Objective:To analyze the results of prenatal diagnosis and outcome of pregnancy for women with a high risk for fetal aneuploidies.Methods:A total of 747 cases of prenatal diagnosis by amniocentesis due to high risks by non-invasive prenatal testing (NIPT) were selected from January 2015 to March 2022 in the Drum Tower Hospital Affiliated to Nanjing University Medical School. The amniotic fluid samples were subjected to chromosomal karyotyping and/or chromosomal microarray analysis. All cases were followed up by searching the birth information or telephone calls, and the results were recorded. 2 test or F test were used for comparing the difference between the groups.Results:Among the 747 pregnant women with a high risk by NIPT, 387 were true positives, and the overall positive predictive value (PPV) was 51.81%. The PPVs for trisomy 21 (T21), trisomy 18 (T18), trisomy 13 (T13) and sex chromosome aneuploidies (SCA) were 80.24% (199/248), 60% (48/80), 14% (7/50) and 38.97% (106/272), respectively. The PPV for T21 was significantly higher than T18 and T13 ( χ2= 85.216, P<0.0001). The PPV for other chromosomal aneuploidies and copy number variations (CNVs) were 11.11% (5/45) and 40.74% (22/52), respectively. The PPV for increased X chromosomes was significantly higher than X chromosome decreases (64.29% vs. 22.22%, χ2= 5.530, P<0.05). The overall PPV for elder women (≥ 35 years old) was significantly higher than younger women (69.35% vs. 42.39%, χ2= 49.440, P<0.0001). For T21 and T18, the PPV of Z ≥ 10 group was significantly higher than that for 3 ≤ Z < 5 group or 5 ≤ Z < 10 group ( P<0.05). Among 52 cases with a high risk for CNVs, the PPV for the ≤ 5 Mb group was significantly higher than the 5 Mb < CNVs < 10 Mb or > 10 Mb groups (60% vs. 30%和60% vs. 23.53%, P<0.05). Among the 387 true positive cases, 322 had opted for induced labor, 53 had delivered with no abnormal growth and development, and 12 were lost during the follow-up. Conclusion:The PPVs for common chromosomal aneuploidies are related to the age and Z value of the pregnant women, which were higher in the elder group and higher Z value group. In addition, the PPV is associated with high risk types. The PPV for T21 was higher than T18 and T13, and that for 45, X was lower than 47, XXX, 47, XYY or 47, XXY syndrome. NIPT therefore has relatively high PPVs for the identification of chromosomal CNVs.

The relevant data reported by laboratories carrying out serological prenatal screening in Jiangsu Province from 2021 to 2022 were retrospectively analyzed, and the provincial inter-ventricular quality assessment was realized by comparing the internal quality control data of laboratories. The quality control analysis platform of prenatal screening in Jiangsu Province was used to collect the determination methods, quality control data results of three concentrations, instrument platform, reagents and other relevant information of maternal serological screening quality control in early and middle pregnancy in each laboratory from 2021 to 2022. Firstly, the test results of each laboratory were statistically analyzed, and the precision of each laboratory was evaluated by the variation coefficient of indoor quality control. Secondly, the consistency was evaluated by comparing laboratories with different experimental platforms. The results showed that the passing rate of PAPP-A(pregnancy-associated plasma protein-A) precision test in 2021 and 2022 was 87.5% (7/8) and 92.3% (12/13), respectively. The accuracy pass rate of the Free β-hCG (free beta-human chorionic gonadotrophin)(early pregnancy) test in 2021 and 2022 was 97.5% (39/40) and 95.5% (42/44), and the accuracy pass rate of the AFP(alpha-fetal protein) test in 2021 and 2022 was 100% (40/40) and 90.9% (40/44), respectively. The accuracy pass rate of Free β-hCG (middle pregnancy) test in 2021 and 2022 was 100% (40/40) and 95.5% (42/44), respectively. The consistency of each index was 100%. In conclusion, the relevant data reported by laboratories carrying out serological prenatal screening in Jiangsu Province has a high consistency, and for a small number of laboratories with poor stability, it is necessary to make periodic evaluation and rectification according to the data analysis results, so as to improve the quality control management of maternal serological prenatal screening.

