Factors associated with low bone mineral density in Turner syndrome: a multicenter prospective observational study

Abstract

Turner syndrome (TS) is associated with a high risk of fracture due to low bone mineral density (BMD). While hypogonadism is known to play a role in decreasing BMD, other factors have not been studied well. Focusing on diet, exercise, and bone metabolism markers, the present, multicentric, prospective, observational study aimed to identify factors contributing to decreased BMD in TS. In total, 48 patients with TS aged between 5 and 49 years comprising a pre-pubertal group (n = 9), a cyclical menstruation group (n = 6), and a hormone replacement therapy (HRT) group (n = 33) were enrolled. The cyclical menstruation group and the HRT group were referred to collectively as the post-pubertal group. The bone mineral apparent density (BMAD) Z-score was higher in the pre-pubertal group than in the post-pubertal group (–0.3 SD vs. –1.8 SD; p = 0.014). Within the post-pubertal group, the median BMAD Z-score was –0.2 SD in the cyclical menstruation group and –2.3 SD in the HRT group (p = 0.016). Spearman’s rank correlation revealed no correlation between the BMAD Z-score and bone metabolism markers. No significant relationship was observed between the BMAD Z-score and either the vitamin D sufficiency rate or the step sufficiency rate. A negative correlation was found between BMAD Z-score and serum sclerostin in the pre-pubertal group and serum FSH in the post-pubertal group. In conclusion, the present study found no relationship between the vertebral BMAD Z-score and diet or exercise habits in TS, indicating that estrogen deficiency is the chief reason for low BMD in TS.

TURNER SYNDROME (TS) is a congenital disorder caused by partial or complete loss of one X-chromosome and has a prevalence of approximately one in 2,000 live born females [1]. TS has various presentations, including short stature, hypogonadism, cardiac malformation, and fractures [1, 2]. Among these, fractures have a prevalence as high as 30.5–32.2% and therefore constitute a significant problem [3-5].

Low bone mineral density (BMD) is one of the factors crucially responsible for increasing the fracture risk in TS [3, 6] although low bone quality, a high risk of falling, X chromosomal abnormalities, and comorbidities are also associated with the fracture risk [7-9]. Previous studies have reported that vertebral areal BMD (aBMD), the most common method of measuring BMD, is lower in patients with TS than in healthy women [10]. Studies using QCT, which directly measures vertebral volumetric BMD (vBMD), have reported similar results [11, 12]. Bone mineral apparent density (BMAD), which is helpful when measuring BMD in children and patients with short stature, is reportedly associated with upper limb and vertebral fractures [13, 14] .

Hypogonadism is the most potent factor leading to low BMD in TS [3, 15]. Evidence supporting the importance of estrogen for BMD in TS is as follows: first, TS with a regular menstrual cycle led to higher BMD acquisition than TS with primary hypogonadism [16-19]; and second, early initiation of estrogen replacement therapy (ERT) resulted in higher BMD in TS [20, 21]. Numerous guidelines and reviews recommend starting ERT between 11 and 12 years of age in patients with TS with hypogonadism [1, 22]. While ERT cannot improve the BMD of patients with TS to a level comparable to that of healthy individuals [10], the outcomes for bone health in TS are likely to improve if ERT is initiated during the pre-pubertal period [23]. Indeed, the vBMD decreases with age during the pre-pubertal period in TS while it increases with age in healthy, pre-pubertal individuals [24].

Although previous reports have highlighted the significance of the relationship of estrogen deficiency to low BMD in TS, only a few reports have examined other factors, such as exercise and diet, which are known to be important for maintaining healthy BMD in the general population [25-27].

Focusing on diet, exercise, and bone metabolism markers, the present study aimed to identify factors contributing to decreased BMD in TS and to investigate the relevance of puberty to BMD using BMAD, an accurate method of evaluating BMD [13, 14].

Method

Study design and participants

The present, multicentric, prospective, observational study enrolled patients with TS aged between 5 and 49 years who visited Tokyo Metropolitan Children’s Medical Center, Yokohama City University Medical Center or Osaka Women’s and Children’s Hospital. The patients were divided into a pre-pubertal group, cyclical menstruation group, and hormone replacement therapy (HRT) group comprising patients aged between 5 and 14 years with no breast development, those with cyclical menstruation without medication, and those with cyclical menstruation with more than two years of estrogen and progesterone therapy (Kaufmann therapy), respectively (Fig. 1). The cyclical menstruation group and the HRT group were referred to collectively as the post-pubertal group (Fig. 1).

