Study design and population
This is a prospective cohort study with up to 12 months of follow-up of TW volleyball athletes (figure 1). The enrolment started in February 2022 and is expected to finalise in September 2023 in São Paulo/Brazil.
Figure 1Selection of transgender women (TW) . (A) Inclusion criteria; (B) exclusion criteria and the control group subjects. BMI, body mass index; CM, cisgender men; CW, cisgender women; DXA, dual-energy X-ray absorptiometry; IPAQ, International Physical Activity Questionnaire.
Individuals will be deemed eligible if they have the following characteristics: (1) classified as gender incongruent according to the International Disease Code 11; (2) aged 18–50 years; (3) body mass index (BMI) between 18.0 and 34.9 kg/m2; (4) classified as having a very high level of physical activity, according to the International Physical Activity Questionnaire score8 9 and (5) if they are engaged in regular volleyball training. Conversely, participants will be excluded if they report or present with signs of acute or chronic cardiovascular, respiratory or orthopaedic comorbidities, which could potentially interfere with exercise capacity if they are pregnant or already taking part in another clinical trial.
CW and CM matched by age, BMI and level of physical activity will undergo a similar evaluation.
Participants are not allowed to change groups throughout the study.
The study will comprise two phases for the TW group: baseline and 6–12 months following regular use of oestrogen and blocked testosterone (<10 nmol/L). At each phase, participants will be required to attend three study visits, during which a full assessment of medical, mental and nutritional health, BC, strength and aerobic capacity will be performed. Several test dates will be made available to facilitate the participant’s choice. The athlete’s participation will be discontinued if requested or if any clinical condition arises, that may interfere with the athlete’s performance or examination findings.
The researchers will not be blinded during the physical tests due to the phenotypic characteristics of the participants.
Research instruments and procedures
All of the following procedures will happen at baseline and at the follow-up phase (6–12 months later).
Basic laboratory tests
Participants will undergo a full haematological and hormonal assessment. The whole metabolic panel will include fasting glucose, glycated haemoglobin (HbA1c), total cholesterol, low-density lipoprotein-cholesterol (LDL-c), high-density lipoprotein-cholesterol (HDL-c), triglycerides. Hormonal profile will include total and free testosterone, estradiol, prolactin, sex hormone binding globulin (SHBG), parathyroid hormone (PTH), 25-hydroxyvitamin D, thyroid stimulating hormone (TSH) and free thyroxine (FT4). Total blood count, renal function (urea, creatinine) and electrolytes (P, Ca2+, Mg2+) will also be measured.
Maximal incremental exercise test
Participants will perform a maximal incremental exercise test to exhaustion on a treadmill (Super ATL, Inbrasport, Porto Alegre, RS, Brazil) to determine peak oxygen uptake (VO2peak), peak heart rate (HRpeak) and other submaximal parameters (such as anaerobic threshold and respiratory compensation point). These parameters will be assessed using a metabolic system (K5, COSMED, Rome, Italy). Following an initial 5 min warm-up, the treadmill incline will be kept at 1%. At the same time, the speed will be increased by 1 km/hour every minute until exhaustion,10 aiming for a total incremental test duration of 8–12 min. Heart rate will be continuously recorded throughout the test with a Garmin heart rate sensor. Blood pressure will be measured at rest, immediately after maximal effort, and 1, 2 and 3 min after exercising by the same examiner. A calibrated upper limb manometer with manual auscultation through a stethoscope will be used.
Vertical jump tests
A 10 min dynamic stretching and warm-up period will be performed, followed by self-administered squatting exercises and submaximal vertical jumps for familiarisation. Before the testing session, the participants will be given instructions on the execution of the squat jump (SQJ) and the countermovement jump (CMJ) without arm swings. At the starting position, for the execution of each type of jump, the arms will be placed on the hips, with feet in full contact with the jump mat (Jump System Pro, Cefise, Nova Odessa, SP, Brazil), and knee angle at approximately 90° (when performing SQJ). The arms will be placed on the hips throughout the jump, flight and landing. Three SQJ attempts will be performed, separated by 10 s. After a rest period of 10 min, three CMJ attempts will be performed, also separated by 10 s.
All jumps will be performed barefoot, and participants will be instructed to jump as high and fast as possible (without a downward movement when performing SQJ). The best attempt of the three trials of each type of jump, using the criterion of maximum jump height achieved, will be selected for further analysis.
