Journal of Assisted Reproduction and Genetics

, Volume 36, Issue 3, pp 433–443 | Cite as

Cumulus-corona gene expression analysis combined with morphological embryo scoring in single embryo transfer cycles increases live birth after fresh transfer and decreases time to pregnancy

  • T. Adriaenssens
  • I. Van Vaerenbergh
  • W. Coucke
  • I. Segers
  • G. Verheyen
  • E. Anckaert
  • M. De Vos
  • J. SmitzEmail author
Assisted Reproduction Technologies



Clinical pregnancy rate after IVF with eSET stagnates between 30 and 40%. In order to increase pregnancy and live birth rates, multiple embryo transfer is still common practice. Providing additional non-invasive tools to choose the competent embryo for transfer could avoid multiple pregnancy and improve time to pregnancy. Cumulus mRNA analysis with quantitative PCR (QPCR) is a non-invasive approach. However, so far, no gene sets have been validated in prospective interventional studies.


A prospective interventional single-center pilot study with two matched controls (day-3 and day-5 eSET) was performed in 96 patients consenting to the analysis of the cumulus-corona of their oocytes. All patients were super-ovulated for ICSI and eSET at day 3. All oocytes were denuded individually and cumulus was analyzed by quantitative PCR using three predictive genes (EFNB2, SASH1, CAMK1D) and two housekeeping genes (UBC and β2M). Patients (n = 62) with 2 or more day-3 embryos (good or excellent morphology) had their embryo chosen following the normalized expression of the genes.


Corona testing significantly increased the clinical pregnancy and live births rates (63% and 55%) compared to single embryo transfer (eSET) on day 3 (27% and 23%: p < 0.001) and day 5 (43% and 39%: p = 0.022 and p = 0.050) fresh transfer cycle controls with morphology-only selection. Time-to-pregnancy was significantly reduced, regardless of the number of good-quality embryos available on day 3.


Combining standard morphology scoring and cumulus/corona gene expression analysis increases day-3 eSET results and significantly reduces the time to pregnancy.

Trial registration number

This is not an RCT study and was only registered by the ethical committee of the University Hospital UZBRUSSEL of the Vrije Universiteit Brussel VUB (BUN: 143201318000).


Cumulus cells Gene expression Single embryo transfer Clinical pregnancy Non-invasive Oocyte quality 



The authors would like to thank their colleagues of the Centre for Reproductive Medicine, UZ Brussel, for their cooperation in this clinical study, the clinical data manager Walter Meul, and Prof. Dr. André Rosenthal for critical reading and suggestions.


This study was funded by IWT/VLAIO Innovation Mandate 130327 and 140568 and by the Vrije Universiteit Brussel IOFPOC26.

Supplementary material

10815_2018_1398_MOESM1_ESM.docx (15 kb)
Supplementary Table 1 (DOCX 14 kb)
10815_2018_1398_MOESM2_ESM.docx (210 kb)
Supplementary Figure 1 (DOCX 210 kb)