Fig. 1

Classification of the study population

The pre-pubertal group and post-pubertal group included patients with TS aged between 5 and 14 years with no breast development and those aged 15 years and older with cyclical menstruation, respectively. The post-pubertal group was further subdivided into a cyclical menstruation group and HRT group. The former comprised TS patients with cyclical menstruation without medication, and the latter comprised TS patients with cyclical menstruation with more than two years of cyclical estrogen and progesterone therapy (Kaufmann therapy).

Protocol and data collection

Blood and urine tests, BMD measurement, nutritional surveys, and step count surveys were performed. Serum sclerostin was measured using ELISA (LSI Medience Corporation, Tokyo, Japan), and vertebral bone mineral content and bone area were measured using dual-energy X-ray absorptiometry (DXA) (HOLOGIC, Toyo Medic Corporation, Tokyo, Japan). The vertebral bone mineral apparent density (BMAD) value and BMAD Z-score were calculated as previously reported [28]. The calcium (Ca), phosphate (P), and vitamin D content of the subjects’ diet were assessed by a nutritionist over three days during the first two weeks of the study. The vitamin D sufficiency rate was calculated by dividing each patient’s vitamin D intake by the required intake for each age group of Japanese children [29] and multiplying the quotient by 100. The number of steps over seven days was measured using a pedometer (HJ-325, OMRON Corporation, Kyoto, Japan) during the same period as the dietary analysis, and the average number of steps taken was calculated. The step sufficiency rate, defined as [the number of steps / the average number of steps*100], was calculated by using the average number of steps for each age group in Japan [30].

Statistical analysis

The background characteristics of the patients were analyzed using the median and interquartile range (IQR) for continuous variables and a frequency distribution and percentage for categorical variables. The Mann-Whitney U test was performed to compare the BMAD Z-score among the groups. Spearman’s rank correlation was used to analyze the relationship between the BMAD Z-score and continuous variables, such as bone metabolism markers, hormones, dietary habits, exercise habits, and duration of growth hormone therapy. Multiple linear regression was performed to examine the relationship between the BMAD Z-score and factors of potential relevance. Two-tailed p < 0.05 was considered to indicate statistical significance. All statistical analyses were performed using SPSS version 27 (IBM Corp., Armonk, NY, US).

Ethical considerations

The present study was conducted in accordance with the ethical standards laid down in the Declaration of Helsinki, the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines, and the Ethical Guidelines for Medical and Biological Research Involving Human Subjects of the Ministry of Health, Labour and Welfare of Japan. This study was approved by the ethics board of Tokyo Metropolitan Children’s Medical Center (ID: 2019b-33), which waived the requirement for written consent. Verbal consent was obtained for the research.

Results

Nine, six, and 33 patients comprising the pre-pubertal, cyclical menstruation, and HRT group, respectively, were included (Fig. 1). The median (IQR) age was 9.8 (8.1–11.0) years, 19.5 (17.5–29.2) years, and 23.6 (20.6–38.0) years for the respective group (Table 1).

Table 1

Patient characteristics

	Prepubertal group (n = 9)	Post-pubertal group (n = 39)
		Cyclical menstruation group (n = 6)	HRT group (n = 33)
Age (y), median, IQR	9.8 (8.1–11.0)	19.5 (17.5–29.2)	23.6 (20.6–38.0)
Karyotype, n (%)
45,X	1 (11)	2 (33)	11 (33)
45,X/46,XX	1 (11)	2 (33)	2 (6)
45.X/47,XXX	1 (11)	1 (17)	0
Other Mosaic	5 (56)	0	8 (24)
Isochromosome	0	0	1 (3)
Deletion	0	0	1 (3)
X ring chromosome	0	0	1 (3)
No information available	1 (11)	1 (17)	9 (27)
Height (cm), median, IQR	120.7 (108.1–131.5)	150.3 (141.3–154.3)	150.3 (145.0–152.0)
Height SD, median, IQR	–1.9 (–2.5, –1.8)	–1.5 (–3.2, –0.8)	–1.5 (–2.5, –1.2)
History of bone fractures, n (%)	1 (11)	0	4 (12)
Age at start of estrogen therapy (y), median, IQR	—	—	14.5 (13.0–16.0)
Age at start of estrogen and progesterone therapy (y), median, IQR	—	—	16.5 (16.0–18.0)
Medical history of GH therapy, n (%)	9 (100)	4 (67)	25 (76)

