Strength Training During Pregnancy: The Trial MOVE4TWO (MOVE4TWO)

Participants assigned to the control group will continue their usual prenatal care and receive general recommendations regarding physical activity and healthy lifestyle behaviors during pregnancy, in accordance with current guidelines. No supervised strength training intervention will be provided as part of the study.

Participants assigned to the intervention group will perform three supervised strength training sessions per week, beginning after baseline assessment and continuing until delivery. Sessions will be conducted in small groups of up to six participants and supervised by qualified exercise professionals. Training intensity will be individually prescribed using repetitions in reserve (RIR), progressing from 4-6 RIR during the first three weeks to 2-3 RIR thereafter.

The intervention will consist of three supervised strength training sessions per week, conducted in person at the Universidad Europea Miguel de Cervantes. Each session will include two exercise circuits targeting major muscle groups through anterior-chain, posterior-chain, pulling, pushing, and core exercises. Exercise intensity will be individualized using repetitions in reserve (RIR), progressing from 4-6 RIR during the first three weeks to 2-3 RIR thereafter. Training loads will be adjusted according to individual performance and tolerance. Sessions will be supervised by qualified professionals, and exercises will be adapted to the participants' gestational stage and physical condition. The intervention will continue until delivery.

The physical component of health-related quality of life will be assessed using the 36-Item Short Form Health Survey (SF-36). The Physical Component Summary (PCS) score will be calculated using the standardized SF-36 scoring algorithm, combining information from the eight health domains through norm-based weighting. PCS scores will be expressed as norm-based T-scores, with a reference population mean of 50 and a standard deviation of 10. Higher scores indicate better physical health-related quality of life. Changes in PCS scores will be compared between the intervention and control groups across the assessment periods.

The mental component of health-related quality of life will be assessed using the 36-Item Short Form Health Survey (SF-36). The Mental Component Summary (MCS) score will be calculated using the standardized SF-36 scoring algorithm, combining information from the eight health domains through norm-based weighting. MCS scores will be expressed as norm-based T-scores, with a reference population mean of 50 and a standard deviation of 10. Higher scores indicate better mental health-related quality of life. Changes in MCS scores will be compared between the intervention and control groups across the assessment periods.

Physical activity will be assessed using the Pregnancy Physical Activity Questionnaire-Long Form (PPAQ-LF). Physical activity energy expenditure will be expressed in metabolic equivalent hours per week (MET-hours/week), with higher values indicating greater physical activity levels.

Moderate-to-vigorous physical activity (MVPA) will be objectively assessed using GT3X+ accelerometers (firmware v1.9.2), initialized at 30 Hz with the normal filter. Participants will wear the device on the right hip, secured by an elastic belt, for seven consecutive days at each assessment. MVPA will be expressed as total minutes per week (min/week), with higher values indicating greater physical activity levels.

Body weight will be measured using a calibrated digital scale, with participants wearing light clothing and no shoes. Measurements will be expressed in kilograms (kg). Changes in body weight will be assessed across the study evaluation points.

Standing height will be measured using a stadiometer and expressed in centimeters (cm).

Body mass index (BMI) will be calculated by dividing body weight in kilograms by the square of height in meters (kg/m²). Changes in BMI will be assessed throughout pregnancy and during postpartum follow-up.

Triceps skinfold thickness will be measured on the posterior aspect of the right upper arm, at the midpoint between the acromion and olecranon, using a skinfold caliper. Three measurements will be obtained following standardized anthropometric procedures, allowing tissue recovery between measurements. The mean value will be expressed in millimeters (mm).

Biceps skinfold thickness will be measured on the anterior aspect of the right upper arm, at the midpoint between the acromion and olecranon, using a skinfold caliper. Three measurements will be obtained following standardized anthropometric procedures, allowing tissue recovery between measurements. The mean value will be expressed in millimeters (mm).

Subscapular skinfold thickness will be measured immediately below the inferior angle of the right scapula, using a skinfold caliper. Three measurements will be obtained following standardized anthropometric procedures, allowing tissue recovery between measurements. The mean value will be expressed in millimeters (mm).

Mid-thigh skinfold thickness will be measured on the anterior aspect of the right thigh, at the midpoint between the inguinal crease and the proximal border of the patella, using a skinfold caliper. Three measurements will be obtained following standardized anthropometric procedures, allowing tissue recovery between measurements. The mean value will be expressed in millimeters (mm).

