A Randomized, Double-blind Placebo-controlled Multi-center Study of Identifying Neural Mechanisms of PTSD Symptom Reduction Induced by Combined Estrogen and Prolonged Exposure Therapy
Phase: PHASE3
Conditions: PTSD
Sponsor: The University of Texas Health Science Center, Houston

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IRB NUMBER: HSC-MS-23-0497 
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A Randomized, Double-blind Placebo-controlled Multi-center Study of Identifying Neural 
Mechanisms of PTSD Symptom Reduction Induced by Combined Estrogen and Prolonged 
Exposure Therapy 
NCT04192266 
Version Date: 03/04/2025 

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Principal Investigator 
Mohammed R. Milad 
 
 
Sponsor 
National Institute of Mental Health (NIMH) 
 
 
 
 
 
Protocol Version 
03MAR2025 
Version 4.4 
 
 
 
 
 
A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, MULTI- 
CENTER STUDY OF IDENTIFYING NEURAL MECHANISMS OF PTSD 
SYMPTOM REDUCTION INDUCED BY COMBINED ESTROGEN AND 
PROLONGED EXPOSURE THERAPY 

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Tool Revision History: 
Version Number 
Version Date 
Summary of Revisions Made 
.01 
10 October 2019 
Original approval 
1.1 
22 October 2019 
Training Protocol changes, adding Bryana Schantz as 
study team member 
1.2 
2 December 2019 
Clarify measures at each visit, clarify discontinuation 
procedures 
1.3 
5 December 2019 
Remove 1-month follow-up visit and offer referrals earlier, 
at post-treatment assessment visit. Add three new study 
team members to the protocol (a 4th year medical student 
and two volunteers). Clarify exclusion criteria; specifically, 
detailing individual diagnoses that will be excluded 
1.4 
17 December 2019 
Revised per IRB requests 
1.5 
16 January 2020 
Fix mistake in Inclusion Criteria, and add Dylan Miller to 
the protocol 
1.6 
4 February 2020 
Corrected visits for which patients take Estradiol to include 
PE session 6, added Adherence Questionnaires to 
Assessments Administered at PE Treatment Sessions 
(Table 2), added Early Withdrawal Form to Other Forms, 
and changed SAE language 
1.7 
27 February 2020 
Updated criteria for additional PE sessions following study 
completion, added biphasic birth control to inclusion 
criteria, clarified procedures for experimental days 1 and 
2, deleted 1-month follow-up to be consistent with rest of 
protocol, corrected amount of blood drawn, granted Emma 
Jennings EPIC access, clarified participation payment 
timing 
1.8 
15 April 2020 
Added Sarah Boukezzi, PhD to protocol, and clarified 
language re: screening/baseline assessment and timing 
1.9 
30 June 2020 
Updated inclusion/exclusion criteria as follows: added 
benzodiazepines to the exclusion criteria, removed right- 
handedness from inclusion criteria. Updated the version of 
the Life Events Checklist (LEC) used to the LEC-5 
Extended version. 
2.0 
10 August 2020 
Updated inclusion/exclusion criteria to remove peanut oil 
allergy. Updated study phase to Phase 3. Removed 
Rebecca Suzuki, Stephanie Wu, and Camille Johnson 
from study staff, and removed Rebecca Suzuki’s EPIC 
access. 
2.1 
23 September 2020 
Added state version of STAI (form Y1) to experimental 
days 1 and 2 both pre- and post-treatment (i.e, visits 2, 3, 
10, & 11). Removed MINI from post-treatment 
assessment (visit 12) and 3-month follow-up (visit 13). 
Updated that electrodes will be placed on the fingers (not 
foot) to record skin conductance data in PE therapy 
sessions. Updated that electrodes will be placed on foot 
(not hand) for electric shocks on experimental days 1 and 
2. Removed Sarah Boukezzi from study staff and 

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discontinued her EPIC access. Added Zhenfu Wen to 
study staff. 
2.2 
8 October 2020 
Granted EPIC access to Katie Eastburn, NP. Clarified 
number of subjects to be enrolled in training portion of 
study. Clarified randomization procedures. Added PDS-5 
measure to baseline assessment, post-treatment 
assessment, 3-month follow-up, and 6-month follow-up. 
2.3 
21 October 2020 
Added that participants who are deemed ineligible during 
or after the Baseline Assessment visit will be paid $10 for 
each hour they spent completing study procedures 
/assessments. Clarified that the lifetime/recent version of 
the CSSRS will be performed at the Baseline 
Assessment, but only the recent (i.e., past 1-3 months) 
version will be administered at the post-treatment 
assessment and 3- and 6-month follow-ups. 
2.4 
4 November 2020 
Added NuvaRing birth control method to study inclusion 
criteria. Added specification to exclusion criteria on self- 
injurious behaviors. 
2.5 
21 December 2020 
Added CAPS-5 Past Week version to assessment visits 
(baseline assessment, post-treatment assessment, and 3- 
and 6-month follow-ups). Removed CAPS-5 Past Month 
version from Post-treatment Assessment and 3-month 
follow-up. Added option to obtain participant consent 
electronically via REDCap. Added ClinCards as payment 
option. Clarified that forms can be filled out either in 
writing or electronically. Updated that the 4 additional PE 
sessions will be offered to eligible patients at the 3-month 
follow-up, not the post-treatment assessment. 
2.6 
23 March 2021 
Added option for pre- and post-experimental day blood 
draws to occur on Day 2 instead of Day 1 if needed due to 
scheduling. Added option for any visit, except for pre- and 
post-experimental days to occur remotely via Webex if 
needed in special circumstances. Updated exclusion 
criteria to include neurological disorders and history of 
seizures or head trauma. 
2.7 
03 June 2021 
Updated inclusion criteria to include the transdermal birth 
control patch. Granted EPIC access to Isabel Moallem, 
PhD. Added Nextdoor to the list of advertisement 
platforms. Updated the name of Dr. Simon’s lab. 
2.8 
14 October 2021 
Added additional recruitment methods; broadened 
inclusion criteria to include certain types of vaginal birth 
control ring (not only brand NuvaRing). Granted Epic 
access to Noor Nassar and Joseph Borelli. 
2.9 
07 December 2021 
Created process regarding positive UTOX for THC during 
the Experimental/Scanning visits: Added saliva THC test 
as additional procedure to implement if participant has 
positive UTOX at the Experimental/Scanning visit. Added 
information about texting participants. 
3.0 
08 February 2022 
Clarified use of updated NEO measure: NEO-FFI-3 form 
3.1 
14 April 2022 
Updated list of team members who have access to EPIC 
results and clarified information for incidental findings. 

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Added information about new recruitment source: 
BuildClinical. 
3.2 
09 June 2022 
Added an exclusion criterion to improve subject screening. 
Included additional NYU MRI scanning location 159 E. 
53rd Street location. Clarified that recruitment strategies 
will including contacting participants through the iConnect 
registry. 
3.3 
15 JULY 2022 
Updated list of study staff who will have access to EPIC. 
Clarified that Adverse Events will be assessed at 
experimental visits (in addition to the clinical visits and 
follow-up assessment visits). 
3.4 
24 AUGUST 2022 
Updated the list of personnel who will have access to Epic 
Updated section 8.8.2 to indicate that any study 
physicians will review AEs/SAEs and handle prescriptions. 
3.5 
11OCTOBER2022 
Updated language to specify blue ink is also allowable. 
Update to list of staff with Epic access 
Updated payment distribution to increase compensation 
for completing the first visit. 
Removal of Payment Method Language 
3.6 
12 DECEMBER 
2022 
Edited inclusion criterion (CAPS score cutoff and including 
subPTSD) 
Update to staff list with Epic access 
Update to incidental finding language 
Clarified language regarding data deletion 
Update to Datacore recruitment practices 
3.7 
19JUNE2023 
Initial submission to UTHealth Houston IRB 
3.8 
21AUG2023 
Replaced Methodist Hospital with Baylor College of 
Medicine, as MRI scanning site. Revised compensation. 
Revised recruitment strategies. Added Labcorp, where 
blood will be drawn and processed. 
3.9 
11SEPT2023 
Updated an exclusion criterion with UTHealth information 
4.0 
05OCT2023 
Added to Recruitment strategies: Info/IRB number for 
BBSB recruitment registry. Edited Study Drug Label. Added 
information on blood draw. 
4.1 
31OCT2023 
Added Jair C. Soares, MD, PhD to the study. 
4.2 
13DEC2023 
Added estradiol test. 
4.3 
08JAN2024 
Updated role of Dr. Mira Milad in the protocol. Added Quest 
Diagnostics as option for blood draw and processing. 
4.4 
03MAR2025 
Added plan for sharing participant contact information with 
protocol HSC-MS-23-1106 

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Study Personnel 
 
Principal Investigator: 
Mohammed R. Milad, Ph.D. 
 
 
 
Additional Investigators: 
Edna Foa, Ph.D. 
 
 
 
 
Key Study Personnel: 
Mohammed R. Milad, PhD 
Principal Investigator 
Mira Milad, M.D. 
Physician 
Ronald E. Acierno, Ph.D. 
Investigator 
Isabel Moallem, Ph.D. 
Clinical Assessor 
Zhenfu Wen, Ph.D. 
Investigator 
Jair C. Soares, M.D., PhD. 
Physician 
UTHealth Houston Study 
Number: 
HSC-MS-23-0497 
Funding Sponsor: 
National Institutes of Health (NIH) 
 
 
IND/IDE Number: 
135357 (IND Exemption received) 
Study Product: 
Estradiol (from Estrace) 
ClinicalTrials.gov Number 
NCT03371654 

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Initial version: 10/10/2019 
 
Amended: 10/30/2019 
Amended: 12/02/2019 
Amended: 12/05/2019 
Amended: 12/17/2019 
Amended: 01/16/2020 
Amended: 02/04/2020 
Amended: 02/27/2020 
Amended: 04/15/2020 
Amended: 06/30/2020 
Amended: 08/10/2020 
Amended: 09/09/2020 
Amended: 09/23/2020 
Amended: 10/08/2020 
Amended: 10/21/2020 
Amended: 11/04/2020 
Amended: 12/21/2020 
Amended: 03/23/2021 
Amended: 06/03/2021 
Amended: 10/14/2021 
Amended: 12/07/2021 
Amended: 02/08/2022 
Amended: 04/14/2022 
Amended: 06/09/2022 
Amended: 07/15/2022 
Amended: 08/24/2022 
Amended: 10/11/2022 
Amended: 12/28/2022 
Amended and initial submission to UTHealth Houston: 06/19/2023 
Amended: 08/21/2023 
Amended: 09/11/2023 
Amended: 10/05/2023 
Amended: 10/31/2023 
Amended: 12/13/2023 
Amended: 01/08/2024 
Amended: 03/03/2025 
 
Statement of Compliance 
This study will be conducted in accordance with the Code of Federal Regulations on the Protection of Human 
Subjects (45 CFR Part 46), 21 CFR Parts 50, 56, 312, and 812 as applicable, any other applicable US 
government research regulations, and institutional research policies and procedures. The International 
Conference on Harmonisation (“ICH”) Guideline for Good Clinical Practice (“GCP”) (sometimes referred to as 
“ICH-GCP” or “E6”) will be applied only to the extent that it is compatible with FDA and DHHS regulations. The 
Principal Investigator will assure that no deviation from, or changes to the protocol will take place without prior 
agreement from the sponsor and documented approval from the Institutional Review Board (IRB), except where 
necessary to eliminate an immediate hazard(s) to the trial participants. All personnel involved in the conduct of 
this study have completed Human Subjects Protection Training. 

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Table of Contents 
STATEMENT OF COMPLIANCE ........................................................................................................................6 
LIST OF ABBREVIATIONS .................................................................................................................................5 
PROTOCOL SUMMARY .....................................................................................................................................7 
SCHEMATIC OF STUDY DESIGN ......................................................................................................................9 
1 
KEY ROLES ............................................................................................................................................. 10 
2 
INTRODUCTION, BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ........................... 11 
2.1 
BACKGROUND INFORMATION AND RELEVANT LITERATURE ................................................................................... 11 
2.1.1 
Introduction to PTSD and the Relevance of Fear Learning and Extinction ................................. 11 
2.1.2 
Prolonged exposure, Estrogen and Extinction Learning ............................................................. 12 
2.2 
NAME AND DESCRIPTION OF THE INVESTIGATIONAL AGENT ................................................................................. 12 
2.2.1 
Preclinical Data ............................................................................................................................ 12 
2.2.2 
Clinical Data to Date .................................................................................................................... 12 
2.2.3 
Dose Rationale ............................................................................................................................ 13 
2.3 
RATIONALE ..................................................................................................................................................................... 13 
2.4 
POTENTIAL RISKS & BENEFITS ................................................................................................................................... 14 
2.4.1 
Known Potential Risks ................................................................................................................. 14 
2.4.2 
Known Potential Benefits ............................................................................................................ 14 
3 
OBJECTIVES AND PURPOSE ............................................................................................................... 14 
3.1 
PRIMARY OBJECTIVE .................................................................................................................................................... 14 
4 
STUDY DESIGN AND ENDPOINTS ........................................................................................................ 15 
4.1 
DESCRIPTION OF STUDY DESIGN ............................................................................................................................... 15 
4.2 
STUDY ENDPOINTS ....................................................................................................................................................... 15 
4.2.1 
Primary Study Endpoints ............................................................................................................. 15 
4.2.2 
Secondary Study Endpoints ........................................................................................................ 15 
5 
STUDY ENROLLMENT AND WITHDRAWAL ........................................................................................ 16 
5.1 
INCLUSION CRITERIA ..................................................................................................................................................... 16 
5.2 
EXCLUSION CRITERIA ................................................................................................................................................... 16 
5.3 
VULNERABLE SUBJECTS .............................................................................................................................................. 17 
5.4 
STRATEGIES FOR RECRUITMENT AND RETENTION ................................................................................................. 17 
5.4.1 
Use of MyChart Information for Recruitment Purposes .............................................................. 18 
5.5 
DURATION OF STUDY PARTICIPATION ....................................................................................................................... 18 
5.6 
TOTAL NUMBER OF PARTICIPANTS AND SITES ........................................................................................................ 18 
5.7 
PARTICIPANT WITHDRAWAL OR TERMINATION ........................................................................................................ 18 
5.7.1 
Reasons for Withdrawal or Termination ...................................................................................... 18 
5.7.2 
Handling of Participant Withdrawals or Termination ................................................................... 19 
6 
STUDY DRUG AND PROCEDURAL INTERVENTION .......................................................................... 19 
6.1 
STUDY AGENT(S) AND CONTROL DESCRIPTION ...................................................................................................... 19 
6.1.1 
Acquisition ................................................................................................................................... 19 
6.1.2 
Formulation, Appearance, Packaging, and Labeling .................................................................. 19 
6.1.3 
Product Storage and Stability ...................................................................................................... 20 
6.1.4 
Preparation .................................................................................................................................. 20 
6.1.5 
Dosing and Administration .......................................................................................................... 20 
6.1.6 
Route of Administration ............................................................................................................... 20 
6.1.7 
Duration of Therapy ..................................................................................................................... 20 
6.1.8 
Tracking of Dose ......................................................................................................................... 20 
6.2 
STUDY AGENT ACCOUNTABILITY PROCEDURES...................................................................................................... 20 

