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Molecular analysis of DNA in blastocoele fluid using next-generation sequencing

  • Embryo Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Background

Preimplantation genetic testing (PGT) requires an invasive biopsy to obtain embryonic material for genetic analysis. The availability of a less invasive procedure would increase the overall efficacy of PGT. The aim of the study was to explore the potential of blastocoele fluid (BF) as an alternative source of embryonic DNA for PGT.

Methods

Collection of BF was performed by aspiration with a fine needle prior to vitrification. BF DNA was subjected to whole-genome amplification (WGA) and analyzed by high-resolution next-generation sequencing (NGS).

Results

A high-quality WGA product was obtained from 8 of 11 (72.7 %) samples. Comparison of matching BF and blastomere samples showed that the genomic representation of sequencing reads was consistently similar with respect to density and regional coverage across the 24 chromosomes. A genome-wide survey of the sample sequencing data also indicated that BF was highly representative of known single gene sequences, and this observation was validated by PCR analyses of ten randomly selected genes, with an overall efficiency of 84 %.

Conclusion

This study provides further evidence that BF is a promising alternative source of DNA for PGT.

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References

  1. Brezina PR, Brezina DS, Kearns WG. Preimplantation genetic testing. BMJ. 2012;345, e5908. doi:10.1136/bmj.e5908.

    Article  PubMed  Google Scholar 

  2. Brezina PR, Kutteh WH. Clinical applications of preimplantation genetic testing. BMJ. 2015;350:g7611. doi:10.1136/bmj.g7611.

    Article  PubMed  Google Scholar 

  3. Yan L, Wei Y, Huang J, Zhu X, Shi X, Xia X, et al. Advances in preimplantation genetic diagnosis/screening. Sci Chin Life Sci. 2014;57(7):665–71. doi:10.1007/s11427-014-4683-5.

    Article  Google Scholar 

  4. Brezina PR, Ke RW, Kutteh WH. Preimplantation genetic screening: a practical guide. Clin Med Insights Reprod Health. 2013;7:37–42. doi:10.4137/CMRH.S10852.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Simpson JL. Preimplantation genetic diagnosis at 20 years. Prenat Diagn. 2010;30(7):682–95. doi:10.1002/pd.2552.

    Article  PubMed  Google Scholar 

  6. SenGupta SB, Delhanty JD. Preimplantation genetic diagnosis: recent triumphs and remaining challenges. Expert Rev Mol Diagn. 2012;12(6):585–92. doi:10.1586/erm.12.61.

    Article  CAS  PubMed  Google Scholar 

  7. Milachich T. New advances of preimplantation and prenatal genetic screening and noninvasive testing as a potential predictor of health status of babies. BioMed Res Int. 2014;2014:306505. doi:10.1155/2014/306505.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Fiorentino F, Biricik A, Nuccitelli A, De Palma R, Kahraman S, Sertyel S, et al. Rapid protocol for pre-conception genetic diagnosis of single gene mutations by first polar body analysis: a possible solution for the Italian patients. Prenat Diagn. 2008;28(1):62–4. doi:10.1002/pd.1905.

    Article  CAS  PubMed  Google Scholar 

  9. Capalbo A, Bono S, Spizzichino L, Biricik A, Baldi M, Colamaria S, et al. Reply: questions about the accuracy of polar body analysis for preimplantation genetic screening. Hum Reprod. 2013;28(6):1733–6. doi:10.1093/humrep/det070.

    Article  PubMed  Google Scholar 

  10. Hou Y, Fan W, Yan L, Li R, Lian Y, Huang J, et al. Genome analyses of single human oocytes. Cell. 2013;155(7):1492–506. doi:10.1016/j.cell.2013.11.040.

    Article  CAS  PubMed  Google Scholar 

  11. Salvaggio CN, Forman EJ, Garnsey HM, Treff NR, Scott Jr RT. Polar body based aneuploidy screening is poorly predictive of embryo ploidy and reproductive potential. J Assist Reprod Genet. 2014;31(9):1221–6. doi:10.1007/s10815-014-0293-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dahdouh EM, Balayla J, Audibert F, Wilson RD, Audibert F, Brock JA, et al. Technical update: preimplantation genetic diagnosis and screening. J Obstet Gynaecol Can: JOGC = J D’obstet Gynecol Du Can: JOGC. 2015;37(5):451–63.

