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Keywords:

  • ANOREXIA NERVOSA;
  • ADOLESCENTS;
  • BONE DENSITY;
  • BONE TURNOVER;
  • BONE METABOLISM;
  • ESTROGEN;
  • TRANSDERMAL;
  • IGF-1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Anorexia nervosa (AN) is prevalent in adolescents and is associated with decreased bone mineral accrual at a time critical for optimizing bone mass. Low BMD in AN is a consequence of nutritional and hormonal alterations, including hypogonadism and low estradiol levels. Effective therapeutic strategies to improve BMD in adolescents with AN have not been identified. Specifically, high estrogen doses given as an oral contraceptive do not improve BMD. The impact of physiologic estrogen doses that mimic puberty on BMD has not been examined. We enrolled 110 girls with AN and 40 normal-weight controls 12 to 18 years of age of similar maturity. Subjects were studied for 18 months. Mature girls with AN (bone age [BA] ≥15 years, n = 96) were randomized to 100 µg of 17β-estradiol (with cyclic progesterone) or placebo transdermally for 18 months. Immature girls with AN (BA < 15 years, n = 14) were randomized to incremental low-dose oral ethinyl-estradiol (3.75 µg daily from 0 to 6 months, 7.5 µg from 6 to 12 months, 11.25 µg from 12 to 18 months) to mimic pubertal estrogen increases or placebo for 18 months. All BMD measures assessed by dual-energy X-ray absorptiometry (DXA) were lower in girls with AN than in control girls. At baseline, girls with AN randomized to estrogen (AN E + ) did not differ from those randomized to placebo (AN E–) for age, maturity, height, BMI, amenorrhea duration, and BMD parameters. Spine and hip BMD Z-scores increased over time in the AN E+ compared with the AN E– group, even after controlling for baseline age and weight. It is concluded that physiologic estradiol replacement increases spine and hip BMD in girls with AN. © 2011 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Anorexia nervosa (AN), a condition characterized by low weight and hypogonadism,1 is prevalent in adolescence2 and is associated with decreased BMD and bone accrual.3, 4 It occurs at a time when normal bone accrual is essential to optimizing peak bone mass, an important determinant of future fracture risk. About 0.2% to 1% of adolescent girls suffer from AN, and 50% have BMD Z-scores of less than −1 at one or more bone sites.5 In contrast to normal-weight girls, AN girls do not increase bone mass prospectively, leading to a continued decrease in Z-scores.3, 6

Low BMD in AN results from nutritional deficiencies and associated hormonal alterations, including hypogonadism, low insulin-like growth factor 1 (IGF-1), and relative hypercortisolemia.5–8 Weight and menstrual recovery can improve BMD, but residual deficits persist.3 Therefore, it is important to develop therapeutic strategies that increase bone accrual while patients and their providers continue to work toward recovery.

Although estrogen deficiency is an important determinant of low BMD in AN, administration of estrogen-progesterone combination pills (such oral contraceptive pills) providing relatively high estrogen doses does not improve BMD.9–11 In addition, oral dehydroepiandrosterone (DHEA)12 or bisphosphonates13 do not increase spine BMD in AN girls after controlling for weight changes, although a study in adults with AN did indicate beneficial effects with bisphosphonates.14 Of importance, the relatively high estrogen doses in oral contraceptives suppress IGF-1 secretion, an important bone trophic hormone.15–17 Therefore, a possible reason for the lack of effectiveness of oral estrogen in increasing BMD in AN is that it further suppresses IGF-1, a hormone already decreased in this condition.18 In contrast, low oral estrogen doses that mimic early pubertal increases in estrogen19, 20 and transdermal replacement doses of estrogen do not suppress IGF-1.15–17 The impact of physiologic estrogen administration (that does not suppress IGF-1) on bone accrual has not been investigated in AN.

We performed a randomized, double-blind, placebo-controlled study to examine the impact of physiologic estrogen replacement on BMD in girls with AN. We hypothesized that physiologic estrogen administration would cause increases in BMD measures in AN girls to approximate changes in normal-weight control girls. Our primary endpoint was the change in spine BMD Z-scores.

