Prenatal Intervention of Maternal Stress - Breastfeeding & Infant Neurodevelopment

This outcome will assess maternal confidence in breastfeeding and actual breastfeeding before childbirth using the Prenatal Breastfeeding Self-Efficacy Scale (PBSES), a validated tool that measures prenatal maternal perceived ability to breastfeed successfully. The PBSES consists of 20 items rated on a 5-point Likert scale, with a SUM score ranging from 20 (minimum) to 100 (maximum). Scores will be recorded without a cutoff point, with higher scores indicating greater prenatal maternal confidence in breastfeeding. Comparisons will be conducted among the three study groups to evaluate differences in PBSES scores based on the intervention received.

Statistical analysis will include the comparisons across study groups using ANOVA or non-parametric equivalents. Regression models to explore potential predictors of higher or lower prenatal self-efficacy.

Maternal breastfeeding self-efficacy will be assessed using the Breastfeeding Self-Efficacy Scale (BSES), a validated questionnaire measuring maternal confidence in breastfeeding. The BSES consists of 14 items, each rated on a 5-point scale (1 = Strongly Disagree, 5 = Strongly Agree). The final score is calculated as the sum of all item scores, with higher scores indicating greater breastfeeding self-efficacy. Minimum Value is 14 (lowest BSES score) and Maximum Value is70 (highest BSES score) Comparative analyses will be conducted across the three study groups to evaluate differences in self-efficacy levels.

Breastfeeding outcomes will be derived from responses to selected PRAMS questions, which indirectly indicate exclusivity. Breastfeeding success will be numerically categorized (1 = Exclusive breastfeeding, 2 = Any breastfeeding, 3 = No breastfeeding) to allow for statistical comparison with Breastfeeding Self-Efficacy Scale (BSES) scores. The analysis will examine whether higher BSES scores are associated with exclusive or any breastfeeding.

The levels of neurotrophins, specifically brain-derived neurotrophic factor (BDNF) and Reelin, will be assessed as neurodevelopmental biomarkers in maternal serum at 15-20 weeks and 36-37 weeks of gestation, and in umbilical cord blood at birth.

Neurotrophin quantification will be performed using the Enzyme-Linked Immunosorbent Assay (ELISA) method, following standardized protocols.

Results will be expressed in picograms per milliliter (pg/mL), and comparative analyses will be conducted to evaluate differences across the three study groups and at different time points.

Neonatal behavior will be assessed using the Neonatal Behavioral Assessment Scale (NBAS), conducted by a personnel trained in administering and scoring the instrumental tool . NBAS contains a large number of individual items scored on a 9-point scale and reflexes scored on a 4-point scale . The evaluated items will be categorized into eight clusters, with scores representing the mean of the item scores within each cluster. The clusters and the number of items per cluster are: 1. Habituation (4 items) , 2. Social -Interactive (7 items) , 3. Motor System (5 items), 4. State Organization(4 items), 5. State Regulation (4 items), 6. Autonomic System (3 items) , 7. Reflexes (18 items), 8. Supplementary Items (7 items)-Additional behavioral observations.

Infant behavior will be assessed using the Infant Behavior Questionnaire-Revised Short Form (IBQ-R SF), completed by parents. The IBQ-R SF consists of 91 items, each rated on a 7-point Likert scale (1 = Never, 7 = Always). Scores are averaged within each of the 14 behavioral subscales, which are further grouped into three broad temperament dimensions. The final score for each subscale or dimension is calculated as the mean of the corresponding item scores, providing a standardized numerical measurement of infant temperament for comparative analyses across the three study groups.

This outcome measure will assess the correlation between maternal age (in years) and breastfeeding success across the three study groups.

Breastfeeding success will be categorized as:

• = No breastfeeding
• = Any breastfeeding
• = Exclusive breastfeeding Data will be collected using structured PRAMS-based questions.

Statistical analysis:

Pearson's or Spearman's correlation coefficient (r-value) will be used to assess the relationship between maternal age and breastfeeding success.

Chi-square tests (χ²) will be used for categorical comparisons.

