Investigating micrornas as biomarkers in disorders of consciousness: a longitudinal multicenter study

ABSTRACT MicroRNAs (miRNAs) are involved in gene regulation and may affect secondary brain injury and recovery in patients with disorders of consciousness (DoC). This study investigated the

role of five miRNAs (150-5p, 132-3p, 23b-3p, 451a, and 16-5p) in prolonged DoC. miRNA levels were assessed in serum samples from 30 patients with unresponsive wakefulness syndrome or

minimally conscious state due to traumatic or hypoxic-ischemic brain injury (TBI, HIBI) at baseline (1–3 months) and 6 months post-injury. Patients’ diagnoses were determined using the Coma

Recovery Scale revised, and functional outcomes were evaluated 6 months after injury with the Glasgow Outcome Scale Extended (GOSE) and the Functional Independence Measure (FIM). Compared to

healthy controls, patients with TBI had lower levels of miRNAs 150-5p, 132-3p, and 23b-3p at baseline, while patients with HIBI had lower levels of miRNA 150-5p at baseline and 6 months

post-injury and a reduction of miRNA 451a at baseline. Higher levels of miRNAs 132-3p and 23b-3p were associated with better outcomes in TBI patients as indicated by GOSE and FIM scores.

This study highlights distinct miRNA dysregulated patterns in patients with prolonged DoC, dependent on etiology and post-injury time, and suggests that miRNAs 132-3p and 23b-3p may serve as

prognostic biomarkers. SIMILAR CONTENT BEING VIEWED BY OTHERS TEMPORAL BRAIN MICRORNA EXPRESSION CHANGES IN A MOUSE MODEL OF NEONATAL HYPOXIC–ISCHEMIC INJURY Article 31 August 2021

MICRORNAS AS BIOMARKERS OF BRAIN INJURY IN NEONATAL ENCEPHALOPATHY: AN OBSERVATIONAL COHORT STUDY Article Open access 19 March 2024 MICRORNA THERAPEUTIC TARGETS IN NEONATAL HYPOXIC–ISCHEMIC

BRAIN INJURY: A NARRATIVE REVIEW Article 19 July 2022 INTRODUCTION The vegetative state, also known as unresponsive wakefulness syndrome (UWS)1, is the worst possible clinical evolution in

comatose survivors of severe brain injuries. Patients with UWS exhibit no behavior indicative of self-awareness or awareness of the external world and they can remain unconscious

indefinitely or progress to a minimally conscious state (MCS), which is characterized by the fluctuating reappearance of minimal signs of awareness2. When UWS or MCS persist for more than 28

 days, they are defined as prolonged disorders of consciousness (DoC)3. Severe brain injuries, such as traumatic brain injuries (TBI) and hypoxic-ischemic brain injuries (HIBI), can cause

long-term structural and functional changes in the brain that may contribute to both neurodegeneration and recovery of brain functions4,5,6; the mechanisms of secondary brain injury and

restorative brain plasticity occurring simultaneously in these patients are still largely unknown. As a result, to make an accurate prognosis for recovery of consciousness and functional

outcomes is a major challenge for physicians treating patients with prolonged DoC. Current prognostic recommendations suggest a multimodal approach that includes serial standardized

behavioral assessments, neurophysiological techniques, and neuroimaging studies7. However, the results are not yet satisfactory, and there is a pressing need for new biomarkers to be

incorporated into current prognostic models to improve their sensitivity. In this context, circulating microRNAs (miRNAs) are good candidates, since each miRNA can reflect large-scale and

long-term genetic reorganizations following acute brain injuries8. miRNAs are a large class of small (18–25 nucleotides) non-coding RNAs that regulate gene expression in both physiological

and pathological contexts8. miRNAs act by degrading messenger RNAs, suppressing translation, or both, by binding to partially complementary sites in the 3′ untranslated region of target

genes9. To date, approximately 2000 miRNAs have been identified, which regulate the expression of about 60% of all genes and play a central role in many biological processes such as cell

cycle, cell metabolism, apoptosis, and immune response10,11. Circulating miRNAs can be detected using digital PCR (dPCR), a highly sensitive technique, and have been proposed as emerging

biomarkers for both acute brain injury and neurodegenerative diseases12,13. The analysis of miRNAs can provide a powerful tool for understanding the pathophysiology of prolonged DoC and

predicting patient outcomes. In particular, the analysis of miRNAs in patients with prolonged DoC can provide valuable insights into the molecular mechanisms occurring during brain

reorganization following a severe brain injury. In this longitudinal multicenter study, we selected five miRNAs involved in acute brain injury or chronic neurodegeneration (miRNAs 16-5p,

