CoChat
Sign Up
Cited by 47Reproductive Biology and FertilityPrenatal Screening and DiagnosticsSingle-cell and spatial transcriptomics

RNA-seq as a tool for evaluating human embryo competence

Open access
Groff, Abigail F., Resetkova, Nina, DiDomenico, Francesca, Sakkas, Denny, Penzias, Alan, Rinn, John L., Eggan, Kevin·2019·Genome Research
DOI 10.1101/gr.252981.119PMC PMC6771404
Open this paper in CoChat — free accountOpen PDF

Abstract

The majority of embryos created through in vitro fertilization (IVF) do not implant. It seems plausible that rates of implantation would improve if we had a better understanding of molecular factors affecting embryo competence. Currently, the process of selecting an embryo for uterine transfer uses an ad hoc combination of morphological criteria, the kinetics of development, and genetic testing for aneuploidy. However, no single criterion can ensure selection of a viable embryo. In contrast, RNA-sequencing (RNA-seq) of embryos could yield high-dimensional data, which may provide additional insight and illuminate the discrepancies among current selection criteria. Recent advances enabling the production of RNA-seq libraries from single cells have facilitated the application of this technique to the study of transcriptional events in early human development. However, these studies have not assessed the quality of their constituent embryos relative to commonly used embryological criteria. Here, we perform proof-of-principle advancement to embryo selection procedures by generating RNA-seq libraries from a trophectoderm biopsy as well as the remaining whole embryo. We combine state-of-the-art embryological methods with low-input RNA-seq to develop the first transcriptome-wide approach for assessing embryo competence. Specifically, we show the capacity of RNA-seq as a promising tool in preimplantation screening by showing that biopsies of an embryo can capture valuable information available in the whole embryo from which they are derived. Furthermore, we show that this technique can be used to generate a RNA-based digital karyotype and to identify candidate competence-associated genes. Together, these data establish the foundation for a future RNA-based diagnostic in IVF.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1101/gr.252981.119

Cites

4 of 4 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Groff and colleagues demonstrated that aneuploidy can be estimated based on significant variations in gene expression in the affected chromosome(s) compared to reference control samples.

    “Groff and colleagues have demonstrated that aneuploidy can be estimated based on significant variations in gene expression in the affected chromosome(s) compared to reference control samples (24).”

    From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [24]

    The Results section, “RNA-seq can provide a digital karyotype” (page unavailable), reports estimating potential aneuploidy from chromosome-level RNA read-count outliers. The Methods section, “RNA digital karyotype” (page unavailable), describes comparing chromosome expression with a mean across samples and testing significant deviations, not comparing affected chromosomes with designated reference control samples. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 63