  1. 1.
    Guerif F, Le Gouge A, Giraudeau B, Poindron J, Bidault R, Gasnier O, et al. Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: a prospective study based on 4042 embryos. Hum Reprod. 2007;22:1973–81.CrossRefPubMedGoogle Scholar
  2. 2.
    Guerif F, Lemseffer M, Leger J, Bidault R, Cadoret V, Chavez C, et al. Does early morphology provide additional selection power to blastocyst selection for transfer? Reprod BioMed Online. 2010;21:510–9.CrossRefPubMedGoogle Scholar
  3. 3.
    Papanikolaou EG, Kolibianakis EM, Tournaye H, Venetis CA, Fatemi H, Tarlatzis B, et al. Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis. Hum Reprod. 2008;23:91–9.CrossRefPubMedGoogle Scholar
  4. 4.
    Glujovsky D, Blake D, Farquhar C, Bardach A. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Glujovsky D, editor. Cochrane database Syst Rev. Chichester, UK: John Wiley & Sons, Ltd; 2012;CD002118.Google Scholar
  5. 5.
    Blondel B, Kogan MD, Alexander GR, Dattani N, Kramer MS, Macfarlane A, et al. The impact of the increasing number of multiple births on the rates of preterm birth and low birthweight: an international study. Am J Public Health. 2002;92:1323–30.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Heino A, Gissler M, Hindori-Mohangoo AD, Blondel B, Klungsøyr K, Verdenik I, et al. Variations in multiple birth rates and impact on perinatal outcomes in Europe. Baud O, editor. PLoS One. 2016;11:e0149252.Google Scholar
  7. 7.
    Lédée N, Gridelet V, Ravet S, Jouan C, Gaspard O, Wenders F, et al. Impact of follicular G-CSF quantification on subsequent embryo transfer decisions: a proof of concept study. Hum Reprod. 2013;28:406–13.CrossRefPubMedGoogle Scholar
  8. 8.
    Scalici E, Traver S, Molinari N, Mullet T, Monforte M, Vintejoux E, et al. Cell-free DNA in human follicular fluid as a biomarker of embryo quality. Hum Reprod. 2014;29:2661–9.CrossRefPubMedGoogle Scholar
  9. 9.
    McKenzie LJ, Pangas SA, Carson SA, Kovanci E, Cisneros P, Buster JE, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod. 2004;19:2869–74.CrossRefPubMedGoogle Scholar
  10. 10.
    Capalbo A, Ubaldi FM, Cimadomo D, Noli L, Khalaf Y, Farcomeni A, et al. MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment. Fertil Steril. 2016;105:225–35.e1–3.Google Scholar
  11. 11.
    Kirkegaard K, Hindkjaer JJ, Grøndahl ML, Kesmodel US, Ingerslev HJ. A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator. J Assist Reprod Genet. 2012;29:565–72.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Goodman LR, Goldberg J, Falcone T, Austin C, Desai N. Does the addition of time-lapse morphokinetics in the selection of embryos for transfer improve pregnancy rates? A randomized controlled trial. Fertil Steril. 2016;105:275–85.e10.CrossRefPubMedGoogle Scholar
  13. 13.
    Armstrong S, Arroll N, Cree LM, Jordan V, Farquhar C. Time-lapse systems for embryo incubation and assessment in assisted reproduction. Cochrane database Syst Rev. 2015;CD011320.Google Scholar
  14. 14.
    Forman EJ, Hong KH, Ferry KM, Tao X, Taylor D, Levy B, et al. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertil Steril. 2013;100:100–7.e1.CrossRefPubMedGoogle Scholar
  15. 15.
    Munné S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod. 1993;8:2185–91.CrossRefPubMedGoogle Scholar
  16. 16.
    Scott RT, Ferry K, Su J, Tao X, Scott K, Treff NR. Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, nonselection study. Fertil Steril. 2012;97:870–5.CrossRefPubMedGoogle Scholar
  17. 17.
    Harper JC, Harton G. The use of arrays in preimplantation genetic diagnosis and screening. Fertil Steril. 2010;94:1173–7.CrossRefPubMedGoogle Scholar
  18. 18.