150-5p, 132-3p, 23b-3p and 451a) and evaluated them in patients with prolonged DoC caused by TBI or HIBI. Following TBI, a reduction in miRNA 16-5p levels has been observed both in plasma

during the acute phase and in the brain of patients with fatal TBI14,15. Changes in 150-5p levels have been observed both in traumatic injuries and neurodegenerative diseases16,17. miRNA

132-3p and 23b-3p have been identified as regulators of inflammatory and apoptotic pathways implicated in HIBI and neurodegenerative diseases18,19,20. Lastly, miRNA 451a has been recognized

for its role in governing apoptotic pathways following ischemia/reperfusion injury21. Our study aimed to provide a better understanding of the long-term genetic reorganization that occurs

after a severe brain injury and to identify new potential biomarkers for patients with prolonged DoC. METHODS PARTICIPANTS This multicenter longitudinal study enrolled patients admitted to

five Italian centers specialized in the rehabilitation of patients with prolonged DoC after an acute brain injury. Patients were included if they met the following criteria: (i) diagnosis of

UWS or MCS according to the Coma Recovery Scale-Revised (CRS-R) at the time of enrollment, (ii) DoC caused by TBI or HIBI, (iii) enrollment at 1–3 months after brain injury, (iv) clinical

and miRNA data availability at baseline and 6 months post-injury, and (v) age ≥ 18 years. Exclusion criteria were (i) previous brain injury and (ii) unstable clinical condition (e.g.,

hemodynamic instability, severe respiratory failure, or acute hydrocephalus). Patients’ data were compared with those of age-matched healthy controls without previous history of neurological

disease who were recruited from blood donors and healthcare personnel at the coordinating center. The study was conducted according to the guidelines of the Declaration of Helsinki and was

approved by the regional ethics review board (Palermo 1 Ethics Committee, Palermo, Italy: verbal no. 10/2021) of the coordinating center (Giuseppe Giglio Foundation, Cefalù, Italy). The

protocol was then ratified by the ethics committees of the other centers involved in the study. Patients’ legal guardians and healthy participants provided written informed consent which was

collected and stored at each participating center. CLINICAL EVALUATION At enrollment, all patients underwent standard neurological examination and assessment with the Italian version of the

Coma Recovery Scale Revised (CRS-R)22 at least 5 times over 3 consecutive days. The CRS-R is the most reliable tool for behavioral assessment in patients with DoC following a coma and is

used to diagnose UWS, MCS, and emergence from MCS23,24. It consists of 23 hierarchically organized items organized into six subscales evaluating auditory, visual, motor, oromotor/verbal,

communication, and arousal functions. The total score ranges from 0 (coma) to 23 (emergence from MCS). To minimize the risk of misdiagnosis, UWS and MCS diagnoses were accepted only if

confirmed at all 5 evaluation timepoints25. Outcomes were assessed 6 months post-injury using the CRS-R, the extended Glasgow Outcome Scale (GOSE) and the Functional Independence Measure

(FIM). The GOSE is a 1–8 global scale with the categories of death (score of 1), vegetative state, severe disability (lower and upper), moderate disability (lower and upper), and good

recovery (lower and upper; scores of 7 and 8)26. The FIM is an 18-item, seven-level ordinal scale that includes measures of independence for self-care, sphincter control, transfers,

locomotion, communication, and social cognition27. TOTAL RNA PURIFICATION At the end of initial and follow-up clinical evaluations, blood samples for miRNAs analysis were collected from

patients into serum-separating tubes. Samples were centrifuged at 1500 rpm for 15 min and stored at − 80 °C until use. Total RNA was purified using the miRNeasy Serum/Plasma Advanced Kit

(cat. No. 217204, QIAGEN) from 200 μL of serum, following the manufacturer’s instructions. This kit maximizes the recovery of small RNAs, including miRNAs, and is optimized for serum

samples. MIRNAS QUANTIFICATION Capillary electrophoresis is considered the most reliable method for accurately determining the integrity and the quantification of miRNAs, that are the major

factors affecting the outcome of downstream expression analyses28. Both quantity and quality of miRNAs have been assessed through chip-electrophoresis using the Agilent Small RNA Kit (cat.

