Estimating zona pellucida hardness under microinjection to assess oocyte/embryo quality: Analytical and experimental studies


The precise determination of zona pellucida (ZP) hardness is largely unknown due to the lack of appropriate measuring and modelling methods. In this study, we have used experimental and theoretical models to describe the mechanical behavior of a single oocyte cell to improve the assisted reproductive technology (ART) outcomes by assessing oocyte/embryo quality. This paper presents the development of: i) a microinjection model to estimate the force of ZP penetration, ii) a micropipette aspiration model to determine the corresponding hardness, and iii) an experimental procedure to generate the required data for these two models. Our results show that the estimated penetration force provides a performance target for the penetration process during intracytoplasmic sperm injection (ICSI), while the estimated corresponding hardness serves as an indicator of the extent of deformation sustained by the oocyte prior to penetration. Evaluation of these results shows that a routine assessment of ZP hardness under microinjection would allow for the identification of certain oocyte pools for which further manipulation is recommended in order to improve injection, hatching and finally ART outcomes.

Share and Cite:

Khalilian, M. , Valojerdi, M. , Navidbakhsh, M. , Chizari, M. and Eftekhari-Yazdi, P. (2013) Estimating zona pellucida hardness under microinjection to assess oocyte/embryo quality: Analytical and experimental studies. Advances in Bioscience and Biotechnology, 4, 679-688. doi: 10.4236/abb.2013.45089.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Lanzendorf, S.E., Maloney, M.K., Veek, L.L., Slusser, J., Hodgen, G.D. and Rosenwaks, Z. (1998) A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertility and Sterility, 49, 835-842.
[2] Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718. doi:10.1016/0140-6736(92)92425-F
[3] Palermo, G.D., Alikani, M., Bertoli, M., Colombero, L.T., Moy, F., Cohen, J. and Rosenwaks, Z. (1996) Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by intracytoplasmic sperm injection. Human Reproduction, 11, 172-176. doi:10.1093/oxfordjournals.humrep.a019012
[4] Van Steirteghem, A.C., Nagy, Z., Joris, H., Liu, J., Staessen, C., Smitz, J., Wisanto, A. and Devroey, P. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Human Reproduction, 8, 10611066.
[5] Mansour, R. (1998) Intracytoplasmic sperm injection: A state of the art technique. Human Reproduction Update, 4, 43-56. doi:10.1093/humupd/4.1.43
[6] Nagy, Z.P., Liu, J., Joris, H., Bocken, G., Desmet, B., Van Ranst, H., Vankelecom, A., Devroey, P. and Van Steirteghem, A.C. (1995) The influence of the site of sperm deposition and mode of oolemma breakage at intracytoplasmic sperm injection on fertilization and embryo development rates. Human Reproduction, 10, 31713177.
[7] Yavas, Y., Roberge, S., Khamsi, F., Shirazi, P., Endman, M.W. and Wong, J.C. (2001) Performing ICSI using an injection pipette with the smallest possible inner diameter and a long taper increases normal fertilization rate, decreases incidence of degeneration and tripronuclear zygotes, and enhances embryo development. Journal of Assisted Reproduction and Genetics, 18, 426-435. doi:10.1023/A:1016634704469
[8] Dumoulin, J.C.M., Coonen, E., Bras. M., Bergers-Janssen, J.M., Ignoul-Vanvuchelen, R.C., van Wissen, L.C., Geraedts, J.P. and Evers, J.L. (2001) Embryo development and chromosomal anomalies after ICSI: Effect of the injection procedure. Human Reproduction, 16, 306-312. doi:10.1093/humrep/16.2.306
[9] Ebner, T., Moser, M., Sommergruber, M., Puchner, M., Wiesinger, R. and Tews, G. (2003) Developmental competence of oocytes showing increased cytoplasmic viscosity. Human Reproduction, 18, 1294-1298. doi:10.1093/humrep/deg232
[10] Zhang, X., Rutledge, J. and Armstrong, D.T. (1991) Studies on zona hardening in rat oocytes that are matured in vitro in a serum-free medium. Molecular Reproduction and Development, 28, 292-296. doi:10.1002/mrd.1080280312
[11] Fong, C.Y., Bongso, A., Sathananthan, H., Ho, J. and Ng, S.C. (2001) Ultrastructural observations of enzymatically treated human blastocysts: Zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties. Human Reproduction, 16, 540-546. doi:10.1093/humrep/16.3.540
[12] Edwards, R.G., Fishel, S.B., Cohen, J., Fehilly, C.B., Purdy, J.M. and Salter, J.M. (1984) Factors influencing the success of in vitro fertilization for alleviating human infertility. Journal in Vitro Fertilization and Embryo Transfer, 1, 3-23. doi:10.1007/BF01129615
[13] Cohen, J., Alikani, M., Trowbridge, J. and Rosenwacks, Z. (1992) Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Human Reproduction, 7, 685-691.
[14] Bertrand, E., Van den Bergh, M. and Englert, Y. (1996) Clinical parameters influencing human zona pellucida thickness. Fertility and Sterility, 66, 408-411.
