Clinical trial NCT02255175
Conditions: Perimenopausal Depression
Keywords: Reproductive Affective Disorder Depression Perimenopause Estrogen Estradiol treatment Mood Disorders Estrogen Replacement Therapy Sex Steroids Depressive disorder Mental Disorders Estradiol Hormones Physiological effects of drugs Reproductive Control Agents
Sponsor: University of North Carolina, Chapel Hill
Start date: 2015-10-01; Completion date: 2018-10-17
Enrollment: 64 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Using neuroimaging, the investigator will study the effects of estrogen on mood and brain function in perimenopausal women either with or without depression.

Detailed description:
Despite decades of research, affective disorders are prevalent and associated with significant morbidity and mortality. Unraveling the pathophysiology of affective disorders has been uniquely challenging because depressive syndromes are heterogeneous and have diverse etiologies. Thus, past studies aimed at identifying neural and genetic biomarkers that would improve the prediction of susceptibility, course of illness, and treatment response have yielded inconsistent results. The investigator proposes to address this problem by studying perimenopausal major depressive disorder (MDD), a depression subtype with a specific endocrine trigger (i.e., ovarian hormone withdrawal). Evidence supporting ovarian hormone withdrawal as a trigger for affective dysfunction in perimenopausal MDD includes the following: perimenopausal women show a temporal association between ovarian hormone withdrawal and the onset of mood symptoms; treatment with estrogen reduces mood symptoms; and blinded estradiol withdrawal re-precipitates depression in women with a history of perimenopausal MDD (manuscript in preparation). Focusing on perimenopausal MDD, a more homogeneous subtype with a specific endocrine trigger, will increase the likelihood of identifying meaningful neurobiological markers.One of the most powerful tools for understanding the neural mediators of MDD is brain imaging. Prior research suggests that the frontostriatal reward system is regulated by estradiol and implicated in MDD. However, neural mechanisms of perimenopausal MDD have never been studied. We will assess the neural reward system in perimenopausal women with and without MDD using functional magnetic resonance imaging (fMRI) at baseline and following estradiol treatment. The central hypothesis is that the neural reward system is hypoactive in perimenopausal MDD, and the antidepressant effects of a three-week transdermal estradiol intervention will be mediated by increased activity in the neural reward system, assessed using fMRI. The investigator will test the hypothesis by executing the following aims:

Aim 1: To measure the frontostriatal response to reward in perimenopausal MDD and test the effects of estradiol on neural activation in perimenopausal women. The investigator will use fMRI at baseline and following estradiol treatment in women with and without MDD to probe frontostriatal reward circuitry.

Aim 2: To quantify motivated behavior at baseline and following estradiol administration in perimenopausal women with and without MDD. Motivated behavior will be operationally defined as the response latency to reward versus non-reward during the fMRI reward task.

Aim 3: To measure the psychological correlates of the frontostriatal response to reward in women with perimenopausal MDD at baseline and following estradiol administration. Depressive symptoms will be assessed at baseline and following estradiol administration.

The results will provide critical information about the neuroendocrine pathophysiology of perimenopausal depression and may subsequently contribute to the development of novel pharmacologic interventions.

Arms: arm 1: Participants will receive transdermal estradiol (100μg/day) for 3 weeks. Participants will receive an additional week of combined estradiol and micronized progesterone (200 mg/day) at the end of the study to precipitate menstruation. arm 2: Participants will receive transdermal estradiol (100μg/day) for 3 weeks. Participants will receive an additional week of combined estradiol and micronized progesterone (200 mg/day) at the end of the study to precipitate menstruation.
Interventions: intervention 1: Estradiol intervention 2: Progesterone
Intervention descriptions: intervention 1: Participants will receive transdermal estradiol (100μg/day) for 3 weeks intervention 2: Participants will receive an additional week of combined estradiol and micronized progesterone (200 mg/day) at the end of the study to precipitate menstruation.

Protocol documents:
Prot_SAP_000.pdf: 
 
Effects of Estradiol on Neural Reward System and Depression in the 
Perimenopause (PEERS) 
 
 
 
NCT number 
NCT02255175 
Document Date 
4/12/2016 


 
CONFIDENTIAL 
CLINICAL RESEARCH PROTOCOL 
 
 
 
 
 
 
 
 
AN OBSERVATIONAL, CASE-CONTROL, SINGLE-CENTER 
STUDY TO DETERMINE THE PERIMENOPAUSAL EFFECTS 
OF ESTRADIOL ON REWARD RESPONSIVENESS (PEERS) 
 
 
 
 
Regulatory Sponsor: 
David R. Rubinow, MD 
UNC Department of Psychiatry 
10514 Neurosciences Hospital, 101 Manning Drive  
Campus Box 7160  
Chapel Hill, NC  
27599-7160  
919-966-4738  
 
Funding Sponsor: 
Eunice Kennedy Shriver National Institute of Child Health and 
Human Development (NICHD) 
31 Center Drive 
Building 31, Room 2A32 
Bethesda, MD 20892-2425 
 
Study Product: 
Estradiol  
Active ingredient: micronized estradiol  
Chemical name: estra-1,3,5,(10)-triene-3, 17β-diol 
 
Progesterone  
Active ingredient: micronized progesterone 
Chemical name: pregn-4-ene-3, 20-dione 
 
Protocol Number: 
13-3572 
IND Number: 
N/A 
 
Verion 0.1: 
9/9/14 
Version 0.2: 
10/17/2014 
Version 1.0: 
9/14/2015 
Version 2.0:       4/12/2016 
 
 
 
 
 
 


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Table of Contents 
 
STUDY SUMMARY ................................................................................................................................. 1 
1 
INTRODUCTION ............................................................................................................................... 3 
1.1 
BACKGROUND .............................................................................................................................. 3 
1.2 
INVESTIGATIONAL AGENT ............................................................................................................... 4 
1.3 
PRECLINICAL DATA ....................................................................................................................... 6 
1.4 
CLINICAL DATA TO DATE................................................................................................................ 7 
1.5 
DOSE RATIONALE AND RISK/BENEFITS ............................................................................................ 8 
2 
STUDY OBJECTIVES ....................................................................................................................... 8 
3 
STUDY DESIGN ................................................................................................................................ 9 
3.1 
GENERAL DESIGN ......................................................................................................................... 9 
3.2 
PRIMARY STUDY ENDPOINTS ....................................................................................................... 12 
3.3 
SECONDARY STUDY ENDPOINTS .................................................................................................. 12 
3.4 
PRIMARY SAFETY ENDPOINTS ...................................................................................................... 12 
4 
SUBJECT SELECTION AND WITHDRAWAL ................................................................................. 12 
4.1 
INCLUSION CRITERIA ................................................................................................................... 12 
4.2 
EXCLUSION CRITERIA .................................................................................................................. 12 
4.3 
SUBJECT RECRUITMENT AND SCREENING...................................................................................... 13 
4.4 
EARLY WITHDRAWAL OF SUBJECTS .............................................................................................. 14 
5 
STUDY DRUG ................................................................................................................................. 15 
5.1 
DESCRIPTION ............................................................................................................................. 15 
5.2 
TREATMENT REGIMEN ................................................................................................................. 15 
5.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ......................................................... 16 
5.4 
PREPARATION AND ADMINISTRATION OF STUDY DRUG .................................................................... 16 
5.5 
SUBJECT COMPLIANCE MONITORING ............................................................................................ 16 
5.6 
PRIOR AND CONCOMITANT THERAPY ............................................................................................ 16 
5.7 
PACKAGING................................................................................................................................ 16 
5.8 
BLINDING OF STUDY DRUG (IF APPLICABLE) ................................................................................... 16 
5.9 
RECEIVING, STORAGE, DISPENSING AND RETURN .......................................................................... 16 
6 
STUDY PROCEDURES .................................................................................................................. 17 
7 
STATISTICAL PLAN ....................................................................................................................... 18 
7.1 
SAMPLE SIZE DETERMINATION ..................................................................................................... 18 
7.2 
STATISTICAL METHODS ............................................................................................................... 18 
7.3 
SUBJECT POPULATION(S) FOR ANALYSIS ....................................................................................... 18 
8 
SAFETY AND ADVERSE EVENTS ................................................................................................. 18 
8.1 
DEFINITIONS .............................................................................................................................. 18 
8.2 
RECORDING OF ADVERSE EVENTS................................................................................................ 20 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS ................................... 20 
8.4 
UNBLINDING PROCEDURES .......................................................................................................... 21 
8.5 
STOPPING RULES ....................................................................................................................... 22 
8.6 
MEDICAL MONITORING ................................................................................................................ 23 
9 
DATA HANDLING AND RECORD KEEPING .................................................................................. 24 
9.1 
CONFIDENTIALITY ....................................................................................................................... 24 
9.2 
SOURCE DOCUMENTS ................................................................................................................. 24 
9.3 
CASE REPORT FORMS (AS APPLICABLE) ........................................................................................ 24 
9.4 
RECORDS RETENTION ................................................................................................................. 24 


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10 
STUDY MONITORING, AUDITING, AND INSPECTING ................................................................ 25 
10.1 
STUDY MONITORING PLAN ......................................................................................................... 25 
10.2 
AUDITING AND INSPECTING ........................................................................................................ 25 
11 
ETHICAL CONSIDERATIONS ...................................................................................................... 24 
12 
STUDY FINANCES ....................................................................................................................... 25 
12.1 
FUNDING SOURCE .................................................................................................................... 25 
12.2 
CONFLICT OF INTEREST ............................................................................................................. 25 
12.3 
SUBJECT STIPENDS OR PAYMENTS ............................................................................................. 26 
13 
PUBLICATION PLAN.................................................................................................................... 26 
14 
REFERENCES .............................................................................................................................. 26 
 
 
 


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List of Abbreviations  
 
AE 
Adverse Event 
BOLD 
Blood-oxygen-level-dependent 
BRIC 
Biomedical Research Imaging Center 
CHD 
Coronary heart disease 
CRF 
Case report form 
DSMB 
Data and Safety Monitoring Board 
DVT 
Deep vein thrombosis 
EC 
Ethics Committee 
EPDS 
Edinburgh Postnatal Depression Scale 
EPT 
Combined estrogen and progestin treatment (EPT) 
FDA 
Food and Drug Administration 
fMRI 
Functional magnetic resonance imaging 
FSL 
Brain imaging analysis software 
GnRH 
Gonadotropin-releasing hormone 
HIPAA 
Health Insurance Portability and Accountability Act 
HRSD 
Hamilton Rating Scale for Depression 
IDAS 
Inventory of Depression and Anxiety Symptoms 
IDS 
Investigational Drug Service 
IM 
Intramuscular 
IRB 
Institutional Review Board  
IVF 
In vitro fertilization 
LH 
Luteinizing hormone 
MASQ-AD 
Mood and Anxiety Symptom Questionnaire – Anhedonic Depression Subscale 
MATLAB 
An interactive environment for numerical computation, visualization, and programming 
MID 
MDD 
Monetary Incentive Delay (fMRI task) 
Major Depressive Disorder 
PHI 
Protected health information 
PPD 
Postpartum depression 
ROI 
Region of interest 
SAE 
Serious Adverse Event 
SCID 
Structured Clinical Interview for DSM-IV-TR Axis-I Disorders 
STRAW 
Stages of Reproductive Aging Workshop 
UNC-CH 
University of North Carolina at Chapel Hill  
WHI 
Women’s Health Initiative 
 