Multivariate regression analysis will adjust for potential confounders.Unit of Measure:

Maternal age: Years (continuous variable) Breastfeeding success: Categorical classification (1, 2, 3)

Higher Scores Mean:

For maternal age: Not applicable (continuous variable) For breastfeeding success: Higher values (3) indicate better outcomes (exclusive breastfeeding)

This outcome measure will assess the correlation between household income and breastfeeding success across the three study groups. Household income will be categorized into six levels based on self-reported data collected between the 15th and 20th week of gestation: 1 = Indigent, 2 = <10,000, 3 = 10,000-20,000, 4 = 20,000-30,000, 5 = 30,000-40,000, and 6 = >40,000. Breastfeeding success will be categorized as 1 = No breastfeeding, 2 = Any breastfeeding, and 3 = Exclusive breastfeeding, based on structured PRAMS-based questions. Statistical analysis will include Spearman's correlation coefficient (r-value) to assess the relationship between household income and breastfeeding success. Chi-square tests (χ²) will be used for categorical comparisons, and multivariate regression analysis for potential confounders. Unit of Measure: Household income will be recorded as an ordinal variable (1-6), and breastfeeding success as a categorical variable (1-3)

Maternal BMI will be calculated as weight (kg) divided by height squared (m²) and recorded at two time points: before pregnancy (self-reported) and at the time of delivery (measured clinically). Maternal BMI: kg/m²

Breastfeeding success will be assessed using structured PRAMS-based questions, categorizing success as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between maternal BMI and breastfeeding outcomes using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders such as maternal age, socioeconomic status, and delivery mode.

Anxiety will be assessed using the State-Trait Anxiety Inventory (STAI), a validated self-report questionnaire measuring state and trait anxiety, with each subscale consisting of 20 items rated on a 4-point Likert scale, leading to a SUM score range of 20-80 per subscale.

Breastfeeding success will be categorized using PRAMS-based structured questions as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between STAI scores and breastfeeding success using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders such as maternal age, socioeconomic status, and delivery mode.

Pregnancy-related anxiety will be assessed using the Pregnancy-Related Anxiety Questionnaire-Revised 2 (PRAQ-R2), a validated self-report questionnaire consisting of 11 items rated on a 5-point Likert scale (1 = Not at all relevant, 5 = Completely relevant), resulting in a SUM score ranging from 11 (lowest anxiety) to 55 (highest anxiety).

Breastfeeding success will be categorized using PRAMS-based structured questionnaires as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between PRAQ-R2 scores and breastfeeding success using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders such as maternal age, socioeconomic status, and delivery mode.

This outcome measure examines how neonatal behavior influences breastfeeding success across study groups. Neonatal behavior will be assessed using the Neonatal Behavioral Assessment Scale (NBAS) between the 3rd and 7th day postpartum, evaluating responses to stimuli, alertness, and sucking reflex. Breastfeeding success will be categorized as exclusive, partial, or no breastfeeding, based on structured breastfeeding questionnaires. Additional data will capture early neonatal feeding behaviors, such as skin-to-skin contact, first latch attempts, and initial feeding difficulties. Comparative analyses will assess differences between study groups, while correlation and regression models will explore whether more adaptive neonatal behaviors are associated with higher breastfeeding success rates.

This outcome measure examines the correlation between neurotrophin levels (BDNF, Reelin) and maternal breastfeeding self-efficacy across the three study groups. Neurotrophin levels will be measured in maternal serum and in umbilical cord blood using ELISA (pg/mL). Breastfeeding self-efficacy will be measured using the Breastfeeding Self-Efficacy Scale (BSES) with SUM score (Range: 14-70) Statistical analyses will include Spearman's or Pearson's correlation coefficients, with regression models adjusting for confounders.

Maternal breastfeeding self-efficacy will be assessed prenatally using the Prenatal Breastfeeding Self-efficacy Scale (PBSES), consisting of 20 items rated on a 5-point Likert scale (SUM score range: 20-100), to assess maternal confidence in breastfeeding during pregnancy. There are no cut-off points. As the scores get higher, the prenatal breastfeeding self-efficacy increases. Breastfeeding success will be categorized numerically as: 1 = No breastfeeding , 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will examine correlations between prenatal breastfeeding self-efficacy, and breastfeeding success.

For PBSES: Higher scores indicate greater prenatal breastfeeding For breastfeeding success: Higher values indicate greater breastfeeding success.

Postnatal breastfeeding self-efficacy will be assessed using the Breastfeeding Self-Efficacy Scale (BSES), a validated questionnaire consisting of 14 items rated on a 5-point Likert scale (SUM score range: 14-70).

Breastfeeding outcomes will be evaluated using structured PRAMS-based questions, categorizing success as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between postnatal BSES scores and breastfeeding outcomes using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders.

