Introduction

China is one of the countries with the highest incidence of birth defects in the world. The incidence of birth defects in China is about 5.6%, and the number of new birth defects is about 900,000 each year, accounting for 20% of the world’s birth defects.1 Birth defects are the main causes of early abortion, stillbirth, perinatal death, infant death, and congenital disability, which not only seriously endangers children’s survival and quality of life but also causes huge life loss and social and economic burden.2,3 Prenatal diagnosis is an effective method to prevent birth defects, and prenatal ultrasound imaging and genetic testing are important means of prenatal diagnosis.4,5

As a routine technique for screening fetal malformation, prenatal ultrasonography can effectively prevent birth defects.6,7 Prenatal ultrasonography is the application of physical characteristics of ultrasound to the fetus and its appendages for imaging examination. It is the most common, non-invasive, and repeatable method to understand the embryo and the main anatomical structure and general shape of the fetus.8,9 Prenatal ultrasonography can not only evaluate fetal anatomy but also observe fetal movement and behavior in utero in real time and evaluate fetal hemodynamic changes.10,11 Transabdominal and transperineal ultrasound also play an important role in assessing the prediction of labor and its progression.12,13 We regard the fetus as an individual, and prenatal ultrasound plays an irreplaceable role in how its phenotype is expressed. Ultrasound screening during pregnancy is mainly divided into 11–13+6 weeks and 20–24+6 weeks.8,14 Among them, 11–13+6 weeks is an important time for ultrasound screening of early fetal structural abnormalities, and more than 80% of abnormalities have been formed at this stage.8 Ultrasound screening during early pregnancy (FTS) can pass: The nasal bone (NB) was observed, the thickness of the nuchal translucency (NT) was measured, and the tricuspid regurgy (TR) and abnormal venous catheter (aDV) Doppler waveform were observed to evaluate the risk of fetal chromosome aneuploidy.15 After 20 weeks, the ultrasound can be used for the fetal growth restriction (FGR) diagnosis and fetal Doppler study, which result is very important for the fetal management.16,17 Fetal ultrasound abnormalities can be classified into fetal structural abnormalities and non-structural abnormalities.18

Chromosome abnormalities account for more than 80% of the genetic causes of birth defects, mainly including chromosome number abnormalities, large fragment deletion/duplication and genome copy number variation (CNV).19,20 Studies have reported that fetal ultrasound abnormalities are caused by about 12.4–35% chromosomal aberrations, about 25% by chromosomal karyotype structure or number abnormalities, and about 10% by chromosomal microstructure abnormalities.21 When the fetal ultrasound is abnormal, genetic examination should be performed. Chromosomal karyotype analysis of fetal specimens has always been the gold standard for prenatal analysis of chromosomal defects in pregnant women, which can detect balanced translocation, inversion, deletion, insertion, and rearrangement of large fragments.20,22 However, karyotype analysis is not sufficient to detect chromosomal submicrostructure abnormalities smaller than 5 mega base (MB).23 Chromosomal microarray analysis (CMA) is the detection of CNV in the whole genome using cloned DNA probe. CMA can detect unbalanced chromosomal CNVs at the whole genome level and can be used to identify chromosomal abnormalities, including those with fragments too small to be detected by karyotyping, as well as minute chromosomal abnormalities.24,25 In 2009, American College of Obstetrics and Gynecology (ACOG) recommended CMA technology for the first time for fetuses with abnormal ultrasound structure and normal karyotype, opening the application of CMA in prenatal diagnosis.26 CMA was detected 3% to 6% more often in fetuses with abnormal ultrasound but normal karyotype.25,27–29

CMA has become a first-line diagnostic technology for fetal chromosome copy number deletion or duplication abnormalities. Studies on the detection rate of structural abnormalities of different fetal systems with CMA technology have different reports. The incidence and characteristics of birth defects and genetic diseases vary from population to region.30,31 There are also great differences in the results of related studies in different regions and different races. In this study, 368 cases of fetal ultrasound abnormalities in the Department of Prenatal Diagnostic Center of Meizhou People’s Hospital were retrospectively analyzed. Interventional prenatal diagnosis and CMA analysis were performed after informed consent, so as to evaluate the clinical significance of CMA in fetal ultrasound structural abnormalities and ultrasound soft indicators.