Medial calf skinfold thickness will be measured on the medial aspect of the right calf, at the level of its maximum circumference, using a skinfold caliper. Three measurements will be obtained following standardized anthropometric procedures, allowing tissue recovery between measurements. The mean value will be expressed in millimeters (mm).

Mid-arm circumference will be measured on the right arm at the midpoint between the acromion and olecranon, using a non-elastic anthropometric tape. Participants will stand upright with their arms relaxed alongside the body. Three measurements will be obtained without compressing the underlying tissues, and the mean value will be expressed in centimeters (cm).

Calf circumference will be measured at the point of maximum circumference of the right calf using a non-elastic anthropometric tape. Participants will stand upright with their weight evenly distributed between both legs. Three measurements will be obtained without compressing the underlying tissues, and the mean value will be expressed in centimeters (cm).

Right rectus femoris muscle thickness will be assessed using real-time B-mode ultrasound with a linear transducer. Measurements will be obtained at the midpoint between the anterior superior iliac spine and the superior border of the patella. Muscle thickness will be calculated as the perpendicular distance between the superficial and deep aponeuroses, averaged across three ultrasound images. Results will be expressed in centimeters (cm), with higher values indicating greater muscle thickness.

Left rectus femoris muscle thickness will be assessed using real-time B-mode ultrasound with a linear transducer. Measurements will be obtained at the midpoint between the anterior superior iliac spine and the superior border of the patella. Muscle thickness will be calculated as the perpendicular distance between the superficial and deep aponeuroses, averaged across three ultrasound images. Results will be expressed in centimeters (cm), with higher values indicating greater muscle thickness.

Right vastus lateralis muscle thickness will be assessed using real-time B-mode ultrasound with a linear transducer. Measurements will be obtained at two-thirds of the distance between the anterior superior iliac spine and the lateral border of the patella. Muscle thickness will be calculated as the perpendicular distance between the superficial and deep aponeuroses, averaged across three ultrasound images. Results will be expressed in centimeters (cm), with higher values indicating greater muscle thickness.

Left vastus lateralis muscle thickness will be assessed using real-time B-mode ultrasound with a linear transducer. Measurements will be obtained at two-thirds of the distance between the anterior superior iliac spine and the lateral border of the patella. Muscle thickness will be calculated as the perpendicular distance between the superficial and deep aponeuroses, averaged across three ultrasound images. Results will be expressed in centimeters (cm), with higher values indicating greater muscle thickness.

Inter-rectus distance (IRD) will be measured using B-mode ultrasound at 5 cm above the umbilicus. Participants will lie in a supine position with their head supported on a pillow, hips and knees flexed, feet flat on the examination table, and arms resting alongside the body. The distance between the medial borders of the rectus abdominis muscles will be measured and expressed in millimeters (mm), with higher values indicating greater abdominal muscle separation.

Inter-rectus distance (IRD) will be measured using B-mode ultrasound at 2 cm above the umbilicus. Participants will lie in a supine position with their head supported on a pillow, hips and knees flexed, feet flat on the examination table, and arms resting alongside the body. The distance between the medial borders of the rectus abdominis muscles will be measured and expressed in millimeters (mm), with higher values indicating greater abdominal muscle separation.

Inter-rectus distance (IRD) will be measured using B-mode ultrasound at 2 cm below the umbilicus. Participants will lie in a supine position with their head supported on a pillow, hips and knees flexed, feet flat on the examination table, and arms resting alongside the body. The distance between the medial borders of the rectus abdominis muscles will be measured and expressed in millimeters (mm), with higher values indicating greater abdominal muscle separation.

Linea alba distortion will be assessed using B-mode ultrasound at 5 cm above the umbilicus. Participants will lie supine with their head supported, hips and knees flexed, and feet flat on the examination table. The distortion index will be calculated by dividing the area between the linea alba trajectory and the straight line connecting the medial borders of the rectus abdominis muscles by the inter-rectus distance. Results will be expressed in millimeters (mm), with higher values indicating greater linea alba distortion.

Linea alba distortion will be assessed using B-mode ultrasound at 2 cm above the umbilicus. Participants will lie supine with their head supported, hips and knees flexed, and feet flat on the examination table. The distortion index will be calculated by dividing the area between the linea alba trajectory and the straight line connecting the medial borders of the rectus abdominis muscles by the inter-rectus distance. Results will be expressed in millimeters (mm), with higher values indicating greater linea alba distortion.

