Prenatal diagnosis and pregnancy outcomes of 1492 fetuses with congenital heart disease: role of multidisciplinary-joint consultation in prenatal diagnosis

Early diagnosis of congenital heart disease (CHD) can improve the prognosis of neonates with CHD. We retrospectively evaluated the value of prenatal diagnosis of CHD by comparing the pregnancy outcomes. Prenatal diagnosis of CHD was established by echocardiographic evaluation of fetal heart. Amniotic fluid and/or cord blood genetic examination, pathological anatomy, casting specimen, and/or multidisciplinary-joint consultation (MDJC) were performed. A total of 1492 fetuses with CHD were diagnosed by prenatal echocardiography from 67834 pregnant women. There were 445, 236, 583, and 228 cases in groups A (simple CHD), B (simple CHD plus extra-cardiac abnormality), C (complex CHD), and D (complex CHD plus extra-cardiac abnormality), respectively. The pregnancy continuation rate in the four groups was 98.67%, 85.71%, 67.65%, and 36.84%, respectively (P < 0.001). The pregnancy termination rate for fetal CHD with extra-cardiac abnormalities was significantly higher than that for fetuses with only CHD (81.24% vs. 53.6%, P < 0.05). Prenatal genetic test revealed chromosomal abnormalities in 20.43% of fetuses with CHD. MDJC significantly decreased the pregnancy termination rate. In 88 cases, the original decision to terminate the pregnancy was changed after consultation and the pregnancy was continued. Of these, 87 cases culminated in live births; 65 of these children had better prognosis. Nine-segment sequential segment analysis method for prenatal fetal echocardiography was compared with the results of pathological anatomy, cast specimen, postoperative diagnosis, and postnatal ultrasound. The accuracy of prenatal ultrasound for diagnosis of fetal complex CHD and fetal simple CHD was 90.5–91.66% and 98.6%, respectively. Prenatal ultrasound is still the most effective method for fetal CHD diagnosis.

Classification. Based on the echocardiography examination, fetuses with CHD were classified into four groups according to the complexity of intracardiac malformation and presence of concomitant extra-cardiac abnormalities. Group A was simple CHD without any concomitant extra-cardiac abnormality; Group B was simple CHD combined with extra-cardiac abnormalities; Group C was complex CHD without any concomitant extra-cardiac abnormality; and Group D was complex CHD with concomitant extra-cardiac abnormalities. The A and B groups were defined as simple fetal CHDs, and the C and D groups were classified as complex fetal CHDs.
Based on the treatment after birth, the progeny affected by CHD were divided into surgery group and observation group.
Fetal heart ultrasound. Ultrasound  Fetal CHD genetic examination. 20-30 mL of amniotic fluid was collected for cell culture and karyotype analysis and prenatal single nucleotide polymorphism (SNP)-Array detection. The specimens were sent to the laboratory for inoculation, production, G-banding, and karyotype analysis using a chromosome automatic analyzer (US PE Company). The karyotype was named according to the International Cytogenetics International Naming System (ISCN). Cord blood (2.5 mL) was obtained percutaneously under ultrasound-guidance in some pregnant women for karyotype analysis and prenatal SNP-Array detection.
Chromosomal microarray chip. Genomic DNA of the fetal amniocytes and/or the cord blood cells were extracted using the QIAamp ® DNA Blood Mini Kit (Qiagen, Germany), analyzed by CytoScan 750 K gene chip (Affymetrix Genome, Santa Clara, California, USA), and evaluated using Chromosome Analysis Suite (ChAS) software (Thermo Fisher Scientific, Waltham, MA, USA).
