Prenatal diagnosis strategies and pregnancy outcomes in the Luohe region of China in the context of increasing maternal age

Abstract

This article aims to explore prenatal diagnostic strategies in the context of increasing maternal age. We calculated the proportion of advanced maternal age (AMA) pregnancies in the Luohe region from 2019 to 2023 and evaluated the indications, abnormal fetal karyotypes, and pregnancy outcomes among 3681 pregnant women (3700 samples) who underwent amniocentesis. The prenatal diagnostic indications, abnormal fetal karyotypes, and pregnancy outcomes of 3681 pregnant women (3700 samples) were determined. The rates of abnormal amniotic fluid karyotypes for different indications and termination of pregnancy (TOP) rates with different types of abnormalities were compared. From 2019 to 2023, the proportion of AMA pregnancies in Luohe region increased from 11.30 to 16.03%. The most common prenatal diagnosis was AMA (54.50%), followed by abnormal maternal serum screening (35.83%). Among the 3700 samples, 97 exhibited numerical abnormalities, 32 structural abnormalities, and 2 had both numerical and structural abnormalities. The highest TOP rates were observed for combined numerical and structural abnormalities, followed by isolated numerical abnormalities and isolated structural abnormalities. Among numerical abnormalities, sex chromosome aneuploidies had a lower TOP rate than common trisomies and rare autosomal aneuploidies. AMA have become the primary prenatal diagnostic indication. In addition to noninvasive prenatal testing (NIPT), fetal karyotyping can also detect mosaicism, structural abnormalities, and triploidy. The combination of karyotyping and molecular detection has increased the detection rate of chromosomal abnormalities. However, NIPT cannot replace amniocentesis.

Introduction

Birth defects are the leading cause of infant mortality and disability¹, with approximately 80% attributed to genetic factors, either alone or in combination with other causes². Chromosomal abnormalities, including common trisomies (T21/T18/T13; trisomy 21/trisomy 18/trisomy 13), sex chromosomal abnormalities (SCAs), copy number variants (CNVs), rare autosomal aneuploidies (RAAs), translocations, inversions, duplications, and deletions, are major causes of failed embryo implantation, embryo growth arrest, miscarriage, pre- and/or postnatal abnormalities, developmental delays, and intellectual disabilities^3–5. Prenatal genetic screening and diagnostic technology are crucial for detecting fetal chromosomal abnormalities⁶.

Common prenatal genetic screening and diagnostic technologies include maternal serum screening (MSS) based on biochemical markers in the mother’s blood, ultrasound screening, noninvasive prenatal testing (NIPT), and invasive prenatal diagnostic procedures (IPDs)⁷. The primary method by which fetal chromosomal abnormalities are diagnosed is chromosome karyotype analysis of amniotic fluid collected via amniocentesis under ultrasonic guidance (Dallaire, 1976; The American College of Obstetricians and Gynecologists Practice Bulletin No. 88, 2007)⁸.Although amniocentesis carries potential risks of miscarriage or infection, these risks are minimal when the procedure is performed by experienced clinicians under ultrasound guidance⁹. While amniotic fluid chromosome karyotype analysis has disadvantages such as a long assessment period (15–30 days) and poor resolution (sensitivity to only chromosomal abnormalities > 10 Mb)¹⁰. Nevertheless, fetal chromosome karyotype analysis is considered the gold standard for the diagnosis of fetal chromosomal disorders¹¹. This testing can detect various chromosomal abnormalities, such as chromosomal numerical abnormalities, chromosomal structural abnormalities, and chromosome mosaicism, and thus this method is irreplaceable¹².

Although previous studies have shown the significance of fetal chromosome karyotype analysis in preventing birth defects^13–15, the incidence of fetal chromosomal abnormalities increases with the average maternal age¹⁶. In recent years, the proportion of pregnant women of advanced maternal age (AMA, 35 years or older at the expected time of delivery) has significantly increased worldwide^17,18. In the Netherlands, the average age of women who gave birth for the first time increased from 24.3 years in 1970 to 31.2 years in 2017¹⁹. Similarly, China is facing the challenges of declining birth rates and increasing maternal age at childbirth²⁰, which has led to a rise in the proportion of AMA pregnancies in the prenatal diagnostic population. With the significant increase in the proportion of AMA pregnant women, the rate of amniotic fluid chromosomal abnormalities may be changing. It is necessary to reevaluate the significance of amniocentesis and fetal chromosome karyotype analysis in the context of rising average maternal age using the latest data. The uniqueness of this study lies in the fact that due to the provision of free amniocentesis and fetal chromosome karyotype analysis for AMA pregnant women by the local government, the proportion of AMA pregnant women receiving amniocentesis in this area reached 54.50%, exceeding the previously reported range of 27.013–39.1%^10,15. The findings of this study represent the occurrence of fetal chromosomal abnormalities in the context of increasing maternal age.

In this study, a statistical analysis was conducted on the clinical data of 3700 amniotic fluid samples from 3681 pregnant women, fetal chromosome karyotype analysis results, and pregnancy outcomes. This study aimed to assess the positive rate (PR) of fetal chromosomal abnormalities by indication and termination of pregnancy (TOP) rates by abnormality type, highlighting the clinical value of karyotype analysis.

Results

Number of newborns and proportion of pregnant women of AMA in the past 5 years in Luohe District, China

As shown in Fig. 1, the number of newborns in the Luohe region has steadily declined over the past five years. In 2019, the region had 25,442 newborns, whereas in 2023, the region had only 13,990 newborns, which represents a 45.01% decrease over the five-year period (11,452 of 25,442). The number of AMA pregnant women decreased by 22.00% over the five-year period (633 of 2,876), but the proportion of pregnant women of AMA increased from 11.30% in 2019 to 16.03% in 2023.

