Bone 74 (2015) 160–165
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Original Full Length Article
The muscle–bone interaction in Turner syndrome Ondrej Soucek a, Jana Matyskova a, Elmar Anliker b, Marco Toigo c, Zdenek Hlavka d, Jan Lebl a, Zdenek Sumnik a,⁎ a
Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic Clinic for Sports Medicine, Lucerne Cantonal Hospital, Lucerne, Switzerland Exercise Physiology Lab, Institute of Human Movement Sciences, ETH Zurich, Zurich, Switzerland d Department of Statistics, Faculty of Mathematics and Physics, Charles University in Prague, Prague, Czech Republic b c
a r t i c l e
i n f o
Article history: Received 28 September 2014 Revised 20 January 2015 Accepted 26 January 2015 Available online 3 February 2015 Edited by Mark Cooper Keywords: Turner syndrome Muscle force Bone strength indices Muscle–bone unit Ovarian function
a b s t r a c t Objectives: Turner syndrome (TS) is associated with an increased fracture rate due to reduced bone strength, which is mainly determined by skeletal muscle force. This study aimed to assess the muscle force–bone strength relationship in TS and to compare it with that of healthy controls. Methods: This study included 39 girls with TS and 67 healthy control girls. Maximum muscle force (Fmax) was assessed through multiple one-legged hopping with jumping mechanography. Peripheral quantitative computerized tomography assessed the bone strength index at the tibial metaphysis (BSI 4) and the polar strength– strain index at the diaphysis (SSI polar 66). The effect of TS on the muscle–bone unit was tested using multiple linear regression. Results: TS had no impact on Fmax (p = 0.14); however, a negative effect on bone strength (p b 0.001 for BSI 4 and p b 0.01 for SSI polar 66) was observed compared with healthy controls. Bone strength was lower in the TS group (by 18%, p b 0.01, for BSI 4 and by 7%, p = 0.027, for SSI polar 66), even after correcting for Fmax. Conclusions: Similar muscle force induces lower bone strength in TS compared with healthy controls, which suggests altered bone-loading sensitivity in TS. © 2015 Elsevier Inc. All rights reserved.
Introduction Turner syndrome (TS) is caused by the complete or partial loss of one X chromosome. Its incidence is 1 in 2000 live female births [39]. Patients with TS are seen primarily by endocrinologists to address their short stature, ovarian failure or autoimmune thyroiditis. However, other specialists may be involved because TS is associated with an increased risk of congenital heart and urinary system malformations and celiac disease [13]. Additionally, an increased fracture rate [15] and decreased bone mineral density (BMD) [36] have been described in TS compared to healthy controls. Several other studies describe osteoporosis in TS [4,14,23], which suggests that skeletal complications are of concern in these patients. The etiology and mechanism of bone fragility in TS have not been completely elucidated. While the results of some studies suggest inappropriate estrogen substitution [7,16,18], other studies claim that either a direct effect of the loss of the short stature homeobox-containing gene
⁎ Corresponding author at: Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, V Úvalu 84, 150 06 Praha 5. Fax: +420 22443 2097. E-mail address: [email protected] (Z. Sumnik).
http://dx.doi.org/10.1016/j.bone.2015.01.017 8756-3282/© 2015 Elsevier Inc. All rights reserved.
(SHOX) [28,37] or an indirect effect of the loss of Xp22.3, which leads to neuromotor impairment [31,44], causes bone fragility in TS. Skeletal muscle force represents a major mechanical stimulus for bone development and determines bone strength [12,33,34]. According to Frost's mechanostat theory [11], every bone should be adapted to withstand the maximal voluntary muscle forces. Ground-reaction forces have been used to calculate that during the multiple one-legged hopping test, the plantarflexor muscle force amounts to approximately 9–10.5 times the body weight [3]. This value is 2.8–3.3 times higher than the dynamometry-derived peak plantarflexor force (approx. 4.8 times the body weight) [3]. Therefore, the mechanical forces at the tibial shaft are much greater during multiple one-legged hopping compared with isokinetic dynamometry, a method that was previously used to assess muscle torque in the pediatric population [19,29]. Moreover, a strong correlation has been observed between maximum force, as assessed using ground-reaction force plate (GRFP) measurements during multiple one-legged hopping, and tibial bone strength, measured using peripheral quantitative computed tomography (pQCT) [2]. This correlation, together with its good reliability [43] and the availability of reference data [6,24,40], make GRFP a suitable method for assessing muscle function in children. We hypothesized that the muscle–bone interaction is impaired in TS patients compared with healthy controls, leading to decreased bone
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strength. Thus, the aims of the present study were 1) to assess the peak force and bone strength using GRFP and pQCT, respectively, in girls with TS and healthy controls; 2) describe the muscle–bone relationships in the participants with TS by implementing the GRFP and pQCT measures; 3) compare these relationships with those of healthy controls; and 4) test the influence of menarcheal status, course of puberty, karyotype, durations of growth hormone (GH) and estrogen (E) therapy and fracture history on muscle–bone relationships in the TS participants. Participants and methods Participants This was a cross-sectional study involving 39 girls with TS from a single university hospital referral center. Eighteen TS participants in the present study were also included in the longitudinal BMD study, and the baseline values of that study have been published previously [36]. Several patients with TS were passed on to adult endocrinologists, while new young girls with TS were also included. However, the present cross-sectional study is based on measurements of the weight-bearing tibia bone, whereas the previous study was based on radius bone measurements. Muscle force data from TS girls reported in this study were published quite recently [35]. However, the novelty of the present study is the concurrent assessment of both peak muscle force and BMD, which allows direct observation of the muscle–bone interaction. Despite the fact that the radiation dose due to pQCT measurement is negligible, only 39 girls with TS (and/or their guardians) consented to be included in the present study (i.e., to have scans at the tibia in addition to the scans at the radius) compared with 60 girls with TS who agreed to the muscle function assessment [35]. The exclusion criteria were any chronic disease (except autoimmune thyroiditis) or medication (except recombinant human growth hormone [GH] and estrogen) with a known effect on the muscles or bones. Body height was measured with a stadiometer to the nearest 1 mm. Weight was measured with an electronic scale to the nearest 100 g. Height, weight and BMI Z-scores were calculated using the most recent national reference data [21]. The girls with TS presented with Tanner stage 1 (N = 13), 2 (N = 3), 3 (N = 2), 4 (N = 6) and 5 (N = 15) breast development. There were 21 pre-menarcheal girls and 18 post-menarcheal girls. Their karyotypes were either monosomy of X (N = 13), the classical mosaic form 45,X/46,XX (N = 4), other double- or triple-line mosaics with at least two aberrant lines (N = 18) or a structural defect of one X chromosome (3 patients with a deletion and 1 patient with an isochromosome). All of the girls with TS were treated with GH at a starting dose of 50 μg/kg/d, which was then adjusted according to the clinical response [32]. The mean age at the start of GH treatment was 7.6 ± 3.9 years, and the mean duration of GH administration was 5.4 ± 3.8 years. On the day of the examination, 3 of the girls had just begun GH treatment, and another 14 girls had already stopped treatment because they had reached their final height. Estrogen replacement was given to 14 patients to initiate their puberty and to 4 patients because of secondary ovarian failure; another 9 patients had a spontaneous pubertal development. The remaining 12 patients were pre-pubertal. Oral estrogen replacement began at the mean age of 13.6 ± 1.7 years, with a starting dose of 5 μg/kg/day of 17-β-estradiol given for 12–18 months. [30]. Afterwards, a dose of 10 μg/kg/day was administered for another 12–18 months, followed by an increase to 20 μg/kg/day. After a mean period of 3 months, the same dose of estradiol was continued from days 1 to 21, with 5 mg of medroxyprogesterone per day added from day 15 to day 21 of every cycle. Thyroxine substitution was necessary in 8 patients with autoimmune thyroiditis. All of these patients had been euthyroid for at least 18 months prior to the study measurements. Altogether, there were 19 fracture events in the histories of 10 patients: 12 in the forearm, 3