Linea alba distortion will be assessed using B-mode ultrasound at 2 cm below the umbilicus. Participants will lie supine with their head supported, hips and knees flexed, and feet flat on the examination table. The distortion index will be calculated by dividing the area between the linea alba trajectory and the straight line connecting the medial borders of the rectus abdominis muscles by the inter-rectus distance. Results will be expressed in millimeters (mm), with higher values indicating greater linea alba distortion.

Right perirenal fat thickness will be assessed using B-mode ultrasound. The perirenal fat deposit will be identified using the renal cortex, hepatic pole, and abdominal wall musculature as anatomical landmarks. Thickness will be measured in millimeters (mm), with higher values indicating greater perirenal fat thickness.

Heart rate variability (HRV) will be assessed using a Polar H10 heart rate monitor (Polar Electro Oy, Kempele, Finland) and the Elite HRV application. Participants will perform a five-minute resting measurement on seven consecutive days at each assessment period. HRV will be quantified using the root mean square of successive differences between normal-to-normal heartbeats (RMSSD), expressed in milliseconds (ms). The mean RMSSD across the seven daily measurements will be used as the outcome measure, with higher values generally indicating greater parasympathetic cardiac modulation.

Functional exercise capacity will be assessed using the Six-Minute Walk Test (6MWT). Participants will be instructed to walk as far as possible during six minutes along a flat, straight corridor. The total distance covered will be recorded in meters (m), with higher values indicating greater functional exercise capacity.

Peak oxygen consumption (VO₂peak) will be assessed during the Six-Minute Walk Test using a portable metabolic analyzer. Participants will wear a face mask connected to the gas analysis system throughout the test. VO₂peak will be expressed in milliliters per kilogram per minute (mL/kg/min), with higher values indicating greater oxygen consumption during exercise.

Functional capacity will be assessed using the Five-Repetition Sit-to-Stand Test (5STS). Participants will be instructed to stand up and sit down five times as quickly as possible from a standard chair without using their arms. The total time required to complete five repetitions will be recorded in seconds (s), with lower values indicating better functional performance.

Functional capacity will be assessed using the 60-Second Sit-to-Stand Test (60STS). Participants will be instructed to complete as many full sit-to-stand repetitions as possible within 60 seconds, without using their arms for assistance. The total number of correctly completed repetitions will be recorded, with higher values indicating better functional performance.

Right-hand grip strength will be assessed using a Jamar Smart hand dynamometer. Participants will perform three maximal voluntary contractions with the right hand, with the elbow flexed at 90 degrees and the forearm supported. The highest recorded value will be expressed in kilograms (kg), with higher values indicating greater handgrip strength.

Left-hand grip strength will be assessed using a Jamar Smart hand dynamometer. Participants will perform three maximal voluntary contractions with the left hand, with the elbow flexed at 90 degrees and the forearm supported. The highest recorded value will be expressed in kilograms (kg), with higher values indicating greater handgrip strength.

Mean concentric velocity will be assessed during squat exercise using a linear position transducer (SmartCoach, Turin, Italy). Participants will perform three maximal-velocity repetitions with an external load corresponding to 50% of their baseline body weight. Mean concentric velocity will be recorded for each repetition, and the average of the three repetitions will be used as the outcome measure. Results will be expressed in meters per second (m/s), with higher values indicating greater concentric movement velocity.

Mean concentric velocity will be assessed during squat exercise using a linear position transducer (SmartCoach, Turin, Italy). Participants will perform three maximal-velocity repetitions with an external load corresponding to 75% of their baseline body weight. Mean concentric velocity will be recorded for each repetition, and the average of the three repetitions will be used as the outcome measure. Results will be expressed in meters per second (m/s), with higher values indicating greater concentric movement velocity.

Mean concentric velocity will be assessed during squat exercise using a linear position transducer (SmartCoach, Turin, Italy). Participants will perform three maximal-velocity repetitions with an external load corresponding to 100% of their baseline body weight. Mean concentric velocity will be recorded for each repetition, and the average of the three repetitions will be used as the outcome measure. Results will be expressed in meters per second (m/s), with higher values indicating greater concentric movement velocity.

