Childhood outcomes of fetal genomic copy number variants: the prenatal microarray cohort study

Childhood outcomes of fetal genomic copy number variants: the prenatal microarray cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Childhood outcomes of fetal genomic copy number variants: the prenatal microarray cohort study Jacqui McCoy, Cecilia Pynaker, Sharon Lewis, David Amor, Fiona Norris, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6952328/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose The long-term developmental outcomes of children with a prenatal diagnosis of a copy number variant of uncertain significance (VUS) remain unclear. This study compared the developmental, social-emotional, and health outcomes of children with and without a prenatal VUS, assessed maternal perceptions of their child’s health and development, and examined the reclassification rate of VUS after more than two years. Materials and Methods Women who underwent prenatal chromosomal microarray testing in Victoria, Australia (2013–2019), were recruited retrospectively (2021–2023). Children with a VUS (cases) were compared with controls without a VUS. Outcomes, including cognitive, developmental, and health measures, were assessed and children had an average age of six years. Statistical analyses compared group outcomes and adjusted for maternal sociodemographic factors. Results The study included 134 mother-child pairs (46 with a VUS and 88 controls). No significant differences were found between groups in intellectual functioning, adaptive behaviour, or social-emotional measures. Maternal perceptions of their child and family well-being were also similar. Reanalysis reclassified 66.0% of VUS as benign and 8.5% as pathogenic. Conclusions Children with a prenatal VUS diagnosis have developmental outcomes and family well-being comparable to those without. These findings contribute valuable evidence to support prenatal genetic counselling and clinical laboratory reporting practices. Observational study registration: ACTRN12620000446965p; Registered on April 6, 2020. Obstetrics & Gynecology Pediatrics Medical Genetics prenatal diagnosis chromosomal microarray analysis copy number variants variant of uncertain significance cognitive function cohort studies paediatric population Figures Figure 1 Introduction Over the past decade, advances in genomics have revolutionised reproductive health and prenatal diagnosis ( 1 ). Chromosomal microarray (CMA) has been the gold standard first line investigation for fetal structural anomalies since 2012 ( 2 ), and is commonly performed for all indications for prenatal diagnosis ( 3 , 4 ). While CMA have enhanced the detection of pathogenic copy number variants (CNVs), they also identify variants of uncertain significance (VUS) ( 5 ). VUS can pose challenges for genetic counselling and decision-making in the prenatal setting ( 6 , 7 ) as they may be associated with an increased chance of developmental disabilities or neuropsychiatric conditions ( 8 ). Unlike the postnatal context, where uncertain genomic findings can be correlated with the child’s phenotype, the disclosure of fetal VUS during pregnancy can introduce significant uncertainty, increasing anxiety and raising ethical dilemmas regarding pregnancy management ( 9 – 11 ). Prenatal genetic counselling for VUS is further complicated by the systemic bias in the literature. Data on CNVs are mostly derived from symptomatic children, skewing the evidence base towards more severe phenotypes that may not be applicable in the prenatal context ( 8 , 12 – 14 ). Only two studies have investigated the outcomes of children born after a prenatal diagnosis of a CNV and neither examine health and development of children diagnosed with a VUS beyond 4 years ( 15 , 16 ). The paucity of comprehensive and long-term outcome data for children with a prenatally diagnosed VUS significantly impedes genetic counselling of expectant couples. We aimed to recruit a cohort of children, with and without a prenatal VUS, to (i) compare the developmental, social-emotional and health status of children with and without a prenatal diagnosis of a VUS; (ii) measure the impact of the CMA results on maternal perceptions of child health, behaviour, and development; and (iii) determine the proportion of prenatal VUS reclassified to benign or pathogenic on reanalysis > 2 years later. Methods Study design We established a population-based cohort of mother-child pairs who had undergone prenatal testing with a CMA in Victoria, Australia between 2013–2019 inclusive. The full study protocol has been published elsewhere ( 15 ). Ethical and governance approval was granted by the Royal Children’s Hospital Human Research Ethics Committee (RCH HREC #60542) and the Mercy Health Human Research Ethics Committee (HREC #2020-046). The study was funded by the National Health and Medical Research Council (#1186862). Recruitment Potential participants were identified from the Victorian Prenatal Diagnosis Database, a statewide research database of all prenatal chromosome testing results ( 16 ). During the study period, chromosome microarrays were used in > 85% of prenatal diagnostic tests, including fetuses with and without structural anomalies ( 17 ). We screened potential cases from singleton pregnancies with a CNV reported on a prenatal microarray result. We defined cases as those with a VUS, inclusive of all indications for prenatal diagnosis including fetal ultrasound abnormality. Controls were singleton pregnancies that had no CNV (i.e., no clinically significant genomic imbalance detected) detected on a prenatal microarray, and no fetal ultrasound abnormality. Medical records of potential cases and controls were screened for full eligibility criteria. Individuals were eligible if they: (i) had a prenatal CMA result reported for a singleton pregnancy between January 2013 and December 2019, (ii) were ≥ 18 years old at enrolment, (iii) could provide informed consent in English, (iv) had a live birth outcome, (v) were the primary caregiver at hospital discharge, (vi) were able to consent on behalf of themselves and their child, (vii) were able to complete questionnaires and attend study assessments, and (viii) lived in Victoria. Participant recruitment and data collection occurred between November 2021 and December 2023. Full details of the recruitment process, perinatal outcomes and participant response rate has been published elsewhere ( 18 ). In brief, 1832 medical records were screened and 1364 (74.5%) mother–child pairs met inclusion criteria. Of the 468 that did not meet inclusion criteria, 282 (60.3%) had ‘no live pregnancy outcome’ (209 terminations of pregnancy (TOP), 73 miscarriages, stillbirths, and infant deaths), 157 (33.5%) required a translator, and 29 (6.2%) had other exclusion criteria). Approximately 77% of study invitation letters were successfully delivered by registered mail (1047/1364). The final recruitment rate was 19.2% (201/1047). All birth parents participating in this study identified as women/mothers, hence these terms are used throughout. Outcome Measures Questionnaires Demographic and health data were gathered at baseline using validated questionnaires. Mother-focussed measures included: the Patient Health Questionnaire ( 19 ), the State-Trait Anxiety Inventory ( 20 ), the Parenting Sense of Competence Scale ( 21 ), the McMaster Family Functioning Subscale ( 22 ), the Revised Scale for Ambiguity Tolerance ( 23 ), the Decision Satisfaction Scale ( 24 , 25 ), the Health Literacy Screening Questions ( 26 ), the Disclosure of Results to Others ( 27 ), the University of North Carolina Genomic Knowledge Scale ( 28 ) and two questions from the Genetics Essentialism scale ( 29 , 30 ) (See Child-focussed measures included: the Strengths and Difficulties Questionnaire ( 31 ), the Vulnerable Child Scale ( 32 ), and the Children with Special Health Care Needs Screener ( 33 ). A description of each questionnaire is available in Supplementary Table 1. Children aged two years and six months (2:6) or older were invited to participate in additional clinical assessments including validated cognitive, behavioural, social, and developmental assessments. These were conducted in person by psychologists and paediatricians Cognitive functioning Age-appropriate Wechsler scales were used to assess intellectual functioning: the Wechsler Preschool & Primary Scale of Intelligence (WPPSI-IV) for children under 7 years and 7 months and the Wechsler Intelligence Scale for Children (WISC-V) for older children. Scores were classified according to test manual norms ( 34 , 35 ). Cognitive assessments for metropolitan participants were conducted in person at The Royal Children’s Hospital (RCH), while regional participants were assessed at local health services. Paediatricians and psychologists conducting assessments were initially blinded to the children's CMA results. However, some parents spontaneously disclosed their experiences during assessments, despite being advised that assessing staff should remain blinded. Standardised assessments were consistently scored and either verified by a second team member or double-scored by the assessor. Behaviour and social functioning The Vineland Adaptive Behaviour Scales – Third Edition, Parent/Caregiver form (Vineland-3) assessed adaptive functioning across five domains: Communication, Socialization, Motor Skills, Daily Living Skills, and Maladaptive Behaviours (for children older than 3). Higher scores indicate better adaptive functioning ( 36 ). The Social Responsiveness Scale – Second Edition, Preschool/School Age forms (SRS-2) assessed social behaviours linked to autism spectrum disorder (ASD). Standardised scores categorised behaviours from normal to severe in relation to ASD symptoms ( 37 ). Child development