Amygdala volume abnormalities and cognitive impairment in major depressive disorder and bipolar disorder II

Abstract

Background

The amygdala, as a crucial brain region, plays a key role in processing emotions and cognitive information. Given that patients with major depressive disorder (MDD) and bipolar disorder II (BD II) experience cognitive impairments, accurate diagnosis becomes a vital research focus. Consequently, we conducted an in-depth investigation into the relationship between the static volume of the amygdala and cognitive functions, aiming to provide valuable insights for future research in this field.

Methods

We collected a total of 42 treatment-naive MDD patients, 38 BD II patients, and 46 healthy controls (HC) from the Third People’s Hospital of Foshan. Using magnetic resonance imaging (MRI) and an automated segmentation tool, we extracted the structural volumes of the amygdala. Pearson correlation analyses were performed with scores from the 17-item Hamilton Depression Rating Scale (HAMD), the Hamilton Anxiety Rating Scale (HAMA), and the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Finally, we compared clinical data, as well as volume changes in the amygdala, among MDD patients, BD patients, and HC.

Results

In terms of depression scores, MDD individuals experience more severe emotional distress than those with BD. Both MDD and BD II patients show significantly higher anxiety levels compared to the HC. BD II is linked to widespread cognitive deficits, indicating poorer cognitive performance compared to both MDD and HC. In MDD, there is an observed amygdala volume increase compared to the HC. Additionally, correlation analysis revealed that the left amygdala volume is significantly correlated with Delayed Memory (List Recall) (r = 0.234, p = 0.010) and Delayed Memory (Story Recall) (r = 0.215, p = 0.018) (after multiple Bonferroni correction).

Conclusion

For medication-naïve individuals with MDD and BD II, we observed a correlation between amygdala volume and cognitive function. From this, changes in amygdala volume may help reflect cognitive impairment in patients during the acute phase.

Introduction

Bipolar disorder (BD) and major depressive disorder (MDD) are two prevalent global mental illnesses [1]. The primary distinction lies in the fact that MDD patients typically experience prolonged and continuous depressive states, whereas BD patients undergo cyclical mood fluctuations, such as depressive or manic episodes [2]. BD encompass both BD I and BD II. BD-I is characterized by recurrent manic, depressive, or mixed episodes, affecting approximately 1% of the population [3]. BD II is characterized by a hypomanic episode and a major depressive episode [4]. The estimated global lifetime prevalence is 0.4–1.1% [4]. The distinction between BD II and MDD relies heavily on identifying hypomania [5]. In addition to the challenge of recognizing mania or hypomania as definitive signs of BD, there are other practical clinical considerations in distinguishing BD II from MDD. These include the need to avoid over-diagnosing MDD, which may result in unnecessary treatment with antidepressants that could be ineffective or destabilizing for BD patients [6], as well as the risk of over-diagnosing BD II, potentially leading to long-term treatment with antimanic, antipsychotic, or mood-stabilizing drugs that may not be needed and could expose patients to unnecessary side effects and increased treatment costs [7, 8]. BD II remains a challenging syndrome to accurately identify and differentiate from MDD, especially in clinical practice [7].

Cognitive deficits frequently accompany psychiatric disorders, notably BD-II and MDD. Characterized by depressive episodes and hypomanic phases without full mania [9], BD-II manifests cognitive impairments spanning psychomotor speed, verbal fluency, memory retention, and executive functions [10, 11]. MDD patients similarly show deficits in attention, processing speed, and verbal learning [12, 13]. Current research presents conflicting findings regarding cross-diagnostic differences. While some studies report greater cognitive impairment in BD-II than MDD, particularly in verbal memory and executive control [14, 15], others suggest preserved cognition in BD-II with broader executive dysfunction in MDD [16]. Notably, several investigations [13, 17, 18] found comparable deficit severity between the two disorders. Overall, research findings on cognitive function in these two disorders remain inconsistent, particularly in the aspect of early cognitive function where studies are relatively limited.

