-2 and enhancing disease resolution following a failure of prevention.