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finger fractures, 2 ankle fractures, 1 wrist fracture and 1 vertebral fracture. As a control group, we selected 67 healthy girls with a wide physical activity range. The control girls were recruited from schools and different sports associations (volleyball, ice hockey, gymnastics and synchronized swimming). The exclusion criteria were any disease or medication affecting the musculoskeletal system. The muscle and the bone measures were obtained using the same methods that were used for the TS group (GRFP and pQCT) and by implementing the same technical settings and maneuvers. These data have been published in part as a subgroup of a larger study comprising 323 children and adults [2]. The present study was approved by the University Ethics Committee and conformed to the Declaration of Helsinki. All of the participants included in this study (or a parent if the patient was younger than 18 years) consented to the testing. Muscle force assessment Maximum ground reaction force (Fmax; N), a surrogate for maximum plantarflexor muscle force, was assessed using the multiple one-legged hopping (M1LH) test performed on a Leonardo Mechanograph® GRFP (Novotec Medical, Pforzheim, Germany). To detect, store and calculate the data, we used the manufacturer's software (Leonardo Mechanography GRFP version 4.2; Novotec Medical). The test consisted of repeated hopping on the forefoot of one leg with a stiff knee and free movement of the arms. The subjects were lightly dressed and wore no shoes. The subject's task was to jump as quickly as possible at the beginning of the test and then, after achieving good balance, to jump higher with every subsequent jump to obtain Fmax. The highest value for Fmax was selected among the three consecutive tests performed with the non-dominant leg. Fmax and its relation to body weight (Fmax/ BW; no unit) were recorded. Bone strength assessment An XCT 2000 scanner (Stratec Medizintechnik, Pforzheim, Germany) was used for pQCT measurements at the non-dominant tibia. The leg dominancy was determined according to handedness, so that the leg on the same side of the body as the dominant hand was considered dominant. A single tomographic 2.0-mm-thick slice was taken at distances corresponding to the 4% and 66% bone lengths, as measured from the medial malleolus to the superior margin of the medial condyle. A voxel size of 0.4 × 0.4 × 2.0 mm was used. The images were processed and the numerical values were calculated using version 6.20 C of the integrated XCT software. At the distal tibia (4% site), the total bone mineral content (BMC 4), total volumetric bone mineral density (vBMD), total bone cross-sectional area (CSA) and trabecular vBMD were measured. The compressional bone strength index (BSI 4) was calculated as the product of the square of the total vBMD and the total bone CSA, as previously described [22]. At the proximal tibia (66% site), the total BMC (BMC 66) and the polar strength–strain index (SSI polar 66) were assessed. The SSI polar 66 was determined using a segmentation threshold of 480 mg/cm3. The precision errors of the pQCT measures were not assessed at the tibia, but they have been found to be low at the radius [36]. The root mean square standard deviations gained from the 3 consecutive measurements with repositioning were as follows: at the distal radius: total BMC 0.009 g/cm, total bone CSA 9.617 mm2, total vBMD 9.353 mg/cm3 and trabecular vBMD 2.905 mg/cm3; at the proximal radius: total BMC 0.007 g/cm and SSI polar 7.685 mm3. Biochemistry Blood samples were obtained from the patients on the day of the muscle and bone assessment and were a part of their regular followup. Serum FSH levels were measured in our accredited hospital
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laboratory by chemiluminiscence immunoassay; the results are given in IU/l. The intra-individual coefficient of variation for the kit was 11%.
Table 2 Summary of the bone and muscle characteristics.
2.5. Statistical analyses The statistical computing environment R [27] was used to conduct all of the statistical analyses. The data are reported as the means (SD). The Z-scores were tested for difference from zero using the onesample t-test. We performed the two-sample t-test with the Welch approximation for the degrees of freedom for two-group comparisons of the anthropometry measures' Z-scores. The difference in the logarithmically transformed muscle force and bone strength parameters between the TS group and the healthy controls was estimated using linear regression with correction for age, weight and height. The influence of the Fmax logarithm on the bone strength parameter logarithms was investigated using linear regression with correction for TS group, age, weight and height. Using this model, we reported a change in the intercept for the TS group (assuming that the slope of the regression line was the same in both groups), i.e., the estimated difference in the bone strength parameter logarithms in the TS group compared with the healthy controls. In the second model, we also investigated the interaction between the effect of log (Fmax) and the TS group, i.e., the estimated change in the slope (of the dependency) of the bone strength parameter logarithms on log (Fmax) in the TS group. Finally, linear regression was used to investigate the effects of GH therapy duration, estrogen substitution duration, course of puberty, menarcheal status, karyotype, fracture history and serum FSH level on bone strength parameters in the TS group after correction for log (Fmax), age, weight and height. For all of the tests, the reported p-values are two-sided. Results The anthropometric characteristics of the TS group and the healthy controls are shown in Table 1. There was no difference in age, but the TS group had lower height and weight Z-scores. The raw muscle and bone data for both groups are presented in Table 2. The apparent differences in the means of some of the raw muscle and bone measures between these two groups need to be interpreted with caution because these measures are age and height dependent, and the two groups differed significantly with respect to height and weight. Linear regression analyses showed that TS had no significant effect on Fmax or Fmax/BW (Table 3). In contrast, TS had a negative effect on bone strength at the metaphysis, amounting to approximately 24%, 17% and 22% decreases for BSI 4, BMC 4 and trabecular vBMD, respectively. TS also had a weaker negative effect on the diaphyseal bone strength (an approximately 1% decrease for the SSI polar 66; Table 3). No effect was observed for BMC 66. TS had a significant effect on the muscle force–bone strength interaction. The intercepts of the linear regression between bone strength measures and Fmax in TS were decreased by approximately 18%
Mechanography Fmax (N) Fmax/BW pQCT Tibia metaphysis (4% site) BMC 4 (g/cm) BSI 4 (g2/cm4) Trabecular vBMD (mg/cm3) Tibia shaft (66% site) BMC 66 (g/cm) SSI polar 66 (mm3)
Turner syndrome (N = 39)
Healthy controls (N = 67)
1291.7 (471.7) 3.1 (0.39)
1402.8 (478.4) 3.4 (0.44)
2.2 (0.70) 0.59 (0.20) 197.8 (30.0)
2.7 (0.74) 0.81 (0.30) 247.4 (49.9)
2.5 (0.64) 1293.8 (430.4)
2.9 (0.8) 1617.9 (645.9)
Group difference (p-value) 0.25 0.006
0.002 b0.001 b0.001
0.016 0.003
Values represent mean (SD). Abbreviations: Fmax = peak muscle force, Fmax/BW = Fmax relative to body weight, BMC = bone mineral content, BSI = bone strength index, vBMD = volumetric bone mineral density, and SSI polar = polar strength–strain index. The two-sample t-test was used for between-group comparisons.
(p b 0.01) and 7% (p = 0.027) for BSI 4 and SSI polar 66, respectively, compared with healthy controls, meaning that for the same Fmax, the bone strength was lower in the TS group. The slope of the regression line was not significantly different between the participants with TS and the healthy controls, meaning that the mechanisms controlling the muscle–bone interaction were the same in both groups (Fig. 1). Menarcheal status, course of puberty, karyotype, the durations of GH and estrogen treatment and fracture history had no significant effect on the four pQCT-derived bone strength indices in the TS group. The only exception was the increased BMC 66 in girls with TS who had undergone spontaneous puberty compared with the girls with TS whose puberty was induced (Table 4). Discussion This study showed that in girls with TS, a) the pQCT-derived estimates of bone strength, particularly at the tibial bone metaphysis, were significantly reduced compared with healthy controls, despite similar maximum ground reaction force during the multiple onelegged hopping test, and b) the karyotype, therapy and fracture history did not affect muscle force–bone strength interaction. We found that the impact of TS on the compressive and torsional strength of the tibial bone (BMC 4 and SSI polar 66, respectively) was significantly negative and that the effect was more apparent at the metaphysis compared with the diaphysis. The published data on bone strength in TS are heterogeneous because of the plethora of methods that are employed to assess this parameter. While Bechtold et al. [5] found a decreased bone strength index (SSI polar) at the radial bone metaphysis and diaphysis in females with TS, our previous study showed that the SSI polar at the radial bone diaphysis in TS girls at all pubertal stages was normal or increased compared with the German
Table 1 Summary of the anthropometric characteristics.
