Cervical Cytokine Profile in High‐Grade Squamous Intraepithelial Lesions

ABSTRACT

Objectives

This study aimed to characterise the local cytokine milieu in the cervicovaginal environment of women with HSIL compared with healthy controls.

Methods

In this case–control study, cervical samples were collected from 84 women aged 20–49 years, including 57 patients with histologically confirmed HSIL and 27 healthy controls. Levels of IL‐2, IL‐4, IL‐6, IL‐10, TNF, IFN‐γ and IL‐17A were quantified in cervicovaginal lavage fluid using a Cytometric Bead Array. Cytokine ratios were calculated to assess the balance between pro‐inflammatory and regulatory immune responses. Statistical comparisons between groups were performed using the Mann–Whitney test.

Results

Women with HSIL exhibited significantly lower concentrations of TNF (p = 0.030) and IL‐2 (p = 0.010) compared with controls. Correspondingly, the TNF/IL‐10 (p = 0.018) and TNF/IL‐4 (p = 0.032) ratios were markedly decreased, suggesting impaired Th1‐mediated immune activation. No significant differences were observed for IL‐17A levels or IL‐17A/IL‐6 ratios, suggesting that Th17‐associated soluble mediators may not be the predominant drivers of the observed immunomodulation in this context, although broader Th17 involvement cannot be excluded.

Conclusions

HSIL is characterised by reduced TNF and IL‐2 expression and a suppressed TNF/IL‐10 and TNF/IL‐4 balance, revealing a shift towards an immunoregulatory profile independent of Th17 activity. This cytokine signature suggests an immune milieu favouring lesion persistence and provides a potential framework for developing cytokine‐based biomarkers and immunotherapeutic strategies in HPV‐associated cervical disease.

Hypothetical mechanism of immune modulation in HSIL associated with high‐risk HPV infection, based on observed cytokine suppression. The diagram illustrates impaired Th1 responses, reflected by reduced production of IL‐2 and TNF, which may compromise the activation and cytotoxic function of CD8⁺ T cells and NK cells, facilitating immune evasion. The figure also highlights immune evasion mechanisms promoted by hrHPV, including downregulation of MHC‐I expression and upregulation of CTLA4, IL‐10, IL‐4 and TGF‐β, which contribute to immune suppression and persistence of the lesion. Red arrows represent the Th17‐independent pathway. Abbreviations: HSIL, high‐grade squamous intraepithelial lesion; hrHPV, high‐risk human papillomavirus; M, macrophages; NK, natural killer; IL, interleukin; TNF, tumour necrosis factor; CTLA4, cytotoxic T‐lymphocyte‐associated protein 4; MHC‐I, major histocompatibility complex class I; TGF‐β, transforming growth factor beta; Th1, T helper 1. Created in https://BioRender.com (accessed on 1 May 2025).

1. Introduction

Chronic inflammation and immunological alterations are increasingly acknowledged as pivotal elements in elucidating the mechanisms that facilitate the progression of cervical dysplasia to malignancy. A notable characteristic of various cancers, including cervical cancer, is the modulation of the host's innate immune system [1, 2]. In the early stages of human papillomavirus (HPV) infection, cervical dysplasia disrupts inflammatory signalling by modulating Toll‐like receptor (TLR) pathways, resulting in altered cytokine production and impaired immune cell differentiation and function [3, 4]. This immunological imbalance fosters an environment conducive to viral persistence and the advancement of lesions.

The interrelationship between inflammation and cancer is intricately linked through both intrinsic and extrinsic pathways. In the intrinsic pathway, genetic mutations that drive oncogenesis concurrently promote the establishment of an inflammatory microenvironment [5]. Conversely, in the extrinsic pathway, chronic unresolved inflammation directly contributes to tumourigenesis [6]. Within the context of cervical cancer, cytokines produced during malignant transformation may either promote or inhibit neoplastic progression, or exert both effects simultaneously, depending on their interactions within the local immune microenvironment.