The relevant data reported by laboratories carrying out serological prenatal screening in Jiangsu Province from 2021 to 2022 were retrospectively analyzed, and the provincial inter-ventricular quality assessment was realized by comparing the internal quality control data of laboratories. The quality control analysis platform of prenatal screening in Jiangsu Province was used to collect the determination methods, quality control data results of three concentrations, instrument platform, reagents and other relevant information of maternal serological screening quality control in early and middle pregnancy in each laboratory from 2021 to 2022. Firstly, the test results of each laboratory were statistically analyzed, and the precision of each laboratory was evaluated by the variation coefficient of indoor quality control. Secondly, the consistency was evaluated by comparing laboratories with different experimental platforms. The results showed that the passing rate of PAPP-A(pregnancy-associated plasma protein-A) precision test in 2021 and 2022 was 87.5% (7/8) and 92.3% (12/13), respectively. The accuracy pass rate of the Free β-hCG (free beta-human chorionic gonadotrophin)(early pregnancy) test in 2021 and 2022 was 97.5% (39/40) and 95.5% (42/44), and the accuracy pass rate of the AFP(alpha-fetal protein) test in 2021 and 2022 was 100% (40/40) and 90.9% (40/44), respectively. The accuracy pass rate of Free β-hCG (middle pregnancy) test in 2021 and 2022 was 100% (40/40) and 95.5% (42/44), respectively. The consistency of each index was 100%. In conclusion, the relevant data reported by laboratories carrying out serological prenatal screening in Jiangsu Province has a high consistency, and for a small number of laboratories with poor stability, it is necessary to make periodic evaluation and rectification according to the data analysis results, so as to improve the quality control management of maternal serological prenatal screening.

Objective:To investigate the influence of maternal autoimmune diseases and anticoagulants, including low-molecular-weight heparin (LMWH) and aspirin, on the fetal fraction of maternal plasma cell-free DNA of non-invasive prenatal testing (NIPT).Methods:A prospective cohort study was conducted on women with singleton pregnancies receiving NIPT in the Nanjing Drum Tower Hospital from March 2021 to July 2022. NIPT was carried out using a polymerase chain reaction (PCR)-free amplification platform. In this study, four types of maternal autoimmune diseases, which were antiphospholipid syndrome, undifferentiated connective tissue disease, Sj?gren's syndrome, and systemic lupus erythematosus (SLE), and two anticoagulants, LMWH and aspirin, were studied. Univariate and multivariate linear regression models were used to analyze the factors influencing fetal fraction of maternal plasma cell-free DNA.Results:A total of 4 102 singleton pregnant women were enrolled in the prospective cohort, and 3 948 were finally included after excluding the cases with unclear dosing time of LMWH or aspirin, other autoimmune diseases, conceiving through ovulation induction alone, and having true positive or failed NIPT result. There were 96 cases with antiphospholipid syndrome, 35 with undifferentiated connective tissue disease, 34 with Sj?gren's syndrome, and 18 with SLE. A total of 108 patients only received LMWH treatment, 121 only received aspirin treatment, and 113 received both LMWH and aspirin treatment. Univariate linear regression analysis showed that maternal body mass index at blood collection ( B=－0.423), conceived by assisted reproductive technology ( B=－0.803), male fetus ( B=－0.458), undifferentiated connective tissue disease ( B=1.774), and SLE ( B=3.467) had influence on the fetal fraction (all P<0.05). Multivariate linear regression analysis showed that maternal body mass index at blood collection ( B=－0.415), conceived by assisted reproductive technology ( B=－0.585), male fetus ( B=－0.322), SLE ( B=3.347) and undifferentiated connective tissue disease ( B=1.336) were factors influencing fetal fraction (all P<0.05). Conclusions:Maternal use of LMWH or aspirin does not affect fetal fraction when performing NIPT on a PCR-free amplification platform, but undifferentiated connective tissue disease and SLE are the influencing factors. Therefore, pregnant women should be informed before the NIPT that the fetal fraction of maternal plasma cell-free DNA may be affected by maternal autoimmune diseases.