Neurophysiology of Postpartum Depression 
 
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Study Summary 
Title 
Perimenopausal Effects of Estradiol on Reward Responsiveness  
Short Title 
PEERS 
Protocol Number 
IRB# 13-3572 
Phase 
N/A  
Methodology 
Experimental, case-control study  
Study Duration 
12-24 months 
Study Center(s) 
Single-center 
Objectives 
There are three practical objectives: 
1. To measure the frontostriatal response to reward in perimenopausal 
MDD and test the effects of estradiol on neural activation in 
perimenopausal women. We will use fMRI at baseline and following estradiol 
treatment in women with and without MDD to probe frontostriatal reward 
circuitry. Implementation of this protocol will provide hands-on training in fMRI 
research, the experimental manipulation of sex steroids in humans, and the 
interpretation of fMRI results based on a pharmacologic manipulation. 
2. To quantify motivated behavior at baseline and following estradiol 
administration in perimenopausal women with and without MDD. 
Motivated behavior will be operationally defined as the response latency to 
reward versus non-reward during the fMRI reward task. Pursuit of this aim will 
provide training in behavioral markers of psychopathology in the context of 
fMRI. 
3. To measure the psychological correlates of the frontostriatal response 
to reward in women with perimenopausal MDD at baseline and following 
estradiol administration. Depressive symptoms will be assessed at baseline 
and following estradiol administration. Training associated with this aim will 
include assessing associations between neural activity and psychological 
symptoms. 
Number of Subjects 
50 
Diagnosis and Main 
Inclusion Criteria 
Group 1: Women with Perimenopausal Depression 
1)    2 skipped cycles and an interval of amenorrhea  60 days, consistent with 
the late menopause transition (stage -1), and who demonstrate an FSH level 
higher than two SDs above the mean for follicular phase premenopausal 
women based on UNC Hospitals laboratory levels (x= 7 IU/mL); 2) 44-55 years 
old; 3) current diagnosis of MDD with an onset associated with menstrual cycle 
irregularity, and no history of psychiatric illness during the 2 years before the 
onset of the current depressive episode as determined by the Structure Clinical 
Interview for DSM-IV-TR for Axis I Disorders (SCID) 
 
Group 2: Healthy Controls 
1) Controls will meet all inclusion criteria specified above except they must not 
have any past or present psychiatric disorder assessed by the SCID 
Study Product, Dose, 
Route, Regimen 
Study Product: 
Estradiol  
Active ingredient: micronized estradiol  
Chemical name: estra-1,3,5,(10)-triene-3, 17β-diol 
Progesterone  
Active ingredient: micronized progesterone 
Chemical name: pregn-4-ene-3, 20-dione 


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Dose:  
Drug 
Dosage 
Formulation 
Estradiol 
100 micrograms/day 
Transdermal  
Progesterone 
200 miligrams/day 
Oral capsule 
 
Route: 
Drug 
Route of Administration 
Estradiol 
Transdermal 
Progesterone 
Oral 
 
Regimen: 
Once determined eligible and physically well, participants will receive 
transdermal estradiol (100 μg/day) for 3 weeks. Prior to estradiol administration 
women will receive a pelvic and breast exam and Papanicolaou test. 
Participants who can provide record of a pelvic exam within the past year and 
normal pap results within the past 3 years will be permitted to decline the GYN 
exam. Women will also have a basic lab panel and serum pregnancy test 
performed, and FSH and LH will be assayed.  Women will be seen in the clinic 
each week during estradiol administration to assess blood levels of estradiol 
and mood symptoms. Women will receive an additional week of combined 
estradiol and micronized progesterone (200 mg/day) at the end of the study to 
precipitate menstruation. 
Duration of 
administration 
3 weeks 
Reference population 
Perimenopausal women, non-depressed  
Statistical 
Methodology 
This study has one between-subjects factor (group: MDD versus control) 
crossed with one within-subjects factor (time: baseline versus estradiol 
treatment) with the outcome (percent signal changed) assessed in response to 
the MID task in the structurally defined frontostriatal ROIs, including the 
nucleus accumbens, caudate, thalamus, frontopolar cortex, and dorsolateral 
prefrontal cortex. Thus, for a task/ROI combination, there is a 2-by-2 repeated 
measures design. We will analyze this design using an ROI analysis and mixed 
models with an unstructured covariance structure. To test whether 
perimenopausal women with MDD show reduced frontostriatal activity and 
motivated behavior in response to reward at baseline, we will examine group 
differences in BOLD signal and response latency at baseline. To measure the 
psychological correlates of the frontostriatal response to reward, we will 
examine correlations between the change in frontostriatal ROI activation and 
change in depressive symptoms in the MDD group between baseline and post-
treatment; also within the MDD group, we will examine the correlation between 
frontostriatal ROI activity at baseline and the change in depressive symptoms 
between baseline and post-treatment. Finally, to examine whether estradiol 
treatment has greater effects on women with MDD than those without, we will 
assess whether the groups differ with respect to the change from baseline to 
post- treatment in percent activation in frontostriatal ROIs and response 
latency. 
 


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1 Introduction 
This document is a protocol for a human research study. This study is to be conducted according to 
US and international standards of Good Clinical Practice (FDA Title 21 part 312 and International 
Conference on Harmonization guidelines), applicable government regulations and Institutional research 
policies and procedures.  
1.1 Background 
Despite decades of clinical research, affective disorders continue to affect 20.9 million Americans 
each year and remain the leading cause of disability worldwide1. Unraveling the Unraveling the 
pathophysiology of affective disorders has been uniquely challenging because depressive syndromes are 
heterogeneous and have diverse etiologies2. Attempts to identify genetic and neural biomarkers that would 
improve the prediction of susceptibility, course of illness, and treatment response have yielded inconsistent 
results. One way to solve the problem of etiological diversity and diagnostic heterogeneity is to identify a 
homogeneous depression subtype with an identified etiology. Reproductive affective disorders (i.e., 
premenstrual dysphoric disorder, postpartum depression, and perimenopausal depression) represent more 
homogeneous subtypes, and a clear role for reproductive steroids has been established in these 
conditions3. Thus, we propose to study the pathophysiology of affective dysfunction by examining neural 
function in women with a specific depression subtype, perimenopausal depression. 
Women with perimenopausal depression represent the ideal population in which to study the neural 
biomarkers of reproductive affective dysfunction because the presumptive etiology—ovarian hormone 
withdrawal—is objectively measurable and amenable to manipulation. Evidence supporting the role of 
ovarian hormone withdrawal in the etiology of perimenopausal depression includes the following: 
perimenopausal women show a temporal association between ovarian hormone withdrawal and the onset 
of mood symptoms in the late perimenopause4; treatment with estrogen reduces mood symptoms5; and 
blinded estradiol withdrawal re-precipitates depression in women with a history of perimenopausal 
depression (manuscript in preparation). Thus, perimenopausal depression represents a relatively pure 
phenotype with respect to the presence of hormone sensitive depression. Focusing on a homogeneous 
etiology will increase the likelihood of identifying meaningful neurobiological markers6.  
One of the most powerful tools for understanding the neural mediators of complex behaviors is brain 
imaging7. Although the mechanisms by which reproductive steroids modulate human behavior is relatively 
unknown, the application of both modern imaging techniques and knowledge of the neurocircuitry 
underlying fundamental human and animal behaviors should permit us to define more precisely the means 
by which changes in reproductive steroids elicit depression in some, but not other, individuals. 
A fundamental behavior that is dysregulated in depression is reward responsiveness8. Reduced 
responsiveness to rewards contributes to the clinical phenomenon of anhedonia (i.e., loss of interest or 
pleasure in rewarding activities)5, a cardinal feature of depression9. In individuals with depression, 
anhedonia is associated with frontostriatal hypoactivity during reward processing10. Frontostriatal reward 
circuit dysregulation may be particularly central to perimenopausal depression. Estradiol modulates reward 
responsiveness in rodents11, 12 and a recent human study demonstrated an association between estradiol 
levels and frontostriatal reactivity to reward during the menstrual cycle in healthy women13. Thus, the 
frontostriatal reward system is regulated by estradiol and implicated in major depression. However, the 
effects of estradiol withdrawal and administration on frontostriatal reward circuitry have never been 
examined directly, and the extent to which frontostriatal dysregulation mediates the effects of estradiol 
withdrawal on depressive symptoms remains unknown.  
Thus, we will examine the effects of estradiol on the neural reward system in perimenopausal 
women. The proposed experimental study will allow us to answer the following questions: 1) Does the 
reward system differ in women with and without perimenopausal depression? 2) Does the reward system 
respond to estradiol differently in those with and without perimenopausal depression? 3) Does reward 
system activity at baseline predict the antidepressant effects of estradiol women with perimenopausal 
depression? Our results will provide critical information about the neuroendocrine pathophysiology of 
perimenopausal depression and may subsequently contribute to the development of novel pharmacologic 
interventions. 
The proposed study design will allow us to examine the role of frontostriatal hypoactivity in the 
pathophysiology of perimenopausal depression (by assessing baseline differences between 
perimenopausal depression and controls). It will also allow us to measure frontostriatal responses to 


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estradiol treatment in both perimenopausal depression and control women, neither of which have been 
previously characterized. This information is critical for understanding the pathophysiology of 
perimenopausal depression and the mechanisms by which estradiol withdrawal and supplementation have 
differential effects on mood across women (i.e., why some but not all women become depressed in the 
perimenopause). These aims are of central relevance to my training goals to gain the skills necessary to 
examine the neural pathophysiology of reproductive mood disorders and conduct experimental hormone 
investigations of the neural circuits contributing to affective illness in women. We have elected not to include 
a placebo control condition, as many studies in the field of affective neuroscience forgo placebo controls 
when examining the effects of drugs on neural circuitry14, 15. If, however, the expense associated with fMRI 
and sample size were not concerns, the inclusion of a placebo condition would allow us to control for the 
potential variance associated with repeated fMRI and to examine the potential placebo effects of medication 
taking on frontostriatal circuitry. Thus, the proposed study will provide pilot data for a larger, randomized, 
placebo-controlled study with sufficient power to compare treatment and placebo responders and non-
responders, which will allow us to further elucidate the precise relationships between changes in 
frontostriatal activation, estradiol, and changes in mood. Nevertheless, results of the proposed study will 
provide critical information about the involvement of frontostriatal circuitry in the pathophysiology of 
perimenopausal depression and the antidepressant effects of estradiol treatment. 
1.2 Investigational Agent 
 
ESTRADIOL  
 
Description 
 
Climara®, estradiol transdermal system, is designed to release estradiol continuously upon application to 
intact skin. Six (6.5, 9.375, 12.5, 15, 18.75 and 25 cm2) systems are available to provide nominal in vivo 
delivery of 0.025, 0.0375, 0.05, 0.06, 0.075 or 0.1 mg respectively of estradiol per day. The period of use 
is 7 days. Each system has a contact surface area of either 6.5, 9.375, 12.5, 15, 18.75 or 25 cm2, and 
contains 2, 2.85, 3.8, 4.55, 5.7 or 7.6 mg of estradiol USP respectively. The composition of the systems per 
unit area is identical. Estradiol USP is a white, crystalline powder, chemically described as estra-1,3,5(10)-
triene-3, 17ß-diol. It has an empirical formula of C18H24O2 and molecular weight of 272.39. The structural 
formula is: 
 
The Climara system comprises three layers. Proceeding from the visible surface toward the surface 
attached to the skin, these layers are (1) a translucent polyethylene film, and (2) an acrylate adhesive 
matrix containing estradiol USP. A protective liner (3) of siliconized or fluoropolymer-coated polyester film 
is attached to the adhesive surface and must be removed before the system can be used. 
 
