Clinical trial NCT00946192
Conditions: Exercise-related Amenorrhea
Keywords: Amenorrhea Adolescent Endurance Athletes Females Osteopenia Osteoporosis Estrogen
Sponsor: Massachusetts General Hospital
Start date: 2009-05-01; Completion date: 2021-04-01
Enrollment: 121 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
One aim of this study is to determine changes in body composition and hormones that differentiate athletes who stop getting their periods versus those who continue to get their periods and non-athletes. The second aim of this study is to determine whether transdermal or oral estrogen (versus no estrogen) is effective in increasing bone density and improving bone microarchitecture in adolescent athletes who are not getting their periods and are thus estrogen deficient. The investigators hypothesize that transdermal estrogen will be more effective than oral estrogen or no estrogen in improving bone health in amenorrheic adolescent athletes.

Detailed description:
As many as 25% of adolescent and young adult endurance athletes develop amenorrhea, and factors that cause amenorrhea to occur in some, but not all, athletes have not been well characterized. Recent data indicate the critical importance of a negative energy balance state and leptin in regulating the Hypothalamic-pituitary-gonadal (H-P-G) axis. However, these factors do not completely account for alterations in this axis, and other contributing factors are unclear. Our preliminary data indicate the importance of low fat mass and fat related hormones in mediating hypogonadism in young athletes. This study will confirm these data and determine whether low fat mass and altered levels of adipokines, such as leptin and adiponectin, and hormones regulated by fat mass, such as ghrelin and peptide YY (PYY), determine alterations in LH pulsatility. A very concerning impact of amenorrhea in athletes is low bone mineral density (BMD). Preliminary data indicate lower BMD in adolescent athletes with amenorrhea (AA) compared with eumenorrheic athletes (EA) and non-athletic controls. The high prevalence of AA in adolescents is particularly concerning, because this population is potentially at greater risk as it is actively accruing bone. Of importance, bone microarchitecture, a better predictor of bone strength than BMD, has not been studied in AA. Because pubertal increases in estrogen are integral to optimizing peak bone mass, and AA is characterized by hypoestrogenism, this randomized study of transdermal estrogen versus oral estrogen or no estrogen will also examine whether estrogen replacement increases BMD and improves bone microarchitecture in adolescent AA 14-21 years old. EA and sedentary controls will be followed without intervention for this period. Despite the prevalent practice of prescribing oral contraceptives in AA, there is a paucity of data regarding benefits of this intervention in teenagers. Because transdermal estrogen, unlike oral estrogen, does not suppress IGF-1, an important bone anabolic factor, we expect effects of transdermal estrogen to exceed those of oral estrogen or no therapy. In addition, preliminary data indicate that low fat mass and alterations in fat related hormones may contribute to decreased bone accrual rates in athletes, and will be confirmed in this study. To summarize, a better understanding of the pathophysiology of reproductive dysfunction is critical to develop therapeutic strategies that will normalize the reproductive axis and bone accrual, and these are the questions that this study aims to answer.

Arms: arm 1: 17Beta-estradiol transdermal patch twice weekly application for 12 months arm 2: One pill containing estrogen and progesterone taken daily for 21 days followed by placebo pills only for 7 days; regimen repeated for 12 months. arm 3: Elemental calcium 1200 mg and Vit D 400 IU taken orally daily
Interventions: intervention 1: Transdermal 17Beta-estradiol, progesterone intervention 2: Ethinyl Estradiol + Desogestrel intervention 3: Sham Comparator
Intervention descriptions: intervention 1: 100 mcg/day 17Beta-estradiol; transdermal twice weekly application for 12 months (with cyclic micronized progesterone pills (Prometrium): 200 mg taken orally daily Day 1 to Day 12 each month) + Elemental calcium 1200 mg and Vit D 400 IU taken orally daily intervention 2: Oral ethinyl estradiol (0.03 mg) + desogestrel (0.15 mg) + Elemental calcium 1200 mg and Vit D 400 IU taken once daily intervention 3: Elemental calcium 1200 mg and Vit D 400 IU taken orally daily

Protocol documents:
Prot_000.pdf: 
PARTNERS HUMAN RESEARCH COMMITTEE 
PROTOCOL SUMMARY 
Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
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Answer all questions accurately and completely in order to provide the PHRC with the relevant 
information to assess the risk-benefit ratio for the study.  Do not leave sections blank. 
 
PRINCIPAL/OVERALL INVESTIGATOR 
Madhusmita Misra, M.D.  
 
PROTOCOL TITLE 
Fat Mediated Modulation of Reproductive and Endocrine Function in Young Athletes 
 
NCT00946192 
 
FUNDING 
NIH 
 
VERSION DATE 
December 4, 2014 
 
 
 


Partners Human Subjects Research Application Form  
 
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Version Date:  June 1, 2005 
 
  
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SPECIFIC AIMS 
Concisely state the objectives of the study and the hypothesis being tested. 
 
Specific Aim 1: We will test the hypothesis that in adolescent athletes  
(i) 
Suboptimal energy availability preferentially lowers fat mass with sparing of lean mass  
(ii) 
Lower fat mass leads to altered levels of adipokines (lower leptin, higher adiponectin) and 
fat regulated hormones (higher ghrelin, PYY), and  
(iii) 
Altered levels of adipokines and fat regulated hormones determine hypogonadism and 
decreased gonadotropin pulsatility. 
In a cross-sectional study, we will specifically investigate whether (i) adolescent athletes in a state of low 
energy availability have lower fat mass than those with optimal energy availability, (ii) lower fat mass 
results in alterations in adipokines (decreases in leptin and increases in adiponectin) and fat regulated 
hormones (increases in ghrelin, PYY), and whether (ii) LH pulsatility patterns are predicted by pulsatility 
patterns of hormones produced (leptin, adiponectin) or regulated (ghrelin, PYY) by fat. 
 
Specific Aim 2: We will test the hypothesis that in adolescent athletes  
(i) 
Bone mass accrual rates (assessed by DXA) are markedly decreased in AA compared with 
EA and controls 
(ii) 
Bone microarchitecture at the ultradistal radius and leg (assessed by X-treme CT) is 
abnormal in AA with decreased trabecular thickness (TbTh) and increased separation (TbSp); 
deterioration occurs over time in AA compared to EA and controls who demonstrate increases 
in healthy bone, and  
(iii) 
Independent of hypogonadism, adipokines and fat regulated hormones are critical 
determinants of bone mass accrual rates and bone microarchitecture  
In a prospective observational study, we will compare changes in bone density, trabecular volume, 
trabecular thickness, number and separation over 12 months in a subset of AA versus EA and controls, 
and will investigate in a regression model whether altered levels of adipokines (lower leptin, higher 
adiponectin) and fat regulated hormones (higher ghrelin, PYY) determine lower bone mass accrual rates 
and altered bone structural parameters independent of gonadal status 
 
Specific Aim 3: In a randomized controlled trial, we will test the hypothesis that in adolescent AA, 
compared to oral estradiol or no treatment, transdermal estradiol replacement will  
(i) 
Increase bone mass accrual rates (DXA)  
(ii) 
Increase TbTh and bone trabecular volume (X-treme CT), and  
(iii) 
Not affect endurance capacity 
In a randomized controlled trial, adolescent AA will be randomized to transdermal estradiol in 
replacement doses, oral estradiol or no therapy for 12 months. Effects on bone accrual rates and 
endurance capacity will be assessed in comparison to EA and controls. In a subset of AA, we will 
examine effects of estradiol replacement on bone structural parameters compared to EA and controls. 
 
Specific Aim 4: We will test the hypothesis that in athletes 14-25 years old 
(i) 
Bone strength [as assessed using Finite Element Analysis] is lower in adolescent and young 
adult AA compared with EA and sedentary controls at baseline and decreases prospectively 
over one-year in AA, whereas bone strength increases in EA over the same duration.  
 
Specific Aim 5: We will test the hypothesis that in athletes 14-25 years old 
(i) 
Greater visceral adipose tissue (VAT) and lower subcutaneous adipose tissue (SAT) are 
associated with lower bone density, impaired microarchitecture and lower bone 
strength, and that  
(ii) 
Greater marrow fat is associated with lower bone density, impaired microarchitecture 
and lower bone strength.  
 
Specific Aim 6: We will test the hypothesis that in amenorrheic athletes 14-25  years old 


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(i) 
Transdermal estradiol, compared to oral estradiol or no treatment will increase bone 
strength to approximate that in controls, with decreases in marrow fat and no changes 
in regional body composition. 
 