Fig. 1.

Number of births (A), number of pregnant women of AMA (B), and proportion of pregnant women of AMA (C) in Luohe District, China, from 2019 to 2023. AMA advanced maternal age.

Distributions of prenatal diagnostic indications and PRs of chromosomal abnormalities for different indications

The 3681 pregnant women had a mean age of 33.62 years (range 15–48) and a mean gestational age of 21.3 weeks (range 18–28). Among the 3681 pregnant women, 3656 were carrying singletons, and 25 were carrying twins, with no cases of triplets. Of the 25 twin pregnancies, 6 were monochorionic monoamniotic and 19 were dichorionic diamniotic. A total of 3700 amniocentesis procedures were performed.

Among the 3681 pregnant women, 3648 (99.10%) had a single prenatal diagnostic indication, whereas 33 (0.90%) had two prenatal diagnostic indications. In this study, none of the women had three or more prenatal diagnostic indications. Among all the indications, the most common was AMA (54.50%, 2006/3681), which indicates that pregnant women of AMA are currently the primary target population for IPD. The next most common indications were AMSS (35.83%, 1319/3681), UA (4.51%, 166/3681), NIPT (2.17%, 79/3681), PFA (1.68%, 62/3681), twin pregnancies (0.71%, 26/3681), EMFCA (0.71%, 26/3681), teratogenic factors (0.38%, 14/3681), voluntary diagnosis (0.22%, 8/3681), family genetic history (0.14%, 5/3681), and IVF (0.08%, 3/3681).

The highest detection rate of abnormal fetal karyotypes was found with EMFCA (38.46%, 10/26), followed by NIPT (10.13%, 8/79), teratogenic factors (7.14%, 1/14), AMA (3.29%, 66/2006), AMSS (3.26%, 43/1319), and UA (3.01%, 5/166). No chromosomal abnormalities were found in the twin pregnancy, PFA, IVF, voluntary diagnosis, or family genetic history groups.

Distributions of abnormal chromosome karyotypes

Of the 3700 amniotic fluid samples, 131 cases of abnormal chromosomal karyotypes were identified, including 97 cases of numerical abnormalities (2.62%), 32 cases of structural abnormalities (0.86%), and 2 cases of numerical and structural abnormalities (0.05%), as shown in Table 1. Among the 131 cases with chromosomal karyotype abnormalities detected in the amniotic fluid, 68 underwent CNV-Seq testing (51.91%).

Table 1.

Abnormal karyotype distribution in 3700 amniotic fluid samples.

Abnormal type	Cases		Abnormal karyotypes
	n	%
Numerical abnormalities	97	2.62	NA
Common trisomies	70	1.89	NA
Trisomy 21	54	1.46	47,XN,+21
Trisomy 18	10	0.27	47,XN,+18
Trisomy 13	1	0.03	47,XN,+13
Mosaicism trisomy 21	5	0.14	47,XN,+21[4]/46,XN[100];47,XN,+21[2]/46,XN[44]; 47,XN,+21[18]/46,XN[55];47,XN,+21[2]/46,XN[36]; 47,XN,+21[5]/46,XN[48]
SCAs	21	0.57	NA
47,XYY	6	0.16	47,XYY
47,XXY	7	0.19	47,XXY
47,XXX	3	0.08	47,XXX
Mosaicism SCAs	5	0.14	46,XX[6]/46,XY[146 ]; 45,X[9]/46,XX[39]; 47,XXX[4]/46,XX[3]; 45,X[2]/46,XY[57]; 45,X[7]/46,XX[45]
Mosaicism RAAs	4	0.11	47,XN,+10[5]/46,XN[37]; 45,XN, -15[2]/46,XN[64]; 47,XN,+20[44]/46,XN, [54]; 47,XN,+12[2]/46,XN[52]
Marker chromosome	1	0.03	47,XN,+mar
Triploidy	1	0.03	69,XXY
Structural abnormalities	32	0.86	NA
Inversion	4	0.11	46,XN, inv(9)(q22.3q32); 46,XN, inv(17)(p11.2q21); 46,XN, inv(4)(p16q21); 46,XN, inv(6)(p21q21)
Translocation	18	0.49	46,XN, t(3;5)(q23;q21); 46,XN, t(2;7)(q11.2;p22); 46,XN, t(3:14)(p21;q22); 46,XN, t(11;22)(q25;q13); 46,XN, t(2;3)(q22;q28)[4]/46,XN[52]; 46,XN, t(10;19)(p10;q10); 46,XN, t(12;21)(q22;q22); 46,XN, t(5;8)(q33;q13); 46,XN, t(4;14)(q31.1;q32); 46,XN, t(5;11)(q32;q21); 46,XN, t(3;5)(p23;q11.2); 46,XN, t(3;6)(p25;p22.2); 46,XN, t(9;16)(q22;q22); 46,XN, t(1;14)(q42;q24); 46,XN, t(4;6)(q21;p23); 46,XN, t(9;20)(q34;q11.2)[7]/46,XN[65]; 46,XN, t(6;18)(q34;q22); 46,XN, t(3;20)(q26.2;q11.2)
Complex translocation	1	0.03	46,XN, t(1:17)(q21:q23),t(5:8)(q35;q21),10ps[8]/46,XN,10ps[92]
Deletion	2	0.05	46,N, del(X)(q26); 46,XN, del(5)(15.2)
Derivative	7	0.19	45,XN, der(13;14)(q10;q10); 46,XN, der(4)t(3;4)(p21;p16); 45,XN, der(21;22)(q10;q10); 46,XN, der(21;21)(q10;q10),+21; 46,XN, der(21;21)(q10;q10),+21[18]/46,XN[20]; 45,XN, der(14;21)(q10;q10)
Numerical and structural abnormalities	2	0.05	46,XN, der(21)r(21;?)(q11.2q22.3;?)[47]/45,XN, -21[34]; 45,XN, der(6)t(6;13)(q27;q10), -13
Total	131	3.54	NA

SCAs sex chromosome aneuploidies, RAAs rare autosomal aneuploidies, NA not applicable.

