Exercise During Pregnancy: Effects on the Maternal-Fetal Unit

The modified cycle protocol will be performed to estimate maximal oxygen uptake (VO2max), and will be used as measure of cardiorespiratory fitness.

Maternal weight gain will be defined as the weight change from baseline measurement to the last measurement

Systolic and diastolic blood pressure (mmHg) will be measured after 5 minutes of rest, on 2 separate occasions (with 2 minutes between trials), with the person seated (Omron Health Care Europe B.V. Hoolddorp). The lowest value of the two trials will be selected for the analysis.

Resting heart rate (bpm), will be measured after 5 minutes of rest, on 2 separate occasions (with 2 minutes between trials), with the person seated (Omron Health Care Europe B.V. Hoolddorp). The lowest value of the two trials will be selected for the analysis.

The handgrip strength and leg press test will be used as measure of strength (upper and lower limbs)

Dual-energy X-ray absorptiometry (DXA) body composition measure

Dual-energy X-ray absorptiometry (DXA) body composition measure

Dual-energy X-ray absorptiometry (DXA) body composition measure

Dual-energy X-ray absorptiometry (DXA) body composition measure

Dual-energy X-ray absorptiometry (DXA) body composition measure

Dual-energy X-ray absorptiometry (DXA) body composition measure

Dual-energy X-ray absorptiometry (DXA) body composition measure

Dual-energy X-ray absorptiometry (DXA) body composition measure

Accelerometry will be used to objectively assess physical activity and sedentary time. Women will be asked to wear a tri-axial accelerometer (ActiSleep+, Pensacola, Florida, United States) for 5-7 consecutive days, starting the same day they receive the monitor (e.g. participants who receive the accelerometer on Monday, will carry the device until Tuesday of the next week).

Accelerometry will be used to objectively assess physical activity and sedentary time. Women will be asked to wear a tri-axial accelerometer (ActiSleep+, Pensacola, Florida, United States) for 5-7 consecutive days, starting the same day they receive the monitor (e.g. participants who receive the accelerometer on Monday, will carry the device until Tuesday of the next week).

We will use the EQ-5D-5L survey, for assessing health-related quality of life, ranging from 0 ("worst imaginable health state") to 100 ("best imaginable health state"), with higher scores representing better perceived health.

The pregnant antenatal depression levels will be assessed by the Center for Epidemiological Studies-Depression Scale questionnaire, which is validated and widely employed in pregnancy. The total score ranges from 0 to 60, with higher scores indicating more severe depressive symptoms.

Low-back pain will be assessed with the Spanish version of the Oswestry Disability Index (ODI) score. The final ODI score ranges from 0 to 100, where 0 represents no disability and 100 represents maximum disability.

Physical Activity will be assessed with the Spanish adaptation and validation of the Pregnancy Physical Activity Questionnaire (PPAQ). The scale ranges from 0 to 400+ MET·h/week, with higher scores indicating greater energy expenditure and physical activity levels.

General or specific pain intensity will also be assessed using the Pain Visual Analogue Scale (VAS), a validated self-reported measure of perceived pain intensity. The VAS consists of a 10-centimeter horizontal line anchored by "no pain" (0) and "worst imaginable pain" (10) at each end. Participants will indicate their current pain level by marking a point along the line, with higher scores representing greater pain intensity.

Global cognitive function will be assessed using the Montreal Cognitive Assessment (MoCA), a validated screening tool that evaluates multiple cognitive domains, including executive function, memory, attention, language, and visuospatial abilities. The total score ranges from 0 to 30, with higher scores indicating better cognitive performance.

Adherence to the Mediterranean dietary pattern will be assessed using the Mediterranean Diet Adherence Screener (MEDAS), a validated 14-item questionnaire designed to evaluate compliance with key components of the Mediterranean diet. Each item is scored 0 or 1, depending on whether the dietary criterion is met, yielding a total score ranging from 0 to 14, where higher scores indicate greater adherence to the Mediterranean diet.