Fetal CHD multi-disciplinary-joint consultation (MDJC). The MDJC team comprised of obstetricians, cardiac surgeons, geneticists, and ultrasound diagnosticians. The key tenets of the MDJC were as follows: 1. Patients in group A who showed no abnormality in genetic test were advised to continue pregnancy. 2. Patients in group B who had simple extra-cardiac malformations with no abnormality detected in genetic test were recommended to continue pregnancy. 3. Patients in group B who had complex extra-cardiac malformations or genetic syndrome tendency were recommended to continue or terminate pregnancy based on the results of genetic test. 4. Patients in group C were advised to continue or terminate pregnancy based on the evaluation of potential development trend (symptoms after birth, feasible treatment strategies, and long-term diagnosis) and genetic test.

5.
Patients who had simple extra-cardiac malformations in group D were advised to continue or terminate pregnancy based on the prognostic assessment and results of genetic test. 6. Patients who had complex extra-cardiac malformations or genetic complex tendency were advised to terminate pregnancy.
Local pathological anatomy of CHD and casting specimen. Cases wherein pregnancy was terminated due to fetal CHD underwent examination of local cardiac pathological anatomy or establishment of casting specimens. Some of the smaller cardiac specimens were observed by removing a part of the atrioventricular wall or vessel wall and opening a window to observe intracardiac morphological abnormalities. Casting specimens were established as described elsewhere 12 .
Statistical analysis. Data analyses were performed using the SPSS statistical package version 19.0.
Quantitative data are expressed as mean ± standard deviation. Qualitative data are presented as frequency (n) and percentage (%). The Chi-squared test was used to assess between-group differences, and the chi-square segmentation method was used for pairwise comparisons. The unconditional logistic regression model was used to calculate the odds ratio (OR) and 95% confidence interval (CI) of the effect of fetal CHD type and presence of  , of which multisystem malformations (23.6%) and central nervous system malformations (22.9%) were the most common extra-cardiac abnormalities (accounting for 46.5%). The rest were edema syndrome (12.4%), skeletal dysplasia (11.2%), urogenital abnormalities (8.5%), facial abnormalities (7.8%), and digestive system abnormalities (7.3%).
Groups of the fetuses with CHD by prenatal ultrasound. Prenatal ultrasound examination showed that 445 cases belonged to group A (including ventricular septal defect, coronary artery fistula, pulmonary artery stenosis, aortic stenosis, etc.); among these, 344 cases were followed-up, 42 pregnancies were terminated, intrauterine death occurred in two cases (all had ventricular septal defects), and two cases died in the neonatal period (ventricular septal defect and pulmonary stenosis, respectively) ( Table 1). 236 cases pertained to group B (simple CHD with extra-cardiac abnormalities). In group B (222 cases were followed-up), 159 pregnancies were terminated, intrauterine death occurred in 6 cases (both ventricular septal defect), 57 culminated in live births, and three cases died during the neonatal period (two with ventricular septal defect and 1 with pulmonary stenosis) ( Table 2). 583 cases were classified into group C (complicated CHD with no extra-cardiac abnormalities, including tetralogy of Fallot, double outlet of right ventricle, transposition of great arteries, hypoplastic left heart syndrome, hypoplastic right heart syndrome, heterotaxy syndrome, persistent truncus arteriosus, atrioventricular septal defect, etc.). In group C (454 cases were followed-up), 386 pregnancies were terminated, intrauterine death occurred in five cases (two cases of tetralogy of Fallot, 1 case of pulmonary atresia), 63 culminated in live birth, and 17 cases died during the neonatal period (six with coarctation of aorta and 3 with tetralogy of Fallot) ( Table 3). 228 cases were classified into group D (complicated CHD with extra-cardiac abnormalities). Among these 215 cases were followed-up, 196 pregnancies were terminated, intrauterine death occurred in nine cases (four cases had atrioventricular septal defects), 10 culminated in live births, and three cases died during the neonatal period (one case each of aortic arch constriction, tetralogy of Fallot, and double outlet of right ventricle) ( Table 4).