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7 
STUDY PROCEDURES AND SCHEDULE ............................................................................................. 20 
7.1 
STUDY PROCEDURES/EVALUATIONS ......................................................................................................................... 21 
7.1.1 
Screening/Baseline Phase .......................................................................................................... 21 
7.1.2 
Experimental, Treatment, and Follow-up Days ........................................................................... 22 
7.1.3 
Allocation to Interventional Group ............................................................................................... 23 
7.2 
STUDY EVALUATIONS AND MEASUREMENTS ............................................................................................................ 24 
7.2.1 
Baseline Evaluation ..................................................................................................................... 24 
7.2.2 
Laboratory Evaluations ................................................................................................................ 24 
7.2.3 
Urine Toxicology Screen and Saliva THC Test ........................................................................... 24 
7.2.4 
Pregnancy Testing....................................................................................................................... 24 
7.2.5 
fMRI Scans .................................................................................................................................. 24 
7.2.6 
Other Evaluations, Measures ...................................................................................................... 24 
7.3 
STUDY SCHEDULE ......................................................................................................................................................... 25 
7.3.1 
Procedures for Training of Clinicians in Prolonged Exposure (PE) Therapy .............................. 26 
TABLE 3: SCHEDULE OF TRAINING STUDY PROCEDURES ..................................................................... 27 
7.3.2 
Standard of Care Study Procedures ........................................................................................... 28 
7.4 
CONCOMITANT MEDICATIONS, TREATMENTS, AND PROCEDURES ....................................................................... 28 
7.5 
JUSTIFICATION FOR SENSITIVE PROCEDURES ......................................................................................................... 28 
7.5.1 
Precautionary Medications, Treatments, and Procedures .......................................................... 29 
8 
ASSESSMENT OF SAFETY ................................................................................................................... 29 
8.1 
SPECIFICATION OF SAFETY PARAMETERS ................................................................................................................ 29 
8.1.1 
Definition of Adverse Events (AE) ............................................................................................... 30 
8.1.2 
Definition of Serious Adverse Events (SAE) ............................................................................... 31 
8.1.3 
Definition of Unanticipated Problems (UP) .................................................................................. 31 
8.2 
CLASSIFICATION OF AN ADVERSE EVENT ................................................................................................................. 31 
8.2.1 
Severity of Event ......................................................................................................................... 31 
8.2.2 
Relationship to Study Agent ........................................................................................................ 31 
8.2.3 
Expectedness .............................................................................................................................. 32 
8.3 
TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP ................................................... 32 
8.4 
REPORTING PROCEDURES – NOTIFYING THE IRB ................................................................................ 33 
8.4.1 
Unanticipated Problem Reporting ............................................................................................... 33 
8.4.2 
Reporting of Pregnancy ............................................................................................................... 33 
8.5 
REPORTING PROCEDURES – NOTIFYING THE STUDY SPONSOR ......................................................................... 33 
8.6 
REPORTING PROCEDURES – PARTICIPATING INVESTIGATORS............................................................................. 34 
8.7 
STUDY HALTING RULES ............................................................................................................................................... 34 
8.8 
SAFETY OVERSIGHT ..................................................................................................................................................... 34 
9 
STATISTICAL CONSIDERATIONS ........................................................................................................ 35 
9.1 
STATISTICAL AND ANALYTICAL PLANS (SAP) ....................................................................................... 35 
9.2 
STATISTICAL HYPOTHESES.......................................................................................................................................... 35 
9.3 
ANALYSIS DATASETS .................................................................................................................................................... 36 
9.4 
DESCRIPTION OF STATISTICAL METHODS ................................................................................................................ 36 
9.4.1 
General Approach ....................................................................................................................... 36 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) .............................................................................. 36 
9.4.3 
Analysis of the Secondary Endpoint(s) ....................................................................................... 36 
9.4.4 
Safety Analyses ........................................................................................................................... 36 
9.4.5 
Adherence and Retention Analyses ............................................................................................ 37 
9.4.6 
Baseline Descriptive Statistics .................................................................................................... 37 
9.4.7 
Planned Interim Analysis ............................................................................................................. 37 
9.4.8 
Safety Review .............................................................................................................................. 37 
9.4.9 
Efficacy Review ........................................................................................................................... 38 
9.4.10 
Multiple Comparison/Multiplicity .............................................................................................. 38 
9.5 
SAMPLE SIZE .................................................................................................................................................................. 38 

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9.6 
MEASURES TO MINIMIZE BIAS ..................................................................................................................................... 38 
9.6.1 
Enrollment/Randomization/Masking Procedures ........................................................................ 38 
10 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS ....................................... 38 
11 
QUALITY ASSURANCE AND QUALITY CONTROL ............................................................................. 39 
12 
ETHICS/PROTECTION OF HUMAN SUBJECTS ................................................................................... 39 
12.1 
ETHICAL STANDARD ...................................................................................................................................................... 39 
12.2 
INSTITUTIONAL REVIEW BOARD .................................................................................................................................. 40 
12.3 
INFORMED CONSENT PROCESS ................................................................................................................................. 40 
12.3.1 
Consent Procedures and Documentation ............................................................................... 40 
12.4 
PARTICIPANT AND DATA CONFIDENTIALITY .............................................................................................................. 41 
12.4.1 
Research Use of Stored Human Samples, Specimens, or Data ............................................ 41 
13 
DATA HANDLING AND RECORD KEEPING ......................................................................................... 41 
13.1 
DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES ................................................................................... 41 
13.2 
STUDY RECORDS RETENTION .................................................................................................................................... 42 
13.3 
PROTOCOL DEVIATIONS ............................................................................................................................................... 42 
13.4 
PUBLICATION AND DATA SHARING POLICY ............................................................................................................... 42 
14 
STUDY FINANCES .................................................................................................................................. 42 
14.1 
FUNDING SOURCE ......................................................................................................................................................... 42 
14.2 
COSTS TO THE PARTICIPANT ....................................................................................................................................... 42 
14.3 
PARTICIPANT REIMBURSEMENTS OR PAYMENTS .................................................................................................... 43 
15 
STUDY ADMINISTRATION ..................................................................................................................... 43 
16 
CONFLICT OF INTEREST POLICY ........................................................................................................ 43 
17 
REFERENCES ......................................................................................................................................... 44 
18 
ATTACHMENTS ...................................................................................................................................... 46 

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List of Abbreviations 
 
ACTH 
Adrenocorticotropic hormone 
AE 
Adverse Event/Adverse Experience 
BOLD 
Blood Oxygen Level Dependent 
CFR 
Code of Federal Regulations 
CR 
Conditioned Response 
CRF 
Case Report Form 
CS 
Conditioned Stimuli 
CSOC 
Clinical Study Oversight Committee 
CTSA 
Center for the Treatment and Study of Anxiety 
CX 
Conditioning Context 
CV 
Coefficient of Variance 
dACC 
Dorsal anterior cingulate cortex 
DCC 
Data Coordinating Center 
DHEA 
Dehydroepiandrosterone 
DHEAS 
Dehydroepiandrosterone Sulfate 
DHHS 
Department of Health and Human Services 
DSMB 
Data and Safety Monitoring Board 
E2 
Estradiol 
FDA 
Food and Drug Administration 
FSH 
Follicle-Stimulating Hormone 
FFR 
Federal Financial Report 
fMRI 
Functional magnetic resonance imaging 
FWA 
Federalwide Assurance 
GAD 
Generalized anxiety disorder 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
LH 
Luteinizing Hormone 
MOP 
Manual of Procedures 
N 
Number (typically refers to participants) 

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NIH 
National Institutes of Health 
OCs 
Oral Contraceptives 
OHRP 
Office for Human Research Protections 
OHSR 
Office of Human Subjects Research 
PHI 
Personal Health Information 
PI 
Principal Investigator 
PTSD 
Post-Traumatic Stress Disorder 
QA 
Quality Assurance 
QC 
Quality Control 
RF 
Radiofrequency coil 
ROI 
Religion of interest 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SCR 
Skin Conductance Response 
SOP 
Standard Operating Procedure 
THC 
Tetrahydrocannabinol, the main psychoactive compound in cannabis 
UIC 
University of Illinois at Chicago 
US 
Unconditioned stimulus 
UTOX 
Urine toxicology screen or urine drug screen 
vmPFC 
Ventromedial prefrontal cortex 

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Protocol Summary 
 
Title 
Estradiol as a Cognitive Enhancer for the Treatment of Post-Traumatic Stress 
Disorder in Women 
Short Title 
The influence of gonadal hormones on fear extinction 
Brief Summary 
The purpose of this research study is to determine if taking a pill of estradiol (E2) 
together with prolonged exposure (PE) therapy can improve this treatment 
outcome in women diagnosed with Post-Traumatic Stress Disorder (PTSD). 80 
subjects will take part in this research study across UTHealth Houston and Penn 
(40 subjects at each site). Participants will be randomized into one of two groups, 
PE + E2 or PE + placebo. The study will include preliminary screening and 
baseline visits, experimental visits, and therapy visits over the course of six 
weeks. Two follow-up visits will take place at 3 and 6 months. 
Phase 
3 
Objectives 
Primary: To examine the effects of E2+PE on the extinction-induced 
psychophysiological responses (SCR), neural responses, and the information flow 
between the fear extinction nodes at post-PE sessions relative to pre-PE. 
Secondary: To examine the impact of E2 administration on PE efficacy at 
reducing PTSD symptom severity. 
Tertiary: To test the interaction between E2+PE induced psychophysiological and 
neuronal changes (pre-to-post) with the improvement of PTSD symptom severity. 
Methodology 
Randomized, double-blind, placebo-controlled trial 
Endpoint 
Primary study endpoint will be Blood Oxygen Level Dependent BOLD fMRI 
signals and Skin Conductance Response (SCR) during recall. The secondary 
study endpoint will be symptom severity. 
Study Duration 
3 years 
Participant 
Duration 
The initial study takes 6 weeks with follow-up visits at 3 and 6 months post- 
completion of the initial study. 
Duration of IP 
administration 
2 mg of Estradiol, as obtained from Estrace, to be administered once over the 
course of 5 sessions. 
Population 
Sample size is 80 enrolled subjects, 40 at UTHealth Houston, 40 at Penn. 
Participants will be females ages 18-45 with chronic (at least 1 month post- 
trauma) DSM-5 FULL PTSD diagnosis and a Clinician-Administered PTSD Scale 
for DSM-5 (CAPS) Past Month version score ≥20 OR chronic (at least 1 month 
post-trauma) subPTSD diagnosis and CAPS Past Month version score of ≥20 
(subPTSD defined as: meeting criterion A, F, G, H, and clusters B, C, and at least 
1 of the clusters D or E.). Trauma needs to fall under Criterion A. Participants will 
be on stable medications (for 3 or more months by the time of study entrance), 
outpatient, and will provide informed consent. For naturally cycling female 
subjects, stage of menstrual cycle will be ascertained by history, and by 
serological measures. For women on oral contraceptives, we will identify those 
using monophasic or biphasic of first, second, third or fourth generation with up to 
35mcg of ethinyl estradiol. 

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Study Sites 
UTHealth Houston 
 
 
 
University of Pennsylvania Perelman School of 
Medicine 
. 
 
 
Number of 
participants 
80 participants expected to be enrolled across 2 sites 
Description of 
Study 
Agent/Procedure 
Study drug will consist of one 2.0 mg dose of estradiol, (obtained from Estrace® 
Tablets, 2.0 mg estradiol tablets, USP, Warner Chilcott [now Allergan, as Warner 
Chilcott was acquired by Allergan]) taken orally. 
Reference 
Therapy 
Placebo 
Key Procedures 
Pregnancy Test, Urine Toxicology Screen, fMRI scan, Blood Draw, 
Estradiol/Placebo Dosage 
Statistical Analysis 
For all aims, analyses of SCR will test for group x condition interactions for the 
different groups in each aim with respect to CS+E vs. CS+U. With respect to the 
fMRI data, the initial analysis will involve assessing for a main effect of group (E 
vs. Plc), post treatment using CS+E vs. CS+U contrast. In addition, we will 
compare post treatment brain activations vs. pre-treatment within each group as 
well as the interactions between time and group also using the same imaging 
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Schematic of Study Design 
 
Process Diagram 
 
Visit 0: Telephone Screening 
• 
Local advertising efforts instruct prospective participants to complete a screener 
• 
The Research Assistant/study team member at UTHealth Houston calls and screens individuals for 
eligibility based off of the screener. Such screening is routine at UTHealth Houston and requires 
about 30 minutes. 
• 
Callers who pass the screen will be scheduled for an in person consent (or consented online via 
Redcap) and clinical assessment where their final eligibility for the study will be determined. 
 
Visit 1: Screening/Baseline Assessment 
• 
Total n=40 
• 
Obtain informed consent 
• 
Screen potential subjects by inclusion and exclusion criteria 
• 
Assessments Administered: CAPS-5 Past Month and Past Week versions, Demographics Form, Day 
to Day Form, MINI, Pregnancy Test, Urine Toxicology Screen, Edinburgh Handedness Survey, LEC-5 
Extended, Safety Assessments, CGI-S, CTQ, BDI-II, Beck Anxiety Inventory, STAI, ASI, NEO-Short 
Form, PSQI, Q-LES-QSF, PDS-5 
• 
Assessments Administered by IRB-approved Medical Professional (MD or NP): Gynecological History 
Form, General Health Form, Prior/Concomitant Medications 
• 
This IRB-approved medical professional will also be available to answer any questions about the 
medication should they arise during the informed consent process. 
• 
The clinical assessment will be audiotaped 
 
Visit 2: Experimental Day 1 
• 
The participant will undergo the experiment in an experimental room with psychophysiological 
instruments at UTHealth Houston 
• 
Habituation, conditioning, and extinction learning procedures will be performed 
• 
Conditioning procedures: subjects will view several presentations of pictorial stimuli and receive a 
shock 
• 
Assessments Administered: Blood draw (4.5ml) conducted to determine level of hormones of interest; 
STAI (state version / form Y1), Shock Questionnaire, Pregnancy Test, Urine Toxicology Screen, 
Saliva THC test (if UTOX positive for THC) (THC is the main psychoactive compound in cannabis) 
 
Visit 3: Experimental Day 2 
• 
Extinction recall and renewal procedure in the scanner at Baylor College of Medicine: subjects will 
view several presentations of similar randomized pictorial stimuli, but with no shock 
• 
Assessments Administered: STAI (state version / form Y1), Shock Questionnaire, Pregnancy Test, 
Urine Toxicology Screen, Saliva THC test (if UTOX positive for THC), (Blood draw could occur on this 
day instead of Visit 2 if needed due to scheduling) 
• 
Subjects randomized: Estrogen (n=20), Placebo (n=20) 
 
Visits 4-9: PE Sessions 1-6 
• 
Treatment Session 1: General overview of treatment and rationale for exposure. A medical 
professional (MD or NP) will prescribe and provide the patient with the study medication bottle, 
confirm the labelling and review the instructions with the participant. 
• 
Blood draw (4.5ml) conducted to determine level of hormones of interest at visits 5 and 9 (PE 
sessions 2 and 6) 
• 
2 mg Estradiol or placebo will be taken by participant via a pill form 5-6 hours before sessions 2-6 of 
PE treatment 
• 
Sessions 2-6: 60 minute sessions of imaginal exposure and processing as well as in vivo exposure 
homework 

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• 
Assessments Administered: PDS-5, PTCI, BDI-II, Skin conductance, Safety Assessments 
• 
All PE sessions are videotaped 
 
Visit 10: Experimental Day 1 
• 
The participant will undergo the experiment in an experimental room with psychophysiological 
instruments at UTHealth Houston 
• 
Habituation, conditioning, and extinction learning procedures will be performed 
• 
Conditioning procedures: subjects will view several presentations of pictorial stimuli and receive a 
shock 
• 
Assessments Administered: Blood draw (4.5ml) conducted to determine level of hormones of interest, 
STAI (state version / form Y1), Shock Questionnaire, Pregnancy Test, Urine Toxicology Screen, 
Saliva THC test (if UTOX positive for THC) 
 
Visit 11: Experimental Day 2 
• 
Extinction recall and renewal procedure in the scanner at Baylor College of Medicine: subjects will 
view several presentations of similar randomized pictorial stimuli, but with no shock 
• 
Assessments Administered: STAI (state version / form Y1), Shock Questionnaire, Pregnancy Test, 
Urine Toxicology Screen, Saliva THC test (if UTOX positive for THC), (Blood draw could occur on this 
day instead of Visit 10 if needed due to scheduling) 
 
Visit 12: Post Treatment Assessment 
• 
An independent evaluator (IE) will administer the CAPS-5 Past Week version immediately after 
therapy. This visit should occur within a week of Visit 11. Visit 12 can occur on the same day as Visit 
11. 
• 
Any participants who ask for referrals or are deemed clinically in need of additional care indicated by 
the clinician for any reason will be provided with them at this visit. 
• 
Assessments Administered: CAPS-5 Past Week version, Day to Day Form, Safety Assessments, 
CGI-S, CTQ, BDI-II, Beck Anxiety Inventory, STAI, ASI, NEO-Short Form, PSQI, Q-LES-QSF, PDS-5 
 
Visits 13 & 14: 3 & 6-month Follow-Ups 
• 
At the 3-month follow-up visit, participants whose pre-treatment to 3-month follow-up CAPS-5 Past 
Week score has not improved by at least 30% and whose CAPS-5 Past Week score is 26 or greater 
at the 3-month follow-up visit will be offered additional PE treatment if they wish. Specifically, 
participants will have the option to receive up to 4 additional sessions of PE delivered for an hour a 
week (10 total sessions of PE). 
• 
All participants regardless of scores interested in additional treatment after their study participation 
will be offered assistance with clinical referrals, as clinically appropriate. 
• 
Assessments Administered: CAPS-5 Past Week versions, Additional Treatment Inventory (ATI), BDI- 
II, Day to Day Form, Safety Assessments, CGI-S, CTQ, BDI-II, Beck Anxiety Inventory, STAI, ASI, 
NEO-Short Form, PSQI, Q-LES-QSF, PDS-5. The MINI and CAPS-5 Past Month will be administered 
at Visit 14 (6-month Follow-Up) only. 
 
 
1 Key Roles 
Dr. Mohammed R. Milad will be the principal investigator on this study. He will oversee the implementation of 
the fMRI experiments, data acquisition and analyses. Dr. Milad will also be in communication with the MRI 
Site at UTHealth Houston for the duration of the study to discuss the implementation of the experiments, 
monitor safe implementation of the paradigm, and review and document any adverse events during that part 
of the study. Dr. Jair C. Soares and/or Dr. Mira Milad will be the clinically responsible physician investigator 
clinical trialists for this treatment trial, and the clinical trial diagnostic, treatment and safety monitoring will 
occur within the clinical research program at UTHealth Houston. Drs. Mohammed R. Milad, Mira Milad, and 
Jair C. Soares will work very closely to coordinate the experimental imaging and clinical trial aspects of this 
translational protocol, and Drs. Mira Milad and Jair C. Soares will support Dr. Mohammed R. Milad in 
reporting requirements. 

