Discordant congenital heart defects in monochorionic twins: Risk factors and proposed pathophysiology.

A six-fold increase in congenital heart defects (CHD) exists among monochorionic (MC) twins compared to singleton or dichorionic twin pregnancies. Though MC twins share an identical genotype, discordant phenotypes related to CHD and other malformations have been described, with reported rates of concordance for various congenital anomalies at less than 20%. Our objective was to characterize the frequency and spectrum of CHD in a contemporary cohort of MC twins, coupled with genetic and clinical variables to provide insight into risk factors and pathophysiology of discordant CHD in MC twins. Retrospective analysis of all twins receiving prenatal fetal echocardiography at a single institution from January 2010 -March 2020 (N = 163) yielded 23 MC twin pairs (46 neonates) with CHD (n = 5 concordant CHD, n = 18 discordant CHD). The most common lesions were septal defects (60% and 45.5% in concordant and discordant cohorts, respectively) and right heart lesions (40% and 18.2% in concordant and discordant cohorts, respectively). Diagnostic genetic testing was abnormal for 20% of the concordant and 5.6% of the discordant pairs, with no difference in rate of abnormal genetic results between the groups (p = 0.395). No significant association was found between clinical risk factors and development of discordant CHD (p>0.05). This data demonstrates the possibility of environmental and epigenetic influences versus genotypic factors in the development of discordant CHD in monochorionic twins.

Introduction

Congenital heart defects (CHD) are the most prevalent group of congenital anomalies, affecting approximately 0.9% of all singleton births [1, 2]. In monochorionic (MC) twins, the prevalence of CHD is six times higher, affecting 59 per 1000 live births [1].

Despite sharing an identical genotype, MC twins can develop discordant phenotypes for congenital malformations including CHD. Studies have not found strong genetic influences on discordant CHD in MC twins, with attributable genetic causes epigenetic in origin as opposed to differences in germline mutations or different phenotypic expression of the same genotype [3, 4]. It has been hypothesized that environmental influences such as teratogens can interfere with epigenetic processes leading to differential gene expression and discordant CHD [5].

In addition to epigenetics, local placental influences have also been identified as significant factors contributing to discordant CHD [5]. A 2011 study reported that 41% of all studied cases of discordant CHD resulted from placenta-related pathophysiologic mechanisms [5]. It has been hypothesized that placental inter-twin vascular connections, cord insertion sites, and relative placental-share contribute to imbalance of blood flow leading to relative hypoperfusion of one twin [5].

A major risk factor unique to MC twin population is the development of twin-to-twin transfusion syndrome (TTTS), an anomaly of placental vascular anastomoses causing an imbalance of blood flow from the donor twin to the recipient twin. The rate of CHD rises to 9.3% in MC pregnancies complicated by TTTS [6]. In the setting of TTTS, the imbalance of blood flow and increased aortic velocity in the recipient twin leads to increased risk of structural CHD in the recipient twin compared to the donor twin [5, 7]. Although several factors have been linked to the development of discordant CHD in MC twins, there is currently an incomplete understanding of the pathophysiology and associated risk factors of this diagnosis [3, 5].

The importance of early identification for CHD has been demonstrated by previous studies [8-11]. Although many cases critical CHD diagnoses occur either prenatally or subsequently through newborn screening, one in five cases of critical CHD are not diagnosed until after the fourth week of life [10]. Despite advances in fetal echocardiography, up to 60% of CHD diagnoses are diagnosed postnatally [12, 13]. Late diagnosis of critical CHD is associated with increased risk of morbidity and mortality, increased hospital length of stay, and 35% higher inpatient costs during infancy [8, 9, 11]. Therefore, understanding the pathophysiology and risk factors associated with discordant CHD in MC twins is critical to allow for early evaluation of an at-risk fetus. Early detection would work to reduce delivery and postnatal complications and aid in prevention through identification of modifiable risk factors.

This study examines fetal and maternal variables comparing MC twin pairs discordant versus concordant for CHD. We aim to describe the spectrum of lesions in this population and to elucidate risk factors and pathophysiology of discordant CHD in a modern cohort of MC twins. The monochorionic twin pair serves as an ideal model to evaluate environmentally triggered errors in cardiac development that contribute to CHD, both in the multifetal pregnancy as well as singleton pregnancies.

Methods

This retrospective cohort study utilized all prenatal screening echocardiograms conducted at the University of California, Los Angeles (UCLA) from January 2010 –March 2020 to identify all MC twins with concordant and discordant CHD with outcome of a livebirth. Dichorionic and conjoined twins were excluded from this study. Institutional Review Board (IRB) was obtained from UCLA (IRB #17–000925). Patient medical record numbers were used to obtain demographic information and were linked to the neonatal medical record numbers. Additional variables related to known risk factors for CHD independent of chorionicity, such as advanced maternal age, family history of CHD, high maternal pre-pregnancy body mass index (BMI), diabetes, and conception with assisted reproductive technology (ART) were also abstracted [14, 15]. All chart-abstracted information was stored in a de-identified research database. Chorionicity was confirmed by both an early perinatal ultrasound and by review of final placental pathology after delivery. CHD diagnoses were determined by postnatal echocardiography along with surgical operative reports, if available.

Concordant CHD was defined as a twin pair with an identical CHD diagnosis. Discordant CHD was defined as twin pairs with one affected and one unaffected fetus or twin pairs with different CHD diagnoses. Chart review was conducted to collect genetic test data, CHD diagnoses, extra-cardiac anomalies, and invasive (clinical) genetic testing results were obtained from prenatal amniocentesis samples or postnatal serum samples. Specifically, genetic test modalities included in analysis were karyotypes, microarrays, and fluorescence in situ hybridization (FISH) collected as an amniocentesis and/or chorionic villus sampling (CVS). Genetic screening, such as non-invasive prenatal screening and state analyte screening were not included.

Categories of CHD diagnoses were assigned based on the International Nomenclature for Congenital Heart Surgery (INCHS) by two providers specialized in fetal echocardiography and pediatric cardiology [16]. CHD were classified as one of the following: septal defects, pulmonary venous anomalies, systemic venous anomalies, right heart lesions, left heart lesions, single ventricle, transposition of the great arteries, and thoracic arteries or veins, which includes aortic arch, coronary artery, and ductus arteriosus anomalies.

