Transgender IndividualsNCT07394400
Effects of Gender-Affirming Hormone Therapy on Cardiovascular, Metabolic, and Mental Health Outcomes in Transgender Adults.
An observational study of Transgender Individuals, run by Milagros Rocha Barajas at 1 site in Spain.
Trial summary
The objective of this observational study is to examine the impact of gender-affirming hormone therapy (GAHT) on mood in transgender individuals and to explore its potential association with early atherosclerotic processes. Read more about this study
- Who is it for?
- Ages 16 years and olderHealthy volunteers can take part
- Conditions the study is for
- Transgender Individuals
- How many people
- 94 participants
- Where
- 1 siteSpain
- Study dates
- 14 Feb 2026to 31 Dec 2029
- How long will I take part?
- 12 Months
- Phase
- Observational
- Are there results?
- Not yet: the study is under way
Key requirements
Who can take part, as listed by the sponsor. Only the study team can confirm whether you are eligible, so take this list to your doctor.
- Ages 16 years and older
- All sexes
- Healthy volunteers accepted
- Child, Adult, Older adult
Study population: Transgender individuals referred to a Gender Identity Unit who are requesting Gender Affirmation Hormone Therapy for the first time. It will consist of three visits: at the start, and after 6 and 12 months of treatment.
Who may be able to join
- Trans men and trans women over the age of 16
Who cannot join
- systemic inflammatory diseases
- uncontrolled infections
- severe autoimmune diseases intersex biological conditions (resistance, severe sexual development or chromosomal abnormalities, congenital adrenal hyperplasia)
- treatment with metformin, statins or oral contraceptives in the previous 6 months
- serious life-threatening diseases
- diseases that compromise follow-up
- BMI higher than 35
Trial locations and contacts
1 site in 1 country. Contact the study team or a site near you to ask about taking part.
Spain1 site
Hospital Universitario Doctor Peset
Valencia, Valencia, 46017
Milagros Rocha, PhD, 963189132 ext. 0034, milagros.rocha@fisabio.es
About this study
Official title: A Comprehensive Assessment of the Impact of Gender-Affirming Hormone Therapy on Cardiovascular Risk, Metabolic Health, and Mood Disorders in Transgender Individuals.
The objective of this observational study is to examine the impact of gender-affirming hormone therapy (GAHT) on mood in transgender individuals and to explore its potential association with early atherosclerotic processes. The main questions it aims to answer are:
- Whether GAHT in transgender individuals has a psychological and functional impact, assessed through mood, stress, and self-esteem. Additionally, the study will analyze whether morphofunctional changes (including body composition, muscle strength, cardiorespiratory fitness, and metabolic flexibility) are related to inflammatory responses and psychological well-being.
- Whether the administration of GAHT poses a cardiovascular risk by influencing biochemical, inflammatory, and oxidative markers, mitochondrial function, and the activation of the NLRP3 inflammasome in leukocytes.
Participants will undergo evaluation of early stages of the atherosclerotic process through the analysis of leukocyte-endothelial interactions, adhesion molecule expression, and ankle-brachial index (ABI), as well as periodontal parameters. Overall, this study aims to contribute to improving healthcare delivery and promoting more equitable and gender-affirming medical services tailored to the needs of transgender individuals.
Read the detailed description
Blood samples will be collected from patients at baseline (before starting gender-affirming hormone therapy), 6 and 12 months after. From fasting blood samples (12h), peripheral blood mononuclear cells (PBMCs) and neutrophil fractions will be isolated using an immunomagnetic method, following the manufacturer's protocol, and plasma and serum will be stored at -80°C until analysis. LUNA-FL will be used to determine cell count and viability (acridine orange and propidium iodide double stain) in cell samples.
For periodontal assessment participants will undergo a comprehensive periodontal examination to determine several gingival parameters, including probing depth (PD), clinical attachment loss (CAL), bleeding index, plaque index, and calculus. Based on these data, the presence or absence of periodontal disease, as well as its extent when present, will be diagnosed.
Biochemical parameters of carbohydrate metabolism - glucose, insulin, glycated hemoglobin (A1c) -, lipid profile - total cholesterol, LDL, HDL, triglycerides (TG), apolipoproteins AI and B -, emerging inflammatory and cardiovascular risk markers - C-Reactive Protein (CRP), C3c, and retinol-binding protein 4 (RBP4) -, and complete blood count will be determined at the hospital's Clinical Analysis Service. Weight, height, and blood pressure will be measured using standardized methods.
Morphofunctional changes will be assessed at baseline and after 12 months of GAHT. Participants will attend the Morphofunctional Assessment Unit after an overnight fast. Body composition and hydration status will be measured using bioelectrical impedance analysis (mBCA 520, SECA, Germany). The quality and distribution of adipose and muscle tissue will be evaluated through Nutritional Ultrasound® using a DP-50 Expert Mobile Ultrasound System (Mindray®) equipped with a multifrequency linear transducer (5-10 MHz). Measurements will be performed in B-mode following established methodological recommendations (10.1016/j.endinu.2022.03.008).
