A retrospective analysis of 6942 amniocentesis cases

Abstract

Objective

The aim of the present study was to advance the understanding of prenatal diagnostic strategies by systematically analyzing gestational age, duration of pregnancy, clinical indications for prenatal testing, and the prevalence of chromosomal abnormalities among pregnant women undergoing amniocentesis.

Materials and methods

This retrospective study involved 6,942 pregnant women with indications for amniocentesis who visited the Maternal and Child Health Hospital in Changzhi, Shanxi Province, between January 2018 and December 2023. Both the overall cohort and the subset of positive cases were stratified according to prenatal indications for amniocentesis into the following categories: advanced maternal age (AMA), abnormal maternal serum screening (MSS), noninvasive prenatal testing (NIPT)-positive, pathological ultrasound finding (PUF), parental chromosomal abnormality carrier (PCAC), and poor obstetric history (POH). The analysis encompassed the detection rate of chromosomal abnormalities via amniocentesis, the proportion and positive predictive value (PPV) of each indication group, the distribution of maternal age and gestational age, and the characteristic patterns of confirmed diagnostic findings. Statistical differences were evaluated via the nonparametric Mann–Whitney U test.

Results

A total of 6,942 samples were included in the study. Of these, 38 samples (0.55%) with completely lost data were excluded. Samples with partially missing data were retained, including 18 cases (0.26%) lacking maternal age information, 23 cases (0.33%) missing gestational age data, and 8 cases (0.12%) without documented indications for prenatal diagnosis. The distribution of valid data was as follows: maternal age was available for 6,886 cases, gestational age was available for 6,882 cases, and prenatal diagnostic indications were available for 6,896 cases. A total of 557 cases of fetal chromosomal abnormalities were diagnosed, with an overall positive rate of 8.07%. Among the 6,882 valid gestational weeks analyzed, the overall range was 15–37 weeks. Specifically, 70.83% fell within 15–20.6 weeks, and 27.08% fell within 21–27.6 weeks, with positivity rates of 6.93% and 10.18%, respectively. A statistically significant difference was observed between the overall and positive groups (P < 0.05). Among the 6,886 cases with valid maternal age data, the overall age range was 17–50 years, with no significant difference observed between the overall population and positive cases. When the patients were stratified into A1–A6 age groups, statistically significant differences were observed among all groups except A1 (P < 0.05). Among the 6,896 valid cases with recorded prenatal indications, the percentages of MSS, AMA, MSS plus NIPT, NIPT, PUF, POH, and PCAC cases were 51.75%, 30.40%, 0.53%, 4.48%, 7.93%, 2.83%, and 0.40%, respectively. The corresponding PPVs were 4.12%, 8.05%, 94.59%, 42.81%, 8.66%, 5.56%, and 32.14%, respectively. Among the 557 positive cases, chromosomal abnormalities were distributed as follows: aneuploidy (65.35%), structural abnormalities (26.57%), mosaicism (7.18%), and marker chromosomes (0.54%). Autosomal aneuploidy was most frequently represented by trisomy 21, whereas 47,XXY was the most common sex chromosome aneuploidy. Structural abnormalities were most frequently represented by the 17p12 microdeletion. Regarding diagnostic approaches, 27.65% of the cases utilized a single method, 66.96% employed two methods, and 5.39% used three or more methods. Among the single-method cases, Chromosomal Microarray Analysis (CMA) was the most frequently selected technique (14.18%). For cases involving two diagnostic methods, the distribution was as follows (in descending order): karyotype plus QF–PCR (33.21%), QF–PCR plus FISH (17.24%), karyotype plus CMA (10.05%), CMA plus QF–PCR (6.28%), and CNV–seq plus QF–PCR (0.18%). The final analysis revealed that across different age and gestational age subgroups, prenatal indications, diagnostic outcomes, and invasive procedures were most concentrated in the 25–29-year age group and 15–20.6-week gestational age cohort. However, no statistically significant differences were observed among the subgroups (P > 0.05).

Conclusion

This study establishes that a risk-stratified approach optimizes prenatal chromosomal diagnosis: combining maternal serum screening with NIPT enhances detection (PPV 94.59%), while karyotyping and CMA respectively address numerical and structural abnormalities. NIPT proves particularly valuable for advanced maternal age, whereas ultrasound anomalies necessitate CMA. Diagnostic yield remains significant across gestational ages, supporting tailored clinical pathways. These findings underscore the importance of integrating multiple modalities for comprehensive prenatal evaluation.

Introduction

The overall incidence of birth defects in China is approximately 5.6%, with chromosomal abnormalities representing one of the leading etiologies [1]. Chromosomal abnormalities involve alterations in chromosome structure or number, typically arising from nondisjunction or chromosomal breakage during anaphase of cell division. These abnormalities often result in clinical manifestations such as intellectual disability, developmental delay, and multiple congenital anomalies, for which there are currently no effective treatments. Consequently, prenatal diagnosis plays a critical role in early detection and clinical decision-making [2–5]. Ultrasound-guided amniocentesis for the collection of amniotic fluid and subsequent karyotype analysis remains the gold standard for the diagnosis of fetal chromosomal abnormalities. However, as an invasive procedure, amniocentesis carries an inherent risk of miscarriage. Since the discovery of cell-free fetal DNA (cffDNA) in maternal peripheral blood in 1997, non-invasive molecular techniques for analyzing cffDNA have gained widespread application in the screening of various chromosomal disorders. Notably, NIPT is now widely accepted as a first-line screening tool for trisomies 13, 18, and 21 [6]. Nevertheless, the role of NIPT in replacing traditional diagnostic modalities such as invasive prenatal testing, MSS, and PUF remains under debate. In the present study, the aim was to conduct a confirmatory analysis of amniotic fluid from 6,942 pregnant women with indications for amniocentesis who were treated at the Women and Children’s Hospital in Changzhi, Shanxi Province from January 2018 to December 2023. By analyzing the gestational age, weeks of pregnancy, indications for prenatal diagnosis, and the frequency of chromosomal abnormalities, diagnostic strategies were further explored.