Abbreviations: HRT, hormone replacement therapy; IQR, interquartile range; GH, growth hormone

The BMAD Z-score was significantly higher in the pre-pubertal group than the post-pubertal group (cyclical menstruation group plus HRT group) (–0.3 SD vs. –1.8 SD; p = 0.014) (Fig. 2). In the post-pubertal cohort, the median (IQR) BMAD Z-score was –0.2 (–1.3, 0.8) in the cyclical menstruation group and –2.3 (–2.7, –0.8) in the HRT group (p = 0.016) (Fig. 2).

Fig. 2

Comparison of the BMAD Z-score among groups

The median BMAD Z-score was significantly higher in the pre-pubertal group than in the post-pubertal group at –0.3 SD and –1.8 SD, respectively (p = 0.014). Within the post-pubertal group, the median BMAD Z-score was higher in the cyclical menstruation group (–0.2 SD) than in the HRT group (–2.3 SD) (p = 0.016).

Spearman’s rank correlation revealed no significant relationship between the BMAD Z-score and bone metabolism markers (Table 2). Moreover, no significant relationship was observed between the BMAD Z-score and either the vitamin D sufficiency rate or the step sufficiency rate (Table 2, Fig. 3). Spearman’s rank correlation revealed a negative correlation between the BMAD Z-score and serum sclerostin in the pre-pubertal group (Table 2, Fig. 4). Spearman’s rank correlation coefficient between the BMAD Z-score and the serum FSH value in the post-pubertal group was high at –0.392 (p = 0.014) (Table 2).

Table 2

Correlation coefficients in the BMAD Z-score in Turner syndrome

	Pre-pubertal group			Post-pubertal group
	Value (n = 9)	BMAD Z-score		Value (n = 39)	BMAD Z-score
		r	p value‡		r	p value‡
Bone formation markers
ALP (U/L)	225.0 (213.9–312.3)†	–0.083	0.831	90.0 (71.8–120.0)†	–0.156	0.344
ALP percent§ (%)	48.7 (43.7–64.8)†	–0.330	0.932	66.5 (53.8–90.8)†	–0.223	0.172
BAP (μg/mL)	60.0 (50.0–93.8)†	–0.200	0.606	15.9 (13.4–20.0)†	–0.207	0.233
P1NP (ng/mL)	469.0 (367.0–986.0)†	0.143	0.760	57.8 (49.2–76.2)†	–0.319	0.051
Bone resorption markers
NTx (nmol BCE/L)	74.0 (48.4–80.0)†	0.424	0.256	16.6 (13.6–24.3)†	–0.117	0.477
TRACP-5b (mU/dL)	1,500 (1,500–1,075)†	–0.044	0.911	250.0 (187.0–315.0)†	–0.232	0.179
Other bone metabolism markers
Sclerostin (pg/mL)	101.0 (90.1–142.0)†	–0.733	0.025	53.5 (32.4–82.0)†	0.219	0.193
Hormones
iPTH (pg/mL)	33.6 (31.5–38.1)†	0.233	0.546	46.3 (35.0–58.3)†	0.069	0.678
25-hydroxyvitamine D (ng/mL)	19.7 (14.9–21.8)†	0.050	0.898	13.3 (10.4–17.2)†	0.050	0.782
FSH (mIU/mL)	17.2 (5.3–58.3)†	0.233	0.546	34.8 (13.8–58.5)†	–0.392	0.014
Diet
Ca sufficiency rate (%)	138.3 (90.0–148.3)†	–0.214	0.610	75.7 (58.5–102.6)†	0.203	0.221
P sufficiency rate (%)	220.5 (171.7–310.8)†	0.405	0.320	102.1 (71.6–113.9)†	0.243	0.141
Vitamin D sufficiency rate (%)	57.5 (33.7–156.2)†	0.119	0.779	61.0 (30.8–113.4)†	0.164	0.326
Exercise habits
Number of steps per day (1,000/unit)	8.3 (7.9–8.7)†	–0.133	0.732	6.6 (5.0–10.0)†	0.270	0.116
Step sufficiency rate (%)	79.8 (76.0–94.7)†	–0.367	0.332	95.3 (67.5–143.8)†	0.254	0.141
Therapy
GH therapy duration, y	3.2 (1.0–4.6)	0.300	0.433	8.3 (5.0–10.8)	0.067	0.736

