Clinical trial NCT06450405
Conditions: Transgenderism Reproductive Issues
Sponsor: University of California, San Diego
Start date: 2025-02-26; Completion date: 2025-03-03
Enrollment: 2 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This research study investigates whether male-level exogenous androgens inhibit the reproductive neuroendocrine axis in otherwise healthy (non-PCOS) females.

Detailed description:
Testosterone Replacement Therapy (TRT) is the mainstay of gender affirming care for transgender men (TGM) who have male gender identity after female sex assignment at birth. TGM receiving TRT over time exhibit irregular menstrual bleeding, however, the mechanism of menstrual disruption is unknown. Therefore, the investigators propose to evaluate the effect of chronic testosterone (T) exposure on ovarian hormones and pituitary gonadotropin release that determines menstrual cyclicity. The investigators will conduct a detailed study of blood reproductive hormone secretion in TGM before and during TRT as well as in untreated cisgender female (CGF) control subjects who report female gender identity congruent with female sex assignment at birth. The investigators will also perform periodic clinical and ultrasonographic evaluations.

Arms: arm 1: Transgender men/non-binary/non-gender-conforming initiating testosterone replacement therapy arm 2: Cisgender females serving as a control group
Interventions: intervention 1: Testosterone Cypionate 50 MG/ML Injectable Solution
Intervention descriptions: intervention 1: Participants in the initiating group will receive weekly doses of testosterone for gender-affirming care.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Office of IRB Administration (OIA) 
Biomedical Protocol for  
Interventional Research 
Version Date 12.08.21 
 
 
      Edited version of NIH-FDA Clinical Trial Template v1.07 
Androgen Effects on the Reproductive Neuroendocrine Axis 
Protocol Number: 808679 
National Clinical Trial (NCT) Identified Number:  NCT06450405 
Principal Investigator: Antoni Duleba, MD 
IND/IDE Number: N/A 
IND/IDE Sponsor: N/A 
Funded by: National Institutes of Health, 1R01HD111650-01 
Version Number:  v.2.1 
September 4, 2024 
 
Summary of Changes from Previous Version: 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
 
 
 
1.2, 1.3, 4.1, 
6.1, 7.2 
Removed at-home urine collection 
Replaced with in-office blood draw for 
hormones 
1.2, 1.3, 6.1 
Changed weekly ultrasound to once only during 
the month of the LH-pulse visits 
Protocol revision 
1.2, 1.3, 4.1 
Renumbered months for all groups 
For consistency and clarity 
1.2, 1.3 
Reinstated ultrasound to initial visit 
Protocol revision 
3, 6.1, 
8.1,9.4.2 
Edited endpoints 
Consistency, simplification, clarity 
All 
Grammar and typos 
Readability 


 
 
  
 
ii 
Table of Contents 
1 
PROTOCOL SUMMARY ............................................................................................................................... 1 
1.1 
Synopsis ................................................................................................................................................ 1 
1.2 
Schema ................................................................................................................................................. 2 
1.3 
Schedule of Activities (SoA) ............................................................................................................... 3 
2 
INTRODUCTION ............................................................................................................................................ 6 
2.1 
Study Rationale .................................................................................................................................... 6 
2.2 
Background ........................................................................................................................................... 6 
2.3 
Risk/Benefit Assessment .................................................................................................................... 6 
2.3.1 
Known Potential Risks ..................................................................................................... 6 
2.3.2 
Known Potential Benefits ................................................................................................ 7 
2.3.3 
Assessment of Potential Risks and Benefits ................................................................ 7 
3 
OBJECTIVES AND ENDPOINTS ................................................................................................................ 8 
4 
STUDY DESIGN ............................................................................................................................................. 9 
4.1 
Overall Design ...................................................................................................................................... 9 
4.2 
Scientific Rationale for Study Design .............................................................................................. 12 
4.3 
Justification for Dose ......................................................................................................................... 12 
4.4 
End of Study Definition ..................................................................................................................... 12 
5 
STUDY POPULATION ................................................................................................................................ 13 
5.1 
Inclusion Criteria ................................................................................................................................ 13 
5.2 
Exclusion Criteria ............................................................................................................................... 13 
5.3 
Lifestyle Considerations .................................................................................................................... 14 
5.4 
Screen Failures .................................................................................................................................. 14 
6 
STUDY INTERVENTION ............................................................................................................................ 14 
6.1 
Study Intervention(s) Administration ............................................................................................... 14 
6.1.1 
Study Intervention Description ..................................................................................... 14 
6.1.2 
Dosing and Administration ............................................................................................ 14 
6.2 
Preparation/Handling/Storage/Accountability ................................................................................ 15 
6.2.1 
Acquisition and accountability ...................................................................................... 15 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ................................................ 15 
6.2.3 
Product Storage and Stability ....................................................................................... 16 
6.2.4 
Preparation ...................................................................................................................... 16 
6.3 
Measures to Minimize Bias: Randomization and Blinding ........................................................... 16 
6.4 
Study Intervention Compliance ........................................................................................................ 16 
6.5 
Concomitant Therapy ........................................................................................................................ 16 
6.5.1 
Rescue Medicine ............................................................................................................ 16 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL .................................................................................................................... 16 
7.1 
Discontinuation of Study Intervention ............................................................................................. 16 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................................................... 17 
7.3 
Lost to Follow-Up ............................................................................................................................... 17 
8 
STUDY ASSESSMENTS AND PROCEDURES ...................................................................................... 17 
8.1 
Efficacy Assessments ....................................................................................................................... 17 
8.2 
Safety and Other Assessments ....................................................................................................... 19 
8.3 
Adverse Events and Serious Adverse Events ............................................................................... 19 
8.3.1 
Definition of Adverse Events (AE) ............................................................................... 19 
8.3.2 
Definition of Serious Adverse Events (SAE) .............................................................. 19 
8.3.3 
Classification of an Adverse Event .............................................................................. 20 
8.3.4 
Time Period and Frequency for Event Assessment and Follow-Up ....................... 21 
8.3.5 
Adverse Event Reporting .............................................................................................. 21 
8.3.6 
Serious Adverse Event Reporting ............................................................................... 21 
8.3.7 
Reporting Events to Participants ................................................................................. 21 


 
 
  
 
iii 
8.3.8 
Events of Special Interest ............................................................................................. 22 
8.3.9 
Reporting of Pregnancy ................................................................................................ 22 
8.4 
Unanticipated Problems .................................................................................................................... 22 
8.4.1 
Definition of Unanticipated Problems (UP) ................................................................. 22 
8.4.2 
Unanticipated Problem Reporting ................................................................................ 22 
8.4.3 
Reporting Unanticipated Problems to Participants ................................................... 22 
9 
STATISTICAL CONSIDERATIONS .......................................................................................................... 22 
9.1 
Statistical Hypotheses ....................................................................................................................... 23 
9.2 
Sample Size Determination .............................................................................................................. 23 
9.3 
Populations for Analyses .................................................................................................................. 23 
9.4 
Statistical Analyses ............................................................................................................................ 23 
9.4.1 
General Approach .......................................................................................................... 23 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) ............................................................. 23 
9.4.3 
Analysis of the Secondary Endpoint(s) ....................................................................... 24 
9.4.4 
Safety Analyses .............................................................................................................. 24 
9.4.5 
Baseline Descriptive Statistics ..................................................................................... 24 
9.4.6 
Planned Interim Analyses ............................................................................................. 24 
9.4.7 
Sub-Group Analyses ..................................................................................................... 24 
9.4.8 
Tabulation of Individual participant Data .................................................................... 24 
9.4.9 
Exploratory Analyses ..................................................................................................... 24 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .............................. 24 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ........................................................... 24 
10.1.1 
Study Discontinuation and Closure ............................................................................. 24 
10.1.2 
Confidentiality and Privacy ........................................................................................... 24 
10.1.3 
Future Use of Stored Specimens and Data ............................................................... 25 
10.1.4 
Key Roles and Study Governance .............................................................................. 25 
10.1.5 
Safety Oversight ............................................................................................................. 25 
10.1.6 
Clinical Monitoring .......................................................................................................... 25 
10.1.7 
Data Handling and Record Keeping ............................................................................ 26 
10.1.8 
Protocol Deviations ........................................................................................................ 26 
10.2 
Additional Considerations ................................................................................................................. 26 
10.3 
Abbreviations ...................................................................................................................................... 26 
10.4 
Protocol Amendment History ........................................................................................................... 29 
11 
REFERENCES ............................................................................................................................................. 30 
 


Androgen Effects, Duleba 
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1 
 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS  
Title: Androgen Effects on the Reproductive Neuroendocrine Axis 
Study Description: Testosterone Replacement Therapy (TRT) is the mainstay of gender affirming care 
for transgender men (TGM) who have male gender identity after female sex assignment at birth 1,2. 
TGM receiving TRT, over time exhibit irregular menstrual bleeding, however, the mechanism of 
menstrual disruption is unknown 3-5. Therefore, we propose to evaluate the effect of chronic 
testosterone (T) exposure on ovarian hormones and pituitary gonadotropin release that determines 
menstrual cyclicity. We will conduct a detailed study of blood reproductive hormone secretion in TGM 
before and during TRT as well as in untreated cisgender female (CGF) control subjects who report 
female gender identity congruent with female sex assignment at birth. We will also perform periodic 
clinical and ultrasonographic evaluations. Furthermore, we will monitor menstrual cycles and 
reproductive hormones in TGM who make the personal decision to discontinue TRT, whether it is to 
pursue fertility or for other reasons.  
Objectives: 
Primary Objective: To determine if male-level exogenous androgens inhibit menstrual 
cycles, affect reproductive hormones, and follicle dynamics in TGM receiving TRT. To 
determine if these effects are reversible in TGM discontinuing TRT. 
Secondary Objectives: (1) To determine if exogenous androgens in TGM suppress 
parameters of pulsatile LH secretion; and (2) To determine if the discontinuation of TRT 
affects the resumption of cyclicity and LH secretion in TGM. 
Endpoints: 
Primary Endpoint: Change in the proportion of ovulatory cycles and menstrual cycle length 
Secondary Endpoints: Measurement of pulsatile LH secretion, ovarian folliculogenesis. 
Study Population: Transgender men starting or stopping TRT, cisgender female control subjects 
Phase: n/a 
Description of Sites/Facilities Enrolling Participants: Department of Obstetrics, Gynecology, and 
Reproductive Sciences, UCSD. Altman Clinical and Translational Research Institute, UCSD.  
   