Specific Aim 7: We will test the hypothesis that in adolescent and young adult male athletes  
(iv) 
Suboptimal energy availability and an energy deficit state preferentially lower fat mass with 
sparing of lean mass  
(v) 
Lower fat mass leads to altered levels of adipokines (lower leptin, higher adiponectin) and 
fat regulated hormones (higher ghrelin, PYY, cortisol)  
(vi) 
Altered levels of adipokines and fat regulated hormones determine a relative hypogonadal 
state 
(vii) 
Hypogonadism and altered levels of adipokines and fat regulated hormones have a 
deleterious effect on bone density, structure (assessed by HRpQCT) and estimated bone 
strength (using FEA) 
(viii) 
Higher visceral adipose tissue (assessed using MRI) and marrow fat (assessed using MRS) 
are associated with impaired bone density, structure and strength 
 
Specific Aim 8: We will test the hypothesis that in adolescent and young adult male athletes  
(i) 
The impact of athletic energy deficit on gonadal status and bone density, structure and 
strength is less marked than in female athletes of comparable maturity 
(ii) 
The lesser impact of athletic energy deficit on gonadal status compared with female athletes 
is attributable to greater lean mass, a lower absolute reduction in fat mass than in females 
(given lower baseline fat in males than in females), and a lesser impact on adipokines and 
fat regulated hormones 
(iii) 
A lesser impact of athletic energy deficit on testosterone secretion in males (compared with 
amenorrhea and hypoestrogenism in females) causes greater preservation of lean mass in 
males and a lesser impact on bone than in females 
 
 
BACKGROUND AND SIGNIFICANCE 
Provide a brief paragraph summarizing prior experience important for understanding the 
proposed study and procedures. 
 
Athletic activities are very common in high school and college, and endurance athletes are at high risk for 
amenorrhea. Athletes with amenorrhea (AA) in turn are at great risk for low bone mineral density (BMD) 
and thus fractures. However, not all athletes develop amenorrhea, and specific factors that determine why 
only some athletes develop amenorrhea are not well understood. Our preliminary data indicate lower fat 
mass in adolescent AA compared with eumenorrheic athletes (EA) and non-athletic controls, despite 
similar levels of physical activity in AA and EA. Hormones produced or regulated by fat may thus be 
critical modulators of the hypothalamo-pituitary-gonadal (H-P-G) axis.  Understanding determinants of 
amenorrhea in athletes is critical to developing future therapeutic strategies to treat or prevent 
amenorrhea, and therefore prevent low BMD. In this prospective study we hypothesize that lower fat 
mass in athletes determines hypogonadotropic hypogonadism through altered levels of specific 
adipokines (lower leptin and higher adiponectin) and fat regulated hormones (higher ghrelin, peptide YY 
(PYY)).  Our preliminary data also indicate markedly lower spine BMD in AA than in EA and controls, 
suggesting that weight-bearing activity cannot protect against deleterious effects of hypogonadism. We 
also aim to prospectively examine bone mass accrual rates (using DXA) and bone structure (using ultra 
high resolution digital flat panel volume CT (X-treme CT)) over 12 months in AA versus EA and 
controls. Because adolescence is critical for bone mass accrual towards attainment of peak bone mass, 
lower bone accrual rates in AA compared with EA and controls would raise serious concerns for 
suboptimal peak bone mass and increased fracture risk. Importantly, our data indicate strong associations 
of fat mass with BMD and microarchitecture (trabecular thickness and separation), and we hypothesize 
that adipokines and fat regulated hormones are important determinants of BMD and microarchitecture 


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independent of hypogonadism. Finally, because hypogonadism is an important cause of low BMD, we 
hypothesize that transdermal estrogen will increase BMD over a year in AA to approximate bone accrual 
rates observed in EA, and will result in improvement in bone structural parameters. In a three-arm 
randomized study, we will compare transdermal to oral estrogen and no treatment, and anticipate that 
transdermal estrogen will have the greatest effects on bone mass accrual because of the IGF-1 sparing 
effects of transdermal estrogen compared with oral estrogen. 
 
It is not known whether an equivalent of the female athlete triad of low energy availability, 
hypogonadism, and low bone density exists in male athletes. In contrast to females, in whom a complex 
and cyclically changing gonadotropin milieu is necessary for normal occurrence of ovulation and a 
normal menstrual cycle, monthly cyclical changes in gonadotropin secretion do not occur in males. 
Whereas alterations in GnRH pulsatility in hyper-exercising females athletes may manifest early as 
subclinical menstrual dysfunction (luteal phase defects or anovulation) (180) and later as oligoamenorrhea 
(178) depending on the extent of energy deficiency, it is possible that sufficient testosterone secretion 
persists in male athletes with preservation of bone density, as long as LH secretion is not completely 
dampened. By better characterizing the consequences of athletic activity on the HPG axis and bone in 
males, we may also better understand the pathogenesis of athletic energy deficit in females.  The proposed 
study will determine whether the equivalent of the female athlete triad exists in male athletes and the 
extent of impairment of bone metabolism in male vs. female endurance athletes in an energy deficient 
state. 
RESEARCH DESIGN AND METHODS 
Briefly describe study design and anticipated enrollment, i.e., number of subjects to be enrolled 
by researchers study-wide and by Partners researchers.  Provide a brief summary of the 
eligibility criteria (for example, age range, gender, medical condition).  Include any local site 
restrictions, for example, “Enrollment at Partners will be limited to adults although the sponsor’s 
protocol is open to both children and adults.”
 
Subjects will be 14-25 year old female athletes with AA (n= 145), EA (n=), and female non-
athletic controls (n= 42). Because different kinds of physical activity have different effects on bone 
metabolism, we will enroll only endurance athletes (please see Inclusion Criteria).  
For Specific Aims 7-8, 40 adolescent and young adult male athletes 14-21 years old, and 20 
adolescent and young adult male non-athletes 14-21 years old will be studied.  Eligibility will be assessed 
over the phone, and the study will include one visit only. 
 
Participation of Children: We will enroll only female adolescents/young adults to optimize power and 
avoid issues arising from deficiencies of different predominant sex hormones in boys (testosterone) vs. 
girls (estrogen). Adolescents and young adults will be studied to determine effects on BMD and its 
predictors in a population that should be actively accruing bone. 
Inclusion Criteria 
(i) Females 14-25 years old: Our pilot data are reassuring in that young women 14-25 years old with 
hypothalamic amenorrhea are not adversely affected with estrogen use. In fact, in our prospective study, 
beneficial effects were observed both in young women 18-25 years old using oral estrogen, and in 14-18 
year old adolescent girls using transdermal estrogen. We therefore feel that including girls in the 14-21 
year age range will not be hazardous to their bone health. In fact, given the lack of data in this age group, 
it is important to study younger women and teenagers rather than extrapolate data from studies in adults 
to this younger population. Hormone dynamics differ in teenagers compared with adults, and bone mass 
accrual is even more dependent on estrogen and IGF-1 in younger than older women who have already 
achieved peak bone mass. 
(ii) Bone age (BA)  14 years: 98% of adult height is achieved at a BA of 14 because only 2% of growth 
potential persists at a BA of 14 years.  Thus, to avoid potential stunting of growth potential with estrogen 
replacement, we have chosen to include girls with BA of  14 years. 
 (iii) BMI between 10th-90th percentiles for age, or BMI of at least 85% of ideal BMI 