Genetic examination of the fetuses with CHD. Interventional prenatal genetic examination was
performed for 235 (19.03%, 235/1235) cases of fetal CHD. Among these, chromosomal abnormalities were detected in 48 cases (20.43%). Of these cases, forty children had abnormal number of chromosomes, including 17 cases of 21-trisomy, 13 cases of 18-trisomy, seven cases of 13 trisomy, three cases of Turner syndrome. Other eight cases were chromosome microdeletions (three cases of 22q11.2 microdeletion syndrome, three cases of Willams-Beuren syndrome, and two cases of Cri-du-chat (cat's cry) syndrome). The abnormal karyotype detection rate in groups B and D (39.8%) (with extra-cardiac abnormalities) was significantly higher than that in groups A and C (5.3%) (without extra-cardiac abnormalities) (Table 5). Overall, the detection rate of chromosomal abnormalities in CHD patients with extra-cardiac abnormalities was significantly higher than those  www.nature.com/scientificreports www.nature.com/scientificreports/ without extra-cardiac abnormalities (χ 2 = 42.375, p < 0.001). However, there was no significant difference in this respect between simple CHD and complex CHD (χ 2 = 2.241, p = 0.134) ( Table 5).

MDJC for management of fetal CHD.
A total of 651 (43.6%) CHD fetuses were assessed by MDJC.
Among these, 580 cases were successfully followed up. Compared with the cases that were not assessed by MDJC, the pregnancy termination rate of cases assessed by MDJC was significantly lower (Table 6), especially in groups B and C (p < 0.001).
Among the pregnant women who participated in MDJC, 88 pregnant women altered their original tendency (termination of pregnancy) after consultation and continued pregnancy (four cases in group A, 21 cases in group B, 45 cases in group C, and 18 cases in group D); of them, 87 cases had live births and 65 children had better prognosis (4 cases in group A, 21 cases in group B, 32 cases in group C, and 8 cases in group D).
Termination of pregnancy was recommended by MDJC in two cases affected by left ventricular dysplasia syndrome. However, the parents did not consent to termination of pregnancy and their infants died during the neonatal period. On the other hand, most pregnancies in groups C and D who continued pregnancy based on the suggestion of MDJC successfully culminated in live births in a hospital with neonatal ICU facility under the care of the MDJC.
Survival rate of the fetuses with CHD. Four hundred and thirty fetuses with CHD were born alive; of these, 25 cases died during the neonatal period. Sixty-five patients with CHD underwent interventional or surgical treatment after birth (surgery group), while 340 patients with CHD received conservative treatment (observation group). Twenty-seven of the 65 patients in the surgery group had complex CHD. Among these, two patients were misdiagnosed by prenatal ultrasound and two cases died of pneumonia after operation. The remaining 25 cases (including two misdiagnosed cases) were followed up for periods ranging from two to five years. Four patients died during the follow-up,the survival rate of patients with complex CHD who underwent operation was    43  41  34  1  6  1   TOF  37  35  33  1  1  1   DORV  24  24  21  1  2  1   HLHS  17  17  16  1  0  0   SV  15  13  12  1     www.nature.com/scientificreports www.nature.com/scientificreports/ survival rate between patients with simple CHD and those with complex CHD (P = 0.101) ( Table 7). Timely surgical treatment of infants with complex CHDs resulted in favorable outcomes.
The observation group had 340 infants with CHD (314 simple and 26 complex CHD). Among the 314 infants with simple CHD, four of the patients had poor quality of life due to extra-cardiac abnormalities. Among the 26 patients with complicated CHD, five patients were lost to follow-up while 21 CHD fetuses were verified by final ultrasound; of these two were misdiagnosed by fetal ultrasound. These cases were not operated or were inoperable.

Accuracy of preneonatal ultrasound for the fetuses with CHD.
