Current study aims to characterize five highly interconnected physiological systems in patients undergoing cross-sex hormone therapy - namely glucose and lipid metabolism, energy balance, eating behavior, functional brain networks involved in the regulation of eating behavior and the cardiovascular system - to gain novel insights into the effects of sex hormones on the human body. Gathered information will help to identify pathophysiological mechanisms for the development of overeating/obesity, insulin resistance, and cardiovascular disease. Secondarily, the relationships between the gut and oral microbiomes and metabolomes and circulating bacterial signatures will be investigated in relation to the other pervasive physiological systems.

Current study is an observational study. The decision if the patient's request for cross-sex hormone therapy can complied with (i.e., if cross-sex hormone therapy is medically indicated) is made prior to the first contact with the study center and with the outpatients clinic for Endocrinology at the University Hospital in Leipzig. Decision ifor treatment is made according to national and international guidelines. Treatment of study participants with testosterone and estradiol/antiandrogens is not affected by the study. During the course of the study no invasive interventions are being performed.

1. Introduction 1.1. Transsexualism Transsexualism is a condition in which an individual's gender identity is incongruent with the assigned sex at birth. Many transsexuals suffer from severe psychological problems caused by this this incongruence, referred to as gender dysphoria. Supporting transsexuals in living in the sex with which they identify leads to amelioration of psychological problems and medical problems. In sex reassignment therapy, cross-sex hormone therapy is an essential element.

   1.2. Cross-sex hormone therapy in transsexual patients In many patients, cross sex hormone therapy is used to conduct sex change. In transmen (biological women with perceived male sex), testosterone is used for gender reassignment. In transwomen (biological men with perceived female sex), the most commonly used medication is estrogen. Successful therapy leads to the development of typical secondary sex characteristics. Data and knowledge regarding the effects of cross-sex hormone therapy on the human body, additionally to the desired sex affirming changes, is scarce.

   1.3. Investigation of the effects of estrogen and testosterone on the human body Investigating the effects of cross-sex hormone therapy provides further information on the effects of testosterone and estradiol on the human body. One of the most recognized differences between men and women regarding health is the increased cardiovascular risk of men as compared to women. In transmen, testosterone therapy leads to an increased risk for myocardial infarction. Data from the diabetes prevention program suggest that in males the probability of developing diabetes mellitus type 2, a major risk factor for cardiovascular disease is positively correlated with serum concentrations of estradiol and negatively correlated with concentrations of testosterone. Furthermore, previous studies pointed to differences in the regulation of eating behavior of men and women potentially contributing to the development of obesity and type-2-diabetes.

   Moreover, while the microbiota, known to affect host homeostasis, immunity, and metabolism, shows sex-related variations, the impact of sex hormones and gender transition on the different microbiome niches and related metabolic processes, remains largely unexplored.

   The above mentioned findings are just examples of plentiful dissimilarities in health and disease of the two sexes with unknown causes. To date studies assessing e.g. markers of cardiometabolic disease in cross-sex hormone therapy remain inconclusive.

   1.4. Purpose of this study To gain novel insights into the effects of sex hormones on the human body, current study aims to characterize four highly interconnected physiological systems in patients undergoing cross-sex hormone therapy: glucose and lipid metabolism, energy balance, eating behavior, and the cardiovascular system as well as several microbiome niches. Gathered knowledge will help to identify pathophysiological mechanisms for the development of overeating/obesity, insulin resistance, and cardiovascular disease.

2. Aims The study is designed to investigate the effects of testosterone and estradiol/cyproteron acetate in patients with gender dysphoria/transsexualism undergoing cross-sex hormone therapy in comparison to a control group without hormone therapy.

   Effects of cross-sex hormone therapy on:

   Part A Glucose and lipid metabolism Part B Body composition and energy balance control, microbiome Part C Psychobehavioral traits with a focus on eating behavior Part D (Cardio-) Vascular system

   To best possibly attribute observed effects to the applied sex hormones, baseline measurements before starting cross-sex hormone treatment will be performed (V0). Measurements will be repeated at five time points during ongoing cross sex hormone treatment (V1-5):

   V0 Baseline assessment before start of cross-sex hormone therapy V1 Three months after start of cross-sex hormone therapy V2 Six months after start of cross-sex hormone therapy V3 Twelve months after start of cross-sex hormone therapy V4 Two years after start of cross-sex hormone therapy V5 Five years after start of cross-sex hormone therapy To be able to exclude unspecific order effects of the measurements and to be able to compare obtained results with non-transsexual persons, controls not undergoing cross-sex hormone therapy matched for age, body mass index, health status (morbidity) and education will be included.

