Association of the Global Alignment and Proportion Score with Locomotive Syndrome and Health-Related Quality of Life in Community-Dwelling Middle-Aged and Older Adults: A Retrospective Cross-Sectional Study

Abstract

Background

The Global Alignment and Proportion score has been validated in surgical cohorts as a predictor of mechanical complications in adult spine deformity, but its significance in the general population is poorly understood. We examined the association of the Global Alignment and Proportion score categories with locomotor syndrome and health-related quality of life in community-dwelling middle-aged and older adults to clarify the role of proportionality of sagittal alignment in musculoskeletal function and quality of life.

Methods

Residents of Hokkaido, Japan, who underwent musculoskeletal health screening in 2019 were included in this retrospective cross-sectional study. Based on their Global Alignment and Proportion score classification, participants were divided into three groups: proportioned, moderately disproportioned (moderate), and severely disproportioned (severe). We compared participants' background, bone mineral density, exercise tests, pain levels, locomotor syndrome stage, and Short Form 36-Item Health Survey score data.

Results

The mean age of the 217 participants was 64.0±9.5 years; 127 were female. The mean Global Alignment and Proportion score was 6.1±3.4 points, and the proportioned, moderate, and severe groups comprised 27, 106, and 84 participants, respectively. A statistically significant trend was observed in which the number of participants at high risk for locomotive syndrome increased as the Global Alignment and Proportion risk state worsened. In the Short Form 36 Health Survey domains of bodily pain and physical component summary scores, statistically significant trends toward lower scores were observed across groups with worsening Global Alignment and Proportion risk status.

Conclusions

We identified an association between poorer Global Alignment and Proportion scores and the presence of locomotive syndrome, as well as lower health-related quality of life, among middle-aged and older adults. These findings suggest that the Global Alignment and Proportion score may be a useful indicator for characterizing musculoskeletal alignment in clinical and public health fields.

Introduction

With the growing prevalence of age-related musculoskeletal decline, understanding the relationship between spine alignment and functional outcomes has become increasingly important^1-3). In Japan, the concept of locomotive syndrome has been established as a key risk factor for loss of motor function, and its association with health-related quality of life (HRQOL) has been widely documented^4-9). Identifying structural factors that contribute to locomotive syndrome and poor HRQOL can provide important insights into the mechanisms linking spine alignment to functional deterioration.

Traditionally, sagittal alignment of the spine has been evaluated using parameters such as the sagittal vertical axis (SVA)^10,11). Although the SVA has proven useful in cases of advanced disease and postoperative patients, it primarily reflects end-stage decompensation once compensatory mechanisms—such as pelvic retroversion, hip extension, and knee flexion—have been exhausted¹²⁾. Thus, the SVA may not adequately capture proportional differences in spinopelvic alignment among relatively healthy individuals.

Importantly, appropriate spine alignment is influenced by an individual's pelvic morphology¹³⁾. The Global Alignment and Proportion (GAP) score was developed to incorporate individual pelvic incidence into a proportional framework, integrating multiple sagittal parameters to define whether alignment is proportional or disproportional¹⁴⁾. Although the GAP score has been validated as a predictor of mechanical complications in surgery for adults with spine deformities, its significance in nonsurgical, community-dwelling populations remains unclear. Applying the GAP score in this context may help clarify whether deviations from proportional alignment are already associated with functional limitations and poorer HRQOL, even in individuals without overt spine deformities.

Therefore, the aim of this study was to investigate the association between GAP score categories and both locomotive syndrome and HRQOL in community-dwelling middle-aged and older adults. By addressing this question, the study sought to deepen the understanding of how spinopelvic alignment relates to musculoskeletal function and quality of life in the general population.

Materials and Methods

Study population

This retrospective cross-sectional study was approved by the affiliated institutions of the authors. All procedures followed the principles of the Declaration of Helsinki, and all participants provided written informed consent before being included in the study.