T‐helper (Th) 1 cytokines, such as interleukin (IL)‐12, IL‐2 and interferon‐γ (IFN‐γ), are instrumental in anti‐tumour immunity by enhancing the functions of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, inhibiting angiogenesis and promoting antigen presentation. For instance, IL‐12 upregulates IFN‐γ production, thereby supporting cytotoxic immune responses, while IL‐2 facilitates the transformation of NK cells into lymphokine‐activated killer cells [7, 8, 9, 10, 11]. In contrast, Th2 cytokines, including IL‐10 and IL‐4, suppress anti‐tumour immunity by downregulating IFN‐γ and IL‐12, reducing major histocompatibility complex (MHC) expression on tumour cells and impairing antigen presentation [11, 12]. These effects contribute to immune evasion and tumour progression, highlighting the classical imbalance between Th1 and Th2 cytokines in immune surveillance and tumour pathogenesis.

Recently, the involvement of Th17 cells and their signature cytokine, IL‐17A, in the progression of HPV‐associated cervical lesions and cancer has garnered considerable attention. IL‐17A, primarily produced by Th17 cells, is associated with chronic inflammation and immune modulation within the tumour microenvironment [13, 14]. Evidence indicates that IL‐17A promotes tumourigenesis by enhancing angiogenesis, recruiting immunosuppressive cells and remodelling the extracellular matrix to facilitate tumour invasion and metastasis [14, 15, 16]. Nonetheless, its dual role is underscored by its capacity to recruit effector immune cells, such as CD8⁺ T cells, to the tumour site [17]. Despite these insights, the specific pathways underlying Th17‐mediated effects remain inadequately understood, reflecting the complexity of Th17 cell plasticity and its context‐dependent functions in cervical carcinogenesis. Cytokine imbalance has been implicated in cancer progression, including cervical cancer, with a shift from pro‐inflammatory to anti‐inflammatory cytokine dominance weakening immune defences and promoting tumour growth [18].

Although growing evidence highlights immune dysregulation as a hallmark of HPV‐associated cervical carcinogenesis, the precise immunological mechanisms operative in the tumour microenvironment of high‐grade squamous intraepithelial lesions (HSIL) remain insufficiently elucidated. HSIL represents a pivotal transitional stage between persistent HPV infection and invasive cervical cancer, offering a critical window to investigate the immune alterations that may facilitate immune evasion and drive neoplastic progression. A more comprehensive understanding of the local immune landscape in HSIL is essential to uncover the pathophysiological basis of lesion persistence and to inform potential immunotherapeutic strategies.

Therefore, this study aimed to evaluate changes in cytokine profiles, focusing on expression levels and functional interactions, in the local immune microenvironment of patients with HSIL compared with healthy controls. Specifically, it sought to identify key immunological dysregulations, such as altered balances between pro‐ and anti‐inflammatory cytokines, that may facilitate immune evasion and drive the progression of cervical lesions.

2. Materials and Methods

2.1. Study Design and Population

A total of 84 adult women were recruited through convenience sampling for this case–control study, conducted at two hospitals in north‐east Brazil from January 2022 to December 2023. Of these participants, 57 women received a diagnosis of high‐grade squamous intraepithelial lesion (HSIL) based on the Bethesda System criteria. All HSIL diagnoses were histologically confirmed via cervical biopsy. HSIL is histologically categorised into cervical intraepithelial neoplasia (CIN) 2 and CIN 3. CIN 2 is characterised by atypical basaloid cells extending into the lower two‐thirds of the epithelial layer, while CIN 3 exhibits proliferation of atypical cells in more than the upper two‐thirds of the epithelium or full‐thickness nuclear atypia. A control group consisting of 27 women with cervical cytology classified as negative for intraepithelial lesion or malignancy (NILM) was also recruited. Clinical and socio‐epidemiological data were extracted from each patient's medical records.

Exclusion criteria included: (a) age younger than 20 years or older than 49 years; (b) pregnancy or postmenopausal status; (c) previous treatment involving chemotherapy, radiation, or surgery; (d) existing cancer; or (e) current medication for sexually transmitted diseases. Additionally, specimens collected during menstrual periods were excluded from the study.

2.2. Ethical Concerns

The study protocol received approval from the institutional ethics committee (protocol number 68615417.0.0000.5293). Written informed consent was obtained from each participant prior to their inclusion in the study.

2.3. Sample Collection and Processing

Cervical cytology was performed using Papanicolaou (Pap) smears on exfoliated cells collected from the surface of the cervix. Results were reported according to the Bethesda System terminology. All participants diagnosed with HSIL subsequently underwent colposcopy and cervical biopsy for histological confirmation. All cervical cytology and histological samples were independently assessed by two experienced cytologists and pathologists from the participating institutions.