The amygdala plays a crucial role in generating, recognizing, and regulating emotions, as well as in controlling learning and memory. It serves as a central hub for emotional processing, especially regarding negative emotions, and is involved in regulating emotions like fear and anxiety [19, 20]. Both BD and MDD patients exhibit abnormal emotional processing, with sustained, abnormal activation in the amygdala [21–23]. Our previous research has shown that an increase in the amygdala volume is sometimes considered a result of impaired regulation and underdevelopment of the prefrontal cortex (PFC) [24]. Additionally, research indicates that changes in amygdala volume can affect cognitive function in patients with BD and MDD [25–27]. Specifically, variations in the volumes of the left and right amygdalae can affect cognitive function to different extents [27, 28].

In MDD, research indicates that patients with MDD show increased activity or enlarged volume in the amygdala compared to healthy individuals [27, 29, 30]. This may explain why individuals with depression often experience heightened emotional responses, particularly in the context of negative emotions. Neuroimaging investigations have demonstrated heightened dynamic functional connectivity between the left medial amygdaloid complex and right medial prefrontal cortical regions in individuals experiencing depressive episodes of BD II [31]. Other research reports suggest that abnormally elevated amygdala activity may serve as a potential biomarker for depressive states [32]. However, there are studies with differing conclusions [33, 34]. These discrepancies in results could be attributed to variations in the stages of BD II studied or the influence of medications. Therefore, alterations in amygdala volume maybe is crucial features of both BD II and MDD.

Due to the certain degree of similarity in cognitive impairments between patients with BD II in the depressive state and those with MDD, our research aims to explore the underlying mechanisms. Therefore, we plan to utilize magnetic resonance imaging (MRI) technology to extract amygdala volume from untreated patients with MDD and BD II, and compare these volume with differences in cognitive function. This will enable us to more comprehensively investigate the mechanisms of cognitive impairments in both severe depression and bipolar disorder.

Method

Participants

Participants in the MDD group (n = 42) and BD II group (n = 38) were recruited from the Third People’s Hospital of Foshan (Foshan Mental Health Center). Inclusion criteria were as follows: 1) Meeting the diagnostic criteria for MDD and BD II as outlined in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5); 2) Age between 18 and 60 years; 3) A minimum of 9 years of education (to ensure the ability to understand assessments); 4) Han Chinese ethnicity, right-handed; 5) No prior use of any psychotropic medications before data collection; 6) All subjects had no contraindications for MRI scanning, no organic brain disorders, no physical illnesses, no history of substance abuse, no history of traumatic brain injury, or neurological diseases; 7) The patients are currently in a depressive state.

The healthy control group (HC) (n = 46) was recruited from the local community and met the following inclusion criteria: 1) Confirmed by a psychiatrist through the Structured Clinical Interview for DSM-5, participants had no personal history of psychiatric disorders or family history of such diseases; 2) Age between 18 and 60 years; 3) At least 9 years of education to ensure the ability to understand the assessments; 4) Han ethnicity, right-handed; 5) All participants had no contraindications for MRI scanning, no evidence of organic brain diseases, physical illnesses, substance abuse history, traumatic brain injury, or neurological disorders.

Procedure

The severity of depression was assessed using the 17-item Hamilton Depression Rating Scale (HAMD), where a higher score indicates a more pronounced level of depressive symptoms [35]. Anxiety symptoms were measured through the Hamilton Anxiety Rating Scale (HAMA), comprising 14 items, each scored from 0 to 4. A higher score on the HAMA reflects a higher severity of anxiety symptoms [36]. Participants’ cognitive abilities were assessed using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), which aimed to evaluate immediate memory, visuospatial construction, language, attention, and delayed memory functions. Higher scores on the RBANS indicate better cognitive functioning [37].

Imaging data acquisition and preprocessing

MRI scanning (3.0 Tesla, General Electric, USA) data processing and analysis were performed as follows: 3D structural MRI scan parameters included a time repetition (TR) of 8.6 milliseconds, an echo time (TE) of 3.3 milliseconds, a flip angle (FA) of 12 degrees, a field of view (FOV) measuring 256 millimeters by 256 millimeters, a matrix size of 256 × 256, slice thickness of 1 mm, no slice gap, and a total of 172 slices. MRIcron software (https://www.nitrc.org/projects/mricron/) was used to convert the raw DICOM format MRI scan data into nii format. VBM data processing was conducted on the Matlab R2021a platform using the CAT12 toolbox (version CAT12.8) in SPM12 for preprocessing each subject. During data processing, age, sex, years of education, and total intracranial volume (TIV) were included as covariates.