Materials and methods

Subjects

A retrospective analysis was conducted on data from 6,942 pregnant women who underwent amniocentesis at the Maternal and Child Health Care Hospital of Changzhi city between January 2018 and December 2023. All procedures were performed after informed consent was obtained. The maternal age ranged from 17 to 50 years, and the gestational age at the time of amniocentesis ranged from 15 to 37 weeks. The indications for prenatal diagnosis included AMA (defined as an expected maternal age ≥ 35 years at delivery), high-risk findings on MSS, positive results from NIPT, PUF (includes fetal structural abnormalities or soft markers detected on prenatal ultrasound), PCAC (refers to the detection of abnormal chromosomal carriage in at least one parent by prior testing before this amniocentesis), and POH (a history of miscarriage, stillbirth, early delivery, birth defects, newborn death, or serious pregnancy problems such as preeclampsia). For cases presenting with multiple indications, classification was based on the primary clinical indication for the invasive procedure, following a predefined hierarchy: PCAC, PUF, POH, and positive prenatal screening results (including MSS and NIPT). With the exception of the AMA group, all patients were under 35 years of age at the time of delivery. Pregnant women classified under the AMA category were included in that group regardless of the presence of additional indications.

Methods

Amniocentesis

Prior to undergoing amniocentesis, all pregnant women received comprehensive genetic counseling and provided informed consent. The procedure was conducted under sterile conditions with real-time ultrasound guidance to ensure safety and accuracy. A total of 20 mL of amniotic fluid was aspirated and subsequently centrifuged at 1,500 r/min for 8 min. Following centrifugation, the supernatant was carefully removed, leaving approximately 1–2 mL of cell suspension for further analysis.

G-banding karyotype analysis

To the prepared cell suspension, 5 mL of amniotic fluid cell culture medium (manufactured by Guangzhou Heneng Biotech and Guangzhou Baiyunshan) was added. The mixture was gently homogenized and transferred into a 25 cm² square culture flask. Cultures were maintained in an incubator at 37 °C with 5% CO₂ for approximately 7 days. Daily monitoring of cell growth was performed. Once multiple colonies and numerous metaphase cells were observed under an inverted microscope, the cultured amniotic fluid cells were harvested. Routine G-banding was performed, and chromosomal analysis was conducted using the Leica GLS-120 automated metaphase scanning system (Leica Biosystems). Two experienced cytogeneticists independently counted 10 metaphase spreads and analyzed 5 karyotypes. Karyotype interpretation was conducted in accordance with the 2020 edition of the International System for Human Cytogenomic Nomenclature (ISCN 2020). Chromosomal polymorphic variations are not classified as chromosomal abnormalities [7].

Verification of other variations

Other detected variations primarily included chromosomal structural abnormalities, copy number variations (CNVs), chromosomal microdeletions and microduplications, low-level chromosomal mosaicism, and uniparental disomy. DNA was extracted from fetal cells present in the amniotic fluid and analyzed via a range of molecular cytogenetic techniques, including copy number variation sequencing (CNV–seq), CMA, quantitative fluorescence–polymerase chain reaction (QF–PCR, abbreviated as QF in the tables), and fluorescence in situ hybridization (FISH). Interpretation and classification of CNVs detected by CMA and CNV-seq strictly adhered to the technical standards and reporting guidelines published by the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) in 2020. On the basis of these criteria, CNVs are categorized into five classes: pathogenic, likely pathogenic (variant of uncertain significance–likely pathogenic), variant of uncertain significance (VUS), likely benign (variant of uncertain significance–likely benign), and benign [8, 9].

Statistical analysis

Excel spreadsheets were used to collect and tabulate data. Statistical analysis and plotting of the data were performed via SPSS (version 27.0) and GraphPad Prism (version 8.0). For continuous data that did not follow a normal distribution, statistical descriptions are presented as the median and interquartile range (IQR). Differences between groups were analyzed via the nonparametric Mann–Whitney U test, with a significance threshold set at P < 0.05. To handle missing data, samples were included in the analysis if they contained valid values for the relevant variables. Samples lacking valid data for the variables under analysis were excluded accordingly.

Results

A total of 6,942 samples were included in the study. Among them, 38 samples with completely missing data were excluded (38/6,942, 0.55%), while samples with partially missing data were retained. This included 18 cases with missing age information (0.26%), 23 cases with missing gestational week information (0.33%), and 8 cases with missing prenatal diagnosis information (0.12%). The distribution of overall valid data was as follows: maternal age information was available for 6,886 cases (6,886/6,942; 99.19%), gestational week data for 6,882 cases (6,882/6,942; 99.14%), and prenatal diagnostic indication data for 6,896 cases (6,896/6,942; 99.34%). A total of 557 cases of fetal chromosomal abnormalities were identified. The overall positive detection rate was 8.07% (557/6,904). Stratified by data availability, the positive detection rate based on age was 8.09% (557/6,886), that based on gestational age was 7.67% (528/6,882), and that based on prenatal diagnostic indications was 8.08% (557/6,896) (Table 1).

Table 1.

Distribution of overall sample size

Item	Missing (n)	Missing (%)	Valid Cases (n)	Valid (%)	Positive Cases (n)	Positive Rate (%)
Aga	18	0.26	6886	99.19	557	8.09
Gestational weeks	23	0.33	6882	99.14	528	7.67
Indications	8	0.12	6896	99.34	557	8.08

The maternal age of the overall cohort ranged from 17 to 50 years, with a median of 31 years, whereas the gestational week ranged from 15 to 37 weeks, with a median of 19 weeks and 1 day. Among the positive cases, maternal age ranged from 19 to 46 years (median: 31 years), and gestational week ranged from 15 to 38 weeks (median: 19 weeks and 5 days). Differences in maternal age and gestational age between the overall cohort and the group of positive cases were analyzed. No statistically significant difference was observed in maternal age, whereas a significant difference was detected in gestational age (Fig. 1).