Each diagnosis was further categorized by severity, with Category 1 indicating low risk of hemodynamic instability in the delivery room, Category 2 minimal risk of hemodynamic instability but requiring postnatal surgical intervention, Category 3 with likely hemodynamic instability requiring immediate specialty care, and Category 4 with expected hemodynamic instability requiring immediate surgical intervention.

Descriptive analysis including frequencies, means, medians, standard deviations, and interquartile range were conducted for all maternal variables, CHD diagnoses, and neonatal variables. Continuous variables were analyzed using the Student t-test and categorical variables were analyzed using the Fisher Exact test.

Results

Of the 163 twin pregnancies identified, 87 were MC twins (53.3%). Twenty-three MC twin pairs (46 neonates) had CHD (n = 5 concordant CHD, n = 18 discordant CHD pairs) (Fig 1). Table 1 displays the spectrum of CHD in discordant and concordant groups for this population. The most common lesions were septal defects (56% and 45.5% in concordant and discordant cohort) and right heart lesions (40% and 18.2% in concordant and discordant cohort). Lesions present in only the discordant pairs were systemic venous anomalies (4.5%), left heart lesions (9.1%), and thoracic arteries and veins (13.6%). There was no difference in the spectrum or severity of CHD between the two groups (p>0.05).

Fig 1

Study population of twins evaluated.

DC, dichorionic; MC, monochorionic; CHD, congenital heart defects; TTTS, twin-to-twin transfusion syndrome.

Table 1

Spectrum of CHD in concordant and discordant MC twins, as diagnosed by prenatal echocardiography.

Diagnosis1,2	Concordant CHD Cohort (n = 10)3	Discordant CHD Cohort (n = 22)3
Septal defects	6 (60%)	10 (45.5%)
Systemic venous anomalies	0 (0%)	1 (4.5%)
Right heart lesions	4 (40%)	4 (18.2%)
Left heart lesions	0 (0%)	2 (9.1%)
Transposition of the great arteries	0 (0%)	2 (9.1%)
Thoracic arteries/veins	0 (0%)	3 (13.6%)
CHD severity category4	1 (0)	1 (0)5

Data are no. (%) or median (IQR).

1Based on the International Nomenclature for Congenital Heart Surgery.

2p = 0.53.

3Includes only neonates affected by CHD.

4Category 1 = low risk of hemodynamic instability in the delivery room, Category 2 = minimal risk of hemodynamic stability but requiring postnatal surgical intervention, Category 3 = likely hemodynamic instability requiring immediate specialty care, Category 4 = expected hemodynamic instability requiring immediate surgical intervention.

5p = 1.

There was no difference in maternal demographic and risk factors in the two groups based on maternal co-morbidities or known risk factors for CHD, including pre-pregnancy BMI, diabetes, hypertensive disorders of pregnancy, and others (p>0.05) (Table 2). Antepartum length of stay (LOS) on labor and delivery was notably longer for the mothers in the discordant group than those in the concordant group, 10.61 versus 5.20 days, but this difference was not significant (p = 0.101). Five (21.7%) in-vitro fertilization (IVF) pregnancies were identified in this population, all of which were diagnosed with discordant heart lesions. Family history of CHD was rare in both cohorts, with none identified in the concordant and 11.1% in the discordant twin pair group.

Table 2

Maternal demographics in concordant and discordant MC twins (p = 0.634).

Variable	Concordant CHD cohort (N = 5)	Discordant CHD cohort (N = 18)	P-value
Maternal age (years)	30.40 ± 3.71	32.39 ± 7.51	0.426
Race			0.745
American Indian or Alaska Native	0 (0%)	0 (0%)
Asian	1 (20%)	1 (5.6%)
Black or African American	0 (0%)	0 (0%)
Native Hawaiian or Other Pacific Islander	0 (0%)	0 (0%)
White	4 (80%)	14 (77.8%)
Other	0 (0%)	2 (11.1%)
Denied	0 (0%)	1 (5.6%)
Ethnicity			1.00
Hispanic or Latino	1(20%)	4 (22.2%)
Not Hispanic or Latino	4 (80%)	13 (72.2%)
Other	0 (0%)	1 (5.6%)
Pre-gravid BMI (kg/m2)	25.70 ± 5.62	24.75 ± 6.26	0.754
Pre-gestational Diabetes Mellitus	0 (0%)	0 (0%)
Gestational Diabetes	0 (0%)	4 (22.2%)	0.539
Chronic Hypertension	0 (0%)	1 (5.6%)	1.00
Hypertensive Disorders of Pregnancy	2 (40%)	4 (22.2%)	0.576
Urinary tract infection	1 (20%)	0 (0%)	0.217
Family History of CHD	0 (0%)	2 (11.1%)	1.00
IVF pregnancy	0 (0%)	5 (27.8%)	0.545
Maternal length of stay (L&D)	5.20 ± 1.30	10.61 ± 13.05	0.101
First trimester exposure to SSRI	1 (20%)	1 (5.6%)	0.395
Illicit drug use in pregnancy	0 (0%)	0 (0%)
Diagnostic genetic testing	4 (80%)	9 (50%)	0.339
Abnormal genetic results	1 (20%)	1 (5.6%)	0.395

BMI, body mass index; IVF, in-vitro fertilization; SSRI, selective serotonin reuptake inhibitor.

Data are mean ± s.d. or no. (%).

Diagnostic genetic testing results were available for 80% of the concordant and 50% of the discordant cohort. There was one abnormal result in each group (20% concordant, 5.6% discordant), with no difference in rate of abnormal genetic results between the two groups (p = 0.395).

