Chromosomal analysis and short-term outcome of prenatally diagnosed complex congenital heart disease

doi:10.21203/rs.3.rs-5038076/v1

Congenital structural heart disease (CHD) is the leading cause of infant death from birth defects. Postnatal survival primarily depends on the type and severity of the defect. In addition, worse cardiac prognosis is observed when extra-cardiac anomalies (ECA) are associated. This retrospective chart review was aimed at finding markers for short-term outcome prediction of prenatally-diagnosed complex CHD, focusing in particular on the impact of CHD category, of CHD severity score and of prenatal or postnatal diagnosis of ECA or chromosomal anomalies on 4 primary outcomes: termination of pregnancy (TOP), intrauterine fetal demise, neonatal mortality and 1-year-survival rate. We reviewed medical files from 381 fetuses, presenting at our center between 2018 and 2021 with CHD for which prenatal advice by a pediatric cardiologist was sought. 341 fetuses met the inclusion criteria for the study. Twin pregnancies (7.62%; OR 4.76 (p<0.001)) and pregnancies resulting from assisted reproductive technology (7.33%; OR 2.44 (p<0.001)) were more prevalent compared to the general population. CHD categories and CHD severity scores, ranging from A (extremely high risk) to D (low risk), were assigned to each fetus. Prenatal or postnatal chromosomal microarray results were available for 232 fetuses (68%) and were abnormal in 30 (12.9%). Logistic regression analysis was used to determine significant predictors for the 4 primary outcomes. TOP was carried out significantly more with: prenatal genetic diagnosis (p<0.001), prenatal extracardiac symptoms (p<0.001), severity score A (p=0.045) and B (p=0.036). Survival was significantly lower with: prenatal extracardiac symptoms (p=0.002), prematurity (p=0.046), severity scores A (p=0.009) and B (p=0.004), and with univentricular heart. CHD severity score, chromosomal anomalies and ECA are determining factors for continuation of the pregnancy, for survival and for overall mortality, underscoring the importance of prenatal ultrasound and genotyping for prognostication of fetuses with CHD.

Health sciences/Cardiology/Cardiovascular biology/Cardiovascular genetics

Biological sciences/Genetics/Cytogenetics

Health sciences/Health care/Diagnosis/Genetic testing

Health sciences/Health care/Health services/Genetic services/Genetic counselling

Health sciences/Diseases/Cardiovascular diseases/Congenital heart defects

congenital heart defects

chromosomal

prenatal

outcome

Congenital heart disease (CHD) is the most common type of birth defect, occurring in about 1% of pregnancies and in 0.8% of live births ^1,2. Although surgical advances have improved the pooled survival rate of CHD to adulthood to about 90% ³, CHD remains the leading cause of mortality from birth defects and imposes a heavy disease burden. Neonatal outcome of prenatally-diagnosed CHD primarily depends on the type and severity of the defect. CHD types are often grouped together in CHD categories based on shared morphology or embryological mechanisms. The most common prenatally diagnosed CHD categories are septal defects (16-48%), conotruncal heart defects (20%), left ventricle outflow tract obstruction (LVOTO, 7-21%), right-sided anomalies (5-7%), univentricular hearts (UVH) (5%) and heterotaxy (6%) ^4–7. Postnatal mortality ranges from 38% in UVH to 2.5% in ventricular septal defects ^8,9. However, outcome varies within each CHD category and even between individuals with the same CHD type. For example individuals with isolated d-transposition of the great arteries (d-TGA) have a better outcome compared to those with complex d-TGA ¹⁰. Neonatal CHD outcome is further determined by perinatal CHD management, by pregnancy complications, such as prematurity, dysmaturity or multiple pregnancies, and by demographic parameters ^5,11,12. In addition, worse cardiac (and extra-cardiac) prognosis is observed when extra-cardiac anomalies (ECA) and/or genetic pathogenic variants are present ^5,7.

The diagnostic yield of genetic testing highly depends on CHD type, family history and co-occurrence of ECA. The majority of CHD are non-syndromic (NS-CHD) (80%) and have a complex background with an interplay of hitherto unknown genetic and environmental factors. Chromosomal or genetic pathogenic variants are found in less than 5% of individuals with sporadic NS-CHD, increasing up to 10-30% for familial CHD, which represents 3-5% of the NS-CHD cohort ^13–15. Syndromic CHD (S-CHD), defined as CHD in association with additional congenital defects and/or abnormal growth (>2SD or <-2SD), development and/or behavior, represents 20% of the CHD cohort. Aneuploidy, including monosomy X and trisomy 21, 13 or 18, is observed in about 14% of individuals with S-CHD ¹⁶, while submicroscopic copy number variants (CNVs) are identified by chromosomal microarray (CMA) or CNV sequencing (CNVseq) in 15-20% ⁴. The spectrum of CHD-related pathogenic CNVs ranges from recurrent clinically recognizable CNV syndromes, like 22q11.2 deletion syndrome or Williams-Beuren syndrome, to partially overlapping CNVs with unique breakpoints comprising dosage-sensitive genes or regulatory elements. Pathogenic or likely pathogenic single nucleotide variants (SNVs), diagnosed by Sanger sequencing, by targeted gene panels or by exome or genome sequencing, are found in about 35-40% of individuals with S-CHD with normal CMA results ^17–19.

