A Randomized Double-blind Placebo Controlled Phase 3 Trial to Evaluate the Efficacy and Safety of Estetrol for the Treatment of Moderate to Severe Vasomotor Symptoms in Postmenopausal Women (E4Comfort Study II)
Phase: PHASE3
Conditions: Vasomotor Symptoms, Menopausal Symptoms
Sponsor: Estetra

Clinical Trial Protocol 
A Randomized Double-blind Placebo Controlled Phase 3 Trial to evaluate the 
Efficacy and Safety of Estetrol for the Treatment of Moderate to Severe Vasomotor 
Symptoms in Postmenopausal Women (E4Comfort Study II) 
Short title: Estetrol for the Treatment of Moderate to Severe Vasomotor Symptoms in 
Postmenopausal Women  
Trial Identification:  
MIT-Do001-C302 
IND Number:  
 
Investigational medicinal  
product:  
Estetrol (E4) 
Indication: 
Treatment of moderate to severe vasomotor symptoms 
associated with menopause  
Clinical phase: 
Phase 3  
Sponsor: 
Estetra SRL 
Rue Saint-Georges 5/7 
4000 Liège, Belgium 
Protocol version and date:  Final Version 1.1, June 11, 2019 
Final Version 2.0, July 18, 2019 (Amendment 1) 
Final Version 3.0, August 13, 2019 (Amendment 2) 
Final Version 4.0, October 08, 2019 (Amendment 3) 
Final Version 5.0, February 03, 2020 (Amendment 4) 
Final Version 6.0, September 23, 2020 (Amendment 5) 
Final Version 7.1, July 30, 2021 (Amendment 6.1) 
Final Version 8.0, June 21, 2022 (Amendment 7) 
  
NCT04090957

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 3 of 150 
 
1. 
ADMINISTRATIVE INFORMATION 
1.1. 
Serious Adverse Event Reporting 
In case of a serious adverse event, including a suspected unexpected serious adverse reaction 
(SUSAR) the Investigator or clinical trial center personnel will send a report within 24 hours 
of notification to: 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 7 of 150 
 
1.4. 
Investigator Approval and Signature Page 
I have read and understood all pages of this clinical trial protocol and relevant appendices. I 
agree that they contain all information required to conduct this trial. I agree to conduct the trial 
as outlined in the protocol and to comply with all terms and conditions set out therein. Any 
modification of the clinical trial protocol must be agreed upon by the Sponsor and the 
investigator and must be documented in writing. 
I confirm that I will conduct the trial in accordance with the local and applicable regulatory 
requirements, The International Council for harmonization of Technical Requirements for 
Pharmaceuticals for Human Use (ICH) - Good Clinical Practice (GCP) guidelines, the 
provisions of the Declaration of Helsinki and the Clinical Trials Directive 2001/20/EC. I will 
also ensure that all relevant members of my staff have access to copies of this protocol and to 
all information relating to preclinical and prior clinical experience (e.g., Investigator’s 
Brochure), local and applicable regulatory requirements, ICH GCP guidelines, and the 
Declaration of Helsinki to enable them to work in accordance with the provisions of these 
documents. 
Name/position/institution 
Date 
Signature 
Investigator 
--------------------------- 
----------------------------------- 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 8 of 150 
 
1.5. 
CRO Approval and Signature Page 
ICON employees have read and understood all pages of this clinical trial protocol and relevant 
appendices. They agree that they have received all information required to conduct this trial. 
They agree to conduct the trial as outlined in the protocol and to comply with all terms and 
conditions set out therein. Any modification of the clinical trial protocol must be agreed upon 
by the Sponsor and the Investigator and must be documented in writing. 
ICON employees confirm that they will conduct the trial in accordance with the local and 
applicable regulatory requirements, The International Council for Harmonization of Technical 
Requirements for Pharmaceuticals for Human Use (ICH) - Good Clinical Practice (GCP) 
guidelines, the provisions of the Declaration of Helsinki and the Clinical Trials Directive 
2001/20/EC. They will also ensure that Investigator(s) and other relevant members have access 
to copies of this protocol and to all information relating to preclinical and prior clinical 
experience (e.g., Investigator’s Brochure), local and applicable regulatory requirements, ICH 
GCP guidelines, and the Declaration of Helsinki to enable them to work in accordance with the 
provisions of these documents. 
Name/position 
Date 
Signature 
Director Project Management, ICON 
Clinical Research Limited  
--------------------------- 
----------------------------------- 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 10 of 150 
 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
DPE has been re-included as an 
endometrial event for which 
administration of P4 200 mg 
(once a day, for 14 consecutive 
days) should be considered by 
the Investigator. 
DPE has been re-included as an 
endometrial event leading to 
discontinuation (see Major Changes). 
2. Study Synopsis 
5.4 Risk-benefit Assessment 
10.4.1 Treatment Emergent 
Adverse Events 
10.4.8 Endometrial biopsy in non-
hysterectomized subjects 
10.4.10 Endometrial safety 
assessment and stopping rules 
(non-hysterectomized subjects 
only) 
A Clinical Event Committee is 
created to support the DSMB in 
the adjudication of the 
cardiovascular and thrombotic 
events that arise during the 
course of the study. 
The DSMB has requested the creation 
of a Clinical Event Committee.  
10.5 Data Safety Monitoring Board 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 11 of 150 
 
Amendment 6.1, leading to version 7.1 of the protocol, date July 30, 2021 
All changes are clearly identified in the track-changes version of the amendment.  
Note: Amendment 6.0, leading to version 7.0 of the protocol, date May 26, 2021, was never 
submitted. All changes of Amendment 6.1 have been tracked against Protocol Version 6.0, 
date September 23, 2020. 
 
Major changes 
Description of changes 
Reason for change 
Sections affected  
Disordered proliferative 
endometrium has been removed 
from the reasons for 
discontinuation. 
To align with regulatory approach and 
based on the available safety 
information. 
2 Study Synopsis 
5.4 Risk-benefit Assessment 
7.1.1 Trial Design (Figure 1) 
8.3. Subject Discontinuation and 
Replacement Procedures 
10.4.1. Treatment Emergent 
Adverse Events 
10.4.8. Endometrial biopsy in non-
hysterectomized subjects. 
10.4.10. Endometrial safety 
assessment and stopping rules 
Disordered proliferative 
endometrium has been removed 
from the Exclusion Criteria. 
To align with regulatory approach and 
based on the available safety 
information. 
2 Study Synopsis 
8.2 Inclusion and Exclusion 
Criteria 
The maximum screening period 
has been extended to 8 weeks 
(instead of 6 weeks) in both 
study parts. 
To limit the screen failure rate, which 
is in part due to the COVID-19 
situation because it limits access to the 
hospital facilities. 
2 Study Synopsis 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
2.2 Schedule of Trial Procedures 
Safety Study part 
7.1.1. Trial Design 
10.1. Experimental Flow 
Text has been added on 
COVID-19 vaccination during 
the trial, including a 
recommendation on the timing of 
vaccination in relation to study 
assessments. 
To provide guidance on COVID-19 
vaccination during the trial.   
9.6.3 COVID-19 vaccination 
The endometrial biopsies will be 
read by four pathologists instead 
of three pathologists. The 
pathologist performing the initial 
reading for safety diagnosis, will 
not be involved in the reading for 
consensus diagnosis.  
According to FDA guidelines 3 
independent pathologist have to read 
slides for Consensus diagnosis. The 
initial reader will only assess the 
safety.   
10.4.8 Endometrial Biopsy in Non-
hysterectomized Subjects 
Table continued on the next page 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 12 of 150 
 
Major changes 
Description of changes 
Reason for change 
Sections affected  
The number of days with 
bleeding and/or spotting and the 
cumulative rates of amenorrhea 
have been added as endpoints to 
the safety objective on vaginal 
bleeding. 
To further define the evaluation of 
vaginal bleeding during treatment with 
E4.   
2 Study Synopsis 
6.1.2 Secondary Objectives and 
Endpoints (Efficacy Study part)   
6.2.2. Secondary Objectives and 
Endpoints (Safety Study part) 
The secondary objective and 
endpoints for frequency and 
severity of VMS have been 
updated. 
To further define the evaluation of the 
secondary efficacy of E4.     
2 Study Synopsis 
6.1.2 Secondary Objectives and 
Endpoints (Efficacy Study part)  
The secondary objectives 1 and 2 
have been added to the timing of 
the final efficacy analysis.     
To clarify that also the final analysis of 
the secondary efficacy objectives 1 
and 2 will be done in parallel with the 
final analysis of the primary efficacy 
objective.    
2 Study Synopsis 
7.4.1 Blinding after analysis of data 
up to Visit 4 in the Efficacy Study 
part. 
12.9 Timing of analysis. 
The interim analysis of safety 
endpoints in the Efficacy Study 
part of the study has been 
deleted.  
No interim analysis will be performed 
on the safety endpoints.  
2 Study Synopsis 
7.4.1 Blinding after analysis of data 
up to Visit 4 in the Efficacy Study 
part. 
12.9 Timing of analysis.  
Sentence on treatment restart has 
been added: “If study treatment 
is interrupted for 4 days and 
more per week, the Investigator 
will need approval from the 
Medical Monitor to restart the 
patient on study treatment.” 
Compliance of treatment intake. 
9.2. Timing of Administration 
Text on the use of 200 mg P4 in 
non-hysterectomized subjects has 
been harmonized throughout the 
Protocol. 
To ensure consistency across protocol 
sections. 
2. Study Synopsis 
5.4. Risk-benefit Assessment 
7.1.1 Trial Design (Figure 1) 
8.3 Subjects Discontinuation and 
Replacement Procedures. 
10.4.8. Endometrial Biopsy in Non-
hysterectomized Subjects 
10.4.10. Endometrial safety 
assessment and stopping rules 
(non-hysterectomized subjects 
only) 
Resolution of endometrial events 
has been defined. 
The definition of the resolution of 
endometrial events was missing. 
2. Study Synopsis 
10.4.1. Treatment Emergent 
Adverse Events 
10.4.10. Endometrial Safety 
Assessment and Stopping Rules 
Exclusion criterion # 3 has been 
updated to specify that high risk 
oncogene HPV includes subtypes 
16 and 18. 
To limit the screen failure due to 
positivity of HPV testing to subtypes 
of lower significance. 
2. Study Synopsis 
8.2.2. Criteria for Exclusion 
Table continued on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 13 of 150 
 
Major changes 
Description of changes 
Reason for change 
Sections affected  
Hierarchy of the pathologic 
diagnoses of endometrial tissue 
has been updated. 
To align with the Statistical Analysis 
Plan. 
10.4.8. Endometrial biopsy in non-
hysterectomized subjects. 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
Exclusion criterion # 23 has been 
updated from “Inadequately 
treated hyperthyroidism with 
abnormal TSH and free T4 at 
screening. Subjects with low 
TSH are allowed if free T4 at 
screening is within normal 
range” to “Inadequately treated 
hyperthyroidism with abnormal 
TSH and free T4 at screening. 
Subjects with low or high TSH 
are allowed if free T4 at 
screening is within normal 
range” 
To clarify that subjects with TSH 
outside normal range at screening, 
including high and low TSH, may be 
allowed in the study if free T4 is 
within normal range. 
2 Study Synopsis 
8.2.2 Criteria for Exclusion 
9.6.2. Concomitant 
Medication/Therapy 
Lactate dehydrogenase enzyme 
and isoenzymes have been 
specified in the list of parameters 
for lab analysis. 
To better describe the analysed 
parameters. 
10.4.5. Routine Clinical Laboratory 
Tests 
The Endometrial Safety Analysis 
set has been added. 
To clarify the definition of the 
different statistical populations. 
2 Study Synopsis 
12.1 Statistical populations 
The pathologic diagnosis 
prompting discontinuation will 
be reported as an AESI and not 
as an SAE.  
Correction in the protocol. The 
pathologic diagnosis prompting 
discontinuation should not be reported 
as an SAE, unless the AESI fulfils the 
criteria of an SAE.   
A clarification letter for this change 
has been implemented, ahead of this 
Amendment.    
10.4 10 Endometrial safety 
assessment and stopping rules 
A sentence was added to clarify 
when the study treatment could 
be restarted. 
To ensure treatment compliance. 
9.2. Timing of Administration 
Start of AESI collection was 
specified. 
To avoid unnecessary retrospective 
reporting.  
10.6. Adverse Events of Special 
Interest 
For the haemostasis parameter 
APCsr ETP the text "in a subset 
of subjects in selected study 
centers" has been removed. 
The APCsr ETP has been collected in 
all subjects in all centers. 
2. Study synopsis 
6.1.2 Secondary Objectives and 
Endpoints 
10.3.3 Laboratory Assessments for 
Efficacy 
Table continued on the next page 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 14 of 150 
 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
The sentence: “The 95% two-
sided CI will be computed on the 
observed frequency of 
hyperplasia/carcinoma of the 
endometrium in order to show 
that the frequency is not superior 
to 2% (upper bound lower than 
the 2% threshold).” has been 
corrected as follows: “The 95% 
two-sided CI will be computed 
on the observed frequency of 
hyperplasia/carcinoma of the 
endometrium in order to show 
that the frequency is not superior 
to 2% (upper bound lower than 
the 2% threshold).” 
The quoted frequency is relevant to 
combined hormone therapy (estrogen 
plus progestin) not for unopposed 
estrogen therapy. 
2. Study synopsis 
12.8 Safety parameters (safety set) 
The section below was added to 
Section 12.9; “The analyses of 
all other efficacy and safety 
objectives from the Efficacy and 
Safety Study parts will be 
triggered when the overall end of 
the trial is reached (i.e. when all 
subjects in the Efficacy Study 
part have completed Visit 8).  
The efficacy endpoints and the 
safety endpoints of the Safety 
Study will be analyzed separately 
from those of the Efficacy Study 
at the end of the Safety Study.” 
To be consistent with the synopsis 
section “Timing of analysis”. 
12.9 Timing of analysis 
The words in bold and italics 
were added: “• 
P4, 200 mg, 
oral, once daily for 14 
consecutive days (after 
completion or discontinuation of 
E4/placebo treatment)” 
For completeness. 
2. Study synopsis 
 
Name and contact details of 
several sponsor study team 
members haven been changed.  
Estetra SPRL has been changed 
to Estetra SRL. 
Name of the storage facilities for 
additional analysis has been 
updated. 
To reflect the changes in the study 
team members, the change in the name 
of the Sponsor and the change in the 
name of the storage facilities for 
additional analysis. 
1.2 Contact Information 
1.3 Sponsor Authorisation 
4.3 List of Abbreviations 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 15 of 150 
 
Amendment 5, leading to version 6.0 of the protocol, date September 23, 2020 
 
Major changes 
Description of changes 
Reason for change 
Sections affected  
Changes in trial design and or trial procedures 
A section on Adverse Events of 
Special Interest (AESI) has been 
added to the protocol.  
Reference to the section on AESI 
has been made in several other 
safety sections. 
A section on AESI was missing in 
earlier version of the protocol and has 
now been added.  
10.4.1 Treatment Emergent 
Adverse Events 
10.4.8 Endometrial biopsy in non-
hysterectomized subjects 
10.4.9 Vaginal bleeding events 
10.6 Adverse Events of Special 
Interest 
 
The footnote belonging to 
inclusion criterion 8 regarding 
the mammography has been 
changed from:   
".. i.e BI-RADS 0, is not 
acceptable" 
".. i.e BI-RADS 0, is not 
acceptable and requires further 
assessment". The text in Section 
10.4.6 has been changed 
accordingly.   
To clarify that a BI-RADS 0 is not 
acceptable, unless further assessment 
is done, demonstrating no clinically 
significant findings.  
2. Study synopsis 
8.2 Inclusion and Exclusion 
Criteria 
10.4.6 Mammography 
An additional TVUS assessment 
has been added to be performed 
after 9 months of treatment 
(Week 41, Visit 6 Efficacy Study 
part and Visit 5 Safety Study 
part). 
The additional TVUS assessment was 
recommended by the DSMB.      
2. Study synopsis 
10.1.5.6 Visit 6: 9 month Safety 
Visit 
10.1.6.5 Visit 5: 9 month Safety 
Visit 
10.4.7 Transvaginal Ultrasound in 
Non-hysterectomized Subjects  
Text has been added in Section 
10.4.8 regarding follow-up of 
subjects in case of disordered 
proliferative endometrium or in 
case of hyperplasia or worse.  
Text was added to provide more 
details on the follow-up and reporting.  
10.4.8 Endometrial Biopsy in Non-
hysterectomized Subjects 
Table continued on the next page 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 16 of 150 
 
 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
CSM has been added to the list 
of contact information.  
To provide a complete overview of all 
facilities. 
1.2 Contact Information 
The text on the additional 
statistical analysis of the daily 
severity score has been moved 
from Secondary Efficacy 
Variables to Primary Efficacy 
Variables: and now reads as 
follows: "The daily severity 
score as defined above will also 
be recomputed for post baseline 
weeks as [((2 x number of 
moderate VMS) + (3 x number 
of severe VMS)) / (Total number 
of moderate + severe VMS)] if at 
least one moderate or severe 
VMS is recorded during the 
day." 
There are differences between the 
FDA and EMA regulatory 
requirements on the assessment of 
efficacy in the treatment of VMS. The 
EMA guidance only includes moderate 
and severe VMS in the primary 
analysis, while in the FDA guidance 
also mild VMS are taken into account.  
 
Both analyses were included in the 
previous version of the protocol, 
although in different subsections.   
Because results of this study will be 
used for submission to the FDA as 
well as to the EMA, both analyses 
have now been placed in the same 
section of the protocol (Primary 
Efficacy Variables), to indicate that 
they are equally important.   
2. Study synopsis (Statistical 
Methods) 
12.4 Primary Efficacy Analysis 
12.6 Secondary Efficacy Analysis 
 
Week and treatment by week 
interaction have been added as 
fixed factors in the MMRM. 
These models factors are necessary to 
get the estimates of the primary 
endpoint.  
2. Study synopsis (Statistical 
Methods) 
12.5. Analysis of Primary Efficacy 
Variables 
The number of subjects has been 
made more flexible throughout 
the protocol. 
For operational purposes this multiple 
centre trial requires some flexibility in 
the number of patients in each study 
part and stratum. 
Throughout the protocol 
Study-specific description of the 
Safety, ITT and PP analysis sets  
To clarify the definition of the 
different statistical populations 
12.1 Statistical Populations 
The text on blood sampling for 
exploratory research has been 
adapted  
Updates have been made for 
clarification purposes. 
10.3.4 Blood Sampling for 
Exploratory Research 
The text on retainment of 
samples for optional additional 
analyses in Section 10.4.5 has 
been reworded and has also been 
added to section 10.3.5. 
Updates have been made for 
clarification purposes. 
10.3.5 Endocrine assessment for 
inclusion and monitoring of 
treatment compliance 
10.4.5 Routine Clinical Laboratory 
Tests 
The text on repeat of BP 
measurements in Section 10.4.3 
at screening has been made 
consistent with the footnote in 
exclusion criteria. 
Update have been made to remove 
inconsistencies. 
10.4.3 Vital signs 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 17 of 150 
 
Amendment 4, leading to version 5.0 of the protocol, date February 03, 2020 
 
Major changes 
Description of changes 
Reason for change 
Sections affected  
Changes in trial design and or trial procedures 
The Safety Study part will also 
include non-hysterectomized 
women. The sample size in the 
Safety Study part has been 
changed from “≤ 400 
hysterectomized subjects” to 
“200 hysterectomized subjects 
and 200 non- hysterectomized 
subjects”.    
Inclusion of non-hysterectomized 
subjects in the safety part of the study 
is requested by the FDA in order to 
gain sufficient safety information for 
the benefit/risk assessment of the 
product in this population.  
2 Study synopsis 
6.2 Objective and Endpoints of the 
Safety Study part 
7.1.1 Trial Design 
7.1.4 Justification of Study 
Population 
8.1.1 Justification of Sample Size 
10.1.6 Visits Safety Study part 
10.4 Safety Assessments 
Inclusion criterion 4 on bi-layer 
endometrial thickness on TVUS 
at screening has been changed 
from “≤ 5 mm” to “≤ 4 mm”.  
At the request of the FDA, the 
endometrial thickness has been 
updated 
2 Study synopsis 
8.2.1 Criteria for Inclusion 
Exclusion criterion 4e has been 
updated. 
Exclusion criterion 4e has been 
updated to clarify that it covers any 
uterine/endometrial abnormality that in 
the judgment of the investigator 
contraindicates the use of estrogen 
and/or progestin therapy.  
2 Study synopsis 
8.2 Inclusion and Exclusion 
Criteria 
Exclusion criterion 8 has been 
changed from: “diabetes mellitus 
with poor glycemic control in the 
last 6 months assessed by fasting 
glucose outside the normal 
ranges and glycated hemoglobin 
above 7%” to “Laboratory values 
of fasting glucose above 125 
mg/dL and/or glycated 
hemoglobin above 7%”. 
Updates have been made to clarify that  
glucose levels and glycated 
hemoglobin levels need to be within 
the set values.  
2 Study synopsis 
8.2.2 Criteria for Exclusion 
Exclusion criterion 9 on 
dyslipoproteinemia has been 
changed from “(LDL >190 
mg/dL and triglycerides >300 
mg/dL)” to  
“(LDL >190 mg/dL and/or 
triglycerides >300 mg/dL) 
Updates have been made for 
clarification purposes. 
2 Study synopsis 
8.2.2 Criteria for Exclusion 
Table continued on the next page 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 18 of 150 
 
Major changes (continued) 
Description of changes 
Reason for change 
Sections affected  
Exclusion criterion 10 on 
smoking in the Efficacy Study 
part has been changed from >5 
cigarettes per day to >15 
cigarettes per day.  
The number of packs per week 
has been deleted for both study 
parts. 
Smoking habits in the Efficacy Study 
part has been made consistent with the 
smoking habits in the Safety Study 
part.  
Number of packs was deleted because 
number of cigarettes per pack may 
vary.   
2 Study synopsis 
8.2.2 Criteria for Exclusion 
Exclusion criterion 23 has been 
changed from “Inadequately 
treated hyperthyroidism at 
screening” to “Inadequately 
treated hyperthyroidism with 
abnormal TSH and free T4 at 
screening. Subjects with low 
TSH are allowed if free T4 at 
screening is within normal range 
Changes were made to further define 
inadequately treated hyperthyroidism.  
2 Study synopsis 
8.2.2 Criteria for Exclusion 
A footnote about reflex T4 
testing was added to exclusion 
criterion 23. 
Footnote was added for clarifying 
purposes. 
2 Study synopsis 
8.2.2 Criteria for Exclusion 
 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
Name and contact details for the 
Clinical Study Leader of  
Estetra SPRL were added to the 
contact information page and 
sponsor authorisation page. 
Addition was made for operational 
purposes.  
1.2 Contact Information 
1.3 Sponsor Authorization 
The QP for pharmacovigilance  
has been replaced on the 
authorisation page.  
Change in study personnel   
1.3 Sponsor Authorization 
The statistician has been replaced 
on the authorisation page. 
Change in study personnel 
1.3. Sponsor Authorization 
The sample size of the Efficacy 
Study part has been updated 
from ± 200 per study arm to 200 
per study arm and a sentence has 
been added that enrollment will 
stop when the minimum required 
number of subjects in each study 
group and study part is enrolled. 
To clarify the number of subject in the 
Efficacy Study part and allow 
sufficient subjects for the objectives of 
the study.  
2 Study Synopsis 
8.1. Source and Number 
The definition of the end of trial 
“the date when the last patient 
has completed the last study 
visit” has been added. 
Update has been made for clarification 
purposes.  
2 Study Synopsis 
7.1.2 Trial Duration 
Table continued on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 19 of 150 
 
Minor changes (continued) 
Description of changes 
Reason for change 
Sections affected  
A footnote “u” in Table 2.1 and 
footnote “s” in Table 2.2 were 
added to clarify when lipid and 
glucose parameters need to be 
assessed at the washout visit or 
during the screening period. 
Updates have been made for 
clarification purposes. 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
2.2 Schedule of Trial Procedures 
Safety Study Part 
Text has been added in footnotes 
“d” of Schedule of Trial 
Procedures Efficacy Study Part 
and “f” of Schedule of Trial 
Procedures Safety Study Part, 
and in Screening Visit section to 
include that the PAP test may be 
repeated once in case of 
inadequate or insufficient 
sample. 
Update has been made allowing a 
repeat of the PAP test at screening. 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
2.2 Schedule of Trial Procedures 
Safety Study Part 
10.1.3 Screening Visit 
Text has been added in footnotes 
“m” and “v” of Schedule of Trial 
Procedures Efficacy Study Part 
and footnotes “a” and “t” of 
Schedule of Trial Procedures 
Safety Study Part, and in 
Screening Visit section to 
include that the laboratory tests 
may be repeated once at the 
discretion of the investigator. 
Update has been made and to allow a 
repeat of the laboratory tests at 
screening. 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
2.2 Schedule of Trial Procedures 
Safety Study Part 
10.1.3 Screening Visit 
A footnote “v” in Schedule of 
Trial Procedures Efficacy Study 
Part and footnote “t” in Schedule 
of Trial Procedures Safety Study 
Part were added to clarify that 
FSH assessment is not required 
for subjects with bilateral 
oophorectomy.  
Updates have been made for 
clarification purposes.  
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
2.2 Schedule of Trial Procedures 
Safety Study Part 
Throughout the protocol the 
pathologic diagnosis based on 
the endometrial biopsy results 
prompting discontinuation from 
study treatment and the start of 
progestin treatment has been 
made consistent to an 
endometrial biopsy showing 
disordered proliferative 
endometrium, hyperplasia or 
worse.  
To ensure consistency throughout the 
protocol on criteria for discontinuation 
based on endometrial biopsy results.  
2 Study Synopsis 
8.3 Subject discontinuation and 
Replacement Procedures 
10.4.8 Endometrial biopsy in non- 
hysterectomized subjects 
10.4.10 Endometrial safety 
assessment and stopping rules 
Wording in section on 
assignment has been changed to 
add assignment to study part.     
Changes have been made to clarify the 
assignment to the study part.  
7.2. Study and Treatment 
Assignment 
Table continued on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 20 of 150 
 
Minor changes (continued) 
Description of changes 
Reason for change 
Sections affected  
Section and Table 3 (Washout 
period) on prior medication have 
been changed to specify use of 
non-hormonal or OTC 
prescriptions for the treatment of 
VMS, and to define criteria for 
use of thyroid treatment. 
Changes have been made to clarify the 
prior use of VMS medication and 
thyroid treatments. 
9.6.1 Prior Medication/Therapy 
9.6.2 Concomitant Medication 
A footnote has been added to 
Table 3 (washout period) for 
tapering off a medication. n 
Footnote has been added to clarify the 
washout period in case of tapering off 
medication 
9.6.1 Prior Medication/Therapy 
 
A footnote has been added in 
Table 4 (Visit schedule) to 
explain flexibility in the Washout 
period and Screening period.  
Updates have been made for 
clarification purposes. 
10.1. Experimental Flow 
Text on screening requirements 
is added in case screening 
procedures cannot be met within 
4 weeks, and the screening 
period needs to be extended. 
However, Screening period may 
not exceed 6 weeks. 
Wording was added to clarify the 
process and requirements in case the 
screening period needs to be extended. 
2 Study Synopsis 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
2.2 Schedule of Trial Procedures 
Safety Study Part  
10.1 Experimental Flow 
10.1.3 Screening Visit 
For re-screening added that 
repeat of the PAP is not needed  
(if a normal written result is 
available within 18 months 
before the start of screening)   
To avoid unnecessary repeats of PAP 
tests. 
10.1.4. Subject Re-screening 
Wording on VMS recording has 
updated to state this VMS will be 
done during screening, and 
during the Efficacy Study part.  
Changes made to clarify when VMS 
will be recorded.  
10.3 Efficacy assessments 
Wording on hyperthyroidism has 
been updated to add 
“inadequately treated” 
hyperthyroidism. 
Change made to be consistent with 
changes made in the inclusion criteria 
regarding hyperthyroidism.    
10.3.5 Endocrine assessment for 
inclusion and monitoring of 
treatment compliance 
Wording on follow-up of 
subjects discontinued due to 
endometrial biopsy results has 
been added. 
Sentence on digital recording has 
been removed. 
Wording was added to define follow-
up process in case of discontinuation. 
Wording on digital recording is 
provided in the Endometrial Biopsy 
Charter. 
10.4.8 Endometrial Biopsy in Non-
hysterectomized Subjects 
Correction made in schedule of 
TVUS assessments: “Visit 1 
(Baseline) changed to  
“screening”.  
Change made in Section 10.4.10 to be 
consistent with schedule of 
assessments.      
10.4.10 Endometrial Safety 
Assessment and Stopping Rules 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 21 of 150 
 
Minor changes (continued) 
Description of changes 
Reason for change 
Sections affected  
In the section on endometrial 
safety wording has been added 
for SAE reporting.  
The sentence on stopping 
enrolment and treatment of non-
hysterectomized subjects in the 
Efficacy Study part due to 
endometrial safety concerns has 
been deleted.   
Additions have been made to clarify 
SAE reporting for endometrial safety.  
The decision to stop (part of) the study 
and/or enrolment NH of subjects will 
be the responsibility of the DSMB, 
based on actual safety data.   
10.4.10 Endometrial Safety 
Assessment and Stopping Rules 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 22 of 150 
 
Amendment 3, leading to version 4.0 of the protocol, date October 08, 2019 
 
Major changes 
Description of changes 
Reason for change 
Sections affected  
Changes in trial design and or trial procedures 
An inclusion criterion was added 
for non-hysterectomized 
subjects: uterus with bi-layer 
endometrial thickness ≤ 5mm on 
TVUS is now also required. 
Inclusion criterion was added to ensure 
an additional safety measure. 
2 Study synopsis 
8.2 Inclusion and Exclusion 
Criteria 
 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
Clarifying Changes 
Footnote in Figure 1 has been 
clarified by including the 
corresponding Visits linked to 
the mentioned follow up times of 
treatment. 
Update was made to ensure ease of 
reading and consistency throughout the 
protocol. 
2 Study Synopsis 
7.1.1 Trial Design 
Wording was updated to state 
“generally up to 8 weeks” 
instead of “up to 8 weeks”. 
Update was made to ensure 
consistency in wording throughout the 
protocol. 
2 Study Synopsis 
7.1.2 Trial Duration 
Footnote “c” of Table 2.1 was 
updated. 
Week 9 was added to footnote “c” of 
Table 2.1 for clarification as it was 
mistakenly omitted. 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
Footnotes “g” of Table 2.1 “f” of 
Table 2.2 have been updated to 
clarify that PAP smear is to be 
done in women with uterus 
and/or cervix. 
Updates have been made for 
clarification purposes and to ensure 
consistency throughout the protocol. 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
2.2 Schedule of Trial Procedures 
Safety Study Part 
Footnotes “a” of Tables 2.1 and 
2.2 was updated to clarify that 
for implantable or injectable 
androgen therapy or 
dehydroepiandrosterone (DHEA) 
containing drugs, a washout 
period of 6 months is required. 
To ensure consistency throughout 
protocol. 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
2.2 Schedule of Trial Procedures 
Safety Study Part 
Table continued on the next page 
 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 23 of 150 
 
Minor changes (continued) 
Description of changes 
Reason for change 
Sections affected  
Clarifying Changes 
Table and text have been updated 
to state treatment allocation 
instead of randomization for the 
safety study part. 
 
To ensure accuracy of procedures in 
safety study part. 
2.2 Schedule of Trial Procedures 
Safety Study Part,  
Section 10.1.6 Visits Safety Study 
part 
8.2 Inclusion and Exclusion 
Criteria (Inclusion 2) 
10.4.5. Routine Clinical 
Laboratory Tests (pregnancy test)  
Reference has been made to 
Table 3 in Section 9.6.1. 
Change was done to improve the flow 
of the reader and help with cross 
referencing. 
7.1.1 Trial Design 
First paragraph was updated to 
state the following: “Treatment 
assignment into any of the study 
parts (i.e. Efficacy and Safety) 
and subgroups (15 mg, 20 mg 
and placebo) will be done by the 
IWRS system.  The treatment 
assignment will take into account 
the modified MENQOL score, 
frequency and severity of hot 
flashes, hysterectomized vs. non-
hysterectomized status, and 
availability of places in each 
study part/subgroup.” 
Wording was added for clarification. 
7.2 Treatment Assignment 
The wording “electronic data 
capture system” has been 
replaced by “Interactive Web 
Response System (IWRS)”. 
Change was done to improve accuracy 
of information. 
7.3 Randomization 
The wording Interactive X 
Response System (IXRS) has 
been replaced by Interactive Web 
Response System (IWRS). 
To ensure consistency throughout 
protocol 
4.3 List of Abbreviations 
7.2 Treatment Assignment 
7.3 Randomization 
Footnote has been added for 
exclusion criterion 8. 
Footnote was added to bring clarity to 
all readers that lab values from past 6 
months should be considered to 
identify a subject with poor glycemic 
control. 
8.2.2 Exclusion Criteria 
Footnote has been added for 
exclusion criterion 1. 
Footnote was added to ensure clarity 
across readers. All malignancies are 
excluded, except for basal cell 
carcinoma and squamous cell 
carcinoma of the skin if these were 
diagnosed more than a year before 
screening. 
8.2.2 Exclusion Criteria 
Last part of the 5th sentence was 
removed.  
Change was done to avoid 
misinterpretation. 
9.4 Packaging and Labelling 
Table continued on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 24 of 150 
 
Minor changes (continued) 
Description of changes 
Reason for change 
Sections affected  
Clarifying Changes 
Wording on P4 treatment was 
added 
Wording has been added for 
completeness. 
9.4 Packaging and Labelling 
Table 1 was updated 
Table 1 was updated to include P4 
treatment information and ensure 
completeness. 
9.4 Packaging and Labelling 
Wording was updated to state 
Safety Study part instead of 
Endometrial and General Safety 
Study Part. 
Change was made to ensure 
consistency in protocol and correct 
sentence with appropriate wording 
9.6.1 Prior Medication/Therapy 
10.1.3 Screening Visit 
TVUS was deleted from the 
assessments needed to be done at 
Baseline visit. 
TVUS is performed at screening, it 
was mistakenly repeated as a 
procedure at Baseline Visit.  Table and 
specific section have been updated. 
2.1 Schedule of Trial Procedures 
Efficacy Study Part 
10.1.5.1 Randomization Visit and 
Baseline Assessments 
It has been clarified that TVUS 
should be performed not only at 
screening but also at End of Trial 
Visit in non-hysterectomized 
subjects or subjects with ovaries 
in both the Efficacy and Safety 
study parts.  
Update was made to ensure 
consistency and clarity throughout the 
protocol. 
2.2 Schedule of Trial Procedures 
Safety Study Part 
10.1.6 6 Visit 6: End of Trial  
Reference was added to the 
Imaging Manual. 
Reference was added to cross refer to 
the appropriate document containing 
detailed information on the 
mammography process and guide sites 
in identifying appropriate source of 
information. 
10.4.6 Mammography  
Wording under “Evaluation of 
endometrial tissue” subtitle has 
been updated. 
Wording was added to clarify the 
decision process in place and to add 
reference to the Charter for 
Endometrial Biopsy Management. 
10.4.8 Endometrial Biopsy in Non-
hysterectomized Subjects 
Section on Country Specific 
Requirements was added to 
accommodate requirements from 
Health Canada. 
Health Canada requested a country 
specific amendment to ensure that 
Canadian sites are aware that only 
hysterectomized subjects are to be 
enrolled in both parts of the study 
(Efficacy and Safety parts). 
16.5 Country Specific 
Requirements 
Administrative changes 
ERT Lab information has been 
deleted 
Since all ECGs have been confirmed 
will be read locally, there is no need 
for ERT central reading. 
1.2 Contact Information 
10.4.4 Electrocardiogram 
Total number of pages was 
added to each footer 
For completeness and avoid 
misinterpretation due to lost pages. 
Throughout the document 
Typographic corrections 
To correct typographical errors. 
Throughout the document 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 25 of 150 
 
Amendment 2, leading to version 3.0 of the protocol, date August 13th 2019 
 
Major changes 
Description of changes 
Reason for change 
Sections affected  
Changes in trial design and or trial procedures 
Exclusion criterion 4c has been 
updated 
At the request of the Central IRB, the 
exclusion criterion 4c has been 
updated to clarify that it also covers 
undiagnosed abnormal uterine 
bleeding. 
2 Study synopsis 
8.2 Inclusion and Exclusion 
Criteria 
Exclusion criterion 14 has been 
updated 
At the request of the Central IRB, the 
exclusion criterion 14 has been 
updated to clarify that it also covers 
the presence or history of severe 
hepatic disease or liver tumors 
2 Study synopsis 
8.2 Inclusion and Exclusion 
Criteria 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
Administrative changes 
 contact information 
has been deleted 
Since PAP test reading will be done 
locally, the information on 
 
central lab has been deleted. 
1.2 Contact Information 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 26 of 150 
 
Amendment 1, leading to version 2.0 of the protocol, dated July 18th 2019 
 
Major changes 
Description of changes 
Reason for change 
Sections affected  
Changes in trial design and or trial procedures 
Addition of an exclusion 
criterion for non-hysterectomized 
subjects regarding peanut 
allergy. 
 
P4 provided as part of this study 
contains peanut oil and should never 
be used by subjects with allergy to 
peanuts. 
2 Study synopsis 
8. 2 Inclusion and Exclusion 
Criteria 
Changes in study evaluation 
In the Efficacy Study part the 
timing of the-efficacy analysis 
has been changed to be done 
after all subjects have completed 
Visit 4. At the same time also an 
interim analysis of the safety will 
be performed.  
To clarify the original intention of the 
protocol, which is to analyse the 
primary efficacy objective when 
efficacy follow up for primary analysis 
is completed (i.e. Visit 4 of the 
Efficacy Study part). 
2 Study Synopsis, (Timing of 
analysis) 
12.9 Timing of Analysis 
Additional wording has been 
added on the process to ensure 
the study blind after analyses in 
the Efficacy Study part are 
conducted while the study is still 
ongoing.  
Because the primary analysis on 
efficacy and an interim analysis on 
safety will be conducted in the 
Efficacy Study part before database 
lock, additional measures are needed 
to keep the study blind.  
Section 7.4.1 Blinding procedures 
Section 12.9 Timing of analysis 
 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
Changes in trial design and or trial procedures 
The Run-in period for VMS 
recording has been incorporated 
into the Screening period and 
wording has been added to make 
clear that the Screening period 
will always start with a Screening 
visit. 
For operational purposes; one 
Screening period, starting with a 
Screening visit, which includes the 
VMS recording, is considered to be  
clearer and more efficient. 
Throughout the protocol "Run-in" 
has been deleted. 
Specific changes in: 
2 Study Synopsis (including 
Schedule of Trial Procedures). 
7.1 Study Design and Justification 
(including Trial Schedule) 
10.1.3: Visit 1 Screening 
Procedures (previous Section 10.1.3 
"Run-in period" combined with 
current Section 10.1.3). 
The duration of the Screening 
period is reduced from 6 weeks 
to 4 weeks. 
By combining the Run-in period with 
the Screening period, the total duration 
of the screening procedures is reduced. 
2 Study Synopsis (Trial Schedule, 
Trial Duration and Schedule of 
Trial Procedures). 
7.1.1 Trial Design 
7.1.2 Trial Duration 
Table continued on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 27 of 150 
 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
Changes in trial design and or trial procedures 
The duration of the Washout 
period is reduced from 10 weeks 
to 8 weeks. 
Usual washout period for most subjects 
is expected to be 8 weeks instead of 10 
weeks. 
2 Study Synopsis (Trial Schedule, 
Trial Duration and Schedule of 
Trial Procedures). 
7.1.1 Trial Design 
7.1.2 Trial Duration 
Addition of pregnancy test at 
Visit 1 at the discretion of the 
Investigator. 
Because of the time interval of 4 weeks 
between the screening Visit and Visit 1 
the pregnancy test may be repeated at 
Visit 1. 
2 Study Synopsis (Schedule of Trial 
Procedures) 
10.1.5.1 Randomization Visit 
(Efficacy Study part) 
10.1.6.1 Randomization visit 
(Safety Study part) 
10.4.5 Routine Clinical Laboratory 
Tests  
Collection of data on Medical 
and Gynecological History and 
Demographic characteristics has 
been moved from Screening Visit 
to Washout Visit.  
 
