Effectiveness and Safety of Different Estradiol Replacement Therapies in Transgender Female
Phase: PHASE2
Conditions: Transgenderism
Sponsor: Washington University School of Medicine

Study Protocol Cover page 
Title:  Effectiveness, safety, and tolerability of different estradiol dosing regimens in transgender 
females. 
NCT Number:  NCT05010707 
Date:  May 26, 2022 

Washington University in St. Louis
Version Date: May 26th, 2022
Principal Investigator: Ginger Nicol, MD
Study Title: Effectiveness, safety, and tolerability of different estradiol dosing regimens in transgender females. 
Faculty Sponsor: Thomas Baranski, MD 
•
Objectives
The purpose of this open label, pilot, randomized clinical trial is to evaluate the effectiveness, safety and 
tolerability of estrogen use in transgender female and the degree of testosterone suppression achieved in this 
population when placed on sublingual 17-beta estradiol in comparison to transdermal 17-beta estradiol for 
gender afﬁrming pharmacological therapy over the ﬁrst two-years of treatment.
This study is important because, to our knowledge, this is the ﬁrst randomized clinical trial evaluating the 
effectiveness, safety, and tolerability of estrogen in transgender female. There are no previous data comparing 
the different presentation of estrogen options in transgender women; therefore, this clinical trial will determine 
the best strategies and methods required to develop a larger randomized clinical trial. This study will also 
provide information useful to close the medical knowledge gap to provide safe and competent care to 
transgender female patients.
Aims
Aim 1. In this randomized clinical trial, we will evaluate the degree of testosterone suppression by measuring 
total testosterone level in transgender female patients undergoing hormonal afﬁrming therapy randomized to 
sublingual 17 beta estradiol or transdermal 17 beta estradiol, in conjunction with spironolactone (antiandrogen), 
for a period of 2 years.
Hypothesis 1a. Transgender females undergoing hormonal afﬁrming therapy randomized to sublingual estradiol 
with multiple daily doses will have higher degree of testosterone suppression. This hypothesis is based on the 
rationale that higher peak levels obtained with sublingual estradiol will suppress the hypothalamic-pituitary axis 
to a greater degree than the more stable levels achieved with transdermal delivery. Multiple studies (15) (18) in 
cisgender women describe the daily biological rhythm of estradiol, and a diurnal cycle of estradiol exhibiting an 
early morning peak followed by two to four recurrent cycles. However, there is no information on transgender 
patients regarding best pharmacological intervention (continuous vs pulsatile) to suppress the hypothalamic-
pituitary-gonadal axis.
Aim 2. In this randomized clinical trial, we will determine the impact on liver enzymes, metabolic proﬁle, and 
thrombogenic factors in transgender female patients undergoing hormonal afﬁrming therapy randomized to 
sublingual 17 beta estradiol or transdermal 17 beta estradiol, in conjunction with spironolactone (an 
antiandrogen), for a period of 2 years.
Hypothesis 2a. Transgender female patients on sublingual estradiol will have higher levels of estrone, which 
will lead to an increase on thrombogenic factors and changes in metabolic factors. It has been determined that 
higher estrone levels in cisgender females, has been linked with a high incidence of venous thromboembolism, 
breast cancer, and endothelial inﬂammation. An explanation for the higher thrombotic risk in postmenopausal 
women includes high production of thrombin due to high levels of estrone (12). Hypothesis 2b. Higher fat mass 
after FHT will have a negative impact on insulin sensitivity. Estradiol therapy in transgender women appears to 
be associated with an increase in fat mass and decrease in lean mass, although prospectively controlled data is 
limited (11). The increase in body fat associated with estrogen therapy raises questions about the effect on 
insulin sensitivity and cardiovascular risk. 
Purpose, Background, and Rationale
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Background and Signiﬁcance
Transgender individuals experience discordance between their self-identiﬁed gender and biological birth sex 
(1).  Some transgender individuals experience gender dysphoria which the Diagnostic and Statistical Manual of 
Mental Disorders (DSM-5) deﬁnes as clinically signiﬁcant distress or impairment in social, occupational, or 

