The influence of physiological and hormonal factors on the composition and stability of the oral microbiota: a microbiological and endocrinological perspective

Introduction

The oral microbiota, a dynamic microbial ecosystem, is subject to various physiological and hormonal influences, including fluctuations in estrogen and progesterone. These hormonal changes, particularly across the menstrual cycle, may alter microbial composition and stability, with potential consequences for oral and systemic health. This study examined samples from women at four phases of the menstrual cycle and from men as a control group, in order to assess differences in microbial dynamics under fluctuating versus stable hormonal conditions. The investigation also explored the influence of salivary flow and hormonal status on microbiota stability. Thyme (Thymus vulgaris L.), known for its bioactive constituents such as thymol and carvacrol, was evaluated as a natural antimicrobial intervention. The findings suggest that hormonal fluctuations in women destabilize the microbial composition, whereas men showed relatively greater microbial stability. Understanding these interactions is essential for developing personalized strategies in oral health management, particularly for women experiencing cyclic hormonal changes.

Research Questions:

1. What physiological and hormonal factors influence the composition and stability of the oral microbiota?
2. How do hormonal fluctuations – particularly those involving estrogen and progesterone–affect oral microbial balance and overall oral health?

Objectives:

1. To evaluate the influence of physiological and environmental factors, including hormonal fluctuations, on the composition and stability of the oral microbiota.
2. To compare the oral microbiota between women during different phases of the menstrual cycle and men, focusing on the impact of estrogen and progesterone levels.
3. To assess the potential consequences of these microbial changes on women's oral health throughout the menstrual cycle.

Significance of the Study

This study highlights the critical interplay between physiological and hormonal factors and the oral microbiota. Estrogen and progesterone fluctuations across the menstrual cycle were found to significantly affect bacterial growth and microbial balance in the oral cavity. The study demonstrated that the highest bacterial load occurred during the luteal phase, while men exhibited more microbial stability due to the absence of hormonal variations. The use of thyme mouthwash (Thymus vulgaris L.), rich in thymol and carvacrol, showed promising antimicrobial effects, especially during hormonally vulnerable phases. These findings underscore the importance of recognizing hormonal influences on oral microbiota in women and support the development of personalized, natural strategies – such as herbal mouthwashes – for maintaining oral health. This research may inform clinical guidelines and preventive measures tailored to women experiencing cyclic hormonal changes.

Materials and Methods

This study was designed to assess the impact of physiological and hormonal factors – specifically fluctuations in estrogen and progesterone – on the composition and stability of the oral microbiota. Additionally, the antimicrobial potential of thyme mouthwash (Thymus vulgaris L.) was evaluated as a natural intervention. A dual-method approach was applied, combining ex vivo sampling and in vitro culturing techniques to provide a comprehensive understanding of oral microbial dynamics. Sample Collection: The study included 20 women and 10 men. Oral microbiota samples were collected from women at four different phases of the menstrual cycle (one per week) and from men once, serving as a control group to represent a hormonally stable baseline. Samples were obtained using sterile oral swabs and processed to assess microbial load before and after using mouthwash. Mouthwash Preparation: Thyme mouthwash was prepared using a 2.5 g/mL concentration of Thymus vulgaris L., containing biologically active components such as thymol and carvacrol. Commercially available Oral-B mouthwash was used for comparison. Both were applied as a 2-minute oral rinse. In Vitro Experiments: In vitro tests were performed to examine the effect of estrogen (10–1 picomol/mL) and progesterone (0.5–0.1 nanomol/mL) on bacterial growth in liquid cultures. The inhibitory effects of thyme and Oral-B mouthwashes – both separately and in combination with hormones – were also evaluated based on colony count measurements. Statistical Analysis: All data were analyzed using appropriate statistical tests, with significance set at p ≤ 0.05. Reductions in microbial load were calculated and compared across menstrual phases and gender to identify statistically meaningful differences.