no. 5067-1548, Agilent Technologies) on the Agilent 2100 Bioanalyzer. CDNA SYNTHESIS Starting from a fixed RNA input of 2.5 μL, cDNA synthesis was carried out using the miRCURY LNA™ RT Kit

(cat. no. 339340, QIAGEN) according to the manufacturer’s instructions. As recommended, cDNAs were diluted 1:30 prior to dPCR. DPCR Absolute quantification (positive particle = copies/μL) of

miRNAs was performed through dPCR with QIAcuity EG PCR Kit (cat. no. 250111, QIAGEN) and 26k-partitions nanoplates (cat. no. 250001, QIAGEN) on QIAcuity One platform. The used primers are

listed in Supplementary Table 1. Thermal cycling and imaging were performed following the manufacturer’s instructions. STATISTICAL ANALYSIS Demographic and clinical data are presented as

medians (interquartile range). The Mann–Whitney _U_ test for continuous variables, and the chi-square test for categorical variables were used to compare demographic and clinical data

between patients and healthy controls, and between groups of patients (TBI vs. HIBI) as well. The Student’s _t_-test was used to compare miRNA levels between patients and healthy controls

and between miRNA levels at 1–3 months (baseline) and 6 months post-injury. Correlations among all miRNA expressions were evaluated in healthy controls and patients (at baseline and

six-month post-injury) using Pearson’s correlation coefficients in correlation matrixes. Correlation coefficients range from − 1 to + 1, where − 1 indicates a total negative correlation

between two miRNAs, 0 no correlation, and + 1 a total positive linear correlation. The R-Squared was also calculated to measure the reliability of the linear relationship among miRNA

expressions included in the model. Its value ranges from 0 (fully correlated miRNAs) to 1 (non-correlated miRNAs). Multiple linear regression was used to determine the effect of each miRNA

and was adjusted for the effects of the other miRNAs. A linear model was constructed, in which the observed (dependent) variables were GOSE, FIM, or CRS-R scores 6 months post-injury in

patients with traumatic and hypoxic-ischemic etiologies, and the explanatory factors (regressors) all five miRNAs. _p_ values < 0.05 were considered significant. Statistical analyses were

performed with GraphPad Prism 8.0.0 (GraphPad Software, San Diego, California, USA; http://www.graphpad.com). RESULTS PARTICIPANT CHARACTERISTICS A total of 30 patients with prolonged DoC

and 35 age-matched healthy controls were included in this study, as shown in Table 1 and Supplementary Table 2. Of the patient cohort, 20 were diagnosed with UWS and 10 with MCS. The

underlying causes of DoC were TBI in 18 patients (15 of whom were involved in road accidents and 3 who suffered falls) and HIBI in 12 patients (10 of whom had cardiac arrests, 1 with opioid

overdose, and 1 with asphyxia). There were no significant differences between TBI and HIBI patients in terms of age, sex, and time since brain injury (Table 1). However, the TBI group had

more patients in MCS compared to the HIBI group. Additionally, patients with TBI had higher CRS-R and FIM scores at 6 months post-injury compared to those with HIBI (Table 1). CHANGES IN

MIRNA EXPRESSION AFTER TBI AND HIBI At the time of study inclusion, miRNA 150-5p expression was reduced both in patients with TBI (_p_ = 0.003) and HIBI (_p_ = 0.01) compared to healthy

controls (Fig. 1A). Six months post-injury, miRNA 150-5p expression returned to normal in patients with TBI, but not in patients with HIBI (_p_ = 0.03). miRNAs 132-3p and 23-b-3p expressions

were reduced only in patients with TBI at the time of study inclusion (_p_ values = 0.04 and 0.001, respectively) (Fig. 1B,C). Conversely, miRNA 451a expression was reduced only in patients

with HIBI at the time of study inclusion (_p_ = 0.001) (Fig. 1D). miRNA 16-5p expression did not change compared to healthy controls (Fig. 1E). No significant differences were found in

miRNA levels between TBI and HIBI patients at either time point. MIRNAS DYSHOMEOSTASIS AFTER TBI AND HIBI Pearson’s correlation matrixes showed that the expression of the selected miRNAs was