Subjects and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Subject selection

The study was performed at the Clinical Research Center of Massachusetts General Hospital (MGH), Boston, MA, USA, and the Clinical Investigation Unit at the Hospital for Sick Children (SickKids), Toronto, Ontario, Canada. We enrolled 150 AN girls (110 at MGH and 40 at SickKids) and 88 normal-weight control girls 12 to 18 years of age. All AN subjects were screened by the study psychiatrist to confirm they met DSM-IV criteria, including having amenorrhea for at least 3 months preceding study participation. All girls with AN were under the care of multidisciplinary treatment teams organized by their primary providers. The study did not assume the clinical care of the subjects. Exclusion criteria included other diseases affecting bone metabolism (including untreated thyroid disease, premature ovarian failure, diabetes, cancer, pituitary disease, renal disease, and bone fracture within the past 6 months), use of prescription medications affecting bone metabolism within 3 months, suicidality, psychosis, substance abuse, hematocrit less than 30%, potassium concentration less than 3.0 mmol/L, or glucose concentration less than 50 mg/dL. No control subject had a past or present history of an eating disorder.

Following the screening visit, 110 AN girls (83 at MGH and 27 at SickKids) and 40 normal-weight control girls (BMI 10th to 90th percentiles) of similar maturity (Tanner stage and bone age [BA]) were enrolled. The remaining either did not qualify or did not agree to participate (Fig. 1).

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Figure 1. Numbers of adolescent girls with anorexia nervosa and normal-weight control girls recruited for the study and attrition over the 18-month course of the study.

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AN subjects were recruited through eating disorder providers and treatment centers and controls through mailings to local pediatricians and advertisements in area periodicals. Informed assent and consent were obtained from subjects younger than 18 years of age and their parents and informed consent from subjects 18 years of age. All subjects with AN were enrolled in active outpatient treatment programs that included behavioral therapy to promote weight gain. The Institutional Review Board of Partners HealthCare, Boston, and the Research Ethics Board at SickKids, Toronto, approved the study.

Experimental protocol

At the screening visit, a history and physical examination were completed, and blood was drawn for complete blood count (CBC) and determinations of potassium, glucose, thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH). A urine pregnancy test was performed. BA was assessed by a single pediatric endocrinologist blinded to the randomization sequence.21 Qualifying AN subjects at MGH and SickKids were randomized, double blind to physiologic estrogen replacement (AN E + ) or placebo (AN E–) by the MGH Research Pharmacy based on a predetermined computer-generated randomization sequence. Mature girls with AN (BA ≥ 15 years, n = 96) were randomized to transdermal 17β-estradiol (100-µg patch applied twice weekly; Novartis Pharmaceuticals, Inc., East Hanover, NJ, USA) continuously over the study duration or identical placebo. Girls randomized to the active estradiol patch also received medroxyprogesterone 2.5 mg daily for 10 days each month, whereas girls randomized to the placebo patch received placebo medroxyprogesterone pills for 10 days each month. Immature girls with AN (BA < 15 years, n = 14) were randomized to escalating doses of oral ethinyl estradiol (3.75 µg daily for the first 6 months, 7.5 µg daily for the second 6 months, and 11.25 µg daily for the last 6 months) or placebo for 18 months (ethinyl estradiol in these small doses was formulated by the Research Pharmacy of MGH). Estrogen doses were based on studies of physiologic estrogen replacement in immature hypogonadal girls to preserve height potential.22, 23 Normal-weight control girls were followed for 18 months without intervention at the same time points and per the same study protocol as randomized subjects with AN. All subjects were given 1200 mg of calcium carbonate and 400 IU of vitamin D daily.

Every 2 months, an interval history, physical examination, and pregnancy test were performed. At these visits, we assessed compliance with study medications with verbal questionnaires and by collecting calendars provided to subjects for recording missed study medication doses. We also collected all used and unused patches and pills. Groups did not differ for compliance with study medications. We used dual-energy X-ray absorptiometry (DXA) to assess BMD (spine and hip) and body composition at baseline and 6, 12, and 18 months. Baseline blood was obtained for 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH), estradiol, IGF-1, leptin, N-terminal propeptide of type 1 procollagen (P1NP), and C-terminal cross-linked peptides (CTX). IGF-1, leptin, P1NP and CTX were also assessed at follow-up. Control girls were studied in the first 10 days of their menstrual cycles. Five girls with AN randomized to placebo resumed menses during the course of the study.

Anthropometric measurements

Subjects were weighed in a hospital gown, and height was measured on a wall-mounted stadiometer (average of three measurements). BMI and percentage of ideal body weight (%IBW) were calculated.

Bone density and body composition measurements

We used DXA (Hologic 4500A densitometer, Version 11.2; Waltham, MA, USA) to assess BMD at the spine (L1–L4) and hip and body composition, including fat and lean mass. We calculated lumbar spine bone mineral apparent density (LBMAD, a height-adjusted measure of spine BMD) using published methods.24 Because of lack of standards, Z-scores for LBMAD are not reported. In addition, given the lack of validated standards for hip BMAD calculations in children, we adjusted hip BMD for height in our statistical analysis as an alternative approach. Coefficients of variation (CVs) for spine and hip BMD are 0.8% to 1.1% and for fat and lean mass are 1.0% to 2.1%.