Details of the CHD diagnoses, neonatal clinical, genetic, and outcome data with associated extra-cardiac malformations for this population are listed in Tables 3–5. CHD severity was similar in both cohorts, with a median CHD severity category of 1 for both concordant and discordant twins. In the concordant cohort, one twin pair with right heart lesions had a NOTCH1 variant c.5720C>T and thumb abnormalities identified for both neonates. In the discordant cohort, renal anomalies were identified in three neonates, one twin pair with a VSD/normal phenotype, and one fetus with a PDA and PFO. One neonate with complete atrioventricular canal and pulmonary atresia had intestinal malrotation and heterotaxy syndrome with asplenia, consistent with right atrial isomerism. Overall, rates of malformations in our discordant cohort were low. None of the patients with malformations and CHD had abnormal family history or genetic testing. Placental malperfusion and/or vascular malformations were common in our study, with 4/5 (80%) of concordant and 9/18 (50%) of discordant pregnancies with either cord or placental findings.

Table 3

Clinical details and outcome data with associated extra-cardiac malformations for monochorionic twins concordant for CHD (excluding those with TTTS).

Chorionicity/amnionicity	Gestation age at birth	Birth weight (g)	Mode of Delivery	APGARs (1 min, 5min)	CHD Category	INCHS detail16	INCHS category16	Extra-cardiac malformations	Number of surgeries in 1st year	Prenatal Genetics*	Postnatal Genetics	Placental Pathology	Outcome
Mono/di	35w1d	2620	Cesarean	9,9	1	VSD, multiple	Septal defect	None	0	Normal karyotype, microarray	None	Foci intervillous thrombohematoma <10% volume	Live birth
		3325	Cesarean	8,9	1	VSD, single	Septal defect	None	0	Normal karyotype, microarray	None	Foci intervillous thrombohematoma <10% volume	Live birth
Mono/di	33w3d	1638	Vag-Spont	8,8	2	Pulmonary atresia, VSD	Right heart lesion	Hypoplastic first metacarpal and proximal phalanx of right thumb	2	Normal karyotype, microarray, FISH 22q11.2	Exome sequencing heterozygous NOTCH1 c.5720C>T variant, maternal allele	None	Live birth
Mono/di	33w3	1742	Vag-Spont	8,9	2	TOF	Right heart lesion	Bifid thumb	2	Normal karyotype, microarray, FISH 22q11.2	Exome sequencing heterozygous NOTCH1 c.5720C>T variant, maternal allele	None	Live birth
Mono/di	32w6d	1700	Cesarean	8,9	1	Tricuspid disease, non Ebstein’s; Mitral regurgitation	Right heart lesion	Right inguinal hernia	0	None	None	None	Live birth
Mono/di	32w6d	1850	Cesarean	8,9	1	Tricuspid disease, non Ebstein’s	Right heart lesion	None	0	None	None	Hypercoiled cord, mild chronic deciduitis	Live birth

*prenatal genetics = diagnostic testing (amniocentesis or chorionic villus sampling).

Table 5

Clinical details and outcome data with associated extra-cardiac malformations for monochorionic twins with TTTS.

Chorionicity/amnionicity	TTTS Stage	Gestation age at birth	Birth weight (g)	Donor or Recipient	Treatment	EGA at treatment	Mode of Delivery	APGARs (1 min/5min)	CHD type	INCHS detail16	INCHS category16	Extra-cardiac malformations	Number of surgeries in 1st year	Prenatal Genetics*	Postnatal Genetics	Placental Pathology	Outcome
Mono/di	Stage 2	34w0d	1983	Recipient	Cerclage and laser ablation	133	Cesarean	9,9	1	ASD, secundum	Septal defect	None	0	Normal karyotype, microarray	None	Lymphoplasmacytic chronic deciduitis—Intervillous fibrin deposition with villous infarction	Live birth
			2105	Donor			Cesarean	5,7	1	ASD, secundum	Septal defect	None	0	Normal karyotype, microarray	None		Live birth
Mono/di	Stage 1	33w4d	1721	Donor	Amnio-reduction	174	Cesarean	7,8	1	ASD, sinus venosus	Septal defects	None	0	Normal karyotype	None	Velamentous insertion, hypercoiled cord, multifocal accelerated villous maturation, chorangioma 0.8 cm, >97%ile size	Live birth
			1393	Recipient			Cesarean	9,9	1	ASD, secundum	Septal defects	None	0	Normal karyotype	None	>97%ile size	Live birth
Mono/di	Stage 1	24w4d	560	Recipient	None	N/A	Vag-Spont	5,7	1	Patent ductus arteriosus	Thoracic arteries and veins	Parietal porencephalic cysts	1	None	None	Vasa previa	Death at 7 weeks of age
			570	Donor			Vag-Spont	6,8	1	Secundum, ASD	Septal defects	None	1	None	None	None	Live birth
Mono/di	Stage 2	34w1d	1785	Recipient	2 Laser ablations	128, 135	Cesarean	9,9	1	Persistent left superior vena cava (PLSV)	Systemic venous anomaly	None	0	Normal karyotype, microarray	None	Unknown	Live birth
			2035	Donor			Cesarean	8,9	1	Tricuspid valve disease, non Ebstein’s	Right heart lesion	None	0	Normal karyotype, microarray	None	Unknown	Live birth
Mono/di	Stage 4	35w4d	2215	Donor	None	N/A	Cesarean	8,8	N/A	N/A	N/A	None	0	None	None	None	Live birth
			3010	Recipient			Cesarean	7,7	1	ASD, secundum	Septal defect	None	0	None	None	None	Live birth
Mono/di	Stage 1	31w0d	1285	Donor	Laser ablation	173	Cesarean	1,7	N/A	N/A	N/A	None	0	Normal karyotype, microarray	None	None	Live birth
			1460	Recipient			Cesarean	1,7	1	Patent ductus arteriosus, PFO	Thoracic arteries and veins	Bilateral hydronephrosis, mild right renal stenosis	0	Normal karyotype, microarray	None	None	Live birth

*prenatal genetics = diagnostic testing (amniocentesis or chorionic villus sampling).

Six (25%) twin pairs in the population were complicated by TTTS: 2 concordant (septal defects) and 4 discordant (thoracic arteries and veins 25%, septal defect 25%, systemic venous anomaly 13%, and right heart lesion 13%) (Table 5). All recipient twins were diagnosed with a heart lesion and 4/6 (67%) donor twins were also affected by CHD. There were no abnormal genetic test results in the twin pairs complicated by TTTS.

