Stress-associated Epigenetic Alterations in Newborns After Fetal Surgery
Condition: Meningomyelocele
MeSH terms: Meningomyelocele; Spina Bifida Cystica
Study type: Observational

Summary: 
      Open spina bifida or myelomeningocele (MMC) is a devastating congenital defect of the central
      nervous system for which there is no cure. The etiology of MMC remains poorly understood.
      Primary failure of neural tube closure at the caudal neuropore in the embryonic period
      results in exposure of the developing spinal cord to the uterine environment. Without
      protective tissue coverage, secondary destruction of the exposed neural tissue by trauma or
      amniotic fluid may occur throughout gestation. In order to protect the spinal cord from this
      secondary destruction, a fetal surgical repair can be performed between gestational weeks 20
      and 26.

      From a psychological point of view fetal repair of MMC constitutes a highly stressful event
      both for the mother and the fetus. To date, however, stress of mothers and children in case
      of prenatal surgery for MMC repair has never been studied. It is therefore unclear, if and to
      what extend the procedure and its consequences are associated with stress, and if there are
      short- or longer-term consequences.

      The aims of this study are threefold:

        1. Do newborns after fetal surgery for MMC show epigenetic alterations in genes that are
           involved in stress regulation?

        2. With which medical and psychosocial variables are epigenetic alterations associated?

        3. At age 3 months, do infants after fetal surgery have a more difficult temperament
           compared to controls?
    

Detailed description: 
      BACKGROUND AND AIMS:

      Open spina bifida or myelomeningocele (MMC) is a devastating congenital defect of the central
      nervous system for which there is no cure. The etiology of MMC remains poorly understood.
      Primary failure of neural tube closure at the caudal neuropore in the embryonic period
      results in exposure of the developing spinal cord to the uterine environment. Without
      protective tissue coverage, secondary destruction of the exposed neural tissue by trauma or
      amniotic fluid may occur throughout gestation. In order to protect the spinal cord from this
      secondary destruction, a fetal surgical repair can be performed between gestational weeks 20
      and 26.

      From a psychological point of view fetal repair of MMC constitutes a highly stressful event
      both for the mother and the fetus. To date, however, stress of mothers and children in case
      of prenatal surgery for MMC repair has never been studied. It is therefore unclear, if and to
      what extend the procedure and its consequences are associated with stress, and if there are
      short- or longer-term consequences.

      The aims of this study are threefold:

        1. Do newborns after fetal surgery for MMC show epigenetic alterations in genes that are
           involved in stress regulation?

        2. With which medical and psychosocial variables are epigenetic alterations associated?

        3. At age 3 months, do infants after fetal surgery have a more difficult temperament
           compared to controls?

      METHODS:

      Sample: The study will include newborns after fetal surgery for MMC at the University
      Hospital Zurich and two control groups. One including healthy newborns and another including
      newborns after prenatal exposure to synthetic glucocorticoids (sGC) in pregnancy. Each group
      shall include n=30 newborns (i.e. total number of study participants: n=90).

      Recruitment and Procedures: Recruitment will take place at the University Children's Hospital
      and the University Hospital Zürich and will be continued until the required sample size is
      reached. For every MMC-patient, secondary recruitment of a control child for group 2 and 3
      will be performed (matched for child sex). Parents of all study groups will be approached by
      the responsible study staff after checking for inclusion and exclusion criteria. They will be
      informed orally about the study and the study information letter (see encl.) will be
      provided. Parents will be contacted again after >24h face to face or by phone by study staff
      to answer any questions, if necessary. If the parents agree to participate in the study, they
      will provide the signed written informed consent form (see encl.). After inclusion in the
      study, the parents will be contacted again in hospital by study staff around 24-36 hours
      after birth to re-explain the collection of the saliva samples. The saliva samples will then
      be collected between 24 and 72h postpartum. Six weeks and 3 months post-partum, the parents
      will receive the study questionnaires by mail and they will be asked to complete and return
      them within 1 week. If the questionnaires are not received after 10 days, study staff will
      contact the parents by phone for a gentle reminder. Parents are not financially compensated
      for taking part in the study.

      Collected data:

      -) DNA and RNA sample collection from the child will take place between 24 and 72 hours of
      life during hospitalization by trained research project staff. In order to obtain DNA and
      RNA, saliva will be collected using special sponge devices designed for use with infants who
      cannot spit independently.

