(2024) 24:536
Pynaker et al. BMC Pediatrics
https://doi.org/10.1186/s12887-024-05012-6

BMC Pediatrics

RESEARCH

Open Access

Perinatal outcomes after a prenatal
diagnosis of a fetal copy number variant:a retrospective population‑based cohort study
Cecilia Pynaker1, Jacqui McCoy1, Jane Halliday1,2, Sharon Lewis1,2, David J. Amor1,2,3, Susan P. Walker4,5,
Lisa Hui1,4,5,6* and On behalf of the PALM cohort study group

Abstract
Background There are no established guidelines for the follow up of infants born after a prenatal diagnosis
of a genomic copy number variant (CNV), despite their increased risk of developmental issues. The aims of this study
were (i) to determine the perinatal outcomes of fetuses diagnosed with and without a CNV, and (ii) to establish
a population-based paediatric cohort for long term developmental follow up.
Methods An Australian state-wide research database was screened for pregnant individuals who had a prenatal
chromosomal microarray (CMA) between 2013–2019 inclusive. Following linkage to laboratory records and clinicalreferrer details, hospital records were manually reviewed for study eligibility. Eligible participants were mother–childpairs where the pregnancy resulted in a livebirth, the mother was able to provide informed consent in English (didnot require a translator) and the mother was the primary caregiver for the child at hospital discharge after birth.
Research invitations were sent by registered post at an average of six years after the prenatal diagnostic test. Statisticalanalysis was performed in Stata17.
Results Of 1832 prenatal records examined, 1364 (74.5%) mother–child pairs were eligible for recruitment
into the follow up cohort. Of the 468 ineligible, 282 (60.3%) had ‘no live pregnancy outcome’ (209 terminationsof pregnancy (TOP) and 73 miscarriages, stillbirths, and infant deaths), 157 (33.5%) required a translator, and 29 (6.2%)
were excluded for other reasons. TOP rates varied by the type of fetal CNV detected: 49.3% (109/221) for pathogenic
CNVs, 18.2% (58/319) for variants of uncertain significance and 3.3% (42/1292) where no clinically significant CNV
was reported on CMA. Almost 77% of invitation letters were successfully delivered (1047/1364), and the subsequent
participation rate in the follow up cohort was 19.2% (201/1047).
Conclusions This study provides Australia’s first population-based data on perinatal outcomes following prenatal
diagnostic testing with CMA. The relatively high rates of pregnancy loss for those with a prenatal diagnosis of a CNV
presented a challenge for establishing a paediatric cohort to examine long term outcomes. Recruiting a mother–childcohort via prenatal ascertainment is a complex and resource-intensive process, but an important step in understanding the impact of a CNV diagnosis in pregnancy and beyond.
Trial registration ACTRN12620000446965p; Registered on April 6, 2020.

*Correspondence:
Lisa Huilisa.hui@unimelb.edu.au
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Pynaker et al. BMC Pediatrics(2024) 24:536

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Keywords Prenatal diagnosis, Chromosomal microarray analysis, Copy number variants, Variant of uncertain
significance, Perinatal outcomes, Cohort studies, Paediatric population, Feasibility studies

Background
Chromosomal microarray analysis (CMA) can interrogate the human genome to a higher resolution thanG-banded karyotyping [1]. This has enabled the detectionof submicroscopic deletions and duplications, termed
copy number variants (CNVs), which can be benign or
pathogenic depending on their location and gene content. CMA is well-established as the gold-standard
first-tier diagnostic test for paediatric patients with an
unexplained developmental disability, intellectual disability or congenital anomalies, providing 15–20% higher
diagnostic yield than G-banded karyotyping [2–4].
Alongside paediatric care, CMA has also revolutionised prenatal care. It has been 10 years since CMA
replaced G-banded karyotyping as the recommended
diagnostic test for fetuses with an ultrasound abnormality because of its superior diagnostic yield [5–7]. CMAs
can detect pathogenic copy number variants (pCNV)
linked to established syndromes, but also CNV of uncertain clinical significance (VUS). VUS are CNVs that often
involve non-disease causing genes, may not have beenpreviously identified or described, or for which there islimited information on genotype–phenotype correlation
due to incomplete penetrance and variable expressivity.
In the Australian state of Victoria, there has been an
increase in the proportion of prenatal diagnostic tests
analysed with CMA, from 39.4% in 2013 to 93.1% in 2021,
regardless of indication for testing [8]. Concurrently, theabsolute number of fetuses diagnosed with a pCNV has
increased over the past decade, from 25 pCNVs in 2013
to 61 pCNVs in 2022 in the background of a declining
number of prenatal diagnostic procedures [8]. The mostfrequent pCNVs in our population are 22q11.2 deletion(DiGeorge syndrome), 4p16.3 deletion (Wolf-Hischhorn
syndrome), and 5p15.33 deletion (cri-du-chat syndrome)[9]. These account for 13.5%, 3.9%, 3.0% of pregnancies
with a pCNV respectively. However, the vast majority ofpCNVs are rare, which makes counselling on long termchildhood outcomes difficult, especially when ascertained before birth when the phenotype is incomplete.
A VUS is diagnosed in approximately 5% of fetuses following chorionic villus sampling or amniocentesis [8].
These VUS can be challenging as there is often no prenatal phenotype to guide CNV interpretation. The limited data available in the literature are commonly skewed
towards cases diagnosed postnatally, possibly biased
towards the more severe end of the phenotypic spectrum[10–12]. In Australia, there are currently no guidelines