The DXA scanners at MGH and SickKids were cross-calibrated using a Hologic spine and whole-body phantom from Synarc, Inc. (San Francisco, CA, USA). Each site completed 10 scans of each phantom. Synarc, Inc., evaluated performance, consistency, and cross-calibration between sites; no adjustments were necessary.

Biochemical analysis

Screening laboratory values (ie, CBC, potassium, glucose, TSH, and FSH) were assessed by the hospital laboratory. We used a chemiluminescent immunoassay to measure PTH (Beckman Coulter, Fullerton, CA, USA; detection limit 1 pg/mL; intraassay CV 1.6% to 2.6%), an IRMA to measure IGF-1 (Diagnostic Systems Laboratories, Inc., Webster, TX, USA; detection limit 2.06 ng/mL; intraassay CV 3.9%), an ELISA to measure leptin (Millipore, St Charles, MO, USA; detection limit 0.5 ng/mL; intraassay CV 2.6% to 4.6%), and an RIA to measure estradiol (Diagnostic Systems Laboratories, Inc.; limit of detection 2.2 pg/mL; intra-assay CV 6.5% to 8.9%) and P1NP (Orion Diagnostica, Espoo, Finland; detection limit 2 ng/mL; intraassay CV of 6.5% to 10.2%). An ISYS Autoanalyzer was used to assess CTX (Immunodiagnostic Systems, Inc., Scottsdale, AZ, USA; detection limit 0.023 ng/mL; intraassay CV 3.2%) and 25(OH)D (Immunodiagnostics Systems, Inc.; limit of detection 3.6 ng/mL; intraassay CV 5.5% to 12.1%). Samples were stored at −80 °C until analysis and run in duplicate.

Statistical analysis

A p value of less than 0.05 on a two-tailed test was used to indicate significance. Our data were normally distributed and did not require transformations. Baseline characteristics of AN girls versus control girls and AN E+ girls versus AN E– girls were compared using Student's t test (reported as mean ± SE). Our primary endpoint was the prospective change in LBMD Z-scores in AN E+ versus AN E– girls. For our primary longitudinal analysis, the first step was to determine models of longitudinal data collected. This applied to BMD and body composition measures. We checked for linearity by examining data from individual subjects and linearity over time held without requiring transformation. Next, LBMD and hip BMD Z-scores were analyzed by a mixed-model analysis of variance (PROC MIXED), a common method for analyzing longitudinal data. We assumed that each subject had a different linear trajectory and tested that the mean trajectory was different in AN E+ versus AN E– girls. This model has a fixed time and time-treatment interaction and a random intercept and time term, the term of primary interest being the time-treatment interaction. An advantage of this method is that it allowed us to use data from subjects dropping out early, who still added data to the analysis. In a subset analysis, we analyzed the mature girls with AN separately, given that this subset consisted of the majority of girls with AN. Given that the immature group included only 14 girls with AN, we did not analyze this subset separately.

For our secondary analyses, we compared absolute and percent changes in BMD measures and in BMD Z-scores in AN E+ versus AN E– girls at 6, 12, and 18 months using Student's t test. In comparing AN E+ versus AN E– girls, we controlled for baseline chronologic age and weight changes using multivariate analysis, given that these are known determinants of prospective changes in BMD measures over time. We confirmed that age and weight changes were predictive of subsequent changes in BMD measures using simple linear (Pearson's) correlation. In addition, we controlled for other covariates, including height, years since menarche, duration of amenorrhea, and duration since diagnosis. We also compared changes in the two AN groups versus normal-weight control girls at 6, 12, and 18 months using ANOVA followed by the Dunnett test to adjust for multiple comparisons. Finally, we used the Fisher exact test to compare proportions of AN girls who did or did not receive estrogen for reported adverse effects.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Baseline characteristics

Baseline characteristics are shown in Table 1. AN girls did not differ from control girls for height and maturity (BA and Tanner stage). Calcium and vitamin D intake, 25(OH)D, and PTH levels were higher, whereas estradiol, IGF-1, and leptin levels were lower in AN girls than in control girls. AN E+ girls did not differ from AN E– girls at baseline for the characteristics reported in Table 1. In addition, girls with AN who completed the study did not differ from noncompleters for age, bone age, weight, height, BMI, percent ideal body weight, pubertal stage, duration of amenorrhea, exercise activity, calcium or vitamin D intake, fat mass, lean mass, percent body fat, spine BMD and spine BMD Z-scores, spine BMAD, and levels of vitamin D, PTH, estradiol, IGF-1, urinary cortisol, and leptin (p > 0.05). The completers had higher hip BMD and corresponding Z-scores than noncompleters (p = 0.03 and 0.01).