other key areas of functioning. Increasing numbers of adolescents seek care at gender identity centers in 
Western Countries (3). 
Studies using short self-reports of gender identity and its variants suggest that 0.17%-1.3% of adolescents and 
young adults identify as transgender (4). Even using the conservative estimate of 0.3%, the number of people 
living in the United States who identify as transgender is nearly 1 million. Health care for this population has 
historically been, and continues to be, overlooked by governmental, health care, and academic establishments 
(16). This has led to a wide range of health disparities and poor health outcomes compared with their cisgender 
counterparts (17). Given the widespread acknowledgment of the health care needs of transgender people, 
priority should be given to those actions that will ensure timely access to appropriate care. Such action includes, 
ﬁrst and foremost, coverage of transition care and a requirement to ensure safe, appropriate, and sensitive care 
in health centers (16) (17).
Transgender women seek feminizing hormone therapy (FHT) to reach feminine physical features consistent 
with their gender identity. The goal of FHT is to achieve feminine features and maintain estradiol levels within 
the normal female range while suppressing endogenous testosterone (5). Both the World Professional 
Association for Transgender Health (WPATH) and the Endocrine Society have created transgender-speciﬁc 
guidelines that serve as a framework for providers caring for gender minority patients. These guidelines are 
mostly based on clinical experience from experts in the ﬁeld. Guidelines for estrogen therapy in transgender 
women are loosely based on treatments used for postmenopausal women (6). Therefore, stronger evidence is 
needed to guide clinicians on best clinical practices for this population.
The current approach to FHT is not uniform or FDA approved and depends on the health care system, cost 
considerations, and differences in the regional availability of estrogens and antiandrogens. A typical regimen 
includes estrogen to provide feminizing effects in conjunction with therapy to block testosterone (antiandrogens 
or gonadotropin-releasing hormone [GnRH] analogs). Estrogen also inhibits testosterone secretion (9)(13). 
Ethinyl estradiol was previously the mainstay of most estrogen-directed therapies; this is no longer the case due 
to its increased risk of cardiovascular death and increased incidence of deep venous thrombosis (7). 17-beta 
estradiol, which can be provided in tablet, patch, and injection, is currently the preferred formulation (8). 
Androgen blockade is usually established in all transgender females undergoing feminizing therapy and is 
achieved by either using gonadotropin blockade or antiandrogens. The latter, deﬁned as medications other than 
estradiol which are used to decrease the synthesis of or actions of androgens.  In the United States, 
spironolactone is commonly prescribed. Cyproterone acetate (CPA) is not licensed for use in the United States; 
however, CPA appears to be favored in many European countries and forms standard care as part of the 
European Network for the Investigation of Gender Incongruence (ENIGI) treatment protocol (10). 
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Innovation
Transgender female patients suffer from poor health outcomes compared with their cisgender peers, mostly because 
feminizing therapy with estrogen has been based on data from postmenopausal women and expert opinion. This represents 
a critical gap in knowledge that prevents the delivery of safe and appropriate care for the transgender population. This 
proposal is innovative because the project is aimed to study the tolerability, safety, and differences in testosterone 
suppression of the most common 17-beta estradiol presentation while using the same antiandrogen (spironolactone). Any 
differences detected between the groups will be attributed to the estrogen effect based on their route of administration. 
Moreover, this randomized clinical trial will allow the collection of valuable preliminary data to determine the best 
strategies and methods required to develop a larger randomized clinical trial. This knowledge will set the stage for future 
feasible and sustainable interventions for transgender youth to improve health related outcomes.
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Methods, Research Plan, and Design
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Study design
This study will be a prospective 2-year randomized, open label, clinical trial, using daily dose sublingual versus 
twice daily dose sublingual versus transdermal 17-beta estradiol in transgender female patients.  Primary 
outcome is degree of testosterone suppression. Secondary outcome to measure safety and tolerability will 