When CHD is diagnosed prenatally, S-CHD is difficult to differentiate from NS-CHD, as assessment of facial features and development is impossible or limited, and some ECA, such as coloboma, minor limb defects or cleft palate, may be missed. Therefore, prenatal genetic testing is gaining importance in the prognostication of fetuses with complex CHD. Non-invasive prenatal testing is increasingly applied to screen for fetal aneuploidies, leading to diagnosis of trisomy 21, 18 or 13, even before CHD is diagnosed on prenatal ultrasound ²⁰. Although sensitivity to detect submicroscopic CNVs by NIPT is increasing, prenatal invasive CNV analysis by CMA or CNVseq is considered the gold standard to identify pathogenic CNV when complex CHD or S-CHD is diagnosed prenatally, with a diagnostic yield of about 10-15%, including fetuses with apparently isolated CHD on ultrasound ^5–7,20. In those with normal CNV results, a pathogenic genetic variant is found by prenatal trio exome sequencing (ES) in 5-21%, varying according to presence of ECA and/or to CHD type ^{5–7,21–23}. In a systematic review, comprising 18 studies, the yield of prenatal ES was 21%, 11% and 37% respectively in all fetuses with CHD, in fetuses with apparently isolated CHD and in fetuses with CHD associated with ECA ²³.

This single-center retrospective chart review is aimed at finding markers for short-term outcome prediction of prenatally-diagnosed complex CHD, focusing in particular on the impact of CHD category, of CHD severity score and of prenatal or postnatal diagnosis of ECA or submicroscopic pathogenic CNVs on 4 primary outcomes: termination of pregnancy (TOP), intrauterine fetal demise (IUM), neonatal mortality and 1-year-survival rate.

Study approval

Ethical approval to study these files was obtained from the ethical commission of KU and UZ Leuven (MP020190). Due to the retrospective nature of the study, the ethical commission of KU and UZ Leuven waived the need of obtaining informed consent. The study was conducted in accordance with the ethical standards of the Helsinki Declaration.

Inclusion criteria

Medical charts were reviewed from all fetuses or children who were diagnosed and/or followed prenatally with CHD at the obstetrics and pediatric cardiology department at University Hospitals Leuven (UZL) between January 1, 2018 and November 22, 2021. Inclusion was restricted to fetuses or children with CHD requiring prenatal counseling by a pediatric cardiologist at UZL, regardless of ethnic background, type of CHD on prenatal ultrasound, course of the pregnancy, neonatal outcome, and regardless of the eventual cardiac diagnosis made postnatally or post-mortem. Fetuses appearing to have a normal heart on first trimester ultrasound and diagnosed with fetal aneuploidy (trisomy 21, 18 or 13, or monosomy X) by non-invasive prenatal screening (NIPS) were excluded. Additional exclusion criteria were: (1) fetuses with prenatal diagnosis of arrhythmia, cardiomyopathy, left-sided superior caval vein or cardiac tumors in the absence of CHD, (2) children with postnatal diagnosis of CHD, (3) fetuses referred to UZL for a second opinion after CHD diagnosis in a different university hospital, (4) fetuses with CHD for whom no prenatal advice by a pediatric cardiologist was sought either because of a low risk CHD (not requiring neonatal intervention), or because of a suspected poor prognosis due to severe ECA (for which termination of pregnancy (TOP) was requested regardless of the cardiac prognosis).

Data collection

Medical information, demographic data and results from diagnostic genetic testing were retrieved from fetal, pediatric and maternal medical files, and were pseudonymized. Familial history of CHD (up to 3rd degree relatives) and/or familial occurrence of known pathogenic CNVs or SNVs for congenital or developmental disorders was recorded. Pregnancy information included: maternal age at the start of pregnancy, gravidity, spontaneous pregnancy versus assisted reproduction, singleton versus multiple pregnancy, and postmenstrual age (PMA) at the time of fetal CHD diagnosis. Pre- and postnatal CHD types were recorded based on the recordings by the pediatric cardiologist. ECA diagnosed either prenatally, at birth or at post-mortem investigation were documented. ECA included additional congenital anomalies, intrauterine growth restriction (IUGR), micro/macrocephaly, increased nuchal translucency and congenital anomalies with little or no functional impact (e.g. facial dysmorphic features, hyperechogenic bowel, single umbilical artery…). Acquired anomalies (e.g. ischemic or infectious brain damage, post-surgical diaphragm paralysis, feeding difficulties…) were not considered as ECA. Primary pregnancy outcomes were (1) termination of pregnancy, (2) intrauterine fetal demise, (3) live birth. Postnatal outcome was documented as (1) 1-year survival, (2) early postnatal demise (<1 month) (3) demise during infancy (>1 month <1 year).

