Abstract
Objective
In children with Prader–Willi syndrome (PWS), growth hormone (GH) treatment has positive effects on bone mineral density (BMD). Two 1-year studies did not show a difference between GH or placebo on BMD in young adults with PWS. However, there are no studies investigating BMD during longer-term GH treatment in young adults with PWS.
Design
Open-label, a prospective study in 43 young adults with PWS.
Methods
BMD of the total body (BMDTBSDS) and lumbar spine (BMADLSSDS) measured by DXA.
Results
In the total group, estimated mean (95% CI) of BMDTB remained similar during 3 years of GH, being −0.76 (−1.11 to −0.41) SDS at start and −0.90 (−1.27 to −0.54) SDS after 3 years (P = 0.11), as did BMADLS, being −0.36 (−0.72 to 0.01) SDS and −0.46 (−0.77 to −0.16) SDS, respectively (P = 0.16). In men, there was a significant decrease in BMDTBSDS during 3 years of GH, while BMADLSSDS remained similar. In women, both BMDTBSDS and BMADLSSDS remained similar. BMDTBSDS was associated with female sex, lean body mass and age. The majority of patients received sex steroid replacement therapy (SSRT).
Conclusions
During 3 years of combined GH and SSRT treatment, BMD remained stable in the normal range in young adults with PWS. However, men showed a decline in BMDTBSDS, probably due to insufficient SSRT. We recommended to continue GH treatment in young adults with PWS and to start SSRT during adolescence unless puberty progresses normally.
Introduction
Prader–Willi syndrome (PWS) is a rare multisystem genetic disorder caused by the lack of expression of paternally inherited imprinted genes on chromosome 15q11–q13, due to a paternal deletion, maternal uniparental disomy (mUPD), imprinting center defect (ICD) or translocation (1, 2). PWS is characterized by muscular hypotonia, short stature, abnormal body composition, developmental delay, behavioral problems and hyperphagia which can result in severe obesity when uncontrolled (2, 3, 4). Many symptoms in PWS may be explained by hypothalamic dysfunction, with endocrinopathies like growth hormone deficiency, hypothyroidism and hypogonadism (5).
Growth hormone (GH) has many beneficial effects in children with PWS (6, 7, 8, 9, 10, 11, 12). Studies have shown that adults with PWS also benefit from GH treatment. GH is able to improve the body composition in previously untreated patients (13, 14, 15). Furthermore, our 3-year study has shown that GH is able to maintain a stable body composition without safety concerns in young adults who continue GH treatment and had been treated with GH during childhood (16, 17). However, GH is currently not a registered treatment for adults with PWS.
Bone mineral density (BMD) is influenced by many factors, including endocrine factors like GH, insulin-like growth factor (IGF-I) and sex steroids. Other factors like body composition, especially lean body mass (LBM) and body weight also influence BMD (18). Adults with PWS have an increased prevalence of osteopenia and osteoporosis and an increased fracture risk (19, 20, 21, 22). Studies in adults with PWS, of which the majority did not receive GH or sex steroid replacement therapy (SSRT), report a history of at least one fracture in almost half of the study population (20, 23, 24). Both GH and sex steroids play an important role in the accrual of peak bone mass (25, 26, 27, 28, 29). Decreased peak BMD is a major determinant for osteoporosis and fracture risk later in life (28). In young adults with childhood-onset growth hormone deficiency (GHD), the continuation of GH after AH attainment was associated with a significantly greater increase in BMD compared to when GH was discontinued (30).
In prepubertal children with PWS, GH has positive effects on BMD (31, 32, 33). However, BMD declined in adolescents with PWS in parallel to incomplete pubertal development and low sex hormone levels (32). Two studies did not show a difference between 1 year of GH and 1 year of placebo on BMD (34, 35). However, 1 year might be too short to find significant differences (30, 36). There are no studies investigating BMD during longer-term GH treatment in young adults with PWS who received GH during childhood.
We, therefore, investigated BMD during 3 years of continuous GH treatment after AH attainment in young adults with PWS who had been treated with GH during childhood. We hypothesized that BMD would remain stable during 3 years of GH treatment. Furthermore, associations of LBM or FM with BMD SDS were investigated, and if other factors like GH dose or IGF-I SDS, 25-OH vitamin D levels and use of SSRT had an additional influence. We hypothesized that only LBM would be associated with BMD SDS in young adults with PWS. Furthermore, we showed the effects of sex steroid replacement therapy (SSRT) on longer-term BMD.
Methods
Patients
For current evaluation, we included 43 young adults participating in the Dutch Young Adult PWS (YAP) study, coordinated by the Dutch Growth Research Foundation. Inclusion criteria: (i) genetically confirmed diagnosis of PWS by positive methylation test; (ii) GH treatment for at least 5 years during childhood; (iii) at least 3 years of continuous GH treatment after AH attainment, which was defined as height velocity less than 0.5 cm per 6 months and complete epiphyseal fusion. Exclusion criteria: (i) medication to reduce weight (fat); (ii) non-cooperative behavior; (iii) obstructive sleep apnea syndrome.
As there is worldwide no approval and reimbursement of GH for adults with PWS, GH treatment has to be discontinued at attainment of AH. The YAP study was started in 2011 to evaluate the longer-term effects and safety of GH treatment in young adults with PWS who had been treated with GH during childhood. Patients were included in the YAP study either after participation in the Dutch PWS cohort study in children (6) or the transition study (13, 34).
For patients who participated in the Dutch PWS cohort study, GH dose was lowered after AH attainment from 1 (~0.035 mg/kg/day) to 0.33 mg/m2/day (~0.012 mg/kg/day). Patients who participated in the transition study had been without GH for a median duration of 1 year before the restart of GH in a dose of 0.33 mg/m2/day. As the aim of the present study was to investigate BMD during 3 years of GH in a stable dose, we did not analyze the 1st year after GH dose lowering or after GH restart. We chose to exclude the 1st year in both groups to eliminate the influence of GH dose lowering or GH restart. Therefore, the study start in the current evaluation refers to the study visit at 1 year after the GH restart or dose lowering (16, 17).
The study was approved by the Medical Ethics Committee of the Erasmus Medical Centre/Sophia Children’s Hospital, Rotterdam, the Netherlands. Written informed consent was obtained from participants and their legal representatives.
Design
Open-label, a prospective study investigating the longer-term effects and safety of 3 years of GH with a stable dose of 0.33 mg/m2/day (~0.012 mg/kg/day). The dose was adjusted based on calculated body surface area and serum IGF-I levels between 1 and 2 SDS. Patients were examined every 6 months by the multidisciplinary PWS team of the Dutch Growth Research Foundation.