Neonatal outcome was analyzed based on concordance of CHD (Table 6). The average gestational age of delivery in both cohorts was ~33 weeks of gestation, with birthweight of 2007g in the concordant and 1960g in the discordant group. The majority of patients, 80% in the concordant cohort and 83% in the discordant cohort, were delivered via cesarean. Almost all neonates were hospitalized in the neonatal intensive care unit (NICU), with a median LOS of 32 (22.75) days in the concordant and 22 (47) days in the discordant cohorts. There was no difference in any neonatal outcomes studied between the two groups (p>0.05) (Table 6). When neonatal outcomes were analyzed to only include neonates affected by CHD in concordant (n = 10) and discordant (n = 22) pairs (Table 7), there was no difference in outcomes, though there was a greater gap between the number of surgeries in the discordant (0.86 ± 1.67) compared to the concordant group (0.40 ± 0.84 surgeries) (Table 7).

Table 6

Neonatal outcomes and clinical details in concordant and discordant CHD groups.

Variable	Concordant CHD cohort (N = 10)	Discordant CHD cohort (N = 36)	P-value
Sex			0.725
Female	4 (40.00%)	18 (50%)
Male	6 (60.00%)	18 (50%)
Gestational age at birth	33w5d ± 5d	33w5d ± 3d	0.919
Birthweight (g)	2007.70 ± 568.34	1959.92 ± 677.32	0.825
Mode of Delivery			1.00
Vaginal	2 (20.00%)	6 (16.7%)
Cesarean delivery	8 (80.00%)	30 (83.3%)
APGAR 1 minute	7.90 ± 1.20	7.20 ± 2.46	0.215
APGAR 5 minute	8.60 ± 0.70	8.20 ± 1.51	0.234
NICU Admission	10 (100.0%)	31 (86.1%)	0.570
NICU LOS	32 (22.75)	22 (47)	0.827
Number of surgeries in first year	0.40 ± 0.84	0.53 ± 1.36	0.718
Outcome			1.00
Live Birth	10 (100%)	34 (94.4%)
Neonatal Death (<28 days)	0 (0%)	0 (0%)
Infant Death (>28 days)	0 (0%)	2 (5.6%)

NICU = neonatal intensive care unit, LOS = length of stay, Data are mean ± s.d., no. (%), or median (IQR).

Table 7

Neonatal outcomes and clinical details in concordant and discordant CHD groups for affected neonates only.

Variable	Concordant CHD cohort (N = 10)	Discordant CHD cohort (N = 22)	P-value
Sex			0.711
Female	4 (40.00%)	11 (50%)
Male	6 (60.00%)	11 (50%)
Gestational age at birth	33w5d ± 5d	33w4d ± 3w5d	0.740
Birthweight (g)	2007.70 ± 568.34	1960.32 ± 792.91	0.850
Mode of Delivery			1.00
Vaginal	2 (20.00%)	5 (22.7%)
Cesarean delivery	8 (80.00%)	17 (77.3%)
APGAR 1 minute	7.90 ± 1.20	6.77 ± 2.60	0.103
APGAR 5 minute	8.60 ± 0.70	7.86 ± 1.78	0.104
NICU Admission	10 (100%)	19 (86.4%)	0.534
NICU LOS	32 (22.75)	23.5 (49.75)	0.471
Number of surgeries in first year	0.40 ± 0.84	0.86 ± 1.67	0.305
Outcome			1.00
Live Birth	10 (100%)	20 (90.9%)
Neonatal Death (<28 days)	0 (0%)	0 (0%)
Infant Death (>28 days)	0 (0%)	2 (9.1%)

NICU = neonatal intensive care unit, LOS = length of stay, Data are mean ± s.d., no. (%), or median (IQR).

Discussion

We compared a modern cohort of MC twins discordant and concordant for CHD to describe the spectrum of heart lesions identified in these two groups and to detail maternal demographic and pregnancy outcomes. The spectrum of CHD described in this study includes septal defects, systemic venous anomalies, right heart lesions, left heart lesions, transposition of the great arteries (TGA), and thoracic arteries/veins, with the most common lesions in both concordant and discordant cohorts being septal defects (60% and 45.5% in concordant and discordant cohort) and right heart lesions (40% and 18.2% in concordant and discordant cohort).

There was similar severity in presentation of CHD, as evidenced by equivalent median CHD severity category scores of affected fetuses in the concordant and discordant pregnancies. AlRais et al. described the spectrum of CHD in a discordant monochorionic cohort of 29 pregnancies, with 10% septal defects and 14% right heart lesions, comparable to our findings. In their cohort, all discordant twin pairs had one affected and one unaffected twin [5]. The majority of our non-TTTS discordant cohort (12/14, 86%) had one normal twin, though in the TTTS discordant subgroup, 50% of pregnancies resulted in both twins with CHD of differing type and severity. These results are consistent with existing data demonstrating that septal defects, are more common in MC twins than in singletons and abnormal blood flow due to TTTS preferentially affects cardiovascular function of the right ventricle in MC twins with associated development of right heart anomalies such as pulmonary atresia [17]. Defects known to be more significantly influenced by genetics, such as Tetralogy of Fallot (TOF), are reported to be equally prevalent in MC twins and singletons [1, 2, 18].

Although somatic mosaicism and somatic chromosomal abnormalities have been described in MC twins, we found no evidence for genetic discordance in twin pairs with discordant CHD [19]. This fact, coupled with the higher rates of discordant (78.2%) versus concordant (21.7%) CHD, low incidence of family history of CHD, and low incidence of genetic abnormalities (8.7%) in this population, suggests that genetic differences in coding regions of the genome are non-contributary in many cases of discordant CHD. There was one abnormal test result in our discordant cohort, a twin pair with a septal defect and left inguinal hernia in one twin and normal finding in the other twin. Their microarray had a 1.5 Mb copy loss of 15q13.2q-13.3 and two copy gains on 7q31.31 and Xp22.2. The copy gains are of unknown significance. The copy loss on chromosome 15 has been described in patients with neurodevelopmental defects but not cardiac abnormalities [20]. The yield of genetic testing in detecting CHD-specific mutations in discordant twins, especially those affected by TTTS, is low, as demonstrated by our negative findings and by studies of copy number variant and exosome sequencing analysis in discordant MC twins [4, 21]. On the contrary, genetic tests with high resolution can be diagnostic in concordant pairs, as evidenced by the NOTCH1 gene variant associated with non-syndromic TOF that was seen in the twin pairs with right heart lesion and thumb malformations [22]. The utility of in-depth genetic analysis has also been demonstrated in singletons, especially in patients with complex and syndromic CHD [23].

