Congenital heart disease (CHD) is the most common birth defect, with an estimation prevalence of one in one hundred of all live born infants.1 The morphologic abnormalities of heart and great vessels resulted from disruption of normal cardiac development. Based on anatomic and physiological phenotypes, diagnosis of cardiac defects can be classified into six exclusive groups as previously described by Gonzalez et al.2 These groups including septal defects (SD), conotruncal defects (CTD), functionally single-ventricle (SV) heart, left-sided obstructive lesions (LSO), right-sided obstructive lesions (RSO), and “others”. About one in three patients with CHD can be categorized as severe, which contains univentricular hearts, atrioventricular canal defects, heterotaxy, CTD, and so on; for this type of patients, medical and surgical interventions were required in the first year of their lives.3
Although CHD is the most common and leading cause of mortality in birth defects, the underlying causes of CHD remain unclear in most cases. Genetic factors are one major contributor to CHD. Aneuploidy, copy number variation (CNV), inherited or de novo point mutations that causally linked to CHD could account for over 30% of cases with CHD.4 CHD is a frequent feature of common aneuploidies including autosomal trisomy21 (T21), trisomy18 (T18), trisomy13 (T13), and sex chromosome aneuploidy (SCA): Turner syndrome (45, X) and Klinefelter syndrome (47, XXY). 40%–50% of T21 individuals are associated with CHD, over 75% of live-born T18 and over 50% of live-born T13 were reported with CHD.5,6 Turner syndrome and Klinefelter syndrome were also linked with 30%–50% incidence of CHD.7 Overall, aneuploidies account for about 9%–18% of live-born CHDs.8
Noninvasive prenatal screening (NIPS) is the widely used screening test for common trisomies, T21, T18, and T13 through fetal cell-free DNA circulating in maternal blood. In singletons, NIPS can detect over 98% of fetus with common trisomies at a combined false-positive rate of 0.13%.9 NIPS have been recommended for all pregnancy, its positive predictive value (PPV) is high among high-risk pregnancies for aneuploidy,10 but the screening yield for population with specific risk such as CHD had not been evaluated. We performed NIPS among a cohort of fetus with CHD detected by ultrasound. We also assessed how NIPS results or ultrasound findings affect parental decision on pregnancy outcomes.
Materials and methods
Study design and subjects
This was a retrospective study of pregnant women with fetuses diagnosed with CHDs by sonographic examination, who willing to underwent NIPS as a side-test for fetal aneuploidies. From August 2016 to October 2017, in the sonographic examination center of Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, a total of 117 women who carried fetuses diagnosed CHDs by sonographically were referred to our central genetic laboratory for NIPS. Genetic counseling and written informed consent for genetic testing was provided. This study was approved by the local ethics committees of the Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region.
Noninvasive prenatal screening
Plasma DNA was prepared from 10 mL maternal blood collected into dipotassium ethylene diamine tetraacetic acid, Insepack TM vacuum tubes (Sekisui Medical Technology, Beijing, Hebei, China). Plasma cell-free DNA extraction, library preparation, sequencing, and data analysis procedures were done as described previously.11 Low pass genome massively parallel sequencing (×0.1 coverage) was performed on the Illumina based NextSeq CN500 platform (Berry Genomics Corporation, Hangzhou, Zhejiang, China). Samples with Z-scores equal or beyond 3 were considered as high-risk for trisomy.11,12 In this methodology, fetal fraction can only be calculated in male pregnancies, using the Y chromosome proportion.
Diagnoses of cardiac defects were classified into six mutually exclusive groups.2 These groups including SD, CTD, functionally SV heart, LSO, RSO, and “others”.13 Our SD group contained fetuses with ventricular SD, and atrial SD. The CTD group contained tetralogy of Fallot (TOF), TOF with hypoplastic aortic arch, TOF with intracardiac pulmonary venous heterotopia, truncus arteriosus, transposition of the great arteries, and double outlet right ventricle. The SV group contained hypoplastic left or right heart syndrome, double inlet and double outlet single right ventricle, and tricuspid atresia. The LSO group contained aortic stenosis, mitral atresia, and hypoplastic aortic arch. The RSO group contained fetuses diagnosed with pulmonary valve stenosis and pulmonary atresia. The “other” group contained dexiocardia and Ebstein anomaly.