Age (year) Height (cm) Height Z-score Weight (kg) Weight Z-score BMI (kg/m2) BMI Z-score
Turner syndrome (N = 39)
Healthy controls (N = 67)
13.6 (4.2) 142.6 (16.1) −1.8 (1.0)*** 42.2 (14.6) −0.7 (1.1)*** 19.9 (3.4) 0.3 (0.9)*
12.7 (3.4) 148.8 (16.3) −0.6 (1.3)*** 42.4 (13.2) −0.2 (1.0) 18.6 (2.5) 0.1 (0.8)
Group difference (p-value) 0.31 b0.001 0.013 0.15
Abbreviation: BMI = body mass index. The Z-scores were tested for difference from zero using the one-sample t-test. *p b 0.05 ***p b 0.001. The two-sample t-test was used for betweengroup comparisons.
O. Soucek et al. / Bone 74 (2015) 160–165 Table 3 The influence of TS on muscle force and bone strength parameters compared with the parameters for healthy controls. Estimated difference of logarithms (mean [SD]) Mechanography Fmax (N) Fmax/BW pQCT Tibial metaphysis (4% site) BMC 4 (g/cm) BSI 4 (g2/cm4) Trabecular vBMD (mg/cm3) Tibial shaft (66% site) BMC 66 (g/cm) SSI polar 66 (mm3)
p-value
−0.051 (0.034) −0.038 (0.032)
0.14 0.24
−0.17 (0.047) −0.24 (0.07) −0.22 (0.045)
b0.001 b0.001 b0.001
−0.039 (0.027) −0.096 (0.035)
0.15 b0.01
Abbreviations: Fmax = peak muscle force, Fmax/BW = Fmax relative to body weight, BMC = bone mineral content, BSI = bone strength index, vBMD = volumetric bone mineral density, and SSI polar = polar strength–strain index. The influence of TS was tested using linear regression models with corrections for age, height and weight.
reference data [36]. The contradictory results of these two studies may be explained by the different comparison methods used: whereas agespecific Z-scores were calculated in the former study, height-specific Z-scores were calculated in the latter. The more appropriate comparison method cannot be unequivocally determined because of the specific growth and pubertal development patterns of patients with TS, which are characterized by short stature, increased body mass index and delayed (mostly induced) puberty. In the present study, we decided to test the impact of TS on bone strength indices by comparing patients with TS with healthy control group using anthropometric measures (age, height and weight) as the covariates in the multiple linear regression models to avoid inadequate matching. In addition, in contrast to previous studies, we selected the load-bearing skeletal site, i.e., the tibia, for bone strength examination.
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In accordance with our observations, a recent high-resolution-pQCT study [17] showed that the bone failure load estimates (obtained by finite element analysis) were 11% and 16% lower in TS patients at the radius and tibia, respectively, compared with healthy controls. The question may arise whether the bone sites we used for the bone strength assessments were the most appropriate. The 4% site was selected based on previous findings of significantly reduced bone mineral content, density and strength at this site in the radius among TS patients [5]. In addition, trabecular vBMD (assessed at the 4% site) has been found to be associated with prevalent fractures in young healthy girls [10]. According to a study on cadaver bones, the tibia's highest propensities to fracture were at the 50% and 85% sites for the 3-point and cantilever bending tests, respectively [42]; thus, choosing the 65% site may be relevant for both sites while decreasing the radiation dose by foregoing the additional scan. Moreover, the association between the maximum muscle force and tibia bone strength at the 65% site was very strong (R2 = 0.833) and was comparable to other sites along the tibia [2]. Therefore, the selected tibia measurement sites appear to be clinically relevant. Importantly, in addition to bone strength, we examined the bone load by assessing Fmax using the M1LH test on the GRFP. TS had no effect on Fmax compared with the control group. This corresponds to the normal Fmax that has been observed in patients with TS [35] when the Z-scores are calculated from reference data comprising 432 healthy females [40]. The findings on grip force tested with hand dynamometry are inconsistent in TS. Both decreased [8] and increased values [25] have been reported, with height being the main determinant of the grip force in TS. Nevertheless, the maximal voluntary forces (those that cause the maximal bone deformation) are detected during lengthening and not isometric muscle actions [3]. Therefore, our results might be more relevant than the results of isokinetic dynamometry studies for the functional evaluation of the muscle–bone unit in TS. If the maximum muscle force is normal but the bone strength is reduced in girls with TS, what are the potential mechanisms? One
Fig. 1. Abbreviations: BMC = bone mineral content, BMD = bone mineral density, BSI = bone strength index, SSI = strength–strain index. The 95% confidence intervals are illustrated, representing the changes in the intercept (left) and the slope (right) of the regression lines for the logarithms of the bone strength surrogates at the tibia in the TS group compared with the healthy controls. The covariates were log (Fmax), age, height and weight.
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Table 4 The influence of selected TS-specific characteristics on tibial bone strength measures in girls with TS.
GH therapy duration (years) E substitution duration (years) Spontaneous puberty (vs. induced) Post-menarche (vs. pre-menarche) 45,X/46,XX karyotype (vs. 45,X karyotype) Positive fracture history (vs. no history) FSH serum level (IU/l)
log BMC 4
p-Value
log BSI 4
p-Value
log BMC 66
p-Value
log SSI polar 66
p-Value
−0.001 (0.008) −0.018 (0.016) 0.179 (0.091) 0.001 (0.071) −0.109 (0.111) −0.002 (0.053) −0.002 (0.002)
0.86 0.28 0.066 0.99 0.35 0.97 0.27
−0.002 (0.011) −0.008 (0.024) 0.236 (0.136) 0.066 (0.102) −0.089 (0.217) −0.090 (0.075) −0.003 (0.002)
0.82 0.74 0.10 0.52 0.69 0.24 0.25
−0.002 (0.006) −0.010 (0.014) 0.169 (0.050) 0.058 (0.059) −0.034 (0.114) 0.019 (0.044) 0.000 (0.002)
0.70 0.46 0.0037 0.33 0.77 0.67 0.80
−0.003 (0.006) −0.010 (0.014) 0.046 (0.068) −0.003 (0.061) 0.020 (0.094) −0.058 (0.044) 0.001 (0.002)
0.65 0.49 0.51 0.96 0.83 0.20 0.65
Abbreviations: GH = growth hormone, E = estrogen, BMC = bone mineral content, BSI = bone strength index, SSI = strength–strain index, and FSH = follicle-stimulating hormone. Beta coefficients (standard errors) are shown. In multivariate linear regression models, Fmax, age, height and weight were used as covariates.