Measurements
Standing height was measured in centimeters with a calibrated Harpenden stadiometer. Body weight was measured in kilograms on an electric calibrated scale (Servo Balance KA-20-150S; Servo Berkel Prior, Katwijk, The Netherlands). Height, weight and BMI were expressed as standard deviations scores (SDS) using Growth Analyser Version 4.0 (available at www.growthanalyser.org, adjusted for sex and age according to Dutch reference values (37, 38).
Bone mineral content (BMC; in g), BMD (in g/cm2) of the total body and lumbar spine at L2–L4 (BMDTB and BMDLS), fat mass (FM) and LBM were measured by the same DXA machine (Lunar Prodigy type; GE Healthcare) in Erasmus MC, with daily quality assurance. BMDTB measurements included the head. The intra-assay coefficients for variation were 0.64% for BMDTB and 1.04% for BMDLS, 0.41–0.88% for FM and 1.57–4.49% for LBM. LBM was calculated as fat-free mass minus bone mineral content. FM% SDS and LBM SDS were calculated according to age- and sex-matched Dutch reference values (39). As BMDLS is underestimated by the areal presentation, we corrected for bone size by calculating the bone mineral apparent density of the lumbar spine (BMADLS). The model BMADLS = BMDLS × (4/(π × width)) was used, with width as the mean width of the second to fourth lumbar vertebral bodies (40). BMDTBSDS, BMDLSSDS and BMADLSSDS were calculated to age- and sex-matched reference values of the Dutch population (41).
Definition of hypogonadism
In girls, hypogonadism was defined as morning serum estradiol levels <100 pmol/L and/or Tanner stage 3 or less from the age of 14 years, and/or no menarche from the age of 16 years. In boys, hypogonadism was defined as morning serum testosterone levels <5 nmol/L and/or Tanner stage 3 or less from the age of 16 years, and/or morning serum testosterone levels <10 nmol/L from the age of 18 years.
Patients received SSRT unless decided otherwise based on patient characteristics like gonadal status and adequate BMD values or concerns regarding behavioral problems. For men, the following SSRT was used: transdermal testosterone, i.m. testosterone or oral testosterone. Transdermal testosterone dose was titrated to early morning serum testosterone levels between 10 and 30 nmol/L. For women, the following SSRT was used: an adult dose of oral 17 beta-estradiol or oral contraceptive medication.
Assays
Blood samples collected after an overnight fast were measured in the Biochemical and Endocrine laboratories of the Erasmus Medical Centre, Rotterdam. Serum IGF-I levels were assessed using the Immulite 2000 (Siemens Health-care Diagnostics) until 2013, with interassay CV of 6.5%. After 2013, IGF-I was measured using the IDS-iSYS (Immunodiagnostic Systems), with an interassay CV of <6.0%. Levels of IGF-I were expressed as SDS, adjusted for age and sex (42). Serum testosterone was measured by UPLC-MS/MS (Waters XEVO TQ-S) and serum estradiol was measured by ECLIA platform (Roche Elecsys). 25-OH vitamin D levels were assayed by immunoassay (DiaSorin Liaison XL).
Statistical analysis
Statistical analyses were performed with SPSS version 24.0 (SPSS Inc.). Variables are expressed as median (interquartile range (IQR)). Changes over time were calculated using linear mixed model analysis with outcomes measured at each time point as a dependent variable with an unstructured covariance matrix. Effects are presented as estimated marginal mean (95% CI). One sample t-test was used to compare values to a healthy reference population (41). Multiple regression analyses were performed to investigate the association of sex, age, height and LBM or FM on BMDTBSDS and BMADLSSDS. Forward selection was used in building the regression model. In addition, GH dose, IGF-I SDS, 25-OH vitamin D levels and use of SSRT were individually added and removed from the model of sex, age and LBM, to investigate if these parameters had an additional influence. We decided to analyze both the effect of LBM and FM since the body composition in patients with PWS is abnormal and, therefore, it could be that in patients with PWS, the FM is more important for BMD than LBM. An independent sample t-test was used to investigate differences between groups. Differences were considered significant if the P-value was <0.05.
Results
Characteristics at start of the 3-year study
Forty-three young adults with PWS (18 males, 25 females) were included (Table 1). Median (IQR) age was 19.5 (18.7–20.7) years for males and 18.4 (16.9–20.8) years for females. Median (IQR) height was −1.0 (−1.7 to −0.3) SDS and BMI 24.5 (21.9 to 27.7) kg/m2, being 0.9 (0.0 to 1.8) SDS. Eighteen (41.9%) patients had a deletion, twenty (46.5%) an mUPD, four (9.3%) an ICD and one (2.3%) a translocation.
Clinical characteristics at start of a 3-year study. Data are expressed as median (IQR).
| Characteristics | Values |
|---|---|
| Total, n | 43 |
| Females, n | 25 |
| Genetic subtype | |
| Deletion | 18 |
| mUPD | 20 |
| ICD | 4 |
| Translocation | 1 |
| Gonadal status | |
| Receiving SSRT | 33 |
| Eugonadal | 3 |
| Hypogonadal | 7 |
| Age at start of childhood GH treatment | 7.6 (5.2 to 10.1) |
| Age at inclusion† | |
| Males | 19.5 (17.5 to 23.3) |
| Females | 18.4 (15.8 to 23.8) |
| Adult height (SDS) | −1.0 (−1.7 to −0.3) |
| BMI (Kg/m2) | 24.5 (21.9 to 27.7) |
| BMI for age (SDS) | 0.9 (0.0 to 1.8) |
| BMI for PWS (SDS) | −1.4 (−2.0 to −0.7) |
| BMD total body (g/cm2) | 1.15 (1.08 to 1.19) |
| BMD total body SDS^ | −0.78 (−1.31 to 0.11) |
| BMD lumbar spine (g/cm2)# | 1.19 (1.09 to 1.26) |
| BMD lumbar spine SDS^# | −0.62 (−1.16 to −0.09) |
| BMAD SDS^# | −0.47 (−1.14 to 0.32) |
| 25-OH vitamin D | 66.0 (55.3 to 88.8) |
| IGF-I SDS* | 1.5 (0.6 to 2.0) |
†Values are presented as mean (range); #n = 34; ^BMD SDS values were calculated according to age- and sex-matched Dutch references (38); *IGF-I SDS was calculated according to age- and sex-matched Dutch references.
