The study was approved by the Institutional Review Board of Nghe An Maternity and Pediatric Hospital (Code 13/QĐ-BVSN dated 16/01/2023). Informed consent was obtained from all participants, detailing the study's purpose, procedures, and potential risks, ensuring informed decision-making. Participant privacy was safeguarded with unique identification codes, maintaining confidentiality. Amniocentesis was performed by certified professionals following clinical standards to minimize risks, with continuous monitoring to address any complications promptly, ensuring participant safety and study integrity.

Table 1

Characteristics	Number (n)	Percentage (%)
Maternal Age
< 35 years	117	66.1
≥ 35 years	60	33.9
Average (years)	Mean ± SD: 31.3 ± 6.9	Range: 15-47 years
Gestational Age
< 20 weeks	97	54.8
≥ 20 weeks	80	45.2
Average (weeks)	Mean ± SD: 20.5 ± 4.0	Range: 16-33 months

Note: SD: indicates Standard deviation

Table 2

Indication	Number (n)	Percentage (%)
Abnormal ultrasound morphology	120	67.8
High-risk maternal serum screening	19	10.7
High-risk NIPT (Non-invasive prenatal test)	5	2.8
History of pregnancies with abnormalities	4	2.3
Abnormal ultrasound + High-risk maternal serum screening	11	6.2
Abnormal ultrasound + High-risk NIPT	14	7.9
Abnormal ultrasound + History of pregnancies with abnormalities	4	2.3
Total	177	100

Note: NIPT refers to Non-invasive prenatal testing, which is a method of determining the risk that the fetus will be born with certain genetic abnormalities.

Table 3

Results	CNV-seq Number (n)	CNV-seq Percentage (%)	Karyotype Number (n)	Karyotype Percentage (%)
Total of abnormal patients	46	26.0%	40	22.6%
Aneuploid chromosomal abnormalities	34		34
47,XXXY,+21	17		17
47,XXXY,+18	12		12
47,XXXY,+13	3		3
47,XXX	1		1
45,X	1		1
Mosaic cases	3		2
Triploidy 69,XXX	0		1
Deletions/duplications ≥ 5 Mb	6#		3
Deletions/duplications < 5 Mb	7#		0
Total of normal patients	131	74.0%	137	77.4%
Total	177	100%	177	100%

Note: ^# indicates a total of 13 Deletions/duplications in 9 patients

Table 4

No.	CNV-seq Result	Karyotype Result	Pregnancy Outcome
1	dup(8)(p22-p21.3)	46,XY (normal)	8 months - Normal development
2	del(4)(p16.3), dup(17)(q24.3-q25.3)	46,XY (normal)	Terminated pregnancy due to the risk of Wolf-Hirschhorn syndrome
3	del(5)(p15.33-p15.31)	46,XY,del(5)(p15.3)	Terminated pregnancy following the diagnosis of Cri-du-chat syndrome
4	dup(1)(p31.1)	46,XX (normal)	Normal development at 2 months
5	del(6)(q27), dup(19)(q13.33-q13.43)	46,XY (normal)	Pregnancy terminated at 21 weeks (The fetus presents with hydrocephalus and cerebellar hypoplasia)
6	del(6)(q27), dup(19)(q13.33-q13.43)	46,XY (normal)	Intrauterine fetal demise at 37 weeks (Previous pregnancy history of abnormalities detected at 26 weeks)
7	Suspected mosaic trisomy 12	47,XX,i(12)(p10),i(12)(q10)[8%]/46,XX[92%]	Terminated pregnancy due to critical heart defects
8	No abnormalities detected	69,XXX	Fetal demise at 20 weeks due to severe growth retardation
9	del(18)(p11.32), dup(9)(q21.11-q34.3)	46,XX,+t(9;15)(q21;q2),-15,+t(15;18)(q21;p11.3),-18	Pregnancy terminated due to severe genetic anomalies
10	dup(6)(q15-q16.1)	46,XY (normal)	Normal development at 9 months
11	dup(18)(p11.32-p11.21): Homozygous	47,XY,+mar	Normal development at 6 months with 6 fingers on the right hand, cryptorchidism, subclinical hypothyroidism, slight aortic arch stenosis
12	Suspected mosaic sex chromosome abnormality	46,XY (normal)	Normal development at 6 months

Table 5

Type of CNV	Pathogenic	Likely pathogenic	Uncertain significance	Total
Deletions	2	0	3	5
Duplications	1	4	3	8
Total	3	4	6	13
Detection rate (%) (n = 177)	1.7% (3/177)	2.3% (4/177)	3.9% (6/177)	7.3% (13/177)