CHD categories and CHD severity scores

CHD types were grouped together in CHD categories based on shared morphology or embryological mechanisms in accordance with the classification that was used by Gowda et al. ²⁴ (Table 1). Only one CHD category was ascribed to each fetus. If several CHD types were diagnosed, CHD classification was based on the most important prognostic and anatomical cardiac defect (e.g. isolated pulmonary valve atresia (PA), tetralogy of Fallot with PA and univentricular heart with PA were classified respectively as a right sided heart defect, conotruncal heart defect and univentricular heart).

In addition, a severity score was assigned to each fetus based on the cardiac phenotype or the co-occurrence of additional major congenital anomalies and/or chromosomal aneuploidy. A 4-class scoring system (A, B, C or D) was applied as described by Gowda et al. ²⁴ (Supplementary Table 1). In summary, severity score A was assigned to CHD associated with severe potentially lethal ECA (e.g. congenital diaphragmatic hernia or hydrops fetalis), with aneuploidy or with genetic disorders associated with severe intellectual impairment (extremely high risk). Score B corresponds to isolated CHD requiring multiple surgeries and associated with high mortality after surgery (high risk). Score C (moderate risk) and score D (low risk) relate to isolated CHD with respectively variable and good prognosis after surgery. For severity scores A and B, the option of TOP was considered whenever legally permissible. For scores C and D continued monitoring of pregnancy with postnatal surgery was emphasized if applicable. Prenatal genetic work-up by CMA was recommended for fetuses with scores A or B, and CMA was offered, either prenatally or postnatally, for all other fetuses ²⁵.

Isolated versus non-isolated CHD

Fetuses were classified as having non-isolated CHD when associated with at least one of the following prenatal ultrasound findings: (1) additional major congenital anomaly, (2) microcephaly and/or IUGR (<-2 SD), (3) facial dysmorphic features, (4) increased nuchal translucency (>3.5 mm at 12 weeks of gestation), cystic hygroma or hydrops fetalis, (5) isomerism/situs anomalies. If prenatal phenotyping was restricted to the cardiac phenotype, fetuses were considered to have CHD with undetermined extracardiac status.

Postnatal classification of non-isolated CHD was defined as CHD in association with at least one of the following criteria: (1) additional major congenital anomaly, (2) microcephaly and/or abnormal growth (<-2.5 SD or >+2.5 SD) taking gestational age at birth into consideration, (3) facial dysmorphic features (defined as the presence of at least 3 minor facial anomalies), (4) severe unexplained hypotonia or motor delay, (5) pathogenic or likely pathogenic CNVs or SNVs associated with developmental disorders. If postnatal development or growth could not be assessed due to intrauterine demise, termination of pregnancy or postnatal loss of follow-up, patients were considered to have CHD with undetermined extracardiac status.

Genetic data

The retrieval of genetic data was restricted to documented pathogenic or likely pathogenic CNV or SNV (in accordance to the ACMG guidelines for CNV or SNV classification) ^26,27 which were identified by prenatal or postnatal CMA or sequencing. Prenatal CMA by OGT 60k array, postnatal CMA by OGT 180k array and NGS by clinical exome sequencing were performed as described ^28–30. NIPS was done as described ³¹. Raw genetic data were not re-analyzed nor were newly generated for this retrospective chart review.

Statistical analyses

One sample t-test was used to compare mean values of continuous variables (e.g. maternal age, gestational age) and chi-square test (or Fisher exact test) to compare categorical variables (e.g. IVF versus spontaneous pregnancy, singleton versus twins) between the patient population and the Belgian reference population ^32–34.

A logistic regression analysis was used to determine significant predictors for outcome. We took the possible influence of multiple variables on the pre- and postnatal outcome into account. This analysis was repeated for three different types of outcomes:

TOP versus non-TOP in the entire prenatal CHD cohort.
postnatal survival versus postnatal death in the subgroup of live births with a prenatally diagnosed CHD.
Mortality (including TOP, IUM and postnatal death) versus survival across the entire prenatal cohort.

Fetuses or live births with missing outcome data were excluded.

Logistic regression was used on the entire prenatal population (n=341) for the dependent variables ‘TOP’ and ‘mortality’:

TOP ~ prenatal genetic diagnosis + CHD category + severity score + prenatal extracardiac abnormalities.
Mortality ~ prenatal genetic diagnosis + CHD category + severity score + prenatal extracardiac abnormalities.