We provide hypothesis generating data that discordant CHD pairs may benefit from tests that provide insight into gene regulation. Although our study did not analyze epigenetic influences, it is important to note that certain post-translational modifications may be significant in the mechanism of discordant CHD development in MC twins. For example, epigenetic analyses found differentially methylated regions in promoters for genes involved in cardiac development in discordant twins [3]. Abu-Halima et al. recently found that microRNAs involved in molecular transport and adrenergic signaling in cardiomyocytes are enriched in CHD compared to non-CHD twins [23].

The etiological question remains how monochorionic twins, presumed to have an identical genotype, can display discordant phenotypes for congenital malformations such as CHD. Our data supports the conclusion that environmental influences likely play a significant role in the pathophysiology of discordant CHD. Factors affecting normal growth and development leading to CHD are hypothesized to occur as early as the first weeks of life, supported by high rates of cardiac looping and laterality defects in discordant CHD as well as the higher prevalence of CHD in twins conceived by assisted reproductive technology [5, 15]. In the discordant cohort, 27.8% had undergone the process of IVF compared to 0% of our concordant cohort, highlighting the influence of gamete manipulation, culture, and embryo implantation on non-familial CHD. In later gestation, although MC twins share a placenta, they do not necessarily experience identical growth environments. Anastomoses between fetal circulations play a significant role in intrauterine development and have the potential to cause syndromes such as TTTS [24]. In this population, the high frequency of TTTS (26%), with incidence of discordance twice that of concordance, indicates the critical role of hemodynamics in the pathophysiology of discordant CHD. The growth conditions for the recipient twin are significantly different than that of the donor twin, subsequently influencing cardiac development. A study analyzing ventricular strain changes in MC twins affected by TTTS shows that at all Quintero stages, recipient left ventricular strain was reduced compared with donors [25]. In stages 3 and 4, recipient right ventricular strain was reduced compared with donors [25]. The relative risk of right ventricular outflow tract obstruction is 70 times that of singletons for MC twins with TTTS, emphasizing the likely importance of hemodynamics on the development of cardiac structures in the second trimester, even after the heart has fully formed [1].

The strengths of this study include a large sample size of discordant twins with a novel comparison to a concordant twin cohort to elucidate environmental risk factors for CHD based on multiple maternal factors and neonatal outcomes. Our study includes a modern cohort with diagnostic testing results achieved using current genetic technology. We were able to follow neonates until at least one year of life, given that all were cared for by a single pediatric cardiology center. Although these results enhance our understanding of the possible mechanism for the development of discordant CHD, it is not possible to establish a temporal relationship between clinical influences and disease development. There are several limitations in our study, including use of a cohort from one specialized center, making this sample subject to selection bias. For example, the prevalence of CHD in our cohort of MC twins is 26.4% compared to literature reporting that about 5.9% of MC twins receive a diagnosis of CHD [1]. The analysis of this specialized cohort, however, also strengthens our findings by allowing for complete data collection with excellent follow up. Additionally, this study details genetic analysis for 80% of the concordant and 50% of the discordant cohort but lacks genetic test results for the remainder of the twin pairs, limiting our ability to draw statistically significant comparisons between the two cohorts with regards to contributing genetic abnormalities. We used abnormal prenatal echocardiography as the criteria for enrollment, and given the low sensitivity of this modality especially in obese patients, CHD diagnosed only postnatally would not be included in our study. Although prenatal fetal echocardiography is effective in early identification of major congenital heart defects, some heart defects with the potential to evolve in utero may be missed on prenatal echocardiography [26]. Therefore, this study errs on the side of capturing more severe CHD or CHD more easily detected on prenatal ultrasound. Future studies analyzing both prenatal and postnatal echocardiography records of twins who did not receive a prenatal diagnosis may be helpful in obtaining a representative population of MC twins with discordant CHD.

Our findings emphasize the importance of evaluating MC twins for CHD, especially those conceived through assisted reproductive technology or affected by TTTS. Increasing availability of genetic tests that are sensitive to discrepancies in epigenetic programming are needed in discordant CHD, given the low yield of traditional sequencing analyses for detecting cardiac specific alterations. Diagnostic evaluation that can detect early changes in twin cardiac hemodynamics that are known risk factors for discordant CHD may also improve prenatal detection of cardiac malformations that have implications for neonatal morbidity and mortality. We describe genetic evaluation and fetal and maternal clinical variables in a modern cohort of MC twins to demonstrate the significance of environmental influences and hemodynamics on the development of discordant CHD, elucidating target areas for early detection and risk modification.

23 Feb 2021

PONE-D-21-01048

Discordant Congenital Heart Defects in Monochorionic Twins: Risk Factors and Proposed Pathophysiology

PLOS ONE

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear Dr. Afshar and colleagues, thanks for submitting your great manuscript to PLOS ONE. Looking forward to see the revise version soon.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Editor,

Thank you for asking me to review this manuscript.

I have following comments and questions, hoping that they will improve the manuscript further.

Methods:

- Please clarify what type(s) of genetic testing was performed.

Results:

- Line 135-137: Please check the claim of LOS being significantly longer. Reported p value is 0.075.

- CHD Category is a categorical data. It should be reported as median with inter quartile range or min-max.

- Line 155: Please classify what is "placental abnormality"

- Line 171-173: Neonatal LOS were 41.23+/-47.16 and 26.83+/-24.17. Standard deviation is very large. Has the data been tested for "normalicy"? Again, reporting median might be appropriate if the data is not normally distributed.