highly interrelated in healthy subjects, but this correlation was generally lost after the brain injury (Fig. 2 and Supplementary Table 3). Six months post-injury, most combinations

realigned with the normal trend, showing positive linear correlations (Fig. 2 and Supplementary Table 3). This trend was observed in both TBI and HIBI patients, but with a more evident

dyshomeostasis in the latter at baseline. Despite some differences related to etiology and evaluation timing, this data indicates a significant disruption in the reciprocal expression of

miRNAs 150-5p, 132-3p, 23b-3p and 451a, with only miRNA16-5p retaining a strong correlation with the other miRNAs both at baseline and 6 months post-injury. CORRELATION BETWEEN MIRNAS LEVELS

AND CLINICAL OUTCOMES Multiple regression analysis showed a positive correlation between miRNA 23b-3p levels 1–3 months post-injury and both FIM (_p_ = 0.01) and GOSE (_p_ = 0.006) scores 6

 months post-injury in patients with TBI (Fig. 3A,B). Additionally, a positive correlation was found between miRNA 132-3p levels and GOSE scores in patients with TBI (_p_ = 0.03) (Fig. 3C).

No other significant correlations were observed. DISCUSSION In this longitudinal study on patients with prolonged DoC, we found that a severe brain injury triggers a reorganization of miRNAs

expression that (i) depends on the time elapsed since injury, (ii) is etiology-independent for some miRNA changes, and (iii) is etiology-specific for other miRNA alterations. In comparison

to healthy controls, patients with TBI exhibited lower levels of miRNAs 150-5p, 132-3p, and 23-b-3p at baseline, while patients with HIBI had lower levels of miRNA 150-5p at baseline and 6 

months post-injury and a reduction of miRNA 451a at baseline. Moreover, higher levels of miRNA 132-3p and miRNA 23b-3p were associated with better outcomes in patients with TBI, as

demonstrated by improved GOSE and FIM scores 6 months post-injury. Our findings concur with previous research indicating a reduction in miRNA 150-5p expression both in a mouse model of TBI29

and in patients with Alzheimer's and Parkinson's diseases30,31, suggesting that miRNA 150-5p is altered in a wide range of pathological conditions from acute brain injury to

neurodegenerative diseases, making it a potential marker of brain injury independently of its etiology. Our study further showed that miRNA 150-5p expression was down-regulated in patients

with DoC, both after TBI and HIBI. One potential explanation for the link between miRNA-150-5p dysregulation and various pathological conditions is its involvement in neuroinflammation.

There is evidence of prolonged microglial activation after severe TBI and HIBI32,33,34 and miRNA-150-5p may inhibit the release of proinflammatory cytokines such as interleukine-1β,

interleukine-6, and tumor necrosis factor-α by binding to the protein kinase AKT331. Additionally, studies in patients with major trauma have shown that miRNA 150-5p targets the mRNA coding

for protein kinase C alpha, a key player in inflammation35. Thus, the down-regulation of miRNA-150-5p may contribute to persistent neuroinflammation following brain injury, and increasing

its expression may offer a new strategy for reducing neuroinflammation. In contrast to patients with TBI, downregulation of miRNA-150-5p persisted up to 6 months post-injury in patients with

HIBI. This suggests that in these patients, whose outcomes were worse than those with TBI, the biochemical relevant pathways dependent on miRNA 150-5p remain dysregulated for an extended

period post-injury. The expression of miRNA 451a was reduced in patients with HIBI 1–3 months post-injury, making it a time- and etiology-specific marker of brain injury that may be related

to inflammation. The down- or up-regulation of miRNA 451a has been shown to either increase or decrease neuroinflammation by modulating the levels of tumor necrosis factor α, interleukin‑1b,

interleukin‑6, and interleukin‑1836. In patients with TBI, miRNAs 132-3p and 23-b-3p were down-regulated 1–3 months after injury, making them specific markers for the timing and etiology of

brain injury. The miRNA132 family is involved in the formation of neuronal connections and in synaptic plasticity. Post-mortem studies have revealed a decrease in miRNA 132-3p in the

hippocampus, prefrontal cortex, and temporal cortex of patients with Alzheimer’s disease, including those in the early stages of disease37,38. miRNA 132 has been linked to various pathways,