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(iv) Amenorrhea (for AA): absence of menses for  three months (74) within a period of oligomenorrhea 
(cycle length > six weeks) for  six months, or absence of menarche at 16 years 
(v)  Eumenorrhea (EA and controls):  nine menses (cycle length 21-35 days) in preceding year.  
(vi) Non-athlete healthy controls will be eligible if weight bearing exercise activity is less than two hours 
a week and if they are not participating in organized team sports. 
(vii) Endurance athletes: severity of low BMD and menstrual dysfunction differ by kind of exercise and 
activity. For example, runners have a higher prevalence of menstrual irregularity than swimmers and 
cyclists (131).  By limiting enrollment to endurance athletes, we will eliminate variability from the type of 
activity.  Endurance training is defined as  4 h of aerobic weight-bearing training of the legs or specific 
endurance training weekly, or  20 miles of running weekly for a period of  6 months in the last year. 
These criteria were developed following discussions with Dr. Gary Skrinar, an expert in exercise 
physiology and a study consultant, and his interactions with athletic coaches at Boston University. Dr. 
Skrinar was until recently a Professor in the Department of Health Sciences at the Sargent College of 
Health & Rehabilitation Sciences, Boston University, a first tier school for competitive athletic activities 
involving endurance training. Dr. Skrinar has been studying athletes and conducting maximal exercise 
testing in Sargent College for the past 28 years on populations that include exercising young women. Our 
criteria differ from those used by a group from Sweden (74) that examined women 16-35 years old. This 
group specified endurance training as  6 h of aerobic weight-bearing training of the legs or   43.75 
miles of running weekly for at least 6 months in the last year or 6 h of specific endurance training weekly. 
These criteria were considered excessive and unrealistic for girls 14-21 years old by Dr. Skrinar and the 
coaches at Boston University, and the criteria modified as described. Cyclists and swimmers will be 
excluded because their training does not include true weight-bearing activities. Rowers (but not 
coxswains) and gymnasts will also be excluded given that these activities have been associated with a 
preservation of bone density even when associated with amenorrhea likely consequent to differences in 
the nature of weight bearing and impact. Track and cross-country runners will be included. Dr. Gary 
Skrinar will review screening information to confirm eligibility.   
Subjects and parents will be counseled regarding deleterious effects of excessive exercise on bone 
at the screening visit, and benefits of behavior modification, namely reduction of activity will be strongly 
recommended. Counseling will continue through the study. We will counsel subjects with athletic 
amenorrhea at the screen and at subsequent visits about the importance of reducing energy expenditure or 
increasing intake. We will also obtain information regarding whether or not they receive such counseling 
from their health care providers, parents or coaches. Of note, most subjects referred to us for our pilot 
studies had received significant counseling but did not respond to this by decreasing activity levels. 
Subjects will also be counseled regarding substance abuse at study visits.  
 
Exclusion Criteria 
• 
Use of medications affecting bone metabolism including estrogen, progesterone, anabolic steroids, 
glucocorticoids except local application of glucocorticoid creams (washout period of three months prior 
to enrollment if medically permissible to discontinue these), phenytoin, phenobarbitone 
• 
Conditions other than endurance training that may cause amenorrhea including PCOS (clinical or 
preceding laboratory evidence of hyperandrogenism with amenorrhea) 
• 
Individuals who have not had their period for at least 2 years (until an endocrine evaluation has ruled 
out other causes of amenorrhea) 
• 
Conditions other than endurance training that may cause bone metabolism to be affected 
• 
Abnormal TSH, elevated FSH, hematocrit < 30% 
For girls with AA (to be randomized to estrogen and progesterone or no treatment) 
• 
Family history or personal history of conditions that may increase risk of thromboembolism: 
a. Myocardial infarction or strokes occurring at <50 years (if not associated with smoking) 
b. Clotting disorders: normal coagulation profile necessary for enrollment in case of family history 
• 
Current history of smoking 
• 
History of thromoboembolism, migraines  
• 
Undiagnosed, abnormal genital bleeding; known, suspected or a history of breast cancer and estrogen-
dependent neoplasia; abnormal LFTs with ALT/AST >2 times the upper limit of normal; known 
hypersensitivity to product ingredients 
 
Males: 


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Inclusion Criteria:  
(i) 
Males 14-21y ≥Tanner stage 4  
(ii) 
BMI 10th-90th %ile  
(iii) 
Endurance athletes engaged in sports that involve weight bearing training of the legs. 
Severity of low BMD and gonadal dys-function may differ by type of exercise (131).  By 
limiting enrollment to weight bearing endurance athletes, we will eliminate variability from 
type of activity.  Endurance training is defined as  4h of aerobic weight-bearing training of 
the legs or specific endurance training weekly, or  20 miles of running weekly for  6 
months in the last year. Cyclists, rowers and swimmers will not be included because these 
activities do not involve lower extremity weight bearing. Gymnasts will not be included (not 
an endurance sport),  
(iv) 
Non-athletes will be eligible if weight bearing exercise activity is <2h a week and they are 
not participating in organized team sports. 
 
Exclusion Criteria:  
(i) 
Use of medications affecting bone metabolism (washout period of 3 months prior to 
enrollment if medically permissible to discontinue these)  
(ii) 
Conditions other than endurance training that may cause hypogonadism or low bone density  
(iii) 
High TSH, FSH or prolactin  
(iv) 
Standard MRI exclusion criteria such as presence of pacemaker, intracranial metal clips, 
severe claustrophobia 
 
 
Briefly describe study procedures.  Include any local site restrictions, for example, “Subjects 
enrolled at Partners will not participate in the pharmacokinetic portion of the study.”  Describe 
study endpoints.
 
Over the proposed 5-year investigation, 145 teenage girls and young adults 14-25 years old with 
AA, 42 EA and 42 non-athletic controls will be studied.  Eligibility will be assessed at the screen, and the 
study will include a baseline visit and follow up visits at 3, 6, 9 and 12 months. The baseline visit will 
occur within 8 weeks of the screen. EA and controls will be followed without intervention (except 
elemental calcium and vitamin D) for 12 months. 
For Specific Aim 3 we anticipate screening 2 times the number of athletes that we will eventually 
enroll in the study to obtain 145 eligible AA adolescents. In addition, for Specific Aims 1 and 2, we 
anticipate screening 2 times the number of EA (n=42) and non-athletic controls (n=42) required for this 
section of the study (total subjects to be screened= 458). 
For Specific Aims 7 and 8 we anticipate screening (over the phone) 2 times the number of 
athletes that we will eventually enroll in the study to obtain 40 eligible male endurance athletes.  In 
addition, we anticipate screening (over the phone) 2 times the number of healthy male non-athletes in 
order to obtain 20 eligible non-athletic controls (total male subjects to be screened = 120). 
 
Screening Visit: Informed consent will be obtained from parents and assent from subjects < 18y old, and 
informed consent from subjects 18y. Eligibility will be determined at this visit, which will include: 
(a) History (including medications, current and past mental health issues), physical examination, height, 
weight, BMI, waist, hip circumference 
(b) Screening labs: complete blood count, ALT, AST, TSH, FSH (to rule out premature/primary ovarian 
failure), 30 cc of blood will be drawn, frozen, and stored for later analysis. 
(c) The Bouchard 3-day activity record (129) (to confirm criteria for endurance training are met). 
(d) Bone age (wrist and hand X-ray) and bone density using DXA: spine, hip and total body. 
(e) Stratification of AA subjects during randomization will be based on Z-scores at the spine ( -1 or > -
1). Body composition will be assessed by DXA. AA dancers will be randomized using a separate 
randomization template than AA non-dancers. 


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(f) Two subtests, Vocabulary and Matrix Reasoning, from the Weschler Abbreviated Scale of 
Intelligence (WASI), a Color-Word Interference Test subtest from the Delis Kaplan Executive 
Functioning Scale (DKEFS), and the California Verbal Learning Test (CVLT).  
 
The DXA, bone age and cognitive tests will be completed at the screening visit, in between the screening 
and baseline visits or at the baseline visit. 
 
Baseline Study Visit: This visit will occur within eight weeks of the screen and will include: 
(a) History and physical examination with anthropometric measurements  
(b) Eating disorder questionnaires (EDI-2), Three-Factor Eating Questionnaire-R18 (TFEQ), and a 4-day 
food diary (to assess caloric intake) 
(c) Resting energy expenditure (REE) using indirect calorimetry 
(d) Salivary cortisol at 11 p.m.  
(e) Fasting (or 2 hour post-prandial) blood samples for leptin, adiponectin, ghrelin, PYY, IGF-1, insulin, 
estradiol, testosterone, SHBG, FSH and LH, bone turnover markers (PINP and N-telopeptide).  
(f) Fasting (or 2 hour post-prandial) calcium, phosphorus and 25(OH) vitamin D  
(g) Indirect calorimetry and handgrip strength. This exam will be performed at the Clinical Research 
Center at Massachusetts General Hospital or at the Charlestown Navy Yard site. This exam will be 
completed sometime in between screening and baseline visits or at the baseline visit. 
(h) Bone microarchitecture at the ultradistal radius and leg (non-dominant) will be assessed using X-
treme CT in a subset of 100 AA (first ~33 in each randomization group), 36 EA, 36 controls sometime 
between the screening and baseline visits or at the baseline visit 
(i) MRI (regional fat and muscle) assessment  
(j) Finite element analysis (FEA) will also be performed. 
(k) Optional fMRI and evaluation through the State Trait Anxiety Inventory (STAI) scores and addictive 
behaviors using the Alcohol Use Disorder Identification Test (AUDIT) and Drug Abuse Screening Test-
10 (DAST-10) in subset of 6 AAs randomized to treatment, 6 EAs, and 6 HC 18-25 years. 
 