Materials and MethodsStudy Cohort

A retrospective study was conducted on 368 fetuses with abnormal ultrasound who received interventional prenatal diagnosis in the Prenatal Diagnosis Center of Meizhou People’s Hospital from October 2022 to December 2023. The inclusion criteria as follows: (1) fetuses conceived by pregnant women with childbearing age; and (2) fetuses with at least one abnormal ultrasound marker. Exclusion criteria were as follows: (1) pregnant women with multiple pregnancies; (2) fetus with threatened abortion; and (3) pregnant women who did not consent to participate in this study.

Chorionic villus sampling (CVS) was conducted at 10 to 12 weeks of gestation; amniotic fluid (10mL) was collected at 15 to 22 weeks; and cord blood (1.0–2.0mL) was sampled at 18 to 28 weeks. This study was performed in accordance with the ethical standards of the Declaration of Helsinki and approved by the Human Ethics Committee of Meizhou People’s Hospital. The written informed consent of pregnant couples for invasive prenatal diagnosis was obtained. The possibility of maternal cell contamination (MCC) of all fetal samples has been ruled out using Short tandem repeat (STR) detection, and the quantity and quality of fetal DNA samples meet the requirements of CMA testing.

Fetal ultrasound abnormalities included:

Fetal ultrasound abnormalities included:

Fetal ultrasound structural abnormalities: cardiovascular system abnormalities, urinary system abnormalities, thoracic abnormalities, cephalic facial abnormalities, nervous system abnormalities, digestive system abnormalities, skeletal system abnormalities, abdominal wall abnormalities, and other malformations.

Fetal ultrasound non-structural abnormalities: Abnormal ultrasound soft indicators: NT thickening, ventriculomegaly, nasal bone dysplasia, choroid plexus cyst, short long bones, pyelic separation, echogenic bowel, single umbilical artery, tricuspid regurgitation, and pyelectasis. Fetal growth restriction (FGR); Abnormalities of amniotic fluid volume.

Main diagnostic criteria of fetal ultrasonographic soft markers:32,33

NT thickening: 11–13+6 weeks of pregnancy, NT ≥ 3.5 mm; Nasal bone dysplasia/loss: no ossified nasal bone can be detected, or the nasal bone is short in length; Thickening of cervical fold (NF), NF ≥6mm after 15 weeks of gestation; Mild lateral ventricular widening (VM), measuring the width of the front or back foot of the lateral ventricle at any gestatory week >10mm; Mild renal dilatation (MP), measured in the short section of the kidney anterior and posterior pelvis diameter, more than 4mm in 14–20 weeks of pregnancy, more than 5mm in 20–30 weeks, more than 7mm after 30 weeks; Ventricular intense light spot (EIF), one side of the heart ventricle of the ventricle appeared spot-like isolated focal echo, similar to fetal bone tissue echo intensity; Enhanced bowel echo (EB), usually defined as a bowel echo equal to or greater than the surrounding fetal skeletal echo; Long bone short, femur/humerus < tenth percentile; Choroid plexus cyst (CPC) refers to the strong echo in the lateral ventricle at 16–24 weeks, and the echoless cystic structure in the choroid plexus; Single umbilical artery (SUA) is most easily displayed in the transverse section of the free umbilical cord, and only one artery is seen on both sides of the bladder; Widening of the posterior cranial fossa means that the diameter of the posterior cranial fossa is greater than 10mm. G-Banding Karyotype Analysis

After cell culture of the collected fetal samples, the G-banding chromosome division phase map was observed under optical microscope after cell collection, section, and Giemsa staining. Twenty divisions were counted, five karyotypes were analyzed, and the chimeric type count was increased to 50–100 divisions. Chromosomal karyotypes were determined according to the International System for human Cytogenetic Nomenclature (ISCN).