 
Because adverse events are collected as 
of signature of Informed Consent 
occurring during Washout Visit, 
collection of Medical and 
Gynecological History and 
Demographic Characteristics should be 
aligned to the same time point. 
2 Study Synopsis (Schedule of Trial 
Procedures) 
10.1.2 Washout Visit (Experimental 
Flow) 
10.1.3 Screening Visit 
10.2 Demographics and Subject 
Characteristics  
 
Changes in study evaluation 
Safety endpoints; for physical 
and gynaecological examination, 
vital signs, electrocardiogram 
(ECG), mammography, and 
breast examination and for 
routine clinical laboratory tests: 
the "frequency of change in 
results" in these parameters will 
be evaluated instead of the 
"frequency of clinically relevant 
results".  
The frequency of clinically relevant 
results in these safety parameters are 
already evaluated as part of the AEs. 
The frequency of changes in results 
these parameters will provide 
additional safety information.  
2 Study Synopsis (Objectives and 
Endpoints) 
6.1.2 Secondary Objectives (for 
Efficacy Study part) 
6.2.1 Primary Objectives (for 
Safety Study part) 
Statistical method section; the 
text on the secondary efficacy 
variables in is reworded. 
To remove inconsistencies and clarify 
the secondary evaluation 
2. Study Synopsis (Secondary 
Efficacy variables 
12.6 Secondary Efficacy Variables 
Clarifying changes  
Changed "Efficacy Study" and 
"Safety Study" in "Efficacy 
Study part" and "Safety Study 
part"  
To clarify that this is one trial with two 
study parts. 
Throughout the protocol 
Addition of "in the last 7 
consecutive days during the 
Screening period" for the 
assessments of VMS symptoms 
during the Screening period.   
For clarity and ensure consistency with 
the in- and exclusion criteria.  
Section 2: Study Synopsis (Trial 
Schedule, Trial Duration and 
Schedule of Trial Procedures). 
7.1.1 Trial Design 
7.1.2 Trial Duration 
Table continued on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 28 of 150 
 
 
Minor changes 
Description of changes 
Reason for change 
Sections affected  
Specified that question number 1 
of the modified MENQOL 
should be used to assess 
bothersome VMS. 
To provide clarity on the definition of 
bothersome VMS.  
2 Study Synopsis (Trial Design and 
Schedule of Trial Procedures) 
7.1.1.  Trial Design 
8. 2 Inclusion and Exclusion 
Criteria 
10.1.3: Visit 1 Screening 
procedures 
Editorial changes to safety 
endpoints 6.2, 7.1 and 7.2 of the 
efficacy study 
 
Clarification of the text to avoid 
misunderstanding. 
 
2 Study Synopsis 
6 Objectives and Endpoints 
Editorial changes in the inclusion 
criteria 2, 6 and 9 and in the 
exclusion criterion 1, and in the 
in- and exclusion criteria 
footnotes  
 
Clarification of the text to avoid 
misunderstanding.  
 
2 Study Synopsis 
8.2 Inclusion and Exclusion Criteria 
Wording added for contacting 
subjects in case of 
discontinuation. 
To provide instructions on the attempts 
to contact a subject in case of 
discontinuation, before considering lost 
to follow up.  
8.3 Subject Discontinuation and 
Replacement Procedures 
Addition of a table with the visit 
schedule.  
To provide additional guidance on the 
visit schedule and permitted time 
window for each visit.  
10.1 Experimental Flow 
An additional section is added on 
the blood sampling for 
exploratory research. 
To provide explanation on the purpose 
of the optional additional samples. 
10.3.4. Blood Sampling for 
Exploratory Research 
QTcFrid has been added as the 
method to be used when 
reporting QT intervals. 
 
To provide clarification and guidance. 10.4.4 Electrocardiogram 
Administrative changes 
Name of the Medical Director of 
Sponsor has been changed. 
Change in Medical Director. 
Section 1.2 Contact information 
Section 1.3 Sponsor Authorisation 
Names of Senior Director Project 
Management CRO and Program 
Manager CRO have been 
changed. 
Change in Project Members. 
Section 1.2 Contact Information 
Names of study personal has 
been added to the Sponsor 
authorisation page. 
For completion.  
Section 1.3 Sponsor Authorisation 
Typographic corrections and 
corrections of inconsistencies.  
To correct typographical errors and 
inconsistencies.  
Throughout the protocol 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 29 of 150 
 
2. 
STUDY SYNOPSIS 
Sponsor: 
Active Ingredient: 
Estetra SRL 
Estetrol 
Study Title: 
A Randomized Double-blind Placebo Controlled Phase 3 Study to evaluate the Efficacy and Safety of Estetrol for 
the Treatment of Moderate to Severe Vasomotor Symptoms in Postmenopausal Women (E4Comfort Study II).  
Study Location: Multiple centers in the USA and Canada 
Study Phase: 3 
Study Design: 
The trial has two parts; an Efficacy Study part (Arms 1-3) and a Safety Study part (Arm 4). 
 
The Efficacy Study part has a randomized, double-blinded placebo-controlled design and evaluates the primary 
efficacy (frequency and severity of vasomotor symptoms [VMS]), secondary efficacy (effect on hemostasis, lipid 
and glucose metabolism, bone turnover, health-related quality of life [HRQoL] and treatment satisfaction [TS]), 
and safety of estetrol (E4) in both hysterectomized and non-hysterectomized postmenopausal women, as well as 
the effect of E4 on the endometrium in non-hysterectomized postmenopausal women. 
Subjects will be randomly allocated (1:1:1) to one of the 3 Arms and will receive E4 15 mg (Arm 1), E4 20 mg 
(Arm 2), or Placebo (Arm 3). During the first 12 weeks of the Efficacy Study part, the effect of E4 on VMS will 
be evaluated. Thereafter, treatment will proceed for a total duration of up to 53 weeks, to continue the evaluation 
of secondary efficacy, safety and the effect on the endometrium. For endometrial protection, all non-
hysterectomized subjects will receive treatment with 200 mg progesterone (P4) once daily for 14 consecutive days, 
after completion of the E4/placebo treatment. 
 
The Safety Study part is conducted in hysterectomized and non-hysterectomized postmenopausal women and has 
an open label design. The Safety Study part evaluates the general safety, secondary efficacy (lipid and glucose 
metabolism, HRQoL and TS) of up to 53 weeks treatment with E4 20 mg (Arm 4). 
 
The trial schedule is summarized in Figure 1. 
Participants in the Efficacy Study part need to have at least 7 moderate to severe bothersome menopausal related 
VMS per day or at least 50 moderate to severe bothersome VMS per week in the last 7 consecutive days during 
the Screening period. Bothersome is defined as a score of 4 to 6 on question 1 of the modified Menopause-specific 
quality of life (MENQOL) questionnaire at screening. The timing and number of VMS and modified MENQOL 
score needed for allocation to the Efficacy Study part will not be disclosed to the study subjects to avoid bias in 
the recording during the Screening process. Participants in the Safety Study part should seek treatment for the 
relief of VMS associated with menopause, with at least 1 moderate to severe menopausal related VMS per week. 
Subjects who do not fulfill the VMS requirements for participation in the Efficacy Study part, may participate in 
the Safety Study part providing that they meet all in- and exclusion criteria for participation in the Safety Study 
part. 
Hysterectomized and non-hysterectomized healthy women will be included in the Efficacy Study part and Safety 
Study part (approximately 50% of the subjects are anticipated to have a history of hysterectomy). 
The target enrollment number of subjects in each of the three arms of the Efficacy Study part is ~200. The target 
number of subjects to be included in the Safety Study part is ~400. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 30 of 150 
 
Figure 1: Trial Schedule 
 
* For non-hysterectomized (NH) subjects, endometrial thickness will be measured after 3, 6, 9 and 12 months of treatment (Visits 4, 5, 6 and 7 of the 
Efficacy Study part and Visits 3, 4, 5 and 6 of the Safety Study part respectively) by transvaginal ultrasound (TVUS) and subjects will record daily the 
vaginal bleeding events. If at any time after the first 4 weeks of treatment a subject presents with persistent and/or recurrent bleeding, confirmed by the 
investigator or presents with a bi-layer endometrial thickness >10 mm as assessed by TVUS, an endometrial biopsy will be performed. If the biopsy 
shows disordered proliferative endometrium, hyperplasia or worse, the study drug will be discontinued, and the subject will be withdrawn from the 
study. The subject will be treated with P4 200 mg once daily for 14 days. If the endometrial event has not resolved, treatment with a progestin will be 
started according to local practice/guidelines. All NH subjects will receive treatment with 200 mg P4 for two weeks after completion of E4/placebo 
treatment.  
  
Blinding:  
Efficacy study part: double-blinded 
Safety Study part: open-label 
Trial Duration: 
Individual subject participation in this trial may be up to 15 months. This includes a washout period of generally 
up to 8 weeks (usual washout period for most subjects), a screening period of generally up to 4 weeks, and a 
treatment period with E4 or placebo of up to 53 weeks. For non-hysterectomized subjects there will be an additional 
follow-up period of approximately 3 weeks. 
For subjects requiring a longer washout (up to 6 months) participation period may be extended. For subjects who 
do not require a washout, the total participation period will be reduced by approximately 8 weeks. 
 
The end of trial is defined as the date when the last patient has completed the last study visit. 
Trial Population: 
A total of ~1000 subjects will be enrolled in the study. Eligible subjects will be postmenopausal women, ≥ 40 up 
to ≤ 65 years of age, seeking treatment for relief of moderate to severe VMS associated with menopause. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 31 of 150 
 
Efficacy Study part: A total of ~600 subjects will be enrolled. Subjects (~300 hysterectomized and ~300 non-
hysterectomized) need to have at least 7 moderate to severe bothersome VMS per day or at least 50 moderate to 
severe bothersome VMS a week in the last 7 consecutive days during the screening. 
Safety Study part: A total of ~400 subjects will be enrolled.  Subjects (~200 hysterectomized and ~200 non-
hysterectomized should seek treatment for the relief of VMS (condition clinically indicated by the investigator) 
associated with menopause and have at least 1 moderate to severe menopausal related VMS per week.  
Investigational Medicinal Product (dose, and mode of administration): 
E4 15 mg (Arm 1) or E4 20 mg (Arm 2 and Arm 4), oral, once daily 
Reference therapy (dose, and mode of administration): 
Placebo, oral, once daily (Arm 3) 
Other trial medication (dose, and mode of administration): 
For non-hysterectomized subjects only:  
• 
P4, 200 mg, oral, once daily for 14 consecutive days (after completion or discontinuation of E4/placebo 
treatment)  
• 
Ad hoc treatment with a progestin according to local practice/guidelines if endometrial biopsy shows 
disordered proliferative endometrium, hyperplasia or worse 
Conduct of the Trial: 
For the Efficacy Study part at least 9 visits are planned: a Washout visit followed by a Screening period which 
will start with a Screening visit, a Baseline visit (Visit 1), and 6 On-treatment visits (Visits 2 to 7). The last 
treatment visit (Visit 7) is also the end of treatment (EoT) Visit, which will also occur in case of early 
discontinuation. For non-hysterectomized women an additional endometrial safety follow-up visit (Visit 8) is 
planned approximately 10 days after completion of the 14 days of P4 treatment, which will also occur in case of 
early discontinuation. 
For the Safety Study part at least 8 visits are planned: a Washout visit followed by a Screening Period which will 
start a screening visit, a Baseline visit (Visit 1), and 5 On-treatment visits (Visits 2 to 6). The last treatment visit 
(Visit 6) is also the end of treatment (EoT) Visit, which will also occur in case of early discontinuation. For non-
hysterectomized women an additional endometrial safety follow-up visit (Visit 7) is planned approximately 10 
days after completion of the 14 days of P4 treatment, which will also occur in case of early discontinuation. 
 
The screening period requires at least one visit, to occur as per protocol, at the beginning of the period, but some 
assessments (e.g., mammography, biopsy, transvaginal ultrasound [TVUS], electrocardiogram [ECG] and 
Papanicolaou [PAP] test) may require additional visits in case these assessments cannot be done during the same 
visit. 
Additional unscheduled visits may also be required during the treatment period for ad hoc safety assessments (e.g., 
TVUS for non-hysterectomized subjects). 
 
Washout and Screening procedures for the Efficacy Study part and Safety Study part 
At the Washout visit, after signing the informed consent, the inclusion and exclusion criteria will be checked as 
well as the prior medication list. If a subject uses prohibited co-medication, then this subject will be asked to stop 
this medication. After a variable washout period (depending on type of prior medication) the subject will return to 
start the screening procedures. For subjects who do not use medication that needs to be stopped and washed out, 
the Washout and Screening visit may occur at the same time. Screening includes a PAP test and mammography 
for all subjects, a TVUS for subjects with ovaries and/or uterus, and an endometrial biopsy for non-
hysterectomized subjects. The length of the Screening period is generally up to 4 weeks, but can be extended (with 
prior Medical Monitor approval) without exceeding 8 weeks. During this period, and in addition to the screening 
procedures, the subject will record VMS in a paper diary. VMS data recorded during the last 7 consecutive days 
of the Screening period will be used to determine eligibility of subjects. If based on VMS symptoms of her current 
medical history a subject is only eligible for the Safety Study part, this subject may be enrolled directly into the 
Safety Study part without the VMS count in the Screening period, providing that all the screening assessments 
have been completed. Subjects who do not fulfill the VMS requirements for participation in the Efficacy Study 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 32 of 150 
 
part, may participate in the Safety Study part, providing that they meet all other in- and exclusion criteria. 
 
 
Treatment period Efficacy Study part  
During the first 12 weeks of treatment, primary efficacy assessments (the number and severity of VMS) will be 
recorded on a daily basis in a paper diary. Subjects will also record vaginal bleeding events (non-hysterectomized 
subjects only) and daily study drug intake in the diary. After completion of the 12 weeks of treatment (Visit 4), 
only non-hysterectomized subjects will keep the diary to record daily vaginal bleeding events and daily study drug 
intake. At baseline (Visit 1) secondary efficacy assessments (hemostasis, lipid and glucose metabolism, and bone 
turnover) will be performed, the MENQOL questionnaire will be completed, and for non-hysterectomized subjects 
endometrial thickness will be assessed. Subjects will visit the trial center after 4 and 8 weeks of treatment (Visit 2 
and Visit 3) for general safety assessments, for review of the diaries, and for completion of the Clinical Global 
Impression (CGI) questionnaire (Visit 2 only). After 12 weeks of treatment, subjects will visit the trial center for 
the final primary efficacy assessment (Visit 4). During this visit also secondary efficacy assessments and general 
safety assessments will be performed and the MENQOL and CGI questionnaires will be completed. For non-
hysterectomized subjects endometrial thickness will be assessed. Subjects will visit the trial center again after 6 
months of treatment (Visit 5) for general safety assessments and endometrial thickness assessment (non-
hysterectomized subjects only) and after 9 months of treatment (Visit 6) for general safety assessments and 
endometrial thickness assessment (non-hysterectomized subjects only). After 12 months of treatment, subjects will 
visit the trial center for the EoT visit (Visit 7) during which secondary efficacy assessments and general safety 
assessments will be performed, the HRQoL and TS questionnaires will be completed and a mammography will be 
made. For non-hysterectomized subjects endometrial thickness will be assessed and, if they have completed at 
least 12 weeks of E4/placebo treatment, an endometrial biopsy will be performed. At Visits 1, 2, 3 and 4 blood 
samples will be taken to monitor estradiol (E2) levels and at Visits 2, 3 and 4 also to monitor E4 levels. 
After the EoT procedures, all non-hysterectomized subjects will receive treatment with 200 mg P4 for 14 days. 
Approximately 10 days after completion of the progestin treatment, endometrial thickness will be measured (Visit 
8). 
 
Treatment period Safety Study part 
At Baseline (Visit 1) secondary efficacy assessments (lipid and glucose metabolism) will be performed and the 
MENQOL questionnaire will be completed. Subjects will visit the trial center after 4 weeks (Visit 2), 12 weeks 
(Visit 3), 6 months (Visit 4) and 9 months (Visit 5) of treatment for safety assessments, and for completion of the 
CGI questionnaire (Visit 2 only). Visit 3 secondary efficacy assessments will also be performed and the MENQOL 
and CGI questionnaires will be completed. For non-hysterectomized subjects endometrial thickness will be 
assessed on visits 3, 4 and 5. During treatment non-hysterectomized subjects will record daily study drug intake 
and vaginal bleeding events in a paper diary. After 12 months of treatment subjects will visit the trial center for 
the EoT visit (Visit 6) during which secondary efficacy assessments and safety assessments will be performed, 
questionnaires will be completed and a mammography will be made. For non-hysterectomized subjects 
endometrial thickness will be assessed and, if they have completed at least 12 weeks of E4, an endometrial biopsy 
will be performed.  
After the EoT procedures, all non-hysterectomized subjects will receive treatment with 200 mg P4 for 14 days. 
Approximately 10 days after completion of the progestin treatment, endometrial thickness will be measured (Visit 
7). 
Endometrial Safety Assessment and Stopping Rules (non-hysterectomized subjects only): 
A baseline endometrial biopsy will be obtained for all non-hysterectomized subjects during the Screening period. 
An end of treatment endometrial biopsy is planned at the EoT Visit (Visit 7 Efficacy Study part, Visit 6 Safety 
Study part) providing that the subject has been treated with E4/placebo for at least 12 weeks. All non-
hysterectomized subjects will receive treatment with 200 mg P4 for two weeks after completion or early 
termination of the E4/placebo treatment. 
Endometrial thickness will be measured after 3, 6, 9 and 12 months (Visits 4, 5, 6 and 7 in the Efficacy Study part 
and Visits 3, 4, 5 and 6 in the Safety Study part) of treatment by TVUS and subjects will record daily vaginal 
bleeding events. If at any time after the first 4 weeks of treatment, a subject presents with persistent and/or recurrent 
bleeding, confirmed by the Investigator or presents with a bi-layer endometrial thickness >10 mm as assessed by 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 33 of 150 
 
TVUS, an endometrial biopsy will be performed. If the biopsy shows disordered proliferative endometrium, 
hyperplasia or worse, the study drug will be discontinued, and the subject will be withdrawn from the study. The 
subject will be treated with P4 200 mg once daily for 14 days. At approximately 10-14 days after completion of 
the progestin treatment, a new TVUS will be performed to measure endometrial thickness and a biopsy will be 
carried out, if deemed necessary by the Investigator, to demonstrate the resolution. If the endometrial event is not 
resolved, the subject will be further treated according to local practice/guidelines and she will be followed up until 
resolution.  
Endometrial safety will be monitored during the trial by an independent Data Safety Monitoring Board (DSMB). 
Objectives and Endpoints of the Efficacy Study part (Arms 1-3): 
Primary Objective and Endpoints:  
1. To measure the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on the frequency and 
severity of moderate to severe VMS in postmenopausal women at 4 and 12 weeks  
Co-primary efficacy endpoints for primary objective #1 
1.1. 
Mean change in weekly frequency of moderate to severe VMS from baseline to week 4 
1.2. 
Mean change in weekly frequency of moderate to severe VMS from baseline to week 12 
1.3. 
Mean change in severity of moderate to severe VMS from baseline to week 4 
1.4. 
Mean change in severity of moderate to severe VMS from baseline to week 12 
 
Secondary Objectives and Endpoints: 
1. To measure the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on the frequency and 
severity of mild, moderate and severe VMS in postmenopausal women weekly up to 12 weeks 
Efficacy endpoints for secondary objective #1  
1.1. 
Change from baseline to week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 in the weekly frequency and 
severity of moderate to severe VMS 
1.2. 
Change from baseline to weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 in the weekly frequency and 
severity of mild, moderate, and severe VMS  
1.3. 
Percentage of subjects with 50% and 75% reduction from baseline in the weekly frequency of moderate 
to severe VMS at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 
1.4. 
Percentage of subjects with 50% and 75% reduction from baseline in the weekly frequency of mild, 
moderate and severe VMS at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 
 
2. To measure the clinical meaningfulness of E4 15 mg or E4 20 mg compared to placebo on the reduction of 
VMS at weeks 4 and 12 
Efficacy endpoint for secondary objective #2  
2.1. 
Percentage of subjects with a clinically important difference (CID) compared to baseline in the weekly 
frequency of moderate to severe VMS after weeks 4 and 12 using the CGI questionnaire  
3. To evaluate the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on hemostasis, lipid and 
glucose metabolism, and bone turnover 
Efficacy endpoint for secondary objective #3 
3.1. Change from baseline to week 12 and week 52 in prothrombin fragment 1 + 2, endogenous thrombin 
potential (ETP)-based activated Protein C sensitivity ratio (APCsr ETP), activated partial thromboplastin 
time (aPTT) based activated Protein C resistance (APCr), anti-thrombin III, Protein-C, free Protein-S, 
Factor VIII, angiotensinogen, and Sex Hormone Binding Globulin (SHBG) 
3.2. Change from baseline to week 12 and week 52 in triglycerides, high-density lipoprotein (HDL)-
cholesterol, low-density lipoprotein (LDL)-cholesterol, total cholesterol, lipoprotein(a), total 
cholesterol/HDL-cholesterol ratio, fasting glycemia, insulin, glycated hemoglobin and homeostasis 
model assessment-estimated insulin resistance (HOMA-IR) 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 34 of 150 
 
3.3. Change from baseline to week 12 and week 52 in procollagen I N-propeptide (PINP), C-terminal 
telopeptide type 1 (CTX-1), calcium, and vitamin D 
4. To evaluate the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on HRQoL and TS 
Efficacy endpoints for secondary objective #4 
4.1. 
Change from baseline to week 12 and week 52 in HRQoL using the MENQOL questionnaire 
4.2. 
Total score in TS after 4, 12, and 52 weeks of treatment using the CGI questionnaire 
 
5. To evaluate the general safety of treatment with E4 15 mg or E4 20 mg compared to placebo 
Safety endpoints for secondary objective #5 
5.1. 
Frequency of treatment emergent adverse events (TEAEs) (including treatment emergent serious 
adverse events [SAEs])  
5.2. 
Frequency of changes in results in physical and, gynecological examination, vital signs, ECG, 
mammography, and breast examination at each measured time point 
5.3. 
Frequency of changes in routine clinical laboratory tests results (hematology and chemistry) at each 
measured time point 
6. To evaluate the effect of treatment with E4 15 mg or E4 20 mg on the endometrium in non-hysterectomized 
subjects compared to placebo 
Safety endpoints for secondary objective #6 
6.1. 
Change from baseline to each measured time point in endometrial thickness measured by ultrasound 
6.2. 
Frequency of subjects in the different endometrial categories according to the Blaustein's pathology 
(see Appendix 16.3) 
 
7. To evaluate the effect of treatment with E4 15 mg or E4 20 mg on vaginal bleeding in non-hysterectomized 
subjects compared to placebo 
Safety endpoints for secondary objective #7 
7.1. 
Frequency of women with vaginal bleeding and/or spotting during each 28-day cycle of treatment with 
E4 based on recording in the patient diary 
7.2. 
Number of days with bleeding and/or spotting during each 28-day cycle of treatment based on 
recording in the patient diary 
7.3. 
Frequency of women with amenorrhea (absence of any bleeding or spotting) during each 28-day cycle 
of treatment with E4 based on recording in the patient diary 
7.4. 
Cumulative rates of amenorrhea defined as the percentage of women who reported consecutive cycles 
of amenorrhea for a given cycle of time 
 
Exploratory Objectives and Endpoints of the Efficacy Study part:  
1. To evaluate the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on breast density  
Safety endpoint for exploratory objective #1 
1.1. 
Change in breast density from digitized mammography readings from baseline to EoT in subjects who 
had a paired digitalized mammography 
 
2. To evaluate the influence of E4 on the changes in the hemostasis parameters compared to placebo in women 
with inherited thrombophilia and women without this genetic disorder  
Efficacy endpoint for exploratory objective #2 
2.1. 
Change in hemostasis parameters in women with and without inherited thrombophilia (Factor V Leiden 
mutation and prothrombin G20210A mutations). 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 35 of 150 
 
Objectives and Endpoints of the Safety Study part (Arm 4): 
Primary Objective and Endpoints: 
1. To evaluate the general safety of treatment with E4 20 mg 
 
Safety endpoints for primary objective #1 
1.1. 
Frequency of TEAE (including SAEs)  
1.2. 
Frequency of changes in results in physical and gynecological examination, vital signs, ECG, 
mammography, and breast examination at each measured time point 
1.3. 
Frequency of changes in routine clinical laboratory tests results (hematology and chemistry) at each 
measured time point  
 
Secondary Objectives and Endpoints:  
1. To evaluate the effect of treatment with E4 20 mg on lipid and glucose metabolism 
 
Efficacy endpoints for secondary objective #1 
1.1. 
Change from baseline to week 12 and week 52 in triglycerides, HDL-cholesterol, LDL-cholesterol, 
total cholesterol, lipoprotein(a), total cholesterol/HDL-cholesterol ratio, fasting glycemia, insulin, 
glycated hemoglobin, and HOMA-IR 
 
2. To evaluate the effect of treatment with E4 20 mg on HRQoL and TS 
 
Efficacy endpoints for secondary objective #2 
2.1. 
Change from baseline to week 12 and week 52 in HRQoL using the MENQOL questionnaire 
2.2. 
Total score in TS after 4, 12, and 52 weeks of treatment using the CGI  
 
3. To evaluate the effect of treatment with E4 20 mg on the endometrium in non-hysterectomized subjects 
Safety endpoints for secondary objective #3 
3.1 
Change from baseline to each measured time point in endometrial thickness measured by ultrasound 
3.2 
Frequency of subjects in the different endometrial categories according to the Blaustein's pathology 
(see Appendix 16.3) 
 
4. 
To evaluate the effect of treatment with E4 20 mg on vaginal bleeding in non-hysterectomized subjects 
Safety endpoints for secondary objective #4 
4.1 Frequency of women with vaginal bleeding and/or spotting during each 28-day cycle of treatment with 
E4 based on recording in the patient diary 
4.2 Number of days with bleeding and/or spotting during each 28-day cycle of treatment based on recording 
in the patient diary 
4.3 Frequency of women with amenorrhea (absence of any bleeding or spotting) during each 28-day cycle of 
treatment with E4 based on recording in the patient diary 
4.4 Cumulative rates of amenorrhea defined as the percentage of women who reported consecutive cycles of 
amenorrhea for a given cycle of time 
Inclusion Criteria: 
Subjects will be allocated to treatment if they meet all of the following inclusion criteria:  
1. Signed and dated written informed consent form and any required privacy authorization prior to the initiation 
of any trial procedure, after the nature of the trial has been explained according to local regulatory 
requirements; 
2. Females, ≥40 up to ≤65 years of age at randomization/treatment allocation;  
3. For hysterectomized subjects: documented hysterectomy must have occurred at least 6 weeks prior to the start 
of screening. Hysterectomy can be total or subtotal (i.e., cervix was not removed); 
4. For non-hysterectomized subjects: uterus with bi-layer endometrial thickness ≤ 4 mm on TVUS; 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 36 of 150 
 
5. For non-hysterectomized subjects: Endometrial biopsy taken during screening that reveals no abnormal 
results, i.e., presence of hyperplasia (simple or complex, with or without atypia), presence of carcinoma, and 
presence of disordered proliferative endometrium findings. The screening biopsy should have sufficient 
endometrial tissue for diagnosis. Biopsies without tissue or with insufficient tissue may be repeated once; 
6. Seeking treatment for relief of VMS associated with menopause; 
a. 
For the Efficacy Study part: at least 7 moderate to severe bothersome VMS per day or at least 50 
moderate to severe bothersome VMS per week in the last 7 consecutive days during the Screening 
period1; 
b. For the Safety Study part: at least 1 moderate to severe VMS per week; 
7. Body mass index ≥18.0 kg/m² up to ≤38.0 kg/m²; 
8. A mammogram that shows no sign of significant disease performed during screening or within 9 months prior 
to the start of screening2; 
9. Postmenopausal status defined as any of the following: 
a) For non-hysterectomized subjects: 
- 
at least 12 months of spontaneous amenorrhea with serum follicle stimulating hormone (FSH) 
>40 mIU/ml (value obtained after washout of estrogen/progestin containing drugs, see exclusion 
criteria 18 and 20); 
- 
or at least 6 months of spontaneous amenorrhea with serum FSH >40 mIU /mL and E2 <20 pg/mL 
(value obtained after washout of estrogen/progestin containing drugs, see exclusion criteria 18 and 
20); 
- 
or at least 6 weeks postsurgical bilateral oophorectomy3; 
b) For hysterectomized subjects: 
- 
serum FSH >40 mIU/mL and E2 <20 pg/mL (values obtained after washout of estrogen/progestin 
containing drug see exclusion criteria 18 and 20); 
- 
or at least 6 weeks postsurgical bilateral oophorectomy3; 
10. Good physical and mental health, in the judgement of the Investigator as based on medical history, physical 
and gynecological examination, and clinical assessments performed prior to Visit 1;  
11. Able to understand and comply with the protocol requirements, instructions, and protocol-stated restrictions;  
12. Able and willing to complete trial daily diaries (if applicable, see Section 10.1.7) and questionnaires. 
Exclusion Criteria: 
Subjects will not be allocated to treatment if they meet one of the following exclusion criteria:  
1. History of malignancy with the exception of basal cell or squamous cell carcinoma of the skin if diagnosed 
more than one year prior to the Screening visit4;  
2. Any clinically significant findings found by the Investigator at the breast examination and/or on 
mammography suspicious of breast malignancy that would require additional clinical testing to rule out breast 
cancer (however, simple cysts confirmed by ultrasound are allowed);  
3. PAP test with atypical squamous cells undetermined significance (ASC-US) or higher (low-grade squamous 
intraepithelial lesion [LSIL], atypical squamous cells- cannot exclude high-grade squamous intraepithelial 
lesion [HSIL] [ASC-H], HSIL dysplastic or malignant cells) in sub-totally hysterectomized and non-
                                                 
1  Bothersome is defined as a score of 4 to 6 on question 1 of the modified MENQOL questionnaire at screening. The timing 
and number of VMS and modified MENQOL score needed for allocation to the Efficacy Study part will not be disclosed to 
the study subjects to avoid bias in the recording during the Screening process. 
2 Subjects must have a Breast Imaging-Reporting And Data System (BI-RADS) score of 1 or 2 to enroll in the study. An 
incomplete mammogram result, i.e., BI-RADS 0, is not acceptable and requires further assessment. The site must obtain a 
copy of the official report for the subject's study file. A digitalized imaging should be obtained if mammography is done as 
part of this study. 
3  A report or a statement on letterhead from the subject's physician documenting both ovaries were removed is needed. 
4  The exception is only for basal cell carcinoma or squamous cell carcinoma of the skin if either of them were diagnosed more 
than one year prior to the screening of the subject. 
Footnote continued on next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 37 of 150 
 
hysterectomized subjects5. Note: ASC-US is allowed if a reflex human papilloma virus (HPV) testing is 
performed and is negative for high risk oncogene HPV subtypes 16 and 18; 
4. For non-hysterectomized subjects:  
a) History or presence of uterine cancer, endometrial hyperplasia, or disordered proliferative 
endometrium; 
b) Presence of endometrial polyp; 
c) Undiagnosed vaginal bleeding or undiagnosed abnormal uterine bleeding; 
d) Endometrial ablation; 
e) Any uterine/endometrial abnormality that in the judgment of the investigator contraindicates the use of 
estrogen and/or progestin therapy. This includes presence or history of adenomyosis or significant 
myoma; 
5. Systolic blood pressure (BP) higher than 130 mmHg, diastolic BP higher than 80 mmHg6 during screening;  
6. History of venous or arterial thromboembolic disease (e.g., superficial or deep vein thrombosis, pulmonary 
embolism, stroke, myocardial infarction, angina pectoris, etc.), or first-degree family history of VTE;  
7. History of known acquired of congenital coagulopathy or abnormal coagulation factors, including known 
thrombophilia’s; 
8. Laboratory values of fasting glucose above 125 mg/dL and/or glycated hemoglobin above 7% 7; 
9. Dyslipoproteinaemia (LDL >190 mg/dL and/or triglycerides >300 mg/dL)8; 
10. Subjects smoking >15 cigarettes per day; 
11. Presence or history of gallbladder disease, unless cholecystectomy has been performed; 
12. Systemic lupus erythematosus; 
13. Any malabsorption disorders including gastric by-pass surgery; 
14. History of acute liver disease in the preceding 12 months before the start of screening or presence or history 
of chronic or severe liver disease [alanine transaminase (ALT) or aspartate transaminase (AST) >2x upper 
limit of normal (ULN), bilirubin >1.5 ULN], or liver tumors; 
15. Chronic or current acute renal impairment (estimated glomerular filtration rate <60 ml/min); 
16. Porphyria; 
17. Diagnosis or treatment of major psychiatric disorder (e.g., schizophrenia, bipolar disorder, etc.) in the 
judgement of the Investigator 
18. Use of estrogen/progestin containing drug(s) up to: 
a) 1 week before screening start for vaginal non-systemic hormonal products (rings, creams, gels); 
b) 4 weeks before screening start for vaginal or transdermal estrogen or estrogen/progestin products; 
c) 8 weeks before screening start for oral estrogen and/or progestin products and/or selective estrogen 
receptor modulator therapy; 
d) 8 weeks before screening start for intrauterine progestin therapy; 
e) 3 months before screening start for progestin implants or estrogen alone injectable drug therapy; 
f) 
6 months before screening start for estrogen pellet therapy or progestin injectable drug therapy; 
19. Use of androgen/DHEA containing drugs: 
a) 8 weeks before screening start for oral, topical, vaginal or transdermal androgen; 
b) 6 months before screening start for implantable or injectable androgen therapy; 
20. Use of phytoestrogens or black cohosh for treatment of VMS up to 2 weeks before the start of screening; 
21. For the women participating in the Efficacy Study part: use of prescription or over-the-counter products used 
for the treatment of VMS, e.g., anti-depressants: paroxetine, escitalopram, methyldopa, opioid and clonidine 
                                                 
5 As indicated by written documentation of a prior test performed within 18 months prior screening or by a test performed at 
screening. 
6 BP measurements at screening may be repeated if values are outside the inclusion criteria after sitting for an additional 5 to 
10 minutes. The last reading will be used for eligibility. Subjects with mild to moderate hypertension who are controlled on 
a stable antihypertension regimen may be enrolled if they meet all inclusion/exclusion criteria. Subjects using methyldopa 
or clonidine containing antihypertensive medication will not be included in the Efficacy Study part. 
7  Laboratory values of fasting glucose outside the normal ranges and glycated hemoglobin  assessed during the last 6 months, 
and during washout and screening should be considered. 
8 Subjects using lipid-lowering therapy should be on a stable dose for at least 1 month before screening. 
Footnote continued on next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 38 of 150 
 
up to 4 weeks before the start of screening, and venlafaxine and desvenlafaxine up to 3 months before the start 
of screening9, and not willing to stop these during their participation in the trial;  
22. Not willing to stop any hormonal products as described in exclusion criteria 18, 19 and 20 during their 
participation in the trial; 
23. Inadequately treated hyperthyroidism with abnormal TSH and free T4 at screening. Subjects with low or high 
TSH are allowed if free T4 at screening is within normal range10; 
24. History or presence of allergy/intolerance to the investigational product or drugs of this class or any 
component of it, or history of drug or other allergy that, in the opinion of the Investigator contraindicates 
subject participation;  
25. For non-hysterectomized subjects: history or presence of allergy to peanuts11; 
26. History of alcohol or substance abuse (including marijuana, even if legally allowed) or dependence in the 
previous 12 months before the start of screening as determined by the Investigator, based on reported 
observations; 
27. Sponsor or CRO employees or employees under the direct supervision of the Investigator and/or involved 
directly in the trial; 
28. Subjects with known or suspected history of a clinically significant systemic disease, unstable medical 
disorders, life-threatening disease or current malignancies that would pose a risk to the subject in the opinion 
of the Investigator; 
29. Participation in another investigational drug clinical trial within 1 month (30 days) or having received an 
investigational drug within the last month (30 days) before the start of screening; 
30. Is judged by the Investigator to be unsuitable for any reason. 
Number of Subjects: 
Efficacy Study part: based on the results observed for the E4 15 mg dose in the Phase 2 study, a sample size of  
~200 subjects per arm provides a power of at least 80% to simultaneously achieve a superiority (upper bound of a 
95% 2-sided confidence interval [CI] lower than zero) over placebo for the four co-primary endpoints together. 
This sample size accounts for a dropout rate during the first 12 weeks of the trial of 35% and 20% for non-
hysterectomized and hysterectomized women, respectively. 
It is planned to enroll approximately 100 hysterectomized and 100 non-hysterectomized subjects per treatment 
arm. 
 
Safety Study part: the target number of ~400 subjects has been selected to fulfill the requirement stated in ICH E1 
guideline (CPMP/ICH/375/95) regarding number of subjects exposed to the drug for safety assessments. 
 
