Copy number variation sequencing detection technology for identifying fetuses with abnormal soft indicators: a comprehensive study

Introduction

Prenatal ultrasound is essential for prenatal screening and diagnosis due to its non-invasive nature, minimal radiation, and safety for the fetus. It helps assess fetal health, identify structural abnormalities, and detect subtle anatomical variations. Ultrasonic soft markers (USM) in prenatal ultrasound help assess fetal health by indicating potential chromosomal abnormalities. These markers are not definitive structural issues but may reflect normal variations or temporary changes during pregnancy. While they can’t diagnose chromosomal abnormalities, they can signal possible risks. Certain soft marker abnormalities are closely associated with specific diseases, and when combined with certain high-risk factors, they can increase the likelihood of chromosomal abnormalities [1], 2].

Research shows a strong link between chromosomal abnormalities and abnormal soft markers. For example, many fetuses with trisomy 21 have high nuchal translucency (NT) values [3], often linked to congenital heart disease. Similarly, trisomy 18 fetuses frequently develop choroid plexus cysts (CPC) [4]. The diagnostic specificity of abnormal ultrasound soft markers for fetal chromosomal abnormalities is low. Therefore, advancing prenatal analysis techniques is essential to improve diagnostic accuracy and support clinical diagnosis and treatment.

Next-generation sequencing (CNV-seq) offers a new, efficient method for prenatal diagnosis. It is user-friendly, highly automated, fast, high-throughput, requires minimal DNA (10 ng), provides high resolution (100 kb accuracy), and can detect low-level chimeras [5], 6]. Notably, experts have suggested that CNV-seq technology, given its capabilities, can serve as a primary prenatal diagnostic tool in clinical settings [7]. CNV-Seq analysis is crucial for prenatal diagnosis, especially in high-risk pregnancies, as it accurately detects chromosomal abnormalities. However, few studies have explored its use in patients with abnormal ultrasound soft markers. This study aims to explore the use of CNV-seq technology to identify chromosomal abnormalities in pregnant women with abnormal ultrasound indicators, addressing a critical need in improving clinical decision-making strategies.

Materials and methods

Between June 2021 and December 2022, 295 pregnant women with abnormal prenatal ultrasound indicators were studied at Yulin Maternal and Child Health Care Hospital. They underwent either amniocentesis (245 cases) or umbilical cord blood sampling (50 cases). Participants’ ages ranged from 16 to 44 years, averaging 30.05 years. Participants’ gestational ages ranged from 16 to 37 weeks, averaging 20 weeks and 8 days. Karyotype analysis, CNV-seq detection, and STR experiments were performed on all samples to confirm fetal origin and exclude maternal contamination. Informed consent was obtained from pregnant women before invasive prenatal diagnosis procedures, including amniocentesis or umbilical cord blood sampling, and was reiterated verbally. The Obstetrical Ethics Committee of Yulin Maternal and Child Health Care Hospital approved the study, ensuring strict confidentiality and protection of patient data.

The study included pregnant women with singleton pregnancies showing abnormal ultrasonic soft indicators, which were reviewed by senior experts in the Ultrasound Department at Yulin Maternal and Child Health Care Hospital. The study excluded pregnant women with fetal structural abnormalities detected by ultrasound, those who had invasive prenatal diagnosis following abnormal noninvasive prenatal testing (NITP) results, and those with multiple pregnancies.

Results

In this study, 295 pregnant women with abnormal fetal ultrasound indicators underwent successful prenatal diagnosis: 50 via umbilical cord blood and 245 via amniotic fluid. Both chromosomal karyotype analysis and CNV-seq detection achieved a 100 % success rate.

Discussion

This study used CNV-seq technology to identify 295 pregnant women with abnormal ultrasound markers, detecting chromosomal abnormalities in 43 cases, yielding a 14.58 % detection rate. In contrast, Wang et al. [9] found a lower detection rate of 3.39 % using CNV-seq technology in a larger study of 737 pregnant women with abnormal ultrasound soft markers. Lan et al. [10] found that CNV-seq technology identified pathogenic copy number variations in 20 out of 400 ultrasound cases with abnormal soft indexes, achieving a 5 % detection rate. In our own investigation, we detected 21 cases of pathogenic CNVs, accounting for 7.12 % of the total sample, surpassing both Lan et al.’s [10] findings and those of another study conducted by Wang et al. [11]. The discrepancy might arise from differences in statistical criteria, with some studies counted only pathogenic CNVs and excluded aneuploidy abnormalities. The large variation in sample sizes of different abnormal soft indexes in the study could also be a factor. Some studies have found a higher chance of chromosomal abnormalities when certain soft markers like NT are abnormal, while most normal non-specific changes like VM have been detected under these conditions.

