Abstract
Purpose:
The study aimed to characterize transgender and nonbinary (TGNB) adolescents and young adults on subcutaneous (SC) estradiol for feminizing gender-affirming hormone therapy and demonstrate efficacy and safety in these individuals.
Methods:
A retrospective single-center chart review of TGNB adolescents and young adults up to 25 years old who received SC estradiol cypionate or SC estradiol valerate and completed laboratory testing between January 1, 2021, and January 1, 2024, was conducted. Mann–Whitney U tests were used for analysis.
Results:
From transgender female (n = 37, 93%) and nonbinary (n = 3, 7%) individuals, 30 (75%) individuals demonstrated therapeutic serum estradiol concentrations (100–300 pg/mL) on SC estradiol cypionate, SC estradiol valerate, or both. Of the 23 patients using SC estradiol cypionate, 52% (n = 12) had therapeutic concentrations, but many were unable to titrate dosing due to nationwide shortage. Of the 32 patients on SC estradiol valerate, 75% (n = 24) achieved therapeutic concentrations. Serum testosterone concentrations were suppressed in all but one instance (97%, n = 35) of therapeutic serum estradiol concentrations; the majority of patients used simultaneous anti-androgens. To achieve therapeutic serum estradiol concentrations, the SC estradiol valerate dose (median 4 mg weekly, interquartile range [IQR]: 4–5.5) was significantly higher than the SC estradiol cypionate dose (median 2 mg weekly, IQR: 2–2.5), p < 0.001. No clinically significant adverse events occurred, including new transaminase elevation, severe dyslipidemia, or thromboembolism.
Conclusion:
Many TGNB adolescents and young adults on SC estradiol administration achieved appropriate serum estradiol concentrations and no adverse events were reported, supporting continued use of SC estradiol in gender-affirming care.
Keywords: estradiol cypionate, estradiol valerate, gender-affirming care, subcutaneous estradiol, transgender youth
Introduction
Transgender and gender diverse (TGD) youth often pursue hormone therapy.1,2 In the setting of gender dysphoria, TGD youth have an increased risk of suicidality with a high burden of depression and anxiety,3 and hormone treatment improves quality of life and decreases anxiety and depression.4–6
TGD youth who desire feminine characteristics may pursue pubertal suppression with gonadotropin-releasing hormone (GnRH) agonists followed by gender-affirming estrogen therapy.7 Estrogen therapy can also reduce testosterone through inhibition of the hypothalamic–pituitary–gonadal axis but is often insufficient to suppress testosterone to physiological female serum testosterone concentrations.8 Thus, many individuals also continue GnRH agonists to suppress testosterone or use spironolactone, a medication with androgen-blocking effects.1 The dose of these medications is titrated to reduce testosterone to the pubertal female range (≤50 ng/dL) and to mimic physiological female serum estradiol concentrations (proceeding through concentrations associated with progressive Sexual Maturity Rating stages to adult serum estradiol concentrations of ∼100–300 pg/mL).1,9
While research supports efficacy and safety of various testosterone formulations, including subcutaneous (SC) testosterone injections in TGD adolescent and young adults, fewer options are available for estradiol.10,11 Studies show TGD individuals prefer SC testosterone as opposed to intramuscular (IM) testosterone.11,12 Estradiol has historically been delivered by transdermal patch, oral tablets, and IM injections.1 However, SC injections have been increasingly used in TGD feminizing treatment with weekly, self-administered dosing.13–18 This affords benefits of increased injection site options, decreased pain and discomfort, and reduced risk of hematoma formation as compared with IM injection. Data demonstrating similar pharmacokinetics and pharmacodynamics of SC estradiol cypionate suspension as compared with IM administration in cisgender females prompted this emerging route of administration.19 Newer literature in adults suggests similar efficacy in TGD adults of SC and IM estradiol valerate.16
With shortages of estradiol cypionate, estradiol valerate has also been prescribed instead for SC administration as it had previously been used intramuscularly by some providers.14–16 Limited data are available regarding the efficacy of SC estradiol cypionate or valerate to maintain serum estradiol concentrations and suppress serum testosterone concentrations in TGD adolescents and young adults. The objective of this study is to present retrospective data on the safety and efficacy of estradiol cypionate and estradiol valerate SC weekly injections in patients who prefer this route of gender-affirming hormone therapy.
