Thymol Reduces Bone Alveolar Loss Caused by Periodontitis and Protects Mice Ovarian Follicles

Abstract

This study aimed to evaluate the effects of thymol on alveolar bone loss and viability of ovarian follicles in mice with induced periodontitis. Female Swiss mice (n=60) were allocated into five groups (n=12 per group): (1) naïve controls, (2) healthy animals treated with vehicle, (3) healthy animals treated with thymol (100 mg/kg), (4) animals with periodontitis, and (5) animals with periodontitis treated with thymol. Following a 30-day period of periodontitis induction and treatment, mice were euthanized and their mandibles were collected for histological analysis to evaluate alveolar bone loss and periodontal integrity. The ovaries were fixed to evaluate collagen fiber organization and follicular morphology. Cumulus-oocyte complexes (COCs) were stained with calcein-AM and ethidium homodimer-1. The percentages of normal follicles and viable COCs were compared by chi-squared or Fisher's exact test. Ovarian collagen levels and alveolar bone loss were compared by ANOVA and Tukey test. The results showed that thymol reduced alveolar bone loss in mice with periodontitis (p<0.0001). Animals with periodontitis exhibited a lower percentage of normal follicles and a higher percentage of degenerated follicles compared to vehicle-treated mice (p<0.05). Periodontitisinduced mice treated with thymol displayed percentages of normal and degenerated follicles similar to those from from naïve group (p>0.05). The oocytes from mice with periodontitis without thymol treatment showed reduced calcein-AM staining, by thymol increased calcein-AM labeling (p<0.05). A higher number of denuded oocytes were observed in animals with periodontitis (p<0.05). In conclusion, thymol attenuated alveolar bone loss associated with induced periodontitis and had protective effect on ovarian follicles.

Keywords
oocyte; periodontitis; ovarian follicle; thymol.

HIGHLIGHTS

• Thymol reduced alveolar bone loss in mice with induced periodontitis.

• Thymol preserved the viability and growth of ovarian follicles.

• Thymol showed protective effects against reproductive damage induced by periodontitis.

INTRODUCTION

Periodontal disease is a chronic inflammatory condition characterized by progressive alveolar bone loss, ultimately culminating in tooth loss [¹]. Beyond its local manifestations, periodontitis has been increasingly associated with systemic repercussions [²], largely attributed to the persistent release of pro-inflammatory cytokines, including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), thereby contributing to a sustained systemic inflammatory state [³]. These mediators may activate the hypothalamic-pituitary-adrenal (HPA) axis, stimulating the secretion of corticotropin-releasing hormone (CRH), promoting adrenocorticotropic hormone (ACTH) release, and consequently increasing glucocorticoid production. Under chronic inflammatory conditions, this sustained activation may result in persistently elevated cortisol levels [⁴]. Excess glucocorticoids have been implicated in alterations of female reproductive physiology, including suppression of gonadotropin-releasing hormone (GnRH) pulsatility, reduced secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and impairment of ovarian steroidogenesis [⁵]. At the ovarian level, cortisol may compromise granulosa cell function, interfere with follicular maturation, and promote follicular atresia, ultimately impairing oocyte viability [⁶,⁷]. In addition, systemic inflammation may directly affect ovarian tissue through cytokine-mediated pathways and enhanced oxidative stress, disrupting stromal organization and follicular survival [⁸,⁹]. Collectively, these mechanisms provide a biologically plausible framework supporting the association between chronic periodontal inflammation and ovarian dysfunction. In light of this scenario, therapeutic strategies aimed at modulating periodontal inflammation and mitigating its systemic consequences have been extensively investigated. Plant-derived compounds exhibiting antioxidant and anti-inflammatory properties have demonstrated promising results in experimental models [¹⁰,¹¹].

Thymol, a phenolic monoterpene predominantly found in the essential oil of Lippia gracilis, exhibits antioxidant and anti-inflammatory activities largely attributed to its phenolic hydroxyl group [¹²,¹³,¹⁴]. In clinical dental practice, thymol is incorporated into essential oil-based mouthrinses, where it acts synergistically with eucalyptol, menthol, and methyl salicylate in reducing biofilm accumulation. It has also been investigated as an intracanal medicament and as a functional component of controlled-release dental biomaterials. Its pharmacological profile is characterized by low cytotoxicity at therapeutic concentrations and a limited propensity to induce microbial resistance [¹⁵]. In human ovarian cells, thymol (50-150 µM) attenuated bleomycin-induced genotoxicity through its antioxidant and anti-inflammatory properties [¹⁶]. These findings suggest that thymol has significant potential to attenuate periodontitis-induced inflammation and, consequently, to protect the ovarian follicles of mice with experimentally induced periodontitis. This study hypothesizes that (1) periodontitis negatively affects the survival and growth of ovarian follicles in mice; (2) thymol reduces alveolar bone loss; and (3) that thymol alleviates the adverse effects of periodontitis on ovarian follicles.

The present study aims to evaluate the effects of periodontitis, either alone or in association with thymol treatment, on the growth and viability of ovarian follicles in female mice. Furthermore, it seeks to investigate the relationship between follicular survival and growth, collagen fiber organization in the ovarian cortex, and oocyte viability.

MATERIAL AND METHODS

Animals

The experiment was conducted using 60 female Swiss mice (Mus musculus) at the Federal University of Ceará and were acclimated for 15 days prior experimental procedures. The animals were randomly allocated into five groups by manual procedure and housed in polyethylene cages, with six animals per cage, lined with wood shavings, and provided with ad libitum access to filtered water and Nuvilab® standard chow. The mice were maintained at a controlled temperature of approximately 22.0°C under a 12-hour light/dark cycle. All procedures involving animals were performed in accordance with the guidelines and normative resolutions established by the National Council for the Control of Animal Experimentation (CONCEA) and the guidelines for in vivo studies. The experimental protocol was approved by the Ethics Committee on the Use of Animals at the Federal University of Ceará (approval number 05/23).

