This retrospective study included 2,321 pregnant women who had undergone invasive prenatal testing at Prenatal Diagnosis Department of Meizhou People’s Hospital, Guangdong Province at a period of January 1th, 2015 to December 31th, 2019. The study was approved by the Ethics Committee of Meizhou People’s Hospital (No. MPH-HEC 2015-A-22). Informed consent was obtained by phone from all participants. 128 women were excluded either without consent or without karyotype results. Follow-up data on pregnancy outcomes and fetal health were collected by phone from all pregnant women.
Invasive prenatal tests were performed by either amniocentesis or CVS. Invasive prenatal testing was conducted for the following indications: abnormal ultrasound, positive serum screening result, positive NIPT outcome, AMA (age ³ 35 years), and obstetric/family history (record of fetus or child with aneuploidy, or parental carriers of chromosomal balance translocations or inversions). Abnormal ultrasound results included increased NT, heart abnormality, choroid plexus cyst, neck lymphatic hydrocele, bone abnormality, brain abnormality, increased NF, kidney abnormality, and multiple abnormality. Positive serum screening was defined as risk value ≥ 1/270 for DS or ≥1/350 for ES. Positive NIPT outcome was defined as absolute value of z-score ≥ 3.
Chorionic villus samples or amniotic fluid were collected from pregnant women by professional gynecologist. Fetus cells derived from chorionic villus samples or amniotic fluid were cultured in Amniotic Cell Medium (Dahui Bio, Guangzhou, China) for 8 to 13 days at 37 ℃ with 5% CO2. Three hours before the endpoint of culture, cells were treated with colcemid solution. Karyotype analysis was performed following GTG banding (13). A Zeiss Axio Imager Z2 system (Zeiss, Wetzlar, Germany) was used to capture microscopic images of metaphase cells for karyotype analysis. For each sample, at least 20 GTG-banded metaphases were counted and at least five metaphases were analyzed. Karyotypes were classified according to the International System for Human Cytogenetic Nomenclature 2013 (13).
Prenatal serological analysis
At 11 to 13+6 weeks of gestation, risk calculation for first-trimester combined screening (FTS) was performed using maternal age, fetal NT thickness, and maternal serum levels of free β-hCG and PAPP-A. At 15 to 20+6 weeks of gestation, risk calculation for second-trimester triple screening was performed using maternal age and maternal serum levels of AFP, free β-hCG, and uE3. Gestational week was determined by crown-rump length or biparietal diameter. Ultrasound testing and blood collection of pregnant women for FTS were performed on the same day. The levels of FTS serum markers were determined by Cobas e601 analyzer (Roche, Basel, Switzerland). The multiples of the median were derived from marker levels and NT thickness and used to calculate the risk of chromosomal abnormalities according to gestational age. Maternal weight, maternal age, and history of smoking were also considered in calculating Down syndrome risk on pregnancy. A risk cut-off value ≥ 1/270 was recognized as positive for trisomy 21 (Down’s syndrome, DS) and a risk rate ≥1/350 was considered positive for trisomy 18 (Edwards syndrome, ES).
5-10 ml of maternal peripheral blood was collected and placed in EDTA-containing tubes (BD Biosciences, Franklin Lakes, NJ, USA). The blood sample was centrifuged at 1600 × g for 10 minutes at 4 ℃ to separated plasma from the peripheral blood cells. Followed by carefully transferred the plasma into a polypropylene tube and centrifuged at 16,000 × g for 10 minutes at 4 ℃ to deposit the remaining cells. Briefly, cell-free DNA was extracted from 600 μL plasma using a nucleic acid extraction kit (CapitalBio Genomics, Beijing, China) according to the manufacturer’s protocol. DNA was used for library construction and semiconductor sequencing, using a fetal aneuploidies (trisomies 21, 18, and 13) detection kit following the manufacturer’s instructions (CapitalBio Genomics). An absolute value of z-score ≥3 in target chromosome were considered positive (14).
Data were analyzed using chi-squared test in SPSS 20.0 software (IBM Corp., Armonk, NY, USA). P < 0.05 was considered statistically significant.