BMAD, bone mineral apparent density; BMD, bone mineral density; ICD, imprinting center defect; mUPD, maternal uniparental disomy.
Median (IQR) BMDTB was 1.15 (1.08 to 1.19) g/cm2, being −0.78 (−1.31 to 0.11) SDS. BMDTBSDS, albeit being within normal ranges, was significantly lower compared to healthy peers (P < 0.01). Nine patients had a BMDTB <−2.0 SDS (20.9%, 4♀/5♂). Patients with BMDTB <−2.0 SDS had a significantly lower mean LBM of −3.0 SDS compared to −1.8 SDS in patients with BMDTB >−2.0 SDS, P = 0.002.
Nine patients had a history of corrective surgery for scoliosis. Since these patients had spondylodesis material in their lumbar spine, the measurement of BMDLS was not reliable. Median (IQR) BMDLS in the remaining 34 patients was 1.19 (1.09 to 1.26) g/cm2, corresponding to a BMADLS of −0.47 (−1.14 to 0.32) SDS, which was similar to BMADLSSDS in healthy pears (P = 0.05). Only one male had a BMADLS <−2.0 SDS (3.0%).
During the study, one patient fractured his elbow after falling down the stairs. There were no other fractures during the study.
Bone mineral density during 3 years of GH
Figure 1 and Table 2 show BMD during 3 years of GH treatment in the total group, and for men and women separately. Estimated mean (95% CI) BMDTB in the total group did not significantly change during the 3-year study, being –0.76 (–1.11 to –0.41) SDS at start and –0.90 (–1.27 to –0.54) SDS after 3 years (P = 0.11). BMADLS also did not significantly change during the 3-year study, being –0.36 (–0.72 to 0.01) SDS and –0.46 (–0.77 to –0.16) SDS, respectively (P = 0.16).
(A, B, C, D, E and F) Bone mineral density during 3 years of GH in the total group of 43 young adults with PWS (A and B) and in the 18 men (C and D) and 25 women (E and F) separately.
Citation: European Journal of Endocrinology 184, 6; 10.1530/EJE-20-1335
(A, B, C, D, E and F) Bone mineral density during 3 years of GH in the total group of 43 young adults with PWS (A and B) and in the 18 men (C and D) and 25 women (E and F) separately.
Citation: European Journal of Endocrinology 184, 6; 10.1530/EJE-20-1335
(A, B, C, D, E and F) Bone mineral density during 3 years of GH in the total group of 43 young adults with PWS (A and B) and in the 18 men (C and D) and 25 women (E and F) separately.
Citation: European Journal of Endocrinology 184, 6; 10.1530/EJE-20-1335
Bone mineral density during the 3-year study in 43 young adults with PWS. Data are expresssed as estimated means (95% CI).
| At start | After 1 year | After 2 years | After 3 years | P-value* | |
|---|---|---|---|---|---|
| Total group | |||||
| BMD | |||||
| Total body (g/cm2) | 1.13 (1.10 to 1.16) | 1.14 (1.11 to 1.18) | 1.14 (1.11 to 1.17) | 1.14 (1.11 to 1.17) | 0.32 |
| Total body SDS | −0.76 (−1.11 to −0.41) | −0.73 (−1.14 to −0.32) | −0.82 (−1.19 to −0.45) | −0.90 (−1.27 to −0.54) | 0.11 |
| Lumbar spine (g/cm2; n = 34) | 1.17 (1.13 to 1.22) | 1.18 (1.13 to 1.22) | 1.20 (1.15 to 1.25) | 1.19 (1.15 to 1.24) | 0.09 |
| Lumbar spine SDS | −0.54 (−0.85 to −0.22) | −0.56 (−0.84 to −0.27) | −0.47 (−0.76 to −0.18) | −0.52 (−0.77 to −0.27) | 0.84 |
| BMAD SDS | −0.36 (−0.72 to 0.01) | −0.43 (−0.78 to −0.08) | −0.40 (−0.77 to −0.03) | −0.46 (−0.77 to −0.16) | 0.16 |
| FM (kg) | 28.5 (25.2 to 31.7) | 28.8 (25.7 to 31.9) | 28.8 (25.5 to 32.0) | 28.5 (25.1 to 32.0) | 0.97 |
| FM% SDS | 2.09 (1.92 to 2.26) | 2.02 (1.86 to 2.19) | 1.98 (1.83 to 2.12) | 1.91 (1.76 to 2.07) | 0.06 |
| LBM (kg) | 39.8 (37.4 to 42.2) | 40.7 (38.4 to 43.0) | 41.2 (38.8 to 43.5) | 40.9 (38.5 to 43.2) | 0.07 |
| LBM SDS | −2.10 (−2.40 to −1.80) | −1.98 (−2.25 to −1.59) | −1.92 (−2.25 to −1.59) | −1.97 (−2.29 to −1.64) | 0.21 |
| 25-OH vitamin D (nmol/L) | 68.2 (60.5 to 75.9) | 67.3 (61.3 to 73.4) | 78.2 (66.9 to 89.6) | 73.8 (69.1 to 78.4) | 0.19 |
| Men (n = 18) | |||||
| BMD | |||||
| Total body (g/cm2) | 1.14 (1.08 to 1.20) | 1.15 (1.08 to 1.21) | 1.14 (1.08 to 1.20) | 1.13 (1.08 to 1.19) | 0.47 |
| Total body SDS | −1.10 (−1.70 to −0.49) | −1.20 (−1.79 to −0.61) | −1.35 (−1.91 to −0.80) | −1.46 (−1.94 to −0.98) | 0.008 |
| Lumbar spine (g/cm2; n = 13) | 1.18 (1.09 to 1.27) | 1.18 (1.09 to 1.26) | 1.20 (1.10 to 1.29) | 1.120 (1.12 to 1.28) | 0.49 |
| Lumbar spine SDS | −0.60 (−1.26 to 0.06) | −0.67 (−1.23 to −0.10) | −0.55 (−1.15 to 0.05) | −0.51 (−1.05 to 0.04) | 0.59 |
| BMAD SDS | −0.11 (−0.86 to 0.65) | −0.30 (−0.99 to 0.39) | −0.18 (−0.93 to 0.57) | −0.17 (−0.76 to 0.43) | 0.73 |
| FM (kg) | 25.4 (20.3 to 30.5) | 26.3 (21.1 to 31.5) | 25.9 (20.5 to 31.2) | 25.1 (20.6 to 29.7) | 0.77 |
| FM% SDS | 2.11 (1.82 to 2.41) | 2.15 (1.85 to 2.44) | 2.14 (1.92 to 2.36) | 2.13 (1.91 to 2.34) | 0.85 |
| LBM (kg) | 46.6 (43.7 to 49.5) | 46.8 (43.9 to 49.8) | 47.2 (44.2 to 50.2) | 47.0 (44.0 to 45.0) | 0.67 |