Among the 97 cases of numerical abnormalities, 70 cases (1.89%) were common trisomies, with 54 cases of T21, 10 cases of T18, 1 case of T13, and 5 cases of mosaic T21; therefore, T21 was the most common fetal chromosomal abnormality. In this study, five cases of mosaic T21 were identified, and in those cases, the percentage of T21 cells ranged from 3.85% (4/104) to 24.65% (18/73). In all, 21 sex chromosome aneuploidies (SCAs) were found, including six cases of 47,XYY (0.16%), seven cases of 47,XXY (0.19%), and three cases of 47,XXX (0.08%). In addition, five cases of mosaic SCAs, including two instances of X/XX, one instance of XX/XY, one instance of XX/XXX, and one instance of X/XY, were found. This study identified four cases of mosaic RAAs involving chromosomes 10, 20, 5, and 12. Additionally, one case of a marker chromosome (47,XN,+mar) and one case of triploidy (69,XXY) were also identified, as shown in Table 1; Fig. 2A and B. Another case of T21 translocation and one case of chimeric and T21 translocation were classified as structural abnormalities in this study.

Fig. 2.

Abnormal chromosome karyotypes identified in this study.

Among the 32 structural abnormalities identified in this study, the most common was translocation (18 cases, 0.49%), including 2 cases of mosaic translocations. Seven cases of derivatives (0.19%), four cases of inversions (0.11%), two cases of deletions (0.05%), and one case of a complex translocation (46,XN, t(1:17)(q21:q23),t(5:8)(q35;q21),10ps[8]/46,XN,10ps[92]) were found. In the case of the complex translocation, the abnormal karyotype was found in only one culture bottle, whereas it was absent in the other culture bottle. This mutation is suspected to have occurred during the cell culture process. The pregnant woman underwent a second amniocentesis and chromosome karyotype analysis, which revealed a result of 46,XN,10ps, as shown in Fig. 2E.

Two cases of numerical and structural abnormalities were found in this study, with karyotypes of 46,XN, der(21)r(21;?)(q11.2q22.3;?)[47]/45,XN,-21[34] and 45,XN, der(6)t(6;13)(q27;q10),-13, as shown in Fig. 2C-D.

The prenatal diagnostic indications corresponding to each type of chromosomal abnormality were counted. Numerical abnormalities were most common in the AMA group (55 cases), followed by AMSS (30), NIPT (7), UA (3), and EMFCA (2). Among the 32 cases of structural abnormalities, 13 occurred in the AMSS group, 10 in the AMA group, 8 in the EMFCA group, 1 in the NIPT group, 1 in the UA group, and 1 in the teratogenic factor group, as shown in Table 2.

Table 2.

Distributions of abnormal karyotypes with different indications.

Abnormal karyotypes	Cases	Indications
		I	II	III	IV	V	VI	VII	VIII	IX	X	XI
Trisomy 21 & Mosaicism Trisomy 21	59	35	17	0	3	0	4	0	0	0	0	0
Trisomy 18	10	8	2	0	0	0	0	0	0	0	0	0
Trisomy 13	1	1	0	0	0	0	0	0	0	0	0	0
47,XYY	6	2	3	0	0	1	0	0	0	0	0	0
47,XXY	7	3	1	0	0	1	2	0	0	0	0	0
47,XXX	3	1	2	0	0	0	0	0	0	0	0	0
Mosaicism SCAs	5	3	1	0	0	0	1	0	0	0	0	0
Mosaicism RAAs	4	2	2	0	0	0	0	0	0	0	0	0
Marker chromosome	1	0	1	0	0	0	0	0	0	0	0	0
Triploidy	1	0	1	0	0	0	0	0	0	0	0	0
Inversion	4	0	2	0	1	1	0	0	0	0	0	0
Translocation	18	8	6	0	0	6	0	0	0	0	0	0
Complex translocation	1	0	1	0	0	0	0	0	0	0	0	0
Deletion	2	1	1	0	0	0	0	0	0	0	0	0
Derivative	7	1	3	0	0	1	1	0	0	1	0	0
Numerical and structural abnormalities	2	1	0	0	1	0	0	0	0	0	0	0
Total	131	66	43	0	5	10	8	0	0	1	0	0

AMA advanced maternal age, AMSS abnormal maternal serum screening, Twin pregnancy, UA ultrasonic anomalies, EMFCA either mother or father with chromosomal abnormality, NIPT noninvasive prenatal testing, PFA previous fetus/child with abnormalities, IVF in vitro fertilization, teratogenic factors, voluntary diagnosis, family genetic history.