Assessment of the estrous cycle

All females, weighing at least 18 g and/or approximately 2 months of age, had their estrous cycles evaluated once daily for 15 consecutive days, between 8:00 and 9:00 a.m., by a single evaluator, following the methodology described by Marcondes, Bianchi, and Tanno [¹⁷]. Based on the cytological evaluation, the stage of the estrous cycle-proestrus, estrus, metestrus, or diestrus-was determined. Only females exhibiting a regular cycle, defined as lasting 4 to 5 days, were selected for the experiments, during which their cycles continued to be monitored. Animal body weights were recorded at the beginning and at the end of the study.

Experimental model for inducing periodontitis

To place the ligature in the periodontal region, the animals were subjected to general anesthesia using ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally. Then, the mice were positioned on an appropriate operating table to allow adequate maintenance of the mouth opening, facilitating access to the posterior teeth of the mandible. The ligation model consisted of placing a polyester braided cotton thread 4.0 with the knot onto the mesial position around the right and left mandible first molars [¹⁸]. After being adapted, the ends of the thread were joined by a knot facing the mesial surface, being located in a supragingival buccal and subgingival position on the lingual surface. The ligatures were maintained in place for 30 days for the induction of experimental periodontitis.

Pharmacological modulation

To investigate pharmacological modulation, the animals were divided into five groups, each comprising twelve animals (n = 60), as follows: (1) naïve; (2) healthy animals treated with vehicle (distilled water containing 1% of Dimethyl sulfoxide (DMSO); (3) healthy animals treated with thymol; (4) animals with experimental periodontitis treated with vehicle; and (5) animals with experimental periodontitis treated with thymol (Figure 1). Thymol was purchased in its pure form from Sigma-Aldrich (St. Louis, MO, USA). The dose of thymol administered was 100 mg/kg, delivered via intraperitoneal injection. This dose was selected based on previous experimental studies demonstrating pronounced anti-inflammatory activity, including significant reduction of edema and inflammatory cell infiltration in rodent models, without reported adverse effects as described by Riella and coauthors [¹⁹]. Thymol was diluted in sterile distilled water containing 1% DMSO. The animals received daily thymol treatment for one month. After the treatment period, mice were euthanized, and the ovaries and mandibles were collected for morphometric and histological analyses. Additionally, the viability of COCs was assessed using calcein-AM/ homodimer ethidium-1 fluorescence staining.

Figure 1
Experimental design. (1) Naive (control); (2) healthy animals treated with vehicle; (3) healthy animals treated with thymol; (4) animals with experimental periodontitis treated with vehicle; and (5) animals with experimental periodontitis treated with thymol.

Measurement of alveolar bone loss

After 30 days of experimental periodontitis, the mandibles were removed and sectioned in half, with the right hemimandible used for morphometric analysis. The hemimandibles were fixed in 10% buffered formalin for 24 hours, then transferred to 70% ethanol for 4 hours. Subsequently, they were dissected and stained with 1% aqueous methylene blue to differentiate bone from teeth. To quantify bone resorption, the samples were photographed, and the images were analyzed using the ImageJ software (version 1.44p; National Institutes of Health, USA). The distance from the cementoenamel junction to the alveolar bone crest was measured and considered as the indicator of alveolar bone loss in animals with induced periodontitis. This measurement was compared to a previously standardized reference area (5 × 5 mm²), as described by Kuhr and coauthors [²⁰]. The area was expressed as square millimeters (mm²). This analysis was performed by a single examiner in a blinded manner. This experiment was repeated six times.

Histopathological analysis of the mandibles

The left hemimandibles, previously fixed in 10% buffered formalin, were demineralized in an EDTA solution for 60 days. Following demineralization, the samples were dehydrated through graded alcohols, cleared in xylene, and embedded in paraffin. Tissue sections were cut at a thickness of 6 µm along the molars on the sagittal plane and subsequently deparaffinized in xylene, followed by rehydration through a series of decreasing alcohol concentrations. The sections were stained with hematoxylin and eosin and processed as described by Santos and coauthors [²¹]. For quantitative analysis, ImageJ software was used to measure the attachment levels at the mesial aspect of the second molar, defined as the distance from the cementoenamel junction to the base of the gingival sulcus or periodontal pocket [²²], in order to assess attachment loss. For standardization, serial mesiodistal sections covering the molar region were screened, and only sections showing the cemento-enamel junction, root surface, and periodontal space in the same plane were included. The mesial aspect of the second molar was adopted as the anatomical reference to ensure intergroup comparability among specimens. The attachment loss was expressed as micrometers (µm).

Histopathological analysis of the ovaries

After the end of the experimental period, the ovaries were collected, fixed in paraformaldehyde, dehydrated in a graded series of ethanol, clarified with xylene and embedded in paraffin wax. For each ovary, 7-μm sections were mounted on slides and stained by the periodic acid-Schiff (PAS)-hematoxylin method. The slides were examined using a microscope (Nikon) at 100x and 400x magnification. To evaluate the effects of treatments on follicular development, ovarian follicles were classified as primordial (a layer of squamous-shaped granulosa cells around the oocyte), primary (a layer of cubic-shaped granulosa cells around the oocyte), secondary (several layers of cubic-shaped granulosa cells around the oocyte) and tertiary (oocyte surrounded by several layers of cumulus cells and the presence of a well-developed antral cavity). Furthermore, follicles were classified as morphologically normal if an intact oocyte was surrounded by wellorganized granulosa cells in one or more layers and did not have pyknotic nuclei [²³]. Degenerated follicles were defined as those with a retracted oocyte and/or a pyknotic nucleus, as well as disorganized granulosa cells that were detached from the basement membrane [²⁴]. In total, approximately 200 follicles were evaluated per group. Follicular categories were determined for each ovary and expressed as percentages for group comparison. The percentages of normal or degenerated follicles, as well as primordial and developing follicles were calculated before and after the experimental protocol. Additionally, the number of corpora lutea present in the ovaries was counted.