A paediatric review with the mother and child collected data on the child’s developmental and medical history, including growth, cognitive, behavioural, and neurological functioning, as well as any medical concerns using a standardised data collection form. Online paediatric assessments were available during COVID-19 pandemic restrictions in 2021–2022. CNV reanalysis The prenatal VUS classifications were reanalysed by the original reporting laboratory. The scientists were blinded to the original classification and outcomes of the research assessments but were provided the indication for prenatal diagnostic testing. Refer to Supplementary Methods for more detail. Statistical methods Data were analysed using Stata Version 18 ( 38 ). Descriptive statistics included counts, percentages, means, standard deviations, and medians with interquartile ranges (IQR) for non-normally distributed data. Continuous outcomes were analysed using t-tests and multivariable linear regression for normally distributed data, while the Mann-Whitney test was used for non-normally distributed data. Categorical outcomes were analysed using the chi-square test. A significance threshold of p < 0.01 accounted for multiple comparisons. Scores from the two age-specific versions of the Strengths and Difficulties Questionnaire (for ages 2:0–3:11, and 4:0–10:0 years) were converted to z-scores and merged for further analysis. Sample sizes vary by measure due to differing participant completion rates across outcome measures. Results From 1047 eligible mothers that were contacted, 218 (20.8%) consented to participate in the study (Fig. 1). Fifty-nine participants were excluded after recruitment due to additional information obtained from the medical record, usually a finding of an ultrasound anomaly in the control group. The final analysis included 134 mother-child pairs, comprising 46 cases with a VUS and 88 controls (Fig. 1). Participant characteristics A higher proportion of mothers in the control group were aged over 35 years at the time of giving birth compared to mothers in the VUS group (75.5%, versus 48.3% respectively, p = 0.02). The VUS group also had a significantly higher proportion of regionally based participants compared to the control group (39.1% versus 19.3%, p = 0.01), due to study recruitment logistics (no controls were recruited from regional areas due to resource allocation). There were no other demographic differences between the VUS case and control cohorts, including those who completed only the online assessments (survey cohort) and those who opted for additional child assessments (clinical cohort, see Table 1). The most common indication for prenatal diagnosis among the pregnancies with a VUS was an ultrasound abnormality (56.6%, 26/46) (see Supplementary Table 2 and Supplementary Table 3 for details of the ultrasound abnormalities and perinatal outcomes). The most common indication for prenatal diagnosis among the controls was an increased probability of aneuploidy on the first trimester combined screen (48.3%, 42/88). Overall, 74.4% of the VUS cases were either maternally or paternally inherited. Perinatal outcomes were compared between the VUS and control groups: there were no significant differences in sex ratio, head circumference, small for gestational age, mode of birth or breast-feeding rates. The VUS group had significantly higher rates of special care nursery or neonatal intensive care admission, and higher rates of hospital readmission within 28 days compared with controls (Supplementary Table 2). Developmental, social-emotional and health outcomes Cognitive functioning General intellectual functioning was formally assessed in 80 out of 134 (60%) children with and without a VUS. The children’s average age at assessment was approximately 5 years and 11 months (range 2 years and 7 months to 9 years and 2 months). There was no significant difference in Full-Scale Intelligence Quotients (FSIQ) between children with a VUS (n = 30, mean 96.3, SD = 14.1) and those without (n = 50, mean 101.5, SD = 16.0; p = .14, see Table 2). Only two participants scored 2 SDs below the mean on the FSIQ (≤ 70), indicating mild intellectual impairment (one each of VUS case and control). A significant difference was observed on the Verbal Comprehension Index, where children with a VUS (VUS cases; mean = 95.5, SD = 13.8) scored lower than children without (controls; mean = 103.7, SD = 14.6; p = .01). However, this difference became non-significant when accounting for maternal age at the child's birth, maternal country of birth, maternal education level, income, locality (metro/regional), and maternal relationship status (p = 0.08). No statistically significant differences were detected in the other cognitive indices after considering the same covariates (see Table 2 and Supplementary Table 4). Adaptive and social functioning Eighty mothers (80/134; 60%) also completed the Vineland-3 Parent/caregiver form. There were no differences in overall adaptive functioning between VUS cases (mean = 93.1, SD = 13.9) and controls (mean = 92.2, SD = 10.5; p = 0.73). There were no differences observed between cases and controls in the other functional domains of the Vineland-3 (see Table 2). Sixty-eight (51%) mothers completed the SRS-2 questionnaire. There were no differences between VUS cases and controls observed in any of the social functioning scores (see Table 2). Child development A total of 78 (58%) mother-child pairs participated in the clinical review conducted by a study paediatrician. Of these, 26% (7/29 VUS cases and 13/49 controls) were seen in person, while the remainder were reviewed via Telehealth with video or telephone. No significant differences were observed between VUS cases and controls in the number of concerns reported across developmental and medical domains. The two exceptions were in the domains of infection and hearing, with more concerns reported in VUS cases than controls. For infection, specific issues within the VUS cohort included 2 cases of pyelonephritis, 7 cases of recurrent otitis media (≥ 3 episodes), and 3 classified as “other” recurrent types of infections. For hearing, there were 6 instances of conductive hearing loss and 1 case of sensorineural hearing loss reported among the VUS cases. See Supplementary Table 5 for detailed results of the paediatric review. BMI was available for 70 (53%) children (42 controls and 28 VUS cases). There was no evidence of a difference in BMI between the two groups, with 75% (21/28) of VUS cases and 60% (25/42, p = .34) of controls having a weight between the 5th and 85th percentiles standardised for age and sex ( 39 ). Maternal perceptions of child health, behaviour, and development No group differences between maternal responses were found on measures of maternal mental health, perceived parental competence, overall family functioning, maternal perceptions of child behaviour and vulnerability, general tolerance of uncertainty, general genetic knowledge, and decisional satisfaction regarding the choice to undergo microarray testing during pregnancy (see Supplementary Table 6). There were no significant differences between the VUS and control groups in the proportion of children who were noted to receive at least one form of additional support including medication, increased health or educational services, functional limitations, specialised therapies, or additional assistance for emotional, behavioural, or developmental issues (27% VUS vs. 20% controls, p = 0.60). Health literacy was also similar, with 74% of VUS mothers and 77% of control mothers reporting no difficulty understanding written medical information, and 91% (VUS cases = 42; controls = 79) in both groups reporting high levels of confidence in completing medical forms independently. Most mothers in both groups disagreed that their child was different from most people (VUS cases: 76% (32/42), controls: 81% (66/81)). Similarly, 72% (31/42) of mothers in the VUS group and 80% (65/81) of control mothers disagreed that their child's genetic makeup made them different. No significant differences were found between the groups on these measures. Chromosomal microarray reanalysis After excluding three participants who withdrew from the study, 56 mothers consented to having their child’s prenatal CNV result reanalysed. An average period of 5.5 years had elapsed since the original prenatal microarray report. Of the 47 participants with a prenatal VUS, 31 (66%) were downgraded to "no clinically significant genomic imbalance detected", four (9%) were upgraded to "pathogenic copy number change with variable expressivity", and 12 (26%) remained unchanged as a VUS. Table 3 provides the details of the reanalysis of VUS. The majority of VUS were inherited variants, accounting for 72% (34/47), while 23.4% (11/47) were undetermined (meaning the parents were not tested, so this could not be evaluated), and 4.3% (2/47) were de novo variants (see Table 3). We conducted a post hoc analysis comparing clinical assessment outcomes for children whose VUS results are no longer reported (n = 19) with the controls (n = 50) and found no significant differences in intellectual functioning, adaptive functioning, or social communication skills associated with autism spectrum disorder (see Supplementary Table 7). Similarly, when comparing these outcomes for children whose VUS results are still reported or have been reclassified as pathogenic with variable expressivity (n = 11) to the controls (n = 50), no differences were observed (see Supplementary Table 8). Discussion We conducted a comprehensive evaluation of the developmental, social-emotional, and health outcomes of children with and without a prenatal diagnosis of a VUS at a mean age of 6 years. The results are reassuring, demonstrating that children