Enrollment will stop when the minimum required number of subjects in each study group and study part is enrolled. 
Statistical populations: 
Analyses will be based on the Enrolled Set, the Safety Analysis Set, the Intent-to-treat (ITT) Set, the Endometrial 
Safety Analysis Set, the Per-protocol (PP) Set and the modified Intent-to-treat (mITT) Set. The definitions of the 
analysis sets, with the exception of the mITT, follow those given in the ICH E9 guideline (CMP/ICH/363/96). The 
mITT Set is defined as the ITT set without subjects presenting at least one of the following features: 
• 
At least one post baseline E4 concentration level below the lower limit of quantification 
• 
At least one post screening E2 concentration level is superior to 40 pg/mL 
Statistical Methods: 
Baseline Values 
                                                 
9 If not used for the treatment of VMS, this medication is allowed if on a stable dose for 3 months prior to the 
Screening visit and no change in the dose regimen is foreseen during the conduct of the trial. 
10 Reflex T4 test to be performed at screening only if TSH at screening is outside normal range 
11 P4 provided as part of this study contains peanut oil and should never be used by subjects with allergy to 
peanuts 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 39 of 150 
 
Unless otherwise noted, baseline is defined as the last non-missing value recorded prior to the first dose of trial 
drug. Unscheduled visits will be used in the determination of baseline values, when applicable. This also applies 
to the primary efficacy variables derived from the VMS data collected in the subject diary, but on a weekly basis 
as described in the definition of the primary efficacy variables. 
Primary Efficacy Variables: 
The weekly frequency of moderate to severe VMS at baseline, week 4 and week 12, respectively, is defined as the 
total number (sum) of all recorded moderate to severe VMS experienced during the last 7 consecutive days during 
the Screening period (baseline), days 22 to 28 (week 4) and days 78 to 84 (week 12), respectively. 
The mean severity score of VMS is defined as the arithmetic mean of the daily severity score values of VMS 
(moderate or severe) observed from days -7 to -1 at baseline and the arithmetic mean of the daily severity score 
values of VMS (mild, moderate or severe) observed during the days 22 to 28 and days 78 to 84 for week 4 and 
week 12 respectively. 
At baseline, the daily severity score is computed as [((2 x number of moderate VMS) + (3 x number of severe 
VMS))/ (total number of moderate + severe VMS)] if at least one moderate to severe VMS was recorded during 
the day. In case of documented absence of moderate to severe VMS during the day, the daily severity will be set 
to zero.  
Post-baseline (days 22 to 28 [Week 4] and days 78 to 84 [Week 12]), the daily severity score is computed as [((1 
x number of mild VMS) + (2 x number of moderate VMS) + (3 x number of severe VMS))/ (total number of mild 
+ moderate + severe VMS)] if at least one mild to severe VMS was recorded during the day. In case of documented 
absence of VMS during the day, the daily severity will be set to zero. 
The primary efficacy variables will be expressed as the changes from baseline computed as the differences between 
the estimated values at week 4 or 12 and the baseline values. 
The daily severity score as defined above will also be recomputed for post baseline weeks as [((2 x number of 
moderate VMS) + (3 x number of severe VMS)) / (Total number of moderate + severe VMS)] if at least one 
moderate or severe VMS is recorded during the day. 
 
The analysis including the mild VMS will be used for submission to the FDA, the analysis without the mild VMS 
will be used for submission to the EMA. 
 
Analysis of Primary Efficacy Variables: 
All statistical tests will be supported by presenting estimates and 95% CIs for the respective treatment effects 
(difference to placebo). These estimates and CIs will be based on the respective statistical models used for the 
analysis. Since all analyses of the efficacy variables are not exploratory in nature, adjustment for multiplicity will 
be performed (Dunnet adjustment for the comparison of each active dose versus placebo). 
The primary efficacy comparison will test the following hypotheses for each treatment arm: 
• 
H0: The average change from baseline in the primary efficacy endpoint in the treatment arm is greater (less 
pronounced) or equal to the average change from baseline in the placebo arm 
• 
H1: The average change from baseline in the primary efficacy endpoint in the treatment arm is lower (more 
pronounced) to the average change from baseline in the placebo arm 
The two dose groups of E4 will be compared to placebo regarding the four co-primary efficacy endpoints. 
 
A comparison between treatment arms of the change from baseline at week 1 to week 12 in the weekly frequency 
of moderate to severe VMS and of the change from baseline in the weekly mean severity of moderate to severe 
VMS will be made by using Mixed-effect Models for Repeated Measures (MMRM). The MMRM models will 
include treatment, week, treatment by week interaction, and pooled trial centers as fixed effects and baseline as a 
covariate. If the pooled center effect included in the model is significant, a treatment by center interaction will be 
included in the model. Treatment effects will be assessed at Week 4 and 12 using two separate MMRM models 
(one on weekly frequency and another on weekly mean severity). 
 
The primary analysis will use the data from the ITT population. Since an MMRM model is used, missing data 
imputation is not necessary. Additional sensitivity analyses will be implemented in order to assess the robustness 
of the results. These may include sensitivity with respect to: 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 40 of 150 
 
 
• 
An imputation of missing data (last observation carried forward [LOCF] or other) 
• 
The modeling strategy [analysis of covariance (ANCOVA) instead of MMRM] 
• 
Confounders (for example ethnicity, hysterectomized status) 
• 
Populations studied (mITT, PP) 
• 
Any combination of the above 
 
In addition, all primary efficacy variables will be tabulated at baseline and at each post-baseline time point. 
Secondary Efficacy Variables: 
In addition to all the secondary efficacy endpoints previously described, all the variables described above for the 
assessment of the primary objectives are also considered as secondary efficacy variables when evaluated at other 
time points than Week 4 and Week 12.  
In addition, three VMS weekly weighted scores, taking into account both the frequency and the severity of VMS, 
will be computed based on the diary data as:  
• 
[(2 x number of moderate VMS) + (3 x number of severe VMS)] for baseline and post baseline visits if at 
least one moderate to severe VMS was recorded and zero otherwise. 
• 
[(1 x number of mild VMS) + (2 x number of moderate VMS) + (3 x number of severe VMS)] for baseline 
and post baseline visits if at least one mild to severe VMS was recorded and zero otherwise.  
• 
 [(2 x number of moderate VMS) + (3 x number of severe VMS)] if at least one moderate to severe VMS was 
recorded for baseline visits and [(1 x number of mild VMS) + (2 x number of moderate VMS) + (3 x number 
of severe VMS)] for post baseline visits if at least one mild to severe VMS was recorded and zero otherwise. 
 
Furthermore, the severity score will also be recomputed as [((1 x number mild VMS) + (2 x number of moderate 
VMS) + (3 x number of severe VMS)) / (total number of mild + moderate + severe VMS)] if at least one mild to 
severe VMS is recorded during the day for baseline and post baseline visits.   
 
In case of documented absence of VMS during the day, the daily severity score will be set to zero. These additional 
efficacy variables will be derived for every week between baseline and week 12 based on the respective 7-day 
periods. All VMS related secondary efficacy variables will be expressed as changes from baseline computed as 
the differences of the estimated values at week 1 to 12 and the baseline values. 
 
The percentage of subjects with a CID in the weekly frequency of moderate to severe VMS at weeks 4 and 12 will 
be assessed according to Gerlinger et. al. (Gerlinger C. et. al. Menopause. 2012 Jul; 19(7):799-803). 
 
Analysis of Secondary Efficacy Variables: 
All VMS related secondary efficacy variables will be analyzed using the same methodology as for the primary 
efficacy variables and will be based on the data from the ITT population. 
All other secondary continuous efficacy variables will be analyzed using either an ANCOVA or a MMRM model 
depending on the type of endpoint considered. These models will be evaluated using data from the ITT, PP and 
mITT populations without imputation of data. Appropriate covariates may be included in the analyses. 
The other categorical efficacy variables (scores, responder analysis) will be analyzed using appropriate statistical 
methods for the comparison of each treatment arm to the placebo arm. 
 
In addition, all secondary efficacy variables will be tabulated by baseline and by each post-baseline time point. 
 
Handling of Dropouts or Missing Data: 
For days with missing VMS diary information, the number and intensity of VMS will be imputed with the mean 
values of the remaining days of the respective 7-day period, but only if at least 4 days with VMS data in this 7 day 
period are available. Otherwise, no calculation of the primary and related secondary efficacy variables will be done 
for this period. 
No method of imputation is planned for the primary efficacy variable analysis since a MMRM model is used. As 
previously stated, sensitivity analyses of the primary efficacy variables may resort to imputation of missing data 
in order to assess the robustness of the MMRM results. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 41 of 150 
 
 
Safety parameters (safety set): 
All safety parameters will be summarized using data from the Safety Analysis Set if not indicated otherwise. No 
formal statistical test will be performed to determine statistical significance between treatment groups. Continuous 
variables will be summarized using descriptive statistics (N, mean, standard deviation, median, minimum, 
maximum), categorical variables will be presented in frequency tables using counts and percentages. The 
frequency of endometrial events will be computed on the evaluable biopsies of the endometrium. The incidence 
of spotting/bleeding will be analyzed by a 28-day period. The incidence rate of endometrial events will be 
computed on the evaluable endometrium. The 95% two-sided CI will be computed on the observed frequency of 
hyperplasia/carcinoma of the endometrium. 
 
Timing of analysis 
The final analysis of the primary efficacy objective and the secondary efficacy objectives 1 and 2 of the Efficacy 
Study part will be triggered when all subjects in the Efficacy Study part have completed Visit 4. The analyses of 
all other efficacy and safety objectives from the Efficacy and Safety Study parts (including a final analysis of the 
safety objectives of the Efficacy Study part) will be triggered when the overall end of the trial is reached (i.e. when 
all subjects in the Efficacy Study part have completed Visit 8).  The efficacy endpoints and the safety endpoints 
of the Safety Study will be analyzed separately from those of the Efficacy Study at the end of the Safety Study. 
Synopsis Final Version 8.0, June 21, 2022 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 
Page 42 of 150 
 
2.1. 
Schedule of Trial Procedures Efficacy Study Part 
 
Washout period a 
Screening period 
Baseline 
(random 
allocation to 
treatment) 
Treatment visits 
EoT /early 
discontinuation 
visit b 
Follow up 
Visit 
NH subjects 
Washout visit 
Screening visit 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Procedures and assessments 
Generally within 8 weeks 
before screening 
Within 4 weeks 
before baseline w 
Day 1 
Week 5 c 
Week 9 c 
Week 13 c 
Week 29 c 
Week 41 c 
Week 53 c 
Week 55/56 d 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
I/E criteria 
X e 
X e 
X 
 
 
 
 
 
 
 
Start washout a 
X 
 
 
 
 
 
 
 
 
 
Med. & gyn. history 
X 
 
 
 
 
 
 
 
 
 
Demographic data 
X 
 
 
 
 
 
 
 
 
 
Physical examination 
 
X 
 
 
 
 
 
 
X 
 
Gyn. examination 
 
X 
 
 
 
 
 
 
X 
 
Breast examination 
 
X 
 
 
 
 
X 
 
X 
 
Vital signs f 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
ECG 
 
X 
 
 
 
 
 
 
X 
 
PAP test g 
 
X 
 
 
 
 
 
 
 
 
Endometrial biopsy h 
 
X 
 
 
 
 
 
 
X i 
 
TVUS h, j 
 
X 
 
 
 
X 
X 
X 
X 
X 
Mammography 
 
X k 
 
 
 
 
 
 
X l 
 
Modified MENQOL questionnaire 
 
X 
 
 
 
 
 
 
 
 
MENQOL questionnaire 
 
 
X 
 
 
X 
 
 
X 
 
CGI questionnaire 
 
 
 
X 
 
X 
 
 
X 
 
Fasted blood sampling for: m  
 
 
 
 
 
 
 
 
 
 
 Hematol./chem.  
 
X v 
X 
 
 
 
 
 
X 
 
 Lipid/glucose parameters for 
 inclusion  
X u, v 
(X) v 
 
 
 
 
 
 
 
 
 Lipid metabolism  
 
 
X 
 
 
X 
 
 
X 
 
 Glucose metabolism  
 
 
X 
 
 
X 
 
 
X 
 
 Table continued on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 
Page 43 of 150 
 
2.1 Schedule of Trial Procedures Efficacy Study Part (continued) 
  
Washout period a 
Screening period 
Baseline 
(random 
allocation to 
treatment) 
Treatment visits 
EoT/early 
discontinuation 
visit b 
Follow up visit 
NH subjects 
Washout visit 
Screening visit 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
 
Generally within 8 
weeks before screening 
Within 4 weeks 
before baseline w 
Day 1 
Week 5 c 
Week 9 c 
Week 13 c 
Week 29 c 
Week 41 c 
Week 53 c 
Week 55/56 d 
Fasting blood sampling for: m 
 
 
 
 
 
 
 
 
 
 
  Hemostasis 
 
 
X 
 
 
X 
 
 
X 
 
  Bone turnover 
 
 
X 
 
 
X 
 
 
X 
 
  Optional additional analysis n 
 
 
X 
 
 
 
 
 
X 
 
Blood sampling for  
 
 
 
 
 
 
 
 
 
 
  FSH, E2 and TSH for inclusion v 
 
X 
 
 
 
 
 
 
 
 
  E2 
 
 
X 
X 
X 
X 
 
 
 
 
  E4 
 
 
 
X 
X 
X 
 
 
 
 
Urinary pregnancy test o 
 
X 
X 
 
 
 
 
 
 
 
Daily completion of diaries by subjects 
  for VMS p 
 
 
 
 
 
 
 
 
 
 
  for medication intake all subjects 
 
 
 
 
 
 
 
 
 
 
  for medication intake h 
 
 
 
 
 
 
 
 
 
 
  for bleeding events h 
 
 
 
 
 
 
 
 
 
 
Weekly reminder q 
 
 
 
 
 
 
 
 
 
 
Dispense paper diary r 
 
X 
X 
 
 
 
 
 
 
 
Return of paper diary  
 
 
X 
X 
X 
X 
X h 
X h 
X h 
X h 
Review of paper diary s 
 
 
X 
X 
X 
X 
X h 
X h 
X h 
X h 
Randomization 
 
 
X 
 
 
 
 
 
 
 
Dispense trial medication 
 
 
X 
X 
X 
X 
X 
X 
X t 
 
Daily intake of trial medication 
 
 
 
 
 
 
 
 
 
 
Return trial medication 
 
 
 
X 
X 
X 
X 
X 
X 
X h 
Drug accountability 
 
 
 
X 
X 
X 
X 
X 
X 
X h 
Prior/concomitant medication 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X h 
Adverse Events (AEs) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X h 
Abbreviations and footnotes are on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 
Page 44 of 150 
 
2.1 Schedule of Trial Procedures Efficacy Study Part (continued) 
CGI=Clinical Global Impression; discont=discontinuation; E2=estradiol; E4=estetrol; ECG=electrocardiogram; EoT=end of treatment; FSH= follicle stimulating hormone; gyn=gynecological; 
Hematol/chem=hematology/chemistry; I/E= in and exclusion criteria; med=medical; MENQOL=Menopause-specific Quality of Life; PAP= Papanicolaou; TSH= Thyroid stimulating hormone; 
TVUS=transvaginal ultrasound; VMS= vasomotor symptoms  
 
a) In case the subject uses medication that is not allowed during the duration of the trial, she is asked to stop this medication. The subject will return for the screening after a variable washout period 
that depends on the type of prior medication. Subjects not using medication that needs to be stopped and washed out can continue with the screening procedures.  
Washout periods are: 1 week for vaginal hormonal products (rings, creams, gels); 4 weeks for transdermal estrogen or estrogen/progestin products; 8 weeks for oral estrogen and/or progestin 
and/or selective estrogen receptor modulator therapy; 8 weeks for intrauterine progestin therapy; 3 months for progestin implants or estrogen alone injectable drug therapy; 6 months for estrogen 
pellet therapy or progestin injectable drug therapy; 8 weeks for oral, topical, vaginal, patch of androgen/DHEA; 6 months for implantable or injectable androgen therapy; 2 weeks for 
phytoestrogens or black cohosh, 3 months for venlafaxine and desvenlafaxine if used for the treatment of VMS and 4 weeks all other non-hormonal prescription or over-the-counter treatments 
used for VMS. 
b) Every effort needs to be made to perform the procedures of the end of trial visit for those subjects that discontinue their participation. 
c) Visits will be scheduled in one of the seven days of week 5, week 9, week 13, week 29, week 41 and week 53, thus allowing a visit window of 7 days. Unscheduled visits may be needed during 
the treatment period for additional ultrasounds, biopsies or any other unforeseen safety assessments. 
d) Visit 8 for non-hysterectomized subjects only at approximately 10 days after completion of the progestin treatment.  
e) At washout visit and during the Screening Period only available I/E criteria at that time point will be checked. 
f) 
Blood pressure, heart rate, body height (during screening only) and body weight (during screening and EoT only). 
g) To be performed in subjects with uterus and/or cervix, unless a written normal result is available within 18 months before screening start. The PAP test may be repeated once in case of inadequate 
or insufficient sample. 
h) Non-hysterectomized subjects only. 
i) 
If the subject was treated for at least 12 weeks 
j) 
TVUS at screening and EoT will be taken for all subjects who still have a uterus and/or ovaries. At all other time points a TVUS will be performed to monitor the endometrium thickness only for 
subjects who still have a uterus. 
k) Unless a written normal result is available within 9 months before screening start. 
l) 
Mammography at EoT visit may only be performed if the subject was treated for at least 12 weeks and the last mammography was taken at least 9 months earlier) 
m) If the subject is not fasting, a new appointment needs to be made as soon as possible. Laboratory tests may be repeated once during the screening period, at the discretion of the investigator. 
n) Additional blood samples including one DNA sample at Visit 1 only, will be collected for optional additional analysis of in the context of this program or any further research on E4. 
o) A pregnancy test may be performed at the discretion of the Investigator. 
p) VMS will be recorded daily in the paper diary during the Screening period and during the treatment period up to Visit 4. 
q) During VMS recording subjects will be reminded weekly (phone, e-mail, SMS etc.) to complete the diary. The first reminder will be sent 5 ± 2 days after start of VMS recording. 
r) 
Subjects are instructed during screening on how to fill in the diary and if needed can be reinstructed. 
s) At each visit the diary will be checked. Completed pages will be collected and will remain at the study site. 
t) 
Progestin treatment for non-hysterectomized subjects only. 
u) If lipid and/or glucose parameters could be a reason of concern in the judgement of the Investigator, fasted blood sampling for lipid and glucose parameters assessment may be done during the 
Washout Visit. Otherwise, this assessment will be done during the screening period. 
v) Laboratory tests may be repeated once during the screening period, at the discretion of the investigator. FSH measurement is not required for subjects with bilateral oophorectomy. 
w) In the event screening requirements cannot be met within 4 weeks, the screening period may be extended with prior Medical Monitor approval, but screening period may not exceed 8 weeks. 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 
Page 45 of 150 
 
2.2. 
Schedule of Trial Procedures Safety Study Part 
  
Washout period a 
Screening period 
Baseline 
(allocation to 
treatment) 
 
Treatment visits 
EoT/early 
discontinuation 
visit b 
Follow up visit 
NH subjects 
Washout visit 
Screening visit 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Procedures and assessments 
Generally within 8 
weeks before screening 
Within 4 weeks 
before baseline u 
Day 1 
Week 5 c 
Week 13 c 
Week 29 c 
Week 41 c 
Week 53 c 
Week 55/56 m 
Informed consent 
X 
 
 
 
 
 
 
 
 
I/E criteria  
X d 
X d 
X 
 
 
 
 
 
 
Start washout a 
X 
 
 
 
 
 
 
 
 
Med. & gyn. history 
X 
 
 
 
 
 
 
 
 
Demographic data 
X 
 
 
 
 
 
 
 
 
Physical examination 
 
X 
 
 
 
 
 
X 
 
Gyn. examination 
 
X 
 
 
 
 
 
X 
 
Vital signs e 
 
X 
X 
X 
X 
X 
X 
X 
 
ECG 
 
X 
 
 
 
 
 
X 
 
PAP test f  
 
X 
 
 
 
 
 
 
 
Endometrial biopsy n 
 
X 
 
 
 
 
 
X o 
 
TVUS l 
 
X 
 
 
X 
X 
X 
X 
X 
Breast examination 
 
X 
 
 
 
X 
 
X 
 
Mammography  
 
X g 
 
 
 
 
 
 X h 
 
Modified MENQOL questionnaire 
 
X 
 
 
 
 
 
 
 
MENQOL questionnaire 
 
 
X 
 
X 
 
 
X 
 
CGI questionnaire 
 
 
 
X 
X 
 
 
X 
 
Fasted blood sampling for: i 
 
 
 
 
 
 
 
 
 
  Hematol./chem.  
 
X t 
X 
 
 
 
 
X 
 
  Lipid/glucose parameters (for 
  inclusion)  
X s, t 
(X) t 
 
 
 
 
 
 
 
  Lipid metabolism 
 
 
X 
 
X 
 
 
X 
 
  Glucose metabolism 
 
 
X 
 
X 
 
 
X 
 
  Optional additional analysis j 
 
 
X 
 
 
 
 
X 
 
 Table continued on the next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 
Page 46 of 150 
 
2.2 Schedule of Trial Procedures Safety Study Part (continued) 
  
Washout period a 
Screening period 
Baseline 
(allocation to 
treatment) 
Treatment visits 
EoT/early 
discontinuation 
visit b 
Follow up visit 
NH subjects 
Washout visit 
Screening visit 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
 
Generally within 8 weeks 
before screening 
Within 4 weeks 
before baseline u 
Day 1 
Week 5 c 
Week 13 c 
Week 29 c 
Week 41 c 
Week 53 c 
Week 55/56 m 
 
 
 
 
 
 
 
 
 
 
Blood sampling for: 
 
 
 
 
 
 
 
 
 
  FSH, E2 and TSH for inclusion t 
 
X 
 
 
 
 
 
 
 
 Urinary pregnancy test k 
 
X 
X 
 
 
 
 
 
 
Daily completion of diaries by 
subjects n  
 
 
 
 
 
 
 
 
 
  for medication intake 
 
 
 
 
 
 
 
 
 
  for bleeding events 
 
 
 
 
 
 
 
 
 
Dispense paper diary n,p 
 
X 
X 
 
 
 
 
 
 
Return of paper diary n 
 
 
X 
X 
X 
X 
X  
X  
X  
Review of paper diary n,q 
 
 
X 
X 
X 
X 
X  
X  
X  
Allocation to Treatment 
 
 
X 
 
 
 
 
 
 
Dispense trial medication 
 
 
X 
X 
X 
X 
X 
X r 
 
Daily intake of trial medication 
 
 
 
 
 
 
 
 
 
Return trial medication 
 
 
 
X 
X 
X 
X 
X 
X 
Drug accountability 
 
 
 
X 
X 
X 
X 
X 
X 
Prior/concomitant medication 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events (AEs) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
CGI= Clinical Global Impression; discont=discontinuation; E2=estradiol; E4=estetrol; ECG=electrocardiogram; EoT=end of treatment; FSH= follicle stimulating hormone; gyn=gynecological; 
Hematol/chem=hematology/chemistry; I/E= in and exclusion criteria; med=medical; MENQOL=Menopause-specific Quality of Life; PAP= Papanicolaou; TSH= Thyroid stimulating hormone; 
TVUS=transvaginal ultrasound; VMS= vasomotor symptoms 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 
Page 47 of 150 
 
a) In case the subject uses medication that is not allowed during the duration of the trial, she is asked to stop this medication. The subject will return to complete the screening after a variable washout period 
that depends on the type of prior medication. Subjects not using medication that needs to be stopped and washed out can continue with the screening procedures.  
Washout periods are: 1 week for vaginal hormonal products (rings, creams, gels); 4 weeks for transdermal estrogen or estrogen/progestin products; 8 weeks for oral estrogen and/or progestin and/or 
selective estrogen receptor modulator therapy; 8 weeks for intrauterine progestin therapy; 3 months for progestin implants or estrogen alone injectable drug therapy; 6 months for estrogen pellet therapy or 
progestin injectable drug therapy; 8 weeks for oral, topical, vaginal, patch of androgen/DHEA; 6 months for implantable or injectable androgen therapy; 2 weeks for phytoestrogens or black cohosh, 3 
months for venlafaxine and desvenlafaxine if used for the treatment of VMS and 4 weeks all other non-hormonal prescription or over-the-counter treatments used for VMS. 
b) Every effort needs to be made to perform the procedures of the end of trial visit for those subjects that discontinue their participation. 
c) Visits will be scheduled in one of the seven days of week 5, week 13, week 29, week 41, and week 53 thus allowing a visit window of 7 days. Unscheduled visits may be needed during the treatment period 
for additional unforeseen safety assessments. 
d) At Washout visit and Screening visit only available I/E criteria at that time point will be checked. 
e) Blood pressure, heart rate, body height (during screening only) and body weight (during screening and EoT only). 
f) 
To be performed in subjects with uterus and/or cervix, unless a written normal result is available within 18 months before screening start. The PAP test may be repeated once in case of inadequate or 
insufficient sample. 
g) Unless a written normal result, including digital imaging, is available within 9 months before screening start. 
h) Mammography at EoT visit may only be performed if the subject was treated for at least 12 weeks and the last mammography was taken at least 9 months earlier 
i) 
If the subject is not fasting, a new appointment needs to be made as soon as possible. Laboratory tests may be repeated once during the screening period, at the discretion of the investigator. 
j) 
Additional blood samples, including one DNA sample at Visit 1 only, will be collected for optional additional analysis of in the context of this program or any further research on E4. 
k) A pregnancy test may be performed at the discretion of the Investigator. 
l) 
TVUS at screening and EoT will be taken for all subjects who still have a uterus and/or ovaries. At all other time points a TVUS will be performed to monitor the endometrium thickness only for subjects 
who still have a uterus. 
m) Visit 7 for non-hysterectomized subjects only at approximately 10 days after completion of the progestin treatment. 
n) Non-hysterectomized subjects only. 
o) If the subject was treated for at least 12 weeks. 
p) Subjects are instructed during screening on how to fill in the diary and if needed can be reinstructed. 
q) At each visit the diary will be checked. Completed pages will be collected and will remain at the study site. 
r) 
Progestin treatment for non-hysterectomized subjects only. 
s) If lipid and/or glucose parameters could be a reason of concern in the judgement of the Investigator, fasted blood sampling for lipid and glucose parameters assessment may be done during the Washout 
Visit. Otherwise, this assessment will be done during the Screening Period 
t) 
Laboratory tests may be repeated once during the screening period, at the discretion of the investigator. FSH measurement is not required for subjects with bilateral oophorectomy. 
u) In the event screening requirements cannot be met within 4 weeks, the screening period may be extended with prior Medical Monitor approval, but screening period may not exceed 8 weeks. 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 48 of 150 
 
3. 
TABLE OF CONTENTS 
1. 
ADMINISTRATIVE INFORMATION .................................................................................. 3 
1.1. 
Serious Adverse Event Reporting ................................................................................ 3 
1.2. 
Contact Information ..................................................................................................... 4 
1.3. 
Sponsor Authorization ................................................................................................. 6 
1.4. 
Investigator Approval and Signature Page .................................................................. 7 
1.5. 
CRO Approval and Signature Page ............................................................................. 8 
1.6. 
List of Changes ............................................................................................................ 9 
2. 
STUDY SYNOPSIS ................................................................................................................. 29 
3. 
TABLE OF CONTENTS ........................................................................................................ 48 
4. 
REFERENCE INFORMATION ........................................................................................... 52 
4.1. 
Trial-Related Responsibilities .................................................................................... 52 
4.2. 
Principal Investigator ................................................................................................. 52 
4.3. 
List of Abbreviations ................................................................................................. 53 
5. 
INTRODUCTION ................................................................................................................... 56 
5.1. 
Background ................................................................................................................ 56 
5.2. 
Prior Clinical Experience in Postmenopausal Women .............................................. 57 
5.3. 
Rationale for the Present Study ................................................................................. 59 
5.4. 
Risk-benefit Assessment ............................................................................................ 60 
6. 
OBJECTIVES AND ENDPOINTS ........................................................................................ 61 
6.1. 
Objectives and Endpoints of the Efficacy Study Part (Arms 1-3) ............................. 61 
6.1.1. Primary Objective and Endpoints ................................................................. 61 
6.1.2. Secondary Objectives and Endpoints ........................................................... 61 
6.1.3. Exploratory Objectives and Endpoints ......................................................... 63 
6.2. 
Objectives and Endpoints of the Safety Study Part (Arm 4) ..................................... 63 
6.2.1. Primary Objective and Endpoints ................................................................. 63 
6.2.2. Secondary Objectives and Endpoints ........................................................... 63 
7. 
CLINICAL TRIAL PLAN ..................................................................................................... 65 
7.1. 
Clinical Trial Design and Justification ...................................................................... 65 
7.1.1. Trial Design .................................................................................................. 65 
7.1.2. Trial Duration ............................................................................................... 68 
7.1.3. Justification of Design, Dose and Endpoints ................................................ 68 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 49 of 150 
 
7.1.4. Justification of Study Population ................................................................. 70 
7.2. 
Study and Treatment Assignment .............................................................................. 70 
7.3. 
Randomization ........................................................................................................... 71 
7.4. 
Blinding Procedures ................................................................................................... 71 
7.4.1. Blinding after analysis of data up to Visit 4 in the Efficacy Study 
part ................................................................................................................ 72 
8. 
SUBJECT SELECTION AND DISCONTINUATION/WITHDRAWAL OF 
SUBJECTS............................................................................................................................... 73 
8.1. 
Source and Number ................................................................................................... 73 
8.1.1. Justification of Sample Size ......................................................................... 73 
8.2. 
Inclusion and Exclusion Criteria ................................................................................ 73 
8.2.1. Criteria for Inclusion .................................................................................... 73 
8.2.2. Criteria for Exclusion ................................................................................... 75 
8.3. 
Subject Discontinuation and Replacement Procedures .............................................. 78 
8.3.1. Criteria for Early Termination of the Study ................................................. 79 
8.3.2. Stopping Criteria for Non-hysterectomized Women Only ........................... 79 
9. 
TRIAL MEDICATION .......................................................................................................... 80 
9.1. 
Study Treatments ....................................................................................................... 80 
9.2. 
Timing of Administration .......................................................................................... 80 
9.3. 
Pharmaceutical Preparations ...................................................................................... 80 
9.3.1. Active Treatment – Investigational Product ................................................. 80 
9.3.2. Placebo (Visually Matching the Active Treatments) ................................... 81 
9.3.3. Progesterone Treatment ................................................................................ 81 
9.4. 
Packaging and Labelling ............................................................................................ 81 
9.5. 
Drug Accountability .................................................................................................. 83 
9.6. 
Prior and Concomitant Medication ............................................................................ 84 
9.6.1. Prior Medication/Therapy ............................................................................ 84 
9.6.2. Concomitant Medication/Therapy ................................................................ 84 
9.6.3. COVID-19 vaccination................................................................................. 85 
9.7. 
Treatment Compliance ............................................................................................... 86 
10. 
TRIAL ASSESSMENTS ........................................................................................................ 87 
10.1. 
Experimental Flow ..................................................................................................... 87 
10.1.1. Informed Consent Procedure ........................................................................ 88 
10.1.2. Washout Visit ............................................................................................... 88 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 50 of 150 
 
10.1.3. Screening Visit (Within 4 weeks before Randomization/Treatment 
allocation) ..................................................................................................... 88 
10.1.4. Subject Re-screening .................................................................................... 90 
10.1.5. Visits Efficacy Study part............................................................................. 90 
10.1.6. Visits Safety Study part ................................................................................ 95 
10.1.7. Subject Diary ................................................................................................ 99 
10.2. 
Demographics and Subject Characteristics .............................................................. 100 
10.3. 
Efficacy Assessments .............................................................................................. 100 
10.3.1. Frequency and Severity of Moderate and Severe VMS ............................. 100 
10.3.2. Health Related Quality of Life and Treatment Satisfaction 
Questionnaires ............................................................................................ 100 
10.3.3. Laboratory Assessments for Efficacy ......................................................... 101 
10.3.4. Blood Sampling for Exploratory Research................................................. 101 
10.3.5. Endocrine Assessment for Inclusion And Monitoring of Treatment 
Compliance ................................................................................................. 102 
10.4. 
Safety Assessments .................................................................................................. 102 
10.4.1. Treatment Emergent Adverse Events ......................................................... 103 
10.4.2. Physical and Gynecological and Breast Examination ................................ 107 
10.4.3. Vital Signs .................................................................................................. 107 
10.4.4. Electrocardiogram ...................................................................................... 108 
10.4.5. Routine Clinical Laboratory Tests ............................................................. 108 
10.4.6. Mammography ........................................................................................... 109 
10.4.7. Transvaginal Ultrasound in Non-hysterectomized Subjects ...................... 109 
10.4.8. Endometrial Biopsy in Non-hysterectomized Subjects .............................. 110 
10.4.9. Vaginal Bleeding Events ............................................................................ 111 
10.4.10. Endometrial Safety Assessment and Stopping Rules ................................. 111 
10.5. 
Data Safety Monitoring Board ................................................................................. 112 
10.6. 
Adverse Events of Special Interest .......................................................................... 112 
11. 
DATA COLLECTION, RECORD KEEPING AND QUALITY ASSURANCE ............ 114 
11.1. 
Data Collection ........................................................................................................ 114 
11.2. 
Protocol Deviations ................................................................................................. 114 
11.3. 
Confidentiality of Subject Records .......................................................................... 115 
11.4. 
Retention of Records ............................................................................................... 115 
11.5. 
Routine Monitoring ................................................................................................. 116 
11.6. 
Site Audits ............................................................................................................... 116 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 51 of 150 
 
11.7. 
Database Management and Quality Control ............................................................ 116 
11.8. 
Archiving of Data .................................................................................................... 117 
12. 
STATISTICAL METHODS................................................................................................. 118 
12.1. 
Statistical populations .............................................................................................. 118 
12.2. 
Baseline Values ....................................................................................................... 118 
12.3. 
Handling of Dropouts or Missing Data .................................................................... 118 
12.4. 
Primary Efficacy Variables ...................................................................................... 119 
12.5. 
Analysis of Primary Efficacy Variables .................................................................. 119 
13. 
ETHICS .................................................................................................................................. 123 
13.1. 
Independent Ethics Committee/Institutional Review Board .................................... 123 
13.2. 
Ethical Conduct of the Study ................................................................................... 123 
13.3. 
Compensation, Insurance, and Indemnity ................................................................ 123 
13.4. 
Subject Information and Consent ............................................................................ 123 
13.5. 
Protocol Amendments.............................................................................................. 124 
14. 
CONFIDENTIALITY AND PUBLICATION POLICY ................................................... 125 
14.1. 
Confidentiality of Information ................................................................................. 125 
14.2. 
Reporting ................................................................................................................. 125 
14.3. 
Publication and Disclosure Policy ........................................................................... 125 
15. 
REFERENCES ...................................................................................................................... 126 
16. 
APPENDICES ....................................................................................................................... 129 
16.1. 
Menopause Specific Quality of Life (MENQOL) Questionnaire ............................ 130 
16.2. 
Modified Clinical Global Impression ...................................................................... 132 
16.3. 
Histologic Descriptions Recommended for use when Reading Endometrial  Biopsy 
Slides........................................................................................................................ 133 
16.4. 
Histologic Descriptions Recommended for use when Reading Endometrial Biopsy 
Slides (Continued) ................................................................................................... 134 
16.5. 
Country Specific Requirements ............................................................................... 135 
16.5.1. Requirements for Canada ........................................................................... 135 
16.6. 
Protocol Amendment – Summary of changes ......................................................... 136 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 52 of 150 
 
4. 
REFERENCE INFORMATION  
4.1. 
Trial-Related Responsibilities 
The Sponsor is responsible for this trial but may transfer any or all trial related activities to a 
CRO. However, the ultimate responsibility for the quality and integrity of the trial data always 
resides with the Sponsor. Any trial-related duties and functions that are transferred to and 
assumed by a CRO will be specified in writing in a separate document. 
4.2. 
Principal Investigator 
The Sponsor may select a Signatory Principal Investigator(s) (PI) from the investigators who 
participate in the trial, according to national law. Selection criteria for this investigator will 
include significant knowledge of the trial protocol, the investigational product, their expertise 
in the therapeutic area and the conduct of clinical research as well as trial participation. The 
Signatory PI(s) will be required to review and sign the clinical study report and by doing so 
agrees that it accurately describes the results of the trial. 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 53 of 150 
 
4.3. 
List of Abbreviations 
AE 
Adverse event 
AESI 
Adverse events of special interest   
ALT 
Alanine transaminase 
ANCOVA 
Analysis of covariance 
ASC-US  
Atypical squamous cells of undetermined significance 
AST 
Aspartate transaminase 
BI-RADS  
Breast imaging-reporting and data system 
BMI 
Body mass index 
BP 
Blood pressure 
CEE 
Conjugated equine estrogens 
CGI 
Clinical global impression  
CI 
Confidence interval 
CID 
Clinically important difference 
CRO 
Contract research organization 
CSR  
Clinical study report 
CTX-1 
C-terminal telopeptide type 1 
DHEA 
Dehydroepiandrosterone 
DSMB 
Data Safety Monitoring Board 
E2 
Estradiol 
E4 
Estetrol 
EC 
Ethics committee 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form  
EMA 
European medicines agency 
EoT 
End of treatment 
ETP 
Endogenous thrombin potential 
FDA 
Food and drug administration 
FSH 
Follicle stimulating hormone 
GCP 
Good clinical practice 
GMP 
Good manufacturing practice 
GSM 
Genitourinary syndrome of menopause  

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 54 of 150 
 
HDL 
High-density lipoprotein  
HF 
Hot flash(es) 
HOMA-IR  
Homeostasis model assessment-estimated insulin resistance 
HPV 
Human papilloma virus 
HRQoL 
Health related quality of life 
IB 
Investigator’s brochure 
IC 
Informed consent 
ICF 
Informed consent form 
ICH 
International council for harmonization of technical requirements for 
pharmaceuticals for human use 
IEC 
Independent ethics committee 
IRB 
Institutional review board 
ITT 
Intention to treat 
IWRS 
Interactive Web Response System 
kg 
Kilogram 
LH 
Luteinizing hormone 
LOCF 
Last observation carried forward 
LDL 
Low-density lipoprotein  
MED 
Minimum effective dose 
MENQOL 
Menopause-specific quality of life 
mg 
Milligram 
MHT 
Menopausal hormone therapy 
mITT 
Modified intention to treat 
mL 
Milliliter 
MMRM 
Mixed-effect model for repeated measures 
MPA 
Medroxyprogesterone acetate 
MRS 
Menopause rating scale 
NAMS 
North American Menopause Society  
ng 
Nanogram 
PI 
Principle Investigator 
PINP 
procollagen I N-propeptide 
PP 
Per protocol 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 55 of 150 
 
SAE 
Serious adverse event 
SHBG 
Sex hormone binding globulin 
SOP 
Standard operation procedure 
SRL 
Société à responsabilité limitée 
SUSAR 
Suspected unexpected serious adverse reaction 
TEAE 
Treatment-emergent adverse event 
TESAE 
Treatment-emergent serious adverse event 
TS 
Treatment satisfaction 
TSH 
Thyroid stimulating hormone 
TVUS 
Transvaginal ultrasound 
ULN 
Upper limit of normal 
VMS 
Vasomotor symptoms 
VTE 
Venous thromboembolism 
VVA 
Vulvovaginal atrophy 
WHI 
World health initiative 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 56 of 150 
 