Materials and Methods
Patients and procedures
A retrospective chart review was performed using electronic medical records to identify patients with gender dysphoria seen at the Connecticut Children’s Gender Program who were prescribed SC estradiol cypionate or SC estradiol valerate and underwent laboratory testing between January 1, 2021, and January 1, 2024. Individuals who met the inclusion criteria were identified using the electronic medical record tool Slicer/Dicer followed by a detailed chart review. Patients included in the study were less than 25 years old at initiation of SC estradiol weekly injections and had at least one laboratory test for serum hormone concentrations (estradiol and testosterone) after initiation of SC estradiol therapy. Patients at our practice were prescribed once-weekly SC injections and taught proper administration technique in person. Patients were instructed to obtain labs 7 days after injection of estradiol cypionate and 5 days after injection of estradiol valerate to estimate mean estradiol concentrations, based on extrapolation from IM data.20 Available estradiol cypionate was in cottonseed oil, and estradiol valerate used a carrier oil of castor oil.
Institutional review board (IRB) exemption through the Connecticut Children’s IRB was received under category 45 CFR 46.104 (d)(4). This study was exempt from patient and guardian consent. All data have been de-identified.
Data collection
Charts were reviewed for patient demographics including race and ethnicity, type of insurance provider, gender identity, medical history including body mass index (BMI) and mental health diagnoses, and age at different stages of gender journey. Gender identity was extracted from both the demographics section and medical provider documentation. Data were collected regarding estradiol formulation and dosage, anti-androgen formulation and dosage, associated lab results (serum estradiol and testosterone concentrations), adverse effects including changes in transaminases or lipid profile, adverse events like thromboembolism, and patient comments, concerns, and reasoning for initiating SC estradiol treatment.
Outcomes
Target serum estradiol concentrations were considered 100–300 pg/mL and serum testosterone concentrations were ≤50 ng/dL to mimic postpubertal adult physiological female ranges (follicular phase 19–144 pg/mL, mid-cycle 64–357 pg/mL, luteal phase 56–214 pg/mL per Quest Diagnostics9). Given the Endocrine Society Guidelines’ recommendations for serum estradiol concentrations of 100–200 pg/mL,1 separate analysis was also completed using this range. Dyslipidemia was defined as low-density lipoprotein (LDL) >100 mg/dL, high-density lipoprotein <40 mg/dL, or triglycerides >150 mg/dL.21 Elevated transaminases were defined as alanine aminotransferase (ALT) >40 U/L.22
For each individual, the lowest dose used to achieve a therapeutic serum estradiol concentration on each SC formulation was used for data analysis if there were multiple doses at which a therapeutic serum estradiol concentration was obtained. The first therapeutic serum estradiol concentration on this lowest dose was used if a patient maintained therapeutic concentrations. Individuals were included in both estradiol cypionate and estradiol valerate groups if they met the inclusion criteria for each group and completed the necessary lab testing on each formulation.
Data analysis
Excel was used for data management and analysis. Means and standard deviations (SD) were used to describe patient characteristic data. Estradiol dose and serum estradiol concentration were assessed for normality using quantile-quantile plot; they were described with median and interquartile range (IQR). Statistical analyses with Mann–Whitney U tests were performed on estradiol dosing and serum estradiol concentration; p < 0.05 was considered statistically significant.
Results
From January 1, 2021, through January 1, 2024, a total of 550 patients with gender dysphoria were seen at the Gender Program clinic at Connecticut Children’s. Of these, 102 individuals received estradiol therapy by route other than SC administration. There were 25 patients who used SC estradiol cypionate and 38 patients who used SC estradiol valerate, with many using both. A total of 40 transgender and nonbinary (TGNB) individuals on SC estradiol for gender-affirming hormone treatment met the inclusion criteria and were included in this study: 23 patients in the SC estradiol cypionate group and 32 patients in the SC estradiol valerate group. Of the 40 patients, the majority identified as transgender (n = 37, 93%) and 3 individuals identified as nonbinary; all patients were assigned male at birth.