Fig. 1.

Analysis of differences in age and number of gestational weeks between overall and positive cases. A: Overall age distribution, A1: Age distribution of positive cases, B: Overall gestational week distribution, B1: Gestational week distribution of positive cases, ns: Not statistically significant (p ≥ 0.05), *p < 0.05, **p < 0.01, ***p < 0.001

The participants were stratified into six age groups (20, 20–24, 25–29, 30–34, 35–39, and ≥ 40 years). The results revealed that women aged 30 years and above (Groups A4–A6, accounting for 4,202/6,886, 61.02%) constituted the primary population undergoing prenatal screening, with this demographic representing 56.91% (317/557) of positive cases. Notably, the ≥ 40 years group presented the highest positive detection rate (10.89%) across all age strata. Additionally, the 25–29 years age group comprised 31.61% (2,177/6,886) of the total screened population and 35.55% (198/557) of the positive cases, yielding a positive detection rate of 9.10% (Table 2).

Table 2.

Positive detection rates by age group

Group	Total (n)	Proportion (%)	Positive Cases (n)	Proportion (%)	Positive Rate (%)
A1	12	0.17	1	0.18	8.33
A2	495	7.19	41	7.36	8.28
A3	2177	31.61	198	35.55	9.10
A4	2082	30.24	146	26.21	7.01
A5	1624	23.58	117	21.01	7.20
A6	496	7.20	54	9.69	10.89
Total	6886	100.00	557	100.00

A1: <20 years, A2: 20–24 years, A3: 25–29 years, A4: 30–34 years, A5: 35–39 years, A6: ≥40 years

We stratified positive cases into four groups according to gestational age (15–20.6 w, 21–27.6 w, 28–32.6 w, and 33–37.6 w). The majority of pregnant women opted for amniocentesis at 15-20.6 weeks (374/528, 70.83%), followed by 21–27.6 weeks (143/528, 27.08%), with positivity rates of 6.93% and 10.18%, respectively. Additionally, 11 cases underwent invasive diagnostic procedures in the third trimester, yielding a positivity rate of 2.08% (11/528) (Table 3).

Table 3.

Positive detection rates by gestational age subgroups

Group	Total (n)	Proportion (%)	Positive Cases (n)	Proportion (%)	Positive Rate (%)
C1	5393.0	78.36	374	70.83	6.93
C2	1405.0	20.42	143	27.08	10.18
C3	76.0	1.10	10	1.89	13.16
C4	8.0	0.12	1	0.19	12.50
Total	6882.0	100.00	528	100.00

C1: 15–20.6w, C2: 21–27.9w, C3: 28–32.6w, C4: 33–37.6w

We further analyzed the significance of differences in age and gestational week distribution within each group. In the age-stratified analysis, significant differences were observed among all subgroups (A6–A2) in the overall cohort, whereas no statistically significant differences were observed between A2 and A1, or between A3 and A1. Similarly, within the positive group, statistically significant differences were observed between groups B6 to B2, while no significant differences were detected between group B1 and the other groups. This outcome is attributable to the small sample sizes in groups A1 and B1, which may have limited the statistical power of the comparisons. However, in terms of gestational weeks, significant within-group differences were observed in both the total sample and positive samples (Fig. 2).

Fig. 2.

Significance of differences among age groups. A1–A2: overall age stratification, B1–B6: age distribution of positive cases (20, 20–24, 25–29, 30–34, 35–39, ≥ 40 years); C1–C2: overall gestational week stratification, B1–B6: gestational week distribution of positive cases (15-20.6, 21-27.6, 28-32.6, 33-37.6 weeks); a-f: Statistically significant difference between the A6–A1 groups, g-l: Statistically significant difference between the B6–B1 groups. m-p: Statistically significant difference between the C1–C4 groups, q-t: Statistically significant difference between the D1–D4 groups. Groups with the same letter are not significantly different (P < 0.05)

When we analyzed the indications for prenatal diagnosis, we found that MSS accounted for the highest proportion, accounting for 51.75% (3,615/6,986) of the cases (Table 4). Notably, three patients initially yielded low-risk results via NIPT but were later classified as high-risk the basis of serum screening. To prevent potential missed or misdiagnoses, karyotype analysis was performed on amniotic fluid and abortive tissues collected from pregnant women. The results revealed that cases 1 and 2 were consistent with trisomy 21, while case 3 was confirmed as trisomy 18. Notably, in case 2, the placental karyotype was entirely normal, whereas the fetal karyotype was T21. In contrast, both cases 1 and 3 presented varying degrees of placental mosaicism, with the maximum mosaicism levels in their placentas being 58% and 56%, respectively (Tables 5 and 6). AMAs were the second most common indication, accounting for 30.40% (2,124/6,986) of the cases, and PUF ranked third (7.93%, 554/6,986).

Table 4.

Clinical indications and their positive predictive values

Indication	Total (n)	Proportion (%)	Positive Cases (n)	Proportion (%)	PPV (%)
MSS	3615	51.75	149	26.75	4.12
NIPT	313	4.48	134	24.06	42.81
MSS + NIPT	37	0.53	35	6.28	94.59
AMA	2124	30.40	171	30.70	8.05
POH	198	2.83	11	1.97	5.56
PUF	554	7.93	48	8.62	8.66
PCAC	28	0.40	9	1.62	32.14
Others	27	0.39	0	0.00	0.00
Total	6986	100.00	557	100.00

Table 5.

Characteristics of 3 false-negative NIPT samples

Case	Age	Gestational (w)	MSS	NIPT	Karyotype	Outcome
Case 1	41	18.2	1: 43	T21 1.686	T21	Labor induction
Case 2	37	18	1: 27	T21 2.548	T21	Labor induction
Case 3	34	20	1: 41	T18 1.89	T18	Labor induction

T21 Trisomy 21, T18 Trisomy 18

Table 6.