The preprocessing steps recommended by CAT12 were as follows: 1) Brain tissue segmentation: The images were segmented into gray matter, white matter, and cerebrospinal fluid based on standard tissue probability maps (TPM). 2) Spatial registration: The East Asian Brains template was chosen for affine transformation, and the DARTEL (Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra) method was used to register the segmented images from individual space to the Montreal Neurological Institute (MNI) standard space using the IXI555_MNI152 template, followed by nonlinear modulation and resampling to 1.5 mm×1.5 mm×1.5 mm. 3) ROI analysis: The Anatomical Automatic Labeling (AAL) template was used to automatically segment the preprocessed gray matter images into 90 regions of interest (ROIs), from which the amygdala volume was extracted for further analysis.

Statistical analysis

Statistical analysis was carried out using IBM SPSS Statistics 23 (SPSS 23, https://www.ibm.com/analytics/spss-statistics-software, IBM, Armonk, New York, USA) to analyze clinical scale scores. The Kolmogorov-Smirnov test (K-S test) indicated that the measurement data for the MDD group, BD group, and HC conformed to a normal distribution. One-way ANOVA, followed by Bonferroni post-hoc t-tests, were employed to evaluate the mean differences in the baseline clinical data among the groups. Chi-square tests were used to compare categorical variables. Additionally, Pearson/Spearman correlation analyses were performed to investigate the relationship between amygdala volume and clinical data. The p-values from the correlation analyses were adjusted for multiple comparisons with the Bonferroni correction.

Result

Demographic and clinical characteristics

The research findings indicate that the HAMD-17 scores of MDD patients are higher than those of BD II patients, highlighting a more negative emotional experience in MDD compared to BD II. Additionally, both MDD and BD II patients exhibit HAMA scores higher than the HC, further confirming significantly elevated anxiety levels in both MDD and BD II patients compared to the healthy control group. In addition, RBANS scores show that BD II patients exhibit widespread cognitive impairments compared to HC, and also perform worse than patients with major depressive disorder. Regarding the amygdala volume, the research results indicate that, compared to the HC, the right amygdala volume is increased in MDD patients. Additionally, there is little difference in the right amygdala volume between MDD and BD II patients (Table 1).

Table 1.

Comparison of clinical and MRI data among HC, MDD, and BD groups

		HC (n = 46)	MDD (n = 42)	BD II (n = 38)	post-hoc t-test	cohen’s d HC vs. MDD	cohen’s d HC vs. BD II	cohen’s d MDD vs. BD II	F/χ2	p
Age		29.369 ± 8.070	26.571 ± 11.607	27.026 ± 7.613	-	0.280	0.299	−0.046	1.154	0.319
Gender (male/female)		19/27	17/25	15/23	-				0.029	0.986
Education (years)		12.695 ± 3.788	12.976 ± 2.373	12.868 ± 2.781	-	−0.089	−0.052	0.042	0.093	0.911
HAMD-17		2.500 ± 3.364	24.682 ± 6.954	19.405 ± 7.679	HC < BD II < MDD	−4.061	−2.852	0.720	155.617	< 0.001*
HAMA		2.021 ± 2.577	15.878 ± 5.455	13.368 ± 7.084	HC < MDD = BD II	−3.248	−2.129	0.397	87.06	< 0.001*
RBANS
Immediate memory (Learning)		27.630 ± 7.034	27.666 ± 6.269	22.315 ± 5.714	HC = MDD > BD II	−0.005	0.829	0.892	9.196	< 0.001*
Immediate memory (Story Memory)		14.413 ± 5.957	13.809 ± 5.836	9.684 ± 4.714	HC = MDD > BD II	0.102	0.880	0.778	8.564	< 0.001*
Visuospatial Construction		17.760 ± 2.414	18.690 ± 2.112	17.815 ± 2.846	-	−0.410	−0.021	0.349	1.897	0.154
Language		18.282 ± 4.344	17.238 ± 4.536	15.973 ± 4.213	-	0.235	0.540	0.289	2.903	0.059
Attention (Digit Span)		14.130 ± 2.176	13.952 ± 2.594	12.921 ± 2.431	-	0.074	0.524	0.410	2.976	0.055
Attention (Coding)		49.804 ± 14.149	47.642 ± 12.664	41.736 ± 13.339	HC = MDD, HC > BD II	0.161	0.587	0.454	3.929	0.022*
Delayed memory (List Recall)		6.608 ± 3.108	6.476 ± 2.940	4.973 ± 2.271	HC = MDD, HC > BD II	0.044	0.601	0.572	4.137	0.018*
Delayed memory (List Recognition)		19.543 ± 1.047	19.547 ± 1.063	18.868 ± 1.298	HC = MDD > BD II	−0.004	0.572	0.572	4.731	0.010*
Delayed memory (Story Recall)		7.521 ± 3.740	7.738 ± 3.728	4.842 ± 2.775	HC = MDD > BD II	−0.036	0.814	0.881	8.557	< 0.001*
Delayed memory (Figure Recall)		14.456 ± 4.707	14.309 ± 4.081	11.026 ± 4.239	HC = MDD > BD II	0.033	0.766	0.789	7.869	0.001*
Volume (Amygdala)	L	0.981 ± 0.113	0.994 ± 0.098	0.974 ± 0.096	-	−0.123	0.067	0.206	0.408	0.666
	R	1.044 ± 0.106	1.106 ± 0.117	1.069 ± 0.104	HC < MDD, MDD = BD II	−0.555	−0.238	0.334	3.535	0.032*