    Munné S, Blazek J, Large M, Martinez-Ortiz PA, Nisson H, Liu E, et al. 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. 2017;108:62–71.e8.CrossRefPubMedGoogle Scholar
  19. 19.
    Magli MC, Pomante A, Cafueri G, Valerio M, Crippa A, Ferraretti AP, et al. Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid? Fertil Steril. 2016;105:676–683.e5.CrossRefPubMedGoogle Scholar
  20. 20.
    Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet. 2012;5:24.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kang H-J, Melnick AP, Stewart JD, Xu K, Rosenwaks Z. Preimplantation genetic screening: who benefits? Fertil Steril. 2016;106:597–602.CrossRefPubMedGoogle Scholar
  22. 22.
    Simon AL, Kiehl M, Fischer E, Proctor JG, Bush MR, Givens C, et al. 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. 2018;110:113–21.CrossRefPubMedGoogle Scholar
  23. 23.
    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. 2018;33:1767–76.CrossRefPubMedGoogle Scholar
  24. 24.
    Zhang S, Luo K, Cheng D, Tan Y, Lu C, He H, et al. Number of biopsied trophectoderm cells is likely to affect the implantation potential of blastocysts with poor trophectoderm quality. Fertil Steril. 2016;105:1222–1227.e4.CrossRefPubMedGoogle Scholar
  25. 25.
    Gleicher N, Orvieto R. Is the hypothesis of preimplantation genetic screening (PGS) still supportable? A review. J Ovarian Res. 2017;10:21.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Eppig JJ. Coordination of nuclear and cytoplasmic oocyte maturation in eutherian mammals. Reprod Fertil Dev. 1996;8:485–9.CrossRefPubMedGoogle Scholar
  27. 27.
    Cakmak H, Franciosi F, Zamah AM, Cedars MI, Conti M. Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells. Proc Natl Acad Sci U S A. 2016;113:2424–9.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Fragouli E, Lalioti MD, Wells D. The transcriptome of follicular cells: biological insights and clinical implications for the treatment of infertility. Hum Reprod Update. 2014;20:1–11.CrossRefPubMedGoogle Scholar
  29. 29.
    Wathlet S, Adriaenssens T, Segers I, Verheyen G, Van de Velde H, Coucke W, et al. Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients. Hum Reprod. 2011;26:1035–51.CrossRefPubMedGoogle Scholar
  30. 30.
    Wathlet S, Adriaenssens T, Segers I, Verheyen G, Van Landuyt L, Coucke W, et al. Pregnancy prediction in single embryo transfer cycles after ICSI using QPCR: validation in oocytes from the same cohort. Lambalk CB, editor. PLoS One. 2013;8:e54226.Google Scholar
  31. 31.
    Buensuceso AV, Deroo BJ. The ephrin signaling pathway regulates morphology and adhesion of mouse granulosa cells in vitro. Biol Reprod. 2013;88:25.CrossRefPubMedGoogle Scholar
  32. 32.
    Dauphinee SM, Clayton A, Hussainkhel A, Yang C, Park Y-J, Fuller ME, et al. SASH1 is a scaffold molecule in endothelial TLR4 signaling. J Immunol. 2013;191:892–901.CrossRefPubMedGoogle Scholar
  33. 33.
    Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al. International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009. Fertil Steril. 2009;92:1520–4.CrossRefPubMedGoogle Scholar
  34. 34.
    Devroey P, Pellicer A, Nyboe Andersen A, Arce J-C, Menopur in GnRH Antagonist Cycles with Single Embryo Transfer Trial Group. A randomized assessor-blind trial comparing highly purified hMG and recombinant FSH in a GnRH antagonist cycle with compulsory single-blastocyst transfer. Fertil Steril. 2012;97:561–71.CrossRefPubMedGoogle Scholar
  35. 35.
    Van Landuyt L, Van de Velde H, De Vos A, Haentjens P, Blockeel C, Tournaye H, et al. Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human day 3 embryos. Hum Reprod. 2013;28:2943–9.CrossRefPubMedGoogle Scholar
  36. 36.