Follow-up Outpatient Study Visits at 6 and 12 months: These will include: 
(a) History and physical examination with anthropometric measurements, adverse event review, changes 
to health status (including mood disorders) 
(b) Bouchard 3-day activity record (daily energy expenditure), 4-day food diary (caloric intake), EDI-2, 
TFEQ, REE (indirect calorimetry) 
(c) Handgrip strength in 60 AA (20 in each treatment group) (after drop-outs), 20 EA, controls 
(d) BMD and body composition using DXA (lumbar spine, hip, total body)  
(l) 12 m only: Bone structure at the ultradistal radius and leg (X-treme CT) in the 90 AA, 30 EA and 30 
controls (after dropouts); MRI (regional fat and muscle) assessment. Finite element analysis (FEA) will 
also be performed. 
(e) Salivary cortisol at 11 p.m.  
(f) Fasting (or 2 hour post-prandial) blood samples for leptin, adiponectin, ghrelin, PYY, IGF-1, insulin, 
estradiol, testosterone, SHBG, FSH and LH, bone turnover markers (PINP and N-telopeptide).  
(g) Color-Word Interference Test subtest from DKEFS and the CVLT. 
(h) Follow up of fMRI and evaluation through the State Trait Anxiety Inventory (STAI) scores and 
addictive behaviors using the Alcohol Use Disorder Identification Test (AUDIT) and Drug Abuse 
Screening Test-10 (DAST-10) at 6 month visit only in the 6AAs scanned at baseline. 
 
 
Follow-up Outpatient Study Visits at 3 and 9 months (abbreviated safety evaluation visits): 
(a) History and physical examination with anthropometric measurements, adverse event review  
(b) Fasting (or 2 hour post-prandial) blood samples for IGF-1, estradiol, testosterone, SHBG and LH, 
bone turnover markers (PINP and N-telopeptide).  
 
Male Study Visit: 
(a) History and physical exam (H&P)  
(b) 7-8 AM Hormones: TSH, FSH, LH, testosterone, SHBG, estradiol, IGF-1, leptin, ghrelin (total), PYY 
(total),  
(c) 11 PM salivary cortisol (to be mailed back);  


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(d) Calciotropic Measures and Markers of Bone Turnover: calcium, phosphorus, 25(OH) vitamin D, 
1,25(OH)2 vitamin D, PTH; P1NP and CTX.   
(e) Activity Assessment: Bouchard 3-day activity record (129). Habitual activity will be further assessed 
using a standardized exercise questionnaire, which provides a good estimate of past year physical activity 
(173, 199),  
(f) 4-day food diary to assess caloric intake (NDS, version 4, Minneapolis, MN) and determine standard 
macronutrient intake,  
(g) Eating disorder questionnaire (EDI-2) (186), Three-Factor Eating Questionnaire-R18 (TFEQ),  
(h) Resting energy expenditure (REE) using indirect calorimetry,  
(i) VO2 max and handgrip strength,  
(j) BMD: Lumbar spine, hip, radius, whole body areal BMD will be measured by DXA,  
(k) Body Composition: Whole body DXA will be used to assess fat and lean mass. In a subset of 10 
athletes and 10 non-athletes, we will use MRI to assess SAT, VAT, thigh muscle area and marrow fat, 
(l98) Bone microarchitecture and strength will be determined at the ultradistal radius and distal tibia using 
HR-pQCT (Xtreme CT, Scanco Medical AG).  Bone strength will be determined using FEA of HR-pQCT 
scan data (187).  
 
 
DNA testing: 
We may ask subjects to participate in DNA testing via a saliva collection and/or blood collection at some 
point during this study in order to determine if there is a genetic basis for the hormonal changes seen in 
non-menstruating athletes.  During one of the 6 visits for the “Fat Mediated Modulation of Reproductive 
and Endocrine Function in Young Athletes” study, we may draw 50cc (slightly more than 3 tablespoons) 
of  blood for DNA testing, and may also ask for a sample of saliva for DNA testing.  This will be an 
optional portion of the study, with a separate section of the consent forms to address this. 
 
MRI:  
We would like to assess SAT, VAT, thigh muscle area and marrow fat of AA prior to treatment and EA 
and non-athlete controls at some point during their 1 year study enrollment using MRI. 
 
Finite Element Analysis:  
FEA is an established analytical engineering technique to estimate stresses and strains induced by 
mechanical loading of complex structures, and is used to study bone strength in humans. Micro-finite 
element techniques applied to data from HR-pQCT strongly predict in vitro femoral and vertebral 
breaking strength independent of DXA-derived BMD (179, 180). Spine FEA differentiates patients with 
and without vertebral fractures (181) and hip FEA predicts new hip fractures independent of DXA-
derived BMD (182).  HR-pQCT is used for FEA using micro-finite element (µFE) techniques (183-185). 
In this context, FEA parameters correlate more strongly with failure load of the distal radius than either 
bone mass or microstructure (161) and clarify the structural mechanisms underlying fragility in subjects 
with fractures (159), including those occurring during the adolescent growth spurt (160). Of note, FEA 
estimates of bone strength have not been reported in female athletes, nor has FEA been utilized to monitor 
response to therapeutic intervention in this population. FEA will be assessed in 66 AA, 22 EA and 22 
controls at baseline and 60 AA, 20 EA and 20 controls (after anticipated drop-outs) at 12m. 
 
fMRI and Evaluation of Cognition, Mood, and Addictive Behaviors: 
Exercise can impact neural functions on many fronts, including decreasing anxiety and addictive 
behavior, and improving cognition. We would like to access a subset of 6 AAs, 6 EAs, and 6 HCs ages 
18-25 years. This will be an optional portion of the study, with a separate section of the consent form to 
address this. fMRI scanning will be performed using (i) the stop-signal task for assessing cognition and 
(ii) estroop task for assessing anxiety. The EAs and AAs will receive one fMRI san at baseline. The AAs 
will receive one scan at baseline and a follow-up scan at the 6 month visit. 
 
 


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Stop signal task: In this task, participants will categorize visual stimuli as fast as possible but are 
instructed to withhold a response when a stop signal appears on a randomly selected 25% of all trials. The 
time delay between the onset of the imperative stimulus and the stop signal will be adapted to the 
participants’ individual response times using a staircase tracking algorithm. A mathematical framework 
allows for estimating the duration of the stop process (“stop signal reaction time”) based on the 
distribution in the go trials as measure of duration of the inhibition process and therefore inhibition ability 
 
Estroop Task: For this task participants will view human faces with happy or fearful expression labeled as 
either ‘happy’ or ’fear’. Subjects then indicate the emotional expression on each face ignoring the words 
written across the faces. Reaction time and accuracy of response will be calculated. 
 
In addition, anxiety will be evaluated using State Trait Anxiety Inventory (STAI) scores and addictive 
behaviors will be assessed using Alcohol Use Disorder Identification Test (AUDIT) and Drug Abuse 
Screening Test-10 (DAST-10). 
 
Combined Screening and Baseline Visit 
 
For subjects who live or attend school far from Boston, we may offer to combine the first two visits, the 
screen and baseline. Because these visits occur within 8 weeks of each other, some subjects have 
difficulty with the time and expense necessary to come to Boston twice within this period. In the case of 
the combined visit, subjects will be phone-screened by a study co-investigator prior to scheduling the visit 
to make sure that inclusion criteria are met and exclusion criteria ruled out.  Some information regarding 
exclusion criteria that we will not be able to gather prior to the combined visit would be bone age < 14 
years, hematocrit<30% and an abnormal TSH level. Subjects with any history of thyroid disorder will not 
be eligible for the combined visit. Because these subjects will not be completing the overnight portion of 
the baseline visit involving frequent sampling, the primary reason for obtaining the CBC prior to this visit 
is no longer applicable. To ensure that bone age of subjects completing the combined visit is mature 
enough to continue study participation, we will restrict the combined visit to subjects who are of a 
chronological age of 16 years or older at the time of the visit, making it very likely that the bone age is > 
14 years.  Additionally, we will not proceed with the baseline visit if the x-ray of the wrist and hand at the 
combined visit indicates a bone age of <14 years. Study drug will be started only after inclusion/exclusion 
criteria have been confirmed at the combined visit. 
 