| LBM SDS | −2.52 (−2.95 to −2.10) | −2.58 (−2.99 to −2.16) | −2.59 (−3.00 to −2.18) | −2.65 (−3.05 to −2.25) | 0.23 |
| 25-OH vitamin D (nmol/L) | 63.4 (50.8 to 76.1) | 72.8 (65.4 to 80.2) | 70.6 (63.4 to 77.8) | 72.0 (65.4 to 78.5) | 0.21 |
| Women (n = 25) | |||||
| BMD | |||||
| Total body (g/cm2) | 1.12 (1.09 to 1.16) | 1.14 (1.09 to 1.19) | 1.14 (1.10 to 1.18) | 1.14 (1.10 to 1.19) | 0.046 |
| Total body SDS | −0.51 (−0.94 to −0.08) | −0.39 (−0.95 to 0.17) | −0.44 (−0.91 to 0.03) | −0.48 (−0.96 to 0.0) | 0.78 |
| Lumbar spine (g/cm2; n = 21) | 1.17 (1.11 to 1.23) | 1.18 (1.12 to 1.24) | 1.20 (1.14 to 1.26) | 1.19 (1.14 to 1.25) | 0.09 |
| Lumbar spine SDS | −0.50 (−0.86 to −0.14) | −0.49 (−0.83 to −0.15) | −0.42 (−0.75 to −0.09) | −0.48 (−0.78 to −0.19) | 0.87 |
| BMAD SDS | −0.51 (−0.91 to −0.11) | −0.51 (−0.93 to −0.09) | −0.53 (−0.97 to −0.10) | −0.59 (−0.95 to −0.22) | 0.29 |
| FM (kg) | 30.7 (26.4 to 34.9) | 30.6 (26.6 to 34.6) | 30.8 (26.7 to 35.0) | 30.9 (26.0 to 35.8) | 0.92 |
| FM% SDS | 2.07 (1.85 to 2.30) | 1.94 (1.74 to 2.14) | 1.86 (1.66 to 2.06) | 1.76 (1.55 to 1.98) | 0.001 |
| LBM (kg) | 34.9 (32.9 to 36.9) | 36.2 (34.2 to 38.2) | 36.8 (34.7 to 39.0) | 36.6 (34.5 to 38.8) | 0.042 |
| LBM SDS | −1.79 (−2.19 to −1.39) | −1.55 (−1.93 to −1.6) | −1.44 (−1.84 to −1.03) | −1.47 (−1.86 to −1.08) | 0.040 |
| 25-OH vitamin D (nmol/L) | 72.1 (62.1 to 82.0) | 63.1 (54.0 to 72.2) | 82.8 (64.1 to 101.4) | 75.2 (68.7 to 81.7) | 0.58 |
*P-value of the change during 3 years of GH treatment.
BMD: bone mineral density. BMAD: bone mineral apperent density. FM%, fat mass percentage; LBM, lean body mass.
In men, there was a significant decrease in BMDTB during 3 years of GH from −1.10 (−1.70 to −0.49) SDS at start to −1.46 (−1.94 to −0.98) SDS after 3 years, P = 0.008, while BMADLS remained similar, being −0.11 (−0.86 to 0.65) SDS and −0.17 (−0.76 to 0.43) SDS, respectively (P = 0.73). In women, both BMDTBSDS and BMADLSSDS remained similar, BMDTB being −0.51 (−0.94 to −0.08) SDS at start and −0.48 (−0.96 to 0.0) SDS after 3 years (P = 0.78), and BMADLS, being −0.51 (−0.91 to −0.11) SDS and −0.59 (−0.95 to −0.22) SDS, respectively (P = 0.29).
Serum 25-OH vitamin D levels remained similar and above 60 nmol/L during all study years, in both sexes.
Bone mineral density in patients with and without SSRT during 3 years of GH
Thirty-three patients received SSRT during the entire study. The majority of men received transdermal testosterone, while the majority of women received oral contraceptive medication. In men receiving transdermal testosterone, median early morning testosterone levels during the study varied between 11 and 14.5 nmol/L. Estimated mean (95% CI) of BMDTB remained similar during the 3-year study in the total group who received SSRT during the study, being −0.77 (−1.19 to −0.35) SDS at start and −0.85 (−1.32 to −0.39) SDS after 3 years (P = 0.37). BMADLS remained also stable during 3 years of GH, being −0.35 (−0.79 to 0.08) SDS and −0.46 (−0.85 to −0.07) SDS, respectively (P = 0.21).
However, in men who received SSRT during the study, there was a significant decrease in BMDTB during 3 years of GH from −1.33 (−1.96 to −0.69) SDS at start to −1.59 (−2.15 to −1.01) SDS after 3 years (P = 0.014), while BMADLSSDS remained stable. In women, both BMDTBSDS and BMADLSSDS remained stable during 3 years of GH, BMDTB being −0.36 (−0.88 to 0.17) SDS at start and −0.31 (−0.91 to 0.30) SDS after 3 years (P = 0.72).
Five patients did not receive SSRT during the study, three were eugonadal (2♀/1♂) and two were hypogonadal (1♀/1♂). Median (IQR) BMDTB at study start was −0.83 (−1.17 to 0.89) SDS in the five patients who did not receive SSRT.
There was no significant difference in BMDTBSDS and BMADLSSDS between the 33 patients who did receive SSRT and the 5 who did not, neither at study start nor after 3 years. All 5 patients who did not receive SSRT had a BMDTB and BMADLS >−2 SDS during the entire study.
Associations
Table 3 shows the results of multiple regression analyses for BMDTBSDS in the total group.
Multiple regression analysis for bone mineral density at study start and after 3 years. Values presented are results of multiple regression.
| BMDTBSDS | ||||
|---|---|---|---|---|
| Study start | After 3 years | |||
| β | P | β | P | |
| Age | −0.215 | 0.005 | −0.223 | 0.077 |
| Sex | 1.956 | <0.001 | 2.100 | <0.001 |
| Height | 0.001 | 0.977 | 0.05 | 0.083 |
| LBM (kg) | 0.135 | <0.001 | 0.065 | 0.04 |
| Model P-value | <0.001 | 0.002 | ||
| Adjusted R square | 0.421 | 0.331 | ||
| R square | 0.478 | 0.404 | ||
β, unstandardized regression coefficient; BMDTB, bone mineral density of the total body.