Logistic regression was used for the dependent variable ‘survival’ on the population of live births (n=277):

Survival ~ prenatal genetic diagnosis + CHD type + prematurity + severity score + prenatal extracardiac abnormalities.

Significance of the severity score was determined among the different CHD types: significance of severity scores A, B or C was determined in comparison to score D. The significance of the 8 different CHD categories was also determined among the different categories where each category was compared to the anomalous venous return category. Prematurity defined as birth <36 weeks PMA was compared to the cohort born ≥36 weeks PMA. P-values of <0.05 were judged as significant.

Fetal cohort

From January 2018 until November 2021 prenatal advice by the pediatric cardiologist was sought for 384 fetuses with prenatally diagnosed CHD. After exclusion of 43 fetuses (25 referrals from other university hospitals for second opinion; 5 without prenatal CHD; 5 with isolated arrhythmia; 4 with isolated cardiomyopathy; 3 with cardiac tumors and 1 with isolated left VCS), maternal and fetal data from 341 fetuses with CHD were reviewed (supplementary figure 1). The average maternal age at the time of CHD diagnosis was 30.93 years (range 18-44), which is equal to the average maternal age of all pregnancies in Belgium in 2020 ³² (supplementary figure 2). The majority of pregnancies involved G1 (N=96) and G2 (N=98) pregnancies (supplementary figure 3). Twin pregnancies represented 7.62% of the fetal CHD cohort (26/341), including 10 DCDA, 14 MCDA and 2 MCMA twins (including one MCDA twin with CHD in both fetuses). At least 25 pregnancies (7.33%) were documented to have occurred after assisted reproduction by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Familial history was positive for CHD (regardless the CHD type) in 40 out of 341 fetuses (11.73%), with respectively 4.99%, 2.34% and 4.4% having at least one first, second or third-degree relative with CHD.

Prenatal CHD diagnosis

The average postmenstrual age (PMA) at CHD diagnosis at our center was 25.29 weeks (Most diagnoses were made between PMA week 20-23, at week 30-31 or at week 36 (supplementary figure 4). CHD categories and CHD severity scores were assigned to each fetus ²⁴. Conotruncal heart defects (33.72%) and LVOTO (18.77%) were the most common CHD categories (Table 1). Severity scores A, B, C and D were assigned respectively to 7%, 40%, 34% and 19% of the fetuses (Table 2).

Table 1. CHD categories (and common CHD types per category). Distribution of CHD categories within this study was compared to that of previously reported cohorts (4-7,27). Abbreviations: Art: arteriosus; AS: aortic stenosis; AVR: anomalous venous return; CoAo: aortic coarctation; CHD: congenital heart defect; DORV: double outlet right ventricle; d-TGA: dextro-transposition of great arteries; IAA: interrupted aortic arch; LVOTO: left ventricle outflow tract obstruction; PS: pulmonary valve stenosis; RAA: right aortic arch; TA: tricuspid atresia; TOF: tetralogy of Fallot; UVH: univentricular heart.

CHD categories and types	number	%	Hureaux et al.	Qiao et al.	van Nisselrooij et al.	Gowda et al.	Stallings et al.	Average literature
conotruncal ToF, d-TGA, truncus art	115	33.72	25%	25%	19.77%	32.85%	39.54%	28.43%
LVOTO AS, CoAo, IAA	64	18.77	21%	7.22%	15.82%	25.55%	33.76%	20.67%
UVH HLHS	39	11.44	21%	NA	9.46%	5.84%	18.28%	13.65%
septal VSD, ASD, AVSD	36	10.56	16%	48.34%	20.34%	32.85%	NA	29.38%
right-sided PS, TA, DORV	31	9.09	5%	6.94%	16.65%	32.85%	25.02%	17.29%
situs anomaly heterotaxy, left/right isomerism	16	4.69	NA	5.83%	1.69%	4.37%	NA	3.96%
AVR TAPVR, PAPVR	9	2.64	NA	NA	2.54%	8.03%	6.64%	5.74%
other RAA	31	9.09	12%	6.67%	13.42%	1.46%	NA	8.39%
total	341	100

Table 2. CHD severity scores (from score A to D) based on the scoring system reported by Gowda et al. (27) and available in supplementary table 1.

CHD severity score	score A	score B	score C	score D	total
Number	24	136	116	65	341
%	7%	40%	34%	19%	100%
Gowda et al.	10%	45.5%	20.5%	24%

Non-isolated CHD

Non-isolated CHD was diagnosed prenatally in 86 out of 341 (25%) fetuses, of which 63% (54/86) were confirmed postnatally or post-TOP to have a syndromic entity, 16% (14/86) were considered to have isolated CHD at birth, 8% (7/86) were diagnosed with situs anomalies and 13% (11/86) could not be assessed due to insufficient phenotypic information after birth or post-TOP.