Subjects will complete all screening and baseline procedures at this visit. The subject will be consented 
and a complete medical history and physical exam will be performed before continuing with further study 
procedures. If subject is found to have any health concern or is found to meet any exclusion criterion 
during the history and physical, further study components will be canceled. Following this portion of the 
visit, the subject will complete the rest of the study procedures occurring at the screening and baseline 
visit, including: 
(a) Screening labs: complete blood count, K, ALT, AST, TSH, FSH (to rule out 
premature/primary ovarian failure), 30 cc of blood will be drawn, frozen and stored for later 
analysis 
(b) Fasting (or 2-hr post prandial)  calcium, phosphorus and 25(OH) vitamin D  
(c) Bone age (wrist and hand X-ray) and bone density using DXA: spine, hip and total body.  
(d) The Bouchard 3-day activity record (129) (to confirm criteria for endurance training are met). 
(e) Eating disorder questionnaires (EDI-2), Three-Factor Eating Questionnaire-R18 (TFEQ), and 
a 4-day food diary (to assess caloric intake) 
(f) Two subtests, Vocabulary and Matrix Reasoning, from the Weschler Abbreviated Scale of 
Intelligence (WASI), a Color-Word Interference Test subtest from the Delis Kaplan 
Executive Functioning Scale (DKEFS), and the California Verbal Learning Test (CVLT).  
(g) Resting energy expenditure (REE) using indirect calorimetry 
(h) Indirect calorimetry and handgrip strength. This exam will be performed at the Clinical 
Research Center at Massachusetts General Hospital or at the Charlestown Navy Yard site.  
(i) Bone microarchitecture at the ultradistal radius and leg (non-dominant) will be assessed using 
X-treme CT in a subset of 100 (~33 in each randomization group)AA,  36 EA, 36 controls 


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sometime between the screening and baseline visits or at the baseline visit. Finite element 
analysis (FEA) will also be performed. 
(j) MRI (regional fat and muscle) assessment. 
(k) Salivary cortisol at 11 p.m. to be sent back to MGH 
  
For the amenorrheic athletes, subjects will be randomized upon receiving results from the DXA scan, and 
verifying eligibility based upon the lab results and bone age x-ray. Subjects will start study medication as 
soon as possible.  
 
Early Termination Visit: This will include procedures performed at the 12 month visit. Subjects will be 
asked to return unused medications and all questionnaires. 
 
For studies involving treatment or diagnosis, provide information about standard of care at 
Partners (e.g., BWH, MGH) and indicate how the study procedures differ from standard care.  
Provide information on available alternative treatments, procedures, or methods of diagnosis.
 
No treatment thus far has been successful in increasing bone density in adolescent athletes with 
amenorrhea. A recent review of the literature for data regarding efficacy of OCPs and hormone 
replacement  in improving BMD in pre- and perimenopausal women (65) found only two randomized 
controlled trials (RCTs) that demonstrated a beneficial role of estrogen replacement on BMD in 
oligo/amenorrheic premenopausal women (66, 67), and one that reported no change (68). However, these 
studies did not target AA in normal weight adolescents/young adults, the population we plan to study, and 
the population where these data are needed. 
In the study by Hergenroeder et al that showed a beneficial effect of estrogen on BMD, only five 
amenorrheic women were randomized to estrogen replacement, and their mean weight was 79% of ideal 
(67), indicating that they were very low weight and likely had anorexia nervosa rather than AA. The 
authors noted an increase in BMD but did not adjust for weight changes over the study duration. Larger 
studies have not demonstrated significant changes in BMD with oral estrogen in low weight amenorrheic 
women with anorexia nervosa (69, 70), particularly after controlling for weight recovery. Therefore, 
results from the study by Hergenroeder et al cannot be extrapolated to teenagers and young women with 
AA without low weight, which is the population we plan to study, and for which there is a real dearth of 
data regarding efficacy of estrogen despite its widespread use. The study by Castelo-Branco et al was 
larger and showed a beneficial effect of estrogen replacement in women with hypothalamic 
oligomenorrhea, but women were self selected into the control arm not receiving estrogen, and the 
authors did not control for weight changes (66). The study by Gibson et al was a pilot study that showed 
small non-significant increases in BMD (68). Most retrospective studies in adult AA suggest a beneficial 
effect of estrogen replacement on BMD (71, 72), and a sparing effect of estrogen replacement on stress 
fractures (72, 73).  Other reported studies are either not controlled, have small numbers of subjects, do not 
specifically address AA, or adjust for confounders such as weight changes (10, 71, 74-77). Of importance, 
estrogen effects in an adolescent and young adult population that should be actively accruing bone are 
likely very different from those in adult AA who have achieved peak bone mass. Despite the importance 
of estrogen in bone accrual in the teenage years, no studies have investigated effects of estrogen 
replacement in estrogen deficient adolescent athletes. 
 
Describe how risks to subjects are minimized, for example, by using procedures which are 
consistent with sound research design and which do not unnecessarily expose subjects to risk or 
by using procedures already being performed on the subject for diagnostic or treatment purposes.
 
(i) Risks from procedures:  
There is a risk of superficial bruising and discomfort at the venepuncture site.  DXA and bone age 
evaluation involve minimal radiation (~0.02 mSv for entire study) - less than 1% of the background 
radiation received/person/year in the US. The effective radiation dose with X-treme CT is well within 


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safety limits of radiation exposure in children (0.027 mSv per measurement compared with 0.1 mSv with 
a chest X-ray). The total effective dose from all exams for the entire study is about 0.3 mSv which is 
about 3% of the effective dose (3mSv) everyone gets from the background radiation per year. 
A pregnancy test will be performed at all visits.  
(ii) Risks from medications: 
Estrogen administration is associated with minimal possible side effects, especially when given 
transdermally. Occasional side effects may occur, the commonest of which are mild headaches, and very 
rarely some irritation or redness at the application site. Adverse reactions that have been reported with 
estrogen therapy include abnormal bleeding patterns, changes in nature and quantity of vaginal discharge, 
tenderness and enlargement of breasts, nausea, vomitting, abdominal pain, bloating, gall bladder disease, 
rashes, intolerance to contact lenses, migraines, dizziness, depression, abnormal movements, and changes 
in weight. These adverse effects are, however, rare, and seen more commonly with the estrogen taken 
orally rather than transdermally. More serious side effects include hypertension, lipid and clotting 
disorders and a higher risk of breast cancer. These are more commonly seen in older women and in 
women with a family history of clotting disrders and cancer. In addition, we plan to administer estrogen 
to hypogoadal adolescents who are at an age when estrogen levels are steadily rising in healthy 
adolescents, a very different physiological state compared to post-menopausal women, a time of life when 
estrogen levels are expected to be low in these women.  Continuous estrogen administration without 
cyclic progesterone can predispose the uterus to cancer.  However, our subjects with AA who receive 
transdermal estrogen will also receive progesterone (Prometrium) 200 mg pills orally daily for the first 12 
days of every month, and subjects with AA receiving oral estrogen (0.030 mg ethinyl estradiol) will also 
receive concomitant progesterone (0.15 mg of desogestrel, a synthetic progesterone) daily for 21/28 days.  
We will monitor the subjects’ blood pressure at each visit and will determine through careful questioning 
and examination the appearance of any side-effect. 
We have carefully considered the possibility that estrogen administration may decrease bone 
density and may thus have negative effects on bone health, based on papers by Hartard et al (88) and Burr 
et al (89). The study by Hartard et al was retrospective (88), and it is possible that girls enrolled in this 
study were more likely to be prescribed oral contraceptives by their providers if they had a longer 
duration of amenorrhea or lower weight, and low weight is an important predictor of low bone density. 
We are planning to study young women and teenagers with normal weight who are amenorrheic, and we 
will monitor weight over the study duration. Burr et al studied healthy women who were not estrogen 
deficient, either exercisers or not exercisers, and examined the effects of exercise and estrogen use on 
bone density in a non-randomized controlled trial (89). Therefore, these data are not applicable to an 
estrogen deficient population. Of note, women receiving estrogen did better than those who exercised but 
did not get estrogen.  It is reassuring that other studies have demonstrated either some or no increase in 
BMD with use of oral estrogen, and none suggest that BMD decreases with estrogen use (12, 66, 72-78). 
In addition, our pilot preliminary data show that in six young women followed prospectively over 12 
months, estrogen use was associated with a significant increase in BMD. 
Bone density information will be presented to the DSMB at each meeting, and study termination 
will be considered for subjects demonstrating a greater than 10% decrease in lumbar spine bone density 
from baseline, if randomized to the estrogen arm. 
Estrogen administration in pregnancy may be teratogenic. We will thus perform a pregnancy test 
prior to administering estrogen and every three months. We will not administer estrogen to subjects with 
contraindications to estrogen therapy such as: 
• 
Known or suspected pregnancy 
• 
Undiagnosed abnormal uterine bleeding 
• 
History of clotting disorders such as thrombophlebitis and thromboembolic disorders 
• 
Known or suspected cancer of the breasts, uterus or ovaries 
• 
History of smoking (please see details described under Exclusion Criteria) 
All study subjects will be counseled regarding risks of pregnancy, and birth control measures will be 
discussed at each study visit (see handout to be given to subjects at study visits regarding contraceptive 
methods that are possible to use). Girls randomized to the arm receiving oral estrogen and progesterone 
(oral contraceptive pill) will also be cautioned about the risks of pregnancy with missed pills. Girls 
randomized to the estrogen patch arm will be cautioned that estrogen patches used in this study do not 
have contraceptive efficacy. We will also discuss with our subjects that study medications will not protect 