Analysis of the extracellular matrix

To assess collagen content of the extracellular matrix (ECM) of the ovarian cortex, sections were stained with Picrosirius Red (Abcam kit), consistent with a workflow previously reported by our group [²⁵]. Picrosirius Red-stained sections were imaged under conventional brightfield microscopy in an area of 100 μm². For each treatment, ten fields per slide were evaluated. Collagen-stained areas exhibited a red coloration, whereas follicles remained unstained. The analyzer software automatically excluded the circumference of unstained follicles from the total red marked area. ImageJ software was used to calculate the percentage of collagenpositive area in the tissue sample. Staining intensity was determined by measuring the average pixel intensity of the total imaged area after background subtraction. This brightfield-based analysis provides a semiquantitative index of collagen-stained content/distribution and does not assess birefringence-based fiber organization or collagen subtypes.

Assessment of the quality of the cumulus-oocyte complex

Immediately after collecting the ovaries, they were placed in TCM-199 and COCs from large tertiary follicles were isolated by mechanical follicular extrusion using 26G needles. Pools of 112-115 COCs per group/treatment of animals were isolated and evaluated for the quality of COCs as follows: Grade I - oocyte with homogeneous cytoplasm surrounded by three or more layers of cumulus cells; Grade II - oocyte with homogeneous cytoplasm surrounded by up to three layers of cumulus cells; Grade III - surrounded by homogeneous or heterogeneous cytoplasm with an incomplete layer of cumulus cells; and Grade IV - oocyte with heterogeneous or degenerate cytoplasm, characterized by the absence of cumulus cells [²⁶,²⁷,²⁸].

Following isolation and selection, the COCs were transferred to 100 µL drops of DMEM⁺ medium mounted on glass slides with 4 mM of calcein-AM and 2 mM of homodimer ethidium-1 (EtdD-1) (Molecular Probes, Invitrogen, Karlsruhe, Germany). The slides were then incubated at 37°C for 15 min, after which the oocytes were examined under a fluorescence microscope. The fluorescent signals emitted by calcein-AM and EtdD-1 were collected at 488 and 568 nanometers, respectively. The initial probe is utilized for the detection of viable cells, while the subsequent probe serves to label the nucleic acids of non-viable cells. The criterion for defining oocytes as viable included the positive labeling of the cytoplasm with calcein-AM (green) and the absence of labeling with EtdD-1 (red) of the chromatin. Conversely, cells with labeled chromatin (red) using EtdD-1 were designated as non-viable [²⁹].

Statistical Analysis

The sample size was determined according to the methodology described by Sá et al. [³⁰]. Statistical analysis was performed using the GraphPad 9.0 software. Data on the percentage of normal and degenerated follicles, as well as in each development category were evaluated using the chi-square test. The analysis of the ECM was performed using the analysis of variance ANOVA test followed by Tukey's multiple comparisons test. The quality assessment of the COCs and viability was conducted using Fisher's exact test. Analysis of bone loss data was performed by ANOVA. After passing the normality test, the data were analyzed using the Tukey test. The results were expressed as mean ± S.E.M. (variables with normal distribution). Differences were considered significant when P<0.05.

RESULTS

In this study, neither animals nor samples were lost during the experimental period, and no adverse reactions were observed during the procedures. The results showed that, prior to the experimental period, all groups exhibited normal estrous cycles (Table 1). In the analysis of the last 7 days of treatment, 100% (12/12) of the mice in the naïve and vehicle groups were cycling normally. In the thymol group, 91.67% (11/12) exhibited a normal cycle. In the periodontitis-induced group treated with vehicle, 75% (9/12) of the animals showed a normal cycle, whereas in the periodontitis-induced group treated with thymol, this percentage increased to 83.33% (10/12). No significant differences were observed among the groups during the final 7 days of the experiment. The body weight of animals in all groups gradually increased from day 0 to day 30 of the experiment, as showed in Table 2.

Table 1
Estrous cycle of animals one week before treatment and in the last week of the 30 days of treatment. Naive group, treated with vehicle (Veh), treated with thymol (Thy), with periodontitis (Perio+Veh), or with periodontitis and treated with thymol (Perio+Thy)

Estrous cycle	Naive	Veh Thy	Perio+Veh	Perio+Thy
		Before treatment
Normal cycle	100% (12/12)	100% (12/12) 100% (12/12)	100% (12/12)	100% (12/12)
Altered cycle	0% (0/12)	0% (0/12) 0% (0/12) Last week of treatment	0% (0/12)	0% (0/12)
Normal cycle	100% (12/12)	100% (12/12) 91.67% (11/12)	75% (9/12)	83.33 (10/12)
Altered cycle	0% (0/12)	0% (0/12) 8.33% (1/12)	25% (3/12)	16.67% (2/12)

Table 2
Weight in grams of mice in the naive group, treated with vehicle (Veh), treated with thymol (Thy), with periodontitis (Perio+Veh), or with periodontitis and treated with thymol (Perio+Thy) on days 0, 15 and 30.

Day 0	22.81 ± 2.32	22.91 ± 3.01	24.09 ± 1.92	22.91 ± 1.70	22.72 ± 2.00
Day 15	23.90 ± 1.78	23.91 ± 2.01	24.41 ± 1.55	23.41 ± 1.18	22.54 ± 1.23
Day 30	24.81 ± 1.85	23.75 ± 2.16	25.00 ± 1.12	23.91 ± 1.44	23.36 ± 1.61
Weight gain	2.00 ± 1.53	0.83 ± 1.51	0.90 ± 1.67	1.00 ± 1.22	0.63 ± 1.66

Alveolar bone loss

The macroscopic appearance of the mandibles (Figure 2 A-E) showed that animals without periodontitis displayed preserved alveolar bone, whereas those with induced periodontitis exhibited evident alveolar bone loss, root exposure, and furcation lesions. Ligature placement on the first lower molar significantly increased alveolar bone loss compared to the groups in which periodontitis was not induced. Thymol treatment did not cause significant changes in animals without periodontitis. In contrast, thymol administration in mice with induced periodontitis reduced alveolar bone loss compared to those treated with vehicle only (Figure 2F).