with a VUS performed similarly to children without a VUS across a wide range of developmental, cognitive, and health measures. Our study found no significant differences in maternal perceptions of child health, behaviour, or development between the VUS and control groups. A key concern with genomic testing and disclosure of uncertain findings is the potential for increased anxiety, which may negatively impact parenting styles and the mother-child relationship ( 40 ). However, mothers in our VUS and control groups reported similar levels of mental health, parental competence, and family functioning. Taken together, these findings suggest no enduring impacts through early childhood of a prenatal diagnosis of a VUS on either child health and development or aspects of family well-being. Our research makes a major contribution to the limited evidence base for this population. Only two other studies have followed up large cohorts with a prenatal diagnosis of a CNV. The Belgian prenatal microarray cohort study used the Ages and Stages Questionnaire to follow up children who had a prenatal diagnosis of a CNV (85 cases, 123 controls). Compared with controls, they identified significant delays in communication and personal-social development among three-year-olds with a susceptibility CNV. However, this study specifically excluded children with a VUS from further analyses, limiting any direct comparisons with this study ( 41 ). More recently, Shi et al. followed 113 infants with a prenatal VUS, conducting follow-up at 2 time points: 0–10 months and 2–4 years of age ( 42 ). They reported 5 with potential disease phenotypes, but the clinical assessments were not described, participant attrition rate was high, and there was no comparator group, thus precluding any firm conclusions about the childhood outcomes for this sample. In contrast, our cohort has been assessed with a broad range of structured, validated tools involving a variety of data sources: in-person assessment of cognitive function by a psychologist, clinical assessment by a paediatrician, and parent-reported measures of adaptive behaviour and function. Our study also has the longest follow up period, with an average age of approximately six years, compared to three years in the Belgium microarray cohort ( 41 ) and four years in Shi et al. ( 42 ). Importantly, our inclusion of a control group mitigated potential pandemic-related factors affecting measured outcomes. We intentionally used many of the same parent-rated measures as Desai et al. ( 43 ), who investigated parental perceptions of their children following a prenatal diagnosis of a VUS. Desai et al. found that parents of VUS children expressed greater concerns about their child’s competence at 12 months, although these concerns resolved by 36 months. Our findings at an average age of 6 years build on their results, providing no evidence of long-term concerns into early childhood. Desai et al. also reported that parents of VUS children had lower satisfaction with their decision to undergo prenatal testing compared to the control group ( 43 ). While our study observed a similar trend, the group differences were not statistically significant and average decisional satisfaction scores were high (> 40/50) across both groups in both studies. Consistent with our reassuring findings, the blinded reanalysis conducted by the original reporting laboratories indicated that two-thirds of the children in the VUS group would be now classified as having "no clinically significant genomic imbalance detected." This corresponds with advances in knowledge and updated reporting practices ( 6 ) and an observed decline in VUS results recorded in the Victorian Prenatal Diagnosis Data collection ( 17 ). Over the past decade in the Australian state of Victoria, the annual number of fetuses identified with a variant of uncertain significance (VUS) has declined from 97 cases (4% of prenatal diagnostic tests) in 2013 to 18 cases (1%) in 2022 ( 17 ). This dramatic decline is due to both a reduction in total prenatal diagnostic procedures (due to the increasing use of prenatal cell-free DNA screening), and a reduction in the rate of VUS reported per CMA. The high rate of reclassification for VUS observed in this study (75%) and Shi et al. ( 42 ) (17%) suggests a potential role of structured reanalysis for children with a fetal VUS, as such reanalysis could impact future care. Reanalysis of VUS cases can provide reassurance to parents when variants are downgraded to benign or non-reportable status. While reclassification offers clear benefits, it also presents ethical, clinical, and logistical challenges, particularly when variants are upgraded. Similar issues arise in fetal exome sequencing, where automated reanalysis is being promoted ( 44 – 46 ). Qualitative research to understand the parental experience of a reclassified prenatal VUS is currently underway among our PALM cohort and will be reported separately in a future publication. The high rate of VUS reclassification to ‘likely benign’ supports a conservative reporting approach in the prenatal setting, given the potential for psychological distress in pregnancy and absence of detectable developmental differences in early childhood. Creating paediatric cohorts from the prenatal diagnostic testing population is valuable for understanding long-term outcomes of genomic information gained before birth. However, there were logistical challenges in this study and multiple unavoidable potential sources of bias. First, our inclusion of only liveborn outcomes likely excluded cases of VUS that contributed to fetal demise or were associated with severe ultrasound abnormalities that prompted termination of pregnancy. Second, the requirement for English proficiency likely excluded families from lower SES or culturally diverse backgrounds, introducing further bias. Third, our recruitment was only 21% among successfully contacted families. This rate is comparable to other international studies, but still potentially contributes to selection bias. We previously reported in detail on the screening process and attrition ( 18 ) and noted that among controls, responders tended to have higher socioeconomic status and lower parity than nonresponders. However, there were no differences in metropolitan/rural status, maternal age, or child’s age at recruitment between responders and non-responders within the control group. No differences in sociodemographic factors were noted between responders and non-responders in the VUS group ( 18 ). Moreover, our prior work has shown that pregnancies with inherited VUS or without ultrasound abnormalities were more likely to proceed to live birth, which may have influenced the composition of the cohort ( 47 ). Although we used standardised developmental assessments with population-based norms, and adjusted for multiple demographic variables, residual confounding cannot be ruled out. These factors should be considered when interpreting the generalisability of our findings. Our recruitment rate of 21%, while modest, compares favourably with similar international cohorts such as the Belgian microarray cohort, which had a recruitment rate of 17% ( 41 ). Given that our study imposed a higher participation burden—requiring in-person child assessments in addition to longer parent questionnaires – we considered the recruitment rate acceptable in the context of the societal disruptions of the COVID-19 pandemic ( 48 ). In total, 29 mother-child pairs with prenatal VUS completed all survey and clinical assessments. While this limits our ability to detect subtle differences, the mean FSIQ scores for both groups were well within the age-normed average range (25th–75th percentiles). Larger cohorts are needed to confirm these findings. We await the results of the US microarray follow-up study by Wapner et al. (Clinical Trials #NCT02160938), which has used the same developmental instruments (WPPSI-IV, WISC-V, and Vineland-3), allowing for future comparative analyses or meta-analysis. Conclusion Children born after a prenatal diagnosis of a VUS have similar intellectual functioning, adaptive living skills, and social behaviours compared to those without a VUS. Likewise, their mothers report comparable levels of mental health, parental competence, and family functioning to controls. Collectively, these findings suggest no lasting impact of a prenatal VUS diagnosis on either child development or family well-being. This contributes valuable evidence to support prenatal genetic counselling and clinical laboratory reporting practices. Declarations Ethics approval and consent to participate Ethics approval was granted by the Royal Children’s Hospital Human Research Ethics Committee (reference number 60542) and Mercy Health Human Research Ethics Committee (reference number 2020-46). All participating parents provided written informed consent. Consent for publication Not applicable Availability of data and materials The data that support the findings of this study are available from the corresponding author to researchers from a recognised academic institution upon reasonable request. Competing interests The authors declare they have no competing interests. Funding This study is funded by an NHMRC Clinical Trials and Cohort Studies grant (2020–2022, APP1186862). The funding body had no role in study design, protocol writing, or publication decisions. LH receives salary support from an MRFF investigator grant (APP1196010) and the University of Melbourne. The PALM study is sponsored by MCRI, with LH as the sponsor contact. CRediT author statement Investigation: J.M., C.P., D.J.A., L.G., F.N., J.H., L.H. Formal analysis: J.M., C.P., S.L., D.J.A., J.H., L.H. Data curation: J.M., C.P. Writing - original draft and review and editing: J.M., C.P., S.L., D.J.A., G.M., S.F., M.R., J.M.S., L.B., N.F., N.Y., R.J.W., B.L., S.P.W., J.H., L.H. Visualization: J.M., C.P. Project administration: J.M., C.P., L.G., F.N., G.M., S.F., M.R., J.M.S., L.B., N.F., N.Y. Conceptualization: S.L., D.J.A., S.P.W., J.H., L.H. Methodology: S.L., D.J.A., R.J.W., B.L., S.P.W., J.H., L.H. Supervision: S.L., D.J.A., J.H., L.H. Funding Acquisition: L.H. Acknowledgements We extend our gratitude to the collaborators and study team of the PALM study: Ms Joanne Kennedy, Dr Marta Arpone, Dr Siobhan Seward-Swann, Dr Stephanie Malarbi, Dr Paulina Stedall, Dr Melissa Visser, Dr Kate Irving, and Dr Megan Ball (Murdoch Children’s Research Institute); Ms Melissa Graetz (Mercy Perinatal); Ms Helen Curd (Monash Genetics); Dr Jayshree Ramkrishna (Eastern Health); Dr Abhijit Kulkarni (Monash Health Pathology); Dr Anand Vasudevan (Royal Women’s Hospital); and Dr Emily Olive (Barwon Health). We also acknowledge the laboratory team for their support with CNV reanalysis: Dr Suzanne Svobodova and Emma Brown (Monash Health Pathology), Ms Amber Burgess, Ms Ellen Casey, Paula Lall, and Dr Meg Wall (Victorian Clinical Genetics Services). Finally, we thank the Syndromes Without A Name (SWAN) community for their early-stage consultation on this project. Additional information The supplementary information in this study includes details on the annotation software used by participating laboratories, maternal baseline questionnaires, and perinatal outcomes for both the full cohort and those who underwent clinical assessment. It also provides a summary of VUS cases with prenatal ultrasound anomalies and presents regression analyses of cognitive (WPPSI-IV/WISC-V), adaptive (Vineland-3), and social communication outcomes (SRS-2). Paediatric assessments are outlined across developmental and medical domains for both VUS and control groups. 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Fam Med 36(8):588–594 Ashida S, Koehly LM, Roberts JS, Chen CA, Hiraki S, Green RC (2009) Disclosing the disclosure: factors associated with communicating the results of genetic susceptibility testing for Alzheimer's disease. J Health Commun 14(8):768–784 Langer MM, Roche MI, Brewer NT, Berg JS, Khan CM, Leos C et al (2017) Development and Validation of a Genomic Knowledge Scale to Advance Informed Decision Making Research in Genomic Sequencing. MDM Policy Pract. ;2(1) Klitzman R, Appelbaum PS, Fyer A, Martinez J, Buquez B, Wynn J et al (2013) Researchers' views on return of incidental genomic research results: qualitative and quantitative findings. Genet Med 15(11):888–895 Phelan JC (2005) Geneticization of deviant behavior and consequences for stigma: the case of mental illness. J Health Soc Behav 46(4):307–322 Goodman R (1997) The Strengths and Difficulties Questionnaire: a research note. J Child Psychol Psychiatry 38(5):581–586 Forsyth BW, Horwitz SM, Leventhal JM, Burger J, Leaf PJ (1996) The child vulnerability scale: an instrument to measure parental perceptions of child vulnerability. J Pediatr Psychol 21(1):89–101 Bethell CD, Read D, Stein REK, Blumberg SJ, Wells N, Newacheck PW (2002) Identifying Children With Special Health Care Needs: Development and Evaluation of a Short Screening Instrument. Ambul Pediatr 2(1):38–48 Wechsler D (2012) Wechsler Preschool and Primary Scale of Intelligence – Fourth Edition (WPPSI-IV), Australian and New Zealand Standardised Edition. Pearson Clinical and Talent Assessment, Sydney, Australia Wechsler D (2016) Wechsler Intelligence Scale for Children – Fifth Edition (WISC-V), Australian and New Zealand Standardised Edition. Pearson Clinical and Talent Assessment, Sydney, Australia Sparrow SS, Cicchetti DV, Saulnier CA (2016) Vineland Adaptive Behavior Scales, Third Edition (Vineland-3) Constantino JN, Gruber CP (2012) Social Responsiveness Scale Second Edition (SRS-2): Manual. Western Psychological Services (WPS) StataCorp (2023) Stata Statistical Software. Release 18 ed. StataCorp LLC, College Station, TX Grummer-Strawn LM, Reinold C, Krebs NF (2010) Use of World Health Organization and CDC growth charts for children aged 0–59 months in the United States. MMWR Recomm Rep 59(Rr–9):1–15 Werner-Lin A, Walser S, Barg F, Bernhardt B (2016) They Can't Find Anything Wrong with Him, Yet: Mothers’ experiences of parenting an infant with a prenatally diagnosed copy number variant (CNV). Am J Med Genet Part A. ;173 Muys J, Jacquemyn Y, Blaumeiser B, Bourlard L, Brison N, Bulk S et al (2020) Prenatally detected copy number variants in a national cohort: A postnatal follow-up study. Prenat Diagn 40(10):1272–1283 Shi P, Liang H, Hou Y, Chen D, Ren H, Wang C et al (2023) The uncertainty of copy number variants: pregnancy decisions and clinical follow-up. Am J Obstet Gynecol 229(2):170. .e1-.e8 Desai P, Haber H, Bulafka J, Russell A, Clifton R, Zachary J et al (2018) Impacts of variants of uncertain significance on parental perceptions of children after prenatal chromosome microarray testing. Prenat Diagn 38(10):740–747 Li LS, Li DZ (2022) Ongoing reanalysis of prenatal exome sequencing data leads to higher diagnostic yield. Ultrasound Obstet Gynecol 59(6):833–834 Mensah NE, Sabir AH, Bond A, Roworth W, Irving M, Davies AC, Ahn JW (2022) Automated reanalysis application to assist in detecting novel gene-disease associations after genome sequencing. Genet Med 24(4):811–820 Peterson B, Hernandez EJ, Hobbs C, Malone Jenkins S, Moore B, Rosales E et al (2023) Automated prioritization of sick newborns for whole genome sequencing using clinical natural language processing and machine learning. Genome Med 15(1):18 Pynaker C, Norris F, Hui L, Halliday J (2023) Perinatal outcomes and genomic characteristics of fetal copy number variants: An individual record linkage study of 713 pregnancies. Prenat Diagn 43(4):516–526 Abdulhussein D, Yap TE, Manzar H, Miodragovic S, Cordeiro F (2022) Factors impacting participation in research during the COVID-19 pandemic: results from a survey of patients in the ophthalmology outpatient department. Trials 23(1):823 Tables Table 1 to 3 are available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files PALMpreprinttable1.docx PALMpreprinttable2.docx PALMpreprinttable3.docx PALMpreprintsupplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6952328","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":474855875,"identity":"4a5d5d06-1c59-428b-a9a9-cd2894c56491","order_by":0,"name":"Jacqui McCoy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACxgYQaQDlfQBiNiCWIFoL4wxitKAAZh4oA68W5vbDD2/zFDBE87cfPiZtm2OXz8fAfPA2D4NdYgMuh/WkGVvzGDDkzjiTliaduy3Zso2BLdmahyEZt5aGHDZpkJYNEjxmQC0HDNgYgAweBmbcWvrfIGmxBGvh/wbUUo9bywxkWxghtgBFGA7j0fLM2HKOgQTIL8mWvduSDdiY2UAix41xaTHsT354480fm9z+9sMHb/zcZmcg394MFKmolsWppQEcC+CIYIFEBzOIMMChHgjkGRARx/wBt7pRMApGwSgYyQAAyoxHrqn6MHsAAAAASUVORK5CYII=","orcid":"","institution":"Murdoch Children's Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Jacqui","middleName":"","lastName":"McCoy","suffix":""},{"id":474855876,"identity":"9167b60e-553a-4827-ae97-a8029a78916f","order_by":1,"name":"Cecilia Pynaker","email":"","orcid":"","institution":"Murdoch Children's Research 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Institute","correspondingAuthor":false,"prefix":"","firstName":"Jane","middleName":"","lastName":"Halliday","suffix":""},{"id":474860515,"identity":"31a2a7a7-be92-4e07-9989-77e0aa6dd12a","order_by":17,"name":"Lisa Hui","email":"","orcid":"","institution":"Murdoch Children's Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Hui","suffix":""}],"badges":[],"createdAt":"2025-06-23 03:25:25","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6952328/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6952328/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85370840,"identity":"e01f117a-1a3b-4d98-85e0-8aa4b2dbe8d7","added_by":"auto","created_at":"2025-06-25 07:29:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106037,"visible":true,"origin":"","legend":"\u003cp\u003ea An additional 17 participants were recruited from private clinical referrers, though the total number of screened private patients is unknown due to clinician-discretionary pre-screening.\u003c/p\u003e\n\u003cp\u003eb Did not meet eligibility criteria after detailed review of medical record.\u003c/p\u003e\n\u003cp\u003ec Questionnaire and developmental assessment outcomes from 9 mother-child pairs with prenatally diagnosed pathogenic CNVs (pCNVs) were excluded in analyses due to small numbers and concerns regarding identifiability.\u003c/p\u003e","description":"","filename":"PALMpreprintfigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6952328/v1/bf006652b6f64a4d0a0e72a2.jpg"},{"id":85373045,"identity":"b119cbeb-6a78-4b37-a33d-156676f07020","added_by":"auto","created_at":"2025-06-25 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07:53:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21111,"visible":true,"origin":"","legend":"","description":"","filename":"PALMpreprinttable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6952328/v1/cc1ad8b5a0a6e15008f67564.docx"},{"id":85372291,"identity":"4606ad17-8273-4052-acb8-f3052f9c9162","added_by":"auto","created_at":"2025-06-25 07:45:47","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20086,"visible":true,"origin":"","legend":"","description":"","filename":"PALMpreprinttable3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6952328/v1/4516b763fa5a515f97d7958d.docx"},{"id":85370848,"identity":"df233b43-4734-443f-97df-d27d051560ee","added_by":"auto","created_at":"2025-06-25 07:29:48","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":83307,"visible":true,"origin":"","legend":"","description":"","filename":"PALMpreprintsupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6952328/v1/de45d5490b35dda6693937af.