Invasive test results of 3 false-negative NIPT samples

Sampling site				Karyotype
Placenta				case 1		case 2		case 3
Fetal surface		0		T21 (10% mos)		46, XN		T18 (40% mos)
		3		T21 (23% mos)		46, XN		T18
		6		T21		46, XN		T18
		9		T21 (58% mos)		46, XN		T18
		Central		T21		46, XN		T18
Maternal surface		0		T21		46, XN		T18 (37% mos)
		3		T21 (31% mos)		46, XN		T18 (56% mos)
		6		T21		46, XN		T18 (41% mos)
		9		T21		46, XN		T18 (49% mos)
		Central		T21		46, XN		T18 (46% mos)
Fetus		Muscle		T21		T21		T18
		Skin		T21		T21		T18

Mos Mosaic, N X or Y, T21 Trisomy 21, T18, Trisomy 18; 0, 3, 6, 9, and central: the tissue sampling sites according to the Clock face notation

In addition, the proportion and PPV of each prenatal diagnostic indication within the positive case group were further evaluated. MSS alone exhibited a relatively low PPV of 4.12%. In contrast, the combination of MSS and NIPT yielded the highest PPV at 94.59%; however, this group comprised a small portion of the positive cases, accounting for only 6.28% (35/557). Although NIPT represented a modest proportion of all prenatal diagnostic indications (4.48%, 313/6,986), it contributed to a markedly higher share of positive cases (24.06%, 134/557). Notably, its PPV (42.81%) was second only to that of MSS combined with NIPT screening. The PPV for PUF (8.66%) was higher than that for AMA (8.05%); the PPVs for POH and PCAC were 5.56% and 32.14%, respectively (Table 4).

As outlined in the Methods section, confirmed positive samples were further classified by the type of chromosomal abnormality. The distributions of the diagnostic results are presented in Table 7. Chromosomal aneuploidies accounted for the majority of cases at 65.35% (364/557), followed by structural abnormalities at 26.57% (148/557), mosaicism at 7.18% (40/557), and marker chromosomes at 0.54% (3/557). Additionally, two cases of polyploidy were identified (69,XXX and 69,XXY). Among the autosomal aneuploidies, trisomy 21 has the highest proportion (244/557, 43.81%), followed by trisomy 18 (55/557, 9.87%) and trisomy 13 (10/557, 1.80%). Among sex chromosome aneuploidies, 47,XXY was the most frequently observed (20/557, 3.59%), followed by 47,XXX (18/557, 3.23%). Furthermore, we further illustrate the types of chromosomal structural abnormalities with relatively high frequencies. Notably, microdeletions and microduplications involving chromosomes 1, 4, 7, 15, 16, 17, 18, 21, 22 and the X chromosome accounted for a greater proportion. Among these, the three most prevalent were 17p12 deletion (5.41%), 16p13.11 deletion (4.73%), and 22q11.2 deletion (4.05%) (Table 8).

Table 7.

Types and proportions of chromosomal abnormalities detected in prenatal diagnosis

Karyotype	AMA	MSS	NIPT	MSS + NIPT	PUF	POH	PCAC	Total	Proportion (%)
Trisomy 21	80	51	72	23	16	2		244	43.81
Trisomy 18	19	22	6	5	3			55	9.87
Trisomy 13	3	1	3		3			10	1.80
47,XXY	7	1	10			2		20	3.59
47,XXX	8	3	6	1				18	3.23
45,X		1	3	1	3			8	1.44
47,XYY	1	2	4					7	1.26
48,XXYY	1		1					2	0.36
69,XXX		1						1	0.18
69,XXY	1							1	0.18
Structural	40	46	23	3	22	6	8	148	26.57
Mosaic	10	20	6	1	1	1	1	40	7.18
Marker	1	1		1				3	0.54
Total	171	149	134	35	48	11	9	557	100.00

Table 8.

Types of structural abnormalities

Type	Number (n)	Proportion(%)
17p12 del	8	(8/148,5.41)
16p13.11 del	7	(7/148,4.73)
22q11.2 del	6	(6/148, 4.05)
22q11.2 dup	5	(5/148,3.38)
16p13.11 dup	5	(5/148,3.38)
16p11.2 del	5	(5/148,3.38)
15q11.2 dup	5	(5/148,3.38)
Xp22.31 dup	5	(5/148,3.38)
4p16.3 del	4	(4/148,2.70)
15q112q13 dup	3	(3/148,2.03)
21q11.2q22.3 dup	3	(3/148,2.03)
1q21.1 del	2	(2/148,1.35)
7q11.23 del	2	(2/148,1.35)
Xp22.33q28 dup	2	(2/148,1.35)
18p11.32p11.31 del	2	(2/148,1.35)

Del Deletion, dup Duplication

We analyzed the distribution of diagnostic items and their results in the positive group (Table 9). The analysis revealed that 27.65% (154/557) of the participants opted for a single diagnostic method, 66.96% (373/557) chose two methods, and only 5.39% (30/557) selected three or more methods. Among the single diagnostic tests, CMA was the primary choice, accounting for 14.18% (79/557), followed by karyotyping at 12.21% (68/557). For dual diagnostic tests, the preferences in descending order were as follows: karyotype plus QF–PCR (185/557, 33.21%), QF–PCR plus FISH (96/557, 17.24%), karyotype plus CMA (56/557, 10.05%), CMA plus QF–PCR (35/557, 6.28%), and CNV–seq plus QF–PCR (1/557, 0.18%). Additionally, karyotyping remains the primary diagnostic method for chromosomal numerical abnormalities, while CMA is predominantly used for structural abnormalities. For the detection of mosaicism, the combination of karyotyping and QF–PCR is the most frequently utilized diagnostic method.

Table 9.