There was diversity in race and ethnicity (Table 1) with 55% White/Caucasian individuals (n = 22), 18% Black/African American individuals (n = 7), and 13% Hispanic/Latino individuals (n = 5). Public insurance was used by 38% of patients (n = 15). There was variety in BMI (range 15.9–47.3 kg/m2), with an overrepresentation of elevated BMI >30 kg/m2 (n = 9, 23%). 70% of individuals (n = 28) were diagnosed with anxiety; many individuals had multiple mental health diagnoses. All patients presented to treatment in their teenage years with an average age of 15 years 5 months (Table 2). Many individuals had experienced long-standing gender dysphoria, with 58% reporting onset by the age of 10 years (range 2–16 years, mean 8 years 8 months). The mean age for initiation of estradiol therapy was 16 years 9 months (range 12 years 11 months to 20 years 0 months); 88% of patients were on patch (n = 29, 73%) or pill (n = 6, 15%) estradiol formulations prior to treatment with SC estradiol (Table 3).
Table 1.
Descriptive Demographics of Patients
| N = 40 (100%) | |
|---|---|
| Race or ethnicity | |
| White/Caucasian | 22 (55%) |
| Black/African American | 7 (18%) |
| Hispanic/Latino | 5 (13%) |
| Unspecified | 4 (10%) |
| Asian | 1 (3%) |
| 2 or more races | 1 (3%) |
| Insurance | |
| Public | 15 (38%) |
| Private | 25 (63%) |
| Body mass index (kg/m2) by category | |
| <19 | 12 (30%) |
| 19–25 | 10 (25%) |
| 25–30 | 9 (23%) |
| >30 | 9 (23%) |
| Any mental health condition | 33 (83%) |
| Anxiety | 28 (70%) |
| Depression | 22 (55%) |
| Suicide attempt/ingestion | 11 (28%) |
| ADHD | 11 (28%) |
ADHD, attention-deficit/hyperactivity disorder.
Table 2.
Quantitative Demographics of Patients
| Mean | Minimum | Maximum | |
|---|---|---|---|
| Age at presentation | 15 years 5 months | 7 years 7 months | 19 years 8 months |
| Age of social transition | 14 years 2 months | 6 years 0 months | 19 years 0 months |
| Age of gender dysphoria | 8 years 8 months | 2 years 0 months | 16 years 0 months |
| Age of initiation of estradiol | 16 years 9 months | 12 years 11 months | 20 years 0 months |
| Age of initiation of SC estradiol | 18 years 4 months | 14 years 8 months | 23 years 7 months |
| Duration of estradiol therapy prior to initiation SC estradiol | 1 years 7 months | 0 years 0 months | 4 years 11 months |
| Age at therapeutic SC estradiol cypionate, n = 12 | 18 years 11 months | 15 years 4 months | 23 years 4 months |
| Age at therapeutic SC estradiol valerate, n = 24 | 19 years 7 months | 15 years 0 months | 24 years 6 months |
SC, subcutaneous.
Table 3.
Distribution of Individuals Receiving Subcutaneous Estradiol and Achieving Therapeutic Serum Estradiol Concentration
| Individuals | Individuals achieving therapeutic serum estradiol concentration |
|
|---|---|---|
|
N = 40 (100%; % of column) |
n = 30 (75%; % of row) |
|
| SC estradiol cypionate | 23 (57%) | 12 (52%) |
| Unable to achieve therapeutic serum estradiol level prior to transition to estradiol valerate | 9 (39%) | |
| SC estradiol valerate | 32 (80%) | 24 (75%) |
| Previously treated with estradiol cypionate | 16 (50%) | On both: 7 (44%) |
| Alternate estradiol prior to SC treatment | 35 (88%) | 25 (71%) |
| Reason for switching— | ||
| Difficulties with patchesa | 25 (72%) | 17 (68%) |
| Better safety profile than estradiol pills (previously with patch difficulties) | 3 (9%) | |
| Desire for improved breast development | 6 (17%) | |
| Other | 4 (11%) |
Irritant dermatitis, patches not staying on for at least 3 days, postpatch hyperpigmentation, subtherapeutic estradiol levels.
