Top

Journal of Assisted Reproduction and Genetics

Open Access 20-04-2024 | Preimplantation Genetic Diagnostics | Genetics

Aneuploidy detection in pooled polar bodies using rapid nanopore sequencing

Authors: Silvia Madritsch, Vivienne Arnold, Martha Haider, Julia Bosenge, Mateja Pfeifer, Beatrix Weil, Manuela Zechmeister, Markus Hengstschläger, Jürgen Neesen, Franco Laccone

Published in: Journal of Assisted Reproduction and Genetics

Abstract

Purpose

Various screening techniques have been developed for preimplantation genetic testing for aneuploidy (PGT-A) to reduce implantation failure and miscarriages in women undergoing in vitro fertilisation (IVF) treatment. Among these methods, the Oxford nanopore technology (ONT) has already been tested in several tissues. However, no studies have applied ONT to polar bodies, a cellular material that is less restrictively regulated for PGT-A in some countries.

Methods

We performed rapid short nanopore sequencing on pooled first and second polar bodies of 102 oocytes from women undergoing IVF treatment to screen for aneuploidy. An automated analysis pipeline was developed with the expectation of three chromatids per chromosome. The results were compared to those obtained by array-based comparative genomic hybridisation (aCGH).

Results

ONT and aCGH were consistent for 96% (98/102) of sample ploidy classification. Of those samples, 36 were classified as euploid, while 62 were classified as aneuploid. The four discordant samples were assessed as euploid using aCGH but classified as aneuploid using ONT. The concordance of the ploidy classification (euploid, gain, or loss) per chromosome was 92.5% (2169 of 2346 of analysed chromosomes) using aCGH and ONT and increased to 97.7% (2113/2162) without the eight samples assessed as highly complex aneuploid using ONT.

Conclusion

The automated detection of the ploidy classification per chromosome and shorter duplications or deletions depending on the sequencing depth demonstrates an advantage of the ONT method over standard, commercial aCGH methods, which do not consider the presence of three chromatids in pooled polar bodies.

Available only for authorised users

Morales C. Current applications and controversies in preimplantation genetic testing for aneuploidies (PGT-A) in in vitro fertilization. Reprod Sci. 2024;31:66–80. https://doi.org/10.1007/s43032-023-01301-0.CrossRefPubMed

Rubio C, Bellver J, Rodrigo L, Castillón G, Guillén A, Vidal C, et al. In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study. Fertil Steril. 2017;107:1122–9. https://doi.org/10.1016/j.fertnstert.2017.03.011.CrossRefPubMed

Handyside AH, McCollin A, Summers MC, Ottolini CS. Copy number analysis of meiotic and postzygotic mitotic aneuploidies in trophectoderm cells biopsied at the blastocyst stage and arrested embryos. Prenat Diagn. 2021;41:525–35. https://doi.org/10.1002/pd.5816.CrossRefPubMed

Sarosiak A, Minota I, Koziol K, Oldak M. 7. Genetic testing for copy number variations in first polar bodies and oocytes by single-cell next-generation sequencing. Reprod Biomed Online. 2019;39:e30–1. https://doi.org/10.1016/j.rbmo.2019.04.060.

Sadecki E, Rust L, Walker DL, Fredrickson JR, Krenik A, Kim T, et al. Comparison of live birth rates after IVF-embryo transfer with and without preimplantation genetic testing for aneuploidies. Reprod Biomed Online. 2021;43:995–1001. https://doi.org/10.1016/j.rbmo.2021.09.011.CrossRefPubMed

Malhotra N, Ahuja M, Loomba P. ISARCON 2017 manual of preimplantation genetic diagnosis & screening, vitrification gametes, embryo and tissues. jaypee; 2018;1/e.

RIS. RIS - Fortpflanzungsmedizingesetz - Bundesrecht konsolidiert, Fassung vom 25.07.2023 2023. https://www.ris.bka.gv.at/GeltendeFassung.wxe?Abfrage=Bundesnormen&Gesetzesnummer=10003046 (accessed July 25, 2023).

ESHRE PGT Consortium and SIG-Embryology Biopsy Working Group, Kokkali G, Coticchio G, Bronet F, Celebi C, Cimadomo D, et al. ESHRE PGT consortium and SIG embryology good practice recommendations for polar body and embryo biopsy for PGT. Hum Reprod Open. 2020;2020:hoaa020. https://doi.org/10.1093/hropen/hoaa020.

Greco E, Litwicka K, Minasi MG, Cursio E, Greco PF, Barillari P. Preimplantation genetic testing: where we are today. Int J Mol Sci. 2020;21:4381. https://doi.org/10.3390/ijms21124381.CrossRefPubMedPubMedCentral

10.