 
 
The active component of the system is estradiol. The remaining components of the system (acrylate 
copolymer adhesive, fatty acid esters, and polyethylene backing) are pharmacologically inactive. 


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Pharmacology 
Endogenous estrogens are largely responsible for the development and maintenance of the female 
reproductive system and secondary sexual characteristics. Although circulating estrogens exist in a 
dynamic equilibrium of metabolic interconversions, estradiol is the principal intracellular human estrogen 
and is substantially more potent than its metabolites, estrone and estriol at the receptor level. 
The primary source of estrogen in normally cycling adult women is the ovarian follicle, which secretes 70 
to 500 mcg of estradiol daily, depending on the phase of the menstrual cycle. After menopause, most 
endogenous estrogen is produced by conversion of androstenedione, secreted by the adrenal cortex, to 
estrone by peripheral tissues. Thus, estrone and the sulfate conjugated form, estrone sulfate, are the 
most abundant circulating estrogens in postmenopausal women. 
Estrogens act through binding to nuclear receptors in estrogen-responsive tissues. To date, two estrogen 
receptors have been identified. These vary in proportion from tissue to tissue. 
Circulating estrogens modulate the pituitary secretion of the gonadotropins, luteinizing hormone (LH) and 
follicle stimulating hormone (FSH), through a negative feedback mechanism. Estrogens act to reduce the 
elevated levels of these hormones seen in postmenopausal women. 
 
PROGESTERONE  
Description 
PROMETRIUM (progesterone, USP) Capsules contain micronized progesterone for oral administration. 
Progesterone has a molecular weight of 314.47 and a molecular formula of C21H30O2. Progesterone 
(pregn-4-ene-3, 20-dione) is a white or creamy white, odorless, crystalline powder practically insoluble in 
water, soluble in alcohol, acetone and dioxane and sparingly soluble in vegetable oils, stable in air, 
melting between 126° and 131°C. The structural formula is: 
 
Progesterone is synthesized from a starting material from a plant source and is chemically identical to 
progesterone of human ovarian origin. PROMETRIUM Capsules are available in multiple strengths to 
afford dosage flexibility for optimum management. PROMETRIUM Capsules contain 100 mg or 200 mg 
micronized progesterone. 
The inactive ingredients for PROMETRIUM Capsules 100 mg include:  peanut oil NF, gelatin NF, glycerin 
USP, lecithin NF, titanium dioxide USP, D&C Yellow No. 10, and FD&C Red No. 40. 
The inactive ingredients for PROMETRIUM Capsules 200 mg include:  peanut oil NF, gelatin NF, glycerin 
USP, lecithin NF, titanium dioxide USP, D&C Yellow No. 10, and FD&C Yellow No. 6. 
Pharmacology 
PROMETRIUM Capsules are an oral dosage form of micronized progesterone which is chemically 
identical to progesterone of ovarian origin. The oral bioavailability of progesterone is increased through 
micronization. 


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Summary of Previous Human Experience 
We will enroll perimenopausal women to undergo estradiol supplementation using an existing 
recruitment infrastructure. Drs. Girdler and Rubinow are currently conducting a 12-month RCT 
(MHR01087619) designed to identify predictors of mood and cardiovascular benefit of transdermal estradiol 
in perimenopausal women. Over the 23 months of recruitment, with ads that specify no current depression, 
2,029 women were screened, 248 (12%) of whom were not eligible based on current MDD symptoms. Our 
data underscore the prevalence of both depressive symptoms for women in this stage of life and the 
likelihood of recruiting women with MDD. These results also show an attrition rate of less than 20% post-
randomization in the context of a 12-month clinical trial. Although attrition may be increased in women with 
MDD, the proposed study is substantially shorter and should be associated with reduced attrition.  
Support for the hypotheses comes from my own research and that of my mentors: 
1. Estradiol withdrawal precipitates anhedonia in rats, and decreasing estradiol levels are 
associated with increasing negative mood symptoms in women11. These results support my 
ability to conduct successful research in the area of reproductive mood disorders. Our current 
hypotheses are consistent with this prior research and suggest that the results may have 
implications for reproductive mood disorders. 
2. Estradiol withdrawal is associated with the onset of perimenopausal MDD, and estradiol 
treatment reduces perimenopausal MDD symptoms compared with placebo. In a 5-year 
longitudinal study of 29 women, a 14-fold increase in MDD was observed in the 24 months 
surrounding menopause4. In a larger cross sectional study (n=116), depressive episodes were 
significantly more likely during the late perimenopause compared with the pre-, early-, and post-
menopause16. Although estradiol levels are highly variable in regularly cycling women and during 
the pre- and early perimenopause, the average level of circulating estradiol declines dramatically 
during the transition from early to late perimenopause20, which is accompanied by an increased 
incidence in MDD4, 16. Depressive symptoms significantly decreased following 3 weeks of estradiol 
treatment in 34 women with perimenopausal MDD compared with baseline and women taking 
placebo5 (Figure 1c). Moreover, despite comparable ovarian hormone levels in women with 
perimenopausal MDD compared with controls4, 17, women with a history of perimenopausal MDD 
(but not those lacking that history) rapidly experience depression recurrence when blindly 
withdrawn from estradiol. Thus, estradiol withdrawal appears to trigger perimenopausal MDD in 
susceptible women, and three weeks of treatment with estradiol is sufficient to treat 
perimenopausal MDD. 
3. Active MDD symptoms and MDD risk are associated with frontostriatal hypoactivity. 
Individuals at high risk for MDD show reduced frontostriatal activation during the proposed 
monetary incentive delay task, and the degree of activation is associated with the severity of self-
reported rumination among those at high risk for MDD18. 
4. Estradiol levels are associated with increased reward-related neural function during the 
menstrual cycle in non-depressed women. Estradiol levels were positively correlated with 
activation of the dorsolateral and frontopolar cortices19, which is consistent with our hypothesis that 
estradiol supplementation will increase activation in frontal regions. 
5. The proposed monetary incentive delay (MID) taskinduces frontostriatal activation during 
monetary reward in control women. At baseline, control women enrolled in our R21 study of the 
effects of a scaled down hormonal model of pregnancy and parturition on mood and brain function 
showed increased activity in frontostriatal regions (specifically the nucleus accumbens, caudate, 
thalamus, dorsolateral prefrontal cortex, and frontopolar cortex) during monetary reward. These 
areas were selectively responsive to reward and not activated during non-reward, indicating that 
the task reliably activates the regions of interest associated with reward in previous studies.  
Status of Drug in Other Countries 
To our knowledge, the proposed drugs have not been withdrawn from investigation or marketing in any 
other country. 
1.3 Preclinical Data 
N/A 


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1.4 Clinical Data to Date 
We do not expect any adverse side effects associated with the hormonal manipulations outlined in 
this protocol for the following reasons: First, we will be administering the physiologically relevant steroid 
hormones (estradiol and progesterone) and not the substituted steroids (such as ethinyl estradiol or 
norethindrone) present in many oral contraceptives and which have been reported to have a potentially 
more serious profile of side effects.  
Estradiol: Nausea is the most common side effect of estrogen administration.  At conventional 
replacement doses, higher than those employed in this protocol, this complaint seldom interferes with 
eating, and no weight loss has been reported.  Breast engorgement, endometrial hyperplasia and 
bleeding are also common side effects of estrogen administration.  Pre-existing fibroid tumors of the 
uterus may enlarge under the effects of estrogen; however, at the dosage and for the duration of estrogen 
administration in this protocol this risk is small. 
The relationship between estrogen, both endogenous and exogenous, and the development of 
endometrial carcinoma has been suggested by several different lines of investigation21. Numerous 
retrospective case control studies published since 1975 have indicated that post-menopausal exposure to 
unopposed estrogens for more than one year results in a two to 12 fold increased relative risk for 
endometrial cancer. A relationship between the dose and duration of estrogen use and the risk for 
endometrial cancer has also been shown, the risk being increased after one to four years of estrogen use 
and rising also with the dosage employed.  However, the addition of progesterone to estrogen 
replacement therapy appears to decrease the risk of endometrial hyperplasia and endometrial cancer to 
equal or below that of women receiving no hormonal treatment. Recent studies suggest that the optimal 
regimen to prevent hyperplasia during long term ERT and thus, inferentially, the risk of carcinoma, 
consists of 12 to 13 days of progesterone treatment each month when estrogens are administered22. 
There is an increase in thromboembolism in women receiving non-contraceptive estrogen therapy23-26. 
Additionally, some but not all studies report an increase in risk of stroke27,28 in older women taking 
estrogen therapy. However, these complications are unlikely at the dose and duration of estrogen 
replacement employed in this protocol, and in the younger age group of women who participate in this 
study.  One study29 reported no effect of the estrogen patch on the four clotting indices previously shown 
to be altered by oral contraceptive use22,30,31 .  Blood pressure, on average, appears to be unaffected by 
estrogen therapy, although both increases and decreases have been reported.  In observational studies, 
post-menopausal estrogen therapy has been observed to lower the relative risk of cardiovascular disease 
in some but not all studies27, 32. In contrast, recent randomized controlled trials in older postmenopausal 
women (e.g., Women’s Health Initiative [WHI]) report an increased risk of cardiovascular disease33. 
Emerging data suggest that these disparities in findings may be related to the timing of initiation of 
estrogen therapy in relation to the proximity of menopause.  Subgroup analyses of the combined estrogen 
and progestin (EPT) arm of the WHI demonstrated a significant interaction between coronary heart 
disease (CHD) risk and time since initiation of EPT, with an increased risk in the early years following 
initiation and a decreased risk in later years. Additionally, the increased risk of CHD was observed in 
older but not younger perimenopausal women34–39. High doses of oral estrogens have been reported to 
elevate hepatocellular enzyme levels and, less commonly, cause cholestatic jaundice. The risk for gall 
stones and hepatocellular adenomas has been reported to be increased in association with oral 
contraceptive use, and although uncommon these complications may also occur with the use of 
replacement doses of estrogen40,41. Estrogen therapy also may increase the risk of urinary incontinence in 
older postmenopausal women42–44. Further, most studies have suggested an increased relative risk of 
breast cancer after four or five years' use45–55, similar to the risk expected if the onset of menopause was 
delayed for a comparable length of time. Due to the publicity surrounding the cancellation of the treatment 
arm of the Women’s Health Initiative study55 that involved the administration of combined conjugated 
estrogens and medroxyprogesterone acetate (Prempro), we have included the following statement in the 
consent documents: 
Adverse Events Related to Combined Hormone Replacement and the Results of the Women's 
Health Initiative (WHI): The WHI study demonstrated that continuous administration of one form of 
estrogen (conjugated estrogens) in combination with one form of progesterone (medroxyprogesterone 
acetate) is associated with an increased risk of dementia, heart attacks, stroke, blood clots, and breast 
cancer.  Estradiol, the form of estrogen that we use in this study, is administered as a sole agent (with the 
exception of one week's combination with progesterone) and, consequently, we do not expect that it will 