Pregnancy outcomes of pregnant women with various fetal chromosomal abnormalities

No abortions or infections resulting from amniocentesis were detected during the one-week follow-up. Figure 3 shows the percentage of TOPs for pregnant women with different fetal chromosomal abnormalities. All 70 pregnant women with common trisomies (including mosaicism) opted for TOP. Among the 21 cases of SCAs in pregnant women, only 7 elected TOP. Among nonmosaic SCAs, the proportions of women who underwent TOP were 33.33% (2/6) for 47,XYY, 57.14% (4/7) for 47,XXY, and 0.00% (0/3) for 47,XXX. In 5 cases of mosaic SCAs, only 1 case with 45,X[9]/46,XX[39] elected to undergo TOP, which indicates that some parents can accept children with SCAs.

Fig. 3.

Rates of TOP for pregnant women with different fetal chromosomal karyotype abnormalities. SCAs sex chromosome aneuploidies, RAAs rare autosomal aneuploidies, TOP termination of pregnancy.

Four pregnant women who had amniotic fluid chromosomal structural abnormalities chose to undergo TOP. Among them, 2 patients had T21 translocations. In one patient, the karyotype of the amniotic fluid was 46,XN, del(5)(15.2), and CNV-seq confirmed that the deletion was a pathogenic CNV. In another case, the karyotype of the amniotic fluid was 46,XN, der(4)t(3;4)(p21;p16), and CNV-seq of the amniotic fluid revealed a pathogenic CNV and a likely pathogenic CNV. Among the seven pregnant women with amniotic fluid chromosomal polymorphisms who chose to undergo TOP, five were identified to have pathogenic CNVs through amniotic fluid CNV-seq. One patient had mosaic 45,X detected by amniotic fluid CNV-seq, and one patient had a normal amniotic fluid CNV-seq result but opted for TOP for personal reasons, as detailed in Table 3.

Table 3.

The cause of pregnancy termination with chromosomal structural abnormalities in 4 patients and chromosomal polymorphisms in 7 patients.

Cases	Amniotic fluid karyotype	Cause of termination of pregnancy
AKA1641	46,XN, del(5)(15.2)	CNV-seq: Del(5)(p15.33p15.2) (20000_13360000); Size:13.34 M; P
AKA1723	46,XN, der(4)t(3;4)(p21;p16)	CNV-seq: Dup(3)(p26.3p21.31) (60000_46560000; Size:46.50 M; LP
		CNV-seq: Del(4)(p16.3p16.3) (80000_2240000); Size:2.16 M; P
AKA2684	46,XN, der(21;21)(q10;q10),+21	Translocation Down Syndrome
AKA2969	46,XN, der(21;21)(q10;q10),+21[18]/46,XN[20]	Translocation Down Syndrome
AKA1967	46,XN,9qh+,14pstk+	Unknown; Amniotic fluid CNV-seq showed no abnormality
AKA1989	46,XN,9qh+	CNV-seq: Dup(3)(p23p22.3)(31660001_33060000); Size:1.4 M; VUS
		CNV-seq: Del(16)(p13.11p12.3)(15210001_18360000); Size:3.15 M; P
AKA2340	46,XN,1qh+	CNV-seq: Del(16)(p11.2)(29640000–30200000); Size:0.56 Mb; P
AKA2409	46,XN,1qh+	CNV-seq: Del(X)(p22.31)(6460000–8140000); Size:1.68 Mb; P
AKA2561	46,XN, inv(9)(p12q13)	CNV-seq:45,X[15%]/46,XN[85%]
AKA2809	46,XN,1qh+	CNV-seq: Del(16)(p13.11)(15120000–16300000); Size:1.18 Mb; P
AKA2987	46,XN,21pstk+	CNV-seq: Del(X)(p22.31)(6460000–8140000); Size:1.68 Mb; P

CNV-seq copy number variant sequencing, Del deletion, Dup duplication, P pathogenic, LP likely pathogenic, VUS variants of uncertain significance.

Discussion

The rising proportion of AMA pregnancies highlights the growing need for effective prenatal diagnostic strategies. We analyzed indications, karyotypes, and outcomes in women undergoing amniocentesis to clarify the clinical value of karyotype analysis. Specifically, this study highlights the advantages of fetal chromosome karyotype analysis over NIPT in the diagnosis of mosaicism, SCAs, and structural abnormalities. Amniotic fluid obtained via amniocentesis contains fetal cells that provide comprehensive genetic information. Besides being able to perform amniotic fluid chromosomal karyotype analysis, various follow-up tests can also be conducted based on the pregnant woman’s situation, such as CNV-Seq, whole exome sequencing, etc. Furthermore, testing the amniotic fluid alpha fetoprotein levels can help diagnose ONTDs²¹.

AMA has become the most common indication for prenatal diagnosis, supporting the continued use of invasive procedures in this population. Among the 11 prenatal diagnostic indications selected for this study, AMA accounted for a high percentage (54.50%), which means that pregnant women of the AMA are the primary target population for prenatal diagnostic testing. In previous reports, the proportion of AMA pregnant women ranged from 27.013% to 39.1%^10,15. The higher percentage of pregnant women of AMA in this study could be attributed to the fact that the local government covers the expenses of amniocentesis and fetal chromosome karyotype analysis for these women. As a result, more pregnant women of AMA may choose IPD over NIPT. Although the PR of chromosomal abnormalities in the fetuses of AMA pregnant women was slightly lower than that of all pregnant women who underwent amniocentesis (3.29% vs. 3.54%), the difference was not statistically significant (p = 0.621). Therefore, women of AMA should prefer IPD as their first choice. Both AMA and AMSS are established indications for IPD. Some studies have reported a higher incidence of fetal chromosomal abnormalities in the AMSS group than in the AMA group^22,23. However, there are also reports of opposite results²⁴, which may be related to differences in study populations and screening protocols. In this study, the PRs of chromosomal abnormalities did not significantly differ between the AMA group and the AMSS group, with rates of 3.29% and 3.26%, respectively (p = 0.842). This finding also demonstrates that AMA pregnant women should choose IPD over NIPT, AMA is an important indication for IPD, which is an important measure for preventing birth defects²⁵. The PR of chromosomal abnormalities in the NIPT group was only 10.13%, which is lower than the reported 50% in the literature¹⁰. This discrepancy may be due to the inclusion of chromosomal aneuploidies and CNVs in the abnormal NIPT results in this study, whereas the statistical analysis of fetal abnormal chromosomal karyotypes did not include CNVs in the count.