In the cohort of 277 liveborn, 60 individuals complied with the criteria of having non-isolated CHD (22%, 60/277)) and 37 individuals could not be assigned to either class due to insufficient information regarding growth or development (13%; 37/277). Forty-three liveborns with non-isolated CHD (72%; 43/60) were diagnosed with ECA prenatally. The remainder (17/60; 28%) presented prenatally with apparently isolated CHD.

NIPS results

NIPS results were available for 154 pregnancies (45%), retrieving 7 abnormal results (trisomy 7 (2x), 18, 20, 21 (2x) and 22q11 deletion), 4 of which (T7 (2x), T20 and T21 (1x)) could not be confirmed by prenatal CMA on amniotic fluid due to false positive results or to confined placental mosaicism. The fetuses with confirmed T18, T21 and 22q11DS had a diagnosis of CHD on prenatal ultrasound prior to performing NIPS. NIPS was not performed or results were not available for the other 187 pregnancies.

CMA results

After excluding fetuses with an abnormal NIPS, CMA results were available for 232 fetuses (68%, 232/341), either performed prenatally (N=126) or after birth/TOP (N=106). Pathogenic or likely pathogenic chromosomal variants were identified for 30 patients either prenatally (N=18; 18/126 (14%)) or postnatally (N=12; 12/106 (11%)) (table 3; supplementary table 2). The most common chromosomal disorders were 22q11.2 deletion syndrome (N=10) and trisomy 21 (N=4) (supplementary table 3). For 27 fetuses with normal prenatal CMA results, CMA was repeated postnatally by 180k OGT array, but did not yield additional pathogenic variants. In accordance with the Belgian guidelines for variant reporting of prenatal genetic testing ²⁸, CNVs of unknown significance (VUS) identified by prenatal CMA were not communicated. These regulations did not apply for postnatal CMA results, reporting back VUS for 21 liveborn children who only had postnatal CMA analysis (N=107) or who had both prenatal and postnatal CMA analysis (N=27) (15.5%; 21/134). None of these VUS were reclassified as pathogenic or likely pathogenic after parental segregation analysis and clinical assessment.

We also compared CMA results between fetuses with and without prenatal diagnosis of ECA. Pre- or postnatal CMA results were available for 135 fetuses with prenatal apparently isolated CHD, for 68 fetuses with non-isolated CHD and for 29 fetuses with undetermined extracardiac status, and identified pathogenic CNVs in respectively 7.4% (10/135), 27.9% (19/68) and 3.4% (1/29) fetuses.

Table 3. Diagnostic yield by prenatal (PREN) or postnatal (POST) chromosomal microarray analysis (CMA) in fetuses with apparently isolated versus non-isolated CHD on prenatal ultrasound. Chromosomal pathogenic variants identified by NIPS were excluded.

ECA status	CMA	total	positive	negative	diagnostic yield (%)
isolated-CHD N=206	PREN CMA	60	5	55
	POST CMA	75	5	70
		135	10	125	7.4%
non-isolated CHD N= 86	PREN CMA	43	13	30
	POST CMA	25	6	19
		68	19	49	27.9%
CHD of unknown ECA status N=49	PREN CMA	23	0	23
	POST CMA	6	1	5
		29	1	28	3.4%
Total CHD cohort N=341	PREN CMA	126	18	108
	POST CMA	106	12	94
		232	30	202	12.9%

NGS results

NGS-based targeted gene panel testing or clinical exome sequencing (ES) was not systematically conducted, but was restricted to 16 individuals with diagnosis of non-isolated CHD and normal CMA results. Pathogenic or likely pathogenic SNVs were retrieved by prenatal NGS in 1 patient (RIT1) and by postnatal (or post-TOP) NGS in 11 patients (KMT2D (2x), PTPN11, RAF1, SON, DYNC2H1, PBX1, FOXF1, SMARCA4, KAT6A, PAH). Variants of unknown significance, inherited from an unaffected parent, were reported in 2 patients (FLT4, SMAD6).

Outcome

Prenatal outcome was not documented for 16 out of 341 pregnancies. The pregnancy in the remaining 325 fetuses ended either in TOP, an intrauterine demise or a live birth in respectively 44 (13.5%), 4 (1.2%) and 277 (85.3%) pregnancies. Early (<1 month) and late (>1 month) postnatal demise were documented for respectively 33 (11.9%) and 15 (5.5%) liveborn children (N=277). Full-term and premature birth (<36 weeks PMA) was recorded for respectively 232 and 31 liveborn children. Extreme prematurity (≤28 weeks PMA) was documented in 7 children. PMA at birth was not available for 14 liveborn children. Prenatal and postnatal outcome according to CMA results, CHD category, CHD severity score and multiple pregnancies are summarized in Table 4.