For evaluation of cardiorrespiratory fitness through an incremental ramp exercise test on a cycle ergometer (Ergoselect 200 P, Ergoline, Germany) performed to voluntary exhaustion, following established recommendations (10.1152/ajpregu.00126.2020.0363-6119/20), to determine VO₂max and ventilatory thresholds. A COSMED K5 metabolic analyzer (COSMED, Italy) will be used for gas exchange analysis. Blood pressure, heart rate (Polar H10 heart rate monitor; Polar Electro Oy, Finland), and oxygen saturation at both systemic and muscular levels (WristOx2 3150, Nonin Medical Inc., USA; and Moxy®, Fortiori Design LLC, USA, respectively) will be continuously monitored. Maximal isometric strength of the knee extensor muscles will be measured using a MicroFET2 dynamometer (Hoggan Scientific LLC, USA), and resting metabolic rate (RMR) will also be assessed with the COSMED K5 metabolic analyzer.
For detection of differences in protein expression, cells will be incubated in lysis buffer with protease and phosphatase inhibitors (RIPA Buffer) for 15 minutes at 4 degrees celsius (°C). The supernatant will be collected after centrifugation for 15 minutes at 16,000g. The total protein concentration will be quantified using a bicinchoninic acid (BCA) protein assay. Aliquots of 25 µg of protein will be resolved on 8-16% gradient SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Target proteins will be detected by incubating the membranes with anti-actin, JNK, NFkB, NLRP3, ASC, caspase 1, NADPH oxidase, catalase, GPX1, SOD1, NRF2, mitochondrial markers such as the different complexes of the electron transport chain and PGC1α. The protein signal will be detected by chemiluminescence and analyzed by densitometry.
To measure the expression of the anti-oxidant enzymes SOD1, NFR2 and the genes dependent on the NRF2 antioxidant pathway (such as GPX1, GCLC, GLCM and TXNRD1) using real-time PCR. Mitochondrial function will be assessed by analysing PBMC oxygen consumption using the Seahorse XF technique (Agilent) as described in 10.1016/j.redox.2025.103516.
Inflammasome activation in PBMCs will be evaluated by detecting ASC specks using fluorescence microscopy. Briefly, PBMCs will be seeded on coverslips coated with Poly-D-Lysine, fixed with paraformaldehyde (PFA) 4% for 20 minutes, permeabilized with Triton X-100 for 20 minutes and blocking with Phosphate-buffered saline buffer-Bovine serum albumn (PBS-BSA) 3% for 1 hour at room temperature. Hybridization with specific primary antibodies (diluted in PBS-BSA 1%) will be carry out overnight at 4 ºC and then, secondary antibodies conjugated with AlexaFluor fluorophores will be incubated for 1 hour in the dark at room temperature. Stained samples will be transferred the coverslip onto microscope slide and conserved in anti-fade fluorescence mounting medium.
Gene expression profiling will be performed using the Nanostring® nCounter® platform to identify transcripts differentially expressed after 12 months of THAG intervention. The multiplex metabolism panel, encompassing 768 genes, will target pathways involved in inflammation (TLR, NF-κB), oxidative stress, cytokine signaling, and mitochondrial respiration. Data analysis will be conducted using the nSolver software (Nanostring Technologies). All experiments will be outsourced to Diagnostica Longwood, S.L.
Circulating levels of cytokines, adhesion molecules and serum oxidative stress markers will be measured in serum samples. This samples will be analyzed with a Luminex® 200 analyzer system following the Milliplex® MAP Kit manufacturer's procedure.
A parallel plate flow chamber, connected to an inverted microscope, will enable the researchers to measure neutrophil-endothelial cell interactions in vitro. Through this system, the leukocyte suspension obtained from patients will be perfused over a monolayer of immortalized endothelial cells (HUVEC/TERT 2) under conditions simulating blood flow. Videos will be analyzed afterward to determine flow, rolling velocity, and firm adhesion of leukocytes to endothelial cells, as previously described (Antioxidants. 2020 Aug 11;9(8):734).
DNA will also be extracted from serum according to the "blood and body fluid protocol" of the QIAamp blood reagent set (QIAgen, Hilden, Germany); 200 μL of plasma will be applied to each column, DNA will be eluted in 100 μL of supplied buffer and will be stored at -20°C until use. mtDNA will be quantified with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using specific primers for obtaining circulating mtDNA levels.
Data analysis will be performed with SPSS 17.0. Groups will be compared using unpaired Student's t-tests or Mann-Whitney U tests for parametric and non-parametric data, respectively. Changes after intervention will be evaluated using paired Student's t-tests or Wilcoxon tests, depending on the variable distribution. Pearson or Spearman correlation coefficients will be used to measure the strength of association between variables. In multivariable regression models, the relationship between two or more explanatory variables (independent variables) and a response variable (dependent variable) will be evaluated by fitting a linear equation to the obtained data. Qualitative data will be expressed in percentages, and proportions will be compared using a Chi-square test. All tests will use a 95% confidence interval, and differences will be considered statistically significant when p <0.05.
- Conditions
- Transgender Individuals
- Keywords
- GAHT, inflammation, atherosclerosis, periodontal disease, Transgender
- Medical subject headings
- Inflammation, Atherosclerosis, Periodontal Diseases
What is being measured
The primary outcome is the main question the study is designed to answer.
Changes in the degree of activation of the inflammasome complex in transgender individuals following gender-affirming hormone therapy.
Relative protein expression of NLRP3, ASC, Caspase-1 and inflammatory mediators NFκB, JNK by Western Blot and normalized to the loading control protein in PBMC. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Changes in endothelial function in the study population.
Evaluation of leucocytes adhesion to the endothelial cell in vitro by means of a parallel flow chamber system coupled to an inverted phase-contrast microscope. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Changes in endothelial function at molecular level in the study population.