5. 
INTRODUCTION 
Detailed background information on estetrol (E4), including previous clinical experience can 
be found in the Investigator’s Brochure (IB).1 A summary is provided in the sections below. 
5.1. 
Background 
Menopausal hormone therapy (MHT) is the broad term used to describe unopposed estrogen 
use (for women who have undergone hysterectomy) or combined estrogen-progestin therapy 
(for women with an intact uterus). The goal of MHT is to relieve menopausal symptoms, most 
importantly vasomotor symptoms (VMS) such as hot flashes (HF). Other symptoms associated 
with perimenopause and menopause that respond to estrogen therapy include vulvovaginal 
atrophy (VVA), sleep disturbances (when related to VMS), etc. 
Vasomotor symptoms occur most often in the late menopausal transition and the early post 
menopause and they are the most common menopausal complaints. Estimates suggest that about 
75% of women who are more than 50 years of age will suffer from VMS.2 Most experience 
VMS for about two to five years, although at least 10% suffer for more than 10 years.3 VMS 
can contribute towards physical and psycho-social impairment, with a consequent reduction in 
health related quality of life (HRQoL), and are one of the main reasons why women may seek 
medical care for the menopause.4 Although there are alternative therapies for VMS, none are as 
effective as estrogen. 
The epithelial linings of the vagina and urethra are very sensitive to estrogen, and estrogen 
deficiency leads to thinning both epithelia. This results in VVA and urinary complaints, causing 
symptoms of vaginal dryness, itching, dyspareunia, dysuria, urinary frequency and an increased 
risk of recurrent urinary infections. In early 2014, the International Society for the Study of 
Women’s Sexual Health (ISSWSH) and the North American Menopause Society (NAMS) 
endorsed the new terminology “genitourinary syndrome of menopause (GSM)” to replace the 
VVA terminology. The rationale for using this new terminology was that VVA term was 
considered to be too restrictive whereas GSM is a more comprehensive term that includes 
symptomatic VVA as well as lower urinary tract symptoms related to low estrogen levels.5 Note 
that because the GSM terminology has not been adopted in guidances for industry issued by 
Food and Drug Administration (FDA) and European Medicines Agency (EMA), the VVA 
terminology will be used throughout this document. 
With current MHT, women with an intact uterus need a progestin in addition to estrogen to 
prevent endometrial changes, which can occur after as little as 6 months of estrogen therapy.6 
Women who have undergone hysterectomy do not require addition of a progestin when 
prescribed estrogen therapy. 
Along with the endometrial safety issue, MHT has been seriously questioned or even abandoned 
by many women and physicians following publication of data from the Women’s Health 
Initiative (WHI) study in 2002. The results of the WHI studies with either conjugated equine 
estrogen (CEE) plus medroxyprogesterone acetate (MPA) or CEE alone 7-9 showed increased 
rates of venous thromboembolism (VTE), breast cancer (CEE+MPA only), gallbladder disease, 
coronary heart disease (CEE+MPA only), in the first year of use, or stroke. 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 57 of 150 
 
Therefore, FDA and EMA guidances encourage pharmaceutical companies to develop the 
lowest doses and exposures for MHT, even though specific relationships between dose, 
exposure, and risk of adverse events (AEs) may not be known. 
Safety concerns that were attributed in the past to MHT are being re-considered following recent 
publications derived from long-term follow-up of the WHI trials. Estrogen alone therapy for 
postmenopausal women who underwent hysterectomy (the target population for the present 
study) showed that: 
1. Treatment with CEE alone for a median of 7.2 years was not associated with an increased 
risk of all-cause, cardiovascular, or cancer mortality during a cumulative follow-up of 18 
years.10 
2. No differences were observed in the time to the first occurrence of coronary heart disease, 
breast cancer, stroke, pulmonary embolism, hip fracture, colorectal cancer, endometrial 
cancer, or death between oral CEE versus oral or transdermal estradiol-containing regimen.11 
3. The risk of cardiovascular disease and cancer was not increased among postmenopausal 
women using vaginal estrogens compared with non-users.12 
E4 is a natural estrogen produced by the fetal liver. In humans, it is only produced during 
pregnancy and reaches the maternal circulation through the placenta. The fetal liver, which 
contains 15 alpha-hydroxylase and 16 alpha-hydroxylase, is the exclusive site of E4 
production.13 E4 is not produced by other species tested so far (mice, rat, and rabbit) except in 
Cynomolgus monkey (unpublished data). Human maternal plasma levels increase during 
pregnancy to high concentrations towards the end of gestation (≥1 ng/mL).13,14 Fetal plasma 
levels have been reported to be nearly 20 times higher than maternal plasma levels at 
parturition.15 
Since early 2000, E4 has been extensively studied in several women’s health care therapeutic 
indications such as contraception, VMS and VVA related to menopause and breast cancer. Both 
preclinical and clinical data obtained to date indicate that E4 may be promising for use in 
women.16 In particular, from Phase 1/2 clinical results, it appears that E4 alone or in 
combination with a progestin minimally impacts the synthesis of liver proteins, and only slightly 
increases triglyceride levels, potentially minimizing the risk of VTE and cardiovascular 
disease.17 
Data from a recently completed Phase 2 study 18 have also shown that E4 15 mg, administered 
to postmenopausal women for 12 consecutive weeks showed proof of concept on the two main 
symptoms associated with the menopause, namely VMS and VVA. In this context, the present 
study in postmenopausal women is planned. 
5.2. 
Prior Clinical Experience in Postmenopausal Women 
When administered orally, E4 is absorbed rapidly (median Tmax of about 0.25 to 0.50 hour) and 
Cmax are dose-dependent. E4 does not bind to sex hormone binding globulin (SHBG) and is 
principally metabolized to glucuronide and sulphate conjugates and eliminated by the kidney. 
Steady-state is reached between 6 to 8 days. The mean T1/2 of E4 following single ascending 
dose administration (from 0.1 mg to 100 mg) in postmenopausal women is about 28 
hours.14,16,19,20 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 58 of 150 
 
Previous clinical studies with E4 alone have shown that it is well tolerated and safe up to 100 
mg given as a single dose and up to 40 mg given once daily for 28 days.20,21 Moreover, data 
from postmenopausal women showed that E4 inhibited secretion of luteinizing hormone (LH) 
and follicle stimulating hormone (FSH) in a dose-dependent manner denoting the central 
inhibiting potency of the compound. Endometrial thickness and proliferation were directly 
related to the dose of E4 administered. The daily oral intake of ascending E4 doses (from 2 mg 
to 20 mg) for 28 days also allowed demonstrating a beneficial effect of E4 for the treatment of 
VMS and for the vaginal epithelium in a possible dose-dependent fashion.21  
Based on this first proof of concept of E4 as MHT, a randomized, placebo-controlled, double-
blinded, dose-finding study was conducted, with the primary objective to establish the oral 
Minimum Effective Dose (MED) of E4 for the treatment of VMS in postmenopausal women 
(hysterectomized and non-hysterectomized).18 E4 doses tested included 2.5 mg, 5 mg, 10 mg, 
and 15 mg for the reduction of VMS at week 4 and week 12 with the greatest improvement 
observed in the E4 15 mg group. Between 47 and 55 subjects were randomized in each treatment 
arm. Statistically significant effects versus placebo were observed for the percentage of change 
in the weekly frequency of VMS and the absolute decrease of the severity of VMS. Absolute 
changes versus placebo in VMS frequency and percentage of change on the severity of VMS 
were borderline significant. In all the other E4 groups, the changes in the VMS endpoints were 
numerically lower than in the E4 15 mg group and did not reach statistical significance versus 
placebo. The greatest response with E4 15 mg was supported with the evaluation of menopause-
related complaints (assessed by the menopause rating scale [MRS]) which resulted in a 
reduction of MRS reported severity of HFs and sweating when compared to placebo. 
All 4 doses of E4 had a positive effect on the vaginal epithelium, as indicated by a substantial 
increase in the percentage of superficial cells at week 12. The incidence and severity of VVA 
symptoms improved from baseline to week 12 in all groups with the greatest reduction in the 
E4 15 mg group compared to placebo. 
Other estrogen responsive endpoints demonstrated potential benefits of E4. All E4 doses (in 
particular 10 mg and E4 15 mg) reduced the level of two bone markers, C-terminal telopeptide 
type 1 (CTX-1) and osteocalcin. Changes in hemostatic, lipid and glucose metabolism markers 
were within normal ranges. E4 did not affect coagulation markers, except for a small but 
statistically significant decrease in free protein S concentration which was observed in the E4 
15 mg group at week 12 while the absolute concentration remained within the normal range. A 
statistically significant increase in SHBG plasma concentrations was observed in the 10 mg and 
E4 15 mg groups, showing only small absolute changes at 12 weeks. Small and potential 
beneficial changes in increased high-density lipoprotein (HDL) cholesterol and decreases 
HbA1c values were observed in the 10 mg E4 and E4 15 mg groups. In non-hysterectomized 
women, vaginal hemorrhage and endometrial thickening, was seen in more subjects from the 
E4 10 and 15 mg groups than from the other groups. Endometrium thickness increased with 
increasing dose of E4 and returned to pretreatment levels at the end of study, i.e., after 2-weeks 
of treatment with Didrogesterone 10 mg once daily for 14 days. None of the biopsies performed 
revealed endometrial hyperplasia or carcinoma. In conclusion vaginal hemorrhage and 
endometrial thickening, in particular, were seen in more subjects from the E4 10 and 15 mg 
groups than from the other groups. Endometrial thickness increased with increasing dose during 
treatment with E4 and returned towards pretreatment levels after 2-week intake of progestin. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 59 of 150 
 
None of the biopsies performed revealed hyperplasia or other abnormalities that would have 
required treatment. 
5.3. 
Rationale for the Present Study 
In the last 2 decades, the fetal estrogen E4 has attracted renewed attention because of its distinct 
pharmacological profile.13,16,22-24 Currently it is in development for several indications. In 
previous trials, E4 has been shown to be effective in reducing VMS in postmenopausal women 
(Refer to Section 5.2). There were no safety concerns arising from these trials. Overall, E4 
appears to be a new treatment that could bring added value to the already existing hormonal 
treatment options. The main purpose of this phase 3 trial is to further evaluate the clinical 
efficacy and safety of E4 in postmenopausal women. 
The trial has two parts: an Efficacy Study part (Arms 1 to 3) and a Safety Study part (Arm 4). 
The Efficacy Study part is conducted in hysterectomized and non-hysterectomized 
postmenopausal subjects and evaluates the primary efficacy (frequency and severity of VMS), 
secondary efficacy (effect on hemostasis, lipid and glucose metabolism, bone turnover, HRQoL 
and treatment satisfaction [TS]), and safety of E4. All subjects will be treated with E4 15 mg, 
E4 20 mg or placebo for a total treatment duration of 12 months. After completion of the 
E4/placebo treatment, all non-hysterectomized subjects will receive treatment with 200 mg 
progesterone (P4) for 14 days for endometrial protection. 
The Safety Study part of this trial evaluates the general safety and secondary efficacy (effect on 
lipid and glucose metabolism, HRQoL and TS) in hysterectomized and non-hysterectomized 
postmenopausal subjects. All subjects will be treated with E4 20 mg for a total treatment 
duration of 12 months. 
The design of this study allows for the assessment of the efficacy of E4 in terms of the relief of 
moderate to severe VMS and for the assessment of the long-term safety of E4 in both 
hysterectomized and non-hysterectomized postmenopausal women. 
The design of the study is consistent with the recommendations given by the FDA and the EMA 
in their respective guidance: 
• 
"Guidance for Industry. Estrogen and Estrogen/Progestin Drug Products to Treat Vasomotor 
Symptoms and Vulvar and Vaginal Atrophy Symptoms - Recommendations for Clinical 
Evaluation" (draft guidance, January 2003), 
• 
"Guideline on clinical investigation of medicinal products for hormone replacement therapy 
of oestrogen deficiency symptoms in postmenopausal women" (EMEA/CHMP/021/97 Rev. 
1, 2005). 
• 
The design is also consistent with the meeting minutes of the End-of-Phase 2 meeting held 
with the FDA (
). 
The results of this study will be part of the planned dossier for submission to regulatory agencies 
to gain approval for E4 for the relief of moderate to severe VMS in postmenopausal women. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 60 of 150 
 
5.4. 
Risk-benefit Assessment 
Benefits of MHT are that it is a highly effective therapy for relief of VMS as well as for vaginal 
dryness and atrophic changes linked to menopause. Additionally, it has dose-related benefit for 
bone health and for prevention of osteoporosis. MHT also has been shown to enhance libido 
and QoL. Lastly, MHT can be administered via multiple routes (oral, transdermal, injection, 
intra-uterine, vaginal) and can thus be provided in a preferred form to meet the needs of most 
women who require this treatment.25,26  
Risks of MHT include estrogen-dependent endometrial neoplasia in women with an intact 
uterus. Both hysterectomized as well as non-hysterectomized postmenopausal women will be 
included in this study. An endometrial biopsy is part of the screening procedures in non-
hysterectomized women and any findings indicating polyps, disordered proliferative 
endometrium, endometrial hyperplasia or cancer will result in exclusion from enrollment. To 
monitor the effect of E4 on the endometrium, transvaginal ultrasounds (TVUS) will be 
performed at multiple time points during E4 treatment to measure the endometrial thickness 
and subjects will record daily the vaginal bleeding pattern. If, after the first 4 weeks of treatment, 
a non-hysterectomized subject presents with a bi-layer endometrial thickness > 10 mm as 
assessed by TVUS or presents with persistent and/or recurrent bleeding, confirmed by the 
investigator, an endometrial biopsy will be performed. If the biopsy shows disordered 
proliferative endometrium, hyperplasia or worse, the study drug will be discontinued, and the 
subject will be withdrawn from the study. The subject will be treated with P4 200 mg once daily 
for 14 days. If the endometrial event has not resolved, treatment with a progestin will be started 
according to local practice/guidelines. Subjects will be followed up until resolution.  
Non-hysterectomized subjects in the trial will receive treatment with 200 mg P4 for 2 weeks 
after completion of the E4/placebo treatment. 
With MHT use, there is a known risk for thromboembolic disease; and this risk increases with 
age and other risk factors (e.g., cardiovascular disease, obesity, fracture, immobilization, 
surgery, cancer, congenital and acquired thrombophilic disorders, smoking). Induction or 
exacerbation of hypertension has also been found in women using MHT, although MHT is 
typically associated with a reduction in blood pressure. Women with cardiovascular disease 
should not initiate or continue MHT for secondary prevention of coronary artery disease. 
Currently, data from controlled trials, as well as observational studies, indicate that when 
initiated before 60 years of age, MHT, used even for long periods of time in postmenopausal 
women, induces lower rates of mortality and coronary heart disease than in comparable women 
who do not use MHT, 10,27,28 although there may be an increased risk of stroke with MHT use.29 
Other minor risks include vaginal bleeding secondary to endometrial stimulation (in women 
with a uterus) which may be mitigated with progestin therapy; nausea or vomiting also has been 
noted in a small percentage of women. 
Contraindications include undiagnosed abnormal genital bleeding; known, suspected, or history 
of estrogen-dependent neoplasm; presence or history of deep vein thrombosis or pulmonary 
embolism; presence or history of arterial thromboembolic disease (stroke or myocardial 
infarction); liver dysfunction or disease; known or suspected pregnancy; and women who may 
have hypersensitivity to MHT preparations. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 61 of 150 
 
6. 
OBJECTIVES AND ENDPOINTS 
This trial has two parts; an Efficacy Study part (Arms 1-3) and Safety Study part (Arm 4). The 
design of the trial is described in more detail in Section 7.1. The objectives and endpoints for 
the two parts of the trial are described in separate sections below. 
6.1. 
Objectives and Endpoints of the Efficacy Study Part (Arms 1-3) 
6.1.1. Primary Objective and Endpoints  
1. To measure the effect of treatment with E4 15 mg or E4 20 mg E4 compared to placebo on 
the frequency and severity of moderate to severe VMS in postmenopausal women at 4 and 
12 weeks  
Co-primary efficacy endpoints for primary objective #1 
1.1. Mean change in weekly frequency of moderate to severe VMS from baseline to week 
4 
1.2. Mean change in weekly frequency of moderate to severe VMS from baseline to week 
12 
1.3. Mean change in severity of moderate to severe VMS from baseline to week 4 
1.4. Mean change in severity of moderate to severe VMS from baseline to week 12 
6.1.2. Secondary Objectives and Endpoints 
1. To measure the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on the 
frequency and severity of mild, moderate and severe VMS in postmenopausal women 
weekly up to 12 weeks 
Efficacy endpoints for secondary objective #1  
1.1. Change from baseline to week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 in the weekly 
frequency of and severity of moderate to severe VMS 
1.2. Change from baseline to weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and12 in the weekly 
frequency and severity of mild, moderate, and severe VMS  
1.3. Percentage of subjects with 50% and 75% reduction from baseline in the weekly 
frequency of moderate to severe VMS at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 
1.4. Percentage of subjects with 50% and 75% reduction from baseline in the weekly 
frequency of mild, moderate and severe VMS at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 
and 12 
 
 
2. To measure the clinical meaningfulness of E4 15 mg or E4 20 mg compared to placebo on 
the reduction of VMS at weeks 4 and 12 
Efficacy endpoint for secondary objective #2  
2.1. Percentage of subjects with a clinically important difference (CID) compared to 
baseline in the weekly frequency of moderate to severe VMS after weeks 4 and 12 
using the Clinical Global Impression (CGI) questionnaire 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 62 of 150 
 
3. To evaluate the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on 
hemostasis, lipid and glucose metabolism, and bone turnover 
Efficacy endpoint for secondary objective #3 
3.1. Change from baseline to week 12 and week 52 in prothrombin fragment 1 + 2, 
endogenous thrombin potential (ETP)-based activated Protein C sensitivity ratio 
(APCsr ETP), activated partial thromboplastin time (aPTT) based activated Protein C 
resistance (APCr), anti-thrombin III, Protein-C, free Protein-S, Factor VIII, 
angiotensinogen, and SHBG 
3.2. Change from baseline to week 12 and week 52 in triglycerides, HDL-cholesterol, low-
density lipoprotein (LDL)-cholesterol, total cholesterol, lipoprotein(a), total 
cholesterol/HDL-cholesterol ratio, fasting glycemia, insulin, glycated hemoglobin 
and homeostasis model assessment-estimated insulin resistance (HOMA-IR) 
3.3. Change from baseline to week 12 and week 52 in procollagen I N-propeptide (PINP), 
CTX-1, calcium, and vitamin D 
4. To evaluate the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on 
HRQoL and TS 
Efficacy endpoints for secondary objective #4 
4.1. Change from baseline to week 12 and week 52 in HRQoL using the Menopause-
specific Quality of Life (MENQOL) questionnaire 
4.2. Total score in TS after 4, 12, and 52 weeks of treatment using the CGI questionnaire 
 
5. To evaluate the general safety of treatment with E4 15 mg or E4 20 mg compared to placebo 
Safety endpoints for secondary objective #5 
5.1. Frequency of treatment emergent adverse events (TEAEs) (including treatment- 
emergent serious adverse events [SAEs])  
5.2. Frequency of changes in results in physical and gynecological examination, vital 
signs, electrocardiogram (ECG), mammography, and breast examination at each 
measured time point 
5.3. Frequency of changes in routine clinical laboratory tests results (hematology and 
chemistry) at each measured time point 
6. To evaluate the effect of treatment with E4 15 mg or E4 20 mg on the endometrium in non-
hysterectomized subjects compared to placebo 
Safety endpoints for secondary objective #6 
6.1. Change from baseline to each measured time point in endometrial thickness measured 
by ultrasound 
6.2. Frequency of subjects in the different endometrial categories according to the 
Blaustein's pathology (see Appendix 16.3) 
 
7. To evaluate the effect of treatment with E4 15 mg or E4 20 mg on vaginal bleeding in non-
hysterectomized subjects compared to placebo 
Safety endpoints for secondary objective #7 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 63 of 150 
 
7.1. Frequency of women with vaginal bleeding and/or spotting during each 28-day cycle 
of treatment with E4 based on data in the patient diary 
7.2. Number of days with bleeding and/or spotting during each 28-day cycle of treatment 
based on data in the patient diary 
7.3. Frequency of women with amenorrhea (absence of any bleeding or spotting) during 
each 28-day cycle of treatment with E4 based on data in the patient diary 
7.4. Cumulative rates of amenorrhea defined as the percentage of women who reported 
consecutive cycles of amenorrhea for a given cycle of time 
6.1.3. Exploratory Objectives and Endpoints 
1. To evaluate the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on 
breast density  
Safety endpoint for exploratory objective #1 
1.1. Change in breast density from digitized mammography readings from baseline to EoT 
in subjects who had a paired digitalized mammography 
 
2. To evaluate the influence of E4 on the changes in the hemostasis parameters compared to 
placebo in women with inherited thrombophilia and women without this genetic disorder 
Efficacy endpoint for exploratory objective #2 
2.1. Change in hemostasis parameters in women with and without inherited thrombophilia 
(Factor V Leiden mutation and prothrombin G20210A mutations). 
6.2. 
Objectives and Endpoints of the Safety Study Part (Arm 4) 
6.2.1. Primary Objective and Endpoints 
1. To evaluate the general safety of treatment with E4 20 mg 
 
Safety endpoints for primary objective #1 
1.1. Frequency of TEAE (including SAEs)  
1.2. Frequency of changes in results in physical and gynecological examination, vital 
signs, ECG, mammography, and breast examination at each measured time point 
1.3. Frequency of changes in routine clinical laboratory tests results (hematology and 
chemistry) at each measured time point  
6.2.2. Secondary Objectives and Endpoints  
1. To evaluate the effect of treatment with E4 20 mg on lipid and glucose metabolism 
 
Efficacy endpoints for secondary objective #1 
1.1. Change from baseline to week 12 and week 52 in triglycerides, HDL-cholesterol, 
LDL-cholesterol, total cholesterol, lipoprotein(a), total cholesterol/HDL-cholesterol 
ratio, fasting glycemia, insulin, glycated hemoglobin, and HOMA-IR 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 64 of 150 
 
2. To evaluate the effect of treatment with E4 20 mg on HRQoL and TS 
 
Efficacy endpoints for secondary objective #2 
2.1. Change from baseline to week 12 and week 52 in HRQoL using the MENQOL 
questionnaire 
2.2. Total score in TS after 4 weeks, 12 weeks, and 52 weeks of treatment using the CGI 
questionnaire 
 
3. To evaluate the effect of treatment with E4 20 mg on the endometrium in non-
hysterectomized subjects 
Safety endpoints for secondary objective #3 
3.1 
Change from baseline to each measured time point in endometrial thickness measured 
by ultrasound 
3.2 
Frequency of subjects in the different endometrial categories according to the 
Blaustein's pathology (see Appendix 16.3) 
 
4. To evaluate the effect of treatment with E4 20 mg on vaginal bleeding in non-
hysterectomized subjects 
Safety endpoints for secondary objective #4 
4.1 
Frequency of women with vaginal bleeding and/or spotting during each 28-day cycle 
of treatment with E4 based on data in the patient diary 
4.2 
Number of days with bleeding and/or spotting during each 28-day cycle of treatment 
based on data in the patient diary 
4.3 
Frequency of women with amenorrhea (absence of any bleeding or spotting) during 
each 28-day cycle of treatment with E4 based on data in the patient diary 
4.4 
Cumulative rates of amenorrhea defined as the percentage of women who reported 
consecutive cycles of amenorrhea for a given cycle of time 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 65 of 150 
 
7. 
CLINICAL TRIAL PLAN 
7.1. 
Clinical Trial Design and Justification 
7.1.1. Trial Design 
The trial has two parts; an Efficacy Study part and a Safety Study part. 
The Efficacy Study part has a randomized, double-blinded placebo-controlled design and 
evaluates the primary efficacy (frequency and severity of VMS), secondary efficacy (effect on 
hemostasis, lipid and glucose metabolism, bone turnover, HRQoL and TS), and safety of (E4) 
in both hysterectomized and non-hysterectomized women, as well as the effect of E4 on 
endometrium in non-hysterectomized women. 
A total of ~600 subjects will be randomly allocated (1:1:1) to one of the 3 Arms and will receive 
E4 15 mg (Arm 1), E4 20 mg (Arm 2), or Placebo (Arm 3). During the first 12 weeks of the 
Efficacy Study part the effect of E4 on VMS will be evaluated. Thereafter treatment will 
proceed for a total duration of 12 months (52 weeks), to continue evaluation of secondary 
efficacy, safety and effect on the endometrium. For endometrial protection, all non-
hysterectomized subjects will receive treatment with 200 mg progesterone (P4) once daily for 
14 consecutive days, after completion of the E4/placebo treatment. 
The Safety Study part of this trial is conducted in ~200 hysterectomized and ~200 non-
hysterectomized women and has an open label design. All subjects will receive treatment with 
E4 20 mg for 12 months (Arm 4). The Safety Study part evaluates the general safety, secondary 
efficacy (lipid and glucose metabolism, HRQoL and TS) of E4 during treatment for 52 weeks. 
For endometrial protection, all non-hysterectomized subjects will receive treatment with 200 
mg progesterone (P4) once daily for 14 consecutive days, after completion of the E4 treatment. 
The trial schedule is provided in Figure 1. 
Participants in the Efficacy Study part need to have at least 7 moderate to severe bothersome 
menopausal related vasomotor symptoms (VMS) per day or at least 50 moderate to severe 
bothersome VMS per week in the last 7 consecutive days during the Screening period. 
Bothersome is defined as a score of 4 to 6 on question 1 of the modified MENQOL 
questionnaire at screening. The number of VMS and modified MENQOL score needed for 
allocation to the Efficacy Study part will not be disclosed to the study subjects to avoid bias in 
the recording during the Screening process.  
Participants in the Safety Study part should seek treatment for the relief of VMS associated with 
menopause, with at least 1 moderate to severe menopausal related VMS per week. Subjects who 
do not fulfill the VMS requirements for participation in the Efficacy Study part, may participate 
in the Safety Study part providing that they meet all in- and exclusion criteria for participation 
the Safety study part. 
Hysterectomized and non-hysterectomized healthy women will be included in the Efficacy 
Study part and in the Safety Study part (approximately 50% of the subjects are anticipated to 
have a history of hysterectomy). 
The target enrollment number of subjects in each of the three arms of the Efficacy Study part is 
~200. The target number of subjects to be included in the Safety Study part is ~400.   

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 66 of 150 
 
 
Figure 1: Trial Schedule 
 * For non-hysterectomized (NH) subjects endometrial thickness will be measured after 3, 6, 9 and 12 months (Visits 4, 5, 6 and 7 of the Efficacy Study 
part and Visits 3, 4, 5 and 6 of the Safety Study part respectively) of treatment by TVUS and subjects will record daily the vaginal bleeding events. If 
after the first 4 weeks of treatment a subject presents with a bi-layer endometrial thickness >10 mm as assessed by TVUS or presents with persistent 
and/or recurrent bleeding, confirmed by the investigator, an endometrial biopsy will be performed. If the biopsy shows disordered proliferative 
endometrium, hyperplasia or worse, the study drug will be discontinued, and the subject will be withdrawn from the study. The subject will be treated 
with P4 200 mg once daily for 14 days. If the endometrial event has not resolved, treatment with a progestin will be started according to local 
practice/guidelines. All NH subjects will receive treatment with 200 mg P4 for two weeks after completion of E4 treatment.  
Study flow 
For the Efficacy Study part at least 9 visits are planned: a Washout visit followed by a Screening 
period, which will start with a Screening visit, a baseline visit (Visit 1), and 6 On-treatment 
visits (Visits 2, 3, 4, 5, 6 and 7). The last treatment visit (Visit 7) is also the end of treatment 
(EoT) Visit, which will also occur in case of early discontinuation. For non-hysterectomized 
women an additional endometrial safety follow-up visit (Visit 8) is planned after completion of 
the 14 days of P4 treatment, which will also occur in case of early discontinuation. 
For the Safety Study part at least 8 visits are planned: a Washout visit followed by a Screening 
period, which will start with a Screening visit, a baseline visit (Visit 1), and 5 On-treatment 
visits (Visits 2, 3, 4, 5 and 6). The last treatment visit (Visit 6) is also the end of treatment (EoT) 
Visit, which will also occur in case of early discontinuation. For non-hysterectomized women 
an additional endometrial safety follow-up visit (Visit 7) is planned approximately 10 days after 
completion of the 14 days of P4 treatment, which will also occur in case of early 
discontinuation. 
The screening period requires at least one visit, to occur as per protocol, at the beginning of the 
period, but some assessments (e.g., mammography, biopsy, transvaginal ultrasound [TVUS], 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 67 of 150 
 
electrocardiogram [ECG] and Papanicolaou [PAP] test) may require additional visits in case 
these assessments cannot be done during the same visit. 
Additional unscheduled visits may also be required for ad hoc safety assessments (e.g., TVUS 
for non-hysterectomized subjects) during the treatment period. 
Washout screening procedures for the Efficacy Study part and Safety Study part 
At the Washout visit, after signing the informed consent, the inclusion and exclusion criteria 
will be checked as well as the prior medication list. If a subject uses prohibited co-medication, 
then this subject will be asked to stop this medication. After a variable washout period 
(depending on type of prior medication, Table 3, Section 9.6.1) the subject will return to start 
the screening. For subjects who do not use medication that needs to be stopped and washed out, 
the Washout visit and screening Visit may occur at the same time. Screening procedures include 
a PAP test and mammography for all subjects, a TVUS for subjects with ovaries and/or uterus, 
and an endometrial biopsy for non-hysterectomized subjects. The length of the Screening period 
is generally up to 4 weeks, but can be extended (with prior Medical Monitor approval) without 
exceeding 8 weeks. During this period, and in addition to screening procedures, the subject will 
record VMS in a paper diary. VMS data recorded during the last 7 consecutive days of the 
Screening period will be used to determine eligibility of subjects. If based on VMS symptoms 
of her current medical history a subject is only eligible for the Safety Study part, this subject 
may be enrolled directly into the Safety Study part without the VMS count in the Screening 
period, providing that all the screening assessments have been completed. Subjects who do not 
fulfill the VMS requirements for participation in the Efficacy Study part, may participate in the 
Safety Study part, providing that they meet all other in- and exclusion criteria. 
Treatment period Efficacy Study part  
During the first 12 weeks of treatment, primary efficacy assessments (the number and severity 
of VMS) will be recorded on a daily basis in a paper diary. Subjects will also record vaginal 
bleeding events (non-hysterectomized subjects only) and daily study drug intake in the diary. 
After completion of the 12 weeks of treatment (Visit 4), only non-hysterectomized subjects will 
keep the diary to record daily vaginal bleeding events and daily study drug intake. At baseline 
(Visit 1) secondary efficacy assessments (hemostasis, lipid and glucose metabolism, and bone 
turnover) will be performed, the MENQOL questionnaire will be completed, and for non-
hysterectomized subjects endometrial thickness will be assessed. Subjects will visit the trial 
center after 4 and 8 weeks of treatment (Visit 2 and Visit 3) for general safety assessments, for 
review of the diaries, and for completion of the CGI questionnaire (Visit 2 only). After 12 weeks 
of treatment, subjects will visit the trial center for the final primary efficacy assessment (Visit 
4). During this visit also secondary efficacy assessments and general safety assessments will be 
performed and the MENQOL and CGI questionnaires will be completed. For non-
hysterectomized subjects endometrial thickness will be assessed. Subjects will visit the trial 
center again after 6 months of treatment (Visit 5) for general safety assessments and endometrial 
thickness assessment (non-hysterectomized subjects only) and after 9 months of treatment 
(Visit 6) for general safety assessments and endometrial thickness assessment (non-
hysterectomized subjects only). After 12 months of treatment, subjects will visit the trial center 
for the EoT visit (Visit 7) during which secondary efficacy assessments and general safety 
assessments will be performed, the HRQoL and TS questionnaires will be completed, and a 
mammography will be made. For non-hysterectomized subjects endometrial thickness will be 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 68 of 150 
 
assessed and, if they have completed at least 12 weeks of E4/placebo treatment, an endometrial 
biopsy will be performed. At Visits 1, 2, 3 and 4 blood samples will be taken to monitor estradiol 
(E2) levels and at Visits 2, 3 and 4 also to monitor E4 levels. 
After the EoT procedures, all non-hysterectomized subjects will receive treatment with 200 mg 
P4 for 14 days. Approximately 10 days after completion of the progestin treatment, endometrial 
thickness will be measured (Visit 8). 
Treatment period Safety Study part 
At baseline (Visit 1) secondary efficacy assessments (lipid and glucose metabolism) will be 
performed and the MENQOL questionnaire will be completed. Subjects will visit the trial center 
after 4 weeks (Visit 2), 12 weeks (Visit 3), 6 months (Visit 4) and 9 months (Visit 5) of treatment 
for safety assessments, and for completion of the CGI questionnaire (Visit 2 only). At Visit 3 
secondary efficacy assessments will also be performed and the MENQOL and CGI 
questionnaires will be completed. For non-hysterectomized subjects endometrial thickness will 
be assessed on visits 3, 4 and 5. During treatment non-hysterectomized subjects will record 
vaginal bleeding events in a paper diary. After 12 months of treatment subjects will visit the 
trial center for the EoT visit (Visit 6) during which secondary efficacy assessments and safety 
assessments will be performed, questionnaires will be completed and a mammography will be 
made. For non-hysterectomized subjects endometrial thickness will be assessed and, if they 
have completed at least 12 weeks of E4, an endometrial biopsy will be performed.  
After the EoT procedures, all non-hysterectomized subjects will receive treatment with 200 mg 
P4 for 14 days. Approximately 10 days after completion of the progestin treatment, endometrial 
thickness will be measured (Visit 7). 
Details on assessments in each visit in the Efficacy Study part and in the Safety Study part are 
provided in Section 10.1. 
7.1.2. Trial Duration 
Individual subject participation in this trial may be up to 15 months. This includes a washout 
period of generally up to 8 weeks (usual washout period for most subjects), a screening period 
of up to 4 weeks, and a treatment period with E4 of up to 53 weeks. For non-hysterectomized 
subjects the trial duration will be 3 weeks longer due to an additional endometrium safety 
follow-up period of approximately 3 weeks. 
For subjects requiring a longer washout (up to 6 months) participation period may be extended 
to allow this washout length. For subjects who do not require a washout, the participation period 
will be reduced by approximately 8 weeks. 
The end of trial is defined as the date when the last patient has completed the last study visit. 
7.1.3. Justification of Design, Dose and Endpoints 
7.1.3.1. Justification of the Design and Primary Efficacy Endpoints 
The design of the trial is consistent with the recommendation given by the FDA and the EMA 
in the respective guidance: 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 69 of 150 
 
• 
"Guidance for Industry. Estrogen and Estrogen/Progestin Drug Products to Treat Vasomotor 
Symptoms and Vulvar and Vaginal Atrophy Symptoms - Recommendations for Clinical 
Evaluation" (draft guidance, January 2003), 
• 
"Guideline on clinical investigation of medicinal products for hormone replacement therapy 
of estrogen deficiency symptoms in postmenopausal women (EMEA/CHMP/021/97 Rev. 
1, 2005). 
The design is also consistent with the meeting minutes of the End-of-Phase 2 meeting held with 
the FDA (
). 
The agencies recommend performing a randomized, placebo-controlled, double-blinded trial to 
evaluate the efficacy and safety of estrogen treatment. For the treatment of moderate to severe 
VMS, the recommended endpoints are the mean change and frequency of moderate to severe 
VMS from baseline to Week 4 and to Week 12. 
The length of the trial is based on the requirements of regulatory bodies in the United States 
(FDA) and the European Union (EMA) and is sufficient to assess the efficacy, general safety 
of treatment with E4 in individual subjects with VMS. A period of 12 weeks is required to 
determine the effect of E4 treatment on the frequency and severity of moderate to severe VMS, 
which is the primary objective in the Efficacy Study part. In addition, the total treatment 
duration of 12 months in the Efficacy and Safety Study parts is required to evaluate the long-
term safety of E4 in menopausal subjects. 
7.1.3.2. Justification of Other Endpoints 
This study will also evaluate other symptoms associated with menopause such as quality of life 
and treatment satisfaction through questionnaires. The parameters will be used to further 
delineate the optimal dose for the treatment of moderate to severe VMS. 
The use of estrogens in the treatment of menopausal complaints has been associated with an 
increased risk for thromboembolic disease, through changes in blood coagulation and 
fibrinolysis. This risk is particularly high for women who are carriers of inherited 
thrombophilia. During the study blood samples will be taken to measure the effect of E4 
treatment on hemostasis parameters. Post-hoc pharmacogenomics studies in a subset of E4 users 
will investigate the relationship between genetic variants and changes in hemostasis parameters 
levels elicited by E4 with the objective to understand any correlation and predictive factors. 
Estrogen treatment may also affect lipid and glucose metabolism and bone turnover, which will 
be monitored during the study. 
In this study a mammogram will be performed at screening (if not performed within 9 months 
prior to screening start) to exclude subjects with any signs of significant symptoms in breast 
tissue. At the end of the study a mammogram will be made for subjects who were treated for at 
least 12 weeks and for whom the last mammography was taken at least 9 months earlier, to 
monitor any effects on breast tissue. Change from baseline to EoT in breast density will also be 
evaluated in subjects who have a paired digitalized mammography. Breast density being 
considered as a risk factor for breast cancer. 
In women with an intact uterus the treatment with unopposed estrogen increases the risk of 
estrogen-dependent endometrial hyperplastic changes that may ultimately result in 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 70 of 150 
 
premalignant lesions and then carcinomas. The effect of E4 on the endometrium in non-
hysterectomized subjects will be monitored by TVUS, bleeding events and endometrial biopsy 
(see Section 7.1.4). 
As some of these efficacy and safety effects may only appear after prolonged exposure, the 
treatment duration in this trial is extended beyond the 3 months to evaluate the effect of E4 
treatment on these endpoints. In order to collect long-term safety data the treatment will 
continue up to 12 months. 
7.1.3.3. Justification of E4 Dose 
The objective of the Phase 2 trial with E4 was to assess the minimum effective dose of E4 for 
the relief of VMS. The results of this trial showed that E4 is safe and well tolerated when 
administered daily up to 15 mg in postmenopausal women over a period of 3 months. The effect 
on VMS was the most pronounced with the highest dose tested, i.e., E4 15 mg. Although this 
dose was associated with a significant reduction in VMS severity compared to placebo, the 
reduction in VMS frequency failed to reach statistical significance. The advantage over placebo 
was nevertheless estimated as clinically relevant given the marked placebo effect observed in 
this trial (65% of VMS reduction compared to baseline for the placebo group versus 84% for 
the E4 15 mg group). The current trial will continue to study the E4 15 mg doses as well as 
evaluate a higher dose (E4 20 mg) in order to further delineate the optimal E4 dose for the 
treatment of moderate to severe VMS. 
7.1.3.4. Justification of Placebo 
The Efficacy Study part of the current trial is designed to evaluate and compare the effect on 
VMS reduction of E4 15 mg and E4 20 mg versus placebo. The use of a placebo to compare 
the effect of E4 on VMS is consistent with the recommendation given by the FDA and the EMA 
in the guidance referenced in Section 7.1.3.1. The use of placebo is considered necessary to 
demonstrate efficacy on VMS relief of an estrogenic product. 
7.1.4. Justification of Study Population  
The trial will be performed in postmenopausal women, which is the intended target population 
of E4 for the treatment of moderate to severe VMS. 
Both hysterectomized and non-hysterectomized postmenopausal women will be included in the 
trial to determine the efficacy and safety of E4 in the total postmenopausal population. 
To further evaluate the safety of 20 mg E4, hysterectomized and non-hysterectomized subjects 
will be included in the Safety Study part. 
7.2. 
Study and Treatment Assignment  
Assignment into any of the study parts (i.e. Efficacy and Safety) and treatment subgroups (15 
mg, 20 mg and placebo) will be done by the Interactive Web Response System (IWRS) system 
(i.e cannot be done by site staff).  This assignment will take into account the modified 
MENQOL score, frequency and severity of hot flashes, hysterectomized vs. non-
hysterectomized status, and availability of places in each study part/subgroup. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 71 of 150 
 
Efficacy Study part 
Subjects eligible for participation in the Efficacy Study part will be randomly allocated to one 
of the three treatment arms [E4 15 mg (Arm 1), E4 20 mg (Arm 2) or placebo (Arm 3)] in a 
1:1:1 ratio using a computer-generated randomization schedule. 
Safety Study part  
Subjects eligible for participation in the Safety Study part will all be allocated to treatment with 
E4 20 mg (Arm 4). 
7.3. 
Randomization 
A randomization schedule will be generated within the Biometrics Department of the designated 
CRO by a statistician not involved in the trial. The randomization schedule will be generated 
by means of the PLAN procedure of SAS®. The computer program uses the method of 
randomly permuted blocks. The block size will not be revealed before unblinding. 
An Interactive Web Response System (IWRS) will be used for the randomization procedure. A 
subject ready for randomization will be entered in the IWRS. The respective next subject 
eligible for randomization will receive the lowest available randomization number at the trial 
center. A randomization confirmation including treatment assignment (i.e., kit number), date 
and time will be sent to the center, to the designated project managers or designee of the CRO 
and to the Sponsor’s project manager. 
The randomization schedule and the complete generation procedure will be filed at a secure 
place by the designated CRO until the trial database is unblinded. A copy of the list will be sent 
to the Pharmaceutical Supply for the purpose of assigning the kits to the subjects. 
7.4. 
Blinding Procedures 
The Efficacy Study part will have a double-blind placebo-controlled design. Subjects will 
receive one of the two dosages of E4 or placebo in a blinded manner. Neither trial center 
personnel, nor the subjects, nor the CRO or Sponsor will know who is receiving E4 15 mg, E4 
20 mg or placebo. Active trial treatments will be supplied as tablets indistinguishable from 
placebo. The Safety Study part will have an open label design; all these subjects will receive 
E4 20 mg. 
The Investigator, trial center personnel, CRO or Sponsor must maintain the blind through 
database lock and must not break the blind via IWRS or code break envelopes without a valid 
reason (e.g., in case of emergency). Emergency unblinding is to be done only when knowing 
the study drug is absolutely necessary for the management of an individual subject and where 
stopping the blinded medication is not sufficient in the judgment of the Investigator. The 
Medical Monitor is to be notified by the Investigator when unblinding of a subject’s treatment 
is being considered or has already occurred. The Investigator has the final decision whether the 
unblinding of a subject’s treatment is in the best interest of the subject. 
The Safety Study part will have an open label design and blinding is not applicable for this part 
of the trial. 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 72 of 150 
 
7.4.1. Blinding after analysis of data up to Visit 4 in the Efficacy Study part  
The final analysis of the primary and secondary efficacy objectives 1 and 2 of the Efficacy 
Study part are to be triggered when all subjects in the Efficacy Study part have completed Visit 
4 and will be conducted by an independent statistician. A firewall group, composed of Estetra 
SRL representatives independent of the E4 project will be appointed. This group will have 
access to the unblinded data of final and interim analyses of the Efficacy Study part. Further 
details regarding the composition and role of the firewall group will be provided in a charter. 
The clinical team, the subjects and the site and sponsor personnel involved in the clinical 
evaluation of the subjects will remain blinded of the treatment assignment in the Efficacy Study 
part until the end of the study. 
 