OBJECTIVE: To explore the genetic etiology and related factors in 1 065 women with spontaneous abortions. METHODS: All patients have presented at the Center of Prenatal Diagnosis of Nanjing Drum Tower Hospital from January 2018 to December 2021. Chorionic villi and fetal skin samples were collected, and the genomic DNA was assayed by chromosomal microarray analysis (CMA). For 10 couples with recurrent spontaneous abortions but normal CMA results for abortive tissues, non-in vitro fertilization-embryo transfer (IVF-ET) pregnancies and no previous history of live births and no structural abnormalities of the uterus, peripheral venous blood samples were collected. Genomic DNA was subjected to trio-whole exome sequencing (trio-WES). Candidate variants were verified by Sanger sequencing and bioinformatics analysis. Multifactorial unconditional logistic regression analysis was carried out to analyze the factors that may affect chromosomal abnormality in spontaneous abortions, such as the age of the couple, number of previous spontaneous abortions, IVF-ET pregnancy and history of live birth. The incidence of chromosomal aneuploidies in spontaneous abortions during the first trimester was compared in young or advanced-aged patients by chi-square test for liner trend. RESULTS: Among the 1 065 spontaneous abortion patients, 570 cases (53.5%) of chromosomal abnormalities were detected in spontaneous abortion tissues, which included 489 cases (45.9%) of chromosomal aneuploidies and 36 cases (3.4%) of pathogenic/likely pathogenic copy number variations (CNVs). Trio-WES results have revealed one homozygote variant and one compound heterozygote variants in two pedigrees, both of which were inherited from the parents. One likely pathogenic variant was detected in the patient from two pedigrees. Multifactorial unconditional Logistic regression analysis suggested that age of patient was an independent risk factor of chromosome abnormalities (OR = 1.122, 95%CI: 1.069-1.177, P < 0.001), the number of previous abortions and IVF-ET pregnancy were independent protective factors for chromosomal abnormalities (OR = 0.791, 0.648; 95%CI: 0.682-0.916, 0.500-0.840; P = 0.002, 0.001), whilst the age of husband and history of live birth were not (P > 0.05). The incidence of aneuploidies in the abortive tissues has decreased with the number of previous spontaneous abortions in young patients (χ² = 18.051, P < 0.001), but was not significantly correlated with the number of previous spontaneous abortions in advanced-aged patients with spontaneous abortions (P > 0.05). CONCLUSION Chromosomal aneuploidy is the main genetic factor for spontaneous abortion, though CNVs and genetic variants may also underlie its genetic etiology. The age of patients, number of previous abortions and IVF-ET pregnancy are closely associated with chromosome abnormalities in abortive tissues.
Pregnancy ; Humans ; Female ; Aged ; Abortion, Spontaneous/genetics* ; DNA Copy Number Variations ; Chromosome Aberrations ; Chromosome Disorders/genetics* ; Aneuploidy ; Abortion, Habitual/genetics*

Objective:To evaluate the value of 99Tc m-methoxyisobutylisonitrile(MIBI) SPECT/CT imaging for the identification of dystonic muscles in patients with primary cervical dystonia (PCD). Methods:A total of 10 patients with PCD (3 males, 7 females, age (47.3±9.9) years) and 10 healthy subjects (4 males, 6 females, age (43.5±9.4) years; control group) between August 2019 and October 2021 in China-Japan Friendship Hospital were enrolled prospectively. All subjects underwent 99Tc m-MIBI SPECT/CT scan. The SUV max of 8 bilateral representative muscles, including rectus capitis posterior major, obliquus capitis inferior, splenius capitis, semispinalis, sternocleidomastoid, trapezius, musculus scalenus muscle and levator scapulae were evaluated in control group. In PCD group, muscles with abnormal uptake were determined. ROI was drawn and SUV max was measured. Independent-sample t test was used to analyze the differences of SUV max between normal and abnormal muscles. The detecting rates of neck MRI and SPECT/CT for abnormal muscles were analyzed by χ2 test. Results:Normal muscles of healthy subjects showed mild symmetrical radioactivity distribution, with the SUV max of 1.10±0.19. A total of 60 muscles with abnormal uptake in 10 patients were found, including 7 rectus capitis posterior major, 10 obliquus capitis inferior, 8 splenius capitis, 8 semispinalis, 10 sternocleidomastoid, 5 trapezius, 3 musculus scalenus muscle and 9 levator scapulae. The SUV max of muscles with abnormal uptake was 1.81±0.43, which was higher than that of normal muscles ( t=17.05, P<0.001). Only 30 pieces abnormal hypertrophy muscle were found by neck MRI, and the detecting rate was much lower than that of SPECT/CT (18.75%(30/160) vs 37.50%(60/160); χ2=28.03, P<0.001). Conclusion:99Tc m-MIBI SPECT/CT may be a useful method for identifying dystonic muscles and a guide to precision therapy in patients with PCD.