The detection rate of chromosomal abnormalities in this study aligns with previous reports, confirming the stability and reliability of karyotype analysis. In this study, the PR of amniotic fluid chromosomal abnormalities was 3.54%, which is similar to the previously reported range of 2.88%-3.46%^13–15. Chromosome karyotype analysis, the gold standard for the diagnosis of fetal chromosomal abnormalities, has relatively stable detection performance. Previous studies have reported that the PR of T21 in amniotic fluid chromosomal karyotype abnormalities is as high as 35.6%-59%, which indicates that T21 is the most common chromosomal abnormality^11,26. In this study, 61 T21 samples (including mosaic T21 and translocation T21) were identified, which accounted for 46.56% of all abnormal karyotypes. The increase in maternal age may lead to a higher incidence of various pregnancy-related disorders. Obstetricians and pregnant women should pay attention to the issue of increased rates of fetal chromosomal abnormalities due to the rise in maternal age. In the Luohe region of China, the government offers free IPD for pregnant women like AMA and AMSS. This initiative has resulted in 54.50% of AMA pregnant women choosing IPD. The rates of fetal chromosomal abnormalities in IPD pregnant women and AMA pregnant women are 3.54% and 3.29%, respectively, consistent with previous studies. In Xiao’s study of 12,365 IPD pregnant women, the AMA ratio was 34.53%, with fetal chromosomal abnormality rates of 3.46% overall and 2.79% for AMA pregnant women¹³. Conversely, Tao’s study of 4,761 IPD pregnant women showed an AMA ratio of 39.10%, with fetal chromosomal abnormality rates of 2.88% overall and 2.10% for AMA pregnant women¹⁵. The importance of IPD and fetal chromosome karyotype analysis increases with maternal age.

Karyotype analysis is particularly valuable for detecting mosaicism, SCAs, and RAAs. This study revealed 21 SCAs, with a PR of 0.57%. The PR for amniotic fluid SCAs in previous studies has shown significant variation, ranging from 0.34 to 3.36%^11,15,27. In this study, a high proportion (23.81%, 5/21) of all SCAs were chimeric SCAs. Accurate detection of low-level chimeras requires counting an adequate number of metaphase cells during karyotype analysis. In this study, the determination of all chimeras was based on a minimum of 50 metaphase cells. This study also identified four cases of chimeric RAAs, which also highlights the importance of counting an adequate number of metaphase cells. RAAs are typically detected as chimeras in amniotic fluid during the second trimester²⁸. This is because their occurrence is low, and they often lead to spontaneous miscarriage or developmental arrest in early pregnancy²⁹, as seen in the four cases reported here. Importantly, in early pregnancy, RAAs are lethal, and only fetuses that undergo “trisomy rescue” can survive²⁹. This “trisomy rescue” may lead to fetal chromosomal uniparental disomy (UPD)³⁰.

The decision to TOP varies significantly depending on the type of chromosomal abnormality. The degree of harm caused by different types of chromosomal abnormalities varies, and the proportion of women who undergo TOP for different fetal chromosomal abnormalities also varies. This is influenced by factors such as the type of abnormality, genetic counseling level, and cultural practices, among others³¹. In this study, all 70 pregnant women with common fetal trisomies chose to undergo TOP, which is similar to the findings by Zhou³¹. According to the literature, 61.1% to 81% of pregnant women carrying fetuses with SCAs choose TOP^31–33. In this study, however, only 33.33% (7/21) opted for TOP. Some pregnant women and their families may accept children with SCAs; despite the developmental or reproductive issues that some of these children may have, they are generally of normal intelligence³⁴. The other pregnant women whose fetuses had chromosomal numerical abnormalities all opted for TOP, including 1 case of triploidy, 4 cases of mosaic RAAs, and two cases of chromosomal numerical and structural abnormalities.

The combination of karyotype analysis and molecular techniques such as CNV-seq improves the detection of pathogenic abnormalities. Notably, our study included 4 cases of fetal chromosomal structural abnormalities and 7 cases of fetal chromosomal polymorphisms, and in those cases, the pregnant women also elected to undergo TOP. Except for one pregnant woman who chose TOP for personal reasons, the remaining women sought TOP due to pathogenic or likely pathogenic CNVs or chromosomal chimeras detected by amniotic fluid CNV-seq. With the increasing popularity of CNV detection, the PR of chromosomal abnormalities and the proportion of pregnant women opting for TOPs have continued to increase.