Hand grip strength test
The Jamar hydraulic hand dynamometer (Patterson Medical, Warrenville, Illinois, USA) will measure handgrip strength.
Participants will comfortably sit on a chair without armrests, with their feet flat on the floor, hips and knees at approximately 90° of flexion. The shoulder of the tested limb will be adducted and neutrally rotated, the elbow flexed at 90°, the forearm in a neutral position, and the wrist in 0°–30° dorsiflexion and 0°–15° adduction. The untested hand will be placed on the thigh. All participants will be evaluated individually.
Before the test, participants will perform submaximal handgrip movements and rest for 1 min. During the test, they cannot look at the dynamometer display to avoid any visual feedback. No command will be given during the test, and the instructions for test execution will be standardised. The volume of the verbal command will remain constant to avoid any influence on the magnitude of muscle contraction.
Three attempts will be performed with both the dominant and non-dominant hands. A maximum contraction lasting 3 s will be performed at each attempt, followed by a 30 s rest period. The mean values of the three attempts of each hand will be used for data analysis.
Echocardiogram
A standard transthoracic Doppler echocardiogram will be performed with the participant lying supine and lateral. Participants’ hearts will be examined for anatomical and functional aspects, including dimensions of the cardiac chambers, the interventricular septum and the left ventricular ejection fraction.
Food intake assessment
Food intake will be verified by 24-hour recalls (R24h) of non-consecutive days applied by trained professionals. Data collection will occur according to the Multiple-Pass Method, which consists of five steps: (1) quick listing of foods and beverages consumed, (2) commonly forgotten foods, (3) time and occasion of consumption, (4) detailing cycle and (5) final review.11
Data collected from the R24h will be converted into standard measurement units (grams) based on tables and reference materials. Subsequently, macronutrients and micronutrients will be calculated in the DietBox. After that, the regular food intake components of interest (eg, carbohydrates, proteins and lipids) will be estimated using the Multiple Source Method software programs and the corrected intraindividual variability.
Dietary practices
The Dietary Practices Measurement Scale will be applied to assess dietary practices according to the Food Guide for the Brazilian Population (GAPB, in Portuguese).12 This tool comprises 24 items that exemplify dietary practices, agreeing or disagreeing with the GAPB recommendations. Possible answers are ‘strongly disagree, disagree, agree and strongly agree’, which express the participant’s level of agreement or accordance with this practice in their daily lives. The score on the scale is computed by the simple sum of the answers (values from 0 to 3), which can vary from 0 to 72 (the maximum). For classification purposes, two cut-off points were defined based on scoring percentiles, generating three score ranges: <P25 (<32 points), P25–P75 (32–41 points) and >P75 (>41 points). The higher the score, the greater the adherence to the GAPB recommendations.
Analysis of REE
REE will be assessed by indirect calorimetry (IC) using the metabolic system K5 (Cosmed, Rome, Italy). The test will be conducted for 20 min in absolute resting conditions in the morning. The IC will be the first test of the day, considering that REE is widely affected by external factors. Thus, on arriving at the laboratory, participants will rest for 10 min in the supine position. Immediately after this, they will be connected to the equipment for REE assessment.
For improved IC reliability, participants will be instructed not to practice physical exercise or consume any food that could modify the resting metabolic rate (eg, coffee, food containing caffeine, high-protein meals) 24 hours before the test and will be asked to fast for 10 hours before the test. Moreover, the laboratory room will remain closed and temperature-controlled to avoid thermal changes that could modify energy expenditure (eg, thermoregulation).
BC and bone mineral density
Measurements of bone mineral density will be taken with dual-energy X-ray absorptiometry (Hologic QDR 4500, Waltham, Massachusetts, USA) at the lumbar spine L1–L4 (LS), total hip (TH) and femoral neck. The analysis of BC will be performed to assess the following parameters: fat mass, body fat percentage, skeletal muscle mass and fat-free mass. To minimise the interference of height, the following indices will be calculated: lean mass/height2 and the Baumgartner Index (appendicular muscle mass/height2). To evaluate the total body fat mass, the fat mass index will be used, which represents the total body fat mass divided by the height squared.