3. Research Design and Methods 3.1. Participants and recruitment This is a five year observational, non-invasive study to evaluate the effect of cross-sex hormone therapy on metabolism, eating behavior, body composition, energy expenditure, oral and the gut microbiomes, and the cardiovascular system. Results will be compared longitudinally on an intra-individual level, as well as on an inter-individual level by comparing transmen with transwomen and both groups with non-treated controls. Transsexual patients will be recruited at the outpatient clinic for Endocrinology at the University Hospital Leipzig. Patients seeking cross-sex hormone therapy will be asked before starting cross-sex hormone therapy for their willingness to participate in the study. Patients willing to participate will be screened for study eligibility. Eligible subjects will be informed about the study procedures and possible risks. Upon receiving written informed consent, the patient will be included into the study. Willingness to participate in the study will not influence the medical treatment.

A control group of men and women matched for age, body mass index, health status (morbidity) and education who are not undergoing cross-sex hormone therapy will be recruited via online advertisements.

3.2. Study procedures Seven days prior to baseline assessment, accelerometers will be handed to participants to measure physical activity. Four days prior to baseline, participants will be asked to complete a 96h activity log to objectify physical activity. Food intake will be self-assessed four days prior to baseline assessment via questionnaire as instructed by a trained dietitian. Dietary restraints will be assessed (e.g., lactose intolerance, etc.) to adjust procedures of ad libitum food intake measurement to individual needs. A tour of our metabolic unit and the MRI facilities will be given to participants and procedures explained. Subjects will be asked to fill out behavioral questionnaires at home, within one hour of completing their breakfast, to decrease variability of answers. Participants will use provided kits to collect stool samples for the gut microbiome analysis.

On the day of baseline assessment, prior to initiation of cross-sex hormone therapy/no therapy, current medical history including current medication, a physical exam including measurement of weight, waist and hip circumferences, blood pressure and heart rate will be performed. In the fasted state, blood will be drawn to measure fasting glucose, HbA1c%, serum insulin, triglycerides, total-, low- and high-density-lipoprotein concentrations. Insulin sensitivity and HOMA-IR will be calculated. The fasting blood draw will further include measurement of 17β-estradiol, progesterone, testosterone, sex-hormone binding globulin (SHBG), blood cell count, creatinine, ALAT, ASAT, GGT, TSH, and FGF21. Saliva samples will be collected in a fasted state for analysis of oral microbiome. All samples will be stored at -80 °C and then sent for commercial sequencing and metabolome analysis. In control women and transmen we will record whether menstrual cycle still persists. From each blood draw, serum will be stored at -80°C for future analyses. In order of procedures performed during baseline assessment, subjects will receive:

• Assessment of body composition: Fat mass and fat-free mass will be assessed using body impedance analysis (BIA).
• Measurement of resting energy expenditure: Baseline indirect calorimetry using a ventilated hood method will be performed in the fasted state resting energy expenditure (REE) prior to hormone substitution therapy/no therapy. The measurement will take 30 min. In brief, a clear plastic hood will be placed over the participants head with a soft collar around the neck to prevent any air leaks. The hood will be securely ventilated with room air and over 30 min. constant fractions of gases flowing out of the hood will be analyzed for carbon dioxide and oxygen concentrations. Via measurement of the respiratory quotient, calorimetric measurements will be performed during 5 min. intervals and over a period of 30 min. the subject's mean REE will be estimated. Using the ventilated hood method, adverse events are scarce and may include claustrophobia. Thus, subjects suffering from claustrophobia will be exempt from this measurement.
• Ultrasound of vessels and microperfusion: Ultrasound will be conducted to evaluate arterial stiffening (abdominal aorta and carotid artery; regional aortic strain and aortic impedance characteristics via LVOT doppler and aortic tonometry, GE Healthcare, Vivid E9 system). Peripheral near-infrared spectroscopy combined with a vascular occlusion test will be used to evaluate microperfusion (near-infrared spectroscopy, Diaspective Vision, Tivita tissue).
• Ad libitum food intake: For breakfast, subjects will undergo an ad libitum food intake situation in which they will be exposed to a buffet and asked to eat as much as they want. The buffet addresses individual dietary retrains (e.g., vegan diet). Caloric and macronutrient intake will be measured. Prior to and immediately after the buffet, participants will rate their mood, hunger, and desire to eat on a 10 cm visual analogue scale.
• Snack Food Taste Test: For assessment of taste preferences and as an additional measure of ad libitum food intake a snack food taste test will be performed. This test will be administered within 30 min. after lunch. This will be a taste test where participants will be asked to rate four types of food that will be placed in front of them in large bowls: potato chips (70 g), peanuts (100 g), Oreo cookies (100 g), and M&M's (100 g). Participants will be asked to provide mood and appetite ratings. They will then be instructed to consume as much or as little as they wish and to judge the taste of the food products using the provided rating scales. The experimenter will then leave the room and return 10 min. later, remove the bowls of food and administer a final rating scale of mood and appetite. The bowls of food will be weighed before and after the taste test as a measure of food and calorie intake.
• Response to thermic stimuli: Via an electrode positioned below the base of the index finger (C6) of the participant's non-dominant hand, thermic stimuli (cold versus warm) will be administered and subjects will be asked to indicate cold/warmth perception. Perception of cold will be measured four times in a row, followed by warmth perception. Then thermal discomfort will be triggered via administration of one cold versus one warm stimulus.
• Measurement of taste perception and smell: Administration of standardized gustatory and olfactory stimuli will enable the assessment of (non)specific taste perception and the subject's ability to perceive certain scents. Taste perception will be assessed one at a time by placing paper slip on the participant's tongue that had been coated with one specific, known flavor. Similarly, the participant's ability to perceive certain scents will be tested. In this case, specific, known scents will be placed in front of the nose of the participant who will be asked to determine the scent and then asked to rate it.
• Sex hormone deposition in hair: Using liquid chromatography-mass spectrometry, deposition of sex hormones in hair will be measured (time points V2-V4). For this purpose, a small amount of hair will be removed from the occipital region of the subject's head and then analyzed.
• Gut, oral microbiome, circulating bacterial signature in the blood and metabolomics: Participants in the study will collect stool samples at home using a provided kit and bring them to the study visit. This kit includes a disposable tray and vials for metagenome sequencing. Additionally, saliva samples will be collected at the study center after fasting, along with blood for microbiome and metabolome analyses. Gut microbiome and oral microbiome will be sent for commercial library preparation and sequencing of whole genome metagenomics. Bacterial DNA in the blood will be analyzed using 16S rRNA Amplicon sequencing and stool, oral and circulating metabolomes will be subjected to commercial metabolomics via gas liquid chromatography-mass spectrometry (GLC-MS).
• Visit to our clinical outpatient unit (transgender subjects only): Based on our unit-specific standards of care, subjects will receive a prescription for hormones intended to induce transformation to the other sex (cyproterone acetate + testosterone/estradiol) and advised to begin administration of their first dose of medication on day 1 of our study. Patients will be advised to continue with treatment according to our unit-specific standards of care for subjects with gender dysphoria undergoing hormone substitution therapy.