Of the 23 patients who received SC estradiol cypionate, only 52% (n = 12) achieved therapeutic serum estradiol concentrations as their therapy was disrupted by a national shortage (Fig. 1). Most individuals not reaching therapeutic concentrations on SC estradiol cypionate switched to SC estradiol valerate prior to dose titration and repeat lab testing (n = 9, 39%), while 2 others were lost to follow up. A total of 32 individuals received SC estradiol valerate and 84% (n = 27) achieved robust serum estradiol concentrations, with 75% (n = 24) in the therapeutic range and another 9% (n = 3) with supratherapeutic concentrations >300 pg/mL requiring further titration. Testosterone was adequately suppressed in all but one instance (97%) of therapeutic serum estradiol concentration using SC estradiol (n = 36) with or without anti-androgen medications (Table 4). The individual with inadequately suppressed testosterone had a serum testosterone concentration of 134 ng/dL while having a therapeutic serum estradiol concentration of 196 pg/mL on a steady SC estradiol cypionate dose for 3 months; once the individual’s histrelin implant was replaced, the serum testosterone concentration was adequately suppressed. One patient was able to achieve adequate testosterone suppression without the use of any anti-androgens.
FIG. 1.
Flowsheet of included individuals on subcutaneous estradiol cypionate and estradiol valerate.
Table 4.
Anti-Androgens Used While on Therapeutic Doses of Subcutaneous Estradiol and Testosterone Suppression
| Anti-androgen | Estradiol cypionate n = 12 |
Estradiol valerate n = 24 |
Suppressed serum testosterone <50 ng/dL n = 35 (97%) |
|---|---|---|---|
| Histrelin acetate implant | 9 | 12 | 20 (95%) |
| Triptorelin injections | 0 | 2 | 2 (100%) |
| Spironolactonea (200 mg/day) | 2 | 9 | 11 (100%) |
| None, s/p orchiectomy | 1 | 2 | 3 (100%) |
| None | 1 | 1 | 2 (100%) |
Participants who were on multiple anti-androgens were included in each appropriate row.
Primary mechanism of action of spironolactone is as an androgen receptor antagonist.
Each individual’s therapeutic SC estradiol cypionate and SC estradiol valerate doses with their associated serum estradiol concentration are displayed (Fig. 2). In order to produce these therapeutic serum estradiol concentrations, the median weekly dose of SC estradiol cypionate was 2 mg (IQR: 2–2.5) with a narrow range from 1.5 to 3 mg (Fig. 3). SC estradiol valerate doses ranged from 2 to 8 mg, with a median of 4 mg (IQR: 4–5.5). The difference in dose was statistically significant by Mann–Whitney U test, p < 0.001. The achieved therapeutic serum estradiol concentrations (100–300 pg/mL; Fig. 4) were not statistically significantly different per Mann–Whitney U test; for patients on SC estradiol valerate, the median was 185 pg/mL with IQR: 125–227 as compared with median 128 pg/mL and IQR: 119–169 on SC estradiol cypionate. After limiting the serum estradiol concentrations range to 100–200 pg/mL per Endocrine Society Guidelines,1 there remains a statistically significant difference in dosing (p < 0.005) without statistically significant difference in serum estradiol concentration; median SC estradiol cypionate dose remained 2 mg (IQR: 2–2.5 mg) and median SC estradiol valerate dose remained 4 mg (IQR: 3–4). This study was not powered to investigate a dose–response relationship.
FIG. 2.
Therapeutic dosing of subcutaneous estradiol and associated serum estradiol concentration achieved for individuals on estradiol cypionate and estradiol valerate.
FIG. 3.
Box and whisker plot of doses of subcutaneous estradiol to reach therapeutic response on estradiol cypionate and estradiol valerate.
FIG. 4.
Box and whisker plots of therapeutic serum estradiol concentrations in individuals on subcutaneous estradiol cypionate and estradiol valerate.