Metabolomic analysis
The untargeted metabolomic analysis will be performed as previously described.13 14 Briefly, blood samples from each participant will be collected and packed in tubes containing EDTA. The extraction of plasma metabolites will be performed by adding 400 µL of methanol and isopropanol (1:1 v/v) and 6 µL of internal standard (3 mg/mL of d27-myristic acid) to 100 µL of plasma. The mixture will be subjected to ultrasound for 5 min, homogenised for 20 min at 4°C and centrifuged at 15 800×g at 4°C for 10 min. Then, the supernatant will be transferred to a microcentrifuge tube and dried for 16 hours in a SpeedVac. The sediment will be derivatised in two steps, beginning with protecting carbonyl functional groups using 50 µL of 40 mg/mL of methoxyamine hydrochloride in pyridine at 25°C for 16 hours. Then, the samples will be derivatised using 100 µL of N-methyl-N-(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane at 25°C for 60 min. The supernatant will be collected by centrifugation at 15 800×g at 4°C for 10 min.
Gas chromatography-mass spectrometry (GC-MS) will analyse the metabolites using a 29 m long (0.25 mm×0.25 µm) DB-5 MS column with a 10 m DuraGuard precolumn. The Agilent Fiehn GC/MS Metabolomics RTL library will be employed for raw data processing and metabolite identification.13 14
Sleep quality analysis
The Portuguese version of the Pittsburgh Sleep Quality Index (PSQI) questionnaire will be used to assess sleep quality.15 The PSQI consists of four open and six multiple-choice questions to assess sleep quality over the last month. It consists of 24 items, 19 of which are self-reported, and their partner or roommate classifies 5. These five items include sleep duration estimates, latency and frequency, and the severity of specific sleep-related problems. Scores are added to generate an overall PSQI score, ranging from 0 to 21 points. Higher scores indicate poorer sleep quality.
Psychological and psychiatric assessments
We will employ the semistructured interview methodology,16 which combines the spontaneity of the interviewee and clarity of the interviewer’s objective with the need for reliable and trustworthy records.
The semistructured interview guide will be as follows:
Sports activity record: talk about the main sports activities linked to you throughout your life, situating them over time; that is, at what age you started practising it and at what age you stopped practising it.
Is volleyball your main sport activity today? Yes/no. Please explain. If yes, can you comment on the reasons that led you to this sport and what sensations and feelings are involved in the choice and the practice?
Up to now, have you always been able to practice the sports activities you want? If not, what were they and why/for what reasons were you unable to practice them? Do you intend, at any point, to try to practice them? Please explain.
Do you believe your gender identity can be associated in some way with your sports choices, whether in terms of access or the possibility of practising it in a pleasurable way? If yes, do you understand this association more properly as an incentive, obstacle or other type of association?
In addition, the Five Digits Test17 will be applied to look at mental processing speed and the ability to direct and switch their attentional control. A formal assessment of depression and suicidal ideation will also be performed using the Patient Health Questionnaire-9.
Statistical analysis
Due to the scarcity of potential subjects (TW volleyball athletes) and lack of previous data to guide the sample size estimation, a convenience sample will be used. We intend to evaluate 20 athletes in each group. Data will be analysed with respect to normality using the Shapiro-Wilk test. Normally distributed variables will be compared between groups using variance analysis (analysis of variance) and Tukey’s post hoc test. In contrast, the Kruskal-Wallis post hoc test of Dunn will be used for non-parametrical variables. Given that muscle strength and peak oxygen uptake (VO2peak) are dependent variables, linear regression models will be applied to verify the relationship between independent variables and the outcomes. The regression models will be elaborated considering biological plausibility criteria, p<0.20, tolerance and multicollinearity. Independent variables are gender, BC, training status and covariables: age, dietary intake and sleep pattern. Thus, first, we will apply the univariate regression model to verify the association between independent variables and outcomes and variables with p<0.20 will be entered into the multiple models.
Statistical analysis for untargeted metabolomics experiments will be performed using MetaboAnalyst V.5.0 software.20 Data will be normalised and scaled for multivariate statistical analyses. Principal component analysis and supervised partial least squares-discriminant analysis will be performed to visualise the differences between groups.
Paired-samples t-test or Wilcoxon test will compare quantitative variables before and after adequate hormonal therapy among TW. All analyses will be performed using the free software R.