Based on the postoperative diagnosis of CHD in the surgery group and the final ultrasound confirmation in the observation group, the diagnostic accuracy of prenatal ultrasound for fetal simple CHD and complex CHD was 98.68% (225 of 228 from both surgery and conservative treatment groups) and 91.66% (44 of 48 from both surgery and conservative treatment groups), respectively. The diagnostic accuracy of prenatal ultrasound for simple CHD was significantly greater than that for complex CHD (P < 0.05) ( Table 8). Among the misdiagnosed cases, one case was prenatally diagnosed as coarctation of aorta; however, the postpartum diagnosis showed ventricular septal defect. Another case was prenatally diagnosed as severe pulmonary stenosis; however, the suggested postoperative diagnosis was pulmonary atresia.     www.nature.com/scientificreports www.nature.com/scientificreports/ In this study population, 783 pregnancies were terminated; of these 147 cases of complex CHD were analyzed by local pathological anatomy. Twenty-one cases of complex CHD were analyzed by casting specimen. The diagnosis based on prenatal ultrasound was compared with the pathological diagnosis (as the standard) ( Table 9); ultrasound diagnosis was completely consistent with the pathological diagnosis in 76.2% cases and approximately consistent with the pathological diagnosis in 14.3% cases. Thus, the diagnostic accuracy of prenatal ultrasound for complex CHD was 90.5%. In other words, the misdiagnosis rate of prenatal ultrasound was 9.5%. Analysis of the pathological anatomy indicated that the misdiagnosis of prenatal ultrasound mainly pertained to pulmonary atresia, heterotaxy syndrome, hypoplastic left heart syndrome, and valvular disease. Analysis of the cast specimens suggested that misdiagnosis of prenatal ultrasound mainly pertained to abnormal branching of aorta and abnormal venous return.
Outcomes of the fetuses with CHD. Among the 1492 cases of fetal CHD, 1235 cases (82.8%) were followed-up. Among these, pregnancy was terminated in 783 cases (63.4%) due to fetal CHD; out of these 783 cases, a pathological diagnosis was obtained in 168 cases by pathological anatomy or vascular casting. Intrauterine death occurred in 22 cases (1.8%). 430 cases (34.8%) were delivered as live births, of which 25 cases died in the neonatal period, 65 cases underwent interventional or surgical treatment, nine cases died after operation, and 340 cases were followed up.
The pregnancy termination rate progressively increased from group A to D (in that order) [12.21%, 71.62%, 85.02%, and 91.16%, respectively (P < 0.001)]. The survival rates of fetuses in cases of continued pregnancy were 98.67%, 85.71%, 67.65%, and 36.84%, respectively (P < 0.001) ( Table 10). After continuity correction χ2 test treatment, the outcomes of pregnancy in the four groups were significantly different (χ2 = 697.942, P < 0.001) ( Table 11). In addition, the Chi-square segmentation method was applied for pair wise comparisons, which indicated significant differences between groups A, B, C, and D (P < 0.0083 for all).

Risk factor analysis of the fetuses with CHD. Unconditional logistic regression analysis suggested that
presence of complex CHD and extra-cardiac abnormalities were two risk factors for termination of pregnancy. Fetuses with complex CHD were at a 3.485 times higher risk of pregnancy termination compared to the fetuses with simple CHD (95% CI: 3.025-4.016). Fetuses with CHD in combination with extra-cardiac abnormalities were at a 1.935 times higher risk of pregnancy termination compared to their counterparts without extra-cardiac abnormalities (95% CI: 1.684-2.223) ( Table 12). The risk of fetal death for fetuses with complex CHD was 2.925 times higher than that of fetuses with simple CHD (95% CI: 1.861-4.598). The risk of intrauterine death in case    www.nature.com/scientificreports www.nature.com/scientificreports/ of CHD fetuses with extra-cardiac abnormalities was 3.407 times higher than that of their counterparts without extra-cardiac abnormalities (95% CI: 2.136-5.436) ( Table 13). In addition, the risk of neonatal death in complex CHD was 5.154 times higher than that in simple CHD (95% CI: 3.093-8.59). Notably, extra-cardiac abnormalities did not affect neonatal death (χ2 = 1.430, P = 0.232) ( Table 14).

