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INTRODUCTION
Chromosomal abnormalities are a common clinical genetic disorder that affects one in every 150 infants 1. Infants with these disorders are often mentally disabled, with a variety of malformations and stunting 2. At present, there is no effective treatment for this type of disease. The primary reliance is on prenatal screening and termination of pregnancy to avoid the birth of such fetuses. The standard prenatal diagnostic methods are divided into two categories: non-invasive prenatal testing and invasive prenatal testing3. Non-invasive prenatal DNA testing (NIPT), ultrasonography and maternal serum screening are commonly used non-invasive screening methods 2,4,5. However, these methods cannot accurately detect genes in fetal cells, and there are cases of missed diagnosis and misdiagnosis 6. Karyotyping after amniocentesis is still the gold standard for detecting fetal chromosomal abnormalities 7.
Amniocentesis is usually performed at 18-24 weeks of pregnancy to detect fetal chromosomal conditions by extracting and culturing amniotic fluid cells 8. Karyotype analysis is highly specific and sensitive, and the diagnostic rate of fetal chromosomal abnormalities is almost 100% 9. Prenatal diagnostic indications for amniocentesis usually include abnormal NIPT, high-risk maternal serum screening, advanced maternal age, abnormal ultrasonographic indications, paternal or maternal carrying chromosome abnormalities, and adverse pregnancy history 10,11.
Despite the wealth of data available on the outcomes of amniocentesis, gaps remain in the literature, particularly concerning the comprehensive analysis of karyotype results by different indications for the procedure. Previous studies have often focused on single indications or a small subset of chromosomal abnormalities. The novelty of the present study lies in its comprehensive approach, analyzing a large, diverse cohort with a wide range of indications for amniocentesis. Therefore, this study aimed to assess the relationship between various prenatal diagnostic indications and fetal chromosomal abnormalities by performing a karyotype analysis of amniotic fluid cells in 1285 instances by amniocentesis in high-risk pregnant women.
PATIENTS AND METHODS
Sample collection
Our study design type is a present retrospective study. One thousand two hundred eighty-five pregnant women admitted to our hospital for amniotic fluid karyotyping from July 2019 to June 2022 were selected as the study population. The pregnant women aged 19 to 46 and 18 to 24 weeks of gestation. Detailed demographic and clinical data were gathered by completing questionnaires, conducting direct interviews with pregnant women, and assessing medical records. The study was approved by the First Affiliated Hospital ethics committee of Soochow University, and all participants were informed and signed the consent form.
Inclusion criteria: presence of prenatal diagnostic indications, including high risk of NIPT, advanced maternal age, high risk of serologic screening, abnormal ultrasonographic indications, history of adverse pregnancy and one parent carrying chromosomal abnormalities; amniocentesis for the first time.
Exclusion criteria: unsuccessful cultures of amniotic fluid cells twice; the presence of threatened abortion.
Amniocentesis
The pregnant women and their families were informed of the risks, and an amniocentesis was performed after signing the informed consent. Twenty mL of amniotic fluid was drawn from the pregnant women and centrifuged at 2000 r/min for 10 minutes. After centrifugation, the supernatant was discarded, and the cell suspension was inoculated in the amniocyte culture medium at 37°C and 5% CO2 for 9 to 10 days. The amniotic fluid cells were collected when multiple clones with various metaphase cells were observed with an inverted microscope. After harvesting the cells, g-band staining was performed and canned by a Leica GLS120 Automated Nuclear Scanning System. Thirty karyotypes were routinely counted, and five karyotypes were analyzed following the International System for Human Cytogenetic Nomenclature (ISCN) standard 12.
RESULTS
Distribution of different diagnostic indications
Among the 1285 pregnant women with diagnostic indications, most cases were prompted by high-risk maternal serum screening, accounting for 573 cases (44.59%), followed by advanced maternal age with 522 cases (40.62%). Less frequent indications included abnormal results from non-invasive prenatal testing (NIPT), which led to 76 cases (5.91%), and abnormal ultrasonographic findings, which accounted for 67 cases (5.21%). The least common reasons for amniocentesis were a history of adverse pregnancy outcomes and parental chromosomal abnormalities, with 26 cases (2.02%) and 21 cases (1.63%) respectively (Table 1).