Note: CNV indicates Copy Number Variation

Figure 1

Figure 2

Figure 3

The study involved a comprehensive analysis of maternal age and gestational age at the time of amniocentesis across a cohort of 177 participants. The distribution of maternal age demonstrated a bimodal pattern, with the majority (66.1%, n=117) of participants under the age of 35. The mean maternal age was 31.3 years (SD ± 6.9; range 15–47 years). The mean gestational age at the time of amniocentesis was 20.5 weeks (SD ± 4.0; range 16–33 weeks), with the majority of procedures (54.8%, n=97) performed before 20 weeks' gestation. These findings are visualized in Figure 1 and Figure 2 and detailed in Table 1.

The primary indication for amniotic fluid testing was abnormal ultrasound morphology, which accounted for 67.8% (120/177) of all cases. Other indications included high-risk maternal serum screening results (10.7%, n=19) and high-risk results from non-invasive prenatal testing (NIPT) (2.8%, n=5). Combined indications included abnormal ultrasound with high-risk maternal serum screening (6.2%, n=11) and abnormal ultrasound with high-risk NIPT results (7.9%, n=14). A history of pregnancies with chromosomal abnormalities was noted in 2.3% (n=4) of cases. These findings are summarized in Table 2.

In the study cohort of 177 amniotic fluid samples, chromosomal anomalies were identified in 46 cases (26.0%) via CNV-sequencing (CNV-seq), slightly surpassing the detection rate of traditional karyotyping, which identified abnormalities in 40 cases (22.6%). Both diagnostic methods demonstrated a complete concordance rate of 100% in detecting specific aneuploidies: trisomy 21 in 17 cases, trisomy 18 in 12 cases, trisomy 13 in three cases, monosomy X in one case, and 47,XXY in one case.

CNV-seq further identified three cases of mosaicism not initially detected by karyotyping. These included a suspected case of low-level trisomy 18 mosaicism, trisomy 2 mosaicism estimated to affect 10–30% of cells, and a case of sex chromosome mosaicism (60% XY and 40% XX), which likely arose from maternal cell contamination. These findings, confirmed by subsequent karyotyping, highlight the sensitivity of CNV-seq in detecting mosaicism.

Moreover, CNV-seq demonstrated its superior resolution by identifying 13 structural chromosomal changes involving deletions and duplications across nine samples, including several alterations greater than 5 Mb—a refinement not achieved by karyotyping. This capability underscores the effectiveness of CNV-seq in capturing submicroscopic chromosomal changes less than 5 Mb, which traditional methods may overlook. These results are detailed in Table 3, which compares the performance of CNV-sequencing and karyotyping in identifying various chromosomal abnormalities.

This section presents a detailed comparative analysis of CNV sequencing (CNV-seq) and traditional karyotyping across twelve cases, showcasing the superior diagnostic capabilities of CNV-seq. Each case provides a factual account of genetic findings and their clinical outcomes, emphasizing the precision of CNV-seq in prenatal diagnostics.

The analysis underscores the diagnostic precision of CNV-seq, particularly in identifying submicroscopic genetic changes, detecting mosaicism, providing precise breakpoints, covering complex abnormalities, and offering comprehensive screening. These attributes demonstrate the comparative advantage of CNV-seq over traditional karyotyping, supporting informed clinical decisions and significantly influencing pregnancy management. Detailed case analyses are presented in Table 4.

In our study, the classification of CNV abnormalities was performed in strict accordance with the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) guidelines, ensuring the accurate categorization of CNV pathogenicity (refer to Table 5). A total of 13 distinct CNVs, including both deletions and duplications, were identified across nine patients. Out of these, seven were classified as pathogenic or likely pathogenic, underscoring their significant potential for clinical impact. Specifically, our analysis recognized two deletions and one duplication as pathogenic, and four duplications as likely pathogenic. The remaining six CNVs were categorized as variants of uncertain significance (VUS), indicating a more ambiguous potential impact.

The pathogenic classification of CNVs, particularly those identified alongside severe morphological anomalies via ultrasound, often guides expectant families toward considering pregnancy termination. This decision is influenced by concerns over the anticipated quality of life and potential severe health challenges for the child. Conversely, CNVs categorized as having uncertain significance pose substantial challenges in prenatal counseling, necessitating a nuanced approach to risk assessment to prevent undue anxiety among prospective parents. Our approach to counseling emphasizes an informed and empathetic methodology, tailored to each case's unique circumstances, ensuring that decisions are made with a comprehensive understanding of all potential outcomes. The impact of CNV classifications on prenatal counseling decisions is depicted in Figure 3.