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Mohammed R. Milad, Ph.D. 
 
 
 
 
Mira Milad, M.D. 
 
 
 
Ronald E. Acierno, Ph.D. 
 
 
 
Jair C. Soares, M.D., Ph.D. 
 
 
 
University of Pennsylvania Study Site (subcontract) 
Edna Foa, Ph.D. (PI at U Penn) 
 
2 Introduction, Background Information and Scientific Rationale 
2.1 Background Information and Relevant Literature 
2.1.1 
Introduction to PTSD and the Relevance of Fear Learning and Extinction 
PTSD is a common chronic condition associated with considerable morbidity and mortality. Approximately 
10% of the US adult population has had a lifetime PTSD (Kessler, 2012). The prevalence rates for women are 
significantly higher than for men, with an estimated 4% of the women in the United States reporting current 
clinically significant PTSD symptoms (McLean, 2011). Natural recovery from PTSD usually occurs within the 
first three months, but studies have found rates of 11-38% of significantly interfering PTSD symptoms in 
women up to 6 years after a traumatic event (Steenkamp, 2012; Arnberg, 2013). 
 
In human fear conditioning paradigms, a neutral cue such as a blue square, is paired with the presentation of 
an aversive cue, such as a mild electric shock. The subjects eventually express conditioned fear responses, 
such as higher skin conductance responses and changes in heart rate when the conditioned cue is 
presented. The subsequent presentation of the conditioned stimulus in the absence of the aversive one (i.e., 
presenting the blue square without the shock) leads to fear extinction learning (Milad, 2012). Milad and 
colleagues, along with several other labs, have identified a network of brain regions involved in the acquisition 
and consolidation of both fear and extinction memories (Graham, 2011). The amygdala, insular cortex, and 
dorsal anterior cingulate cortex (dACC) typically show increased activation during the fear learning phase. As 
extinction learning takes place, the ventromedial prefrontal cortex (vmPFC) exhibits increased activation, 
suggesting that it may be involved in the encoding and/or consolidation of the fear extinction memory. After a 

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delay (typically 24 hours later), successful recall of the extinction memory trace has been associated with 
increased activations of the vmPFC and the hippocampus (Graham, 2011; Milad, 2007; Phelps, 2004; 
Kalisch, 2006; Pitman, 2012). Milad et al. (2009) have shown that the delayed extinction memory recall is 
impaired in PTSD as measured by SCR. Importantly, this SCR deficit during recall has been associated with 
dysfunctional activation patterns of the fear extinction network in this patient population. PTSD patients were 
able to form associations between neutral stimuli and aversive stimuli and were able to learn to extinguish the 
conditioned fear responses during extinction learning. After a 24-hour delay, however, the PTSD patients 
showed very high conditioned fear responses to the extinguished cues, despite the apparent successful 
extinction training. These data suggest that the consolidation or retrieval of extinction or safety memories may 
be impaired in PTSD patients. Moreover, this failure to store and express the extinction memory was 
associated with altered functioning of the vmPFC, dACC, amygdala, and hippocampus in those patients 
(Milad, 2009a). 
 
2.1.2 
Prolonged exposure, Estrogen and Extinction Learning 
Prolonged exposure (PE) is a treatment for PTSD that has the most evidence for its efficacy (McLean, 2013; 
Powers, 2010; Foa, 2013; Foa, 2007). Exposure-based treatments for anxiety related disorders including PE 
have cogent parallels with fear extinction paradigms (Foa, 2000; Gillihan, Foa, 2011). PE involves 
confrontation with safe but feared situations, objects, and memories, which result in fear reduction to the 
formerly feared stimuli. This fear reduction is thought to be a result of an active process in the brain, leading 
to the formation of a new memory (safety memory), which updates or modifies the original fear memories that 
caused the expression of PTSD symptoms (Bouton, 2004). Notably, while most patients benefit from PE, 
some remain symptomatic. Given the parallel between PE and fear extinction, neuroscience laboratories that 
are in search of pharmacological agents to strengthen extinction memory consolidation have used fear 
conditioning paradigms to test the effectiveness of their interventions. 
 
2.2 Name and Description of the Investigational Agent 
Study drug will consist of either one 2.0 mg dose of estradiol, (obtained from Estrace® Tablets, 2.0 mg 
estradiol tablets, USP, Warner Chilcott [now Allergan, as Warner Chilcott was acquired by Allergan]) orally or 
look-alike placebo capsules. These doses was selected based on studies of micronized estradiol in the 
literature suggesting that peak plasma levels are reached 5-6 hr post-administration. 
 
2.2.1 
Preclinical Data 
Female rats as well as women who undergo extinction training while their estrogen (E) levels are naturally 
high show significantly lower fear during the extinction recall test, indicating enhanced extinction memory 
consolidation (Milad, 2009b; Milad, 2010; Zeidan, 2011). Estrogens are female sex hormones that are 
primarily produced by the ovaries and include estrone (E1), estradiol (E2) and estriol (E3). E2 is the 
predominant and most potent circulating estrogen produced during the reproductive years in non-pregnant 
women and is commonly prescribed in pill and transdermal form to treat postmenopausal symptoms. 
Henceforth, when we use the term estradiol (E2), we are referring to the exogenously administered estradiol 
and when we use the term estrogen (E), we are referring more broadly to all estrogens, still noting E2 as the 
most potent one. Elevated E levels significantly increased activation of brain regions associated with fear 
extinction in both women and female rats, including the vmPFC (IL in rodents) along with other brain regions 
such as the hippocampus in humans (Zeidan, 2011). Notably, women using oral contraceptives (OC), which 
induce tonically low E levels, exhibit extinction memory deficits (Graham, 2013). Women diagnosed with 
PTSD exhibit increased fear acquisition compared to men with PTSD (Inslicht, 2013). Moreover, low levels of 
estrogen in women diagnosed with PTSD are associated with impaired fear extinction and more severe PTSD 
symptoms (Glover, 2012). Taken together, these facts not only support the epidemiological data showing 
higher levels of PTSD in women but also implicate low E levels in PTSD pathophysiology, suggesting a novel 
potential target for PTSD prevention and intervention. 
 
2.2.2 
Clinical Data to Date 
In humans, neuroimaging studies have illustrated differential, sex-specific activity in several limbic regions, 
including the vmPFC, during the processing of emotional stimuli. These sex-specific responses are modulated 
by natural variance in gonadal hormones during the menstrual cycle. These data indicate that gonadal 
hormones could regulate fear extinction and thereby potentially account for sex-differences in the prevalence 
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In humans, we recently conducted a study examining the psychophysiological responses of fear extinction 
and extinction recall in healthy males vs. females in two different phases of their menstrual cycle: mid 
follicular, where estrogen levels are highest, and early follicular, where estrogen levels are at their lowest. We 
observed that males show higher conditioned responses relative to females. Moreover, we observed that 
males and females in early follicular phase expressed similar levels of extinction memory whereas females 
tested during late follicular phase showed relatively impaired extinction memory recall (Milad et al., 2006). 
However, in the above pilot data, we did not obtain blood samples to specifically measure the levels of 
gonadal hormones at test. In addition, other phases of the menstrual cycle, early and late luteal phases, were 
not tested. These latter phases have different hormonal characteristics that may have different effects on fear 
conditioning and extinction. Rising levels of both estrogen and progesterone characterize early luteal phase, 
where progesterone levels tend to be slightly higher than those of estrogen. In the late luteal phase, both 
estrogen and progesterone levels are declining. See figure 1 below for graphical representation of the levels 
of estrogen and progesterone at the four different phases of the menstrual cycle. 
 
Figure 1. The levels of estrogen and progesterone during the four different phases of the menstrual cycle. 
 
2.2.3 
Dose Rationale 
2.0 mg Estradiol tablets or pill placebo will be administered orally 5-6 hours prior to each PE treatment 
session. 
 
2.3 Rationale 
In a double-blind placebo-controlled study, we recently found that a single administration of 2mg of estradiol 
(E2) (which is an active form of Estrogen) to healthy women before extinction learning significantly enhanced 
extinction memory recall when tested the next day. Naturally cycling women participated in a 3-day paradigm 
while their endogenous estradiol levels were low (early follicular phase of their menstrual cycle). On day 1, 
they underwent a fear conditioning procedure without hormonal manipulations. On day 2, women received 
either a pill of E2 (2mg) or placebo prior to extinction training. Extinction recall was then assessed on day 3 in 
a drug-free state. On day 2, extinction was successful for both groups and no between-group differences 
were noted during this phase. On day 3, however, the percent of fear recovered in women who have received 
E2 was significantly lower compared to women receiving placebo. Thus, a single pill of E2 administered 
before extinction learning significantly enhanced consolidation of the extinction memory trace in healthy 
naturally cycling women. 
 
We will recruit women with PTSD symptoms and who are taking low-dose monophasic or biphasic oral 
contraceptives. All participants will receive 6 Prolonged Exposure (PE) sessions (2 sessions/week for 3 
weeks). During the last 5 of the 6 PE sessions, participants will receive either PE combined with 2mg of E2 or 
PE combined with Placebo. Note that the typical PE usually involved 10-12 sessions. In this case, we have 
decided to use a suboptimal level of PE to avoid ‘floor’ effects and thus be able to examine if E2 used in 
conjunction with PE will accelerate PTSD symptom reduction relative to Placebo given in conjunction with PE. 
All women will undergo a 2 day-fear conditioning and extinction paradigm at baseline and after having 
completed the 6 PE sessions. They will then receive follow up assessments at 1, 3 and 6 months. 
 
In the current study, we propose to examine how 2 mg doses of estrogen might modulate the neural circuitry 
of fear extinction in conjunction with Prolonged Exposure therapy. Delineating how sex and gonadal 

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hormones modulate fear extinction could set the stage for the development of novel and sex-specific 
treatments for anxiety disorders, specifically PTSD. 
2.4 Potential Risks & Benefits 
2.4.1 
Known Potential Risks 
Risks are as follows: a) Participants could develop mild to moderate emotional discomfort or frustration 
associated with psychiatric interviewing or filling out questionnaires; b) participants may experience subjective 
distress during treatment; c) potential side effects of E2 include headache, breast pain, irregular vaginal 
bleeding, stomach cramps, nausea, or hair loss. These side effects, however, are documented with the long- 
term use of E2 and there are no known side effects of short-term use of E2 as proposed in this application; e) 
noise from the fMRI machine can cause discomfort. Participants will be wearing earplugs to minimize this 
discomfort and they can stop the study at any time; f) the electrical shock will be applied to the foot and is 
uncomfortable but not painful but may cause discomfort in the participants; g) participants may find blood 
withdrawal at different time points discomforting or slightly painful; h) the association between the electric 
shocks and the pictures to be presented during the fear conditioning and extinction paradigm might induce 
some anxiety when viewing the pictures in anticipation of the shock. 
 
It is anticipated that subjects with derive benefit from receiving well-delivered treatment for PTSD. The 
findings from these studies will help to advance psychiatric research generally, and knowledge of the role of 
estrogen in augmenting existing treatments for PTSD. Results of this study will also help us to identify 
important information regarding fear extinction and learning, which could advance clinical knowledge of 
anxiety disorders. By addressing these important questions, the findings from the proposed study will help 
shape further research on fear extinction, thereby improving our understanding of fear learning. 
 
2.4.2 
Known Potential Benefits 
Participants may not directly benefit as a result of participating in this study. Participants may benefit from 
receiving careful diagnostic assessment, clinical monitoring and well-delivered first line evidence based 
targeted psychotherapy treatment for PTSD. Participants may also benefit from knowing that they have 
contributed to the scientific understanding of the role of estrogen in augmenting existing treatments for PTSD 
and that this may help other women with similar problems. The data to be ascertained will contribute to 
enhancing our knowledge of PTSD psychopathology and the neural mechanisms associated with treatment 
outcomes in PTSD. 
 
3 Objectives and Purpose 
In this proposal we will examine the mechanisms by which estradiol (E2) administration in conjunction with 
Prolonged Exposure therapy (PE) could enhance extinction recall and activation of the extinction network in 
individuals with significant PTSD symptoms; and to examine how such an enhancement may be associated 
with PTSD symptom reduction 
 
3.1 Primary Objective 
The primary objective is to delineate the influence of exogenous administration of estrogen on conditioned 
responses during extinction recall in women with PTSD. 
 
Specific Aim 1.1: To examine the effects of E2+PE on the extinction-induced psychophysiological responses 
(SCR), neural responses, and the information flow between the fear extinction nodes at post-PE sessions 
relative to pre-PE. 
 
Hypothesis 1.1: During extinction recall, both groups will show lower SCR at post- relative to pre-PE. A 
general improvement is expected over time in both groups given the anticipated benefits of PE alone. 
However, we expect a significant Time x Group interaction, where the Estradiol-treated group should exhibit 
significantly lower fear levels (indexed via SCR) at the follow-up assessment relative to the Placebo group. 2) 
For both groups, extinction recall-induced activations will be higher in brain regions associated with extinction 
learning and recall (i.e. vmPFC and hippocampus) and lower in regions associated with fear expression (i.e. 
dACC and amygdala) at post- relative to pre-treatment. Note that a general improvement from pre to post is 
expected in both groups, given the anticipated benefits of PE alone. We do expect, however, a significant 

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Time x Group interaction, with most pronounced effects at post-PE in the estradiol-treated group. 3) 
Information flow between the extinction nodes will improve (indexed by DCM models), with stronger effects in 
E2+PE after treatment. 
 
Specific Aim 2: To examine the impact of E2 administration on PE efficacy at reducing PTSD symptom 
severity. 
 
Hypothesis 2: PTSD symptom severity will be significantly lower in the E2+PE group relative to the 
Placebo+PE group following acute treatment, as well as at the 3- and 6-month follow-up assessments. 
 
 
Specific Aim 3: To test the interaction between E2+PE induced psychophysiological and neuronal changes 
(pre-to-post) with the improvement of PTSD symptom severity. 
 
Hypothesis 3: PTSD symptom reduction will correlate with BOLD and SCR changes observed during 
extinction recall following PE. 
 
4 Study Design and Endpoints 
Section 4 will provide a brief overview of the phases of the clinical protocol (screening, baseline, experimental 
days). More details on procedures occurring during each phase will be included in subsequent sections of the 
protocol. 
 
Therapist Training Procedures: 
In addition, prior to beginning work with randomized study participants as an approved study therapist, all 
study therapists at UTHealth Houston will undergo a training procedure with a study participant recruited to 
participate in this training phase of the study. The details of this are outlined in 7.3.1. and a separate consent 
form explaining the training procedures with the treatment but not imaging components of the study is 
included. 
 
4.1 Description of Study Design 
This is a randomized, double-blind, placebo-controlled trial where 80 participants will undergo fear 
conditioning, fear extinction, and fear recall experiments, fMRI scans, as well as receive Prolonged Exposure 
treatment. Participants will be randomly assigned to additionally receive a pill placebo or 2 mg Estradiol. 
 
The study includes a) Psychiatric intake evaluation to determine study eligibility, b) 2 consecutive day visits 
that occur at visits 2 and 3 and again at visits 10 and 11 with fMRI scanning on the second day, c) prolonged 
exposure therapy sessions. The 4 consecutive visits include roughly 1 hour fMRI scans, as well as fear 
conditioning through a mild shock stimulus from a battery-powered stimulator, the Biopac STM100C 
Stimulator, and the use of psychophysiological measures (SCR). There are clinical follow up assessments at 
3 and 6 months post treatment. 
 
4.2 Study Endpoints 
4.2.1 
Primary Study Endpoints 
Primary study endpoint will be Blood Oxygen Level Dependent BOLD fMRI signals and Skin Conductance 
Response (SCR) during recall. 
 
4.2.2 
Secondary Study Endpoints 
The secondary study endpoint will be the evaluation of PTSD symptom severity using DSM-5 PTSD 
symptoms measured with the Clinician-Administered PTSD Scale for DSM-5 (CAPS) Past Week version at 
the Post-treatment assessment. In addition to the experimental primary and secondary end points, there will 
be primary safety endpoints. Subjects will not receive any stimulation beyond what they consider highly 
annoying to them. Subjects will be thoroughly screened prior to starting the fMRI component of the study to 
make sure that they have no metal implants of any kind that would be considered a hazard to their health in 
the fMRI. We will use a metal detector to screen all subjects and make sure that they carry no metals to the 

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scanner room. Pregnancy tests will be done for all female participants with child-bearing potential prior to the 
initiation of the experiment to rule out pregnancy. If pregnancy is suspected, subjects will not be allowed to 
participate in the study. If any excess movement by the subjects is observed (which could be indicative of 
discomfort), the study staff will check with the subjects and inquire whether they would like to stop the 
experiment. After estrogen administration, all subjects will be monitored for any adverse events. This risk, 
however, is low given that estrogen is a commonly used agent with known side effect profiles, a component of 
many oral and non-oral birth controls and given only for 5 total 2mg/ dose twice weekly doses in the study. 
Reasons for study discontinuation for any participants who stop the treatments or study prematurely will also 
be recorded and reported. 
 