Table 1 Distribution of different diagnostic indications.
| Clinical indicator | Cases | Proportion (%) |
|---|---|---|
| High-risk maternal serum screening | 573 | 44.59 |
| Advanced maternal age | 522 | 40.62 |
| Abnormal ultrasonographic Indications | 67 | 5.21 |
| Abnormal NIPT | 76 | 5.91 |
| Paternal/maternal carrying chromosome Abnormality | 21 | 1.63 |
| Adverse pregnancy History | 26 | 2.02 |
| Total | 1285 | 100 |
Classification and detection rate of abnormal karyotypes
The study examined 1,285 cases of amniotic fluid samples from high-risk pregnant women. Of these, 96 (7.47%) cases showed chromosomal abnormalities. Numerical abnormalities were the most common, accounting for 68 (70.83%) abnormal karyotypes. The most frequent numerical abnormality was Trisomy 21, which was observed in 43 (44.79%) cases, followed by Trisomy 18 in 10 (10.42%) cases, Trisomy 13 in 2 (2.08%) cases, 47 XXX (trisomy X) in 2 (2.08%) cases, 47 XYY (Jacob’s syndrome) in 1 (1.04%) case, and 47 XXY (Klinefelter syndrome) in 2 (2.08%) cases. Additionally, 1 (1.04%) case of 45 X (Turner Syndrome or TS) was detected. Structural abnormalities were observed in 28 (29.17%) cases, including 6 (6.25%) translocations, 13 (13.54%) inversions, and 9 (9.38%) chromosome polymorphisms (Table 2).
Table 2 Classification and detection rate of abnormal karyotypes.
| Chromosomal karyotype | Number (n) | Occupancy% (n/96) | Detection rate% (n/1285) |
|---|---|---|---|
| Numerical abnormalities | 68 | 70.83 | 5.29 |
| Trisomy 21 | 43 | 44.79 | 3.35 |
| Trisomy 18 | 10 | 10.42 | 0.78 |
| Trisomy 13 | 2 | 2.08 | 0.16 |
| 47, XXX | 2 | 2.08 | 0.16 |
| 47, XYY | 1 | 1.04 | 0.08 |
| 47, XXY | 2 | 2.08 | 0.16 |
| 45, X | 1 | 1.04 | 0.08 |
| Mosaicism | 7 | 7.29 | 0.54 |
| Structural abnormalities | 28 | 29.17 | 2.18 |
| Translocation | 6 | 6.25 | 0.47 |
| Inversion | 13 | 13.54 | 1.01 |
| Chromosome polymorphism | 9 | 9.38 | 0.70 |
| Total | 96 | 100.00 | 7.47 |
Distribution of diagnostic indications in pregnant women with amniotic fluid karyotype abnormalities
The abnormal karyotype detection rate of one parent carrying a chromosome abnormality was 42.86% (9/21; calculation formula of detection rate (%): n/number of pregnant women per diagnostic indication in 1285 cases (Table 3)), which was the highest detection rate of all indications, followed by high-risk NIPT (27.63%) and abnormal ultrasonographic indications (14.93%). The more significant number of karyotype abnormalities was found in advanced maternal age, accounting for 30.21% (29/96), followed by high-risk maternal serum (26.04%) and abnormal NIPT (21.88%).
Table 3 Distribution of diagnostic indications in pregnant women with amniotic fluid karyotype abnormalities.
| Clinical indication | Abnormal karyotype(n) | Detection rate (%) | Proportion (%) |
|---|---|---|---|
| High-risk maternal serum screening | 25 | 4.36 | 26.04 |
| Advanced maternal age | 29 | 5.56 | 30.21 |
| Abnormal ultrasonographic indications | 10 | 14.93 | 10.42 |
| Abnormal NIPT | 21 | 27.63 | 21.88 |
| Paternal/maternal carrying chromosome abnormality | 9 | 42.86 | 9.38 |
| Adverse pregnancy history | 2 | 7.69 | 2.08 |
| Total | 96 | 7.47 | 100.00 |
Distribution of abnormal karyotypes for different prenatal diagnostic indications
As shown in Table 4, trisomy 21 was most common in pregnant women with advanced maternal age. Trisomy 21 also has the highest proportion in other abnormal karyotypes of older pregnant women. Fetal chromosomal structural abnormalities are mainly distributed in abnormal serological screening, and one parent carries the abnormal chromosome. Fetal chromosomal numerical abnormalities are mainly distributed in advanced maternal age, abnormal NIPT, and high-risk maternal serum screening. However, in terms of the total number, fetal chromosomal abnormalities are mainly distributed between advanced maternal age and high-risk maternal serum screening.
Table 4 Distribution of abnormal karyotypes for different prenatal diagnostic indications.
| Diagnosis indicator | Chromosomal karyotype | ||||||
|---|---|---|---|---|---|---|---|
| a | b | c | d | e | f | Total | |
| Trisomy 21 | 7 | 23 | 5 | 8 | 43 | ||
| Trisomy 18 | 2 | 3 | 2 | 3 | 10 | ||
| Trisomy 13 | 1 | 1 | 2 | ||||
| 47, XXX | 1 | 1 | 2 | ||||
| 47, XYY | 1 | 1 | |||||
| 47, XXY | 2 | 2 | |||||
| 45, X | 1 | 1 | |||||
| Mosaicism | 3 | 4 | 7 | ||||
| Translocation | 1 | 2 | 2 | 1 | 6 | ||
| Inversion | 3 | 2 | 2 | 6 | 13 | ||
| Chromosome polymorphism | 5 | 2 | 2 | 9 | |||
| Total | 25 | 29 | 10 | 21 | 9 | 2 | 96 |
a, High-risk maternal serum screening; b, Advanced maternal age; c, Abnormal ultrasonographic indications; d, Abnormal NIPT; e, Paternal/maternal carrying chromosome abnormality; f, Adverse pregnancy history.
