Diagnostic accuracy and value of chromosomal microarray analysis for chromosomal abnormalities in prenatal detection: a prospective clinical study

Chromosomal microarray analysis (CMA) has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. The aim of this study was to compare the accuracy and diagnostic value of CMA and karyotyping on chromosomal abnormalities in Fujian province of South China. In the study, 410 samples were obtained from pregnant women between March 2015 and December 2016, including 3 villus (0.73%, 3/410), 296 amniotic uid (72.20%, 296/410), and 111 umbilical cord blood (27.07%, 111/410). Each sample was screening for chromosomal abnormalities by both using CMA and karyotyping. (7.56%, 31/410) characteristic showed that


Introduction
Chromosomal abnormalities has focused commonly on detection of the aneuploidy in human trisomy 21 and 18 in prenatal diagnosis (PND) [1,2]. Currently, the mainly means of PND are to apply a combination of diagnostic procedures in the one and two-trimester based on concentrations of serum analytes, genetic history, maternal age, and ultrasound-detected data from pregnant women [3][4][5]. Karyotyping is commonly technique in screen chromosome abnormalities from individuals with congenital malformations, including chromosome deletion, inversion, duplication, translocation, aneuploidy, and polyploidy [6]. Because karyotyping is reliably method for the detection of chromosome aneuploidies and large rearrangements, which has been the preferred method for prenatal diagnosis of chromosomal abnormalities for a long time in the past. However, karyotyping has considerable limitations, especially for the lack of detection of many unbalanced structural abnormalities from submicroscopic chromosomal aberrations. In recent years, molecular cytogenetic methods, including multiplex ligationdependent probe ampli cation (MLPA), quantitative uorescence polymerase chain reaction (QF-PCR), and uorescence in situ hybridization (FISH), are gradually applied to evaluate submicroscopic chromosomal aberrations [7,8]. However, these techniques are not feasible to detect all possible chromosome deletion and duplication.
Chromosomal microarray analysis (CMA) is known as array-based comparative genomic hybridization (array CGH) [9,10]. At present, the advantages of CMA in PND are gradually presented with the rapid development of chip technology. CMA has the ability to disclose a wide range of chromosomal abnormalities with length from 50 kb to 100 kb, which produces 100 times better resolution than karyotyping [11]. More and more evidence has indicated that CMA can improve the diagnostic accuracy by approximately 15-20% over that of karyotyping when applied for the evaluation of fetuses with unexplained developmental delay, mental retardation, and autism [12]. CMA raises the diagnostic rates from 0.5-16% for screening commonly chromosomal abnormalities in PND [13]. In addition, CMA also obviously increases the success rates for diagnosing fetuses with chromosomal structural anomalies compared with karyotyping [14,15]. Up to now, there is still no systematic study on the diagnostic accuracy of CMA for chromosomal abnormalities in PND in Fujian province of South China.
Here, our study aimed to compare the diagnostic accuracy of CMA and karyotyping for screening chromosomal abnormalities in PND, and to analyze theirs diagnostic values. Firstly, we collected 410 samples were obtained from pregnant women. Second, the CMA and karyotyping were performed to determine the rate of chromosomal abnormalities from all samples. Finally, the sensitivity and speci city of CMA and karyotyping in the diagnosis of chromosomal abnormalities was calculated and compared using ROC analysis. Our data con rmed that CMA is e cient to improve the diagnostic accuracy of chromosomal abnormalities in PND. CMA has a better diagnostic value than karyotyping, especially for pregnant women with normal karyotypes.

Karyotyping Analysis
Karyotyping was performed using G-banding analysis as previous literatures [16,17]. G-banding was conducted according to the manufacturer operational protocols. Each samples were digested mechanically with collagenase II (TIANGEN, Beijing, China) at 37 °C for 20 min. After that, the metaphases were tretaed with the CytoVision computer assisted karyotyping system version 2.7 (Santa Clara, CA, USA). Karyotyping was then observed based on the criterion of the International System for Human Cytogenetics Nomenclature in 1995 (ISCN). Five metaphase cells were detailed examined by two experienced diagnostic specialists to determine chromosomal structural abnormalities, and at least fteen metaphase cells were used to de ne chromosomal numerical abnormalities.
Chromosomal Microarray Analysis (CMA) CMA test was carried out as previous literatures [18,19]. Brie y, genomic DNA from each samples was isolated using a commonly DNA extraction kit (Qiagen, Hilden, Germany), according to the manufacturer's procedures. Then the isolated DNA was quanti ed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scienti c, MA, USA). When the concentration of DNA from sample was > 100 ng/µl and optical densities were 1. According to the deletion and duplication in chromosome location, the clinical signi cances of each abnormalities were evaluated. Chromosomal abnormalities were de ned as ve types of properties, including pathogenicity, possible pathogenicity, benign, possible benign, and unclear.

Statistical analysis
The statistical analyses were conducted with the SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). Data were shown as mean ± SD (standard deviation) from three independent assays with each measured in triplicate. Differences among the groups were estimated using Chi-square test. Receiver operating characteristic (ROC) curves were drawn, and the area under curve (AUC) was analyzed to determine the speci city and sensitivity of CMA and karyotyping. A value of P < 0.05 was considered to be a statistically signi cant difference.