5 Study Enrollment and Withdrawal 
5.1 Inclusion Criteria 
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
1. Female, 18-45 years of age 
2. Chronic (at least one month post-trauma) DSM-5 FULL PTSD diagnosis OR subPTSD diagnosis 
(subPTSD defined as: meeting criterion A, F, G, H, and clusters B, C, and at least 1 of the clusters D 
or E.) 
3. CAPS-5 Past Month score ≥ 20 
4. Criterion A traumatic event 
5. Stable medications for 3 or more months by the time of study entrance (with the exception of 
benzodiazepines) 
6. Women on oral contraceptives, specifically those using monophasic or biphasic of first, second, third 
or fourth generation with up to 35mcg of ethinyl estradiol; OR using etonogestrel / ethinyl estradiol 
0.120mg/0.015mg per day vaginal ring birth control; OR using the norelgestromin / ethinyl estradiol 
0.150mg/0.035mg per day transdermal patch birth control. 
7. Willing and able to provide informed consent 
 
5.2 Exclusion Criteria 
An individual who meets any of the following criteria will be excluded from participation in this study: 
1. Diagnosis of bipolar I disorder with a past year manic episode 
2. Diagnosis of a psychotic disorder or psychotic symptoms that would interfere with the ability to focus 
on posttraumatic stress disorder (PTSD) in clinic, as determined by clinical judgment. 
3. Diagnosis of moderate or severe substance use disorder that would interfere with the ability to focus 
on posttraumatic stress disorder (PTSD) in clinic, as determined by clinical judgment. 
4. Cognitive impairment that would interfere with the ability to focus on posttraumatic stress disorder 
(PTSD) in clinic, as determined by clinical judgment. 
5. History of neurological disease (that involves the brain), seizure, or significant head trauma (i.e., 
extended loss of consciousness, neurological sequelae, or known structural brain lesion). 
6. Suicidal ideation with imminent risk that warrants a higher level of care. 
7. Concurrent trauma focused psychotherapy 
8. Pregnancy (to be ruled out by urine ß-HCG). 
9. Metallic implants or devices contraindicating magnetic resonance imaging by interfering with patient 
safety or fMRI data collection. Cases will be cleared by the Principal Investigator and/or Baylor 
College of Medicine (Imaging). 
10. History of breast cancer or hormone-responsive cancer. 
11. Use of benzodiazepines 
12. Self-injurious behavior that involves suicidal intent, requires medical attention, or occurs daily. 
13. High risk of adverse emotional or behavioral reaction, and/or an inability to understand study 
procedures or the informed consent process, based on investigator/clinician clinical evaluation (e.g., 
evidence of serious personality disorder, antisocial behavior, serious current stressors, lack of 
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5.3 Vulnerable Subjects 
This study does not involve the enrollment of pregnant women, prisoners, employees, people with cognitive 
impairment or any decisional incapacity, fetuses, neonates or elderly or children under the age of 18. 
 
Confidentiality for all study participants will be strictly adhered to. Only group level de-identified outcomes will 
be shared outside the study team and UTHealth Houston approved functions with access to clinical research 
participant level data (e.g. IRB, UTHealth Houston research pharmacy). All participants will undergo formal 
informed consenting in person (or online via Redcap) with an IRB approved clinical study investigator with as 
much time for questions as needed. 
 
5.4 Strategies for Recruitment and Retention 
Subjects will be recruited from the local community via advertisement as well as social media such as: 
Craigslist, Reddit, Instagram, LinkedIn, Twitter, Facebook, Research Match, and Nextdoor, as well as 
UTHealth Houston‘s MyChart, and BuildClinical. Research Match is a nonprofit program funded by the NIH 
that connects people interested in participating in research studies with applicable studies. It will specify 
location, the study’s inclusion criteria, and be linked with the approved prescreen. BuildClinical is a data- 
driven software platform that helps academic researchers recruit participants for research studies more 
efficiently using social media, software, and machine learning. They have worked with IRBs in the US to 
ensure they adhere to all the appropriate guidelines and procedures. They utilize study-specific 
advertisements to engage participants on digital platforms such as Facebook, Google, WebMD, etc., and 
redirect them to a study-specific landing page should they click it. On the landing page, the person can 
complete an online pre-screen questionnaire that gets routed into BuildClinical's platform. BuildClinical's 
Secure Socket Layer (SSL) software, which encrypts all inputted information, keeps subject information 
private and HIPAA compliant. Their backend servers are stored in the USA at some of the most secure data 
centers in the world. 
 
The study team will work with the UTHealth Houston communications department to create a webpage 
describing our research study on the websites that the institution uses to recruit participants for studies and will 
work with UTHealth Houston‘s MyChart to recruit from UTHealth Houston patients. In addition, we will post 
flyers at UTHealth locations, show ads on the internal UTHealth Houston tv monitors, and use UTHealth 
Houston’s website and newsletters. 
 
Subjects will also be recruited from the public through local media advertisements and UTHealth Houston 
clinical sites, including Behavioral Sciences Research Institute registry/recruitment (BSRI). We will approach 
patients in clinic, via posted flyers in the outpatient buildings, post signs on campus, and we will also recruit 
from the Department of Psychiatry Recruitment Registry: HSC-MS-23-0768. Flyers will be posted at local 
university campuses, hospitals, and community clinics. Local advertising efforts will instruct prospective 
participants to complete a brief online confidential screening questionnaire. The Research Assistant, or study 
team member, at UTHealth Houston will then call and screen individuals for eligibility based on results of the 
screening questionnaire. Individuals who contact our centers will be screened initially by phone for interest in 
the proposed research, as well as general diagnostic and study eligibility, and interest and availability for 
research study participation. Such screening is routine at UTHealth Houston and requires about 30 minutes. 
Callers who meet initial potential eligibility based on the phone screen will be scheduled for an in-person 
assessment with a study clinician. At this visit they will review and sign the informed consent form (if not 
already consented online via Redcap), and assessments will occur to determine their final eligibility for the 
study. 
 
There is a plan for sharing contact information of the previously consented participants interested in future 
research studies. We will share a list of participant contact information (name, email and phone number) to Dr. 
Li’s team through a secure process (i.e., OneDrive) for the study HSC-MS-23-1106. 
 
Communicating with the Research Team: We added the following information to our informed consent, 
regarding texting research participants and give participants the option to opt in or out of receiving text 
communications from the research team. 

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Researchers may need to communicate with you about information relevant to the research study. The 
research team will usually contact you for these purposes by phone, but if you have given the Researchers 
your email address and mobile/cell phone number and permission to send a text message, the research 
team may contact you that way. When the research team sends email messages that include identifiable 
health information, they will use encrypted messaging (e.g., SendSafe). When the research team uses 
texting over mobile/cell phones there is no way to encrypt the message. This means that information you 
send or receive by text message is unencrypted and could be intercepted or viewed by an unintended 
recipient, or by your mobile/cell phone provider or carrier. Therefore, text messages carry security and 
privacy risks. 
 
Below are some important points about texting in this research study. 
• 
Text messages are not encrypted, and therefore are unsecure and may result in a breach of your 
confidentiality. 
• 
You will be responsible for all fees charged by your carrier’s service plan for text messaging. 
This research study and UTHealth Houston will not cover the cost related to any increased 
charges, data usage against plan limits or changes to data fees from the research texts. 
• 
Text messages will only be read during regular business hours. However, if you have a scheduled 
visit outside of business hours, you may receive a text in relation to this visit outside of regular 
business hours. 
• 
Text messaging should not be used in case of an emergency. If you experience a medical 
emergency, call 911 or go to the nearest hospital emergency department. 
• 
You may decide to not send or receive text messages with staff associated with this research 
study at any time. You can do this in person or by sending the research number a text message 
that says “Stop Research Text.” 
• 
Your agreement applies to this research study only. Agreeing to other texts from UTHealth 
Houston, for example appointment reminders, is a separate process. Opting out of other texts 
from UTHealth Houston is a separate process as well. 
 
5.4.1 
Use of MyChart for Recruitment Purposes 
The study will also use UTHealth Houston‘s MyChart to identify subjects. Data gathered will be used to 
identify potentially eligible subjects who did not opt out of being contacted for research. We will also use this 
data to gain contact information about the patient. 
A research coordinator, or study team member, will contact potential participants identified through MyChart. 
Any recruitment information containing sensitive information will be sent by secure email (e.g., Send Safe) 
email or will be done through MyChart. Once contact is made, approved recruitment language will be used to 
communicate the reason for contact and subjects will be asked if they are interested in participating in this 
specific study. If the potential subjects agree, the study team will provide the potential participants with 
information regarding the next steps for participation. If a subject requests information regarding opting out of 
further recruitment for all research, subjects will be directed to contact the UTHealth Houston 
department/contact that is responsible for processing such requests. 
 
5.5 Duration of Study Participation 
We will ask participants to make 14 visits to our facilities at UTHealth Houston. After the first assessment visit, 
participants will go through the 2-day visit for the emotional learning task. Following this, the participants will 
go through 6 therapy sessions over 3 weeks: 2 sessions per week. On visits 10 and 11 participants will go 
through the emotional learning task again and complete clinical outcomes assessments. Participants will 
return at 3 and 6 months for follow ups. 
 
5.6 Total Number of Participants and Sites 
Recruitment will end when approximately 80 subjects complete the screening, meet eligibility criteria and are 
randomized. It is expected that approximately 80 subjects will be randomized between both UTHealth 
Houston and Penn. 
 
5.7 Participant Withdrawal or Termination 

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5.7.1 
Reasons for Withdrawal or Termination 
Participants are informed they are free to withdraw from participation in the study at any time upon request. 
Patients’ participation in the treatment trial may be discontinued at any time because of serious medication 
side effects or if continued participation is considered a danger to their welfare. Specific reasons for treatment 
discontinuation include: 1) Serious adverse event; 2) Extreme laboratory values confirmed with a repeat test; 
3) discontinuation would be in the patient's best interest; 4) Participant no longer wants to continue. 
 
5.7.2 
Handling of Participant Withdrawals or Termination 
Participants deemed to be candidates for discontinuation will be reviewed by the principal investigator and 
other relevant project personnel (e.g., study psychiatrist or study treatment provider). Patients who require 
discontinuation of study treatments for any reason will be scheduled for a final evaluation and given 
appropriate treatment referrals. The reason patients are discontinued from the clinical trial will be clearly 
specified. 
 
6 Study Drug and Procedural Intervention 
6.1 Study Agent(s) and Control Description 
Study drug will consist of either one 2.0 mg dose of estradiol (obtained from Estrace® Tablets, 2.0 mg 
estradiol tablets, USP, Warner Chilcott [now Allergan, as Warner Chilcott was acquired by Allergan]) orally or 
look-alike placebo capsules. The IND number is 135357; this study has received an IND Exemption from the 
FDA given that our use of Estrace has met all 5 IND regulations set forth by the FDA (please see FDA letter 
included in the IRB application for details). 
 
6.1.1 
Acquisition 
UTHealth Houston Investigational Pharmacy will dispense study medication in patient-specific kits. The 
research assistant assigned to this study will pick up the medication from the pharmacy with the study 
psychiatrist or nurse, will account for all study medication received. Any damaged or unusable study pills in a 
given shipment (active drug or placebo) will be documented in the study files. Study medication will be 
provided to the participants only by approved study clinicians who will review the medication prescription, 
instructions and answer any questions with the participant. The investigator will notify the pharmacy of any 
damaged or unusable study medication received. 
 
6.1.2 
Formulation, Appearance, Packaging, and Labeling 
The Estradiol 2mg tablets and Placebo tablets will be prepared by a compounding pharmacy at both sites so 
the active and placebo pills are indistinguishable from one another in terms of appearance of the capsules. 
This will permit the appropriate concealment of the intervention from participants and study personnel alike, 
thereby enabling the double-blind. The capsules will be placed into a sealed plastic container and labeled 
clearly with the contents (drug, strength, quantity). 
 
At the time of dispense, five capsules will be placed into an Amber vial with a child-resistant, tightly sealing 
screw-cap. The vial will be labeled as follows: 
UTHealth Houston -Investigational Pharm 
 
RX # XXXXXXXXX 
Pt#: ###### 
Date:  
 
Dr. 
 
PATIENT NAME 
TAKE ONE CAPSULE BY MOUTH ONCE AS INSTRUCTED 
ESTRADIOL 2MG OR PLACEBO CAPSULES 
Quantity: 5 Capsules 
Refills: 0 
Do Not Use After: MM/DD/YYYY PROTOCOL: HSC-MS-23-0497 
Caution: New Drug Limited by 
Federal (United States) Law to 
Investigational Use Only 

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6.1.3 
Product Storage and Stability 
Medication will be managed and stored in a double-locked medication closet at UTHealth Houston’s 
Investigational Pharmacy. Study medication will be stored at ambient temperature. Temperature is monitored 
in the Investigational pharmacy daily. It remains at ambient temperature at the study site. 
 
6.1.4 
Preparation 
The study drug is prepared and dispensed from UTHealth Houston’s BBSB Pharmacy. 
 
6.1.5 
Dosing and Administration 
A single dose of estradiol 2mg or placebo will be taken at home by the study participant 5-6 hours before 
each of 5 PE treatment sessions (sessions 2 to 6). 
 
The study medication, 5 pills, will be given to the participant to take home after the first PE session by an 
approved study clinician (MD or NP) who will confirm the labelling and review the instructions with the 
participant. The subject will be instructed and reminded through a text or phone call to take the medication 5 
hours before the time of each of their next 5 therapy sessions. 
 
6.1.6 
Route of Administration 
The study medication will be taken orally. 
 
6.1.7 
Duration of Therapy 
Subjects will take one pill 5-6 hours before each PE session 2-6, which will take place over the course of 
approx. 3 weeks. Typically, a minimum of 8-12 PE sessions are required for the treatment of PTSD. In our 
study design, we aim to examine the effectiveness of only 6 PE sessions that would be strengthened by the 
administration of estradiol. The interaction between exposure/extinction learning that occurs while estradiol is 
elevated (due to the pill) during 5 PE sessions is predicted to be sufficient to induce a significant reduction in 
PTSD symptoms in the active drug group compared to placebo. 
 
Primary analyses will be based on a modified intent to treat sample including all participants who took at least 
one dose of the study medication. 
 
6.1.8 
Tracking of Dose 
Medication records will be completed on a regular basis with the following information: date administered, 
amount administered and to which subjects, product remaining, product damaged/destroyed and date of 
damage/destruction, product returned and date of return, etc. A standard form will be utilized to document this 
information throughout the study period. 
 
6.2 Study Agent Accountability Procedures 
The Pharmacy will destroy remaining study medication, after the study research assistant, under the direction 
of the study nurse and/or physician, returns it to them from this site. 
 
At the completion of the study, there will be a final reconciliation of drug dispensed, shipped, consumed, 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. 
 
7 Study Procedures and Schedule 
The screening/baseline assessment must be completed within 4 weeks of the first experimental days. If more 
than 4 weeks have passed since the screening/baseline assessment, the study PI/clinician will advise if 
participant will be rescreened during a separate visit and/or the CAPS will be repeated at Experimental Day 1. 

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The 2-day experimental visits must occur on 2 consecutive days. All study treatment visits can occur within a 
window of 7 +/- 5 days from the previous visit. 
 
Randomization to study medication will occur after the participant has come for Visit 3, and the participant will 
receive the medication and instructions for its use at visit 4 (the first PE session) so they can initiate 
medication at home 5 hours prior to visit 5 (the second PE session) 
 
In order to have the study intervention and assessments be as complete as possible, any visit, with the 
exception of the pre- and post-experimental days, may be conducted remotely via Webex if needed at the 
discretion of the research team, such as in the circumstance of weather or a possible participant COVID-19 
exposure. Webex will only be utilized if the research team has determined that the participant will be able to 
tolerate remote exposures safely. 
 
7.1 Study Procedures/Evaluations 
 
7.1.1 
Screening/Baseline Phase 
Subjects will complete a pre-screening survey on REDCap. The link to the initial REDCap survey will be 
included in our advertisements and potential participants will be screened out if they meet for any exclusion 
criteria. Once participants get through this survey, they will be contacted by a research coordinator to 
schedule the initial phone screen. An initial phone contact will be arranged with the study coordinator, or study 
team member, at UTHealth Houston based off of the initial pre-screening survey. During the initial contact, 
entry criteria will be reviewed and the study procedures will be outlined. Female subjects will be asked 
regarding their use of oral contraceptives as well as initial approved phone screening questions. Those 
women using oral contraceptives will be asked what kind to initially check if they are using 1st, 2nd, 3rd, or 
4th generation, monophasic or biphasic oral contraceptives containing up to 35mcg of ethinyl estradiol as part 
of the phone screen (to be confirmed by the in-person screening clinician). For candidates who are interested 
in participating and appear to be potentially appropriate for enrollment, an onsite visit at UTHealth Houston for 
formal consenting (or consenting will be completed online via Redcap), and in person psychiatric assessment 
with an IRB approved study clinical evaluator will be arranged. 
 