  1. Ao A, Erickson RP, Winston RM, Handyside AH. 1994 Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage. Zygote 2: 281– 287. 8665158
  2. Bainbridge SA, Minhas A, Whiteley KJ, Qu D, Sled JG, Kingdom JCP, Adamson SL. 2012 Effects of reduced Gcm1 expression on trophoblast morphology, fetoplacental vascularity, and pregnancy outcomes in mice. Hypertension 59: 732– 739. 22275534
  3. Baumann CG, Morris DG, Sreenan JM, Leese HJ. 2007 The quiet embryo hypothesis: molecular characteristics favoring viability. Mol Reprod Dev 74: 1345– 1353. 10.1002/mrd.20604 17342740 · DOI
  4. Bera TK, Das S, Maeda H, Beers R, Wolfgang CD, Kumar V, Hahn Y, Lee B, Pastan I. 2004 NGEP, a gene encoding a membrane protein detected only in prostate cancer and normal prostate. Proc Natl Acad Sci 101: 3059– 3064. 14981236 PMC365744
  5. Blakeley P, Fogarty NME, del Valle I, Wamaitha SE, Hu TX, Elder K, Snell P, Christie L, Robson P, Niakan KK. 2015 Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142: 3151– 3165. 10.1242/dev.123547 26293300 PMC4582176 · DOI
  6. Briggs SF, Dominguez AA, Chavez SL, Reijo Pera RA. 2015 Single-cell XIST expression in human preimplantation embryos and newly reprogrammed female induced pluripotent stem cells. Stem Cells 33: 1771– 1781. 25753947 PMC4441606
  7. Capalbo A, Rienzi L. 2017 Mosaicism between trophectoderm and inner cell mass. Fertil Steril 107: 1098– 1106. 10.1016/j.fertnstert.2017.03.023 28433375 · DOI
  8. Chen C-P, Chen C-Y, Yang Y-C, Su T-H, Chen H. 2004 Decreased placental GCM1 (glial cells missing) gene expression in pre-eclampsia. Placenta 25: 413– 421. 15081636
  9. Dahdouh EM, Balayla J, García-Velasco JA. 2015 Comprehensive chromosome screening improves embryo selection: a meta-analysis. Fertil Steril 104: 1503– 1512. 10.1016/j.fertnstert.2015.08.038 26385405 · DOI
  10. Daniels R, Zuccotti M, Kinis T, Serhal P, Monk M. 1997 XIST expression in human oocytes and preimplantation embryos. Am J Hum Genet 61: 33– 39. 10.1086/513892 9245982 PMC1715845 · DOI
  11. Disteche CM, Berletch JB. 2015 X-chromosome inactivation and escape. J Genet 94: 591– 599. 10.1007/s12041-015-0574-1 26690513 PMC4826282 · DOI
  12. Duijf PHG, Schultz N, Benezra R. 2013 Cancer cells preferentially lose small chromosomes. Int J Cancer 132: 2316– 2326. 10.1002/ijc.27924 23124507 PMC3587043 · DOI
  13. Eggan K, Rode A, Jentsch I, Samuel C, Hennek T, Tintrup H, Zevnik B, Erwin J, Loring J, Jackson-Grusby L, 2002 Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation. Nat Biotechnol 20: 455– 459. 10.1038/nbt0502-455 11981557 · DOI
  14. Fiddler M, Abdel-Rahman B, Rappolee DA, Pergament E. 1995 Expression of SRY transcripts in preimplantation human embryos. Am J Med Genet 55: 80– 84. 7702103
  15. Fragouli E, Spath K, Alfarawati S, Kaper F, Craig A, Michel C-E, Kokocinski F, Cohen J, Munne S, Wells D. 2015 Altered levels of mitochondrial DNA are associated with female age, aneuploidy, and provide an independent measure of embryonic implantation potential. PLoS Genet 11: e1005241 10.1371/journal.pgen.1005241 26039092 PMC4454688 · DOI
  16. Gardner DK, Schoolcraft WB. 1999 In vitro culture of human blastocysts. In Towards reproductive certainty: fertility and genetics beyond 1999 (ed. Jansen R, Mortimer D), pp. 378– 388 Parthenon Press, Carnforth, UK.
  17. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. 2000 Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 73: 1155– 1158. 10856474
  18. Gardner DK, Sakkas D, Seli E, Wells D, eds. 2013 Human gametes and preimplantation embryos. Springer, New York.
  19. Griffin DK, Ogur C. 2018 Chromosomal analysis in IVF: Just how useful is it? Reproduction 156: F29– F50. 10.1530/REP-17-0683 29945889 · DOI
  20. Hassold TJ, Jacobs PA. 1984 Trisomy in man. Annu Rev Genet 18: 69– 97. 10.1146/annurev.ge.18.120184.000441 6241455 · DOI
  21. International Human Genome Sequencing Consortium. 2001 Initial sequencing and analysis of the human genome. Nature 409: 860– 921. 10.1038/35057062 11237011 · DOI · Paper page
  22. Islam S, Kjällquist U, Moliner A, Zajac P, Fan J-B, Lönnerberg P, Linnarsson S. 2011 Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res 21: 1160– 1167. 10.1101/gr.110882 21543516 PMC3129258 · DOI
  23. Kinde I, Papadopoulos N, Kinzler KW, Vogelstein B. 2012 FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing. PLoS One 7: e41162 10.1371/journal.pone.0041162 22815955 PMC3399813 · DOI
