Effects of Exercise During Gestation on Maternal and Foetal Health

Insulin sensitivity will be derived from the homeostatic model assessment for insulin resistance (HOMA-IR), which will be calculated using the formula [fasting insulin (μIU/mL) x fasting glucose (mg/dL)]/405.

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

Neonatal insulin sensitivity will be assessed through the ratio glucose/insulin.

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 Mediterranean Diet Score in a version adapted to the specific needs during pregnancy for Fe, Ca and folic acid will be used to assess the adherence to the traditional Mediterranean dietary pattern.

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

We perform the 6-minute walk test, which measures the maximum distance (in meters) each participant can walk in 6 minutes along a 45.7 m rectangular course.

The handgrip strength test will be used as measure of strength

The back-scratch test will be used as measure of flexibility

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 9 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). Participants will be instructed to wear the accelerometer during the whole day (24 hours) on their wrist attached by an elastic belt. Time (in minutes) engaged in light, moderate, and moderate-vigorous intensity PA and sedentary time will be calculated. This accelerometer has been previously used in pregnancy with similar methodology as described in the present protocol.

Behaviours related to sleep, such as sleep onset, sleep latency, total sleep time, number and duration of awakenings and sleep efficiency will be objectively calculated by using a triaxial accelerometer (ActiSleep+, Pensacola, Florida, United States). The data analysis of such ActiSleep accelerometry data will be carried out through its specific software (Actilife).

It will be used to assess sleep quality and disturbances over a l-month time interval.

The presence of symptoms of insomnia will be measured with the Insomnia Symptoms Questionnaire, validated in pregnant women.

We will use the Short-Form Health Survey 36, for assessing health-related quality of life.

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.

Anxiety levels will be assessed with the State Trait Anxiety Index.

The "Restless Legs Syndrome" largely determines the quality of life for many pregnant and is related to their levels of depression and sleep quality. The Spanish version of the Detention of Restless Legs Syndrome questionnaire will be employed to assess the presence and severity of this syndrome .

Low-back pain will be assessed with the Spanish version of the Oswestry Disability Index score

Low-back pain intensity will be also assessed with the Pain Visual Analogue Scale

The 6-item Female Sexual Function Index will be used to assess sex function. This instrument is composed of six questions: desire, arousal, lubrication, orgasm, satisfaction and pain. Each question can be scored from 0 to 5 and then summed up to provide a total score.

"Positive health" will be evaluated through the following questionnaires:

• The Trait Meta-Mood Scale
• The Positive and Negative Affect Schedule
• The Satisfaction with Life Scale
• The 10-item Connor-Davidson Resilience Scale

Erythrocyte count, haematocrit, haemoglobin, platelets, leukocytes and erythrocyte mean corpuscular volume will be quantified by Coulter (Brand).

Plasma total, high-density lipoprotein and low-density lipoprotein cholesterol and triglycerides (all in mg/dL) will be assessed using an autoanalyzer (Hitachi-Roche p800, F. Hoffmann-La Roche Ltd. Switzerland)

Plasma glucose, insulin and glycosylated haemoglobinwill be assessed using an autoanalyzer (Hitachi-Roche p800, F. Hoffmann-La Roche Ltd. Switzerland)

It will be measured by using commercial kit (spectrophotometry).

Plasma vitamin E, retinol, carotene, coenzyme Q10 and coenzyme Q9 will be measured through mass spectrometry

Eritrocite membrane catalase, glutathione peroxidase and superoxide dismutase enzymes will be by measured by spectrophotometry.

Measurement of carbonyl proteins will be done by using commercial kit (spectrophotometry).

Measurement of 4-hydroxynonenal and isoprostanes in urine and plasma and hydroperoxides in plasma and erythrocyte membrane by using commercial kits (spectrophotometry and ELISA)

measurement of 8-hydroxyguanosine in urine and plasma by commercial kits (ELISA).

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.

Various 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.

Glucocorticoid receptor; peroxisome proliferation activated receptor (PPAR); the beta-11 hydroxysteroid dehydrogenase type I (11βHSD1), and beta-11 hydroxysteroid dehydrogenase type II (11βHSD2) will be assessed in maternal and umbilical cord blood and in oral mucosa from the mother and newborn.

Moreover, 4 genes highly involved in foetal and postnatal development of adipose tissue have been selected; two are receptors: the Glucocorticoid receptor and the PPAR-γ, and two are metabolic enzymes: beta-11 hydroxysteroid dehydrogenase type I or 11βHSD1, and 11 beta-hydroxysteroid dehydrogenase type II or 11βHSD2. Of great importance are PPAR-γ1 and PPAR-γ2, which are generated from the same gene by alternative promoter usage and mRNA. We will measure such proteins as well as paraoxonase/arylesterase 1 (PON1) concentrations in maternal and umbilical cord plasma by Polymerase Chain Reaction and Western Blot techniques.