Description of Study Intervention: Initiation of testosterone replacement therapy and discontinuation 
of testosterone replacement therapy 
 
Study Duration: 5 years 
Participant Duration: 1 to 10 months, dependent on group assignment 
 
 


Androgen Effects, Duleba 
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1.2 
SCHEMA 
TGM Initiating TRT 
Screening Visit/Study Visit 1a: 
Informed consent, blood test. If qualified physical exam, medical history, u/s. 
Month 1 (pre-testosterone):  
Weekly visitsb | Daily diary | LH-pulse blood sampling visitc | Ultrasoundd 
Month 2 (testosterone): 
Weekly visitsb | Daily diary | 
Month 3 (testosterone): 
Weekly visitsb | Daily diary |  
Month 4 (testosterone): 
Weekly visitsb | Daily diary | LH-pulse blood sampling visitc | Ultrasoundd 
Month 5 (testosterone): 
Weekly visitsb | Daily diary |  
Month 6 (testosterone): 
Weekly visitsb | Daily diary | 
Month 7 (testosterone): 
Weekly visitsb | Daily diary | LH-pulse blood sampling visitc | Ultrasoundd 
 
TGM Discontinuing TRT 
Screening Visit/Study Visit 1a: 
Informed consent, blood test. If qualified physical exam, medical history, u/s. 
Month 1 (testosterone):  
Weekly visitsb | Daily diary | LH-pulse blood sampling visitc  | Ultrasoundd 
Month 2 (post-testosterone): 
Weekly visitsb | Daily diary |  
Month 3 (post-testosterone): 
Weekly visitsb | Daily diary | 
Month 4 (post-testosterone): 
Weekly visitsb | Daily diary | LH-pulse blood sampling visitc | Ultrasoundd 
Month 5 (post-testosterone): 
Weekly visitsb | Daily diary |  
Month 6 (post-testosterone): 
Weekly visitsb | Daily diary |  
Month 7 (post-testosterone): 
Weekly visitsb | Daily diary | LH-pulse blood sampling visitc | Ultrasoundd 
Month 8 (post-testosterone): 
Weekly visitsb | Daily diary |  
Month 9 (post-testosterone): 
Weekly visitsb | Daily diary |  
Month 10 (post-testosterone): Weekly visitsb | Daily diary | LH-pulse blood sampling visitc | Ultrasoundd 
 
CGF Control Group 
Screening Visit/Study Visit 1a: 
Informed consent, blood test. If qualified physical exam, medical history, u/s. 
Participation Month:  
Weekly visitsb | Daily diary | LH-blood blood sampling visitc | Ultrasound 
 
 
a The Screening Visit and Study Visit 1 may be performed on the same day. The participant will have a 
choice to think about participation and come back on another day. If the participant is ready to begin 
and is qualified, they can continue with the study protocol after passing the screening portion. 
b Weekly visits will include a blood test for progesterone, estradiol, LH, and FSH. 
c  LH-pulse visits will sample blood for 8 hours, with one hour of prep and discharge (total 9 hours) 
d Ultrasound will measure ovarian volume, all follicles, uterine volume, and endometrial thickness. 
 
 
 
 
 
 
 
 
 
 
 
 


Androgen Effects, Duleba 
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1.3 
SCHEDULE OF ACTIVITIES (SOA) 
TGM INITIATING TRT 
Procedures 
Screening Visit 
Study Visit 1 
 
Month 1 
Pre-Testosterone 
Testosterone Month 2  
 
Testosterone Month 3 
Testosterone Month 4 
Testosterone Month 5 
Testosterone Month 6 
Testosterone Month 7 
Final Study Visit 
Informed consent 
X 
 
 
 
 
 
 
 
 
Screening blood test 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
Medical history 
 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Randomization 
N/A 
 
 
 
 
 
 
 
 
Testosterone 
Administration 
 
 
 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Concomitant 
medication review 
 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Physical exam 
(including height and 
weight, thyroid, hip and 
waist measurement) 
 
 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Vital signs 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Bleeding Log Survey 
(home) 
 
 
Daily 
Daily 
Daily 
Daily 
Daily 
Daily 
Daily 
Hematology  
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Serum chemistry 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Pregnancy test 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
 
Pelvic ultrasound 
 
 
X 
X 
 
 
X 
 
 
X 
Adverse event review 
and evaluation 
 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
LH Pulses visit 
 
 
X 
 
 
X 
 
 
X 
Complete Case Report 
Forms (CRFs) 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly 
 
 
 


Androgen Effects, Duleba 
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TGM DISCONTINUING TRT 
Procedures 
Screening Visit 
Study Visit 1 
Month 1  
Still on testosterone 
Month 2 
Post-testosterone 
Month 3  
 Post-testosterone 
Month 4 
Post-testosterone 
Month 5 
Post-testosterone 
Month 6 
Post-testosterone 
Month 7 
Post-testosterone 
Month 8 
Post-testosterone 
Month 9 
Post-testosterone 
Final Study Visit 
Month 10 
Post-testosterone 
Final Study Visit 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Screening blood test 
X 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
Randomization 
N/A 
 
 
 
 
 
 
 
 
 
 
 
Testosterone 
Administration 
 
 
Weekly 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant 
medication review 
 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
Physical exam 
(including height and 
weight, thyroid, hip and 
waist measurement) 
 
 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
Vital signs 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
Bleeding Log Survey 
(home) 
 
 
Daily 
Daily 
Daily 
Daily 
Daily 
Daily 
Daily 
Daily 
Weekly 
Daily 
Hematology  
 
X 
Daily 
Daily 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
Serum chemistry 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
Pregnancy test 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
 
Pelvic ultrasound 
 
 
X 
X 
 
 
X 
 
 
X 
 
 
X 
Adverse event review 
and evaluation 
 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
LH Pulses visit 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
Complete Case Report 
Forms (CRFs) 
 
X 
Weekly 
Weekly 
Weekly 
Weekly 
Weekly Weekly Weekly 
Weekly 
Weekly 
Weekly 
 
 
 


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CONTROL GROUP 
Procedures 
Screening Visit 
Study Visit 1 
 
Participation Month 
Informed consent 
X 
 
 
Screening blood test 
X 
 
 
Demographics 
X 
 
 
 
Medical history 
 
 
X 
Weekly 
Randomization 
NA 
 
 
Testosterone 
Administration 
 
 
 
Concomitant 
medication review 
 
 
X 
Weekly 
Physical exam 
(including height and 
weight, thyroid, hip and 
waist measurement) 
 
 
 
X 
Weekly 
Vital signs 
 
X 
Weekly 
Bleeding Log Survey 
(home) 
 
 
Daily 
Hematology  
 
X 
Weekly 
Serum chemistry 
 
X 
Weekly 
Pregnancy test 
 
X 
Weekly 
 
Pelvic ultrasound 
 
 
X 
X 
Adverse event review 
and evaluation 
 
 
X 
Weekly 
LH Pulses visit 
 
 
X 
Complete Case Report 
Forms (CRFs) 
 
X 
Weekly 
 
 