possible explanation is suggested by a study in which the surrogates of bone strength were decreased in oligoamenorrheic athletes compared with eumenorrheic athletes [1]. Additionally, patients with hypogonadism have been found to have decreased trabecular BMD [26]. Both studies point towards estrogen insufficiency. In contrast, we detected no significant impact of estrogen substitution duration (β = 0.0035 ± 0.016, p = 0.83), spontaneous puberty (β = 0.047 ± 0.095, p = 0.63) or serum FSH levels (β = −0.002 ± 0.002, p = 0.63) on the relationship between Fmax and the trabecular BMD in the TS group. We also found no significant effect of estrogen status on BSI 4 or SSI polar 66 in girls with TS. In young adult women with TS, transdermal estradiol replacement seemed to induce more physiological pharmacokinetics than oral pills [41], and subcutaneous estrogen implants given every 6 months for three years lead to significant and sustained increases in serum estradiol, BMD Z-score and trabecular bone volume [20]. We may thus speculate that there are generally insufficient amounts of estradiol available to the bone tissue in TS. However, one limitation of this study is that we did not randomize our participants with TS to receive different means of estradiol administration, and we lacked data on serum estradiol and/or FSH in the healthy controls. The relatively low number of TS participants and their limited age span could also have caused our non-significant observations. We were therefore unable to explore whether estradiol bioavailability impacts the muscle–bone interaction in TS. It has been shown that immobility decreases trabecular vBMD by 73% in the tibia bones of patients after spinal cord injury [9], a consequence of the loss of muscle mass and activity. In contrast, long-term tennis players have been shown to gain 6% more trabecular bone in the playing arm compared with the contralateral arm [22]. These two reports clearly show the existing relationship between muscle activity and trabecular bone density. The present study found that despite having similar Fmax values, the TS group had lower bone strength (BMC4 and BSI 4) and trabecular vBMD compared with healthy controls. Despite being appropriately substituted with oral 17-β-estradiol, our patients with TS may not have been adequately treated relative to physiological estrogen activity in healthy controls. Therefore, one cannot rule out the possibility that estrogen “inadequacy” increases the threshold for disuse-driven endocortical bone resorption, the result of which is low trabecular vBMD (and thus decreased bone strength). The second possible mechanism of decreased bone strength in TS may be based on SHOX deficiency, which is a common feature in patients with TS [28]. Patients with isolated SHOX deficiency share a similar radial bone geometry phenotype with girls with TS [38]. Unfortunately, we have no data on the muscle function in the patients with isolated SHOX deficiency, and we could not test for possible differences in the muscle force–bone strength relationship between these patients and the participants with TS. Another limitation of our study is the lack of data regarding dietary calcium intake, serum vitamin D levels and physical activity in the study participants. This lack of data prevented us from testing the possible influences of these measures on bone strength parameters. Because of the cross-sectional design of this study and the relatively low number of participants with TS in certain subgroups (9 with
spontaneous puberty vs. 18 with estrogen substitution; 13 with X monosomy vs. 4 with the 45,X/46,XX karyotype), the non-significant results regarding the influence of hypogonadism or karyotype on bone strength cannot not be considered definitive. Conclusions Bone strength in girls with TS was significantly reduced despite normal maximum muscle force in terms of absolute and relative force. These results point to a primary bone defect in TS. The mechanisms leading to the abnormal bone load–bone strength relationship in TS remain to be elucidated. Acknowledgments We would like to thank Marta Snajderova, Stanislava Kolouskova and Stepanka Pruhova for their cooperation in recruiting their patients with TS. We acknowledge all of the study participants and their parents for their kind consent with the study protocol. The paper was partially supported by the Czech Ministry of Health's Project for Conceptual Development of Research Organization no. 00064203 (University Hospital Motol, Prague, Czech Republic). References [1] Ackerman KE, Pierce L, Guereca G, Slattery M, Lee H, Goldstein M, et al. Hip structural analysis in adolescent and young adult oligoamenorrheic and eumenorrheic athletes and nonathletes. J Clin Endocrinol Metab 2013;98:1742–9. [2] Anliker E, Rawer R, Boutellier U, Toigo M. Maximum ground reaction force in relation to tibial bone mass in children and adults. Med Sci Sports Exerc 2011;43: 2102–9. [3] Anliker E, Toigo M. Functional assessment of the muscle–bone unit in the lower leg. J Musculoskelet Neuronal Interact 2012;12:46–55. [4] Bakalov VK, Chen ML, Baron J, Hanton LB, Reynolds JC, Stratakis CA, et al. Bone mineral density and fractures in Turner syndrome. Am J Med 2003;115:259–64. [5] Bechtold S, Rauch F, Noelle V, Donhauser S, Neu CM, Schoenau E, et al. Musculoskeletal analyses of the forearm in young women with Turner syndrome: a study using peripheral quantitative computed tomography. J Clin Endocrinol Metab 2001;86:5819–23. [6] Busche P, Rawer R, Rakhimi N, Lang I, Martin DD. Mechanography in childhood: references for force and power in counter movement jumps and chair rising tests. J Musculoskelet Neuronal Interact 2013;13:213–26. [7] Carrascosa A, Gussinye M, Terradas P, Yeste D, Audi L, Vicens-Calvet E. Spontaneous, but not induced, puberty permits adequate bone mass acquisition in adolescent Turner syndrome patients. J Bone Miner Res 2000;15:2005–10. [8] Clark C, Klonoff H, Hayden M. Regional cerebral glucose metabolism in Turner syndrome. Can J Neurol Sci 1990;17:140–4. [9] Eser P, Frotzler A, Zehnder Y, Wick L, Knecht H, Denoth J, et al. Relationship between the duration of paralysis and bone structure: a pQCT study of spinal cord injured individuals. Bone 2004;34:869–80. [10] Farr JN, Tomas R, Chen Z, Lisse JR, Lohman TG, Going SB. Lower trabecular volumetric BMD at metaphyseal regions of weight-bearing bones is associated with prior fracture in young girls. J Bone Miner Res 2011;26:380–7. [11] Frost HM. The mechanostat: a proposed pathogenic mechanism of osteoporoses and the bone mass effects of mechanical and nonmechanical agents. Bone Miner 1987;2: 73–85. [12] Frost HM. Muscle, bone, and the Utah paradigm: a 1999 overview. Med Sci Sports Exerc 2000;32:911–7. [13] Gravholt CH, Juul S, Naeraa RW, Hansen J. Morbidity in Turner syndrome. J Clin Epidemiol 1998;51:147–58. [14] Gravholt CH, Lauridsen AL, Brixen K, Mosekilde L, Heickendorff L, Christiansen JS. Marked disproportionality in bone size and mineral, and distinct abnormalities in
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[29] Rauch F, Neu CM, Wassmer G, Beck B, Rieger-Wettengl G, Rietschel E, et al. Muscle analysis by measurement of maximal isometric grip force: new reference data and clinical applications in pediatrics. Pediatr Res 2002;51:505–10. [30] Rosenfield RL, Devine N, Hunold JJ, Mauras N, Moshang Jr T, Root AW. Salutary effects of combining early very low-dose systemic estradiol with growth hormone therapy in girls with Turner syndrome. J Clin Endocrinol Metab 2005;90:6424–30. [31] Ross JL, Stefanatos GA, Kushner H, Bondy C, Nelson L, Zinn A, et al. The effect of genetic differences and ovarian failure: intact cognitive function in adult women with premature ovarian failure versus Turner syndrome. J Clin Endocrinol Metab 2004;89:1817–22. [32] Saenger P, Wikland KA, Conway GS, Davenport M, Gravholt CH, Hintz R, et al. Recommendations for the diagnosis and management of Turner syndrome. J Clin Endocrinol Metab 2001;86:3061–9. [33] Schiessl H, Frost HM, Jee WS. Estrogen and bone–muscle strength and mass relationships. Bone 1998;22:1–6. [34] Schoenau E. From mechanostat theory to development of the “Functional Muscle– Bone-Unit”. J Musculoskelet Neuronal Interact 2005;5:232–8. [35] Soucek O, Lebl J, Matyskova J, Snajderova M, Kolouskova S, Pruhova S, et al. Muscle function in Turner syndrome: normal force but decreased power. Clin Endocrinol (Oxf) 2015;82:248–53. [36] Soucek O, Lebl J, Snajderova M, Kolouskova S, Rocek M, Hlavka Z, et al. Bone geometry and volumetric bone mineral density in girls with Turner syndrome of different pubertal stages. Clin Endocrinol (Oxf) 2011;74:445–52. [37] Soucek O, Lebl J, Zapletalova J, Novotna D, Plasilova I, Kolouskova S, et al. Bone geometry and volumetric bone density at the radius in patients with isolated SHOX deficiency. Exp Clin Endocrinol Diabetes 2013;121:109–14. [38] Soucek O, Zapletalova J, Zemkova D, Snajderova M, Novotna D, Hirschfeldova K, et al. Prepubertal girls with Turner syndrome and children with isolated SHOX deficiency have similar bone geometry at the radius. J Clin Endocrinol Metab 2013;98:E1241–7. [39] Stochholm K, Juul S, Juel K, Naeraa RW, Gravholt CH. Prevalence, incidence, diagnostic delay, and mortality in Turner syndrome. J Clin Endocrinol Metab 2006;91: 3897–902. [40] Sumnik Z, Matyskova J, Hlavka Z, Durdilova L, Soucek O, Zemkova D. Reference data for jumping mechanography in healthy children and adolescents aged 6–18 years. J Musculoskelet Neuronal Interact 2013;13:297–311. [41] Torres-Santiago L, Mericq V, Taboada M, Unanue N, Klein KO, Singh R, et al. Metabolic effects of oral versus transdermal 17beta-estradiol (E(2)): a randomized clinical trial in girls with Turner syndrome. J Clin Endocrinol Metab 2013;98:2716–24. [42] Varghese B, Short D, Hangartner T. Development of quantitative computedtomography-based strength indicators for the identification of low bone-strength individuals in a clinical environment. Bone 2012;50:357–63. [43] Veilleux LN, Rauch F. Reproducibility of jumping mechanography in healthy children and adults. J Musculoskelet Neuronal Interact 2010;10:256–66. [44] Zinn AR, Roeltgen D, Stefanatos G, Ramos P, Elder FF, Kushner H, et al. A Turner syndrome neurocognitive phenotype maps to Xp22.3. Behav Brain Funct 2007;3:24.