PBold values represent significance at P<0.05
In the total group, female sex was associated with higher BMDTBSDS, both at study start and after 3 years. Age was inversely associated with BMDTBSDS but only at study start (β = −0.22, P = 0.005), indicating a lower BMDTB of 0.22 SDS with each 1 year increase in age. LBM was associated with BMDTBSDS at study start (β = 0.14, P < 0.001) and after 3 years (β = 0.07, P = 0.04), indicating a higher BMDTB of 0.14 SDS at study start, with each 1 kg increase in LBM, when age, sex and height were the same. A model with age, sex, height and LBM explained 42% of variability in BMDTBSDS at study start and 33% after 3 years. Adding SSRT use (y/n), IGF-I or GH dose, or vitamin D levels to the model did not show significant associations of these variables and did not improve adjusted R square values. Substituting LBM for FM or weight did not show significant associations of FM or weight with BMDTBSDS.
We did not find significant associations of age, sex, height, LBM, vitamin D levels, IGF-I or GH dose with BMADLSSDS, neither at start nor after 3 years.
There was no difference in BMDTBSDS between patients with an mUPD or deletion neither at study start nor after 3 years. Mean (SD) BMADLS after 3 years was significantly higher in patients with an mUPD, being −0.10 (0.91) SDS, than in patients with a deletion, being −0.78 (0.62) SDS, P = 0.03 but there was no difference in BMADLSSDS at study start.
Discussion
This study confirms our hypothesis that BMADLSSDS remains stable in the normal range during the 3-year GH-study in young adults with PWS who received GH for several years during childhood. However, in men, we found a significant decrease in BMDTBSDS during the 3-year study, while in women BMDTBSDS remained stable. During the entire study, BMDTBSDS was significantly higher in women than in men. This study also confirms our hypothesis that LBM was significantly associated with BMDTBSDS both at study start and after 3 years. As the majority of the patients received a stable dose of SSRT during the study, the results of this study show the effects of 3 years of combined GH and SSRT treatment on BMD in young adults with PWS who were treated with GH during childhood.
Data regarding the long-term effects of GH on BMD in adults with PWS are very scarce. Our previous cross-over trial in GH-treated young adults and a study by Jørgensen et al. in GH-naïve adults did not show a difference between 1 year of placebo or 1 year of GH on BMD (34, 35). However, 1 year might be too short to find significant differences (30, 36). The study by Jørgensen et al. also investigated the effects of 2 years of GH and did not find an improvement in BMD during these years. However, patients had a lower BMDLS at start of that study and were older (mean age of 28.5 years) than in our study. In addition, patients were GH-naïve for at least 1 year before inclusion and about half of the patients received SSRT, which might explain the lower BMD found in that study.
Our previous cross-over study showed that GH alone is not able to prevent the decline in BMD SDS in hypogonadal young adults with PWS unless it is combined with SSRT (34). In the current study, the majority of the patients received a stable dose of SSRT during the 3 years and BMD remained stable. Only five patients did not receive SSRT of which two were hypogonadal. Since this number was small, it was not possible to analyze if BMD decreased in patients not receiving SSRT. However, average BMD was not lower in these five patients during the study compared to the total group. This is very likely the result of the fact that, in clinical practice, a low BMD or decrease in BMD is a strong reason to start SSRT in hypogonadal patients.
LBM was strongly associated with BMDTBSDS, which is in accordance with other studies investigating the influence of LBM on BMD both in patients with and without PWS (18, 32, 43). Continuation of GH is able to maintain a stable LBM, while discontinuation leads to a deterioration of body composition in young adults with PWS (13, 16). In the total group, there was a non-significant increase in LBM of 1.1 kg (relative change of +2.8%). In women, there was a significant increase in LBM, with an increase in mean LBM of 1.7 kg (relative change of +4.9%), while in men LBM remained stable. The association between LBM and BMD might suggest that a decline in LBM following discontinuation of GH could result in a decrease in BMD in the following years.
In children with PWS, we previously found a decline in BMDTBSDS and BMADLSSDS during adolescence in parallel to incomplete pubertal development and low sex hormone levels (32). Present study shows that during 3 years of combined GH and SSRT treatment, BMDTB and BMADLS remain stable in the normal range in young women, which is reassuring. However, we found a decline in BMDTB of −0.4 SDS during 3 years in men with SSRT. In the previous study in children, BMDTB declined −1.5 SDS during 4 years in adolescent boys who did not receive SSRT (32). Therefore, the combination of SSRT and GH appears to be able to slow the decline in BMD in young men with PWS.
During the entire study, BMDTBSDS was lower in men than in women. The study by Jørgensen et al. also found that BMD was significantly lower in men (35). There are several possible explanations for this difference. As LBM is an important predictor for BMDTBSDS, one explanation could be that LBM SDS in men with PWS is lower than in women. Indeed, in our study, mean LBM SDS was significantly lower in men, with LBM at the end of the study being −2.7 SDS in men and −1.5 SDS in women. Another explanation could be that SSRT in men is suboptimal compared to women. In men, clinicians and parents are more hesitant to start SSRT and to titrate to adult doses because of concerns about the influence of testosterone on (aggressive) behavior. In our experience, however, when SSRT is started in low doses and gradually increased, (increase in) behavioral problems rarely occur. Most men received transdermal testosterone and doses were titrated to early morning serum testosterone levels >10 nmol/L. However, in current study median levels were <15 nmol/L. Based on our data, we suggest that higher dosages are probably necessary for men to normalize BMD. In women, there are often less concerns about SSRT and the wish for contraception is also a reason to start oral contraceptive medication. Most women in our study indeed received oral contraceptive medication as SSRT, likely resulting in similar estradiol levels during SSRT as levels in healthy women.
BMADLSSDS after 3 years of GH was significantly higher in patients with an mUPD than in patients with a deletion. There was no difference in sex, SSRT use and LBM SDS between these patients. The significance of this finding is unknown, as there are no other reports about the difference in BMD between genetic subtypes.