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2 
 
INTRODUCTION 
2.1 
STUDY RATIONALE  
In the U.S. alone, ~1.4 million adults self-identify as transgender 7-10 and this prevalence is not only 
growing11 but is likely higher than reported due to selection bias and social stigma9,12-14. Among 
transgender (TG) individuals, approximately 1/2 report masculine gender identity after female sex 
assignment at birth and identify as TGM or gender non-binary people (collectively designated TGM). TRT 
(provided to typically achieve serum T in the male physiological range) is the mainstay of gender-
affirming medical care in TGM 2,15. In TGM, TRT is used to masculinize physical characteristics and 
suppress menstrual cycles4,5,16-19. Yet, TRT effects on the neuroendocrine brain and pituitary hormone 
function have been largely overlooked. Importantly, TGM may desire childbearing20,21 and many engage 
in vaginal intercourse with the potential to conceive spontaneously21-27. Yet, most TGM undergoing TRT 
become amenorrheic4,5,17 and cannot spontaneously conceive. 
Furthermore, no clinical report to date has prospectively evaluated the time-course or mechanisms of 
reversibility of TRT-induced effects on the HPO axis, including the time-course and pattern of restoration 
of normal LH pulse secretion, ovarian measures, and ovulation. Our study will systematically evaluate 
the sequence of reproductive/endocrine events following discontinuation of TRT in TGM planning to 
pursue fertility or stopping TRT for other personal reasons. 
2.2 
BACKGROUND  
High anovulation rates were recently reported in TGM undergoing TRT28, but it is unknown whether this 
ovulatory dysfunction is due, fully or in part, to altered preovulatory LH surge secretion or how T 
contributes to such pathophysiology. Aim 1 will clinically study how TRT in TGM impacts pituitary 
gonadotropin secretion and ovarian function to better understand T effects on HPG axis functioning in 
healthy individuals with ovaries, an understudied area of investigation. 
To date, little is known regarding endocrine/reproductive effects of TRT, and the limited reports have 
yielded inconsistent results, in large part due to very small sample sizes and variability in TRT doses and 
durations studied16,19,29-31. Some evidence suggests that TGM receiving prolonged T in the typical male 
range show inhibited LH16,19,32. Even less is known regarding effects of TRT on LH pulsatility. Although 
one study reported no change in LH pulse pattern after short-term (6 weeks) low-dose oral TRT30, 
another study by the same group reported a trend for reduced LH pulse frequency after longer TRT (6 
mos.; given IM) at a higher T dose considered standard for TGM16. While promising, the data in both LH 
pulse studies was variable, likely due to inadequate sample sizes (n = just 5-6/group), and hence 
statistical analyses were underpowered. In fact, most prior TGM studies unfortunately had low 
statistical power owing to very small sample sizes and, hence, even moderate variability precluded 
definitive conclusions. Our study design will rectify this with larger, statistically appropriate sample sizes 
and enhanced power. 
2.3 
RISK/BENEFIT ASSESSMENT   
2.3.1 KNOWN POTENTIAL RISKS  
Risks of this study include risk of testosterone replacement therapy, discomfort and/or swelling due to 
blood draws, and the risk of a loss of confidentiality.  
1. Testosterone: While testosterone replacement therapy is medically appropriate and considered safe 
for transgender men, treatment with testosterone carries some risks and side effects. Those undergoing 
testosterone therapy may notice thicker, oilier skin and larger pores, acne, more masculine facial 
appearance, growth of facial hair, deepening voice, hair growth on the arms, back and chest, thinning 
hair on the head, reduction in fat around hips and thighs, an increase in stomach fat, increased muscle in 


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the arms and legs, more prominent veins, an increase of sweat production, and changes in the odors of 
sweat and urine.  
Reproductive effects include menstrual changes such as lighter or heavier periods or discontinuation of 
periods as well as difficulty releasing eggs from the ovaries and a reduction in the ability to get pregnant. 
In case of pregnancy, testosterone may pose risks to the fetus.  
Other complications may include abnormal level of lipids (such as cholesterol) salt retention, increased 
blood pressure, cardiovascular disease, production of too many red blood cells, sleep apnea, and weight 
gain. 
Emotional changes may include emotional shifts or reduction of emotions; when a person receives 
testosterone for gender dysphoria, they may begin to feel more like themselves and may become more 
comfortable with their bodies. Sexual changes typically include changes in libido, an enlarged clitoris as 
well as changes in orgasms, arousal, and sexual interests. 
2. Blood draws: Placement of the IV for blood draws and single stick blood drawing can cause pain, 
discomfort, swelling, bruising, a small risk of getting an infection, and occasionally dizziness and fainting.  
3. Loss of Confidentiality: There exists the chance of loss of confidentiality associated with both 
participants and study data/specimens.  
4. Pelvic ultrasound: Either done as transabdominal or transvaginal, there is risk of discomfort during 
the procedure.  
2.3.2 KNOWN POTENTIAL BENEFITS  
We plan to enroll three groups of subjects. Each have different potential benefit profiles. 
1. TGM subjects who initiate testosterone replacement therapy will be provided medication 
(testosterone replacement therapy) for the duration of the trial (up to six months of 
testosterone replacement per subject) at no charge to the subject.  
2. Cisgender subjects will have no direct benefit from participation in the study. 
3. Transgender subjects discontinuing testosterone replacement therapy will also have no direct 
benefit from the participation in the study. 
 
Although not all individual subjects will benefit directly from participating in this research study, there is 
a significant likelihood that this research will help society by advancing knowledge of the effects of 
testosterone replacement and discontinuation of testosterone replacement on reproductive physiology.  
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
As stated in above, the risks to subjects in this study are: testosterone replacement therapy for those 
receiving it, blood draws, loss of confidentiality, and pelvic ultrasound. 
1. Testosterone. The dose of testosterone proposed in this study follows the most recently 
published standards for the health care of transgender people 1. Safety of this TRT has been 
well-documented 33,34. Testosterone replacement therapy is well tolerated. This study will enroll 
subjects who have made the decision along with their medical providers to initiate TRT for 
gender-affirming care.  
2. Blood Draws will be performed by qualified/credentialed personnel using aseptic technique to 
prevent infection. Hemoglobin will be checked in all subjects prior to enrollment to ensure that 
there is no baseline anemia nor polycythemia; individuals with hemoglobin of less than 11 g/dL 
or higher than 16g/dL will not be studied. We will test hemoglobin before each LH-pulse visit 
and disenroll subjects with hemoglobin less than 11 g/dL or higher than 16 g/dL; this timing and 
frequency of testing is consistent with recommendations to check hemoglobin every three 
months for people during their first year on testosterone therapy. They will be advised to 
follow-up with their primary care physician for complete work-up and treatment. The United 
States Health and Human Services Office for Human Research Protections recommends that in 


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healthy, nonpregnant individuals who weigh at least 110 pounds. For these subjects, the 
amount of blood drawn for clinical research purposes may not exceed 550 ml in an 8-week 
period and collection may not occur more frequently than 2 times per week. The maximum 
blood draw volume in this protocol will be 240 ml per study day and will be performed no more 
frequently than every 3 months (for a total of 3 study days in our transgender/non-binary group 
initiating group and a total of 4 study days in our transgender/non-binary discontinuing group).  
3. Loss of Confidentiality. Risks associated with a loss of confidentiality could feasibly include risks 
to employability, insurability, reputation, ability to adopt, or other social risks. These risks will be 
disclosed during the consenting process in verbal and written form. Risk will be minimized by 
following good research practices: subjects will be assigned a study number. Most study 
documents will not contain subjects’ names or other identifying information. The Consent Forms 
will contain name and signature. One database will contain the linkage information to connect 
study participant identities to study data for cross-referencing. In order to protect subjects’ 
identity and protected information safe, the investigators will keep the list of subjects and study 
numbers in a locked and protected area and/or in HIPAA-secured databases such as REDCap, 
and in password-protected servers using password-protected computers. Research records may 
be seen by the American Society of Reproductive Medicine, the National Institutes of Health 
(NIH), other governmental agencies, and UCSD Human Research Protections Program. The local 
resources available ensure this study is conducted in a way that assures protection of the rights 
and welfare of participants are adequate. Such resources include availability of the research 
staff and facilities at UCSD; protected research time for the investigational team members to 
conduct and complete the study; and availability of medical resources that participants may 
need as a consequence of the research. Procedures are in place for reporting protocol 
deviations/violations and local unanticipated problems involving risks to participants or others 
to the UCSD HRPP. The Principal Investigator will be responsible for monitoring and reporting of 
Adverse Events (AEs) to the IRB. 
4. Pelvic ultrasound: Either done as transabdominal or transvaginal, there is risk of discomfort 
during the procedure. These procedures will be done by experienced technicians and/or 
physicians. 
The potential benefits of this study to society and to gender-diverse individuals considering testosterone 
replacement therapy are huge and outweigh the risks. Scientists, clinicians, and gender-diverse 
individuals all seek more answers to their questions about the ability to preserve fertility after TRT. This 
research has a high potential for obtaining important clinically relevant findings on preserving fertility 
after TRT.  
3 
OBJECTIVES AND ENDPOINTS 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
The primary objective of this study 
is to evaluate effects of 
testosterone replacement therapy 
on reproductive physiology. 
 
The primary endpoints are: 
Aim 1: Evidence of Luteal Activity 
(ELA) as defined by serum 
progesterone level above 3 ng/mL 
in transgender men initiating 
testosterone replacement therapy 
Aim 2: LH pulsatility (amplitude 
and frequency)  
The primary endpoints 
were selected to provide 
detailed information 
regarding effects of 
testosterone replacement 
therapy on reproductive 
physiology and especially 
on ovulatory function. 


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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
 
Secondary 
 
 
The secondary objective is to 
comprehensively evaluate serum 
reproductive hormones. 
Aim 1: Mean serum 
FSH/LH/Estradiol. 
Aim 2: Details of LH pulses 
including total LH secreted in 
pulses, basal LH secretion rate, 
and LH half-life 
Aim 3:  Serum gonadotropin levels 
and sex steroid levels. 
 
Measurements of these 
endpoints will provide 
comprehensive evaluation 
of the secretion of 
reproductive hormones in 
the presence and in the 
absence of testosterone 
replacement therapy. 
Tertiary/Exploratory  
 
 
The tertiary objective is to monitor 
ovarian and uterine appearance via 
pelvic ultrasonography 
Pelvic sonography of ovaries 
including sizes and number of 
follicles, presence or absence of 
corpus luteum and evaluation of 
uterine endometrium. 
These endpoints will 
provide correlation of 
reproductive hormones 
with their effects on the 
reproductive organs. 
4 
STUDY DESIGN  
4.1 
OVERALL DESIGN 
Study Design  
We will perform a prospective, interventional study of TGM who are initiating or discontinuing gender-
affirming testosterone replacement therapy (TRT) as part of their medical care. We will also study a 
control group of cisgender females who are not receiving testosterone. The interventions in this study 
will include venipuncture/phlebotomy, questionnaires, and vital sign collection. We will enroll three 
groups: (1) TGM initiating TRT will be enrolled prior to initiation of TRT; they will undergo one 
observational month (Month 1) with weekly evaluations and then will undergo weekly testosterone 
injections and weekly evaluations for 6 months (Months 2-7) while on TRT. (2) CGF controls will be 
enrolled and undergo weekly evaluations for one menstrual cycle (approximately one month). (3) TGM 
discontinuing TRT will be enrolled before their final month of TRT; they will undergo one observational 
month (Month 1) with weekly evaluations while on TRT and then will undergo weekly evaluations for 9 
months (Months 2-10) after their final dose of TRT.  
Pre-Screening 
We will use a pre-screening questionnaire over the phone to see if patients are interested and meet the 
bare minimums for participating in the research study. Patients will be provided with basic information 
about the research study and then asked to assent to a pre-screening questionnaire to establish 
preliminary eligibility. If they meet the inclusion criteria and none of the exclusion criteria, they will be 
deemed eligible to continue and instructed on how to come in for a screening visit. 
Screening 
Before or during the first visit, potential participants will make an appointment to be consented. If they 
agree to the consent, we will do an initial blood draw to check hemoglobin levels. If the hemoglobin is 
less than 11 g/dL or over 16 g/dL, the subject will not be enrolled in the study.  
They will have a history and physical examination performed which will include a urine pregnancy test 
and thyroid studies.  
Subjects will be included in one of three groups: TGM Initiating TRT, TGM Discontinuing TRT, and CGF.  