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against sexually transmitted diseases and that appropriate measures need to be adopted (use of barrier 
contraception) to prevent these diseases. 
Oral progesterone has been associated with clotting abnormalities, dizziness, nausea, abdominal 
pain, fatigue, headaches, insomnia, nervousness, sleepiness and breast tenderness and secretion.  Rare 
cases of breast cancer have been reported in women taking combined estrogen and progesterone.  Rare 
instances of abnormal liver function have also been reported.  The doses of progesterone that will be 
given are small and are less likely to cause these side effects. Particularly micronized progesterone 
(Prometrium) is associated with few adverse effects. The oral estrogen-progesterone preparation we plan 
to use is the generic form used for birth control, and most commonly used amongst adolescents. 
Oral calcium can cause constipation and a metallic taste in the mouth. 
Vitamin D in the doses being administered does not cause any side effects. 
Subjects will be instructed to call us if they develop any side-effects of the medication.  
Subsequent management will be decided based on the severity of the side-effect.  Serious side effects 
such as thrombosis will necessitate protocol discontinuation (described under ‘Criteria for discontinuing 
the protocol’).  If a subject develops a minor side effect like breakthrough bleeding, she will be asked to 
continue medications as per the protocol.  Worsening of symptoms may necessitate temporarily 
withholding medication until other potential causes of symptoms are ruled out.  Withholding medication 
for longer than two months will necessitate discontinuation of the study. 
A physician will be available by pager at all times during the study to answer any questions or address 
any concerns that a subject may have. All efforts will be made to protect the confidentiality of study 
subjects who will be referred to by an enrollment number only. 
 
Describe explicitly the methods for ensuring the safety of subjects.  Provide objective criteria for 
removing a subject from the study, for example, objective criteria for worsening disease/lack of 
improvement and/or unacceptable adverse events.  The inclusion of objective drop criteria is 
especially important in studies designed with placebo control groups.
 
One central Data Safety Monitoring Board (DSMB) will be convened at MGH every six months to 
review all adverse events.  Moreover, additional meetings may be called to consider urgent issues.  Bone 
density information will be presented to the DSMB at each meeting, and study termination will be 
considered for subjects demonstrating a greater than 10% decrease in lumbar spine bone density from 
baseline, if randomized to the estrogen arm. 
 
1. A serious adverse event related to use of estrogen, e.g. thrombosis.  Details will be reported to the 
DSMB and to the primary care provider of the subject.  The safety of estrogen use in this population will 
be reassessed. 
2. Pregnancy 
3. Development of other disorders that may affect bone metabolism during the study 
4. Therapeutic use of medications that may affect bone metabolism during the study period 
5. > 10% decrease in BMD in subjects randomized to estrogen as determined by the DSMB 
 
FORESEEABLE RISKS AND DISCOMFORTS 
Provide a brief description of any foreseeable risks and discomforts to subjects.  Include those 
related to drugs/devices/procedures being studied and/or administered/performed solely for 
research purposes.  In addition, include psychosocial risks, and risks related to privacy and 
confidentiality.  When applicable, describe risks to a developing fetus or nursing infant.
Please see page 6 
 
EXPECTED BENEFITS 
Describe both the expected benefits to individual subjects participating in the research and the 
importance of the knowledge that may reasonably be expected to result from the study.  Provide 


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a brief, realistic summary of potential benefits to subjects, for example, “It is hoped that the 
treatment will result in a partial reduction in tumor size in at least 25% of the enrolled subjects.”  
Indicate how the results of the study will benefit future patients with the disease/condition being 
studied and/or society, e.g., through increased knowledge of human physiology or behavior, 
improved safety, or technological advances. 
 
Potential Benefits include an increase in BMD in AA girls who are randomized to estrogen and a 
better understanding of the risk for low BMD from prolonged amenorrhea in other AA girls. In addition, 
use of calcium and vitamin D may cause an increase in BMD in all subjects. 
Given that (i) there is no available treatment for low BMD in AA, (ii) oral estrogen is not effective 
in increasing BMD in adult with AA, and (iii) no studies have examined the effect of transdermal 
estrogen (not IGF-I suppressive) on BMD in AA, we believe that equipoise does exist in this study.
 
EQUITABLE SELECTION OF SUBJECTS 
The risks and benefits of the research must be fairly distributed among the populations that stand 
to benefit from it.  No group of persons, for example, men, women, pregnant women, children, 
and minorities, should be categorically excluded from the research without a good scientific or 
ethical reason to do so.  Please provide the basis for concluding that the study population is 
representative of the population that stands to potentially benefit from this research.
 
Inclusion of Women and Minorities:  We will enroll only female adolescents and young adult 
women to optimize power and to avoid issues rising from deficiencies of different predominant sex 
hormones in boys (testosterone) vs. girls (estrogen). Adolescents will be studied in order to determine 
effects on bone density and its predictors in a population that should be actively accruing bone. 
 Inclusion of Children: During adolescence and the young adult years, more than 90% of bone 
mass is formed, making the study of bone accretion and factors which interfere with normal bone accretion 
during this time particularly important. We are therefore investigating changes in bone and whole body 
metabolism in normal girls, EA and girls with AA ages 14-25 years, and the effects of estrogen 
administration on bone in AA.  Because 99% of adult height has been achieved by the time the bone age is 
15 years or more, estrogen administration will not compromise adult height in the group receiving this.  
Our preliminary data indicate that estrogen use is not harmful in young adult women 14-25 years old. All 
subjects will be included in the study only after consent forms have been signed (further details are 
described under ‘Study Population’).  
 
When people who do not speak English are excluded from participation in the research, provide 
the scientific rationale for doing so.  Individuals who do not speak English should not be denied 
participation in research simply because it is inconvenient to translate the consent form in 
different languages and to have an interpreter present.
 
People who do not speak English will not be excluded from study participation. If a patient who does not 
speak English should wish to participate in our studies, a consent form in the patient’s language will be 
prepared and an interpreter will be made available at the study visits. 
 
For guidance, refer to the following Partners policy: 
          Obtaining and Documenting Informed Consent of Subjects who do not Speak English
          http://healthcare.partners.org/phsirb/nonengco.htm 
 
 


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RECRUITMENT PROCEDURES 
Explain in detail the specific methodology that will be used to recruit subjects.  Specifically 
address how, when, where and by whom subjects will be identified and approached about 
participation.  Include any specific recruitment methods used to enhance recruitment of women 
and minorities.
 