The participants were healthy volunteers who participated in a basic health checkup supported by the local government in 2019. The participants consisted mainly of residents who were physically active and able to complete activities of daily living independently. This checkup has been performed annually in Yakumo, Hokkaido, Japan, for >30 years and is familiar to the local people. The program consists of residents who voluntarily undergo orthopedic and physical function tests, internal medicine checkups, and psychological tests. Every year, a letter explaining the purpose of the health-screening program is sent to residents by post, and their intention to participate is confirmed by return mail¹⁵⁾. Yakumo has a population of approximately 15,000, with 35% of residents aged ≥65 years. The region is prosperous, with primary industries such as dairy farming. We enrolled individuals aged 40-87 years. Individuals with missing spine radiographs and those with insufficient locomotor syndrome data were excluded.

Variables and grouping

Age, sex, body mass index, bone mineral density (T-score), back muscle strength, walking speed (10-Meter Walk Test), and pain (visual analog scale for back pain and lower extremity pain) were investigated. A water-bath ultrasound system (Lunar A-1000 Plus II; GE Healthcare, Chicago, IL, USA) was used to measure bone status parameters of the calcaneus. T-scores were recorded using the standard protocol provided by the manufacturer¹⁶⁾.

The risk stage for locomotive syndrome was evaluated for each individual based on the criteria of the Japanese Orthopedic Association¹⁷⁾ using the stand-up test, Two-Step test, and 25-question Geriatric Locomotive Function Scale (GLFS-25). Locomotive syndrome risk stage 1 is diagnosed as difficulty standing on one leg (either leg) at a height of 40 cm from a sitting position, but the ability to stand up from a 20-cm platform on both legs, with a Two-Step test score of ≥1.1 and <1.3 points and a GLFS-25 score of ≥7 and <16 points. Participants meeting any of these criteria were diagnosed as starting to lose motor ability. Locomotive syndrome risk stage 2 is diagnosed as difficulty standing on both legs at a height of 20 cm from a sitting position, but the ability to stand up from a 30-cm platform on both legs, with a Two-Step test score of ≥0.9 and <1.1 points and a GLFS-25 score of ≥16 and <24 points. Participants meeting any of these criteria were diagnosed with progressive decline in mobility. Locomotive syndrome risk stage 3 is diagnosed as difficulty standing on both legs at a height of 30 cm from a sitting position, with a Two-Step test score of <0.9 points and GLFS-25 score of ≥24 points. Participants meeting any of these criteria were diagnosed with progressive decline in mobility and impediments to social participation. The tests were carried out by several examiners—orthopedic surgeons who fully understood these tests—at the health checkup venue. Because of the nature of the screening, each person performed the tests once, similar to the procedures described in previous studies^18,19).

HRQOL was assessed using the Medical Outcomes Study Questionnaire Short Form 36 Health Survey (SF-36, Japanese version 2.0). The physical component, mental component, and role/social component summaries, as well as eight subscales including physical functioning, role physical, bodily pain, general health, vitality, social functioning, role-emotional, and mental health, were assessed.

The GAP score is a pelvic incidence-based proportional method for analyzing sagittal alignment using whole-spine x-ray. We evaluated spine alignment parameters such as the SVA, thoracic kyphosis, lumbar lordosis, pelvic tilt, sacral slope, and pelvic incidence. Whole-spine radiographs were obtained with the participant in a freestanding position, with their fingers resting on their clavicles while looking straight ahead. The GAP score consists of the sum of five parameters: relative pelvic version (measured minus the ideal sacral slope [pelvic incidence×0.59+9]), relative lumbar lordosis (measured minus the ideal lumbar lordosis [pelvic incidence×0.62+29]), lordosis distribution index (L4-S1 lordosis÷the L1-S1 lordosis×100), relative spinopelvic alignment (measured minus the ideal global tilt [pelvic incidence×0.48−15]), and age¹⁴⁾. Participants were classified as proportioned (0-2 points; proportioned group), moderately disproportioned (3-6 points; moderate group), and severely disproportioned (7-13 points; severe group) based on the sum of these five parameters. Participants were stratified into these three groups according to their GAP scores, and their data were compared.