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The longest study visits will be conducted at the University of California, San Diego- Altman Clinical and 
Translational Research Institute or at the _____________________ [site TBD] and will last 
approximately 9 hours (8 hours of sampling, plus 1 hour of prep and discharge). All subjects will undergo 
the following during each 9-hour study visit: 
1. Baseline vital signs (Height, weight, waist circumference, hip circumference, urine pregnancy 
test)  
2. An intravenous (IV) line placement in a vein in each subjects’ non-dominant forearm. The IV line 
will be flushed with 1 ml sterile saline after placement. 
3. Frequent serum LH sampling- 3 mL blood draw every 10 minutes between 08:30 to 16:20 (8 
hours) 
o Prior to each sample collection, 1 ml of “waste” comprised of sterile saline in the line, 
mixed with a small amount of blood will be removed from the IV line and disposed in a 
standardized, deidentified manner compliant with hospital protocols. 
o After each sample collection, the IV line will be “flushed” with 1 ml sterile saline. 
The total amount of blood drawn at each study visit will be about 140 - 180 ml over an 8-hour period, 
with most being <150ml. All blood samples will be processed to collect serum for hormonal testing. 
 
 
 
 
CGF subjects will be studied at:  
• 
one 1-hour initial visit 
• 
4 weekly visits (3 will be 30 minutes, 1 will last one hour and include ultrasound) 
• 
one 9-hour (8 hours of sampling, plus 1 hour of prep and discharge) LH-pulse monitoring study 
visit.  
Including time spent completing surveys, total study-participation time will be 13 hours. 
TGM Initiating TRT will be studied at: 
• 
one 1-hour initial visit 
• 
28 weekly visits (25 30-minute visits, 3 will last one hour and include ultrasound) 
• 
three 9-hour LH-pulse monitoring study visits (8 hours of sampling, plus 1 hour of prep and 
discharge)  
Including time spent completing surveys, total study-participation time will be 47 hours.  
 
LH-pulse visits in TGM Initiating TRT will be scheduled as follows: 
- 
LH-pulse Visit #1 during the mid-follicular phase of a menstrual cycle (cycle day 5-7) prior 
to initiating T 
- 
LH-pulse Visit #2 timed 3 months (± 14 days) after initiation of T 
- 
LH-pulse Visit #3 timed 6 months (± 14 days) after initiation of T 


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Research Materials/Data and Specimen Storage 
Serum, vital signs, and questionnaire responses collected for this study will be stored using unique study 
number, without personally identifying information. Research data (demographics, screening data, vital 
signs, and questionnaire responses) will be stored in REDCap, a secure, web-based, HIPAA-compliant, 
data collection platform with a user management system allowing project owners to grant and control 
varying levels of access to data collection instruments and data (e.g. read only, de-identified-only data 
views) for other users. 
De-identified serum specimens will be stored in a freezer in the UCSD Biomedical Sciences Building on a 
floor with restricted access until they are sent for hormone level analysis at the National Institutes of 
Health Ligand Assay & Analysis Core of the Center for Research in Reproduction at University of Virginia. 
No PHI will be disclosed to entities beyond the PI and key personnel.  
All research material and data will be obtained specifically for research purposes and no use will be 
made of existing specimens, records, or data. 
 
 
Data collection, Data analysis, Significance, and Sample Size 
At each 9-hour study visit, each subject will have blood drawn for measurement of serum LH, FSH, 
testosterone, estradiol, and progesterone levels, immediately followed by initiation of frequent blood 
sampling at 10-minute intervals for an additional 8 hours beginning at 08:30 and ending at 16:20. During 
the 8-hour frequent sampling period, blood samples will be used for measurement of LH concentration. 
Testosterone dose, date of the last T injection and compliance will be assessed and documented. 
The data analysis for this study will be very similar to that of previous studies conducted by Drs. Dan 
Apter and Sam Yen [18]. Analysis of LH pulsatility will be performed using the Cluster 7 computer 
program of Veldhuis and Johnson [19]. This program uses deconvolution analysis to take into account 
changes in secretory burst number, amplitude, mass, duration and/or alterations in the hormone half-
life.  Mean pulse frequency between groups will be compared using ANOVA with post hoc testing.  
Based on the findings by Spinder, et al. in 1989, we anticipate LH pulse frequencies of 69 ± 35 
minutes before testosterone therapy and every 90 ± 31 minutes after 6 months on therapy, resulting in 
an effect size of 0.63. Thus, a sample size of 15 subjects per group is estimated to have 90% power to 
detect an LH pulse frequency effect size of 0.63 between treated TGM and controls when employing 
Wilcoxon’s rank test at a significance level P = 0.05. Assuming a 20% drop out rate, 20 subjects will be 
TGM Discontinuing TRT will be studied at:  
• 
one 1-hour initial visit 
• 
40 weekly visits (36 30-minute visits, 4 will last one hour and include ultrasound) 
• 
four 9-hour LH-pulse monitoring study visits (8 hours of sampling, plus 1 hour of prep and 
discharge) 
Including time spent completing surveys, total study-participation time will be 64 hours.  
 
LH-pulse visits in TGM Discontinuing TRT will be scheduled as follows: 
- LH-pulse Visit #1 as soon as possible after enrollment, no later than 7 days after their final 
dose of T 
- LH-pulse Visit #2 timed 3 months (± 14 days) after final dose of T 
- LH-pulse Visit #3 timed 6 months (± 14 days) after final dose of T 
- LH-pulse Visit #4 timed 9 months (± 14 days) after final dose of T 
 


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enrolled per group in an effort to complete study of 15 individuals per group. Therefore, a total of 40 
individuals will be enrolled (20 per group). 
Inclusion of Women and Minorities 
This study will include 30 cisgender female and 50 transgender male/non-binary/gender non-
conforming subjects. All participants will have had female sex assignment at birth, given that we are 
interested in disrupted ovulatory signals, which only occur in people with ovaries.  The prevalence of 
gender incongruence in the United States is 0.4-1.2%, and not reported to be significantly affected by 
race or ethnicity. We suspect that ethnic make-up of our study cohort will reflect the diverse population 
of San Diego. 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
In the U.S. alone, ~1.4 million adults self-identify as transgender 7-10 and this prevalence is not only 
growing11 but is likely higher than reported due to selection bias and social stigma9,12-14. Among 
transgender (TG) individuals, approximately 1/2 report masculine gender identity after female sex 
assignment at birth and identify as TGM or gender non-binary people (collectively designated TGM). TRT 
(provided to typically achieve serum T the male physiological range) is the mainstay of gender-affirming 
medical care in TGM 2,15. In TGM, TRT is used to masculinize physical characteristics and suppress 
menstrual cycles4,5,16-19. Yet, TRT effects on the neuroendocrine brain and pituitary hormone function 
have been largely overlooked. Importantly, TGM may desire childbearing20,21 and many engage in vaginal 
intercourse with the potential to conceive spontaneously21-27. Yet, most TGM undergoing TRT become 
amenorrheic4,5,17 and cannot spontaneously conceive. 
Furthermore, no clinical report to date has prospectively evaluated the time-course or mechanisms of 
reversibility of TRT-induced effects on the HPO axis, including the time-course and pattern of restoration 
of normal LH pulse secretion, ovarian measures, and ovulation. Our study will systematically evaluate 
the sequence of reproductive/endocrine events following discontinuation of TRT in TGM planning to 
pursue fertility or stopping TRT for other personal reasons. 
4.3 
JUSTIFICATION FOR DOSE 
Testosterone replacement therapy proposed in this study follows the most recently published standards 
for the health care of transgender people 1. Safety of this therapy has been well-documented 33,34. 
TGM will receive intramuscular or subcutaneous administration of testosterone cypionate or enanthate 
(TC) 50 mg4 (standard dose used clinically) every 7 days for six months.   
TRT proposed in this study follows the most recently published standards for the health care of 
transgender people 1. Safety of this TRT has been well-documented 33,34. 
Note: This investigation of effects of testosterone on TGM is not intended to be reported to FDA as a 
well-controlled study in support of a new indication for use. This investigation is not intended to support 
any other significant change in the labeling of the drug. Testosterone is lawfully marketed as a 
prescription drug product. This investigation is not intended to support a significant change in the 
advertising of testosterone. This investigation does not involve a route of administration that 
significantly increases the risks (or decreases the acceptability of the risks) associated with the use of 
testosterone. This investigation does not involve a dosage level that significantly increases the risk (or 
decreases the acceptability of the risks) associated with the use of testosterone. This investigation does 
not involve use in a patient population that significantly increases the risks (or decreases the 
acceptability of the risks) associated with the use of testosterone. This investigation does not involve 
any other factor that significantly increases the risks (or decreases the acceptability of the risks) 
associated with the use of testosterone. 
4.4 
END OF STUDY DEFINITION 