The height of the mother and child will be recorded using a scale (Seca 22, Hamburg). A comprehensive analysis of body composition will be carried out for both the mother and child (DXA, Hologic Discovery QDR, Nasdaq). We will also record weight (Inbody R20, Biospace, Korea) as well as waist and neck circumference in both the mother and child.

Systolic and diastolic blood pressure, along with heart rate, will be measured three times after 5 minutes of rest (Omron Hoolddorp) in both mother and child.

A food frequency questionnaire and a Mediterranean Diet Adherence questionnaire will be administered. The use of nutritional supplements will also be recorded.

We will assess physical activity, sedentary behavior, and sleep-related behaviors (latency, efficiency, etc.) using accelerometers (ActigraphGT3X+, Pensacola, Florida, USA) worn on the wrist for nine consecutive days. Acceleration changes will be recorded in 5-second epochs for children and 60-second epochs for mothers. Data will be collected on total volume and intensity of activity (mean physical activity intensity), energy expenditure, and the number of minutes per day spent at various intensities: low (sedentary), light, moderate, vigorous, and moderate-to-vigorous. Sleep quality will also be recorded using the specific Actisleep software.

Muscular strength, cardiorespiratory fitness, balance, speed, and motor coordination-agility will be assessed using the following PREFIT battery tests: 20m shuttle run test, handgrip dynamometry, standing long jump test, one-leg balance test, and 4x10m shuttle run test, respectively. The PREFIT battery is a feasible, reliable, maximal, and valid tool to assess physical fitness in preschool-aged children.

MRI will be conducted at the Brain, Mind and Behavior Research Center (CIMCYC) at the University of Granada. Dr. Irene Esteban-Cornejo, a team member, has coordinated the neuroimaging area of previously funded studies (e.g., the ACTIVEBRAINS project, a randomized controlled trial assessing the effect of exercise on the brain in children). She has completed two research stays at the Monash Institute of Cognitive and Clinical Neurosciences in Australia, holds a Master's degree in advanced MRI analysis, and is the first author of the first two brain articles from the ACTIVEBRAINS project, both published in the prestigious journal Neuroimage. Dr. Esteban-Cornejo has also worked with leading experts in exercise and brain health (Erickson KI, Kramer AF, and Hillman CH) at Northeastern University and the University of Pittsburgh. Dr. Erickson and neuroscientist Marina López-Solà are also part of the current project team.

Three key aspects of executive function will be assessed:

• Cognitive Inhibition: Assessed via the Stroop test, which involves three conditions evaluating interference when identifying the ink color of words that do not match their meaning.
• Cognitive Flexibility: Assessed with two independent tests:

Design Fluency Test: Consists of three progressive conditions where participants create unique designs under different rules in 60 seconds, assessing flexibility, creativity, and visual pattern generation.

Trail Making Test: Five conditions assessing visual search speed, attention, mental flexibility, working memory, and motor function.

• Working Memory: Assessed with a computerized n-back task (see MRI section), which evaluates the ability to simultaneously store and process information.
• Intelligence: Assessed with the Kaufman Brief Intelligence Test (KBIT), which includes subtests for verbal and non-verbal intelligence.

• Woodcock-Muñoz III Battery: Spanish adaptation of the Woodcock-Johnson III (WJ III), composed of 22 subtests that assess language, mathematics, and science. Selected subtests will be used to assess math fluency, calculation, comprehension, and writing.
• School Grades: Parents will also be asked to provide the child's school grades from the academic year(s) during which the assessments take place.

Other Motor, Cognitive, and Language Development Scales:

• Bayley Scales of Infant Development, Third Edition (BSID-III)
• Wechsler Intelligence Scale for Children
• BENCI Battery (Computerized Neuropsychological Assessment Battery for Children)

Buccal mucosa samples will be collected from children to analyze the expression of the FTO gene polymorphism rs9939609 and MC4R gene polymorphism rs17782313. DNA will be extracted from saliva using Whatmann® paper supports and organic extraction methods optimized for quality and quantity. Samples will be quantified using a NanoDrop 2000 spectrophotometer (ThermoScientific) and normalized to ~5 ng/µL before being genotyped with TaqMan probes (Applied Biosystems) on a RealTime ABI PRISM® 7900 HT. Unsuccessful samples will be amplified and sequenced using specific primers and analyzed with the 3130 HID Genetic Analyzer (Applied Biosystem). DNA and RNA will be extracted using the QIAamp DNA Mini Kit and DNeasy Blood & Tissue Kit (Qiagen), and processed with the Qiacube robot.

The content in placenta of fat-soluble antioxidants (tocopherol, coenzyme Q10 and Q9 and retinol) will be analysed by HPLC, and nitric oxide and the total antioxidant capacity by the ABTS method.