Evaluation of circulating levels of adhesion molecules - P-selectin, ICAM-1, and VCAM-1, levels of the pro-oxidant enzyme MPO and circulating cytokines (L1β, IL18, IL6, IL10 and TNFα) in serum using Luminex technique. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Changes in the systemic inflammation status in the study population.
Evaluation of circulating levels of mtDNA in serum samples using RT-PCR. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Changes in the expression of genes related to inflammatory pathways in PBMC in the study population.
Identification of altered messengers using a metabolic multiplex panel, evaluating genes of interest in inflammatory pathways (TLR and NFkB), oxidative stress, cytokine signalling, and mitochondrial respiration. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Changes in the expression of genes related to oxidative stress levels in PBMC in the study population.
Relative expression of genes related to oxidative stress (SOD1, NRF2, GSR, GPX1, GCLC, GCLM and TXNRD1) by PCR real time. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Changes in the expression of genes related to cellular respiration in PBMC in the study population.
Relative expression of pro and antioxidants enzimes (NADPH oxidase, catalase, GPX1, SOD1, and NFR2) and genes related to mitochondrial respiration (I, II, III, IV and V complexes) and PGC1α in PBMC by WB. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Changes in the degree of activation of the inflammasome complex in transgender individuals following gender-affirming hormone therapy.
Study of inflammasome assembly by confocal microscopy in PBMC. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Secondary outcomes (2)
Evaluation of redox status (PBMCs and PMNs) in the study population.
Determination of redox status using flow cytometry with fluorescent probes. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Evaluation of mitochondrial function of leukocytes (PBMCs and PMNs) in the study population.
Determination of mitochondrial function by analysing oxygen consumption in PBMCs using the Seahorse XF technique (Agilent). This measurement will be taken at the start, and at 6 and 12 months of THAG.
Measured: At recruitment
Timeline
From the start of the study to its completion; the mark is today. Estimated dates are the sponsor’s plan.
- First posted6 Feb 2026
- Study start14 Feb 2026 (estimated)
- Primary completion30 Sep 2029 (estimated)
- Study completion31 Dec 2029 (estimated)
Resources
- Official record on ClinicalTrials.govThe registry entry this page is built from, with its full change history.
- Publications (11)Papers the sponsor has linked to this study.
- Other trials for Transgender IndividualsStudies of the same condition, open and under way.
- What the trial phases meanHow a treatment moves from first tests in people to approval.
More details
For doctors and researchers: how the study is designed, who runs it, and its registry IDs.
Study design
- Study type
- Observational
- Observational model
- Cohort
- Time perspective
- Prospective
- Enrolment
- 94 participants (estimated)
- Target duration
- 12 Months
Sponsor and investigators
- Lead sponsor
- Milagros Rocha Barajas
- Sponsor type
- University, hospital or other
- Collaborators
- Instituto de Salud Carlos III
- Responsible party
- Sponsor-investigator, Milagros Rocha Barajas, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana
- Investigators
- Milagros Rocha, PhD, Principal investigator, FISABIO-HOSPITAL DR PESET
IDs, dates and publications
- ClinicalTrials.gov ID
- NCT07394400
- Sponsor's protocol ID
- PI25/01841
- Status last verified
- Oct 2025
- First submitted
- 13 Jan 2026
- Last updated
- 6 Feb 2026
- Results posted
- No
- Data monitoring committee
- No
- FDA-regulated drug
- No
- FDA-regulated device
- No
Publications
Koukoulis GN, Filiponi M, Gougoura S, Befani C, Liakos P, Bargiota Alpha. Testosterone and dihydrotestosterone modulate the redox homeostasis of endothelium. Cell Biol Int. 2022 Apr;46(4):660-670. doi: 10.1002/cbin.11768. Epub 2022 Jan 30.
PubMed 35066972Exposito-Campos P, Gomez-Balaguer M, Hurtado-Murillo F, Morillas-Arino C. Evolution and trends in referrals to a specialist gender identity unit in Spain over 10 years (2012-2021). J Sex Med. 2023 Feb 27;20(3):377-387. doi: 10.1093/jsxmed/qdac034.
PubMed 36763946Banuls C, Rovira-Llopis S, Martinez de Maranon A, Veses S, Jover A, Gomez M, Rocha M, Hernandez-Mijares A, Victor VM. Metabolic syndrome enhances endoplasmic reticulum, oxidative stress and leukocyte-endothelium interactions in PCOS. Metabolism. 2017 Jun;71:153-162. doi: 10.1016/j.metabol.2017.02.012. Epub 2017 Feb 27.
PubMed 28521868Zhang Q, Goodman M, Adams N, Corneil T, Hashemi L, Kreukels B, Motmans J, Snyder R, Coleman E. Epidemiological considerations in transgender health: A systematic review with focus on higher quality data. Int J Transgend Health. 2020 Apr 15;21(2):125-137. doi: 10.1080/26895269.2020.1753136. eCollection 2020.
PubMed 33015664Victor VM, Rovira-Llopis S, Banuls C, Diaz-Morales N, Castello R, Falcon R, Gomez M, Rocha M, Hernandez-Mijares A. Effects of metformin on mitochondrial function of leukocytes from polycystic ovary syndrome patients with insulin resistance. Eur J Endocrinol. 2015 Nov;173(5):683-91. doi: 10.1530/EJE-15-0572. Epub 2015 Aug 28.