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A participant is considered to have completed the study if they have completed all phases of the study 
including the last visit or the last scheduled procedure shown in the Schedule of Activities (SoA), Section 
1.3 and as follows: 
1. TGM subjects initiating TRT will complete the study upon completion of last evaluation at the 
end of month 7. 
2. CGF will complete the study upon completion of one menstrual cycle. 
3. TGM discontinuing TRT will complete the study upon the completion of last evaluation at the 
end of month 10.  
The study will be considered complete when full enrollment has been achieved in each study group and 
all participants have completed their participation. 
5 
STUDY POPULATION 
5.1 
INCLUSION CRITERIA 
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
All 
1. Provision of signed and dated informed consent form 
2. Stated willingness to comply with all study procedures and availability for the duration of the 
study 
3. Aged 18-35 
Transgender/Non-binary Group - Initiators 
4. Plan to initiate testosterone therapy  
5. History of regular menstrual cycles (every 24-35 days) at baseline, before beginning TRT 
Transgender/Non-binary Group – Discontinuers 
4. Plan to discontinue testosterone therapy  
5. History of regular menstrual cycles (every 24-35 days) before beginning TRT 
Cisgender Female Group 
4.  Having regular menstrual cycles (every 24-35 days) 
5.2 
EXCLUSION CRITERIA 
An individual who meets any of the following criteria at baseline evaluation will be excluded from 
participation in this study: 
All 
1. Pregnant 
2. Incarcerated 
3. Known cognitive impairment or institutionalized 
4. Hemoglobin <11 g/dL or >16g/dL  
5. Weight less than 110 pounds 
6. BMI <18 or >35 
7. Current endocrine disease- including untreated thyroid abnormalities, pituitary or adrenal 
disease, polycystic ovary syndrome, or androgen producing tumor 
8. Current or recent pregnancy within two months of study enrollment 
9. Current or recent breast feeding within two months of study enrollment  
10. Diabetes, or renal, liver, or heart disease 
11. History of oophorectomy or hysterectomy 
12. History of radiation or surgery involving brain structures 
13. Currently taking any medications that may affect their reproductive hormones, such as 
contraceptive medications, androgens, estrogens, progestins, GnRH antagonists, GnRH agonists, 
insulinomimetics, and metformin.  


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For TRT Initiation Group and Cisgender Female group: 
14. History of prior testosterone therapy 
5.3 
LIFESTYLE CONSIDERATIONS 
Not Applicable 
5.4 
SCREEN FAILURES 
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently randomly assigned to the study intervention or entered in the study. A minimal set of 
screen failure information is required to ensure transparent reporting of screen failure participants, to 
meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes demography, screen 
failure details, eligibility criteria, and any serious adverse event (SAE). Individuals who do not meet the 
criteria for participation in this trial (screen failure) because of hemoglobin less than 11 g/dL or higher 
than 16 g/dL will not be enrolled.  
6 
STUDY INTERVENTION 
6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
6.1.1 STUDY INTERVENTION DESCRIPTION 
Investigators will conduct a prospective, controlled clinical trial of ovarian and menstrual 
cyclicity in TGM initiating TRT, TGM discontinuing TRT, and cisgender female control subjects who have 
never used TRT. Enrollment will include 30 TGM initiating, 20 TGM discontinuing and 30 CGF control 
subjects who report female gender identity congruent with female sex assignment at birth. All subjects 
will be at least 18 years old and no older than 35, with female sex assignment at birth, regular menstrual 
cycles, and body mass index 18-35 kg/m2. Subjects with history of prior Testosterone use, cancer, 
chemotherapy, or radiation of the brain, abdomen, or pelvis, current use of hormonal medications 
(including, but not limited to, metformin, insulin, progestins, or estrogenic medications), current 
endocrinopathy (including, but not limited to, PCOS, androgen secreting tumor, diabetes, or pituitary, 
thyroid, or adrenal disease), and renal, hepatic, cardiac, or hematologic disease will be excluded.  
TGM subjects in the initiating group will undergo baseline endocrine and menstrual cycle 
evaluation, followed by intramuscular or subcutaneous administration of testosterone cypionate (TC) 50 
mg (standard dose) every 7 d for 24 wks.  
TGM in the discontinuing group will undergo baseline evaluation during up to a month before 
discontinuing TRT.  
All subjects will complete a daily uterine bleeding log using REDCap® and undergo weekly 
measurement of serum FSH, LH, estradiol (E2), and P4 (ELISA), and T (LC-MS/MS. Subjects will also 
undergo interval ultrasound (US) during the month(s) of their LH-pulse visit(s), either transvaginally or 
transabdominally, using a 4- to 9-MHz probe to obtain 3D pelvic imaging to track corpus luteum (CL) 
formation, as well as regression analysis of hormone secretion patterns, endometrial thickness, antral 
follicle count (AFC), and volume of uterus and ovaries. Changes in follicle dynamics will be analyzed via 
3D U/S of the mean diameter of each antral follicle in 1-mm increments from 2 to 9 mm171,172.  
 
A primary endpoint will be Evidence of Luteal Activity (ELA), as defined by a serum progesterone 
level above 3 ng/mL in transgender men initiating testosterone replacement therapy. Secondary 
endpoints will include mean serum FSH, LH, and estradiol levels. 
 
UCSD Investigators have already demonstrated successful recruitment and retention of TGM and 
CF participants in longitudinal studies involving daily bleeding diaries and serial pelvic ultrasound (U/S) in 
our pilot study and others171,172. TGM subjects will be serially evaluated for adverse effects of TRT per 
Endocrine Society recommendations1. Safety monitoring details are in the Human Subjects Section. We 
calculate that 20 subjects/group will have >95% power to detect a 30% decrease in the proportion of 


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subjects with ELA at baseline compared to the final 4-wk TC study interval (wks 21-24). Although we 
anticipate a larger, more clinically meaningful decrease, we utilized a conservative target to maximize 
the study’s power. While we had no dropouts in our TGM pilot study, a 1/3 dropout rate is factored into 
our enrollment target of 30 subjects/initiating and CGF group and a 1/2 dropout rate is factored into our 
enrollment target of 20 subjects in the discontinuing group to ensure achieving sufficient power. We aim 
to recruit 30 TGM initiating (20 complete), 20 TGM discontinuing (10 complete), 30 CGF controls (20 
complete). 
6.1.2 DOSING AND ADMINISTRATION 
No enrolled subjects will receive experimental products or doses. The group initiating TRT will receive 
TRT per standard of care. The form and dose of testosterone used in this study is FDA approved to treat 
medical conditions associated with low levels of naturally produced testosterone. It is also commonly 
prescribed off-label to help transgender people transition. The effects of these hormones are not well 
documented on the transitioning body, so the researchers are interested in learning about the effects of 
testosterone on participants’ reproductive hormones and reproductive organs. 
Justification for IND exemption: This investigation of effects of testosterone on TGM is not intended to 
be reported to FDA as a well-controlled study in support of a new indication for use. This investigation is 
not intended to support any other significant change in the labeling of the drug. Testosterone is lawfully 
marketed as a prescription drug product. This investigation is not intended to support a significant 
change in the advertising of testosterone. This investigation does not involve a route of administration 
that significantly increases the risks (or decreases the acceptability of the risks) associated with the use 
of testosterone. This investigation does not involve a dosage level that significantly increases the risk (or 
decreases the acceptability of the risks) associated with the use of testosterone. This investigation does 
not involve use in a patient population that significantly increases the risks (or decreases the 
acceptability of the risks) associated with the use of testosterone. This investigation does not involve 
any other factor that significantly increases the risks (or decreases the acceptability of the risks) 
associated with the use of testosterone.1, 33, 34 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
Testosterone replacement therapy for the initiating group will be given in the same injection mode and 
dose as is the standard of care. We will obtain testosterone from the USCD pharmacy, and it will be 
injected, per standard of care by licensed clinical practitioners. There is no investigational drug being 
used in this research study. The UCSD pharmacy is experienced in storing, handling, and dispensing TRT.  
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
There is no investigational drug being used in this research study. We are giving testosterone to the 
initiating group per standard of care.  
TESTOSTERONE CYPIONATE INJECTION 
Injection: 200 mg/mL in a single-dose vial  
Testosterone Cypionate Injection, USP for intramuscular or subcutaneous injection, contains 
testosterone cypionate which is the oil-soluble 17 (beta)-cyclopentylpropionate ester of the androgenic 
hormone testosterone. 
Testosterone cypionate is a white or creamy white crystalline powder, odorless or nearly so and stable 
in air. It is insoluble in water, freely soluble in alcohol, chloroform, dioxane, ether, and soluble in 
vegetable oils. 
The chemical name for testosterone cypionate is androst-4-en-3-one, 17-(3-cyclopentyl-1- oxopropoxy)-, 
(17ß)-. Its molecular formula is C27H40O3, and the molecular weight 412.61. 
Testosterone Cypionate Injection, USP is provided as sterile, clear colorless to pale yellow solution 


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containing 200 mg/mL testosterone cypionate in vials. 
Each mL of solution contains: 
Testosterone cypionate: 
200 mg 
Benzyl alcohol:  
 
20 mg 
Benzyl benzoate: 
 
0.2 mL 
Cottonseed oil:  
 
542 mg 
6.2.3 PRODUCT STORAGE AND STABILITY 
Testosterone Cypionate Injection is supplied as a sterile, clear colorless to pale yellow solution in 
single-dose vials as 200 mg/mL testosterone cypionate. 
NDC Number Package Size 71225-127-01 1 mL vials 
Store at 15°C to 25°C (59°F to 77°F); excursions permitted to 2°C to 30°C (36°F to 86°F). Store 
product in carton to protect contents from light. 
6.2.4 PREPARATION 
There is no special preparation required. TRT will be prepared as per standard clinical care.  
6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
We have no plans for randomization or blinding.  
6.4 
STUDY INTERVENTION COMPLIANCE 
Subjects will be expected to come in for weekly evaluations and, if in the TRT initiator group, for weekly 
testosterone injections.  
Study staff will send appointment reminders and follow-up calls for no-shows. Study staff will help 
participants to minimize barriers to study completion. Study staff will closely monitor each participant 
for compliance with the protocol.   
6.5 
CONCOMITANT THERAPY 
For this protocol, a prescription medication is defined as a medication that can be prescribed only by a 
properly authorized/licensed clinician. Medications to be reported in the Case Report Form (CRF) are 
concomitant prescription medications, over-the-counter medications and supplements.  
 