For cytokine measurement, cervicovaginal lavage samples were collected and diluted in 2 mL of saline. In control women, lavage was performed at the time of the routine cervical cancer screening visit, concurrent with the Pap smear collection. In HSIL patients, lavage was collected at the colposcopy visit, prior to colposcopic examination and cervical biopsy, following referral based on a prior cytological result of at least high‐grade squamous intraepithelial lesion or a positive previous biopsy. Cervical samples were then centrifuged at 800 g for 10 min at room temperature and subsequently stored at −70°C until analysis.

2.4. Measurement of Cytokine Levels

Levels of IL‐2, IL‐4, IL‐6, IL‐10, TNF, IFN‐γ and IL‐17A in the cervical microenvironment were quantified using a Th1/Th2/Th17 Cytometric Bead Array (CBA) (BD Biosciences, San Jose, CA), following the manufacturer's guidelines and protocols. Sample acquisition was performed using a CytoFLEX FACS flow cytometer (Beckman Coulter, 3B1R0 V). Cytokine levels were subsequently analysed using FCAP Array software (version 3.0.1, BD).

2.5. Statistical Analyses

All data were evaluated for normal distribution using the Shapiro–Wilk test. Continuous variables were presented as medians and interquartile ranges (IQR = third quartile–first quartile). Differences between groups were assessed using the Mann–Whitney nonparametric test. Statistical analyses and graphical representations were executed with GraphPad Prism software (version 8.0, San Diego, CA, USA). To evaluate the balance between pro‐inflammatory and anti‐inflammatory cytokines, the cytokine ratio was computed for each individual, and the results were compared using the Mann–Whitney non‐parametric test. p values of < 0.05 were deemed statistically significant.

3. Results

3.1. Characteristics of Included Participants

The demographic and clinical characteristics of the study population are presented in Table 1. The mean age was comparable between cases (39.3 ± 11.5 years) and controls (37.8 ± 7.47 years). Most participants in both groups self‐identified as Brown (70.2% of cases and 77.8% of controls), followed by individuals identifying as White (15.8% of cases and 14.8% of controls). A significant proportion of participants completed high school (45.6% of cases and 51.9% of controls). Marital status exhibited a similar distribution across groups, with single women representing the majority (54.4% of cases and 55.6% of controls).

TABLE 1.

Demographic and clinical characteristics of included women.

Variable	Case (n = 57)	Control (n = 27)	p
Age, mean (SD)	39.3 (11.5)	37.8 (7.47)	0.0534
Race, n (%)			< 0.0001
Black	1 (1.8)	2 (7.4)
Brown	40 (70.2)	21 (77.8)
White	9 (15.8)	4 (14.8)
Not informed	7 (12.3)	0
Education level, n (%)			0.3037
Elementary school	18 (31.6)	10 (37.0)
High school	26 (45.6)	14 (51.9)
Undergraduate	6 (10.5)	3 (11.1)
Not informed	7 (12.2)	0
Marital status, n (%)			0.2396
Single	31 (54.4)	15 (55.6)
Married	15 (26.3)	11 (40.7)
Widowed	2 (3.5)	1 (3.7)
Stable union	2 (3.5)	0
Not informed	7 (12.3)	0

3.2. Comparison of Cytokine Expression Between HSIL Patients and Controls

Cytokine expression profiles were assessed and compared between women diagnosed with HSIL (n = 57) and healthy controls (n = 27). Overall, most cytokines did not demonstrate statistically significant differences between the groups; however, notable exceptions were identified (Figure 1). Tumour necrosis factor (TNF) levels were significantly reduced in the HSIL group compared with controls (7.66 ± 0.53 vs. 7.87 ± 0.50; p = 0.030). Additionally, IL‐2 levels were significantly lower in the HSIL group (7.54 ± 0.28) than in controls (7.63 ± 0.19; p = 0.010). Conversely, no significant differences were observed in IL‐17A levels between the HSIL group (5.99 ± 0.86) and controls (6.08 ± 0.79; p = 0.926), nor in IFN‐γ (5.63 ± 0.35 vs. 5.58 ± 0.44; p = 0.952), IL‐10 (6.28 ± 0.69 vs. 6.31 ± 0.40; p = 0.543), IL‐6 (7.04 ± 2.27 vs. 8.47 ± 5.08; p = 0.127), or IL‐4 (5.77 ± 0.34 vs. 5.81 ± 0.25; p = 0.877).

FIGURE 1.