PubMed 26320144Villar CC, Sloniak MC, de Assis JB, Porto RC, Romito GA. Unveiling sex-disparities and the impact of gender-affirming hormone therapy on periodontal health. Front Dent Med. 2024 Sep 16;5:1430193. doi: 10.3389/fdmed.2024.1430193. eCollection 2024.
PubMed 39917660Nokoff NJ, Nemkov T, Bothwell S, Cree MG, Fuller KNZ, Keller AC, Kelsey MM, Nadeau KJ, Moreau KL. Differences in cardiorespiratory fitness by gonadotropin-releasing hormone agonist treatment before and after testosterone in transgender adolescents. J Appl Physiol (1985). 2024 Nov 1;137(5):1470-1483. doi: 10.1152/japplphysiol.00629.2024. Epub 2024 Oct 17.
PubMed 39417821Cheung AS, Zwickl S, Miller K, Nolan BJ, Wong AFQ, Jones P, Eynon N. The Impact of Gender-Affirming Hormone Therapy on Physical Performance. J Clin Endocrinol Metab. 2024 Jan 18;109(2):e455-e465. doi: 10.1210/clinem/dgad414.
PubMed 37437247Lakshmikanth T, Consiglio C, Sardh F, Forlin R, Wang J, Tan Z, Barcenilla H, Rodriguez L, Sugrue J, Noori P, Ivanchenko M, Pinero Paez L, Gonzalez L, Habimana Mugabo C, Johnsson A, Ryberg H, Hallgren A, Pou C, Chen Y, Mikes J, James A, Dahlqvist P, Wahlberg J, Hagelin A, Holmberg M, Degerblad M, Isaksson M, Duffy D, Kampe O, Landegren N, Brodin P. Immune system adaptation during gender-affirming testosterone treatment. Nature. 2024 Sep;633(8028):155-164. doi: 10.1038/s41586-024-07789-z. Epub 2024 Sep 4.
PubMed 39232147Glintborg D, Christensen LL, Andersen MS. Transgender healthcare: metabolic outcomes and cardiovascular risk. Diabetologia. 2024 Nov;67(11):2393-2403. doi: 10.1007/s00125-024-06212-6. Epub 2024 Jul 3.
PubMed 38958699Coleman E, Radix AE, Bouman WP, Brown GR, de Vries ALC, Deutsch MB, Ettner R, Fraser L, Goodman M, Green J, Hancock AB, Johnson TW, Karasic DH, Knudson GA, Leibowitz SF, Meyer-Bahlburg HFL, Monstrey SJ, Motmans J, Nahata L, Nieder TO, Reisner SL, Richards C, Schechter LS, Tangpricha V, Tishelman AC, Van Trotsenburg MAA, Winter S, Ducheny K, Adams NJ, Adrian TM, Allen LR, Azul D, Bagga H, Basar K, Bathory DS, Belinky JJ, Berg DR, Berli JU, Bluebond-Langner RO, Bouman MB, Bowers ML, Brassard PJ, Byrne J, Capitan L, Cargill CJ, Carswell JM, Chang SC, Chelvakumar G, Corneil T, Dalke KB, De Cuypere G, de Vries E, Den Heijer M, Devor AH, Dhejne C, D'Marco A, Edmiston EK, Edwards-Leeper L, Ehrbar R, Ehrensaft D, Eisfeld J, Elaut E, Erickson-Schroth L, Feldman JL, Fisher AD, Garcia MM, Gijs L, Green SE, Hall BP, Hardy TLD, Irwig MS, Jacobs LA, Janssen AC, Johnson K, Klink DT, Kreukels BPC, Kuper LE, Kvach EJ, Malouf MA, Massey R, Mazur T, McLachlan C, Morrison SD, Mosser SW, Neira PM, Nygren U, Oates JM, Obedin-Maliver J, Pagkalos G, Patton J, Phanuphak N, Rachlin K, Reed T, Rider GN, Ristori J, Robbins-Cherry S, Roberts SA, Rodriguez-Wallberg KA, Rosenthal SM, Sabir K, Safer JD, Scheim AI, Seal LJ, Sehoole TJ, Spencer K, St Amand C, Steensma TD, Strang JF, Taylor GB, Tilleman K, T'Sjoen GG, Vala LN, Van Mello NM, Veale JF, Vencill JA, Vincent B, Wesp LM, West MA, Arcelus J. Standards of Care for the Health of Transgender and Gender Diverse People, Version 8. Int J Transgend Health. 2022 Sep 6;23(Suppl 1):S1-S259. doi: 10.1080/26895269.2022.2100644. eCollection 2022.
PubMed 36238954
Study at a glance
From the registry record, last updated by the sponsor on 6 Feb 2026. Recruitment status is as the sponsor reported it: confirm with the study team.
- Official title
- A Comprehensive Assessment of the Impact of Gender-Affirming Hormone Therapy on Cardiovascular Risk, Metabolic Health, and Mood Disorders in Transgender Individuals.
- ClinicalTrials.gov ID
- NCT07394400
- Acronym / sponsor ID
- PI25/01841
- Conditions
- Transgender Individuals
- Keywords
- GAHT, inflammation, atherosclerosis, periodontal disease, Transgender
- Study type
- Observational
- Recruitment status
- Not yet recruiting
- Enrolment
- 94 participants (estimated)
- Lead sponsor
- Milagros Rocha BarajasUniversity, hospital or other
- Collaborators
- Instituto de Salud Carlos III
- Study start
- 14 Feb 2026 (estimated)
- Primary completion
- 30 Sep 2029 (estimated)
- Study completion
- 31 Dec 2029 (estimated)
- First posted
- 6 Feb 2026 (actual)
- Results
- Not posted in the source record
Clinical summary
The research question as the sponsor registered it. This describes what the study plans to test, not what it found.