Prohibited therapies, medications, or supplements include:  
• 
FOR ALL GROUPS:  
o Contraceptive medications, androgens, estrogens, progestins, GnRH antagonists, GnRH 
agonists, insulinomimetics, and metformin.  
 
Justification: these medications may affect their reproductive hormones. 
o Medications for Diabetes, or renal, liver, or heart disease.  
 
Justification: We are excluding subjects with significant systemic diseases. If 
they start medications for these diseases during the study, they will be 
withdrawn from the study. 
6.5.1 RESCUE MEDICINE 
No applicable rescue medications.  
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
Discontinuation from testosterone, if in the initiating group, should be done in consultation with a 
participant’s personal physician. If a participant in the initiating group discontinues testosterone 
voluntarily, they will be disenrolled from the study. There will be no useful knowledge gained from 
completing remaining study procedures. If a clinically significant finding is identified (including, but not 


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limited to changes from baseline) after enrollment, the investigator or qualified designee will determine 
if any change in participant management is needed. Any new clinically relevant finding will be reported 
as an adverse event (AE). 
Participants in all groups are welcome to withdraw from the study at any time. 
The data to be collected at the time of study intervention discontinuation will include the following: 
• 
Reason(s) for discontinuing testosterone 
• 
Reason for discontinuing study. 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may discontinue or withdraw a participant from the study for the following reasons: 
• 
Pregnancy 
• 
If the patient starts a hormonal birth control method 
• 
Significant study intervention non-compliance, e.g., subject does not complete daily diaries, 
subject misses weekly visits. 
• 
If any clinical adverse event (AE), laboratory abnormality, or other medical condition or situation 
occurs such that continued participation in the study would not be in the best interest of the 
participant 
• 
A new disease or condition that requires treatment with medications that are contraindicated to 
this study or disease progression which requires discontinuation of any study tasks including, if 
applicable, taking testosterone.  
• 
If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
• 
If the patient misses 2 weekly visits and/or is unable to receive testosterone for two weeks. 
The reason for participant discontinuation or withdrawal from the study will be recorded on the Case 
Report Form (CRF). Research participants who sign the informed consent form but do not complete 
tasks, ask to withdraw, or are withdrawn or discontinued from the study may be replaced.   
7.3 
LOST TO FOLLOW-UP 
The following actions must be taken if a participant fails to return to the clinic for a required study visit: 
• 
The site will attempt to contact the participant and reschedule the missed visit within 2 weeks of 
a planned visit and counsel the participant on the importance of maintaining the assigned visit 
schedule and ascertain if the participant wishes to and/or should continue in the study. 
• 
Before a participant is deemed lost to follow-up, the investigator or designee will make every 
effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, 
a certified letter to the participant’s last known mailing address or local equivalent methods). 
These contact attempts should be documented in the participant’s medical record or study file.  
• 
Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
• 
A participant will be considered lost to follow-up if they fail to return for two weekly scheduled 
visits and is unable to be contacted by the study site staff.  
8 
STUDY ASSESSMENTS AND PROCEDURES 
8.1 
EFFICACY ASSESSMENTS  
Study procedures and evaluations to be done as part of this study to support the determination of 
efficacy therein are outlined and described in Section 1.3 Schedule of Activities.  
 


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• 
A pre-screening telephone call will be conducted prior to any interventions. Subjects will have 
time to consider their options before coming to a screening visit. 
• 
During the screening visit, the study investigators will discuss the protocol in detail and the 
medical aspects including potential risks and benefits.  
• 
Once the patient has all their questions answered satisfactorily and has agreed to participate in 
the study, the participant will be asked to sign the informed consent in English.  
• 
The participant will be given a copy of the consent form and other applicable forms. Research 
personnel who may obtain consent include the principal and co-investigators, research 
coordinators, medical students, and UCSD resident physicians in the Department of Obstetrics, 
Gynecology, and Reproductive Sciences. All research personnel associated with this study will 
have completed proper training and certification and will be named in the IRB Kuali module.  
• 
The Investigators will retain the signed consent form in either REDCap or as a paper pen-and-ink 
signature (control subjects may sign electronically whereas subjects on Testosterone must sign 
in a method that is FDA Part 11 compliant, either Part 11-compliant Docusign or Paper). Other 
associated forms will be stored in the HIPAA-compliant REDCap system.  
• 
After signing the consent forms, the subject may undergo screening. Screening may be 
conducted within a week or more after signing the consent forms.  
• 
Actual study participation may begin between 1 and 5 weeks thereafter, depending on timing of 
menstrual cycle, timing of TRT initiation, and/or timing of TRT discontinuation. These waiting 
periods will allow potential subjects sufficient time to consider whether to participate and 
minimize the possibility of coercion or undue influence.  
 
All procedures and exams will be conducted by licensed and experienced personnel. All instruments 
used will be calibrated and routinely maintenanced. 
• 
Physical examination by physician: height, weight, waist circumference, hip circumference 
• 
Radiographic or other imaging assessments by qualified personnel: Pelvic ultrasound, either 
transabdominal or transvaginal, according to participant’s preference. 
• 
Biological specimen collection and laboratory evaluations: Blood test for hemoglobin, urine 
pregnancy test. Blood tests for LH, FSH, estrone (E1) conjugates, and pregnanediol glucuronide 
(PdG). Serum tests for FSH, LH, estradiol (E2), and P4 (ELISA), and T (LC-MS/MS) for confirmation 
of hormone dynamics.  
• 
Frequent serum LH sampling:  3 mL blood draw every 10 minutes for 8 hours. 
o An intravenous (IV) line will be placed in a vein in each participant’s non-dominant 
forearm.  
o Prior to each sample collection, 1 ml of “waste” comprised of sterile saline in the line, 
mixed with a small amount of blood will be removed from the IV line and disposed in a 
standardized, deidentified manner compliant with hospital protocols. 
o After each sample collection, the IV line will be “flushed” with 1 ml sterile saline. 
• 
Daily bleeding diaries: surveys will be delivered via text message every morning and participants 
will complete these electronically.  
• 
Procedures that will be completed during the study as part of regular standard of clinical care: 
weekly testosterone injections for TGM participants initiating TRT 
 
Evaluations of weight, blood tests evaluating reproductive hormones, and pelvic ultrasounds are 
expected to provide information relevant to understanding the effects of testosterone on reproductive 
physiology. Participants will be notified of any irregularities that could affect their health and/or safety. 
All tests and procedures will be done for and paid by this study.  


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Serum, medical history data, vital signs, and questionnaire responses collected for this study will be 
stored using unique study numbers, without personally identifiable information. De-identified serum 
specimens will be stored in a freezer in the UCSD Biomedical Sciences Building or in the 
UCSD____________ (TBD office building) on a floor with restricted access until they are sent for 
hormone level analysis at the National Institutes of Health Ligand Assay & Analysis Core of the Center 
for Research in Reproduction at University of Virginia. No PHI will be disclosed to entities beyond the PI 
and key personnel.  
Research data (demographics, screening data, vital signs, and questionnaire responses) will be 
stored in REDCap, a secure, web-based, HIPAA-compliant, data collection platform with a user 
management system allowing project owners to grant and control varying levels of access to data 
collection instruments and data (e.g. read only, de-identified-only data views) for other users. 
All research material and data will be obtained specifically for research purposes and no use will be 
made of existing specimens, records, or data. 
 