    Van Landuyt L, De Vos A, Joris H, Verheyen G, Devroey P, Van Steirteghem A. Blastocyst formation in in vitro fertilization versus intracytoplasmic sperm injection cycles: influence of the fertilization procedure. Fertil Steril. 2005;83:1397–403.CrossRefPubMedGoogle Scholar
  37. 37.
    Segers I, Mateizel I, Van Moer E, Smitz J, Tournaye H, Verheyen G, et al. In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising &quot;ex vivo&quot; method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe. J Assist Reprod Genet. 2015;32:1221–31.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Gardner D, Schoolcraft W. In-vitro culture of human blastocysts. In: Jansen R, Mortimer D, editors. Towar reprod certain fertil genet beyond 1999. Carnforth: Parthenon Press; 1999. p. 378–88.Google Scholar
  39. 39.
    Utsunomiya T, Ito H, Nagaki M, Sato J. A prospective, randomized study: day 3 versus hatching blastocyst stage. Hum Reprod. 2004;19:1598–603.CrossRefPubMedGoogle Scholar
  40. 40.
    Ziebe S, Lundin K, Janssens R, Helmgaard L, Arce J-C. MERIT (Menotrophin vs Recombinant FSH in vitro Fertilisation Trial) Group. Influence of ovarian stimulation with HP-hMG or recombinant FSH on embryo quality parameters in patients undergoing IVF. Hum Reprod. 2007;22:2404–13.CrossRefPubMedGoogle Scholar
  41. 41.
    Sfontouris IA, Kolibianakis EM, Lainas GT, Petsas GK, Tarlatzis BC, Lainas TG. Blastocyst development in a single medium compared to sequential media: a prospective study with sibling oocytes. Reprod Sci. 2017;24:1312–8.CrossRefPubMedGoogle Scholar
  42. 42.
    Rienzi L, Gracia C, Maggiulli R, LaBarbera AR, Kaser DJ, Ubaldi FM, et al. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update. 2017;23:139–55.PubMedGoogle Scholar
  43. 43.
    Veeck LL, Bodine R, Clarke RN, Berrios R, Libraro J, Moschini RM, et al. High pregnancy rates can be achieved after freezing and thawing human blastocysts. Fertil Steril. 2004;82:1418–27.CrossRefPubMedGoogle Scholar
  44. 44.
    Stoop D, Ermini B, Polyzos NP, Haentjens P, De Vos M, Verheyen G, et al. Reproductive potential of a metaphase II oocyte retrieved after ovarian stimulation: an analysis of 23 354 ICSI cycles. Hum Reprod. 2012;27:2030–5.CrossRefPubMedGoogle Scholar
  45. 45.
    Adriaenssens T, Mazoyer C, Segers I, Wathlet S, Smitz J. Differences in collagen expression in cumulus cells after exposure to highly purified menotropin or recombinant follicle-stimulating hormone in a mouse follicle culture model. Biol Reprod. 2009;80:1015–25.CrossRefPubMedGoogle Scholar
  46. 46.
    Grøndahl ML, Borup R, Lee YB, Myrhøj V, Meinertz H, Sørensen S. Differences in gene expression of granulosa cells from women undergoing controlled ovarian hyperstimulation with either recombinant follicle-stimulating hormone or highly purified human menopausal gonadotropin. Fertil Steril. 2009;91:1820–30.CrossRefPubMedGoogle Scholar
  47. 47.
    Adriaenssens T, Wathlet S, Segers I, Verheyen G, De Vos A, Van der Elst J, et al. Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics. Hum Reprod. 2010;25:1259–70.CrossRefPubMedGoogle Scholar
  48. 48.
    Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR, et al. The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil Steril. 2014;101:656–663.e1.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • T. Adriaenssens
    • 1
  • I. Van Vaerenbergh
    • 1
  • W. Coucke
    • 2
  • I. Segers
    • 3
  • G. Verheyen
    • 3
  • E. Anckaert
    • 1
  • M. De Vos
    • 3
  • J. Smitz
    • 1
    Email author
  1. 1.Follicle Biology LaboratoryVrije Universiteit Brussel (VUB)BrusselsBelgium
  2. 2.Department of Clinical BiologyScientific Institute of Public HealthBrusselsBelgium
  3. 3.Centre for Reproductive MedicineUniversitair Ziekenhuis Brussel (UZ Brussel)BrusselsBelgium

Personalised recommendations