Diagnostic methods and result characteristics of confirmed cases

Method		T 21	T 18	T 13	SCAs	Polyploidy	Structural	Mosaic	Mar ker	Total	Proportion (%)
1	CMA	18	3	1	10		47			79	14.18
	Karyo	34	8	2	4		15	5		68	12.21
	CNV-seq		1				4			5	0.90
	FISH	2								2	0.36
2	Karyo + QF	78	23	3	14	2	40	23	2	185	33.21
	QF + FISH	76	8	1	7		1	3		96	17.24
	Karyo + CMA	19	5	1	9		14	7	1	56	10.05
	CMA + QF	9	2	2	7		15			35	6.28
	CNV-seq + QF	1								1	0.18
≥ 3		7	5		4		12	2		30	5.39
Total		244	55	10	55	2	148	40	3	557	100.00

Karyo Karyotype, QF QF–PCR.

The “+” symbol indicates “combined with”

Finally, our comprehensive analysis of prenatal indications, diagnostic outcomes, and invasive diagnostic methods across different age groups and gestational stages revealed several key patterns (Table 10). The majority of cases occurred in women aged 25–29 years (accounting for 48.3–60.4% across various indications) at 15–20.6 weeks of gestation (representing 50.0–87.4% of cases). Trisomy 21 was the most common chromosomal abnormality (34.8% prevalence in the 25–29 age group), while structural anomalies constituted 36.5% of all cases. Diagnostic approaches vary by age, with karyotyping combined with QF–PCR being the most frequently employed method (27.0–80.8% across different age groups). Notably, among the 7 late pregnancy diagnoses, 5 were established following the detection of abnormal ultrasound findings. No statistically significant differences were observed between the subgroups (P > 0.05).

Table 10.

Diagnostic methods and result characteristics of confirmed cases

	Gestation (w)					Age
	15-20.6	21-27.6	28-32.6	33-37.6	Total	<20	20-24	25-29	30-34	35-39	≥40	Total
Indication
MSS	125(125/143, 87.41%)	18(18/143, 12.59%)			143	1(1/149, 0.67%)	11(11/149, 7.38%)	72(72/149, 48.32%)	65(65/149, 43.62%)			149
NIPT	72(72/126, 57.14%)	51(51/126, 40.48%)	2(2/126, 1.59%)	1(1/126, 0.79%)	126		14(14/134, 10.45%)	65(65/134, 48.51%)	55(55/134, 41.04%)			134
MSS+NIPT	17(17/33, 51.52%)	16(16/33, 48.48%)			33		8(8/35, 22.86%)	20(20/35, 57.14%)	7(7/35, 20.00%)			35
PUF	20(20/40, 50.00%)	15(15/40, 37.50%)	5(5/40, 12.50%)		40		8(8/48, 16.67%)	29(29/48, 60.42%)	11(11/48, 22.92%)			48
Outcome
T21	160(160/236, 67.80%)	75(75/236, 31.78%)		1(1/236, 0.42%)	236		23(23/244, 9.43%)	85(85/244, 34.84%)	56(56/244, 22.95%)	57(57/244, 23.36%)	23(23/244, 9.43%)	244
T18	38(38/52, 73.08%)	14(14/52, 26.92%)			52		7(7/55, 12.73%)	18(18/55, 32.73%)	11(11/55, 20.00%)	11(11/55, 20.00%)	8(8/55, 14.55%)	55
T13	7(7/8, 87.50%)	1(1/8, 12.50%)			8			5(5/10, 50.00%)	2(2/10, 20.00%)	3(3/10, 30.00%)		10
47, XXY	14(14/19, 73.68%)	5(5/19, 26.32%)			19		1(1/20, 5.00%)	4(4/20, 20.00%)	8(8/20, 40.00%)	4(4/20, 20.00%)	3(3/20, 15.00%)	20
47,XXX	14(14/18, 77.78%)	4(4/18, 22.22%)			18			8(8/18, 44.44%)	2(2/18, 11.11%)	5(5/18, 27.78%)	3(3/18, 16.67%)	18
Structural	90(90/131, 68.70%)	36(36/131, 27.48%)	5(5/131, 3.82%)		131	1(1/148, 0.68%)	7(7/148, 4.73%)	54(54/148, 36.49%)	44(44/148, 29.73%)	30(30/148, 20.27%)	12(12/148, 8.11%)	148
Mosaicism	34(34/38, 89.47%)	3(3/38, 7.89%)	1(1/38, 2.63%)		38		1(1/40, 2.50%)	11(11/40, 27.50%)	18(18/40, 45.00%)	5(5/40, 12.50%)	5(5/40, 12.50%)	40
Invasive
CMA	41(41/65, 63.08%)	22(22/65, 33.85%)	2(2/65, 3.08%)		65		5(5/79, 6.33%)	39(39/79, 49.37%)	18(18/79,22.78%)	11(11/79, 13.92%)	6(6/79, 7.59%)	79
Karyo	40(40/59, 67.80%)	19(19/59, 32.20%)			59	1(1/185, 0.54%)	3(3/68, 4.41%)	30(30/68, 44.12%)	15(15/68,22.06%)	16(16/68, 23.53%)	4(4/68, 5.88%)	68
Karyo+QF	147(147/182, 80.77%)	34(34/182, 18.68%)		1(1/182, 0.55%)	182		8(8/185, 4.32%)	50(50/185, 27.03%)	51(51/185,27.57%)	48(48/185, 25.95%)	27(27/185, 14.59%)	185
QF+FISH	52(52/92, 56.52%)	39(39/92, 42.39%)	1(1/92, 1.09%)		92		18(18/96, 18.75%)	28(28/96, 29.17%)	26(26/96,27.08%)	19(19/96, 19.79%)	5(5/96, 5.21%)	96
Karyo+CM	42(42/56, 75.00%)	11(11/56, 19.64%)	3(3/56, 5.36%)		56		4(4/56, 7.14%)	23(23/56, 41.07%)	15(15/56,26.79%)	8(8/56, 14.29%)	6(6/56, 10.71%)	56

