The study is a case-controlled investigational assessment of the long-term impacts of SARS-CoV2 in utero exposure on children from birth to 15 years old. Ethics approval has been obtained through Monash Health Human Research Ethics Committee RES-20-0000-801A (protocol #6, 17/03/2022) and the National Council of Research Ethics (CONEP, acronym in Portuguese) with protocol number 5.234.055. The study aligns with the SPIRIT guidelines. Women infected with SARS-CoV-2 during pregnancy are recruited from Monash Health, the Royal Women’s Hospital and Sunshine Hospital (Melbourne sites), and Londrina Municipal Maternity Hospital Lucilla Ballalai and PUCPR Medical Clinic (Londrina, Brazil). A control group in a 2:1 ratio is also recruited of women who gave birth in the same month of delivery, and are of similar age (within a 5 year age bracket) but who did not contract SARS-CoV-2 during their pregnancy. Demographic information is collected from the mother at the first visit. See Table 1 for the complete list of demographic data collected. Exclusion criteria are loss or death of the child for the current pregnancy. Assessments are planned at birth, 3 months, 1, 2, 3, 4, 5, 10 and 15 years (see Figure 1 Timeline diagram).
Table 1. Demographics
Number of previous pregnancies
Educational attainment: High school or less / Diploma/TAFE / University degree or higher
Language spoken at home
Migration status (last 5 years)
Past medical conditions
Medical conditions of 1st degree relatives
Gestation of infection: 1st, 2nd or 3rd trimester
Mode of delivery: Normal vaginal delivery / Caesarean section
History of mental illness
History of substance abuse
History of domestic violence
Vaccination status: 1, 2 or 3 doses; brand of vaccine; adverse reactions to vaccine
Dominant variant at time of infection
Medications received for infection
Gestational age at birth
Neonatal Intensive Care Unit (NICU) admission
Only child or siblings
Medical conditions of siblings
Maternal study specific data
1. For women who tested positive for COVID-19: timing of the illness (weeks of pregnancy), highest temperature recorded during illness, duration of illness and symptom severity (World Health Organisation, seven-point ordinal scale) , as well as disease modifying treatments received are recorded. COVID-19 vaccination status at the first appointment is also recorded. If the participant is vaccinated, the date of each vaccination and brand of vaccine is recorded.
2. All mothers complete the Edinburgh Postnatal Depression Scale (EPDS). The EPDS is a questionnaire designed to screen women for symptoms of emotional distress during pregnancy and the postnatal period . This is a 10-question survey which takes approximately 5-10 minutes to complete. This test will be completed at the initial birth assessment as well as the 3 and 12-month follow up assessments.
3. All mothers complete the Maternal Postnatal Attachment Scale (MPAS). The MPAS is 19-item self-report questionnaire to measure a mother’s subjective feelings of attachment to her infant . The MPAS will be completed at 3 month and 12-month time points and should take approximately 10-15 minutes to complete.
Parent-completed questionnaires about the child
At birth, 3 and 12 months the following questionnaires are administered:
1. The Vineland adaptive behaviour scale - Third Edition (VABS-3) is an assessment of the child’s adaptive functioning . It assesses 4 domains: communication, daily living, socialisation and motor skills. The VABS takes approximately 15-20 minutes to complete and is administered through Q Global web-based platform for test administration (Pearson Clinical Assessment, Sydney, NSW, Australia).
2. Sensory Profile-2 (SP-2 questionnaire) : an assessment of the child’s sensory processing patterns to understand how they may be impacting their participation in home, school and community-based activities. It takes approximately 5-20 minutes to complete and is again administered through the Q global web-based platform (Pearson Clinical Assessment, Sydney, NSW, Australia).
At 2 and 3 years of age the VABS-3 and SP-2 will be administered as well as the Child Behaviour Checklist (CBCL) and the Repetitive Behavior Scale-Revised (RBS-R).
The CBCL: Preschool Version assesses specific kinds of behavioural, emotional and social difficulties that can be experienced by pre-school and school-aged children . The questionnaire takes approximately 10-20 minutes to complete.