The analyses to be done after completion of Visit 4 will be performed by an independent 
statistician. Only the overall outcome of the results will be provided to the clinical team. The 
clinical team, the subjects and the site and sponsor personnel involved in the clinical evaluation 
of the subjects will remain blinded of the treatment assignment until the end of the study. 
Blinding for the data at subject and treatment group levels will be kept through a firewall 
process that will be put in place. No individual listings with the subject numbers’ information 
or tables per treatment group will be disseminated at the time of final or interim triggered 
analysis of the Efficacy Study part. 
 
No changes in the study conduct will be undertaken regardless of the primary efficacy analysis 
results. The study will continue until its completion in order to contribute to the safety profile 
of the study medication. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 73 of 150 
 
8. 
SUBJECT SELECTION AND DISCONTINUATION/WITHDRAWAL 
OF SUBJECTS 
8.1. 
Source and Number 
This will be a multicenter trial performed in multiple study sites in the United States of America 
and Canada. Subjects will be recruited by the investigator from his/her patients or will be 
recruited by recruitment campaigns. Subjects must seek treatment for the relief of menopausal 
symptoms, or subject may already be treated for menopausal symptoms and willing to 
discontinue their current treatment. 
All investigators must keep a record of subjects who were considered for enrollment and were 
consented, but were never enrolled e.g., subject screening log to address concerns over selection 
bias. 
This trial will enroll a total of approximately 1,000 subjects; ~600 subjects ~200 per treatment 
arm) in the Efficacy Study part and ~400 subjects in the Safety Study part of the trial. 
Enrollment will stop when the minimum required number of subjects in each study group and 
study part is enrolled.   
The number of completers after three months of treatment in the Efficacy Study part is 
anticipated to be around 435, anticipating a drop-out rate of 35% and 20% for non-
hysterectomized and hysterectomized women, respectively. The number of completers after 12 
months of treatment is anticipated to be ~300, anticipating a drop-out rate of 50% after one year 
of treatment. 
The number of completers in Safety Study part after 12 months of treatment is expected to be 
~200, anticipating a drop-out rate of 50%. 
8.1.1. Justification of Sample Size 
Efficacy Study part: based on results observed for the E4 15 mg dose in the Phase 2 study, a 
sample size of ~200 subjects per arm provides a power of at least 80% to simultaneously achieve 
a superiority (upper bound of a 95% 2-sided confidence interval [CI] lower than zero) over 
placebo for the four co-primary endpoints together. This sample size accounts for a dropout rate 
during the first 12 weeks of the study of 35% and 20% for non-hysterectomized and 
hysterectomized women, respectively. 
It is planned to enroll approximately 100 hysterectomized and 100 non-hysterectomized 
subjects per treatment arm. 
Safety Study part: the target number of ~400 subjects has been selected to fulfill the requirement 
stated in International council for harmonization of technical requirements for pharmaceuticals 
for human use (ICH) E1 guideline (CPMP/ICH/375/95) regarding number of subjects exposed 
to the drug for safety assessments. 
8.2. 
Inclusion and Exclusion Criteria 
8.2.1. Criteria for Inclusion 
Subjects will be allocated to treatment if they meet all of the following inclusion criteria:  

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 74 of 150 
 
1. Signed and dated written informed consent form and any required privacy authorization 
prior to the initiation of any trial procedure, after the nature of the trial has been explained 
according to local regulatory requirements; 
2. Females, ≥40 up to ≤65 years of age at randomization/treatment allocation;  
3. For hysterectomized subjects: documented hysterectomy must have occurred at least 6 
weeks prior to the start of screening. Hysterectomy can be total or subtotal (i.e., cervix was 
not removed); 
4. For non-hysterectomized subjects: uterus with bi-layer endometrial thickness ≤ 4 mm on 
TVUS; 
5. For non-hysterectomized subjects: endometrial biopsy taken during screening that reveals 
no abnormal result, i.e., presence of hyperplasia (simple or complex, with or without 
atypia), presence of carcinoma, and presence of disordered proliferative endometrium 
findings. The screening biopsy should have sufficient endometrial tissue for diagnosis. 
Biopsies without tissue or with insufficient tissue may be repeated once; 
6. Seeking treatment for relief of VMS associated with menopause; 
a. For the Efficacy Study part: at least 7 moderate to severe bothersome VMS per day or 
at least 50 moderate to severe bothersome VMS per week in the last 7 consecutive days 
during the Screening period12; 
b. For the Safety Study part: at least 1 moderate to severe VMS per week; 
7. Body mass index ≥18.0 kg/m² up to ≤38.0 kg/m²; 
8. A mammogram that shows no sign of significant disease performed during screening or 
within 9 months prior to the start of screening13; 
9. Postmenopausal status defined as any of the following: 
a. For non-hysterectomized subjects: 
- At least 12 months of spontaneous amenorrhea with serum follicle stimulating 
hormone (FSH) >40 mIU/mL (value obtained after washout of estrogen/progestin 
containing drugs, see exclusion criteria 18 and 20); 
- or at least 6 months of spontaneous amenorrhea with serum FSH >40 mIU /mL and 
E2 <20 pg/mL (value obtained after washout of estrogen/progestin containing drugs, 
see exclusion criteria 18 and 20); 
- or at least 6 weeks postsurgical bilateral oophorectomy3; 
                                                 
12   Bothersome is defined as a score of 4 to 6 on question 1 of the modified MENQOL questionnaire at screening. The 
timing and number of VMS and modified MENQOL score needed for allocation to the Efficacy Study part will not be 
disclosed to the study subjects to avoid bias in the recording during the screening process. 
13  Subjects must have a Breast Imaging-Reporting And Data System (BI-RADS) score of 1 or 2 to enroll in the study. An 
incomplete mammogram result, i.e., BI-RADS 0, is not acceptable and requires further assessment. The site must obtain a 
copy of the official report for the subject's study file. A digitalized imaging should be obtained if mammography is done 
as part of this study. 
Footnote continued on next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 75 of 150 
 
b. For hysterectomized subjects: 
- serum FSH >40 mIU/mL and E2 <20 pg/mL (values obtained after washout of 
estrogen/progestin containing drug, see exclusion criteria 18 and 20); 
- or at least 6 weeks postsurgical bilateral oophorectomy14; 
10. 
Good physical and mental health, in the judgement of the Investigator as based on medical 
history, physical and gynecological examination, and clinical assessments performed 
prior to Visit 1;  
11. 
Able to understand and comply with the protocol requirements, instructions, and protocol-
stated restrictions;  
12. 
Able and willing to complete trial daily diaries (if applicable, see Section 10.1.7) and 
questionnaires. 
8.2.2. Criteria for Exclusion 
Subjects will not be allocated to treatment if they meet one of the following exclusion criteria:  
1. History of malignancy, with the exception of basal cell or squamous cell carcinoma of the 
skin if diagnosed more than 1 year prior to the Screening visit15;  
2. Any clinically significant findings found by the Investigator at the breast examination and/or 
on mammography suspicious of breast malignancy that would require additional clinical 
testing to rule out breast cancer (however, simple cysts confirmed by ultrasound are 
allowed);  
3. PAP test with atypical squamous cells undetermined significance (ASC-US) or higher (low-
grade squamous intraepithelial lesion [LSIL], atypical squamous cells cannot exclude high-
grade squamous intraepithelial lesion [HSIL] [ASC-H], HSIL dysplastic or malignant cells) 
in sub-totally hysterectomized and non-hysterectomized subjects 16. Note: ASC-US is 
allowed if a reflex human papilloma virus (HPV) testing is performed and is negative for 
high risk oncogene HPV subtypes 16 and 18; 
4. For non-hysterectomized subjects:  
a. History or presence of uterine cancer, endometrial hyperplasia, or disordered 
proliferative endometrium; 
b. Presence of endometrial polyp; 
c. Undiagnosed vaginal bleeding or undiagnosed abnormal uterine bleeding; 
d. Endometrial ablation; 
                                                 
14  A report or a statement on letterhead from the subject's physician documenting both ovaries were removed is needed. 
15  The exception is only for basal cell carcinoma or squamous cell carcinoma of the skin if either of them were diagnosed 
more than one year prior to the screening of the subject. 
16  As indicated by written documentation of a prior test performed within 18 months prior screening or by a test performed 
at screening. 
 
Footnote continued on next page 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 76 of 150 
 
e. Any uterine/endometrial abnormality that in the judgment of the investigator 
contraindicates the use of estrogen and/or progestin therapy. This includes presence or 
history of adenomyosis or significant myoma; 
5. Systolic blood pressure (BP) higher than 130 mmHg, diastolic BP higher than 80 mmHg17 
during screening;  
6. History of venous or arterial thromboembolic disease (e.g., superficial or deep vein 
thrombosis, pulmonary embolism, stroke, myocardial infarction, angina pectoris, etc.), or 
first-degree family history of VTE;  
7. History of known acquired of congenital coagulopathy or abnormal coagulation factors, 
including known thrombophilia’s; 
8. Laboratory values of fasting glucose above 125 mg/dL and/or glycated hemoglobin above 
7%18;  
9. Dyslipoproteinemia (LDL >190 mg/dL and/or triglycerides >300 mg/dL)19; 
10. Subjects smoking >15 cigarettes per day; 
11. Presence or history of gallbladder disease, unless cholecystectomy has been performed; 
12. Systemic lupus erythematosus; 
13. Any malabsorption disorders including gastric bypass surgery; 
14. History of acute liver disease in the preceding 12 months before the start of screening or 
presence or history of chronic or severe liver disease [alanine transaminase (ALT) or 
aspartate transaminase (AST) >2x upper limit of normal (ULN), bilirubin >1.5 ULN], or 
liver tumors; 
15. Chronic or current acute renal impairment (estimated glomerular filtration rate <60 ml/min); 
16. Porphyria; 
17. Diagnosis or treatment of major psychiatric disorder (e.g., schizophrenia, bipolar disorder, 
etc.) in the judgement of the Investigator; 
18. Use of estrogen/progestin containing drug(s) up to: 
a. 1 week before screening start for vaginal non-systemic hormonal products (rings, 
creams, gels); 
b. 4 weeks before screening start for vaginal or transdermal estrogen or estrogen/progestin 
products; 
c. 8 weeks before screening start for oral estrogen and/or progestin products and/or 
selective estrogen receptor modulator therapy; 
                                                 
17  BP measurements at screening may be repeated if values are outside the inclusion criteria after sitting for an additional 5 
to 10 minutes. The last reading will be used for eligibility. Subjects with mild to moderate hypertension who are 
controlled on a stable antihypertension regimen may be enrolled if they meet all inclusion/exclusion criteria. Subjects 
using methyldopa or clonidine containing antihypertensive medication will not be included in the Efficacy Study part. 
18  Laboratory values of fasting glucose and glycated hemoglobin assessed during the last 6 months, and during washout and 
screening should be considered. 
19  Subjects using lipid-lowering therapy should be on a stable dose for at least 1 month before screening. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 77 of 150 
 
d. 8 weeks before screening start for intrauterine progestin therapy; 
e. 3 months before screening start for progestin implants or estrogen alone injectable drug 
therapy; 
f. 6 months before screening start for estrogen pellet therapy or progestin injectable drug 
therapy; 
19. Use of androgen/DHEA containing drugs: 
a. 8 weeks before screening start for oral, topical, vaginal or transdermal androgen; 
b. 6 months before screening start for implantable or injectable androgen therapy; 
20. Use of phytoestrogens or black cohosh for treatment of VMS up to 2 weeks before the start 
of screening; 
21. For the women participating in the Efficacy Study part: use of prescription or over-the-
counter products used for the treatment of VMS, e.g., anti-depressants: paroxetine, 
escitalopram, methyldopa, opioid and clonidine up to 4 weeks before the start of screening, 
and venlafaxine and desvenlafaxine up to 3 months before the start of screening20, and not 
willing to stop these during their participation in the trial;  
22. Not willing to stop any hormonal products as described in exclusion criteria 18, 19 and 20 
during their participation in the trial; 
23. Inadequately treated hyperthyroidism with abnormal TSH and free T4 at screening. Subjects 
with low or high TSH are allowed if free T4 at screening is within normal range21; 
24. History or presence of allergy/intolerance to the investigational product or drugs of this 
class or any component of it, or history of drug or other allergy that, in the opinion of the 
Investigator contraindicates subject participation;  
25. For non-hysterectomized subjects: history or presence of allergy to peanuts22; 
26. History of alcohol or substance abuse (including marijuana, even if legally allowed) or 
dependence in the previous 12 months before the start of screening as determined by the 
Investigator, based on reported observations; 
27. Sponsor or CRO employees or employees under the direct supervision of the Investigator 
and/or involved directly in the trial; 
28. Subjects with known or suspected history of a clinically significant systemic disease, 
unstable medical disorders, life-threatening disease or current malignancies that would pose 
a risk to the subject in the opinion of the Investigator; 
29. Participation in another investigational drug clinical trial within 1 month (30 days) or having 
received an investigational drug within the last month (30 days) before the start of screening; 
                                                 
20  If not used for the treatment of VMS, this medication is allowed if on a stable dose for 3 months prior to the 
Screening visit and no change in the dose regimen is foreseen during the conduct of the trial. 
21  Reflex T4 test to be performed at screening only if TSH at screening is outside normal range 
22  P4 provided as part of this study contains peanut oil and should never be used by subjects with allergy to 
peanuts 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 78 of 150 
 
30. Is judged by the Investigator to be unsuitable for any reason. 
8.3. 
Subject Discontinuation and Replacement Procedures 
Subjects may withdraw their consent and may discontinue from trial treatment and assessments 
at any time and for any reason. Subjects are free to discontinue their participation in the trial, 
without prejudice to further treatment. 
In addition, a subject may be withdrawn by the investigator or Sponsor in the case of emerging 
effects of such a nature that the risk/benefit ratio is unacceptable, in the case of poor compliance 
or protocol violation, if enrollment into the trial is inappropriate or for administrative and/or 
other safety reasons. 
If a subject does not complete the trial for any reason (including investigator discretion), the 
reason and circumstances for the subject’s early termination must be fully documented in the 
electronic case report form (eCRF). Possible reasons for a subject discontinuing participation 
in the trial are (non-exhaustive list): 
• 
SAE(s) that endanger the health of subjects, making it ethically unacceptable to continue or 
any SAE(s) that the subject finds intolerable 
• 
Deterioration 
of 
the 
subject’s 
clinical 
condition(s) 
that 
requires 
appropriate 
therapy/treatment during the trial period as per Investigator’s or Sponsor decision 
• 
Endometrium biopsy showing a disordered proliferative pattern, hyperplasia or worse, 
requiring treatment with a progestin 
• 
Endometrial safety findings that preclude further treatment of non-hysterectomized subjects 
• 
Withdrawal of consent due to lack of efficacy 
• 
Withdrawal of consent for another reason 
• 
Non-compliance to trial protocol: VMS count compliance 
• 
Non-compliance to trial protocol: study drug intake compliance 
• 
Non-compliance to trial protocol: others 
• 
Lost to follow up 
• 
Death 
• 
Other 
Wherever possible, subjects who discontinue should be seen and assessed by the investigator. 
Subjects should complete the early termination visit (Visit 7 Efficacy Study part /Visit 6 Safety 
Study part). Non-hysterectomized subjects who discontinue early, should also receive treatment 
with 200 mg P4 for 14 days and return to the clinic for the additional endometrial safety visit 
(Visit 8 Efficacy study part). If the endometrial event has not resolved by then, treatment with 
a progestin will be started according to local practice/guidelines. For any subject withdrawn or 
discontinuing the trial prematurely because of an adverse event (AE) related to one of the trial 
treatments, the investigator must strive to follow the subject until the AE has either resolved, 
becomes clinically insignificant, the event is stabilized, or the subject is lost to follow up. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 79 of 150 
 
Study site personnel will attempt to follow the progress of every subject admitted to the study 
through to study completion. If a subject fails to return for a scheduled visit, a reasonable effort 
should be made to contact the subject and ascertain the reason(s) for not returning. As a 
minimum, the site staff will contact the subject twice by phone and, if no response, will send a 
letter by registered mail. 
Subjects who are discontinued prematurely from the trial may be replaced at the discretion of 
Sponsor, in case the total number subjects becomes too low for the evaluation of the primary 
objective of this trial (see Section 8.1). Investigational products and trial materials should be 
returned by the subject. 
8.3.1. Criteria for Early Termination of the Study 
The clinical trial may be terminated early if the extent (incidence and/or severity) of emerging 
effects/clinical endpoints or new information regarding the safety of the product becomes 
available that results in an unacceptable risk/benefit ratio for the trial population. 
During the trial an independent Data Safety Monitoring Board (DSMB) will monitor the safety 
of E4 treatment (see Section 10.5). The study may be terminated early based on the advice of 
the DSMB. 
In addition, further recruitment in the trial or at (a) particular trial center(s) may be stopped due 
to insufficient compliance with the protocol, good clinical practice (GCP) and/or other 
applicable regulatory requirements, procedure-related problems or the number of 
discontinuations for administrative reasons is considered to be too high. 
If it becomes necessary to consider termination of the trial for any reason (e.g., safety, 
overestimation of the sample size, etc.) dosing may be suspended pending discussion between 
the investigator and Sponsor. Dosing will be stopped immediately on safety grounds. 
The trial may also be terminated or suspended at the request of the local authorities or ethics 
committee (EC)/ institutional review board (IRB). 
8.3.2. Stopping Criteria for Non-hysterectomized Women Only  
Monitoring of endometrial safety is part of the ongoing safety monitoring by the DSMB. More 
details on this may be found in the DSMB charter. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 80 of 150 
 
9. 
TRIAL MEDICATION  
9.1. 
Study Treatments 
Efficacy Study part: 
Once daily oral treatment with: 
• 
E4 15 mg (Arm 1) 
• 
E4 20 mg (Arm 2) 
• 
Placebo (Arm 3)  
Safety Study part 
Once daily treatment with:  
• 
E4 20 mg (Arm 4) 
9.2. 
Timing of Administration 
The trial medication should be taken once daily. Subjects will be instructed to take the trial 
medication at approximately the same time each day. 
If a subject has missed a dose, she should be instructed to take the dose as soon as she 
remembers. It is acceptable to take a dose up to 12 hours late. If taken more than 12 hours late 
the subjects should omit dosing for that day and continue with the next dose as usual. 
If study treatment is interrupted for 4 days and more per week, the Investigator will need 
approval from the Medical Monitor to restart the patient on study treatment if the subject has 
not received P4 200 mg (or other progestins). 
9.3. 
Pharmaceutical Preparations 
9.3.1. Active Treatment – Investigational Product 
Active substance:  Estetrol monohydrate (E4) 
Strength:  
Efficacy Study part: E4 15 mg or E4 20 mg 
 
Safety Study part: E4 20 mg 
Dosage form:  
Film coated tablet 
Excipients:  
Lactose monohydrate 
 
Sodium starch glycolate type A 
 
Maize starch 
 
Purified Water 
 
Povidone 30LP 
 
Magnesium Stearate 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 81 of 150 
 
 
 (containing hypromellose, 
hydroxypropylcellulose, stearic acid, talc, titanium dioxide, Brilliant 
Blue, Indigo Carmine and Tartrazine) 
Manufacturer: 
  
Blinding primary 
packaging:  
Blister pack (PVC/Aluminum) containing 28 tablets per blister 
Supplier/secondary 
packaging:  
Carton Wallet 
9.3.2. Placebo (Visually Matching the Active Treatments) 
Active substance 
No active substance 
Dosage form:  
Film coated tablet 
Excipients:  
 (co-processed excipient containing lactose monohydrate and 
 
maize starch) 
 
Magnesium stearate 
 
 (containing hypromellose, 
hydroxypropylcellulose, stearic acid, talc, titanium dioxide, Brilliant 
Blue, Indigo Carmine and Tartrazine) 
Manufacturer: 
 
Blinding primary 
packaging:  
Blister pack (PVC/Aluminum) containing 28 tablets per blister 
Supplier/secondary 
packaging:  
Carton Wallet 
 
9.3.3. Progesterone Treatment 
The 200 mg P4 treatment for the follow-up treatment of the non-hysterectomized subjects will 
be supplied by the Sponsor as commercially available medication in the original packaging. 
For ad hoc treatment with a progestin, according to local practice/guidelines, the choice of the 
progestin is left at the discretion of the investigator and treatment will be supplied by the local 
pharmacy. 
9.4. 
Packaging and Labelling 
The E4 and placebo trial medication will be supplied as tablets. The once daily dose consists of 
one E4 or placebo tablet. All tablets will have a similar weight, shape and color. The tablets 
will be provided in blisters (28 tablets in one blister) and packed in wallets each containing one 
blister. The E4 or placebo trial medication will be provided at the study visits as shown in Table 
1 (Efficacy Study part) and Table 2 (Safety Study part). One additional blister will be provided 
to the subjects at Visit 1 in a separate wallet as spare medication, in case the subject loses her 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 84 of 150 
 
At the conclusion of the trial and as appropriate during the course of the trial, the Investigator 
will return all used and unused drug containers, drug labels, and a copy of the completed drug 
accountability form to Sponsor. Some sites may destroy medications locally at an appropriate 
time point, if previously agreed with the Sponsor. 
9.6. 
Prior and Concomitant Medication 
9.6.1. Prior Medication/Therapy 
A washout period is required for the use of estrogens, progestins, androgens, and DHEA 
containing products for all subjects. For subjects in the Efficacy Study part a washout period is 
also required for the use of non-hormonal medication or OTC products that are used for the 
treatment of VMS (see also exclusion criteria, Section 8.2.2). These treatments need to be 
washed out before the start of screening. 
Table 3 provides details on the washout period of each of these drugs. 
Table 3 Washout of prior medication 
Medication 
Washout period before 
screening start 3, 4 
Estrogen/progestin containing drugs: 
 
- vaginal hormonal non-systemic products (rings, creams, gels) 
1 week 
- vaginal or transdermal estrogen or estrogen/progestin products 
4 weeks 
- oral estrogen and/or progestin and/or selective estrogen receptor modulator 
therapy 
8 weeks 
- intrauterine progestin therapy 
8 weeks 
- progestin implants and estrogen alone injectable drug therapy 
3 months 
- estrogen pellet therapy or progestin injectable drug therapy 
6 months 
Androgen or dehydroepiandrosterone (DHEA) containing drugs:  
 
- oral, topical, vaginal or transdermal androgen 
8 weeks 
- implantable or injectable androgen 
6 months 
- phytoestrogens, black cohosh used for the treatment of VMS 
2 weeks 
Non-hormonal prescription or over-the-counter treatments that are used for the 
treatment of VMS1, 2:  
- paroxetine, escitalopram, methyldopa, opioids, clonidine or others; 
- venlafaxine, desvenlafaxine 
 
 
 
4 weeks 
3 months 
 1 If used for other indications (e.g., for depressive disorders) this medication is allowed if on a stable dose for 
3 months prior to the screening visit and no changes in the dose regimen is foreseen during the trial. 
2 In case it is clear that the subject will participate in the Safety Study part, washout of these medications is 
not required. 
3 If tapering off a medication is required per Investigator’s medical judgement, the washout period should 
only start after last dose of such medication. 
4 When a subject starts a washout period, it’s important to ensure that the subject will have sufficient time to 
randomize within the enrolment period. 
9.6.2. Concomitant Medication/Therapy 
The use of any concomitant medication taken during the trial will be recorded in the eCRF and 
will include the name of the drug, treatment regimen (dose, units, frequency, and route of 
administration), the date of initiation and discontinuation of the therapy, and the indication. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 85 of 150 
 
Lipid lowering drugs 
If a subject is receiving a lipid-lowering therapy, then her dyslipoproteinemia has to be in line 
with the exclusion criteria, with LDL ≤190 mg/dL and triglycerides ≤300 mg/dL. The subject 
should be on a stable dose of lipid-lowering therapy for at least 1 month before the start of 
screening. 
Thyroid treatment  
Thyroid treatments are allowed as long as the subject has normal TSH levels or low or high 
TSH levels with normal free T4 at screening. 
Treatment for VMS symptoms 
Estrogen and/or progestin therapies are not allowed during the trial. A washout period is 
necessary as described in Section 9.6.1. 
The use of non-hormonal medication (prescription medication or OTC) for the treatment of 
VMS is not allowed in the Efficacy Study part. A washout period is necessary as described in 
Section 9.6.1. If the medication is used for other indications (e.g., for depressive disorders) then 
participation is allowed if on a stable dose for at least 3 months prior to the start of screening 
and no changes in the dose regimen is foreseen during the study period. 
To confirm compliance with the rules of unauthorized concomitant therapy, blood samples for 
the analysis of E2 concentrations will be taken during the treatment visits in the Efficacy Study 
part. 
Antihypertensives 
Subjects with mild to moderate hypertension who are controlled on a stable antihypertension 
regimen may be enrolled if they meet the blood pressure criteria at screening (systolic BP ≤130 
mmHg, diastolic BP ≤80 mmHg). Subjects using methyldopa or clonidine containing 
antihypertensive medication may not be included in the Efficacy Study part. 
9.6.3. COVID-19 vaccination 
Subjects cannot be excluded from the trial based on vaccination status for COVID-19 consistent 
with restriction for other vaccinations, which may happen in the subjects (influenza, tetanus and 
other).  
Subjects can be vaccinated with a regulatory approved COVID-19 vaccine according to national 
guidelines but should not participate in a COVID-19 clinical trial. 
Recommendation on the timing of the COVID-19 vaccination in relation to study assessments, 
due to possible acute reaction on vaccination: 
- 
The vaccination should be performed preferably after regular visits with the planned 
blood collections. 
- 
Otherwise minimum one week before regular visits with blood samples collections. 
Rationale for timing restriction: the acute reaction on vaccination may cause transitory changes 
in biochemistry (liver enzymes, LDH etc.), it is not a common reaction but may happen. The 
possible transitory increase in temperature may cause changes in absorption of study drug, PK 
fluctuations etc.  

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 86 of 150 
 
In case of any acute condition, the scheduled visit should be postponed (if no safety issues) until 
the status of the subject improves. 
The vaccination during the trial should be reported as concomitant medication. Any AE that 
occurs after vaccination should be properly reported. 
9.7. 
Treatment Compliance  
Subjects will be asked to bring all packages of used and unused trial medication, including 
empty blisters/bottles and wallets to each visit. Treatment compliance will be assessed by 
counting the returned trial medication at each visit. The counts of the returned trial medication 
will be recorded by the clinical staff in the subject drug accountability log. Any discrepancies 
between actual and expected amount of returned trial medication must be recorded and 
discussed with the subject at the time of the visit. 
Additionally, for some subjects (see Section 10.1.7), intake of the trial medication will be 
recorded in a paper diary to check on treatment compliance. The diaries will be checked at each 
visit for any discrepancies in medication intake. 
To confirm treatment compliance in the Efficacy Study part, blood samples for the analysis of 
E4 concentrations will be taken during Visits 2, 3 and 4. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 87 of 150 
 
10. 
TRIAL ASSESSMENTS 
10.1. Experimental Flow 
For the Efficacy Study part at least 9 visits are planned: a Washout visit followed by a Screening 
period which will start with a Screening visit, a Baseline visit (Visit 1), and 6 On-treatment 
visits (Visits 2 to 7). The last treatment visit (Visit 7) is also the end of treatment (EoT) Visit, 
which will also occur in case of early discontinuation. For non-hysterectomized women an 
additional endometrial safety follow-up visit (Visit 8) is planned after completion of the 14 days 
of P4 treatment, which will also occur in case of early discontinuation. 
For the Safety Study part at least 8 visits are planned: a Washout visit followed by a Screening 
period which will start with a Screening visit), a Baseline visit (Visit 1), and 5 On-treatment 
visits (Visits 2 to 6). The last treatment visit (Visit 6) is also the end of treatment (EoT) Visit, 
which will also occur in case of early discontinuation. For non-hysterectomized women an 
additional endometrial safety follow-up visit (Visit 7) is planned approximately 10 days after 
completion of the 14 days of P4 treatment, which will also occur in case of early 
discontinuation. 
The screening period requires at least one visit, to occur as per protocol, at the beginning of the 
period, but some assessments (e.g., mammography, biopsy, TVUS, ECG, and PAP test) may 
require additional visits to another clinic in case these assessments cannot be done by the study 
site. 
An overview of the trial visits and time windows for each visit is presented in Table 4 
 
Table 4 Visit schedule and permitted time window 
Study part 
Visit number 
Week number 
Time window 
Efficacy Study part 
Washout visit 
Week - 12 to week - 4 1 
n.a.  
Screening period 
Week - 4 to week -1 1 
n.a.  
Visit 1 
Week 1 
n.a.  
Visit 2  
Week 5 
Day 29 - 35 
Visit 3  
Week 9 
Day 57 - 63 
Visit 4 
Week 13 
Day 85 - 91  
Visit 5 
Week 29 
Day 197 - 203 
Visit 6 
Week 41  
Day 281 - 287 
Visit 7 
Week 53 
Day 365 - 371 
Visit 8 NH subjects only 
Week 55/56 
Day 379 - 392 
Safety Study part 
Washout visit 
Week - 12 to week - 4 1 
n.a.  
Screening period 
Week - 4 to week - 1 1 
n.a.  
Visit 1 
Week 1 
n.a.  
Visit 2 
Week 5 
Day 29 - 35 
Visit 3 
Week 13 
Day 85 - 91 
Visit 4 
Week 29 
Day 197 - 203 
Visit 5  
Week 41 
Day 281 - 287 
Visit 6 
Week 53 
Day 365 - 371 
Visit 7  NH subjects only 
Week 55/56 
Day 379 - 392 
1  
The week numbers for the Washout visit and Screening period may deviate. The washout visit is generally up to 8 weeks 
prior to the screening visit. The Screening period is up to 4 weeks, but may be extended to a maximum of 8 weeks. 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 88 of 150 
 
An overview of the trial procedures and the timing of the assessments is provided in the 
Schedule of Trial Procedures (Section 2.1 for the Efficacy Study part and Section 2.2 for the 
Safety Study part). An overview of the procedures per visit is provided in the next sections. 
Details on the procedures can be found in Section 10.2 (demographics), Section 10.3 (efficacy 
assessments) and Section 10.4 (safety assessments). 
10.1.1. Informed Consent Procedure  
When a suitable candidate is identified by the Investigator among the outpatients coming to the 
trial site, the investigator or designated healthcare professional will ask the subject about her 
willingness to be included in the clinical trial. The subject is to be informed verbally and in 
writing about the nature, risks, benefits, and expectations of participating in the clinical trial 
and a copy of the subject informed consent form (ICF) is to be given to the subject in the 
appropriate language. Following this, subjects will be allowed sufficient time, in their own 
opinion, to consider trial entry, and will be offered the opportunity to ask any further questions 
prior to completing the ICF. The subject ICF is to be signed by the subject and countersigned 
by the attending investigator or designee prior to proceeding with the Washout/Screening visit 
and a copy of the signed ICF is provided to the subject. 
10.1.2. Washout Visit 
All subjects will be pre-screened at the Washout visit and Informed Consent (IC) is taken at 
this visit. After signing the ICF the following procedures will be performed:  
• 
Check of already available inclusion/exclusion criteria  
• 
Review of the drug treatments/therapy 
• 
If lipid and/or glucose parameters could be a reason of concern in the judgement of the 
investigator, fasted blood sampling for lipid and glucose parameters assessment may be 
done during this visit. Otherwise, this assessment will be done during the Screening 
Period. 
• 
Medical and gynecological history  
• 
Obtain demographic data  
 
In case the subject uses medication for which a washout is necessary (see Section 9.6.1) the 
screening procedures will be halted. The subject is asked to stop taking this medication and 
will return for screening after the required washout period (see Section 9.6.1). 
In case no washout period is required the subject will immediately continue the procedures of 
the Screening Visit. For these subjects the Washout visit and Screening visit may occur at the 
same time. 
10.1.3. Screening Visit (Within 4 weeks before Randomization/Treatment allocation) 
After the washout period (if applicable) the participant will come to the Screening visit marking 
the beginning of the Screening period. The Screening period will last up to 4 weeks and the 
visit needs to occur within 4 weeks prior to Visit 1). The following procedures will be performed 
as screening procedures:  

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 89 of 150 
 
• 
Review of already available inclusion and exclusion criteria 
• 
Physical examination 
• 
Gynecological examination 
• 
Breast examination 
• 
Vital signs 
• 
12-lead ECG  
• 
PAP test (unless a written normal result is available within 18 months before the start of 
screening) 
• 
TVUS (for women who still have uterus and/or ovaries) 
• 
Endometrial biopsy (for non-hysterectomized women only)  
• 
Digitalized mammography (unless a written normal result is available within 9 months 
before the start of screening) 
• 
Modified MENQOL questionnaire 
• 
Fasted blood sampling for:  
- 
Routine hematology and chemistry 
- 
Lipid and glucose parameters for inclusion (if not done as Washout visit) 
- 
FSH, E2 and TSH for inclusion (fasting not required) 
• 
Urinary pregnancy test (at the discretion of the Investigator) 
• 
Dispense and explain paper diary  
• 
Record prior medication and start recording concomitant medication. 
• 
Start recording AEs. 
The screening period may require additional visits for some assessments (i.e., mammography, 
biopsy, TVUS, ECG, PAP test) in case these assessments cannot be done during the Screening 
visit.  
During screening the subjects will receive a paper diary and are instructed to record the severity 
and frequency of VMS until return for Visit 1. The study personnel will remind the subjects 
weekly (by phone, SMS, e-mail etc.) to complete the diary. The first reminder will be sent 5 ± 
2 days after start of VMS recording (i.e. screening visit). 
In case it is clear during screening that a subject will participate in the Safety Study part, either 
because she does not present a score of 4 to 6 on question 1 of the modified MENQOL 
questionnaire at screening, or based on current medical history, she will not reach the VMS 
requirements to participate in the Efficacy Study part or because the enrollment in the Efficacy 
Study part has been completed, the recording of VMS symptoms in the paper diaries may be 
skipped. The subject will return for Visit 1 after all screening results are available. 
Subjects are required to come to the next visit in a fasted state (fasting for at least 8 hours), and 
take the diary with them. 
Subjects presenting the required number of bothersome moderate to severe VMS/day or per 
week may participate in the Efficacy Study part. Bothersome is defined as a score of 4 to 6 on 
question 1 of the modified MENQOL questionnaire at screening. Women with at least 1 
moderate to severe VMS/week may participate in the Safety Study part. The timing and number 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 90 of 150 
 
of VMS and modified MENQOL score needed for allocation to the Efficacy Study part will not 
be disclosed to the study subjects to avoid bias in the VMS recording during the screening 
process. 
Laboratory tests may be repeated once during the screening period, at the discretion of the 
investigator.   
Cervical smears and endometrial biopsies may only be repeated once during the screening 
period in case of insufficient samples. 
In the event screening requirements cannot be met within 4 weeks, the screening period may be 
extended with prior Medical Monitor approval, but screening period may not exceed 8 weeks. 
In case the screening period is extended, the site will provide additional/sufficient diary cards 
to the subject, to ensure completeness of diary cards until the end of the extended screening 
period.  Repeat of entry laboratory parameters will need to be considered in case of extended 
screening period. 
10.1.4. Subject Re-screening 
This trial permits the re-screening of a subject who has discontinued the trial as a pre-treatment 
failure (i.e., subject has not been treated with the study medication); the reason for failure must 
be temporary and expected to resolve and not related to the primary endpoint (frequency of 
VMS). If re-screened, all assessments/procedures from the screening period need to be redone 
except: 
• 
Gynecological examination, including breast examination (if done 2 months before re-
screening) 
• 
Mammography (if done 9 months before re-screening) 
• 
Endometrial biopsy (if done 2 months before re-screening) 
• 
PAP test (if a normal written result is available within 18 months before the start of 
screening) 
10.1.5. Visits Efficacy Study part 
10.1.5.1. 
Randomization Visit and Baseline Assessments (Visit 1 – Day 1) 
Subjects will return to the trial site at the end of the Screening period (Study Day 1). The 
following observations/procedures are to be performed and checked at Randomization 
visit/Visit 1: 
• 
Review of inclusion and exclusion criteria 
• 
Vital signs 
• 
MENQOL questionnaire 
• 
Fasted blood sampling for:  
- 
Routine hematology and chemistry 
- 
Lipid metabolism 
- 
Glucose metabolism 
- 
Hemostasis  