    Google Scholar 

  13. Scott KL, Hong KH, Scott Jr RT. Selecting the optimal time to perform biopsy for preimplantation genetic testing. Fertil Steril. 2013;100(3):608–14. doi:10.1016/j.fertnstert.2013.07.004.

    Article  PubMed  Google Scholar 

  14. Scott Jr RT, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertil Steril. 2013;100(3):624–30. doi:10.1016/j.fertnstert.2013.04.039.

    Article  PubMed  Google Scholar 

  15. Goossens V, De Rycke M, De Vos A, Staessen C, Michiels A, Verpoest W, et al. Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis. Hum Reprod. 2008;23(3):481–92. doi:10.1093/humrep/dem327.

    Article  PubMed  Google Scholar 

  16. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod. 2006;21(12):3246–52. doi:10.1093/humrep/del285.

    Article  CAS  PubMed  Google Scholar 

  17. Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, Magnani M, et al. Genomic DNA in human blastocoele fluid. Reprod Biomed Online. 2013;26(6):603–10. doi:10.1016/j.rbmo.2013.02.012.

    Article  CAS  PubMed  Google Scholar 

  18. Gianaroli L, Magli MC, Pomante A, Crivello AM, Cafueri G, Valerio M, et al. Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertil Steril. 2014;102(6):1692–9. doi:10.1016/j.fertnstert.2014.08.021. e6.

    Article  CAS  PubMed  Google Scholar 

  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. 2015. doi:10.1016/j.fertnstert.2015.11.018.

    Google Scholar 

  20. Tobler KJ, Zhao Y, Ross R, Benner AT, Xu X, Du L, et al. Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis. Fertil Steril. 2015;104(2):418–25. doi:10.1016/j.fertnstert.2015.04.028.

    Article  CAS  PubMed  Google Scholar 

  21. Cohen J, Grudzinskas G, Johnson MH. Embryonic DNA sampling without biopsy: the beginnings of non-invasive PGD? Reprod Biomed Online. 2013;26(6):520–1. doi:10.1016/j.rbmo.2013.03.001.

    Article  CAS  PubMed  Google Scholar 

  22. Liang D, Lv W, Wang H, Xu L, Liu J, Li H, et al. Non-invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing. Prenat Diagn. 2013;33(5):409–15. doi:10.1002/pd.4033.

    Article  CAS  PubMed  Google Scholar 

  23. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril. 2000;73(6):1155–8.

    Article  CAS  PubMed  Google Scholar 

  24. Lukaszuk K, Pukszta S, Wells D, Cybulska C, Liss J, Plociennik L, et al. Routine use of next-generation sequencing for preimplantation genetic diagnosis of blastomeres obtained from embryos on day 3 in fresh in vitro fertilization cycles. Fertil Steril. 2015;103(4):1031–6. doi:10.1016/j.fertnstert.2014.12.123.

    Article  PubMed  Google Scholar 

  25. Chang LJ, Huang CC, Tsai YY, Hung CC, Fang MY, Lin YC, et al. Blastocyst biopsy and vitrification are effective for preimplantation genetic diagnosis of monogenic diseases. Hum Reprod. 2013;28(5):1435–44. doi:10.1093/humrep/det048.

    Article  PubMed  Google Scholar 

  26. Vanderzwalmen P, Bertin G, Debauche C, Standaert V, van Roosendaal E, Vandervorst M, et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod. 2002;17(3):744–51.

    Article  PubMed  Google Scholar 

  27. Hardy K, Handyside AH, Winston RM. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development. 1989;107(3):597–604.

    CAS  PubMed  Google Scholar 

  28. Hardy K, Stark J, Winston RM. Maintenance of the inner cell mass in human blastocysts from fragmented embryos. Biol Reprod. 2003;68(4):1165–9. doi:10.1095/biolreprod.102.010090.

    Article  CAS  PubMed  Google Scholar 

  29. Mouliere F, Rosenfeld N. Circulating tumor-derived DNA is shorter than somatic DNA in plasma. Proc Natl Acad Sci U S A. 2015;112(11):3178–9. doi:10.1073/pnas.1501321112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Taylor TH, Gitlin SA, Patrick JL, Crain JL, Wilson JM, Griffin DK. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans. Hum Reprod Update. 2014;20(4):571–81. doi:10.1093/humupd/dmu016.

    Article  CAS  PubMed  Google Scholar 

  31. Zong C, Lu S, Chapman AR, Xie XS. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Sci (New York, NY). 2012;338(6114):1622–6. doi:10.1126/science.1229164.