include liver enzymes, thrombogenic, and metabolic factors. Patients will be evaluated every 6 months during 
the length of the study 
b. Subjects 
Inclusion criteria: Female transgender patients between the ages of 18 to 45 years of age who are seen at the 
Washington University Transgender Center. Patients must have met the eligibility and readiness criteria for 
gender-afﬁrming hormone therapy.
Exclusion criteria: Use of GnRH agonist for the last 12 months, history of liver disease, dyslipidemia requiring 
treatment, drug use, alcohol use (>2drinks per day), cigarette smoking, history of blood clot (venous 
thromboembolism, pulmonary embolism), history of hyperkalemia, history of Addison’s disease, concomitant 
use of eplerenone.
Patients who have been referred to the Washington University Transgender Center for feminizing hormonal 
therapy and meet the inclusion criteria, will undergo consent process before initial evaluation. Methods for 
recruitment includes phone calls, emails, letters, and EPIC slicer dicer. For consent, physical or electronic 
consent form will be provided. 
 If consent is obtained, patients will be assigned to either daily sublingual vs twice daily sublingual vs 
transdermal 17-beta estradiol. Assignment will be done through block randomization.. Using the inclusion 
criteria, our center provides care to 150-200 new transgender female patients in a year for initiation of female 
hormone therapy.
We aim to enroll a total of 70 patients. This is a pilot study, which results will guide the feasibility, design, and 
implementation of a larger clinical trial. To account for 15% of potential dropout, we have planned to enroll a 
total of 80 patients.
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Research Plan
The proposed clinical trial involves 2 research aims to be conducted over two years (Table 1). Subjects will be recruited 
from the Washington University Transgender Center. The Center is projected to see more than 1,000 patients every year, 
of which 150-200 are new transgender female patients for initiation of FHT. 
Patients who agree to participate in the study and meet inclusion criteria will undergo the evaluation described in Table 2. 
Data of interest will be collected on all patients enrolled and will be saved on a database created in Research Electronic 
Data Capture (REDCap). Information to be collected will include:
1. Demographic and anthropometric variables: Parameters will be routinely collected through chart review.
2. Laboratory testing: During each visit, we will obtain hormone levels, coagulation factors, metabolic factors, 
electrolytes level. A hematologist will review the lab results and communicate any recommended interventions 
(including stopping medication) to the participant’s treatment team.
Initial evaluation will include patient history, physical examination, and baseline labs. During the initial visit, 
patients will be started on spironolactone and 17-beta estradiol transdermal or or daily sublingual or twice daily 
sublingual based on their treatment group assignment. Our team will check hormonal levels every 4 weeks (+/- 
2 weeks) until estradiol level is within goal (see dosing guidelines) as established by standard of care. Once 
patients are started on estradiol, they will be asked to attend 6 more visits for the 2 years duration of the study as 
described in table 2. 
Dosing guidelines:
Per the current standard of treatment, all study medications are initiated at the lowest available clinical dose and titrated to 
effectiveness at the study clinician’s discretion by 6 months of study participation, using plasma total testosterone level in 
the cisgender female range (<50ng/dL) with serum estradiol level that should not excess the peak physiologic range 
100-200 pg/mL. In some cases, medication doses will be titrated at monthly study follow up visits to optimize treatment 
response.