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 91 of 150 
 
- 
Bone turnover  
- 
Additional optional analysis (including a DNA sample)  
- 
E2 (fasting not required) 
• 
Urinary pregnancy test (at the discretion of the Investigator) 
• 
Collect diary for the Screening period 
• 
Review of diary  
• 
Randomization 
• 
Dispense diary for treatment period 
• 
Dispense trial medication  
• 
Record prior medication 
• 
Record AEs  
 
Subjects who fulfil all of the inclusion and exclusion criteria will be randomized to trial 
treatment. 
After randomization, the trial medication (E4 or placebo) for the first 8 weeks will be dispensed 
(see Section 9.4) and subjects need to start treatment on the day of randomization visit (= day 1 
of week 1). The next visit will be scheduled and subjects are instructed to come to the next visit 
with all packages of used and unused trial medication, including empty blisters and wallets. 
Subjects are instructed to record daily the severity and frequency of VMS and medication intake 
in the paper diary. During VMS recording the study personnel will remind the subjects weekly 
(by phone, SMS, e-mail etc.) to complete the diary. The first reminder will be sent 5 ± 2 days 
after start of treatment. Non-hysterectomized subjects are instructed to also record the vaginal 
bleeding events. Subjects need to take the diary to the next visit. 
10.1.5.2. 
Visit 2: Primary Efficacy Visit (week 5)  
Subjects will return to the trial site in week 5, after completion of 4 weeks of treatment. The 
following observations/procedures are to be performed and checked at Visit 2: 
• 
Vital signs 
• 
CGI questionnaire 
• 
Blood sampling for E2 and E4 
• 
Review of diary and collect completed pages 
• 
Dispense trial medication  
• 
Review of returned trial medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs  
After completion of Visit 2 procedures, trial medication for weeks 9 to 12 will be dispensed 
(see Section 9.4) and the next visit will be scheduled. Subjects are instructed to come to the next 
visit with all packages of used and unused trial medication, including empty blisters and wallets. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 92 of 150 
 
Subjects are instructed to record daily the severity and frequency of VMS and medication intake 
in the paper diary. Non-hysterectomized subjects are instructed to also record the vaginal 
bleeding events. Subjects need to take the diary to the next visit. 
10.1.5.3. 
Visit 3: Primary Efficacy Visit (week 9)  
Subjects will return to the trial site in week 9, after completion of 8 weeks of treatment. The 
following observations/procedures are to be performed and checked at Visit 3: 
• 
Vital signs 
• 
Blood sampling for E2 and E4 
• 
Review of diary and collect completed pages  
• 
Dispense trial medication  
• 
Review of returned trial medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs  
After completion of Visit 3 procedures, trial medication for weeks 9 to 16 will be dispensed 
(see Section 9.4) and the next visit will be scheduled. Subjects are instructed to come to the next 
visit with all packages of used and unused trial medication, including empty blisters and wallets. 
Subjects are instructed to record daily the severity and frequency of VMS and medication intake 
in the paper diary. Non-hysterectomized subjects are instructed to also record the vaginal 
bleeding events. Subjects are required to come to the next visit in a fasted state (fasting for at 
least 8 hours), and take the diary with them. 
10.1.5.4. 
Visit 4: End of Primary Efficacy Period (week 13)  
Subjects will return to the trial site in week 13 for Visit 4. This will be the end of the primary 
efficacy period. The following observations/procedures are to be performed and checked at 
Visit 4: 
• 
Vital signs 
• 
TVUS (for non-hysterectomized women only) 
• 
MENQOL and CGI questionnaires 
• 
Fasted blood sampling for:  
- 
Lipid metabolism 
- 
Glucose metabolism 
- 
Hemostasis parameters 
- 
Bone markers 
- 
E2 and E4 (fasting not required) 
• 
Review of diary and collect completed pages 
• 
Dispense trial medication 
• 
Review returned unused medication/drug accountability 
• 
Record prior/concomitant medication 
• 
Record AEs  

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 93 of 150 
 
After completion of the Visit 4 procedures, trial medication for weeks 17 to 32 will be dispensed 
(see Section 9.4) and the next visit will be scheduled. Subjects are instructed to come to the next 
visit and take all packages of used and unused trial medication, including empty blisters and 
wallets. Non-hysterectomized subjects are instructed to continue daily recording of study drug 
intake and vaginal bleeding events in the diary and to take the diary to the next visit. 
Hysterectomized subjects will return their diary. 
10.1.5.5. 
Visit 5: 6 Month Safety Visit (week 29) 
Subjects will return to the trial site in week 29 after completion of 28 weeks of treatment. The 
following observations/procedures are to be performed and checked at Visit 5: 
• 
Vital signs 
• 
Breast examination 
• 
TVUS (for non-hysterectomized subjects only) 
• 
Review of diary and collect completed pages 
• 
Dispense trial medication  
• 
Review returned unused medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs 
After completion of the Visit 5 procedures trial medication for weeks 33-44 will be dispensed 
(see Section 9.4) and the next visit will be scheduled. Subjects are instructed to come to the next 
visit and take all packages of used and unused trial medication, including empty blisters and 
wallets. Non-hysterectomized subjects are instructed to continue daily recording of study drug 
intake and vaginal bleeding events in the diary and to take the diary to the next visit. 
10.1.5.6. 
Visit 6: 9 Month Safety Visit (week 41) 
Subjects will return to the trial site in week 41 after completing 40 weeks of treatment. The 
following observations/procedures are to be performed and checked at Visit 6: 
• 
Vital signs 
• 
TVUS (for non-hysterectomized subjects only) 
• 
Review of diary and collect completed pages 
• 
Dispense trial medication 
• 
Review returned unused medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs  
After completion of the Visit 6 procedures, trial medication for weeks 45 to 56 will be dispensed 
(see Section 9.4). Only medication of week 45 to 53 is to be used as the next visit (the EoT 
visit) will be scheduled in week 53. Subjects are instructed to come to the next visit in a fasted 
state (fasting for at least 8 hours) and take all packages of used and unused trial medication, 
including empty blisters and wallets. Non-hysterectomized subjects are instructed to continue 
daily recording of study drug intake and vaginal bleeding events in the diary and to take the 
diary to the next visit. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 94 of 150 
 
10.1.5.7. 
Visit 7: End of Treatment/Early Termination Visit (week 53 or earlier)  
Subjects will return to the trial site in week 53 for Visit 7. This will be the EoT and follow-up 
procedures will be performed. Subjects will take their last daily dose of the trial medication on 
the evening before or in the morning of the day of the EoT visit. 
For subjects who terminate early, every reasonable effort should be made by the investigator to 
complete the final assessment procedures as specified in this section (see also Section 8.3). In 
case of early termination, treatment may have been stopped earlier. 
The following observations/procedures are to be performed and checked at Visit 7: 
• 
Physical examination 
• 
Gynecological examination 
• 
Breast examination 
• 
Vital signs 
• 
12-lead ECG 
• 
Endometrial biopsy if the subject has completed at least 12 weeks of treatment (for non-
hysterectomized women only)  
• 
TVUS (for women who still have uterus and/or ovaries) 
• 
Digitalized mammography if the subject has completed at least 12 weeks of treatment and 
the last mammography was taken at least 9 months earlier 
• 
MENQOL and CGI questionnaires 
• 
Fasted blood sampling for:  
- 
Routine hematology, chemistry 
- 
Lipid metabolism 
- 
Glucose metabolism 
- 
Hemostasis parameters 
- 
Bone markers 
- 
Additional optional analysis 
• 
Review of diary and collect completed pages (non-hysterectomized subjects) 
• 
Dispense trial medication (P4 200 mg for non-hysterectomized subjects only) 
• 
Review returned unused medication/drug accountability 
• 
Record prior/concomitant medication 
• 
Record AEs  
• 
Complete end of trial form (hysterectomized subjects only) 
For hysterectomized subjects: after completion of the Visit 7 procedures in the Efficacy Study 
part, the subject has completed the trial. 
For non-hysterectomized subjects: after completion of Visit 7 procedures the subjects will start 
treatment with 200 mg P4 once daily for 14 days. Subjects should take the progestin medication 
as per investigator's instructions. Subjects are instructed to record the vaginal bleeding events 
and P4 intake in the diary. Subjects will return to the clinic approximately 10 days after 
completion of the progestin treatment for the endometrial safety follow-up visit. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 95 of 150 
 
10.1.5.8. 
Visit 8: Non-hysterectomized Subjects Only: Endometrial Safety Follow-up Visit 
(week 55/56 or earlier) 
The following observations/procedures are to be performed and checked at Visit 8: 
• 
TVUS 
• 
Return of diary 
• 
Review of diary 
• 
Review returned unused medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs 
• 
Complete end of trial form  
After completion of the Visit 8 procedures in the Efficacy Study part, the subject has completed 
the trial. 
10.1.6. Visits Safety Study part 
10.1.6.1. 
Visit 1: Treatment Allocation Visit and Baseline Assessments (Day 1) 
Subjects will return to the trial site at the end of the Screening period. The following 
observations/procedures are to be performed and checked at the Treatment allocation visit/Visit 
1: 
• 
Review of inclusion and exclusion criteria 
• 
Vital signs 
• 
MENQOL questionnaire 
• 
Fasted blood sampling for:  
- 
Routine hematology and chemistry 
- 
Lipid metabolism 
- 
Glucose metabolism 
- 
Additional optional analysis (including a DNA sample)  
• 
Urinary pregnancy test (at the discretion of the Investigator) 
• 
Collect diary for Screening period 
• 
Review of diary  
• 
Allocation to treatment 
• 
Dispense trial medication 
• 
Dispense diary for treatment period (non-hysterectomized subjects only) 
• 
Record prior medication 
• 
Record AEs  
Subjects who fulfil all of the inclusion and exclusion criteria will be allocated to trial treatment. 
After treatment allocation, the trial medication (20 mg E4) for the first 8 weeks will be dispensed 
(see Section 9.4) and subjects need to start treatment on the day of treatment allocation visit (= 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 96 of 150 
 
day 1 of week 1). The next visit will be scheduled and subjects are instructed to come to the 
next visit with all packages of used and unused trial medication, including empty blisters and 
wallets. Non-hysterectomized subjects are instructed to daily recording study drug intake and  
vaginal bleeding events in the diary and to take the diary to the next visit. 
10.1.6.2. 
Visit 2: 4 Weeks Safety Visit (week 5)  
Subjects will return to the trial site in week 5, after completion of 4 weeks of treatment. The 
following observations/procedures are to be performed and checked at Visit 3: 
• 
Vital signs 
• 
CGI questionnaire  
• 
Review of diary and collect completed pages (non-hysterectomized subjects only) 
• 
Dispense trial medication 
• 
Review of returned trial medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs  
After completion of Visit 2 procedures, trial medication for weeks 9 to 16 will be dispensed 
(see Section 9.4) and the next visit will be scheduled. 
Subjects are instructed to come to the next visit in a fasted state (fasting for at least 8 hours) and 
take all packages of used and unused trial medication, including empty blisters and wallets. 
Non-hysterectomized subjects are instructed to daily recording of study drug intake and vaginal 
bleeding events in the diary and to take the diary to the next visit. 
 
10.1.6.3. 
Visit 3: 12 Weeks Safety Visit (week 13)  
Subjects will return to the trial in week 13 after completion of 12 weeks of treatment. The 
following observations/procedures are to be performed and checked at Visit 3: 
• 
Vital signs 
• 
TVUS (non-hysterectomized subjects only) 
• 
MENQOL and CGI questionnaires 
• 
Fasted blood sampling for:  
- 
Lipid metabolism 
- 
Glucose metabolism 
• 
Review of diary and collect completed pages (non-hysterectomized subjects only) 
• 
Dispense trial medication 
• 
Review of returned trial medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs  
After completion of the Visit 3 procedures, trial medication for weeks 17 to 32 will be dispensed 
(see Section 9.4) and the next visit will be scheduled. Subjects are instructed to come to the next 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 97 of 150 
 
visit and take all packages of used and unused trial medication, including empty blisters and 
wallets. Non-hysterectomized subjects are instructed to daily recording of study drug intake and 
vaginal bleeding events in the diary and to take the diary to the next visit. 
10.1.6.4. 
Visit 4: 6 Month Safety Visit (week 29) 
Subjects will return to the trial site in week 29 after completion of 28 weeks of treatment. The 
following observations/procedures are to be performed and checked at Visit 4: 
• 
Vital signs 
• 
Breast examination 
• 
TVUS (non-hysterectomized subjects only) 
• 
Review of diary and collect completed pages (non-hysterectomized subjects only) 
• 
Dispense trial medication  
• 
Review of returned trial medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs 
After completion of the Visit 4 procedures trial medication for weeks 33-44 will be dispensed 
(see Section 9.4) and the next visit will be scheduled. Subjects are instructed to come to the next 
visit and take all packages of used and unused trial medication, including empty blisters and 
wallets. Non-hysterectomized subjects are instructed to daily recording of study drug intake and 
vaginal bleeding events in the diary and to take the diary to the next visit. 
10.1.6.5. 
Visit 5: 9 Month Safety Visit (week 41) 
Subjects will return to the trial site in week 41 after completing 40 weeks of treatment. The 
following observations/procedures are to be performed and checked at Visit 5: 
• 
Vital signs 
• 
TVUS (for non-hysterectomized subjects only) 
• 
Review of diary and collect completed pages (non-hysterectomized subjects only) 
• 
Dispense trial medication 
• 
Review of returned trial medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs  
After completion of the Visit 5 procedures, trial medication for weeks 45 to 56 will be dispensed 
(see Section 9.4). Only medication of week 45 to 53 is to be used as the next visit (the EoT 
visit) will be scheduled in week 53. Subjects are instructed to come to the next visit in a fasted 
state (fasting for at least 8 hours) and take all packages of used and unused trial medication, 
including empty blisters and wallets. Non-hysterectomized subjects are instructed to daily 
recording of study drug intake and vaginal bleeding events in the diary and to take the diary to 
the next visit. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 98 of 150 
 
10.1.6.6. 
Visit 6: End of Trial/Early Termination Visit (week 53 or earlier) 
Subjects will return to the trial site in week 53 for Visit 6. This will be the EoT and follow-up 
procedures will be performed. Subjects will take their last daily dose of the trial medication on 
the evening before the EoT visit. 
For subjects who terminate early every reasonable effort should be made by the investigator to 
complete the final assessment procedures as specified in this section (see also Section 8.3). In 
case of early termination treatment may have been stopped earlier. 
The following observations/procedures are to be performed and checked at Visit 6: 
• 
Physical examination 
• 
Gynecological examination 
• 
Breast examination 
• 
Digitalized mammography (in case of early termination, mammography may only be 
performed if subjects was treated for at least 12 weeks and last mammography was taken 
at least 9 months earlier) 
• 
Vital signs 
• 
12-lead ECG 
• 
Endometrial biopsy if the subject has at least completed 12 weeks of treatment (for non-
hysterectomized women only)  
• 
TVUS (for women who still have uterus and/or ovaries) 
• 
MENQOL and CGI questionnaires 
• 
Fasted blood sampling for:  
- 
Routine hematology, chemistry 
- 
Lipid metabolism 
- 
Glucose metabolism 
- 
Additional optional analysis 
• 
Review of returned trial medication/drug accountability 
• 
Dispense trial medication (P4 200 mg for non-hysterectomized subjects only) 
• 
Record prior/concomitant medication 
• 
Record AEs  
• 
Complete end of trial form (hysterectomized subjects only) 
For hysterectomized subjects: after completion of the Visit 6 procedures in the Safety Study 
part, the subject has completed the trial. 
For non-hysterectomized subjects: after completion of Visit 6 procedures the subjects will start 
treatment with 200 mg P4 once daily for 14 days. Subjects should take the progestin medication 
as per investigator's instructions. Subjects are instructed to record the vaginal bleeding events 
and P4 intake in the diary. Subjects will return to the clinic approximately 10 days after 
completion of the progestin treatment for the endometrial safety follow-up visit (Visit 7). 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 99 of 150 
 
10.1.6.7. 
Visit 7: Non-hysterectomized Subjects Only: Endometrial Safety Follow-up Visit 
(week 55/56 or earlier)  
The following observations/procedures are to be performed and checked at Visit 7: 
• 
TVUS 
• 
Return of diary 
• 
Review of diary 
• 
Review returned unused medication/drug accountability 
• 
Record concomitant medication 
• 
Record AEs 
• 
Complete end of trial form  
After completion of the Visit 7 procedures in the Safety Study part, the non-hysterectomized 
subject has completed the trial. 
10.1.7. Subject Diary  
In the Efficacy Study part, subjects will complete a paper diary for: 
• 
Daily VMS recording during the Screening period and during the 12 weeks treatment 
period (until Visit 4):  
o Amount of VMS  
o Severity of each VMS (mild, moderate, severe) (see Section 10.3.1 for 
definitions) 
• 
Daily recording of trial drug intake, including P4 intake during the follow-up period for 
the non-hysterectomized subjects 
• 
For non-hysterectomized subjects only: daily recording of the vaginal bleeding events (see 
Section 10.4.9 for definitions), until the end of study: 
o None 
o Spotting 
o Bleeding 
During the VMS recording period subjects will be reminded weekly (by phone, e-mail, SMS 
etc.) of daily completion of the diary. The first reminder will be sent 5 ± 2 days after start of 
VMS count in the Screening period. 
In the Safety Study part, only non-hysterectomized subjects will complete a paper diary for:  
• 
Daily recording of the trial drug intake (including P4 intake) 
• 
Daily recording of vaginal bleeding events (see Section 10.4.9 for definitions), including 
during the follow-up period: 
o None 
o Spotting 
o Bleeding 
The subject will be instructed to bring the diary to the site at each subject visit. Review of the 
diary by the Investigator or designee for completeness and consistency is one of the procedures 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 100 of 150 
 
to be followed on ongoing basis and at each subject’s visit. Discrepancies and/or missing data 
need to be followed-up. 
Completed sections of the subject diaries will be retrieved at each visit and the remaining pages 
will be collected at the end of the study and data will be entered in the eCRFs. 
The hysterectomized subjects who continue into the safety follow-up or who are enrolled in the 
Safety Study part will not complete any diary. 
10.2. Demographics and Subject Characteristics 
Demographic data will be collected during washout visit and includes age, race, education level, 
and medical, surgical and gynecological history. Subject characteristics include body weight, 
height, BMI and smoking habits. BMI will be calculated from body weight and height [BMI = 
weight (kg)/height (m²)] and will be rounded to one decimal point according to standard 
convention to judge inclusion. 
10.3. Efficacy Assessments  
VMS recording will be done as part of the screening, before assignment to the Efficacy Study 
part or the Safety Study part and during the Efficacy Study part.   
The assessment of hemostatic parameters and bone markers are applicable for subjects in the 
Efficacy Study part only. Research on genetic coagulopathies is only planned in subjects in the 
Efficacy Study part. 
The other efficacy assessments (lipid and glucose metabolism, HRQoL and TS) are applicable 
for subjects in the Efficacy Study part and subjects in the Safety Study part. 
10.3.1. Frequency and Severity of Moderate and Severe VMS 
VMS in postmenopausal women are commonly known as VMS or hot flashes/night sweats. 
The primary efficacy variables in this trial are the frequency and severity of moderate and severe 
VMS. Subjects will receive a paper diary to record the severity of any VMS on a daily basis 
during the Screening period (all subjects) and during the first 12 weeks of the treatment period 
in the Efficacy Study part. The severity scoring system of the VMS will be documented by the 
subjects in the diary using the following scores:  
1. Mild = Sensation of heat without sweating. 
2. Moderate = Sensation of heat with sweating. Able to continue activity. 
3. Severe = Sensation of heat with sweating. Causes cessation of activity. 
10.3.2. Health Related Quality of Life and Treatment Satisfaction Questionnaires  
A modified MENQOL questionnaire will be completed by the subjects during the Screening 
visit in order to define if the subjects are suitable for the Efficacy Study part or not. 
After that, the MENQOL questionnaire will be completed at Visit 1 (baseline), Visit 4 and Visit 
7 in the Efficacy study part and at Visit 1 (baseline), Visit 3 and Visit 6 in the Safety Study part 
to assess the HRQoL. The CGI questionnaire for TS will be completed at Visit 2, Visit 4 and 
Visit 7 in the Efficacy Study part and at Visit 2, Visit 3 and Visit 6 in the Safety Study part. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 101 of 150 
 
Examples of the questionnaires are provided in Appendix16.1 (MENQOL) and Appendix 16.2 
(GCI). 
10.3.3. Laboratory Assessments for Efficacy  
Samples for lipid and glucose metabolism will be taken for all subjects. Samples for 
hemostasis parameters and bone markers will be taken in the Efficacy Study part only. 
Methods for blood sample collection, processing, and shipment are described in instructional 
materials provided to investigational sites. Leftover samples will be retained after this trial for 
optional additional analyses in the context of this program or any further research on E4; details 
of the sample retention for future research as well as subject’s rights in regard to these samples 
are presented in the ICF. 
Blood samples for the assessments mentioned below need to be taken in fasting conditions (at 
least 8 hours fast). 
The following samples for lipid and glucose metabolism are to be collected at Visit 1 (baseline), 
Visit 4, and Visit 7 (EoT) in the Efficacy Study part and at Visit (baseline), Visit 3, and Visit 6 
(EoT) in the Safety Study part. 
• 
Lipid metabolism: triglycerides, HDL-cholesterol, LDL-cholesterol, total cholesterol, 
lipoprotein (a) and total cholesterol/HDL-cholesterol ratio. 
• 
Glucose metabolism: fasting glycemia, insulin, glycated hemoglobin, and HOMA-IR. 
The following samples for hemostasis and bone markers are to be collected at Visit 1 (Baseline) 
Visit 4 and Visit 7 (EoT) for subject in the Efficacy Study part:  
• 
Hemostasis parameters: prothrombin fragment 1 + 2, ETP-based activated Protein C 
sensitivity ratio (APCsr ETP), activated aPTT based activated Protein C resistance (APCr), 
anti-thrombin III, Protein-C, free Protein-S, Factor VIII, angiotensinogen and SHBG.  
For subjects who gave consent for the DNA sampling only: Factor V Leiden mutation and 
prothrombin G20210A mutations.  
• 
Bone markers: PINP, CTX-1, calcium and vitamin D 
All samples will be processed in the central laboratory for analysis.  
The results of these tests will not be reported or provided during the trial but will be included 
in the Clinical Study Report (CSR). After the end of the study, results will be provided to the 
investigator upon request. 
10.3.4. Blood Sampling for Exploratory Research 
In the context of future exploratory research on E4, three additional blood samples (including a 
DNA sample) will be taken. These extra samples are optional and will be taken only in subjects 
who provide their consent. 
One of these samples is intended to be used as DNA sample (taken at Visit 1). The second and 
third samples (taken at Visit 1 and End of Treatment Visit) are intended to be used, at sponsor 
discretion, to perform any further analyses needed in the context of this program or any further 
research on E4, in order to better understand and assess how E4 works.  

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 102 of 150 
 
The results of these future possible exploratory analyses will be shared with the investigators, 
who may inform subjects of their results if requested by them. 
These samples will be retained in a coded manner during storage and analysis process and will 
not be labeled with personal identifiers. Privacy and confidentiality of data generated in the 
future on stored samples is protected by the same standards applicable to all other clinical data 
as well as the long-term retention of samples for additional future research. 
Details of the sample retention for future research as well as subjects' rights in regard to these 
samples are presented in the ICF. 
10.3.5. Endocrine Assessment for Inclusion And Monitoring of Treatment Compliance 
During screening a blood sample will be taken for assessment of FSH and E2 to confirm 
postmenopausal status and for the assessment of TSH to exclude inadequately treated 
hyperthyroidism. If TSH is outside the normal ranges, a reflex T4 will be done. 
During the first 12 weeks of treatment with trial medication in the Efficacy Study part blood 
samples for E2 will be taken at each visit to continue monitoring the E2 levels and assess 
compliance with study requirements in terms of unauthorized concomitant therapies. 
During the first 12 weeks of treatment with trial medication in the Efficacy Study part blood 
samples for E4 analysis will be taken at each visit (except at Visit 1) to confirm treatment 
compliance. E4 analyses will only be performed at the end of the study to avoid unblinding. 
Methods for blood sample collection, processing, and shipment of these samples are described 
in instructional materials provided to investigational sites. Leftover samples will be retained 
after this trial for additional analyses in the context of this program or any further research on 
E4; details of the sample retention for future research as well as subject’s rights in regard to 
these samples are presented in the ICF. 
 
10.4. Safety Assessments 
Safety evaluations will include the following: 
• General safety will be assessed in terms of occurrence of TEAS (including SAE monitoring) 
• Physical and Gynecological examination (including TVUS for safety assessment) 
• Breast examination 
• Vital signs 
• ECG 
• Routine Clinical Laboratory Tests 
• Mammography 
• Endometrium thickness measured by TVUS (non-hysterectomized subjects only) 
• Endometrial biopsies (non-hysterectomized subjects only) 
• Vaginal bleeding events (non-hysterectomized subjects only) 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 103 of 150 
 
10.4.1. Treatment Emergent Adverse Events 
• All AEs that occur during the trial after the subject has signed the informed consent are to 
be documented in the eCRF, regardless of whether they are reported by the subject, elicited 
by Investigator questioning, detected through physical examination, or by other means. 
For adverse events of special interest see Section 10.6. 
As far as possible, each AE is described by: 
• Duration (start and end dates); 
• Start/end of trial medication; 
• Severity grade (mild, moderate, severe); 
• Investigation causality (relationship to the trial product); 
• Action(s) taken (concomitant medication, change of trial medication etc.) including start 
and end of respective action; 
• Concomitant diseases and respective medication in general; 
• Start, end and dosage of rescue medication; 
• Outcome. 
Definition of terms: 
AE 
An AE is any untoward medical occurrence (change in anatomical, physiological, or metabolic 
function) in a subject, which does not necessarily have any causal relationship with the product 
under investigation. An AE may be an exacerbation of a pre-existing medical condition that 
was present before the subject was assigned to a treatment group. 
TEAE  
Treatment emergent AEs (TEAEs) are those AEs occurring from time point of first ingestion 
of investigational product until last visit or any event already present that worsens in either 
intensity or frequency following exposure to the treatment. 
SAE or TESAE 
An SAE is any untoward medical occurrence that at any dose: 
• Results in death; 
• Is life-threatening (Note: the term “life-threatening” refers to an event in which the subject 
is at risk of death at the time of the event; it does not refer to an event which hypothetically 
may cause death if it is more severe); 
• Requires subject hospitalization or prolongation of existing hospitalization (for the purpose 
of this trial, a hospitalization is defined as a hospital stay of at least 8 hours and/or an 
overnight stay); 
• Results in persistent or significant disability/incapacity;  
• Is a congenital anomaly/birth defect; 
• Other medically important condition. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 104 of 150 
 
Events that require intervention to prevent one or more of the outcomes listed in the definition 
above are also to be considered as serious. Examples of such events are intensive treatment in 
an emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsion that 
does not result in hospitalization, or development of drug dependency or drug abuse. 
However, medical judgment has to be exercised in deciding whether an event is serious in any 
other situations considered medically relevant. 
The evaluation of the AE as serious or not serious is made independently of any attribution of 
causality. 
Events NOT considered to be SAEs are those that require: 
• Treatment, which is elective or pre-planned, for a pre-existing condition that is unrelated to 
the indication under trial and does not worsen; 
• Treatment of an emergency on outpatient basis for an event NOT fulfilling any of the 
definitions of serious given above and NOT resulting in hospital admission for the purpose 
of this trial, a hospitalization is defined as a hospital stay of at least 8 hours and/or an 
overnight stay). 
 
Expected AE 
The determination of whether an AE is expected is based on previous experience with the 
investigational product, as follows: 
1. Expected AEs are AEs that have already been identified during the investigational product’s 
use, or are a known complication of standard medical procedures that may be conducted as 
part of the clinical study. Expected AEs are described in the IB. If an event increases in 
intensity or severity from that described in the IB, it will be considered unexpected. 
2. Unexpected AEs are all other AEs which are not deemed anticipated. 
 
Suspected Unexpected Serious Adverse Reaction (SUSAR) 
A SUSAR is any unexpected AEs assessed as related to treatment that at any dose: 
1. Results in death; 
2. Is life-threatening (Note: the term “life-threatening” refers to an event in which the subject 
is at risk of death at the time of the event; it does not refer to an event which hypothetically 
may cause death if it is more severe); 
3. Requires subject hospitalization or prolongation of existing hospitalization (for the purpose 
of this trial, a hospitalization is defined as a hospital stay of at least 8 hours and/or an 
overnight stay); 
4. Results in persistent or significant disability/incapacity;  
5. Is a congenital anomaly/birth defect; 
6. Other medically important condition. 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 105 of 150 
 
AE intensity 
AE intensity is determined by the clinical Investigator based on his/her direct observations or 
the subject’s reporting: 
- 
Mild: causes no limitation of usual activities; the subject may experience slight 
discomfort 
- 
Moderate: causes some limitation of usual activities; the subject may experience 
annoying discomfort 
- 
Severe: causes inability to carry out usual activities; the subject may experience 
intolerable discomfort or pain. 
AE causality (relationship guide) 
Any AE has to be judged for causality (relationship to trial medication and relationship to trial 
procedure). 
The relationship of an AE to the trial product is to be graded by the Investigator based on the 
following: 
Table 5: Definitions for Investigator Assessment of Adverse Event Relationship to Study 
Drug 
Category 
Criteria 
Final Reporting of 
Relationship to Study 
Drug 
Highly probable 
• Event or laboratory test abnormality, with plausible time 
relationship to drug intake. 
• Cannot be explained by disease or other drugs. 
• Response to withdrawal plausible (pharmacologically, 
pathologically). 
• Event pharmacologically and/or phenomenologically 
related. 
• Rechallenge satisfactory, if necessary. 
Related 
Probable 
• Event or laboratory test abnormality, with reasonable time 
relationship to investigational drug intake. 
• Unlikely to be attributed to disease or other drugs. 
• Response to withdrawal clinically reasonable. 
• Rechallenge not required. 
Possible 
• Event or laboratory test abnormality, with reasonable time 
relationship to investigational drug intake. 
• Could also be explained by disease or other drugs. 
• Information on drug withdrawal may be lacking or unclear. 
Unlikely 
• Event or laboratory test abnormality, with a time to 
investigational product intake that makes a relationship 
improbable (but not impossible). 
• Disease or other drugs provide plausible explanations. 
Not Related 
Not Related 
• Event due to underlying or concurrent illness, 
complications, concurrent treatments or effect of another 
concurrent drug/therapy and is not associated to the 
investigational drug. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 106 of 150 
 
Handling of AEs 
If an AE occurs, appropriate diagnostic and therapeutic measures are to be taken and the trial 
product must be discontinued if appropriate. Follow up evaluations of the subject are to be 
performed until the subject recovers or until the clinical Investigator considers the situation to 
be no longer clinically significant, at the maximum of 30 days following completion of the trial. 
If clinically significant laboratory abnormalities appear at the final visit, appropriate additional 
tests may to be performed to clarify the nature of any clinically significant laboratory 
abnormalities that occur. 
AEs are monitored and registered on the AE form of the eCRF at each visit. The Investigator 
must make a determination of relatedness, seriousness, and intensity. All AEs during the study 
will be documented in the subject’s medical record. This information will then be transcribed 
on the AEs form of the eCRF by designated study personnel. Required information includes a 
description of the event, date of onset, date of resolution, the outcome, the action taken to 
manage the AE, the impact on the study drug intake, as well as relatedness, seriousness and 
intensity. In the absence of a specific diagnosis, an individual AE form has to be filled in for 
each sign or symptom. 
Persistent AEs will be entered once in the eCRF until they are resolved or if a new event has to 
be documented due to deterioration. These AEs will be carefully monitored; further details of 
monitoring of persistent AEs will be provided in the monitoring plan. If an AE is still not 
resolved at the end of the trial, this will be documented as ongoing. 
For recurrent AEs, i.e., AEs of the same nature, but with a different date of onset, an individual 
AE form must be completed for each of them. 
AEs occurring after the termination of the trial individually and/or of the trial in total are to be 
reported to the Sponsor even after the clinical trial has been finished if, in the judgment of the 
Investigator, there is an association between the event and the previous use of the product under 
investigation. 
If the AE is classified as serious, Investigators or designated study site personnel must inform 
the appropriate CRO representatives of any SAEs that occur (whether or not related to the study 
drug) in the course of the study, within 24 hours of when he/she becomes aware of it. 
All cases of VTE or arterial thromboembolism should be treated as SAE and reported in the 
same manner as SAEs. 
SAEs will be entered by the site members into the eCRF, which contains specific questions for 
serious events. The CRO Drug Safety Department and the Sponsor will receive the alert through 
automated e-mail notification. 
In case of technical problems in transmitting the SAE through the eCRF within the 24-hour 
timeline, the investigator/site representative shall complete the SAE form and e-mail to: 
 
E-mail:
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 107 of 150 
 
Follow-up information on SAEs must also be reported by the investigator by entering this 
updated information into the eCRF within the same time frames. 
If a non-serious AE becomes serious, this and other relevant follow-up information must also 
be provided to the CRO within 24 hours as described above. 
At the earliest possible date, the SAE report must be followed by a detailed report and any 
documentation that may be available, e.g., hospital case records, autopsy reports, and/or other 
pertinent documents. No private and/or confidential data that allow identification of trial 
subjects are to be transmitted. 
All the above documents will be sent by email to the Drug Safety Department of the designated 
CRO within 24 hours of receipt. The Investigator will be responsible for reporting the SAE to 
ethics committees or Institutional Review Board; the Sponsor will be responsible for reporting 
the SAE requiring expedited reporting to the respective health authorities, according to the 
national regulatory requirements. Serious adverse events are to be followed up until the subject 
recovers or until the clinical Investigator considers the situation to be no longer clinically 
significant, at the maximum of 30 days following completion of the trial. Subjects with 
disordered proliferative endometrium, hyperplasia or worse will be followed up until resolution. 
Resolution of DPE and hyperplasia should be documented by TVUS with endometrial thickness 
≤ 4 mm or an endometrial biopsy, if deemed necessary by the Investigator, repeated no earlier 
than 10-14 days after oral treatment with P4 200 mg/day for 14 days given after discontinuation 
of the subject. 
 