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eChildhood outcomes of fetal genomic copy number variants: the prenatal microarray cohort study\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOver the past decade, advances in genomics have revolutionised reproductive health and prenatal diagnosis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Chromosomal microarray (CMA) has been the gold standard first line investigation for fetal structural anomalies since 2012 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), and is commonly performed for all indications for prenatal diagnosis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). While CMA have enhanced the detection of pathogenic copy number variants (CNVs), they also identify variants of uncertain significance (VUS) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). VUS can pose challenges for genetic counselling and decision-making in the prenatal setting (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) as they may be associated with an increased chance of developmental disabilities or neuropsychiatric conditions (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Unlike the postnatal context, where uncertain genomic findings can be correlated with the child\u0026rsquo;s phenotype, the disclosure of fetal VUS during pregnancy can introduce significant uncertainty, increasing anxiety and raising ethical dilemmas regarding pregnancy management (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Prenatal genetic counselling for VUS is further complicated by the systemic bias in the literature. Data on CNVs are mostly derived from symptomatic children, skewing the evidence base towards more severe phenotypes that may not be applicable in the prenatal context (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Only two studies have investigated the outcomes of children born after a prenatal diagnosis of a CNV and neither examine health and development of children diagnosed with a VUS beyond 4 years (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe paucity of comprehensive and long-term outcome data for children with a prenatally diagnosed VUS significantly impedes genetic counselling of expectant couples. We aimed to recruit a cohort of children, with and without a prenatal VUS, to (i) compare the developmental, social-emotional and health status of children with and without a prenatal diagnosis of a VUS; (ii) measure the impact of the CMA results on maternal perceptions of child health, behaviour, and development; and (iii) determine the proportion of prenatal VUS reclassified to benign or pathogenic on reanalysis\u0026thinsp;\u0026gt;\u0026thinsp;2 years later.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eWe established a population-based cohort of mother-child pairs who had undergone prenatal testing with a CMA in Victoria, Australia between 2013\u0026ndash;2019 inclusive. The full study protocol has been published elsewhere (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Ethical and governance approval was granted by the Royal Children\u0026rsquo;s Hospital Human Research Ethics Committee (RCH HREC #60542) and the Mercy Health Human Research Ethics Committee (HREC #2020-046). The study was funded by the National Health and Medical Research Council (#1186862).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRecruitment\u003c/h3\u003e\n\u003cp\u003ePotential participants were identified from the Victorian Prenatal Diagnosis Database, a statewide research database of all prenatal chromosome testing results (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). During the study period, chromosome microarrays were used in \u0026gt;\u0026thinsp;85% of prenatal diagnostic tests, including fetuses with and without structural anomalies (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). We screened potential cases from singleton pregnancies with a CNV reported on a prenatal microarray result. We defined cases as those with a VUS, inclusive of all indications for prenatal diagnosis including fetal ultrasound abnormality. Controls were singleton pregnancies that had no CNV (i.e., no clinically significant genomic imbalance detected) detected on a prenatal microarray, and no fetal ultrasound abnormality.\u003c/p\u003e \u003cp\u003eMedical records of potential cases and controls were screened for full eligibility criteria. Individuals were eligible if they: (i) had a prenatal CMA result reported for a singleton pregnancy between January 2013 and December 2019, (ii) were \u0026ge;\u0026thinsp;18 years old at enrolment, (iii) could provide informed consent in English, (iv) had a live birth outcome, (v) were the primary caregiver at hospital discharge, (vi) were able to consent on behalf of themselves and their child, (vii) were able to complete questionnaires and attend study assessments, and (viii) lived in Victoria. Participant recruitment and data collection occurred between November 2021 and December 2023. Full details of the recruitment process, perinatal outcomes and participant response rate has been published elsewhere (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In brief, 1832 medical records were screened and 1364 (74.5%) mother\u0026ndash;child pairs met inclusion criteria. Of the 468 that did not meet inclusion criteria, 282 (60.3%) had \u0026lsquo;no live pregnancy outcome\u0026rsquo; (209 terminations of pregnancy (TOP), 73 miscarriages, stillbirths, and infant deaths), 157 (33.5%) required a translator, and 29 (6.2%) had other exclusion criteria). Approximately 77% of study invitation letters were successfully delivered by registered mail (1047/1364). The final recruitment rate was 19.2% (201/1047). All birth parents participating in this study identified as women/mothers, hence these terms are used throughout.\u003c/p\u003e\n\u003ch3\u003eOutcome Measures\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuestionnaires\u003c/h2\u003e \u003cp\u003eDemographic and health data were gathered at baseline using validated questionnaires. Mother-focussed measures included: the Patient Health Questionnaire (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), the State-Trait Anxiety Inventory (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), the Parenting Sense of Competence Scale (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), the McMaster Family Functioning Subscale (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), the Revised Scale for Ambiguity Tolerance (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), the Decision Satisfaction Scale (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), the Health Literacy Screening Questions (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), the Disclosure of Results to Others (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), the University of North Carolina Genomic Knowledge Scale (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and two questions from the Genetics Essentialism scale (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) (See\u003c/p\u003e \u003cp\u003eChild-focussed measures included: the Strengths and Difficulties Questionnaire (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), the Vulnerable Child Scale (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), and the Children with Special Health Care Needs Screener (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). A description of each questionnaire is available in Supplementary Table\u0026nbsp;1. Children aged two years and six months (2:6) or older were invited to participate in additional clinical assessments including validated cognitive, behavioural, social, and developmental assessments. These were conducted in person by psychologists and paediatricians\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCognitive functioning\u003c/h3\u003e\n\u003cp\u003eAge-appropriate Wechsler scales were used to assess intellectual functioning: the Wechsler Preschool \u0026amp; Primary Scale of Intelligence (WPPSI-IV) for children under 7 years and 7 months and the Wechsler Intelligence Scale for Children (WISC-V) for older children. Scores were classified according to test manual norms (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Cognitive assessments for metropolitan participants were conducted in person at The Royal Children\u0026rsquo;s Hospital (RCH), while regional participants were assessed at local health services. Paediatricians and psychologists conducting assessments were initially blinded to the children's CMA results. However, some parents spontaneously disclosed their experiences during assessments, despite being advised that assessing staff should remain blinded. Standardised assessments were consistently scored and either verified by a second team member or double-scored by the assessor.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBehaviour and social functioning\u003c/h2\u003e \u003cp\u003eThe Vineland Adaptive Behaviour Scales \u0026ndash; Third Edition, Parent/Caregiver form (Vineland-3) assessed adaptive functioning across five domains: Communication, Socialization, Motor Skills, Daily Living Skills, and Maladaptive Behaviours (for children older than 3). Higher scores indicate better adaptive functioning (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Social Responsiveness Scale \u0026ndash; Second Edition, Preschool/School Age forms (SRS-2) assessed social behaviours linked to autism spectrum disorder (ASD). Standardised scores categorised behaviours from normal to severe in relation to ASD symptoms (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChild development\u003c/h3\u003e\n\u003cp\u003eA paediatric review with the mother and child collected data on the child\u0026rsquo;s developmental and medical history, including growth, cognitive, behavioural, and neurological functioning, as well as any medical concerns using a standardised data collection form. Online paediatric assessments were available during COVID-19 pandemic restrictions in 2021\u0026ndash;2022.