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pose the increased risks observed with the chronic combination of the conjugated estrogens and 
medroxyprogesterone administered in the WHI study.  Indeed, while the estrogen alone arm of the WHI 
trial was shown to be associated with an increased risk of stroke, no increased risk of either heart disease 
or breast cancer was observed55,56. Estrogens may precipitate migraine headaches, and depression has 
also been reported to occur with the use of estrogens. In general, considering the dose and duration of 
treatment that we propose to use in this protocol, the risk of developing such side effects is negligible. 
Progesterone: Progesterone and the synthetic progestins are widely prescribed, with indications 
including dysfunctional uterine bleeding, endometriosis, mastodynia, galactorrhea, and precocious 
puberty57.  Side effects reported in women taking progestins may include breakthrough bleeding, edema, 
change in weight (increase or decrease), cholestatic jaundice, rash (with or without pruritus), depression, 
easy fatigue and sedation, lack of initiative, and chloasma.  Since progestins are often used in women 
with antecedent menstrual irregularity, it is not clear whether the breakthrough bleeding represents an 
effect of the medication or refractoriness to treatment.  In the large majority of patients, menstruation 
occurs predictably following withdrawal of progestins and is usually more regular than in spontaneous 
cycles.  In a recent study, an average dose of 1750 mg of oral micronized progesterone was given to 
59 women with PMS for a period of three months and was well tolerated by this sample.  The side effects 
reported on progesterone were lightheadedness, fatigue, forgetfulness, and headaches.  These were very 
mild and caused no dropouts. 
For the sake of completeness we will also describe the side effects reported when estradiol and 
progesterone are combined in the form of oral contraceptives.  Side effects observed in patients receiving 
combined oral contraceptives include nausea, breast soreness, vaginal discharge, fluid retention, 
hypertension, and clotting abnormalities that have been associated with the estradiol component of the 
oral contraceptive. Thromboembolic disorders including thrombophlebitis, pulmonary embolism, and 
cerebral and coronary thrombosis appear to occur with greater frequency in women taking oral 
contraceptives.  While the increased incidence of these disorders has been associated with the estradiol 
component of the oral contraceptives, it is now believed that the progestogen component may, to a lesser 
extent, contribute to the increased risk.  There are relatively few reports associating oral contraceptives 
with the development of carcinomas (vaginal, uterine, hepatic, and mammary) despite the vast use of 
these agents, although this may reflect the latent period needed for cellular transformation.  Finally, 
several reports suggest an association between intrauterine exposure to female sex hormones and 
congenital anomalies. 
1.5 Dose Rationale and Risk/Benefits 
As previously stated Drs. Girdler and Rubinow are currently conducting a 12-month RCT (MHR01087619) 
designed to identify predictors of mood and cardiovascular benefit of transdermal estradiol in 
perimenopausal women. This study will enroll perimenopausal women to undergo estradiol 
supplementation using their existing infrastructure. Many studies have identified perimenopausal women 
as being at high risk for mood disorders, especially during the late menopause transition, but only a few 
studies have systematically assessed this biological vulnerability. The benefits of this study are three-fold: 
1) increased knowledge of the neuroendocrine mechanisms underlying both the triggering of and 
susceptibility to depression in women, 2) improved identification of perimenopausal women for whom 
hormone replacement therapy is likely to have beneficial neuroregulatory effects, and 3) increased 
information regarding novel targets for pharmacologic intervention. While there are scientific benefits to this 
study, the benefit to patients will be minimal. Estradiol administration may reduce depressive symptoms in 
some of the participants, and this information may be helpful to women as they plan their future health care 
in relation to menopause symptom management. 
2 Study Objectives 
 
The long-term research objectives are to 1) advance our understanding of the effects of ovarian 
hormones on the female brain and how these effects contribute to the triggering of and susceptibility to 
mood disorders; and 2) to identify neural and endocrine markers of depression risk, thereby improving our 
ability to prevent affective illness in women. 
 


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The central hypothesis is that the neural reward system is hypoactive in perimenopausal MDD, and the 
antidepressant effects of a three-week transdermal estradiol intervention will be mediated by increased 
activity in the neural reward system, assessed using fMRI. We will test our hypothesis by executing the 
following aims: 
 
Aim 1: To measure the frontostriatal response to reward in perimenopausal MDD and test the 
effects of estradiol on neural activation in perimenopausal women. We will use fMRI at baseline and 
following estradiol treatment in women with and without MDD to probe frontostriatal reward circuitry. 
Implementation of this protocol will provide hands-on training in fMRI research, the experimental 
manipulation of sex steroids in humans, and the interpretation of fMRI results based on a pharmacologic 
manipulation. 
H1a: We expect women with perimenopausal MDD to show reduced frontostriatal reactivity to 
reward at baseline compared with perimenopausal women without MDD. Specific regions of 
interest include the nucleus accumbens, caudate, and putamen. 
H1b: We also expect that estradiol administration will be associated with greater increases in 
frontostriatal responsivity to reward in those with MDD compared with those without MDD. 
Aim 2: To quantify motivated behavior at baseline and following estradiol administration in 
perimenopausal women with and without MDD. Motivated behavior will be operationally defined as the 
response latency to reward versus non-reward during the fMRI reward task. Pursuit of this aim will 
provide training in behavioral markers of psychopathology in the context of fMRI.  
H2a: Women with perimenopausal MDD will show greater response latencies during reward trials 
but not non-reward trials compared with those without MDD.  
H2b: We also expect that response latency to reward will be attenuated with estradiol 
administration in women with MDD. 
Aim 3: To measure dysphoria at baseline and following estradiol administration. Dysphoria will be 
assessed at baseline and following estradiol administration. Training associated with this aim will include 
assessing associations between neural activity and psychological symptoms.  
H3a: Women with perimenopausal MDD will report a significant reduction in depressive 
symptoms following estradiol administration, and the degree of symptom improvement will be 
associated with the change in frontostriatal responsivity to reward.  
3 Study Design 
3.1 General Design 
Overview. This is an observational, case-control, single-center study using estradiol and progesterone to 
examine the role of frontostriatal hypoactivity in the pathophysiology or perimenopausal MDD (by assessing 
baseline differences between perimenopausal MDD and controls.) It will also allow us to measure 
frontostriatal responses to estradiol treatment in both perimenopausal MDD and control women. This study 
will include 6 study visits that will take place over the course of approximately 6 weeks. Participants will 
receive transdermal estradiol for 3 weeks and FMRI sessions will occur at baseline and at the end of the 
third week of estradiol administration. The procedures that will take place are outlined in Table 1 and 
detailed below. 
Table 1. Study Procedures and Timeline 
Study visit 
1 
2 
3 
4 
5 
6 
Consent 
x 
 
 
 
 
 
OBGYN Exam  
 
x 
 
 
 
 
fMRI 
 
 
x 
 
 
x 
PRT 
 
 
x 
 
 
x 
Venipuncture 
 
x 
x 
x 
x 
x 
SCID 
x 
 
 
 
 
 
SNAP 
x 
 
 
 
 
 


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HAM-D 
x 
 
x 
x 
x 
x 
IDAS 
x 
 
x 
x 
x 
x 
PANAS 
x 
 
x 
x 
x 
x 
MASQ 
x 
 
x 
x 
x 
x 
GCS 
x 
 
x 
x 
x 
x 
PSQI 
 
 
x 
 
 
x 
Estradiol Rx provided 
 
 
x 
x 
x 
x 
Progesterone Rx provided 
 
 
 
 
 
x 
Payment 
$100 
$30 
$150 
$35 
$35 
$150 
 
Participants. Participants will include healthy, unmedicated perimenopausal women ages 44-55 either 
with (n=20) or without (n=20) current major depression. 
 
Hormone Administration.  
In the OBGYN visit, women will have a basic lab panel and serum pregnancy test performed, and FSH and 
LH will be assayed. Prior to estradiol administration women will receive a pelvic exam, breast exam and 
Papanicolaou test. Participants who can provide record of a pelvic exam within the past year and a normal 
pap results within the past 3 years will be permitted to decline the GYN exam and. Women will be seen in 
the clinic each week during estradiol administration to assess blood levels of estradiol and mood symptoms. 
Women will receive an additional week of combined estradiol and micronized progesterone (200 mg/day) 
at the end of the study to precipitate menstruation.  Blood will be batched until the end of data collection, at 
which point estradiol will be assayed. 
 
Clinical Assessments 
The Structured Clinical Interview for DSM-IV-TR Axis-I Disorders (SCID)58 and the Schedule for The SCID1 
will be administered at baseline to determine study eligibility. The following standard measures will be 
administered at baseline, both fMRI sessions, and weekly clinic visits: 
• 
The Positive and Negative Affect Schedule59 is a 20-item questionnaire that measures both positive 
affect (PA) and negative affect (NA) and is sensitive to subtle changes in affect over time. 
• 
The Hamilton Rating Scale for Depression (HRSD)60 is a 21-item researcher-rated standardized 
scale that assesses depressive symptoms and is widely used as a measure of symptom change 
over time.  
• 
The Inventory of Depression and Anxiety Symptoms (IDAS)61 is a 64-item self-report questionnaire 
that comprehensively assesses anxiety and depression symptoms on 10 subscales.The IDAS has 
excellent psychometric properties and has been validated for assessing reproductive mood 
disorders.  
• 
The Mood and Anxiety Symptom Questionnaire  Anhedonic Depression Subscale (MASQ-AD)62 
is a 22-item scale that assesses anhedonia (i.e., loss of interest and low positive affect). MASQ-
AD scores have been associated with blunted striatal response to reward in previous studies of 
patients with depression63.  
• 
The Greene Climacteric Scale (GCS)64 is a 21-item scale that is the gold-standard measure of four 
domains of climacteric symptoms: vasomotor, somatic, anxiety, and depression. 
• 
The Pittsburgh Sleep Quality Index (PSQI)65 is a 9-item questionnaire that measures the quality 
and sleep patterns in older adults. *This measure will only be administered at the two fMRI 
sessions. 
 