Statistical analysis

Easy R software was used to perform statistical analyses (Saitama Medical Center, Jichi Medical University, Japan)²⁰⁾. Among the three GAP groups, continuous and categorical variables were compared using the Jonckheere–Terpstra and Cochran–Armitage trend tests, respectively. Continuous and categorized variables were compared between participants in the analytic cohort and those with missing GAP scores using the Mann–Whitney U and chi-square test, respectively. Statistical significance was set at p<0.05. Furthermore, the association between SF-36-derived QOL component summary scores and eight subscales, and the three groups stratified by GAP score were evaluated using multivariable linear regression analysis. The following models were developed to adjust for confounding factors: (i) unadjusted model, (ii) age and sex adjusted model, (iii) fully adjusted model. The fully adjusted model includes age, sex, body mass index, T-score, pain visual analog scale (lumbar, lower extremity), and locomotive syndrome status (Grade 0 or others) as covariates. Residuals were checked for normal distribution using histograms. The Variance Inflation Factor metric showed that all adjustment covariates had variance inflation factor values below 3.0, indicating only moderate multicollinearity.

Results

Of the 536 volunteers who took part in the health checkups in Yakumo, Japan, in 2019, 217 participants were included in the analytic cohort of this study (Figure 1). The mean age of participants was 64.0±9.5 years, and 127 (58.5%) were female. The mean back muscle strength was 81.0±32.3 kg. The mean visual analog scale score was 17.4±20.3 mm at the lumbar position and 14.9±21.1 mm at the lower extremity position. The distribution of locomotive syndrome stage was as follows: Stage 0, 40.6%; Stage 1, 45.2%; Stage 2, 7.8%; and Stage 3, 6.4% (Table 1). No statistically significant differences were observed between participants in the analytic cohort and those excluded (Table S1). The following means were noted: SVA, 7.8±34.1 mm; thoracic kyphosis, 23.4±11.0°; lumbar lordosis, 44.2±15.3°; pelvic tilt, 18.5±8.9°; sacral slope, 30.7±10.4°; pelvic incidence, 48.9±14.0°; and GAP score, 6.1±3.4 points (Table 2). The proportioned group comprised 27 participants, the moderate group 106 participants, and the severe group 84 participants.

Figure 1.

Flowchart of participant selection.

GAP, Global Alignment and Proportion

Table 1.

Participants’ Demographic Characteristics.

	Analytic cohort (n=217)
Age (years) [range]	64.0±9.5 [40 – 91]
Sex, female	127 (58.5%)
BMI (kg/m2)	23.8±3.5
T-score, QUS	−1.6±1.5
Back muscle strength (kg)	81.0±32.3
10-m walk test (seconds)	4.9±0.8
Pain (VAS, mm)
Back	17.4±20.3
Lower extremity	14.9±21.1
LS grade
Stage 0	88 (40.6%)
Stage 1	98 (45.2%)
Stage 2	17 (7.8%)
Stage 3	14 (6.4%)

BMI: body mass index; LS: locomotive syndrome; QUS: quantitative ultrasonography; VAS: visual analogue scale

Table 2.

Participants’ Global Alignment Parameters.

	Analytic cohort (n=217)
Spinal sagittal parameters
Sagittal Vertical Axis (mm)	7.8±34.1
Thoracic Kyphosis (degrees)	23.4±11.0
Lumbar Lordosis (degrees)	44.2±15.3
Pelvic Tilt (degrees)	18.5±8.9
Sacral Slope (degrees)	30.7±10.4
Pelvic Incidence (degrees)	48.9±14.0
GAP score	6.1±3.4
Relative pelvic version	−7.3±7.1
Relative lumbar lordosis	−15.1±11.5
Lordosis distribution index	110.0±218.0
Relative spinopelvic alignment	9.2±10.3

GAP: Global Alignment and Proportion

The mean age of the severe group was higher than that of the other groups (61.1±8.8 vs. 62.4±9.2 vs. 66.9±9.5 years; p<0.001). Body mass index was higher in the severe group than in the other groups (22.8±2.7 vs. 23.5±3.5 vs. 24.5±3.5 kg/m²; p=0.010). The severity of back pain was greater in the severe group (p=0.013) (Table 3). A statistically significant trend was observed in which the number of participants at high risk for locomotive syndrome increased as the GAP risk stage worsened. The distribution of GAP score categories based on locomotive syndrome risk stage was as follows: for the proportioned, moderate, and severe groups, the locomotive syndrome risk stage percentages were Stage 0: 15.9%, 54.5%, and 29.6%; Stage 1: 10.2%, 51.0%, and 38.8%; Stage 2: 5.9%, 29.4%, and 64.7%; and Stage 3: 14.3%, 21.4%, and 64.3%, respectively (Figure 2; Table S2).