Table 1. Baseline Characteristics of Adolescent Girls With Anorexia Nervosa and Normal-Weight Control Girls
 Controls (n = 40)Anorexia nervosa (n = 110)p Value (Controls versus AN)
  1. Ns = not significant.

Age (years)15.6 ± 0.216.5 ± 0.20.001
Bone age (years)15.8 ± 0.216.2 ± 0.1Ns
Weight (kg)57.9 ± 1.447.2 ± 0.5<0.0001
Height (cm)164.3 ± 0.9164.3 ± 0.6Ns
BMI (kg/m2)21.4 ± 0.517.4 ± 0.1<0.0001
% Ideal body weight (BMI)106.3 ± 2.484.6 ± 0.6<0.0001
Tanner stage (breasts)4.7 ± 0.14.6 ± 0.1Ns
Tanner stage (pubic hair)4.6 ± 0.14.5 ± 0.1Ns
Amenorrhea duration (years)0.90 ± 0.08
Exercise activity (hours)16.5 ± 1.716.8 ± 1.3Ns
Calcium intake (mg)1180 ± 852040 ± 84<0.0001
Vitamin D intake (IU)191 ± 28554 ± 28<0.0001
DXA measures
 Fat mass (kg)15.9 ± 0.88.9 ± 0.3<0.0001
 Lean mass (kg)42.0 ± 0.837.7 ± 0.4<0.0001
 Percent body fat26.2 ± 0.818.2 ± 0.5<0.0001
 Lumbar BMD (g/cm2)0.972 ± 0.0160.907 ± 0.0100.0009
 Lumbar BMD Z-score0.136 ± 0.1476−0.623 ± 0.098<0.0001
 Lumbar BMAD (g/cm3)0.152 ± 0.00250.140 ± 0.001<0.0001
 Hip BMD (g/cm2)0.974 ± 0.01890.887 ± 0.011<0.0001
 Hip BMD Z-score0.277 ± 0.1793−0.644 ± 0.098<0.0001
Biochemical parameters
 Vitamin D (ng/mL)23.1 ± 1.631.8 ± 0.9<0.0001
 PTH (pg/mL)11.2 ± 1.820.3 ± 1.90.004
 Estradiol (pg/mL)66.2 ± 13.339.6 ± 5.40.03
 IGF-1 (ng/mL)361.6 ± 16.2239.3 ± 11.3<0.0001
 Leptin (ng/mL)12.4 ± 1.04.6 ± 0.4<0.0001
 P1NP (ng/mL)200.8 ± 23.8110.6 ± 13.90.0007
 CTX (ng/mL)1.01 ± 0.060.78 ± 0.040.002

Changes in bone density parameters

AN E+ girls had greater increases in BMD Z-scores at the spine and hip than AN E– girls (p = 0.044 and 0.040, respectively; Table 2) in our primary analysis, an intention-to-treat analysis, using a mixed-model ANOVA to analyze longitudinal data. Differences between the groups persisted after controlling for covariates, including age and weight changes. In a subset analysis, when only mature girls with AN were examined (who received transdermal estradiol versus placebo), AN E+ girls had greater increases in BMD Z-scores at the spine than AN E– girls (p = 0.03) and trended to have greater increases in hip BMD Z-scores (p = 0.07).

Table 2. Mixed-Model Analysis of Variance for Longitudinal Data (BMD Z-Scores at the Different Sites): AN E+ Versus AN E−
 EffectGroupParameter estimateStandard errort Valuep Valuep Valuea
  • a

    p Value after controlling for covariates (age and weight).

LBMD Z-scoresVisit × groupAN E−−0.06950.0342−2.030.0450.046
 Visit × groupAN E+0  
Hip BMD Z-scoresVisit × groupAN E−−0.06600.0318−2.080.0410.037
 Visit × groupAN E+0  

BMD changes were predicted inversely by baseline age and positively by weight changes (data not shown). In addition, BMD changes were predicted inversely by height and years since menarche. On secondary analyses to quantify prospective changes, AN E+ girls had greater increases in absolute LBMD, percent change in LBMD, percent change in LBMAD, and absolute LBMD Z-scores at all skeletal sites at all time points measured compared with AN E– girls, even after adjusting for baseline age and change in weight. In addition, AN E+ girls had greater increases in absolute and percent change hip BMD and absolute hip BMD Z-scores at 18 months compared with AN E– girls (Table 3). Changes in LBMD, LBMAD, and hip BMD at 6, 12, and 18 months were lower in AN E– girls than in normal-weight control girls (p < 0.05) but comparable in AN E+ girls versus control girls (percent change in spine BMD in the three groups is shown in Fig. 2).