Handyside AH. Polar body analysis for preimplantation genetic testing. Reprod Biomed Online. 2019;39:e9. https://doi.org/10.1016/j.rbmo.2019.04.028.CrossRef

11.

van der Ven K, Montag M, van der Ven H. Polar body diagnosis – a step in the right direction? Dtsch Ärztebl Int. 2008;105:190–6. https://doi.org/10.3238/arztebl.2008.0190.CrossRefPubMedPubMedCentral

12.

Geraedts J, Montag M, Magli MC, Repping S, Handyside A, Staessen C, et al. Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results. Hum Reprod Oxf Engl. 2011;26:3173–80. https://doi.org/10.1093/humrep/der294.CrossRef

13.

Christopikou D, Tsorva E, Economou K, Shelley P, Davies S, Mastrominas M, et al. Polar body analysis by array comparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age. Hum Reprod. 2013;28:1426–34. https://doi.org/10.1093/humrep/det053.CrossRefPubMed

14.

Verpoest W, Staessen C, Bossuyt PM, Goossens V, Altarescu G, Bonduelle M, et al. Preimplantation genetic testing for aneuploidy by microarray analysis of polar bodies in advanced maternal age: a randomized clinical trial. Hum Reprod Oxf Engl. 2018;33:1767–76. https://doi.org/10.1093/humrep/dey262.CrossRef

15.

Verdyck P, Altarescu G, Santos-Ribeiro S, Vrettou C, Koehler U, Griesinger G, et al. Aneuploidy in oocytes from women of advanced maternal age: analysis of the causal meiotic errors and impact on embryo development. Hum Reprod. 2023;38:2526–35. https://doi.org/10.1093/humrep/dead201.CrossRefPubMed

16.

Fabian D, Čikoš Š, Rehák P, Koppel J. Do embryonic polar bodies commit suicide? Zygote Camb Engl. 2014;22:10–7. https://doi.org/10.1017/S0967199412000159.CrossRef

17.

Delhanty JD, SenGupta SB, Ghevaria H. How common is germinal mosaicism that leads to premeiotic aneuploidy in the female? J Assist Reprod Genet. 2019;36:2403–18. https://doi.org/10.1007/s10815-019-01596-6.CrossRefPubMedPubMedCentral

18.

Capalbo A, Bono S, Spizzichino L, Biricik A, Baldi M, Colamaria S, et al. Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Hum Reprod. 2013;28:509–18. https://doi.org/10.1093/humrep/des394.CrossRefPubMed

19.

Ghevaria H, SenGupta S, Sarna U, Sargeant S, Serhal P, Delhanty J. The contribution of germinal mosaicism to human aneuploidy. Cytogenet Genome Res. 2015;144:264–74. https://doi.org/10.1159/000381073.CrossRef

20.

Feichtinger M, Stopp T, Göbl C, Feichtinger E, Vaccari E, Mädel U, et al. Increasing live birth rate by preimplantation genetic screening of pooled polar bodies using array comparative genomic hybridization. PLoS ONE. 2015;10:e0128317. https://doi.org/10.1371/journal.pone.0128317.CrossRefPubMedPubMedCentral

21.

Shi Q, Qiu Y, Xu C, Yang H, Li C, Li N, et al. Next-generation sequencing analysis of each blastomere in good-quality embryos: insights into the origins and mechanisms of embryonic aneuploidy in cleavage-stage embryos. J Assist Reprod Genet. 2020;37:1711–8. https://doi.org/10.1007/s10815-020-01803-9.CrossRefPubMedPubMedCentral

22.

Ye Y, Ma J, Cui L, Lu S, Jin F. A rapid NGS-based preimplantation genetic testing for chromosomal abnormalities in day-3 blastomere biopsy allows embryo transfer within the same treatment cycle. Front Genet. 2021;12:636370. https://doi.org/10.3389/fgene.2021.636370.

23.

Alyafee Y, Alam Q, Tuwaijri AA, Umair M, Haddad S, Alharbi M, et al. Next-generation sequencing-based pre-implantation genetic testing for aneuploidy (PGT-A): first report from Saudi Arabia. Genes. 2021;12:461. https://doi.org/10.3390/genes12040461.CrossRefPubMedPubMedCentral

24.

Tong J, Niu Y, Wan A, Zhang T. Next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) of trophectoderm biopsy for recurrent implantation failure (RIF) patients: a retrospective study. Reprod Sci. 2021;28:1923–9. https://doi.org/10.1007/s43032-021-00519-0.CrossRefPubMed

25.

Ghevaria H, SenGupta S, Naja R, Odia R, Exeter H, Serhal P, et al. Next generation sequencing detects premeiotic errors in human oocytes. Int J Mol Sci. 2022;23:665. https://doi.org/10.3390/ijms23020665.CrossRefPubMedPubMedCentral

26.

Viotti M. Preimplantation genetic testing for chromosomal abnormalities: aneuploidy, mosaicism, and structural rearrangements. Genes. 2020;11:602. https://doi.org/10.3390/genes11060602.CrossRefPubMedPubMedCentral

27.

