Noninvasive Evaluation of Fetal Zygosity in Twin Pregnancies Involving a Binary Analysis of Single-Nucleotide Polymorphisms

Twin pregnancy constitutes significant risks for maternal and fetal health, which is usually detected by ultrasound examination at early gestation. However, the imaging-based approach may not accurately identify all twins confounded practical or clinical variables. The analysis of cell-free DNA in noninvasive prenatal screening assays can completement method twin detection, differentiates fraternal identical based on their distinct genotypes. Here, a new employing high-coverage next-generation sequencing targeted nucleotide polymorphisms was developed detection zygosity determination fraction pregnancies. This utilizes binary both number allelic fetus-specific single-nucleotide to infer zygosity. In 323 samples collected from 215 singleton, 90 dizygotic, 18 monozygotic pregnancies, dizygotic were correctly detected, with 100% sensitivity specificity. addition, this detect complex such as egg donors, contamination, complete hydatidiform mole. change monitored nine demonstrated highly variable dynamics turnover up 7 weeks after reduction. Overall, study provides strategy accurate identification quantification fraction, has important implications management Approximately 3% pregnant women have multiple pregnancy, causes including gestational diabetes, intrauterine growth restriction, preeclampsia, premature delivery.1Collins J. Global epidemiology birth.Reprod Biomed Online. 2007; 15 Suppl 3: 45-52Abstract Full Text PDF Google Scholar,2Norwitz E.R. Edusa V. Park J.S. Maternal physiology complications pregnancy.Semin Perinatol. 2005; 29: 338-348Crossref PubMed Scopus (124) Scholar overall rate been increasing use ovulation drugs assisted reproductive technology.1Collins Scholar,3Thilaganathan B. Khalil A. Multiple pregnancy: preface.Best Pract Res Clin Obstet Gynaecol. 2014; 28: 189-190Crossref Scholar, 4Sebghati M. Reduction counselling techniques.Best 2021; 70: 112-122Crossref (15) 5Committee S.R. Grantz K.L. Kawakita T. Lu Y.L. Newman R. Berghella Caughey SMFM special statement: state science multifetal gestations: unique considerations importance.Am J Gynecol. 2019; 221: B2-B12Abstract (42) accounts 97% 98% associated either (MZ) (DZ) determined zygosities.2Norwitz Scholar,6Fellman Aspects history research: statistical congresses 19th century Hellin's law.Twin Hum Genet. 2018; 21: 57-66Crossref (3) 7Bai Z. Zhao H. Lin S. Huang L. He Wang Ou X. Evaluation microhaplotype-based test paternity, zygosity, fraction.Genes (Basel). 2020; 12: 26Crossref (9) 8Benn P. Rebarber Non-invasive testing pregnancies.Prenat Diagn. 41: 1233-1240Crossref (14) MZ result division single zygote into two embryos essentially embryonic structures, whereas DZ are derived fertilization pairs ova sperm.7Bai pregnancies detectable gestation, followed care.8Benn Scholar,9Sfakianaki A.K. Han C.S. Ultrasound evaluation pregnancy.Minerva Ginecol. 2009; 61: 127-139Google chorionicity most risk factor adverse outcome pregnancies.10ACOG: Practice bulletin No169 summary: twin, triplet, higher-order pregnancies.Obstet 2016; 128: 926-928Google Monochorionic an approximately four times higher perinatal morbidity mortality than dichorionic pregnancies.11Van Mieghem Abbasi N. Shinar Keunen Seaward G. Windrim Ryan monoamniotic pregnancies.Am 2022; 4100520Google 12Oger A.S. Robillard P.Y. Barau Randrianaivo Bonsante F. Iacobelli Boukerrou Perinatal monochorionic 775 Reunion Island.J Gynecol Biol Reprod. 2013; 42: 655-661Crossref (2) 13Al Riyami Al-Rusheidi Al-Khabori comparison pregnancies.Oman Med 173-177Crossref (12) Because dichorionic, establishing prognostic value proper care.14Bajoria Kingdom case routine pregnancy.Prenat 1997; 17: 1207-1225Crossref (129) Chorionicity be identified first trimester, but its accuracy affected age time assessment, clinician's experience, sonographic variables used examination.15Blumenfeld Y.J. Momirova Rouse D.J. Caritis S.N. Sciscione Peaceman A.M. Reddy U.M. Varner M.W. Malone F.D. Iams J.D. Mercer B.M. Thorp Jr., J.M. Sorokin Y. Carpenter Lo Ramin S.M. Harper Eunice Kennedy Shriver National Institute Child Health Human Development Maternal-Fetal Medicine Units NetworkAccuracy classification gestations.J Med. 33: 2187-2192Crossref (30) 16Lee Y.M. Cleary-Goldman Thaker H.M. Simpson