Figure 2
Representative images of average resorption in mm² of alveolar bone in animals from the naïve group (A), vehicle (B), thymol (C), periodontitis (D) and periodontitis with thymol (E). The average resorption in mm² of alveolar bone from different treatments is shown in (F). Lowercase letters indicate difference between different groups. a, b Differences between groups (P<0.0001).

Histopathological analysis of mandibles

The periodontitis-induced group treated with thymol did not significantly reduce or prevent the progression of attachment loss when compared to periodontitis-induced animals treated with vehicle. The histological features associated with attachment loss are illustrated in Figures 3 A-E. A significant increase in periodontal attachment loss in the first molars of animals with induced periodontitis compared to those without periodontitis was observed (Figure 3F).

Figure 3
Histological section illustrating alveolar bone loss of the 1st molars of the naïve group (A), treated with vehicle (B), treated with Thymol (C), with periodontitis treated with vehicle (D), or with periodontitis and treated with thymol (E). Scale bar = 100μm (40x). The Loss of attachment of the 1st molars in animals from different treatments is shown in (F). a, b Differences between groups (P<0.0001).

Ovarian histology

The morphology of ovarian follicles, classified as normal or degenerated within the different categories (primordial, primary, and secondary), is shown in Figure 4 A-F. Figure 4G shows that health animals (naïve) and those treated with vehicle solution or thymol animals had a higher percentage of normal follicles than those with periodontitis that had been treated with thymol or vehicle solution. However, animals with periodontitis and treated with thymol had significantly higher percentage of normal follicles than those with periodontitis and treated with vehicle solution. Additionally, animals from the periodontitis group displayed higher percentage of degenerated follicles when compared to the other groups. Notably, animals with periodontitis and treated with thymol showed a percentage of normal and degenerated follicles similar to the naïve group.

Figure 4
Representative images of mice ovaries showing morphological analysis. A, Normal and (D) degenerated primordial follicle; B, Normal and (E) degenerated primary follicle; C, Normal and (F) degenerated secondary follicle. GC: Granulosa cells; O: Oocyte; N: Oocyte nucleus; dGC: disorganized granulosa cells; dO: degenerated oocyte. Scale bar = 100μm (40x). (G) Percentage of normal follicles in ovaries of mice from the naïve group, vehicle (Veh), thymol (Thy), periodontitis (Perio + Veh) and periodontitis with thymol (Perio + Thy) on day 30. Different lowercase letters indicate difference in normal follicles percentage between different groups. Different capital letters indicate difference in degenerated follicles percentage between different groups. (H) Percentage of developing follicles in ovaries of mice from the naïve group, vehicle (Veh), thymol (Thy), periodontitis (Perio + Veh) and periodontitis treated with thymol (Perio + Thy) on day 30. Different lowercase letters indicate difference in primordial follicles percentage between different groups. Different capital letters indicate difference in development follicles percentage between different groups (P<0.05).

Regarding follicular development, Figure 4H shows that health mice treated with thymol and those periodontitis with vehicle exhibited a reduction in the number of primordial follicles compared to those from naïve group or with periodontitis and treated with thymol. Conversely, animals with periodontitis treated with thymol presented a similar percentage of primordial follicles than to those from naïve group or treated with vehicle solution. Animals from thymol and periodontitis groups treated with vehicle showed a higher percentage of developing follicles compared to those with periodontitis and treated with thymol group was similar to that seen in animals from naïve and vehicle groups.

Extracellular matrix analysis

The collagen-stained pattern/content in the ovaries varied according to the different treatments (Figure 5 A-E). It was observed that mice from the vehicle, thymol, periodontitis groups had a higher percentage of collagen fibers than those from naive group (Figure 5F).

Figure 5
Percentage of total collagen area of female mice in the Naïve (A), Vehicle (Veh) (B), Thymol (Thy) (C), Periodontitis (Perio+Veh) (D), and Periodontitis treated with thymol (Perio+Thy) (E) groups. Statistical graph of percentage of total collagen area is shown in Figure F. *Asterisk represents significant difference between naïve and treatments. *P<0.05.

Analysis of oocytes viability after ovarian culture by fluorescence microscopy

Oocytes exhibited a higher percentage of calcein-AM fluorescence intensity compared to EthD-1 (Figure 6 A-J). Moreover, oocytes from mice with periodontitis treated with vehicle showed reduced calceinAM intensity compared to those from the other groups. In contrast, in the periodontitis groups, the one treated with thymol showed greater calcein-AM intensity. Additionally, animals in the periodontitis group treated with vehicle displayed oocytes with higher EthD-1 fluorescence intensity compared to animals from naïve, vehicle, and thymol-treated groups (Figure 6 K).

Figure 6
Calcein-AM (green) and ethidium homodimer-1 (EthD-1) (red) staining in oocytes from ovarian follicles of female mice in the Naïve (A-B), Vehicle (C-D), Thymol (E-F), Periodontitis plus vehicle (G-H), and Periodontitis plus thymol (I-J) groups. Statistical graph of fluorescence intensity is shown in Figure K. Data: mean ± SEM, (P<0.05). *Represents differences between calcein-AM and EthD-1 fluorescence within each group; Different capital letters indicate difference in calcein-AM fluorescence intensity between different groups; Different lowercase letters indicate difference in EthD-1 fluorescence intensity between different groups. Scale bars = 50 μm.

Assessment of the quality of the cumulus-oocyte complex

Table 3 shows that animals in from periodontitis group treated with vehicle had a lower number of grade III COCs, when compared to other groups. On the other hand, a higher number of grade IV COCs was noted in animals from the periodontitis group treated with vehicle when compared to other groups. No significant differences were observed among the groups for grades I and II. Representative images of COCs are shown in Figure 7.

