Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
권호 표지
DOI QR코드

DOI QR Code

Enhancement of preimplantation mouse embryo development with optimized in vitro culture dish via stabilization of medium osmolarity

  • Hyejin Yoon (Department of Senior Healthcare, Graduate School of Eulji University) ;
  • Jongwoo Lee (CNC Biotech Incorporated) ;
  • Inyoung Kang (Department of Biomedical Laboratory Science, Graduate School of Eulji University) ;
  • Kyoo Wan Choi (CNC Biotech Incorporated) ;
  • Jaewang Lee (Department of Biomedical Laboratory Science, Graduate School of Eulji University) ;
  • Jin Hyun Jun (Department of Senior Healthcare, Graduate School of Eulji University)
  • Received : 2023.09.01
  • Accepted : 2023.11.06
  • Published : 2023.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Objective: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Methods: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. Results: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Conclusion: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

Keywords

Acknowledgement

This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare of the Republic of Korea (grant numbers: HI21C135003 and HI21C1639).

References

  1. Wale PL, Gardner DK. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Reprod Update 2016;22:2-22.
  2. Scarica C, Monaco A, Borini A, Pontemezzo E, Bonanni V, De Santis L, et al. Use of mineral oil in IVF culture systems: physico-chemical aspects, management, and safety. J Assist Reprod Genet 2022;39:883-92.
  3. Wiley LM, Yamami S, Van Muyden D. Effect of potassium concentration, type of protein supplement, and embryo density on mouse preimplantation development in vitro. Fertil Steril 1986;45:111-9.
  4. Ruiz M, Santamaria-Lopez E, Blasco V, Hernaez MJ, Caligara C, Pellicer A, et al. Effect of group embryo culture under low-oxygen tension in benchtop incubators on human embryo culture: prospective, randomized, controlled trial. Reprod Sci 2020;27:1522-33.
  5. Stojanov T, Alechna S, O'Neill C. In-vitro fertilization and culture of mouse embryos in vitro significantly retards the onset of insulin-like growth factor-II expression from the zygotic genome. Mol Hum Reprod 1999;5:116-24.
  6. Gopichandran N, Leese HJ. The effect of paracrine/autocrine interactions on the in vitro culture of bovine preimplantation embryos. Reproduction 2006;131:269-77.
  7. Salahuddin S, Ookutsu S, Goto K, Nakanishi Y, Nagata Y. Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos. Hum Reprod 1995;10:2382-5.
  8. Johnson MH, Nasr-Esfahani MH. Radical solutions and cultural problems: could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro? Bioessays 1994;16:31-8.
  9. Joo BS, Kim MK, Na YJ, Moon HS, Lee KS, Kim HD. The mechanism of action of coculture on embryo development in the mouse model: direct embryo-to-cell contact and the removal of deleterious components. Fertil Steril 2001;75:193-9.
  10. Paria BC, Dey SK. Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci U S A 1990;87:4756-60.
  11. Kane MT, Carney EW, Ellington JE. The role of nutrients, peptide growth factors and co-culture cells in development of preimplantation embryos in vitro. Theriogenology 1992;38:297-313.
  12. Gandolfi F. Autocrine, paracrine and environmental factors influencing embryonic development from zygote to blastocyst. Theriogenology 1994;41:95-100.
  13. Christianson MS, Zhao Y, Shoham G, Granot I, Safran A, Khafagy A, et al. Embryo catheter loading and embryo culture techniques: results of a worldwide Web-based survey. J Assist Reprod Genet 2014;31:1029-36.
  14. Fancsovits P, Pribenszky C, Lehner A, Murber A, Kaszas Z, Nemes A, et al. Prospective-randomized study comparing clinical outcomes of IVF treatments where embryos were cultured individually or in a microwell group culture dish. Biol Futur 2022;73:229-36.
  15. Reed ML, Woodward BJ, Swain JE. Single or group culture of mammalian embryos: the verdict of the literature. J Reprod Biotechnol Fertil 2011;2:77-87.
  16. Herreros M, Marti L, Diaz N, Tio MC, Rodriguez-Arnedo A, Guerrero J, et al. Impact of group embryo culture vs individual embryo culture strategies on blastocyst rate and quality. Hum Reprod 2022;37(Supplement 1):deac104.077.
  17. De Munck N, Santos-Ribeiro S, Mateizel I, Verheyen G. Reduced blastocyst formation in reduced culture volume. J Assist Reprod Genet 2015;32:1365-70.