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Bone journal homepage: www.elsevier.com/locate/bone
Original Full Length Article
The muscle–bone interaction in Turner syndrome Ondrej Soucek a, Jana Matyskova a, Elmar Anliker b, Marco Toigo c, Zdenek Hlavka d, Jan Lebl a, Zdenek Sumnik a,⁎ a
Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic Clinic for Sports Medicine, Lucerne Cantonal Hospital, Lucerne, Switzerland Exercise Physiology Lab, Institute of Human Movement Sciences, ETH Zurich, Zurich, Switzerland d Department of Statistics, Faculty of Mathematics and Physics, Charles University in Prague, Prague, Czech Republic b c
a r t i c l e
i n f o
Article history: Received 28 September 2014 Revised 20 January 2015 Accepted 26 January 2015 Available online 3 February 2015 Edited by Mark Cooper Keywords: Turner syndrome Muscle force Bone strength indices Muscle–bone unit Ovarian function
a b s t r a c t Objectives: Turner syndrome (TS) is associated with an increased fracture rate due to reduced bone strength, which is mainly determined by skeletal muscle force. This study aimed to assess the muscle force–bone strength relationship in TS and to compare it with that of healthy controls. Methods: This study included 39 girls with TS and 67 healthy control girls. Maximum muscle force (Fmax) was assessed through multiple one-legged hopping with jumping mechanography. Peripheral quantitative computerized tomography assessed the bone strength index at the tibial metaphysis (BSI 4) and the polar strength– strain index at the diaphysis (SSI polar 66). The effect of TS on the muscle–bone unit was tested using multiple linear regression. Results: TS had no impact on Fmax (p = 0.14); however, a negative effect on bone strength (p b 0.001 for BSI 4 and p b 0.01 for SSI polar 66) was observed compared with healthy controls. Bone strength was lower in the TS group (by 18%, p b 0.01, for BSI 4 and by 7%, p = 0.027, for SSI polar 66), even after correcting for Fmax. Conclusions: Similar muscle force induces lower bone strength in TS compared with healthy controls, which suggests altered bone-loading sensitivity in TS. © 2015 Elsevier Inc. All rights reserved.
Introduction Turner syndrome (TS) is caused by the complete or partial loss of one X chromosome. Its incidence is 1 in 2000 live female births [39]. Patients with TS are seen primarily by endocrinologists to address their short stature, ovarian failure or autoimmune thyroiditis. However, other specialists may be involved because TS is associated with an increased risk of congenital heart and urinary system malformations and celiac disease [13]. Additionally, an increased fracture rate [15] and decreased bone mineral density (BMD) [36] have been described in TS compared to healthy controls. Several other studies describe osteoporosis in TS [4,14,23], which suggests that skeletal complications are of concern in these patients. The etiology and mechanism of bone fragility in TS have not been completely elucidated. While the results of some studies suggest inappropriate estrogen substitution [7,16,18], other studies claim that either a direct effect of the loss of the short stature homeobox-containing gene
⁎ Corresponding author at: Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, V Úvalu 84, 150 06 Praha 5. Fax: +420 22443 2097. E-mail address: [email protected] (Z. Sumnik).
http://dx.doi.org/10.1016/j.bone.2015.01.017 8756-3282/© 2015 Elsevier Inc. All rights reserved.
(SHOX) [28,37] or an indirect effect of the loss of Xp22.3, which leads to neuromotor impairment [31,44], causes bone fragility in TS. Skeletal muscle force represents a major mechanical stimulus for bone development and determines bone strength [12,33,34]. According to Frost's mechanostat theory [11], every bone should be adapted to withstand the maximal voluntary muscle forces. Ground-reaction forces have been used to calculate that during the multiple one-legged hopping test, the plantarflexor muscle force amounts to approximately 9–10.5 times the body weight [3]. This value is 2.8–3.3 times higher than the dynamometry-derived peak plantarflexor force (approx. 4.8 times the body weight) [3]. Therefore, the mechanical forces at the tibial shaft are much greater during multiple one-legged hopping compared with isokinetic dynamometry, a method that was previously used to assess muscle torque in the pediatric population [19,29]. Moreover, a strong correlation has been observed between maximum force, as assessed using ground-reaction force plate (GRFP) measurements during multiple one-legged hopping, and tibial bone strength, measured using peripheral quantitative computed tomography (pQCT) [2]. This correlation, together with its good reliability [43] and the availability of reference data [6,24,40], make GRFP a suitable method for assessing muscle function in children. We hypothesized that the muscle–bone interaction is impaired in TS patients compared with healthy controls, leading to decreased bone
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strength. Thus, the aims of the present study were 1) to assess the peak force and bone strength using GRFP and pQCT, respectively, in girls with TS and healthy controls; 2) describe the muscle–bone relationships in the participants with TS by implementing the GRFP and pQCT measures; 3) compare these relationships with those of healthy controls; and 4) test the influence of menarcheal status, course of puberty, karyotype, durations of growth hormone (GH) and estrogen (E) therapy and fracture history on muscle–bone relationships in the TS participants. Participants and methods Participants This was a cross-sectional study involving 39 girls with TS from a single university hospital referral center. Eighteen TS participants in the present study were also included in the longitudinal BMD study, and the baseline values of that study have been published previously [36]. Several patients with TS were passed on to adult endocrinologists, while new young girls with TS were also included. However, the present cross-sectional study is based on measurements of the weight-bearing tibia bone, whereas the previous study was based on radius bone measurements. Muscle force data from TS girls reported in this study were published quite recently [35]. However, the novelty of the present study is the concurrent assessment of both peak muscle force and BMD, which allows direct observation of the muscle–bone interaction. Despite the fact that the radiation dose due to pQCT measurement is negligible, only 39 girls with TS (and/or their guardians) consented to be included in the present study (i.e., to have scans at the tibia in addition to the scans at the radius) compared with 60 girls with TS who agreed to the muscle function assessment [35]. The exclusion criteria were any chronic disease (except autoimmune thyroiditis) or medication (except recombinant human growth hormone [GH] and estrogen) with a known effect on the muscles or bones. Body height was measured with a stadiometer to the nearest 1 mm. Weight was measured with an electronic scale to the nearest 100 g. Height, weight and BMI Z-scores were calculated using the most recent national reference data [21]. The girls with TS presented with Tanner stage 1 (N = 13), 2 (N = 3), 3 (N = 2), 4 (N = 6) and 5 (N = 15) breast development. There were 21 pre-menarcheal girls and 18 post-menarcheal girls. Their karyotypes were either monosomy of X (N = 13), the classical mosaic form 45,X/46,XX (N = 4), other double- or triple-line mosaics with at least two aberrant lines (N = 18) or a structural defect of one X chromosome (3 patients with a deletion and 1 patient with an isochromosome). All of the girls with TS were treated with GH at a starting dose of 50 μg/kg/d, which was then adjusted according to the clinical response [32]. The mean age at the start of GH treatment was 7.6 ± 3.9 years, and the mean duration of GH administration was 5.4 ± 3.8 years. On the day of the examination, 3 of the girls had just begun GH treatment, and another 14 girls had already stopped treatment because they had reached their final height. Estrogen replacement was given to 14 patients to initiate their puberty and to 4 patients because of secondary ovarian failure; another 9 patients had a spontaneous pubertal development. The remaining 12 patients were pre-pubertal. Oral estrogen replacement began at the mean age of 13.6 ± 1.7 years, with a starting dose of 5 μg/kg/day of 17-β-estradiol given for 12–18 months. [30]. Afterwards, a dose of 10 μg/kg/day was administered for another 12–18 months, followed by an increase to 20 μg/kg/day. After a mean period of 3 months, the same dose of estradiol was continued from days 1 to 21, with 5 mg of medroxyprogesterone per day added from day 15 to day 21 of every cycle. Thyroxine substitution was necessary in 8 patients with autoimmune thyroiditis. All of these patients had been euthyroid for at least 18 months prior to the study measurements. Altogether, there were 19 fracture events in the histories of 10 patients: 12 in the forearm, 3