Diagnostic Values Of CMA And Karyotyping For Chromosomal Abnormalities
To investigate the potential diagnostic value of CMA and karyotyping for chromosomal abnormalities in PND, ROC curves were plotted on data from 410 samples. As presented in Fig. 4, representation of the data revealed the AUC of CMA was 0.93 (95% CI: 0.90 to 0.95), the sensitivity and speci city was 90.68% and 94.40%, respectively. The AUC of karyotyping was 0.90 (95% CI: 0.87 to 0.93) with 87.56% sensitivity and 91.22% speci city. Compared with karyotyping, the diagnostic value of CMA was remarkable for chromosomal abnormalities in PND.

Analysis of the relationships between the chromosomal abnormalities and clinical indications
As shown in Table 2, the rates of chromosomal abnormalities by karyotyping in high age group was 5.8%, in abnormal ultrasound group was 8.24%, in fetuses with abnormal karyotypes group was 76.92%, in patients with abnormal karyotypes group was 41.67%, in adverse pregnancy history group was 8.70%, in high risk of NIPT group was 20%, in two kinds of abnormal indications group was 11.43%, and in three kinds of abnormal indications group was 66.67%. The rates of chromosomal abnormalities by CMA in high age group was 1.45%, in abnormal ultrasound group was 14.84%, in high risk of serological screening in early or middle pregnancy group was 8.00%, in fetuses with abnormal karyotypes group was 61.54%, in patients with abnormal karyotypes group was 16.67%, in adverse pregnancy history group was 17.39%, in high risk of NIPT group was 20%, and in two kinds of abnormal indications group was 14.29%, in three kinds of abnormal indications group was 66.67%, and in others groups was 50.00%. There were no signi cant differences in chromosomal abnormalities of clinical indication groups by CMA and karyotyping.  [20,21]. Recently, a mostly proportion of chromosomal abnormalities has be con rmed with the promotion of CMA in PND, in addition to some balanced rearrangements, triploidies, and uniparental disomy [22,23]. The resolution of detectable chromosomal abnormalities has heightened from 10 Mb or larger-sized rearrangements to a few kb in size, thus signi cantly improves the diagnostic accuracy of abnormalities in PND.
Here, the purpose of our study was to assess the diagnostic accuracy of CMA and karyotyping on chromosomal abnormalities, and to analyze the diagnostic value of CMA as a routine inspection for chromosomal abnormalities in PND. The 410 cases of villus, amniotic uid, and umbilical cord blood samples were obtained and cultured, and karyotyping and CMA were conducted in all samples in parallel.
In the 410 samples, the success rate of CMA was 100%. 61 samples were found to show chromosomal abnormalities by CMA. Furthermore, 31 samples with normal karyotypes were presented with chromosomal abnormalities using CMA. The overall abnormal rate of chromosomal abnormalities by CMA (14.88%) in this study was higher than several reports at recent studies (2-7.1%) [24,25]. The causes mainly focused on larger proportion of women with high risk of serological screening in early or middle pregnancy accounted for 6.10% of our cohort.
Further, 47 samples were shown with chromosomal abnormalities by karyotyping, including 13 cases of numerical abnormalities, 26 cases of structural abnormalities, and 8 cases of chimeras. 61 samples were presented with chromosomal abnormalities by CMA, including 10 cases of CNVs, 9 cases of large fragment abnormality, 38 cases of small fragment abnormality, and 4 cases of heterozygous abnormality. The overall abnormal rate of chromosomal abnormalities by CMA was 14.88%, which re ected 14 more cases than identi ed by karyotyping (11.46%), for an additional diagnostic yield of 3.42%. The samples with chromosomal abnormalities by CMA were exhibited as small fragment abnormality, deletion, and duplication. These results were consistent with a recent meta-analysis (3-5.2%) by Hillman et al [26]. Moreover, our data indicated that CMA have a high diagnostic value for chromosomal abnormalities in PND. The ROC curve of CMA showed 90.68% sensitivity and 94.40% speci city. The AUC of CMA was signi cantly larger than that of karyotyping, indicating that CMA may have excellent diagnostic value for chromosomal abnormalities in PND.
Besides, we also analyzed the relationships between chromosomal abnormalities and clinical indications.
In terms of single clinical indication, the rates of chromosomal abnormalities had not obviously differences by CMA and karyotyping in simple high age, abnormal ultrasound, fetuses and patients with abnormal karyotypes, adverse pregnancy history, and high risk of NIPT groups. However, the rates of chromosomal abnormalities by CMA had an increased tendency in high risk of serological screening in early or middle pregnancy group. The diagnostic yield of CMA is related to the particular population, clinical indications, fetuses from selective terminations, and spontaneous miscarriages [27,28].

Conclusions
Our study demonstrated that CMA is e cient to improve diagnostic accuracy of chromosomal abnormalities in PND. CMA has a higher diagnostic value for chromosomal abnormalities, especially for pregnant women with normal karyotypes. The limitation of this study is that clinical samples were relative small. Further researches with larger population should be conducted. Figure 1 The representative karyotyping of chromosomal numerical abnormality. The karyotypes of a sample with 47, XY, +21 were shown.