Effects of a Multiple Sclerosis Relapse Therapy on Offspring Neurocognitive Development and Behaviour

Introduction: Multiple Sclerosis (MS) affects 1 in 1,000 people in industrialised countries, mainly women of childbearing age. To prevent MS relapses, most patients receive disease-modifying therapies (DMTs). However, most DMTs - with the exception of interferons and glatiramer acetate - are not approved during pregnancy. Consequently, DMTs are regularly discontinued during pregnancy with a subsequently increased risk of relapses. Thus, relapses during pregnancy are a common phenomenon. The average relapse rate per year in pregnant MS patients not taking DMTs during the 1st and 2nd trimester is nearly identical to the pre-partum period after which it significantly declines during the 3rd Trimester. The German Neurologic Society estimates that approximately 25% of all pregnant women with MS will suffer a relapse during their pregnancy period(s).

It is generally recommended by European and US guidelines to treat MS relapses during pregnancy with the synthetic glucocorticoid (GC) methylprednisolone (MP), which is thought to be harmless for the unborn. However, regardless of the undoubtedly positive effects of GCs for MS relapse treatment, there is considerable doubt as to whether synthetic GCs are as low in side effects for the child as MS guidelines assume. The fetus develops in the presence of very low GC levels because it is unable to produce cortisol until the end of pregnancy and about 90% of maternal cortisol is being inactivated by the placental enzyme 11ß-HSD2. In contrast to maternal cortisol, synthetic GCs are no substrate for the placental enzyme 11ß-HSD2 and, thus, pass the placenta without being inactivated. Once within the fetal circulation, GCs interfere with the development of the fetal central nervous system (CNS). The effects of synthetic GCs on fetal development and health and disease in later life as well as the mechanisms mediating these effects are best characterised for betamethasone exposure to induce fetal lung maturation in babies threatening premature labour as this treatment is used in nearly 10% of all pregnancies. In various studies excess exposure to synthetic GCs is associated with reduced birth weight, altered fetal brain development e.g., by direct effect on neuro- and gliogenesis via anti-proliferative effects on neural stem/progenitor cells, and persisting functional changes of the stress axis with its two arms, the hypothalamus-pituitary-adrenal axis (HPAA) and the autonomic nervous system (ANS). Supraphysiological GC concentrations are also thought to reset the set point of the negative feedback regulation of the fetal HPAA which results in an increase of HPAA activity in later life. Increased HPAA activity has been associated with increased stress sensitivity and a number of behavioural changes and neuropsychiatric disorders including anxiety, depressive-like disorders, and attention deficit hyperactivity disorder (ADHD). The relationship between adverse environmental influences during critical periods of fetal life, such as exposure to excess GC, and offspring health in later life is the basis of the 'Fetal Programming' hypothesis.

A predecessor study of the investigators' work group tested the hypothesis that fetal betamethasone treatment impairs structural and functional brain development as well as stress sensitivity leading to disturbances in overall neurocognitive performance and behaviour in later life. It is noteworthy, that the dosage of betamethasone (2 x 12mg, 24h apart) was approximately 300-fold lower than the dosage used to treat MS relapses (1000mg over 3-5 days). As a major study outcome, the investigators noticed detrimental effects of prenatal betamethasone exposure on general cognitive ability at the age of 8 - 9 years. Intelligence quotient (IQ)-scores in children who were prenatally exposed to betamethasone were on average 10.5 points lower than in controls, with a large effect size of d = 0.68. Even though IQ-scores of betamethasone-exposed children were still within the expected population range, the investigators assume that this difference is clinically relevant and impedes future life success of the participants. In addition, children exposed to betamethasone showed a significant increase in ADHD related symptoms on the International Classification of Diseases (ICD)-10-based rating scale, the Diagnostik-System für psychische Störungen nach ICD-10 und DSM-5 für Kinder und Jugendliche-III, with a medium effect size of d = 0.51. Electroencephalographic (EEG) analysis showed that BM-exposed children had a higher spectral edge frequency at rest and during stress suggesting a reduced overall neuroelectric relaxation capacity. Heart Rate Variability (HRV) analysis revealed a resetting of the ANS in betamethasone-exposed children.

Objectives: The primary goal of this study is to examine the general cognitive ability as a marker of brain development of children of MS patients who received MP to treat an MS relapse during pregnancy. The secondary goals are to determine the effects of MP administered during pregnancy on offspring structural and functional brain development and neuropsychiatric and behavioural performance. Stress sensitivity and epigenetic changes of GC receptor function will be measured to determine underlying mechanisms.