-Table 1: CHD Severity is a categorical variable. It should be reported as median

- Please clarify terms/groups "affected" and "all"

- Please clarify group numbers. Are these number of fetuses or number of twins? Flow diagram indicated 6 pregnancies with concordant anomaly and 18 pregnancies with discordant anomalies.

- Table3a: I am not sure if a conjoint twin should be included in this study and if it is included it should be counted as concordant. Twins are thoracopagus. I suspect there is only one heart.

- Please clarify outcomes column. Please consider rewording "death at 3 months". I think authors meant "3 months of age".

Reviewer #2: Afshar et al studied frequency and spectrum of CHD in a cohort of MC twins as well as risk factors and proposed pathophysiology in discordant CHDs in MC twins. This study was a retrospective single institution study from 1/2010-3/2020. The most common lesions were septal defects and right heart lesions with no significant difference between the concordant and discordant pairs. Interestingly, there was also no difference in rate of abnormal genetic results between the groups and there was no association between clinical risk factors and development of discordant CHD. Authors concluded that their data may demonstrate possibility of environmental and epigenetic influences in the development of discordant CHD in MC twins.

I first would like to congratulate the authors for nicely structured and well-written manuscript. I agree with the authors that some of their data may be influenced by selection bias given the study was from one specialized center and those who were diagnosed postnatally could not be included in the study, but I have found their study findings thought provoking and promising for a larger, potentially multi-center, study. Genetic testing results were available in 67% of the concordant and 50% of the discordant cohort. Although having genetic testing data >50% of their cohort is impressive, not having genetic testing for the entire cohort, especially for the discordant pairs should also be included as a limitation of the study. I have no other comments for the authors.

Reviewer #3: This is an observational study describing the prevalence of congenital heart defects among monochorionic (MC) twin pregnancies according to whether the CDHs were concordant or discordant, and according to genetic results, assisted reproductive technology and the presence or absence of twin-to-twin transfusion syndrome. The authors reported that. genetic testing was abnormal in 17% of the concordant and 6% of the discordant pairs, with no difference in the rates of abnormal genetic results between the groups. The authors did not find a significant association between clinical risk factors and development of discordant CHD, and should be congratulated in their efforts

Additional comments:

1. The rates of abnormal genetic testing was almost three times higher in the concordant CHD group vs. the discordant one. The difference was not significant most likely because of the sample size (type II error). This finding is intuitive and supports the notion that in concordant MC twins the role of genetic abnormalities in CHDs is important

2. The prevalence of CHD in the cohort of MC pregnancies is almost 28% (24/87). This is not representative of the general population and suggests an important selection bias in the study, which needs to be reemphasized in the limitation section of the study

3. Lines 238 to 240, the mechanisms by which TTTS could increase the prevalence of CHD is unlikely to be related with abnormalities in ventricular strain in the recipient twin, otherwise the prevalence of left-sided lesions would be higher that right-sided lesions. However, it usually is the opposite in the recipient twin.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

15 Mar 2021

Also included in the cover letter.

March 6, 2021

Dear PLOS Editorial Team Editorial team,

Please find enclosed our REVISED manuscript entitled Discordant Congenital Heart Defects in Monochorionic Twins: Risk Factors and Proposed Pathophysiology.

As you know, in this manuscript we describe discordant congenital heart disease (CHD) in genetically identical monochorionic twin pairs and propose implications for etiological causes in a large referral center over 10 years. We are so appreciative of the thoughtful comments from all reviewers which has improved this submission significantly. All comments have been addressed in the revised version we are submitting. We believe this study provides hypothesis generating data on the possibility of environmental and epigenetic influences versus genotypic factors in the development of discordant CHD in monochorionic twins.

All authors had access to relevant aggregated study data and other information and all authors take responsibility for the way in which research findings are presented and published, were fully involved at all stages of publication and presentation development. The author list accurately reflects all substantial intellectual contributions to the research, data analyses, and publication or presentation development. No co-author has any disclosures or any relationships or competing interests relating to the research and its publication or presentation.

As part of PLOS One data availability, aggregate level data is and will be available and, in the event, that individual patient level will be needed, we expect a formal material transfer agreement (MTA) to be submitted with the UCLA IRB as part of the human research board, https://ohrpp.research.ucla.edu/. The UCLA IRB and MTA can be contacted at mirb@research.ucla.edu or (310) 825-5344.

We have read the instruction for authors and below is a detailed point by point response to each comment, with our responses below.

We affirm that this manuscript is an honest, accurate, and transparent account of these patients’ clinical course, and that no important aspects of the clinical course have been omitted. We look forward to a favorable response of our work in PLOS One.

Yours sincerely,

Yalda Afshar, MD, PhD

Division of Maternal-Fetal Medicine

Department of Obstetrics and Gynecology

University of California, Los Angeles

200 Medical Plaza, Suite 430; Los Angeles, CA 90095

Phone number: (310) 794-8492; YAfshar@mednet.ucla.edu

Below is a detailed point by point response to each comment, with our responses follow and are in red

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

We have confirmed that our manuscript matches the PLOS One style requirements and utilized the templates.

2. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

As part of PLOS One data availability, aggregate level data is and will be available and, in the event, that individual patient level will be needed, we expect a formal material transfer agreement (MTA) to be submitted with the UCLA IRB as part of the human research board, https://ohrpp.research.ucla.edu/. The UCLA IRB and MTA can be contacted at mirb@research.ucla.edu or (310) 825-5344.

3. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables should be uploaded as separate "supporting information" files.

Thank you. We have done this in the revision.

Additional Editor Comments:

Dear Dr. Afshar and colleagues, thanks for submitting your great manuscript to PLOS ONE. Looking forward to see the revise version soon.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Editor,

Thank you for asking me to review this manuscript.

I have following comments and questions, hoping that they will improve the manuscript further.

Methods:

- Please clarify what type(s) of genetic testing was performed.

Thank you for this clarifying point. We have updated this in our methodology to say: “Specifically, genetic test modalities included in this analysis were karyotypes, microarrays, and fluorescence in situ hybridization (FISH)” (Line 162-164)

Results:

- Line 135-137: Please check the claim of LOS being significantly longer. Reported p value is 0.075.