Discussion

The aim of the present study was to perform a diagnostic analysis of amniocentesis in 6,942 pregnant women with indications for invasive testing at a maternal and child health hospital in Changzhi, Shanxi Province, from January 2018 to December 2023. By examining gestational age, pregnancy weeks, prenatal diagnostic indications, and chromosomal abnormality rates, optimal diagnostic strategies were further explored. Among the total samples analyzed, 557 cases (8.07%) were diagnosed with fetal chromosomal abnormalities. After excluding cases with missing data, analysis of the 6,986 valid prenatal indications revealed the following distributions: MSS (51.75%), AMA (30.40%), MSS + NIPT (0.53%), NIPT (4.48%), PUF (7.93%), POH (2.83%), and PCAC (0.40%). The corresponding positive predictive values (PPVs) were 4.12%, 8.05%, 94.59%, 42.81%, 8.66%, 5.56%, and 32.14%, respectively. Among the 557 confirmed positive cases, the distribution of chromosomal abnormalities was as follows: aneuploidy (65.35%), structural abnormalities (26.57%), mosaicism (7.18%), and marker chromosomes (0.54%). Trisomy 21 was the most frequently observed autosomal aneuploidy, while 47,XXY was the most common sex chromosome aneuploidy. Among structural abnormalities, microdeletions and microduplications involving chromosomes 1, 4, 7, 15, 16, 17, 18, 21, 22 and the X chromosome are particularly common. The three most prevalent variants were 17p12 deletion (5.41%), 16p13.11 deletion (4.73%), and 22q11.2 deletion (4.05%).

Selection of amniocentesis analysis methods

Numerous studies from China and other countries have reported the indications and outcomes of amniocentesis. In Wang’s study [10], the detection rate of fetal chromosomal abnormalities was 6.52%, while Li [11] reported a rate of 8.54%. At present, amniocentesis combined with karyotype analysis remains the gold standard for the clinical diagnosis of fetal chromosomal disorders. As a prenatal diagnostic procedure, amniocentesis involves the extraction of a small volume of amniotic fluid surrounding the fetus. This fluid is then subjected to a range of analytical techniques, which may include chromosomal karyotyping, CMA, FISH, and QF–PCR, among others.

As shown in Table 9, which presents the distribution of diagnostic approaches and corresponding results in the positive case group, the combined use of the two methods was the predominant strategy, accounting for 66.96% of the cases—most notably the combination of karyotyping and QF–PCR (33.21%). However, the number of combined protocols with three or more tests was relatively low, which we attributed to the imbalance between cost and practicality. Further analysis indicated that karyotyping was primarily utilized for diagnosing chromosomal numerical abnormalities, whereas CMA was more frequently employed in cases involving structural abnormalities. This preference likely reflects the inherent strengths of karyotyping, particularly its ability to provide direct visual assessment of both chromosomal number and structure, thereby offering immediate and reliable evidence for the diagnosis of chromosomal abnormalities. Nonetheless, its detection capacity is limited to structural abnormalities exceeding 10 Mb. To identify smaller structural abnormalities, supplementary diagnostic techniques such as CMA and CNV–seq should be considered primary alternatives. Chau [12] reported a 5.30% detection rate of structural abnormalities in their CMA analysis of 1,510 fetal samples from a single center, which was higher than the 3.08% rate reported by Shi [13] in their CNV–seq analysis of 17,994 fetal samples. This is because CMA can detect a wide spectrum of chromosomal abnormalities, including aneuploidy, mosaicism, microdeletions and microduplications, as well as more complex anomalies such as polyploidy and uniparental disomy. Consequently, for fetuses with prenatal indications suggestive of imprinting disorders or other conditions associated with uniparental disomy, CMA or alternative high-resolution methods are recommended to minimize the risk of missed diagnoses that may occur with CNV–seq. However, this does not imply that CNV–seq should be abandoned in clinical diagnosis, as it can more accurately detect chromosomal copy number variations (CNVs) in special scenarios (such as chromosomal balanced translocations). Notably, it also has the ability to detect low-level mosaics, highlighting the need to recommend optimal diagnostic modalities tailored to each patient’s specific requirements. Although FISH provides valuable single-cell resolution within metaphase chromosomes—enabling the detection of balanced chromosomal rearrangements in parents, mosaicism, and complex structural abnormalities—and because QF–PCR allows for rapid, quantitative detection of CNVs in targeted genomic regions, neither technique is typically recommended as a standalone diagnostic tool. FISH is constrained by its reliance on locus-specific probes, which typically span at least 10 kb and cannot identify unknown genomic alterations. Similarly, QF–PCR faces limitations in the design of specific and reliable primer pairs, optimization of PCR conditions, and its reduced sensitivity in detecting low-level mosaicism. These technical constraints highlight the necessity of using FISH and QF–PCR in conjunction with complementary methods for comprehensive chromosomal evaluation. Therefore, The results from both methods require validation through CMA, CNV–seq, or other techniques [14]. CNV–seq and karyotype analysis demonstrate comparable detection capabilities for numerical abnormalities and structural abnormalities larger than 10 Mb. However, CNV–seq is capable of detecting pathogenic or likely pathogenic CNVs ranging in size from approximately 100 kilobases (kb) to 10 megabases (Mb). In contrast, conventional karyotyping can identify large-scale structural chromosomal abnormalities, including translocations, inversions, and ring chromosomes, which are typically beyond the resolution of CNV–seq. When abnormalities are identified by both CNV–seq and karyotyping, comprehensive genetic counseling is strongly advised. In such cases, confirmatory testing via orthogonal methodologies may be warranted to ensure diagnostic accuracy [13].