L.L. Antenatal prediction chorionicity.Am 2006; 195: 863-867Abstract (78) 17Baud D. Van Twin-twin transfusion syndrome: frequently missed diagnosis consequences.Ultrasound 44: 205-209Crossref (18) 18Shetty Smith A.P. chorionicity.Prenat 25: 735-739Crossref (84) Another limitation reduced vanishing syndrome one stops development utero, leading spontaneous reduction singleton pregnancy.19Gjerris A.C. Loft Pinborg Christiansen Tabor effect “vanishing twin” biochemical trimester markers Down's conceived technology.Hum 24: 55-62Crossref (0) 20Sankaran Rozette C. Dean Kyle Spencer K. Screening presence vanished twin: nuchal translucency combined test?.Prenat 2011; 31: 600-601Crossref (8) 21Landy H.J. Keith twin.Acta Genet Gemellol. 1982; 179-194Crossref Vanishing occurs 36% sacs, increased preterm birth, low birth weight, mortality.22Zhou Gao Wu Zhang Analysis outcomes survivors vitro embryo transfer.Eur Reprod Biol. 203: 35-39Abstract (24) 23Chasen S.T. Luo Perni S.C. Kalish R.B. Are high risk?.Am 814-817Abstract 24Li Y.X. Sun T.Z. Lv M.Q. Zhou Ge Li H.N. D.X. Is obstetric ART singletons? systematic review meta-analysis.J Assist 37: 2783-2796Crossref (5) 25Zhu Chen Liu late co-twin contributes surviving singleton.Hum 35: 1553-1561Crossref (6) 26Dickey R.P. Taylor Sartor Storment Rye P.H. Pelletier W.D. Zender J.L. Matulich E.M. Spontaneous incidence outcome.Am 2002; 186: 77-83Abstract (182) With advances technologies, (NIPS) involving sequence (cfDNA) proved effective fetuses aneuploidies.27Gil M.M. Accurti Santacruz Plana M.N. Nicolaides K.H. blood aneuploidies: updated meta-analysis.Ultrasound 2017; 50: 302-314Crossref (440) (FF) reduce NIPS accuracy.28Futch Spinosa Bhatt de Feo E. Rava Sehnert A.J. Initial laboratory experience aneuploidy plasma samples.Prenat 569-574Crossref (141) FF inferred examining (SNPs) through low-depth whole-genome sequencing, also useful female-male twins.29Dang Xu W. Bai Fang Liang Q. Guan Inferring fractions read heterozygosity empowers screening.Genet 22: 301-308Abstract using determine >12,000 SNPs.8Benn Scholar,30Pergament Cuckle Zimmermann Banjevic Sigurjonsson Hall M.P. Dodd Lacroute Stosic Chopra Hunkapiller Prosen D.E. McAdoo Demko Siddiqui Hill Rabinowitz Single-nucleotide polymorphism-based high-risk low-risk cohort.Obstet 124: 210-218Crossref (224) 31Hedriana Martin Saltzman Billings Benn Cell-free gestations screening.Prenat 40: 179-184Crossref (16) 32Norwitz McNeill Kalyan Rivers Ahmed Meng Vu Egbert Shapira Kobara Parmar Goel Prins S.A. Aruh I. Persico Robins J.C. Kirshon Z.P. P.R. K.A. Hedriana H.L. Validation non-invasive individual sex, aneuploidy.J 8: 937Crossref fetus aneuploidies lower produce false results when it fails recognize pregnancy.33Chaveeva Wright Syngelaki Konstantinidou First-trimester trisomies twin.Ultrasound 55: 326-331Crossref 34Curnow K.J. Wilkins-Haug Kirkizlar Gross S.J. Detection triploid, molar, test.Am 2015; 212: 79.e1-79.e9Abstract 35Niles K.M. Murji Chitayat Prolonged duration persistent 52: 547-548Crossref Similarly, demise interfere assay because cfDNA nondeveloping remains circulation portion total cfDNA.36Chen Su Chai Yuan Cen Temporal persistence residual deceased cotwin selective diamniotic 1602-1610Crossref (1) Scholar,37Soster Boomer Hicks Caldwell Dyr Chibuk Almasri Three years genome-wide copy-number variants.Genet 23: 1349-1355Abstract (19) current understanding limited, prevents improvement pregnancies.34Curnow 36Chen Scholar,38Bevilacqua Doshi White Marchin Conotte Jani Schmid pregnancy.Ultrasound 132-133Crossref study, SNP-based calculation pregnancy. Comprehensive analyses SNPs performed monitored, provided guidance how should these A 499 peripheral 407 ≥12 included study. Singleton confirmed ultrasound. protocol approved institutional board China International Peace Maternity Hospital (GKLW2019-72 GKLW2019-52). Informed consent obtained participants. For disease-related chromosome copy variations, >2600 probes designed loci frequent chromosomal abnormalities. >200 target without common (≥1% population frequency) variation reported Database Genomic Variants (http://dgv.tcag.ca/dgv/app/home, last accessed August 5, 2019). above had minor allele frequency 30% 70% East Asian, African, non-Finnish European individuals ensure sufficient polymorphism across different populations. To probe hybridization bias between reference alternative alleles, possible nucleotides (A, C, G, T) selected locus corresponding SNP minimum melting temperature difference pairing allele.39Xu Cai Jing Qin Pan Ma Yang Tang Song Qiu Ying H.