While the SCID and HAM-D are researcher administered, the other measures will be collected through self-
report questionnaires using Qualtrics or paper and pencil questionnaires.  
 
Despite the use of exclusion criteria designed to reduce risk to participants, depressive symptoms are 
monitored closely. Any subject who develops severe depressive symptoms (e.g., suicidal ideation) will be 
discontinued from the study and offered treatment paid for by the study.  If inpatient hospitalization becomes 


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necessary as a result of the study, UNC Health Care will cover the cost.  The following language has been 
added to the study consent form to reflect this policy:  
 
“All research involves a chance that something bad might happen to you.  This may include the risk of 
personal injury. In spite of all safety measures, you might develop a reaction or injury from being in 
this study. 
 
“If you become depressed or anxious as a direct result of participating this study, the UNC 
Department of Psychiatry will provide outpatient medical treatment. If vaginal bleeding or any other 
gynecological problem occurs, we will arrange for a visit with a UNC gynecologist. The research study 
will cover the costs of such outpatient exams and treatment at UNC Health Care. 
 
“If you become sick or injured as a direct result of participating in this study, and your condition 
cannot be addressed with outpatient treatment, UNC Health Care will provide all needed inpatient 
medical treatment. UNC Health Care will reimburse you for reasonable and necessary costs of such 
inpatient medical treatment not covered by your insurance company. No other form of reimbursement 
for study-related injury or illness is offered by UNC Health Care. You do not give up any legal rights 
by signing this consent.  
 
“If you receive Medicare benefits, UNC Health Care is required by law to report payments made to 
you for treatment, complications, and injuries that arise from this study. Information that you are 
taking part in this study, medical treatments received, Medicare claims, and other personal 
information about your such as your name, social security number, and date of birth, will be provided 
to the Centers of Medicare and Medicaid Services and its agents and/or contractors for this purpose. 
 
“If you seek treatment outside of UNC Health Care, you will be responsible for the costs of your 
treatment.” 
 
FMRI Tasks 
The following task will be included in each of the two fMRI sessions: 
The Monetary Incentive Delay (MID) Task66 engages the reward circuitry during monetary incentive 
anticipation and outcomes. Each of two MID runs consists of 90 6-second trials during which 
subjects are presented with of one of nine cue shapes, a fixation crosshair (for a variable duration), 
the target, and performance (win/loss/neutral) feedback. The cue indicates whether it is an 
incentive (gain, loss) or non-incentive trial. During incentive trials, participants can either gain or 
lose money by pressing a button during target presentation. Task difficulty is based on participant 
reaction times. This task elicits reliable frontostriatal dysregulation in people with remitted MDD67. 
Resting state scans will also be conducted at the beginning and end of each session.  
The Hariri Emotional Faces Task is an emotional face-matching task that engages neuronal regions 
implicated in emotional processing, including the amygdala and dorsomedial PFC. This block 
design paradigm consists of 4 blocks of a perceptual face-processing task interleaved with 5 blocks 
of a sensorimotor control task. Participants are presented with a target stimulus and asked to select 
one of 2 images, presented on the low half of the screen, which match the target. Each face-
processing block consists of fear and anger sub-blocks. During the sensorimotor control blocks, 
subjects view a trio of simple geometric shapes. Notably, this task elicited differences in amygdala 
activation between women with PPD and euthymic postpartum women in a previous study of PPD. 
 
FMRI Data Acquisition and Image Processing 
Scanning is performed using a Siemens Magnetom 3T TIM Trio scanner. High-resolution, T1-weighted 
anatomical images will be acquired using an MPRAGE sequence. Whole-brain functional images will be 
acquired using a single-shot, gradient recalled echoplanar pulse sequence sensitive to blood-oxygen-level-
dependent (BOLD) contrast. Each of 2 runs will consist of the acquisition of 195 successive brain volumes. 
FMRI image preprocessing, processing, and analysis will be conducted using FSL and custom MATLAB 
scripts. FMRI analyses will include an event-related BOLD response analysis within a priori structurally 
defined frontostriatal regions of interest (ROIs). Contrasts of interest will include win versus non-win 
outcomes68, although the anticipation of wins will also be explored. 


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Other Tasks 
Probabilistic Reward Task (PRT)69 will be conducted outside of the scanner. This task allows us to test the 
additional hypothesis that perimneopausal depression is characterized by blunted reward learning. During 
this task, correct identifications of two ambiguous stimuli are differentially rewarded. The task will consist of 
300 trials, divided into 3 blocks of 100 trials. Each trial starts with the presentation of a fixation cross for 500 
msec followed by a mouthless face. After a delay of 500 msec, either a short mouth (11.5 mm) or a long 
mouth (13 mm) is presented for 100 msec. Participants will be asked to identify which type of mouth was 
presented. For each block, the long and short mouths are presented equally often in a pseudorandomized 
sequence and 40 correct trials are followed by reward feedback immediately after the correct response. An 
asymmetrical reinforcer ratio will be used in this task, such that for half of the participants, correct 
identification of the short mouth will be associated with three times more positive feedback (30 of 40) than 
correct identification of the long mouth (10 of 40). For the other half of the participants, the contingencies 
will be reversed. 
 
Participants will be paid same-day for the amount earned on the PRT and the MID.  Participants will sign 
a form at that time to confirm they received payment and to confirm the amount they received. The money 
earned during the PRT and MID will be part of the $500 compensation.   
3.2 Primary Study Endpoints 
1. Frontiostriatal reactivity to reward during the MID fMRI task 
2. Response latency to reward versus non-reward during the MID fMRI task 
3. Depressive symptoms  
3.3 Secondary Study Endpoints 
1. Neural connectivity measured during resting-state fMRI 
3.4 Primary Safety Endpoints 
N/A 
4 Subject Selection and Withdrawal 
4.1 Inclusion Criteria 
Participants will include healthy, unmedicated perimenopausal women ages 44-55 either with (n=20) or 
without (n=20) current major depression.Thus, only participants capable of giving informed consent will be 
enrolled. Participants will be compensated upon completion of the study.   
 
Inclusion Criteria. 
1. Perimenopause Status: We will employ the Stages of Reproductive Aging Workshop (STRAW) 
criteria70 to confirm perimenopausal status. The stages are primarily based on the characteristics 
of the menstrual cycle and secondarily on follicle stimulating hormone (FSH) levels. The anchor 
for the staging system is the final menstrual period (FMP). We will enroll women who have ≥ 2 
skipped cycles and an interval of amenorrhea ≥ 60 days, consistent with the late menopause 
transition (stage -1), and who demonstrate an FSH level higher than two SDs above the mean for 
follicular phase premenopausal women based on UNC Hospitals laboratory levels (x= 7 IU/mL). 
Because extremes of body weight (BMI < 18 or > 30 kg/m2) or a history of chronic menstrual 
cycle irregularity can contribute to inaccurate reproductive staging70, these will serve as additional 
exclusion criteria; 
2. MDD Group Eligibility Criterion: current diagnosis of MDD with an onset associated with 
menstrual cycle irregularity, and no history of psychiatric illness during the 2 years before the 
onset of the current depressive episode as determined by the Structure Clinical Interview for 
DSM-IV-TR for Axis I Disorders (SCID); 


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3. Control Group Eligibility Criterion: absence of any past or present psychiatric disorder as 
assessed by the SCID. 
4.2 Exclusion Criteria 
 
. Patients will not be permitted to enter this protocol if they have any of the following: 
1. 
current medication use (i.e., psychotropics, anti-hypertensives, statins, hormonal preparations, or frequent 
use of anti-inflammatory agents (> 10 times/month)). Women will be allowed to enroll who take medications 
without known mood effects (e.g. stable thyroid hormone replacement and occasional (< 5 times/month) use 
of Ambien)*; 
2. 
pregnant, breastfeeding or trying to conceive; 
3. 
FMP more than 12 months prior to enrollment; 
4. 
history of undiagnosed vaginal bleeding; 
5. 
undiagnosed enlargement of the ovaries; 
6. 
polycystic ovary syndrome; 
7. 
history of breast or ovarian cancer; 
8. 
first degree relative with ovarian cancer; 
9. 
first degree relative with premenopausal or bilateral breast cancer; 
10. 2+ first degree relatives with breast cancer (regardless of onset); 
11. 3+ relatives with postmenopausal breast cancer; 
12. abnormal finding in a provider breast exam and/or mammogram; 
13. known carrier of BRCA1 or 2 mutation; 
14. endometriosis; 
15. blood clots in the legs or lungs; 
16. porphyria; 
17. diabetes mellitus; 
18. malignant melanoma; 
19. Hodgkin's disease; 
20. recurrent migraine headaches that are preceded by aura; 
21. gallbladder or pancreatic disease**; 
22. heart or kidney disease**; 
23. liver disease; 
24. cerebrovascular disease (stroke); 
25. first degree relative with history of heart attack or stroke; 
26. current cigarette smoking; 
27. current suicidal ideation or psychosis; 
28. past suicide attempts or psychotic episodes requiring hospitalization; 
29. chronic depression (i.e., episode(s) lasting 3+ years); 
30. recurrent depression (i.e., more than 1 prior episode, not including episodes with postpartum onset) 
31. depressive episode(s) within 2 years of enrollment; 
32. self-reported claustrophobia 
33. peanut allergy 
*all reported prescription medications will be reviewed and cleared by a study physician prior to a participant's 
enrollment; 
**participants will be given the opportunity to describe these conditions in the online screening survey.  Reported 
conditions that are acute in nature and/or benign will be reviewed by a study physician and exclusions will be decided 
case-by-case.  All chronic conditions will be exclusionary.  
4.3 Subject Recruitment and Screening 
 
Methods of recruiting for this study include:  
• 
Letters 
• 
Flyers 
• 
E-mail announcements 


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• 
Facebook advertisements 
• 
Craigslist advertisements  
 
Recruitment materials (e.g., advertisements) will also be placed in university buildings and local businesses, 
and a university wide email will be used to recruit participants. Advertisements, including the letter, flyer, 
and email, will instruct participants to call our research lab to complete a phone screen or follow a link to 
our online eligibility survey. The phone screen and online eligibility survey include same questions (see 
attached).  
 