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finger fractures, 2 ankle fractures, 1 wrist fracture and 1 vertebral fracture. As a control group, we selected 67 healthy girls with a wide physical activity range. The control girls were recruited from schools and different sports associations (volleyball, ice hockey, gymnastics and synchronized swimming). The exclusion criteria were any disease or medication affecting the musculoskeletal system. The muscle and the bone measures were obtained using the same methods that were used for the TS group (GRFP and pQCT) and by implementing the same technical settings and maneuvers. These data have been published in part as a subgroup of a larger study comprising 323 children and adults [2]. The present study was approved by the University Ethics Committee and conformed to the Declaration of Helsinki. All of the participants included in this study (or a parent if the patient was younger than 18 years) consented to the testing. Muscle force assessment Maximum ground reaction force (Fmax; N), a surrogate for maximum plantarflexor muscle force, was assessed using the multiple one-legged hopping (M1LH) test performed on a Leonardo Mechanograph® GRFP (Novotec Medical, Pforzheim, Germany). To detect, store and calculate the data, we used the manufacturer's software (Leonardo Mechanography GRFP version 4.2; Novotec Medical). The test consisted of repeated hopping on the forefoot of one leg with a stiff knee and free movement of the arms. The subjects were lightly dressed and wore no shoes. The subject's task was to jump as quickly as possible at the beginning of the test and then, after achieving good balance, to jump higher with every subsequent jump to obtain Fmax. The highest value for Fmax was selected among the three consecutive tests performed with the non-dominant leg. Fmax and its relation to body weight (Fmax/ BW; no unit) were recorded. Bone strength assessment An XCT 2000 scanner (Stratec Medizintechnik, Pforzheim, Germany) was used for pQCT measurements at the non-dominant tibia. The leg dominancy was determined according to handedness, so that the leg on the same side of the body as the dominant hand was considered dominant. A single tomographic 2.0-mm-thick slice was taken at distances corresponding to the 4% and 66% bone lengths, as measured from the medial malleolus to the superior margin of the medial condyle. A voxel size of 0.4 × 0.4 × 2.0 mm was used. The images were processed and the numerical values were calculated using version 6.20 C of the integrated XCT software. At the distal tibia (4% site), the total bone mineral content (BMC 4), total volumetric bone mineral density (vBMD), total bone cross-sectional area (CSA) and trabecular vBMD were measured. The compressional bone strength index (BSI 4) was calculated as the product of the square of the total vBMD and the total bone CSA, as previously described [22]. At the proximal tibia (66% site), the total BMC (BMC 66) and the polar strength–strain index (SSI polar 66) were assessed. The SSI polar 66 was determined using a segmentation threshold of 480 mg/cm3. The precision errors of the pQCT measures were not assessed at the tibia, but they have been found to be low at the radius [36]. The root mean square standard deviations gained from the 3 consecutive measurements with repositioning were as follows: at the distal radius: total BMC 0.009 g/cm, total bone CSA 9.617 mm2, total vBMD 9.353 mg/cm3 and trabecular vBMD 2.905 mg/cm3; at the proximal radius: total BMC 0.007 g/cm and SSI polar 7.685 mm3. Biochemistry Blood samples were obtained from the patients on the day of the muscle and bone assessment and were a part of their regular followup. Serum FSH levels were measured in our accredited hospital
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laboratory by chemiluminiscence immunoassay; the results are given in IU/l. The intra-individual coefficient of variation for the kit was 11%.
Table 2 Summary of the bone and muscle characteristics.
2.5. Statistical analyses The statistical computing environment R [27] was used to conduct all of the statistical analyses. The data are reported as the means (SD). The Z-scores were tested for difference from zero using the onesample t-test. We performed the two-sample t-test with the Welch approximation for the degrees of freedom for two-group comparisons of the anthropometry measures' Z-scores. The difference in the logarithmically transformed muscle force and bone strength parameters between the TS group and the healthy controls was estimated using linear regression with correction for age, weight and height. The influence of the Fmax logarithm on the bone strength parameter logarithms was investigated using linear regression with correction for TS group, age, weight and height. Using this model, we reported a change in the intercept for the TS group (assuming that the slope of the regression line was the same in both groups), i.e., the estimated difference in the bone strength parameter logarithms in the TS group compared with the healthy controls. In the second model, we also investigated the interaction between the effect of log (Fmax) and the TS group, i.e., the estimated change in the slope (of the dependency) of the bone strength parameter logarithms on log (Fmax) in the TS group. Finally, linear regression was used to investigate the effects of GH therapy duration, estrogen substitution duration, course of puberty, menarcheal status, karyotype, fracture history and serum FSH level on bone strength parameters in the TS group after correction for log (Fmax), age, weight and height. For all of the tests, the reported p-values are two-sided. Results The anthropometric characteristics of the TS group and the healthy controls are shown in Table 1. There was no difference in age, but the TS group had lower height and weight Z-scores. The raw muscle and bone data for both groups are presented in Table 2. The apparent differences in the means of some of the raw muscle and bone measures between these two groups need to be interpreted with caution because these measures are age and height dependent, and the two groups differed significantly with respect to height and weight. Linear regression analyses showed that TS had no significant effect on Fmax or Fmax/BW (Table 3). In contrast, TS had a negative effect on bone strength at the metaphysis, amounting to approximately 24%, 17% and 22% decreases for BSI 4, BMC 4 and trabecular vBMD, respectively. TS also had a weaker negative effect on the diaphyseal bone strength (an approximately 1% decrease for the SSI polar 66; Table 3). No effect was observed for BMC 66. TS had a significant effect on the muscle force–bone strength interaction. The intercepts of the linear regression between bone strength measures and Fmax in TS were decreased by approximately 18%
Mechanography Fmax (N) Fmax/BW pQCT Tibia metaphysis (4% site) BMC 4 (g/cm) BSI 4 (g2/cm4) Trabecular vBMD (mg/cm3) Tibia shaft (66% site) BMC 66 (g/cm) SSI polar 66 (mm3)
Turner syndrome (N = 39)
Healthy controls (N = 67)
1291.7 (471.7) 3.1 (0.39)
1402.8 (478.4) 3.4 (0.44)
2.2 (0.70) 0.59 (0.20) 197.8 (30.0)
2.7 (0.74) 0.81 (0.30) 247.4 (49.9)
2.5 (0.64) 1293.8 (430.4)
2.9 (0.8) 1617.9 (645.9)
Group difference (p-value) 0.25 0.006
0.002 b0.001 b0.001
0.016 0.003
Values represent mean (SD). Abbreviations: Fmax = peak muscle force, Fmax/BW = Fmax relative to body weight, BMC = bone mineral content, BSI = bone strength index, vBMD = volumetric bone mineral density, and SSI polar = polar strength–strain index. The two-sample t-test was used for between-group comparisons.