In a group of 295 pregnant women with abnormal ultrasound soft markers, the seven most common were thickened nuchal translucency, nasal bone issues, multiple abnormal markers, enhanced or dilated intestinal echo, lateral ventricular dilatation, CPC, and single umbilical artery. Fetal NT is the fluid buildup in the back of a fetus’s neck between 11 and 13+6 weeks of pregnancy. An NT measurement below 2.5 mm is normal, while 2.5 mm or more indicates thickening, often linked to chromosomal abnormalities [12], [13], [14]. Leung et al. noted that around 10 % of fetuses with high NT values had chromosomal variations undetectable by standard karyotyping. In our study, CNV-seq identified abnormal chromosomes in 13 out of 120 such cases, yielding a 10.83 % detection rate, aligning with Leung et al.’s findings [15]. Fetal Ventriculomegaly (VM) is characterized by ventricular chambers measuring ≥10 mm in width. Our retrospective analysis found a 14.29 % (3/21) detection rate of chromosomal abnormalities using CNV-seq in lateral VM cases, comparable to the incidence reported in isolated mild VM cases in foreign studies [16], 17]. The CNVs found in the three isolated VM cases in our study were deemed benign or possibly benign, aligning with prior research showing most fetuses with mild Ventriculomegaly had normal variations [18]. Nasal bone dysplasia is a prevalent ultrasound manifestation observed in trisomy 21 fetuses. In our study, CNV-seq detected abnormalities in five of 36 cases (13.89 %) of isolated nasal bone dysplasia, with 3 cases linked to trisomy 21, highlighting a significant correlation between nasal bone dysplasia and trisomy 21. This finding aligned with the results reported by Cicero et al. [19]. Enhanced bowel sounds (EB) were often noted in the second trimester of pregnancy, with detection rates between 0.6 and 2.4 % [20]. Using CNV-seq, our study identified six chromosome copy number variations, achieving a 20.69 % detection rate, potentially affected by sample selection bias and other factors. Notably, only one copy number variation was found to be pathogenic, and all pregnancies led to live births with no noticeable abnormalities in the first month. This result was consistent with existing literature on abnormal fetal intestinal echogenicity, though its clinical importance was unclear [21]. Additionally, Ronin et al. [22] noted that isolated intestinal echogenicity and late-pregnancy intestinal echogenicity were both strong, independent indicators of good neonatal outcomes. CPC are hemangioma-like lesions in the choroid plexus capillaries, containing cerebrospinal fluid. Research showed no significant link between isolated CPC and chromosomal abnormalities, with few studies on non-isolated CPC and such abnormalities [23], 24]. In line with existing literature, this study found no abnormal chromosomes in isolated CPC when analyzed using CNV-seq and karyotype analysis. Several studies had indicated [25] CPC were significantly linked to trisomy 18, occurring in about half of affected fetuses. In our study, we found one trisomy 18 case, marked by a CPC, increased nuchal translucency, and an abnormal single umbilical artery. Furthermore, Our study found two abnormalities in seven cases of posterior cranial fossa widening, two in five cases of renal pelvis dilation, one in three cases of ventricular hyperechoic plaque, and one in six cases of vagal right subclavian artery. These findings differ significantly from previous research, particularly due to the limited number of abnormal soft indexes liked ventricular hyperechoic plaque, observed in fewer than 10 cases. Consequently, the potential for bias in the results was substantial. Additionally, NF thickening, single umbilical artery, short femur, and tricuspid valve regurgitation were rare, and no abnormal chromosomes were found via CNV-seq or karyotype analysis, so they were not thoroughly analyzed. Our study found that when multiple abnormal ultrasound soft markers were present, CNV-seq detected abnormal chromosomes in 29.41 % of cases, compared to 12.64 % with a single soft marker. These findings suggested using CNV-seq detection when multiple ultrasound soft markers were found in fetuses, as numerous studies had linked multiple soft marker abnormalities to a higher risk of chromosomal issues. Additionally, Lu et al. [26] suggested considering invasive prenatal diagnosis for multiple soft index evaluations, but recommended avoiding unnecessary invasive tests when only a single soft index abnormality was present without high-risk factors.