10.4.2. Physical and Gynecological and Breast Examination 
A physical examination will be performed for each subject during the Screening Period and at 
the EoT (Visit 7 Efficacy Study part/Visit 6 Safety Study part). 
This evaluation will include an examination of general appearance, head, eyes, ears, nose, 
throat, skin neck, lungs, breast, lymph nodes, abdomen, and the cardiovascular musculoskeletal 
and neurological systems. 
A gynecological and breast examination will be performed for each subject during the 
Screening Period and at the EoT (Visit 7 Efficacy Study part/Visit 6 Safety Study part). This 
evaluation will include a manual pelvic and breast. Additional breast examinations will be 
performed after 6 months of treatment (Visit 5 Efficacy Study part/Visit 4 Safety Study part). 
Gynecological and breast examinations will be done by trained and experienced personnel as 
per to local regulations. 
All abnormal findings in the physical, gynecological and breast examinations that are 
considered by the Investigator to be clinically significant will be recorded as AEs (Section 
10.4.1). 
10.4.3. Vital Signs 
Vital signs will include height, body weight, BMI, sitting systolic and diastolic BPs, and heart 
rate. BP will be taken at all visits; height will only be measured during screening, weight will 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 108 of 150 
 
only be measured at during screening and at the EoT visit. The same equipment (e.g., scale) 
should be used for all measurements. BMI will be calculated from the subjects height and 
weight using the formula BMI = kg/m2; where kg is the body weight in kilograms and m2 is the 
body height in meters squared. BP measurements will be made using a sphygmomanometer 
with an appropriate cuff size for the individual subject. Measurements will be taken while the 
subject is seated after at least 5 minutes at rest. Other procedures should not be performed during 
the time of the blood pressure and pulse rate measurements. BP measurements at screening may 
be repeated if values are outside the inclusion criteria after sitting for an additional 5 to 10 
minutes. The last reading will be used for eligibility. 
All abnormal findings in vital signs that are considered by the Investigator to be clinically 
significant will be recorded as AEs (Section 10.4.1). 
10.4.4. Electrocardiogram  
A 12-lead ECG will be recorded for each subject during screening and at the EoT Visit (Visit 7 
Efficacy Study part/Visit 6 Safety Study part). ECGs will be read locally. The ECG 
interpretation scheme will include the analysis of the morphology, rhythm, conduction, ST 
segment, PR, QRS, QT and QTc (according to QTcFrid) intervals, T waves, U waves and the 
presence or absence of any pathological changes. 
All abnormal findings in ECG that are considered by the Investigator to be clinically significant 
will be recorded as AEs (Section 10.4.1). 
10.4.5. Routine Clinical Laboratory Tests 
Methods for blood collection, processing, and shipment are described in instructional materials 
provided to investigational sites. Leftover blood samples will be retained after this trial for 
optional additional analyses in the context of this program or any further research on E4; details 
of the sample retention for future research as well as subject’s rights in regard to these samples 
are presented in the ICF.  
Blood samples for the assessments lipids and glucose need to be taken in fasting conditions (at 
least 8 hours fast). 
Samples for clinical blood tests will be taken during the Screening Period, at Visit 1 (baseline) 
and at the EoT (Visit 7 Efficacy Study part/Visit 6 Safety Study part). Samples will be processed 
in the central laboratory for analysis of the following parameters:  
• 
Hematology: hematocrit, hemoglobin, erythrocytes, platelets, leukocytes, neutrophils, 
lymphocytes, basophils, eosinophils and monocytes 
• 
Chemistry: urea, creatinine, total bilirubin, alkaline phosphatase, ALT, AST, sodium, 
potassium, chloride, bicarbonate, calcium, albumin, total protein, lactate dehydrogenase 
(LDH), LDH isoenzymes (I, II, III, IV, V), troponin, and blood glucose 
• 
A urinary pregnancy tests may be performed at the discretion of the Investigator during 
the Screening visit and during the Randomization/Treatment allocation visit (Visit 1) 
A sample for lipid and glucose parameters will be taken at washout visit or at screening visit 
and will be processed in the central laboratory for analysis of the following parameters: 
• 
Lipid parameters: triglycerides, HDL-cholesterol, LDL-cholesterol, total cholesterol 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 109 of 150 
 
• 
Glucose parameters: fasting glucose, glycated hemoglobin 
The results of the above mentioned hematology, chemistry, lipid and glucose parameters will 
be analyzed during the trial on an ongoing basis and will be reported during the trial to the 
Investigator for safety monitoring. After review of the laboratory data, the Investigator must 
sign and date each laboratory report. 
The laboratory will provide normal reference ranges for the laboratory results, and the 
laboratory report will flag all abnormal values. The Investigator will assess the clinical 
relevance of values outside the normal range and repeat, if needed, any clinically significant 
abnormal laboratory test. 
Laboratory results should only be recorded and reported as AEs if they are considered as 
clinically significant, i.e., if they are associated with clinical symptoms, require active 
management, constitute SAEs or lead to discontinuation of treatment. As far as possible, the 
clinically significant abnormal laboratory value, as such, should not be recorded on the eCRF 
pages as an AE; rather, the etiology of the abnormality should be identified, and any clinical 
signs, symptoms or diagnoses which correspond to the laboratory abnormality should be 
recorded as an AE (Section 10.4.1). 
Abnormal clinically significant laboratory values detected during screening are not considered 
AEs, but the underline disease needs to be reported in the medical history page, as appropriate. 
10.4.6. Mammography  
A digitalized mammogram will be performed during the Screening period unless a written 
report, preferably with digital imaging, demonstrating a normal result is available within 9 
months before the Screening visit. Subjects must have a Breast Imaging-Reporting And Data 
System (BI-RADS) score of 1 or 2 to enroll in the study. An incomplete mammogram result, 
i.e., BI-RADS 0, is not acceptable and requires further assessment. The site must obtain a copy 
of the official report for the subject's study file, including the digital imaging if available. 
An additional digitalized mammography will be performed at the EoT (Visit 7 Efficacy Study 
part/Visit 6 Safety Study part) or at early termination. In case of early termination, 
mammography may only be performed if the subject was treated for at least 12 weeks and the 
last mammography was taken at least 9 months earlier. 
The mammogram will be used for exploratory evaluation of the change from baseline to EoT 
in breast density in subjects who have a paired digitalized mammography. 
More information is to be found in the Imaging Manual. 
10.4.7. Transvaginal Ultrasound in Non-hysterectomized Subjects 
A TVUS will be performed during screening and at the EoT visit in all subjects who still have 
uterus and/or ovaries for general safety assessment. 
In addition TVUS will be performed to monitor the endometrium thickness during the treatment 
at Visit 4, Visit 5 and Visit 6 in the Efficacy Study part and Visit 3, Visit 4 and Visit 5 in the 
Safety Study part. The TVUS will be done in the sagittal section showing the cervix including 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 110 of 150 
 
the cervical canal. The TVUS will be assessed by the Investigator, gynecologist or designee and 
bi-layer endometrial thickness will be measured. 
10.4.8. Endometrial Biopsy in Non-hysterectomized Subjects 
Endometrial biopsies will be used for the evaluation of endometrial safety of non-
hysterectomized subjects participating in the trial. An endometrial biopsy will be performed for 
all non-hysterectomized subjects at screening (Visit 1), at the EoT Visit (Visit 7 in the Efficacy 
Study part and Visit 6 in the Safety Study part) or upon early termination providing that the 
subject has been treated with E4 or placebo for at least 12 weeks. 
Additional unscheduled endometrial biopsies will be performed in case a bi-layer endometrial 
thickness of >10 mm is detected on TVUS and/or in the presence of persistent and/or recurrent 
bleeding, confirmed by the investigator. 
Detailed procedures for obtaining endometrial biopsies, tissue preparation and endometrial 
biopsy assessment will be described in separate study manuals to be provided to the sites, a 
summary is provided below. 
Procedure for obtaining endometrial biopsy 
The endometrial biopsy should be taken according to local procedures by a board-certified 
gynecologist or designee, preferably using the Pipelle technique. The “Pipelle de Cornier” is 
internationally used for endometrial biopsies, because it is adequate and safe. However, 
comparable devices may exist and be used, providing that they are adequate for sampling and 
not traumatizing. The screening biopsy should have sufficient endometrial tissue for diagnosis. 
If insufficient tissue specimen or if no tissue is obtained, the endometrial biopsy may be 
repeated once. 
Subjects can be given sedation and/or pain relief prior to the procedure under standard local 
care providing that the medications are not listed as unauthorized therapies in Section 9.6.2. and 
as per Investigator judgment. Women will receive counselling to include discharge instructions 
and to expect mild to moderate bleeding that can last for a few days and will be advised to call 
the site if they experience fever, pain or increased or prolonged bleeding. If an abnormal uterine 
bleeding occurs within 10 weeks after a first normal endometrial biopsy, a thorough 
gynecological examination, a TVUS, and – if necessary – a second endometrial biopsy will 
have to be performed. 
Evaluation of endometrial tissue 
The endometrial tissue obtained by endometrial biopsy at screening, during the conduct of the 
study, and at the end-of-study will be processed in the same manner by a central laboratory. 
Biopsies obtained during screening and during the conduct of the study, including end-of-study 
biopsies, will be initially read by one safety pathologist for safety diagnosis only. The 
Investigator will decide inclusion or discontinuation of the subject from the study based on the 
initial reading. Screening biopsies will also be read by two other expert pathologists. Biopsy 
samples collected during the conduct of study and at the end-of-study will be read by three 
expert pathologists for the final (consensus) diagnosis. These three expert pathologists are 
independent and belong to different institutions. They are blinded to treatment and to each 
other’s readings. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 111 of 150 
 
The investigator will be informed if subsequent readings impact the initial decision. A reading 
of disordered proliferative endometrium, hyperplasia (simple or complex, with or without 
atypia) or carcinoma from any of the pathologists providing the safety readings will prompt the 
exclusion or discontinuation of the subject from the study. 
The concurrence of two of the three pathologists who read the biopsy samples for final 
(consensus) diagnosis will be accepted as the Final Diagnosis. If there is no agreement among 
the three pathologists, the most severe pathologic diagnosis, i.e., carcinoma > atypical complex 
hyperplasia > atypical simple hyperplasia > complex hyperplasia > simple hyperplasia > benign 
endometrium will be used as the final diagnosis. The Final Diagnosis will be used for the 
analysis of the endometrial safety objective.   
Standardized criteria as provided in Blaustein’s pathology text (Pathology of the Female Genital 
Tract 8, see Appendix 16.3) will be used for the characterization of the endometrial tissue. 
Endometrial polyps will be fully characterized as to the glandular proliferation and atypia (see 
Appendix 16.3 for additional histologic characteristics of the specimen). 
Procedure in case of disordered proliferative endometrium, hyperplasia or worse 
If a biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study drug 
will be discontinued, and the subject will be withdrawn from the study. The subject will be 
treated with P4 200 mg once daily for 14 days. If the endometrial event has not resolved, 
treatment with a progestin will be started according to local practice/guidelines. The subject 
will be followed up (i.e. collect diagnostic and therapeutic assessment(s)) until resolution, even 
if after study termination. The diagnostic assessment which proves resolution must be reported 
to the Sponsor even after study termination. In case of disordered proliferative endometrium, 
hyperplasia or worse, the event must be reported as an Adverse Event of Special Interest (AESI) 
(Section 10.6) and the Sponsor will be notified. 
10.4.9. Vaginal Bleeding Events 
The non-hysterectomized subjects will record daily the vaginal bleeding events in the paper 
diary for the evaluation of the bleeding pattern (spotting, presence, absence). 
Absence or occurrence of vaginal bleeding/ spotting on a daily basis will be assessed using the 
scale below: 
0 =  Absence of vaginal bleeding or spotting; 
1 =  Spotting: evidence of minimal blood loss requiring none or at most one pad, tampon  or 
panty liner per day; 
2 =  Bleeding: evidence of blood loss requiring more than one pad, tampon or panty liner per 
day. 
Subjects are advised to inform the clinic in case of abnormal uterine bleeding. 
Vaginal bleeding events grade 2 must be reported as AESIs (Section 10.6). 
10.4.10. Endometrial Safety Assessment and Stopping Rules 
A baseline endometrial biopsy will be obtained for all the non-hysterectomized subjects during 
the Screening period. An end-of-treatment endometrial biopsy is planned at the EoT Visit (Visit 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 112 of 150 
 
7 in the Efficacy study part and Visit 6 in the Safety Study part) providing that the subject has 
been treated with E4 for at least 12 weeks. 
Endometrial thickness will be measured at screening, Visit 4, Visit 5, Visit 6 and Visit 7 (EoT) 
in the Efficacy Study part and at screening, Visit 3, Visit 4, Visit 5 and Visit 6 (EoT) in the 
Safety Study part and subjects will record daily the vaginal bleeding events. If after the first 4 
weeks of treatment, subject presents with a bi-layer endometrial thickness >10 mm as assessed 
by TVUS and/or presents with persistent and/or recurrent bleeding, confirmed by the 
investigator, an endometrial biopsy will be performed. If the biopsy shows disordered 
proliferative endometrium, hyperplasia or worse, the study drug will be discontinued, and the 
subject will be withdrawn from the study. The subject will be treated with P4 200 mg once daily 
for 14 days. At approximately 10-14 days after completion of the progestin treatment, a new 
TVUS will be performed to measure endometrial thickness and a biopsy will be carried out if 
deemed necessary by the Investigator to demonstrate the resolution. If the endometrial event 
has not resolved, treatment will be started according to local practice/guidelines. The pathologic 
diagnoses of DPE, hyperplasia and worse will be reported as AESI and the subject will be 
followed up until the resolution of the event. Resolution of disordered proliferative 
endometrium and hyperplasia should be documented by TVUS with endometrial thickness ≤4 
mm or an endometrial biopsy repeated no earlier than 10-14 days after oral treatment with P4 
200 mg/day for 14 days given after discontinuation of the subject. 
Information on the treatment (including surgical treatment) and any additional pathology results 
will be reported.   
Endometrial safety will be monitored during the trial by a DSMB (see Section 10.5).  
10.5. Data Safety Monitoring Board  
An independent DSMB will be established to perform ongoing safety surveillance. The voting 
members on the DSMB are external to the Sponsor and are not involved with the trial in any 
other way and have no competing interests that could affect their roles with respect to the trial. 
During the conduct of the study, the safety data of the study will be collected on an ongoing 
basis and provided to the DSMB. The DSMB will monitor the general safety and the 
endometrial safety of the trial at an appropriate frequency as provided in the DSMB charter and 
will make recommendations to the Sponsor regarding steps to ensure the safety of the trial 
subjects. The DSMB will be supported by a Clinical Event Committee who will adjudicate 
cardiovascular and thrombotic events in a consistent and unbiased manner throughout the 
course of the study. 
More details on the DSMB, including the composition, responsibilities, timing and organization 
of the meetings and administrative structure will be described in a separate DSMB charter. 
10.6. Adverse Events of Special Interest  
AESIs are a subset of AEs that include the events listed below: 
• Vaginal bleeding events grade 2 as assessed using the vaginal bleeding events scale (see 
section 10.4.9);  
• Events resulting from endometrial biopsy reading limited to: 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 113 of 150 
 
o Disordered proliferative endometrium;  
o Simple hyperplasia without atypia;  
o Complex hyperplasia without atypia; 
o Simple hyperplasia with atypia; 
o Complex hyperplasia with atypia; 
o Carcinoma. 
In order to facilitate the documentation of AESIs in the eCRF, the event term on the report 
should be listed as above. In addition, a standard questionnaire will be available to investigators 
in the eCRF in the SAE screen as guidance on the information to be reported.   
The standard time period for collecting and recording of AESIs will begin at the first intake of 
study drug/placebo and will end at EoT visit. AESIs will be reported to the Sponsor within the 
same timeframes described for SAEs. AESI collection should start as of the Ministry of Health 
and/or IRB/EC approval date of Protocol Version 6.0 (Amendment 5). There is no need for 
retrospective reporting.  
Once an onset of a new AESI or exacerbation of a pre-existing AESI is identified (serious or 
non-serious) in a study subject, the investigator (or designee) must complete the information in 
the SAE screens of the eCRF WITHIN 24 HOURS after he/she becomes aware of the diagnosis. 
A field on the SAE screen allows to specify that the event is an AESI and whether it is serious 
or non-serious. These screens will always be completed as thoroughly as possible with all 
available details of the event, in accordance with the AESI standard questionnaire provided in 
the eCRF. Even if the investigator does not have all information regarding an AESI, the screens 
should still be completed within 24 hours. Once additional relevant information is received 
regarding the AESI, the eCRF should be updated WITHIN 24 HOURS. The investigator will 
always provide an assessment of causality at the time of the initial report. 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 114 of 150 
 
11. 
DATA COLLECTION, RECORD KEEPING AND QUALITY 
ASSURANCE 
Detailed procedures will be separately provided in the data management, monitoring, and 
quality plans. 
11.1. Data Collection 
All subject data have to be reported on the eCRFs in an anonymous fashion. Subjects are 
identified only by screening number. 
The Investigator will be responsible for the completeness, accuracy, and legibility of the 
information in the eCRF and other trial documents. In line with Good Documentation Practice, 
the source data should be accurate, legible, attributable, original, and contemporaneous. For 
documents other than eCRF, only ballpoint pen is to be used and any change of data is to be 
done by striking out the incorrect data with a single line and dating and initialing the changes 
made to provide an audit trail. Corrections should be explained in writing, where applicable. 
Correction fluid or pencils should not be used. 
The trial monitors must check the eCRFs against the source documents for accuracy and validity 
as per the monitoring schedule, as applicable, which includes any data recorded directly on the 
eCRF (for example, no prior written or electronic record of data) which are considered source 
data. Also, any step in the creation of source data is to be identified, such as the computerized 
system used to create, modify, maintain, archive, retrieve, or transmit source data. The subject 
diary will remain as source at site and will only be source data verified by the monitors. Source 
data verification will include the eCRF data, and drug accountability. In addition, the diary data 
entered into the eCRF will be cross-checked against any additional documentation in the 
subjects’ source data. 
Upon completion of each visit, prompt eCRF completion is expected from each site to ensure 
quality of data and subject safety. Once eCRFs are completed, they will be available for remote 
review by the monitor and the designated CRO Clinical Data Management department. 
Completed eCRFs will be reviewed remotely for logical discrepancies. Missing data will be 
verified through comparison of eCRF and source data at on-site monitoring visits. If necessary, 
data queries will be used to make updates to the eCRF. The monitor will ensure that responses 
to all data queries and subsequent amendments in the eCRF documentation are made according 
to GCP guidelines. 
After termination of the trial, the Investigator will be provided with an electronic copy of the 
eCRF to be archived together with the trial documents, source data, and laboratory records for 
the time required by the national regulation. 
11.2. Protocol Deviations 
A protocol deviation is defined as an instance of failure to follow, intentionally or 
unintentionally, the requirements of the protocol. 
The Investigator has to conduct the study in accordance with the current protocol. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 115 of 150 
 
In the event that a protocol deviation occurs, the Investigator should promptly report any 
deviations that might have an impact on patient safety and data integrity to the Sponsor and to 
the IRB/EC in accordance with established IRB/EC policies and procedures. In no instance 
should this increase the subject’s risk or affect the validity of the study data. 
11.3. Confidentiality of Subject Records 
The collection and processing of personal data from subjects enrolled in this study will be 
limited to those data that are necessary to fulfil the objectives of the study. 
All subject data will be collected and processed with adequate precautions to ensure 
confidentiality and compliance with applicable data privacy protection laws and regulations. 
The confidentiality of each record with subject identification is to be guaranteed by the clinical 
Investigator. This means that the clinical Investigator must respect subject confidentiality 
concerning all data collected, and that he has to code the data so that the subject identity will be 
replaced by a unique code (containing no element such as name initials, date of birth, etc.) 
before any type of data transmission outside the investigational site. 
The IC obtained from the subject includes explicit consent for the processing of personal data 
and for the investigator/institution to allow direct access to his or her original medical records 
(source data/documents) for study- related monitoring, audit, independent ethics committee 
(IEC)/IRB review, and regulatory inspection. The IC should also address the transfer of the data 
to other entities and to other countries. 
11.4. Retention of Records 
In compliance with the ICH/GCP guidelines, the investigator/institution will maintain all eCRF 
and all source documents that support the data collected from each subject, as well as all study 
documents as specified in ICH/GCP Section 8, Essential Documents for the Conduct of a 
Clinical Trial, and all study documents as specified by the applicable regulatory requirement(s) 
in a secure place as long as needed to comply with national and international regulations. The 
investigator/institution will take measures to prevent accidental or premature destruction of 
these documents. In the event the Investigator retires, relocates, or for any other reason 
withdraws from the responsibility for maintaining records for the period of time required, 
custody of the records must be transferred to any other person who accepts responsibility for 
the records, e.g., the Sponsor, an EC, or another Investigator/institution. The sponsor must be 
notified in writing of the name and address of the new custodian prior to the transfer. Under no 
circumstance the investigator/institution shall relocate or dispose of any study documents 
before having obtained written approval from the Sponsor. 
Essential documents must be retained until at least 2 years after the last approval of a marketing 
application in an ICH region and until there are no pending or contemplated marketing 
applications in an ICH region or until at least 2 years have elapsed since the formal 
discontinuation of clinical development of the investigational product. These documents will 
be retained for a longer period if required by the applicable regulatory requirements or by an 
agreement with the sponsor. It is the responsibility of the sponsor to inform the 
investigator/institution as to when these documents no longer need to be retained. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 116 of 150 
 
These records must be made available for inspection upon reasonable request by a 
representative of the Sponsor or regulatory authorities. 
11.5. Routine Monitoring 
To provide a clear understanding of the trial protocol monitoring and to ensure protocol 
compliance, a Site Initiation Visit will be performed by a monitor prior to the start of subject 
enrollment. The aims of the trial, the trial protocol, eCRFs/patient diary, trial treatment supplies, 
and trial procedures and monitoring requirements will be explained in detail. 
The purpose of monitoring is to verify the rights and well-being of human subjects are 
protected; that trial data are accurate, complete, and verifiable with source data; and that the 
trial is conducted in compliance with the protocol, GCP, and the applicable regulatory 
requirements. A monitor assigned by the CRO will conduct regular site visits for the purpose 
of trial monitoring. The monitoring strategy will be based on the importance of the data and 
will be performed on site and centrally. Risk based monitoring will be described in the 
monitoring plan and/or other appropriate documentation according ICH E6(R2) guideline 
(EMA/CHMP/ICH/135/1995). 
The Investigator must agree to allow the trial monitor and authorized representatives of the 
CRO or the Sponsor to inspect all eCRFs and corresponding source documents (e.g., original 
medical records, subject records and laboratory raw data); to allow access to the clinical 
supplies, dispensing, and storage areas; and to agree to assist with their activities, if requested. 
The Investigator should provide adequate time and space for monitoring visits and visits of 
other Sponsor representatives. 
The monitor will query any missing or spurious data with the Investigator, which should be 
resolved in a timely manner. A monitoring log will be maintained to record each visit, the reason 
for the visit, the monitor’s signature, and the Investigator or designee’s confirmation signature. 
11.6. Site Audits 
The Sponsor or its designee may carry out a site audit at any time. Investigators will be given 
adequate notice before the audit occurs. The purpose of an audit is to confirm that the trial is 
conducted as per protocol, GCP and applicable regulatory requirements, that the rights and well-
being of the subjects enrolled have been protected, and that the data relevant for the evaluation 
of the investigational product have been captured, processed and reported in compliance with 
the planned arrangements. The Investigator will permit direct access to all trial documents, drug 
accountability records, medical records, and source data. 
Regulatory authorities may perform an inspection of the trial, even up to several years after its 
completion. If an inspection is announced, the Investigator must inform the Sponsor 
immediately. 
11.7. Database Management and Quality Control 
The CRO will be responsible for the activities associated with the data management of this trial, 
including the production of an eCRF, setting up a relevant database, along with appropriate 
validation of data and generation of queries. The eCRF will be completed by site using the EDC 
system. Automated and manual checks will be made against the data entered into eCRF to 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 117 of 150 
 
ensure completeness and consistency. Resolution of queries will be implemented in the 
database. The data will be converted into a Clinical Data Interchange Standards Consortium 
compliant format. 
AEs will be standardized for terminology and classification, using the last version of the 
Medical Dictionary for Regulatory Activities (MedDRA). Concomitant medications will be 
classified by site of action and therapeutic and clinical characteristics using the World Health 
Organization Medical Dictionary for Regulatory Activities. Versions of the dictionaries to be 
used will be documented in the Data Management Plan and the Statistical Analysis Plan (SAP). 
11.8. Archiving of Data 
The investigator/institution should maintain the trial documents according to the GCP 
guidelines and as required by the applicable regulatory requirements. The investigator must 
arrange for the storage of the trial file for the duration of at least 15 years after the completion 
or discontinuation of the trial or for at least two years after the granting of the last marketing 
authorization in an ICH region (when there are no pending or contemplated marketing 
applications) or for at least two years after formal discontinuation of clinical development of 
the investigational product. Storage of trial file may be prolonged if required by local 
regulations. Subject files and other source data must be kept for the maximum period of time 
permitted by the hospital, institution, or private practice. The investigator should notify the 
Sponsor if archiving cannot be maintained for the required archiving period. 
All data and documents must be made available if requested by relevant authorities. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 118 of 150 
 
12. 
STATISTICAL METHODS 
12.1. Statistical populations 
Analyses will be based on the Enrolled Set, the Safety Analysis Set, the Intent-to-treat (ITT) 
Set, the Endometrial Safety Analysis Set, the Per-protocol (PP) Set and the modified Intent-to-
treat (mITT) Set. The definitions of the analysis sets, with the exception of the mITT, follow 
those given in the ICH E9 guideline (CMP/ICH/363/96).  
Enrolled Set: All subjects who sign the ICF form the Enrolled Set. 
Safety Analysis Set: Randomized subjects who receive at least one dose of randomized study 
medication form the Safety Set. The Safety Analysis Set will be used for all analyses of safety, 
tolerability, and background characteristics and all analyses on this set will be based on 
treatment received. 
Endometrial Safety Analysis Set: All randomized subjects who received at least one dose of 
randomized study medication, had an evaluable biopsy at Baseline and at Month 12 (defined 
by a Visit window as on or after Day 326) or had a diagnosis of endometrial hyperplasia prior 
to Month 12. 
Intent-to-treat (ITT) Set: The ITT Set consists of all randomized subjects who receive at least 
one dose of randomized study medication. The ITT Set will be the primary analysis population 
for the efficacy analyses and all analyses on this set will be based on randomized treatment. 
Modified Intent-to-treat (mITT) Set: The mITT Set consists of all randomized subjects in the 
ITT Set excluding subjects presenting at least one of the following features: 
• 
At least one post baseline E4 concentration level below the lower limit of quantification 
• 
At least one post screening E2 concentration level is superior to 40 pg/mL 
Per-protocol (PP) Set: Subjects in the ITT Set who do not have major protocol deviations will 
form the PP Set. The major protocol deviations will be defined at the time of the evaluability 
assessment between the database soft lock and hard lock before unblinding. The PP Set will be 
used for efficacy sensitivity analyses and all analyses on this set will be based on randomized 
treatment. 
12.2. Baseline Values  
Unless otherwise noted, baseline is defined as the last non-missing value recorded prior to the 
first dose of trial drug. Unscheduled visits will be used in the determination of baseline values, 
when applicable. This also applies to the primary efficacy variables derived from the VMS data 
collected in the subject diary, but on a weekly basis as described in the definition of the primary 
efficacy variables. 
12.3. Handling of Dropouts or Missing Data  
For days with missing VMS diary information, the number and intensity of VMS will be 
imputed with the mean values of the remaining days of the respective 7-day period, but only if 
at least 4 days with VMS data in this 7-day period are available. Otherwise, no calculation of 
the primary and related secondary efficacy variables will be done for this period. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 119 of 150 
 
No method of imputation is planned for the primary efficacy variable analysis since an MMRM 
model is used. As previously stated, sensitivity analyses of the primary efficacy variables may 
resort to imputation of missing data in order to assess the robustness of the MMRM results. 
12.4. Primary Efficacy Variables 
The weekly frequency of moderate to severe VMS at baseline, week 4 and week 12, 
respectively, is defined as the total number (sum) of all recorded moderate to severe VMS 
experienced during the last 7 consecutive days during the Screening period, days 22 to 28 (week 
4) and days 78 to 84 (week 12), respectively. 
The mean severity score of VMS is defined as the arithmetic mean of the daily severity score 
values of VMS (moderate or severe) observed from days -7 to -1 at baseline and the arithmetic 
mean of the daily severity score values of VMS (mild, moderate or severe) observed during the 
days 22 to 28 and days 78 to 84 for week 4 and week 12 respectively.  
At baseline, the daily severity score is computed as [((2 x number of moderate VMS) + (3 x 
number of severe VMS))/ (total number of moderate + severe VMS)] if at least one moderate 
to severe VMS was recorded during the day. In case of documented absence of moderate to 
severe VMS during the day, the daily severity will be set to zero.  
Post-baseline (days 22 to 28 [Week 4] and days 78 to 84 [Week 12]), the daily severity score is 
computed as [((1 x number of mild VMS) + (2 x number of moderate VMS) + (3 x number of 
severe VMS))/ (total number of mild + moderate + severe VMS)] if at least one mild to severe 
VMS was recorded during the day. In case of documented absence of VMS during the day, the 
daily severity will be set to zero. 
The daily severity score as defined above will also be recomputed for post baseline weeks as 
[((2 x number of moderate VMS) + (3 x number of severe VMS)) / (total number of moderate 
+ severe VMS)] if at least one moderate or severe VMS is recorded during the day.  
The primary efficacy variables will be expressed as the changes from baseline computed as the 
differences between the estimated values at week 4 or 12 and the baseline values. 
The analysis including the mild VMS will be used for submission to the FDA, the analysis 
without the mild VMS will be used for submission to the EMA.  
12.5. Analysis of Primary Efficacy Variables 
All statistical tests will be supported by presenting estimates and 95% CIs for the respective 
treatment effects (difference to placebo). These estimates and CIs will be based on the 
respective statistical models used for the analysis. Since all analyses of the efficacy variables 
are not exploratory in nature, adjustment for multiplicity will be performed (Dunnet adjustment 
for the comparison of each active dose versus placebo). 
The primary efficacy comparison will test the following hypotheses for each treatment arm: 
• 
H0: The average change from baseline in the primary efficacy endpoint in the treatment 
arm is greater (less pronounced) or equal to the average change from baseline in the 
placebo arm  
• 
H1: The average change from baseline in the primary efficacy endpoint in the treatment 
arm is lower (more pronounced) to the average change from baseline in the placebo arm 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 120 of 150 
 
The two dose groups of E4 will be compared to placebo regarding the four co-primary 
endpoints. 
  
A comparison between treatment arms of the change from baseline at week 1 to week 12 in the 
weekly frequency of moderate to severe VMS and of the change from baseline in the weekly 
mean severity of moderate to severe VMS will be made by using Mixed-effect Models for 
Repeated Measures (MMRM). The MMRM models will include treatment, week, treatment by 
week interaction and pooled trial centers as fixed effects and baseline as a covariate. If the 
pooled center effect included in the model is significant, a treatment by center interaction will 
be included in the model. Treatment effects will be assessed at week 4 and 12 using two separate 
MMRM models (one on weekly frequency and another on weekly mean severity). 
  
The primary analysis will use the data from the ITT population. Since an MMRM model is 
used, missing data imputation is not necessary. Additional sensitivity analyses will be 
implemented in order to assess the robustness of the results. These may include sensitivity with 
respect to: 
• 
An imputation of missing data (last observation carried forward [LOCF] or other) 
• 
The modelling strategy [analysis of covariance (ANCOVA) instead of MMRM] 
• 
Confounders (for example ethnicity, hysterectomized status) 
• 
Populations studied (mITT, PP) 
• 
Any combination of the above  
In addition, all primary efficacy variables will be tabulated at baseline and at each post-
baseline time point. 
12.6. Secondary Efficacy Variables 
In addition to all the secondary efficacy endpoints previously described, all the variables 
described above for the assessment of the primary objectives are also considered as secondary 
efficacy variables when evaluated at other time points than Week 4 and Week 12.  
In addition, three VMS weekly weighted scores, taking into account both the frequency and the 
severity of VMS, will be computed based on the diary data as:  
• 
[(2 x number of moderate VMS) + (3 x number of severe VMS)] for baseline and post 
baseline visits if at least one moderate to severe VMS was recorded and zero otherwise. 
• 
[(1 x number of mild VMS) + (2 x number of moderate VMS) + (3 x number of severe 
VMS) for baseline and post baseline visits if at least one mild to severe VMS was 
recorded and zero otherwise.  
• 
[(2 x number. of moderate VMS) + (3 x number of severe VMS)] if at least one moderate 
to severe VMS was recorded for baseline visits and [(1 x number of mild VMS) + (2 x 
number of moderate VMS) + (3 x number of severe VMS)] for post baseline visits if at 
least one mild to severe VMS was recorded and zero otherwise. 
Furthermore, the severity score will also be recomputed as [((1 x number mild VMS) + (2 x 
number of moderate VMS) + (3 x number of severe VMS)) / (total number of mild + moderate 
+ severe VMS)] if at least one mild to severe VMS is recorded during the day for baseline and 
post baseline visits.   

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 121 of 150 
 
In case of documented absence of moderate to severe VMS during the day, the daily severity 
score will be set to zero. These additional efficacy variables will be derived for every week 
between baseline and week 12 based on the respective 7-day periods. All VMS related 
secondary efficacy variables will be expressed as changes from baseline computed as the 
differences of the estimated values at week 1 to 12 and the baseline values. 
The percentage of subjects with a CID in the weekly frequency of moderate to severe VMS at 
weeks 4 and 12 will be assessed according to Gerlinger et al. (31). 
12.7. Analysis of Secondary Efficacy Variables 
All VMS related secondary efficacy variables will be analyzed using the same methodology as 
for the primary efficacy variables and will be based on the data from the ITT population. 
All other secondary continuous efficacy variables will be analyzed using either an ANCOVA 
or an MMRM model depending on the type of endpoint considered. These models will be 
evaluated using data from the ITT, PP and mITT populations without imputation of data. 
Appropriate covariates may be included in the analyses. 
The other categorical efficacy variables (scores, responder analysis) will be analyzed using 
appropriate statistical methods for the comparison of each treatment arm to the placebo arm. 
In addition, all secondary efficacy variables will be tabulated at baseline and at each post-
baseline time point. 
12.8. Safety parameters (safety set) 
All safety parameters will be summarized using data from the Safety Analysis Set if not 
indicated otherwise. No formal statistical test will be performed to determine statistical 
significance between treatment groups. Continuous variables will be summarized using 
descriptive statistics (N, mean, standard deviation, median, minimum, maximum), categorical 
variables will be presented in frequency tables using counts and percentages. The frequency of 
endometrial events will be computed on the evaluable biopsies of the endometrium. The 
incidence of spotting/bleeding will be analyzed by a 28-day period. The incidence rate of 
endometrial events will be computed on the evaluable endometrium. The 95% two-sided CI 
will be computed on the observed frequency of hyperplasia/carcinoma of the endometrium. 
12.9. Timing of analysis 
The final analysis of the primary efficacy objective and the secondary efficacy objectives 1 and 
2 of the Efficacy Study part will be triggered when all subjects in the Efficacy Study part have 
completed Visit 4 and will be conducted by an independent statistician. The analyses of all other 
efficacy and safety objectives from the Efficacy and Safety Study parts will be triggered when 
the overall end of the trial is reached (i.e. when all subjects in the Efficacy Study part have 
completed Visit 8). The efficacy endpoints and the safety endpoints of the Safety Study will be 
analyzed separately from those of the Efficacy Study at the end of the Safety Study. 
A firewall group, composed of Estetra SRL representatives independent of the E4 project will 
be appointed. This group will have access to the unblinded data of final and interim analyses of 
the Efficacy Study part. Further details regarding the composition and role of the firewall group 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 122 of 150 
 
will be provided in a charter. The clinical team, the subjects and the site and sponsor personnel 
involved in the clinical evaluation of the subjects will remain blinded of the treatment 
assignment in the Efficacy Study part until the end of the study. 
 
The analyses to be done after completion of Visit 4 will be performed by an independent 
statistician. Only the overall outcome of the results will be provided to the clinical team. The 
clinical team, the subjects and the site and sponsor personnel involved in the clinical evaluation 
of the subjects will remain blinded of the treatment assignment until the end of the study. 
Blinding for the data at subject and treatment group levels will be kept through a firewall 
process that will be put in place. No individual listings with the subject numbers’ information 
or tables per treatment group will be disseminated at the time of final or interim triggered 
analysis of the Efficacy Study part. 
 
No changes in the study conduct will be undertaken regardless of the primary efficacy analysis 
results. The study will continue until its completion in order to contribute to the safety profile 
of the study medication. 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 123 of 150 
 
13. 
ETHICS 
13.1. Independent Ethics Committee/Institutional Review Board  
Site initiations will be undertaken only after the IEC/IRB has given full approval of the 
study including approval for all study related documents according to GCP and applicable local 
regulatory requirements, and the sponsor has received a copy of this approval. This approval 
letter must be dated and must clearly identify the IEC/IRB and the documents being approved. 
At the end of the study, the investigator (or sponsor where required) will notify the 
IEC/IRB about the study completion. 
13.2. Ethical Conduct of the Study 
This trial will be conducted in accordance with the Declaration of Helsinki, the ICH Guidelines 
for GCP and regulatory requirements as applicable. The Investigator agrees that the study will 
be conducted according to the protocol and these principles. The Investigator will conduct all 
aspects of this study in accordance with all national, state, and local laws of the pertinent 
regulatory authorities. Study documents will be maintained in accordance with applicable 
regulations. During the pre-trial activities, the investigator and his staff must have confirmed a 
working knowledge of the regulatory requirements of the clinical trial. 
13.3. Compensation, Insurance, and Indemnity 
Information regarding compensation, insurance, and indemnity will be provided to the 
Investigator in the Clinical Trial Agreement. Country-specific insurance will be obtained in 
accordance with local regulations. 
13.4. Subject Information and Consent 
The investigator is responsible for ensuring that the subject fully understands the nature and 
purpose of the trial. Each subject must give written consent according to local requirements 
after the nature of the study has been fully explained. The ICF(s) that is/are used must be 
approved by both the sponsor and by the reviewing IEC/IRB and be in a language that the 
subject can read and understand. The informed consent should be in accordance with principles 
that originated in the Declaration of Helsinki, current ICH and GCP guidelines, applicable 
regulatory requirements, and sponsor policy. 
Subjects will be informed that their participation is voluntary and that they may withdraw 
consent to participate at any time without giving any reason. Subjects will be told that 
alternative treatments are available if they refuse to take part and that such refusal will not 
prejudice future treatment. The subject or legally acceptable representative will be given 
sufficient time to read the ICF and the opportunity to ask questions. 
The ICFs and any subsequent amended ICFs, as well as any other written information provided 
to the subjects must be submitted for review to the EC/IRB and a favorable opinion must be 
received in advance of use. The ICF(s) must be signed before performance of any study-related 
activity. The original signed ICFs will be retained by the investigator and made available (for 
review only) to the trial monitor and auditor on request. Subjects will be given a signed copy of 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 124 of 150 
 
the informed consent. Source documentation will also document that IC was obtained prior to 
study related activity. 
The investigator is responsible for ensuring that the subject fully understands the nature and 
purpose of the trial. In general, information should be given in both oral and written form 
whenever possible. No subject should be obliged to participate in the trial. Subjects, their 
relatives, guardians (or if applicable legal representatives) must be given ample opportunity to 
inquire about the details of the trial. The information must make clear that refusal to participate 
or withdrawal from the trial at any stage is without any prejudice to any subsequent care. 
Subjects must be allowed sufficient time to decide whether or not they wish to participate. 
The ICFs and any subsequent amended ICFs, as well as any other written information provided 
to the subjects must be submitted for review to the EC/IRB and a favorable opinion must be 
received in advance of use. Informed consent will be obtained prior to the conduct of any trial-
related procedures. The original signed ICFs will be retained by the investigator and made 
available (for review only) to the trial monitor and auditor on request. Subjects will be given a 
signed copy of the informed consent. Source documentation will also document that IC was 
obtained prior to the subject’s participation in the trial. 
13.5. Protocol Amendments 
Any change of the clinical trial must be written and formally documented as an amendment to 
this protocol. Such amendments will be made jointly by the Sponsor and the CRO. No change 
to the protocol will be implemented until the Sponsor, have reviewed and approved the 
amendment. Amendments will be submitted to the EC/IRB as required by local regulations. If 
the subject information sheet and ICF are updated as a result of the amendment, the new 
approved versions will be used to re-consent currently enrolled subjects and must be provided 
to additional subjects prior to their entry into the trial. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 125 of 150 
 
14. 
CONFIDENTIALITY AND PUBLICATION POLICY 
14.1. Confidentiality of Information 
This protocol and other study documents contain trade secrets and commercial information that 
is privileged and confidential. Such information is not to be disclosed unless required by laws 
or regulations. The Investigator/CRO agrees to use this information only in conducting this 
study and is not allowed to use it for other purposes without written consent from the Sponsor. 
Results obtained from this study are the property of the Sponsor. 
By signing this clinical trial protocol, the investigator/CRO reaffirms to the Sponsor that he/she 
will maintain in confidence all information provided to him/her during the present trial or 
resulting from this trial. The investigator will only divulge such information as may be 
necessary to the EC, the members of the staff and the subjects who are involved in this trial. 
14.2. Reporting 
The CRO will perform the statistical analysis and provide the Sponsor and the investigator with 
a written CSR. Except for compelling legal reasons, neither the Sponsor nor the investigator 
will communicate to third parties any result of the clinical trial before they have both agreed on 
the results of the analysis and its interpretation. 
The final CSR of this clinical trial will be prepared and released by the Sponsor. A statement 
will be required of the investigator to confirm that the report accurately describes the conduct 
and results of the trial. This released final report may also be submitted to relevant health 
authorities to support a request for registration. 
14.3. Publication and Disclosure Policy 
The data that will be obtained in this trial will be the property of the Sponsor, who will make 
reasonable efforts to assure that the results are published in a peer-reviewed journal. As some 
of the information concerning the investigational product and development activities at the 
Sponsor may be of strictly confidential nature, any manuscript or other presentation of data 
must first be reviewed by the Sponsor before its submission. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 126 of 150 
 
15. 
REFERENCES 
1. 
Donesta Bioscience. Investigator's Brochure Estetrol. Version 9.0; March 29, 2019. 
2. 
Utian WH, Speroff L, Ellman H, Dart C. Comparative controlled trial of a novel oral 
estrogen therapy, estradiol acetate, for relief of menopause symptoms. Menopause 2005; 
12(6): 708-15. 
3. 
Rodstrom K, Bengtsson C, Lissner L, Milsom I, Sundh V, Bjorkelund C. A longitudinal 
study of the treatment of hot flushes: the population study of women in Gothenburg 
during a quarter of a century. Menopause 2002; 9(3): 156-61. 
4. 
Santoro N. Symptoms of menopause: hot flushes. Clin Obstet Gynecol 2008; 51(3): 
539-48. 
5. 
Portman DJ, Gass ML. Genitourinary syndrome of menopause: new terminology for 
vulvovaginal atrophy from the International Society for the Study of Women's Sexual 
Health and the North American Menopause Society. Menopause 2014; 21(10): 1063-8. 
6. 
Woodruff JD, Pickar JH. Incidence of endometrial hyperplasia in postmenopausal 
women taking conjugated estrogens (Premarin) with medroxyprogesterone acetate or 
conjugated estrogens alone. The Menopause Study Group. Am J Obstet Gynecol 1994; 
170(5 Pt 1): 1213-23. 
7. 
Anderson GL, Limacher M, Assaf AR, et al. Effects of conjugated equine estrogen in 
postmenopausal women with hysterectomy: the Women's Health Initiative randomized 
controlled trial. JAMA 2004; 291(14): 1701-12. 
8. 
Chlebowski RT, Hendrix SL, Langer RD, et al. Influence of estrogen plus progestin on 
breast cancer and mammography in healthy postmenopausal women: the Women's 
Health Initiative Randomized Trial. JAMA 2003; 289(24): 3243-53. 
9. 
Rossouw JE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus 
progestin in healthy postmenopausal women: principal results From the Women's 
Health Initiative randomized controlled trial. JAMA 2002; 288(3): 321-33. 
10. 
Manson JE, Aragaki AK, Rossouw JE, et al. Menopausal Hormone Therapy and Long-
term All-Cause and Cause-Specific Mortality: The Women's Health Initiative 
Randomized Trials. JAMA 2017; 318(10): 927-38. 
11. 
Crandall CJ, Hovey KM, Andrews C, et al. Comparison of clinical outcomes among 
users of oral and transdermal estrogen therapy in the Women's Health Initiative 
Observational Study. Menopause 2017; 24(10): 1145-53. 
12. 
Crandall CJ, Hovey KM, Andrews C, et al. Breast cancer, endometrial cancer, and 
cardiovascular events in participants who used vaginal estrogen in the Women's Health 
Initiative Observational Study. Menopause 2018; 25(1): 11-20. 
13. 
Visser M, Coelingh Bennink HJ. Clinical applications for estetrol. J Steroid Biochem 
Mol Biol 2009; 114(1-2): 85-9. 
14. 
Tulchinsky D, Frigoletto FD, Ryan KJ, Fishman J. Plasma estetrol as an index of fetal 
well-being. J Clin Endocrinol Metab 1975; 40: 560-7. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 127 of 150 
 
