From CRONOS to CRONOS-Kids: Investigating Neurological Development in Toddlers After Maternal SARS-CoV-2 Infection During Pregnancy – A Study Protocol For A Prospective Cohort Study

Abstract

Introduction

Infection with SARS-CoV-2 during pregnancy increases the risk for adverse events in mothers and newborns. Research is increasingly exploring potential consequences of intrauterine exposure to the developing nervous system. However, there is still a lack of high-quality studies on long-term outcomes. The CRONOS-Kids study aims to investigate the global development of children after exposure to SARS-CoV-2 in utero in comparison to an unexposed control group. This paper outlines the CRONOS-Kids study protocol.

Methods

The study is designed as a prospective population-based cohort study and will recruit children aged 36–60 months at the time of assessment. The exposed cohort will be recruited from the German CRONOS registry and will include only children who have been exposed to the Alpha and Delta variants of SARS-CoV-2. The unexposed cohort will be recruited by local pediatricians and via social media. For clinical evaluations, the Movement Assessment Battery for Children, 2 ^nd Edition will be used. Parental self-reports on development will use the Ages and Stages Questionnaire, 3 ^rd Edition. Additional questionnaires cover information about maternal and child health, vaccinations, healthcare usage, media consumption, and maternal quality of life. Until April 2026, we aim to recruit 125 exposed and 125 unexposed children across five study sites.

Discussion

The CRONOS-Kids study will explore the impact of intrauterine SARS-CoV-2 exposure to specific virus variants on long-term neurologic outcomes in children as well as broader consequences of the pandemic.

Introduction

SARS-CoV-2 infection during pregnancy has been shown to increase the risk of severe maternal COVID-19, stillbirth, and preterm delivery ^{1 2 3 4 5 6} . Subsequent neonatal SARS-CoV-2 infection is rather rare, typically presenting asymptomatically and mostly acquired during the postnatal period ^{7 8 9} .

Although predominantly affecting the pulmonary system, SARS-CoV-2 is a virus with multisystemic symptoms, including neurological manifestations. Neurological symptoms are often nonspecific, such as lethargy or hyperexcitability in infants and headache and myalgia in older children. Other symptoms, such as encephalitis or seizures, occur mainly in severely ill or already neurologically compromised children ^{10 11 12 13} .

Several studies have discussed the potential effects of SARS-CoV-2 on brain development, particularly in cases of intrauterine exposure ^{14 15 16} . Potential mechanisms by which the virus could affect infant development are either a direct impact of the virus due to transplacental SARS-CoV-2 transmission through maternal and secondary fetal immune activation, or in the context of virus-induced pregnancy complications such as premature births ^{17 18} .

Studies investigating intrauterine SARS-CoV-2 exposure have produced equivocal results. Several have demonstrated the presence of neurological abnormalities in newborns and infants following in utero exposure to SARS-CoV-2 or postnatal SARS-CoV-2 infection ^{14 19 20 21 22} . Other studies have not identified a clear association ^{23 24 25} . These controversial results could be explained by variability in sample size and study quality. Furthermore, details of the control group have been documented inconsistently ^{26 27 28} . Most prospective investigations were based on parental questionnaires and reported concerns about the infant’s neurodevelopment ^{21 22 25 29} . Standardized clinical assessments have been carried out rarely, and if so, solely on infants ¹⁴ . To date, only three studies report on outcomes in older children, using parental self-reporting in 24-month-old children ^{25 30 31} .

Previously, no study has distinguished between the intrauterine exposures of different SARS-CoV-2 variants of concern (VOC). In contrast to the wild-type and the Omicron variant, infections with the Alpha and Delta variants have been associated with a higher rate of placentitis and a concomitant increased risk of stillbirth and preterm delivery ^{32 33 34} , thus possibly impacting fetal and neonatal neurodevelopment to a greater degree than the other variants.

As there is a need for screening for long-term neurodevelopmental deficits of children who were exposed to SARS-CoV-2 prenatally, we have established this multisite national initiative based on the COVID-19 Related Obstetric and Neonatal Outcome Study (CRONOS) ^{35 36} . We hypothesize that prenatal exposure to the Alpha and Delta variants increases the risk for neurodevelopmental disorders in terms of changes in motor development, social-emotional development, problem-solving, and communication skills in toddlers and preschoolers. In addition, we assess the same parameters of neurodevelopment in an unexposed control group, thereby also investigating collateral effects of the pandemic on mothers and their offspring.