Cytokine expression levels in patients with HSIL and healthy controls. Swarm plots illustrate the expression levels of selected cytokines: (A) IL‐17A; (B) IFN‐γ; (C) TNF; (D) IL‐10; (E) IL‐6; (F) IL‐4; (G) IL‐2, comparing controls and HSIL patients. Each point represents an individual sample, with horizontal lines denoting median values. Statistical significance was established at p < 0.05. HSIL, high‐grade squamous intraepithelial lesion.

3.3. Comparison of Cytokine Ratios Between HSIL Patients and Controls

Significant reductions were documented in the TNF/IL‐10 (p = 0.01) and TNF/IL‐4 (p = 0.03) ratios in the HSIL group compared with controls (Table 2). However, most cytokine ratios, including IFN‐γ/IL‐17 (p > 0.999), IFN‐γ/TNF (p = 0.256), IFN‐γ/IL‐10 (p = 0.984), and IFN‐γ/IL‐4 (p = 0.871), did not reveal significant differences between the two groups. Similarly, ratios involving IL‐17A, such as TNF/IL‐17A (p = 0.593), IL‐17/IL‐10 (p = 0.690), IL‐17A/IL‐6 (p = 0.122), IL‐17A/IL‐4 (p = 0.810), and IL‐17A/IL‐2 (p = 0.663), also exhibited no significant changes.

TABLE 2.

Comparison of cytokine ratios between case and control groups.

	Case (n = 57)	Control (n = 27)	p
IFNg/IL‐17	0.924 (0.104)	0.920 (0.09)	> 0.999
IFNg/TNF	0.729 (0.045)	0.730 (0.041)	0.256
IFNg/IL‐10	0.895 (0.041)	0.893 (0.063)	0.984
IFNg/IL‐4	0.969 (0.045)	0.972 (0.049)	0.871
IFNg/IL‐2	0.742 (0.045)	0.733 (0.047)	0.259
TNF/IL‐17	1.283 (0.137)	1.281 (0.139)	0.593
TNF/IL‐10	1.227 (0.058)	1.252 (0.032)	0.01
TNF/IL‐4	1.327 (0.071)	1.353 (0.082)	0.03
TNF/IL‐2	1.012 (0.05)	1.021 (0.05)	0.143
IL‐17/IL‐10	0.962 (0.102)	0.980 (0.88)	0.690
IL‐17/IL‐6	0.837 (0.293)	0.748 (0.399)	0.122
IL‐17/IL‐4	1.042 (0.117)	1.045 (0.116)	0.810
IL‐17/IL‐2	0.788 (0.096)	0.796 (0.084)	0.663
IL‐6/IL‐10	1.122 (0.374)	1.384 (0.803)	0.211
IL‐6/IL‐4	1.203 (0.398)	1.518 (0.806)	0.260
IL‐6/IL‐2	0.935 (0.315)	1.100 (0.609)	0.391

4. Discussion

This study elucidates a distinct cervical cytokine profile in HSIL patients, characterised by reduced levels of TNF and IL‐2, alongside reduced TNF/IL‐10 and TNF/IL‐4 ratios, which appear to operate without detectable dysregulation of IL‐17A or related Th17‐associated soluble mediators in cervicovaginal lavage. These findings imply a complex immune environment in HSIL patients, marked by a blunted pro‐inflammatory response and a potential shift towards immune tolerance. Such dynamics may represent early mechanisms of immune escape, in which the downregulation of Th1‐associated cytokines impairs effective anti‐tumour surveillance, potentially facilitating lesion persistence and progression.

Previous research has indicated that patients with squamous intraepithelial lesions (SIL) may exhibit an elevated Th1/Th2 ratio independent of HPV infection [19]. TNF serves as a crucial mediator in orchestrating complex multicellular processes, functioning as both a pro‐inflammatory and anti‐tumour cytokine. It plays a pivotal role in downregulating HPV gene transcription while enhancing host inflammatory responses to control infection [20]. Furthermore, TNF contributes to immune defence by promoting the antigen presentation of HPV‐derived proteins via antigen‐presenting cells to effector T cells [21], as well as inducing apoptosis in cervical cancer cells [22]. Additionally, TNF facilitates the degradation of tumour stroma through CTLs and tumour‐infiltrating macrophages [23]. However, the expression and functionality of TNF can be influenced by various factors, including single nucleotide polymorphisms (SNPs), epigenetic modifications, environmental influences and interactions with other molecular pathways.

