Epigenetics and Protective Factors in the Preterm Infant
Epigenetics and Protective Factors in the Preterm Infant
Preterm infants (PT) spend their first weeks of life in the Neonatal Intensive Care Unit
 (NICU) where they are exposed to unfavorable conditions with different effects on child
 development including long-term alterations in epigenetic regulation (DNA methylation).
 Recent studies document that these epigenetic changes are associated with behavioral
 modifications, such as altered stress reactivity at 3 months and 4 years. A growing number of
 studies suggest that protective Developmental Care (DC) procedures (e.g., breastfeeding,
 skin-to-skin contact (SSC), maternal holding) positively impact neurophysiological and
 behavioral adaptation of PT with long-term effects. Additionally, a neuro-imaging study
 reported that parental support in the NICU is associated with improved brain connectivity.
 While in term (FT) infants, parental interpersonal touch (breastfeeding, affectionate touch)
 is associated with reduced methylation and activation of specific brain areas associated with
 affective interpersonal touch, to date no study has investigated whether DC practices and
 maternal care in NICU (specifically, SSC) buffer methylation and support the brain response
 to affectionate physical touch in PT. The present study investigates the association between
 DC procedures in NICU, DNA methylation, and brain responses to affectionate touch,
 investigated through the use of MRI, at 2 months of age (corrected for prematurity),
 controlling for: (1) birth status (PT vs FT); (2) the duration of SSC during the NICU stay;
 (3) parental affectionate touch in the home environment and during mother-child interaction.
Background: preterm infants (PT) spend their first weeks of life in the Neonatal Intensive
 Care Unit (NICU) where they are exposed to conditions with different effects on child
 development including long-term alterations in epigenetic regulation. Recent studies document
 that these epigenetic changes are associated with behavioral modifications, such as altered
 stress reactivity at 3 months and 4 years. A growing number of studies suggest that
 protective Developmental Care (DC) procedures (e.g., breastfeeding, skin-to-skin contact
 (SSC), maternal containment) positively impact neurophysiological and behavioral adaptation
 of PTs with long-term effects. Moreover, structural factors of the NICU, such as the
 organization of the unit space based on Open-Bay rather than Single Family Rooms could impact
 the child's neurobehavioral development and the parent's well-being. For example, a study on
 the impact of the Single Family Room shows improved medical and neurobehavioral outcomes for
 the infant at discharge and increased maternal involvement. However, the Single Family Room
 literature also reports mixed results with some studies finding increased parent involvement
 but no effect on child growth and some showing increased parent stress.

 Primary aim: to evaluate the methylation status of target genes (e.g., BDNF, SLC6A4, OXTR,
 NR3C1) in association with exposures to DC practices during NICU hospitalization in PT
 children, compared with a sample of FT children.

 Secondary aim: to investigate the relationship between preterm/term birth, DC practices (PT
 only), maternal touch in the postnatal period, epigenetic status and brain response to
 soft/soft stimulation during fMRI, at 2 months of age (corrected for prematurity in PT
 children), controlling for characteristics of the NICU of provenance (NICU Hospital of Monza:
 Single Family Room vs NICU Hospital of Lecco: Open-Bay).

 Planned Activities:

 Methods

 The project is characterized as observational, micro-longitudinal and is structured in two
 phases:

 - PHASE 1: FROM BIRTH TO DISCHARGE FOR PT CHILDREN; TIME OF BIRTH FOR FT CHILDREN)

 At the time of birth, both PT and FT will have their cord blood collected (methylation at
 birth) using non-invasive methods. Only for PTs at the time of discharge will be performed a
 second blood draw at the same time as the routine pre-discharge checks. In addition, at this
 stage, the mother will fill out some questionnaires about her mood and will be obtained
 information on pregnancy and childbirth (gestational age, birth weight, length and head
 circumference of the child at birth, type of delivery, duration of hospitalization, presence
 of perinatal diseases) and socio-anagraphic variables. Finally, only for the PT group, during
 hospitalization in the NICU, the following data will be collected on the negative and
 positive experiences to which PTs are exposed during hospitalization, specifically:

 1. number of pain-related stress procedures (e.g.: number of withdrawals, central
 insertions, invasive and non-invasive mechanical ventilation) obtained from the
 patient's medical record;

 2. time spent doing DC procedures (e.g.:, total time spent in which the child is attached
 to the mother's breast, total time spent in holding, total time spent in kangaroo care)
 obtained through the use of the "closeness diary" implemented in electronic format, in
 the form of an APP.

 At the end of the NICU admission, only for PT children, a second peripheral blood sampling
 (methylation at discharge) will be performed at the same time as routine pre-discharge
 checks. At the end of hospitalization, mothers of PTs will complete the same questionnaires
 completed at birth and in addition will be asked to complete questionnaires on the experience
 of stress and support received from staff during hospitalization in the NICU.

 - PHASE 2: AROUND 2 MONTHS OF AGE OF THE CHILD (CORRECTED AGE FOR PT) The children of both
 groups (PT and FT) and their mothers will go to the IRCCS "E. Medea" for a Functional
 Magnetic Resonance Imaging (fMRI) examination. The fMRI session will be conducted according
 to a recent study on the neural correlates of maternal affective touch in FT infants. The
 fMRI scan will be conducted by researchers and clinicians experienced in the use of MRI in
 pediatric settings. Prior to the fMRI session, all parents will be adequately briefed with
 respect to the MRI-related procedure and the scanning protocol will be viewed with the
 parents and staff will confirm the absence of safety risks. To minimize the potential risk
 and harm to infants caused by unnecessary sedation, fMRI will be performed during natural
 sleep and no sedation will be performed. Infants will be wrapped, fed, and scanned. To
 promote falling asleep, infants will be changed and fed by their mother and given time for
 them to fall asleep and then moved into the scanner. All infants will be fitted with hearing
 protection. In the event that the infant wakes up during the scan, the mother will be asked
 to attempt to put the infant back to sleep and if this occurs, the scan will be attempted
 again. Standard hearing protection will be given to the parents who will be allowed to remain
 in the scanning room for the duration of the acquisition session. The scan will be observed
 by control room staff. The entire procedure was structured in accordance with the guidelines
 for conducting fMRI in healthy child samples. After the fMRI, a 10-minute video recording of
 the mother-child interaction will be made. During the video recording, the child will be
 placed in a comfortable infant seat and the mother will be asked to sit in front of the child
 and interact with him/her for 5 min. Baby and mother's behavior and interaction will be coded
 using the Global Rating Scales mother-child interaction coding system and maternal touch
 coding using the Maternal Touch Coding System.
Inclusion criteria for PT children are:

 -  gestational age: 26+0 to 31+6 weeks;

 -  absence of documented neurological pathology;

 -  absence of sensory deficits;

 -  absence of malformative syndromes and/or major malformations.

 Inclusion criteria for FT infants are:

 -  gestational age ≥ 37weeks;

 -  birth weight ≥ 2,500g;

 -  APGAR 5' ≥ 7 - delivery without any complications for the child and/or mother;

 -  no pre/postnatal/postnatal clinical conditions;

 -  no hospitalizations at the time of birth or postpartum;

 -  absence of malformative syndromes and/or major malformations.

 Inclusion criteria for mothers are:

 -  mothers of Italian nationality;

 -  mother over 18 years of age;

 -  mother with absence of manifest psychiatric and/or cognitive pathologies (must be
 previously diagnosed major psychiatric pathologies);

 -  non-addicted/no habitual use of psychotropic medications, drugs, alcohol no smoking;

 -  non-single-parent families.

 Exclusion criteria: refer to inclusion criteria.