  18. Minasi MG, Fabozzi G, Casciani V, Lobascio AM, Colasante A, Scarselli F, et al. Improved blastocyst formation with reduced culture volume: comparison of three different culture conditions on 1128 sibling human zygotes. J Assist Reprod Genet 2015;32:215-20.
  19. Tagawa M, Matoba S, Narita M, Saito N, Nagai T, Imai K. Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system. Theriogenology 2008;69:574-82.
  20. Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, et al. New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system. Mol Reprod Dev 2000;55:256-64.
  21. Swain JE, Pool TB. ART failure: oocyte contributions to unsuccessful fertilization. Hum Reprod Update 2008;14:431-46.
  22. Biggers JD, Summers MC. Choosing a culture medium: making informed choices. Fertil Steril 2008;90:473-83.
  23. Wood SA, Allen ND, Rossant J, Auerbach A, Nagy A. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 1993;365:87-9.
  24. Vajta G. Cloning: a sleeping beauty awaiting the kiss? Cell Reprogram 2018;20:145-56.
  25. Ieda S, Akai T, Sakaguchi Y, Shimamura S, Sugawara A, Kaneda M, et al. A microwell culture system that allows group culture and is compatible with human single media. J Assist Reprod Genet 2018;35:1869-80.
  26. Park JC, Kim JA, Kim DJ, Bae JG, Kim JI, Rhee JH. Effect of human hydrosalpingeal fluid on the development of mouse embryo. Korean J Reprod Med 2010;37:125-34.
  27. Kim HJ, Yoon HJ, Jang JM, Lee WD, Yoon SH, Lim JH. Evaluation of human embryo development in in vitro fertilization- and intracytoplasmic sperm injection-fertilized oocytes: a time-lapse study. Clin Exp Reprod Med 2017;44:90-5.
  28. Hur YS, Ryu EK, Hyun CS, Yang SH, Yoon SH, Lim KS, et al. Retrospective study of single vitrified-warmed blastocyst transfer cycles according to the presence of morphokinetic variables. Clin Exp Reprod Med 2018;45:52-5.
  29. Lee SC, Seo HC, Lee J, Jun JH, Choi KW. Effects of dynamic oxygen concentrations on the development of mouse pre- and peri-implantation embryos using a double-channel gas supply incubator system. Clin Exp Reprod Med 2019;46:189-96.
  30. Kim J, Lee J, Jun JH. Non-invasive evaluation of embryo quality for the selection of transferable embryos in human in vitro fertilization-embryo transfer. Clin Exp Reprod Med 2022;49:225-38.
  31. Morbeck DE, Krisher RL, Herrick JR, Baumann NA, Matern D, Moyer T. Composition of commercial media used for human embryo culture. Fertil Steril 2014;102:759-66.
  32. Hegele-Hartung C, Piegsa K, Fischer B. Effect of osmolarity of the fixative on the ultrastructure of preimplantation rabbit embryos. Acta Anat (Basel) 1989;136:79-88.
  33. Lawitts JA, Biggers JD. Overcoming the 2-cell block by modifying standard components in a mouse embryo culture medium. Biol Reprod 1991;45:245-51.
  34. Biggers JD, Lawitts JA, Lechene CP. The protective action of betaine on the deleterious effects of NaCl on preimplantation mouse embryos in vitro. Mol Reprod Dev 1993;34:380-90.
  35. Lim JM, Kim JH, Okuda K, Niwa K. The importance of NaCl concentration in a chemically defined medium for the development of bovine oocytes matured and fertilized in vitro. Theriogenology 1994;42:421-32.
  36. Hwang IS, Park MR, Moon HJ, Shim JH, Kim DH, Yang BC, et al. Osmolarity at early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) in pre-implantation porcine NT embryos. Mol Reprod Dev 2008;75:464-71.
  37. Lane M, Gardner DK. Understanding cellular disruptions during early embryo development that perturb viability and fetal development. Reprod Fertil Dev 2005;17:371-8.
  38. Valojerdi MR, Karimian L, Yazdi PE, Gilani MA, Madani T, Baghestani AR. Efficacy of a human embryo transfer medium: a prospective, randomized clinical trial study. J Assist Reprod Genet 2006;23:207-12.
  39. Meseguer M, Herrero J, Tejera A, Hilligsoe KM, Ramsing NB, Remohi J. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod 2011;26:2658-71.
  40. Rubio I, Kuhlmann R, Agerholm I, Kirk J, Herrero J, Escriba MJ, et al. Limited implantation success of direct-cleaved human zygotes: a time-lapse study. Fertil Steril 2012;98:1458-63.
  41. 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;2:CD011320.
  42. Kirkegaard K, Kesmodel US, Hindkjaer JJ, Ingerslev HJ. Time-lapse parameters as predictors of blastocyst development and pregnancy outcome in embryos from good prognosis patients: a prospective cohort study. Hum Reprod 2013;28:2643-51.