for the routine follow up and assessment of children witha prenatally diagnosed VUS. Furthermore, as genomic
databases and clinical interpretation guidelines are
updated some prenatal VUS are subsequently reclassified as either pathogenic or benign [13]. This highlights
the challenge of providing appropriate long term care, as
children may not only be lost to follow up after the newborn period, but may also carry a nonextant genetic diagnosis throughout childhood due to changes in scientificknowledge [12, 14].
The PrenatAL Microarray (PALM) is a nationallyfunded cohort study of mother–child pairs who have
had a prenatal diagnosis with a chromosomal microarray (CMA) from 2013 to 2019 in the Australian state ofVictoria. In brief, this cohort study of children- with andwithout a prenatally-ascertained CNV- aims to examine
their developmental, social-emotional and health outcomes in early childhood through a range of parent completed questions, in person cognitive assessments, andclinical paediatric review [15]. The full protocol has beenpreviously published in this journal [15].
In this paper, we report the perinatal outcomes offetuses that had a prenatal chromosomal microarray(potential PALM participants, with and without a CNV),
including rates of termination of pregnancy (TOP) andspontaneous perinatal losses following prenatal diagnosis. We also present the challenges of creating a representative paediatric cohort of children from a prenatal
cohort.

Methods
Study population

This was a population-based study set in the Australianstate of Victoria. Victoria has approximately 78,000 births
per year with a median maternal age of 31.5 years [16].
All pregnant individuals are offered screening for fetal
structural anomalies and chromosomal conditions, withan uptake of 83.6% state-wide [17]. Between 2013–2021,
80.9% of prenatal diagnostic tests were analysed by CMA[8]. TOP is lawful on maternal request up to 24 weeks,
and after 24 weeks if two medical practitioners deem it“appropriate in all the circumstances” [18].
Maternity healthcare in Australia operates througha dual public–private system. Public care, funded by
Medicare, offers subsidized services in public hospitals.
Private care, covered by private health insurance or outof-pocket, provides additional options such as choosing

Pynaker et al. BMC Pediatrics(2024) 24:536

obstetricians and amenities in private hospitals. In Australia, approximately 75% of births occur in a public
hospital.
Eligibility criteria for paediatric cohort

Participants were eligible if: the pregnancy resulted in a
livebirth, they were the primary-caregiver for the childat hospital discharge, resident in the Australian state ofVictoria, and able to provide informed consent in English(did not require a translator).
Data sources

Multiple sources were utilised to identify and pre-screen
potential study participants.
Victorian prenatal diagnosis database

The Victorian Prenatal Diagnosis Database (VPDD) is a
population-based research dataset that collects all chromosome testing results from amniotic fluid and chorionic villus samples (CVS) [8]. The VPDD was screened
for pregnant individuals who underwent prenatal diagnosis with CMA from January 2013 to December 2019.
Clinical laboratories classified prenatal CNVs in accordance with the guidelines of the American College ofMedical Genetics (ACMG) and other established guidelines [19–21]. CNVs were classified as ‘pathogenic’
when they encompassed a region implicated in a welldescribed abnormal phenotype, as documented in multiple peer-reviewed publications. ‘Likely pathogenic’variants were CNVs that met the ACMG definitions ofa CNV ‘described in a single case report but with welldefined breakpoints and phenotype, both specific andrelevant to the patient findings’, or CNV interval ‘withvery compelling gene function that is relevant and specific to the reason for patient referral’. ‘Cases’ included
pregnancies with a pathogenic or likely pathogenic CNV(pCNV) and variant of uncertain significance (VUS).
‘Controls’ were pregnancies that had a primary clinicalindication other than an ultrasound abnormality and had‘no clinically significant genomic imbalance’ reported on
CMA. These included positive (‘high chance) screening
result (non-invasive prenatal testing, combined first trimester screening, or second trimester serum screening),and other testing indications (such as advanced maternalage, maternal request).
Due to the modifier of a structural anomaly or known
genetic condition on childhood outcomes, ‘controls’ witha clinical indication of a fetal structural abnormality, family history of a chromosomal condition or a single genecondition were excluded. However, there were some
controls where the ultrasound abnormality was a secondary indication after a primary indication of a positive
screening result. These were predominantly soft markers

