*2.2. Measurements*

In all patients, we evaluated bone mineral density (BMD) of the lumbar spine (LS: L2–L4), femoral neck (FN), and total hip (TH), using dual-energy X-ray absorptiometry (DXA: Horizon DXA System; Hologic Inc., Santa Clara, CA, USA), and serum levels of bone turnover markers including bone-specific alkaline phosphatase (BAP; Beckman Coulter Inc. Brea, CA, USA) and tartrate-resistant acid phosphatase 5b (TRACP5b; DS Pharma Biomedical Inc., Osaka, Japan).

#### 2.2.1. Radiographical Evaluation

X-ray images taken in frontal and lateral views of the whole spine, including the hip joints, in the standing position were reviewed to evaluate spinal sagittal alignment and VFs. For the evaluation of spinal sagittal alignment, three spinal sagittal alignment parameters were measured. To assess pelvic tilt (PT), the angle between the line joining the midpoint of the bilateral center of the femoral head to the center of the S1 endplate and a vertical reference line was measured. Pelvic incidence (PI) was determined by measuring the angle between a line joining the midpoint of the bilateral center of the femoral head to the center of the S1 endplate, and a line orthogonal to the S1 endplate, as previously reported [7]. To measure lumbar lordosis (LL), we assessed the angles between the first line parallel to the upper endplate of L1 and the second line parallel to the superior endplate of the sacral base on lateral views of the whole-spine radiograph. Then, PI-LL was used to evaluate spinal sagittal alignment. For measuring the sagittal vertical axis (SVA), the horizontal distance between the posterior-superior corner of the sacrum and a vertical line from the center of C7 was measured, as previously reported [8]. In accordance with the SRS-Schwab classification scheme [9], patients were categorized using three sagittal spinopelvic modifiers, including PT, PI-LL, and SVA. SVA > 40 mm, PT > 20◦, or PI-LL > 10◦ was defined as spinal sagittal malalignment. Based on these data, subjects were divided into a normal alignment group and a sagittal malalignment group.

#### 2.2.2. Clinical Outcome Evaluation

LBP was evaluated using the Japanese Orthopedic Association Back Pain Evaluation Questionnaire (JOABPEQ), the Oswestry Disability Index (ODI), and the visual analogue scale (VAS). JOABPEQ consists of five functional scores: pain-related disorders, lumbar spine dysfunction, gait disturbance, social life dysfunction, and psychological disorders. Each domain score ranges from 0 to 100, and higher scores corresponded to an improved patient condition.

In addition, HRQoL was evaluated using the MOS 36-Item Short-Form Health Survey (SF-36). SF-36 consists of 8 subscales, as follows: physical function, PF; role physical, RP; body pain, BP; general health, GH; vitality, VT; social functioning, SF; role emotional, RE; and mental health, MH. The score for each domain ranged from 0 to 100, and higher scores indicated a better condition.

#### *2.3. Statistical Analysis*

First, the prevalence of a spinal sagittal malalignment was evaluated based on the number of VFs identified. Factors including age, BMD, serum levels of bone turnover markers, and parameters of spinal sagittal alignment of the three groups of patients with 0, 1, and ≥2 VFs were compared using the Bonferroni test for multiple comparisons. Sex and the spinal sagittal alignment differences were compared using the chi-squared test.

Characteristics of spinal sagittal malalignment evaluated in sagittal malalignment and normal alignment groups in patients without VFs were compared. Leven's test was used to assess variance for variables of interest. To assess data with unequal variance, the Mann–Whitney U test was applied. An unpaired *t*-test was used to assess data with equal variance. All data were analyzed using IBM SPSS Statistics version 26 (IBM, Atmonk, NY, USA), and *p* < 0.05 were considered significant.

## *2.4. Ethics*

Ethical approval from Institutional Review Board in Kitasato University was obtained for this study (Approval code, #B17–197), which was conducted in accordance with the ethical principles specified in the 1964 Declaration of Helsinki and its later amendments.