Although NIPT has high sensitivity for common trisomies, it cannot replace invasive prenatal diagnosis due to its limitations in detecting mosaicism, structural abnormalities, and SCAs. NIPT has a high positive predictive value (PPV) and an extremely low false-negative rate (FNR) in screening for common trisomies³⁵. The reported PPV ranges in the literature are 71–100%, 48–85%, and 11–54%, for T21, T18, and T13, respectively^33,36,37. In this study, of 79 pregnant women with positive NIPT results, only 8 cases were confirmed through amniotic fluid chromosome karyotype analysis, which resulted in a PPV of only 10.13%. The lower PPV of the NIPT group is due to IPD being conducted based on NIPT indications, including: (1) a high risk of fetal chromosomal aneuploidy detected by NIPT; (2) a high risk of fetal CNVs detected by NIPT; (3) IPD performed due to failed NIPT results. Actually, positive NIPT results are associated with a high risk for chromosomal aneuploidies or CNVs³⁸, whereas traditional chromosome karyotype analysis is unable to detect CNVs. In previous reports, the PPVs of NIPT or NIPT-plus for T21, T18, and T13 were found to be 71%-100%, 48%-85%, and 11%-54%, respectively^33,36,37,39. In another study conducted by Luohe Central Hospital of China from January 2019 to June 2023, it was found that out of 6792 cases of NIPT and 5237 cases of NIPT-plus, the composite PPVs for T21/T18/T13 were 71.43% and 73.53%, respectively⁴⁰. This suggests that the lower PPV in the NIPT group in this study was not attributed to any shortcomings in the NIPT testing process. According to the results of this study, the advantages of chromosome karyotype analysis over NIPT are as follows: (1) Chromosome karyotype analysis revealed 32 cases of chromosomal structural abnormalities, which cannot be detected by NIPT; (2) the PPV of NIPT is influenced by various factors, as different sequencing platforms lead to different results, whereas the PR of chromosome karyotype analysis remains stable; (3) NIPT testing for SCAs and RAAs has a lower PPV that ranges from 38.46% to 68.00% for screening SCAs^33,41. NIPT may not effectively screen for SCAs, especially 45,X, possibly because of the presence of many homologous sequences between the X and Y chromosomes, which can lead to sequencing errors due to the shorter read lengths of NIPT⁴². The PPV of NIPT screening for RAAs is only 4.88%³⁹. (4) NIPT is not able to detect mosaicism effectively⁴³, as this study revealed that mosaicism accounts for 13.74% of abnormal chromosomal karyotypes (18 of 131). (5) One main drawback of amniocentesis is its invasive nature; however, amniotic fluid can be used not only for chromosome karyotype analysis but also for the detection of CNVs.

Rare and complex karyotypic findings require careful interpretation and, in some cases, repeated amniocentesis or additional testing. In this study, several abnormal chromosomal karyotypes that require special attention were discovered. A 32-year-old pregnant woman with a high risk of T21 based on MSS results underwent amniocentesis. The amniotic fluid chromosome karyotype revealed 46,XN, t(1:17)(q21:q23),t(5:8)(q35;q21),10ps[8]/46,XN,10ps[92], as shown in Fig. 2E. Notably, all abnormal cells (46,XN, t(1:17)(q21:q23),t(5:8)(q35;q21),10ps) were found in one culture bottle, whereas none were found in the other bottle. Furthermore, abnormal cells accounted for only 8% of the total number of cells. Due to the unavailability of cordocentesis in the local area, the pregnant woman underwent a second amniocentesis one month after the first amniocentesis, and the amniotic fluid chromosome karyotype was 46,XN,10ps[60]. She eventually gave birth to a healthy baby with no abnormalities. For rare abnormal chromosomal karyotypes, it is necessary to perform a second amniocentesis or umbilical cord blood sampling for further examination. In a study conducted by Sun involving 3387 cases of umbilical cord blood chromosomal karyotype analysis, 1.54% (52 cases) of pregnant women underwent cordocentesis to verify chromosome karyotypes found in the amniotic fluid. Ultimately, the chromosomal karyotype analysis of umbilical cord blood was consistent with that of amniotic fluid in only 23 patients⁴⁴. Another pregnant woman who was 29 years of age with an amniotic fluid chromosome karyotype of 47,XN,+mar (Fig. 2A), underwent amniocentesis due to a high risk of T21 based on the MMS result. The extra + mar chromosome closely resembled chromosome 21 morphologically, and thus it could have been easily misdiagnosed as T21. However, the C-banding results revealed that the extra chromosome was not chromosome 21, and the CNV-seq results did not reveal any abnormalities. This finding highlights the value of special banding techniques, such as C-banding and N-banding, in the diagnosis of special abnormal chromosomal karyotypes, as well as the advantages of combining CNV-seq and other molecular testing techniques with chromosome karyotype analysis.

Amniocentesis remains a safe and reliable procedure when performed by experienced clinicians under ultrasound guidance. According to a recent meta-analysis, the estimated miscarriage rate after amniocentesis is between 0.3 and 0.6%^45,46. In this study, of 3681 pregnant women who underwent amniocentesis, no fetal losses occurred due to the procedure, which indicates that amniocentesis is a mature and relatively safe procedure. The success rate of amniotic fluid culture ranges from 95.03% to 98.8% in the literature^15,47. In this study, out of all 3700 amniotic fluid samples, there were 4 cases where one culture bottle was successful while the other one failed, indicating the importance of using two culture bottles for the same amniotic fluid simultaneously.

This study has two limitations: (1) the sample size is small, which may have resulted in data bias in some of the detection indicators; (2) in this study, karyotype analysis was used to detect chimerism, but the determination of the chimeric ratio may not be accurate. In vitro cell culture may lead to changes in the chimeric ratio due to differences in the proliferation of cells with different karyotypes. For example, it has been reported that diploid cells may grow faster than nondiploid cells during the cell culture process⁴⁸.