Table 4. Pregnancy outcomes in accordance to severity score, CHD category, genetic diagnosis and singleton versus multiple pregnancy. Abbreviations: AVR: anomalous venous return; IUM: intra-uterine demise; LVOTO: left ventricle outflow tract obstruction; TOP: termination of pregnancy; UVH: univentricular heart.

		numbers	TOP	IUM	unknown	live birth	mortality < 1 month	mortality 1-12 months	survival to 12 months
pregnancy	singleton	315	43 (13.65%)	4 (1.27%)	15 (4.76%)	253 (80.32%)	29 (9.21%)	14 (4.44%)	210 (66.67%)
pregnancy	multiple	26	1 (3.85%)	0	1 (3.85%)	24 (92.31%)	4 (15.38%)	1 (3.85%)	19 (73.08%)
genetic diagnosis	yes	45	12 (26.7%)	0	1 (2.2%)	32 (71%)	8 (17.8%)	1 (2.2%)	23 (51%)
genetic diagnosis	no	296	32 (10.8%)	4 (1.35%)	15 (5%)	245 (82.7%)	25 (8.4%)	14 (4.7%)	206 (69.6%)
severity score	A	24	9 (37.50%)	0	2 (8.33%)	13 (54.17%)	3 (12.50%)	3 (12.50%)	7 (29.17%)
	B	136	30 (22.06%)	4 (2.94%)	10 (7.35%)	92 (67.65%)	24 (17.65%)	9 (6.62%)	59 (43.38%)
	C	116	4 (3.45%)	0	2 (1.72%)	110 (94.83%)	6 (5.17%)	2 (1.72%)	102 (87.93%)
	D	65	1 (1.54%)	0	2 (3.08%)	62 (95.38%)	0	1 (1.54%)	61 (93.85%)
CHD category	conotruncal	115	6 (5.22%)	1 (0.87%)	3 (2.61%)	105 (91.30%)	7 (6.09%)	5 (4.35%)	93 (80.87%)
	LVOTO	64	5 (7.81%)	1 (1.56%)	3 (4.69%)	55 (85.94%)	7 (10.94%%)	3 (4.69%)	45 (70.31%)
	UVH	39	18 (46.15%)	0	3 (7.69%)	18 (46.15%)	10 (25.64%)	3 (7.69%)	5 (12.82%)
	Septal	36	6 (16.67%)	0	2 (5.56%)	28 (77.78%)	2 (5.56%)	0	26 (72.22%)
	Right-sided	31	5 (16.13%)	1 (3.23%)	2 (6.45%)	23 (74.19%)	4 (12.90%)	0	19 (61.29%)
	Situs anomaly	16	2 (12.5%)	1 (6.25%)	3 (18.75%)	10 (62.50%)	0	4 (25.00%)	6 (37.50%)
	AVR	9	0	0	0	9 (100.00%)	3 (33.33%)	0	6 (66.67%)
	other	31	2 (6.45%)	0	0	29 (93.55%)	0	0	29 (93.55%)
Total	all	341	44 (12.9%)	4 (1.17%)	16 (4.69%)	277 (81.23%)	33 (9.68%)	15 (4.40%)	229 (67.16%)

Markers for outcome

Logistic regression was performed to find variables predisposing to ‘TOP’, to overall ‘mortality’ (fetal death, TOP or postnatal demise (<1 year)) and to postnatal ‘survival’ beyond the age of 1 year.

Logistic regression analysis with outcome TOP determined that prenatal genetic diagnosis (p<0.001), prenatal extracardiac symptoms (p=0.045), severity score A (p=0.021) and severity score B (p=0.036) were significant contributing factors in the decision for TOP. Significance of the severity scores A, B and C was determined versus severity score D. Scores A and B were significantly more associated with TOP than score D. There was no significant difference between scores C and D. When using the CHD category ‘anomalous venous return’ as a reference, none of the 8 CHD categories had sufficient power in this model to determine significant association with TOP. No significant results were obtained if other CHD categories were used as a reference.
Logistic regression analysis for postnatal survival determined that prenatal extracardiac symptoms (p=0.002), prematurity of <36 weeks PMA (p=0.046) and severity score A (p=0.009 and B (p=0.004) had a significant negative impact on survival of a life birth with a complex CHD. When using univentricular heart as a reference for each other CHD category, we determined that there is a lower chance of postnatal survival in live births with a univentricular heart compared to patients with conotruncal abnormalities (p=0.009) or right sided cardiac abnormalities (p=0.02)
Logistic regression analysis for total mortality determined significance for prenatal extracardiac symptoms (p<0.001) and severity score A (p<0.001), B (p<0.001) and C (p=0.046) in the full cohort (n=341). These factors significantly increase the chances of mortality. Furthermore, the CHD categories conotruncal abnormalities (p=0.003) and LVOTO (p=0.041) had a significantly lower chance of mortality if we normalized versus the univentricular heart category.