CNV-seq accurately identified abnormal karyotype fragments, improving prenatal diagnosis and genetic counseling. It detected all chromosomal aneuploidies, including 13 trisomy 21 cases, one trisomy 18 case, and 1 Turner syndrome case, consistent with chromosomal karyotyping results. Notably, in the case of A221322 (see Appendix, Table A1), chromosomal karyotyping showed a 47, XYY/46, XY pattern, while CNV-seq indicated a diploid Y chromosome, resulting in a 47, XYY syndrome diagnosis. This is likely due to the nearly equal presence of two cell lines, 47, XYY[51]/46, XY[49]. Other studies [7], 27] had shown that CNV-seq results for chimeras with balanced multiple cell lines or 50 % of two aneuploid lines can vary. We suggest verifying these results with additional methods like fluorescence in situ hybridization.

CNV-seq can accurately pinpoint the source of abnormal chromosome fragments and detect chromosomal microdeletions and microduplications that karyotyping cannot. Wang et al. [11] used CNV-seq to detect abnormal soft markers in pregnant women via ultrasound, achieving a 0.97 % higher detection rate of pathogenic CNVs than karyotyping. In our study, CNV-seq identified five more pathogenic variants, outperforming karyotyping by 1.69 % and slightly surpassing Wang et al.’s results. Although CNV-seq technology has improved the detection of pathogenic copy number variations (pCNVs), it also has identified more copy number variations of uncertain significance (VOUS). This complicates genetic counseling and can cause psychological distress in pregnant women [28]. In our study, a total of 13 cases of VOUS were identified in pregnant women exhibiting abnormal soft index. The detection rate of VOUS was found to be 4.4 %, which aligns with the findings reported internationally [8], 29]. Considering various factors, including cost, only seven confirmed pathogenic and VOUS cases agreed to parental traceability analysis. Results showed 6 cases were genetically related: four maternal transmissions, two paternal transmissions, and 1 de novo mutation. Of these, 2 cases led to induced labor due to pathogenic diseases, while the rest resulted in live births. One live birth (case A222045 in Appendix Table A1, Figure A1) involved left hydronephrosis and left ureteral dilatation. The mother had a normal phenotype, and the father had a repaired congenital cleft lip. The other cases showed no abnormalities. Of the 13 cases with variants of uncertain significance (VOUS), one led to induced labor, and the other 12 resulted in live births without noticeable physical abnormalities. Using CNV-seq to detect parental DNA provides crucial insights into fetal CNV pathogenicity and variants of uncertain significance (VOUS). This method helps identify parental DNA origins, assess recurrence risks, and determines if a case is novel, aiding genetic counseling and informed decision-making for pregnant women and their families. It also supplies essential data for clinical evaluation of soft indicators.

CNV-seq cannot effectively detect balanced translocations and inversions, which are better identified through traditional karyotyping. However, it can identify chromosomal microdeletions or duplications associated with balanced translocations. In this study, CNV-seq missed a balanced translocation in one case, despite an ultrasound showing a short nasal bone abnormality in the pregnant woman. Further examination of the parents’ chromosomes revealed that the balanced translocation chromosome was inherited from the mother. Research showed that balanced translocations and inversions of chromosomes significantly contributed to reproductive issues. Additionally, about 10 % of full-term fetuses from couples with balanced translocation carriers had chromosome abnormalities, a rate higher than in the general population [30]. Additionally, balanced translocation had been linked to recurrent miscarriages [31]. In our study, a pregnant woman with balanced translocation chose to continue her pregnancy, leading to a live birth. Follow-up checks showed no abnormalities in the newborn. Due to the rare detection of balanced translocation in ultrasound soft markers, a detailed examination was not conducted.

Advanced maternal age significantly impacts the need for prenatal diagnosis, as research showed a higher rate of fetal birth defects and chromosomal abnormalities in older pregnant women compared to younger ones [32]. The present study further supports these findings, revealing a greater incidence of chromosomal abnormalities in the advanced age group compared to the young age group. Hu et al. [33] found a 21.07 % detection rate of abnormal chromosomes with an abnormal isolated soft sexual index. This rate rose to 31.16 % when combined with advanced age, a statistically significant increase compared to the non-advanced age group. This study found no significant differences (p>0.05) between older adults with an abnormal isolated soft sexual index and younger adults, unlike Hu’s [33] findings. However, a significant difference was found between older individuals with multiple soft index abnormalities and younger individuals with the same condition (p<0.05). This finding indicated a higher detection rate of chromosome abnormalities in older individuals with multiple soft index abnormalities, consistent with previous studies. Additionally, CNV-seq detected three additional chromosomal abnormalities in this age group, including a pathogenic NT thickening. Genetic counseling led to an abortion to prevent congenital anomalies. This demonstrated CNV-seq’s superior accuracy in identifying chromosomal microdeletions and microduplications.

Conclusions

CNV-seq technology is valuable for identifying fetuses with abnormal soft markers, guiding its future use in perinatal diagnosis and aiding clinical genetic counseling.