CMA Detection and Data Analysis

DNA extraction of fetal sample was performed in strict accordance with the operating instructions (Qiagen, Valencia, CA, USA). The extracted DNA samples were cleaved, ligated, amplified, pure, quantified, fragmented, labeled, hybridized, washed, stained, scanned, and analyzed according to standard operating procedures (SOP). The chip used for CMA detection is Affymetrix Cytoscan 750K Array chip (Affymetrix, USA). Finally, the obtained original data is analyzed by the corresponding software. Test data that meets the data quality standards set by the test platform were analyzed and interpreted.

The CMA test results were analyzed and interpreted in combination with public databases commonly used internationally, such as the University of California Santa Cruz Database (UCSC) (https://genome.ucsc.edu), Phenotype in Humans using Ensembl Resources (DECIPHER) (http://decipher.sanger.ac.uk), Clinical Genome Resource (ClinGen) (https://www.clinicalgenome.org/), Database of Genomic Variants (DGV) (http://dgv.tcag.ca/dgv/app/homr), and Online Mendelian Inheritance Database in Man (OMIM) (https://www.omim.org). According to the American College of Medical Genetics and Genomics (ACMG) guidelines, the clinical significance of CNVs is divided into 5 grades: pathogenic (P) CNV, likely pathogenic (LP) CNV, variants of uncertain significance (VUS) CNV, likely benign (LB) CNV, and benign (B) CNV.34,35 Deletion or duplication regions are defined as P/LP CNV if they contain key regions with known microdeletion/microduplication syndromes, carry OMIM pathogenic genes, or are clinically significant in relatives inherited from phenotypic abnormalities that have been reported in multiple literature or databases.

ResultsBaseline Characteristics of Study Cohort

Of the 368 fetuses included in the study, there were 314 (85.3%) fetuses of pregnant women under the age of 35 years old, and 54 (14.7%) fetuses of pregnant women aged >35 years old. At the time of inclusion in this study, there were 93 cases (25.3%) with gestational weeks ≤13 weeks, 228 cases (61.9%) with gestational weeks 14–28 weeks, and 47 cases (13.0%) with gestational weeks >28 weeks. Among the fetuses undergoing interventional prenatal diagnosis due to abnormal ultrasound, there were 55 (14.9%), 312 (84.8%) and 1 (0.3%) samples were villus, amniotic fluid, and cord blood, respectively. Among the fetuses with abnormal ultrasound, 114 cases (31.0%) had structural abnormalities, 225 cases (61.1%) had non-structural abnormalities, and 29 cases (7.9%) had both structural and non-structural abnormalities (Table 1).

Table 1 Demographic Characteristics of Pregnant Women and General Characteristics of Fetuses

The Relationship Between Fetal Ultrasonic Structural Abnormalities and Chromosomal Abnormalities

There were 110 (29.9%, 110/368) fetuses with single ultrasound structural abnormality and 4 (1.1%, 4/368) fetuses with multiple ultrasonic structural aberrations. In the fetuses with single structural abnormality of ultrasound, in order from the most to the least number of cases: 38 (33.33%) with cardiovascular system abnormality, 22 (19.30%) with urinary system abnormality, 11 (9.65%) with thoracic abnormality, 10 (8.77%) cephalic facial abnormality, 9 (7.89%) with nervous system abnormality, 6 (5.26%) with digestive system abnormality, 6 (5.26%) with skeletal system abnormality, and 2 (1.75%) with abdominal wall abnormality. The detection rate of aneuploidy and P/LP CNVs in fetuses with ultrasonic structural abnormalities was 5.26% (6/114), the detection rate of chromosome karyotype was 2.63% (3/114), and the additional diagnosis rate of CMA was 2.63%. In the fetuses with single structural abnormality of ultrasound, the detection rate of aneuploidy and P/LP CNVs in fetuses with ultrasonic structural abnormalities was 4.55% (5/110), the detection rate of chromosome karyotype was 1.82% (2/110), and the additional diagnosis rate of CMA was 2.73% (Table 2).