Recruitment for the study will be carried out through mailings to pediatricians, nutritionists, 
therapists, sports medicine specialists, high schools and colleges in the New England area, and through 
advertisements in local newspapers and magazines.   
Subjects will be recruited over a four year period, thus at the rate of about ~ 40 subjects per year 
(27 AA, 12 EA and healthy adolescents). The accrual goal is reasonable given systems of referral already 
in place from our studies in girls with anorexia nervosa. We have collaborations ongoing with primary 
care providers, psychiatrists, therapists, dieticians and adolescent medicine physicians in the New 
England area for patients with eating disorders and the female athlete triad. Our group informs these 
providers about new studies, and providers then present studies to their patients who contact our group if 
interested in study participation. We have been very successful in our recruitment efforts in girls with 
anorexia nervosa, and expect recruitment of AA to be easier given that prevalence of female athletes is 
higher than that of anorexia nervosa, and because AA girls have fewer psychiatric co-morbidities 
compared with girls with anorexia nervosa. In addition, a co-investigator in this grant is Dr. Gary Skrinar, 
who is a very well known for his work with athletes. Dr. Skrinar was until recently a Professor in the 
Department of Health Sciences at the Sargent College of Health & Rehabilitation Sciences, Boston 
University, a member and Fellow of the American College of Sports Medicine and a Certified ACSM 
program Director. Given Dr. Skrinar’s expertise in this field and access to providers looking after athletes, 
we anticipate recruitment to be steady and to be completed in the specified period. Mailings will also be 
sent out to our regional high schools. In addition to mailing lists already in place, we are preparing 
mailing lists of area Sports Medicine specialists who are most likely to see young athletes. We anticipate 
completing enrollment in four years; such that the last subject will complete the one-year follow up by the 
end of the fifth year. We anticipate another month will be required by the Core Laboratory to run samples 
for secondary endpoints, and two months for data analysis. Three months will be necessary to write up 
results of the study. Study duration is thus anticipated to be 5.5 years. 
Recruitment of males will be carried out through mailings to pediatricians, therapists, 
dieticians, sports and adolescent medicine specialists, high schools and colleges in New England, and 
advertisements in local newspapers and magazines.  Subjects will be recruited over 11 months at the 
rate of 5-6 subjects/month. The accrual goal is reasonable given referral systems in place from our 
studies in young athletes. We have collaborations ongoing with primary care providers, psychiatrists, 
therapists, dieticians and sports and adolescent medicine physicians in New England for patients with 
eating disorders and athletes. Our group informs providers about new studies, and providers then 
present the studies to patients who contact our group if interested in participation. We will inform our 
referral pool of this addition to the original study. We will also send information to local teams and 
coaches. We anticipate that a month will be required to run blood samples for secondary endpoints, 
and for data analysis. Study duration is anticipated to be 1 year. 
 
Provide details of remuneration, when applicable.  Even when subjects may derive medical 
benefit from participation, it is often the case that extra hospital visits, meals at the hospital, 
parking fees or other inconveniences will result in additional out-of-pocket expenses related to 
study participation.  Investigators may wish to consider providing reimbursement for such 
expenses when funding is available
 
Subjects will receive a $125 stipend for the baseline, 6 month, and 12 month visits and a $75 stipend 
for the 3 month and 9 month visits, $50 for baseline and 12 month MRI ($100 total), and $100 for 
optional fMRI visit ($100 for EAs and HCs, $200 for AAs completing both scans).  Parking at MGH 


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will be offered at no cost; transportation costs to Massachusetts General Hospital will be reimbursed 
up to $25. 
 
 
For guidance, refer to the following Partners policies: 
          Recruitment of Research Subjects 
          http://healthcare.partners.org/phsirb/recruit.htm
 
          Guidelines for Advertisements for Recruiting Subjects
          http://healthcare.partners.org/phsirb/advert.htm
 
          Remuneration for Research Subjects
          http://healthcare.partners.org/phsirb/remun.htm
 
 
CONSENT PROCEDURES 
Explain in detail how, when, where, and by whom consent is obtained, and the timing of consent 
(i.e., how long subjects will be given to consider participation).  For most studies involving more 
than minimal risk and all studies involving investigational drugs/devices, a licensed physician 
investigator must obtain informed consent.  When subjects are to be enrolled from among the 
investigators’ own patients, describe how the potential for coercion will be avoided.
 
Written, informed consent will be obtained from subjects ≥ 18 years old. For subjects 14-17 years 
old, consent will be obtained from one parent, and assent from the subject. Consent forms, data safety 
monitoring plan and protocol will be per Institutional Review Board guidelines. 
 
NOTE: When subjects are unable to give consent due to age (minors) or impaired decision-
making capacity, complete the forms for Research Involving Children as Subjects of Research 
and/or Research Involving Individuals with Impaired Decision-making Capacity, available on 
the New Submissions page on the PHRC website: 
http://healthcare.partners.org/phsirb/newapp.htm#Newapp
 
For guidance, refer to the following Partners policy: 
          Informed Consent of Research Subjects
          http://healthcare.partners.org/phsirb/infcons.htm
 
 
 
DATA AND SAFETY MONITORING 
Describe the plan for monitoring the data to ensure the safety of subjects.  The plan should 
include a brief description of (1) the safety and/or efficacy data that will be reviewed; (2) the 
planned frequency of review; and (3) who will be responsible for this review and for determining 
whether the research should be altered or stopped.  Include a brief description of any stopping 
rules for the study, when appropriate.  Depending upon the risk, size and complexity of the 
study, the investigator, an expert group, an independent Data and Safety Monitoring Board 
(DSMB) or others might be assigned primary responsibility for this monitoring activity.        
 


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NOTE: Regardless of data and safety monitoring plans by the sponsor or others, the principal 
investigator is ultimately responsible for protecting the rights, safety, and welfare of subjects 
under his/her care. 
 
One central Data Safety Monitoring Board (DSMB) will be convened at MGH every six months to 
review all adverse events.  Moreover, additional meetings may be called to consider urgent issues.  Bone 
density information will be presented to the DSMB at each meeting, and study termination will be 
considered for subjects demonstrating a greater than 10% decrease in lumbar spine bone density from 
baseline, if randomized to the estrogen arm. 
• Ellen O’Donnell, Ph.D., Licensed Clinical Psychologist, Massachusetts General Hospital, 
Instructor, Harvard Medical School  
• Soja Park-Bennett, M.D., Pediatric Endocrine Unit, Massachusetts General Hospital 
• Mariette Murphy, MD, Adolescent Medicine physician, Massachusetts General Hospital 
• Jean Mulder, MD, (expert on metabolic bone disease), Division of Endocrinology, Brigham and 
Women’s Hospital 
• Brian Healy, PhD, Biostatistician, Massachusetts General Hospital 
All data will be recorded on specified Case Report Forms.   
 
Adverse events or other unanticipated problems will be reported to the PHRC as described in the PHRC policy 
on ‘Adverse Event Reporting and Unanticipated Problems Involving Risks to Subjects or Others.  
 
In addition, semi-annual data review and evaluation of side effects will be conducted. Results of the semi-annual 
review will be forwarded to the Partners IRB. 
 
Describe the plan to be followed by the Principal Investigator/study staff for review of adverse 
events experienced by subjects under his/her care, and when applicable, for review of sponsor 
safety reports and DSMB reports.  Describe the plan for reporting adverse events to the sponsor 
and the Partners’ IRB and, when applicable, for submitting sponsor safety reports and DSMB 
reports to the Partners’ IRBs.  When the investigator is also the sponsor of the IND/IDE, include 
the plan for reporting of adverse events to the FDA and, when applicable, to investigators at 
other sites.   
 
NOTE: In addition to the adverse event reporting requirements of the sponsor, the principal 
investigator must follow the Partners Human Research Committee guidelines for Adverse Event 
Reporting
 
Adverse events or other unanticipated problems will be reported to the PHRC as described in the PHRC policy 
on ‘Adverse Event Reporting and Unanticipated Problems Involving Risks to Subjects or Others. 
 
MONITORING AND QUALITY ASSURANCE 
Describe the plan to be followed by the principal investigator/study staff to monitor and assure 
the validity and integrity of the data and adherence to the IRB-approved protocol.  Specify who 
will be responsible for monitoring, and the planned frequency of monitoring.  For example, 
specify who will review the accuracy and completeness of case report form entries, source 
documents, and informed consent.   
 
NOTE: Regardless of monitoring plans by the sponsor or others, the principal investigator is 
ultimately responsible for ensuring that the study is conducted at his/her investigative site in 


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accordance with the IRB-approved protocol, and applicable regulations and requirements of the 
IRB.
 
Specimens: Blood specimens and all other testing will be obtained from subjects for research purposes 
only. From the study visits, most blood samples will be frozen and stored in freezers in the 
Neuroendocrine Unit of Massachusetts General Hospital. These freezers are accessible to no one but the 
Neuroendocrine staff. Some blood samples will be sent to the chemistry laboratories of MGH for 
analysis, and left over samples will be disposed of as per institutional guidelines. 
 