HC Healthy control group, MDD Major depressive disorder, BD II Bipolar disorder II, HAMD-17 17-item Hamilton Depression Rating Scale, HAMA Hamilton Anxiety Rating Scale, RBANS Repeatable Battery for the Assessment of Neuropsychological Status

*Indicated p<0.05; Independent sample t-test, two-tailed

Correlation between amygdala volume and cognitive functions

We conducted a correlation analysis between the amygdala volume and gender, age, education, HAMD, HAMA and cognitive functions in all HC, MDD, and BD II groups.

After multiple Bonferroni corrections, the results indicate that amygdala volume is positive correlated with education and negative correlated with gender and age. Also, the right amygdala volume is positive correlated with HAMD (taking gender, age, and education as covariates) (Table 2).

Table 2.

Correlation between amygdala volume and gender, age, education, HAMD, HAMA in HC, MDD, and BD II

Amygdala			Left	Right
Gendera	r	−0.568		−0.419
	p	< 0.001*		< 0.001*
	p’	< 0.001*		< 0.001*
Ageb	r	−0.269		−0.288
	p	0.002*		0.001*
	p’	0.010*		0.005*
Educationb	r	0.249		0.270
	p	0.005*		0.002*
	p’	0.025*		0.010*
HAMDb	r	0.030		0.246
	p	0.744		0.006*
	p’	-		0.030*
HAMA b	r	0.012		0.220
	p	0.898		0.014*
	p’	-		0.072

HAMD Hamilton Depression Rating Scale, HAMA Hamilton Anxiety Rating Scale

^aPoint-biserial Correlation analysis

^bPearson correlation analysis (n = 88)

*Indicated p<0.05; Independent sample t-test, two-tailed, p' represents the result after multiple Bonferroni correction

Additionally, taking gender, age, and education as covariates, the results reveal that the left amygdala volume is significantly correlated with Delayed Memory (List Recall) and Delayed Memory (Story Recall) (Table 3). No correlation was found after multiple Bonferroni corrections.

Table 3.

Correlation between amygdala volume and cognitive functions in HC, MDD, and BD II following multiple bonferroni correction

Amygdala		Left	Right
Immediate memory (Learning)	r	0.149	0.069
	p’	0.103	0.453
Immediate memory (Story Memory)	r	0.145	0.040
	p’	0.114	0.661
Visuospatial Construction	r	0.073	0.092
	p’	0.424	0.317
Language	r	0.120	0.072
	p’	0.190	0.436
Attention (Digit Span)	r	0.094	0.040
	p’	0.307	0.665
Attention (Coding)	r	0.140	0.130
	p’	0.126	0.154
Delayed memory (List Recall)	r	0.234	0.149
	p’	0.010*	0.103
Delayed memory (List Recognition)	r	0.034	−0.003
	p’	0.713	0.976
Delayed memory (Story Recall)	r	0.215	0.092
	p’	0.018*	0.315
Delayed memory (Figure Recall)	r	0.118	0.076
	p’	0.199	0.406

*Indicated p'<0.05; p' represents the result after multiple Bonferroni correction

Discussion

The primary aim of this study is to explore the differences in amygdala volume and cognitive function in medication-free individuals with MDD and BD II. The research findings indicate that, in comparison to individuals with BD II, those with MDD experience more negative mood. Both MDD and BD II patients exhibit significantly higher levels of anxiety compared to the HC. Furthermore, BD II patients demonstrate extensive cognitive impairments, performing worse than both MDD and HC. Regarding the amygdala volume, the research results indicate that, compared to the HC, the right amygdala volume is increased in MDD. Additionally, there is little difference in the right amygdala volume between MDD and BD II patients.