Table 3
Quantification of COCs according to the grade of classification in the groups naïve group, vehicle, thymol, periodontitis and periodontitis with thymol on day 30

	Grade I	Grade II	Grade III	Grade IV
Naive	7.01% (8/114)A	10.52% (12/114)B	47.36% (54/114)C	35.08% (40/114)D
Veh	4.42% (5/113)A	20.35% (23/113)b	42.47% (48/113)C	32.74% (37/113)D
Thy	5.35% (6/112)A	14.28% (16/112)Bb	47.32% (53/112)C	33.03% (37/112)D
Perio + Veh	1.73% (2/115)A	10.43% (12/115)B	23.47% (27/115)c	64.34% (74/115)d
Perio + Thy	6.25% (7/112)A	13.39% (15/112)Bb	37.50% (42/112)C	42.85% (48/112)D

Figure 7
Representation of cumulus oocyte complexes classified in grades I, II, III and IV. Scale bars = 100 μm.

DISCUSSION

This is the first report to demonstrate the effects of thymol on alveolar bone loss and its influence on the development and viability of ovarian follicles and COCs in mice with induced periodontitis. Our findings indicate that thymol not only reduced alveolar bone loss but also mitigated periodontitis-induced damage to ovarian follicles and COCs. Alveolar bone resorption is a direct consequence of chronic inflammatory processes associated with the progression of periodontitis [³¹]. In our study, thymol treatment effectively reduced alveolar bone loss. During the progression of periodontitis, cytokines play a critical role in regulating homeostasis and inflammatory responses, particularly during the initial immune response to pathogens and stimuli at barrier sites. These cytokines also mediate interactions among tissue cells, lymphocytes, and accessory immune cell populations [³²]. Key cytokines involved in periodontitis include tumor necrosis factoralpha (TNF-α), prostaglandin E2 (PGE2), and interleukins 1β and 6 (IL-1β and IL-6) [³]. Conversely, thymol has been shown to downregulate the expression of pro-inflammatory cytokines such as TNF-β, IL-6 [¹³], TNF-α, IL-8, and IL-1β [³³]. In addition, thymol may promote the expression of anti-inflammatory cytokines such as interleukin-10 (IL-10) and interleukin-4 (IL-4) [¹³]. Although micro-computed tomography could provide additional three-dimensional structural information, the present study focused on marginal inflammatory resorption at the cemento-enamel junction-alveolar crest interface; thus, future studies using micro-CT may further complement bone characterization.

The apparent divergence between the reduction in alveolar bone loss and the absence of significant improvement in periodontal attachment loss may reflect distinct biological events evaluated [¹]. In ligatureinduced periodontitis, marginal bone resorption is primarily associated with inflammatory osteoclast activation, whereas attachment loss involves epithelial and connective tissue alterations [³¹]. Thus, attenuation of inflammatory bone resorption does not necessarily imply concomitant recovery of the periodontal attachment apparatus within the experimental period. In this context, thymol likely limited osteoclast-mediated marginal bone loss without promoting structural reestablishment of the attachment tissues during the 30-day observation period, consistent with the biological progression described in ligature models [²⁰]. The histological analysis was performed to document structural periodontal alterations and to complement the morphometric findings. Additional parameters such as inflammatory infiltrate quantification, epithelial downgrowth assessment, or bone remodeling markers may provide further mechanistic perspective and should be explored in future investigations. Further studies directly assessing inflammatory pathways are required to determine whether the observed effects involve immunomodulatory mechanisms. This method is widely established in the literature because it reproduces key features of human periodontal disease by promoting biofilm accumulation and the subsequent host inflammatory response to a dysbiotic oral microbiome. The mechanical presence of the ligature facilitates rapid periodontal tissue destruction, resulting in pronounced alveolar bone resorption and leukocyte infiltration. Therefore, this model represents a reliable experimental tool for evaluating the effects and mechanisms of pharmacological agents on periodontal inflammatory responses [¹⁸].

In recent decades, some studies have suggested a potential association between chronic periodontitis and fertility impairment, as well as adverse pregnancy outcomes such as preterm birth, preeclampsia, and low birth weight [³⁴,³⁵]. However, the relationship between periodontitis and female infertility remains unclear. Despite numerous investigations, the results remain inconclusive [³⁶]. To address this gap, we examined the effects of periodontitis and thymol on the development and viability of ovarian follicles and COCs. Our results showed that all periodontitis groups exhibited reduced rates of normal follicles. Thymol administration, however, protected the follicles against degeneration. These observations reflect preservation of follicular structural integrity as assessed by morphological criteria. Recently, Caetano Filho and coauthors [¹²] demonstrated that thymol preserved the integrity of ovarian follicles in bovine ovarian tissue during in vitro culture.

Beyond its anti-inflammatory effects, thymol has been recognized as a potent antioxidant. It effectively scavenges hydroxyl radicals, forming phenoxyl radicals as major transient species [¹³]. Thymol also enhances the activity of endogenous antioxidant enzymes, including catalase [¹²], superoxide dismutase, glutathione peroxidase, and glutathione-S-transferase, as well as non-enzymatic antioxidants such as vitamins C and E and reduced glutathione [³⁷]. Based in these previously reported properties of thymol, the observed outcomes may be compatible with anti-inflammatory and antioxidant actions. Nonetheless, additional research is warranted to explore thymol’s antioxidant effects in the context of periodontitis.

Regarding follicular development, our data indicates that thymol treatment increased the number of primordial follicles while decreasing the number of developing follicles, thereby supporting the maintenance of the follicular reserve in mice with induced periodontitis. Previous studies have shown that thymol protects the ovarian reserve primarily by neutralizing oxidative stress and modulating the crosstalk between IGF-1 and TNF-α in Wistar rats with radiation-induced ovarian insufficiency [³⁸]. Proper growth and maintenance of ovarian cells are critically dependent on the integrity of the ECM, which is primarily composed of collagen [³⁹].

