Identification of Chromosomal Abnormalities in Early Pregnancy Loss Using a High-Throughput Ligation-Dependent Probe Amplification–Based Assay

Embryonic chromosomal abnormalities are the major cause of miscarriage. An accurate, rapid, and cheap method chromosome analysis in miscarriage is warranted clinical practice. Thus, a high-throughput ligation-dependent probe amplification (HLPA)-based detecting aneuploidies copy number variations was developed. A total 1060 cases were assessed. Each specimen subjected to quantitative fluorescence (QF)-PCR/HLPA microarray (CMA) parallel. All samples successfully analyzed using both methods; these samples, 1.7% (18/1060) identified as having significant maternal cell contamination. Among remaining 1042 without contamination, QF-PCR/HLPA reached diagnostic yield 59.6% (621/1042), which comparable 60.3% (628/1042) with CMA. Compared CMA results, sensitivity specificity identification pathogenic 98.9% 100%, respectively. Furthermore, overall prevalence spontaneous abortion not significantly different from that recurrent (61.3% versus 58.5%). 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Borrell Added value systematic review meta-analysis.Ultrasound 51: 453-462Crossref (18) Nonetheless, technologies has some limitations, costs challenges interpretation variants uncertain significance.22Westerfield Darilek van den Veyver I.B. Counseling Challenges significance incidental findings prenatal screening diagnosis.J 3: 1018-1032Crossref (50) High-throughput (HLPA), new modified been demonstrated inexpensive miscarriage.23Yang Tang Lu Yang Xiao Xiang Novel loss.Acta Gynecol Scand. 95: 1433-1440Crossref (4) Scholar,24Chen Fan Qian Huang Zhu variation specimens.Prenat Diagn. 176-183Crossref (8) previous studies,23Yang microdeletions microduplications (<10 Mb), well partial (?10 HLPA. Therefore, aneuploidies, and/or duplications, based an improved HLPA method, developed, its efficacy power evaluated parallel >1000 consecutive prospective, double-blind study conducted Prenatal Center, Nanjing Maternity Child Health Care Hospital (Jiangsu, China), April 2018 January 2020. Women who had before 13 weeks' gestation requested enrolled. Informed consent obtained patients. RM defined two losses. mean age 30.7 years (range, 18 46 years), gestational 9.2 6 weeks). Chorionic villi collected specimens method.25Lathi Milki A.A. Tissue sampling affects karyotype missed abortions.J Assist Reprod 2002; 536-538Crossref (46) Specimens clearly excluded. protocol approved Medicine Ethics Committee Hospital. performed abnormalities. strategies summarized Figure 1. DNA isolated QIAamp MiniKit (Qiagen, Hilden, Germany). Subsequently, each qualified sample divided into equal parts For strategy, investigated QF-PCR triploidy, disomy (UPD), contamination (MCC). Cases MCC subsequently (CNVs). Tetraploidy cannot detected QF-PCR/HLPA. directly (SNP) array, can used polyploidies, CNVs, UPD, MCC. (>30%) excluded study. CytoScan 750K (Affymetrix, Santa Clara, CA), comprising 550,000 oligonucleotide probes 200,000 probes, applied scan. experiments previously reported,4Wang ChAS software version 3.2 (Affymetrix). CNVs effective minimal 100 Kb, regions allelic homozygosity reported threshold 5 Mb. Mosaicism ?5 Mb when it above 30%. levels exceeded CNV classification according American College Medical guideline.26Kearney H.M. K.K. Quintero-Rivera South S.T. Working Group Laboratory Quality Assurance CommitteeAmerican standards guidelines reporting postnatal variants.Genet 680-685Crossref (582) Human Personal Identification Detection Kit (catalog R1006; Genesky, Suzhou, China) reported.24Chen 17 markers MCC, including 16 autosomes (D2S1338, D5S818, D7S820, D8S1179, TH01, Vwa, D13S317, D16S539, D18S51, D8S588, G4S0001, G2S0002, G15S0001, G7S0005, G10S0001, G5S0001) 