A limitation of the current study is the fact that our study did not include a control group of patients who did not receive GH and/or patients who did not receive SSRT. About half of the patients participated previously in our randomized, placebo-controlled cross-over study investigating 1 year of GH vs 1 year of placebo. GH vs placebo resulted in a significant and clinically relevant improvement of body composition, with less FM and more LBM (13). In addition, the majority of patients and their parents reported a major subjective improvement in condition, strength and quality of life during GH vs placebo. Therefore, we did not want to withhold GH treatment in these patients for a longer period. Also, the Growth Hormone Research Society Workshop published a consensus guideline in 2013 for GH treatment in adults with PWS stating that there are data regarding clear benefits to GH treatment in transition to adulthood. It is possible that BMD would have remained stable without GH treatment. There are currently no studies describing the course of BMD during several years in young adults with PWS without GH treatment. However, studies in GHD patients show beneficial effects of GH continuation after AH attainment on BMD (30, 44, 45, 46). Furthermore, an increased prevalence of osteoporosis and increased fracture risk are reported in GH and SSRT untreated patients with PWS (19, 20, 21, 22, 23, 24). The fact that BMD remained stable in our study group suggests that the combination of GH and SSRT might protect against the development of osteoporosis in young adults with PWS.
We found that serum 25-OH vitamin D levels were >60 nmol/L during the current study and, therefore, vitamin D levels were normal in the majority of our study population. Previous studies have suggested that vitamin levels lower than 50 nmol/L are suboptimal for skeletal health (47, 48, 49). Vitamin D levels were checked annually and supplementation was advised when vitamin D levels were <50 nmol/L. The fact that we did not find an association between vitamin D levels and BMD is likely explained by the adequate 25-OH vitamin D levels in our study population.
In conclusion, our study shows that during 3 years of combined GH and SSRT treatment, BMD remains stable within the normal range in young adults with PWS who have been treated with GH during childhood. However, there is a decline in BMDTB in young men with PWS, possibly due to insufficient SSRT. Given the other positive effects of GH treatment in adults with PWS, we recommend to continue GH treatment when adolescents have attained AH. Furthermore, we recommend to start SSRT during adolescence unless puberty progresses normally. More studies are needed to investigate optimal SSRT in young adult men with PWS.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study.
Funding
This study was an investigator-initiated study, supported by an independent research grant from Pfizer. Pfizer was not involved in conception or design of the study, nor in collection, analysis or interpretation of data, writing the manuscript, or decision to submit the manuscript for publication.
Data availability
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
Acknowledgements
The authors express their gratitude to all children and parents for their enthusiastic participation in this study and thank Mariëlle van Eekelen, Laura Schafthuizen and Ezra Piso, research nurses, for all their work. The authors thank all collaborating pediatric-endocrinologists, pediatricians and other health care providers.
References
- 1↑
Cassidy SB & Driscoll DJ Prader-Willi syndrome. European Journal of Human Genetics 2009 17 3–13. (https://doi.org/10.1038/ejhg.2008.165)
- 2↑
Goldstone AP, Holland AJ, Hauffa BP, Hokken-Koelega AC, Tauber Mspeakers contributors at the Second Expert Meeting of the Comprehensive Care of Patients with PWS. Recommendations for the diagnosis and management of Prader-Willi syndrome. Journal of Clinical Endocrinology and Metabolism 2008 93 4183–4197. (https://doi.org/10.1210/jc.2008-0649)
- 3↑
Holm VA, Cassidy SB, Butler MG, Hanchett JM, Greenswag LR, Whitman BY & Greenberg F Prader-Willi syndrome: consensus diagnostic criteria. Pediatrics 1993 91 398–402.
- 4↑
Cassidy SB Prader-Willi syndrome. Journal of Medical Genetics 1997 34 917–923. (https://doi.org/10.1136/jmg.34.11.917)
- 5↑
Eiholzer U, Bachmann S & l’Allemand D Is there growth hormone deficiency in Prader-Willi syndrome? Six arguments to support the presence of hypothalamic growth hormone deficiency in Prader-Willi syndrome. Hormone Research 2000 53 (Supplement 3) 44–52. (https://doi.org/10.1159/000023533)
- 6↑
Bakker NE, Kuppens RJ, Siemensma EP, Tummers-de Lind van Wijngaarden RF, Festen DA, Bindels-de Heus GC, Bocca G, Haring DA, Hoorweg-Nijman JJ & Houdijk EC et al.Eight years of growth hormone treatment in children with Prader-Willi syndrome: maintaining the positive effects. Journal of Clinical Endocrinology and Metabolism 2013 98 4013–4022. (https://doi.org/10.1210/jc.2013-2012)
- 7↑
Festen DA, Wevers M, Lindgren AC, Bohm B, Otten BJ, Wit JM, Duivenvoorden HJ & Hokken-Koelega AC Mental and motor development before and during growth hormone treatment in infants and toddlers with Prader-Willi syndrome. Clinical Endocrinology 2008 68 919–925. (https://doi.org/10.1111/j.1365-2265.2007.03126.x)
- 8↑
Siemensma EP, Tummers-de Lind van Wijngaarden RF, Festen DA, Troeman ZC, van Alfen-van der Velden AA, Otten BJ, Rotteveel J, Odink RJ, Bindels-de Heus GC & van Leeuwen M et al.Beneficial effects of growth hormone treatment on cognition in children with Prader-Willi syndrome: a randomized controlled trial and longitudinal study. Journal of Clinical Endocrinology and Metabolism 2012 97 2307–2314. (https://doi.org/10.1210/jc.2012-1182)
- 9↑
Lo ST, Siemensma E, Collin P & Hokken-Koelega A Impaired theory of mind and symptoms of autism spectrum disorder in children with Prader-Willi syndrome. Research in Developmental Disabilities 2013 34 2764–2773. (https://doi.org/10.1016/j.ridd.2013.05.024)