  24. Levin JZ, Yassour M, Adiconis X, Nusbaum C, Thompson DA, Friedman N, Gnirke A, Regev A. 2010 Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat Methods 7: 709– 715. 10.1038/nmeth.1491 20711195 PMC3005310 · DOI · Paper page
  25. Li B, Dewey CN. 2011 RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12: 323. 21816040 PMC3163565
  26. Licciardi F, Lhakhang T, Kramer YG, Zhang Y, Heguy A, Tsirigos A. 2018 Human blastocysts of normal and abnormal karyotypes display distinct transcriptome profiles. Sci Rep 8: 14906 10.1038/s41598-018-33279-0 30297919 PMC6175822 · DOI
  27. Loftfield E, Zhou W, Graubard BI, Yeager M, Chanock SJ, Freedman ND, Machiela MJ. 2018 Predictors of mosaic chromosome Y loss and associations with mortality in the UK Biobank. Sci Rep 8: 12316 10.1038/s41598-018-30759-1 30120341 PMC6098142 · DOI
  28. Long PA, Zimmermann MT, Kim M, Evans JM, Xu X, Olson TM. 2016 De novo RRAGC mutation activates mTORC1 signaling in syndromic fetal dilated cardiomyopathy. Hum Genet 135: 909– 917. 27234373 PMC4947566
  29. Love MI, Huber W, Anders S. 2014 Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15: 550 10.1186/s13059-014-0550-8 25516281 PMC4302049 · DOI · Paper page
  30. Maluenda J, Manso C, Quevarec L, Vivanti A, Marguet F, Gonzales M, Guimiot F, Petit F, Toutain A, Whalen S, 2016 Mutations in GLDN, encoding gliomedin, a critical component of the nodes of Ranvier, are responsible for lethal arthrogryposis. Am J Hum Genet 99: 928– 933. 27616481 PMC5065655
  31. Mangs AH, Morris B. 2007 The human pseudoautosomal region (PAR): origin, function and future. Curr Genomics 8: 129– 136. 10.2174/138920207780368141 18660847 PMC2435358 · DOI
  32. Marusawa H, Matsuzawa S-I, Welsh K, Zou H, Armstrong R, Tamm I, Reed JC. 2003 HBXIP functions as a cofactor of survivin in apoptosis suppression. EMBO J 22: 2729– 2740. 10.1093/emboj/cdg263 12773388 PMC156760 · DOI
  33. McCoy RC. 2017 Mosaicism in preimplantation human embryos: when chromosomal abnormalities are the norm. Trends Genet 33: 448– 463. 10.1016/j.tig.2017.04.001 28457629 PMC5484399 · DOI · Paper page
  34. Moreira de Mello JC, Fernandes GR, Vibranovski MD, Pereira LV. 2017 Early X chromosome inactivation during human preimplantation development revealed by single-cell RNA-sequencing. Sci Rep 7: 10794 10.1038/s41598-017-11044-z 28883481 PMC5589911 · DOI
  35. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. 2008 Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5: 621– 628. 10.1038/nmeth.1226 18516045 PMC13303166 · DOI · Paper page
  36. Munné S, Blazek J, Large M, Martinez-Ortiz PA, Nisson H, Liu E, Tarozzi N, Borini A, Becker A, Zhang J, 2017 Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing. Fertil Steril 108: 62– 71.e8. 10.1016/j.fertnstert.2017.05.002 28579407 · DOI
  37. Nichols J, Silva J, Roode M, Smith A. 2009 Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo. Development 136: 3215– 3222. 10.1242/dev.038893 19710168 PMC2739140 · DOI
  38. Ohnishi YN, Sakumi K, Yamazaki K, Ohnishi YH, Miura T, Tominaga Y, Nakabeppu Y. 2008 Antagonistic regulation of cell-matrix adhesion by FosB and ΔFosB/Δ2ΔFosB encoded by alternatively spliced forms of fosB transcripts. Mol Biol Cell 19: 4717– 4729. 10.1091/mbc.e07-08-0768 18753407 PMC2575163 · DOI
  39. Pedemonte N, Galietta LJV. 2014 Structure and function of TMEM16 proteins (anoctamins). Physiol Rev 94: 419– 459. 24692353
  40. Petropoulos S, Edsgärd D, Reinius B, Deng Q, Panula SP, Codeluppi S, Reyes AP, Linnarsson S, Sandberg R, Lanner F. 2016 Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos. Cell 167: 285 10.1016/j.cell.2016.08.009 27662094 PMC5628172 · DOI
  41. Picelli S, Faridani OR, Björklund ÅK, Winberg G, Sagasser S, Sandberg R. 2014 Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9: 171– 181. 10.1038/nprot.2014.006 24385147 · DOI · Paper page
  42. Ramsköld D, Luo S, Wang Y-C, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, 2012 Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol 30: 777– 782. 22820318 PMC3467340
  43. Rapaport F, Khanin R, Liang Y, Pirun M, Krek A, Zumbo P, Mason CE, Socci ND, Betel D. 2013 Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data. Genome Biol 14: R95 10.1186/gb-2013-14-9-r95 24020486 PMC4054597 · DOI · Paper page
  44. Robinson WP, McFadden DE, Stephenson MD. 2001 The origin of abnormalities in recurrent aneuploidy/polyploidy. Am J Hum Genet 69: 1245– 1254. 10.1086/324468 11675616 PMC1235536 · DOI