including astrocyte-related inflammation39, synaptic structure and plasticity40,41, and cholinergic signaling42, all of which play key roles in Alzheimer's disease. TBI increases the

risk of developing dementia, including Alzheimer's disease, likely via mechanisms of long-term neurodegeneration43,44. Thus, the decrease in miRNA 132-3p levels may affect brain

plasticity and contribute to neurodegeneration in patients with prolonged post-traumatic DOC, impacting their outcomes. After 6 months, miRNA 132-3p levels returned to normal values when

most patients with post-traumatic DOC showed improvements in their level of consciousness. Therefore, the upregulation of miRNA 132-3p could have potential implications for brain plasticity

and the recovery of consciousness in post-traumatic DoC, as demonstrated by its association with improved cognitive abilities in animal models45. The expression of miRNA 23-b-3p exhibited a

similar pattern to miRNA 132-3p, with a decrease in patients with TBI at baseline. miRNA 23-b-3p plays a role in regulating neuronal apoptosis and is involved in various cellular pathways in

both physiological and pathological conditions20. A previous study on TBI patients and animal models has shown that miRNA 23-b levels decrease after TBI, and that overexpression of miRNA

23-b reduces neuronal autophagy in the hippocampus by targeting autophagy-related genes46. Our study expands upon this previous work by investigating patients with more severe injuries and a

different temporal window (1–3 months post-injury compared to 24 h post-injury) and demonstrating that miRNA 23-b-3p levels recover to normal after 6 months. This finding is significant

because it provides new insights into the dynamic changes in miRNA 23-b-3p levels after TBI and highlights the potential for miRNA 23-b-3p as a biomarker for post-traumatic DoC. The levels

of miRNA 16-5p, which have been linked to apoptosis induction and cell proliferation47,48, did not differ between healthy controls and patients with TBI or HIBI at 1–3 months and 6 months

post-injury. However, a previous study reported reduced levels of miRNA 16-5p in plasma of patients with severe TBI 25–72 h after injury compared to healthy controls or patients with

orthopedic injury14. Therefore, our findings suggest that the downregulation of miRNA 16-5p occurs only in the early phase after TBI. In terms of clinical relevance, the most noteworthy

finding was the correlation between the levels of miRNAs 132-3p and 23-b-3p 1–3 months post-injury and GOSE and FIM scores. Patients with higher expression of these miRNAs displayed better

functional outcomes 6 months after injury. Previous studies have linked decreased levels of miRNAs 132-3p and 23-b-3p with neurodegeneration in both animal models and patients with

Alzheimer's disease, while increased levels have been related to enhanced cognitive function37,45,46,49. Mechanisms of neurodegeneration and impaired plasticity have been described in

patients with prolonged DoC, such as amyloid-β deposition50, gray and white matter atrophy51,52,53, and decreased expression of brain-derived neurotrophic factor54. Elevated levels of miRNAs

132-3p and 23-b-3p may counteract neurodegeneration and promote brain plasticity in patients with DoC, leading to better outcomes. So, miRNAs 132-3p and 23-b-3p are promising candidates for

inclusion in prognostic models for patients with prolonged DoC and warrant further investigation as potential targets for therapeutic intervention. Studying the overall changes in miRNA

expression after a brain injury can provide a more comprehensive view of the impact of severe brain injuries on the miRNA regulatory network. We observed a loss of reciprocal correlations

among miRNAs, which was more pronounced 1–3 months post-injury and in patients with HIBI. The loss of reciprocal correlations among miRNAs may reflect a disruption of the complex molecular

network regulating neuronal survival and plasticity, and this dysregulation is more profound in patients with HIBI. Furthermore, the more pronounced dysregulation in the acute phase after

injury suggests that this is a critical time window for therapeutic intervention. The complex and simultaneous regulation of multiple biochemical pathways by each miRNA makes it challenging

to predict the final effects on neuronal survival and plasticity. Our understanding of this field is still limited, and further research is needed, such as developing computational models to

predict the combined effect of multiple miRNAs on patient outcomes. Two recent studies have investigated changes in miRNA expression in patients with prolonged DoC55,56. Although they

focused on different miRNAs or employed different methodologies compared to our study, their findings are consistent in suggesting that miRNAs represent an intriguing target for evaluation