Though most patients tolerated SC injections well, some (n = 9, 23%) noted difficulty with the technique, with the majority of these (n = 7) noting loss of a few drops of their dose. One patient had local irritation with a red bump and itchiness that resolved once the type of needle used was changed. Another individual complained of pain and difficulty remembering injections and opted to switch to oral estradiol. Dyslipidemia was present in 23% (n = 9) of patients using SC estradiol, but only 2 patients (5%) had new dyslipidemia when starting SC estradiol as compared with previous lipid panels. All cases were mild, and no patients required pharmacologic treatment for dyslipidemia. Of those with elevated LDL (n = 7), 3 individuals had BMI >30 kg/m2, and another 2 had BMI 25–29 kg/m2. Elevation in ALT was seen in only four patients while on SC estradiol, and all individuals had abnormal transaminases prior to SC estradiol use. There were no reported thrombotic complications documented.
Discussion
This was a small, single-center retrospective analysis of SC estradiol cypionate and SC estradiol valerate to achieve adequate serum estradiol and testosterone concentrations in gender-affirming hormone therapy. The demographics highlight the significant mental health burden, as well as elevated BMI, in TGD adolescents and young adults.5,23,24 This is the first study to show that both SC estradiol cypionate and SC estradiol valerate can be used to achieve therapeutic serum estradiol concentrations in adolescents and young adults.
The increasing data available on SC estradiol use in TGD individuals comes from adults.13–18 In one study, adults cited similar reasons for transitioning to SC estradiol, including subtherapeutic serum estradiol concentrations or inadequate estradiol concentrations for preferred feminizing effects, and patient preference.14 A study on estradiol for gender-affirming care in youth only included six individuals on SC estradiol.15 An abstract of 23 adult transwomen on SC estradiol in a single reproductive endocrinology clinic suggested efficacy in achieving appropriate serum estradiol and testosterone concentrations with minimal adverse events.13 The largest study commenting on adverse events in TGD individuals using SC estradiol by Herndon et al.14 reported no cardiovascular or venous thromboembolism adverse events in their sample of 74 individuals.
In that study, 82.4% (n = 61) of patients on SC estradiol, predominantly estradiol valerate, achieved serum estradiol concentrations >100 pg/mL, which included supratherapeutic concentrations >300 pg/mL.14 Another study showed only 25% (n = 33) of individuals on SC estradiol achieved serum estradiol concentrations of 100–200 pg/mL.16 The success in reaching therapeutic serum estradiol concentrations of 100–300 pg/mL when using SC estradiol cypionate was only 52% in our study, with the lower efficacy most likely due to inadequate supply and opportunity for dose titration. With 75% of patients on SC estradiol valerate in our study achieving therapeutic serum estradiol concentrations, our study showed similar or superior efficacy in our sample of adolescents and young adults compared with the adult samples studied.
While Herndon et al. showed median weekly SC estradiol cypionate dose was statistically significantly lower (3 mg) than estradiol valerate (4 mg),14 larger studies suggest there may not be a difference; however, these larger studies do not analyze data from individuals receiving SC estradiol separately from those on IM estradiol.16 Nonetheless, in our study less SC estradiol cypionate (median 2 mg) was required than SC estradiol valerate (median 4 mg), p < 0.005, for both therapeutic ranges 100–300 pg/mL9 and 100–200 pg/mL1. Similar to their study, there were no differences in serum estradiol concentrations achieved with this differing dosing of SC estradiol formulations. The finding that low doses <5 mg of estradiol cypionate and valerate typically achieve therapeutic serum estradiol concentrations is congruent with recent literature suggesting that recommendations for initial dosing of 2–10 mg weekly by World Professional Association for Transgender Health (WPATH) and the Endocrine Society Guidelines may lead to supratherapeutic serum estradiol concentrations.16,17
While doses of SC estradiol valerate in this study were similar to those used in six TGD youth in a recent Sumerwell et al. study,15 the serum estradiol concentrations differed likely due to differences in timing of laboratory testing. In adults, data show a significant difference in serum estradiol concentrations of 19 pg/mL on average per day after injection.16
Since estrogen production is cyclical in individuals with ovaries with normal values extending >300 pg/mL, there is controversy about appropriate guidelines in that serum estradiol concentrations up to 300 pg/mL may be an appropriate target. There was no evidence of adverse events, including the individuals that achieved therapeutic serum estradiol concentrations of 200–300 pg/mL, suggesting adequate short-term safety at higher serum estradiol concentrations. Data in adults show many individuals on IM and SC estradiol achieve serum estradiol concentrations >200 pg/mL; however, the largest study does not report safety outcomes.16 While this study could not comment on bone health effects, higher serum estradiol concentrations may be beneficial for bone health.