Eligibility screening will include: 
• An initial phone or online screening that includes questions about past medical and mental health 
history to assess potential participants’ eligibility based on the criteria listed in sections 4.1 and 4.2. 
• Participants will undergo a Clinical and Health Screening process to determine whether they are 
healthy enough to participate in this study. This screening will include past medical and mental health 
history and physical exam. During this evaluation, they will be asked questions about past and present 
psychiatric symptoms. They will also be asked to complete questionnaires about psychiatric 
symptoms. They may choose not to answer any or all of the questions for any reason. 
• Participants will complete a safety questionnaire to determine whether they have any foreign iron or 
steel metal objects in their bodies, such as a pacemaker, shrapnel, metal plate, or metal debris. If they 
have any such objects in their bodies, they cannot participate in the MRI session. 
• All participants will receive a pregnancy test. No pregnant women will be entered into the study, 
because the study drugs (estrogen and progesterone) may be associated with birth defects. 
4.4 Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
 
Participants with significant clinical or laboratory abnormalities will be discontinued from the study prior to 
estrogen administration. 
 
Adverse mood symptoms will be monitored by administering the the Hamilton Rating Scale for 
Depression (HRSD) at each study visit. If suicidal thoughts or severe mood symptoms are 
observed at any period in the medication phase, then the suicide item of the HRSD will be 
administered on consecutive days for three days. Anyone who expresses suicidal thoughts with 
plans or intent, or who expresses concern about her ability to continue in the study, will be 
considered to have severe mood symptoms and be discontinued from the protocol. In the event of 
the occurrence of severe mood symptoms, the protocol will be terminated. Should this step prove to be 
unsuccessful, conventional medication will be prescribed as needed. Although we do not anticipate 
severe adverse reactions, we have arranged for inpatient hospital admission in the Psychiatry 
Department if symptoms are otherwise unmanageable. 
 
Any patient experiencing clinically significant side effects such as nausea, hypertension, vomiting or 
extreme fluid retention from the medication will have the dose titrated to achieve relief of the symptoms. If 
adequate relief cannot be achieved in this manner, drug treatment will be discontinued.  
 
The determination of when and how to withdrawal subjects will be overseen by the study’s principal 
investigator, co-investigators, and research coordinators and will be reported to the UNC Biomedical IRB. 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
 
Participants who elect to discontinue the hormone protocol or are discontinued for safety reasons will be 
asked to continue to complete self-report ratings through the end of the proposed study period. However, 
fMRI exams will not be done if participants discontinue the hormone protocol.  
 


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5 Study Drug  
5.1 Description 
Drug 
Dosage 
Formulation 
Estradiol 
100 micrograms/day 
Transdermal  
Progesterone 
200 miligrams/day 
Oral capsule 
 
5.2 Treatment Regimen 
 
Regimen 
 
Once determined eligible and physically well, participants will receive transdermal estradiol (100 μg/day) 
for 3 weeks. Women will receive an additional week of combined estradiol and micronized progesterone 
(200 mg/day) at the end of the study to precipitate menstruation. 
5.3 Method for Assigning Subjects to Treatment Groups 
This is a case-control study design, and all subjects will receive the same drug protocol. 
5.4 Preparation and Administration of Study Drug 
All study drugs will be stored, prepared and dispensed from the UNC Investigational Drug Service (IDS). 
 
Contact: 
Sue Pope, Manager 
Investigational Drug Service 
Department of Pharmacy 
UNC Hospitals 
CB 7600, Room 3001 
101 Manning Drive 
Chapel Hill, NC 27514 
Office: 919-966-1766 
Fax: 919-966-6359 
5.5 Subject Compliance Monitoring 
We will monitor participants’ compliance with the drug regimen by assaying blood levels of estradiol 
collected at weekly study visits. The drug protocol will be reviewed at each study session, and participants 
who are significantly non-compliant with the study treatment regimen will be discontinued from the study. 
5.6 Prior and Concomitant Therapy 
Women are required to be free of medications that have known CNS effects to enroll in this study; however, 
prior medication usage will not preclude participation in the study. 
5.7 Packaging 
The UNC Investigational Drug Service will receive the active drug from their Pharmacy storeroom and will 
provide the capsules for blinding. Estradiol patches will be provided to participants at weekly study visits. 
The patches release estradiol for a week. Progesterone capsules will be provided to participants at the final 
study visit.   
5.8 Blinding of Study Drug (if applicable) 
N/A 


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5.9 Receiving, Storage, Dispensing and Return 
The UNC Investigational Drug Service will receive the study drugs from the UNC Pharmacy Storeroom and 
will dispense the drug to the PI or research coordinator to deliver to participants. Any unused drug will be 
returned to the UNC Investigational Drug Service by the PI or research coordinator. 
5.9.1 Receipt of Drug Supplies 
Upon receipt of the of the study treatment supplies, an inventory will be performed and a drug receipt log 
filled out and signed by the person accepting the shipment.  The designated study staff will count and verify 
that the shipment contains all the items noted in the shipment inventory.  Any damaged or unusable study 
drug in a given shipment (active drug or comparator) will be documented in the study files.  The investigator 
will notify the UNC Pharmacy Storeroom of any damaged or unusable study treatments that were supplied 
to the Investigational Drug Service. 
5.9.2 Storage 
Estradiol and Progesterone will be stored at 20° to 25° C in a temperature-controlled facility.  
5.9.3 Dispensing of Study Drug 
Drugs will be dispensed in tight, light-resistant containers as defined n the USP, with a child-resistant 
closure. Regular study drug reconciliation will be performed to document drug assigned, drug consumed, 
and drug remaining. This reconciliation will be logged on the drug accountibility form, and signed and dated 
by the study team. 
5.9.4 Return or Destruction of Study Drug 
At the completion of the study, there will be a final reconciliation of drug shipped, drug consumed, and drug 
remaining.  This reconciliation will be logged on the drug reconciliation form, signed and dated.  Any 
discrepancies noted will be investigated, resolved, and documented prior to return or destruction of unused 
study drug.  Drug destroyed on site will be documented in the study files. 
6 Study Procedures 
 
The flowchart outlining the procedures at each study visit it attached.  
 
OBGYN Exam  
Participants will receive a gynecological exam prior to drug administration to rule out exclusionary 
health problems. Results of the exam will be added to participants’ UNC Hospital medical record.  However, 
participants who can provide documentation of a normal gynecological exam within the last year will not 
receive the gynecological exam.  
 
Venipuncture 
Participants will undergo venipuncture at 5 study visits. Initial blood tests will include a pregnancy 
test, FSH, LH, BUN, creatinine, and a hepatic panel to rule out exclusionary health problems. Lab results 
will be added to participants’ medical records.  Subsequent venipuncture will be for the purpose of assaying 
estradiol levels to ensure participant compliance.  
 
 
Drug Administration 
Participants will receive estradiol at study visits 3-5. Participants will receive Progesterone at visit 
6.  
 
Clinical Assessments 
The Structured Clinical Interview for DSM-IV-TR Axis-I Disorders (SCID)58 and the Schedule for The 
SCID1 will be administered at baseline to determine study eligibility. The following standard measures will 
be administered at baseline, both fMRI sessions, and weekly clinic visits: 


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• 
The Positive and Negative Affect Schedule59 is a 20-item questionnaire that measures both positive 
affect (PA) and negative affect (NA) and is sensitive to subtle changes in affect over time. 
• 
The Hamilton Rating Scale for Depression (HRSD)60 is a 21-item researcher-rated standardized 
scale that assesses depressive symptoms and is widely used as a measure of symptom change 
over time.  
• 
The Inventory of Depression and Anxiety Symptoms (IDAS)61 is a 64-item self-report questionnaire 
that comprehensively assesses anxiety and depression symptoms on 10 subscales.The IDAS has 
excellent psychometric properties and has been validated for assessing reproductive mood 
disorders.  
• 
The Mood and Anxiety Symptom Questionnaire  Anhedonic Depression Subscale (MASQ-AD)62 
is a 22-item scale that assesses anhedonia (i.e., loss of interest and low positive affect). MASQ-
AD scores have been associated with blunted striatal response to reward in previous studies of 
patients with depression63.  
• 
The Greene Climacteric Scale (GCS)64 is a 21-item scale that is the gold-standard measure of four 
domains of climacteric symptoms: vasomotor, somatic, anxiety, and depression. 
• 
The Pittsburgh Sleep Quality Index (PSQI)65 is a 9-item questionnaire that measures the quality 
and sleep patterns in older adults. *This measure will only be administered at the two fMRI 
sessions. 
 
While the SCID and HAM-D are researcher administered, the other measures will be collected through self-
report questionnaires using Qualtrics or paper and pencil questionnaires.  
 
Despite the use of exclusion criteria designed to reduce risk to participants, depressive symptoms are 
monitored closely. Any subject who develops severe depressive symptoms (e.g., suicidal ideation) will be 
discontinued from the study and offered treatment, including inpatient hospitalization. 
 
FMRI Tasks 
The following task will be included in each of the two fMRI sessions: 
The Monetary Incentive Delay (MID) Task66 engages the reward circuitry during monetary incentive 
anticipation and outcomes. Each of two MID runs consists of 90 6-second trials during which 
subjects are presented with of one of nine cue shapes, a fixation crosshair (for a variable duration), 
the target, and performance (win/loss/neutral) feedback. The cue indicates whether it is an 
incentive (gain, loss) or non-incentive trial. During incentive trials, participants can either gain or 
lose money by pressing a button during target presentation. Task difficulty is based on participant 
reaction times. This task elicits reliable frontostriatal dysregulation in people with remitted MDD67. 
Resting state scans will also be conducted at the beginning and end of each session.  
The Hariri Emotional Faces Task is an emotional face-matching task that engages neuronal regions 
implicated in emotional processing, including the amygdala and dorsomedial PFC. This block 
design paradigm consists of 4 blocks of a perceptual face-processing task interleaved with 5 blocks 
of a sensorimotor control task. Participants are presented with a target stimulus and asked to select 
one of 2 images, presented on the low half of the screen, which match the target. Each face-
processing block consists of fear and anger sub-blocks. During the sensorimotor control blocks, 
subjects view a trio of simple geometric shapes. Notably, this task elicited differences in amygdala 
activation between women with PPD and euthymic postpartum women in a previous study of PPD. 
 