Our study included 177 participants, highlighting a trend toward delayed childbearing with a mean maternal age of 31.3 years, reflecting broader implications for prenatal diagnostic strategies. This age profile is slightly older compared to findings from Guangdong, China, where the mean maternal age was 29.84 years11, yet similar to data from Yunnan, China, with ages ranging from 27 to 36 years25. The increased risk of chromosomal anomalies associated with advanced maternal age was significant, demonstrating a higher incidence of chromosome number abnormalities as maternal age increased (p<0.001). However, no significant trends were noted for structural chromosomal abnormalities26, 27, 28. Additionally, the rapid results delivery by CNV-sequencing offers crucial benefits for timely and informed clinical decision-making, especially crucial when anomalies are detected at advanced gestational stages28.

CNV-Sequencing has proven to be a significant advancement over traditional karyotyping, detecting a wider range of submicroscopic deletions and duplications. Our findings demonstrate a 3.4% increase in the detection rate of chromosomal abnormalities with CNV-Seq, identifying abnormalities in 46 cases (26.0%), compared to 22.6% by karyotyping. This capability reflects the diagnostic limitations of traditional methods, especially in identifying smaller genetic changes.

In a comparative context, a study from Guangzhou, China, using different collection methods including chorionic villus sampling and amniocentesis, found abnormalities in 13.47% of cases11. This contrasts with our exclusive use of amniocentesis, collecting 20-30 mL of amniotic fluid, which may explain the differences in detection rates. Furthermore, our CNV-Seq detected a higher incidence of pathogenic microdeletions/duplications (1.7%) compared to a Yunnan study (0.7%), highlighting the impact of sample source differences on detection rates25.

Our study's higher yield of CNVs and detection rates of pathogenic anomalies (3.95%) also compares favorably with a large Sichuan study, which reported lower rates (2.83%) and a smaller proportion of variants of uncertain significance (1.43%)29. These results suggest that the specific clinical indications for our cohort, primarily abnormal ultrasound morphology, contributed to a more targeted and thus higher yield of detectable abnormalities.

Such findings underline the importance of considering the specific clinical and methodological contexts when interpreting comparative studies. The selection bias inherent in focusing on high-risk populations, as in our study, emphasizes the need for careful generalization of findings to broader populations.

Our study, alongside those conducted in Jiangsu and Sichuan, uniformly applied amniocentesis, extracting similar quantities of amniotic fluid during the same gestational stages, adhering to protocols typical in China. Notably, the participant profiles in our and the Jiangsu studies were closely matched, involving high-risk pregnancies, unlike the Sichuan study which included low-risk voluntary participants, potentially diluting its detection rates29, 30. This methodological variance underscores the necessity of considering cohort characteristics when evaluating detection capabilities.

Our study marks a significant advancement in prenatal diagnostics in Vietnam by integrating CNV-Seq, which has substantially improved the detection of chromosomal anomalies. CNV-Seq excels particularly in identifying submicroscopic deletions and duplications, areas where traditional karyotyping falls short. This capability not only enriches prenatal diagnostic insights but also enhances clinical decision-making, potentially decreasing the prevalence of genetic disorders.

Despite these advancements, the study faces limitations. Primarily, CNV-Seq cannot detect certain structural and numerical chromosomal aberrations such as balanced translocations and polyploidies, underscoring the continued necessity for traditional karyotyping. Additionally, focusing predominantly on a high-risk Vietnamese population may restrict the broader applicability of our findings, potentially introducing a selection bias that inflates the observed detection rates.

A significant aspect of our study involves the interpretation of variants of uncertain significance (VUS), which presents ongoing challenges in prenatal counseling and patient management. The complexity of interpreting VUS necessitates advanced methodological approaches and a dynamic adaptation to evolving genomic sciences. Current interpretative frameworks, although based on the latest guidelines, may not fully capture the rapidly changing landscape of genomic data.

There is a critical need for future studies to include more diverse genetic cohorts, extending research to varied ethnic backgrounds to ensure the generalizability of our findings. Enhancing counseling protocols and developing robust support systems are essential for maximizing the benefits of advanced genomic technologies like CNV-Seq in prenatal diagnostics.