• Oxidative damage to proteins: carbonyl proteins using commercial kits (spectrophotometry).
• Oxidative damage to lipids: 4-hydroxynonenal and isoprostanes using commercial kits (spectrophotometry and ELISA). Oxidative damage to DNA: 8-hydroxyguanosine by commercial kits (ELISA).

Changes inmitochondrial dynamics and their quality control are a key aspect to explain the observed findings.

Therefore, using Western-blotting or RT-PCR, the mitochondrial processes of fission (Drp1, Fis1 and OPA1), fusion (Mfn1 and Mfn2), biogenesis (PGC1alpha, Tfam, Sirt1, Nrf1, Nrf2) and autophagy (PARKIN2, PINK1, Beclin-1, ATG8, ATG5, ATG12, VDAC1).

The determination of total content of these essential (Ca, Mg, Mn, Fe, Cu, Zn, Se) or toxic elements (Hg, V, Cr, As, Cd, Pb, Ni) in placenta samples will be performed by an ICP-MS instrument (Agilent 7500, Agilent Technologies, Tokyo, Japan) fitted with a Meinhard type nebulizer (Glass Expansion, Romainmotier) and equipped with a He collision cell.

Samples will be processed as it is previously described in the "Genetic analyses section". Genomic and Methylation will be developed by pyrosequencing method. This will be developed for detection of FTO and MC4R genes. Genomic DNA from placentas will be isolated using DNeasy Blood&Tissue Kit following the recommended protocol (Qiagen N.V, Hi.GE). After checking the quality and quantity of the DNA, the promoters of the FTO and MC4R genes will be amplified by means of predesigned PyroMark CpG Assays. Region amplification using these primers will allow us to directly quantify the methylation status of the CGs within the region. First, we will amplify FTO and MC4R regions of interest by PCR using the PyroMark PCR Kit (QIAGEN N.V, Hi.GE), which enables labelling CG nucleotides susceptible of being methylated. Reaction products (one per sample and gene) will be purified and used for the sequencing reaction.

For a better understanding of the metabolic pathways involved, we will carry out the study of candidate genes, as well as the determination of miRNAs that will allow us to establish a genotype-expression-environment relationship (with Small RNA Sequencing analysis).

For mRNA and miRNA analysis, placental RNA will be obtained through the kit "RNeasy Mini" (Qiagen) following the manufacturer instructions. RNA integrity will be analyzed by a "RNA 6000 Nano chip" (Agilent) using a Bionalalyzer 2100 (Agilent) and the concentration will be calculated by NanoDrop 2000 (ThermoFisher Scientific).

Predesigned TaqMan Gene Expression assays (ThermoFisher Scientific) will be used to study the expression of candidate genes: IL6, CXCL8, TNF-alpha, CSF3, BDNF; FTO, MC4R and KL klotho (KL).

Targeted metabolomics analyses will be performed using liquid chromatography coupled to mass spectrometry. Lipid species extraction will be extracted from plasma samples with MTBE. Ten μL of the extracted sample will be applied onto UPLC 1290 (Agilent Technologies, Barcelona, Spain) equipped with a reversed-phase column coupled to QTOF mass spectrometer model 6545 (Agilent Technologies, Barcelona, Spain). Data will be collected in positive and negative electrospray modes. MassHunter (Agilent Technologies, Barcelona, Spain) and R software will be used to collect, extract, align, filter and normalize the data using quality controls. Untargeted metabolomics analyses will be performed using liquid chromatography coupled to mass spectrometry. Metabolite extraction will be extracted from plasma samples with methanol.

Concentrations of free bioactive phenols will be determined by multiclass ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The following phenols will be assessed:

bisphenol A; parabens: methylparaben, ethylparaben, propylparaben, butylparaben; benzofenonas (BzP): BzP-1, BzP-2, BzP-3, BzP-6, BzP-8, 4-hydroxy-BzP. Concentrations of perfluoroalkyl substances in placenta will be assessed by coupling salt assisted liquid-liquid extraction with dispersive liquid-liquid microextraction and UHPLC-MS/MS. The following perfluoroalkyl substances will be measured: PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA, PFTrA, PFHxS and PFOS.

We will use Luminex xMAP technology (Millipore, Darmstadt, Germany) kits to assess placenta Fractalkine, IL-1α, IL1β, IL-RA, IL-2, IL-6, IL-8, IL-10, IFN-γ, TNF-α, TNF-β, Brain-derived neurotrophic factor, Epidermal growth factor, Fibroblast growth factor 2, Granulocyte-colony stimulating factor, Granulocyte-macrophage colony-stimulating factor, Platelet-derived growth factor-AA, Monocyte chemoattractant protein-1, Vascular endothelial growth factor. For these determination we will use Human Sepsis Magnetic Bead Panel 3 Multiplex Assay (cat. No. HTH17MAG-14K).