Other Tasks 
Pizzagalli's Probabilistic Reward Task (PRT)69 will be conducted outside of the scanner. This task allows 
us to test the additional hypothesis that perimenopausal depression is characterized by blunted reward 
learning. During this task, correct identifications of two ambiguous stimuli are differentially rewarded.  The 
task will consist of 300 trials, divided into 3 blocks of 100 trials. Each trial starts with the presentation of a 
fixation cross for 500 msec followed by a mouthless face. After a delay of 500 msec, either a short mouth 
(11.5 mm) or a long mouth (13 mm) is presented for 100 msec. Participants will be asked to identify which 
type of mouth was presented. For each block, the long and short mouths are presented equally often in a 
pseudorandomized sequence and 40 correct trials are followed by reward feedback immediately after the 
correct response. An asymmetrical reinforcer ratio will be used in this task, such that for half of the 


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participants, correct identification of the short mouth will be associated with three times more positive 
feedback (30 of 40) than correct identification of the long mouth (10 of 40). For the other half of the 
participants, the contingencies will be reversed. 
 
7 Statistical Plan 
7.1 Sample Size Determination 
We approximated power and sample size using a model comparing the two groups on change-scores from 
baseline to post-treatment. With 20 subjects per group, we have 80% power to detect effect sizes of 0.9, 
which represents a large effect size. Given our strong preliminary evidence and the experimental nature of 
the proposed research, we expect to detect statistically significant results. Previous clinical research 
supports our hypothesized effect size. Studies comparing participants with MDD and controls have yielded 
effect sizes (Cohens d) ranging from 1.0 to 1.56 in ventral striatum responsivity to reward 5153. We will 
enroll an additional 10 participants to account for attrition, increasing the total N to 50.  
 
 
7.2 Statistical Methods 
 
This study has one between-subjects factor (group: MDD versus control) crossed with one within-subjects 
factor (time: baseline versus estradiol treatment) with the outcome (percent signal changed) assessed in 
response to the MID task in the structurally defined frontostriatal ROIs, including the nucleus accumbens, 
caudate, thalamus, frontopolar cortex, and dorsolateral prefrontal cortex. Thus, for a task/ROI 
combination, there is a 2-by-2 repeated measures design. We will analyze this design using an ROI 
analysis and mixed models with an unstructured covariance structure. To test whether perimenopausal 
women with MDD show reduced frontostriatal activity (H1) and motivated behavior (H2) in response to 
reward at baseline, we will examine group differences in BOLD signal and response latency at baseline. 
To measure the psychological correlates of the frontostriatal response to reward, we will examine change 
in depressive symptoms between baseline and post-treatment in MDD relative to controls (H3).  
 
7.3 Subject Population(s) for Analysis 
Given that we are interested in understanding the effect of estradiol on brain function, only data from 
protocol-compliant participants will be subjected to the study analysis. 
8 Safety and Adverse Events 
8.1 Definitions 
Unanticipated Problems Involving Risk to Subjects or Others 
Any incident, experience, or outcome that meets all of the following criteria:  
• Unexpected in nature, severity, or frequency  (i.e. not described in study-related documents such 
as the IRB-approved protocol or consent form, the investigators brochure, etc) 
• Related or possibly related to participation in the research (i.e. possibly related means there is a 
reasonable possibility that the incident experience, or outcome may have been caused by the 
procedures involved in the research, 
• Serious (as defined below) “Serious” is different than “severe” as reported in the CTC criteria 
that applies a grade to the AE. 
 
Adverse Event 


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An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in severity 
during the course of the study.  Intercurrent illnesses or injuries should be regarded as adverse events.  
Abnormal results of diagnostic procedures are considered to be adverse events if the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance 
 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  A serious adverse event is any AE that is:  
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event 
 
Important medical events are those that may not be immediately life threatening, but are clearly of major 
clinical significance.   They may jeopardize the subject, and may require intervention to prevent one of the 
other serious outcomes noted above.  For example, drug overdose or abuse, a seizure that did not result 
in in-patient hospitalization, or intensive treatment of bronchospasm in an emergency department would 
typically be considered serious.  
 
All adverse events that do not meet any of the criteria for serious should be regarded as non-serious 
adverse events.  
 
Adverse Event Reporting Period 
The study period during which adverse events will be reported is normally defined as the period from the 
initiation of any study procedures to the end of the study treatment follow-up.  For this study, the study 
treatment follow-up is defined as 30 days following the last administration of study treatment.  
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting condition will be 
recorded as an adverse event if the frequency, intensity, or the character of the condition worsens during 
the study period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality will be recorded as a preexisting condition.  At the end 
of the study, any new clinically significant findings/abnormalities that meet the definition of an adverse event 
will also be recorded and documented as an adverse event.  
 
Post-study Adverse Event 
All unresolved adverse events will be followed by the investigator until the events are resolved, the subject 
is lost to follow-up, or the adverse event is otherwise explained.  At the last scheduled visit, the investigator 
will instruct each subject to report any subsequent event(s) that the subject, or the subject’s personal 
physician, believes might reasonably be related to participation in this study.  The investigator will notify the 
study sponsor of any death or adverse event occurring at any time after a subject has discontinued or 
terminated study participation that may reasonably be related to this study.  The sponsor will also be notified 
if the investigator should become aware of the development of cancer or of a congenital anomaly in a 
subsequently conceived offspring of a subject that has participated in this study.  
 
Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event if any one of the following 
conditions is met:  


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• The laboratory abnormality is not otherwise refuted by a repeat test to confirm the abnormality 
• The abnormality suggests a disease and/or organ toxicity 
• The abnormality is of a degree that requires active management; e.g. change of dose, 
discontinuation of the drug, more frequent follow-up assessments, further diagnostic investigation, 
etc. 
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization will be documented and 
reported as a serious adverse event unless specifically instructed otherwise in this protocol.  Any condition 
responsible for surgery will be documented as an adverse event if the condition meets the criteria for and 
adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an adverse 
event in the following circumstances: 
• Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures for a 
preexisting condition.  Surgery should not be reported as an outcome of an adverse event if the 
purpose of the surgery was elective or diagnostic and the outcome was uneventful. 
• Hospitalization or prolonged hospitalization required to allow efficacy measurement for the study. 
• Hospitalization or prolonged hospitalization for therapy of the target disease of the study, unless it 
is a worsening or increase in frequency of hospital admissions as judged by the clinical investigator. 
8.2 Recording of Adverse Events 
At each contact with the subject, the investigator will seek information on adverse events by specific 
questioning and, as appropriate, by examination.  Information on all adverse events will be recorded 
immediately in the source document, and also in the appropriate adverse event module of the case report 
form (CRF).  All clearly related signs, symptoms, and abnormal diagnostic procedures results will be 
recorded in the source document, though will be grouped under one diagnosis. 
 
All adverse events occurring during the study period will be recorded.  The clinical course of each event will 
be followed until resolution, stabilization, or until it has been determined that the study treatment or 
participation is not the cause.  Serious adverse events that are still ongoing at the end of the study period 
will be followed up to determine the final outcome.  Any serious adverse event that occurs after the study 
period and is considered to be possibly related to the study treatment or study participation will be recorded 
and reported immediately. 
8.3 Reporting of Serious Adverse Events and Unanticipated Problems 
 
Investigators and the protocol sponsor must conform to the adverse event reporting timelines, formats and 
requirements of the various entities to which they are responsible, but at a minimum those events that must 
be reported are those that are: 
• related to study participation, 
• unexpected, and  
• serious or involve risks to subjects or others  
(see definitions, section 8.1).   
 
If the report is supplied as a narrative, the minimum necessary information to be provided at the time of the 
initial report includes: 
• 
Study identifier 
• 
Study Center 
• 
Subject number 
• 
A description of the event 
• 
Date of onset 
• 
Current status 
• 
Whether study treatment was discontinued 
• 
The reason why the event is classified as serious 
• 
Investigator assessment of the association 
between the event and study treatment 
 


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8.3.1 Investigator reporting: notifying the study sponsor 
 
Any study-related unanticipated problem posing risk of harm to subjects or others, and any type of serious 
adverse event, will be reported to the PI and IRB by telephone within 24 hours of the event.  To report such 
events, an FDA Form 3500A will be completed by the investigator, signed by the sponsor, and faxed to the 
IRB within 24 hours.  The investigator will keep a copy of this FDA Form 3500A on file at the study site.   
 
Within the following 48 hours, the investigator must provide further information on the serious adverse event 
or the unanticipated problem in the form of a written narrative.  This should include a copy of the completed 
FDA Form 3500A, and any other diagnostic information that will assist the understanding of the event.  
Significant new information on ongoing serious adverse events should be provided promptly to the study 
sponsor and the FDA. 
8.3.2 Investigator reporting 
 
For reportable deaths, the initial submission to the UNC IRB may be made by contacting the IRB Director 
or Associate Director. The AE/Unanticipated Problem Form is required as a follow up to the initial 
submission. 
 
Other Reportable events: 
For clinical drug trials, the following events are also reportable to the UNC IRB: 
• Any adverse experience that, even without detailed analysis, represents a serious unexpected adverse 
event that is rare in the absence of drug exposure (such as agranulocytosis, hepatic necrosis, Stevens-
Johnson syndrome). 
• Any adverse event that would cause the sponsor to modify the investigators brochure, protocol or 
informed consent form, or would prompt other action by the IRB to assure protection of human subjects. 
• Information that indicates a change to the risks or potential benefits of the research, in terms of severity 
or frequency. For example:  
– An interim analysis indicates that participants have a lower rate of response to treatment than 
initially expected. 
– Safety monitoring indicates that a particular side effect is more severe, or more frequent than 
initially expected. 
– A paper is published from another study that shows that an arm of your research study is of no 
therapeutic value. 
• Change in FDA safety labeling or withdrawal from marketing of a drug, device, or biologic used in a 
research protocol. 
• Breach of confidentiality 
• Change to the protocol taken without prior IRB review to eliminate apparent immediate hazard to a 
research participant. 
• Incarceration of a participant when the research was not previously approved under Subpart C and 
the investigator believes it is in the best interest of the subject to remain on the study. 
• Complaint of a participant when the complaint indicates unexpected risks or the complaint cannot 
be resolved by the research team. 
• Protocol violation (meaning an accidental or unintentional deviation from the IRB approved protocol) 
that in the opinion of the investigator placed one or more participants at increased risk, or affects 
the rights or welfare of subjects. 
8.3.3 Investigator reporting: Notifying UNC if affiliate site 
Investigators who are not UNC faculty or affiliated with a UNC research site are responsible for safety 
reporting to their local IRB.  Investigators are responsible for complying with their local IRB’s reporting 


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requirements, though must submit the required reports to their IRB no later than 10 working days.  Copies 
of each report and documentation of IRB notification and receipt will be kept in the investigator’s study file. 
8.3.4 Sponsor reporting: Notifying the FDA 
 
The study sponsor is required to report certain study events in an expedited fashion to the FDA.  These 
written notifications of adverse events are referred to as IND safety reports. The following describes the 
safety reporting requirements by timeline for reporting and associated type of event: 
 
• Within 7 calendar days 
Any study event that is: 
– associated with the use of the study drug 
– unexpected,  
– fatal or life-threatening, and  
 
• Within 15 calendar days 
Any study event that is: 
– associated with the use of the study drug, 
– unexpected, and 
– serious, but not fatal or life-threatening 
-or- 
– a previous adverse event that was not initially deemed reportable but is later found to fit the 
criteria for reporting (reporting within 15 calendar days from when event was deemed 
reportable). 
Any finding from tests in laboratory animals that:  
– suggest a significant risk for human subjects including reports of mutagenicity, teratogenicity, 
or carcinogenicity. 
 