## **3. Results**

Characteristics of the patient population and BMD, serum levels of bone turnover markers, and parameters of spinal sagittal alignment, are listed in Table 1. Spinal malalignment was observed in 205 of 259 (79.2%) patients. BMDs of the FN and TH in the group with ≥2 VF were significantly lower than those of the 0 group. The BMD of the TH in the group with 1 VF was significantly lower than that of the 0 VF group (*p* < 0.05).


**Table 1.** The patient population of this study.


**Table 1.** *Cont.*

BMD: body mass index, LS: lumbar spine, FN: femoral neck, TH: total hip, BAP: bone-specific alkaline phosphatase, TRACP5b: tartrateresistant acid phosphatase 5b, PT: pelvic tilt, PI-LL: pelvic incidence minus lumbar lordosis, SVA: sagittal vertical axis, VF: vertebral fracture, JOABPEQ: Japanese Orthopedic Association Back Pain Evaluation Questionnaire, ODI: Oswestry Disability Index, VAS: visual analogue scale of low back pain, SF-36: MOS 36-Item Short-Form Health Survey, PF: physical function, RP: role physical, BP: body pain, GH: general health, VT: vitality, SF: social functioning, RE: role emotional, MH: mental health.

> With regard to spinal sagittal alignment parameters, PT and SVA values of the ≥2 VF group was significantly higher than that of the 0 VF group (*p* < 0.05). No significant differences between 0, 1, ≥2 VF groups were observed with regard to BMD, bone turnover markers, and PI-LL (*p* > 0.05). The prevalence of a spinal sagittal malalignment in patients with 0, 1, or ≥2 VFs was 72.1%, 86.0%, and 86.3%, respectively, and differences among the three groups were determined as significant (*p* < 0.05) (Table 1).

> With regard to LBP and the HRQoL score, all five domains of JOABPEQ, ODI, and VAS, as well as all eight subscales of the SF-36 of the ≥2 VF group, were significantly worse than those of the 0 VF group (*p* < 0.05). In addition, the values of all five domains of JOABPEQ, ODI, RP, BP, SF, RE, and MH of SF-36 in the 1 VF group were significantly worse than those in the 0 VF group (*p* < 0.05) (Table 1).

> In a sub-analysis of patients without VFs, no significant differences were observed between the sagittal malalignment group and the normal alignment group with regard to age; LS, FN, and TH of BMD; or bone turnover markers, including BAP and TRACP5b (*p* > 0.05) (Figure 1). In contrast, all five JOABPEQ functional scores (including painrelated disorders, lumbar spine dysfunction, gait disturbance, social life dysfunction, and psychological disorders) of the sagittal malalignment group were significantly lower than those of the normal alignment group (*p* < 0.05) (Figure 2A). Furthermore, ODI and the VAS values determined for LBP in patients without VFs were significantly higher in the sagittal malalignment group than in the normal alignment group (*p* < 0.05) (Figure 2B,C). Additionally, PF, RP, VT, RE, and MH of SF-36 values of the sagittal malalignment group were significantly lower than those of the normal alignment group (*p* < 0.05) (Figure 3).

**Figure 1.** In patients without VFs, comparisons of ( **A**) age, (**B**) BMD, ( **C**) BAP, and ( **D**) TRACP5b values determined in patients of the normal alignment and sagittal malalignment groups are shown. VF, vertebral fracture; N.S.: not significant; BMD, bone mineral density; BAP, bone-specific alkaline phosphatase; TRACP5b, tartrate-resistant acid phosphatase 5b.

**Figure 2.** In patients without VFs, comparisons of (**A**) JOABPEQ, (**B**) ODI, and (**C**) VAS of LBP for patients of the normal alignment and sagittal malalignment groups are shown. VF, vertebral fracture; LBP, low back pain; ODI, Oswestry Disability Index; JOABPEQ, Japanese Orthopedic Association Back Pain Evaluation Questionnaire; VAS, Visual Analogue Scale, \* *p* < 0.05.

**Figure 3.** A comparison of MOS 36-Item Short-Form Health Survey scores of normal alignment and sagittal malalignment groups in patients without VFs is shown. VF, vertebral fracture; PF: Physical function, RP: Role physical, BP: Body pain, GH: General health, VT: Vitality, SF: Social functioning, RE: Role emotional, MH: Mental health, N.S.: not significant, \* *p* < 0.05.