A primary endpoint will be Evidence of Luteal Activity (ELA), as defined by serum progesterone 
level above 3 ng/mL. Secondary endpoints will include mean serum FSH, LH, and estradiol levels 
8.2 
SAFETY AND OTHER ASSESSMENTS 
Subjects are encouraged to ask any questions concerning the research study both before agreeing 
to participate and at any time during the course of the study.  
Subjects will be seen weekly and will be encouraged to report any unexpected or undesired side-
effects. We will closely monitor all subjects during the study and will address any discomforts or side 
effects as warranted. The most common testosterone-related effects include acne, hair thinning, and 
mood changes. If a participant reports displeasure from unwanted side effects greater than the 
subjective benefits of testosterone treatment, they can elect to disenroll from the study. They will be 
advised to consult with their personal physician/treatment team. The testosterone used in this study 
will be the same dose for all participants; if they choose to disenroll, they can modify dose of 
testosterone with their primary care physician if desired.  
The specific timing of procedures/evaluations to be done at each study visit are captured in 
Section 1.3, Schedule of Activities (SoA). All procedures/evaluations will be performed by qualified 
personnel. 
8.3 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
This study does not include the use of experimental drugs or devices. TRT is being studied per 
the same dose and mode of administration per standard of care.  The FDA definition of an Adverse 
event is any untoward medical occurrence associated with the use of an intervention in humans, 
whether or not considered intervention-related (21 CFR 312.32 (a)).  
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of 
either the investigator or sponsor, it results in any of the following outcomes: death, a life-threatening 
adverse event, inpatient hospitalization or prolongation of existing hospitalization, a persistent or 
significant incapacity or substantial disruption of the ability to conduct normal life functions, or a 
congenital anomaly/birth defect. Important medical events that may not result in death, be life-
threatening, or require hospitalization may be considered serious when, based upon appropriate 
medical judgment, they may jeopardize the participant and may require medical or surgical intervention 
to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or 


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convulsions that do not result in inpatient hospitalization, or the development of drug dependency or 
drug abuse. 
8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
8.3.3.1 SEVERITY OF EVENT 
For adverse events (AEs) not included in the protocol defined grading system, the following guidelines 
will be used to describe severity.  
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• 
Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”.] 
8.3.3.2 
RELATIONSHIP TO STUDY INTERVENTION 
All adverse events (AEs) must have their relationship to study intervention assessed by the clinician who 
examines and evaluates the participant based on temporal relationship and his/her clinical judgment. 
The degree of certainty about causality will be graded using the categories below. In a clinical trial, the 
study product must always be suspect.  
• 
Related – The AE is known to occur due to the study procedures/exams/sample 
collections/testosterone, there is a reasonable possibility that something related to being in the 
study caused the AE, or there is a temporal relationship between the study and the event. 
Reasonable possibility means that there is evidence to suggest a causal relationship between 
the study and the AE. 
• 
Not Related – There is not a reasonable possibility that participation in the study caused the 
event, there is no temporal relationship between the study and event onset, or an alternate 
etiology has been established. 
OR 
• 
Definitely Related – There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal laboratory test 
result, occurs in a plausible time relationship to study participation and cannot be explained by 
concurrent disease or other drugs or chemicals. The response to withdrawal from the study 
should be clinically plausible. The event must be pharmacologically or phenomenologically 
definitive, with use of a satisfactory rechallenge procedure if necessary. 
• 
Probably Related – There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs within 
a reasonable time after study participation, is unlikely to be attributed to concurrent disease or 
other drugs or chemicals, and follows a clinically reasonable response. 
• 
Potentially Related – There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of testosterone or participating in a 
blood draw). However, other factors may have contributed to the event (e.g., the participant’s 
clinical condition, other concomitant events). Although an AE may rate only as “possibly related” 
soon after discovery, it can be flagged as requiring more information and later be upgraded to 
“probably related” or “definitely related”, as appropriate. 
• 
Unlikely to be related – A clinical event, including an abnormal laboratory test result, whose 
temporal relationship to study participation makes a causal relationship improbable (e.g., the 
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or chemicals or underlying disease provides plausible explanations (e.g., the participant’s clinical 
condition, other concomitant treatments). 
• 
Not Related – The AE is completely independent of study participation, and/or evidence exists 
that the event is definitely related to another etiology. There must be an alternative, definitive 
etiology documented by the clinician. 
8.3.3.3 EXPECTEDNESS  
Principal investigator, any co-investigators, and key personnel will be responsible for 
determining whether an adverse event (AE) is expected or unexpected.  An AE will be considered 
unexpected if the nature, severity, or frequency of the event is not consistent with the risk information 
previously described for the study events. 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the 
attention of study personnel during study visits and interviews of a study participant presenting for 
medical care, or upon review by a study monitor. 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured 
on the appropriate case report form (CRF). Information to be collected includes event description, time 
of onset, clinician’s assessment of severity, relationship to testosterone or study procedures (assessed 
only by those with the training and authority to make a diagnosis), and time of resolution/stabilization 
of the event. All AEs occurring while on the study must be documented appropriately regardless of 
relationship. All AEs will be followed to adequate resolution. 
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant’s condition 
deteriorates at any time during the study, it will be recorded as an AE.  
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode. 
Study staff will record all reportable events with start dates occurring any time after informed 
consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study 
participation.  At each study visit, the investigator will inquire about the occurrence of AE/SAEs since the 
last visit.  Events will be followed for outcome information until resolution or stabilization. 
8.3.5 ADVERSE EVENT REPORTING  
Adverse events will be identified during weekly visits with subjects and if the events reach the 
threshold of an unanticipated problem will be reported to IRB in accordance with the requirements. 
8.3.6 SERIOUS ADVERSE EVENT REPORTING  
The study clinician(s) will immediately report to the sponsor any serious adverse event, whether 
or not considered study intervention related, including those listed in the protocol or investigator 
brochure and must include an assessment of whether there is a reasonable possibility that testosterone 
or any of the study procedures caused the event. Study endpoints that are serious adverse events (e.g., 
all-cause mortality) must be reported in accordance with the protocol unless there is evidence 
suggesting a causal relationship between the study procedures and the event. In that case, the 
investigator must immediately report the event to the sponsor. 
All serious adverse events (SAEs) will be followed until satisfactory resolution or until the site 
investigator deems the event to be chronic or the participant is stable. Other supporting documentation 
of the event may be requested by the Data Coordinating Center (DCC)/study sponsor and should be 
provided as soon as possible. 
8.3.7 REPORTING EVENTS TO PARTICIPANTS  


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In the event of identifying adverse findings (for example abnormal blood test results), subjects 
will be contacted, and the findings will be discussed. 
8.3.8 EVENTS OF SPECIAL INTEREST  
Not applicable 
8.3.9 REPORTING OF PREGNANCY  
In the event of pregnancy, subjects will be withdrawn from the study and the possible effects of 
testosterone on pregnancy will be discussed. 
8.4 
UNANTICIPATED PROBLEMS 
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
participant population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
8.4.2  UNANTICIPATED PROBLEM REPORTING  
The investigator will report unanticipated problems (UPs) to the reviewing Institutional Review Board 
(IRB) and to the Data Coordinating Center (DCC)/lead principal investigator (PI). The UP report will 
include the following information: 
• 
Protocol identifying information: protocol title and number, PI’s name, and the IRB project 
number; 
• 
A detailed description of the event, incident, experience, or outcome;  
• 
An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP;  
• 
A description of any changes to the protocol or other corrective actions that have been taken or 
are proposed in response to the UP. 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline:   
• 
UPs that are serious adverse events (SAEs) will be reported to the IRB and to the DCC/study 
sponsor within 7 calendar days –per NIH policy—of the investigator becoming aware of the 
event.  
• 
Any other UP will be reported to the IRB and to the DCC/study sponsor within 14 calendar days 
of the investigator becoming aware of the problem.  
• 
All UPs should be reported to appropriate institutional officials (as required by an institution’s 
written reporting procedures), the supporting agency head (or designee), and the Office for 
Human Research Protections (OHRP) within 30 days of the IRB’s receipt of the report of the 
problem from the investigator. 
8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
In the event of unanticipated problems, participants will be notified of any that affect their safety or 
confidentiality.  
9 
STATISTICAL CONSIDERATIONS  


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9.1 
STATISTICAL HYPOTHESES 
Our overall hypothesis is that in otherwise healthy (non-polycystic ovary syndrome) females, male-level 
exogenous androgens inhibit the reproductive neuroendocrine axis through androgen-receptor (AR) 
signaling in hypothalamic Kiss1 neurons to alter luteinizing hormone (LH) pulse secretion and LH surges 
and inhibit neural circuits controlling these modes of LH secretion. This work will greatly improve 
understanding of the impact of high androgens on female reproductive physiology and be of high clinical 
importance to the rapidly growing, yet currently understudied, TGN/TGM population.   
9.2 
SAMPLE SIZE DETERMINATION 
Primary Efficacy Endpoint(s):  
Aim 1, comparing TRT initiators to CGF: We calculate that 20 subjects/group will have >95% power to 
detect a 30% decrease in the proportion of subjects with evidence of luteal activity at baseline 
compared to the final month of the study. We plan to enroll 30 subjects/these groups (30 TRT Initiators 
and 30 CGF) assuming a loss of 1/3 of the subjects in course of the study. 
Aim 2, TRT Initiators: We anticipate that 20 subjects will provide >95% statistical power to detect a 30% 
decrease in LH pulse frequency among testosterone treated transgender men. Assuming a 1/3 drop-out, 
30 subjects in the TRT Initiators group will be enrolled to ensure a final n=20. 
Aim 3, TRT Discontinuers: Based on a reported ovulatory rate of 4.5% in patients with a median 11 
months on testosterone replacement, we calculate that 10 subjects will provide >95% power to detect 
an increase in the proportion of subjects with ELA to 60% in the final month interval after 
discontinuation of testosterone replacement therapy. Assuming a 1/2 drop-out, 20 subjects in the TRT 
discontinuation group will be enrolled to ensure a final n=10. We assume the drop-out rate for this 
group might be higher because subjects in this group may be very eager to discontinue testosterone and 
may withdraw at a higher rate during the first month.  
9.3 
POPULATIONS FOR ANALYSES 
Group 1: Transgender men who are initiating gender-affirming testosterone replacement therapy 
Group 2: Transgender men who are discontinuing gender-affirming testosterone replacement therapy 
Group 3: Cisgender women who are not and have never been on testosterone replacement therapy 
 
We intend to analyze the data from all subjects who successfully complete the study per protocol. 
9.4 
STATISTICAL ANALYSES 
9.4.1 GENERAL APPROACH 
Data will be analyzed with the aid of statistical packages such as JMP. Continuous data will be 
summarized as means, standard deviations and ranges. Categorical data will be presented as 
percentages. 
P-values will be considered significant at 0.05 threshold.  
9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) 
Aim 1: primary endpoint (Evidence of Luteal Activity ) will be analyzed with the aid of Chi-Square test 
(for comparison of proportion of ovulatory cycles in cis-gender subjects with transgender subjects) and 
by McNemar test (for paired comparison of proportion of ovulatory cycles before and during 
testosterone treatment in subjects undergoing testosterone replacement therapy).  
Aim 2: primary endpoints (LH pulse amplitude and frequency) will be analyzed with the aid of t-test 
(comparing cisgender subjects with transgender subjects) or by paired t-test comparing transgender 
subjects before and during testosterone replacement therapy). Normality of distribution will be 
assessed by the Shapiro-Wilk W test. In the absence of normality and/or unequal variance, data were 
appropriately transformed and/or non-parametric testing will be performed. 