Conclusions

From 2019 to 2023, the proportion of AMA pregnant women in the Luohe region of China increased from 11.30% to 16.03%, making AMA the primary indication for prenatal diagnosis. The detection rate of fetal chromosomal abnormalities in the AMA group was 3.29%, which showed no significant difference compared to the AMSS group. This indicates that AMA is a key independent risk factor for fetal chromosomal abnormalities, suggesting that IPD should be recommended as the first-choice prenatal diagnostic approach for AMA pregnant women. This study highlights the irreplaceable value of combining amniocentesis with fetal chromosome karyotype analysis. The overall detection rate of chromosomal abnormalities in the study was 3.54%. Karyotype analysis successfully identified a variety of abnormalities, including structural abnormalities, mosaicism, and triploidy, which are often missed by NIPT and cannot replace IPD. The proportion of pregnant women opting for TOP varies depending on the type of fetal chromosomal abnormality. The termination rate for trisomy 21/18/13 is 100%, whereas for sex chromosome aneuploidies, it is significantly lower at 33.33%. This highlights the crucial role of comprehensive genetic counseling in parental decision-making. Combining fetal chromosome karyotype analysis with CNV-Seq can increase the detection rate of chromosomal abnormalities, providing more precise information for clinical management and genetic counseling. This represents the direction of development in prenatal diagnosis. In conclusion, this study emphasizes the value of conducting IPD and fetal chromosome karyotype analysis in the context of increasing maternal age now and in the future.

Materials and methods

Subjects

We obtained data on the number, age, and fertility status of pregnant women from hospitals in the Luohe region of Henan Province, China. Using this information, we calculated the proportion of AMA pregnancies from 2019 to 2023. From November 2019 to May 2024, a total of 3901 pregnant women underwent amniocentesis at Luohe Central Hospital in China. The inclusion criteria were: (1) Amniocentesis performed between 18 and 26 weeks of gestation; (2) Chromosomal karyotype analysis of amniotic fluid conducted after amniocentesis. The exclusion criteria were: failure to obtain amniotic fluid. Eventually, 3681 pregnant women were included in this study. Luohe Central Hospital is the only hospital in the Luohe area that offers IPD services. This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and complied with the relevant Chinese regulations, including the Measures for the Ethical Review of Life Science and Medical Research Involving Humans (2023). The research protocol was reviewed and approved by the Medical Ethics Committee of Luohe Central Hospital (Approval No. MEC-2018-087; Date of approval: November 12, 2018). All participants provided written informed consent for the use of their clinical data and amniotic fluid samples for research purposes.

The prenatal diagnostic indications among the included pregnant women were classified into 11 categories: AMA, abnormal maternal serum screening (AMSS), twin pregnancy, ultrasonic anomalies (UA), either mother or father with chromosomal abnormality (EMFCA), high risk for NIPT (NIPT), previous fetus/child with abnormalities (PFA), in vitro fertilization (IVF), teratogenic factors, and voluntary diagnosis. The details for each indicator are listed in Table 4.

Table 4.

Positive rates of abnormal amniotic fluid chromosome karyotypes according to different indications.

Indications	Total cases		Numerical abnormalities		Structural abnormalities		Total abnormalities
	n	%	n	%	n	%	n	%
AMA	2006	54.50	56	2.79	11	0.55	66	3.29
AMSS	1319	35.83	30	2.27	13	0.99	43	3.26
Twin pregnancy	26	0.71	0	0.00	0	0.00	0	0.00
UA	166	4.51	4	2.41	2	1.20	5	3.01
EMFCA	26	0.71	2	7.69	8	30.77	10	38.46
NIPT	79	2.15	7	8.86	1	1.27	8	10.13
PFA	62	1.68	0	0.00	0	0.00	0	0.00
IVF	3	0.08	0	0.00	0	0.00	0	0.00
Teratogenic factors	14	0.38	0	0.00	1	7.14	1	7.14
Voluntary diagnosis	8	0.22	0	0.00	0	0.00	0	0.00
Family genetic history	5	0.14	0	0.00	0	0.00	0	0.00
Total	3681	NA	99	2.68 (99/3700)	34	0.92 (34/3700)	131	3.54 (131/3700)

Indications	Details of indications
AMA	Advanced maternal age, 35 years or older, at the expected time of delivery
AMSS	Abnormal maternal serum screening. (1) high risk: trisomy 21 ≥ 1/270, trisomy 18 ≥ 1/350; (2) intermediate risk: 1/1000 ≤ trisomy 21 ≤ 1/270, 1/1000 ≤ trisomy 18 ≤ 1/350
Twin pregnancy	(1) Twin pregnancy; (2) Twin pregnancy became single pregnancy
UA	Ultrasonic anomalies. (1) Nuchal Translucency ≥ 3.0 mm; (2) Abnormal ultrasound soft markers; (3) Abnormal ultrasound structural findings
EMFCA	Either mother or father with chromosomal abnormality
NIPT	(1) High risk of fetal chromosomal aneuploidy detected by NIPT; (2) High risk of fetal CNVs detected by NIPT; (3) IPD performed due to failed NIPT results.
PFA	Previous fetus/child with abnormalities
IVF	In vitro fertilization
Teratogenic factors	Exposed to teratogenic factors or treatment with medication during pregnancy
Voluntary diagnosis	Prenatal diagnosis without a high-risk factor
Family genetic history	(1) Family genetic history; (2) Pregnant women with intellectual disability

Pregnant women with two amniocentesis indications were included in different groups for repeated statistical analysis. According to local government policy, pregnant women with the following indications have their prenatal diagnostic expenses covered by the government: AMA, AMSS, EMFCA, PFA. Two cases of numerical and structural abnormalities were included in both the numerical abnormality group and the structural abnormality group for repeated statistical analysis. NA, not applicable.