    Article  CAS  Google Scholar 

  32. Liu D, Liu C, DeVries S, Waldman F, Cote RJ, Datar RH. LM-PCR permits highly representative whole genome amplification of DNA isolated from small number of cells and paraffin-embedded tumor tissue sections. Diagn Mol Pathol: Am J Surg Pathol, Part B. 2004;13(2):105–15.

    Article  CAS  Google Scholar 

  33. Lam CW, Mak CM. Allele dropout caused by a non-primer-site SNV affecting PCR amplification—a call for next-generation primer design algorithm. Clinica Chimica Acta. Int J Clin Chem. 2013;421:208–12. doi:10.1016/j.cca.2013.03.014.

    CAS  Google Scholar 

  34. Natesan SA, Bladon AJ, Coskun S, Qubbaj W, Prates R, Munne S, et al. Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro. Genet Med: Off J Am Coll Med Genet. 2014;16(11):838–45. doi:10.1038/gim.2014.45.

    Article  CAS  Google Scholar 

  35. Gimenez C, Sarasa J, Arjona C, Vilamajo E, Martinez-Pasarell O, Wheeler K, et al. Karyomapping allows preimplantation genetic diagnosis of a de-novo deletion undetectable using conventional PGD technology. Reprod Biomed Online. 2015;31(6):770–5. doi:10.1016/j.rbmo.2015.08.017.

    Article  PubMed  Google Scholar 

  36. Konstantinidis M, Prates R, Goodall NN, Fischer J, Tecson V, Lemma T, et al. Live births following Karyomapping of human blastocysts: experience from clinical application of the method. Reprod Biomed Online. 2015;31(3):394–403. doi:10.1016/j.rbmo.2015.05.018.

    Article  PubMed  Google Scholar 

  37. Berger VK, Baker VL. Preimplantation diagnosis for single gene disorders. Semin Reprod Med. 2014;32(2):107–13. doi:10.1055/s-0033-1363552.

    Article  PubMed  Google Scholar 

  38. Wang L, Cram DS, Shen J, Wang X, Zhang J, Song Z, et al. Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos. Biol Reprod. 2014;91(2):37. doi:10.1095/biolreprod.114.120576.

    Article  PubMed  Google Scholar 

  39. D’Alessandro A, Federica G, Palini S, Bulletti C, Zolla L. A mass spectrometry-based targeted metabolomics strategy of human blastocoele fluid: a promising tool in fertility research. Mol BioSyst. 2012;8(4):953–8. doi:10.1039/c1mb05358b.

    Article  PubMed  Google Scholar 

  40. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10–2.

    Article  Google Scholar 

  41. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25:1754–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26(6):841–2. doi:10.1093/bioinformatics/btq033.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The study was supported by the grant awarded to Yuanqing Yao by the Key Program of the “Twelfth Five-Year Plan” of the People’s Liberation Army (No. BWS11J058) and the National High Technology Research and Development Program (2015AA020402).

Author information

Authors and Affiliations

  1. School of Medicine, Nankai University, Tianjin, 300071, China

    Yixin Zhang

  2. Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing, 100853, China

    Yixin Zhang, Li Wang, Minyue Ma, Hui Wang, Xiaofei Xu, Wenke Zhang, Yingyu Liu & Yuanqing Yao

  3. Department of Obstetrics and Gynecology, Affiliated Hospital of Academy of Military Medical Sciences, Beijing, 100071, China

    Na Li

  4. Center for Reproductive Medicine, The First Hospital of Kunming, Kunming, 650011, China

    Li Wang

  5. Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China

    Huiying Sun & Baofa Sun

  6. Berry Genomics, Co., Ltd., Beijing, 100015, China

    David S. Cram

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  1. Yixin Zhang
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Correspondence to Baofa Sun or Yuanqing Yao.

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Ethical approval

This study was approved by the Institutional Review Board of Chinese PLA General Hospital (S2013-092-02). All embryos donated to research were obtained after obtaining informed written consent by couples undertaking PGS.

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Zhang, Y., Li, N., Wang, L. et al. Molecular analysis of DNA in blastocoele fluid using next-generation sequencing. J Assist Reprod Genet 33, 637–645 (2016). https://doi.org/10.1007/s10815-016-0667-7

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  • DOI: https://doi.org/10.1007/s10815-016-0667-7

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