Thank you for this comment. We have adjusted this claim to say: “Antepartum length of stay (LOS) on labor and delivery was notably longer for the mothers in the discordant group…” (Line 200-203)

- CHD Category is a categorical data. It should be reported as median with inter quartile range or min-max.

Thank you for bringing this to our attention. We have adjusted Table 1 and the body of the text to report CHD Severity as a median with an interquartile range.

- Line 155: Please classify what is "placental abnormality"

We included all placental vascular malformations and/or mal-perfusions noted on the pathology report to be a placental abnormality.

- Line 171-173: Neonatal LOS were 41.23+/-47.16 and 26.83+/-24.17. Standard deviation is very large. Has the data been tested for "normalicy"? Again, reporting median might be appropriate if the data is not normally distributed.

Thank you for this comment. We have adjusted Table 4a and 4b to report Neonatal LOS as a median with an interquartile range. These values have also been reported in the text “Almost all neonates were hospitalized in the neonatal intensive care unit (NICU), with a median LOS of 32 (22.75) days in the concordant and 22 (47) days in the discordant cohorts” (Line 247-249)

-Table 1: CHD Severity is a categorical variable. It should be reported as median

Thank you for bringing this to our attention. We have adjusted Table 1 and the body of the text to report CHD Severity as a median with an interquartile range.

- Please clarify terms/groups "affected" and "all"

We have removed the term “affected” from the last row of this table and instead stated in the footnotes that this table reflects data from only affected neonates. We have also removed the column labeled “CHD severity category (all)” as we felt this data point did not add to our discussion.

- Please clarify group numbers. Are these number of fetuses or number of twins? Flow diagram indicated 6 pregnancies with concordant anomaly and 18 pregnancies with discordant anomalies.

Thank you for this comment. We have updated our footnotes in Table 1 to clarify that the sample described is neonates affected by CHD.

- Table3a: I am not sure if a conjoint twin should be included in this study and if it is included it should be counted as concordant. Twins are thoracopagus. I suspect there is only one heart.

Thank you for this clarifying point. Yes, as a thoracopagus there was a single heart with CHD, and we agree with the reviewer that this patient should be excluded. We have excluded this patient, updated our statistics and tables, and included a sentence in the methods section to clarify this exclusion: “Dichorionic and conjoined twins were excluded from this study” (Line 148)

- Please clarify outcomes column. Please consider rewording "death at 3 months". I think authors meant "3 months of age".

We have changed the wording of the outcome for Table 3b row 17 to “death at 3 months of age” and Table 3c row 5 to “death at 7 weeks of age”.

Reviewer #2: Afshar et al studied frequency and spectrum of CHD in a cohort of MC twins as well as risk factors and proposed pathophysiology in discordant CHDs in MC twins. This study was a retrospective single institution study from 1/2010-3/2020. The most common lesions were septal defects and right heart lesions with no significant difference between the concordant and discordant pairs. Interestingly, there was also no difference in rate of abnormal genetic results between the groups and there was no association between clinical risk factors and development of discordant CHD. Authors concluded that their data may demonstrate possibility of environmental and epigenetic influences in the development of discordant CHD in MC twins.

I first would like to congratulate the authors for nicely structured and well-written manuscript. I agree with the authors that some of their data may be influenced by selection bias given the study was from one specialized center and those who were diagnosed postnatally could not be included in the study, but I have found their study findings thought provoking and promising for a larger, potentially multi-center, study. Genetic testing results were available in 67% of the concordant and 50% of the discordant cohort. Although having genetic testing data >50% of their cohort is impressive, not having genetic testing for the entire cohort, especially for the discordant pairs should also be included as a limitation of the study. I have no other comments for the authors.

Thank you for the comment about the work and for the summary. We agree that a larger multi-center study would be paramount to draw stronger conclusion and we are working on this work in collaboration with multiple centers now that we have obtained results from our own center. So, thank you for this comment. Agree one of the biggest limitations is the lack of universal testing. We have included the lack of available genetic test results for the entire cohort in the limitations section of our discussion (Line 384-387)

Reviewer #3: This is an observational study describing the prevalence of congenital heart defects among monochorionic (MC) twin pregnancies according to whether the CDHs were concordant or discordant, and according to genetic results, assisted reproductive technology and the presence or absence of twin-to-twin transfusion syndrome. The authors reported that. genetic testing was abnormal in 17% of the concordant and 6% of the discordant pairs, with no difference in the rates of abnormal genetic results between the groups. The authors did not find a significant association between clinical risk factors and development of discordant CHD, and should be congratulated in their efforts

Additional comments:

1. The rates of abnormal genetic testing was almost three times higher in the concordant CHD group vs. the discordant one. The difference was not significant most likely because of the sample size (type II error). This finding is intuitive and supports the notion that in concordant MC twins the role of genetic abnormalities in CHDs is important

Thank you for this comment and we agree that we would have loved to have universal genetic testing in the entire cohort to draw a stronger conclusion. In light of your comment, re: role of type 2 error b/c of limitation in the difference in testing availability we have added language to the limitations about the lack of genetic testing in the entire population (Line 384-387). Thank you.

2. The prevalence of CHD in the cohort of MC pregnancies is almost 28% (24/87). This is not representative of the general population and suggests an important selection bias in the study, which needs to be reemphasized in the limitation section of the study

Thank you so much for this observation. We have emphasized the higher rates of CHD in our cohort of MC twins in the limitation section of the study and compared this prevalence to literature (Line 381-383).

3. Lines 238 to 240, the mechanisms by which TTTS could increase the prevalence of CHD is unlikely to be related with abnormalities in ventricular strain in the recipient twin, otherwise the prevalence of left-sided lesions would be higher that right-sided lesions. However, it usually is the opposite in the recipient twin.

We have reviewed literature in support of the reviewers comment and our comment (#25) and because of this discrepancy we have decided to remove this theoretical framework from our discussion and we believe it provides clarity to the discussion without this controversial proposed pathophysiology. Thank you.