Table 3. Changes in BMD Measures in Treated AN (AN E + ), Untreated AN (AN E–), and Normal-Weight Control Girls
Bone density changesAN E+AN E–AN E+ versus E–Controlsp (ANOVA) three groups
papbpcpdpe
  • Δ = difference in; 6-0 = at 6 months compared with baseline; 12-0 = at 12 months compared with baseline; 18-0 =  at 18 months compared with baseline.

  • a

    p Value after controlling for baseline age and weight changes.

  • b

    p Value after controlling for baseline age, weight changes, and height.

  • c

    p Value after controlling for baseline age, weight changes, and years since menarche.

  • d

    p Value after controlling for baseline age, weight changes, and duration of amenorrhea.

  • e

    p Value after controlling for baseline age, weight changes, height, years since menarche, and duration of amenorrhea.

  • *

    p < 0.05 compared with AN E–.

ΔLBMD 6-0 (g/cm2)0.015 ± 0.004−0.006 ± 0.0040.0030.0030.00030.00020.00030.021 ± 0.003*<0.0001
%ΔLBMD 6-01.772 ± 0.530−0.529 ± 0.4970.0040.0050.00030.00030.00032.283 ± 0.361*<0.0001
ΔLBMD Z 6-00.043 ± 0.044−0.155 ± 0.0400.0040.0060.00030.00030.00020.079 ± 0.030*<0.0001
ΔLBMD 12-0 (g/cm2)0.020 ± 0.006−0.002 ± 0.0070.0040.0020.0030.00070.00040.030 ± 0.006a0.002
%ΔLBMD 12-02.513 ± 0.796−0.072 ± 0.8210.0040.0020.0030.0010.00063.315 ± 0.623a0.005
ΔLBMD Z 12-00.027 ± 0.060−0.218 ± 0.0590.0030.0020.0020.0010.00070.077 ± 0.048*0.0005
ΔLBMD 18-0 (g/cm2)0.021 ± 0.0090.002 ± 0.0110.020.020.030.040.020.042 ± 0.008a0.01
%ΔLBMD 18-02.611 ± 1.0470.307 ± 1.1440.010.010.020.030.014.483 ± 0.890a0.02
ΔLBMD Z 18-0−0.026 ± 0.078−0.236 ± 0.0910.050.050.050.050.030.099 ± 0.068a0.01
ΔLBMAD 6-0 (g/cm3)0.003 ± 0.001−0.001 ± 0.0010.0040.0020.0030.0010.0020.004 ± 0.001a<0.0001
%ΔLBMAD 6-01.988 ± 0.580−0.560 ± 0.5500.0040.0020.0020.0010.0012.390 ± 0.369a0.0001
ΔLBMAD 12-0 (g/cm3)0.003 ± 0.001−0.000 ± 0.0010.0080.0050.010.0080.0050.004 ± 0.001a0.02
%ΔLBMAD 12-02.226 ± 0.802−0.109 ± 0.8320.010.0070.020.010.0082.727 ± 0.657*0.02
ΔLBMAD 18-0 (g/cm3)0.003 ± 0.0010.000 ± 0.0010.070.070.0060.010.0040.006 ± 0.001a0.01
%ΔLBMAD 18-01.919 ± 0.9440.232 ± 0.8120.0490.050.0040.010.0033.987 ± 0.944a0.02
ΔHip BMD 6-0 (g/cm2)−0.001 ± 0.004−0.006 ± 0.0040.120.110.070.080.070.012 ± 0.003a0.006
%ΔHip BMD 6-0−0.027 ± 0.438−0.559 ± 0.4920.160.160.090.100.0981.221 ± 0.321a0.02
ΔHip BMD Z 6-0−0.002 ± 0.037−0.077 ± 0.0410.080.070.060.060.060.096 ± 0.032a0.006
ΔHip BMD 12-0 (g/cm2)0.005 ± 0.008−0.004 ± 0.0070.080.060.120.040.040.016 ± 0.004a0.06
%ΔHip BMD 12-00.617 ± 0.870−0.299 ± 0.7340.100.070.160.060.031.715 ± 0.3790.12
ΔHip BMD Z 12-0−0.080 ± 0.069−0.193 ± 0.0550.080.060.120.0490.04−0.009 ± 0.034*0.048
ΔHip BMD 18-0 (g/cm2)−0.001 ± 0.008−0.013 ± 0.0110.020.020.060.040.040.021 ± 0.006*0.02
%ΔHip BMD 18-00.004 ± 0.837−1.178 ± 1.930.0470.020.080.060.062.175 ± 0.650*0.04
ΔHip BMD Z 18-0−0.177 ± 0.063−0.331 ± 0.0890.030.020.070.0450.049−0.016 ± 0.056*0.01
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Figure 2. Percent change in lumbar spine bone mineral density (LBMD) in adolescent girls with anorexia nervosa (AN) randomized to placebo (AN E–; black bars), girls with AN randomized to estrogen (AN E + ; gray bars), and normal-weight control girls (C; white bars). AN E+ girls had significant increases in LBMD at 6, 12, and 18 months compared with AN E– girls. When compared with control girls, AN E– girls had significant decreases in LBMD at 6, 12, and 18 months, whereas AN E+ girls did not differ from control girls for changes in BMD over time. Analysis was performed for differences between means for pairs. *p < 0.05.