15. 
Kundu N, Wachs M, Iversen GB, Petersen LP. Comparison of serum unconjugated 
estriol and estetrol in normal and complicated pregnancies. Obstet Gynecol 1981; 58: 
276-81. 
16. 
Coelingh Bennink HJ, Holinka CF, Diczfalusy E. Estetrol review: profile and potential 
clinical applications. Climacteric 2008; 11 Suppl 1: 47-58. 
17. 
Mawet M, Maillard C, Klipping C, Zimmerman Y, Foidart JM, Coelingh Bennink HJ. 
Unique effects on hepatic function, lipid metabolism, bone and growth endocrine 
parameters of estetrol in combined oral contraceptives. Eur J Contracept Reprod Health 
Care 2015; 20(6): 463-75. 
18. 
Donesta SPRL. A multicentre dose-finding, randomised, double-blind, placebo-
controlled study to select the daily oral dose of estetrol (E4) for the treatment of 
vasomotor symptoms in postmenopausal women (E4Relief). ClinicalTrials.gov. 
NCT02834312. 
19. 
Coelingh Bennink F, Holinka CF, Visser M, Coelingh Bennink HJ. Maternal and fetal 
estetrol levels during pregnancy. Climacteric 2008; 11 Suppl 1: 69-72. 
20. 
Visser M, Holinka CF, Coelingh Bennink HJ. First human exposure to exogenous 
single-dose oral estetrol in early postmenopausal women. Climacteric 2008; 11 Suppl 
1: 31-40. 
21. 
Coelingh Bennink HJT, Zimmerman Y, Verhoeven C, Visser M, Foidart JM, Gemzell-
Danielsson K. Clinical effects of the fetal estrogen estetrol in a multiple-rising-dose 
study in postmenopausal women. Maturitas 2016; 91: 93-100. 
22. 
Holinka CF, Diczfalusy E, Coelingh Bennink HJ. Estetrol: a unique steroid in human 
pregnancy. J Steroid Biochem Mol Biol 2008; 110(1-2): 138-43. 
23. 
Abot A, Fontaine C, Buscato M, et al. The uterine and vascular actions of estetrol 
delineate a distinctive profile of estrogen receptor alpha modulation, uncoupling nuclear 
and membrane activation. EMBO Mol Med 2014; 6(10): 1328-46. 
24. 
Arnal JF, Lenfant F, Metivier R, et al. Membrane and Nuclear Estrogen Receptor Alpha 
Actions: From Tissue Specificity to Medical Implications. Physiol Rev 2017; 97(3): 
1045-87. 
25. 
Baber RJ, Panay N, Fenton ATIWG. 2016 IMS Recommendations on women's midlife 
health and menopause hormone therapy. Climacteric 2016; 19(2): 109-50. 
26. 
NAMS. The 2017 hormone therapy position statement of The North American 
Menopause Society. Menopause 2017; 24(7): 728-53. 
27. 
Hodis HN, Mack WJ. Postmenopausal hormone therapy and cardiovascular disease in 
perspective. Clin Obstet Gynecol 2008; 51(3): 564-80. 
28. 
Lobo RA, Pickar JH, Stevenson JC, Mack WJ, Hodis HN. Back to the future: Hormone 
replacement therapy as part of a prevention strategy for women at the onset of 
menopause. Atherosclerosis 2016; 254: 282-90. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 128 of 150 
 
29. 
Canonico M, Carcaillon L, Plu-Bureau G, et al. Postmenopausal Hormone Therapy and 
Risk of Stroke: Impact of the Route of Estrogen Administration and Type of 
Progestogen. Stroke 2016; 47(7): 1734-41. 
30. 
Maclennan AH, Broadbent JL, Lester S, Moore V. Oral oestrogen and combined 
oestrogen/progestogen therapy versus placebo for hot flushes. Cochrane Database Syst 
Rev 2004; (4): CD002978. 
31 
Gerlinger C, Gude K, Hiemeyer F, Schmelter T, Schäfers M. An empirically validated 
responder definition for the reduction of moderate to severe hot flushes in 
postmenopausal women. Menopause. 2012 Jul;19(7):799-803. 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 129 of 150 
 
16. 
APPENDICES 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
Page 130 of 150 
 
16.1. Menopause Specific Quality of Life (MENQOL) Questionnaire 
Modified MENQOL for screening 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 132 
16.2. Modified Clinical Global Impression  
 
The Modified CGI to be used in this study is presented below 
 
 
 
 
 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 133 
16.3. Histologic Descriptions Recommended for use when Reading Endometrial 
 
Biopsy Slides  
 
Histologic Characteristics of the Endometrium 
 
0. No tissue 
1. Tissue insufficient for diagnosis 
2. Atrophic 
3. Inactive 
4. Proliferative 
a. Weakly proliferative 
b. Active proliferative 
c. Disordered proliferative 
5. Secretory 
a. Cyclic type 
b. Progestational type (including stromal decidualization) 
6. Menstrual type 
7. Simple hyperplasia without atypia 
8. Simple hyperplasia with atypia 
9. Complex hyperplasia without atypia 
10. Complex hyperplasia with atypia 
11. Carcinoma (specify type) 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 134 
16.4. Histologic Descriptions Recommended for use when Reading Endometrial 
Biopsy Slides (Continued) 
 
Additional Histologic Characteristics 
 
If there are any polyps, please specify the type or types. 
Functional 
Atrophic 
Hyperplastic without atypia 
Hyperplastic with atypia 
Carcinomatous 
 
If there is any stromal tissue, please specify the type or types. 
Smooth muscle tissue, normal 
Features suggestive of adenomyoma 
Features suggestive of stromal nodule 
Sarcoma (specify type) 
 
If there is any metaplasia, please specify the type or types. 
Squamous 
Papillary 
Eosinophilic 
Ciliated 
Mucinous 
Syncytial 
Other type (specify type) 
 
If there is any cervical tissue, please specify the type or types. 
Fragments of negative cervical epithelium 
Endocervical polyp 
Atypical endocervical glandular epithelium 
Atypical squamous metaplasia 
Squamous dysplasia 
Cervical carcinoma 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 135 
16.5. Country Specific Requirements 
16.5.1. Requirements for Canada 
This appendix includes specific changes to the study protocol as required by Health 
Canada (
). These changes are to 
be implemented and taken into account in Canadian sites conducting the MIT-Do001-
C302 study (E4Comfort Study II). 
Concerning the STUDY POPULATION 
In Canadian sites, only Hysterectomized women will be eligible to participate in both 
parts of the study, Efficacy and Safety study parts. 
 
Concerning Section 6.1.3 on Exploratory Analyses of the Efficacy Study Part, 
second exploratory analysis 
Subjects from Canadian sites will not participate in the second exploratory analysis of 
the Efficacy Study part intending to evaluate the influence of E4 on the changes in the 
hemostasis parameters compared to placebo in women with inherited thrombophilia 
and women without this genetic disorder. 
Therefore, the following parameter of the efficacy endpoint for exploratory objective 
#2 will not be assessed in subjects from Canadian sites: 
• Change in hemostasis parameters in women with and without inherited 
thrombophilia (Factor V Leiden mutation and prothrombin G20210A 
mutations). 
The DNA sample taken at Visit 1, as mentioned in Section 10.3.4, will therefore not be 
collected.  
Local ICF will reflect this change to the study protocol. 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 136 
16.6. Protocol Amendment – Summary of changes 
Modified sections in Protocol: MIT-Do001-C302 Amendment 7 
This section summarizes the changes that have been performed between Protocol Version 7.1, 
date July 30, 2021, and Protocol Version 8, date June 21, 2022. 
 
Note: wherever applicable, deleted text has been crossed out and added text has been 
printed in bold and italic characters. 
Minor changes involving grammar, wordsmithing, punctuation, and other editorial 
changes have been made throughout the document. All are clearly identified in the track-
changes version of the amendment but are not shown in the Summary of changes.  
[[Updates in Section “1. ADMINISTRATIVE INFORMATION” are also absent from the 
Summary of changes but are clearly identified in the track-changes version of the 
amendment.]] 
 
2. 
STUDY SYNOPSIS 
 
Study Design: 
[…] 
Figure 1: Trial Schedule 
[…] 
* […] If the biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study drug will be discontinued, and the subject will be 
withdrawn from the study. […] 
Other trial medication (dose, and mode of administration): 
For non-hysterectomized subjects only:  
• 
P4, 200 mg, oral, once daily for 14 consecutive days (after completion or discontinuation of E4/placebo 
treatment)  
• 
Ad hoc treatment with a progestin according to local practice/guidelines if endometrial biopsy shows 
disordered proliferative endometrium, hyperplasia or worse 
Endometrial Safety Assessment and Stopping Rules (non-hysterectomized subjects only): 
[…] If the biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study drug will be 
discontinued, and the subject will be withdrawn from the study. The subject will be treated with P4 200 mg 
once daily for 14 days. At approximately 10-14 days after completion of the progestin treatment, a new TVUS 
will be performed to measure endometrial thickness and a biopsy will be carried out, if deemed necessary by 
the Investigator, to demonstrate the resolution. If the endometrial event is not resolved, the subject will be 
further treated according to local practice/guidelines and she will be followed up until resolution. In case of an 
AESI of DPE, the subject will be followed up in the trial with regular TVUS according to the Protocol. If a 
DPE persists at the end-of-study biopsy, treatment with P4 200 mg for 14 days will be given according to the 
Protocol and resolution should be documented by an endometrial biopsy repeated 10 14 days after treatment 
with P4. TVUS is to be performed before biopsy to measure endometrial thickness. […] 
Inclusion Criteria: 
[…] 
5. For non-hysterectomized subjects: Endometrial biopsy taken during screening that reveals no abnormal 
results, i.e., presence of hyperplasia (simple or complex, with or without atypia), and presence of carcinoma, 
and presence of disordered proliferative endometrium findings. The screening biopsy should have sufficient 
endometrial tissue for diagnosis. Biopsies without tissue or with insufficient tissue may be repeated once; 
[…] 
Exclusion Criteria: 
[…] 
4. For non-hysterectomized subjects:  
a) History or presence of uterine cancer or, endometrial hyperplasia, or disordered proliferative 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 137 
endometrium; 
[…] 
Synopsis Final Version 7.1, July 30, 20218.0, June 21, 2022 
[…] 
5.4. 
Risk-benefit Assessment 
[…] An endometrial biopsy is part of the screening procedures in non-hysterectomized women 
and any findings indicating polyps, disordered proliferative endometrium, endometrial 
hyperplasia or cancer will result in exclusion from enrollment. […] If the biopsy shows 
disordered proliferative endometrium, hyperplasia or worse, the study drug will be 
discontinued, and the subject will be withdrawn from the study.  
[…] 
7. 
CLINICAL TRIAL PLAN 
7.1. 
Clinical Trial Design and Justification 
7.1.1. Trial Design 
[…] 
Figure 1: Trial Schedule 
[…] 
* […] If the biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study drug will be discontinued, and the subject will be withdrawn 
from the study. […] 
[…] 
8.2. 
Inclusion and Exclusion Criteria 
8.2.1. Criteria for Inclusion 
[…] 
5. 
For non-hysterectomized subjects: endometrial biopsy taken during screening that 
reveals no abnormal result, i.e., presence of hyperplasia (simple or complex, with or without 
atypia), and presence of carcinoma, and presence of disordered proliferative endometrium 
findings. The screening biopsy should have sufficient endometrial tissue for diagnosis. Biopsies 
without tissue or with insufficient tissue may be repeated once; 
[…] 
 
8.2.2. Criteria for Exclusion 
[…] 
4. 
For non-hysterectomized subjects:  
a. 
History or presence of uterine cancer or, endometrial hyperplasia, or disordered 
proliferative endometrium; 
[…] 
8.3. 
Subject Discontinuation and Replacement Procedures 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 138 
[…] 
• 
Endometrium biopsy showing a disordered proliferative pattern, hyperplasia or worse, 
requiring treatment with a progestin 
• 
[…] 
 
10.4. Safety Assessments 
[…] 
10.4.1. Treatment Emergent Adverse Events 
[…] Subjects with disordered proliferative endometrium, hyperplasia or worse will be 
followed up until resolution. 
Resolution of DPE and hyperplasia should be documented by TVUS with endometrial 
thickness ≤ 4 mm or an endometrial biopsy, if deemed necessary by the Investigator, repeated 
no earlier than 10-14 days after oral treatment with P4 200 mg/day for 14 days given after 
discontinuation of the subject. 
In case of an AESI of DPE, the subject will be followed up in the trial with regular TVUS 
according to the Protocol. If a DPE persists at the end of study biopsy, treatment with P4 200 
mg for 14 days will be given according to the Protocol and resolution should be documented 
by an endometrial biopsy repeated 10 14 days after treatment with P4. 
[…] 
10.4.8. Endometrial Biopsy in Non-hysterectomized Subjects 
[…] 
The investigator will be informed if subsequent readings impact the initial decision. A reading 
of disordered proliferative endometrium, hyperplasia (simple or complex, with or without 
atypia) or carcinoma from any of the pathologists providing the safety readings will prompt 
the exclusion or discontinuation of the subject from the study. 
[…] 
Procedure in case of disordered proliferative endometrium, hyperplasia or worse 
If a biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study drug 
will be discontinued, and the subject will be withdrawn from the study. […] 
10.4.10. 
Endometrial Safety Assessment and Stopping Rules 
[…] If the biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study 
drug will be discontinued, and the subject will be withdrawn from the study. The subject will 
be treated with P4 200 mg once daily for 14 days. At approximately 10-14 days after completion 
of the progestin treatment, a new TVUS will be performed to measure endometrial thickness 
and a biopsy will be carried out if deemed necessary by the Investigator to demonstrate the 
resolution. If the endometrial event has not resolved, treatment will be started according to local 
practice/guidelines. The pathologic diagnoses of DPE, hyperplasia and worse will be reported 
as an AESI and the subject will be followed up until the resolution of the event. Resolution of 
disordered proliferative endometrium and hyperplasia should be documented by TVUS with 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 139 
endometrial thickness ≤4 mm or an endometrial biopsy repeated no earlier than 10-14 days 
after oral treatment with P4 200 mg/day for 14 days given after discontinuation of the subject. 
In case of an AESI of DPE, the subject will be followed up in the trial with regular TVUS 
according to the Protocol. If a DPE persists at the end of study biopsy, treatment with P4 200 
mg for 14 days will be given according to the Protocol and resolution should be documented 
by an endometrial biopsy repeated 10-14 days after treatment with P4. TVUS is to be performed 
before biopsy to measure endometrial thickness. […] 
10.5. Data Safety Monitoring Board 
[…] During the conduct of the study, the safety data of the study will be collected on an ongoing 
basis and provided to the DSMB. The DSMB will monitor the general safety and the 
endometrial safety of the trial at an appropriate frequency as provided in the DSMB charter and 
will make recommendations to the Sponsor regarding steps to ensure the safety of the trial 
subjects. The DSMB will be supported by a Clinical Event Committee who will adjudicate 
cardiovascular and thrombotic events in a consistent and unbiased manner throughout the 
course of the study. 
[…] 
 
 
 
 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 140 
Modified sections in Protocol: MIT-Do001-C302 Amendment 6.1 
This section summarizes the changes that have been performed between Protocol Version 6.0, 
date September 23, 2020, and Protocol Version 7.1, date July 30, 2021. 
 
Note: wherever applicable, deleted text has been crossed out and added text has been 
printed in bold and italic characters. 
Minor changes involving grammar, wordsmithing, punctuation, and other editorial 
changes have been made throughout the document. All are clearly identified in the track-
changes version of the amendment but are not shown in the Summary of changes.  
Updates in Section “1. ADMINISTRATIVE INFORMATION” are also absent from the 
Summary of changes but are clearly identified in the track-changes version of the 
amendment. 
 
 
2. 
STUDY SYNOPSIS 
Study Design: 
[…] 
Figure 1: Trial Schedule 
[…] 
* […] If the biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study drug will be discontinued, and the subject will be 
withdrawn from the study. The subject will be treated with P4 200 mg once daily for 14 days. If the endometrial event has not resolved, 
treatment with a progestin will be started according to local practice/guidelines and the subject will be withdrawn from the study. 
Other Trial Medication (dose, and mode of administration): 
 For non-hysterectomized subjects only:  
• 
P4, 200 mg, oral, once daily for 14 consecutive days (after completion or discontinuation of E4/placebo 
treatment)  
Ad hoc treatment with a progestin according to local practice/guidelines if endometrial biopsy shows disordered 
proliferative endometrium, hyperplasia or worse 
Conduct of the Trial: 
[…] 
Washout and screening procedures for the Efficacy Study part and Safety Study part 
[…] 
The length of the Screening period is generally up to 4 weeks, but can be extended (with prior Medical 
Monitor approval) without exceeding 68 weeks. 
[…] 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 141 
Endometrial Safety Assessment and Stopping Rules (non-hysterectomized subjects only): 
[…] 
If the biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study drug will be 
discontinued, and the subject will be withdrawn from the study. The subject will be treated with P4 200 mg 
once daily for 14 days treatment with a progestin will be started according to local practice/guidelines and the 
subject will be withdrawn from the study. At approximately 10-14 days after completion of the progestin 
treatment, a new TVUS will be performed to measure endometrial thickness and a biopsy will be carried out 
to demonstrate the resolution. In case of disordered proliferative endometrium, hyperplasia or worseIf the 
endometrial event is not resolved, the subject will be further treated according to local practice/guidelines and 
she will be followed up until resolution. In case of an AESI of DPE, the subject will be followed up in the 
trial with regular TVUS according to the Protocol. If a DPE persists at the end-of-study biopsy, treatment 
with P4 200 mg for 14 days will be given according to the Protocol and resolution should be documented by 
an endometrial biopsy repeated 10-14 days after treatment with P4. TVUS is to be performed before biopsy 
to measure endometrial thickness. 
[…] 
Objectives and Endpoints of the Efficacy Study part (Arms 1-3): 
[…] 
Secondary Objectives and Endpoints: 
1. To measure the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on the frequency and 
severity of mild, moderate toand severe VMS in postmenopausal women weekly up to 12 weeks  
Efficacy endpoints for secondary objective #1  
1.1. […] 
1.2. […] 
1.3. Percentage of subjects with 50% and 75% reduction from baseline in the weekly frequency of 
moderate to severe VMS at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12  
1.4. Percentage of subjects with 50% and 75% reduction from baseline in the weekly frequency of mild, 
moderate and severe VMS at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12  
[…] 
 
3. […] 
3.1. Change from baseline to week 12 and week 52 in prothrombin fragment 1 + 2, endogenous thrombin 
potential (ETP)-based activated Protein C sensitivity ratio (APCsr ETP) (in a subset of subjects in 
selected study centers), […] 
w) 
 
7. To evaluate the effect of treatment with E4 15 mg or E4 20 mg on vaginal bleeding in non-hysterectomized 
subjects compared to placebo 
Safety endpoints for secondary objective #7 
7.1. […] 
7.2. Number of days with bleeding and/or spotting during each 28-day cycle of treatment based on 
recording in the patient diary  
7.3. 7.2 […] 
7.4. Cumulative rates of amenorrhea defined as the percentage of women who reported consecutive 
cycles of amenorrhea for a given cycle of time  
Objectives and Endpoints of the Safety Study part (Arm 4): 
[…] 
Secondary Objectives and Endpoints: 
[…] 
4. To evaluate the effect of treatment with E4 20 mg on vaginal bleeding in non-hysterectomized subjects 
Safety endpoints for secondary objective #4 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 142 
4.1. […] 
4.2. Number of days with bleeding and/or spotting during each 28-day cycle of treatment based on 
recording in the patient diary  
4.3. 4.2. […] 
4.4. Cumulative rates of amenorrhea defined as the percentage of women who reported consecutive 
cycles of amenorrhea for a given cycle of time 
Inclusion Criteria: 
[…] 
5. For non-hysterectomized subjects: Endometrial biopsy taken during screening that reveals no abnormal 
results, i.e., presence of hyperplasia (simple or complex, with or without atypia), and presence of 
carcinoma, and presence of disordered proliferative findings. The screening biopsy should have sufficient 
endometrial tissue for diagnosis. […] 
Exclusion Criteria: 
[…] 
3. PAP test with atypical squamous cells undetermined significance (ASC-US) or higher (low-grade 
squamous intraepithelial lesion [LSIL], atypical squamous cells- cannot exclude high-grade squamous 
intraepithelial lesion [HSIL] [ASC-H], HSIL dysplastic or malignant cells) in sub-totally hysterectomized 
and non-hysterectomized subjects1. Note: ASC-US is allowed if a reflex human papilloma virus (HPV) 
testing is performed and is negative for high risk oncogene HPV subtypes 16 and 18; 
x)  
4. For non-hysterectomized subjects: 
f) 
History or presence of uterine cancer, or endometrial hyperplasia, disordered proliferative findings; 
[…] 
23. Inadequately treated hyperthyroidism with abnormal TSH and free T4 at screening. Subjects with low or 
high TSH are allowed if free T4 at screening is within normal range10; 
[…] 
Statistical populations: 
Analyses will be based on the Enrolled Set, the Safety Analysis Set, the Intent-to-treat (ITT) Set, the 
Endometrial Safety Analysis Set, the Per-protocol (PP) Set and the modified Intent-to-treat (mITT) Set.  
[…] 
Statistical Methods: 
[…] 
Safety parameters (safety set): 
[…] The 95% two-sided CI will be computed on the observed frequency of hyperplasia/carcinoma of the 
endometrium in order to show that the frequency is not superior to 2% (upper bound lower than the 2% 
threshold). 
 
Timing of analysis 
The final analysis of the primary efficacy objective and the secondary efficacy objectives 1 and 2 of the 
Efficacy Study part will be triggered when all subjects in the Efficacy Study part have completed Visit 4. An 
interim analysis of the safety objectives of the Efficacy Study part will be triggered at the same time (i.e. when 
all subjects in the Efficacy Study part have completed Visit 4). […] 
 
2.1 Schedule of Trial Procedures Efficacy Study Part 
[…] 
y) 
In the event screening requirements cannot be met within 4 weeks, the screening period may be extended with prior Medical Monitor 
approval, but screening period may not exceed 86 weeks. 
[…] 
 
2.2  
Schedule of Trial Procedures Safety Study part 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 143 
[…] 
u) 
In the event screening requirements cannot be met within 4 weeks, the screening period may be extended with prior Medical Monitor 
approval, but screening period may not exceed 8 weeks. 
 
5.4. 
Risk-benefit Assessment 
[…] 
To monitor the effect of E4 on the endometrium, transvaginal ultrasounds (TVUS) will be 
performed at multiple time points during E4 treatment to measure the endometrial thickness 
and subjects will record daily the vaginal bleeding pattern. If, after the first 4 weeks of 
treatment, a non-hysterectomized subject presents with a bi-layer endometrial thickness > 10 
mm as assessed by TVUS or presents with persistent and/or recurrent bleeding, confirmed by 
the investigator, an endometrial biopsy will be performed. In case of disordered proliferative 
endometrium, If the biopsy shows hyperplasia or worse, the study drug will be discontinued, 
and the subject will be withdrawn from the study. The subject will be treated with P4 200 
mg once daily for 14 days. If the endometrial event has not resolved, treatment with a 
progestin will be started according to local practice/guidelines and study drug will be 
discontinued. Subjects will be followed up until resolution.  
[…] 
 
6. OBJECTIVES AND ENDPOINTS 
[…] 
 
6.1.2 Secondary Objectives and Endpoints: 
 
1. To measure the effect of treatment with E4 15 mg or E4 20 mg compared to placebo on the 
frequency and severity of mild, moderate and to severe VMS in postmenopausal women 
weekly up to 12 weeks  
Efficacy endpoints for secondary objective #1  
1.1. […] 
1.2. […] 
1.3. Percentage of subjects with 50% and 75% reduction from baseline in the weekly 
frequency of moderate to severe VMS at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12  
1.4. Percentage of subjects with 50% and 75% reduction from baseline in the weekly 
frequency of mild, moderate and severe VMS at weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 
and 12  
[…] 
3. […] 
3.1. Change from baseline to week 12 and week 52 in prothrombin fragment 1 + 2, 
endogenous thrombin potential (ETP)-based activated Protein C sensitivity ratio 
(APCsr ETP) (in a subset of subjects in selected study centers), […] 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 144 
7. To evaluate the effect of treatment with E4 15 mg or E4 20 mg on vaginal bleeding in non-
hysterectomized subjects compared to placebo 
Safety endpoints for secondary objective #7 
7.1.   […] 
7.2. Number of days with bleeding and/or spotting during each 28-day cycle of treatment 
based on data in the patient diary 
7.3. 7.2 […] 
7.4. Cumulative rates of amenorrhea defined as the percentage of women who reported 
consecutive cycles of amenorrhea for a given cycle of time 
 
[…] 
 
6.2 Objectives and Endpoints of the Safety Study Part (Arm 4) 
[…] 
Secondary Objectives and Endpoints: 
[…] 
2. To evaluate the effect of treatment with E4 20 mg on vaginal bleeding in non-
hysterectomized subjects 
Safety endpoints for secondary objective #4 
4.1 
Frequency of women with vaginal bleeding and/or spotting during each 28-day cycle 
of treatment with E4 based on data in the patient diary 
4.2 
Number of days with bleeding and/or spotting during each 28-day cycle of treatment 
based on data in the patient diary 
4.3 
Frequency of women with amenorrhea (absence of any bleeding or spotting) during 
each 28-day cycle of treatment with E4 based on data in the patient diary 
4.4 
Cumulative rates of amenorrhea defined as the percentage of women who reported 
consecutive cycles of amenorrhea for a given cycle of time 
 […] 
 
7. CLINICAL TRIAL PLAN 
 
7.1 Clinical Trial Design and Justification 
 
7.1.1 Trial Design 
[…] 
Figure 2: Trial Schedule 
[…] 
* […] If the biopsy shows disordered proliferative endometriumhyperplasia or worse, the study drug will be discontinued, and the subject will be 
withdrawn from the study. The subject will be treated with P4 200 mg once daily for 14 days. If the endometrial event has not resolved, treatment 
with a progestin will be started according to local practice/guidelines and the subject will be withdrawn from the study. […] 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 145 
[…] 
Washout screening procedures for the Efficacy Study part and Safety Study part 
[…] 
The length of the Screening period is generally up to 4 weeks, but can be extended (with prior 
Medical Monitor approval) without exceeding 68 weeks. […] 
 
7.4.1 Blinding after analysis of data up to Visit 4 in the Efficacy Study part 
 
The final analysis of the primary and secondary efficacy objective of the Efficacy Study part 
and the interim analysis of the safety endpointsobjectives 1 and 2 of the Efficacy Study part are 
to be triggered when all subjects in the Efficacy Study part have completed Visit 4 and will be 
conducted by an independent statistician. […] 
 
8. 
SUBJECT SELECTION AND 
DISCONTINUATION/WITHDRAWAL OF SUBJECTS 
[…] 
8.2. 
Inclusion and Exclusion Criteria 
8.2.1. Criteria for Inclusion 
[…] 
5. For non-hysterectomized subjects: endometrial biopsy taken during screening that reveals 
no abnormal result, i.e., presence of hyperplasia (simple or complex, with or without 
atypia), and presence of carcinoma, and presence of disordered proliferative findings. […] 
 
8.2.2. Criteria for Exclusion 
[…] 
3. PAP test with atypical squamous cells undetermined significance (ASC-US) or higher (low-
grade squamous intraepithelial lesion [LSIL], atypical squamous cells cannot exclude high-
grade squamous intraepithelial lesion [HSIL] [ASC-H], HSIL dysplastic or malignant cells) 
in sub-totally hysterectomized and non-hysterectomized subjects1. Note: ASC-US is 
allowed if a reflex human papilloma virus (HPV) testing is performed and is negative for 
high risk oncogene HPV subtypes 16 and 18; 
4. For non-hysterectomized subjects: 
a) History or presence of uterine cancer, or endometrial hyperplasia, disordered 
proliferative findings; 
[…] 
23. Inadequately treated hyperthyroidism with abnormal TSH and free T4 at screening. 
Subjects with low or high TSH are allowed if free T4 at screening is within normal range21; 
[…] 
 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 146 
8.3. 
Subject Discontinuation and Replacement Procedures 
[…] Possible reasons for a subject discontinuing participation in the trial are (non-exhaustive 
list): 
• 
[…] 
• 
[…] 
• 
Endometrium biopsy showing disordered proliferative pattern, hyperplasia or worse, 
requiring treatment with a progestin  
[…] 
Wherever possible, subjects who discontinue should be seen and assessed by the investigator. 
Subjects should complete the early termination visit (Visit 7 Efficacy Study part /Visit 6 Safety 
Study part). Non-hysterectomized subjects who discontinue early, should also receive treatment 
with 200 mg P4 for 14 days and return to the clinic for the additional endometrial safety visit 
(Visit 8 Efficacy study part). If the endometrial event has not resolved by then, treatment with 
a progestin will be started according to local practice/guidelines. […] 
 
9.2. 
Timing of Administration 
[…] 
If study treatment is interrupted for 4 days and more per week, the Investigator will need 
approval from the Medical Monitor to restart the patient on study treatment if the subject has 
not received P4 200 mg (or other progestins).  
[…] 
 
9.6.2. Concomitant Medication/Therapy 
[…] 
Thyroid treatment  
Thyroid treatments are allowed as long as the subject has normal TSH levels or low or high 
TSH levels with normal free T4 at screening. 
[…] 
 
9.6.3 COVID-19 vaccination 
Subjects cannot be excluded from the trial based on vaccination status for COVID-19 
consistent with restriction for other vaccinations, which may happen in the subjects 
(influenza, tetanus and other).  
Subjects can be vaccinated with a regulatory approved COVID-19 vaccine according to 
national guidelines but should not participate in a COVID-19 clinical trial. 
Recommendation on the timing of the COVID-19 vaccination in relation to study 
assessments, due to possible acute reaction on vaccination: 
- 
The vaccination should be performed preferably after regular visits with the planned 
blood collections. 
- 
Otherwise minimum one week before regular visits with blood samples collections. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 147 
Rationale for timing restriction: the acute reaction on vaccination may cause transitory 
changes in biochemistry (liver enzymes, LDH etc.), it is not a common reaction but may 
happen. The possible transitory increase in temperature may cause changes in absorption of 
study drug, PK fluctuations etc.  
In case of any acute condition, the scheduled visit should be postponed (if no safety issues) 
until the status of the subject improves. 
The vaccination during the trial should be reported as concomitant medication. Any AE that 
occurs after vaccination should be properly reported. 
 
10.1. Experimental Flow 
[…] 
Table 4 Visit Schedule and Washout of prior medication  
[…] 
1  The week numbers for the Washout visit and Screening period may deviate. The washout visit is generally up 
to 8 weeks prior to the screening visit. The Screening period is up to 4 weeks, but may be extended to a maximum 
of 6 8 weeks.  
[…] 
 
10.1.3. Screening Visit (Within 4 weeks before Randomization/Treatment Allocation) 
[…] 
In the event screening requirements cannot be met within 4 weeks, the screening period may 
be extended with prior Medical Monitor approval, but screening period may not exceed 6 8 
weeks. 
[…] 
 
10.3.3. Laboratory Assessments for Efficacy 
[…] 
The following samples for hemostasis and bone markers are to be collected at Visit 1 (Baseline) 
Visit 4 and Visit 7 (EoT) for subject in the Efficacy Study part:  
• 
Hemostasis parameters: prothrombin fragment 1 + 2, ETP-based activated Protein C 
sensitivity ratio (APCsr ETP) (in a subset of subjects in selected study centers), […] 
 
10.4.1. Treatment Emergent Adverse Events 
[…] 
Handling of AEs 
[…] 
Subjects with disordered proliferative endometrium, hyperplasia or worse will be followed up 
until resolution. 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 148 
Resolution of hyperplasia should be documented by an endometrial biopsy repeated 10-14 
days after oral treatment with P4 200 mg/day for 14 days given after discontinuation of the 
subject. 
In case of an AESI of DPE, the subject will be followed up in the trial with regular TVUS 
according to the Protocol. If a DPE persists at the end-of-study biopsy, treatment with P4 200 
mg for 14 days will be given according to the Protocol and resolution should be documented 
by an endometrial biopsy repeated 10-14 days after treatment with P4. 
 
10.4.5. Routine Clinical Laboratory Tests 
[…] 
Samples will be processed in the central laboratory for analysis of the following parameters:  
[…] 
• Chemistry: urea, creatinine, total bilirubin, alkaline phosphatase, ALT, AST, sodium, 
potassium, chloride, bicarbonate, calcium, albumin, total protein, lactate dehydrogenase 1 
(LDH1), LDH2 isoenzymes (I, II, III, IV, V), troponin, and blood glucose 
[…] 
 
10.4.8 Endometrial biopsy in Non-hysterectomized subjects 
[…] 
Evaluation of endometrial tissue 
The endometrial tissue obtained by endometrial biopsy during screening, during the conduct of 
the study, and at the end-of-study will be processed in the same manner by a central laboratory 
and will be evaluated by three independent expert pathologists from different institutions, 
blinded to treatment group and to each other’s readings. 
Biopsies obtained during screening and during the conduct of the study, including end-of-study 
biopsies, will be initially read by one safety expert pathologist for safety diagnosis only. The 
Investigator will decide inclusion or discontinuation of the subject from the study based on the 
initial reading. Screening biopsies will also be read by two other expert pathologists. Biopsy 
samples collected during the conduct of study and at the end-of-study will then be read by the 
two other three expert pathologists for the final (consensus) diagnosis. These three expert 
pathologists are independent and belong to different institutions. They are blinded to 
treatment and to each other’s readings.  
and tThe investigator will be informed if subsequent readings impact the initial decision. A 
reading of disordered proliferative endometrium, hyperplasia (simple or complex, with or 
without atypia) or carcinoma from any of the three pathologists will prompt the exclusion or 
discontinuation of the subject from the study.  
The concurrence of two of the three pathologists who read the biopsy samples for final 
(consensus) diagnosis will be accepted as the Final Diagnosis. If there is no agreement among 
the three pathologists, the most severe pathologic diagnosis, i.e, carcinoma > atypical complex 
hyperplasia > atypical simple hyperplasia > complex hyperplasia > simple hyperplasia > 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 149 
benign endometrium, will be used as the final diagnosis. The Final Diagnosis will be used for 
the analysis of the endometrial safety objective. 
[…] 
Procedure in case of disordered proliferative endometrium or in case of hyperplasia or worse 
If a biopsy shows disordered proliferative endometrium, hyperplasia or worse, the study drug 
will be discontinued, and the subject will be withdrawn from the study. The subject will be 
treated with P4 200 mg once daily for 14 days. If the endometrial event has not resolved, 
treatment with a progestin will be started according to local practice/guidelines and the 
subject will be withdrawn from the study. […] 
 
10.4.10. 
Endometrial Safety Assessment and Stopping Rules 
[…] If the biopsy shows disordered proliferative endometriumhyperplasia or worse, the study 
drug will be discontinued, and the subject will be withdrawn from the study. The subject will 
be treated with P4 200 mg once daily for 14 days. At approximately 10-14 days after 
completion of the progestin treatment, a new TVUS will be performed to measure 
endometrial thickness and a biopsy will be carried out to demonstrate the resolution. If the 
endometrial event has not resolved, treatment will be started according to local 
practice/guidelines and the subject will be withdrawn from the study. The pathologic diagnosis 
prompting discontinuation diagnoses of DPE, hyperplasia and worse will be reported as an 
AESI and the subject will be followed up until the resolution of the event. Resolution of 
hyperplasia should be documented by an endometrial biopsy repeated 10-14 days after oral 
treatment with P4 200 mg/day for 14 days given after discontinuation of the subject. 
In case of an AESI of DPE, the subject will be followed up in the trial with regular TVUS 
according to the Protocol. If a DPE persists at the end-of-study biopsy, treatment with P4 
200 mg for 14 days will be given according to the Protocol and resolution should be 
documented by an endometrial biopsy repeated 10-14 days after treatment with P4. TVUS is 
to be performed before biopsy to measure endometrial thickness. 
[…] 
 
10.6. Adverse Events of Special Interest 
[…] 
The standard time period for collecting and recording of AESIs will begin at the first intake of 
study drug/placebo and will end at EoT visit. AESIs will be reported to the Sponsor within the 
same timeframes described for SAEs. AESI collection should start as of the Ministry of 
Health and/or IRB/EC approval date of Protocol Version 6.0 (Amendment 5). There is no 
need for retrospective reporting. 
[…] 
 
12.1. Statistical populations 

 
 
Protocol Final Version 8.0 
E4Comfort Study II 
MIT-Do001-C302 
June 21, 2022 
 
 
Confidential 
 150 
Analyses will be based on the Enrolled Set, the Safety Analysis Set, the Intent-to-treat (ITT) 
Set, the Endometrial Safety Analysis Set, the Per-protocol (PP) Set and the modified Intent-to-
treat (mITT) Set. 
 
[…] 
Endometrial Safety Analysis Set: All randomized subjects who received at least one dose of 
randomized study medication, had an evaluable biopsy at Baseline and at Month 12 (defined 
by a Visit window as on or after Day 326) or had a diagnosis of endometrial hyperplasia prior 
to Month 12. 
[…] 
 
12.8. Safety parameters (safety set) 
[…] The 95% two-sided CI will be computed on the observed frequency of 
hyperplasia/carcinoma of the endometrium in order to show that the frequency is not superior 
to 2% (upper bound lower than the 2% threshold). 
 
12.9. Timing of analysis 
The final analysis of the primary efficacy objective and the secondary efficacy objectives 1 
and 2 of the Efficacy Study part and the interim analysis of the safety endpoints of the Efficacy 
Study part are towill be triggered when all subjects in the Efficacy Study part have completed 
Visit 4 and will be conducted by an independent statistician. The analyses of all other efficacy 
and safety objectives from the Efficacy and Safety Study parts will be triggered when the 
overall end of the trial is reached (i.e. when all subjects in the Efficacy Study part have 
completed Visit 8). The efficacy endpoints and the safety endpoints of the Safety Study will 
be analyzed separately from those of the Efficacy Study at the end of the Safety Study. 
[…]