Objective:To investigate the RHD genotypes of RhD-negative pregnant women and explore the optimum strategy for fetal RHD screening among this population in the region. Methods:This prospective study recruited 33 cases of RhD-negative singleton pregnancies at ≥12 weeks of gestation in Nanjing Drum Tower Hospital from March to November 2021. On the basis of RHD genotyping, quantitative real-time polymerase chain reaction (PCR) was used to amplify the exons 5 and 10 of RHD gene in the circulating cell-free DNA of RhD-negative pregnant women harboring whole RHD gene deletion and RHD-CE(2-9)- D. High-throughput sequencing was performed to detect chr1:25648453 locus from circulating cell-free DNA in plasma of RhD-negative pregnant women harboring RHD 1227A mutation to screen the fetal RhD blood group. Neonatal umbilical cord blood samples were collected for verifying fetal RHD genotyping. Descriptive statistical analysis was used. Results:Whole RHD gene deletion homozygous genotype ( n=20, 60.6%), RHD-CE(2-9) -D/whole RHD gene deletion heterozygous genotype ( n=5, 21.2%), RHD 1227A/whole RHD gene deletion heterozygous genotype ( n=7, 15.2%) and RHD 711delC/whole RHD gene deletion heterozygous genotype ( n=1) were identified in the 33 RhD-negative pregnant women. In the 25 cases with whole RHD gene deletion homozygous genotype or RHD-CE(2-9)- D/whole RHD gene deletion heterozygous genotype, 22 fetuses were RhD-positive and three were RhD-negative based on prenatal screening, which were confirmed by the neonatal serological test results after birth. In the seven cases carrying RHD 1227A/whole RHD gene deletion heterozygous genotype, all fetuses were RhD-positive, which were consistent with the results of serological detection after delivery. The case harboring RHD 711delC/whole RHD gene deletion heterozygous genotype did not receive fetal RHD screening. Conclusions:This study suggests that whole RHD gene deletion homozygous genotype is the most common allele in RhD-negative population in this area, followed by RHD 1227A/whole RHD gene deletion heterozygous genotype and RHD- CE(2-9)- D/whole RHD gene deletion heterozygous genotype. For women with whole RHD gene deletion homozygous genotype, RHD- CE(2-9)- D, or RHD 1227A mutation, fetal RHD screening with quantitative real-time PCR and high-throughput sequencing are important for the management of RhD-negative pregnant women.

OBJECTIVE To study the components of ethyl acetate fraction in Qubai tablet ，and its pharmacodynamics on de melanocyte model ，and explore the material basis for anti-vitiligo effect of Qubai tablet. METHODS The ethyl acetate fraction of Qubai tablets was obtained by extraction ，and its components were analyzed by ultra performance liquid chromatography-mass spectrometry（UPLC-MS）. Model control group ，vehicle control group and 8-methoxypsoralen（8-MOP）administration groups （10，50，100，150，200 μmol/L），ethyl acetate fraction administration groups of Qubai tablet （10，50，100，150，200 μg/mL） were set up in the experiment. By establishing the de melanocyte model ，the effects of ethyl acetate fraction of Qubai tablet on de melanocyte were studied from four aspects ：cell number ，cell viability ，melanin formation and tyrosinase activity. RESULTS UPLC-MS component analysis preliminarily determined the structure of 64 compounds in the ethyl acetate fraction of Qubai tablet ， of which 14 compounds were detected in positive and negative ion mode ；psoralen compounds accounted for the largest proportion ， and the content of psoralen chromone chalcone was the highest in positive and negative ion mode. The results of pharmacodynamic study showed that the ethyl acetate fraction of Qubai tablet could increase the number of de melanocytes ，and significantly improve the cell proliferation rate ，the rate of promoting melanin formation and the rate of promoting tyrosinase activity in the process of melanin formation （P＜0.01）. CONCLUSIONS Psoralen compounds may be the material basis for the anti-vitiligo effect of ethyl acetate fraction of Qubai tablet ；good anti-vitiligo effect of ethyl acetate fraction of Qubai tablet may be related to the promotion of tyrosinase activity.