The ovarian cortical extracellular matrix (ECM) provides structural support and contributes to the local microenvironment that sustains follicle growth and oocyte competence, with collagen as a major stromal component. In the present study, Picrosirius Red staining was quantified under conventional brightfield microscopy as a semi-quantitative index of collagen-stained content/distribution (collagen-positive area fraction and mean pixel intensity after background subtraction and exclusion of unstained follicular areas). The quantitative analysis indicated that naïve animals presented a lower collagen-positive area than animals from other experimental groups, while no significant differences were detected among vehicle-treated mice, thymol-treated mice, and mice with periodontitis. These findings suggest that, within the experimental window, ligature-induced periodontitis did not produce a robust remodeling of ovarian stromal collagen that could be detected by our brightfield-based Picrosirius readout, and thymol did not measurably alter collagenstained content compared with the corresponding controls. Importantly, because this approach does not assess birefringence-based fiber organization or collagen subtypes, the collagen data should be interpreted as changes in collagen-stained content rather than definitive evidence of fiber reorganization or fibrosis. The main limitations of this study include the absence of collagen subtype-specific markers and/or birefringencebased imaging to clarify periodontitis and thymol influence on ovarian ECM remodeling.

We also assessed oocyte viability using fluorescence staining with calcein-AM and EtdD-1, as well as the morphology of COCs by evaluating cumulus expansion and cytoplasmic integrity. Thymol treatment increased calcein-AM fluorescence and decreased EtdD-1 fluorescence, suggesting enhanced oocyte viability. In terms of COCs morphology, the thymol-treated group showed outcomes comparable to those of the naïve, vehicle, and periodontitis groups. Given the known anti-inflammatory and antioxidant properties of thymol [¹³,³³], we suggest that the observed benefits on follicular maintenance and oocyte viability may be attributed to thymol's ability to alleviate cellular damage, as previously demonstrated in human fibroblasts [⁴⁰].

CONCLUSION

The thymol effectively improved alveolar bone loss and protected ovarian follicles and COCs in female mice with periodontitis. Elucidating the mechanisms underlying its antioxidant and anti-inflammatory effects in periodontitis is crucial for exploring its potential biotechnological applications.

• Funding:
  This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant number 407992/2021-9).
• Institutional Review Board Statement: “The animal study protocol was approved by Ethics Committee of Federal University of Ceará, Sobral, CE Animal Experiments Local Ethics Committee (protocol code nº 05/23 and 30.05.2023 date of approval)” for studies involving animals.
• Informed Consent Statement: Not applicable.

Use of Generative Artificial Intelligence

The authors declare that large language models and other generative artificial intelligence (AI) or AIassisted technologies cannot be credited as authors and have not been listed as authors of this paper.

The author declare that did not use the artificial intelligence.

Acknowledgments:

We would like to thank the collaboration of the scientific initiation students, technicians and staff of the Federal University of Ceará, Research Center of Animal Experimentation (NUPEX), for their support during the experimental conduct of the study.

Data Availability Statement:

Research data are available in the body of the manuscript.