Additional reporting requirements 
Sponsors are also required to identify in IND safety reports all previous reports concerning similar adverse 
events and to analyze the significance of the current event in light of the previous reports. 
 
Reporting Process 
Adverse events may be submitted on FDA Form 3500A or in a narrative format. If supplied as in a narrative 
format, the minimum information to be supplied is noted above at the beginning of section 8.3. All adverse 
events will be reported to the FDA. 
8.3.5 Sponsor reporting: Notifying participating investigators 
It is the responsibility of the study sponsor to notify all participating investigators, in a written IND safety 
report, of any adverse event associated with the use of the drug that is both serious and unexpected, as 
well as any finding from tests in laboratory animals that suggest a significant risk for human subjects.  
Additionally, sponsors are also required to identify in IND safety reports all previous reports concerning 
similar adverse events and to analyze the significance of the current event in light of the previous reports. 
8.4 Unblinding Procedures 
N/A 
8.5 Stopping Rules  
The protocol only includes rules for discontinuing individual participant, but not rules for stopping the entire 
study: 
 
Participants with significant clinical or laboratory abnormalities will be discontinued from the study prior 
to estrogen administration. 


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Adverse mood symptoms will be monitored by administering the the Hamilton Rating Scale for 
Depression (HRSD) at each study visit. If suicidal thoughts or severe mood symptoms are 
observed at any period in the medication phase, then the suicide item of the HRSD will be 
administered on consecutive days for three days. Anyone who expresses suicidal thought 
withplans or intent, or who expresses concern about her ability to continue in the study, will 
be considered to have severe mood symptoms and be discontinued from the protocol. In the 
event of the occurrence of severe mood symptoms, the protocol will be terminated. Should this step 
prove to be unsuccessful, conventional medication will be prescribed as needed. Although we do not 
anticipate severe adverse reactions, we have arranged for inpatient hospital admission in the 
Psychiatry Department if symptoms are otherwise unmanageable. 
 
Any patient experiencing clinically significant side effects such as nausea, hypertension, vomiting or 
extreme fluid retention from the medication will have the dose titrated to achieve relief of the 
symptoms. If adequate relief cannot be achieved in this manner, drug treatment will be discontinued.  
 
The determination of when and how to withdrawal subjects will be overseen by the study’s principal 
investigator, co-investigators, and research coordinators and will be reported to the UNC Biomedical 
IRB. 
8.6 Medical Monitoring 
The principal investigator will oversee the safety of the study at his/her site.  This safety monitoring will 
include careful assessment and appropriate reporting of adverse events as noted above, as well as the 
construction and implementation of a site data and safety-monitoring plan.  Medical monitoring will include 
a regular assessment of the number and type of serious adverse events.  
 
The Principal Investigator’s responsibilities are to: 
 
• 
review the research protocol, informed consent documents, and plans for data safety and 
monitoring; 
 
• 
evaluate the progress of the trial, including periodic assessments of data quality and timeliness, 
participant recruitment, accrual and retention, participant risk versus benefit, performance of the 
trial site, and other factors that can affect study outcome; 
 
• 
consider factors external to the study when relevant information becomes available, such as 
scientific or therapeutic developments that may have an impact on the safety of the participants or 
the ethics of the trial; 
 
• 
protect the safety of the study participants; 
 
• 
report on the safety and progress of the trial; 
 
• 
make recommendations to the UNC-CH IRB, the PI, and, if required, to the Food and Drug 
Administration (FDA) concerning continuation, termination, or other modifications of the trial based 
on the observed beneficial or adverse effects of the treatment under study; 
 
• 
if appropriate, conduct interim analysis of efficacy in accordance with stopping rules which are 
clearly defined in advance of data analysis and have the approval of the DSMB; 
 
• 
ensure the confidentiality of the trial data and the results of monitoring; and, 
 
• 
assist the UNC-CH IRB by commenting on any problems with study conduct, enrollment, and 
sample size and/or data collection. 
 


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8.6.1 Independent Data and Safety Monitoring Board 
N/A 
9 Data Handling and Record Keeping 
9.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the requirements of 
the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a signed 
subject authorization informing the subject of the following:  
• What protected health information (PHI) will be collected from subjects in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, retains 
the ability to use all information collected prior to the revocation of subject authorization.  For subjects that 
have revoked authorization to collect or use PHI, attempts should be made to obtain permission to collect 
at least vital status (i.e. that the subject is alive) at the end of their scheduled study period. 
 
In the event that the research team is unable to contact a subject (either by 3 phone calls, or over 2 days’ 
time), Dr. Schiller will contact the subject’s emergency contact, thereby breaching confidentiality.  This will 
be communicated to the subject at the time of consent, and she will be encouraged to inform her emergency 
contact person of this plan.   
9.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other activities in a 
clinical trial necessary for the reconstruction and evaluation of the trial.  Source data are contained in source 
documents.  Examples of these original documents, and data records include: hospital records, clinical and 
office charts, laboratory notes, memoranda, subjects’ diaries or evaluation checklists, pharmacy dispensing 
records, recorded data from automated instruments, copies or transcriptions certified after verification as 
being accurate and complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, 
subject files, and records kept at the pharmacy, at the laboratories, and at medico-technical departments 
involved in the clinical trial. 
9.3 Case Report Forms (as applicable) 
The study case report form (CRF) is the primary data collection instrument for the study.  All data requested 
on the CRF must be recorded.  All missing data must be explained.  If a space on the CRF is left blank 
because the procedure was not done or the question was not asked, write “N/D”.  If the item is not applicable 
to the individual case, write “N/A”.  All entries should be printed legibly in black ink.  If any entry error has 
been made, to correct such an error, draw a single straight line through the incorrect entry and enter the 
correct data above it.  All such changes must be initialed and dated.  DO NOT ERASE OR WHITE OUT 
ERRORS.  For clarification of illegible or uncertain entries, print the clarification above the item, then initial 
and date it. 
9.4 Records Retention 
It is the investigator’s responsibility to retain study essential documents for at least 2 years after the last 
approval of a marketing application in their country and until there are no pending or contemplated 
marketing applications in their country or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the investigational product.  These documents should be retained for a longer period 
if required by an agreement with the sponsor.  In such an instance, it is the responsibility of the sponsor to 
inform the investigator/institution as to when these documents no longer need to be retained.  
 


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10 Study Monitoring, Auditing, and Inspecting 
10.1 Study Monitoring Plan 
This study will be monitored according to the monitoring plan in Appendix ___.  The investigator will allocate 
adequate time for such monitoring activities.  The Investigator will also ensure that the monitor or other 
compliance or quality assurance reviewer is given access to all the above noted study-related documents 
and study related facilities (e.g. pharmacy, diagnostic laboratory, etc.), and has adequate space to conduct 
the monitoring visit. 
10.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, the sponsor, 
government regulatory bodies, and University compliance and quality assurance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study data etc.).  
The investigator will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable University compliance and quality assurance offices. 
11 Ethical Considerations 
This study is to be conducted according to US and international standards of Good Clinical Practice (FDA 
Title 21 part 312 and International Conference on Harmonization guidelines), applicable government 
regulations and Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted independent Ethics 
Committee (EC) or Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study conduct.  The decision of the EC/IRB concerning the conduct of the study will be 
made in writing to the investigator and a copy of this decision will be provided to the sponsor before 
commencement of this study.  The investigator should provide a list of EC/IRB members and their affiliate 
to the sponsor. 
 
All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study.  See Attachment 
___ for a copy of the Subject Informed Consent Form.  This consent form will be submitted with the protocol 
for review and approval by the EC/IRB for the study.  The formal consent of a subject, using the EC/IRB-
approved consent form, must be obtained before that subject undergoes any study procedure.  The consent 
form must be signed by the subject or legally acceptable surrogate, and the investigator-designated 
research professional obtaining the consent.  
12 Study Finances 
12.1 Funding Source 
This study is financed through a grant from the Eunice Kennedy Shriver National Institute of Child Health 
and Human Development (NICHD).  
12.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or financial gain 
greater than the minimum allowable by their institution, etc.) will have the conflict reviewed by a properly 
constituted Conflict of Interest Committee with a Committee-sanctioned conflict management plan that has 
been reviewed and approved by the study sponsor prior to participation in this study.  All UNC investigators 
will follow the University conflict of interest policy. 


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12.3 Subject Stipends or Payments 
Participants will be compensated the amount specified below upon completion of the study for a total of 
$500.00. If a subject withdraws or is withdrawn from the study as a result of an adverse event, her 
compensation will be prorated based on the following schedule: 
 
Initial evaluation and Psychological Interview (Visit 1; 2.5 hours) $100.00 
Physical Exam (Visit 2; 1-2 hours) $30.00 
FMRI Imaging combined with clinic visits (Visits 3 and 6) $150.00/visit -- $300.00 total 
Clinic visits with venipuncture (Visits 4 and 5) $35.00/visit -- $70.00 
 
Payment is processed through the UNC Department of Psychiatry.  
13 Publication Plan 
Neither the complete nor any part of the results of the study carried out under this protocol, nor any of the 
information provided by the sponsor for the purposes of performing the study, will be published or passed 
on to any third party without the consent of the study sponsor.  Any investigator involved with this study is 
obligated to provide the sponsor with complete test results and all data derived from the study. 
14 References 
1. 
Kessler, R. C., Chiu, W. T., Demler, O., Merikangas, K. R. & Walters, E. E. Prevalence, severity, and 
comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication. Arch. 
Gen. Psychiatry 62, 617627 (2005) 
2. 
Krishnan, V. & Nestler, E. J. The molecular neurobiology of depression. Nature 455, 894902 
 
(2008). 
3. 
Rubinow, D. R. & Schmidt, P. J. Gonadal steroids, brain, and behavior: role of context. Dialogues 
Clin. Neurosci. 4, 123137 (2002). 
4. 
Schmidt, P. J., Haq, N. & Rubinow, D. R. A longitudinal evaluation of the relationship between 
reproductive status and mood in perimenopausal women. Am. J. Psychiatry 161, 22382244 (2004). 
5.  
Schmidt, P. J. et al. Estrogen replacement in perimenopause-related depression: A preliminary report. 
Am. J. Obstet. Gynecol. 183, 414420 (2000). 
6. 
 Hasler, G. & Northoff, G. Discovering imaging endophenotypes for major depression. Mol. 
 
Psychiatry 16, 604619 (2011). 
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