-F. Genetic deconvolution enables screening.Cell Discov. 109Crossref More 99.0% required ≥200× depth meet coverage quality control requirement assay. perform assay, 5 10 mL each participant. undergoing procedure, points before centrifuged 4°C twice, then extracted Magnetic Serum/Plasma Circulating Maxi Kit (catalog YDP710; TIANGEN, Beijing, China). construct library, end repaired ligated adapters following manufacturer’s 1002212; Nanodigmbio, Nanjing, Sample barcode introduced PCR, PCR products quantified Qubit 1X dsDNA HS Assay Kits Q33231; Thermo Fisher Scientific, Invitrogen, Waltham, MA). After enrichment 1072281; Integrated Technologies, Coralville, IA) HS2008; Heristar, Houston, TX), recovered washed purified Dynamag-270 magnetic beads 65306; Invitrogen). generate 12 cycles amplification Nanodigmbio). Next, concentration library ssDNA Q10212; Finally, sequenced MGISEQ-2000 2 × 100 paired-end mode 1000012554; MGI, Shenzhen, Detailed procedures previous study.39Xu There (170 329 validation) sample size chosen allow authors 10% margin error 90% confidence level data sets. estimated SD given 0.25 mean empirical data. At any biallelic locus, probability carrying informative allele, where mother homozygous heterozygous, calculated (Supplemental Table S1). Let particular p, queried N, N0, N1, N2. expressed follows. Lowercase letters observed values, capital italicized notations only.N0=p2+1−p2∗N(1) N1=p21−p+p1−p2∗N=p1−p∗N(2) N2=p21−p2+p1−p2+p1−p+1−p2p2+p1−p2+p1−p∗N=p1−pp−122+54∗N(3) N2N1=p−122+54≥54(4) N1N0=p1−pp+1−p2≤12(5) N2N0=p1−pp−122+54p2+1−p2≤58(6) Therefore, ≥25% more that twins. B-allele (BAF) heterozygous expected FF/2 (1 – FF/2), being respectively Figure S1, B, Supplemental let FF1 FF2 1 2, respectively; BAF centered around FF1/2, FF2/2 (FF1 + FF2)/2, FF2/2, FF2)/2 C–F, values divided groups genotype (BB AA) among median M1 M2. Then, calculate those BAFs group, σ1 σ2. Take weighted average σave σ2, group. denoted Mdep. corrected variation, σcor, hypergeometric distribution.σcor=σave∗MdepM1∗M2(7) algorithm established utilizing (Figures 2A). (either variant allele) FF. Confirmed validation imaging consistent sex. imaging, showed completely concordant profile singletons respect pattern, described above.Figure 2The (SNPs). A: donor samples. x axis relative abundance SNPs, ratio variants loci; y axis, SNPs. B: An C: 5% contamination. D: molar fetus. (Chr)–specific marked colors. ChrRef, Chr.View Large Image ViewerDownload Hi-res image Download (PPT) X Y reads respective female male respectively. haploid genome copies 1, R1, R2, M, respectively, present sample.FF1=R1R1+M(8) FF2=R2R2+M(9) autosome i, rdai. j, rdxj rdyk, f, paternally inherited rdxff. Now, depths autosome, X, chromosomes RDa, RDx, RDy, Last, RDxf (the twin). rdai, rdxj, These functions Ka, Kx, Ky.RDa=Ka∗2∗R1+R2+M(10) RDx=Kx∗2∗R1+R22+M(11) RDy=Ky∗R2(12) RDxf=Kx∗R1(13) Ky euploid ra, rx, ry nonpregnant training samples, respectively.KxKa=2∗rxra=C1(14) KyKa=2∗ryra=C2(15) On basis Equations 14 15, Kx follows:Kx=C1∗Ka(16) Ky=C2∗Ka(17) computed follows:FF1=2∗C2∗RDxfC1∗C2∗RDa−2∗C1∗RDy(18) FF2=2∗C1∗RDyC1∗C2∗RDa−2∗C2∗RDxf(19) Unlike discriminative same calculation. least heterozygous. combinations genotypes S2), calculated. twins' theoretically six levels discernible C–F). As BB AA loci, twin-specific depth. clusters symmetrically distributed, >0.5, substituted BAF. Now three, BAF1, BAF2, BAF3 ascending order. twins, loss generality, assuming R1 ≤ below. FF, obtain BAF3, especially close other. end, grouped Bayesian-Gaussian mixture model, Gaussian model.40Blei D.M. Jordan M.I. Variational inference Dirichlet process mixtures.Bayesian Analysis. 1: 121-144Crossref 41Attias Advances Neural Information Processing Systems Bayesian Framework Graphical Models. MIT Press, Cambridge, MA2000: 209-215Google 42Pedregosa Varoquaux Gramfort Michel Thirion Grisel O. Blondel Prettenhofer Weiss Dubourg Vanderplas Passos Cournapeau Brucher Perrot Duchesnay É. Scikit-learn: machine learning python.J Mach Learn Res. 2825-2830Google assigned groups, cluster Clusters less five omitted suppress outliers. When there three left, take medians clustered bafm1, bafm2, bafm3.BA