in prolonged DoC. In patients with TBI, HIBI, or subarachnoid hemorrhage, Petrova and colleagues identified significant differences in the expression of miRNAs 93-5p, 21-5p, and 191-5p in

both cerebrospinal fluid and plasma, depending on the specific etiologies55. Zilliox and colleagues examined the whole blood miRNA profile in six patients with post-traumatic DoC lasting an

average of 1.5 years, discovering 48 miRNAs that were differentially expressed compared to controls56. While the findings of this study provide valuable insights into the potential role of

miRNAs as biomarkers for prognosis and targets for therapeutic intervention in patients with prolonged DoC, there are several limitations to consider. One of the primary limitations is the

small sample size, especially concerning HIBI and female patients, which may limit the generalizability of the results. Moreover, we evaluated miRNAs in the blood only, while analyzing

miRNAs in the cerebrospinal fluid may possibly provide more accurate results regarding the changes occurring in the central nervous system55,57. Additionally, our assessment of outcomes was

limited to a follow-up period of 6 months post-injury. While this time frame may be adequate to capture most of the recovery in patients with non-TBI etiologies, it may not fully evaluate

the complete recovery of patients with TBI, who may continue to improve up to 1-year post-injury3. Finally, while the study focused on miRNA dysregulation, it must be added that miRNAs are

just one of the many potential molecular mechanisms dysregulated in DoC, and further research is needed to fully understand the particularly complex pathophysiology of this condition. In

conclusion, this study offers important insights into the potential of miRNAs for understanding the molecular mechanisms of DoC as well as for enhancing prognosis and treatment in patients

with prolonged DoC. However, the limited sample size and the need for further research to fully understand the complex interactions of miRNAs and their effects on neuronal survival and

plasticity should be considered. Further studies with larger sample sizes and longer follow-up periods could help validate and expand upon these findings. DATA AVAILABILITY The data

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https://doi.org/10.3390/brainsci13020364 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references AUTHOR INFORMATION Author notes * These authors contributed

equally: Nicolò Musso and Dalida Bivona. AUTHORS AND AFFILIATIONS * Department of Biomedical and Biotechnological Sciences, University of Catania, 95123, Catania, Italy Nicolò Musso, Dalida

Bivona, Carmelo Bonomo, Paolo Bonacci & Stefania Stefani * Molecular Biology Laboratory, Giuseppe Giglio Foundation, 90015, Cefalù, Italy Maria Enza D’Ippolito * Unit of Neurophysiology

and Unit for Severe Acquired Brain Injuries, Giuseppe Giglio Foundation, 90015, Cefalù, Italy Cristina Boccagni, Francesca Rubino & Sergio Bagnato * Cardinal Ferrari Center, 43012,

Fontanellato, Italy Antonio De Tanti * RAN (Research in Advanced Neuro-Rehabilitation), S. Anna Institute, 80067, Crotone, Italy Lucia Francesca Lucca * Neurorehabilitation and Spinal Units,

Istituti Clinici Scientifici Maugeri IRCCS, 27100, Pavia, Italy Valeria Pingue * Montecatone Rehabilitation Institute, 40026, Imola, Italy Valentina Colombo * Don Gnocchi Foundation IRCCS,

50124, Florence, Italy Anna Estraneo * Clinical Pathology Laboratory, Provincial Health Authority of Caltanissetta, 93100, Caltanissetta, Italy Maria Andriolo Authors * Nicolò Musso View

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inPubMed Google Scholar CONTRIBUTIONS N.M., S.S., M.A., and S.B. designed this work. N.M., D.B., Ca.B., and P.B. performed miRNA analyses. M.E.D.I., Cr.B., F.R., A.D.T., L.F.L., V.P., and

V.C. collected blood samples and clinical data. N.M. performed the statistical analysis. N.M. and S.B. wrote the manuscript. Cr.B., A.E., and S.B. revised the manuscript. CORRESPONDING

AUTHOR Correspondence to Sergio Bagnato. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE Springer Nature

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Bonomo, C. _et al._ Investigating microRNAs as biomarkers in disorders of consciousness: a longitudinal multicenter study. _Sci Rep_ 13, 18415 (2023).

https://doi.org/10.1038/s41598-023-45719-7 Download citation * Received: 27 February 2023 * Accepted: 23 October 2023 * Published: 27 October 2023 * DOI:

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