REFERENCES

• ¹ Kinane DF, Stathopoulou PG, Papapanou PN. Periodontal diseases. Nat Rev Dis Primers. 2017;3:17038. https://doi.org/10.1038/nrdp.2017.38
  » https://doi.org/10.1038/nrdp.2017.38
• ² Slots J. Periodontitis: facts, fallacies and the future. Periodontol. 2000. 2017;75:7-23. https://doi.org/10.1111/prd.12221
  » https://doi.org/10.1111/prd.12221
• ³ Ramadan DE, Hariyani N, Indrawati R, Ridwan RD, Diyatri I. Cytokines and chemokines in periodontitis. Eur J Dent. 2020;14(3):483-95. https://doi.org/10.1055/s-0040-1712718
  » https://doi.org/10.1055/s-0040-1712718
• ⁴ Rosalin YE, Santos TMM, Araújo CSA, Campos EBV, Campos MLG, Saraiva PP. Periodontal disease relation to stress and depression indicators. Health Sci J. 2018;20(2). Available from: https://periodicoseletronicos.ufma.br/index.php/rcisaude/article/view/7695/9395
  » https://periodicoseletronicos.ufma.br/index.php/rcisaude/article/view/7695/9395
• ⁵ Bhaumik S, Lockett J, Cuffe J, Clifton VL. Glucocorticoids and their receptor isoforms: roles in female reproduction, pregnancy, and foetal development. Biology (Basel). 2023;12(8):1104. https://doi.org/10.3390/biology12081104
  » https://doi.org/10.3390/biology12081104
• ⁶ Varley M. Stress and reproduction. Pig News Inf. 1991;12(4):567-71. Available from: https://cabidigitallibrary.org by 200.129.42.33, on 07/03/26.
  » https://cabidigitallibrary.org
• ⁷ Mariz ALF, Albuquerque FQ, dos Santos HC, Segundo JCCF, de Sousa Filho JMT, Cunha RCSP, et al. The effect of stress on glucocorticoid secretion as a cause of infertility. Braz J Health Rev. 2022;5(4):14508-14. https://doi.org/10.34119/bjhrv5n4-210
  » https://doi.org/10.34119/bjhrv5n4-210
• ⁸ Shang J, Liu H, Zheng Y, Zhang Z. Role of oxidative stress in the relationship between periodontitis and systemic diseases. Front Physiol. 2023;14:1210449. https://doi.org/10.3389/fphys.2023.1210449
  » https://doi.org/10.3389/fphys.2023.1210449
• ⁹ Grosbois J, Bailie EC, Kelsey TW, Anderson RA, Telfer EE. Spatio-temporal remodelling of the composition and architecture of the human ovarian cortical extracellular matrix during in vitro culture. Hum Reprod. 2023;38(3):44458. https://doi.org/10.1093/humrep/dead008
  » https://doi.org/10.1093/humrep/dead008
• ¹⁰ Zuo X, Gu Y, Wang C, Zhang J, Zhang J, Wang G, et al. A systematic review of the anti-inflammatory and immunomodulatory properties of 16 essential oils of herbs. Evid Based Complement Alternat Med. 2020;2020:8878927. https://doi.org/10.1155/2020/8878927
  » https://doi.org/10.1155/2020/8878927
• ¹¹ Chen X, Shang S, Yan F, Jiang H, Zhao G, Tian S, et al. Antioxidant activities of essential oils and their major components in scavenging free radicals, inhibiting lipid oxidation and reducing cellular oxidative stress. Molecules. 2023;28(11):4559. https://doi.org/10.3390/molecules28114559
  » https://doi.org/10.3390/molecules28114559
• ¹² Caetano Filho FF, Paulino LRF, Bezerra VS, Azevedo VAN, Barroso PAA, Costa FC, et al. Thymol increases primordial follicle activation, protects stromal cells, collagen fibers and down-regulates expression of mRNA for superoxide dismutase 1, catalase and peroxiredoxin 6 in cultured bovine ovarian tissues. Anim Reprod Sci. 2024;266:107514. https://doi.org/10.1016/j.anireprosci.2024.107514
  » https://doi.org/10.1016/j.anireprosci.2024.107514
• ¹³ Meeran MFN, Javed H, Al Taee H, Azimullah S, Ojha SK. Pharmacological properties and molecular mechanisms of thymol: prospects for its therapeutic potential and pharmaceutical development. Front Pharmacol. 2017;8:380. https://doi.org/10.3389/fphar.2017.00380
  » https://doi.org/10.3389/fphar.2017.00380
• ¹⁴ Sheorain J, Mehra M, Kumari S. Characterization and an assessment of anti-inflammatory effect of an optimized thymol loaded sodium alginate-chitosan copolymeric nanoformulation. Int Multidiscip Res J. 2020;7(3):487-92. https://doi.org/10.1016/j.ijbiomac.2018.12.095
  » https://doi.org/10.1016/j.ijbiomac.2018.12.095
• ¹⁵ Potocka W, Assy Z, Bikker FJ, Laine ML. Current and potential applications of monoterpenes and their derivatives in oral health care. Molecules. 2023;28(20):7178. https://doi.org/10.3390/molecules28207178
  » https://doi.org/10.3390/molecules28207178
• ¹⁶ Arab HA, Fathi M, Mortezai E, Hosseinimehr SJ. Chemoprotective effect of thymol against genotoxicity induced by bleomycin in human lymphocytes. Pharm Biomed Res. 2015;1(1):26-31. https://doi.org/10.18869/acadpub.pbr.1.1.26
  » https://doi.org/10.18869/acadpub.pbr.1.1.26
• ¹⁷ Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol. 2002;62:609-14. https://doi.org/10.1590/S1519-69842002000400008
  » https://doi.org/10.1590/S1519-69842002000400008
• ¹⁸ Tavares SJS, Pereira CR, Fortes RAM, Alves BES, Fonteles CSR, Wong DVT, et al. Umbelliferone reduces inflammation and ligature-induced osteoclastic alveolar bone resorption in mice. J Periodontal Res. 2024;59(5):982-92. https://doi.org/10.1111/jre.13277
  » https://doi.org/10.1111/jre.13277
• ¹⁹ Riella KR, Marinho RR, Santos JS, Pereira-Filho RN, Cardoso JC, Albuquerque-Junior RLC, et al. Antiinflammatory and cicatrizing activities of thymol, a monoterpene of the essential oil from Lippia gracilis, in rodents. J Ethnopharmacol. 2012;143(2):656-63. https://doi.org/10.1016/j.jep.2012.07.028
  » https://doi.org/10.1016/j.jep.2012.07.028
• ²⁰ Kuhr A, Popa-Wagner A, Schmoll H, Schwahn C, Kocher T. Observations on experimental marginal periodontitis in rats. J Periodontal Res. 2004;39(2):101-6. https://doi.org/10.1111/j.1600-0765.2004.00710.x
  » https://doi.org/10.1111/j.1600-0765.2004.00710.x
• ²¹ Santos KRP, Aguiar Júnior FCA, Antonio EA, Silva FC, Silva KT, Marinho KSN, et al. Manual of routine histological techniques and staining methods. Recife: Federal University of Pernambuco; 2021: 32 p.