Oxford Nanopore Technologies. How nanopore sequencing works 2024. https://nanoporetech.com/platform/technology (accessed January 5, 2024).

28.

Wei S, Weiss ZR, Gaur P, Forman E, Williams Z. Rapid preimplantation genetic screening using a handheld, nanopore-based DNA sequencer. Fertil Steril. 2018;110:910-916.e2. https://doi.org/10.1016/j.fertnstert.2018.06.014.CrossRefPubMedPubMedCentral

29.

Tan VJ, Liu T, Arifin Z, Pak B, Tan ASC, Wong S, et al. Third-generation single-molecule sequencing for preimplantation genetic testing of aneuploidy and segmental imbalances. Clin Chem. 2023;69:881–9. https://doi.org/10.1093/clinchem/hvad062.CrossRefPubMed

30.

Oxford Nanopore Technologies. Analysis solutions for nanopore sequencing data. Available online. 2024. https://nanoporetech.com/nanopore-sequencing-data-analysis (accessed January 3, 2024).

31.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Availability: http://samtools.sourceforge.net. Bioinformatics. 2009;25:2078–9. https://doi.org/10.1093/bioinformatics/btp352.

32.

Bakker B, Taudt A, Belderbos ME, Porubsky D, Spierings DCJ, de Jong TV, et al. Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies. GitHub repository, https://github.com/ataudt/aneufinder. Genome Biol. 2016;17:115. https://doi.org/10.1186/s13059-016-0971-7.

33.

Taudt A. A quick introduction to AneuFinder 2023. https://bioconductor.org/packages/release/bioc/vignettes/AneuFinder/inst/doc/AneuFinder.pdf (accessed April 25, 2023).

34.

Gel B, Serra E. karyoploteR: an R/Bioconductor package to plot customizable genomes displaying arbitrary data. GitHub repository, https://github.com/bernatgel/karyoploteR. Bioinformatics. 2017;33:3088–90. https://doi.org/10.1093/bioinformatics/btx346.

35.

Toolkit for processing sequences in FASTA/Q formats 2024. https://github.com/lh3/seqtk (accessed January 4, 2024).

36.

Normand E, Qdaisat S, Bi W, Shaw C, Van den Veyver I, Beaudet A, et al. Comparison of three whole genome amplification methods for detection of genomic aberrations in single cells. Prenat Diagn. 2016;36:823–30. https://doi.org/10.1002/pd.4866.CrossRefPubMed

37.

Demko ZP, Simon AL, McCoy RC, Petrov DA, Rabinowitz M. Effects of maternal age on euploidy rates in a large cohort of embryos analyzed with 24-chromosome single-nucleotide polymorphism–based preimplantation genetic screening. Fertil Steril. 2016;105:1307–13. https://doi.org/10.1016/j.fertnstert.2016.01.025.CrossRefPubMed

38.

Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR, et al. Aneuploidy across individual chromosomes at the embryonic level in trophectoderm biopsies: changes with patient age and chromosome structure. J Assist Reprod Genet. 2014;31:1501–9. https://doi.org/10.1007/s10815-014-0333-x.CrossRefPubMedPubMedCentral

39.

Fragouli E, Alfarawati S, Spath K, Jaroudi S, Sarasa J, Enciso M, et al. The origin and impact of embryonic aneuploidy. Hum Genet. 2013;132:1001–13. https://doi.org/10.1007/s00439-013-1309-0.CrossRefPubMed

40.

Kallioniemi A. Comparative genomic hybridization (CGH). In: Brenner S, Miller JH, editors. Encycl. Genet., New York: Academic Press; 2001, pp. 424–426. https://doi.org/10.1006/rwgn.2001.1560.

41.

Xiao M, Lei C-X, Xi Y-P, Lu Y-L, Wu J-P, Li X-Y, et al. Next-generation sequencing is more efficient at detecting mosaic embryos and improving pregnancy outcomes than single-nucleotide polymorphism array analysis. J Mol Diagn. 2021;23:710–8. https://doi.org/10.1016/j.jmoldx.2021.02.011.CrossRefPubMed

Title: Aneuploidy detection in pooled polar bodies using rapid nanopore sequencing
Authors: Silvia Madritsch
Vivienne Arnold
Martha Haider
Julia Bosenge
Mateja Pfeifer
Beatrix Weil
Manuela Zechmeister
Markus Hengstschläger
Jürgen Neesen
Franco Laccone
Publication date: 20-04-2024
Publisher: Springer US
Keywords: Preimplantation Genetic Diagnostics
In Vitro Fertilisation
In Vitro Fertilisation
Published in: Journal of Assisted Reproduction and Genetics
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI: https://doi.org/10.1007/s10815-024-03108-7

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Aneuploidy detection in pooled polar bodies using rapid nanopore sequencing

Abstract

Purpose

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusion

Please log in to get access to this content