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We also examined differences in AN E+ girls versus AN E– girls after controlling for (1) baseline age, height, and weight changes, for (2) baseline age, years since menarche, and weight changes, for (3) baseline age, duration of amenorrhea, and weight changes, and finally for (4) baseline age, height, years since menarche, duration of amenorrhea, and weight changes (Table 3). Differences between AN E+ and AN E– girls became even stronger for changes in LBMD and LBMAD measures and for most hip BMD measures after controlling for baseline age, height, years since menarche, duration of amenorrhea, and weight changes. Data did not differ when duration of amenorrhea was replaced by duration since diagnosis of AN. Similarly, addition of changes in lean mass to the multivariate model did not change the results and are not reported.

Changes in biochemical parameters

AN E+ girls did not differ from AN E– girls over 18 months for changes in levels of IGF-1 (8.4 ± 32.6 versus 6.2 ± 26.0 ng/mL, p = 0.96) and leptin (3.0 ± 1.8 versus 1.7 ± 0.8 ng/mL, p = 0.36). Levels of CTX, a marker of bone resorption, decreased more in AN E+ girls than in AN E– girls over the 18 months, although the difference was not statistically significant (−0.25 ± 0.09 versus −0.12 ± 0.10 ng/mL, p = 0.35). Changes in P1NP, a marker of bone formation, also did not differ in AN E+ girls versus AN E– girls (−2.9 ± 22.5 versus −10.9 ± 27.0 ng/mL, p = 0.83).

Changes in weight and body composition parameters

AN E+ girls did not differ from AN E– girls for prospective changes in weight (p = 0.50), BMI (p = 0.22), or lean mass (p = 0.20) or percent fat mass (p = 0.25) in our primary analysis, an intention-to-treat analysis, using a mixed-model ANOVA. Similarly, when mature girls with AN were analyzed, AN E+ girls did not differ from AN E– girls for changes in weight (p = 0.46), BMI (p = 0.26), or lean mass (p = 0.21) or percent fat mass (p = 0.24). No differences were observed in AN E+ girls versus AN E– girls for changes in height over the study duration within mature girls and immature girls. Additionally, on secondary analysis (Table 4), AN E+ girls did not differ from AN E– girls for changes in weight, BMI, and fat and lean mass over 6, 12, and 18 months.

Table 4. Changes in Body Composition Parameters in Treated AN (AN E + ) Versus Untreated AN (AN E–)
 AN E+AN E–p Value
  1. Δ = difference in; 6-0 = at 6 months compared with baseline; 12-0 = at 12 months compared with baseline; 18-0 = at 18 months compared with baseline.

ΔWeight 6-0 (kg)0.27 ± 0.110.29 ± 0.140.90
ΔWeight 12-0 (kg)3.35 ± 0.974.83 ± 0.770.23
ΔWeight 18-0 (kg)4.63 ± 1.024.18 ± 0.990.75
ΔBMI 6-0 (kg/m2)0.98 ± 0.231.14 ± 0.200.59
ΔBMI 12-0 (kg/m2)1.07 ± 0.361.63 ± 0.290.23
ΔBMI 18-0 (kg/m2)1.57 ± 0.381.47 ± 0.360.84
ΔFat mass 6-0 (kg)1.11 ± 0.381.76 ± 0.410.26
ΔFat mass 12-0 (kg)1.31 ± 0.682.92 ± 0.530.06
ΔFat mass 18-0 (kg)2.01 ± 0.882.90 ± 0.630.41
ΔLean mass 6-0 (kg)1.63 ± 0.311.02 ± 0.330.18
ΔLean mass 12-0 (kg)1.60 ± 0.492.00 ± 0.450.55
ΔLean mass 18-0 (kg)1.82 ± 0.561.72 ± 0.600.90

Adverse effects of study medications

Adverse events did not differ among the groups (Table 5).

