Stress-associated Epigenetic Alterations in Newborns After Fetal Surgery

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?

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.

Eligibility Criteria:
Inclusion Criteria:

Group 1: Newborns after successful fetal surgery for MMC, delivered by ECS (elective c-section) at weeks 350 - 370.

Group 2: Healthy newborns after exposure to synthetic glucocorticoids for lung maturity during pregnancy, delivered by ECS between 350 - 400 weeks, matched for child sex with group 1. This group is needed to control for the effects of sGC exposure.

Group 3: Healthy newborns, uncomplicated pregnancy, delivered at term by ECS between 360 - 390 weeks (healthy controls). Matched for child sex with group 1.

Exclusion Criteria for all 3 groups:

* Multiple pregnancies
* Apgar 5 min \<=7, or significant postpartal health problems such as respiratory distress syndrome
* ph\<7.15
* General anaesthesia for ECS
* Insufficient knowledge of German or English by the mother
* Mother exposed to traumatic stress during pregnancy (other than fetal surgery)
* Non-Caucasian origin
* Egg donation