Participants that had NIPS negative and positive results were informed by phone and interviewed in 7–12 days and 5–7 days, respectively after blood sampling. Interview questions including pregnancy decision based on what information the participants come to such pregnancy decision, date of pregnancy termination if applicable, and any invasively confirmed genetic result. If the participants did not come to a final pregnancy decision, a second round of phone interview with the same questionnaire will take place in 30 days. For participants who decided to continue their pregnancy, we performed after birth follow-up (18 months or longer after the participant's expecting due day) to see if they eventually completed their pregnancy and had their babies delivered, also to see how their babies behaved functionally in daily life.
The statistical calculations were performed using Excel 2007 (Microsoft, Redmond, WA, USA).
From August 2015 to October 2016, maternal blood serum cell-free DNA screening was offered free of charge to 117 pregnant women with fetuses that were diagnosed sonographically with CHDs, and recorded the participants’ decision of pregnancy and based on what information they made such decision (Fig. 1). The demographic information of the studied group is shown in Table 1.
NIPS result and birth decision
A total of 13 NIPS positive cases (T21/T18/T13/SCA, 13/117, 11.1%) was detected. This positive rate is much higher compared to that for general cohort done at the same time period in our hospital (489/30 923, 1.6%). All NIPS positive pregnancies were terminated, 76.9% (10/13) of those decisions were made mainly based on ultrasound results (Fig. 1). Among the 13 NIPS positive cases, six were true positive confirmed by subsequent karyotype or microarray test, one was false-positive. The remaining six cases terminated the pregnancy without confirmation (Table 2). Therefore the PPV is 85.7% (6/7) in our NIPS towards pregnancies with fetal CHDs. Two of T21 and one of T18 NIPS positive participants wait for genetic confirmation then terminated their pregnancy; the rest of positive cases terminated before confirmation results. In the NIPS negative group (n = 104), two participants lost contact, 23 continued pregnancies, and 79 terminated pregnancies. A total of 77.2% (61/79) of cases made the decision on terminating pregnancy based on ultrasound results.
Types of CHDs and birth decision
CHDs were classified into six groups: SD, CTD, functionally SV heart, LSO, RSO, and “others”. Each fetus may be affected by one or multiple types of CHD. Table 3 listed the statistics of affected status. In 50 cases of fetuses with single type of CHDs (simplex CHD), 66.0% (33/50) were terminated whereas in 66 cases with two or more types of CHDs (complex CHD), 93.9% (62/66) were terminated. The termination rate is significantly higher in pregnancies with complex CHD (P < 0.001). In cases that terminated pregnancies without considering the NIPS results (71/115), 73.2% (52/71) of these were with complex CHDs (Fig. 1), whereas those considered NIPS result and continued pregnancy (23/115), 78.3% (18/23) carried fetuses with simplex CHD.
After birth follow-up
We obtained after birth information on 20 out of 23 cases that continued pregnancy. One case terminated pregnancy at 34 weeks of gestation due to other medical condition, and the rest 19 completed their pregnancies. One baby died 32 days after birth due to pneumonia combined with CHD, this baby was prenatally diagnosed with SD and SV; the rest of the babies were functionally normal in their daily lives with (4/19) or without (15/19) noticeable heart problem needing medical management.
In our cohort, a total of 33 participants opt to have their fetal genotype examined by single nucleotide polymorphism (SNP) array, after the blood sampling for NIPS. Six SNP array confirmed trisomy samples were all correctly screened out by NIPS, and four NIPS negative samples found to be associated with CNVs (Table 4). One of those four CNV positive samples contained two pathogenic CNVs: an 18-Mb duplication in chromosome 11q23.3q25 and a4-Mb duplication in chromosome 22q11.1q11.21. Partial duplication on 11q is known to relate with CHDs and other malformation.14 Three other samples’ CNVs were variants with uncertain significance, their sizes range between 1.1-Mb and 2.2-Mb. Within this selected subgroup, we yield a positive rate of 18.2% (6/33) for NIPS and 30.3% (10/33) for SNP array.