The RBS-R is a 43 item questionnaire that assesses presence and severity of stereotyped behaviour, self-injurious behaviour, compulsive behaviour, routine behaviour, sameness behaviour, and restricted behaviours, which are associated with autism . The questionnaire takes approximately 5-15 minutes to complete and will be administered through the Q Global web-based platform (Pearson Clinical Assessment, Sydney, NSW, Australia).
At 4 years of age, the VABS-3, SP2, CBCL and RBS-R will be administered as well as the Children’s Communication Checklist – Second Edition (CCC-2). The CCC-2 screens children who are likely to have communication difficulties and pragmatic language impairments . The questionnaire takes approximately 5-15 min to complete.
At 5 years of age, the VABS-3, SP2, CBCL, RBS-R and CCC-2 will be administered as well as the Behaviour Rating Inventory of Executive Functioning (BRIEF) (child version). The BRIEF assesses aspects of executive functioning as observed in the home environment
At 10 and 15 years of age, the VABS-3, SP2, CBCL, RBS-R, CCC-2 and BRIEF (child version) will be administered as well as the Connors 3rd Edition-Parent assessment of Attention Deficit / Hyperactivity disorder . The Connors assessment is commonly used to assess for ADHD and its common comorbidities in children aged 6 to 18 years.
Study-specific data collected from the infant
Birth time point: The following information will be collected at birth (or within gestational ages 40-44 weeks):
1. Anthropometry: weight, length, and head circumference.
2. Hammersmith Neonatal Neurological Examination (HNNE). The HNNE is a 34-item examination assessing tone, motor patterns, observation of spontaneous movements, reflexes, visual and auditory attention and behaviour . This assessment will be scored by a health professional trained in the administration of the HNNE and takes approximately 10-15 minutes.
3. General movements assessment (GMA). The GMA is used to identify normal writhing, or abnormal cramped synchronised, poor repertoire or chaotic movements . The assessment is scored from a 3-5 minute video of the infant while they are lying on their back in a calm but alert state. This assessment will be scored by a health professional trained in the administration of GM’s.
3 months (corrected age) time point: At 3 months of age (corrected for prematurity) anthropometry (weight, length, head circumference) will be recorded as well as the GMA and the Hammersmith Infant Neurological Examination (HINE). At 3 months of age the GMA is used to assess normal fidgety or absent or abnormal movement. The HINE is a neurological assessment for infants aged between 2 and 24 months. The assessment includes a neurological examination which is scored, developmental milestones and behaviour (which are not scored) . The neurological examination consists of 26 items from 5 domains, including cranial nerve function, posture, quality and quantity of movements, muscle tone, and reflexes and reactions. The GMA and HINE at 3 months will be scored by a health professional trained to administer these assessments.
12 months (corrected age) time point: At 12 months of age, anthropometric data are collected. In addition, the following scales are administered by a trained allied health professional:
1. The Bayley’s Scale of Infant and Toddler Development Fourth Edition (BSID IV), which is a test of development quotient .
The Ages and Stages Questionnaire (ASQ-3) as well as the ASQ: social and emotional 2. This questionnaire is a developmental screening tool for children aged between one month to 5 1/2 years .
24 months (corrected age) time point: At 24 months of age anthropometric data will be collected and a medical examination for vision, hearing and cerebral palsy is conducted. In addition, the following scales are administered by a trained health professional:
1. Bayley’s Scale of Infant and Toddler Development Fourth Edition (BSID IV).
2. The Autism Diagnostic Observation Schedule-Second Edition (ADOS-2) 
3. Preschool Language Scales-Fourth Edition (PLS-4) , a test for communication skills.
3-year time point: At 3 years of age, the BSID IV, ADOS-2 and PLS-4 will be administered (as above at the 2-year time point).
4-year time point: At 4 years of age, the ADOS-2 and PLS-4 will be administered as well as the Stanford-Binet Intelligence Scale (SBIS)  – intelligence quotient.
5-year time point: At 5 years of age, the ADOS-2 and SBIS will be administered as well as the Clinical Evaluation of Language Fundamentals- Fourth Edition (CELF-4) . The CELF tests for communication and language skills for children 5 years and older.
10 and 15-year time point: At 10 years and at 15 years of age, the ADOS-2, SBIS and CELF-4 will be administered.