The privacy of subjects and confidentiality of data obtained from subjects will be maintained strictly. In 
any reports or publications resulting from the study, the privacy and anonymity of individuals in the 
report will be protected. The subject's name will be removed as markers on any clinical reports submitted 
as attachments to an adverse event report. Subjects will be assigned a code, which will be used in all data 
used for analysis. 
 
Adverse Events: Adverse events or other unanticipated problems will be reported to the PHRC as 
described in the PHRC policy on ‘Adverse Event Reporting and Unanticipated Problems Involving Risks 
to Subjects or Others 
 
Data Quality Control:  Accuracy of data entry will be checked using different methods at the beginning 
of the study and later when site monitoring has started. At the start of the study, before the first site 
monitoring data is returned, the coordinator will check 25 random data items on the database against the 
case records each month. If the error rate of data entry is worse than 2.5% overall or appears in crucial 
variables we will consider that a “data entry problem” exists and initiate whatever corrective action is 
necessary. One corrective action would be to initiate double entry of all data.   
 
For guidance, refer to the following Partners policies: 
          Data and Safety Monitoring Plans and Quality Assurance
          http://healthcare.partners.org/phsirb/datasafe.htm
 
          Adverse Event Reporting Guidelines
          http://healthcare.partners.org/phsirb/adverse.htm
 
 
 
PRIVACY AND CONFIDENTIALITY 
Describe methods used to protect the privacy of subjects and maintain confidentiality of data 
collected.  This typically includes such practices as substituting codes for names and/or medical 
record numbers; removing face sheets or other identifiers from completed 
surveys/questionnaires; proper disposal of printed computer data; limited access to study data; 
use of password-protected computer databases; training for research staff on the importance of 
confidentiality of data, and storing research records in a secure location.   
 
NOTE: Additional measures, such as obtaining a Certificate of Confidentiality, should be 
considered and are strongly encouraged when the research involves the collection of sensitive 
data, such as sexual, criminal or illegal behaviors.
 
Most blood samples will be frozen and stored in freezers in the Neuroendocrine Unit of Massachusetts 
General Hospital. These freezers are accessible to no one but the Neuroendocrine staff.  Some blood 


Partners Human Subjects Research Application Form  
 
Filename: Protocol Summary 
Version Date:  June 1, 2005 
 
  
18 
samples will be sent to the chemistry and hematology laboratories of MGH for analysis, and left over 
samples will be disposed of as per institutional guidelines.  
 
The privacy of subjects and confidentiality of data obtained from subjects will be maintained strictly. In 
any reports or publications resulting from the study, the privacy and anonymity of individuals in the 
report will be protected. The subject's name will be removed as markers on any clinical reports submitted 
as attachments to an adverse event report. Subjects will be assigned a code, which will be used in all data 
used for analysis. 
 
SENDING SPECIMENS/DATA TO RESEARCH COLLABORATORS OUTSIDE 
PARTNERS 
Specimens or data collected by Partners investigators will be sent to research collaborators 
outside Partners, indicate to whom specimens/data will be sent, what information will be sent, 
and whether the specimens/data will contain identifiers that could be used by the outside 
collaborators to link the specimens/data to individual subjects.
 
Not applicable 
 
Specifically address whether specimens/data will be stored at collaborating sites outside 
Partners for future use not described in the protocol.  Include whether subjects can withdraw 
their specimens/data, and how they would do so.  When appropriate, submit documentation of 
IRB approval from the recipient institution.
 
Specimens and data will not be stored at collaborating sites outside Partners. 
 
RECEIVING SPECIMENS/DATA FROM RESEARCH COLLABORATORS OUTSIDE 
PARTNERS 
When specimens or data collected by research collaborators outside Partners will be sent to 
Partners investigators, indicate from where the specimens/data will be obtained and whether the 
specimens/data will contain identifiers that could be used by Partners investigators to link the 
specimens/data to individual subjects.  When appropriate, submit documentation of IRB 
approval and a copy of the IRB-approved consent form from the institution where the 
specimens/data were collected.
 
No specimens or data will be collected by research collaborators outside Partners.
 

SAP_001.pdf: 
STATISTICAL ANALYSIS PLAN 
Title: Fat Mediated Modulation of Reproductive and Endocrine Function in Young Athletes 
 
NCT00946192 
 
Version Date: December 4, 2014 
 
 
 


Statistical Analysis Plan 
Biostatistical analysis: Hang Lee, Ph.D., a collaborator on this grant, is our CRC statistician and has 
co-authored many papers with us and other members of the endocrine division. Dr. Hang Lee and Dr. 
David Schoenfeld, Head of the Biostatistics Core, have been instrumental in study design and power 
analysis. Dr. Lee will also analyze the data at study completion. Analysis and interpretation of specific 
sub-aims are included in each section of the grant. Data generated will be largely longitudinal. Measures 
of hormone function, bone turnover, nutrition, activity and body composition will be collected every 6 
months. In addition, there will be baseline variables.  Subject groups include: AA randomized to 
transdermal estrogen, AA randomized to oral estrogen, AA randomized to observation, EA and controls.  
Randomization will be performed centrally by our research pharmacy. 
The first step in data analysis is to determine models for longitudinal data collected. These models 
are usually based on our knowledge of how these data behave and details of the study. These models can 
also be checked to see whether they fit the data by viewing data for each patient without regard to 
patient group.  For instance, BMD can be assumed to increase linearly over the study duration. We 
hypothesize that this linear increase will be slowest in untreated AA patients and intermediate in those 
on oral estrogen, and the smallest effect size would be the difference between groups randomized to 
transdermal versus oral estrogen. We would check this linearity by looking at data from individual 
patients and checking that actual measurements are normally distributed around the patient’s linear 
trajectory by looking at residuals (difference between observed value and trend line).  It is possible that 
in some cases linearity and normality might hold after a log or other transformation. If linearity holds for 
actual or transformed values, variables will be analyzed by mixed model ANOVA, a common method 
for analyzing longitudinal data.  One advantage of this method is that it allows us to use data from 
subjects that drop out early. The linear model is appropriate for BMD, but will not be appropriate for 
variables that may be relatively constant during treatment. These might include measurements of bone 
turnover and hormones. These would be modeled assuming that each patient has a constant level. After 
examining how well the assumed model fits the data, repeated measures ANOVA will be used for 
analysis.  In this model there is a fixed treatment term of primary interest. Measurements at each time 
are assumed to be a multivariate observation with an unspecified variance covariance matrix.  
Power calculation for primary endpoint: Power analysis is based on the primary endpoint, i.e. 
average change in BMD over one year in a randomized trial comparing mean change in spine BMD in 
young women 14-25 years old with AA randomized to transdermal estrogen versus oral estrogen or no 
therapy for one year.  Girls with AA will be stratified based on spine BMD Z-scores ( -1 or > -1).   
Our preliminary data indicate a relative increase in spine BMD of 4.7% over one year with 
transdermal estrogen vs. no therapy. In addition, one study in AA women has reported a 1.5% relative 
increase in BMD in AA women following oral estrogen vs. placebo. Our preliminary data indicate that 
the SD for change in lumbar BMD over a year is 2.7%. Based on these data, using the Dunnett’s test, a 
total of 99 AA (33 each in the two treatment arms no-treatment arm) will be needed to detect a 
difference of 3.2% (4.7%-1.5%) in mean %increase in spine BMD in groups receiving transdermal vs. 
oral estrogen, with a power of 80%, using a two sided p=0.05 level test.  
The estimated sample size of 99 subjects with AA does not take into account study drop-outs. Based 
on previous studies, we expect an 8- 10% drop-out rate over one year. We thus estimate that we will 
need to recruit 109 subjects with AA (37 in the transdermal estrogen arm, and 36 each in the oral 
estrogen and no-treatment arms) to have 99 evaluable subjects at the end of the study. An independent 
Data and Safety Monitoring Board (consisting of an adolescent medicine physician, a statistician, a bone 


expert, a pediatric endocrinologist and a psychiatrist) will review data regarding adverse events, study 
deviations and violations at study initiation and every six months. BMD information will be presented to 
the DSMB at each meeting, and study termination considered for subjects demonstrating >10% decrease 
in spine BMD from baseline, if randomized to estrogen.