The Kessler Psychological Distress Scale (K10) will be used to assess nonspecific psychological distress, including symptoms of anxiety and depression, experienced over the past four weeks. The K10 consists of 10 items, each rated on a 5-point Likert scale ranging from 1 (none of the time) to 5 (all of the time), resulting in a total score between 10 and 50. Higher scores indicate greater psychological distress.

Erythrocyte count (×10⁶ cells/µL), will be measured using an automated Coulter haematology analyser (Brand, City, Country).

Plasma total, high-density lipoprotein and low-density lipoprotein cholesterol and triglycerides (all in mg/dL) will be assessed using an autoanalyzer.

Plasma glucose concentrations will be determined using the glucose oxidase enzymatic colorimetric method.

Total plasma antioxidant capacity (in maternal and cord blood) will be measured using a commercial colorimetric assay kit based on spectrophotometry, following the manufacturer's instructions. Results will be expressed in Trolox equivalents (mmol Trolox/L), where higher values indicate greater antioxidant capacity.

Erythrocyte membrane catalase, glutathione peroxidase, and superoxide dismutase enzyme activities will be measured by spectrophotometry using standard biochemical assays. Results will be expressed in units per milligram of hemoglobin (U/mg Hb), where higher values indicate greater enzymatic antioxidant activity.

Some maternal and umbilical cord plasma pro-inflammatory and anti-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α, IL-1ra and TNF Srii α), some adipokines (adiponectin, adipsin, resistin, PAI-active, insulin and leptin) and myokines (irisin) will be measured by the employment of Luminex xMAP technology. Other relevant biomarkers related to bone metabolism (ACTH, DKK-1, FGF-23, Osteocalcin, OPN-Osteopontin, Osteoprotegerin, PTH and SOST) will be measured with Luminex xMAP technology. Each outcome will be reported separately, with concentrations expressed in pg/mL or ng/mL, as appropriate for the specific analyte.

Birth weight will be recorded in grams (g).

Mode of delivery (vaginal or caesarean section) and any maternal or neonatal complications occurring during delivery will be obtained from hospital medical records and classified according to the attending obstetrician's report.

Apgar score will be evaluated at 1 and 5 minutes after delivery by trained obstetric or neonatal staff, following standard clinical procedures. Scores range from 0 to 10, with higher values indicating better neonatal condition.

Polypharmacy will be defined as the concurrent use of five or more medications.

Resting energy expenditure is measured in the fasting and fed state by indirect calorimetry

Haemoglobin concentration (g/dL) will be measured using an automated Coulter haematology analyser (Brand, City, Country).

Platelet count (×10³ cells/µL) and leukocyte count (×10³ cells/µL) will be measured using an automated Coulter haematology analyser (Brand, City, Country).

Mean corpuscular volume (fL) will be measured using an automated Coulter haematology analyser (Brand, City, Country).

Plasma insulin levels will be quantified by enzyme-linked immunosorbent assay (ELISA).

Glycosylated haemoglobin (HbA1c) will be determined using the enzymatic colorimetric method.

Length in centimeters (cm), and head circumference in centimeters (cm).

Hospitalization will be recorded as the number and duration of inpatient stays.

Cost-effectiveness analyses will be conducted based on health service utilization and associated costs extracted from the hospital database. Each outcome will be reported separately, with appropriate units (e.g., number of admissions, days hospitalized, number of medications, EQ-5D-5L index score, euros): Count (episodes, days, or medications), EQ-5D-5L index (score), and euros (€).

Mitochondrial oxygen (O₂) flux will be measured in placental tissue using high-resolution respirometry (Oroboros O₂k, Oroboros Instruments, Innsbruck, Austria). Tissue samples will be analyzed in fresh homogenates under controlled substrate-uncoupler-inhibitor titration (SUIT) protocols to evaluate basal, leak, and maximal respiratory states. Oxygen flux will be expressed as picomoles of O₂ consumed per second per milligram of tissue (pmol O₂·s-¹·mg-¹).

Changes in proteome will be analyzed using the Olink® proximity extension assay (PEA) technology, following the manufacturer's standardized protocol (Olink Proteomics, Uppsala, Sweden). Protein abundance will be expressed as normalized protein expression (NPX) units, where higher NPX values indicate greater relative protein concentration.