Reduced TNF levels may hinder HPV antigen presentation, thus promoting viral persistence and contributing to cervical carcinogenesis. Notably, elevated TNF levels have been documented in patients with cervical carcinoma, irrespective of HPV status [24, 25, 26]. Conversely, significantly lower levels of both TNF and IL‐2 have been observed in low‐grade lesions when compared with HSIL [27], indicating a dynamic shift in immune response as cervical lesions progress.

In this context, our findings of markedly decreased TNF in the HSIL cohort, accompanied by a reduction in the TNF/IL‐10 and TNF/IL‐4 ratios, suggest a shift towards an immunosuppressive microenvironment favouring lesion persistence and immune evasion. The diminished pro‐inflammatory capacity, particularly of TNF, likely reflects impaired activation of Th1 cells, which play a critical role in antiviral defence and cytotoxic T‐cell priming. TNF is known to enhance the expression of adhesion molecules and MHC class I on epithelial cells, thereby facilitating the recruitment and antigen‐specific response of CD8⁺ T cells. Its downregulation, therefore, may attenuate effective immune surveillance.

The role of IL‐2 in the development of SIL remains ambiguous, with previous studies yielding conflicting results [28, 29]. The anti‐tumour potential of IL‐2 is primarily attributed to its capacity to stimulate cell‐mediated immunity and exert direct effects on tumour cells. Some studies have demonstrated an inverse association between IL‐2 expression and SIL progression [29], with higher IL‐2 levels correlating with a reduced risk of SIL [30]. Evidence suggests that diminished IL‐2 levels may contribute to tumour growth and progression [31, 32]. Intriguingly, our findings of decreased IL‐2 levels in the HSIL group may indicate a weakened cell‐mediated immune response, potentially impairing the activation of CTLs and NK cells and thereby facilitating lesion persistence and progression [33].

However, this decrease may also be associated with a shift towards immunoregulatory processes that mitigate excessive pro‐inflammatory responses, such as the Treg clone expansion or T cell functional exhaustion [34, 35], contributing to an immune microenvironment that is less effective in controlling lesion progression. Thus, reduced IL‐2 levels in HSIL may reflect a dysfunctional or suppressed cell‐mediated immune response rather than an active anti‐tumor effort.

The presence of HPV‐infected epithelial cells in HSIL lesions appears to trigger both immune evasion and dysregulated immune activation. While HPV enhances tumour immune escape through downregulation of major histocompatibility complex class I (MHC‐I) [36] and the induction of immunosuppressive factors such as transforming growth factor (TGF)‐β, IL‐10 and cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) [37], we hypothesize that, in parallel, there is suppression of innate immune cell activity, including natural killer (NK) cells, macrophages and dendritic cells, or a shift towards an immunoregulatory phenotype that undermines Th1 polarisation and antiviral cytotoxic responses. It remains unclear whether this altered immune profile reflects a viral strategy to escape immune control or a host‐driven adaptation to limit tissue damage; nevertheless, both mechanisms may contribute to lesion persistence and progression.

IL‐17‐producing cells exhibit a dual role in tumour immunity, participating in both pro‐tumorigenic and anti‐tumorigenic processes, including angiogenesis. Elevated levels of IL‐17 and IL‐23 have been documented in patients with HPV‐related cervical epithelial hyperplasia, suggesting an immunosuppressive role in early lesions [38]. The differentiation of Th17 cells is regulated by IL‐6 in conjunction with TGF‐β, which promotes the development of Th17 cells from naïve T cells. Conversely, IL‐6 inhibits TGF‐β‐induced Treg differentiation, thereby disrupting immune tolerance and potentially exacerbating inflammation [39].

Increased levels of Th17 cells in cervical cancer have been correlated with poor clinical outcomes, while other studies report anti‐tumour effects of IL‐17 through immune activation and angiogenesis. Notably, IL‐17A is elevated in low‐grade SIL and may serve as a prognostic marker in cervical cancer [41], although its precise role in the SIL microenvironment remains unclear. Our findings indicate that the cervical cytokine signature in HSIL does not show detectable differences in IL‐17A or IL‐6 levels, nor in the IL‐17A/IL‐6 ratio, suggesting that Th17‐associated soluble mediators may not be the predominant drivers of the immunomodulation observed in our sample. However, this should not be interpreted as definitive evidence against Th17 involvement; our study assessed only soluble cytokines in cervicovaginal lavage and did not evaluate Th17 cell frequencies, tissue‐level Th17 markers or related upstream regulators (e.g., IL‐23, RORγt). The potential contribution of Th17‐mediated immunity to HSIL pathogenesis therefore warrants further investigation using cellular and tissue‐based approaches [40].