The study is based on the following hypotheses:

Fetal exposure to MP during relapse treatment in pregnant MS patients leads to

• disturbances in structural and functional brain development due to the maturational potential of MP which induces asymmetries in the complex sequence of the maturation of cerebral functional systems with lifelong changes in brain function,
• permanent alterations in HPAA sensitivity and autonomic function which are associated with neuropsychiatric and behavioural disturbances in later life, and
• alterations in GC receptor function and methylation of the GC receptor gene (NR3C1) and the H19/IGF2 locus as the underlying mechanisms linking MP exposition during pregnancy and offspring cognitive and behavioural functioning in later life.

Methods: The investigators will carry out a multicentre, observational, cross-sectional study in children and adolescents aged 8 to 18 years. The study design is retrospective due to ethical considerations of withholding the standard relapse treatment and the long study duration of at least 10 years. Following recruitment, all participants will undergo the same examination procedures in one study visit consisting of two days. By extending the study visit to two days, the investigators ensure stress-free examinations. In general, day 1 and day 2 of the study visits take place in direct succession. However, in case of scheduling issues or if the children and their parents live close by and do not want to stay overnight, there may be a few days in between.

The sequence of examinations was chosen in such a way that blood sampling and neuropsychological examinations induce as little stress as possible for the participants.

1. Functional brain development: The investigators will apply state-of-the-art neurocognitive, behavioural and mental health measures to determine the stage of functional brain development: General cognitive ability (primary endpoint) is determined using German age-appropriate versions of the Reynolds Intellectual Assessment Scales and Screening (RIAS) intelligence test. Behaviour is measured using three external assessment questionnaires which are routinely used in clinics and the scientific community. The German rating scale Fremdbeurteilungsbogen für Aufmerksamkeitsdefizit-/Hyperaktivitätsstörungen (FBB-ADHS) is based on the ICD-10 criteria for ADHD and assesses inattentiveness, hyperactivity, and impulsivity. The child behaviour checklist (CBCL), an established diagnostic screening procedure, measures psychopathological abnormalities in children and adolescents. The strengths and difficulties questionnaire (SDQ) assesses children's strengths and weaknesses. The Continuous Performance Test (CPT) is a clinically and scientifically established instrument to detect selective attention, vigilance, and impulsive behaviour with sufficient reliability and validity. Motor development is assessed using the Movement Assessment Battery for Children - Second Edition (M-ABC 2), which allows a reliable and time-efficient assessment of motor-coordinative deficits.
2. Structural brain development: The stage of structural brain development will be quantified based on a volumetric T1-MRI standard sequence using markers such as gyrification and cortical thickness as well as the BrainAge Score, which was development by the investigators' research group. The investigators expect that prenatal MP has a significant effect on brain age since GCs accelerate tissue differentiation and maturation at the expense of tissue growth.
3. Stress sensitivity and autonomic function: The individual stress sensitivity including the function of the HPAA and the autonomic nervous system is examined using the Trier Social Stress Test (TSST). This is an established and commonly used tool for children to generate a reproducible psychophysiological stress response using a simulated examination situation. It comprises a 10 minute rest period and a task period consisting of five-minute story-telling and mental arithmetic periods, which is completed in front of an "examination committee". Saliva samples for measurement of cortisol levels and alpha-amylase are taken before and during the stress test to determine HPAA and sympathetic activity at rest and during activation. In addition, continuous electrocardiography (ECG) and EEG leads are performed. The autonomic activity is also determined using HRV analysis from the derived ECG based on established HRV standards as well as on innovative nonlinear information based approaches developed at the department. Autonomic activity reflected in HRV is a sensitive marker of disturbances in the neurodevelopmental trajectory. Neurophysiological activation is measured using power spectral and nonlinear EEG analysis techniques.
4. Mechanism: The investigators will determine GC receptor sensitivity and epigenetic changes of the NR3C1 promoter and the H19/IGF2 locus.

Expected Impact: The results of this study will improve the ability of clinicians to make an evidence-based benefit-risk assessment regarding the continuation of a DMT during pregnancy under consideration of its safety under 'real world' conditions in relation to the potential long-term risks of an acute relapse therapy with MP for the health of the unborn child in later life.

The investigators expect that the results will provide an additional argument (1) to continue immunomodulatory relapse prophylaxis, e.g. with interferons during pregnancy, especially in light of their recently recognized relative safety for the child, or (2) to choose a long-acting DMT such as cladribine that provides lasting protection from relapses during pregnancy.