Mean concentric power will be assessed during squat exercise using a linear position transducer (SmartCoach, Turin, Italy). Participants will perform three maximal-velocity repetitions with an external load corresponding to 50% of their baseline body weight. Mean concentric power will be determined from the force and velocity data obtained during the concentric phase. The average power across the three repetitions will be used as the outcome measure and expressed in watts (W), with higher values indicating greater concentric power production.

Mean concentric power will be assessed during squat exercise using a linear position transducer (SmartCoach, Turin, Italy). Participants will perform three maximal-velocity repetitions with an external load corresponding to 75% of their baseline body weight. Mean concentric power will be determined from the force and velocity data obtained during the concentric phase. The average power across the three repetitions will be used as the outcome measure and expressed in watts (W), with higher values indicating greater concentric power production.

Mean concentric power will be assessed during squat exercise using a linear position transducer (SmartCoach, Turin, Italy). Participants will perform three maximal-velocity repetitions with an external load corresponding to 100% of their baseline body weight. Mean concentric power will be determined from the force and velocity data obtained during the concentric phase. The average power across the three repetitions will be used as the outcome measure and expressed in watts (W), with higher values indicating greater concentric power production.

Maximum dynamic concentric force will be estimated from the force-velocity relationship obtained during squat exercise. Participants will perform three maximal-velocity repetitions at each of three external loads corresponding to 50%, 75%, and 100% of their baseline body weight, with three minutes of rest between sets. Movement velocity will be recorded using a linear position transducer (SmartCoach, Turin, Italy). A single maximum dynamic concentric force value will be derived from the force-velocity profile at each assessment and expressed in newtons (N), with higher values indicating greater estimated force production.

Postural balance will be assessed using a force platform (Hawkin Dynamics) during the Romberg test. Participants will stand upright for 30 seconds with their eyes open. Center of pressure (CoP) sway area will be calculated from the recorded postural sway data and expressed in square millimeters (mm²). Higher values will indicate greater postural sway and reduced postural stability.

Postural balance will be assessed using a force platform (Hawkin Dynamics) during the Romberg test. Participants will stand upright for 30 seconds with their eyes closed. Center of pressure (CoP) sway area will be calculated from the recorded postural sway data and expressed in square millimeters (mm²). Higher values will indicate greater postural sway and reduced postural stability.

Postural balance will be assessed using a force platform (Hawkin Dynamics) during the Romberg test. Participants will stand upright for 30 seconds with their eyes open. Mean center of pressure (CoP) velocity will be calculated as the total CoP displacement divided by the recording duration. Results will be expressed in millimeters per second (mm/s), with higher values indicating greater postural sway velocity.

Postural balance will be assessed using a force platform (Hawkin Dynamics) during the Romberg test. Participants will stand upright for 30 seconds with their eyes closed. Mean center of pressure (CoP) velocity will be calculated as the total CoP displacement divided by the recording duration. Results will be expressed in millimeters per second (mm/s), with higher values indicating greater postural sway velocity.

Perceived exertion will be assessed using the Borg Category-Ratio 10 (CR-10) Rating of Perceived Exertion Scale after each training session. Scores range from 0 (no exertion at all) to 10 (maximal exertion), with higher scores indicating greater perceived exercise intensity. The scores will be recorded throughout the strength training intervention.

Fetal crown-rump length (CRL) will be measured by obstetric ultrasound as the distance between the top of the fetal head (crown) and the bottom of the torso (rump). Measurements will be expressed in millimeters (mm). CRL will be used to assess fetal size and growth during early pregnancy.

Fetal biparietal diameter (BPD) will be assessed by obstetric ultrasound as the transverse diameter of the fetal head between the two parietal bones, using standardized anatomical landmarks. Measurements will be expressed in millimeters (mm). BPD will be used to assess fetal head growth and development.

Fetal head circumference (HC) will be measured by obstetric ultrasound in a standardized axial plane of the fetal head. The circumference will be determined using ultrasound measurement tools and expressed in millimeters (mm). HC will be used to assess fetal head growth and development.

Fetal femur length (FL) will be assessed by obstetric ultrasound by measuring the length of the ossified femoral diaphysis, excluding the epiphyses. Measurements will be expressed in millimeters (mm). FL will be used to assess fetal skeletal growth and development.

Fetal abdominal circumference (AC) will be measured by obstetric ultrasound in a standardized transverse plane of the fetal abdomen, at the level of the stomach and portal sinus. Measurements will be expressed in millimeters (mm). AC will be used to assess fetal abdominal growth and identify potential abnormalities in fetal growth.