The objective of this observational study is to examine the impact of gender-affirming hormone therapy (GAHT) on mood in transgender individuals and to explore its potential association with early atherosclerotic processes. The main questions it aims to answer are:
- Whether GAHT in transgender individuals has a psychological and functional impact, assessed through mood, stress, and self-esteem. Additionally, the study will analyze whether morphofunctional changes (including body composition, muscle strength, cardiorespiratory fitness, and metabolic flexibility) are related to inflammatory responses and psychological well-being.
- Whether the administration of GAHT poses a cardiovascular risk by influencing biochemical, inflammatory, and oxidative markers, mitochondrial function, and the activation of the NLRP3 inflammasome in leukocytes.
Participants will undergo evaluation of early stages of the atherosclerotic process through the analysis of leukocyte-endothelial interactions, adhesion molecule expression, and ankle-brachial index (ABI), as well as periodontal parameters. Overall, this study aims to contribute to improving healthcare delivery and promoting more equitable and gender-affirming medical services tailored to the needs of transgender individuals.
Detailed description
Blood samples will be collected from patients at baseline (before starting gender-affirming hormone therapy), 6 and 12 months after. From fasting blood samples (12h), peripheral blood mononuclear cells (PBMCs) and neutrophil fractions will be isolated using an immunomagnetic method, following the manufacturer's protocol, and plasma and serum will be stored at -80°C until analysis. LUNA-FL will be used to determine cell count and viability (acridine orange and propidium iodide double stain) in cell samples.
For periodontal assessment participants will undergo a comprehensive periodontal examination to determine several gingival parameters, including probing depth (PD), clinical attachment loss (CAL), bleeding index, plaque index, and calculus. Based on these data, the presence or absence of periodontal disease, as well as its extent when present, will be diagnosed.
Biochemical parameters of carbohydrate metabolism - glucose, insulin, glycated hemoglobin (A1c) -, lipid profile - total cholesterol, LDL, HDL, triglycerides (TG), apolipoproteins AI and B -, emerging inflammatory and cardiovascular risk markers - C-Reactive Protein (CRP), C3c, and retinol-binding protein 4 (RBP4) -, and complete blood count will be determined at the hospital's Clinical Analysis Service. Weight, height, and blood pressure will be measured using standardized methods.
Morphofunctional changes will be assessed at baseline and after 12 months of GAHT. Participants will attend the Morphofunctional Assessment Unit after an overnight fast. Body composition and hydration status will be measured using bioelectrical impedance analysis (mBCA 520, SECA, Germany). The quality and distribution of adipose and muscle tissue will be evaluated through Nutritional Ultrasound® using a DP-50 Expert Mobile Ultrasound System (Mindray®) equipped with a multifrequency linear transducer (5-10 MHz). Measurements will be performed in B-mode following established methodological recommendations (10.1016/j.endinu.2022.03.008).
For evaluation of cardiorrespiratory fitness through an incremental ramp exercise test on a cycle ergometer (Ergoselect 200 P, Ergoline, Germany) performed to voluntary exhaustion, following established recommendations (10.1152/ajpregu.00126.2020.0363-6119/20), to determine VO₂max and ventilatory thresholds. A COSMED K5 metabolic analyzer (COSMED, Italy) will be used for gas exchange analysis. Blood pressure, heart rate (Polar H10 heart rate monitor; Polar Electro Oy, Finland), and oxygen saturation at both systemic and muscular levels (WristOx2 3150, Nonin Medical Inc., USA; and Moxy®, Fortiori Design LLC, USA, respectively) will be continuously monitored. Maximal isometric strength of the knee extensor muscles will be measured using a MicroFET2 dynamometer (Hoggan Scientific LLC, USA), and resting metabolic rate (RMR) will also be assessed with the COSMED K5 metabolic analyzer.
For detection of differences in protein expression, cells will be incubated in lysis buffer with protease and phosphatase inhibitors (RIPA Buffer) for 15 minutes at 4 degrees celsius (°C). The supernatant will be collected after centrifugation for 15 minutes at 16,000g. The total protein concentration will be quantified using a bicinchoninic acid (BCA) protein assay. Aliquots of 25 µg of protein will be resolved on 8-16% gradient SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Target proteins will be detected by incubating the membranes with anti-actin, JNK, NFkB, NLRP3, ASC, caspase 1, NADPH oxidase, catalase, GPX1, SOD1, NRF2, mitochondrial markers such as the different complexes of the electron transport chain and PGC1α. The protein signal will be detected by chemiluminescence and analyzed by densitometry.
To measure the expression of the anti-oxidant enzymes SOD1, NFR2 and the genes dependent on the NRF2 antioxidant pathway (such as GPX1, GCLC, GLCM and TXNRD1) using real-time PCR. Mitochondrial function will be assessed by analysing PBMC oxygen consumption using the Seahorse XF technique (Agilent) as described in 10.1016/j.redox.2025.103516.