This study presents several limitations. The relatively small sample size may restrict statistical power and the generalisability of the findings. Furthermore, the difference in timing between lavage collection in controls (at routine screening) and HSIL patients (at the colposcopy visit, following prior abnormal cytology or biopsy) may have introduced variability in the local immune milieu between groups, which could influence cytokine levels independently of lesion grade. Additionally, the adoption of a convenience sample compromises the extrapolation of results to a broader population. We did not assess functional immune cell subsets or intracellular signalling pathways. Importantly, the study was limited to the analysis of soluble cytokines in cervicovaginal lavage fluid; tissue‐based cellular immune analysis, such as immunohistochemistry or flow cytometry of HSIL biopsy specimens compared with normal cervical tissue, was not feasible, as biopsy material was collected exclusively for routine histopathological diagnosis and was not available for research‐grade immunophenotyping. Such cellular analysis would provide important complementary insights into the local immune landscape and is identified as a priority for future work. Additionally, the absence of HPV typing and viral load data constitutes a significant limitation, especially since the immune response was compared between groups and the presence of the virus strongly influences this response. Moreover, the study design precludes the evaluation of lesion progression or regression over time, limiting our capacity to ascertain the prognostic significance of reduced TNF and IL‐2 levels. Future prospective studies with larger cohorts and longitudinal follow‐up are required to elucidate the role of these cytokines in immune surveillance, tumour escape and disease outcomes, potentially guiding the development of immunological biomarkers for cervical cancer prevention.

5. Conclusion

This study demonstrates that HSIL is characterised by reduced levels of TNF and IL‐2, alongside diminished TNF/IL‐10 and TNF/IL‐4 ratios, suggesting a shift towards an immunoregulatory profile within the cervical microenvironment. Notably, no significant differences in IL‐17A levels or IL‐17A‐related ratios were observed between groups. While this finding does not establish that Th17‐mediated pathways are uninvolved in HSIL pathogenesis, it suggests that Th17‐associated soluble mediators may not be the predominant drivers of the immunomodulation detected in this study.

These findings reveal the potential utility of TNF and IL‐2 as biomarkers for HSIL progression and provide a rationale for further investigation into targeted immunotherapeutic strategies. Longitudinal studies with larger cohorts and cellular immune analyses are needed to determine the prognostic value of these cytokines and to further elucidate HPV‐specific immune interactions, addressing key gaps in our understanding of immune evasion and tumour progression in cervical carcinogenesis.

Author Contributions

Talles Henrique de Araújo Pontes, Carolina de O. Mendes‐Aguiar, Deyse de Souza Dantas, and Janaina Cristiana de Oliveira Crispim conceived and designed the study. Talles Henrique de Araújo Pontes, Amaxsell Thiago Barros de Souza, Carolina de O. Mendes‐Aguiar, Maria do Perpétuo Socorro Nobre Medeiros e Silva, and George Alexandre Lira performed the experiments and collected the data. Juliana Dantas de Araújo Santos Camargo and Carolina de O. Mendes‐Aguiar analyzed the data. Talles Henrique de Araújo Pontes, Amaxsell Thiago Barros de Souza, Carolina de O. Mendes‐Aguiar, Maria do Perpétuo Socorro Nobre Medeiros e Silva, Juliana Dantas de Araújo Santos Camargo, George Alexandre Lira, Ricardo Ney Cobucci, Deyse de Souza Dantas, and Janaina Cristiana de Oliveira Crispim interpreted the data. Talles Henrique de Araújo Pontes, Amaxsell Thiago Barros de Souza, Carolina de O. Mendes‐Aguiar, Ricardo Ney Cobucci, and Janaina Cristiana de Oliveira Crispim wrote the manuscript. Deyse de Souza Dantas and Janaina Cristiana de Oliveira Crispim supervised the study. All authors approved the final version of the manuscript.

Funding

This research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES—Finance Code 001).

Ethics Statement

The study protocol received approval from the institutional ethics committee (protocol number 68615417.0.0000.5293).

Consent

Written informed consent was acquired from each participant prior to their inclusion in the study.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.