Optional biospecimen collection
Maternal biospecimen collection: For mothers who consent to biospecimen sample collection we will access their bio-banked samples collected during their infectious period. Blood samples and nasal mucosa will be assessed for viral load and inflammatory and cytokine marker analysis. If the mother has recovered prior to study participation biospecimens, including blood, saliva and buccal swabs, will be collected upon first visit. Blood samples will be collected by a health professional and assessed for levels of inflammatory markers . Saliva will be collected to assess levels of cortisol . Saliva samples are collected by the participant as soon as they wake, on the morning of their first assessment, in order to capture the waking cortisol response. Buccal swabs will be collected by a health professional and DNA will be extracted for epigenetic analysis.
Infant biospecimen collection: Parents may consent to provide a buccal swab sample from the infant. Biospecimens will be collected by a health professional at birth (or near the expected due date if born preterm). DNA will be extracted from buccal swabs for epigenetic analysis. At the time of birth, mothers who have a caesarean birth will also be given the option to consent to the collection of the umbilical cord blood and placental tissue. In cord blood and placental tissue, we will assess viral load, inflammatory and cytokine markers, mitochondrial function, and indices of mitochondrial structure and function. Additionally, placental morphology will be assessed using routine histopathological methods. Umbilical cord blood and stem and progenitor cell composition will be determined using flow cytometry.
STATISTICAL ANALYSIS AND POWER CALCULATIONS
The data collected at each assessment will be compared between SARS-CoV-2 exposed and control groups longitudinally using a separate linear mixed effects analysis for each outcome measure. Given the number and frequency of measures there are likely to be missing datapoints, thus a mixed modelling approach will avoid the need for listwise deletion of incomplete data. Sociodemographic and clinical characteristics will be compared between groups using t-tests, Mann-Whitney U tests, or Chi-square tests as appropriate. Machine learning approaches will be used to determine risk profiles based on demographic and biological data.
Power analysis using G*Power for an ANOVA repeated measure, between factor approach (similar to our proposed analysis) with 2 groups and 4 measures (child assessments at birth, 3 months, 1, and 2 years) suggests a sample size of 132 is required to have 95% power to detect a medium effect size of Cohen’s f = 0.25. Therefore, we aim to recruit a minimum of 150 mother-infant dyads exposed to SARS-CoV-2 and 300 matched controls.
DATA MANAGEMENT PLAN
Biospecimens will be stored and analysed in the laboratories at Monash Health, Monash Medical Centre, Monash University Clayton. Samples collected at Sunshine Hospital or at the Royal Women’s hospital will be stored short-term at these facilities before being transferred as a cohort to Monash Health (Behavioural neuroscience laboratory, Monash University). Samples collected will be de-identified at the time of collection and allocated a study code. This means that any information which could identify the participant, such as name, address, date of birth and hospital record number will be removed before the specimen is sent to the laboratory for analysis. We expect that all the blood, saliva and buccal swabs that we collect will be used for laboratory analysis. However, after the laboratory work has been completed, if there is any sample left over, it will either be stored at MMC or discarded depending on the consent completed by the participant. Here we will give the participant the option (tick box) to either consent to immediate use, then any left over to be discarded, or to long-term storage of the samples for future unspecified use related to the study.
Maternal demographics and questionnaires will be stored in a password protected file, or in a locked cabinet held at Monash Medical Centre. Demographic and questionnaire data will be de-identified at the time of collection and allocated a study code. This means that any information which could identify the participant, such as name, address, date of birth and hospital record number will be removed prior to analysis. Data may only be accessed by researchers listed on the proposal.
Child developmental outcomes will be stored in a password protected file, or in a locked cabinet held at Monash Medical Centre. All assessment data will be de-identified at the time of collection and allocated a study code. This means that any information which could identify the participant, such as name, address, date of birth and hospital record number will be removed prior to analysis. Data may only be accessed by researchers listed on the proposal.
Plans for return of results of research to participants
We will generate a short summary report in lay terms following each assessment displayed as a ‘strengths and difficulties’ framework. Scores will not be shared with the parents/caregivers as assessment scores may be misinterpreted. We will ask for parent/caregivers’ permission to share data with professionals on an ‘as requested’ basis – as required for health, disability and/or education purposes.