Page 3 of 11(increased nuchal translucency, hypoplastic nasal bone).
Further details will be available in the next phase of the
PALM study and any controls with a major structuralabnormality on antenatal ultrasound excluded fromanalysis.
Results of single gene testing were not available for theentire cohort. These results were only available for the
final consented PALM study participants and will not bereported here.
Clinical laboratories that submitted the cases and controls to the VPDD internally reidentified records andobtained the name of the public maternity hospital or
private clinical referrer. Follow up was different for these
two groups:a) Public hospital medical record review
Hospital medical records were manually reviewed for
perinatal outcome and study eligibility. Perinatal outcomes were coded as either miscarriage (spontaneous
pregnancy loss < 20 weeks’ gestation), stillbirth (infantborn with no signs of life ≥ 20 weeks’ gestation), TOP,
neonatal death (death within 28 days of birth), infant
death (death within 2 years of birth), or live birth.
A minimum de-identified dataset was collected for all
individuals screened containing: hospital name, maternal postcode, test date, gestational age, clinical indication, CNV classification, perinatal outcome, parity, andstudy eligibility status. Maternal postcode was mapped
to the corresponding local government area and assigned
the relevant Index of Relative Socioeconomic Advantage
and Disadvantage (IRSAD) allocated by the Australian
Bureau of Statistics from 2016 Census data [22].
b) Private clinical referrers
Private clinicians were contacted by phone and/or
email and asked to pre-screen their patients for study
eligibility and send an invitation letter to eligible participants. No minimum dataset was collected for thesepatients as pre-screening was performed at the clinician’s
discretion.
Study recruitment

Study invitation letters were sent by registered post
between November 2021 and June 2023. Each contained
a participant information and consent form, hard-copyquestionnaire booklet, and replied paid envelope. Registered post enabled tracking of research letters, including proof of mailing and signature on delivery. The publicroll of the Australian Electoral Commission (AEC) wasused to check the details of participants whose post
were returned to sender. Participants had the option toPynaker et al. BMC Pediatrics(2024) 24:536

complete a hard copy or online consent form and questionnaire. Completed hard copies were returned using
the provided replied paid envelope, while the online version, hosted in Research Electronic Data Capture (REDCap), were accessed via a QR code in the invitation letter[23, 24].
Protocol amendments
Pandemic impacts

In response to the COVID-19 pandemic in 2020, thestudy protocol was updated to include alternative online
child assessments. Due to pandemic-related disruptions, a 12-month extension was requested due to delays
in recruitment, participant assessments, and obtaining
approvals for new regional sites.
Low recruitment rate

In response to a low recruitment rate, the study protocol
was amended. Participants were: (i) sent two reminder
letters, and (ii) offered an AUD$110 gift card in appreciation of their time. The first reminder was sent three
weeks after the initial study invitation (if successfully
delivered), and the second reminder three weeks after the
first reminder. Ethics Committee approval for all amendments was obtained.
Statistical analysis

Statistical analysis was performed in Stata17 using chisquared test for proportions with p < 0.05 considered significant [25]. Wilson score method was used to calculate
95% confidence intervals using Epitools [26].
Ethics approval

This study received Human Research Ethics Committee
approval from the Royal Children’s Hospital on April 8,2020 (Reference no. 60542) and Mercy Health on September 15, 2020 (Reference no. 2020–046).

Results
Pre‑screening of potential participants

During the 7-year study period 8184 prenatal diagnostic tests were performed by CMA; a fetal CNV wasreported for 1029 samples (12.6%, 95%CI: 11.9–13.3%),and ‘no clinically significant CNV’ reported in 7155 samples (87.4%, 95%CI: 85.7–88.1%). Of these, 4316 with ‘noclinically significant CNV’ were excluded due to an indication of an ultrasound abnormality, family history of achromosomal condition, or a single gene condition.
Figure 1 illustrates the pre-screening and recruitmentof participants with and without a copy number variant.
The personal identifiers of the 2458 potential casesand controls were provided to the eight relevant hospitals and were manually reviewed for eligibility criteria.