Table 3.

Background Characteristics, Stratified According to the Global Alignment and Proportion Scores.

	Proportioned (n=27)	Moderately disproportioned (n=106)	Severely disproportioned (n=84)	p trend
Age (years)	61.1±8.8	62.4±9.2	66.9±9.5	<0.001*
Sex, female	18 (66.7%)	69 (65.1%)	40 (47.6%)	0.030*
BMI (kg/m2)	22.8±2.7	23.5±3.5	24.5±3.5	0.01*
T-score, QUS	−1.4±1.4	−1.5±1.7	−1.8±1.4	0.296
Pain (VAS, mm)
Back	12.6±16.3	15.7±19.6	21.2±21.8	0.013*
Lower extremity	16.1±22.5	10.5±17.6	20.4±23.5	0.004*
Back muscle strength (kg)	82.3±35.1	81.9±31.8	79.5±32.5	0.774

BMI: body mass index; QUS: quantitative ultrasonography; VAS: visual analogue scale

* p<0.05

Figure 2.

Risk distribution of participants for the development of locomotive syndrome, based on their Global Alignment and Proportion scores.

Statistically significant trends were found toward lower scores across groups with worsening GAP risk status in the SF-36 subdomains of physical functioning (proportioned: 89.8±11.6; moderate: 89.7±12.1; severe: 84.5±15.4; p=0.020), bodily pain (proportioned: 76.6±19.4; moderate: 71.7±22.9; severe: 64.3±23.9; p=0.009), and physical component summary score (proportioned: 51.9±8.5; moderate: 50.1±9.8; severe: 45.2±10.4; p<0.001) (Figure 3; Table S2).

Figure 3.

Participants’ Short Form 36 Health Survey summary scores and subscales, based on the Global Alignment and Proportion scores.

In the multivariable linear regression analysis, adjusted models 2 and 3 showed that moderately and severely disproportioned GAP status was significantly correlated with worse bodily pain and physical component summary score (Table 4).

Table 4.

Multivariable Linear Regression Analysis of the Difference on the SF-36 Scores for each Group Categorized by GAP Score.