Table 5. Adverse Effects in Treated AN (AN E + ) and Untreated AN (AN E–)
 AN E+AN E–
  1. *p < 0.05.

Admissions to the hospital for low weight and/or bradycardia21.2%29.6%
Nausea/vomiting25.0%14.8%
Dizziness11.5%18.5%
Headaches17.3%25.9%
Bloating32.7%29.6%
Constipation7.7%7.4%
Breast tenderness25.0%16.7%
Premenstrual symptoms1.9%0%
Mood swings1.9%0%
Increased vaginal discharge23.1%16.7%
Excessive or irregular bleeding23.1%16.7%
Irritation from contact lenses3.9%11.1%
Perceived increase in facial hair5.8%1.9%
Perceived loss of scalp hair13.5%9.3%
Worsening depression5.8%9.3%
Erythema at patch application site31.1%35.4%
Vasovagal symptoms0%1.9%
Calf pain (not associated with deep vein thrombosis)1.9%0%

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We demonstrate for the first time that physiologic estrogen replacement increases spine and hip BMD in girls with AN in a randomized, placebo-controlled study.

Adolescents with AN lack estrogen at a time when physiologic estrogen secretion is critical for optimizing bone accrual. Low BMD is common in AN,5 and bone accrual is profoundly impaired,3, 6 resulting in lower than optimal peak bone mass, which may impair future bone health. Adolescence is a relatively narrow window in time in which to maximize bone accrual, and deficits incurred at this time may lead to permanent deficits in peak bone mass. In fact, adult women who develop AN during adolescence have lower BMD values than those developing the condition in adult life, even when duration of amenorrhea is comparable.25 Consequently, it is important to identify therapeutic strategies to improve bone accrual in AN during adolescence.

Consistent with previous studies, AN girls had lower spine and hip BMD values and corresponding Z-scores and lower levels of bone turnover markers than normal-weight control girls. Importantly, these differences were observed despite higher calcium and vitamin D intake and higher 25(OH)D levels in AN girls, as reported in an earlier study.26 AN is associated with a nutritionally acquired resistance to growth hormone (GH) with low IGF-1,7 in contrast to the high IGF-1 levels typically seen during puberty.27 Another hormonal alteration in AN that may have an impact on BMD and bone accrual is low leptin concentrations.28, 29 AN girls in this study had lower lean mass and lower estradiol, IGF-1, and leptin levels than control girls.

Although recovery of weight and menses is the optimal strategy for improving bone accrual in AN, studies indicate that weight gain and menses recovery are not sufficient to normalize bone accrual.3, 26, 30 Importantly, recovery can be difficult to attain and sustain despite the concentrated efforts of a multidisciplinary treatment team. In this study, only 5 girls in the placebo-treated group resumed regular menses.

Oral estrogen has been used effectively as replacement therapy in normal-weight hypogonadal adolescents (eg, girls with Turner syndrome) for many years.22, 31 However, multiple studies show that oral estrogen (given as an oral contraceptive) is not effective in increasing BMD in AN girls.9–11 Although the reason for lack of beneficial effects of oral estrogen on BMD in AN remains speculative, possibilities include nonphysiologic dosing and/or suppression of systemic IGF-1 by oral estrogen, as in postmenopausal women.15 This is particularly an issue in AN, a condition already associated with low IGF-1.7 Use of transdermal estrogen, which does not suppress IGF-1, should be a more effective replacement strategy.15 In fact, a recent exploratory study in girls with Turner syndrome demonstrated that transdermal estrogen caused greater increases in BMD than did oral estrogen.31 Additionally, small, incremental doses of oral estrogen in early puberty (to mimic the early pubertal rise in estrogen) do not suppress IGF-119, 20 and could be beneficial to bone.

Based on these data, we hypothesized that administration of replacement doses of transdermal estrogen to mature AN girls (BA ≥ 15 years) and small, incremental doses of oral estrogen to younger AN girls (with the potential to grow based on BA < 15 years) should be effective in increasing BMD. Our results support our hypothesis, and we show that physiologic estrogen replacement over 18 months is effective in increasing spine and hip BMD Z-scores in AN girls 12 to 18 years of age. We also demonstrate that spine and hip BMD changes are lower in AN E– girls than in normal-weight control girls, whereas AN E+ girls do not differ from control girls in bone accrual. Overall, AN E+ girls had greater increases in spine and hip BMD values and corresponding Z-scores than AN E– girls. These results became even stronger after controlling for important covariates, including baseline age, weight changes, height, years since menarche, duration of amenorrhea, and duration since diagnosis.