OBJECTIVE To stud y the chemical cons tituents of n-butanol part of Qubai tablet and its pharmacodynamic effect on the model of de melanocyte. METHODS The n-butanol part of Qubai tablet was prepared. The chemical constituents were analyzed by ultra-high performance liquid chromatography-mass spectrometry （UPLC-MS）. Taking mice B 16 melanoma cells as the research object ，the de melanocyte model was established and divided into model group ，positive control different concentration groups（8-methoxypsoralen 10，50，100，150，200 μmol/L），solvent group （diluted with DMSO ）and Qubai tablet n-butanol part different concentration groups （10，50，100，150，200 μmol/L）. The number of cells were observed by inverted microscope ，and the cell proliferation rate ，the rate of melanin production and promotion rate of tyrosinase activity were also detected. RESULTS In the positive and negative ion mode ，53 compounds in the n-butanol part of Qubai tablet were preliminarily determined （29 in the positive ion mode ，33 in the negative ion mode ，overlapping 9），of which coumarins accounted for the largest proportion ， followed by flavonoids. The n-butanol part of Qubai tablet could significantly increase the number of cells ，which was positively correlated with the action time and administration concentration. It could significantly increase the proliferation rate of cells ，the rate of melanin production and promotion rate of tyrosinase activity （P＜0.01）. CONCLUSIONS Coumarins and flavonoids may be the material basis for the anti-vitiligo effect of n-butanol part from Qubai tablet ；anti-vitiligo effect of n-butanol part of Qubai tablet may be realized by promoting tyrosinase activity.

OBJECTIVE: To assess the application value of noninvasive prenatal testing (NIPT) based on cell-free fetal DNA. METHODS: The results of 2777 cases of basic and extended NIPT were retrospectively analyzed. The clinical data and outcome of pregnancy were analyzed, in addition with the diagnosis rate and testing efficiency. RESULTS: Among the 2777 pregnant women, 1192 (42.9%) had accepted basic NIPT and 1585 (57.1%) accepted extended NIPT. With a failure rate of 0.1%, 8 and 6 cases were reported respectively as high-risk pregnancies for trisomy 21 and sex chromosomal abnormalities. Other genetic abnormalities were detected in 32 cases. The positive predictive value for trisomy 21 was 85.7%, and one case of 47,XXX was diagnosed among 3 women with high risks for sex chromosomal abnormalities. For those with a high risk for other genetic abnormalities, pregnant diagnosis rates of basic and extended NIPT were 71.4% (5/7) and 68.2% (15/22), respectively. Seven copy number variations (CNVs) were confirmed, including one pathogenic CNV, one likely pathogenic CNV and 5 variants of unknown significance. Among 6 cases with high-risk of maternal CNVs, 5 fetuses and the mothers were confirmed to be carriers. No CNV was detected in the remainder fetus by chromosomal microarray analysis, while its mother was a carrier of the corresponding CNV. CONCLUSION NIPT has shown a relatively high positive predictive value for the screening of trisomy 21 and maternal CNVs but with a limited efficiency for the discovery of fetal CNVs. For other genetic abnormalities signaled by NIPT, informed choice by the pregnant women during pre-testing consultation is recommended. Invasive prenatal diagnosis should be considered in the combination of NIPT reports and fetal ultrasound, while the residual risks should be fully informed.
Aneuploidy ; Cell-Free Nucleic Acids/genetics* ; DNA/genetics* ; DNA Copy Number Variations ; Female ; Fetus ; Humans ; Noninvasive Prenatal Testing ; Pregnancy ; Retrospective Studies