Inflammasome activation in PBMCs will be evaluated by detecting ASC specks using fluorescence microscopy. Briefly, PBMCs will be seeded on coverslips coated with Poly-D-Lysine, fixed with paraformaldehyde (PFA) 4% for 20 minutes, permeabilized with Triton X-100 for 20 minutes and blocking with Phosphate-buffered saline buffer-Bovine serum albumn (PBS-BSA) 3% for 1 hour at room temperature. Hybridization with specific primary antibodies (diluted in PBS-BSA 1%) will be carry out overnight at 4 ºC and then, secondary antibodies conjugated with AlexaFluor fluorophores will be incubated for 1 hour in the dark at room temperature. Stained samples will be transferred the coverslip onto microscope slide and conserved in anti-fade fluorescence mounting medium.
Gene expression profiling will be performed using the Nanostring® nCounter® platform to identify transcripts differentially expressed after 12 months of THAG intervention. The multiplex metabolism panel, encompassing 768 genes, will target pathways involved in inflammation (TLR, NF-κB), oxidative stress, cytokine signaling, and mitochondrial respiration. Data analysis will be conducted using the nSolver software (Nanostring Technologies). All experiments will be outsourced to Diagnostica Longwood, S.L.
Circulating levels of cytokines, adhesion molecules and serum oxidative stress markers will be measured in serum samples. This samples will be analyzed with a Luminex® 200 analyzer system following the Milliplex® MAP Kit manufacturer's procedure.
A parallel plate flow chamber, connected to an inverted microscope, will enable the researchers to measure neutrophil-endothelial cell interactions in vitro. Through this system, the leukocyte suspension obtained from patients will be perfused over a monolayer of immortalized endothelial cells (HUVEC/TERT 2) under conditions simulating blood flow. Videos will be analyzed afterward to determine flow, rolling velocity, and firm adhesion of leukocytes to endothelial cells, as previously described (Antioxidants. 2020 Aug 11;9(8):734).
DNA will also be extracted from serum according to the "blood and body fluid protocol" of the QIAamp blood reagent set (QIAgen, Hilden, Germany); 200 μL of plasma will be applied to each column, DNA will be eluted in 100 μL of supplied buffer and will be stored at -20°C until use. mtDNA will be quantified with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using specific primers for obtaining circulating mtDNA levels.
Data analysis will be performed with SPSS 17.0. Groups will be compared using unpaired Student's t-tests or Mann-Whitney U tests for parametric and non-parametric data, respectively. Changes after intervention will be evaluated using paired Student's t-tests or Wilcoxon tests, depending on the variable distribution. Pearson or Spearman correlation coefficients will be used to measure the strength of association between variables. In multivariable regression models, the relationship between two or more explanatory variables (independent variables) and a response variable (dependent variable) will be evaluated by fitting a linear equation to the obtained data. Qualitative data will be expressed in percentages, and proportions will be compared using a Chi-square test. All tests will use a 95% confidence interval, and differences will be considered statistically significant when p <0.05.
Study design
- Observational model
- Cohort
- Time perspective
- Prospective
- Target follow-up
- 12 Months
Who may qualify
- Ages
- 16 years and older
- Sex
- All
- Healthy volunteers
- Accepted
- Age groups
- Child, Adult, Older adult
- Study population
- Transgender individuals referred to a Gender Identity Unit who are requesting Gender Affirmation Hormone Therapy for the first time. It will consist of three visits: at the start, and after 6 and 12 months of treatment.
Key inclusion criteria (1 listed)
- Trans men and trans women over the age of 16
Key exclusion criteria (7 listed)
- systemic inflammatory diseases
- uncontrolled infections
- severe autoimmune diseases intersex biological conditions (resistance, severe sexual development or chromosomal abnormalities, congenital adrenal hyperplasia)
- treatment with metformin, statins or oral contraceptives in the previous 6 months
- serious life-threatening diseases
Eligibility is decided by the study team after screening; this overview does not establish that a patient qualifies. Full inclusion and exclusion criteria.
Primary outcome
Changes in the degree of activation of the inflammasome complex in transgender individuals following gender-affirming hormone therapy.
Time frame: At recruitment
Relative protein expression of NLRP3, ASC, Caspase-1 and inflammatory mediators NFκB, JNK by Western Blot and normalized to the loading control protein in PBMC. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Changes in endothelial function in the study population.
Time frame: At recruitment
Evaluation of leucocytes adhesion to the endothelial cell in vitro by means of a parallel flow chamber system coupled to an inverted phase-contrast microscope. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Changes in endothelial function at molecular level in the study population.
Time frame: At recruitment
Evaluation of circulating levels of adhesion molecules - P-selectin, ICAM-1, and VCAM-1, levels of the pro-oxidant enzyme MPO and circulating cytokines (L1β, IL18, IL6, IL10 and TNFα) in serum using Luminex technique. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Changes in the systemic inflammation status in the study population.
Time frame: At recruitment
Evaluation of circulating levels of mtDNA in serum samples using RT-PCR. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Changes in the expression of genes related to inflammatory pathways in PBMC in the study population.
Time frame: At recruitment
Identification of altered messengers using a metabolic multiplex panel, evaluating genes of interest in inflammatory pathways (TLR and NFkB), oxidative stress, cytokine signalling, and mitochondrial respiration. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Changes in the expression of genes related to oxidative stress levels in PBMC in the study population.
Time frame: At recruitment
Relative expression of genes related to oxidative stress (SOD1, NRF2, GSR, GPX1, GCLC, GCLM and TXNRD1) by PCR real time. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Changes in the expression of genes related to cellular respiration in PBMC in the study population.