  45. Sekiguchi T, Hirose E, Nakashima N, Ii M, Nishimoto T. 2001 Novel G proteins, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B. J Biol Chem 276: 7246– 7257. 11073942
  46. Setton R, Melnick AP, Rosenwaks Z, Spandorfer SD. 2015 Natural cycle frozen embryo transfer after IVF-PGS may double live birth rates compared to programmed cycles. Fertil Steril 104: e50 10.1016/j.fertnstert.2015.07.150 · DOI
  47. Simon AL, Kiehl M, Fischer E, Proctor JG, Bush MR, Givens C, Rabinowitz M, Demko ZP. 2018 Pregnancy outcomes from more than 1,800 in vitro fertilization cycles with the use of 24-chromosome single-nucleotide polymorphism-based preimplantation genetic testing for aneuploidy. Fertil Steril 110: 113– 121. 29908770
  48. Sousa EJ, Stuart HT, Bates LE, Ghorbani M, Nichols J, Dietmann S, Silva JCR. 2018 Exit from naive pluripotency induces a transient X chromosome inactivation-like state in males. Cell Stem Cell 22: 919– 928.e6. 29804891 PMC5989057
  49. Steinman HA, Hoover KM, Keeler ML, Sands AT, Jones SN. 2005 Rescue of Mdm4-deficient mice by Mdm2 reveals functional overlap of Mdm2 and Mdm4 in development. Oncogene 24: 7935– 7940. 10.1038/sj.onc.1208930 16027727 · DOI
  50. Sun Y, Boyd K, Xu W, Ma J, Jackson CW, Fu A, Shillingford JM, Robinson GW, Hennighausen L, Hitzler JK, 2006 Acute myeloid leukemia-associated Mkl1 (Mrtf-a) is a key regulator of mammary gland function. Mol Cell Biol 26: 5809– 5826. 10.1128/MCB.00024-06 16847333 PMC1592762 · DOI
  51. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, 2009 mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods 6: 377– 382. 19349980
  52. Titus AJ, Way GP, Johnson KC, Christensen BC. 2017 Deconvolution of DNA methylation identifies differentially methylated gene regions on 1p36 across breast cancer subtypes. Sci Rep 7: 11594 10.1038/s41598-017-10199-z 28912426 PMC5599639 · DOI
  53. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. 2010 Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28: 511– 515. 10.1038/nbt.1621 20436464 PMC3146043 · DOI · Paper page
  54. Treff NR, Zhan Y, Tao X, Olcha M, Han M, Rajchel J, Morrison L, Morin SJ, Scott RT. 2017 Levels of trophectoderm mitochondrial DNA do not predict the reproductive potential of sibling embryos. Hum Reprod 32: 954– 962. 10.1093/humrep/dex034 28333210 PMC5400072 · DOI
  55. van Echten-Arends J, Mastenbroek S, Sikkema-Raddatz B, Korevaar JC, Heineman MJ, van der Veen F, Repping S. 2011 Chromosomal mosaicism in human preimplantation embryos: a systematic review. Hum Reprod Update 17: 620– 627. 10.1093/humupd/dmr014 21531753 · DOI
  56. Veeriah S, Brennan C, Meng S, Singh B, Fagin JA, Solit DB, Paty PB, Rohle D, Vivanco I, Chmielecki J, 2009 The tyrosine phosphatase PTPRD is a tumor suppressor that is frequently inactivated and mutated in glioblastoma and other human cancers. Proc Natl Acad Sci 106: 9435– 9440. 19478061 PMC2687998
  57. Wang J, Sauer MV. 2006 In vitro fertilization (IVF): a review of three decades of clinical innovation and technological advancement. Ther Clin Risk Manag 2: 355– 364. 10.2147/tcrm.2006.2.4.355 18360648 PMC1936357 · DOI
  58. Wang TL, Maierhofer C, Speicher MR, Lengauer C, Vogelstein B, Kinzler KW, Velculescu VE. 2002 Digital karyotyping. Proc Natl Acad Sci 99: 16156– 16161. 10.1073/pnas.202610899 12461184 PMC138581 · DOI
  59. Weissbein U, Schachter M, Egli D, Benvenisty N. 2016 Analysis of chromosomal aberrations and recombination by allelic bias in RNA-Seq. Nat Commun 7: 12144 10.1038/ncomms12144 27385103 PMC4941052 · DOI · Paper page
  60. Xue Z, Huang K, Cai C, Cai L, Jiang C-Y, Feng Y, Liu Z, Zeng Q, Cheng L, Sun YE, 2013 Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature 500: 593– 597. 10.1038/nature12364 23892778 PMC4950944 · DOI · Paper page
  61. Yamaguchi S, Asanoma K, Takao T, Kato K, Wake N. 2009 Homeobox gene HOPX is epigenetically silenced in human uterine endometrial cancer and suppresses estrogen-stimulated proliferation of cancer cells by inhibiting serum response factor. Int J Cancer 124: 2577– 2588. 10.1002/ijc.24217 19173292 · DOI
  62. Yan L, Yang M, Guo H, Yang L, Wu J, Li R, Liu P, Lian Y, Zheng X, Yan J, 2013 Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biology 20: 1131– 1139. 10.1038/nsmb.2660 23934149 · DOI · Paper page
  63. Zhu Q, Tan Z, Zhao S, Huang H, Zhao X, Hu X, Zhang Y, Shields CB, Uetani N, Qiu M. 2015 Developmental expression and function analysis of protein tyrosine phosphatase receptor type D in oligodendrocyte myelination. Neuroscience 308: 106– 114. 26341907 PMC4600676