The present study aims to investigate the neurodevelopmental effects of intrauterine SARS-CoV-2 exposure in children aged 36 to 60 months and to compare the results to an unexposed age-matched control group born in the same time frame.

The primary outcome measure is

• Clinical assessment of motor skills using the Movement Assessment Battery for Children, 2 ^nd Edition (M-ABC-2).

Secondary outcome measures are

• Clinical assessment of motor skills using the Movement Assessment Battery for Children, 2 ^nd Edition (M-ABC-2).

• Screening for global development in terms of communication, problem-solving, and social-emotional skills using the Ages and Stages Questionnaire, 3 ^rd Edition (ASQ-3);

• Timing of vaccinations and preventive medical examinations; and

• Healthcare system usage in the first year of life.

Methods

Study design and participants

The study is designed as a prospective population-based cohort study, with participants recruited from the ages of 36 to 60 months at the time of assessment. The identification of exposed children will be conducted through the CRONOS registry. A control group will be recruited through local pediatric practices and targeted outreach via social media platforms (Instagram, LinkedIn, and WhatsApp). Data collection will comprise the administration of questionnaires, including parental self-report measures, in addition to clinical assessments conducted by researchers who are unaware of the children's exposure status. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist will guide reporting ^{37 38} .

Setting

CRONOS (COVID-19 Related Obstetric and Neonatal Outcome Study) was a prospective German registry including women with SARS-CoV-2 infection during their pregnancy, with 130 obstetric hospitals actively participating, comprising one-third of all German births ³⁶ . Between April 2020 and February 2023, more than 8500 women and their newborns were included ⁷ . Maternal SARS-CoV-2 infection was defined as a detection of viral RNA by PCR testing of a nasopharyngeal or oropharyngeal swab, regardless of symptoms or symptom severity, or detection of SARS-CoV-2 antibodies in an antibody-based serum assay in symptomatic women. In the case of PCR testing, some hospitals differentiated between variants of concern (VOC). If this information was not available, the most likely VOC was assigned to the case depending on the time of infection, with the wild-type virus being predominant until February 2021, Alpha from March 2021 to July 2021, Delta from August 2021 to December 2021, and Omicron since January 2022 ³⁹ .

In addition to the CRONOS-Kids principal investigator's research team, four university hospitals were selected as consortium partners based on their previous recruitment rates in the CRONOS registry and the number of eligible patients.

Sampling and recruitment

Exposure cohort

As the objective of the study is to assess the possible impact of the Alpha and Delta variants on the neurodevelopment of the offspring, families are eligible for study inclusion when the pregnant woman was infected between 1 March 2021 and 31 December 2021, or when infection with Alpha or Delta was confirmed via PCR test, regardless of the timing of infection. Pregnant women are required to have agreed during initial enrollment in CRONOS to be re-contacted. After identifying eligible women by the CRONOS study center, families are contacted by the local study sites and invited to participate in the CRONOS-Kids study.

Unexposed control group

A cohort of children without known or documented intrauterine exposure to SARS-CoV-2 is recruited using probability sampling. Local pediatricians in outpatient practices are invited to provide study information to families during routine preventive medical exams, which are mandatory for four-year-old children in Germany. Additional recruitment efforts include the dissemination of study materials through local kindergartens and nurseries as well as via personal networks. To enhance public engagement, a dedicated Instagram account featuring a study mascot has been established to share information about the study with a broader audience.

A potential SARS-CoV-2 infection in childhood will be recorded in both groups and considered in secondary analyses.

Written consent is obtained from all participants.

Recruitment

Recruitment started on 1 April 2025 and will continue through April 2026. Families interested in participating are invited to contact the CRONOS-Kids coordinating team via an official study website to schedule an appointment at one of the five study sites. Pseudonymisation is carried out centrally by the coordination team using an eight-digit code. The research team conducting developmental assessments remains blinded to the exposure status of the children, as they do not have access to the pseudonymization key. Parents accompanying the children are informed of the blinding procedure and asked not to disclose any information regarding SARS-CoV-2 infection during pregnancy.

We aim to recruit at least 125 children who were exposed in utero and 125 unexposed children.