Multivariable regression models	Proportioned (n=27)			Moderately disproportioned (n=106)			Severely disproportioned (n=84)
	β	95% CI	p-Value	β	95% CI	p-Value	β	95% CI	p-Value
Physical Functioning
Model 1: Unadjusted	Reference	Reference	n/a	−0.218	−1.457 to −0.280	0.004*	−0.288	−2.140 to −0.150	0.024*
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	−0.196	−1.380 to −0.177	0.012	−0.247	−1.980 to 0.013	0.053
Model 3:Fully adjusted1	Reference	Reference	n/a	−0.075	−0.864 to 0.269	0.301	−0.133	−1.444 to 0.391	0.259
Role Physical
Model 1: Unadjusted	Reference	Reference	n/a	−0.091	−1.276 to 0.320	0.239	−0.103	−1.887 to 0.809	0.431
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	−0.070	−1.192 to 0.462	0.385	−0.073	−1.750 to 0.986	0.583
Model 3:Fully adjusted1	Reference	Reference	n/a	0.005	−0.822 to 0.879	0.947	−0.005	−1.409 to 1.351	0.967
Bodily Pain
Model 1: Unadjusted	Reference	Reference	n/a	−0.240	−2.648 to −0.624	0.002*	−0.298	−3.747 to -0.328	0.020*
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	−0.288	−3.004 to −0.993	<0.001*	−0.342	−4.048 to -0.619	0.008*
Model 3:Fully adjusted1	Reference	Reference	n/a	−0.201	−2.388 to −0.367	0.008*	−0.239	−3.272 to -0.001	0.049*
General Health
Model 1: Unadjusted	Reference	Reference	n/a	−0.021	−0.923 to 0.702	0.789	0.065	−1.022 to 1.719	0.617
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	−<0.001	−0.843 to 0.842	0.999	0.094	−0.888 to 1.896	0.476
Model 3:Fully adjusted1	Reference	Reference	n/a	0.058	−0.551 to 1.167	0.481	0.154	−0.577 to 2.204	0.250
Vitality
Model 1: Unadjusted	Reference	Reference	n/a	0.072	−0.387 to 1.074	0.355	0.026	−1.110 to 1.360	0.842
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	0.007	−0.708 to 0.779	0.925	−0.035	−1.400 to 1.064	0.789
Model 3:Fully adjusted1	Reference	Reference	n/a	0.062	−0.451 to 1.036	0.438	0.008	−1.165 to 1.244	0.948
Social Functioning
Model 1: Unadjusted	Reference	Reference	n/a	0.023	−0.653 to 0.887	0.766	0.121	−0.685 to 1.912	0.353
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	0.028	−0.657 to 0.944	0.724	0.134	−0.644 to 1.999	0.313
Model 3:Fully adjusted1	Reference	Reference	n/a	0.062	−0.482 to 1.161	0.438	0.151	−0.576 to 2.082	0.265
Role Emotional
Model 1: Unadjusted	Reference	Reference	n/a	0.026	−0.654 to 0.924	0.736	0.046	−1.097 to 1.555	0.734
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	0.048	−0.572 to 1.063	0.555	0.075	−0.957 to 1.732	0.570
Model 3:Fully adjusted1	Reference	Reference	n/a	0.108	−0.284 to 1.388	0.195	0.141	−0.627 to 2.073	0.292
Mental Health
Model 1: Unadjusted	Reference	Reference	n/a	0.066	−0.412 to 1.006	0.409	0.023	−1.104 to 1.308	0.868
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	0.055	−0.488 to 0.982	0.508	0.003	−1.216 to 1.244	0.982
Model 3:Fully adjusted1	Reference	Reference	n/a	0.049	−0.553 to 0.983	0.582	−0.001	−1.265 to 1.260	0.997
Physical Component Summary
Model 1: Unadjusted	Reference	Reference	n/a	−0.315	−1.382 to −0.508	<0.001*	−0.391	−1.905 to −0.441	0.002*
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	−0.301	−1.350 to −0.452	<0.001*	−0.362	−1.822 to −0.347	0.004*
Model 3:Fully adjusted1	Reference	Reference	n/a	−0.193	−1.019 to −0.145	0.009*	−0.245	−1.442 to −0.032	0.041*
Mental Component Summary
Model 1: Unadjusted	Reference	Reference	n/a	0.111	−0.107 to 0.663	0.156	0.136	−0.304 to 0.990	0.297
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	0.050	−0.269 to 0.518	0.532	0.073	−0.465 to 0.830	0.579
Model 3:Fully adjusted1	Reference	Reference	n/a	0.065	−0.240 to 0.564	0.427	0.081	−0.445 to 0.853	0.536
Role Component Summary
Model 1: Unadjusted	Reference	Reference	n/a	0.099	−0.159 to 0.739	0.204	0.145	−0.327 to 1.179	0.266
Model 2:Age- and sex-adjusted	Reference	Reference	n/a	0.125	−0.100 to 0.832	0.123	0.175	−0.252 to 1.278	0.188
Model 3:Fully adjusted1	Reference	Reference	n/a	0.141	−0.076 to 0.911	0.097	0.081	−0.272 to 1.322	0.536

GAP: Global Alignment and Proportion; n/a: not applicable

¹The Fully adjusted model for multivariable regression analysis includes age, sex, body mass index, T-score, pain visual analogue scale (lumbar, lower leg), and locomotive syndrome (grade 0 or others) as covariates.

* p<0.05

Discussion

In this study involving a community-dwelling cohort of middle-aged and older adults, we used the GAP score to investigate the distribution of spine alignment and the association between the GAP score and locomotor syndrome, as well as HRQOL. Stratification of the GAP score revealed statistically significant trends between poor GAP scores and increased risk of locomotor syndrome, as well as between poorer GAP scores and lower HRQOL. These findings suggest that the Global Alignment and Proportion score may be a useful indicator for characterizing musculoskeletal alignment in clinical and public health fields.

In the current study, a high GAP risk status was associated with poor HRQOL. Poor spine alignment, such as that indicated by an increased SVA, reduces HRQOL²¹⁾. However, the mean SVA of this study's analytic cohort was only 7.8 mm. Because the GAP score is a tailored indicator based on an individual's pelvis, it may potentially serve as a representative tool for a comprehensive understanding of an individual's overall alignment. Indeed, we identified a negative correlation between GAP risk status and HRQOL components or subdomains, including the physical component summary and bodily pain subdomains, among community residents who had not undergone spine surgery. Although the GAP score includes age, the age component is a binary assessment using 60 years as the threshold. Therefore, multicollinearity between age treated as a continuous covariate in the adjusted model and the age component embedded in the GAP score is minimal. Furthermore, the correlation was maintained even after full adjustment, supporting the robustness of these findings.