\u003c/p\u003e\n\u003ch3\u003eCNV reanalysis\u003c/h3\u003e\n\u003cp\u003eThe prenatal VUS classifications were reanalysed by the original reporting laboratory. The scientists were blinded to the original classification and outcomes of the research assessments but were provided the indication for prenatal diagnostic testing. Refer to Supplementary Methods for more detail.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical methods\u003c/h2\u003e \u003cp\u003eData were analysed using Stata Version 18 (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Descriptive statistics included counts, percentages, means, standard deviations, and medians with interquartile ranges (IQR) for non-normally distributed data. Continuous outcomes were analysed using t-tests and multivariable linear regression for normally distributed data, while the Mann-Whitney test was used for non-normally distributed data. Categorical outcomes were analysed using the chi-square test. A significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 accounted for multiple comparisons. Scores from the two age-specific versions of the Strengths and Difficulties Questionnaire (for ages 2:0\u0026ndash;3:11, and 4:0\u0026ndash;10:0 years) were converted to z-scores and merged for further analysis. Sample sizes vary by measure due to differing participant completion rates across outcome measures.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e From 1047 eligible mothers that were contacted, 218 (20.8%) consented to participate in the study (Fig.\u0026nbsp;1). Fifty-nine participants were excluded after recruitment due to additional information obtained from the medical record, usually a finding of an ultrasound anomaly in the control group. The final analysis included 134 mother-child pairs, comprising 46 cases with a VUS and 88 controls (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eParticipant characteristics\u003c/h2\u003e \u003cp\u003eA higher proportion of mothers in the control group were aged over 35 years at the time of giving birth compared to mothers in the VUS group (75.5%, versus 48.3% respectively, p\u0026thinsp;=\u0026thinsp;0.02). The VUS group also had a significantly higher proportion of regionally based participants compared to the control group (39.1% versus 19.3%, p\u0026thinsp;=\u0026thinsp;0.01), due to study recruitment logistics (no controls were recruited from regional areas due to resource allocation). There were no other demographic differences between the VUS case and control cohorts, including those who completed only the online assessments (survey cohort) and those who opted for additional child assessments (clinical cohort, see Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThe most common indication for prenatal diagnosis among the pregnancies with a VUS was an ultrasound abnormality (56.6%, 26/46) (see Supplementary Table\u0026nbsp;2 and Supplementary Table\u0026nbsp;3 for details of the ultrasound abnormalities and perinatal outcomes). The most common indication for prenatal diagnosis among the controls was an increased probability of aneuploidy on the first trimester combined screen (48.3%, 42/88). Overall, 74.4% of the VUS cases were either maternally or paternally inherited.\u003c/p\u003e \u003cp\u003ePerinatal outcomes were compared between the VUS and control groups: there were no significant differences in sex ratio, head circumference, small for gestational age, mode of birth or breast-feeding rates. The VUS group had significantly higher rates of special care nursery or neonatal intensive care admission, and higher rates of hospital readmission within 28 days compared with controls (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDevelopmental, social-emotional and health outcomes\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eCognitive functioning\u003c/h2\u003e \u003cp\u003eGeneral intellectual functioning was formally assessed in 80 out of 134 (60%) children with and without a VUS. The children\u0026rsquo;s average age at assessment was approximately 5 years and 11 months (range 2 years and 7 months to 9 years and 2 months). There was no significant difference in Full-Scale Intelligence Quotients (FSIQ) between children with a VUS (n\u0026thinsp;=\u0026thinsp;30, mean 96.3, SD\u0026thinsp;=\u0026thinsp;14.1) and those without (n\u0026thinsp;=\u0026thinsp;50, mean 101.5, SD\u0026thinsp;=\u0026thinsp;16.0; p\u0026thinsp;=\u0026thinsp;.14, see Table\u0026nbsp;2). Only two participants scored 2 SDs below the mean on the FSIQ (\u0026le;\u0026thinsp;70), indicating mild intellectual impairment (one each of VUS case and control).\u003c/p\u003e \u003cp\u003e A significant difference was observed on the Verbal Comprehension Index, where children with a VUS (VUS cases; mean\u0026thinsp;=\u0026thinsp;95.5, SD\u0026thinsp;=\u0026thinsp;13.8) scored lower than children without (controls; mean\u0026thinsp;=\u0026thinsp;103.7, SD\u0026thinsp;=\u0026thinsp;14.6; p\u0026thinsp;=\u0026thinsp;.01). However, this difference became non-significant when accounting for maternal age at the child's birth, maternal country of birth, maternal education level, income, locality (metro/regional), and maternal relationship status (p\u0026thinsp;=\u0026thinsp;0.08). No statistically significant differences were detected in the other cognitive indices after considering the same covariates (see Table\u0026nbsp;2 and Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAdaptive and social functioning\u003c/h2\u003e \u003cp\u003eEighty mothers (80/134; 60%) also completed the Vineland-3 Parent/caregiver form. There were no differences in overall adaptive functioning between VUS cases (mean\u0026thinsp;=\u0026thinsp;93.1, SD\u0026thinsp;=\u0026thinsp;13.9) and controls (mean\u0026thinsp;=\u0026thinsp;92.2, SD\u0026thinsp;=\u0026thinsp;10.5; p\u0026thinsp;=\u0026thinsp;0.73). There were no differences observed between cases and controls in the other functional domains of the Vineland-3 (see Table\u0026nbsp;2). Sixty-eight (51%) mothers completed the SRS-2 questionnaire. There were no differences between VUS cases and controls observed in any of the social functioning scores (see Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChild development\u003c/h2\u003e \u003cp\u003eA total of 78 (58%) mother-child pairs participated in the clinical review conducted by a study paediatrician. Of these, 26% (7/29 VUS cases and 13/49 controls) were seen in person, while the remainder were reviewed via Telehealth with video or telephone. No significant differences were observed between VUS cases and controls in the number of concerns reported across developmental and medical domains. The two exceptions were in the domains of infection and hearing, with more concerns reported in VUS cases than controls. For infection, specific issues within the VUS cohort included 2 cases of pyelonephritis, 7 cases of recurrent otitis media (\u0026ge;\u0026thinsp;3 episodes), and 3 classified as \u0026ldquo;other\u0026rdquo; recurrent types of infections. For hearing, there were 6 instances of conductive hearing loss and 1 case of sensorineural hearing loss reported among the VUS cases. See Supplementary Table\u0026nbsp;5 for detailed results of the paediatric review.\u003c/p\u003e \u003cp\u003eBMI was available for 70 (53%) children (42 controls and 28 VUS cases). There was no evidence of a difference in BMI between the two groups, with 75% (21/28) of VUS cases and 60% (25/42, p\u0026thinsp;=\u0026thinsp;.34) of controls having a weight between the 5th and 85th percentiles standardised for age and sex (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMaternal perceptions of child health, behaviour, and development\u003c/h2\u003e \u003cp\u003eNo group differences between maternal responses were found on measures of maternal mental health, perceived parental competence, overall family functioning, maternal perceptions of child behaviour and vulnerability, general tolerance of uncertainty, general genetic knowledge, and decisional satisfaction regarding the choice to undergo microarray testing during pregnancy (see Supplementary Table\u0026nbsp;6).\u003c/p\u003e \u003cp\u003eThere were no significant differences between the VUS and control groups in the proportion of children who were noted to receive at least one form of additional support including medication, increased health or educational services, functional limitations, specialised therapies, or additional assistance for emotional, behavioural, or developmental issues (27% VUS vs. 20% controls, p\u0026thinsp;=\u0026thinsp;0.60). Health literacy was also similar, with 74% of VUS mothers and 77% of control mothers reporting no difficulty understanding written medical information, and 91% (VUS cases\u0026thinsp;=\u0026thinsp;42; controls\u0026thinsp;=\u0026thinsp;79) in both groups reporting high levels of confidence in completing medical forms independently.\u003c/p\u003e \u003cp\u003eMost mothers in both groups disagreed that their child was different from most people (VUS cases: 76% (32/42), controls: 81% (66/81)). Similarly, 72% (31/42) of mothers in the VUS group and 80% (65/81) of control mothers disagreed that their child's genetic makeup made them different. No significant differences were found between the groups on these measures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eChromosomal microarray reanalysis\u003c/h2\u003e \u003cp\u003eAfter excluding three participants who withdrew from the study, 56 mothers consented to having their child\u0026rsquo;s prenatal CNV result reanalysed. An average period of 5.5 years had elapsed since the original prenatal microarray report.