Results

The findings revealed significant differences in oral microbial load across menstrual phases in women and between sexes. In women, the microbial load fluctuated in correlation with hormonal phases, while men – whose hormonal levels remained stable – exhibited more consistent microbial profiles. Ex Vivo Findings: In women, the lowest reduction in microbial load after using thyme mouthwash was observed during the follicular phase (Week 1), with a 37.03% decrease. The highest reduction occurred in the late luteal phase (Week 4), reaching 57.69%. In men, thyme mouthwash produced a stable microbial reduction of 48.1%. Oral-B mouthwash showed higher inhibition across all groups, with the most significant effect (78%) observed in men. In women, it achieved a 66% reduction during Week 1 and 50.93% during Week 3. In Vitro Findings: Hormonal stimulation significantly promoted bacterial growth. Estrogen at 10–1 picomol/mL and progesterone at 0.5–0.1 nanomol/mL both led to increased bacterial proliferation. However, thyme mouthwash at 2.5 g/mL and Oral-B mouthwash at commercial concentration each demonstrated complete bacterial growth inhibition (100%) in vitro. When combined with hormones, both mouthwashes neutralized the stimulatory effects and maintained full inhibitor Reduction in Microbial Load by Mouthwashy activity.

Tables and Graphs – Oral Microbiota Study

Figure and table represented Reduction in Microbial Load by Mouthwash.

Table

Reduction in Microbial Load by Mouthwash

Fig. Reduction in Microbial Load by Mouthwash

Discussion

This study contributes to a deeper understanding of how physiological and hormonal factors, particularly estrogen and progesterone fluctuations, influence the composition and stability of the oral microbiota. By integrating both ex vivo and in vitro approaches, the findings demonstrate that hormonal changes significantly affect bacterial growth and microbial load in the oral cavity. The increased bacterial proliferation observed during the luteal phase of the menstrual cycle aligns with previous reports suggesting that high levels of sex hormones promote microbial adhesion and biofilm formation. Estrogen and progesterone appear to create a favorable environment for bacterial growth, potentially by altering salivary flow and immune responses. This may explain the pronounced microbial instability observed in women compared to the relatively stable profiles in men. Thyme mouthwash exhibited potent antimicrobial effects, particularly during hormonally sensitive phases. Its bioactive components – thymol and carvacrol – are known to disrupt bacterial cell membranes and metabolic pathways. The observed 57.69% reduction in microbial load during the late luteal phase highlights thyme’s capacity to counteract hormone-induced bacterial overgrowth. Furthermore, the complete inhibition seen in vitro confirms its therapeutic potential. In contrast, Oral-B mouthwash demonstrated a consistent and strong inhibitory effect, regardless of hormonal context. This is likely due to its synthetic antimicrobial agents such as chlorhexidine, which offer broad-spectrum activity independent of hormonal fluctuations. However, this uniformity may come with side effects or resistance risks not observed with natural alternatives like thyme. The results support the notion that hormonal cycles must be considered in personalized oral health strategies. Women may benefit from phase-specific interventions – such as enhanced oral hygiene or targeted use of natural antimicrobials – especially during the luteal phase, when the risk of microbial imbalance is highest.

Conclusion

This study confirms that hormonal and physiological factors – particularly fluctuations in estrogen and progesterone – have a significant impact on the oral microbiota. The findings from both ex vivo and in vitro experiments demonstrate that hormonal peaks, such as those during the luteal phase, can stimulate bacterial growth and destabilize microbial balance in the oral cavity. Thyme mouthwash, containing natural bioactive compounds, showed strong antimicrobial properties, especially during hormonally sensitive phases. It achieved the highest microbial load reduction during the late luteal phase in women, and maintained full inhibitory activity in vitro even when combined with hormonal stimuli. In men, the microbial load remained more stable, reflecting the absence of hormonal variability. These results suggest that thyme-based interventions may offer an effective, natural alternative for managing oral health – particularly in women with cyclic hormonal changes. Personalized oral hygiene strategies that account for hormonal fluctuations could improve preventive care and reduce the risk of periodontal disease and dysbiosis. Future studies should explore the long-term effects of thyme use, optimal dosing, and its effectiveness across diverse populations and hormonal conditions. The integration of microbiological and endocrinological perspectives may enhance oral healthcare outcomes and support tailored clinical protocols.

Recommendations:

1. Conduct long-term studies to evaluate the sustained antimicrobial efficacy of thyme mouthwash, especially across multiple menstrual cycles.
2. Investigate the effectiveness of different thyme concentrations and combinations with other natural agents to optimize its clinical use.
3. Assess thyme’s impact on oral microbiota in larger, demographically diverse populations to enhance generalizability of findings.
4. Explore the role of hormonal fluctuations in other age groups, including postmenopausal women and adolescents, to broaden understanding.
5. Develop personalized oral care protocols that incorporate hormonal phase-specific interventions for women, particularly during the luteal phase.
6. Compare thyme mouthwash to synthetic commercial products in terms of user compliance, side effects, and microbiota resilience.