Page 4 of 11

Only 74.5% (n = 1832) had a known birth outcome documented in the hospital records. The remaining missing
birth outcome data was due to patients delivering in adifferent hospital to the one in which the prenatal diagnostic procedure was performed.
Of those with a known birth outcome (n = 1832), 1364
(74.5%) were eligible for recruitment (‘potential participants’) and 468 (25.5%) were ineligible. Of the 468 ineligible, 282 (60.3%) had ‘no live pregnancy outcome’ (209terminations of pregnancy (TOP) and 73 miscarriages, stillbirths, and infant deaths), 157 (33.5%) required a translator,and 29 (6.2%) were excluded for other reasons (Table 1).
Perinatal outcomes by CMA result

Birth outcomes varied significantly by the type of fetal
CNV detected and are presented in Table 2. Fetuses witha pathogenic CNV had a higher TOP rate compared withthose with a VUS (49.3% vs. 18.2%, p < 0.05) or ‘no clinically significant’ CNV (3.3%, p < 0.05).
Perinatal outcome by indication for prenatal diagnosis

The perinatal outcomes of pregnancies with and without a CNV varied by indication for prenatal diagnosis(Table 3). For pregnancies with a pCNV or VUS the mostcommon indication for prenatal diagnosis was an ultrasound abnormality, 54.8% (121/221) and 62.4% (199/319)
respectively. A TOP occurred in 56.2% (68/121) of caseswith a pCNV and an ultrasound abnormality. In comparison, when there was a VUS and an ultrasound abnormality, a TOP occurred in 30.6% (61/199) of cases. Almost
two percent of pregnancies with ‘no clinically significant
genomic imbalance’ and a high chance screening result
resulted in a TOP (17/1067). These did not have an ultrasound abnormality.
Responders and non‑responders

A total of 3304 research letters were sent to 1364 eligible patients over the 20-month study period (1607 invitations (including repeats), 893 first reminders, 804 s
reminders) (Fig. 1). Initially, a third of all study invitations
were returned to sender due to incorrect address (31.9%,
435/1364). An updated address was found for 43.2%(188/435) of these using the AEC public electoral roll.
Overall, 1047 participants were successfully contacted(1047/1364, 76.8%).
The rate of informed consent to participate in the
PALM study among those who were successfully contacted (‘responders’) was 19.2% (201/1047) (Fig. 1).
Comparison of cases and controls

Data collected through examination of hospital medical records were used to compare the 1832 ‘potential

Pynaker et al. BMC Pediatrics(2024) 24:536

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Fig. 1 Study flowchart of the pre-screening and recruitment of participants. Abbreviations: CNV, copy number variant; pCNV, pathogenic copy
number variant; VUS, copy number variant of uncertain significance. † In total, 14 participants were recruited into the study through a private
obstetrician or general practitioner (data not shown)participants’ (with a known birth outcome) stratified by
CNV status (cases vs. controls).
There were no consistent patterns of difference in sociodemographic characteristics between cases and controls in their study eligibility or ability to be contacted
via mail (see Supplementary Table 1 and Supplementary
Table 2 for details). A slight difference in response rate

was observed with 23.8% of cases responding comparedwith 17.8% of controls (Table 4).
Compared with non-responding controls, participating controls were significantly more likely to be of higher
socioeconomic status and lower parity. There was no evidence of any differences between responders and nonresponding cases (Table 4).

Pynaker et al. BMC Pediatrics(2024) 24:536

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Table 1 Reasons for study ineligibility
Reason for study ineligibility

Pathogenic copy
number variant

Variant of uncertain
significanceNo clinically significant
genomic imbalanceTotaln (% pCNVs)n (% VUS)n (% NAD)n (% total)

Perinatal/infant death

128 (90.1)

75 (72.8)

79 (35.4)

282 (60.3)

Translator required

12 (8.5)

22 (21.4)

123 (55.2)

157 (33.5)

Mother (gestational carrier) not the intended
primary caregiver

1 (0.7)

1 (1.0)7 (3.1)9 (1.9)Multifetal pregnancy

0 (0.0)0 (0.0)2 (0.9)2 (0.4)

Unable to provide informed consent

0 (0.0)1 (1.0)0 (0.0)1 (0.2)Othera1 (0.7)4 (3.9)12 (5.4)

17 (3.6)

Total

142 (100.0)

103 (100.0)

223 (100.0)

468 (100.0)

Abbreviations: pCNV pathogenic copy number variant, VUS variant of uncertain significance, NAD no clinically significant genomic imbalanceaOther reasons for study ineligibility were made at the discretion of the site Principle Investigator