\u003c/p\u003e \u003cp\u003eOf the 47 participants with a prenatal VUS, 31 (66%) were downgraded to \"no clinically significant genomic imbalance detected\", four (9%) were upgraded to \"pathogenic copy number change with variable expressivity\", and 12 (26%) remained unchanged as a VUS. Table\u0026nbsp;3 provides the details of the reanalysis of VUS. The majority of VUS were inherited variants, accounting for 72% (34/47), while 23.4% (11/47) were undetermined (meaning the parents were not tested, so this could not be evaluated), and 4.3% (2/47) were de novo variants (see Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eWe conducted a post hoc analysis comparing clinical assessment outcomes for children whose VUS results are no longer reported (n\u0026thinsp;=\u0026thinsp;19) with the controls (n\u0026thinsp;=\u0026thinsp;50) and found no significant differences in intellectual functioning, adaptive functioning, or social communication skills associated with autism spectrum disorder (see Supplementary Table\u0026nbsp;7). Similarly, when comparing these outcomes for children whose VUS results are still reported or have been reclassified as pathogenic with variable expressivity (n\u0026thinsp;=\u0026thinsp;11) to the controls (n\u0026thinsp;=\u0026thinsp;50), no differences were observed (see Supplementary Table\u0026nbsp;8).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe conducted a comprehensive evaluation of the developmental, social-emotional, and health outcomes of children with and without a prenatal diagnosis of a VUS at a mean age of 6 years. The results are reassuring, demonstrating that children with a VUS performed similarly to children without a VUS across a wide range of developmental, cognitive, and health measures. Our study found no significant differences in maternal perceptions of child health, behaviour, or development between the VUS and control groups. A key concern with genomic testing and disclosure of uncertain findings is the potential for increased anxiety, which may negatively impact parenting styles and the mother-child relationship (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). However, mothers in our VUS and control groups reported similar levels of mental health, parental competence, and family functioning. Taken together, these findings suggest no enduring impacts through early childhood of a prenatal diagnosis of a VUS on either child health and development or aspects of family well-being.\u003c/p\u003e \u003cp\u003eOur research makes a major contribution to the limited evidence base for this population. Only two other studies have followed up large cohorts with a prenatal diagnosis of a CNV. The Belgian prenatal microarray cohort study used the Ages and Stages Questionnaire to follow up children who had a prenatal diagnosis of a CNV (85 cases, 123 controls). Compared with controls, they identified significant delays in communication and personal-social development among three-year-olds with a susceptibility CNV. However, this study specifically excluded children with a VUS from further analyses, limiting any direct comparisons with this study (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). More recently, Shi et al. followed 113 infants with a prenatal VUS, conducting follow-up at 2 time points: 0\u0026ndash;10 months and 2\u0026ndash;4 years of age (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). They reported 5 with potential disease phenotypes, but the clinical assessments were not described, participant attrition rate was high, and there was no comparator group, thus precluding any firm conclusions about the childhood outcomes for this sample.\u003c/p\u003e \u003cp\u003eIn contrast, our cohort has been assessed with a broad range of structured, validated tools involving a variety of data sources: in-person assessment of cognitive function by a psychologist, clinical assessment by a paediatrician, and parent-reported measures of adaptive behaviour and function. Our study also has the longest follow up period, with an average age of approximately six years, compared to three years in the Belgium microarray cohort (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) and four years in Shi et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Importantly, our inclusion of a control group mitigated potential pandemic-related factors affecting measured outcomes.\u003c/p\u003e \u003cp\u003eWe intentionally used many of the same parent-rated measures as Desai et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), who investigated parental perceptions of their children following a prenatal diagnosis of a VUS. Desai et al. found that parents of VUS children expressed greater concerns about their child\u0026rsquo;s competence at 12 months, although these concerns resolved by 36 months. Our findings at an average age of 6 years build on their results, providing no evidence of long-term concerns into early childhood. Desai et al. also reported that parents of VUS children had lower satisfaction with their decision to undergo prenatal testing compared to the control group (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). While our study observed a similar trend, the group differences were not statistically significant and average decisional satisfaction scores were high (\u0026gt;\u0026thinsp;40/50) across both groups in both studies.\u003c/p\u003e \u003cp\u003eConsistent with our reassuring findings, the blinded reanalysis conducted by the original reporting laboratories indicated that two-thirds of the children in the VUS group would be now classified as having \"no clinically significant genomic imbalance detected.\" This corresponds with advances in knowledge and updated reporting practices (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and an observed decline in VUS results recorded in the Victorian Prenatal Diagnosis Data collection (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Over the past decade in the Australian state of Victoria, the annual number of fetuses identified with a variant of uncertain significance (VUS) has declined from 97 cases (4% of prenatal diagnostic tests) in 2013 to 18 cases (1%) in 2022 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). This dramatic decline is due to both a reduction in total prenatal diagnostic procedures (due to the increasing use of prenatal cell-free DNA screening), and a reduction in the rate of VUS reported per CMA.\u003c/p\u003e \u003cp\u003eThe high rate of reclassification for VUS observed in this study (75%) and Shi et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) (17%) suggests a potential role of structured reanalysis for children with a fetal VUS, as such reanalysis could impact future care. Reanalysis of VUS cases can provide reassurance to parents when variants are downgraded to benign or non-reportable status. While reclassification offers clear benefits, it also presents ethical, clinical, and logistical challenges, particularly when variants are upgraded. Similar issues arise in fetal exome sequencing, where automated reanalysis is being promoted (\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Qualitative research to understand the parental experience of a reclassified prenatal VUS is currently underway among our PALM cohort and will be reported separately in a future publication. The high rate of VUS reclassification to \u0026lsquo;likely benign\u0026rsquo; supports a conservative reporting approach in the prenatal setting, given the potential for psychological distress in pregnancy and absence of detectable developmental differences in early childhood.\u003c/p\u003e \u003cp\u003eCreating paediatric cohorts from the prenatal diagnostic testing population is valuable for understanding long-term outcomes of genomic information gained before birth. However, there were logistical challenges in this study and multiple unavoidable potential sources of bias. First, our inclusion of only liveborn outcomes likely excluded cases of VUS that contributed to fetal demise or were associated with severe ultrasound abnormalities that prompted termination of pregnancy. Second, the requirement for English proficiency likely excluded families from lower SES or culturally diverse backgrounds, introducing further bias. Third, our recruitment was only 21% among successfully contacted families. This rate is comparable to other international studies, but still potentially contributes to selection bias. We previously reported in detail on the screening process and attrition (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) and noted that among controls, responders tended to have higher socioeconomic status and lower parity than nonresponders. However, there were no differences in metropolitan/rural status, maternal age, or child\u0026rsquo;s age at recruitment between responders and non-responders within the control group. No differences in sociodemographic factors were noted between responders and non-responders in the VUS group (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Moreover, our prior work has shown that pregnancies with inherited VUS or without ultrasound abnormalities were more likely to proceed to live birth, which may have influenced the composition of the cohort (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Although we used standardised developmental assessments with population-based norms, and adjusted for multiple demographic variables, residual confounding cannot be ruled out. These factors should be considered when interpreting the generalisability of our findings.