In accordance with the subsidy policy of the local government, pregnant women with the following specified indications have expenses for amniocentesis and fetal chromosome karyotype analysis fully funded by the government: AMA, high risk for maternal serum screening (T18 ≥ 1/350, T21 ≥ 1/270), EMFCA, and PFA.

Amniocentesis

Genetic counseling physicians recommend that all pregnant women who undergo amniotic fluid chromosome karyotype analysis should also undergo amniotic fluid copy number variant sequencing (CNV-seq) to detect fetal copy number variants (CNVs). The use of fluorescent quantitative PCR to conduct rapid diagnosis of common chromosome aneuploidies (13/18/21/XY) in all amniotic fluid samples ensures no contamination by maternal cells. After 18 weeks of pregnancy, the pregnant women underwent a procedure to extract approximately 20 mL of amniotic fluid with the help of ultrasound guidance in a sterile environment. For amniotic fluid chromosome karyotype analysis, the amniotic fluid should be cultured on the day of amniocentesis. However, for CNV-seq, the amniotic fluid should be stored at 4 °C, and genomic DNA should be extracted within 48 h.

Amniotic fluid chromosome karyotype analysis

After approximately 15 mL of amniotic fluid was centrifuged, it was cultured in at least two culture bottles. The cells were cultured in complete medium supplemented with 20% fetal bovine serum at 37 °C in a 5% CO₂ atmosphere for 10–11 days. Following cultivation, collection and G-banding, the chromosome images were captured via a Carl Zeiss automated chromosome scanning system Imager Z2 (Carl Zeiss, Jena, Germany), and chromosome karyotype analysis was performed via the accompanying MetaSystems MetaClient chromosome analysis software (Version 5.0, MetaSystems, Altlussheim, Germany; URL: https://metasystems-international.com/). For each sample of amniotic fluid, at least 30 metaphase cells were counted, and a minimum of five karyotypes were analyzed. In the case of chimeras, 30–100 metaphase cells were counted according to the chromosomes involved in the chimerism. The karyotypes were described according to the International System for Human Cytogenomic Nomenclature (2020) (ISCN 2020)⁴⁹. In this study, a strict two-line operation was followed for each amniotic fluid sample during the culture process, which involved culture in two separate bottles, using two different batches of culture media, two incubators, and two personnel who performed the procedure.

CNV-seq

After genomic DNA was extracted from amniotic fluid via the QIAamp DNA Mini Kit (Qiagen, NY, USA), quantification was performed using the Invitrogen Qubit 5.0 (Thermo Fisher Scientific, MA, USA). CNV detection kits, next-generation sequencing (NGS) library construction kits, and DNA purification kits from Berry Genomics in Beijing, China, were used for CNV sequencing library construction, purification, quality control, and other related procedures. Library preparation was performed according to the manufacturer’s instructions, and sequencing was carried out on the Illumina NextSeq CN500 platform with an average depth of ≥ 0.1× and at least 8 million unique reads per sample. The Xromate analysis system (Berry Genomics, Beijing, China) was used to analyze the sequencing data along with the human reference genome sequence GRCh37 (hg19). In accordance with the guidelines set forth by the American College of Medical Genetics (ACMG), the pathogenicity of the CNVs was assessed by referencing public databases (ClinGen, DECIPHER, DGV, OMIM) to determine their classification as benign, likely benign, variants of uncertain significance (VUS), likely pathogenic, or pathogenic⁵⁰.

Clinical follow‑up assessment

If the amniotic fluid chromosome karyotype of the fetus was abnormal or if it was normal but the amniotic fluid CNV-seq results were pathogenic/likely pathogenic/VUS, the pregnant woman would receive genetic counseling and decide whether to proceed with the TOP. All pregnant women received at least two telephone follow-ups, with one follow-up scheduled one week after amniocentesis to check for any abortions or infections and another follow-up after giving birth to assess the delivery situation, newborn outcomes, physical examination of the newborn, and developmental details. Pregnancy outcomes were divided into four categories: pregnancy loss, TOP, live birth, and loss to follow-up.

Statistical analysis

Data analysis was conducted via SPSS 25.0 software (SPSS Inc., Chicago, IL, USA; URL: https://www.ibm.com/analytics/spss-statistics-software). Descriptive statistics are presented as the means and minimum-maximum values, whereas categorical data are presented as rates. When two or more sets of rates were compared, a chi-square test was used to determine if there was a statistically significant difference, with a significance level of p < 0.05. Pregnant women who had two indications for amniocentesis were divided into different groups for repeated statistical analysis.

Author contributions

Conceptualization, Xiuhong Fu and Shaozhe Yang; methodology, Rongxiang Li and Zhenhua Gong; writing—original draft preparation, Shuwen Xin, Yanqi He and Yanan Gao; writing—review and editing, Shaozhe Yang and Shaoxia Teng; funding acquisition, Xiuhong Fu. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Henan Key Laboratory of Fertility Protection and Aristogenesis (No. SYLBHYSSYS).

Data availability

The datasets generated and/or analysed during the current study are available in the Science Data Bank repository, [https://www.scidb.cn/anonymous/QkJmVVZy] .

Declarations

Competing interests

The authors declare no competing interests.