Table 2 Birth outcome of fetuses with and without a copy number variant
Pregnancy result

Pathogenic copy
number variant

Variant of uncertain
significanceNo clinically significant
genomic imbalanceTotaln (% pCNVs)n (% VUS)n (% NAD)n (% total)

Livebirth

93 (42.1)

244 (76.5)

1213 (93.9)

1550 (84.6)

Termination of pregnancy

109 (49.3)

58 (18.2)

42 (3.3)

209 (11.4)

Spontaneous stillbirth (≥ 20 weeks)

9 (4.1)

10 (3.1)

19 (1.5)

38 (2.1)

Miscarriage (< 20 weeks)

7 (3.2)2 (0.6)

10 (0.8)

19 (1.0)

Neonatal death (death within 28 days of birth)

2 (0.9)4 (1.3)8 (0.6)14 (0.8)

Infant death (death within 2 years of birth)

1 (0.5)1 (0.3)0 (0.0)2 (0.1)

Total

221 (100.0)

319 (100.0)

1292 (100.0)

1832 (100.0)

Abbreviations: pCNV pathogenic copy number variant, VUS variant of uncertain significance, NAD no clinically significant genomic imbalancePrivate practice referrers

A substantial proportion of patients were referred fortheir diagnostic procedure by a private referrer rather
than a public hospital (1132/3868, 29.3%, Fig. 1). These
1132 patients were referred by 497 private clinicians. Of
these, 304 clinicians (associated with 874 patients) could
be contacted by phone and/or email; 28 clinicians (9.2%;
associated with 119 patients (13.6%)) agreed to prescreen their patients for study eligibility; 14 patients were
ultimately recruited through this method. Data fromthe 119 patients screened by private referrers were notcollected and were excluded from this analysis as prescreening of potential participants was performed at thediscretion of the private referrer.
Regional participants

One in five patients who had a prenatal diagnostic procedure in a tertiary hospital was referred from a regional
hospital (175/850, 20.6%). To assist recruitment andminimise selection bias by location, the protocol wasamended to include data collection from the three largest
referring regional health services (representing 156/175

regional patients). It took on average 20-months fromethics amendment approval to site governance approval.
The additional burden of adding these three sites resulted
in an additional 9 participants (all cases).
Recruitment rate

Overall, the 201 mother–child pairs were recruited
into the PALM childhood outcome study, comprising
144 controls and 57 cases (10 pCNVs, 47 VUS). This
cohort represented 2.5% of the initial 8184 prenatal
diagnosis cases pre-screened from the Victorian Prenatal Diagnosis Data Collection. This was lower than 8.7%
(719/8184) recruitment rate estimated in the original
study protocol [15].

Discussion
Principle findings

CMA has been instrumental in improving the diagnostic
yield of prenatal diagnosis, but published data on the perinatal and paediatric outcomes of fetal CNVs are scarcedue to their rarity. We conducted a lengthy and thoroughmanual linkage process to retrospectively recruit 201

Pynaker et al. BMC Pediatrics(2024) 24:536

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Table 3 Birth outcomes of pregnancies with and without a copy number variant by indication for prenatal diagnosis
Indications forprenatal diagnosis

Livebirth

Termination ofpregnancyn (% indication) n (% indication)

Spontaneous
stillbirth(≥ 20 weeks)

Miscarriage(< 20 weeks)

Neonatal/infant
death

Total

n (% indication)n (% indication)n (% indication)n (% indication)

Pathogenic copy number variant
Ultrasound­abnormalitya

39 (32.2)

68 (56.2)

7 (5.8)4 (3.3)3 (2.5)121 (100.0)

Positive screening­resultb

35 (64.8)

16 (29.6)

1 (1.9)2 (3.7)0 (0.0)54 (100.0)

All other testing­indicationsc

19 (41.3)

25 (54.3)

1 (2.2)0 (0.0)0 (0.0)46 (100.0)

Total

93 (42.1)

109 (49.3)

9 (4.1)7 (3.2)3 (1.4)221 (100.0)

Variant of uncertain significanceUltrasound­abnormalitya

138 (68.3)

46 (23.1)

9 (4.5)2 (1.0)4 (2.0)199 (100.0)

Positive screening­resultb

74 (93.7)

3 (3.8)1 (1.3)0 (0.0)1 (1.3)79 (100.0)

All other testing­indicationsc

32 (78.0)

9 (22.0)0 (0.0)0 (0.0)0 (0.0)41 (100.0

Total

244 (76.5)

58 (18.2)

10 (3.1)

2 (0.6)5 (1.6)319 (100.0)