The value of mid-trimester amniocentesis

Traditionally, amniocentesis is performed between 16 and 24 weeks of gestation, which is consistent with our study, and most existing data on its safety, efficacy, diagnostic capability, complications, and patient acceptance are associated with this timeframe [15]. A recent study by Drukker [16] demonstrated that, despite normal findings in fetal anomaly screening performed before 24 weeks of gestation, 11.4% of fetal abnormalities (474 out of 15,244 cases; 3.1%) may only become apparent during subsequent ultrasound evaluations conducted at later gestational stages. These findings may necessitate invasive prenatal diagnostic procedures to confirm suspected anomalies. Although data regarding the safety and efficacy of amniocentesis performed beyond 24 weeks are limited, existing evidence suggests its potential clinical value. Consistent with this, the present findings reveal a difference in gestational age distribution between cases with positive findings and the total cohort, with a discernible trend toward later gestational ages (Fig. 1). Moreover, 11 pregnant women underwent invasive diagnosis in late pregnancy, 5 of whom were confirmed due to abnormal ultrasound findings (Tables 3 and 10).

However, this procedure may pose certain adverse pregnancy risks to the fetus. In a study by Tanisha [15], amniocentesis was associated with a 5% rate of preterm delivery, 0.91% incidence of placental abruption, and 3.66% rate of intrauterine fetal death. Research by Rezagholi [17] demonstrated that the most common early and late complications of amniocentesis were amniotic fluid leakage (4.6%) and delivery before 37 weeks of gestation, respectively. Additionally, the procedure itself can impose psychological stress on pregnant women. Therefore, the development of noninvasive techniques is particularly crucial.

Significance of serological screening

In the present study, the primary indication for prenatal diagnosis was MSS (51.75%), which aligns with the findings of Ekin [18]. The PPV of MSS in the present study was 4.12%, which was lower than the domestic reported range of 5.0–10.2% [13], potentially due to regional variations or differences in detection methodologies. Studies have shown that second-trimester triple screening, which is widely implemented in clinical practice, achieves a detection rate of approximately 65–70%. However, its diagnostic performance is influenced by several factors, including the accuracy of gestational age estimation and maternal weight. These limitations contribute to a relatively high false positive rate, estimated at approximately 5% [19, 20]. However, these findings do not diminish the clinical utility of MSS. In our cohort, three cases with low-risk results from NIPT but high-risk MSS findings were subsequently confirmed through invasive diagnostic procedures to be consistent with the MSS outcomes. This phenomenon can be attributed to the fact that placental mosaicism ranks among the primary factors contributing to false negatives and false positives in NIPT. Accordingly, the clinical relevance of serological screening should not be overlooked, nor can NIPT fully supplant serological screening. Studies have demonstrated a strong correlation between elevated inhibin A levels and increased risks of fetal growth restriction, preeclampsia, and preterm delivery. Specifically, each unit increase in inhibin A levels was associated with a 1.83-fold greater probability of adverse pregnancy outcomes, while abnormal human chorionic gonadotropin (hCG) levels were associated with a 3.12-fold greater risk. Individuals with elevated alpha-fetoprotein (AFP) levels are more likely to require neonatal intensive care unit admission [21]. Considering both simplicity and cost-effectiveness, serological screening continues to serve as the primary modality for prenatal risk assessment. In cases with abnormal screening results, a comprehensive evaluation integrating additional clinical indicators and, when appropriate, further diagnostic testing is recommended to refine risk stratification and guide clinical decision-making.

Recommended prenatal diagnosis for advanced maternal age

AMAs accounted for 30.40% of all prenatal indications and represented 30.70% of positive cases. Among these, trisomy 21 had the highest prevalence (43.81%), followed by structural abnormalities (26.57%). The PPV for AMA was 8.05% (Tables 4 and 7). These findings correlate with China’s two-child policy implementation and increasing trends of delayed childbearing, as age-related ovarian decline elevates the risk of meiotic nondisjunction during oogenesis [22]. However, the age-stratified analysis revealed that a considerable proportion of cases were under 35 years old. The age groups A5–A6, A4, and A1–A3 accounted for 30.79%, 30.24%, and 38.98% of the total valid samples, respectively. Among the positive cases, 30.70%, 26.21%, and 43.09% were positive, with positivity rates of 8.07%, 7.01%, and 8.94%, respectively (Table 2). Multiple studies have indicated that a maternal age of 35 years should not be used as the sole criterion for recommending invasive diagnostic testing [23, 24]. Research by Rezagholi [17] demonstrated that approximately 70% of infants with Down syndrome were born to mothers under the age of 35. This trend may be attributed to improved socioeconomic conditions and increased awareness of prenatal care, which have contributed to broader adoption of prenatal diagnostic technologies among younger, traditionally low-risk populations. Our findings further support the diagnostic efficacy of screening in this demographic population, underscoring the importance of extending genetic screening efforts to younger maternal age groups. In alignment with these observations, the ACOG recommends that all pregnant individuals, irrespective of maternal age, be offered serum-based prenatal screening. For pregnancies classified as AMA, NIPT is considered a preferable option, as it provides high diagnostic accuracy while markedly reducing the reliance on invasive diagnostic procedures. However, in China, despite the increasing availability and demonstrated effectiveness of NIPT, which has contributed to a decline in amniocentesis rates, a significant proportion of AMA pregnant women continue to choose invasive testing. This may reflect cultural preferences, perceived diagnostic certainty, or differing clinical practices that warrant further investigation [25]. Concurrently, prenatal diagnostic technologies are undergoing significant transformation. In addition to conventional chromosomal karyotyping, an increasing number of expectant mothers are opting for advanced molecular diagnostic techniques, reflecting a shift toward more comprehensive and precise prenatal evaluation. Given these evolving trends, further in-depth analysis of clinical data specific to AMA pregnancies is essential. Such analyses will support the optimization of prenatal diagnostic strategies and guide the selection of appropriate technologies tailored to individual risk profiles and clinical needs.