Table 2 The Relationship Between Fetal Ultrasonic Structural Abnormalities and Chromosomal Abnormalities

The Relationship Between Fetal Ultrasonic Non-Structural Abnormalities and Chromosomal Abnormalities

In this study, the fetuses with non-structural ultrasound abnormalities included 212 (57.6%, 212/368) fetuses with abnormal ultrasound soft markers, 30 (8.2%, 30/368) fetuses with growth restriction, 8 (2.2%, 8/368) fetuses with abnormal amniotic fluid volume, and 5 (1.4%, 5/368) fetuses with multiple non-structural abnormalities. In fetuses with abnormal ultrasound soft markers, there were 81 (36.00%) fetuses with NT thickening, 29 (12.89%) fetuses with ventriculomegaly, 24 (10.67%) fetuses with nasal bone dysplasia, 9 (4.00%) fetuses with choroid plexus cyst, 6 (2.67%) fetuses with short long bones, 6 (2.67%) fetuses with NT thickening + choroid plexus cyst, and 5 (2.22%) fetuses with pyelic separation. In the fetuses with abnormal ultrasound soft markers, the detection rate of chromosome karyotype was 7.69% (14/182), the detection rate of aneuploidy and P/LP CNVs in fetuses with ultrasonic structural abnormalities was 9.89% (18/182), and the additional diagnosis rate of CMA was 2.2%. In the fetuses with ultrasonic non-structural abnormalities, the detection rate of chromosome karyotype was 6.22% (14/225), the detection rate of aneuploidy and P/LP CNVs in fetuses with ultrasonic structural abnormalities was 9.33% (21/225), and the additional diagnosis rate of CMA was 3.11% (Table 3).

Table 3 The Relationship Between Fetal Ultrasonic Non-Structural Abnormalities and Chromosomal Abnormalities

The Relationship Between Fetuses with Structural Abnormalities Combined with Non-Structural Abnormalities and Chromosomal Abnormalities

In this study, there were 29 fetuses with structural abnormalities combined with non-structural abnormalities. In fetuses with structural abnormalities combined with non-structural abnormalities, NT thickening combined with structural abnormalities (2.17%, 8/368) and ventriculomegaly combined with structural abnormalities (1.63%, 6/368) were common. In the fetuses with structural abnormalities combined with non-structural abnormalities, the detection rate of chromosome karyotype was 6.90% (2/29), the detection rate of aneuploidy and P/LP CNVs in fetuses with ultrasonic structural abnormalities was 13.79% (4/29), and the additional diagnosis rate of CMA was 6.89% (Table 4).

Table 4 The Relationship Between Fetuses with Structural Abnormalities Combined with Non-Structural Abnormalities and Chromosomal Abnormalities

Comparison of Chromosome Abnormality Rate Among Different Abnormal Fetal Ultrasound Types and Groups of Different Number of Abnormal Ultrasonic Items

The rates of chromosomal abnormalities were compared between fetuses with different types of ultrasound abnormalities. There was no significant difference in chromosome abnormality detection rate of CMA among structural abnormality, non-structural abnormality, and structural abnormality combined with non-structural abnormality groups (all p > 0.05). According to the different ultrasonic abnormalities, these fetuses were divided into single ultrasonic abnormality group and multiple ultrasonic abnormalities group. The chromosome abnormality detection rate of CMA and karyotype in multiple abnormalities group was higher than those in single abnormality group, but the differences were not statistically significant (p>0.05) (Table 5).