(p b 0.01) and 7% (p = 0.027) for BSI 4 and SSI polar 66, respectively, compared with healthy controls, meaning that for the same Fmax, the bone strength was lower in the TS group. The slope of the regression line was not significantly different between the participants with TS and the healthy controls, meaning that the mechanisms controlling the muscle–bone interaction were the same in both groups (Fig. 1). Menarcheal status, course of puberty, karyotype, the durations of GH and estrogen treatment and fracture history had no significant effect on the four pQCT-derived bone strength indices in the TS group. The only exception was the increased BMC 66 in girls with TS who had undergone spontaneous puberty compared with the girls with TS whose puberty was induced (Table 4). Discussion This study showed that in girls with TS, a) the pQCT-derived estimates of bone strength, particularly at the tibial bone metaphysis, were significantly reduced compared with healthy controls, despite similar maximum ground reaction force during the multiple onelegged hopping test, and b) the karyotype, therapy and fracture history did not affect muscle force–bone strength interaction. We found that the impact of TS on the compressive and torsional strength of the tibial bone (BMC 4 and SSI polar 66, respectively) was significantly negative and that the effect was more apparent at the metaphysis compared with the diaphysis. The published data on bone strength in TS are heterogeneous because of the plethora of methods that are employed to assess this parameter. While Bechtold et al. [5] found a decreased bone strength index (SSI polar) at the radial bone metaphysis and diaphysis in females with TS, our previous study showed that the SSI polar at the radial bone diaphysis in TS girls at all pubertal stages was normal or increased compared with the German
Table 1 Summary of the anthropometric characteristics.
Age (year) Height (cm) Height Z-score Weight (kg) Weight Z-score BMI (kg/m2) BMI Z-score
Turner syndrome (N = 39)
Healthy controls (N = 67)
13.6 (4.2) 142.6 (16.1) −1.8 (1.0)*** 42.2 (14.6) −0.7 (1.1)*** 19.9 (3.4) 0.3 (0.9)*
12.7 (3.4) 148.8 (16.3) −0.6 (1.3)*** 42.4 (13.2) −0.2 (1.0) 18.6 (2.5) 0.1 (0.8)
Group difference (p-value) 0.31 b0.001 0.013 0.15
Abbreviation: BMI = body mass index. The Z-scores were tested for difference from zero using the one-sample t-test. *p b 0.05 ***p b 0.001. The two-sample t-test was used for betweengroup comparisons.
O. Soucek et al. / Bone 74 (2015) 160–165 Table 3 The influence of TS on muscle force and bone strength parameters compared with the parameters for healthy controls. Estimated difference of logarithms (mean [SD]) Mechanography Fmax (N) Fmax/BW pQCT Tibial metaphysis (4% site) BMC 4 (g/cm) BSI 4 (g2/cm4) Trabecular vBMD (mg/cm3) Tibial shaft (66% site) BMC 66 (g/cm) SSI polar 66 (mm3)
p-value
−0.051 (0.034) −0.038 (0.032)
0.14 0.24
−0.17 (0.047) −0.24 (0.07) −0.22 (0.045)
b0.001 b0.001 b0.001
−0.039 (0.027) −0.096 (0.035)
0.15 b0.01
Abbreviations: Fmax = peak muscle force, Fmax/BW = Fmax relative to body weight, BMC = bone mineral content, BSI = bone strength index, vBMD = volumetric bone mineral density, and SSI polar = polar strength–strain index. The influence of TS was tested using linear regression models with corrections for age, height and weight.
reference data [36]. The contradictory results of these two studies may be explained by the different comparison methods used: whereas agespecific Z-scores were calculated in the former study, height-specific Z-scores were calculated in the latter. The more appropriate comparison method cannot be unequivocally determined because of the specific growth and pubertal development patterns of patients with TS, which are characterized by short stature, increased body mass index and delayed (mostly induced) puberty. In the present study, we decided to test the impact of TS on bone strength indices by comparing patients with TS with healthy control group using anthropometric measures (age, height and weight) as the covariates in the multiple linear regression models to avoid inadequate matching. In addition, in contrast to previous studies, we selected the load-bearing skeletal site, i.e., the tibia, for bone strength examination.
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In accordance with our observations, a recent high-resolution-pQCT study [17] showed that the bone failure load estimates (obtained by finite element analysis) were 11% and 16% lower in TS patients at the radius and tibia, respectively, compared with healthy controls. The question may arise whether the bone sites we used for the bone strength assessments were the most appropriate. The 4% site was selected based on previous findings of significantly reduced bone mineral content, density and strength at this site in the radius among TS patients [5]. In addition, trabecular vBMD (assessed at the 4% site) has been found to be associated with prevalent fractures in young healthy girls [10]. According to a study on cadaver bones, the tibia's highest propensities to fracture were at the 50% and 85% sites for the 3-point and cantilever bending tests, respectively [42]; thus, choosing the 65% site may be relevant for both sites while decreasing the radiation dose by foregoing the additional scan. Moreover, the association between the maximum muscle force and tibia bone strength at the 65% site was very strong (R2 = 0.833) and was comparable to other sites along the tibia [2]. Therefore, the selected tibia measurement sites appear to be clinically relevant. Importantly, in addition to bone strength, we examined the bone load by assessing Fmax using the M1LH test on the GRFP. TS had no effect on Fmax compared with the control group. This corresponds to the normal Fmax that has been observed in patients with TS [35] when the Z-scores are calculated from reference data comprising 432 healthy females [40]. The findings on grip force tested with hand dynamometry are inconsistent in TS. Both decreased [8] and increased values [25] have been reported, with height being the main determinant of the grip force in TS. Nevertheless, the maximal voluntary forces (those that cause the maximal bone deformation) are detected during lengthening and not isometric muscle actions [3]. Therefore, our results might be more relevant than the results of isokinetic dynamometry studies for the functional evaluation of the muscle–bone unit in TS. If the maximum muscle force is normal but the bone strength is reduced in girls with TS, what are the potential mechanisms? One
Fig. 1. Abbreviations: BMC = bone mineral content, BMD = bone mineral density, BSI = bone strength index, SSI = strength–strain index. The 95% confidence intervals are illustrated, representing the changes in the intercept (left) and the slope (right) of the regression lines for the logarithms of the bone strength surrogates at the tibia in the TS group compared with the healthy controls. The covariates were log (Fmax), age, height and weight.
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Table 4 The influence of selected TS-specific characteristics on tibial bone strength measures in girls with TS.