21 Apr 2021

Discordant Congenital Heart Defects in Monochorionic Twins: Risk Factors and Proposed Pathophysiology

PONE-D-21-01048R1

Dear Dr. Afshar,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Alireza Abdollah Shamshirsaz

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Well done and please send us more good quality manuscript.

Reviewers' comments:

27 Apr 2021

PONE-D-21-01048R1

Discordant congenital heart defects in monochorionic twins: risk factors and proposed pathophysiology

Dear Dr. Afshar:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Alireza Abdollah Shamshirsaz

Academic Editor

PLOS ONE

Table 4

Clinical details and outcome data with associated extra-cardiac malformations for monochorionic twins discordant for CHD (excluding those with TTTS).

Chorionicity/amnionicity	Gestation age at birth	Birth weight (g)	Mode of Delivery	APGARs (1 min/5min)	CHD Category	INCHS detail16	INCHS category16	Extra-cardiac malformations	Number of surgeries in 1st year	Prenatal Genetics*	Postnatal Genetics	Placental Pathology	Outcome
Mono/mono	28w3d	820	Cesarean	1,6	1	VSD, multiple	Septal defect	Duplicated left renal collecting system, mild hydronephrosis	0	Normal karyotype, FISH (13, 18, 21)	Normal karyotype, microarray	None	Live birth
		1160	Cesarean	8,8	N/A	N/A	N/A	Mild L hydronephrosis	0	Normal karyotype, FISH (13, 18, 21)	Normal karyotype, microarray	None	Live birth
Mono/di	31w0d	1385	Vag-Spont	6,8	2	Aortic stenosis, valvar; Mitral stenosis, supravalvar mitral ring	Left heart lesion	None	2	Normal karyotype, microarray	Normal karyotype, microarray	420 g, <10%le weight for GA	Live birth
		1740	Vag-Spont	6,8	N/A	N/A	N/A	None	0	Normal karyotype, microarray	None	420 g, <10%le weight for GA	Live birth
Mono/di	32w2d	1595	Cesarean	8,9	N/A	N/A	N/A	None	0	Normal karyotype	Microarray 1.5Mb copy loss involving 15q13.2q-13.3 and two copy gains on 7q31.31 and Xp22.2	Hypocoiled cord	Live birth
		850	Cesarean	7,8	1	ASD, secundum	Septal defect	Left inguinal hernia	0	Normal karyotype	Microarray 1.5Mb copy loss involving 15q13.2q-13.3 and two copy gains on 7q31.31 and Xp22.2	Hypocoiled cord	Live birth
Mono/di	32w6d	2230	Cesarean	9,9	1	ASD, secundum; pulmonary artery stenosis, branch central	Septal defect	None	0	None	None	None	Live birth
		2120	Cesarean	9,9	N/A	N/A	N/A	None	0	None	None	None	Live birth
Mono/di	34w0d	1330	Cesarean	9,10	N/A	N/A	N/A	None	0	Microarray uniparental disomy 2 but PCR of C2 normal biparental inheritance	None	75% vascular distribution	Live birth
		2065	Cesarean	8,9	1	Tricuspid valve disease, non Ebstein’s related	Right heart lesion	None	0	Microarray uniparental disomy 2 but PCR of C2 normal biparental inheritance	None	25% vascular distribution, hypercoiled, velamentous insertion, infarction and intervillous thrombohematoma	Live birth
Mono/di	34w1d	1970	Cesarean	9,9	N/A	N/A	N/A	None	0	None	None	Bivascular cord	Live birth
		1760	Cesarean	6,8	1	VSD, single	Septal defect	None	0	None	None	Normal	Live birth
Mono/di	34w6d	2300	Cesarean	8,7	1	VSD, single	Septal defect	None	0	None	None	None	Live birth
		2315	Cesarean	8,9	N/A	N/A	N/A	None	0	None	None	None	Live birth
Mono/di	35w4d	2380	Cesarean	9,9	N/A	N/A	N/A	None	0	None	None	None	Live birth
		2350	Cesarean	9,9	1	Tricuspid valve disease, non Ebstein’s related	Right heart lesion	None	0	None	None	Hypocoiled cord	Live birth
Mono/di	36w4d	2375	Cesarean	8,8	3	DORV, VSD type	Transposition of the great arteries	None	5	Unknown	Normal karyotype, microarray, exome sequencing	Size >90%ile	Death at 3 months of age
		2680	Cesarean	9,9	N/A	N/A	N/A	None	0	Unknown	None	Size >90%ile	Live birth
Mono/di	36w5d	2220	Cesarean	8,8	2	AVC, complete; pulmonary atresia	Septal defect	Intestinal malrotation, hydronephrosis, heterotaxy syndrome with asplenia	6	Normal karyotype, microarray	Normal karyotype, microarray, exome sequencing	None	Live birth
		2260	Cesarean	8,9	N/A	N/A	N/A	None	0	Normal karyotype, microarray	None	None	Live birth
Mono/di	37w4d	2610	Vag-Spont	1,1	1	Systemic venous anomalies	Thoracic arteries and veins	None	0	None	None	Single UA, marginal insertion, 30% chorionic plate	Live birth
		3660	Vag-Spont	8,9	1	Tricuspid valve disease, non Ebstein’s related	Right heart lesion	None	0	None	None	70% chorionic plate	Live birth
Mono/di	38w2d	2380	Cesarean	9,9	N/A	N/A	N/A	None	0	None	None	None	Live birth
		2995	Cesarean	9,9	1	VSD, multiple	Septal defect	None	0	None	None	None	Live birth
Mono/mono	34w0d	1990	Cesarean	8,9	1	AVC, complete	Septal defect	None	0	None	Normal karyotype, microarray	Single UA	Live birth
		1930	Cesarean	8,9	2	DORV, VSD type	Transposition of the great arteries	None	2	None	Normal karyotype, microarray	None	Live birth
Mono/di	35w4d	2167	Cesarean	9,9	2	HLHS	Left heart lesion	None	2	None	None	None	Live birth
		2000	Cesarean	9,9	N/A	N/A	N/A	None	0	None	None	None	Live birth

*prenatal genetics = diagnostic testing (amniocentesis or chorionic villus sampling).