Discussion
In this study, septal defect and conus arterial malformations were the major types of fetal CHD. The ten most common CHDs were ventricular septal defect (31.3%), tetralogy of Fallot (9.8%), vascular ring (9.2%), atrioventricular septal defect (7.0%), aortic coarctation (6.1%), heterotaxy syndrome (4.3%), pulmonary stenosis (4.1%), double outlet of right ventricle (3.8%), transposition of the great arteries (3.7%), and pulmonary atresia (3.6%), respectively. The frequency of various abnormalities is slightly different from that reported by previous studies 14,15 . In this study, we found more cases of heterotaxy syndrome, this may be attributable to the focus on comprehensive sonographic evaluation rather than the detection of a single deformity,Four structures connection in the transverse section of the upper abdomen is conducive to the rapid diagnosis of heterotaxy syndrome 13 . Increased number of the cases with vascular rings is associated with the application of bilateral subclavian artery sections 16 . Our hospital is the provincial referral centre for prenatal diagnosis and consultation. Therefore, most cases pertained to complex CHD, which may have affected the statistical distribution of the disease types.
In this study, the accuracy of prenatal ultrasound for diagnosis of fetal CHD showed slight differences from the previous reports in which the diagnostic coincidence rate was 87.89-95.35% [17][18][19] . Here, we performed nine-segment sequential segment analysis method of prenatal fetal echocardiography and compared with the pathological anatomy, cast specimen, postoperative diagnosis, and the postnatal ultrasound. The accuracy of prenatal ultrasound for diagnosis of fetal complex CHD and fetal simple CHD was 90.5-91.66% and 98.6%, respectively (Table 9).
Fetal CHD includes a wide variety of anomalies with varying degrees of severity, often associated with hereditary diseases and extra-cardiac malformations, or may occur as part of certain syndromes 20,21 . Although there has been considerable improvement in the accuracy of ultrasound diagnosis of CHD, it may not detect all concomitant extra-cardiac abnormalities 22 . Indeed, in our study, six CHD fetuses with simple ventricular septal defect detected by prenatal ultrasound were finally diagnosed as CHD with cleft palate after postnatal follow-up. Therefore, cases of fetal CHD should be rigorously evaluated to exclude extra-cardiac abnormalities.
The prevalence of chromosomal abnormalities in CHD patients with extra-cardiac abnormalities was significantly higher than that in CHD patients without extra-cardiac abnormalities (p ˂ 0.05), which is consistent with other reports 23,24 . Genetic testing of CHD fetus can reveal fetal genetic information, provide evidence for pregnancy decision-making, and even help guide the next pregnancy.
The complexity of CHD is significantly associated with fetal termination rates 25 . In this study, the pregnancy termination rate in cases with complex fetal CHD was as high as 87.0%. The pregnancy termination rate, fetal death rate, and neonatal mortality associated with fetal complex CHD were obviously higher than those with simple CHD (p < 0.05), which is consistent with a previous study 26 . The pregnancy termination rate and fetal death rate in the cases of fetal CHD with extra-cardiac abnormalities were higher than those without extra-cardiac abnormalities (P < 0.05). However, there was no significant difference in the effect of extra-cardiac abnormalities on neonatal mortality.   Table 14. The effect of associated extra-cardiac anomalies and type of CHD on neonatal mortality.
The pregnancy termination rates in groups A, B, C, and D showed a significant progressive increase in that order, while the pregnancy continuation rate showed a significant progressive decrease (P < 0.05). Both the pregnancy termination rate and the neonatal mortality rate in group C were higher than that in group B, which suggests that the severity of cardiac malformation has a greater impact on prognosis. This is likely attributable to the more severe disease and hemodynamic changes in cases with complicated CHD. Moreover, these cases are less amenable to surgical correction and are more likely to have impaired quality of life after surgery.