Early and accurate prenatal diagnosis of CHD is crucial to optimize perinatal care to reduce CHD-related mortality and to improve quality of survival. However, risk stratification after prenatal diagnosis of CHD remains challenging ³⁵. Heart anatomy and coexisting congestive heart failure, reflected by ultrasound markers such as cardiomegaly, hydrops, abnormal myocardial function or abnormal venous Doppler, play the most important role in fetal and postnatal survival, underlying the importance of longitudinal prenatal echocardiographic examination for individualized outcome prediction of fetuses with CHD ^36,37. In this single-center retrospective chart review of 341 fetuses with CHD, we showed that in addition to high CHD severity scores (A or B) other factors such as prenatal diagnosis of pathogenic CNVs and prenatal co-occurrence of extracardiac anomalies were significantly associated with a decision to terminate pregnancy as well. This is in line with the study of Qiu et al. showing that more complex CHD and presence of ECA were inversely correlated with continuation of the pregnancy ³⁶. In addition, prenatal diagnosis of ECA and high CHD severity score, as well as prematurity <36 weeks PMA, were negatively correlated with postnatal survival beyond the age of 1 year. These results showed that prenatal cardiac and extracardiac phenotyping, complemented with prenatal genotyping, aid to provide adequate counseling to parents of fetuses with CHD with respect to postnatal survival rates. In this cohort of prenatally diagnosed complex CHD, the pregnancy was terminated in 13% and was complicated by fetal death in 1%. Pregnancy outcome was not documented in 4.5%. The overall postnatal mortality in our cohort was 17% (<4wk in 33/277 and >4wk in 15/277 liveborn children), which is similar to previous studies ²⁴ (table 4). Survival was significantly lower in fetuses with UVH compared to conotruncal CHD and right sided-CHD. Larger sample sizes per CHD category are required to attain sufficient statistical power to accurately associate CHD categories to TOP decisions or to short- or long-term survival. Ultrasound markers for outcome prediction, as described by Wieczorek et al. ³⁷ and Qiu et al. ³⁶, were not available and therefore the use of ultrasound parameters, such as the cardiovascular profile score, could not be applied.

The distribution of CHD categories in this study aligns well with the average distribution based on previously reported prenatal CHD cohorts ^4–7. (Table 1). Differences between individual reports is likely due to differences in classification of CHD categories. In this study, each fetus was attributed to only one CHD category based on the CHD type that was anatomically or prognostically most prominent, explaining the relatively low prevalence of septal defects in our cohort. Prenatal counseling by pediatric cardiologists in our center is less frequently requested in fetuses with CHD of lower complexity (CHD severity score D) or in fetuses with chromosomal aneuploidy en/or life-threatening ECA (CHD severity score A). Therefore, a shift towards CHD severity score C was observed, in comparison to the CHD severity distribution reported by Gowda et al. ²⁴. (Table 2).

As expected, TOP and postnatal mortality were significantly higher among fetuses with severity scores A and B compared to severity score D, while scores A, B and C are associated with higher overall mortality. We used the same parameters to score CHD severity as those introduced by Gowda et al. ²⁴. Although the distribution of CHD severity scores was comparable between both cohorts (except for score C which was more represented in our study population), we observed in our CHD cohort a higher survival rate beyond 6 months: overall (67% versus 40%) and across the subgroups with scores A (29% versus 7%), B (44% versus 9.7%) and C (88% versus 55.6%). These differences were related to a lower fetal death rate (1% versus 11.7%) and lower neonatal mortality (9.6% versus 24.1%), which may be due to differences in TOP policy as a more restrictive TOP policy may add to higher mortality rates. Large sample size follow-up studies are required to confirm these findings.

A significantly higher rate of multiple pregnancies (7.62%;p<0.001) and of IVF/ICSI pregnancies (7.33%;p<0.001) was observed in this CHD cohort compared to the general population (twinning rate of 1.6% and IVF/ICSI rate of 5.1% according to the 2019 report of the Flemish Center for Study of Perinatal Epidemiology). Compared to singletons, multiple pregnancies are known to be at increased risk of CHD, particularly among monochorionic twins due to altered intra-uterine hemodynamics ^38–41. In addition, we confirm the findings of Giorgione et al. showing that assisted human reproduction by IVF or ICSI was associated with a higher CHD incidence as well (odds ratio 1,45) ^40,42. CHD (of any type) in first-degree relatives was documented in 4.99% fetuses with CHD, which is similar to previous reports on familial CHD ⁴³.