Time frame: At recruitment
Relative expression of pro and antioxidants enzimes (NADPH oxidase, catalase, GPX1, SOD1, and NFR2) and genes related to mitochondrial respiration (I, II, III, IV and V complexes) and PGC1α in PBMC by WB. This measurement will be taken at the start, and at 6 and 12 months of THAG.
Changes in the degree of activation of the inflammasome complex in transgender individuals following gender-affirming hormone therapy.
Time frame: At recruitment
Study of inflammasome assembly by confocal microscopy in PBMC. This measurement will be taken at the start, and at 6 and 12 months of THAG.
2 secondary outcomes
Evaluation of redox status (PBMCs and PMNs) in the study population.
Time frame: At recruitment
Evaluation of mitochondrial function of leukocytes (PBMCs and PMNs) in the study population.
Time frame: At recruitment
Results and safety
This study is still under way. Results are not available in the source record yet.
Research links
- Official record on ClinicalTrials.govLast updated by the sponsor 6 Feb 2026.
- Background: PubMed 35066972Koukoulis GN, Filiponi M, Gougoura S, Befani C, Liakos P, Bargiota Alpha. Testosterone and dihydrotestosterone modulate the redox homeostasis of endothelium. Cell Biol Int. 2022 Apr;46(4):660-670. doi: 10.1002/cbin.11768. Epub 2022 Jan 30.
- Background: PubMed 36763946Exposito-Campos P, Gomez-Balaguer M, Hurtado-Murillo F, Morillas-Arino C. Evolution and trends in referrals to a specialist gender identity unit in Spain over 10 years (2012-2021). J Sex Med. 2023 Feb 27;20(3):377-387. doi: 10.1093/jsxmed/qdac034.
- Background: PubMed 28521868Banuls C, Rovira-Llopis S, Martinez de Maranon A, Veses S, Jover A, Gomez M, Rocha M, Hernandez-Mijares A, Victor VM. Metabolic syndrome enhances endoplasmic reticulum, oxidative stress and leukocyte-endothelium interactions in PCOS. Metabolism. 2017 Jun;71:153-162. doi: 10.1016/j.metabol.2017.02.012. Epub 2017 Feb 27.
- Background: PubMed 33015664Zhang Q, Goodman M, Adams N, Corneil T, Hashemi L, Kreukels B, Motmans J, Snyder R, Coleman E. Epidemiological considerations in transgender health: A systematic review with focus on higher quality data. Int J Transgend Health. 2020 Apr 15;21(2):125-137. doi: 10.1080/26895269.2020.1753136. eCollection 2020.
- Background: PubMed 26320144Victor VM, Rovira-Llopis S, Banuls C, Diaz-Morales N, Castello R, Falcon R, Gomez M, Rocha M, Hernandez-Mijares A. Effects of metformin on mitochondrial function of leukocytes from polycystic ovary syndrome patients with insulin resistance. Eur J Endocrinol. 2015 Nov;173(5):683-91. doi: 10.1530/EJE-15-0572. Epub 2015 Aug 28.
- Background: PubMed 39917660Villar CC, Sloniak MC, de Assis JB, Porto RC, Romito GA. Unveiling sex-disparities and the impact of gender-affirming hormone therapy on periodontal health. Front Dent Med. 2024 Sep 16;5:1430193. doi: 10.3389/fdmed.2024.1430193. eCollection 2024.
- Background: PubMed 39417821Nokoff NJ, Nemkov T, Bothwell S, Cree MG, Fuller KNZ, Keller AC, Kelsey MM, Nadeau KJ, Moreau KL. Differences in cardiorespiratory fitness by gonadotropin-releasing hormone agonist treatment before and after testosterone in transgender adolescents. J Appl Physiol (1985). 2024 Nov 1;137(5):1470-1483. doi: 10.1152/japplphysiol.00629.2024. Epub 2024 Oct 17.
- Background: PubMed 37437247Cheung AS, Zwickl S, Miller K, Nolan BJ, Wong AFQ, Jones P, Eynon N. The Impact of Gender-Affirming Hormone Therapy on Physical Performance. J Clin Endocrinol Metab. 2024 Jan 18;109(2):e455-e465. doi: 10.1210/clinem/dgad414.
- Background: PubMed 39232147Lakshmikanth T, Consiglio C, Sardh F, Forlin R, Wang J, Tan Z, Barcenilla H, Rodriguez L, Sugrue J, Noori P, Ivanchenko M, Pinero Paez L, Gonzalez L, Habimana Mugabo C, Johnsson A, Ryberg H, Hallgren A, Pou C, Chen Y, Mikes J, James A, Dahlqvist P, Wahlberg J, Hagelin A, Holmberg M, Degerblad M, Isaksson M, Duffy D, Kampe O, Landegren N, Brodin P. Immune system adaptation during gender-affirming testosterone treatment. Nature. 2024 Sep;633(8028):155-164. doi: 10.1038/s41586-024-07789-z. Epub 2024 Sep 4.
- Background: PubMed 38958699Glintborg D, Christensen LL, Andersen MS. Transgender healthcare: metabolic outcomes and cardiovascular risk. Diabetologia. 2024 Nov;67(11):2393-2403. doi: 10.1007/s00125-024-06212-6. Epub 2024 Jul 3.