No clinically significant genomic imbalance
Ultrasound­abnormalitya

53 (68.8)

18 (23.4)

4 (5.2)

1 (1.3)

1 (1.3)77 (100.0)dPositive screening­resultb

1024 (96.0)

17 (1.6)

11 (1.0)8 (0.7)7 (0.7)1067 (100.0)

All other testing­indicationsc

136 (91.9)

7 (4.7)4 (2.7)1 (0.7)0 (0.0)148 (100.0)

Total

1213 (93.9)

42 (3.3)

19 (1.5)

10 (0.8)8 (0.6)1292 (100.0)aUltrasound abnormality included soft marker on ultrasound such as increased nuchal translucency and hypoplastic nasal boneb

Positive (‘high chance’ or ‘high risk’) screening result included non-invasive prenatal testing, first trimester combined screening and second trimester serum
screeningc

All other testing indications” included family history of chromosomal abnormality, single gene condition, and advanced maternal age

d70/77 had a concurrent indication of a positive (‘high chance’ or ‘high risk’) screening result

mother–child pairs for a prospective childhood outcome
study from a starting data source of re-identifiable prenatal diagnostic test results. This complex process was necessary to overcome the challenges of maintaining patient
privacy and traversing siloed medical record information
across health services and research institutes.
Results in the context of what is known

Overall, the rates of TOP for the pCNVs in our cohort
were at the lower end of the range reported in the literature. We observed a 49.3% TOP rate for pCNVs overall and a 56.2% TOP rate for pCNVs with an ultrasoundabnormality. This compares with TOP rates for pCNV
ranging from 50–100% in other studies [13, 14, 27–32].
Higher rates have been report for pCNVs with an ultrasound abnormality (either structural or soft marker)(76.2–100%) in China, France, and Israel [13, 29–32].
Similarly, for VUS we observed a 18.2% TOP rate for
VUS overall and a 23.1% rate for VUS with an ultrasoundabnormality. Again, this aligns with the lower end ofreported TOP rates for VUS from 11.0–44.9% [14, 29–31,
33, 34], with higher rates for de novo VUS (50.8–58.0%)[13, 34] and fetuses with an ultrasound abnormality(17.1–37.5%) [13, 29–31, 34]. There are many methodological factors that may contribute to the different TOP
rates for pCNV and VUS that preclude meaningful directcomparisons between studies. These factors include
highly variable cohort sizes, study inclusion criteria, clinical testing pathways, CNV classification systems, healthsystem and patient factors, availability of TOP, and timing of prenatal diagnosis.
Clinical and research implications

Paediatrics cohorts established from prenatal genomic
testing populations are rare due to logistical and ethicalchallenges, yet they are crucial for obtaining long-term
outcome data. A previous cohort of this nature examinedthe childhood outcomes of children prenatally diagnosed
with confined placental mosaicism in the Australian stateof Victoria [35]. By utilising the same prenatal diagnosis

Pynaker et al. BMC Pediatrics(2024) 24:536

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Table 4 Potential sources of participation bias
Cases
Variable

Controls

Responders(participants)

Non-responders

n = 57 (%)

n = 182 (%)

P value

Responders(participants)

Non-responders

n = 144 (%)

n = 664 (%)

P value

IRSAD quintile
1 (most disadvantaged)

9 (15.8)

29 (15.9)

24 (7.0)25 (13.7)

0.40

6 (4.2)75 (11.3)

22 (15.3)

77 (11.6)

3

13 (22.8)

52 (28.6)

29 (20.1)

184 (27.7)

4

17 (29.8)

46 (25.3)

48 (33.3)

190 (28.6)

5 (most advantaged)

14 (24.6)

20 (16.5)

39 (27.1)

138 (20.8)

0.012

Remoteness area
Metropolitan

45 (78.9)

158 (86.8)

Regional/remote

12 (21.1)

24 (13.2)

0.15

127 (88.2)

613 (92.3)

17 (11.8)

51 (7.7)

0.11

Mother’s age at recruitment
35 years

13 (22.8)

48 (26.4)

8 (5.6)67 (10.1)

35 – 39 years

20 (35.1)

45 (24.7)

0.31

35 (24.3)

155 (23.3)≥ 40 years

24 (42.1)

89 (48.9)

101 (70.1)

442 (66.6)

0.24

Parity022 (38.6)

58 (31.9)55 (38.2)

205 (30.9)

1

21 (36.8)

68 (37.4)

0.83

50 (34.7)

251 (37.8)

29 (15.8)

37 (20.3)

30 (20.8)

115 (17.3)

