Prediction of the blastulation onset in one day behind embryos by the expression profile of small non-coding RNAs


Y.S. Drapkina, A.V. Timofeeva, V.V. Chagovets, N.P. Makarova, A.M. Frolova, E.A. Kalinina Kulakov National Medical Research Center of Obstetrics, Gynecology, and Perinatology, Akademika Oparina str., 4, Moscow, 117997, Russian Federation E-mail: [email protected]

Introduction. Successful embryonic development immediately after fertilization depends on the maternal messenger RNAs (mRNAs) destruction and zygotic genome transcription activation during maternal to zygotic transition (MZT). Key regulators of MZP are small non-coding RNAs (sncRNAs) – microRNAs and piwiRNAs. These molecules can be identified in embryo culture medium and used as a marker of embryo quality, blastulation ability, as well as its implantation potential. The aim this study. To analyze blastulation rate in embryos, which are one day behind in development, according to microRNAs and piwiRNAs expression profile in their culture medium. Methods. The RT-PCR method was used for the quantitative analysis of microRNAs and piwiRNAs in 22 samples of embryo culture media obtained on day 4 after fertilization. Embryos included were one day behind in development and were at the 8-cells stage. The samples were divided into three groups according to morphological and functional characteristics of the embryos on day 6: group I – average/excellent blastocysts (1 – 4AA, 1 – 4BB, 1 – 6BB, 1 – 3AA, 1 – 3BB), group II – poor quality blastocysts (3 – 4СС, 4 – 3СС, 1 – 2СС, 1 – 3ВС, and 1 – 4СВ) and group III – degraded embryos (7). Results. The culture media from embryos which degrade or become poor quality blastocysts on day 6 has almost the same level of microRNA let-7i-5p, but microRNA let-7i-5p expression level is statistically significantly higher in culture medium of the embryos which are capable to develop into average/excellent quality blastocyst. In culture media of embryos which develop into average/excellent quality blastocyst, the level of microRNA let-7b-5p and piwiRNA piR020401 is also statistically significantly higher then in culture media of degraded embryos and embryos which developed into bad quality blastocysts. Conclusion. The data obtained confirm the level of microRNA let-7b-5p, microRNA let-7i-5p and piwiRNA piR020401 to show embryo quality and development potential. Assessment of their expression level can be used for diagnostic and prognostic purposes in selecting the best quality embryo.
ART, culture medium, embryo, microRNAs, piwiRNAs, pregnancy

Список литературы: 
  1. Gardner D., Balaban B. Assessment of human embryo development using morpho-logical criteria in an era of time-lapse, algorithms and ‘OMICS’: is looking good still important? Mol. Hum. Reprod. 2016; 22 (10): 704–18.
  2. Blake D., Farquhar C., Johnson N., Proctor M. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database Syst. Rev. 2016; 30 (6): CD002118.
  3. Papanikolaou E., Kolibianakis E., Tournaye H., Venetis C., Fatemi H., Tarlatzis B., Devroey P. 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 (1): 91–9.
  4. Kang S., Lee S., Jeong H., Yoon S., Koh M., Lim J., and Lee S. Clinical outcomes of elective single morula embryo transfer versus elective single blastocyst embryo transfer in IVF-ET. J. Assist. Reprod. Genet. 2012; 29 (5): 423–8.
  5. De Vos A., Van Landuyt L., Santos-Ribeiro S., Camus M., Van de Velde H., Tournaye H., Verheyen G. Cumulative live birth rates after fresh and vitrified cleavage-stage versus blastocyst-stage embryo transfer in the first treatment cycle. Hum. Reprod. 2016; 31 (11): 2442–9.
  6. Haas J., Meriano J., Bassil R., Barzilay E., Zilberberg E., Casper R. Developmental potential of slow-developing embryos: day-5 morulae compared with day-5 cavitating morulae. Fertil. Steril. 2019; 111 (1): 105–11.
  7. Timofeeva A.V., Kalinina E.A., Drapkina Ju.S., Chagovets V.V., Makarova N.P., Suhih G.T. Otsenka kachestva embriona po profilju ekspressii malyh nekodirujuschih RNK v kul'tural'noj srede embriona v programmah vspomogatel'nyh reproduktivnyh tehnologij. Akusherstvo i ginekologija. 2019; 6: 79–86. [Timofeeva A.V., Kalinina E.A., Drapkina Yu.S., Chagovec V.V., Makarova N.P., Suhih G.T. Embryo quality assessment with the help of small non-coding RNAs in embryo culture medium during IVF programme. Akusherstvo i ginekologiya. 2019; 6: 79–86.]
  8. Drapkina Ju.S., Timofeeva A.V., Chagovets V.V., Kononihin A.S., Frankevich V.E., Kalinina E.A. Primenenie omiksnyh tehnologij v reshenii problem reproduktivnoj meditsiny. Akusherstvo i ginekologija. 2018; 9: 24–32. [Drapkina Yu.S., Timofeeva A.V., Chagovec V.V., Kononihin A.S., Frankevich V.E., Kalinina E.A. OMICS technology in reproductive medicine. Akusherstvo i ginekologiya. 2018; 9: 24–32.]
  9. Chua J., Armugam A., Jeyaseelan K. MicroRNAs: biogenesis, function and applications. Curr. Opin. Mol. Ther. 2009; 11 (2): 189–99.
  10. Timofeeva A., Chagovets V., Drapkina Y., Makarova N., Kalinina E., Sukhikh G. Cell-Free, Embryo-Specific sncRNA as a Molecular Biological Bridge between Patient Fertility and IVF Efficiency. Int. J. Mol. Sci. 2019; 20 (12).
  11. Siomi M., Sato K., Pezic D., Aravin A. PIWI-interacting small RNAs: the vanguard of genome defence. Nat. Rev. Mol. Cell. Biol. 2011; 12 (4): 246–58.
  12. Giraldez A., Mishima Y., Rihel J., Grocock R., Van Dongen S., Inoue K., Enright A., Schier A. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science. 2006; 312 (5770): 75–9.
  13. Svoboda P., Flemr M. The role of miRNAs and endogenous siRNAs in maternal-to-zygotic reprogramming and the establishment of pluripotency. EMBO Rep. 2010; 11 (8): 590–7.
  14. Han B., Wang W., Li C., Weng Z., Zamore P. PiRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production. Science. 2015; 6236: 817–21.
  15. Liying Y., Mingyu Y., Hongshan G., Lu Y., Jun W., Rong L., Ping L., Ying L., Xiaoying Z., Jie Y., Jin H., Ming L., Xinglong W., Lu W., Kaiqin L., Ruiqiang L., Jie Q., Fuchou T. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat. Struct. Mol. Biol. 2013; 20 (9): 1131–9.
  16. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum. Reprod. 2011; 26 (6): 1270–83.
  17. R Core Team. A language and environment for statistical computing. Sci. Rep. 2017; 7: 17598.
  18. RStudio Team. RStudio: Integrated Development for R. RStudio, Inc., Boston, MA URL.
  19. Schier A. The Maternal-Zygotic Transition: Death and Birth of RNAs. Science. 2007; 316 (5823): 406–7.
  20. Yuan S., Schuster A., Tang C., Yu T. Ortogero N., Bao J., Zheng H., Yan W. Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development. Development. 2016; 143 (4): 635–47.