Salivary cortisol will be measured prenatally, using Salivette (Sarstedt) devices and analyzed via Chromatography/Mass Spectrometry (nmol/L).

Breastfeeding outcomes will be evaluated using structured PRAMS-based questions, categorizing success as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between prenatal maternal cortisol levels and breastfeeding outcomes using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders.

Infant behavior will be assessed at 6 months postpartum using the Infant Behavior Questionnaire-Revised Short Form (IBQ-R SF), a validated parental-report measure of temperament. Breastfeeding success will be categorized as 1 = No breastfeeding, 2 = Partial breastfeeding, 3 = Exclusive breastfeeding, based on structured breastfeeding questionnaires. Unit of Measure: IBQ-R SF score: Mean score per subscale (temperament domains) Breastfeeding success: Categorical classification (1 = No breastfeeding, 2 = Partial breastfeeding, 3 = Exclusive breastfeeding) Statistical analyses will assess correlations between IBQ-R SF scores and breastfeeding success using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders.

This outcome measure examines the correlation between neonatal hospitalization (Yes/No) and breastfeeding success across the three study groups. Neonatal hospitalization will be defined as admission to the Neonatal Intensive Care Unit (NICU) or other specialized care units after birth, and recorded as a binary variable (Yes = 1, No = 0).

Breastfeeding success will be categorized based on structured PRAMS-based questions as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess associations between neonatal hospitalization and breastfeeding success using Chi-square tests (χ²) for categorical comparisons, while logistic regression models will adjust for potential confounders such as gestational age, birth weight, and maternal health factors. For neonatal hospitalization: Not applicable (binary variable) For breastfeeding success: Higher values indicate greater breastfeeding success

Breastfeeding difficulties will be assessed using 11 PRAMS-based questions, where each item is answered as Yes (2) or No (1), resulting in a SUM score ranging from 11 (minimum difficulties) to 22 (maximum difficulties).

Postpartum breastfeeding support from maternity hospital staff will be evaluated using 12 PRAMS-based questions, where each item is answered as Yes (2) or No (1), leading to a SUM score ranging from 12 (lowest support) to 24 (highest support).

Breastfeeding success will be categorized based on PRAMS-based structured questionnaires, as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between breastfeeding difficulties, postpartum support, and breastfeeding success using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders such as maternal age, socioeconomic status, and delivery mode.

This outcome measure examines the correlation between mode of delivery and breastfeeding success across the three study groups. Mode of delivery will be categorized as follows:

1 = Cesarean section 2 = Vaginal delivery 3 = Vaginal delivery with induction 4 = Induction attempted for vaginal delivery but resulted in cesarean section 5 = Cesarean section with general anesthesia 6 = Emergency cesarean section Breastfeeding success will be categorized using PRAMS-based structured questionnaires as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between mode of delivery and breastfeeding success using Spearman's or Pearson's correlation coefficients, while logistic regression models will adjust for confounders such as maternal age, socioeconomic status, and gestational age at birth.

Units of Measure are Mode of delivery: Categorical classification (1-6) Breastfeeding success: Categorical classification (1 - 3)

Postpartum depression symptoms will be assessed using the Edinburgh Postnatal Depression Scale (EPDS), a 10-item self-report questionnaire, where each item is rated on a 4-point Likert scale (0-3), resulting in a SUM score ranging from 0 (no depressive symptoms) to 30 (severe depressive symptoms). Cutoff point will be 10 .

Breastfeeding success will be categorized using PRAMS-based structured questionnaires as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between EPDS scores and breastfeeding success using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders such as maternal age, socioeconomic status, and mode of delivery.

Stressful life events will be assessed using the Holmes and Rahe Stress Scale, a validated tool that assigns weighted scores to 43 life events during the last year based on their relative stress impact. The total score is calculated as a SUM score, with higher values indicating greater exposure to stress and increased health risk. Breastfeeding success will be categorized using PRAMS-based structured questionnaires as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding. Statistical analyses will assess correlations between Holmes and Rahe Stress Scale scores and breastfeeding success using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders such as maternal age, socioeconomic status, and mode of delivery.Unit of Measure: Holmes and Rahe Stress Scale score: SUM score (higher values indicate greater exposure to stress) and Breastfeeding success: Categorical classification (1-3)

Maternal attachment will be assessed using the Maternal Postnatal Attachment Scale (MPAS). The MPAS consists of 19 items, with different item scoring ranging from two-point to five-point scaling. Items are re-coded prior to analysis for equal weighting, with final item scores ranging from 1 to 5. The maximum score is 95, and the theoretical minimum is 19. Lower scores indicate more problematic responses. The MPAS does not have validated cut off points. Higher scores indicate more optimal mother-infant bonding. Breastfeeding success will be categorized as 1 = No breastfeeding, 2 = Partial breastfeeding, 3 = Exclusive breastfeeding, based on structured breastfeeding questions. Statistical analyses will assess correlations between MPAS scores and breastfeeding success using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders.