- 10↑
Lo ST, Festen DA, Tummers-de Lind van Wijngaarden RF, Collin PJ & Hokken-Koelega AC Beneficial effects of long-term growth hormone treatment on adaptive functioning in infants with Prader-Willi syndrome. American Journal on Intellectual and Developmental Disabilities 2015 120 315–327. (https://doi.org/10.1352/1944-7558-120.4.315)
- 11↑
Carrel AL, Myers SE, Whitman BY, Eickhoff J & Allen DB Long-term growth hormone therapy changes the natural history of body composition and motor function in children with Prader-Willi syndrome. Journal of Clinical Endocrinology and Metabolism 2010 95 1131–1136. (https://doi.org/10.1210/jc.2009-1389)
- 12↑
Lindgren AC & Lindberg A Growth hormone treatment completely normalizes adult height and improves body composition in Prader-Willi syndrome: experience from KIGS (Pfizer International Growth Database). Hormone Research 2008 70 182–187. (https://doi.org/10.1159/000145019)
- 13↑
Kuppens RJ, Bakker NE, Siemensma EP, Tummers-de Lind van Wijngaarden RF, Donze SH, Festen DA, van Alfen-van der Velden JA, Stijnen T & Hokken-Koelega AC Beneficial effects of GH in young adults with Prader-Willi syndrome: a 2-year crossover trial. Journal of Clinical Endocrinology and Metabolism 2016 101 4110–4116. (https://doi.org/10.1210/jc.2016-2594)
- 14↑
Sode-Carlsen R, Farholt S, Rabben KF, Bollerslev J, Schreiner T, Jurik AG, Frystyk J, Christiansen JS & Hoybye C Growth hormone treatment for two years is safe and effective in adults with Prader-Willi syndrome. Growth Hormone and IGF Research 2011 21 185–190. (https://doi.org/10.1016/j.ghir.2011.05.002)
- 15↑
Butler MG, Smith BK, Lee J, Gibson C, Schmoll C, Moore WV & Donnelly JE Effects of growth hormone treatment in adults with Prader-Willi syndrome. Growth Hormone and IGF Research 2013 23 81–87. (https://doi.org/10.1016/j.ghir.2013.01.001)
- 16↑
Damen L, Donze SH, Kuppens RJ, Bakker NE, de Graaff LCG, van der Velden JAEM & Hokken-Koelega ACS Three years of growth hormone treatment in young adults with Prader-Willi syndrome: sustained positive effects on body composition. Orphanet Journal of Rare Diseases 2020 15 163. (https://doi.org/10.1186/s13023-020-01440-6)
- 17↑
Damen L, Grootjen LN, Donze SH, Juriaans AF, de Graaff LCG, van der Velden JAEM & Hokken-Koelega ACS Three years of growth hormone treatment in young adults with Prader-Willi syndrome previously treated with growth hormone in childhood: effects on glucose homeostasis and metabolic syndrome. Clinical Endocrinology 2020 93 439–448. (https://doi.org/10.1111/cen.14274)
- 18↑
Ho-Pham LT, Nguyen UD & Nguyen TV Association between lean mass, fat mass, and bone mineral density: a meta-analysis. Journal of Clinical Endocrinology and Metabolism 2014 99 30–38. (https://doi.org/10.1210/jc.2014-v99i12-30A)
- 19↑
Butler MG, Haber L, Mernaugh R, Carlson MG, Price R & Feurer ID Decreased bone mineral density in Prader-Willi syndrome: comparison with obese subjects. American Journal of Medical Genetics 2001 103 216–222. (https://doi.org/10.1002/ajmg.1556)
- 20↑
Sinnema M, Maaskant MA, van Schrojenstein Lantman-de Valk HM, van Nieuwpoort IC, Drent ML, Curfs LM & Schrander-Stumpel CT Physical health problems in adults with Prader-Willi syndrome. American Journal of Medical Genetics: Part A 2011 155A 2112–2124. (https://doi.org/10.1002/ajmg.a.34171)
- 21↑
Hoybye C Endocrine and metabolic aspects of adult Prader-Willi syndrome with special emphasis on the effect of growth hormone treatment. Growth Hormone and IGF Research 2004 14 1–15. (https://doi.org/10.1016/j.ghir.2003.09.003)
- 22↑
Brunetti G, Grugni G, Piacente L, Delvecchio M, Ventura A, Giordano P, Grano M, D’Amato G, Laforgia D & Crino A et al.Analysis of circulating mediators of bone remodeling in Prader-Willi syndrome. Calcified Tissue International 2018 102 635–643. (https://doi.org/10.1007/s00223-017-0376-y)
- 23↑
Butler JV, Whittington JE, Holland AJ, Boer H, Clarke D & Webb T Prevalence of, and risk factors for, physical ill-health in people with Prader-Willi syndrome: a population-based study. Developmental Medicine and Child Neurology 2002 44 248–255. (https://doi.org/10.1017/s001216220100202x)
- 24↑
Kroonen LT, Herman M, Pizzutillo PD & Macewen GD Prader-Willi syndrome: clinical concerns for the orthopaedic surgeon. Journal of Pediatric Orthopedics 2006 26 673–679. (https://doi.org/10.1097/01.bpo.0000226282.01202.4f)
- 25↑
Cadogan J, Blumsohn A, Barker ME & Eastell R A longitudinal study of bone gain in pubertal girls: anthropometric and biochemical correlates. Journal of Bone and Mineral Research 1998 13 1602–1612. (https://doi.org/10.1359/jbmr.1998.13.10.1602)
- 26↑
Compston JE Sex steroids and bone. Physiological Reviews 2001 81 419–447. (https://doi.org/10.1152/physrev.2001.81.1.419)
- 27↑
Matkovic V, Jelic T, Wardlaw GM, Ilich JZ, Goel PK, Wright JK, Andon MB, Smith KT & Heaney RP Timing of peak bone mass in Caucasian females and its implication for the prevention of osteoporosis. Inference from a cross-sectional model. Journal of Clinical Investigation 1994 93 799–808. (https://doi.org/10.1172/JCI117034)
- 28↑
Boot AM, de Ridder MA, van der Sluis IM, van Slobbe I, Krenning EP & Keizer-Schrama SM Peak bone mineral density, lean body mass and fractures. Bone 2010 46 336–341. (https://doi.org/10.1016/j.bone.2009.10.003)
- 29↑
Giustina A, Mazziotti G & Canalis E Growth hormone, insulin-like growth factors, and the skeleton. Endocrine Reviews 2008 29 535–559. (https://doi.org/10.1210/er.2007-0036)
- 30↑
Conway GS, Szarras-Czapnik M, Racz K, Keller A, Chanson P, Tauber M, Zacharin M1369 GHD to GHDA Transition Study Group. Treatment for 24 months with recombinant human GH has a beneficial effect on bone mineral density in young adults with childhood-onset GH deficiency. European Journal of Endocrinology 2009 160 899–907. (https://doi.org/10.1530/EJE-08-0436)