      For DNA, 2x ORAcollect for pediatrics (OC-175; DNA Genotek Inc) will be used to collect
      saliva in order to obtain enough DNA sample.

      For RNA, 2x saliva collection devices for pediatrics (CP-190; DNA Genotek Inc) will be used
      to collect saliva in order to obtain enough RNA sample from the newborns.

        -  Medical data: a) Child: Gestational age at birth; gender; birth length and percentile;
           birth weight and percentile; head circumference at birth and percentile; Apgar score;
           umbilical artery ph and lactate; meconium-stained amniotic fluid; Gestational age at
           fetal surgery; time and length of fetal surgery; positioning of the fetus necessary;
           transfer to neonatology (group 2 and 3). b) Mother: Maternal age; parity; gestational
           age at glucocorticoid treatment; treatment of preterm labour; medications during
           pregnancy; surgery and perinatal variables; history in terms of smoking, alcohol,
           nicotine, marihuana, other illicit drugs; Indication for ECS (group 2 and 3).

        -  Psychosocial data: The following variables shall be assessed from mothers by
           standardized and validated questionnaires: country of birth (parents, grandparents);
           Socio-economic status, maternal and parental education; current job situation (no,
           part-time, full-time); maternal and paternal trauma history (Childhood Trauma
           Questionnaire - Short Form from Bernstein & Fink, 1998; Karos et al. 2014); significant
           life events during the pregnancy (Life Event Scale from Landolt & Vollrath, 1998);
           maternal and paternal subjective stress during pregnancy (Perceived Stress Scale PSS-10
           from Cohen, Kamarck, & Mermelstein (1983) and Klein et al. (2016); maternal and paternal
           mental health both retrospectively during pregnancy and currently at the assessment
           points after birth: anxiety and depression by means of two modules of the Patient Health
           Questionnaire PHQ from Spitzer, Kroenke, & Williams (1999); posttraumatic stress
           disorder by means of the International Trauma Questionnaire ITQ (Cloitre et al., 2018)
           and the infant's temperament by means of the Infant Behaviour Questionnaire IBQ - Very
           Short Form by Rothbart (1981). All measures are standardized and available in validated
           German versions.

      Statistical Analyses: Aim 1 will be examined with ANCOVAs to compare total methylation /
      gene-expression (mRNA level) for the studied gene regions across the 3 groups. The
      correlation between methylation levels and gene-expression will be investigated, expecting
      that hypermethylation induces reduced gene-expression and vice versa.

      Aim 2 will be examined by (partial) correlational analyses between predictor variables and
      total methylation. If requirements are met, multivariate analyses will be performed.

      Aim 3 will be examined with ANCOVAs comparing IBQ-scores across the three groups.

      Data will not be transformed before analysis. If the data doesn't meet the requirements for
      the selected statistical tests, then modified tests (e.g. one-way testing) or appropriate
      tests (e.g. non-parametric methods) will be used.

      Statistical analysis will be performed either with the program Statistical Package for Social
      Sciences (SPSS, Version 25) or the statistical program R. Statistical significance will be
      set to a significance level of α= <.05.

      Sample size: According to a power-analysis calculation with the program G*Power 3.1 (Faul,
      Erdfelder, Buchner & Lang, 2009), the required sample size for an ANCOVA with 3 groups, an
      effect size of d=0.4 (small effect) and a power of .90 is n=84.
    

Intervention name(s): Fetal surgery for MMC repair
Intervention type(s): Procedure
Intervention description(s): Fetal surgery for MMC repair
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        The study will include newborns after fetal surgery for MMC at the University Hospital
        Zurich and two control groups. One including healthy newborns and another including
        newborns after prenatal exposure to synthetic glucocorticoids (sGC) in pregnancy. Each
        group shall include n=30 newborns (i.e. total number of study participants: n=90). In the
        following paragraph you can find a detailed description of the groups as well as the
        inclusion and exclusion criteria for all groups.
      
Biospecimen description: 
      DNA and RNA sample collection from the child will take place between 24 and 72 hours of life
      during hospitalization by trained research project staff. In order to obtain DNA and RNA,
      saliva will be collected using special sponge devices designed for use with infants who
      cannot spit independently.

      For DNA, 2x ORAcollect for pediatrics (OC-175; DNA Genotek Inc) will be used to collect
      saliva in order to obtain enough DNA sample.

      For RNA, 2x saliva collection devices for pediatrics (CP-190; DNA Genotek Inc) will be used
      to collect saliva in order to obtain enough RNA sample from the newborns.