Table 5 Comparison of Chromosome Abnormality Rate Among Different Abnormal Fetal Ultrasound Types and Groups of Different Number of Abnormal Ultrasonic Items

Discussion

Fetal structural development abnormalities are congenital birth defects characterized by morphological and structural abnormalities, often accompanied by changes in genetic material. Fetal structural abnormalities can be transmitted to offspring by genetic material of germ cells such as chromosomal abnormalities, gene mutations, environmental factors, and other unknown causes.36,37 The fetus may also have some minor anatomical changes and abnormalities (known as non-structural abnormalities), and associated with an increased risk of fetal genetic abnormalities.38 At present, prenatal ultrasonography can detect the vast majority of fetal morphological and structural abnormalities, as well as some minor anatomical changes.39 Studies have shown that 2–4% of fetuses during pregnancy have structural abnormalities.40 Ultrasound can detect most fetal structural abnormalities, and the detection rate varies according to different stages of pregnancy and different tissue systems.

In this study, in the group of single ultrasonic structural abnormalities, the number of cardiovascular system abnormalities was the largest (38 cases, 33.3%), which was consistent with the main types of fetal congenital structural abnormalities.41 NT thickening is an independent marker of fetal chromosome aneuploidy and an indicator for further interventional prenatal diagnosis and genetic analysis.42 Bromley et al reported that the detection rate of trisomy 21 was 41.7% in fetuses with NT thickening but no other structural malformations detected by ultrasound.43 In this study, a total of 10 fetuses with chromosome aneuploidy were detected by karyotype analysis, and 6 of them showed NT thickening. Ventriculomegaly means that the width of the anterior or posterior foot of the lateral ventricle is measured between 10–15mm at any gestational week, and more than 50% of the non-isolated mild lateral ventricular widening is often associated with central nervous system abnormalities.44,45 Nasal bone dysplasia/deletion refers to undetectable ossification of the nasal bone or short length of the nasal bone, which is closely related to trisomy 21 syndrome, trisomy 18 syndrome, and trisomy 13 syndrome and is an indication of interventional prenatal diagnosis.46 CMA can not only detect chromosome aneuploidy but also detect chromosome microdeletion and microduplication.47 Pan et al48 conducted CMA analysis on fetuses with nasal bone abnormalities and found that 17.7% of fetuses had chromosomal abnormalities, and the detection rate was higher when nasal bone abnormalities combined with other soft markers or structural abnormalities. In this study, a total of 10 fetuses with chromosome aneuploidy were detected by karyotype analysis, and 3 of them showed nasal bone dysplasia. There are many kinds of fetal ultrasound soft indicators, the incidence of chromosomal abnormalities varies among different isolated ultrasound soft indicators.33,49

Compared with traditional karyotype analysis, CMA can improve the detection rate. The study of Ronald et al showed that 6.0% pCNVs were detected by CMA in fetuses with abnormal ultrasound but normal karyotype analysis.27 Liao C has detected pCNVs in 11.4% of 446 fetuses with abnormal ultrasound but normal karyotype analysis.50 In Malgorzata I Srebniak’s study, aneuploid abnormalities were excluded in 1033 fetuses with abnormal ultrasound, and 7.3% pCNVs were detected by CMA.51 Therefore, in the prenatal diagnosis of abnormal fetuses with ultrasound, in addition to chromosome karyotype analysis, it is recommended to further perform CMA detection, which can detect chromosome microdeletions and microduplications, and improve the detection rate of fetal chromosome abnormalities.