Abbreviations: ALP, Alkaline phosphatase; BAP, bone alkaline phosphatase; P1NP, procollagen type 1 N-terminal-propeptide; NTx, N-telopeptide of type I collagen; TRACP-5b, tartrate-resistant acid phosphate 5b; iPTH, intact parathyroid hormone; FSH, follicle-stimulating hormone; GH, growth hormone; BMAD, bone mineral apparent density

^† Values are expressed as the median and interquartile range.

^‡ p < .05 was considered to indicate statistical significance.

^§ ALP percent = ALP value of patient / upper limit of the normal value of ALP * 100

Fig. 3

Correlation between BMAD Z-score and diet and exercise habits in patients with TS

No significant relationship was observed between the BMAD Z-score and either the vitamin D sufficiency rate or step sufficiency rate.

Fig. 4

Correlation between BMAD Z-score and serum sclerostin in patients with TS

Spearman’s rank correlation revealed a negative correlation between the BMAD Z-score and serum sclerostin in the pre-pubertal group (r = –0.733; p = 0.025). However, no significant relationship was observed between these in the post-pubertal group (r = 0.219; p = 0.193).

Multiple regression analysis performed to identify factors associated with the BMAD Z-score demonstrated that the pre-pubertal period contributed to increasing the BMAD Z-score in TS, but that the step sufficiency rate and the vitamin D sufficiency rate were not associated with the BMAD Z-score (Table 3).

Table 3

Multiple regression analysis of factors associated with the BMAD Z-score

Independent variable	B	95% CI		p value‡
		Lower	Upper
Post-pubertal group†	–1.359	–2.425	–0.293	0.014
Step sufficiency rate	0.004	–0.006	0.014	0.475
Vitamin D sufficiency rate	–0.001	–0.008	0.006	0.827

Abbreviations: B, unstandardized regression coefficient; 95% CI, 95% confidence interval

^† The independent variable was the subjects’ classification into either the pre-pubertal group or post-pubertal group.

^‡ p < .05 was considered to indicate statistical significance.

Discussion

The present, multicentric, prospective, observational study found no relationship between the vertebral BMAD Z-score and either diet or exercise habits in patients with TS. The diet was evaluated by a nutritionist, and the exercise habits were measured using a pedometer.

In contrast to our findings, one previous study found that physical fitness, measured by the maximal oxygen uptake during the use of a bicycle ergometer, was a significant determinant of aBMD in TS [31]. Regarding diet, no studies have reported any relationship between vitamin D intake and BMD in TS although low serum vitamin D is known to be associated with low BMD [32, 33]. The failure of the present study to assess long-term habits, as discussed below, may account for the lack of significant findings since BMD can change over time. To clarify the relationship between BMD and diet and exercise habits in TS, future studies may consider evaluating the impact of patients’ long-term habits on their bone health.

In the present study, the vertebral BMAD Z-score was higher in the pre-pubertal group than in the post-pubertal group. This result does not indicate that the BMAD value was greater in the pre-pubertal group than in the post-pubertal group but rather that the difference between the BMAD of patients with TS and that of healthy individuals was smaller in the pre-pubertal period than in the post-pubertal period. Although age-based trends in BMD values in TS have been reported, no studies have, as of yet, compared the BMAD Z-score in pre-pubertal and post-pubertal individuals. In the present study, the differences in the BMAD Z-scores appeared during these periods; in the pre-pubertal period, the lower BMD value of the patients with TS contributed to this difference. Nanao et al. demonstrated that vertebral vBMD measured by QCT in patients with TS aged 4–12.9 years decreased with age [11] in line with the findings of other studies [34].

In the post-pubertal period, estrogen deficiency accounts for the significantly greater difference in the BMD value between patients with TS and their healthy counterparts. Indeed, our data demonstrated that the vertebral BMAD Z-score was higher in the cyclical menstruation group than the HRT group. Numerous studies have demonstrated similar findings [16-19], indicating the importance of estrogen for maintaining BMD in TS. These findings also suggested that the current HRT regimen is insufficient to increase BMD. The estrogen dosage, timing of ERT initiation, and administration route may be further refined to improve outcomes. Considering the BMD decline during early childhood in TS [11, 31] and the small amounts of estrogen secreted during the pre-pubertal period in healthy individuals [35], earlier initiation of transdermal 17β estradiol may help increase BMD [23].