Our study results indicate that the amygdala volume in males is larger than that in females. Animal studies have shown that the medial amygdala volume is larger in male guinea pigs, which may be a key factor in the overall gender differences in amygdala volume [38]. This finding is consistent with earlier human studies, which concluded that male amygdala volume is generally larger than that of females [39]. Our study further reveals a negative correlation between amygdala volume and age, possibly due to an increase in cortisol secretion with age leading to a decrease in amygdala volume [40]. Additionally, it may also be associated with age-related shrinkage of brain regions.

Abundant evidence indicates that individuals with both MDD and BD exhibit symptoms of pleasure deficits compared to HC [41, 42]. Previous studies have shown that in over half of the patients with BD experiencing a depressive episode, there are noticeable symptoms of pleasure deficits [43]. Simultaneously, nearly 75% of patients with MDD also exhibit symptoms of pleasure deficits [44]. This demonstrates a higher occurrence rate of depressive episodes in MDD patients compared to those with BD. Several previous prospective studies have indicated that, compared to MDD, the treatment of depressive symptoms in BD often progresses more rapidly [45, 46]. Our study results also indicate that individuals with MDD experience more negative mood compared to those with BD. Compared to patients with MDD, those with BD experience milder depressive symptoms, possibly due to the concurrent presence of manic symptoms in BD. However, this does not imply a reduction in the overall risk of the disorder. As for anxiety symptoms, both MDD and BD exhibit significantly higher levels of anxiety compared to the HC, a result that was expected. The research indicates that anxiety symptoms in both MDD and BD can lead to impaired cognitive function in these two disorders [47].

BD exhibits extensive cognitive deficits, performing worse than both MDD and the HC. This is consistent with clinical observations and previous research findings [48, 49]. Previous studies have shown, compared to the HC, individuals with BD exhibit specific domain deficits in cognitive impairments, including areas such as attention, language learning, and working memory, even after controlling for demographic and clinical variables [50]. Recent meta-analyses have indicated social cognitive impairment in BD, encompassing deficits in theory of mind and emotion recognition [51]. Furthermore, some longitudinal studies have reported that over time, individuals with BD experience declines in cognitive abilities, particularly in measures of verbal memory and executive functions [52, 53]. BD is associated with extensive neurocognitive impairments, which may manifest in the early stages of the illness and persist even during euthymic periods [54]. This poses serious damage to individuals’ social and psychological functioning and significantly increases the burden associated with the disease [55]. Consequently, alleviating neurocognitive impairments and correspondingly improving patients’ psychosocial functioning and quality of life has become a key goal in the treatment of BD [56]. Interestingly, our study results show that there is no significant difference in cognitive function between patients with MDD and the HC. This may be attributed to the fact that the enrolled patients are newly diagnosed, and their cognitive function has not yet shown significant impairments, despite the presence of compromised delayed memory.