Cited by

Sorted by citations

Authors
Groff, A. F., Resetkova, N., DiDomenico, F., Sakkas, D., Penzias, A., Rinn, J. L., & Eggan, K.
Affiliations
  • Harvard University (US)
  • Beth Israel Deaconess Medical Center (US)
  • Boston IVF (US)
ORCID
Venue
Genome Research
Publisher
Cold Spring Harbor Laboratory
Published
2019-09-23
Volume
29
Issue
10
Pages
1705-1718
ISSN
  • 1088-9051 (ISSN-L)
  • 1549-5469
DOI
10.1101/gr.252981.119
PMC
PMC6771404
OpenAlex
W2977229130
Cited by
47
References
4
Citations by year
2026: 3 · 2025: 7 · 2024: 8 · 2023: 12 · 2022: 8 · 2021: 5 · 2020: 4
Type
posted content
Language
en
Open access
Locations
  • Genome Research (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HighWire Press Open Archive (submittedVersion)
Funders
  • Harvard Stem Cell Institute
  • Howard Hughes Medical Institute
  • Stowers Medical Institute
  • HHMI
  • Integramed National Innovation
Topics
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Reproductive Biology and Fertility
  • Health Sciences › Medicine › Pediatrics, Perinatology and Child Health › Prenatal Screening and Diagnostics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
embryo developmental competence, embryo selection, in vitro fertilization, trophectoderm biopsy, embryo transcriptome, preimplantation genetic testing for aneuploidy, embryo implantation, embryo quality assessment
MeSH terms
Exome Sequencing, Biopsy, Blastocyst, Female, Fertilization in Vitro, Genetic Testing, Humans, Karyotyping, Embryo Implantation, Pregnancy, Sequence Analysis, RNA, Preimplantation Diagnosis, Embryonic Development, Karyotype

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review