All participants will meet with a study clinician for consent (or complete consent online via Redcap) prior to the 
baseline in person screening evaluation and baseline assessments, consisting of a diagnostic interview and 
completion of the battery of self-report measures. The study clinical rater will describe the study in detail (i.e., 
the nature of the treatments and random assignment, duration of the treatment, fMRI component, payments 
for assessments, measurements collected, and follow-up procedures) and answer any questions. After 
signing the consent form, the participant will complete the clinician rated assessments and eligible 
participants will complete the self-report forms. 
 
Participants who are deemed ineligible after baseline screening will be offered assistance with clinical referrals, 
as clinically appropriate. Individuals who have consented and are found ineligible after baseline screening, will 
have all identifiers related to their clinical screening maintained, unless the participant requests for such 
identifiers to be destroyed. 
 
The initial evaluation will be conducted by a masters or doctorate-level clinician (or Independent Evaluator- 
IE) who is trained in psychiatric assessment. All study staff with the exception of the research pharmacy will 
be blind to medication treatment assignment. During the evaluation, the clinician or IE will interview the 
participant to assess current and lifetime disorders that meet exclusion criteria using the MINI, and to 
determine whether the participant meets the cutoff score using the CAPS-5 Past Month version. All efforts will 
be made to have the same independent evaluator assess the same patient throughout the course of the study 
when possible. Audio consent will be obtained to have all assessments audiotaped for reliability purposes. 
 
If a participant does not meet PTSD/subPTSD inclusion criteria, the clinician or IE may offer to repeat the 
CAPS-5 Past Month assessment one week or more following the original baseline assessment visit. This will 
be offered at the clinician’s discretion for participants whose scores are close to the cutoff score because 
symptoms may vary with time and/or changed circumstances. Participants will not be told the specific reason 

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for repeating the CAPS, but rather that further assessment is needed to determine eligibility. All baseline 
assessment measures must still be completed within 4 weeks of the initial experimental days. 
Participants will also complete a pregnancy test before each scan to ensure safety of fMRI scanning and will 
be scheduled to complete the first (of a total of two) fMRI paradigm (see below) and the treatment sessions. 
 
 
7.1.2 
Experimental, Treatment, and Follow-up Days 
Experimental Procedure: Fear conditioning and extinction paradigm 
To assess the effects of the combined E2 and PE on the neurobiology of fear regulation, all participants will 
undergo our 2 day-fear conditioning and extinction paradigm before the commencement of PE and after the 
completion of the 6 PE sessions with double blind E2 or pill placebo. Psychophysiological experiments will be 
performed either in an experimental room or in the scanner. 
 
Prior to the conditionability procedure, the subject will be reminded that a mild electric unconditioned stimulus 
(US) will be used and that if necessary she will be free to terminate the experiment at any time. Stimulating 
and recording electrodes will be attached. The stimulating electrodes will be attached to only one foot. Prior to 
setting the US level, the experimenter will give the following instructions: "For this experiment, you will set 
your own level of electric stimulation. You should choose a level that is highly annoying but not painful. I will 
start the stimulation at a very low level and gradually increase the level until you say ‘stop’. The level that you 
set will then be used throughout the remainder of the experiment." After the US level is established, the 
experiment will begin. 
 
Prior to the initiation of the experiment, subjects will view pictures of two rooms presented on a monitor, both 
of which differ in color and content (e.g., an office and a conference room). The conditioned stimuli (CSs) will 
be three different cues in the room (e.g. a light that differs in color: yellow, blue and red). Two of these lights 
will be followed by the US at a partial reinforcement rate of 62.5% (CS+s). The third CS will never be followed 
by the US. The CS+s, CS- and the US will only be presented in one context (conditioning context; CX+). The 
other context will be used for extinction training (CX-). Neither CS+ presented in the context of the CX-, nor a 
CS- presented in either context, will ever be paired with the US. The experiment will be composed of five 
sequential phases. The initial Habituation phase will consist of 4 presentations of the to-be CS+ (i.e. blue), 4 
presentations of the other to-be-CS+ (i.e. red) and 4 presentations of the to-be-CS- (i.e. yellow) in pseudo- 
random order (there will be no more than two consecutive presentations of the same type), all in the context 
of the CX+. Each CS will last 6 sec. The inter-trial-interval will be 15 + 3 sec., determined at random. 
 
Immediately after will be the Conditioning phase, which will consist of presentations of each stimulus type in 
pseudo-random order, again all in the context of the CX+. At the offset of 5 out of 8 trials CS+ (but not CS-); a 
500-msec. electrical current US will be administered. There will be a total of 8 CS+ (blue), 8 CS+ (red) and 16 
CS- (yellow) trials during the conditioning phase. After a rest period, the Extinction phase will begin. During 
extinction, the CS- and only one of the CS+s (Extinguished CS+, CS+E) will be presented in the context of 
the CX-, and no US will be delivered. There will be a total of 16 CS+E trials and 16 CS- trials. 
 
One day after extinction training, the subject will return for the Extinction Recall phase (extinction memory 
test). During this test, the CS+, CS-, and the Unextinguished CS+ (CS+U) will be presented. There will be 8 
CS+E, 8 CS+U, and 16 CS- trials presented during this phase in the extinction context (CX-). Following a brief 
rest period, the Renewal phase will be administered. The Renewal phase will be identical to the Extinction 
Recall phase except that all stimuli will be presented in the conditioning context (CX+). No shocks will be 
delivered during either phase during the retention test. 
 
Treatment with Prolonged Exposure combined with E2 or placebo: 
To test the hypothesis that E2 will augment the efficacy of PE in reducing PTSD symptoms, we propose to 
conduct a double-blind placebo controlled study in which we will administer E2 or placebo to women with 
significant PTSD symptoms who undergo PE. 2mg of synthetic estradiol (Estrace) will be administered 5 
hours prior to each of the 5 PE therapy sessions in which imaginal exposure will be administered (PE 
sessions 2 to 6). An automatic reminder will be sent to the participant when medication has to be taken in 
order to increase compliance. After the first introductory PE session, 5 imaginal exposure sessions will occur, 
such that there will be 2 sessions per week conducted for a total of 6 sessions over 3 weeks (more details in 

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the next section). The timing of the E2 administration was selected using our preliminary data that was based 
on the pharmacokinetics obtained from a recent publication. These data show that peak estrogen levels are 
achieved approximately 6 hours after ingestion (therapy session would be ending or about to end). This timing 
would allow for maximum estrogen levels to be present to strengthen the consolidation of the safety memory 
acquired during the therapy. 
 
Therapy Sessions and Assessment During Treatment: 
Session 1. This session provides an overview of the PE treatment and a general rationale for exposure, along 
with the proposed mechanism of action of estrogen on the exposure process. The therapist (same for all 
sessions) gathers information about the patient’s symptoms, details of the trauma, history of previous trauma, 
and functioning, and provides an explanation of PTSD and discusses common reactions to trauma and the 
rationale for treatment. In addition, the therapist will construct an in vivo hierarchy with the patient, which is 
why this session is extended to 120 minutes. The study medication, 5 pills, will be given to the participant to 
take home after the first PE session by an approved study clinician (MD or NP) who will confirm the labelling 
and review the instructions with the participant. 
 
Sessions 2-5. These sessions are 60 minutes long and consist of imaginal exposure. Specifically, the patient 
is instructed to imagine the trauma and recount it aloud for about 25-30 minutes. In the following 15 minutes, 
the patient processes her reactions to revisiting the traumatic event by discussing related thoughts and 
feelings. The session ends with in vivo exposure homework to be completed that same day as the session. 
 
Session 6. In this last session, imaginal exposure is conducted one last time for the same duration as 
previous sessions. The therapist and patient review treatment progress and discuss applications of treatment 
principles to daily life. 
 
Participants will complete the PDS-5 and BDI-II at each session and the PTCI at baseline, Visit 5, 7, and 9. A 
non-invasive skin conductance monitor (purchased from Mindfield eSense Skin Response) will be used to 
measure skin conductance throughout the imaginal exposure sessions. Electrodes will be placed on the 
fingers of the participant and will be connected to an iPad to record the skin conductance throughout each PE 
session. This will not cause discomfort or any additional risk to the subject. 
 
Post-treatment Assessment: 
Lastly, an independent evaluator, blind to treatment assignment, will assess PTSD severity after therapy (Visit 
12) (following same procedures as before therapy (Visit 1), and at 3 and 6 month follow-up visits) using the 
CAPS-5 Past Week version to assess whether changes in symptom severity are stable over time, and to 
detect any effects of the experimental manipulation on reduction of fear. 
 
Follow-Up Assessments (3 and 6 months post-treatment): 
These assessments will be identical to the post-treatment clinical and self-rated questionnaire assessments, 
and the 6-month follow-up will also include the CAPS-5 Past Month assessment. There is no additional 
experimental conditioning, fMRI or pregnancy test in follow up. In addition, participants will be asked to report 
any psychological, psychiatric and medical treatment they received since the last assessment. This 
information will be recorded on the Additional Treatment Inventory (ATI). Each follow up visit will include a 
clinical safety assessment for suicidality, and consider the potential need for clinical referrals, as above. 
 
Participants who do not improve at least 30% from the pre-treatment to 3-month follow-up CAPS-5 Past Week 
score and who have a CAPS-5 Past Week score of 26 or greater at the 3-month follow-up assessment will be 
offered additional PE treatment at UTHealth Houston if they wish. Specifically, participants will have the 
option to receive up to 4 additional sessions of PE delivered for an hour a week (for a total number of 10 
sessions of PE) immediately following the 3-month follow-up, consistent with prior study designs utilizing PE 
and to mimic real world clinical practice where patients receive a variable number of sessions of PE. The 
need for clinical referrals will be discussed and assessed with all participants by the study clinician regardless 
of endpoint improvement scores, and referrals made when indicated. 

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7.1.3 
Allocation to Interventional Group 
Participant randomization will be done by the UTHealth Houston Investigational pharmacy, from which we will 
be obtaining Estradiol and placebo. We will employ block randomization (with blocks size TBD to retain the 
blind), and randomization will be stratified by severity score at baseline. 
 
7.2 Study Evaluations and Measurements 
 
7.2.1 
Baseline Evaluation 
Baseline evaluations including: 
• 
Trauma history 
• 
Medical (including gynecological), psychiatric, and neurological history 
• 
Characteristics (age, gender, race) 
• 
Drug and medication use 
• 
Life history such as any stressful life events 
• 
Information regarding the subject’s OCP use and associated menstrual cycle 
 
7.2.2 
Laboratory Evaluations 
Approximately 18ml of blood will be drawn from each participant over the course of the study (see Table 1 
below) using venipuncture. Hormone measures examined will include gonadal (total estrogens, estradiol and 
progesterone), hormones. The blood samples will be drawn and processed at LabCorp or Quest Diagnostics. 
If needed, due to scheduling constraints, blood may also be drawn by phlebotomist at UTHealth Houston and 
then processed by LabCorp/Quest Diagnostics. There are no plans to store any samples for prospective 
processing. 
 
7.2.3 
Urine Toxicology Screen and Saliva THC Test 
If, on the day of any scanning visit, the participant’s UTOX is positive for THC (and negative for all other 
subtances), a saliva THC test will be administered in order to determine recency of THC use. If saliva THC test 
is negative; the scan will proceed as scheduled. If the saliva test is positive; the scan will be rescheduled. Saliva 
THC tests are rapid tests, with low participant burden, that can indicate recent THC use. 
 
7.2.4 
Pregnancy Testing 
A urine pregnancy test will be performed for all female subjects. See section 7.2.5 for more detail. 
 
7.2.5 
fMRI Scans 
Research subjects will undergo a 3T MRI scan during the second of the two experimental days at baseline 
and again at treatment endpoint. The MRI does include the use of experimental pulse sequences and/or RF 
coils. Some of the pulse sequences and/or RF coils are not FDA approved but are considered to pose no 
more than minimal risk. 
 
Pregnancy Clause -- Although there are no known risks related to MRI on pregnant women or a fetus, there 
is a possibility of yet undiscovered pregnancy related risks. Since there is no possible benefit from 
participating in this protocol for a pregnant woman, we will exclude pregnant women. A negative urine 
pregnancy test will be required before a woman of child-bearing potential can participate in this study, and all 
participants will be on an oral contraceptive. Should a pregnancy be identified, a separate modification 
request including information on whether or not the pregnancy will be followed will occur. 
 
7.2.6 
Other Evaluations, Measures 
 
Clinician Rated Measures 
• 
MINI International Neuropsychiatric Interview for DSM-5 (MINI; Hergueta, Baker, & Dunbar, 1998) 
• 
Clinician-Administered PTSD Scale for DSM-5 Past Month and Past Week versions (CAPS) 
• 
Medical health: the general health form 

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• 
Gynecological/Menstrual history form 
• 
Columbia Suicide Severity Rating Scale (C-SSRS; Posner et al., 2007) 
• 
Clinical Global Impressions Severity and Improvement Scale (CGI-S; Guy 1976) 
• 
Life Events Checklist Extended (LEC-5 Extended; Weathers et al., 2013) 
 
Self-Rated Measures 
• 
The Beck Depression Inventory (BDI-II: Beck et al., 1961) to quantify depressive symptoms 
• 
The Beck Anxiety Inventory (Beck and Steer, 1990) to quantify general anxiety symptoms 
• 
The Speilberger State Trait Anxiety Inventory (STAI: Spielberger, 1983) to quantify trait aspects of 
anxiety 
• 
The Anxiety Sensitivity Index (ASI) 
• 
Shock Expectancy Questionnaire 
• 
Day-to-Day Experiences Form 
• 
NEO-FFI-3-Short form (to assess big five dimensions of personality) 
• 
Pittsburg Sleep Quality Index (PSQI) 
• 
PTSD Diagnostic Scale for DSM-5 (PDS-5) 
• 
Posttraumatic Cognitions Inventory (PTCI) 
• 
Childhood Trauma Questionnaire (CTQ) 
• 
Quality of Life Enjoyment and Satisfaction Questionnaire Short Form (Q-LES-Q-SF) 
 
Safety Assessments: 
All clinical visits and follow up assessments and experimental visits will include the following: 
• 
Adverse Events (AE): clinicians/study team will track any adverse events that patients have 
experienced since their last visit. AEs will be documented and reviewed at all study visits. 
• 
Concomitant Medications and Therapy: The study clinician will record the use of both study 
medication and concomitant medications on the Concomitant Medication log at each study visit. In 
addition any other psychosocial treatment will be recorded. 
• 
Suicide Checklist: A modified CSSRS derived checklist to enable the clinician to assess, track and 
record any suicidal ideation, intent or behavior as part of the safety assessment. 
 
Other Forms 
• 
Demographics Form 
• 
Edinburgh Handedness Survey 
• 
Shock Expectancy Questionnaire 
• 
Prior/Concomitant Medications Form 
• 
Early Withdrawal Form 
 
All study assessments and measures can be completed as written forms, filled out electronically using 
fillable PDFs, or completed by participants as surveys via the REDCap database system. 
 