1 marker sex (Amelo). PCR ABI 3730XL automated sequencer (Applied Biosystems, Foster City, results GeneMapper 5.0 Biosystems). On basis published contained 170 pairs targeting 24 aneuploidies23Yang (Supplemental Table S1), addition 171 targeted terminal achieve higher S2). As result, 341 included assay. distribution shown Supplemental S1. them, 3 (with approximately distance probe), 2 pericentromeric regions, Meanwhile, added target range 0 chromosome, per experiment described.24Chen brief, denatured, hybridized, ligated, amplified Chromosome Copy Number N1002S; Genesky). Similar comprised adapter sequence binding universal primers target-specific complementary regions. introduced lengthening ligation system four types 5? labeled fluorophores 3? throughput.24Chen sequencer, data (both Applied calculated study, values 1.7 2.3 considered normal. three one below 1.5 2.5 representative deletions <3.5 3.5 <4.5 determined copies copies, suggested mosaic deletions, while those indicated duplications. Comparisons groups ?2 test Fisher exact test. P <0.05 statistically significant. tissues Significant (1.7%) (Figure 1). no negligible 628 CMA, providing (Table comparison, 621 (59.6%) Seven (0.7%) QF-PCR/HLPA, (0.3%) 4 (0.4%) 100.0%, respectively 2). remarkably ($114.30 $514.30 case). turnaround time day, shorter (3 days).Table 1Diagnostic Yields Miscarriage QF-PCR/HLPAChromosomal abnormalityCMAQF-PCR/HLPATotalSARMP valueTotalSARMSingle aneuploidy488 (46.8)312 (47.9)176 (45.1)0.394488 (45.1) Autosomal trisomy391 (37.5)248 (38)143 (36.7)0.658391 (36.7) monosomy2 (0.2)2 (0.3)00.5312 (0.3)0 Monosomy X93 (8.9)61 (9.4)32 (8.2)0.52893 (8.2) Trisomy (sex chromosome)2 (0.2)1 (0.3)1.0002 (0.3)Multiple aneuploidy18 (1.7)12 (1.8)6 (1.5)0.71718 (1.5) Double (1.5)Polyploidy80 (7.7)55 (8.4)25 (6.4)0.23580 (6.4) Triploidy66 (6.3)49 (7.5)17 (4.4)0.04366 (4.4) Hypertriploidy9 (0.9)3 (0.5)6 (1.5)0.1409 Hypotriploidy5 (0.5)3 (0.5)2 (0.5)1.0005 (0.5)Partial aneuploidy30 (2.9)17 (2.6)13 (3.3)0.49827 (2.6)16 (2.5)11 (2.8) Terminal deletion duplication11 (1.1)7 (1.1)4 (1.0)1.00011 (1.0) + duplication (suggestive unbalanced translocation)8 (0.8)3 (0.5)5 (1.3)0.2698 (1.3) Interstitial duplication3 (0.3)1 (0.5)0.652000 Multiple duplications8 (0.8)6 (0.9)2 (0.5)0.7178?Two HLPA, case coupled duplication. (0.5)Pathogenic microdeletion duplication7 (0.7)2 (0.3)5 (1.3)0.1413 (0.3)2 (0.3) (0.3)1.0003 duplication4 (0.4)04 (1.0)0.038000UPD5 (0.3)3 (0.8)0.5605 (0.8) Whole-genome UPD5 (0.8)Total628 (60.3)400 (61.3)228 (58.5)0.357621 (59.6)399 (61.2)222 (56.9)Data expressed n (%).CMA, analysis; PCR/high-throughput amplification; RM, miscarriage; SA, abortion; disomy.? Two Open table tab 2Efficiency MiscarriageChromosomal abnormalityCMA reference, nQF-PCR/HLPA, nSensitivity, %Specificity, %Numeric abnormalities586586100100Partial aneuploidy302790.0100Pathogenic duplication7342.9100Whole-genome UPD55100100Total62862198.9100CMA, disomy. Data (%). 100% consistent identifying UPD Numeric observed 586 (56.2%), 506 (48.6%) 80 (7.7%) triploidies. represented 96.4% (488/506) cases, multiple composed 3.6% (18/506). Aneuploidy except 19. trisomies (393/488, 80.5%), others monosomies (95/488, 19.5%). trisomies, trisomy (128/393, 32.6%), followed 22 (67/393, 17.0%) (31/393, 7.9%) With respect monosomies, monosomy X accounted 97.9% (93/95) whereas autosomal 2.1% (2/95) (2 21). Of 16, 5) other (1 22, 7, X) found nonmosaic owing proportion aneuploidies. suggestive molar pregnancy, (0.5%). Partial (CNVs ?10 Mb) (2.9%) S3). These 11 (1.1%) duplication, 8 (0.8%) (unbalanced translocation), interstitial duplications (two involving chromosomes). 27 90.0%. Because false-positive 100%. completely concordant incompletely (consistent but HLPA) diagnosed reason u