Upon baseline assessment, cross-sex hormone treatment versus no treatment will commence. Study procedures for V2 through V5 are the same as the above mentioned.

Inclusion Criteria:

• transsexual patients undergoing cross-sex hormone therapy versus controls
• able to give informed consent, if <18 years from all legal guardians
• only for part B (patients will participate in all other study parts): weight stable (± 5%) for last 3 months, BMI ≤ 30 kg/m2

Exclusion Criteria:

• severe medical impairments (e.g., uncontrolled cardiovascular disease, severe heart failure, uncontrolled hypertension, cerebral insult, active malign disease, etc.)
• self-initiated cross-sex hormone therapy before study start only for part B (patients will participate in all other study parts): Insufficiently controlled endocrine disorders (Cushing's disease, other uncontrolled pituitary disorders, uncontrolled hypothyroidism, hyperthyroidism, etc.)
• Chronic pulmonary disorders, including chronic obstructive pulmonary disease that would limit ability to follow the protocol (investigator judgment) and obstructive sleep apnea syndrome; only subjects with mild or exercise-induced asthma on no medications or on beta-adrenergic agonists only will be allowed to enter the study (provided use of these agents is not required for 1 week before
• Diagnosed gastrointestinal diseases, including inflammatory bowel diseases (e.g. Crohn's disease and ulcerative colitis), malabsorption syndromes (e.g. celiac disease), gastric ulcer (active); only subjects with gastro-esophageal reflux will be allowed to enter the study entry).
• History of HIV infection or ongoing chronic infection (such as tuberculosis)
• only for part D (patients will participate in all other study parts): Contraindication against performance of an MRI scan (i.e., presence of metal in body, tattoos in head/neck region, claustrophobia etc.)

Transsexual patients will be recruited in the outpatient clinic for Endocrinology at the University Hospital Leipzig. Patients seeking cross-sex hormone therapy will be asked before starting cross-sex hormone therapy for their willingness to participate in the study. If the patients would like to participate, a screening of in- and exclusion criteria will be performed. If the patient matches the criteria, the patient will be informed about the study procedures and eventual risks and if written consent will be given, the patient will be included into the study. Willingness to participate in the study will not influence the medical treatment. A control group of men and women matched for age, body mass index, health status and education who are nor undergoing cross-sex hormone therapy will be recruited through advertisements in the internet.

Individual participant data on primary outcomes of all study arms (A-E) will be shared upon personal and reasonable request in an anonymized manner.

Up to two years post termination of recruitment.

Personal and reasonable request.