7.3 Study Schedule 
TABLE 1: SCHEDULE OF STUDY PROCEDURES 
 
Study Phase 
Phone 
Screen 
Screening/ 
Baseline 
Assessment 
Experimental 
Days (I) 
PE 
Session 1 
PE 
Sessions 2- 
6 
Experimental 
Days (II) 
Post-Treatment 
Assessment 
Follow- 
ups 
Visit Number 
n/a 
1 
2 
3 
4 
5-9 
10 
11 
12 
13-14 
Informed Consent 
X 
Review 
Inclusion/Exclusion 
Criteria 
X 
X 
Demographics/Medic 
al History 
X 

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Pregnancy Test 
X 
X 
X 
X 
X 
Urine 
 Toxicology 
Screen, Saliva THC 
test (if positive UTOX 
for 
THC 
on 
experimental/scannin 
g days) 
X 
X 
X 
X 
X 
Concomitant 
Medications 
and 
treatments 
X 
X 
X 
X 
X 
X 
Blood Draw 
X (or 
Visit 3) 
X (at visit 5 
and 9) 
X (or 
Visit 
11) 
Randomization 
X 
Psychiatric evaluation 
measures 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Estradiol/Placebo 
dosage 
X 
Adverse Event / 
Unanticipated 
Problems 
Assessment 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Habituation/Extinction 
Procedures 
X 
X 
Recall/Renewal 
Procedures 
X 
X 
PE Treatment 
Procedures 
X 
X 
Resting Skin 
Conductance 
X 
X 
 
 
TABLE 2: Schedule and list of evaluations and self-reports completed by participants. 
Assessments Administered at Visit 1 
Assessments Administered 
at PE Treatment Sessions 
Assessments Administered 
at Experimental Visits 
Assessments Administered at Post 
Treatment and Follow-ups 
Clinician Administered Forms: LEC-5 
Extended, MINI, CAPS-5 Past Month and 
Past Week versions, CGI-S, CSSRS (lifetime 
and recent) 
Psychiatric Evaluation 
Measures: PDS-5, PTCI (PE 
Session 2, 4, and 6), BDI-II, 
Safety Assessments, 
Adherence Questionnaires 
Shock Questionnaire, 
Speilberger State Trait 
Anxiety Inventory (STAI; 
state version / form Y1 only), 
AE Form 
Clinician Administered Forms: CAPS-5 Past 
Week version, CAPS-5 Past Month version at 6- 
month follow-up only, MINI at 6-month f/u only, 
CGI-S, CSSRS (recent) 
Psychiatric Evaluation Measures: CTQ, Beck 
Depression Inventory (BDI-II), Beck Anxiety 
Inventory, Speilberger State Trait Anxiety 
Inventory (STAI), Anxiety Sensitivity Index 
(ASI), Day-to-Day Experiences Form, NEO- 
Short Form, PSQI, PTCI, Quality of Life 
Enjoyment and Satisfaction Questionnaire 
Short Form (Q-LES-QSF), PDS-5 
Pregnancy test, Urine 
Toxicology Test, Saliva THC 
Test (if UTOX is positive for 
THC) 
Psychiatric Evaluation Measures: CTQ, Beck 
Depression Inventory (BDI-II), Beck Anxiety 
Inventory, Speilberger State Trait Anxiety 
Inventory (STAI), Anxiety Sensitivity Index (ASI), 
Day-to-Day Experiences Form, NEO-Short 
Form, PSQI, PTCI, Quality of Life Enjoyment 
and Satisfaction Questionnaire Short Form (Q- 
LES-QSF), PDS-5 
MD/NP Administered Forms: Medical 
History/General Health Form, Gynecological 
History Form, Safety Assessments 
MD/NP Administered Forms: Safety 
Assessments, Additional Treatment Inventory 
Other Self-Rated Patient Forms: Edinburgh 
Handedness Survey, Demographics Form 
Pregnancy Test, Urine Toxicology Test 

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7.3.1 
Procedures for Training of Clinicians in Prolonged Exposure (PE) Therapy 
 
Training Specific Aim: To train psychotherapists who will be involved in the main study to administer 
Prolonged Exposure (PE) therapy for posttraumatic stress disorder (PTSD) prior to the initiation of the main 
study. 
 
Training Enrollment: Each clinician performing PE therapy on the study needs to pass on performance for 6 
sessions of PE therapy for 2 eligible participants in order to receive their PE certification. If new clinicians are 
hired onto the study, more training participants will be recruited in order to certify these clinicians. We 
estimate this will take 2-3 participants treated per trainee. We also estimate a 50% screen fail rate on 
eligibility. Thus, we estimate that in total 25 participants will be enrolled in order to train 5 clinicians over the 
course of the study. These participants will only be included for training purposes and their data and 
participation will not contribute to the total participants included, or the analyses in the main part of the study 
 
Consent for Training Participation: Participants will undergo a specific consent for the training protocol 
included in this submission. This consent will clarify the training aspects of the PE training and will not include 
the study medication or imaging components of the study (which will not be delivered to training participants). 
All other questionnaire assessments will be identical. 
 
Recruitment and screening procedures, safety monitoring and post treatment referrals as needed for training 
participants will be identical to the procedures outlined in the main trial. Screening procedures will identify 
adults with a primary diagnosis of PTSD for whom treatment with PE is appropriate with the same entry 
criteria with the exception of concerns related to undergoing MRIs (e.g. metal implants) or testing estrogen. 
Thus the entry criteria for training participants is as follows: 
 
Training Participant Inclusion Criteria 
• 
Male or Female, 18-70 years of age 
• 
Criterion A traumatic event 
• 
At least one month post-trauma DSM-5 PTSD symptoms 
• 
PDS-5 score ≥ 28 
• 
Stable medications (for 3 or more months by the time of study entrance), with the exception of 
benzodiazepines). Benzodiazepine use will be excluded. 
• 
Provide informed consent 
 
Training Participant Exclusion Criteria 
• 
Other primary diagnoses (e.g., pervasive developmental disorders, psychotic disorder, thought 
disorder, or presence of other disorder that is primary relative to PTSD symptoms) 
• 
Imminent suicide risk in need of a higher level of care 
• 
Concurrent trauma focused psychotherapy 
• 
Pregnancy (to be ruled out by urine ß-HCG). 
 
Training procedures: Participants will receive the identical PE intervention described above with therapists 
being trained as approved study therapists. In addition, they will consent to videotapes of their therapy and 
assessment sessions being used for training purposes to certify the study therapist, as below. 
 
TABLE 3: SCHEDULE OF TRAINING STUDY PROCEDURES 
 
Study Phase 
Screening 
Baseline Assess 
PE Session 1 
PE Sessions 2-6 
Visit Number 
n/a 
1 
2 
3-7 
Informed Consent/Assent 
X 
Review Inclusion/Exclusion 
Criteria 
X 
X 
Demographics/Medical History 
X 

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Pregnancy Test 
X 
Urine Toxicology Screen 
X 
Prior/Concomitant Medications 
X 
X 
Psychiatric evaluation measures 
X 
Adverse Event / Unanticipated 
Problems Assessment 
X 
X 
X 
PE Treatment Procedures 
X 
X 
 
Training protocol risks and discomforts are limited to those associated with assessments and PE therapy, with 
the addition of potential discomfort having sessions videotaped and reviewed by trainers. 
 
Benefits include that training participants undergo expert diagnostic assessments and receive a gold standard 
psychotherapy treatment of PTSD at no cost. Prolonged Exposure is an evidence based first line treatment 
designed to reduce symptoms of PTSD. 
 
Participants will receive free, evidence-based, first-line treatment for PTSD as part of the training study. 
Monetary compensation in the amount of $100 will be provided to compensate for their time and any travel 
expenses. This amount will be given to participants upon completion of the PE therapy sessions. 
 
Recording of Therapy Sessions: 
All therapy sessions will be digitally video-recorded. This will allow us to 1) provide supervision for the 
clinicians, and 2) monitor the treatment and assessment information to be sure that we can evaluate whether 
the sessions are carried out according to the therapy guidelines. These recordings will be kept on an 
electronically secure, password protected hard drive in a locked room for no longer than 5 years. We will 
provide participants with a separate video consent form to review and complete. 
 
People who will watch the recordings include the participant’s study therapist and study-approved supervisors 
at UTHealth Houston and the University of Pennsylvania. Other study clinicians and/or people who are 
learning the study protocol may also hear some segments of the recording. Videos will be accessed only 
through secure channels that give access for viewing within the UTHealth Houston firewall. 
 
Post-Endpoint: 
Following the end of the treatment phase, patients interested in further treatment for residual PTSD symptoms 
will be given appropriate clinical referrals. 
 
7.3.2 
Standard of Care Study Procedures 
All procedures will be specific to the study protocol. 
 
 
7.4 Concomitant Medications, Treatments, and Procedures 
See Inclusion/Exclusion Criteria above. 
All concomitant prescription medications taken during study participation will be recorded on the case report 
forms (CRFs). For this protocol, a prescription medication is defined as a medication that can be prescribed 
only by a properly authorized/licensed clinician. Medications to be reported in the CRF are concomitant 
prescription medications, over-the-counter medications and non-prescription medications. 
In addition, all psychosocial interventions will be recorded. 
 
7.5 Justification for Sensitive Procedures 
Assessment of trauma is critical to the study aims and needed for the diagnosis and treatment of PTSD with 
prolonged exposure. Details of trauma will be asked only by trained approved study assessors and clinical 
providers, and all clinical study staff will be supervised by [study physician] at UTHealth Houston. See 
procedures to minimize risk below. 

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The use of placebo in our experiments is very important to reduce experimental bias. All participants will be 
receiving an active evidence-based treatment for PTSD, PE, in addition to the study medication, and non- 
responders with significant PTSD symptoms at the 3-month follow-up timepoint (who did not improve at least 
30% in their CAPS-5 Past Week score from pre-treatment to the 3-month follow-up and have a CAPS-5 Past 
Week score of 26 or greater post-treatment) will be offered additional PE sessions after the 3-month follow-up 
visit. 
 
7.5.1 
Precautionary Medications, Treatments, and Procedures 
The serum peak concentration of 2mg doses 1-2X/week of estradiol is expected to result in levels that are 
lower than that reached during ovulation in naturally cycling women (which is on average 400pg/ml). 
Therefore, the dose we are using will induce E2 levels that still fall within a normal physiological range for 
women of reproductive age. Thus, we do not anticipate any medical complications or concerning drug 
interactions from administration of the dose chosen in our study. 
 
8 Assessment of Safety 
8.1 Specification of Safety Parameters 
Procedures to Minimize Risk. Participants’ reactions to study assessments and treatment will be closely 
monitored, and negative reactions will be addressed therapeutically. Participants could develop mild to 
moderate emotional discomfort or frustration associated with psychiatric interviewing or filling out 
questionnaires. Participants will be allowed any breaks in the testing that are needed. Additionally, the Intake 
Assessment can be spaced over visits, if needed. 
 
Risks associated with PE therapy are mild to moderate discomfort when exposed to anxiety-provoking images 
and situations in treatment sessions and at home for homework and discomfort about being in treatment for 
victims of a traumatic event. The exposure sessions of PE are intended to elicit some emotional reactions 
(fear/anxiety) in order to engage the client in processing the traumatic memories. All exposures will be carried 
out with the full knowledge and consent of the client. Exposure therapy has produced considerable benefit for 
thousands of participants with anxiety disorders. While some participants fail to benefit from this therapy, 
there are only a handful of reports of negative side effects from it. None of the participants in our past or most 
recent treatment studies, sustained prolonged negative reactions to exposure. Potential side effects from PE 
(e.g., exacerbation of PTSD and associated symptoms) will be addressed by reassuring the client if 
necessary that these are temporary and are often associated with improvement. The therapists will also be 
trained to titrate response to exposure. In vivo exposure homework assignments will be gradual, beginning 
with situations that elicit moderate anxiety and progressing to increasingly anxiety-provoking situations. 
Participants will be informed by their therapist that they may call him/her between sessions should they 
experience crisis or are distressed enough to need psychological support. 
 
In addition, any participants who have not improved at least 30% from their pre-treatment the 3-month follow- 
up CAPS-5 Past Week score and have a CAPS-5 Past Week score of 26 or greater at the 3-month follow-up 
will be offered additional treatment if they wish. Participants will then have the option to receive up to 4 
additional sessions of PE delivered for an hour a week (for a total number of 10 sessions of PE) immediately 
following the 3-month follow-up visit. All participants will receive safety assessments throughout the study and 
at 3 and 6 month follow ups, and will be assisted with clinical referrals if desired and/or indicated. 
 
Monitoring of Mental Status and Mood. Subjects’ mental status will be closely monitored throughout the 
course of the study. As previously stated, it is not uncommon for treatment of trauma-related symptoms to be 
associated with temporary exacerbation in participants’ symptoms of PTSD and depression. However, some 
participants may experience symptom exacerbations or psychiatric emergencies that require treatment 
beyond that provided by the study. In all cases, the client’s mental status will be the utmost priority and 
immediate action will be taken to provide appropriate care. The intake evaluation for the study will include the 
Columbia-Suicide Severity Rating Scale to adequately assess suicide risk before study enrollment, and study 
therapists will routinely evaluate mood over the course of treatment, including the presence or absence of 
suicidal thoughts (using the BDI-II followed by clinical interview with a modified suicide check list at each 

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visit), and if any suicidal thoughts, plans or actions are present, these will be dealt with using the detailed plan 
outlined in Protection against Suicidality in the section immediately below. 
 
Management of Suicide Risk 
Several strategies will be implemented to assist in early identification and appropriate management of 
suicide risk. This protocol will utilize the Columbia Suicide Scale (C-SSRS) at screening and a suicide 
check list at each clinical and psychiatric assessment follow up visit to routinely assess suicidal ideation, 
impulses and behaviors. In addition, the BDI will be completed and reviewed by the study therapist who will be 
working closely with each participant. 
 
Further the primary clinical supervisors on the project a study clinician serving as her designate will be on call 
and available to discuss urgent and emergent clinical issues. Study therapist training will include identification 
of signs of depression and suicidality in patients. As noted above, clinicians will assess mood symptoms and 
suicidality on an ongoing basis and will specifically inquire about it at every visit. 
 
Participants deemed to be at acute risk for suicide based on the C-SSRS, checklist or clinical 
assessment will be referred for emergency care (e.g., hospital emergency room or urgent care) and, if 
necessary, transferred to an appropriate level of clinical care (i.e., inpatient hospitalization) until 
stabilized. Once stable, the need for study treatment termination will be determined on a case by case 
basis in coordination with the care team, site PI, and the study PI. However, no prohibition against 
necessary treatment will be in place, if needed. 
 
Safe Use of Estrogen. Common side effects of chronic administration of this drug include headache, breast 
pain, irregular vaginal bleeding, stomach cramps, nausea, or hair loss. However, there are no known side 
effects of acute single doses of estrogen, as it is the case in the current application. Importantly, all women 
will already be using oral contraceptives. A total of 5 doses of 2mg each over 3 weeks will be administered for 
this study. These acute E2 doses do not pose a significant risk for the participants. In fact, OC use plus these 
additional exogenous estrogen doses act through a negative feedback loop. Therefore, pregnancy is even 
less likely than with OCs alone, as is spotting. The exogenously administered E2 will mimic estrogen levels 
that are observed during ovulation in naturally cycling women. Importantly, at each appointment, participants 
will be asked how they are feeling and if they had encountered any of the potential side effects. If that is the 
case, participants will discuss with the MD in charge of the study and a clinical decision will be made whether 
or not the subject should stop her participation in the study. 
 
Minimizing risk during fMRI. No subjects will have metallic implants contraindicating MRI. All female 
subjects will have pregnancy ruled out via a urine pregnancy (beta-HCG) test. Subjects may ask to have a 
scan stopped and discontinue participation in the study at any time. Every effort will be made to reassure the 
patient and minimize any discomforts while in the fMRI scanner. Subjects will be able to converse with a staff 
member via a microphone and speaker system throughout. To reduce the discomfort caused by the MRI loud 
noises, subjects will wear earphones to enable presentation of auditory stimuli while minimizing ambient 
scanner noise. 
 
Participant Study Discontinuation 
Participants are free to discontinue study treatments or assessments at any time. 
 
An individual patient’s participation in the treatment trial may be discontinued at any time because of serious 
medication and/or therapy side effects or if continued participation is considered a danger to their welfare. 
 
Patients who require discontinuation of both of the study treatments (PE and double blind Estrace) for any 
reason will be scheduled for a final evaluation and given appropriate treatment referrals. The reason the 
patients are discontinued from the clinical trial and any referrals made will be clearly specified. 
 
8.1.1 
Definition of Adverse Events (AE) 
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: 

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• 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 
 
8.1.2 
Definition of Serious Adverse Events (SAE) 
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. 
 
8.1.3 
Definition of Unanticipated Problems (UP) 
 
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) 
• Suggests that the research places subjects or others at greater risk of harm (including physical, 
psychological, economic, or social harm). 
 
8.2 Classification of an Adverse Event 
 
8.2.1 
Severity of Event 
For AEs not included in the protocol defined grading system, the following guidelines will be used to describe 
severity. 
• Mild – Events require minimal or no treatment and do not interfere with the participant’s daily activities. 
• Moderate – Events result in a low level of inconvenience or concern with the therapeutic measures. 
Moderate events may cause some interference with functioning. 
• Severe – Events interrupt a participant’s usual daily activity and may require systemic drug therapy or 
other treatment. Severe events are usually potentially life-threatening or incapacitating. 
 
8.2.2 
Relationship to Study Agent 
For all collected AEs, the clinician who examines and evaluates the participant will determine the AE’s 
causality based on temporal relationship and his/her clinical judgment. The degree of certainty about causality 
will be graded using the categories below. 