In addition, the detection rate of chromosome abnormalities in fetuses with non-isolated structural abnormalities is significantly higher than that in fetuses with isolated abnormalities.52–54 In Shaffer’s study, the detection rate of CMA in the group with multiple fetal structural abnormalities was higher than that in the group with single structural abnormalities, and the difference was statistically significant.55 Mastromoro et al found that the detection rate of pathogenic CNVs was 19.47% for single structural abnormality and 27.47% for multiple structure abnormalities after CMA was performed on abnormal fetal ultrasound.56 Lee et al found that the detection rate of pCNVs was 10.5% in fetuses with a single ultrasound structural abnormality and 15.4% in fetuses with multiple ultrasound structural abnormalities.57 In this study, there was no significant difference in chromosome abnormality detection rate among structural abnormality group, non-structural abnormality group, and structural abnormality combined with non-structural abnormality group. However, the chromosome abnormality detection rate of the group with structural abnormality combined with non-structural abnormality was higher than that of the other two groups. According to the different ultrasonic abnormalities, they were divided into single ultrasonic abnormality group and multiple ultrasonic abnormality group. The detection rate of chromosome abnormalities in the multiple ultrasonic abnormality group was higher than that in the single ultrasonic abnormality group, but the difference was not statistically significant. It may be related to the small sample size of this study.

Moreover, the detection rate of chromosome abnormality in FGR alone was 6.67%, but no chromosome abnormality was detected in amniotic fluid alone. The study by Dap et al found that the rate of chromosomal abnormalities in fetuses with FGR was 10.5%.58 Zhu et al found that the rate of chromosomal abnormalities in fetuses with FGR was 10.5% detected by CMA.59 Result from a multicenter study in Spain showed that chromosome abnormality detection rate of CMA in fetuses with FGR was 6.8%.60 One study from Israel showed that the detection rate of P/LP variants was 6.3% in 174 FGR fetuses.61 The differences between these findings may be due to the different genetic backgrounds of the population and the different sample sizes included in the studies.

Providing appropriate testing of genetic material for fetuses with abnormal ultrasound can help to discover the genetic causes of fetal abnormalities, at the same time, to evaluate the prognosis of the fetuses, formulate appropriate delivery methods and neonatal management plans, and provide re-fertility risk assessment.62 It is believed that with the continuous improvement of CNV database, CMA will play a greater value in clinical application. The limitation of CMA is that it is difficult to detect low proportion of chromosome mosaicism and balance structural abnormalities. It is suggested that chromosome karyotype analysis combined with CMA detection should be performed on fetuses with ultrasound abnormalities. The two methods complement each other to improve the detection rate and accuracy of abnormal results.

There were some limitations in this study. First, because the sample size included in the study was not large, this study did not summarize the chromosomal abnormalities of various structural abnormalities. Because some types of structural abnormalities have their associated chromosomal abnormalities.63,64 Second, due to the relatively small number of cases with multiple structural abnormalities in this study, it did not compare the chromosomal abnormalities of fetuses with single and multiple structural abnormalities. Some studies generally believe that the incidence of chromosomal aberrations in fetuses with multiple structural abnormalities is higher than that in fetuses with single structural abnormalities.65 Third, due to the limitation of sample size, this study did not summarize the variation regions with significant directional characteristics for different fetal ultrasound structural abnormalities and fetal ultrasound non-structural abnormalities. Therefore, we need to conduct a larger sample size study to enrich the relevant data.

Conclusions

Compared with the traditional chromosome karyotype analysis, CMA can significantly improve the detection rate of genetic abnormalities in prenatal diagnosis of fetuses with ultrasound abnormalities. There was no significant difference in chromosome abnormality detection rate among structural abnormality, non-structural abnormality, and structural abnormality combined with non-structural abnormality groups, also among multiple ultrasonic abnormality and single ultrasonic abnormality groups, suggesting the necessity of CMA detection for all fetuses with abnormal ultrasound.

Data Sharing Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics Approval

All participants were informed on the study procedures and goals and the study obtained written informed consent from all the participants. This study was approved by the Human Ethics Committees of Meizhou People’s Hospital.

Acknowledgments

The author would like to thank other colleagues who were not listed in the authorship for their helpful comments on the manuscript.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This study was supported by and the Scientific Research Cultivation Project of Meizhou People’s Hospital (Grant No.: PY-C2024018), and the Science and Technology Program for Social Development of Meizhou (Grant No.: 2023B37, and 2023B38).

Disclosure

The authors declare that they have no competing interests in this work.

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