Sample size calculation

We calculated that 64 patients per group are needed, i. e., 128 patients in total, to detect a difference of 0.5 standard deviation in an unpaired t-test in mean M-ABC-2 total scores (Cohen’s d=0.5), with an alpha level of 0.05 and a power of 0.8 ⁴⁰ .

Exclusion criteria

Children with major congenital anomalies, regardless of intrauterine infection status, are excluded from analysis. Children are eligible for assessment regardless of whether they are preterm or not singletons.

Study instruments

Primary outcome

The primary objective of this study is to clinically assess the motor skills of all participating children. For this, the M-ABC-2 will be used ⁴¹ . It consists of three movement domains: manual dexterity (MD), aiming and catching (AC), and balance (B), with two (AC) or three (MD and B) different tasks each. There are three age bands available, the first (3 to 6 years) being used in this study. The M-ABC-2 was chosen because of its validation in Western European children, including German children, the availability of a German translation, an appropriate age range, internal reliability (r=.97), and short administration time (20 to 30 minutes). We will use the total score as the primary outcome. The total score ranges from 14 to≥108 (total scores above 108 points do not result in further improvement of the percentile rank).

Rater training was provided prior to the start of the study, based on a previously published rater training program for the assessment of observations ⁴² using a four-step approach. 1) Conveying expertise: Expertise in child development was provided by shadowing pediatricians in outpatient care and by accompanying psychologists and physiotherapists in developmental assessments. 2) Self-study: The research team familiarized themselves with the assessment tool and the theoretical aspects of rater error. 3.) First assessment with experts: The research team shadowed experts as they performed two clinical assessments using the M-ABC-2 and simultaneously scored the results with the experts. 4) Pilot testing: Two clinical assessments and consecutive scoring were conducted by the research team under the supervision of experts. All consecutive tests are performed by the same research team to ensure a high interrater reliability.

Secondary outcome

To screen global development, the age-appropriate questionnaires of the ASQ-3 will be used. Each questionnaire contains five domains: communication, gross motor, fine motor, problem-solving, and personal-social development, with six questions each. Every question can be rated according to the child’s abilities with “yes”, “sometimes”, and “not yet”, scored 10, 5, or 0 points, respectively. Each domain is analyzed separately with total scores ranging from 0 to 60. Age-specific thresholds are defined and validated, indicating potentially increased risk for neurodevelopmental delay ⁴³ .

For screening social-emotional skills, the parent questionnaire for supplementary developmental assessment (EEE – “Elternfragebogen zur ergänzenden Entwicklungsbeurteilung”) will be used ⁴⁴ . The EEE can be used in preventive medical examinations (PMEs) that are mandatory in Germany at different ages of a child ⁴⁵ . They compile specific sets of social-emotional and practical life skills that are expected to be acquired at that age. Each EEE comprises a different number of questions, ranging from 27 (10 to 12 months) to 35 (60 to 64 months), which can be rated with “yes” or “no”.

A web-based general questionnaire was developed on REDCap (Research Electronic Data Capture) ^{46 47} , comprising sections on current and previous pregnancies, birth, child health, postnatal SARS-CoV-2 infection, the child’s media consumption, the maternal quality of life, socioeconomic status, and social desirability. Questions on pregnancy and birth focus on potential confounders of child development, such as pre-existing maternal illness, smoking, drug use, and preterm birth. Questions on child health include postnatal complications, feeding practices, and health care visits. Socioeconomic status is measured using a multidimensional index score consisting of three metric components: education and occupational qualification, occupational status, and net income ⁴⁸ . The German version of the EuroQoL quality of life questionnaire (EQ-5D-5L) is used to evaluate the perceived quality of life at the time of the analysis ⁴⁹ . To assess potential confounding in self-reported outcomes, we included questions on social desirability using the SDS-CM scale, consisting of 23 items ⁵⁰ .

Timing of vaccinations and PMEs will be extracted from official vaccination certificates and specific PME documents (so-called “U-Heft”) and entered into a designated web-based database.

Data collection

Data will be collected solely by one research team on all study sites, thus ensuring high internal consistency.

ASQ-3 and EEE questionnaires are sent to parents at least one week before their clinical appointments, thus allowing time for self-assessing their children.