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• Definitely Related – There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal laboratory test result, 
occurs in a plausible time relationship to drug administration and cannot be explained by concurrent 
disease or other drugs or chemicals. The response to withdrawal of the drug (dechallenge) should be 
clinically plausible. The event must be pharmacologically or phenomenologically definitive, with use of 
a satisfactory rechallenge procedure if necessary. 
• Probably Related – There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs within a 
reasonable time after administration of the drug, is unlikely to be attributed to concurrent disease or 
other drugs or chemicals, and follows a clinically reasonable response on withdrawal (dechallenge). 
Rechallenge information is not required to fulfill this definition. 
• Possibly Related – There is some evidence to suggest a causal relationship (e.g., the event occurred 
within a reasonable time after administration of the trial medication). However, other factors may have 
contributed to the event (e.g., the participant’s clinical condition, other concomitant events). Although 
an AE may rate only as “possibly related” soon after discovery, it can be flagged as requiring more 
information and later be upgraded to “probably related” or “definitely related,” as appropriate. 
• Unlikely to be related – A clinical event, including an abnormal laboratory test result, whose temporal 
relationship to drug administration makes a causal relationship improbable (e.g., the event did not 
occur within a reasonable time after administration of the trial medication) and in which other drugs or 
chemicals or underlying disease provides plausible explanations (e.g., the participant’s clinical 
condition, other concomitant treatments). 
• Not Related – The AE is completely independent of study drug administration, and/or evidence exists 
that the event is definitely related to another etiology. There must be an alternative, definitive etiology 
documented by the clinician. 
 
8.2.3 
Expectedness 
Study clinicians will be responsible for determining whether an AE is expected or unexpected. An AE will be 
considered unexpected if the nature, severity, or frequency of the event is not consistent with the risk 
information previously described for the study intervention. 
 
8.3 Time Period and Frequency for Event Assessment and Follow-Up 
The occurrence of an AE or SAE may come to the attention of study personnel during study visits and 
interviews of a study participant. All AEs including local and systemic reactions not meeting the criteria for 
SAEs will be captured on the appropriate RF. Information to be collected includes event description, time of 
onset, clinician’s assessment of severity, relationship to study intervention (assessed only by those with the 
training and authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs occurring 
while on study must be documented appropriately regardless of relationship. All AEs will be followed to 
adequate resolution, as appropriate. 
 
Any medical condition that is present at the time that the participant is screened will be considered as 
baseline and not reported as an AE. However, if the study participant’s condition deteriorates at any time 
during the study, it will be recorded as an AE. UPs will be recorded in the data collection system throughout 
the study. 
 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the event at 
each level of severity. AEs characterized as intermittent require documentation of onset and duration of each 
episode. 
 
Study staff will record all reportable events with start dates occurring any time after informed consent is 
obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study participation. At each 
study visit, the investigator will inquire about the occurrence of AE/SAEs since the last visit. Events will be 
followed for outcome information until resolution or stabilization. All unresolved adverse events should be 
followed by the investigator until the events are resolved, the subject is lost to follow-up, or the adverse event 
is otherwise explained or addressed. At the last scheduled visit, the investigator should instruct each subject 

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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 should notify the study sponsor, IRB and DSMB of any death or serious adverse event 
occurring at any time after a subject has discontinued or terminated study participation that may reasonably 
be related to this study. 
 
8.4 Reporting Procedures – Notifying the IRB 
All AEs will be reported to the IRB in the yearly continuing review process. SAEs that are at least probably 
related to the study and unexpected will be reported to the IRB and DSMB within 72 hours of the study 
clinician/PI learning of the SAE. 
 
SAE and UP reporting to the NIH sponsor will follow NIMH reporting guidelines (ie deaths as soon as possible 
but within 5 days of PI learning, related SAEs and UPs within 10 days of PI learning: 
https://www.nimh.nih.gov/funding/clinical-research/nimh-reportable-events-policy.shtml). 
 
Reports will be completed by a study staff member. 
 
8.4.1 
Unanticipated Problem Reporting 
Incidents or events that meet the OHRP criteria for UPs require the creation and completion of an UP report 
form. It is the site investigator’s responsibility to report any UPs to their IRB and the study PI who will be 
responsible for reporting to the DSMB and to the NIH. The UP report will include the following information: 
 
• Protocol identifying information: protocol title and number, PI’s name, and the IRB project number; 
• A detailed description of the event, incident, experience, or outcome; 
• An explanation of the basis for determining that the event, incident, experience, or outcome represents 
an UP; 
• A description of any changes to the protocol or other corrective actions that have been taken or are 
proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline: 
 
• UPs that are study related SAEs will be reported to the IRB and to the DCC/study sponsor within 72 
hours of the investigator becoming aware of the event. 
• Any other UP will be reported to the IRB, DSMB and NIH at regular reporting or continuing review. 
 
8.4.2 
Reporting of Pregnancy 
Pregnancy tests will be done for all female participants with child-bearing potential prior to the initiation of the 
experiment to rule out pregnancy. If pregnancy is suspected, subjects will not be allowed to participate in the 
study. Further, participants will all be on oral contraceptives, making pregnancy unlikely. 
 
Although there are no known risks related to MRI on pregnant women or a fetus, there is a possibility of yet 
undiscovered pregnancy related risks. Since there is no possible benefit from adding estrogen to treatment as 
being tested in this protocol for a pregnant woman and potential risks with MRI, we will exclude pregnant 
women. A negative urine pregnancy test will be required before a woman of child-bearing potential can 
participate in this study. If the subject is post-menopausal for at least one year or has undergone a 
hysterectomy, the subject will not be required to undergo a urine pregnancy test prior to obtaining the MRI 
scan. Should a pregnancy be identified after initiation of study drug, study medication will be discontinued and 
this will be reported to the IRB, DSMB and NIH in regular reporting with a separate IRB event report including 
information on how this may alter study treatment and follow up will occur. 

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8.5 Reporting Procedures – Notifying the Study Sponsor 
Any study-related unanticipated problem posing risk to subjects or others, and any type of serious adverse 
event, will be reported to the NIH sponsor and will follow NIMH reporting guidelines (ie deaths as soon as 
possible but within 5 days of PI learning, related SAEs and UPs within 10 days of PI learning: 
https://www.nimh.nih.gov/funding/clinical-research/nimh-reportable-events-policy.shtml). 
 
To report such events, a Serious Adverse Event (SAE) form will be completed by the investigator which will 
be shared with the NIMH program officer (as well as the IRB and DSMB). The investigator will keep a copy of 
this SAE form on file at the study site. 
 
The investigator will provide any 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 Serious Adverse Event 
form, 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 IRB, DSMB and NIMH PO. 
 
8.6 Reporting Procedures – Participating Investigators 
Investigators at the second study site, University of Pennsylvania, who are not UTHealth Houston faculty or 
affiliated with an UTHealth Houston research site are responsible for safety reporting to their local IRB. 
Investigators are responsible for complying with their local IRB’s reporting 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. 
 
8.7 Study Halting Rules 
Estrace is a safe widely used medication and it is being used at low dose with an evidence based trauma 
therapy in a population of women aged 18 to 45. Extensive safety data is available for this population for each 
intervention alone. Thus, the need for halting of the study itself is not anticipated. Nonetheless, the DSMB will 
review study continuation at yearly DSMB meetings considering study progress with guidelines included in the 
DSMP. Should any new concerns about the use of estrogen at this dose in women age 18 to 45 emerge they 
will be reported to the DSMB and IRB for consideration. 
 
Nonetheless, this study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause. Written notification, documenting the reason for study suspension or termination, will be 
provided by the suspending or terminating party to the necessary parties including investigators, the NIH, the 
FDA, and/or other regulatory authorities. If the study is prematurely terminated or suspended, the PI will 
promptly inform the IRB and will provide the reason(s) for the termination or suspension. 
 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping 
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination of futility 
 
Study may resume once concerns about safety, protocol compliance, data quality is addressed and satisfy 
the sponsor, IRB and/or FDA. 
 
8.8 Safety Oversight 
 
8.8.1 
Data and Safety Monitoring Plan 
The investigator will allocate adequate time for monitoring activities. The Investigator will also ensure that any 
compliance or quality assurance reviewers are given access to all the above noted study-related documents 
and study related facilities at UTHealth Houston if assigned. A detailed strategy outline can be found in the 
attached Data and Safety Monitoring Plan. 

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8.8.2 
Data Safety Monitoring Board 
Consistent with NIMH guidelines on data and safety monitoring, in advance of trial inception we will establish 
a DSMB. Details on DSMB role, composition, and operating procedures can be found in the attached Data 
and Safety Monitoring Plan. Clinical Monitoring. Dr. Milad will serve as Principal Investigator (PI) of the study. 
It is the responsibility of the PI to oversee the safety of the study at UTHealth Houston. 
 
Imaging procedures will be conducted either at UTHealth Houston. The investigators for this project are not 
trained to perform radiological diagnosis, and the MRI scans performed in this study are not designed to find 
abnormalities and are being conducted only for research purposes. If the investigators notice a finding on an 
MRI scan that is of clinical significance and medically actionable, the scan will be shared with a licensed 
radiologist for further evaluation. Should this occur, a neuroradiologist would be consulted and the MRI scan 
will be sent to the UTHealth Houston. If the neuroradiologist thinks that further investigation of the finding is 
called for, the participant will be contacted about the finding and next steps. A copy of the radiology report will 
also be provided to the participant for their records. The decision as to whether to proceed with further 
examination or treatment lies solely with the participant and their physician. The images collected in this study 
do not comprise a proper clinical MRI study these images will not be made available for diagnostic purposes. 
 
For this study, Dr. Soares and/or Dr. Mira Milad, licensed physicians, or a delegate (MD or NP) will be 
responsible for the prescription administration and all procedures related to the study treatments. All 
participants will be monitored for any adverse events related to Estrogen use and PE by an approved study 
physician, nurse practitioner, and psychologists involved in the study. A study Physician will be in charge of 
managing AEs, SAEs, and unanticipated problems related to study treatment and psychiatric risks to 
participants. The psychotherapy will be delivered by approved study clinicians but all medication related 
procedures will be overseen by IRB-approved, licensed MD or NP staff. Dr. Mira Milad and Dr. Soares will 
work with the PI Dr. Mohammed Milad to support all necessary reporting. 
 
Dr. Mohammed R. Milad serves as the overall PI of the proposal. He will oversee the implementation of fMRI- 
related tasks and aspects of the project and the fMRI data acquisition before and after treatment. Dr. Milad 
will monitor the safe implementation of the protocol at the center for brain imaging. Dr. Jair C. Soares and/or 
Dr. Mira Milad serve as the overseeing, licensed physician(s). They or their delegates will be responsible for 
the prescription administration and all procedures related to the study medication. The delegates will consult 
with Dr. Soares/Dr. Mira Milad as needed/indicated. Clinical safety will be monitored by the psychologists, 
psychiatrists, and nurse, and all will consult with Dr. Soares/Dr. Mira Milad as needed/indicated. The 
psychotherapy will be delivered by approved study clinicians but all medication related procedures will be 
overseen by IRB-approved, licensed MD or NP staff. The PI and study clinicians will meet as needed to 
discuss recruitment and retention, data acquisition and management and all matters related to the 
prescription and therapy. They will review any unanticipated emerging problems or serious adverse events in 
real time with each other or a delegated, responsible, licensed, IRB-approved medical clinician. The PI will be 
responsible for reporting to the IRB and DSMB. 
 
9 Statistical Considerations 
9.1 Statistical and Analytical Plans (SAP) 
The data to be gathered from this study is multimodal: neurobiological responses (fMRI) before and after 
therapy, psychophysiological data obtained before, during, and after therapy, self-assessment questionnaires, 
and clinical data pertaining to PTSD symptoms ascertained before and after therapy. Our analytic approach 
will examine the impact of using estradiol as an adjunct to PE on all of these data. The major focus of the 
analytic approach will be to examine change in each of the above noted metrics before and after treatment 
and how the use of estradiol may have improved/changed these multimodal data. 
 
9.2 Statistical Hypotheses 
Hypothesis 1: Compared to the placebo-PE group (Plc+PE): 1) the E2+PE group will exhibit faster reduction 
of SCR between PE sessions, and 2) after 5 E2+PE sessions, the extinction recall deficit indexed by elevated 
SCR during extinction retention test will be rescued (i.e., SCR in the E2+PE group will be significantly lower 

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relative to the Plc+PE group). 2) Extinction-induced activations will be higher in the vmPFC and lower in the 
dACC and amygdala in post relative to pre E2+PE treatment. When comparing E2+PE vs. Plc+PE, similar 
differences are expected, but to a lesser degree given the anticipated benefits of PE alone. 
 
Hypotheses 2: PTSD symptom severity will be significantly lower in the E+PE group relative to the Plc+PE 
group following treatment, as well as at the 3 and 6-month follow-up assessments. 
Hypothesis 3: The degree of PTSD symptom reduction post- compared to pre-PE will be associated with 
BOLD changes in the fear extinction network and reduction in SCR during the extinction recall test after PE. 
The magnitude of BOLD and SCR changes will be significantly larger in the E2+PE group compared to the 
Plc+PE group. 
 
9.3 Analysis Datasets 
The proposed experiments in this project are aimed to examine the effects of E2+PE on the extinction- 
induced SCR and neural responses at post-PE sessions relative to pre-PE. SCR will be recorded as a 
psychophysiological index of fear extinction and extinction recall. We will conduct standard fMRI analyses to 
examine information flow changes in the extinction network. These data will test hypothesis 1 stated above. 
 
Another objective of the project is to examine the impact of E2 administration on PE efficacy at reducing 
PTSD symptom severity. Here, we will use all clinical data (CAPS) before and after treatment as well as 3, 6 
months follow up data. These data will be used to test hypothesis 2 stated above. 
 
The combined data sets will then be used to test hypothesis 3 above. 
 
Primary analyses will be based on a modified intent to treat sample including all participants who took at least 
one dose of the study medication. 
 
9.4 Description of Statistical Methods 
 
9.4.1 
General Approach 
Statistical Methods 
SCR Analyses 
For all experimental phase, SCR will be computed for each trial by subtracting the mean skin conductance 
level observed during the last two seconds of context presentation from the maximal skin conductance level 
reached during CS presentation. All SCR values will be square-root transformed prior to any statistical 
analyses. To evaluate extinction recall, an extinction retention index (ERI) will be computed for each individual 
using the following formula: 100 – (mean SCR to the first 4 CS+E trials during recall / maximum SCR reached 
during conditioning for this same cue) * 100. 
 
Functional MRI analysis 
The specific emphasis of the fMRI analysis will be on the extinction recall phase (first four trials of the CS+E 
vs. first four trials of the CS+U contrast). This contrast has been chosen given the literature demonstrating a 
significant deficit of PTSD patients during that specific phase of the experimental protocol. BOLD signal will 
be extracted from the four ROIs (amygdala, dACC, vmPFC and hippocampus). For each of these ROIs, we 
will use mixed model ANOVAs with time (pre vs. post PE) and group (E2+PE vs. Plc+PE) to investigate main 
effect of each of these variables as well as group by time interaction. Whole-brain analyses will also be 
conducted as supplemental analyses for the sake of completeness. We will also conduct regression analyses 
comparing the change in BOLD responses in each of our ROIs from post- to pre-treatment with PTSD 
symptom reduction in each of the two groups (E2+PE and Plc+PE). 
 
 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) 
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9.4.3 
Analysis of the Secondary Endpoint(s) 
Details of the analyses for the secondary efficacy endpoints are provided above in sections 10.1, 10.2, and 
10.3. 
 
9.4.4 
Safety Analyses 
See Protection against risks and safety assessments under procedures above. 
 
Participants’ reactions to study assessments and treatment will be closely monitored, and negative reactions 
will be addressed therapeutically. Participants could develop mild to moderate emotional discomfort or 
frustration associated with psychiatric interviewing or filling out questionnaires. Participants will be allowed 
any breaks in the testing that are needed. Any events related to safety during assessment period will be 
documented as they occur. 
 
PE is considered very safe and therefore risks associated with its delivery are mild to moderate. Patients 
might experience discomfort when exposed to anxiety-provoking images and situations in treatment sessions 
and at home for homework and discomfort about being in treatment for victims of a traumatic event. Based on 
our extensive experience in PE, only a handful report negative side effects from it. None of the participants in 
our past or most recent treatment studies, sustained prolonged negative reactions to exposure. Any negative 
events associated with PE will documented as they occur. 
 
Regarding safety analyses associated with Estradiol use, common side effects of chronic administration of 
this drug include headache, breast pain, irregular vaginal bleeding, stomach cramps, nausea, or hair loss. As 
noted above, there are no known side effects of acute single doses of estrogen, as it is the case in the current 
application. All of the women to be recruited will already be using oral contraceptives. A total of 5 doses over 
3 weeks will be administered for this study. These acute E2 doses do not pose a significant risk for the 
participants. To assess for safety, at each PE appointment, participants will be asked how they are feeling 
and if they had encountered any of the potential side effects. If that is the case, these will be documented and 
reported. An MD from our study staff will discuss and review the symptoms with the participants and a clinical 
decision will be made whether or not the subject should stop her participation in the study. 
 
9.4.5 
Adherence and Retention Analyses 
The adherence to the protocol will be noted based on the absence or decision made by participants not 
complete the study procedures during the 6 PE sessions. Adherence to medication consumption will be 
assessed based on the subjective reports of the participants. Note that while blood samples will be collected 
to measure hormonal levels in our participants, these cannot be used to analyze adherence given that some 
participants will be using the placebo pills and thus no change in serum estradiol would be expected. 
 