Spironolactone:
Starting dose will be 50 mg daily
Plan to increase 50 mg monthly to standard dose of 200 mg daily
For patients randomized to transdermal (patch) estradiol:
Starting dose will be 100 mcg/24hrs
Plan to increase by 100 mcg/24hrs every month 
Max dose 400 mcg/24hrs
Goal is to achieve a serum estradiol level between 100-200 pg/mL and to suppress testosterone to 
cisgender female levels
Estradiol levels will be measured in the morning (fasting sample)
For patients randomized to daily sublingual estradiol:
Starting dose will be 2 mg daily
Plan to increase every month by 2 mg daily
Goal is to achieve serum estradiol level between 100-200 pg/mL mL and to suppress testosterone to 
cisgender female levels.
Estradiol levels will be measured in the morning (fasting sample).
For patients randomized to twice daily dose sublingual estradiol
Starting dose will be 1 mg twice daily
Plan to increase every month by 2 mg daily
Goal is to achieve serum estradiol level between 100-200 pg/mL mL and to suppress testosterone to 
cisgender female levels
Estradiol levels will be measured in the morning (fasting sample).
Table 1. Planned Research Timeline
Task
Year 1
Year 2
Year 3
1
2
3
4
1
2
3
4
1
2
3
4
Obtain 
I R B 
approv
al
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Databa
s
e 
creatio
n 
(REDC
ap)
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Recruit
ment
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•
•
Collect
ion of 
b l o o d 
sample
s
•
•
•
•
•
•
•
•
•
D a t a 
entry
•
•
•
•
•
•
•
•
•

D a t a 
cleanin
g and 
analysi
s
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•
•
•
G r a n t 
applica
tion
•
•
•
•
F i n a l 
results 
manus
cript
•
•
•
Present
a t i o n 
prepara
tion
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•
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Table 2. Description of the evaluation and laboratory testing in each visit during the clinical trial
Study visit
Baseline
6 months
+/- 2 months
12 months
+/- 2 months
18 months
+/- 2 months
24 months
+/- 2 months
Anthropometric 
measurement 
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•
•
•
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Metabolic factors
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•
•
•
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Hormone levels
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•
•
•
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Electrolytes
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•
•
•
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Liver function test
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•
•
•
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Coagulation  
factors 
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•
•
•
 
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Lipid Panel
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•
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Beneﬁt/Risk assessment
i. Beneﬁts
Direct beneﬁts from this study to participants are anticipated. During the research study, their tolerability and 
side effects will be monitored closely so any changes can be detected earlier. Also, participants may feel 
gratiﬁed by contributing to the ﬁeld. Patients will have the opportunity to help us improve services and 
treatment options for them and future generations. 
ii. Risks
Potential risks with this study are no different than the risks that may occur while receiving gender afﬁrming 
therapy. This may include changes in their lipoprotein proﬁle and triglycerides level (14) and increase risk of 
venous thromboembolism, diabetes, high blood pressure, coronary heart disease, hepatotoxicity. While the 
patient is receiving gender afﬁrming therapy, the investigators will provide careful attention to laboratory 
results. 
Laboratory testing will be obtained every 3 months for the ﬁrst year, and every 6 months on the second year. 
For this purpose, placement of an intravenous (IV) access may be required.  IV access or venipuncture may be 
associated with possible minor discomfort or pain when inserted. 
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Statistical methods and Data Analysis

Baseline characteristics and laboratory results will be reported as mean± SD, median (interquartile range), or 
percentages. Independent T-test will be used to analyze the difference between treatment groups. We may use 
Wilcoxon rank sum test in case of non-normal distribution.
g. Limitations and contingent plans 
A potential limitation to this study is the difﬁculty of recruitment since the PI is a First Year Clinical Fellow 
with a heavy clinical rotation. Our project is also supported by the director of the Washington University 
Transgender Center which will improve the recruitment of patients. Also, the co-directors of the Washington 
University Transgender Center at St Louis Children’s Hospital are supporting the project, therefore, we could 
increase our enrollment numbers by recruitment patients seen in their clinic. Another limitation is the cost of the 
entire panel of thrombogenic factors. We could address this by just testing the most valuable test (thrombin) 
which has been used in previous research to assess the thrombotic risk in postmenopausal women on hormone 
replacement therapy.
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