Our study evaluated the clinical application of NIPS in fetuses with CHDs among a cohort of 117 pregnancies. Through NIPS we detected 13 positive cases, which represent a positive rate of 11.1%. Due to the fact that most of our participants terminate their pregnancies before the NIPS results became available or before the NIPS positive results were confirmed, we can only have confirmatory data for about half of our NIPS positive cases (7/13). Based on this small number, the PPV for pregnancy with CHD was 85.7%. The documented incidence of aneuploidy, including 22q11.2 deletion syndrome, in neonates with CHDs ranging from 9% to 19%, and a 33% of aneuploidy among prenatally diagnosed CHD had been reported in selected pregnancies.8,13,15 False negative is not likely contributing to the relatively low positive detection rate in our cohort, rather the fact that we only detected T21, T18, T13, and SCA and the recruited pregnancies were less-selected may be main reasons. This perspective of view was supported by a subgroup of our recruitments: 33 out of the 115 participants with higher proportion of fetal multiplex CHDs chose to have SNP array test for their fetus and yielded a NIPS positive rate of 18.2% and SNP array positive rate of 30.3%. Therefore, one way to further increase the prenatal screening positive rate is to include CNVs into our screening targets, especially the CHD related CNVs.16–18
American Heart Association recommended that decision on terminating pregnancy with CHD should consider the genetic basis of CHDs, especially genetic abnormalities associated with functional or neurodevelopment defects which may require intensive postnatal management.19 In our study, 76.9% and 77.2% of the pregnancies were terminated based on sonographic results alone in NIPS positive and negative groups, respectively, indicating that there is very little impact of NIPS results on pregnancy outcomes. This is partly due to the delayed availability of genetic screening/testing results, partly because of the lack of counseling for parents with CHD fetus. We anticipate that fetus with CHD will undergo more extensive and accurate genetic testing, such as microarray, whole genome sequencing or targeted next generation sequencing on fetal tissue would help to discover more CHD relevant pathogenic variants,20–22 and well counseling the pregnancy couples as revealing their ultrasound findings, before the decision be made.
In this study, we observed significantly high percentage of birth termination in groups with complex CHDs (92.5%, 62/67) compared to that in fetuses with simplex CHD (68.8%, 33/48); we also observed participants who considered NIPS result and continued pregnancy, 78.3% (18/23) carried fetuses with simplex CHD. Depends on the types of CHDs, patients will have varied manifestations, for example, patients with SD alone are usually asymptomatic at infancy and childhood; as they aged, a small percentage will develop serious condition.23 Manifestation and prognosis of the patient with complex CHD are more complicated. Our after birth follow-up showed 75.0% (3/4) of babies with prenatally diagnosed complex CHDs reported heart condition that required continue treatment. Meanwhile 91.7% (11/12) of babies with prenatally diagnosed simplex CHD reported with no noticeable heart condition. With a better prognosis, participants with fetal simplex CHD tend to take different fetal tests, for example, NIPS, into account when they making the pregnancy decision. As the medical technology evolved, prognosis for fetus with complex CHD may be better than used to be. We anticipate a thorough counseling section including suggestion for fetal genetic diagnosis and available fetal intervention/prognosis analysis toward the specific CHD will help to counsel the parents or pregnant women with fetal CHDs appropriately, before they come to any decision of their pregnancy.
In conclusion, from this study, we reported a positive rate of 11.1% and a PPV of 85.7% of NIPS in fetuses with CHDs. The NIPS results, particularly the negative ones, played some roles in continuing pregnancy for fetuses with simplex CHD, yet the majority of decision on pregnancy termination was made on the basis of ultrasound finding, which is a phenomenon that requires appropriate counseling.
We thank our sonographic examination center for providing ultrasound details, and our genetic counseling department for participants’ before and after NIPS test counseling, also data files collection.
Yun Chen, Yun-Li Lai, Yi-Ping Shen, and Xiao-Xian Tian carried out the experiment/examination, data collection, and data analysis. Chen-Guang Zheng and Hong-Wei Wei designed and supervised the project. Yun Chen wrote the article with support and reviewed by Yi-Ping Shen. All authors discussed the results and approved the manuscript for submission.
This study was supported by the Guangxi Zhuang Autonomous Region Health Commission (project number S2015 43 and Z20190827).
Conflicts of Interest
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