GH therapy duration (years) E substitution duration (years) Spontaneous puberty (vs. induced) Post-menarche (vs. pre-menarche) 45,X/46,XX karyotype (vs. 45,X karyotype) Positive fracture history (vs. no history) FSH serum level (IU/l)
log BMC 4
p-Value
log BSI 4
p-Value
log BMC 66
p-Value
log SSI polar 66
p-Value
−0.001 (0.008) −0.018 (0.016) 0.179 (0.091) 0.001 (0.071) −0.109 (0.111) −0.002 (0.053) −0.002 (0.002)
0.86 0.28 0.066 0.99 0.35 0.97 0.27
−0.002 (0.011) −0.008 (0.024) 0.236 (0.136) 0.066 (0.102) −0.089 (0.217) −0.090 (0.075) −0.003 (0.002)
0.82 0.74 0.10 0.52 0.69 0.24 0.25
−0.002 (0.006) −0.010 (0.014) 0.169 (0.050) 0.058 (0.059) −0.034 (0.114) 0.019 (0.044) 0.000 (0.002)
0.70 0.46 0.0037 0.33 0.77 0.67 0.80
−0.003 (0.006) −0.010 (0.014) 0.046 (0.068) −0.003 (0.061) 0.020 (0.094) −0.058 (0.044) 0.001 (0.002)
0.65 0.49 0.51 0.96 0.83 0.20 0.65
Abbreviations: GH = growth hormone, E = estrogen, BMC = bone mineral content, BSI = bone strength index, SSI = strength–strain index, and FSH = follicle-stimulating hormone. Beta coefficients (standard errors) are shown. In multivariate linear regression models, Fmax, age, height and weight were used as covariates.
possible explanation is suggested by a study in which the surrogates of bone strength were decreased in oligoamenorrheic athletes compared with eumenorrheic athletes [1]. Additionally, patients with hypogonadism have been found to have decreased trabecular BMD [26]. Both studies point towards estrogen insufficiency. In contrast, we detected no significant impact of estrogen substitution duration (β = 0.0035 ± 0.016, p = 0.83), spontaneous puberty (β = 0.047 ± 0.095, p = 0.63) or serum FSH levels (β = −0.002 ± 0.002, p = 0.63) on the relationship between Fmax and the trabecular BMD in the TS group. We also found no significant effect of estrogen status on BSI 4 or SSI polar 66 in girls with TS. In young adult women with TS, transdermal estradiol replacement seemed to induce more physiological pharmacokinetics than oral pills [41], and subcutaneous estrogen implants given every 6 months for three years lead to significant and sustained increases in serum estradiol, BMD Z-score and trabecular bone volume [20]. We may thus speculate that there are generally insufficient amounts of estradiol available to the bone tissue in TS. However, one limitation of this study is that we did not randomize our participants with TS to receive different means of estradiol administration, and we lacked data on serum estradiol and/or FSH in the healthy controls. The relatively low number of TS participants and their limited age span could also have caused our non-significant observations. We were therefore unable to explore whether estradiol bioavailability impacts the muscle–bone interaction in TS. It has been shown that immobility decreases trabecular vBMD by 73% in the tibia bones of patients after spinal cord injury [9], a consequence of the loss of muscle mass and activity. In contrast, long-term tennis players have been shown to gain 6% more trabecular bone in the playing arm compared with the contralateral arm [22]. These two reports clearly show the existing relationship between muscle activity and trabecular bone density. The present study found that despite having similar Fmax values, the TS group had lower bone strength (BMC4 and BSI 4) and trabecular vBMD compared with healthy controls. Despite being appropriately substituted with oral 17-β-estradiol, our patients with TS may not have been adequately treated relative to physiological estrogen activity in healthy controls. Therefore, one cannot rule out the possibility that estrogen “inadequacy” increases the threshold for disuse-driven endocortical bone resorption, the result of which is low trabecular vBMD (and thus decreased bone strength). The second possible mechanism of decreased bone strength in TS may be based on SHOX deficiency, which is a common feature in patients with TS [28]. Patients with isolated SHOX deficiency share a similar radial bone geometry phenotype with girls with TS [38]. Unfortunately, we have no data on the muscle function in the patients with isolated SHOX deficiency, and we could not test for possible differences in the muscle force–bone strength relationship between these patients and the participants with TS. Another limitation of our study is the lack of data regarding dietary calcium intake, serum vitamin D levels and physical activity in the study participants. This lack of data prevented us from testing the possible influences of these measures on bone strength parameters. Because of the cross-sectional design of this study and the relatively low number of participants with TS in certain subgroups (9 with
spontaneous puberty vs. 18 with estrogen substitution; 13 with X monosomy vs. 4 with the 45,X/46,XX karyotype), the non-significant results regarding the influence of hypogonadism or karyotype on bone strength cannot not be considered definitive. Conclusions Bone strength in girls with TS was significantly reduced despite normal maximum muscle force in terms of absolute and relative force. These results point to a primary bone defect in TS. The mechanisms leading to the abnormal bone load–bone strength relationship in TS remain to be elucidated. Acknowledgments We would like to thank Marta Snajderova, Stanislava Kolouskova and Stepanka Pruhova for their cooperation in recruiting their patients with TS. We acknowledge all of the study participants and their parents for their kind consent with the study protocol. The paper was partially supported by the Czech Ministry of Health's Project for Conceptual Development of Research Organization no. 00064203 (University Hospital Motol, Prague, Czech Republic). References [1] Ackerman KE, Pierce L, Guereca G, Slattery M, Lee H, Goldstein M, et al. Hip structural analysis in adolescent and young adult oligoamenorrheic and eumenorrheic athletes and nonathletes. J Clin Endocrinol Metab 2013;98:1742–9. [2] Anliker E, Rawer R, Boutellier U, Toigo M. Maximum ground reaction force in relation to tibial bone mass in children and adults. Med Sci Sports Exerc 2011;43: 2102–9. [3] Anliker E, Toigo M. Functional assessment of the muscle–bone unit in the lower leg. J Musculoskelet Neuronal Interact 2012;12:46–55. [4] Bakalov VK, Chen ML, Baron J, Hanton LB, Reynolds JC, Stratakis CA, et al. Bone mineral density and fractures in Turner syndrome. Am J Med 2003;115:259–64. [5] Bechtold S, Rauch F, Noelle V, Donhauser S, Neu CM, Schoenau E, et al. Musculoskeletal analyses of the forearm in young women with Turner syndrome: a study using peripheral quantitative computed tomography. J Clin Endocrinol Metab 2001;86:5819–23. [6] Busche P, Rawer R, Rakhimi N, Lang I, Martin DD. Mechanography in childhood: references for force and power in counter movement jumps and chair rising tests. J Musculoskelet Neuronal Interact 2013;13:213–26. [7] Carrascosa A, Gussinye M, Terradas P, Yeste D, Audi L, Vicens-Calvet E. Spontaneous, but not induced, puberty permits adequate bone mass acquisition in adolescent Turner syndrome patients. J Bone Miner Res 2000;15:2005–10. [8] Clark C, Klonoff H, Hayden M. Regional cerebral glucose metabolism in Turner syndrome. Can J Neurol Sci 1990;17:140–4. [9] Eser P, Frotzler A, Zehnder Y, Wick L, Knecht H, Denoth J, et al. Relationship between the duration of paralysis and bone structure: a pQCT study of spinal cord injured individuals. Bone 2004;34:869–80. [10] Farr JN, Tomas R, Chen Z, Lisse JR, Lohman TG, Going SB. Lower trabecular volumetric BMD at metaphyseal regions of weight-bearing bones is associated with prior fracture in young girls. J Bone Miner Res 2011;26:380–7. [11] Frost HM. The mechanostat: a proposed pathogenic mechanism of osteoporoses and the bone mass effects of mechanical and nonmechanical agents. Bone Miner 1987;2: 73–85. [12] Frost HM. Muscle, bone, and the Utah paradigm: a 1999 overview. Med Sci Sports Exerc 2000;32:911–7. [13] Gravholt CH, Juul S, Naeraa RW, Hansen J. Morbidity in Turner syndrome. J Clin Epidemiol 1998;51:147–58. [14] Gravholt CH, Lauridsen AL, Brixen K, Mosekilde L, Heickendorff L, Christiansen JS. Marked disproportionality in bone size and mineral, and distinct abnormalities in
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