Thirty-eight patients with simple CHD who underwent cardiac surgery were followed up till date, 35 patients have shown good growth and development, while three patients died after operation, the survival rate was 92.10%. Of the 314 patients with simple CHD (A group), 310 had a good quality of life, suggesting that simple CHD is associated with a good overall prognosis. Therefore, continuation of pregnancy should be actively recommended in case of simple CHD after excluding chromosomal abnormalities and severe extra-cardiac abnormalities. Twenty-seven patients with complex CHD (group B) who accepted cardiac surgery were followed up till date, 21 patients have shown good growth and development, while six patients died after operation,the survival rate was 77.78%. In this study, good surgical outcomes were achieved in patients with tetralogy of Fallot, atrioventricular septal defect, coarctation of aorta, transposition of the great arteries, double outlet of right ventricle, pulmonary atresia, and interruption of aortic arch.
Local pathological anatomy of CHD is the most definitive method to verify the prenatal diagnosis of CHD. In situ observation allows for visual assessment of the cardiac morphology. Local necropsy allows for detailed assessment of the structure of heart chambers, myocardial tissue, and heart valves. Comparison of prenatal ultrasound diagnosis with results of pathological anatomy in 147 complex CHDs indicated that the misdiagnosis with ultrasound mainly pertained to pulmonary atresia, heterotaxy syndrome, hypoplastic left heart syndrome, and valvular disease; these findings are consistent with previous reports 16,24,27 .
Examination of cast specimens of 21 cases suggested that the misdiagnosis with ultrasound mainly pertained to the abnormal branching of the aorta and the abnormal reflux of the body and pulmonary veins, which is consistent with previous reports 16,28 . Local pathological anatomy can facilitate accurate post-mortem diagnosis of intracardiac malformations while cast specimens allow for assessment of the branches of the aorta and the abnormalities of body and pulmonary venous return. In addition, pathological anatomy or casting can provide real specimens for clinical and teaching purposes.
Due to the vacant positions of pediatric cardiologists in most women and children hospitals, it is difficult to accurately determine the subtype and long-term prognosis of fetal CHD, and provide authoritative treatment and prognosis counseling. Therefore, the MDJC is an effective model adopted by maternal and child specialist hospitals for perinatal management of fetal CHD. Subsequent follow-up found that there was a difference in the rate of induction of labor of the CHD patients in each group with MDJC. On comparing the pregnancy outcomes of patients with MDJC in each group, we found that the pregnancy termination rates in groups B and C were significantly lower than those without MDJC (50.65% vs. 82.76% in group B; 78.41% vs. 94.21% in group C).
In this study, the overall pregnancy termination rate due to fetuses with CHD was 64%; of them, the termination rates for simple CHD, complex CHD, and CHD with extra-cardiac abnormalities were 35.51%, 87.00%, and 81.23%, respectively. MDJC consultation and subsequent counseling of family members facilitated their understanding of the actual situation regarding the fetal CHD. Among them, 88 pregnant women and their families who participated in MDJC altered their original tendency to continue pregnancy; 65 of them achieved a better prognosis. Thus, MDJC management mode offered a distinct clinical leverage by optimizing the diagnosis and treatment of CHD.

conclusion
The accuracy rate of prenatal ultrasound diagnosis of complex and simple CHDs was 90.50-91.66% and 98.68%, respectively. Prenatal ultrasound nine-segment sequential analysis method is an effective method for the diagnosis of fetal CHD. Interventional prenatal genetic diagnosis to exclude chromosomal abnormalities is recommended for fetuses affected by CHD. Local pathological anatomy and establishment of cast specimens of fetal CHD are effective methods to verify the prenatal ultrasound diagnosis. MDJC significantly decreased the pregnancy termination rate. MDJC is an effective strategy for perinatal management of fetal CHD.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.