DISCUSSION
This study aimed to assess the relationship between various prenatal diagnostic indications and fetal chromosomal abnormalities by performing a karyotype analysis of amniotic fluid cells in 1,285 high-risk pregnant women. The most common indications for amniocentesis were high-risk maternal serum screening (44.59%) and advanced maternal age (40.62%), followed by abnormal NIPT results (5.91%) and abnormal ultrasonographic findings (5.21%). Karyotype analysis detected chromosomal abnormalities in 96 cases, resulting in an abnormality rate of 7.47%. Numerical chromosomal abnormalities were more common, accounting for 70.83% of the abnormalities, with trisomy 21 being the most frequent (44.79%). Structural chromosomal abnormalities comprised 29.17% of the abnormalities, with inversions being the most common (13.54%).
The findings of this study are consistent with previous research on the distribution of prenatal diagnostic indications and the detection rates of chromosomal abnormalities. A study by Grgić et al. reported that the most common indication for amniocentesis was advanced maternal age 13. Additionally, a study by Golshahi et al. reported that the most common indication for amniocentesis was abnormal serum screening, similar to the results of the current study 14. Also, this study’s overall chromosomal abnormality detection rate of 7.47% is similar to other studies 15,16. However, the study of Sun et al. reported the detection rate of abnormal karyotypes to be 2.02%, which may be due to the sample size and methods used 17.
The high detection rate of numerical chromosomal abnormalities, particularly trisomy 21, is well-documented in the literature. A review by Liu et al. found that trisomy 21 was the most common chromosomal abnormality detected prenatally, accounting for 46.77% of all abnormalities, which aligns with the 44.79% reported in the current study 18. Additionally, the study of Ocak et al. confirms this finding with 46% of all abnormalities 19. Regarding structural chromosomal abnormalities, the findings of this study are also consistent with previous research. A study by Liu et al. reported that deletion, duplication, inversion, and translocation were the most common structural abnormalities detected prenatally 18.
The distribution of chromosomal abnormalities among the different prenatal diagnostic indications also aligns with previous studies. Advanced maternal age and abnormal serum screening have been consistently associated with higher rates of chromosomal abnormalities, particularly numerical abnormalities like trisomy 21 20. The higher detection rate of structural abnormalities in cases with parental chromosomal abnormalities or abnormal NIPT results has also been reported in the literature 21,22. The abnormal NIPT group, a new kind of prenatal screening in which fetal DNA was extracted from maternal serum for testing, had the second-highest detection rate. It is commonly used in pregnancy screening because of its excellent safety and specificity 23. NIPT mainly targets the detection of autosomal aneuploidy, and 12 cases of autosomal aneuploidy were detected in the NIPT high-risk group, including 8 cases of trisomy 21, 3 cases of trisomy 18 and 1 case of trisomy 13. However, according to the statistical results, the high risk of NIPT also implied the possibility of mosaicism, inversion, and translocation.
Prenatal ultrasonography cannot directly detect fetal chromosomal abnormalities, but it can detect some ultrasound soft markers associated with genetic abnormalities. These soft indicators include thickened nuchal fold, echogenic focus in the heart, choroid plexus cyst and others 24. Positive ultrasound soft indicators showed an increased risk of aneuploidy in the fetus. In our study, abnormal karyotypes were detected in 10 pregnant women with abnormal ultrasound findings, with a detection rate of 14.93% (10/67), 8 of which were aneuploid. Ultrasonography in the middle of pregnancy is essential for prenatal screening of chromosomal abnormalities, especially in fetuses with chromosomal aneuploidy.
While traditional karyotyping via amniocentesis remains the gold standard for prenatal chromosomal analysis, new technologies are emerging that offer alternatives or supplements to this invasive procedure. Non-invasive prenatal testing (NIPT) using cell-free fetal DNA from the mother’s blood has become an increasingly common screening tool, with an abnormal NIPT result prompting 5.91% of the amniocenteses in this study. NIPT has high detection rates for common aneuploidies like trisomies 21, 18, and 13, though it has limitations in identifying structural chromosomal abnormalities 25. Additionally, chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) are newer technologies offering higher resolution and the capability to detect submicroscopic chromosomal alterations that karyotyping might miss 26.
In conclusion, 96 karyotype abnormalities were detected in 1285 high-risk pregnant women, with an abnormality rate of 7.47%. NIPT, ultrasound, and serological screening help detect fetal chromosomal abnormalities. Nevertheless, karyotype analysis is still irreplaceable. Karyotype analysis of amniotic fluid cells is recommended for all pregnant women with indications of prenatal diagnosis. However, this study has limitations in that only the main indications were included in the statistics for pregnant women who met several indications. The sample size was small, and more research is required in future work.
