Study design and participants
A total of 2422 singleton-pregnant women accepting amniocentesis at the Deyang People’s Hospital from September 2019 to May 2022 were included in our study. The gestational age of these women ranged from 15+ 4th to 35+ 6th weeks, while their ages were 17 to 53 years old. Pregnant women at advanced maternal age and with normal ultrasound results were included in the control group, while fetuses with ultrasound soft markers were included in ultrasound soft markers group (USM group).
In our study, ultrasound soft markers included increased NT thickness (≥ 2.5mm), absence or dysplasia of nasal bone (absence or less than 2.5th percentile for gestational age), ventriculomegaly (≥ 10mm and < 15mm), echogenic bowel, single umbilical artery, shortened humerus and/or femur (below the 5th percentile for gestational age), echogenic intracardiac focus, choroid plexus cysts, renal abnormalities and abnormal amniotic fluid volume (more than 18cm or less than 5cm). Renal abnormalities included hydronephrosis (> 10mm), pyelectasis (≥ 4 mm and < 10 mm), hyperechogenicity and duplex kidney. Informed consent was obtained from all individual participants included in the study. All methods were performed following the relevant guidelines and regulations and approved by the Ethical Committee of Deyang People’s Hospital.
Quantitative fluorescent polymerase chain reaction (QF-PCR) assay
The STR Genotyping Kit for Chromosomes 13/18/21/X/Y (DaRui Biotech Co., Guangzhou, China) was used to detect common fetal chromosome aneuploidy according to the manufacturer’s instructions. The TIANamp Genomic DNA kit (Tiangen Biotech Co., Beijing, China) was used to extract genomic DNA from 8ml of amniotic fluid according to the manufacturer’s instructions. The concentrations of genomic DNA were measured using Qubit 1× dsDNA High Sensitivity (HS) and Broad Range (BR) Assay Kits (Thermo Fisher Scientific Inc., Rockford, USA) and then diluted to 5-10ng/µl. Then, 20 highly polymorphic short tandem repeats (STRs) (Table 1) were amplified using a Bio–Rad PTC 200 PCR system (Bio–Rad Laboratories, Hercules, USA). The PCR profile was pre-denaturation at 95°C for 5 minutes followed by 95°C for 30 seconds, 58°C for 40 seconds, and 72°C for 50 seconds, for 25 cycles with a final extension at 72°C for 10 minutes. Then, capillary electrophoreses were performed using the 3500 ABI Genetic Analyser (Applied Biosystems, Waltham, USA) after mixing 1µl PCR product with 13.5µl HiDi formamide (Thermo Fisher) and 1µl LIZ600 (Thermo Fisher). Finally, the electrophoresis results were interpreted according to the manufacturer’s instructions.
Table 1
STR markers analyzed by QF-PCR
Chromosome | STR | Length (bp) |
21 | 21q11.2 | 170–220 |
21 | DS21S1411 | 270–325 |
21 | DS21S1412 | 380–450 |
21 | DS21S1414 | 315–370 |
21 | DS21S1433 | 140–190 |
21 | DS21S1445 | 470–530 |
18 | DS18S1002 | 108–140 |
18 | DS18S386 | 305–375 |
18 | DS18S391 | 180–220 |
18 | DS18S535 | 235–280 |
13 | DS13S305 | 370–430 |
13 | DS13S628 | 140–190 |
13 | DS13S634 | 320–365 |
13 | DS13S742 | 220–275 |
X/Y | AMXY | 102/108 |
X | DXS1187 | 130–170 |
X | DXS6809 | 255–300 |
X | DXS8377 | 180–254 |
X | DXS981 | 310–370 |
Y | SRY | 248 |
Karyotyping
For each sample, two independent cultures were established. Twenty milliliters of amniotic fluid were collected from each pregnant woman and then equally divided into two parts. After centrifugation at 1000 rpm for 10 minutes, the amniotic supernatant was discarded and the precipitated amniocytes were resuspended in 3ml BIO-AMF-3 complete medium (Biological Industries, Cromwell, USA). Then, amniocytes were incubated at 37°C in a Thermo 3111 CO2 incubator (Thermo Fisher). After 9–14 days of culturing, amniocytes were harvested for G banding. Zeiss automatic karyotyping scanning system (Carl Zeiss, Jena, Germany) was used to capture 20 metaphase images for each sample. Five of these metaphase images were analyzed using IKAROS software (Carl Zeiss). Karyotypes were described according to ISCN 2020 guidelines.
CNV-seq
CNV-seq was performed to detect chromosome abnormalities and CNVs. Briefly, the experimental process included genomic DNA extraction, library construction, quality control, pooling, sequencing, bioinformatics analysis and results interpretation. Firstly, 2-4ml of amniotic fluid was centrifuged at 10000 rpm for 5 minutes, then the supernatant was discarded. According to the manufacturer's instructions, the TIANamp Genomic DNA kit (Tiangen) was used to extract genomic DNA from precipitated amniocytes. The concentrations of genomic DNA were measured using Qubit 1× dsDNA High Sensitivity (HS) and Broad Range (BR) Assay Kits (Thermo Fisher) and then diluted to 2ng/µl. For each sample, 10µl diluted genomic DNA was fragmented by NEBNext dsDNA Fragmentase (New England Biolabs, Ipswich, USA) at 37°C for 50 minutes. Then, Foetal Chromosome Aneuploidy (T21, T18, and T13) Testing Kits (Annoroad, Beijing, China) were used for library construction via end filling, adaptor ligation and PCR amplification. Quality control of libraries included measuring their concentrations by Qubit 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay Kits (Thermo Fisher) and capillary electrophoresing using Agilent 2100 Bioanalyzed (Agilent Technologies, Palo Alto, USA). Subsequently, qualified libraries were pooled together and subjected to massively parallel sequencing using NextSeq 550AR (Illumina, San Diego, USA). At least 4.5 million raw reads with a length of 40 bp were generated for each sample. The quality control criteria of the sequencing results were as follows: reads > 4.5 Mb, GC content: 38.5%-45.5%, Q30 ratio > 85%, alignment ratio > 62.5%, unique read ratio > 60%, and duplication ratio < 10%. Qualified sequencing results were mapped to the grch37 version of the human genome using the Burrows–Wheeler's algorithm[18], and CNVs were detected by bioinformatics analysis.
According to American College of Medical Genetics guidelines[19], the pathogenicity of CNVs was classified as pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign and benign. CNVs were described according to ISCN 2020 guidelines and their pathogenicity was interpreted using public databases including ClinGen, Database of Genomic Variation and Phenotype in Humans Using Ensembl Resources (DECIPHER), Online Mendelian Inheritance in Man (OMIM), 1000 Genomes, and the Database of Genomic Variants.
Statistics
SPSS 19.0 (IBM, New York, USA) was used for statistical analysis. The chi-square test was used to analyze qualitative data, while the t-test and one-way ANOVA were used to analyze normally distributed quantitative data. A P < 0.05 indicated that the difference was statistically significant.