A pathogenic variant was identified by pre- or postnatal CMA in 27.9% of fetuses presenting prenatally with non-isolated CHD. Nine additional fetuses from this subgroup were diagnosed by NGS with a pathogenic SNV. The diagnostic yield of chromosomal testing in fetuses with apparently isolated CHD (N=206) or with CHD of undetermined extracardiac status (N=49) was significantly lower (5.8%; 1 by NIPS, 5 by prenatal CMA, 6 by postnatal or post-TOP CMA, 3 by WES). All these patients presented with ECA at birth or post-mortem, despite having an isolated CHD on prenatal ultrasound (supplementary table 3). Our results are in line with those by van Nisselrooij et al. reporting chromosomal or genetic pathogenic variants in 15%: in 28.7% of fetuses with non-isolated CHD and in 11% of fetuses with apparently isolated CHD on prenatal ultrasound ⁷. The most common chromosomal and genetic syndromes in fetuses with CHD identified by us and others ⁷ were 22q11 deletion syndrome and Noonan syndrome. We also confirmed that CHD categories ‘septal defects’ (AVSD and VSD) and ‘LVOTO’ (interrupted aortic arch and CoAo) were associated with the highest yield of genetic testing, respectively in 19.4% and 20.3% (supplementary Table 2). Results from NIPS were available for 45% of fetuses (154/341), which is lower compared to the uptake of NIPS in the general population of pregnant women in Belgium (78.7%), where NIPS is available as a publicly funded nationwide first-tier screening as of July 2017 ⁴⁴. Prenatal invasive testing is preferred over NIPS when first trimester ultrasound is abnormal, which explains the lower uptake in the CHD cohort. Since fetal aneuploidy (T21, T18 or T13) weighs heavily on the decision to terminate pregnancy regardless of the presence of CHD ⁴⁵ potentially causing a bias with respect to outcome prediction in this study, we excluded fetuses with abnormal non-invasive aneuploidy screening prior to the diagnosis of CHD on prenatal ultrasound. NGS was performed in only a small subset of patients, either presenting with prenatal features of a rasopathy as described ⁴⁶, explaining the high incidence of Noonan syndrome in our cohort, or presenting postnatally or post-mortem with unexplained non-isolated CHD.

The outcome measures of this retrospective study were restricted to pregnancy continuation, overall mortality and postnatal survival. Long-term follow-up data on growth, development, behavior or survival into infancy were not available. ‘CHD category’, ‘extracardiac anomalies’ and ‘CHD severity score’ were included in the logistic regression outcome prediction model. However, as CHD severity scores were based on CHD type and on the presence of ECA, these variables are not independent, potentially impacting the results. As previously mentioned, CMA analysis was offered, either prenatally or postnatally, for all fetuses, but CMA results were available for only 68%, failing us to provide insight into chromosomal anomalies in the full cohort. NGS was not offered systematically, and was restricted to fetuses or live born children with non-isolated CHD and normal CMA results. This NGS policy is a limitation of this study. As NGS technology becomes broadly available and sequencing costs are dropping, ultrarapid trio exome or genome sequencing emerges as the standard-of-care prenatal genetic test when non-isolated CHD is diagnosed, and could be considered in fetuses with apparently isolated complex CHD despite the low diagnostic yield. However, when moving from postnatal to prenatal genetic testing, some challenges need to be taken into consideration, including technology availability, access to health care, health insurance issues and sociocultural differences. Moreover, short turn-around-times should be guaranteed and adequate pre-test counseling is required, dealing with diagnostic yield, risks of invasive prenatal procedures and the reporting of incidental findings. Expectations and preferences of the parents should be prioritized when offering prenatal genomic testing.

In conclusion, we showed that cardiac and extracardiac phenotyping by prenatal ultrasound and elaborate prenatal genetic testing are crucial to provide adequate counseling to parents of fetuses with CHD with respect to postnatal survival rates.

Data availability

The dataset generated and analysed during this retrospective study is not publicly available due to the sensitive nature of the data. However, inquiries regarding the dataset and its use can be directed to the corresponding author upon reasonable request.

Acknowledgements

The authors would like to thank the patients and their families for their participation.

Author contributions

J.B. and M.V. conceptualized the study, J.B., K.D., K.V.d.B, B.C., L.D.C. and M.G. retrospectively included patients. M.V. reviewed medical files and collected relevant data. J.B., M.V. and L.H. analyzed the data and wrote the main text of the manuscript. All authors reviewed the manuscript.

Funding sources

J.B. is funded by a clinical investigator fellowship by FWO-Flanders.

Conflict of interest

The authors declare no conflicts of interest.

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No competing interests reported.

supplementalmaterial.docx

Chromosomal analysis and short-term outcome of prenatally diagnosed complex congenital heart disease

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Abstract

Introduction

Patients and methods

Results

Discussion

Conclusion

References

Additional Declarations

Supplementary Files

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