- Background: PubMed 36238954Coleman E, Radix AE, Bouman WP, Brown GR, de Vries ALC, Deutsch MB, Ettner R, Fraser L, Goodman M, Green J, Hancock AB, Johnson TW, Karasic DH, Knudson GA, Leibowitz SF, Meyer-Bahlburg HFL, Monstrey SJ, Motmans J, Nahata L, Nieder TO, Reisner SL, Richards C, Schechter LS, Tangpricha V, Tishelman AC, Van Trotsenburg MAA, Winter S, Ducheny K, Adams NJ, Adrian TM, Allen LR, Azul D, Bagga H, Basar K, Bathory DS, Belinky JJ, Berg DR, Berli JU, Bluebond-Langner RO, Bouman MB, Bowers ML, Brassard PJ, Byrne J, Capitan L, Cargill CJ, Carswell JM, Chang SC, Chelvakumar G, Corneil T, Dalke KB, De Cuypere G, de Vries E, Den Heijer M, Devor AH, Dhejne C, D'Marco A, Edmiston EK, Edwards-Leeper L, Ehrbar R, Ehrensaft D, Eisfeld J, Elaut E, Erickson-Schroth L, Feldman JL, Fisher AD, Garcia MM, Gijs L, Green SE, Hall BP, Hardy TLD, Irwig MS, Jacobs LA, Janssen AC, Johnson K, Klink DT, Kreukels BPC, Kuper LE, Kvach EJ, Malouf MA, Massey R, Mazur T, McLachlan C, Morrison SD, Mosser SW, Neira PM, Nygren U, Oates JM, Obedin-Maliver J, Pagkalos G, Patton J, Phanuphak N, Rachlin K, Reed T, Rider GN, Ristori J, Robbins-Cherry S, Roberts SA, Rodriguez-Wallberg KA, Rosenthal SM, Sabir K, Safer JD, Scheim AI, Seal LJ, Sehoole TJ, Spencer K, St Amand C, Steensma TD, Strang JF, Taylor GB, Tilleman K, T'Sjoen GG, Vala LN, Van Mello NM, Veale JF, Vencill JA, Vincent B, Wesp LM, West MA, Arcelus J. Standards of Care for the Health of Transgender and Gender Diverse People, Version 8. Int J Transgend Health. 2022 Sep 6;23(Suppl 1):S1-S259. doi: 10.1080/26895269.2022.2100644. eCollection 2022.
Related trials for Transgender Individuals
Recruiting
Adapting and Pilot Testing a Skills-based Smoking Cessation Intervention to Address Developmental and Health-related Stress Among Adults Who Smoke
Smoking Cessation, Depression
Not yet recruitingPhase 2
A Clinical Study of Alimatravir (MK-8527) in Transgender (Trans) and Gender Diverse Individuals on Gender-affirming Hormone Therapy and at Low Likelihood of Human Immunodeficiency Virus (HIV)-1 Exposure (MK-8527-018)
HIV
Recruiting
Effects of Resistance Exercise Among TGD Individuals Initiating Estrogen-Based Gender-Affirming Hormone Therapy
Muscle Mass and Strength
RecruitingObservational study
Swiss ProspectivE Cohort of TRAnsgender and Gender Diverse Individuals - The SPECTRA Study
Transgender Individuals, Gender Incongruence
Recruiting
Effects of Combined Neuromuscular Electrical Stimulation Robot and Trans-Spinal Electrical Stimulation in Poststroke Rehabilitation
Stroke
Referral and contact
AzTrials does not enrol participants or take referrals. Contact the study team or a site listed below; they decide who can take part.
Milagros Rocha, PhD principal investigator
FISABIO-HOSPITAL DR PESET
1 site in 1 country: see every site and its status.
Questions about this trial
What is this clinical trial studying?
An observational study of Transgender Individuals, run by Milagros Rocha Barajas at 1 site in Spain.
The full registered description is in the About this study section and the official record.
Who may be eligible to take part?
The sponsor lists 1 inclusion and 7 exclusion criteria. Eligibility is decided by the study team after screening, so read the full criteria and contact a listed site.
Is this clinical trial currently recruiting?
The record says: opening soon, as of the sponsor's last update on 6 Feb 2026. Status can change between updates, so confirm with the study contact before making plans.
Where is the study taking place?
The record lists 1 site in Spain. Whether a site is taking part right now should be confirmed with that site. See every site.
What are the possible benefits and risks?
They depend on the study and the treatment, and no study can promise a benefit. The study team explains the known risks during informed consent, before anyone joins; the record itself does not list them.
Are results available for this study?
This study is still under way. Results are not available in the source record yet.
How can a doctor refer a patient or contact the research team?
Contact Miagros Rocha, PhD (963189132 ext. 0034, milagros.rocha@fisabio.es), or a site near the patient. AzTrials does not enrol participants or take referrals.
What does the primary outcome measure?
Changes in the degree of activation of the inflammasome complex in transgender individuals following gender-affirming hormone therapy. (time frame: At recruitment). The study lists 8 primary outcomes in all. It is what the study was designed to measure, not a finding.
Where can I read the original study information?
On the ClinicalTrials.gov record for NCT07394400, which also lists 11 linked publications.
Does a listing mean the treatment is approved or proven to work?
No. A listing means the study is registered. It does not show that the treatment is safe, works, or is approved by a regulator.
How often is this information updated?
AzTrials copies changed records from ClinicalTrials.gov every night and reloads the whole registry every week. The sponsor last updated this record on 6 Feb 2026.