3

3 (5.3)14 (7.7)

7 (4.9)43 (6.5)

4+2 (3.5)5 (2.7)0 (0.0)34 (5.1)

Missing

0 (0.0)0 (0.0)2 (1.4)16 (2.4)

0.0042Child’s age at recruitment≥ 5 years

25 (43.9)

58 (31.9)

5 years

32 (56.1)

124 (68.1)

0.097

35 (24.3)

173 (26.1)

109 (75.7)

491 (73.9)

0.66

IRSAD Index of Relative Socioeconomic Advantage and Disadvantage

dataset this paediatric cohort was not biased towards
children with an established clinical phenotype. Despite
contacting participants 5.5 years after prenatal diagnosis, the study achieved a recruitment rate of 76%. This isalmost four times the response rate achieved in our study(19.1%). It is unknown what factors contributed to thehigher recruitment rate but one factor could be the clinician-patient relationship as the treating doctor facilitatedthe contact between the participant and the researchteam, rather than a hospital departmental representative
as in our study. Moreover, despite modifying the study
protocol to minimise exposure to COVID-19 (such asoffering virtual assessments), the pandemic might havenegatively influenced patient attitudes towards participating in research studies [36].
There are only two other studies that have reported
paediatric outcomes of children with a prenatal diagnosis of a CNV [13, 37]. Shi et al. prospectively followed up
109 children with a prenatally diagnosed VUS up the ageof 2–4 years. Five children apparently showed clinicalsigns or phenotypic features of disease, but the clinicalassessments were not described in detail. Muys et al.
retrospectively recruited 85 mother–child pairs with apCNV or VUS (‘cases’) and 123 with no or a benign CNV(‘controls’). The response rate of 15.8% (208/1312) was
lower than our results, even though their study had lower
participant burden (parental questionnaire only). However, these response rates may not be comparable. Muys
et al. did not perform extensive pre-screening to determine perinatal outcomes: several participants were ineligible due to a perinatal or neonatal death. Had we not
performed our extensive pre-screening step, 282 women
who experienced a perinatal loss would have been invited
to participate in our childhood outcome study. Also,
Muys et al. did not report the number of research invitation letters successfully delivered, only the total number sent. Use of registered post and manual follow up at
the AEC public roll enabled us to determine the numberof potential participants successfully contacted. Of note
21.3% (43/201) of our final cohort were contacted following use of the AEC to update contact addresses, demonstrating the value of this additional step.

Pynaker et al. BMC Pediatrics(2024) 24:536

Strengths and limitations

This is one of the largest studies to manually determine
the perinatal outcomes of pregnancies with and without aprenatally diagnosed CNV. Through this process, we havesuccessfully established a paediatric cohort and prospectively conducted detailed cognitive and clinical assessments on each child. Data analysis is currently underway,
with public dissemination of results expected in 2024.
Another one of our strengths was the attention paid
to patient psychological safety through manual record
review and the exclusion of patients who had experienced a perinatal loss or infant death. We thereby averted
potential distress for 282 families by removing themfrom our study invitation list. However, the extensive
pre-screening procedures at multiple maternity hospitals
and the siloed nature of Australian health records pose
substantial resource and administrative barriers to future
research of this type.
One of the limitations of our cohort is the missing birth
data on one quarter of potentially eligible patients due toprivate and regional referral patterns. This finding highlights the challenges in tracing participants and engaging
with hospitals for population-based research studies.
Another limitation of this study is that our ethics
approval did not permit us to retain data on perinatal
outcomes by specific CNVs due privacy concerns withpotentially identifying information. We have previously
reported the most common CNVs in our populationfrom 2012 to 2018 [9]. The three most frequent pathogenic CNVs were 22q11.2 deletion syndrome, 4p16.3
deletion, and 5p15.33 deletion. The three most frequent
VUS were 15q11.2 del, 22q11.2 duplication and 1q21.1
duplication. Further details including perinatal outcomes and ultrasound abnormalities are available in thatpublication.
Future directions

Establishing systems that enable the routine paediatricfollow up of prenatally diagnosed genetic abnormalities
are essential for understanding the full phenotypic spectrum of CNVs. In particular, there are no standard recommendations regarding long term follow up of children
with VUS. Our cohort will provide long term outcome
data on the developmental outcomes of children with
VUS that may help guide future clinical care.
One of the barriers to collecting long term outcomesfrom genomic CNVs is the limitations of current congenital anomaly coding systems such as the International Classification of Diseases ­10th Revision (ICD-10).
The ICD-10 has very few specific genetic diagnostic
codes as it is based on phenotypes and organ systems.