Bone health is an area of particular interest in TGD individuals who received GnRH agonist treatment, which may cause decreased bone mineral density due to disruption of pubertal sex steroid secretion.7,25 Data show subsequent catch-up bone development occurs with hormonal therapy; however, optimal serum estradiol concentrations for bone health have not been established despite early research that higher estradiol doses and potentially higher serum estradiol concentrations may be beneficial for bone health.7,25–27 Literature regarding linear dose dependence of serum estradiol concentrations for IM estradiol is variable,14,26 but some dose dependence has been identified in larger adult studies of TGD individuals on SC estradiol.14,16 Our study was not powered to investigate this. Regardless, the SC estradiol doses used in our sample can provide guidance to future providers in starting dosage of SC estradiol, with initiation of SC estradiol followed by close titration of dose based on serum estradiol concentration and desired feminizing effects.
None of the research into the use of SC estradiol, including this study, documented significant adverse events including any episodes of venous thromboembolism.13–15 In this study, clinically significant dyslipidemia and elevated transaminases that could be directly attributed to SC estradiol were rare and mild in nature; this is similar to the lack of increased risk of cardiometabolic outcomes seen in TGD youth receiving estradiol once data were adjusted for age, duration in study, overweight/obesity, depression, and antipsychotic prescription.23
Limitations
While this study provides a reasonably diverse sample of TGNB adolescents and young adults receiving SC estradiol treatment, it is retrospective and primarily descriptive in nature. Clinical outcomes of feminization were not available; this outcome may be of particular interest to patients as some cited desire for further breast development as a reason to transition to SC estradiol injections. Limited sample size prevents rigorous statistical analysis as well as generalizability. The national shortage of estradiol cypionate prevented dosing titration and achievement of therapeutic serum estradiol concentrations on SC estradiol cypionate for many patients. This, paired with delayed (or missed) lab draws, makes it hard to draw an accurate assessment of the proportion of patients who can achieve appropriate serum estradiol concentrations on SC estradiol injections. Continued titrations may allow more patients to eventually achieve therapeutic serum estradiol concentrations, especially given a portion of individuals had supratherapeutic concentrations at the conclusion of this study. While it was requested that patients obtained labs at specific intervals after injection, it was not possible to verify this for all samples through chart review.
Conclusion
Despite the limitations, this study demonstrates that SC estradiol cypionate and SC estradiol valerate can be used to achieve therapeutic serum estradiol concentrations with an acceptable safety profile, supporting continued use of this modality of gender-affirming hormone therapy with titration to meet the needs of TGD adolescents and young adults.
Acknowledgments
The authors extend many thanks to Dr. Emily Germain-Lee for her guidance and mentorship, including article review. The authors also extend special thanks to Samuel Aaron for his help with Excel management and data formatting and analysis, as well as his endless patience and support.
Abbreviations Used
- ADHD
Attention-deficit/hyperactivity disorder
- ALT
Alanine aminotransferase
- BMI
Body mass index
- GnRH
Gonadotropin-releasing hormone
- IM
Intramuscular
- IQR
Interquartile range
- IRB
Institutional review board
- LDL
Low-density lipoprotein
- SC
Subcutaneous
- SD
Standard deviation
- TGD
Transgender and gender diverse
- TGNB
Transgender and non-binary
- WPATH
World Professional Association for Transgender Health
Authors’ Contributions
M.S.A. contributed to methodology, data acquisition and curation, formal analysis, visualization, and writing—original draft and review and editing. P.P. led conceptualization and contributed to methodology and writing—review and editing.
Author Disclosure Statement
No competing financial or other interests exist
Funding Information
No funding sources were used for this study.
Cite this article as: Aaron MS, Phulwani P (2025) Single-center retrospective analysis of safety and efficacy of subcutaneous estradiol use in transgender and nonbinary adolescents and young adults, Transgender Health 10:4, 316–324, DOI: 10.1089/trgh.2024.0264.
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