• ²² Sima C, Aboodi GM, Lakschevitz FS, Sun C, Goldberg MB, Glogauer M. Nuclear factor erythroid 2-related factor 2 down-regulation in oral neutrophils is associated with periodontal oxidative damage and severe chronic periodontitis. Am J Pathol. 2016;186(6):1417-26. https://doi.org/10.1016/j.ajpath.2016.01
  » https://doi.org/10.1016/j.ajpath.2016.01
• ²³ Lins TLBG, Gouveia BB, Barberino RS, Silva RLS, Monte APO, Pinto JGC, et al. Rutin prevents cisplatin-induced ovarian damage via antioxidant activity and regulation of PTEN and FOXO3a phosphorylation in mouse model. Reprod Toxicol. 2020;98:209-17. https://doi.org/10.1016/j.reprotox.2020.10.001
  » https://doi.org/10.1016/j.reprotox.2020.10.001
• ²⁴ Gouveia BB, Barberino RS, Silva RLS, Lins TLBG, Guimarães VS, Do Monte APO, et al. Involvement of PTEN and FOXO3a proteins in the protective activity of protocatechuic acid against cisplatin-induced ovarian toxicity in mice. Reprod Sci. 2021;28(3):865-76. https://doi.org/10.1007/s43032-020-00305-4
  » https://doi.org/10.1007/s43032-020-00305-4
• ²⁵ Rittié L. Method for picrosirius red-polarization detection of collagen fibers in tissue sections. In: Rittié L, editor. Fibrosis. Methods Mol Biol. 2017;1627:395-407. https://doi.org/10.1007/978-1-4939-7113-8_26
  » https://doi.org/10.1007/978-1-4939-7113-8_26
• ²⁶ De Loos F, Kastrop P, Van Maurik P, Van Beneden TH, Kruip TAM. Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes. Mol Reprod Dev. 1991;28(3):255-59. https://doi.org/10.1002/mrd.1080280307
  » https://doi.org/10.1002/mrd.1080280307
• ²⁷ Zarcula SM, Godja G, Milovanov C, Tulcan C, Cernescu H, Otavă G, et al. Morphological aspects of cumulusoocyte complexes in different species. Lucr Stiint USAMV Iasi Med Vet. 2014;57(3-4):91-7. Available from: https://ibn.idsi.md/sites/default/files/j_nr_file/vol_57_2014_3-4.pdf?__cf_chl_f_tk=ulJu.eIL2x.DMbb0UrGx_bynweG6XEg11a4EaNlXAtM-1783101611-1.0.1.1ypxTldnyRE5lemFN5p_eXrCneREmR0s5fCJ9PI4n3Oo
  » https://ibn.idsi.md/sites/default/files/j_nr_file/vol_57_2014_3-4.pdf?__cf_chl_f_tk=ulJu.eIL2x.DMbb0UrGx_bynweG6XEg11a4EaNlXAtM-1783101611-1.0.1.1ypxTldnyRE5lemFN5p_eXrCneREmR0s5fCJ9PI4n3Oo
• ²⁸ Hasbi H, Sonjaya H, Gustina S. Oocytes population and development competence of Bali cattle embryo in vitro with different ovarian reproductive statuses. Trop J Anim Sci. 2022;45(4):389-96. https://doi.org/10.5398/tasj.2022.45.4.389
  » https://doi.org/10.5398/tasj.2022.45.4.389
• ²⁹ De Assis EIT, Azevedo VAN, De Lima Neto MF, Costa FC, Paulino LRFM, Barroso PAA, et al. Protective effect of Cimicifuga racemosa (L.) Nutt extract on oocyte and follicle toxicity induced by doxorubicin during in vitro culture of mice ovaries. Animals (Basel). 2022;13(1):18. https://doi.org/10.3390/ani13010018
  » https://doi.org/10.3390/ani13010018
• ³⁰ Sá AF, Gomes DF, Araujo IML, Lima RBA, Vasconcelos ALC, Monteiro JC, et al. The effects of dexamethasone on periodontitis and the influence of this disease in development and survival of mice ovarian follicles. Arch Curr Res Int. 2025;25(4):273-86. https://doi.org/10.9734/acri/2025/v25i41157
  » https://doi.org/10.9734/acri/2025/v25i41157
• ³¹ Alvarez C, Suliman S, Almarhoumi R, Vega ME, Rojas C, Monasterio G, et al. Regulatory T cell phenotype and anti-osteoclastogenic function in experimental periodontitis. Sci Rep. 2020;10(1):19018. https://doi.org/10.1038/s41598-020-76038-w
  » https://doi.org/10.1038/s41598-020-76038-w
• ³² Pan W, Wang Q, Chen Q. The cytokine network involved in the host immune response to periodontitis. Int J Oral Sci. 2019;11(3):30. https://doi.org/10.1038/s41368-019-0064-z
  » https://doi.org/10.1038/s41368-019-0064-z
• ³³ Pandur E, Micalizzi G, Mondello L, Horváth A, Sipos K, Horváth G. Antioxidant and anti-inflammatory effects of thyme (Thymus vulgaris L.) essential oils prepared at different plant phenophases on Pseudomonas aeruginosa LPS-activated THP-1 macrophages. Antioxidants (Basel). 2022;11(7):1330. https://doi.org/10.3390/antiox11071330
  » https://doi.org/10.3390/antiox11071330
• ³⁴ Boggess KA, Beck JD, Murtha AP, Moss K, Offenbacher S. Maternal periodontal disease in early pregnancy and risk for a small-for-gestational-age infant. Am J Obstet Gynecol. 2006;194:1316-22. https://doi.org/10.1016/j.ajog.2005.11.059
  » https://doi.org/10.1016/j.ajog.2005.11.059
• ³⁵ Saadaoui M, Singh P, Al Khodor S. Oral microbiome and pregnancy: a bidirectional relationship. J Reprod Immunol. 2021;145:103293. https://doi.org/10.3892/br.2022.1569
  » https://doi.org/10.3892/br.2022.1569
• ³⁶ Ricci E, Ciccarelli S, Mauri PA, Gerli S, Favilli A, Cipriani S, et al. Periodontitis, female fertility and conception. Biomed Rep. 2022;17(5):86. https://doi.org/10.1002/jbt.21431
  » https://doi.org/10.1002/jbt.21431
• ³⁷ Meeran MF, Prince PS. Protective effects of thymol on altered plasma lipid peroxidation and nonenzymic antioxidants in isoproterenol-induced myocardial infarcted rats. J Biochem Mol Toxicol. 2012;26(9):368-73. https://doi.org/10.1002/jbt.21431
  » https://doi.org/10.1002/jbt.21431
• ³⁸ Mahran YF, Badr AM, Aldosari A, Bin-Zaid R, Alotaibi HN. Carvacrol and thymol modulate the cross-talk between TNF-alpha and IGF-1 signaling in radiotherapy-induced ovarian failure. Oxid Med Cell Longev. 2019;2019:3173745. https://doi.org/10.1155/2019/3173745
  » https://doi.org/10.1155/2019/3173745
• ³⁹ Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci. 2010;123(Pt 24):4195-200. https://doi.org/10.1242/jcs.023820
  » https://doi.org/10.1242/jcs.023820
• ⁴⁰ Günes-Bayir A, Kocyigit A, Guler EM, Dadak A. In vitro hormetic effect investigation of thymol on human fibroblast and gastric adenocarcinoma cells. Molecules. 2020;25(14):3270. https://doi.org/10.3390/molecules25143270
  » https://doi.org/10.3390/molecules25143270

• Editor-in-Chief: Paulo Vitor Farago

• Associate Editor:
  Fábio André dos Santos

Publication Dates

• Publication in this collection
  17 Aug 2026
• Date of issue
  2026

History

• Received
  04 Dec 2025
• Accepted
  13 Apr 2026