At the time of clinical assessment, M-ABC-2 will be performed by the research team, blinded to the exposure status of the children. ASQ-3 and EEE questionnaires are collected and checked for completeness. The general questionnaire will be completed during the clinical assessment using a dedicated CRONOS-Kids study tablet (Microsoft Surface 2.0). Data on vaccination and PMEs are also collected on this study tablet.

Bias

A potential sampling bias is anticipated, as families with higher educational levels and German language proficiency may be more likely to participate in the study. However, since this tendency is expected in both the exposed and unexposed groups, and detailed information on socioeconomic status is being collected, potential confounding will be addressed through statistical adjustment.

To minimize performance and observer bias, the research team conducting the assessments is separate from the coordination team responsible for recruitment and appointment logistics. Furthermore, accompanying parents are instructed not to reveal any information regarding SARS-CoV-2 infection during pregnancy to ensure assessor blinding.

Ethics approval and registration

The study was approved by ethics committees at all local study sites. Research will be conducted in accordance with the Declaration of Helsinki.

The study was registered at clinicaltrials.gov in April 2025.

Statistical analysis

The research team will be responsible for data acquisition and validation and conducting the statistical analysis.

The cohort’s baseline sociodemographic and clinical characteristics will be compared between the exposure and unexposed control cohorts using appropriate tests depending on variable categories: chi-square tests will be used for categorical variables, and t-tests for continuous variables. There will be no adjustments for study sites.

Logistic regression models will be employed to examine the exposure status and total scores in M-ABC-2, ASQ-3, and the EEE. Adjusting for potential confounders is planned for maternal age, social status, parental education, children's sex, and gestational age of delivery. Further analysis on ASQ-3 and EEE-scores will include social desirability measured by the SDS-CM scale as a potential confounder. An exploratory subgroup analysis will be performed for the trimester of infection, infection severity, maternal quality of life, and duration of breastfeeding. The timing of vaccinations and PMEs will be analyzed descriptively as well as the healthcare system usage in the first year of life.

We will summarize the amount of missing data in each of the variables for both cohorts separately. Cohorts will be compared using complete cases for the specific outcome parameter. Based on the proposed data collection, we expect the proportion of missing data to be small. In case of a high amount of missing data, we may consider multiple imputation as a sensitivity analysis.

P values<0.05 will be considered statistically significant. For all analyses, the statistical software SPSS (IBM Corp., Armonk, NY, USA) will be used.

Discussion

To our knowledge, this is the first prospective cohort study to clinically assess neurodevelopment in 3 to 5-year-old children after intrauterine exposure to the Alpha or Delta variant of SARS-CoV-2. We will conduct a multicenter cohort study based on the CRONOS registry using a carefully designed set of study instruments developed within the CRONOS-Kids study group, incorporating two internationally recognized and validated tools. Furthermore, the exposed children will be compared to a control group of unexposed children of the same age, controlling for potential confounding parameters.

Clinical standardized assessments have been rarely used in previous studies investigating the effects of intrauterine exposure to SARS-CoV-2 on children, especially not in children older than 12 months ¹⁴ . These will be conducted by specifically trained researchers in this study to minimize the possibility of confounding bias. Furthermore, we will systematically examine social desirability, a potential confounding factor in parental assessments of children's development.

We will exclude children who were exposed to the wild-type or Omicron variant in utero. Previous research on intrauterine exposure to SARS-CoV-2 has yielded inconsistent findings, potentially due to the failure to distinguish between different viral variants. Given that the Alpha and Delta variants have been associated with an elevated risk of stillbirth, preterm birth, placentitis, and neonatal SARS-CoV-2 infection ^{32 33 34} , their impact on fetal neurodevelopment may be more pronounced than that of the wild-type or Omicron variant. Moreover, since neurodevelopmental delay is generally uncommon, aggregating data from multiple virus variants may obscure potential differences in their effects.

Conclusion

The findings of this study will contribute to a more comprehensive understanding of the long-term postnatal effects of SARS-CoV-2 infection during pregnancy, as well as the broader consequences of the pandemic, including social isolation or reduced access to healthcare. A better understanding of these factors could improve care for children exposed to the virus in utero. Insights gained from the long-term effects of SARS-CoV-2 infection and pandemic disruptions could help to prepare for future crises, ensuring better maternal and child health.

Data availability statement

The original dataset will not be made available due to ethical reasons.