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Aim 3: primary endpoint (Evidence of Luteal Activity) will be analyzed with the aid of McNemar test (for 
paired comparison of proportion of ovulatory cycles before and after discontinuation of testosterone 
treatment in subjects undergoing testosterone replacement therapy). 
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
Secondary endpoints will be analyzed in identical fashion to primary endpoints whereby categorical 
variables will be tested using Chi-Square test or McNemar tests, as appropriate, while continuous 
variables will be evaluated using t-test or paired t-test, as appropriate. In the absence of normal 
distribution, nonparametric tests will be used. 
9.4.4 SAFETY ANALYSES 
Not applicable 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
Cisgender group and transgender group (Aims 1 and 2) will be compared with regard to demographics 
and laboratory measurements using identical tests to tests described in 9.4.3. 
9.4.6 PLANNED INTERIM ANALYSES  
Not applicable 
9.4.7 SUB-GROUP ANALYSES 
Not applicable 
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
Individual participant data will be listed by measure and time point. 
9.4.9 EXPLORATORY ANALYSES 
Not applicable 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
10.1.1 STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  Written notification, documenting the reason for study suspension or termination, 
will be provided by the suspending or terminating party to study participants, investigators, staff, and 
the funding agency.  If the study is prematurely terminated or suspended, the Principal Investigator (PI) 
will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will 
provide the reason(s) for the termination or suspension.  Study participants will be contacted, as 
applicable, and be informed of changes to study visit schedule. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping    
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, 
and satisfy the sponsor, IRB and/or Food and Drug Administration (FDA).] 
10.1.2 CONFIDENTIALITY AND PRIVACY  
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover testing of 
biological samples in addition to the clinical information relating to participants. Therefore, the study 


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protocol, documentation, data, and all other information generated will be held in strict confidence. No 
information concerning the study or the data will be released to any unauthorized third party without 
prior written approval of the sponsor.  
All research activities will be conducted in as private a setting as possible. 
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), or regulatory agencies may inspect all documents and records required 
to be maintained by the investigator, including but not limited to, medical records (office, clinic, or 
hospital) and pharmacy records for the participants in this study. The clinical study site will permit 
access to such records. 
The study participant’s contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a secure 
location for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor 
requirements. 
To further protect the privacy of study participants, a Certificate of Confidentiality will be issued 
by the National Institutes of Health (NIH).  This certificate protects identifiable research information 
from forced disclosure. It allows the investigator and others who have access to research records to 
refuse to disclose identifying information on research participation in any civil, criminal, administrative, 
legislative, or other proceeding, whether at the federal, state, or local level. By protecting researchers 
and institutions from being compelled to disclose information that would identify research participants, 
Certificates of Confidentiality help achieve the research objectives and promote participation in studies 
by helping assure confidentiality and privacy to participants. 
10.1.3 FUTURE USE OF STORED SPECIMENS AND DATA  
Data collected for this study will be analyzed and stored at UCSD. De-identified data pertinent to 
Aim 2 will be shared with our collaborator, Dr. Chris McCartney (University of Virginia).  
With the participant’s approval and as approved by local Institutional Review Boards (IRBs), de-
identified biological samples will be stored at the ACTRI Biorepository with the same goal as the sharing 
of data with the University of Virginia Biorepository. The ACTRI Biorepository will also be provided with a 
code-link that will allow linking the biological specimens with the phenotypic data from each participant, 
maintaining the blinding of the identity of the participant.  
During the conduct of the study, an individual participant can choose to withdraw consent to 
have biological specimens stored for future research. However, withdrawal of consent with regard to 
biosample storage may not be possible after the study is completed.  
When the study is completed, access to study data and/or samples will be provided through the 
ACTRI Biorepository. 
10.1.4 KEY ROLES AND STUDY GOVERNANCE 
Provide the name and contact information of the Principal Investigator and the Medical Monitor. 
Principal Investigator 
Medical Monitor 
Antoni J Duleba, MD, Professor 
N/A  
University of California, San Diego   
9500 Gilman Drive, 0633 
La Jolla, CA  92093-0633 
 
203-804-7123 
 
Antoni.duleba@health.ucsd.edu 
 
10.1.5 SAFETY OVERSIGHT 
Adverse events will be monitored by the investigators. 
10.1.6 CLINICAL MONITORING 


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Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of 
the trial is in compliance with the currently approved protocol/amendment(s), with International 
Conference on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory 
requirement(s).  
• 
No external monitoring is anticipated and the study will comply with all UCSD requirements. 
• 
Independent audits will not be conducted by the study sponsor to ensure monitoring practices 
because this is the only site.  
10.1.7 DATA HANDLING AND RECORD KEEPING  
10.1.7.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
investigator. The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. 
All source documents should be completed in a neat, legible manner to ensure accurate interpretation 
of data.   
Hardcopies of the study visit worksheets will be provided for use as source document worksheets for 
recording data for each participant enrolled in the study.  Data recorded in the electronic case report 
form (eCRF) derived from source documents should be consistent with the data recorded on the source 
documents.  
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse reactions 
data) and clinical laboratory data will be entered into RedCAP, a 21 CFR Part 11-compliant data capture 
system provided by the ACTRI. The data system includes password protection and internal quality 
checks, such as automatic range checks, to identify data that appear inconsistent, incomplete, or 
inaccurate. Clinical data will be entered directly from the source documents. 
10.1.7.2 STUDY RECORDS RETENTION  
Study documents should be retained according to UCSD and NIH requirements. 
10.1.8 PROTOCOL DEVIATIONS  
A protocol deviation is any noncompliance with the clinical trial protocol, International Conference on 
Harmonisation Good Clinical Practice (ICH GCP), or Manual of Procedures (MOP) requirements. The 
noncompliance may be either on the part of the participant, the investigator, or the study site staff. As a 
result of deviations, corrective actions are to be developed by the site and implemented promptly.  
These practices are consistent with ICH GCP:  
• 
4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
• 
5.1 Quality Assurance and Quality Control, section 5.1.1  
• 
5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
It is the responsibility of the site investigator to use continuous vigilance to identify and report 
deviations in accordance with IRB requirements. All deviations must be addressed in study source 
documents. Protocol deviations must be sent to the reviewing Institutional Review Board (IRB) per their 
policies. The investigator is responsible for knowing and adhering to the reviewing IRB requirements.  
10.2 ADDITIONAL CONSIDERATIONS 
Not applicable 
10.3 ABBREVIATIONS 
The list below includes abbreviations utilized in this template.  However, this list should be customized for 
each protocol (i.e., abbreviations not used should be removed and new abbreviations used should be 
added to this list). 
 


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AE 
Adverse Event 
AFC 
Antral Follicle Count 
ANCOVA 
Analysis of Covariance 
AR 
Androgen Receptor 
AUC 
Atypical Urothelial Cells 
CGF 
Cisgender Female 
CFR 
Code of Federal Regulations 
CL 
Corpus Luteum 
CRF 
Case Report Form 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
E1 
Estrone 
E2 
Estradiol 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
ELA 
Evidence of Luteal Activity 
ELISA 
Enzyme-Linked Immunosorbent Assay 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
FSH 
Follicle-Stimulating Hormone 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act  
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation  
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
IV 
Intravenous 
LC-MS/MS 
Liquid Chromatography Tandem Mass Spectrometry 
LH 
Luteinizing Hormone 
LSMEANS 
Least-squares Means 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 


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NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
PCOS 
Polycystic Ovary Syndrome 
PdG 
Pregnanediol Glucuronide  
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SoA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
T 
Testosterone 
TC 
Testosterone Cypionate or Enanthate 
TG 
Transgender 
TGM 
Transgender Male 
TGN 
Transgender Non-Binary 
TRT 
Testosterone Replacement Therapy 
UP 
Unanticipated Problem 
US 
United States 
U/S 
Ultrasound 
 
 


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10.4 PROTOCOL AMENDMENT HISTORY 
The table below is intended to capture changes of IRB-approved versions of the protocol, including a 
description of the change and rationale. A Summary of Changes table for the current amendment is 
located in the Protocol Title Page.  
Version 
Date 
Description of Change  
Brief Rationale 
1.0 
November 
1, 2023 
 
 
1.1 
May 7, 2024 
Revisions in response to IRB pre-
review 
To clarify protocol. 
1.3 
June 29, 
2024 
Changed consent form protocol to 
paper collection. 
To comply with FDA Part 11 
regulations. 
1.4 
July 1, 2024 
Added option for subcutaneous TRT 
injections 
Similar bioavailability, may be 
more palatable to some 
participants. 
1.5 
August 6, 
2024 
Revisions in response to IRB 
conditional approval. 
To clarify protocol. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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