Neonatal anthropometric measurements, including birth weight, body length, and head circumference, will be recorded at birth. Infant anthropometric parameters, including weight, length, and head circumference, will be assessed at 6 months postpartum during routine clinical visit. Units of Measure are for Birth weight & infant weight in kilograms (kg), Body length & head circumference in Centimeters (cm) Breastfeeding success will be categorized as 1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding, based on structured PRAMS-based questions. Statistical analyses will assess correlations between anthropometric parameters and breastfeeding outcomes using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders such as maternal BMI, gestational age, and birth complications.

Breastfeeding success: Categorical classification (1 = No breastfeeding, 2 = Any breastfeeding, 3 = Exclusive breastfeeding)

This outcome measure examines the correlation between maternal cortisol levels and perceived stress as measured by the Perceived Stress Scale (PSS-14) pre and postnatally across the three study groups and between the time frames. Salivary cortisol will be measured prenatally using Salivette (Sarstedt) devices and analyzed via Chromatography/Mass Spectrometry (nmol/L). PSS-14, is a validated self-report questionnaire that evaluates maternal stress levels on a 5-point Likert scale (from Never = 0 to Very often = 4).Total score of the scale is the sum of all 14 item scores with range 0 (lowest stress) to 56 (highest stress).

Higher scores indicate greater perceived stress. Statistical analyses will assess correlations between cortisol levels and PSS-14 scores using Pearson's or Spearman's correlation coefficients, while regression models will adjust for potential confounders.

Τhis outcome measure examines the relationship between maternal cortisol levels and prenatal stress as measured by the Pregnancy Experience Scale Brief (PES-brief) across the three study groups. Salivary cortisol will be measured using Salivette (Sarstedt) devices and analyzed via Chromatography/Mass Spectrometry (nmol/L). Prenatal stress will be assessed using the PES-brief, which consists of two factors measuring distinct positive and negative aspects of pregnancy stress, each each item rated from 0 (not at all) to 3(a great deal). Scoring yields six scores. These are: the frequency of hassles and the frequency of uplifts, intensity of hassles and the intensity of uplifts and two ratio scores Hassles/Uplifts frequencies and Hassles/Uplifts Intensities. Statistical analyses will assess correlations between cortisol levels and PES scores using Pearson's or Spearman's correlation coefficients, while regression models will adjust for confounders.

This outcome measure examines the correlation between neurotrophin levels (BDNF, Reelin) and infant behavior at 6 months postpartum across the three study groups. Neurotrophin levels will be measured in maternal serum and in umbilical cord blood , using ELISA (pg/mL). Infant behavior will be assessed using the Infant Behavior Questionnaire-Revised Short Form (IBQ-R SF). Statistical analyses will include Spearman's or Pearson's correlation coefficients, with regression models adjusting for confounders.

Neurotrophin levels will be measured in maternal serum and in umbilical cord blood , using ELISA (pg/mL. Neonatal behavior will be assessed using the Neonatal Behavioral Assessment Scale (NBAS) during the first week of life. Correlations between neurotrophin levels and NBAS scores will be analyzed using Pearson's or Spearman's correlation coefficients, while regression models will adjust for confounders.Unit of Measure: Neurotrophin levels: pg/mL (ELISA method) Neonatal behavior: NBAS scores

Maternal attachment will be assessed using the Maternal Postnatal Attachment Scale (MPAS), while infant behavior will be evaluated using the Infant Behavior Questionnaire-Revised Short Form (IBQ-R SF). The MPAS consists of 19 items each one ranging from 1 to 5, divided in three constellations : pleasure in interaction, Absence of hostility and Quality of attachment. maximum total score of the scale is 95. Statistical analyses will assess correlations between MPAS and IBQ-R SF scores using Spearman's or Pearson's correlation coefficients, while regression models will adjust for confounders. Unit of Measure: for MPAS score is the sum of the three factors . and for IBQ-R SF score is the Mean score per subscale (temperament domains)