Mode of delivery will be reported by participants during the postpartum assessment and, when available, verified using clinical documentation voluntarily provided by participants. Delivery will be classified as spontaneous vaginal delivery, assisted vaginal delivery, or cesarean section. Results will be reported as the number of participants in each category.

Duration of labor will be reported by participants during the postpartum assessment and, when available, verified using clinical documentation voluntarily provided by participants. Duration will be calculated as the time elapsed between the documented onset of labor and delivery. Results will be expressed in hours (h), with higher values indicating longer labor duration.

The use of instrumental assistance during delivery will be reported by participants during the postpartum assessment and, when available, verified using clinical documentation voluntarily provided by participants. Instrumental assistance will be classified according to the type of instrument used (forceps or vacuum extraction). Results will be reported as the number of participants requiring each type of instrumental assistance.

Newborn sex will be reported by participants during the postpartum assessment and, when available, verified using clinical documentation voluntarily provided by participants. Results will be reported as the number of newborns in each recorded sex category.

Newborn birth weight will be reported by participants during the postpartum assessment and, when available, verified using clinical documentation voluntarily provided by participants. Birth weight will be expressed in grams (g) and used as an indicator of fetal growth and neonatal status at delivery.

Placental weight will be reported by participants during the postpartum assessment and, when available, verified using clinical documentation voluntarily provided by participants. Placental weight will be expressed in grams (g) and used to assess placental characteristics at delivery.

The Apgar score at 1 minute after birth will be reported by participants during the postpartum assessment and, when available, verified using clinical documentation voluntarily provided by participants. The score evaluates five components: heart rate, respiratory effort, muscle tone, reflex irritability, and skin color. Total scores range from 0 to 10, with higher scores indicating better neonatal condition.

The Apgar score at 5 minutes after birth will be reported by participants during the postpartum assessment and, when available, verified using clinical documentation voluntarily provided by participants. The score evaluates five components: heart rate, respiratory effort, muscle tone, reflex irritability, and skin color. Total scores range from 0 to 10, with higher scores indicating better neonatal condition.

Interleukin-1 (IL-1) concentration will be measured in maternal plasma samples obtained from fasting venous blood. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in IL-1 concentration will be assessed across the study evaluation points.

Interleukin-6 (IL-6) concentration will be measured in maternal plasma samples obtained from fasting venous blood. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in IL-6 concentration will be assessed across the study evaluation points.

Interleukin-8 (IL-8) concentration will be measured in maternal plasma samples obtained from fasting venous blood. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in IL-8 concentration will be assessed across the study evaluation points.

Interleukin-10 (IL-10) concentration will be measured in maternal plasma samples obtained from fasting venous blood. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in IL-10 concentration will be assessed across the study evaluation points.

Interferon-gamma (IFN-γ) concentration will be measured in maternal plasma samples obtained from fasting venous blood. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in IFN-γ concentration will be assessed across the study evaluation points.

Tumor necrosis factor-alpha (TNF-α) concentration will be measured in maternal plasma samples obtained from fasting venous blood. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in TNF-α concentration will be assessed across the study evaluation points.

C-reactive protein (CRP) concentration will be measured in maternal plasma samples obtained from fasting venous blood. Concentrations will be expressed in milligrams per liter (mg/L). Changes in CRP concentration will be assessed across the study evaluation points.

Fasting glucose concentration will be measured in maternal plasma obtained from venous blood samples collected after an overnight fast. Glucose concentrations will be expressed in milligrams per deciliter (mg/dL). Changes in fasting plasma glucose will be assessed across the study evaluation points.

Fasting insulin concentration will be measured in maternal plasma obtained from venous blood samples collected after an overnight fast. Insulin concentrations will be expressed in micro-international units per milliliter (µIU/mL). Changes in fasting plasma insulin will be assessed across the study evaluation points.

Insulin resistance will be estimated using the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), calculated from fasting plasma glucose and insulin concentrations using the formula: fasting insulin (µIU/mL) × fasting glucose (mg/dL) / 405. HOMA-IR is a dimensionless index, with higher values indicating greater insulin resistance. Changes in HOMA-IR will be assessed across the study evaluation points.