Changes in IGF-1 levels did not differ in AN E+ versus AN E– girls, consistent with our hypothesis that physiologic estrogen replacement would not suppress IGF-1 levels. Levels of CTX, a marker of bone resorption, decreased in girls with AN randomized to estrogen, but the change was not significant when compared with girls with AN randomized to placebo, likely because levels of bone turnover markers were already very suppressed in girls with AN compared with normal-weight girls at baseline, as also reported in earlier studies.32 A further suppression of bone-resoprtion markers following estrogen administration may be difficult to appreciate in a maximally suppressed state of bone turnover. Levels of P1NP, a bone-formation marker, did not differ between the groups.

Importantly, we found no difference in prospective changes in weight, lean or fat mass, or leptin levels in AN E+ versus AN E– girls, indicating that physiologic estrogen replacement does not cause weight or body composition changes, which may be reassuring to AN girls and enhance compliance. Additionally, adverse events did not differ in AN E+ versus AN E– girls. Of note, attrition in our study population was high but consistent with other treatment studies in adolescents with AN.33, 34

In order to normalize and “catch up” BMD over time, girls with AN not only may need to gain bone mass at a rate comparable with control girls (as in this study) but also may need to surpass control girls. In order for complete catch-up, other hormonal alterations in AN may need to be addressed as well. For example, it may be important to raise IGF-1 through weight recovery or recombinant human IGF-1 administration. One study in normal-weight women with hypothalamic amenorrhea reported that leptin replacement caused menses resumption in 5 of 8 women, with associated increases in bone-formation markers.35 However, these women had subjective reductions in appetite and lost significant weight. Leptin replacement thus is not a good strategy for improving BMD in AN patients, in whom low leptin levels are likely adaptive.

Another possible strategy to increase BMD in AN is to use bisphosphonates. Our group recently has reported an improvement in BMD in adults with AN randomized to bisphosphonates.14 However, caution is necessary in considering bisphosphonates in adolescents given their long half-life and also because bisphosphonates are associated with marked reductions in bone turnover, already low in adolescents with AN. In one trial of 12 months of alendronate in AN girls, there was no improvement in spine BMD after controlling for weight changes, although some effect at the femoral neck was reported.13

Our study indicates that physiologic estrogen replacement is effective in prospectively halting BMD reductions in AN girls and may be considered a therapeutic option in girls refractory to weight and menstrual recovery despite ongoing multidisciplinary therapy. Further studies are necessary to determine strategies that result in complete catch-up of BMD and normalization of peak bone mass acquisition in young women with AN.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

All the authors state that they have no conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We thank the skilled nursing and bionutrition staff of the Clinical Research Center of Massachusetts General Hospital, Boston, MA, USA, and Clinical Investigation Center of the Hospital for Sick Children, Toronto, Ontario, Canada, for their help with carrying out this study. Finally, we thank our subjects, without whom this study would not have been possible. This work was supported by National Institutes of Health Grants R01 DK 062249, K23 RR018851, M01-RR-01066, and 1 UL1 RR025758-03.

Author's roles: Dr. Misra worked on the concept and design of the study; acquisition, analysis, and interpretation of data; the writing of the manuscript; and approved the final version of the submitted manuscript. Dr. Katzman worked on the design of the study; acquisition, analysis, and interpretation of data; revised the submitted manuscript for intellectual contentl; and approved the final version of the submitted manuscript. Dr. Miller worked on the concept and design of the study, analysis and interpretation of data, revised the submitted manuscript for intellectual content, and approved the final version of the submitted manuscript. Ms. Mendes, Ms. Snelgrove, Dr. Russell, Dr. Goldstein, Dr. Ebrahimi, Ms. Clauss, Dr. Weigel, Dr. Mickley, and Dr. Herzog worked on data acquisition for this study, revised the submitted manuscript for intellectual content, and approved the final version of the submitted manuscript. Dr. Schoenfeld worked on data analysis for this study, revised the submitted manuscript for intellectual content, and approved the final version of the submitted manuscript. Dr. Klibanski worked on the concept and design of the study; acquisition, analysis, and interpretation of data; revised the submitted manuscript for intellectual content; and approved the final version of the submitted manuscript. Dr. Misra, Dr. Schoenfeld, and Dr. Klibanski accept responsibility for the integrity of the data analysis.

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  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
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