9.4.6 
Baseline Descriptive Statistics 
We will perform descriptive statistics prior to testing our hypotheses. These statistics will include means (or 
frequencies and proportions for categorical variables), standard deviations, skewness and kurtosis, and odds- 
ratios. Variables with distributions violating assumptions of normality will be transformed. If this fails to 
improve the normality of the distribution, distribution-free statistics will be employed. This study has baseline, 
post-treatment, and two follow-up assessments, creating clustered data due to repeated measures. Thus, we 
will use established methods for such study designs to examine treatment differences, including the two most 
popular approaches for longitudinal data modeling: the generalized estimating equations (GEE) and linear 
mixed-effects model approach (LMM). We will apply these models for both the intent-to-treat and completer 
analyses. Inference based on GEE or LMM is valid provided that missing data are non-informative or 
“missingness” does not depend on the value of the unobserved outcome. Although missing data in many 
studies in mental health research are non-informative, we will perform sensitivity analyses to ensure that this 
is the case. In particular, we will entertain both parametric and semi-parametric models that assume some 
missingness mechanism and compare estimates obtained from such models with those from GEE and LMM. 
Such analyses will inform us whether the non-informative missingness assumption is violated and if so, to 
what extent. If the non-informative missingness assumption is deemed to be severely violated, we will report 
treatment effects using the methods that account for informative missingness. 

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9.4.7 
Planned Interim Analysis 
Not applicable 
 
9.4.8 
Safety Review 
See above re safety monitoring, reporting and DSMB including individual participant and study halting 
guidelines. 
 
9.4.9 
Efficacy Review 
Interim analyses will not be conducted. All analyses will be conducted after breaking the blind at the end of 
the study unless a blinded review is requested by the DSMB. 
 
9.4.10 Multiple Comparison/Multiplicity 
As noted above, the study has baseline, post-treatment, and two follow-up assessments, creating clustered 
data due to repeated measures. We describe the plan of analysis and adjustments in section 10.4.6. 
 
9.5 Sample Size 
Pre to post treatment assessment using our experimental approach has not been previously conducted, and 
therefore it is challenging to calculate power to estimate potential sample or effect size comparing the pre to 
post time-points. The following sample size calculations pertain to the expected changes related the 
psychophysiological and neurobiological indices to be obtained. The sample size selected for this proposal is 
based on the detection of between-group significant differences at an alpha of .05 using data collected in our 
previous study of fear conditioning and extinction recall in controls and patients (PTSD) and using our 
preliminary fMRI data in women with high (HE) vs. low estradiol (LE) (Zeidan et al, 2011). We have calculated 
our power to detect between-group differences with a sample size of 25 subjects per group. Based on our 
previous experience with this population and this paradigm, we expect that (at each of the two time points) it 
is possible that we will have unusable data in approximately 20% of the subjects due to data of insufficient 
quality for at least one phase of the experiment. Reasons for this include poor SCR responses, head motion 
during scanning, or technical difficulties with SCR or fMRI. In addition, we anticipate dropout of ~20 % of 
patients after the initiation of the study. Thus out of the total of 80 patients, we expect to obtain a full data set 
(from all time points) from 50 patients. By using the effect size obtained from our previous study (see above), 
an alpha level of 0.05 and 50 patients, we will have 85% power to detect a significant between-group 
difference. Regarding the clinical measures, we do not have data to calculate power because of the novelty, 
but since it is a preliminary proof-of-concept aim, the results to be obtained will help inform future power 
analyses and calculations for this line of research. 
 
9.6 Measures to Minimize Bias 
 
9.6.1 
Enrollment/Randomization/Masking Procedures 
The study randomization table is created by the study statistician and provided to the UTHealth Houston and 
U Penn site pharmacies to be used during the study. A research assistant will be responsible for randomizing 
the participant by entering the CAPS Severity in REDCap. A randomization number is generated, which is 
then reported to the Investigational Pharmacy which has the key indicating which randomizations numbers 
correlate to estradiol or placebo pills. The research team and the subject will be blinded and will not know the 
investigational product contained in the prescription bottle. The pharmacy has the names of participants in 
their log. The blind may be broken in the case of an emergency. To request unblinding, after permission to do 
so is granted by the PI or the study clinical lead, a member of the study team will contact the Investigational 
Pharmacy to receive the condition of the subject. 
 
10 Source Documents and Access to Source Data/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 
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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 blue or black ink or recorded in any 
direct entry forms. 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. 
 
Access to study records will be limited to IRB-approved members of the study team. The investigator will 
permit study-related monitoring, audits, and inspections by the IRB/EC, 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 Quality Assurance and Quality Control 
QC procedures will be implemented beginning with the data entry system and data QC checks that will be run 
on the database will be generated. Any missing data or data anomalies will be communicated to the site(s) for 
clarification/resolution. 
 
Following written SOPs, the study team will verify that the clinical trial is conducted and data are generated, 
documented (recorded), and reported in compliance with the protocol, GCP, and the applicable regulatory 
requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). 
 
The investigational site will provide direct access to all trial related sites, source data/documents, and reports 
for the purpose of monitoring and auditing by the sponsor, and inspection by local and regulatory authorities. 
 
The investigative team has extensive experience conducting clinical psychotherapy and medication studies for 
PTSD (Dr. Foa) and fMRI methodologies (Dr. Milad). They will train and oversee the overall conduct of the staff 
in clinical trials methodology. They will ensure that on-going supervision is provided, study assessments are 
completed, and there is good compliance of staff with all research procedures. They will assume responsibility to 
see that project staff is sufficiently trained to administer project interviews, review assessments with participants, 
carry out laboratory/scanning procedures, and maintain participants in the study throughout follow-ups. Every 
quarter, participants’ casebooks will be checked by Dr. Milad for accuracy with regard to inclusion/exclusion 
criteria; staff adherence to study procedures; completeness; and documentation of participant attrition, adverse 
events, and sources of potential protocol violations. Discrepancies will be noted, brought to the attention of the 
PI and project staff, investigated and resolved. IEs and RAs will be given specific training on follow-up methods 
similar to those of our numerous prior studies, and a set of MINI and CAPS recordings will be reviewed to 
assess inter-rater reliability for each independent evaluator prior to the start of independent evaluations. The 
criterion of each tape in the set will be 80% agreement, with agreement on the CAPS defined as the scores of 
the rater within 10% of the total scale range. 
 
Before beginning any experimental procedures, Dr. Milad provide extensive training to all team members 
regarding data acquisition and maintenance as well as safety of participants. Dr. Milad will also run data 
quality checks every 3 months to ensure adequate quality and consistency of fMRI and SCR data. 

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12 Ethics/Protection of Human Subjects 
12.1 Ethical Standard 
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 The UTHealth Houston Institutional Review Board 
(IRB), in agreement with local legal prescriptions, for formal approval of the study conduct. The decision of the 
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 NIH before commencement of this study. 
 
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 A 
for a copy of the Subject Informed Consent Forms. These consent forms will be submitted with the protocol 
for review and approval by the IRB for the study. The formal consent of a subject, using the 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. 
 
12.2 Institutional Review Board 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be submitted 
to the IRB for review and approval. Approval of both the protocol and the consent form must be obtained 
before any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB 
before the changes are implemented to the study. All changes to the consent form will be IRB approved; a 
determination will be made regarding whether previously consented participants need to be re-consented. 
 
12.3 Informed Consent Process 
Consent forms describing in detail the study intervention, study procedures, and risks are given to the 
participant and written documentation of informed consent is required prior to starting intervention. The 
following consent materials are submitted with this protocol: 
1. Consent for adult participants 
2. Video consent for adult participants 
3. Audio consent for adult participants 
4. Consent for adult participants for training study 
5. Video consent for adult participants for training study 
 
12.3.1 Consent Procedures and Documentation 
 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the study and 
continues throughout the individual’s study participation. Extensive discussion of risks and possible benefits of 
participation will be provided to the participants (and their families, if requested). Consent forms will be IRB- 
approved and the participant will be asked to read and review the document. The investigator will explain the 
research study to the participant and answer any questions that may arise. All participants will receive a 
verbal explanation in terms suited to their comprehension of the purposes, procedures, and potential risks of 
the study and of their rights as research participants. Participants will have the opportunity to carefully review 
the written consent form and ask questions prior to signing. The participants should have the opportunity to 
discuss the study with their surrogates or think about it prior to agreeing to participate. 
 
Written or electronic (via REDCap) informed consent will be obtained by one of the IRB approved UTHealth 
Houston study investigators prior to participation in the study. Subjects must be competent to provide 
informed consent. Study procedures and potential risks and benefits will be described to subjects at the time 
participation in the study is sought. Ample opportunity will be provided for potential subjects to carefully read 
the consent form and to ask any questions they may have. The PI will retain copies of the signed consent 

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form patients will be given a copy of the consent form. A subject may end their study participation at any time 
if they chose to do so. 
 
A copy of the signed informed consent document will be stored in the subject’s research record. The consent 
process, including the name of the individual obtaining consent, will be thoroughly documented in the 
subject’s research record. Any alteration to the standard consent process (e.g. use of a translator, consent 
from a legally authorized representative, consent document presented orally, etc.) and the justification for 
such alteration will likewise be documented. 
 
12.4 Participant and Data 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. 
 
To further protect the privacy of study participants, a Certificate of Confidentiality will be obtained from the 
NIH. This certificate protects identifiable research information from forced disclosure. It allows the investigator 
and others who have access to research records to refuse to disclose identifying information on research 
participation in any civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, 
or local level. By protecting researchers and institutions from being compelled to disclose information that 
would identify research participants, Certificates of Confidentiality help achieve the research objectives and 
promote participation in studies by helping assure confidentiality and privacy to participants. 
 
Facial images in the video files will not be obscured because the emotional expression of the participants’ 
faces during the training PE sessions are an essential component for the training. Thus, blurring the images 
may hinder some aspects of the training and therefore will not be done. To ensure confidentiality, all files 
containing video recordings will be password protected and stored on UTHealth Houston computers that are 
encrypted and password protected. Those computers will be located in locked offices and only viewed by 
IRB-approved personnel for clinician training purposes. Videos that need to be viewed by personnel located 
at UPenn will remain within the UTHealth Houston firewall and accessed only after approvals for the UPenn 
personnel to access the UTHealth Houston system through creating an ID and gaining access to a shared 
network server. 
 
12.4.1 Research Use of Stored Human Samples, Specimens, or Data 
• Intended Use: Samples and data collected under this protocol may be used to study PTSD and its 
treatment. No blood samples will be stored and no genetic testing will be performed. 
• Storage: Data will be kept in password-protected computers within the UTHealth Houston firewall. 
Only investigators will have access to the study data. 
 
13 Data Handling and Record Keeping 
13.1 Data Collection and Management Responsibilities 
Paper-based records will be kept in a secure location and only be accessible to personnel involved in the 
study. Computer-based files will only be made available to personnel involved in the study through the use of 
access privileges and passwords. Prior to access to any study-related information, personnel will be required 
to sign statements agreeing to protect the security and confidentiality of identifiable information. Wherever 

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feasible, identifiers will be removed from study-related information. A Certificate of Confidentiality will be 
obtained, because the research could place the subject at risk of criminal or civil liability or cause damage to 
the subject's financial standing, employability, or liability. Precautions are in place to ensure the data is secure 
by using passwords and encryption, because the research involves web-based surveys. We will use 
Research Electronic Data Capture (UTHealth Houston’s REDCap) to support direct data entry by patients and 
study staff. RedCap will be used for data collection and management. RedCap is a secure web platform for 
delivery of surveys and management of databases. For this study we will take additional steps outlined by 
UTHealth to ensure that it is 21 CFR Part 11 compliant for data collection and management. These 
requirements/steps are outlined in the Attachment labeled UTHealth Redcap 21CRFPart11. Lastly, any 
electronic data collected will be stored on the UTHealth Houston’s secured and managed network servers 
and devices. Only cleared study personnel will have access to the server and devices. 
 
Data and image recordings will be accessed by IRB-approved Penn personnel only through the UTHealth 
Houston firewall. This will ensure data and image recordings are not being transferred outside of the 
UTHealth Houston firewall to maintain subject confidentiality. For neuroimaging and psychophysiological data, 
UPenn personnel will de-identify the data and then transfer data via secure servers into UTHealth Houston 
computers. This will be done once a month. Once deposited into UTHealth Houston secure servers, the PI 
and the team will maintain the security of the data storage as described. For clinical data to be input into 
RedCap, UPenn will use UTHealth Houston’s RedCap, located within UTHealth Houston’s secure firewall. 
 
13.2 Study Records Retention 
Study essential documents and data for this project will be retained for at least 5 years after the publication of 
the last findings from this study. These documents may be retained for a longer period if required for 
additional research activities. 
 
13.3 Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or Manual of Procedures 
(MOP) requirements. The noncompliance may be either on the part of the participant, the investigator, or the 
study site staff. As a result of deviations, corrective actions are to be developed by the site and implemented 
promptly. 
 
These practices are consistent with ICH E6: 
• 4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
• 5.1 Quality Assurance and Quality Control, section 5.1.1 
• 5.20 Noncompliance, sections 5.20.1, and 5.20.2. 
 
It is the responsibility of the site to use continuous vigilance to identify and report deviations. Protocol 
deviations must be reported to the IRB per their guidelines. The site PI/study staff is responsible for knowing 
and adhering to their IRB requirements. Further details about the handling of protocol deviations will be 
included in the MOP. 
 
13.4 Publication and Data Sharing Policy 
The PI (Mohammed Milad) holds the primary responsibility for publication of the results of the study and 
oversight of the data storage and utilization. 
 
This study will comply with the NIH Public Access Policy, which ensures that the public has access to the 
published results of NIH funded research. It requires scientists to submit final peer-reviewed journal 
manuscripts that arise from NIH funds to the digital archive PubMed Central upon acceptance for publication. 
 
14 Study Finances 
14.1 Funding Source 
This study is financed through a grant from the National Institute of Mental Health. 

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14.2 Costs to the Participant 
Participants will not incur any costs associated with their participation in the study. Participants will benefit 
from receiving well-delivered treatment for PTSD. Each subject will be compensated for completing the study 
procedures and will be reimbursed for their parking expenses. In the event of premature termination, subjects 
will receive partial reimbursement for the procedures they have completed. 
 
14.3 Participant Reimbursements or Payments 
Participants will receive $60 for visit 1, $20 for visits 2, 5 and 10, $15 for visits 3, 4, 6-9 and 11, to help defray 
costs of travel and time. Participants will then receive $235 upon completion of visit 12. Participants will 
receive $50 at each remaining follow-up visit (visits 13 and 14). The total compensation for all completed 
study visits will be $560. Participants who drop out of the study will be paid for the visits they attended. 
Participants who are deemed ineligible at /after the baseline assessment visit will be paid $60 for completing 
the initial assessment. 
 
15 Study Administration 
The study will be overseen and managed by the PI Dr. Milad with the clinical trial additionally overseen by Dr. 
Mira Milad, Dr. Jair C. Soares and Dr. Foa. 
 
16 Conflict of Interest Policy 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical. Therefore any actual conflict of interest of persons who have a role in the design, conduct, 
analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who 
have a perceived conflict of interest will be required to have such conflicts managed in a way that is 
appropriate to their participation in the trial. The study leadership has established policies and procedures for 
all study group members to disclose all conflicts of interest and will establish a mechanism for the 
management of all reported dualities 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.) must have the conflict reviewed by the UTHealth 
Houston Conflict of Interest Management Unit (CIMU) with a Committee-sanctioned conflict management plan 
that has been reviewed and approved by the study sponsor prior to participation in this study. All UTHealth 
Houston investigators will follow the applicable conflict of interest policies. 

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17 References 
1. Arnberg FK, Bergh Johannesson K, Michel P-O. Prevalence and duration of PTSD in survivors 6 
years after a natural disaster. J Anxiety Disord. 2013;27:347–52. 
2. Bouton ME. Context and behavioral processes in extinction. Learn Mem. 2004;11:485–94. 
3. Foa EB. Psychosocial treatment of posttraumatic stress disorder. J Clin Psychiatry. 2000;61:43–51. 
4. Foa EB, Gillihan SJ, Bryant RA. Challenges and successes in dissemination of evidence-based 
treatments for posttraumatic stress: Lessons learned from prolonged exposure therapy for PTSD. 
Psychol Sci Public Interest. 2013;14:65–111. 
5. Foa EB, Hembree E, Rothbaum BO. Prolonged Exposure Therapy for PTSD: Emotional Processing 
of Traumatic Experiences Therapist Guide (Treatments That Work). New York, NY: Oxford University 
Press; 2007. 
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18 Attachments 
These documents are relevant to the protocol, but they are not considered part of the protocol. They are 
stored and modified separately. As such, modifications to these documents do not require protocol 
amendments. 
• 
Consent Form 
• 
Consent Form for Training Study 
• 
Video Consent Form for Training Study 
• 
Audio consent for Assessments Primary Study 
• 
Questionnaire assessments 
• 
Recruitment materials