A high GAP risk status was associated with the risk of developing locomotor syndrome. Locomotive syndrome is a state of diminished physical capabilities, including mobility, that carries a high likelihood of requiring nursing care⁸⁾. Therefore, a high GAP score may be associated with physical problems such as trunk function issues²²⁾, resulting in the hypothesis that a poor GAP score is associated with low physical quality of life and a high risk of developing locomotive syndrome. These factors may influence each other, and the association between the GAP score and physical HRQOL could be a spurious correlation due to the strong link between the GAP score and locomotor syndrome. This issue warrants further investigation. Nevertheless, the fact that the GAP score correlated with physical HRQOL and risk of developing locomotor syndrome suggests that the GAP score can be used to estimate physical risk states in community-dwelling residents.

Previous studies investigating sagittal spine alignment and physical function in community-dwelling middle-aged and older adults have focused on partial elements of both alignment and motor function. For example, trunk forward shift and forward bend have been associated with poorer performance in the single-leg stance, stand-up test, and two-step tests³⁾, and lumbar lordosis has been associated with walking ability²³⁾. When a spinal inclination angle ≥6° was used to diagnose locomotive syndrome, the sensitivity for diagnosing Stage 2 locomotive syndrome was 52% and the specificity 87%²⁴⁾. However, reduced lumbar lordosis has been correlated with a higher stage of locomotive syndrome⁴⁾. Although these indicators are easy to measure, the relatively small threshold of the spinal inclination angle raises concerns that measurement errors could distort results. Lumbar lordosis represents local alignment and may therefore have limitations when used to assess whole-body balance. By contrast, the GAP score reflects multiple items through which alignment of the entire spine can be considered. Therefore, this score is expected to provide a more accurate assessment than single indicators. The next phase of our research involves verifying whether the GAP score enables early detection of deterioration in physical function or risk of developing locomotor syndrome; intervention effects will also be examined. As a result, we need to investigate whether the findings of the present study can be applied to other populations, particularly those with different lifestyles. Because improvements in physical function and spine alignment have been reported with locomotor training exercises such as the single-leg stance and squats^25,26), future research should verify whether such interventions are effective in improving GAP scores, risk of developing locomotor syndrome, and HRQOL.

This study had several limitations. First, as a cross-sectional study, the findings demonstrate independent associations at a single time point. Thus, there are limitations in inferring causality between GAP scores and HRQOL or life satisfaction. Second, assessments of muscle strength and physical flexibility were limited. Third, this cohort represented a specific population in Japan that was relatively active and independent, which may introduce selection bias. Thus, the findings may have limited generalizability. Despite these limitations, this study identified an association between global alignment and HRQOL among community-dwelling middle-aged and older adults from a unique perspective.

Conclusions

This study suggested that poor GAP scores in middle-aged and older adults were associated with the presence of locomotor syndrome as well as lower HRQOL. These findings indicated that the GAP score may be a useful indicator for characterizing musculoskeletal alignment in clinical and public health fields.

Author Contributions: Supervision, H.N., Shiro. Imagama.; resources, Y.H.; visualization, Y.N.; methodology, Y.N., N.S., and Shiro. Imagama.; project administration, H.N., Shiro. Imagama.; data curation, J.O. and I.Y.; conceptualization, Shiro Imagama; writing-original draft, Y.N.; writing-review and editing, H.N.; formal analysis, Sadayuki. Ito., T.S., Y.T.; validation, Sadayuki. Ito., J.O.; investigation, Y.N., Shinya. Ishizuka., J.O., I.Y. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest: The authors declare that there are no relevant conflicts of interest.

Availability of Data and Materials: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval: The study protocol was approved by the Human Research Ethics Committee and Institutional Review Board of Nagoya University (approval number 2022-0328). The study procedures were performed according to the principles of the Declaration of Helsinki. Informed consent was obtained from all participants included in the study. This research was approved by the authors' affiliated institutions.