Genetic counseling is recommended for NIPT-positive pregnancies

NIPT has been widely adopted in clinical testing for T21, T18, and T13 due to its safety, accuracy, and rapid turnaround time, demonstrating high detection rates. Although NIPT constituted a relatively small proportion of all prenatal indications in the present study (4.48%), it accounted for a disproportionately high percentage of positive cases (24.06%). Notably, its PPV reached 42.81%, ranking second only to the combined use of MSS and NIPT (Table 4). At present, NIPT has become the primary screening modality for Down syndrome, exhibiting high diagnostic performance with an overall sensitivity of 99.61% and specificity of 99.91%. The corresponding positive and negative predictive values are 89.74% and 99.99%, respectively [26–29]. ACOG Practice Bulletin No. 226 (2020) stated that cell-free fetal DNA analysis represents the most sensitive and specific screening method for common fetal aneuploidies [30]. Recent studies have further indicated that NIPT also has significant screening potential for sex chromosome abnormalities, rare chromosomal aneuploidies, and structural chromosomal abnormalities [31]. Nevertheless, NIPT is not without limitations, including false-negative and false-positive results. One study reported assay failure rates of 2%-6% [32], whereas another reported a 27:1 ratio of false-positive to false-negative results [33]. Therefore, families receiving normal NIPT results may develop a false sense of reassurance. False-negative outcomes can arise from factors such as a low fetal DNA fraction, placental mosaicism, or the presence of a vanishing twin. Consequently, a negative NIPT result should not preclude ongoing prenatal surveillance; routine ultrasound monitoring remains essential to detect structural anomalies or evolving abnormalities. Conversely, a positive NIPT result warrants confirmation through invasive diagnostic procedures to ensure diagnostic accuracy and inform appropriate clinical management.

The risk of chromosomal abnormalities in fetuses with abnormal ultrasound results

The present analysis revealed that ultrasound indications accounted for 7.93% of the total samples and 8.62% of the positive cases, with a PPV of 8.66% (Table 4). These findings demonstrate that ultrasonography remains a crucial tool for preventing births with chromosomal abnormalities. Even with rapid advances in molecular testing technologies, prenatal ultrasound examination remains an indispensable component of pregnancy care. Research by Irene [34] demonstrated that “abnormal ultrasound” serves as a primary indicator for X chromosome monosomy, triploidy, unbalanced chromosomal rearrangements, and rare trisomies. However, the detection rates vary significantly depending on the inclusion criteria for abnormal ultrasound findings. Notably, when Nicolaides [35] restricted their “abnormal ultrasound” category to include only fetuses with structural malformations, growth restrictions, or both, they reported a high abnormality detection rate of 14%. Tanisha [15] demonstrated that abnormal ultrasound findings constitute the most common indication for amniocentesis after 24 weeks of gestation. Mingjing [36] further demonstrated that the presence of multiple structural anomalies detected via prenatal ultrasound is strongly associated with an increased incidence of chromosomal abnormalities, with trisomy 18 being the most frequently observed. In contrast, isolated structural anomalies are more commonly linked to trisomy 21, sex chromosome abnormalities, and unbalanced chromosomal rearrangements. With continued advancements in ultrasound imaging resolution, there has been a notable rise in the prenatal detection of soft markers and subtle fetal microstructural anomalies. Empirical studies have established a positive correlation between the number of abnormal ultrasound findings and the likelihood of underlying chromosomal abnormalities [37]. ACOG recommends CMA as the preferred diagnostic tool over conventional karyotyping when one or more fetal ultrasound abnormalities are identified, warranting invasive prenatal diagnosis [38].

Personalized diagnostic approach for high-Risk families (with adverse obstetric history/parental chromosomal abnormalities)

Yang [39] reported that among 5,309 couples with a history of adverse obstetric outcomes, 3.0% had previously conceived offspring with chromosomal abnormalities, and 3.5% of the individuals themselves exhibited numerical or structural chromosomal anomalies. In the present study, the PPVs for POH and PCAC were 5.56% and 32.14%, respectively (Table 4). Given the complexity of adverse reproductive outcomes, prompt genetic counseling is recommended to assess recurrence risk and establish individualized prenatal diagnostic protocols.

Limitations

This study has the inherent limitations of a single-center retrospective design. First, all cases were from Changzhi Women and Children’s Hospital in Shanxi Province. Owing to regional disparities, population characteristics, and medical resource variations, these results cannot fully represent the distribution patterns in other regions. Second, although the data validity rate exceeded 99%, partial missing information existed (e.g., 0.33% missing gestational weeks), and only 11 cases of late-pregnancy invasive diagnosis might have compromised the statistical power for analyzing large-gestational-age cases. Additionally, while restricted by single-center sampling, these data provide critical references for regional prenatal diagnosis. Future multicenter studies are needed to validate the generalizability of detection technologies, construct more precise prenatal diagnostic pathways, and enhance the efficiency of birth defect prevention.

Conclusion

This large-scale retrospective study demonstrated that optimal prenatal diagnosis of chromosomal abnormalities requires a stratified approach: maternal serum screening serves as a cost-effective first-line tool, with NIPT providing high predictive value (PPV 94.59% when combined with MSS) for high-risk cases, while karyotyping remains essential for numerical abnormalities and CMA proves superior for structural anomalies (26.57% positive). For advanced mothers, NIPT may reduce the number of invasive procedures, whereas ultrasound abnormalities warrant direct CMA. Although most diagnoses occurred at 15–20.6 weeks (70.83%), late-gestation interventions retain clinical value. These findings support personalized diagnostic pathways based on risk stratification, with future multicenter studies needed to validate generalizability.

Author contributions

Conceptualization, H.Y.X and A.Q.S.; methodology, Y.F.F.,T.Y.L and L.J.; software, T.Y.L.; writing-original draft preparation, A.Q.S.; writing-review and editing, L.X.Z., and A.Q.S., All the authors contributed to the article and approved the final manuscript.

Funding

General Project of Natural Science Research in Shanxi Province (202403021221345).

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Review Committee of Changzhi Maternal and Child Health Hospital (CZSFYLL2025-008). Patient consent was waived by the Maternal and Child Health Hospital, due to the retrospective nature of the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.