Dysbiosis of the Human Oral Microbiome During the Menstrual Cycle and Vulnerability to the External Exposures of Smoking and Dietary Sugar

Abstract

Physiological hormonal fluctuations exert endogenous pressures on the structure and function of the human microbiome. As such, the menstrual cycle may selectively disrupt the homeostasis of the resident oral microbiome, thus compromising oral health. Hence, the aim of the present study was to structurally and functionally profile the salivary microbiome of 103 women in reproductive age with regular menstrual cycle, while evaluating the modifying influences of hormonal contraceptives, sex hormones, diet, and smoking. Whole saliva was sampled during the menstrual, follicular, and luteal phases (n = 309) of the cycle, and the participants reported questionnaire-based data concerning their life habits and oral or systemic health. No significant differences in alpha-diversity or phase-specific clustering of the overall microbiome were observed. Nevertheless, the salivary abundances of genera Campylobacter, Haemophilus, Prevotella, and Oribacterium varied throughout the cycle, and a higher species-richness was observed during the luteal phase. While the overall community structure maintained relatively intact, its functional properties were drastically affected. In particular, 11 functional modules were differentially abundant throughout the menstrual cycle, including pentose phosphate metabolism, and biosynthesis of cobalamin and neurotransmitter gamma-aminobutyric acid. The menstrual cycle phase, but not oral contraceptive usage, was accountable for greater variations in the metabolic pathways of the salivary microbiome. Further co-risk factor analysis demonstrated that Prevotella and Veillonella were increased in current smokers, whereas high dietary sugar consumption modified the richness and diversity of the microbiome during the cycle. This is the first large study to systematically address dysbiotic variations of the oral microbiome during the course of menstrual cycle, and document the additive effect of smoking and sugar consumption as environmental risk factors. It reveals the structural resilience and functional adaptability of the oral microbiome to the endogenous hormonal pressures of the menstrual cycle, while revealing its vulnerability to the exogenous exposures of diet and smoking.

Keywords: diet; hormonal contraceptives; menstrual cycle; oral microbiome; saliva; shotgun sequencing; sugar; women’s health.

Figure 1

Bar plot displaying the taxonomic composition of samples, sorted by phase of the menstrual cycle and contraceptive method.

Figure 2

Violin plots representing the distribution in relative abundance for each phase of the menstrual cycle for the four genera found to vary across the cycle. Pink: menstrual. Yellow: follicular. Green: luteal.

Figure 3

(A) Richness (observed species) and (B) Diversity (inverted Simpson's index) in each contraceptive group and phase of the menstrual cycle. Pink: menstrual. Yellow: follicular. Green: luteal.

Figure 4

Principal Coordinates Analysis (PCoA) of each sample, based on Bray-Curtis distance. The left panel depicts PC1 and PC2, while the right panel depicts PC1 and PC5. Other principal components are depicted in Supplementary Figure 3. The species with highest impact on the depicted principal components are overlaid as grey arrows. Red: menstrual phase. Yellow: follicular phase. Blue: luteal phase. Circle: non-hormonal contraceptives. Square: combined oral contraceptives. Triangle: intra-uterine levonegestrel system. H. parainfluenzae, Haemophilus parainfluenzae; N. flavescens, Neisseria flavescens; N. mucosa, Neisseria mucosa; N. subflava, Neisseria subflava; oral taxon 306, Prevotella sp. oral taxon 306; oral taxon 313, Prevotella sp. oral taxon 313; P. histicola, Prevotella histicola; P. melaninogenica, Prevotella melaninogenica; P. pallens, Prevotella pallens; R. dentocariosa, Rothia dentocariosa; R. mucilaginosa, Rothia mucilaginosa; S. mitis, Streptococcus mitis; S. parasanguinis, Streptococcus parasanguinis; S. salivarius, Streptococcus salivarius; V. atypica, Veillonella atypica; V. dispar, Veillonella dispar.

Figure 5

Violin plot representing the distribution in relative abundance for each phase of the menstrual cycle method for species of the Socransky complexes. The blue, yellow, green, and purple complexes are associated with periodontal health, whereas the orange and red complexes are correlated with periodontal disease.

Figure 6

Violin plot representing the distribution in relative abundance for each phase of the menstrual cycle according to the subgingival core microbiome classification (green: health-associated; red: periodontitis-associated).

Figure 7

Shifts of functional gene composition. Boxplots presenting pathways that are differentially abundant during the menstrual cycle or according to contraceptive usage. Detailed results are presented in Supplementary Table 5.