The present study revealed no significant relationship between the BMAD Z-score and bone metabolism markers in either the pre-pubertal or the post-pubertal group. A previous study reported a negative correlation of vertebral vBMD with bone formation and resorption markers, such as alkaline phosphatase (ALP), bone alkaline phosphatase (BAP), procollagen type 1 N-terminal-propeptide (P1NP), and urine N-telopeptide of type I collagen (NTx) [36]. However, this correlation may not be entirely accurate due to the inclusion of children and adults in the study, as generally, the vBMD is lower and the markers are higher during childhood [37, 38]. The present study analyzed data on the pre-pubertal and the post-pubertal groups separately and used the BMAD Z-score instead of BMAD values as a measure of BMD. This approach enabled a more precise assessment of the association between BMD and bone metabolism markers in TS.

The present study also found a significant relationship between the BMAD Z-score and FSH in the post-pubertal group. The correlation between the BMAD Z-score and FSH is believed to stem from two mechanisms (Fig. 5). First, estrogen deficiency affects both FSH and bone resorption; it stimulates FSH production via negative feedback [39] while regulating bone resorption by modulating RANKL expression [40], leading to activated bone resorption. Second, FSH directly stimulates bone resorption. Sun et al. demonstrated that neither FSHβ nor FSH receptor-null mice had bone loss despite severe hypogonadism [41]. Hypergonadotropic, amenorrheic women with high FSH had lower BMD than hypogonadotropic, eumenorrheic women, suggesting that FSH had a direct effect on bone resorption [42].

Fig. 5

Relationship between FSH, E₂, and BMAD Z-score

Previous studies have reported a negative and positive impact of E₂ on FSH and the BMAD Z-score, respectively, as well as a negative impact of FSH on the BMAD Z-score. The present study also found a negative correlation between the BMAD Z-score and FSH.

The present study found a negative correlation between the vertebral BMAD Z-score and serum sclerostin, an inhibitor of the Wnt/beta-catenin pathway, in the pre-pubertal group. However, no correlation was observed between these factors in the post-pubertal group. A previous study including both pediatric and adult patients with TS reported a negative correlation between the lumbar BMAD Z-score and serum sclerostin [43] in line with our findings in the pre-pubertal group. The study also demonstrated that the serum sclerostin level was higher in TS patients than in controls [43]. Furthermore, another study indicated that estrogen therapy in postmenopausal women resulted in reduced serum sclerostin [44]. It is possible that earlier initiation of ERT decreases serum sclerostin, potentially preventing the loss of BMD in the pre-pubertal period.

The present study has some limitations. First, the number of cases, especially in the pre-pubertal period, was small. Second, the assessment period for diet and exercise habits was not long enough. Diet was assessed for only three days, and the number of steps was measured for only seven days. A longer assessment period would have allowed a more accurate evaluation. Third, bone metabolism markers in the pre-pubertal period were unable to be adjusted age-specifically due to the lack of previous data. However, the serum sclerostin value is independent of age and therefore does not require adjustment [45].

In conclusion, the present study failed to find any significant relationship between the vertebral BMAD Z-score and either diet or exercise habits in TS. Estrogen deficiency is a potent factor contributing to low BMD in TS. Further research with a larger cohort and longer or multiple analyses of diet and exercise habits is needed to gain a more comprehensive understanding of bone health in TS.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

K.I., T.I., and Y.H. designed the study. K.I., E.K., and M.K. collected the samples and historical data. K.I. conducted the statistical analyses. H.S., M.K., and Y.H. contributed to the interpretation of the results. K.I. wrote the first draft of the manuscript. Y.H. reviewed the manuscript.

Funding

This study was funded by The Japanese Society for Pediatric Endocrinology Future Development Grant supported by Novo Nordisk Pharma, Ltd.

Acknowledgments

We would like to thank Shiori Yumino for her valuable contribution to the dietary assessment in this study. We are also indebted to Mr. James R. Valera for his assistance with editing this manuscript.

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