Page 9 of 11

More locally, our state-wide Victorian Congenital
Anomalies Report reports the population prevalenceand birth outcomes for common autosomal trisomies
but not CNVs (pathogenic or VUS) [38]. Expanding
the reporting of congenital anomalies to encompass
a broader spectrum of genomic variants would offer
a more comprehensive understanding of the impact
of prenatal diagnosis and enable us to perform better
quality linkage studies for long term outcome data.

Conclusion
This study provides Australia’s first population-based
data on perinatal outcomes including termination ofpregnancy following prenatal diagnostic testing with
CMA. Three-quarters of fetuses with a VUS and lessthan half of fetuses with a pCNV resulted in a livebirth.
Our establishment of a mother–child cohort via prenatal ascertainment was a complex and resource-intensive
process, but an important step in understanding theimpact of a CNV diagnosis in pregnancy and beyond.
Abbreviations
AEC	Australian Electoral Commission
CMA	Chromosomal microarray
CNV	Copy number variant
CVS	Chorionic villus sampling
HREA	Human Research Ethics Application
HREC	Human Research Ethics Committee
MCRI	Murdoch Children’s Research Institute
NHMRC	National Health and Medical Research Council
PALM	PrenatAL Microarray
pCNV	Pathogenic copy number variantTOP	Termination of pregnancy
VPDD	Victorian Prenatal Diagnosis Data collection
VUS	Variants of uncertain/unknown significanceSupplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12887-​024-​05012-6.
Supplementary Material 1: Supplementary Table 1. Sociodemographic
characteristics of eligible and ineligible participants. IRSAD, Index of Relative Socioeconomic Advantage and Disadvantage.
Supplementary Material 2: Supplementary Table 2. Sociodemographic
characteristics of cases and controls by ability to be contacted by mail.
IRSAD, Index of Relative Socioeconomic Advantage and Disadvantage.
Acknowledgements
We thank the associate and site investigators in the PALM study group: Ms
Joanne Kennedy (Murdoch Children’s Research Institute, Royal Women’s Hospital), Ms Fiona Norris (Victorian Clinical Genetics Services), Ms Lucy Gugasyan(Monash Pathology), Ms Emma Brown (Monash Pathology), Dr Suzanne Svobodova (Monash Pathology), Dr Matthew Regan (Monash Medical Centre), Ms
Helen Kincaid (Monash Medical Centre), Dr Anand Vasudevan (Royal Women’s
Hospital and Western Health), Ms Susan Fawcett (Royal Women’s Hospital), Ms
Melissa Graetz (Mercy Hospital for Women), Prof Joanne Said (Western Health),
Dr Lisa Begg (Box Hill Hospital, Eastern Health), Dr Nicole Yuen (Bendigo
Health), Dr Natasha Frawley (Grampians Health Ballarat), and Dr Geraldine
Masson (Barwon Health Geelong).

Pynaker et al. BMC Pediatrics(2024) 24:536

Authors’ contributions
LH and JH conceived the original concept for this study. LH was the lead writerfor the study protocol and funding application, with intellectual input from all
members of the chief investigator team (JH, DA, SL, SP). CP and JM are salaried
members of the PALM study team. Data collection was performed by CP andJK; CP and LH prepared the first draft of the manuscript. All authors read, provided intellectual input and approved the final version of this manuscript.
Funding
This study is being funded by a NHMRC Clinical Trials and Cohort Studies grantfor 2020–2022 (NHMRC APP1186862). The funding body has had no role in the
study design, in the writing of the protocol or in the decision to submit the
paper for publication.
LH receives salary support from a Medical Research Future Fund investigator
grant (APP1196010) and the University of Melbourne.
The PALM study is sponsored by MCRI. The contact on behalf of the sponsor is LH.
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.

Page 10 of 11

6.

7.
8.

9.
10.
11.

12.

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.
Competing interests
The authors declare no competing interests.
Author details
1
Reproductive Epidemiology Group, Murdoch Children’s Research Institute,
Parkville, VIC, Australia. 2 Department of Paediatrics, University of Melbourne,
Parkville, VIC, Australia. 3 Neurodisability and Rehabilitation Group, MurdochChildren’s Research Institute, Parkville, VIC, Australia. 4 Department of Obstetrics, Gynaecology and Newborn Health, University of Melbourne, Parkville, VIC,
Australia. 5 Mercy Hospital for Women, Heidelberg, VIC, Australia. 6 Northern
Health, Melbourne, VIC, Australia.

13.
14.
15.
16.
17.

18.
19.

Received: 7 February 2024 Accepted: 13 August 2024
20.

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