- 31↑
Myers SE, Carrel AL, Whitman BY & Allen DB Sustained benefit after 2 years of growth hormone on body composition, fat utilization, physical strength and agility, and growth in Prader-Willi syndrome. Journal of Pediatrics 2000 137 42–49. (https://doi.org/10.1067/mpd.2000.105369)
- 32↑
Bakker NE, Kuppens RJ, Siemensma EP, Tummers-de Lind van Wijngaarden RF, Festen DA, Bindels-de Heus GC, Bocca G, Haring DA, Hoorweg-Nijman JJ & Houdijk EC et al.Bone mineral density in children and adolescents with Prader-Willi syndrome: a longitudinal study during puberty and 9 years of growth hormone treatment. Journal of Clinical Endocrinology and Metabolism 2015 100 1609–1618. (https://doi.org/10.1210/jc.2014-4347)
- 33↑
Nakamura Y, Murakami N, Iida T, Asano S, Ozeki S & Nagai T Growth hormone treatment for osteoporosis in patients with scoliosis of Prader-Willi syndrome. Journal of Orthopaedic Science 2014 19 877–882. (https://doi.org/10.1007/s00776-014-0641-0)
- 34↑
Donze SH, Kuppens RJ, Bakker NE, van Alfen-van der Velden JAEM & Hokken-Koelega ACS Bone mineral density in young adults with Prader-Willi syndrome: a randomized, placebo-controlled, crossover GH trial. Clinical Endocrinology 2018 88 806–812. (https://doi.org/10.1111/cen.13567)
- 35↑
Jorgensen AP, Ueland T, Sode-Carlsen R, Schreiner T, Rabben KF, Farholt S, Hoybye C, Christiansen JS & Bollerslev J Two years of growth hormone treatment in adults with Prader-Willi syndrome do not improve the low BMD. Journal of Clinical Endocrinology and Metabolism 2013 98 E753–E760. (https://doi.org/10.1210/jc.2012-3378)
- 36↑
Jorgensen AP, Fougner KJ, Ueland T, Gudmundsen O, Burman P, Schreiner T & Bollerslev J Favorable long-term effects of growth hormone replacement therapy on quality of life, bone metabolism, body composition and lipid levels in patients with adult-onset growth hormone deficiency. Growth Hormone and IGF Research 2011 21 69–75. (https://doi.org/10.1016/j.ghir.2011.01.001)
- 37↑
Fredriks AM, van Buuren S, Burgmeijer RJ, Meulmeester JF, Beuker RJ, Brugman E, Roede MJ, Verloove-Vanhorick SP & Wit JM Continuing positive secular growth change in the Netherlands 1955–1997. Pediatric Research 2000 47 316–323. (https://doi.org/10.1203/00006450-200003000-00006)
- 38↑
Fredriks AM, van Buuren S, Wit JM & Verloove-Vanhorick SP Body index measurements in 1996–1997 compared with 1980. Archives of Disease in Childhood 2000 82 107–112. (https://doi.org/10.1136/adc.82.2.107)
- 39↑
Boot AM, Bouquet J, de Ridder MA, Krenning EP & de Muinck Keizer-Schrama SM Determinants of body composition measured by dual-energy X-ray absorptiometry in Dutch children and adolescents. American Journal of Clinical Nutrition 1997 66 232–238. (https://doi.org/10.1093/ajcn/66.2.232)
- 40↑
Kroger H, Vainio P, Nieminen J & Kotaniemi A Comparison of different models for interpreting bone mineral density measurements using DXA and MRI technology. Bone 1995 17 157–159. (https://doi.org/10.1016/s8756-3282(95)00162-x)
- 41↑
van der Sluis IM, de Ridder MA, Boot AM, Krenning EP & de Muinck Keizer-Schrama SM Reference data for bone density and body composition measured with dual energy x ray absorptiometry in white children and young adults. Archives of Disease in Childhood 2002 87 341–347; discussion 341–347. (https://doi.org/10.1136/adc.87.4.341)
- 42↑
Bidlingmaier M, Friedrich N, Emeny RT, Spranger J, Wolthers OD, Roswall J, Korner A, Obermayer-Pietsch B, Hubener C & Dahlgren J et al.Reference intervals for insulin-like growth factor-1 (IGF-I) from birth to senescence: results from a multicenter study using a new automated chemiluminescence IGF-I immunoassay conforming to recent international recommendations. Journal of Clinical Endocrinology and Metabolism 2014 99 1712–1721. (https://doi.org/10.1210/jc.2013-3059)
- 43↑
Viardot A, Purtell L, Nguyen TV & Campbell LV Relative contributions of lean and fat mass to bone mineral density: insight from Prader-Willi syndrome. Frontiers in Endocrinology 2018 9 480. (https://doi.org/10.3389/fendo.2018.00480)
- 44↑
Baroncelli GI, Bertelloni S, Sodini F & Saggese G Longitudinal changes of lumbar bone mineral density (BMD) in patients with GH deficiency after discontinuation of treatment at final height; timing and peak values for lumbar BMD. Clinical Endocrinology 2004 60 175–184. (https://doi.org/10.1046/j.1365-2265.2003.01949.x)
- 45↑
Tritos NA, Hamrahian AH, King D, Greenspan SL, Cook DM, Jonsson PJ, Wajnrajch MP, Koltowska-Haggstrom M & Biller BM A longer interval without GH replacement and female gender are associated with lower bone mineral density in adults with childhood-onset GH deficiency: a KIMS database analysis. European Journal of Endocrinology 2012 167 343–351. (https://doi.org/10.1530/EJE-12-0070)
- 46↑
Underwood LE, Attie KM, Baptista JGenentech Collaborative Study Group. Growth hormone (GH) dose-response in young adults with childhood-onset GH deficiency: a two-year, multicenter, multiple-dose, placebo-controlled study. Journal of Clinical Endocrinology and Metabolism 2003 88 5273–5280. (https://doi.org/10.1210/jc.2003-030204)
- 47↑
Giustina A, Adler RA, Binkley N, Bouillon R, Ebeling PR, Lazaretti-Castro M, Marcocci C, Rizzoli R, Sempos CT & Bilezikian JP Controversies in vitamin D: summary statement from an International Conference. Journal of Clinical Endocrinology and Metabolism 2019 104 234–240. (https://doi.org/10.1210/jc.2018-01414)
- 48↑
Kuchuk NO, Pluijm SM, van Schoor NM, Looman CW, Smit JH & Lips P Relationships of serum 25-hydroxyvitamin D to bone mineral density and serum parathyroid hormone and markers of bone turnover in older persons. Journal of Clinical Endocrinology and Metabolism 2009 94 1244–1250. (https://doi.org/10.1210/jc.2008-1832)
- 49↑
Lips P & van Schoor NM The effect of vitamin D on bone and osteoporosis. Best Practice and Research: Clinical Endocrinology and Metabolism 2011 25 585–591. (https://doi.org/10.1016/j.beem.2011.05.002)