Pancreatic beta-cell function will be estimated using the Homeostatic Model Assessment of Beta-Cell Function (HOMA-B), calculated from fasting plasma glucose and insulin concentrations using the formula: 360 × fasting insulin (µIU/mL) / [fasting glucose (mg/dL) - 63]. Results will be expressed as a percentage (%), with higher values indicating greater estimated beta-cell function. Changes in HOMA-B will be assessed across the study evaluation points.

Total cholesterol (TC) concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in milligrams per deciliter (mg/dL). Changes in total cholesterol concentration will be assessed across the study evaluation points.

Triglyceride (TG) concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in milligrams per deciliter (mg/dL). Changes in triglyceride concentration will be assessed across the study evaluation points.

High-density lipoprotein cholesterol (HDL-C) concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in milligrams per deciliter (mg/dL). Changes in HDL-C concentration will be assessed across the study evaluation points.

Low-density lipoprotein cholesterol (LDL-C) concentration will be assessed in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in milligrams per deciliter (mg/dL). Changes in LDL-C concentration will be assessed across the study evaluation points.

Human chorionic gonadotropin (hCG) concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in milli-international units per milliliter (mIU/mL). Changes in hCG concentration will be assessed across the study evaluation points.

Pregnancy-associated plasma protein A (PAPP-A) concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in milli-international units per liter (mIU/L), subject to the analytical method used. Changes in PAPP-A concentration will be assessed across the study evaluation points.

Placental growth factor (PlGF) concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in PlGF concentration will be assessed across the study evaluation points.

Soluble fms-like tyrosine kinase-1 (sFlt-1) concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in sFlt-1 concentration will be assessed across the study evaluation points.

The ratio of soluble fms-like tyrosine kinase-1 (sFlt-1) to placental growth factor (PlGF) will be calculated from their concentrations measured in maternal plasma samples obtained from fasting venous blood. The sFlt-1/PlGF ratio is dimensionless, with higher values indicating greater angiogenic imbalance. Changes in the ratio will be assessed across the study evaluation points.

Leptin concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in nanograms per milliliter (ng/mL). Changes in leptin concentration will be assessed across the study evaluation points.

Resistin concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in nanograms per milliliter (ng/mL). Changes in resistin concentration will be assessed across the study evaluation points.

Irisin concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in nanograms per milliliter (ng/mL). Changes in irisin concentration will be assessed across the study evaluation points.

Brain-derived neurotrophic factor (BDNF) concentration will be measured in maternal plasma obtained from fasting venous blood samples. Concentrations will be expressed in picograms per milliliter (pg/mL). Changes in BDNF concentration will be assessed across the study evaluation points.

Hemoglobin (Hb) concentration will be measured in maternal venous whole blood samples using a validated hematological analyzer. Blood samples will be collected under fasting conditions. Hemoglobin concentrations will be expressed in grams per deciliter (g/dL). Changes in hemoglobin concentration will be assessed across the study evaluation points.

Interleukin-1 (IL-1) concentration will be measured in colostrum samples collected manually after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interleukin-6 (IL-6) concentration will be measured in colostrum samples collected manually after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interleukin-8 (IL-8) concentration will be measured in colostrum samples collected manually after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interleukin-10 (IL-10) concentration will be measured in colostrum samples collected manually after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Fractalkine (CX3CL1) concentration will be measured in colostrum samples collected manually after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Tumor necrosis factor-alpha (TNF-α) concentration will be measured in colostrum samples collected manually after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interferon-gamma (IFN-γ) concentration will be measured in colostrum samples collected manually after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interleukin-1 (IL-1) concentration will be measured in mature breast milk samples collected manually four weeks after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interleukin-6 (IL-6) concentration will be measured in mature breast milk samples collected manually four weeks after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interleukin-8 (IL-8) concentration will be measured in mature breast milk samples collected manually four weeks after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interleukin-10 (IL-10) concentration will be measured in mature breast milk samples collected manually four weeks after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Fractalkine (CX3CL1) concentration will be measured in mature breast milk samples collected manually four weeks after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Tumor necrosis factor-alpha (TNF-α) concentration will be measured in mature breast milk samples collected manually four weeks after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).

Interferon-gamma (IFN-γ) concentration will be measured in mature breast milk samples collected manually four weeks after delivery. Samples will be stored at -80 °C until analysis. Concentrations will be expressed in picograms per milliliter (pg/mL).