\u003c/p\u003e \u003cp\u003eOur recruitment rate of 21%, while modest, compares favourably with similar international cohorts such as the Belgian microarray cohort, which had a recruitment rate of 17% (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Given that our study imposed a higher participation burden\u0026mdash;requiring in-person child assessments in addition to longer parent questionnaires \u0026ndash; we considered the recruitment rate acceptable in the context of the societal disruptions of the COVID-19 pandemic (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). In total, 29 mother-child pairs with prenatal VUS completed all survey and clinical assessments. While this limits our ability to detect subtle differences, the mean FSIQ scores for both groups were well within the age-normed average range (25th\u0026ndash;75th percentiles). Larger cohorts are needed to confirm these findings. We await the results of the US microarray follow-up study by Wapner et al. (Clinical Trials #NCT02160938), which has used the same developmental instruments (WPPSI-IV, WISC-V, and Vineland-3), allowing for future comparative analyses or meta-analysis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eChildren born after a prenatal diagnosis of a VUS have similar intellectual functioning, adaptive living skills, and social behaviours compared to those without a VUS. Likewise, their mothers report comparable levels of mental health, parental competence, and family functioning to controls. Collectively, these findings suggest no lasting impact of a prenatal VUS diagnosis on either child development or family well-being. This contributes valuable evidence to support prenatal genetic counselling and clinical laboratory reporting practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval was granted by the Royal Children\u0026rsquo;s Hospital Human Research Ethics Committee (reference number 60542) and Mercy Health Human Research Ethics Committee (reference number 2020-46). All participating parents provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author to researchers from a recognised academic institution upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is funded by an NHMRC Clinical Trials and Cohort Studies grant (2020\u0026ndash;2022, APP1186862). The funding body had no role in study design, protocol writing, or publication decisions. LH receives salary support from an MRFF investigator grant (APP1196010) and the University of Melbourne. The PALM study is sponsored by MCRI, with LH as the sponsor contact.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u003c/strong\u003e J.M., C.P., D.J.A., L.G., F.N., J.H., L.H.\u003cstrong\u003e\u0026nbsp;Formal analysis:\u0026nbsp;\u003c/strong\u003eJ.M., C.P., S.L., D.J.A., J.H., L.H. \u003cstrong\u003eData curation:\u0026nbsp;\u003c/strong\u003eJ.M., C.P. \u003cstrong\u003eWriting - original draft and review and editing:\u0026nbsp;\u003c/strong\u003eJ.M., C.P., S.L., D.J.A., G.M., S.F., M.R., J.M.S., L.B., N.F., N.Y., R.J.W., B.L., S.P.W., J.H., L.H. \u003cstrong\u003eVisualization:\u0026nbsp;\u003c/strong\u003eJ.M., C.P.\u003cstrong\u003e\u0026nbsp;Project administration:\u0026nbsp;\u003c/strong\u003eJ.M., C.P., L.G., F.N., G.M., S.F., M.R., J.M.S., L.B., N.F., N.Y.\u003cstrong\u003e\u0026nbsp;Conceptualization:\u0026nbsp;\u003c/strong\u003eS.L., D.J.A., S.P.W., J.H., L.H.\u003cstrong\u003e\u0026nbsp;Methodology:\u0026nbsp;\u003c/strong\u003eS.L., D.J.A., R.J.W., B.L., S.P.W., J.H.,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eL.H.\u003cstrong\u003e\u0026nbsp;Supervision:\u0026nbsp;\u003c/strong\u003eS.L., D.J.A., J.H., L.H.\u003cstrong\u003e\u0026nbsp;Funding Acquisition:\u0026nbsp;\u003c/strong\u003eL.H.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to the collaborators and study team of the PALM study: Ms Joanne Kennedy, Dr Marta Arpone, Dr Siobhan Seward-Swann, Dr Stephanie Malarbi, Dr Paulina Stedall, Dr Melissa Visser, Dr Kate Irving, and Dr Megan Ball (Murdoch Children\u0026rsquo;s Research Institute); Ms Melissa Graetz (Mercy Perinatal); Ms Helen Curd (Monash Genetics); Dr Jayshree Ramkrishna (Eastern Health); Dr Abhijit Kulkarni (Monash Health Pathology); Dr Anand Vasudevan (Royal Women\u0026rsquo;s Hospital); and Dr Emily Olive (Barwon Health). We also acknowledge the laboratory team for their support with CNV reanalysis: Dr Suzanne Svobodova and Emma Brown (Monash Health Pathology), Ms Amber Burgess, Ms Ellen Casey, Paula Lall, and Dr Meg Wall (Victorian Clinical Genetics Services). Finally, we thank the Syndromes Without A Name (SWAN) community for their early-stage consultation on this project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supplementary information in this study includes details on the annotation software used by participating laboratories, maternal baseline questionnaires, and perinatal outcomes for both the full cohort and those who underwent clinical assessment. It also provides a summary of VUS cases with prenatal ultrasound anomalies and presents regression analyses of cognitive (WPPSI-IV/WISC-V), adaptive (Vineland-3), and social communication outcomes (SRS-2). Paediatric assessments are outlined across developmental and medical domains for both VUS and control groups. Between-group comparisons are presented, including controls versus VUS cases no longer reported and those still reported or upgraded. Maternal questionnaire data and key clinical outcomes are also summarised in the context of genetic findings.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBodurtha J, Strauss JF 3 (2012) Genomics and perinatal care. N Engl J Med 366(1):64\u0026ndash;73\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWapner RJ, Martin CL, Levy B, Ballif BC, Eng CM, Zachary JM et al (2012) Chromosomal microarray versus karyotyping for prenatal diagnosis. 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Ultrasound Obstet Gynecol 59(6):833\u0026ndash;834\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMensah NE, Sabir AH, Bond A, Roworth W, Irving M, Davies AC, Ahn JW (2022) Automated reanalysis application to assist in detecting novel gene-disease associations after genome sequencing. Genet Med 24(4):811\u0026ndash;820\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeterson B, Hernandez EJ, Hobbs C, Malone Jenkins S, Moore B, Rosales E et al (2023) Automated prioritization of sick newborns for whole genome sequencing using clinical natural language processing and machine learning. Genome Med 15(1):18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePynaker C, Norris F, Hui L, Halliday J (2023) Perinatal outcomes and genomic characteristics of fetal copy number variants: An individual record linkage study of 713 pregnancies. Prenat Diagn 43(4):516\u0026ndash;526\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdulhussein D, Yap TE, Manzar H, Miodragovic S, Cordeiro F (2022) Factors impacting participation in research during the COVID-19 pandemic: results from a survey of patients in the ophthalmology outpatient department. Trials 23(1):823\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"prenatal diagnosis, chromosomal microarray analysis, copy number variants, variant of uncertain significance, cognitive function, cohort studies, paediatric population","lastPublishedDoi":"10.21203/rs.3.rs-6952328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6952328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe long-term developmental outcomes of children with a prenatal diagnosis of a copy number variant of uncertain significance (VUS) remain unclear. This study compared the developmental, social-emotional, and health outcomes of children with and without a prenatal VUS, assessed maternal perceptions of their child’s health and development, and examined the reclassification rate of VUS after more than two years.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWomen who underwent prenatal chromosomal microarray testing in Victoria, Australia (2013–2019), were recruited retrospectively (2021–2023). Children with a VUS (cases) were compared with controls without a VUS. Outcomes, including cognitive, developmental, and health measures, were assessed and children had an average age of six years. Statistical analyses compared group outcomes and adjusted for maternal sociodemographic factors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study included 134 mother-child pairs (46 with a VUS and 88 controls). No significant differences were found between groups in intellectual functioning, adaptive behaviour, or social-emotional measures. Maternal perceptions of their child and family well-being were also similar. Reanalysis reclassified 66.0% of VUS as benign and 8.5% as pathogenic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChildren with a prenatal VUS diagnosis have developmental outcomes and family well-being comparable to those without. These findings contribute valuable evidence to support prenatal genetic counselling and clinical laboratory reporting practices.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObservational study registration:\u003c/strong\u003eACTRN12620000446965p; Registered on April 6, 2020.\u003c/p\u003e","manuscriptTitle":"Childhood outcomes of fetal genomic copy number variants: the prenatal microarray cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 07:29:43","doi":"10.21203/rs.3.rs-6952328/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"196e62dd-aeb0-4c06-9c13-7288362562b3","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50418081,"name":"Obstetrics \u0026 Gynecology"},{"id":50418082,"name":"Pediatrics"},{"id":50418083,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2025-06-25T07:29:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-25 07:29:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6952328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6952328","identity":"rs-6952328","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}