Effects of Gender-Affirming Hormone Therapy on Cardiovascular, Metabolic, and Mental Health Outcomes in Transgender Adults.

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.

• 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]