The amygdala is a crucial brain region responsible for processing emotional and cognitive information, particularly in the context of emotional stimuli and executive functions [57]. Studies have also shown that changes in the structure and function of the amygdala are closely related to changes in cognitive function. In particular, the amygdala plays a crucial role in basic brain functions such as memory, social cognition, emotion, and consciousness, and may serve as a key hub in cognitive regulation [58]. Previous studies have identified an imbalanced interaction between the amygdala and functional networks involved in various emotional and cognitive processes. This model helps explain potential neural mechanism abnormalities and provides a basis for understanding the functional impairments observed across different domains in individuals with MDD [59]. Preclinical data suggest that chronic stress may cause distinct changes in the volumes of the amygdala and hippocampus, leading to an enlargement of the amygdala and a reduction in the hippocampus. These alterations are primarily attributed to dendritic remodeling [60–62]. In our study, the observed increase in the volume of the right amygdala in individuals with MDD compared to the HC further substantiates this observation, indicating that individuals with MDD may consistently be in a state of chronic stress. Compared to HC, individuals experiencing their first episode of psychosis (including schizophrenia, psychotic depression, and other psychotic disorders) show a significantly larger average amygdala volume [63]. Clinical studies indicate a lateralization of amygdala function [64], where electrical stimulation in the right hemisphere may elicit more irritability/negative responses compared to the left hemisphere and be involved in the rapid assessment of aversive stimuli [65, 66]. Stimulation of the right amygdala can lead to pronounced fear and anxiety in patients, resulting in alterations in their cognitive levels [67]. Additionally, an increase in cortisol levels is associated with a larger amygdala volume [68]. Elevated cortisol levels may negatively impact cognitive function, as cortisol can cross the blood-brain barrier and bind to specific receptors primarily located in the hippocampus, amygdala, and frontal lobe—regions essential for learning, memory, and executive functions [69]. Therefore, an enlargement of the amygdala volume often correlates with changes in cognitive levels in patients. A study employing automated segmentation for amygdala substructure volume analysis revealed that, compared to the HC, individuals with MDD exhibited a larger volume in the right medial subnucleus. Furthermore, in comparison to the left side, the overall and substructure volumes on the right side in MDD showed a relative increase [27]. Moreover, research has demonstrated a positive correlation between amygdala enlargement and the severity of depressive symptoms [29].

While our study results do not definitively indicate that amygdala volume can differentiate between BD II, MDD, and HC, we observed an enlargement of the amygdala in individuals with untreated MDD. Previous research suggests that the amygdala not only plays a crucial role in emotional processing but also significantly influences cognitive functions.

This study has several limitations that should be acknowledged. First, the relatively small sample size may have limited the statistical power and the generalizability of the results. Second, the analysis was restricted to the amygdala, without accounting for potential contributions from other brain regions involved in emotion and cognition. Third, only medication-naïve patients were included, and the absence of a longitudinal design prevented evaluation of treatment-related changes in amygdala volume. Fourth, all participants were assessed during a depressive episode, and the potential presence of hypomanic symptoms was not examined. Fifth, head size was not controlled for in the sex-based analysis of amygdala volume, which may have influenced the findings. Finally, due to the cross-sectional nature of the study, causal inferences cannot be drawn. Future longitudinal studies are warranted to clarify whether amygdala enlargement reflects a consequence of chronic stress or represents a pre-existing vulnerability, and to examine broader clinical phenotypes and additional brain regions to enhance the robustness of these findings.

Conclusion

In summary, under the condition of untreated initial presentation, both patients with depression and bipolar disorder show increased amygdala volume, contrary to the chronic atrophy observed in some other diseases. For medication-naïve individuals with MDD and BD II, we observed a correlation between amygdala volume and cognitive function. In summary, changes in amygdala volume may help reflect cognitive impairment in patients during the acute phase.

Abbreviations

MDD

Major depressive disorder

BD

Bipolar disorder

HC

Healthy controls

MRI

Magnetic resonance imaging

HAMD

17-item Hamilton Depression Rating Scale

HAMA

Hamilton Anxiety Rating Scale

RBANS

Repeatable Battery for the Assessment of Neuropsychological Status

Authors’ contributions

Bin Li, Chunguo Zhang: Writing - original draft, Writing - review & editing, Methodology, Software.Wensheng Chen, Guojun Xie: Validation, Investigation, Resources.Jiaquan Liang: Supervision, Project administration, Funding acquisition.

Funding

This research was supported by the project of Foshan Science and Technology Bureau (2320001006110, 2220001004473) and the Foshan “14th five-year plan” medical high level key psychiatric specialty construction project (FSGSP145069).

Data availability

The data used or analyzed during the current study are available from the Corresponding Author on reasonable requests.

Declarations

Ethics approval and consent to participate

The studies involving human participants were reviewed and approved by ethics committee of the third people’s hospital of Foshan (FSSY-LS202312). This study has secured informed consent from all participants and/or their legal guardians, and specifically excludes the participation of minors or individuals with illiteracy.

Consent for publication

No individual data is presented, and consent to publication is therefore not applicable.

Competing interests

The authors declare no competing interests.