[15] Huang, T., Kimura, Y. and Yanagimachi, R. (1996) The use of piezo micromanipulation for intracytoplasmic sperminjection of human oocytes. Journal of Assisted Reproduction and Genetic, 13, 320-328. doi:10.1007/BF02070146
[16] Yanagida, K., Katayose, H., Yazawa, H., Kimura, Y., Konnai, K. and Sato, A. (1999) The usefulness of a Piezo-micromanipulator in intracytoplasmic sperm injection in humans. Human Reproduction, 14, 448-453. doi:10.1093/humrep/14.2.448
[17] Rienzi, L.B., Greco, E., Ubaldi, F., Iacobelli, M., Martinez, F. and Tesarik, J. (2001) Laser-assisted intracytoplasmic sperm injection. Fertility and Sterility, 76, 10451047. doi:10.1016/S0015-0282(01)02861-8
[18] Nagy, Z.P., Oliveira, S.A., Abdelmassih, V. and Abdelmassih, R. (2002) Novel use of laser to assist ICSI for patients with fragile oocytes: A case report. Reproductive and Biomedicine Online, 4, 27-31. doi:10.1016/S1472-6483(10)61911-6
[19] Abdelmassih, S., Cardoso, J., Abdelmassih, V., Dias, J.A., Abdelmassih, R. and Nagy, Z.P. (2002) Laser-assisted ICSI: A novel approach to obtain higher oocyte survival and embryo quality rates. Human Reproduction, 17, 26942699. doi:10.1093/humrep/17.10.2694
[20] Moser, M., Ebner, T., Sommergruber, M., Gaisswinkler, U., Jesacher, K., Puchner, M., Wiesinger, R. and Tews, G. (2004) Laser-assisted zona pellucida thinning prior to routine ICSI. Human Reproduction, 19, 573-578. doi:10.1093/humrep/deh093
[21] Sun, Y., Wan, K., Roverts, K., Bischof, J. and Nelson, B.J. (2003) Mechanical property characterization of mouse zona pellucida. IEEE Transaction on Nanobioscience, 2, 279-286. doi:10.1109/TNB.2003.820273
[22] Murayama, Y., Constantinou, C.E. and Omata, S. (2004) Micro-mechanical sensing platform for the characterization of the elastic properties of the ovum via uniaxial measurement. Journal of Biomechanics, 37, 67-72. doi:10.1016/S0021-9290(03)00242-2
[23] Murayama, Y. , Mizuno, J. , Kamakura, H., Fueta, Y. , Nakamura, H. , Akaishi, K, Anzai, K., Watanabe, A., Inui, H. and Omata, S. (2006) Mouse zona pellucida dynamically changes its elasticity during oocyte maturation, fertilization and early embryo development. Human Cell, 19, 119-125. doi:10.1111/j.1749-0774.2006.00019.x
[24] Murayama, Y., Yoshida, M., Mizuno, J., Nakamura, H., Inoue, S., et al. (2008) Elasticity Measurement of Zona Pellucida Using a Micro Tactile Sensor to Evaluate Embryo Quality. Journal of Mammalian Ova Research, 25, 8-16. doi:10.1274/jmor.25.8
[25] Kim, D.H., Hwang, C.N., Sun, Y., Lee, S.H., Kim, B. and Nelson, B.J. (2006) Mechanical analysis of chorion softening in prehatching stages of zebrafish embryos. IEEE Transaction on Nanobioscience, 5, 89-94. doi:10.1109/TNB.2006.875054
[26] Khalilian, M., Navid-bakhsh, M., Rezazadeh-Valojerdi, M., Chizari, M. and Eftekhari-Yazdi, P. (2009) Estimating young’s modulus of zona pellucida by micropipette aspiration in combination with theoretical models of ovum. Journal of the Royal Society Interface, 7, 689-694. doi:10.1098/rsif.2009.0380
[27] Khalilian, M., Navidbakhsh, M., Rezazadeh-Valojerdi, M., Chizari, M. and Eftekhari-Yazdi, P. (2010) Alteration in the mechanical properties of human ovum zona pellucida following fertilization: Experimental and analytical studies. Experimental Mechanics, 51, 175-182. doi:10.1007/s11340-010-9357-z
[28] Wan, K.T., Chan, V. and Dillard, D. (2002) Constitutive equation for elastic indentation of a thin-walled bio-mimetic microcapsule by an atomic force microscope tip. Colloids and Surfaces B: Biointerfaces, 27, 241-248. doi:10.1016/S0927-7765(02)00073-5
[29] Porter, R.N., Smith, W., Craft, I.L., Abdulwahid, N.A. and Jacobs, H.S. (1984) Induction of ovulation for in vitro fertilization using buserelin and gonadotropins. Lancet, 2, 1284-1285. doi:10.1016/S0140-6736(84)92840-X
[30] Evans, E.A. and Skalak, R. (1980) Mechanics and thermodynamics of biomembranes. CRC Press Inc., Boca Raton.
[31] Cheng, L.Y. (1987) Deformation analyses in cell and developmental biology. Part I-Format methodology. Journal of Biomechanical Engineering, 109, 10–17. doi:10.1115/1.3138634
[32] Cheng, L.Y. (1980) Deformation analyses in cell a developmental biology. Part II: Mechanical experiments on cell. Journal of Biomechanical Engineering, 109, 18-24. doi:10.1115/1.3138635
[33] Smith, A.E., Moxham, K.E. and Middelberg, A.P.J. (1998) On uniquely determining cell-wall material properties with the compression experiment. Chemical Engineering Science, 53, 3913-3922. doi:10.1016/S0009-2509(98)00198-5
[34] Zhu, C., Bao, G. and Wang, N. (200) Cell Mechanics: Mechanical response, cell adhesion, and molecular deformation. Annual Review of Biomedical Engineering, 2, 189-226. doi:10.1146/annurev.bioeng.2.1.189
[35] Foo, J.J., Chan, V. and Liu, K.K. (2006) Coupling bending and shear effects on liposome deformation. Journal of Biomechanics, 39, 2338-2343. doi:10.1016/j.jbiomech.2005.07.008
[36] Liu, K.K. (2006) Deformation behaviour of soft particles: A review. Journal of Physics D: Applied Physics, 39, 189-199. doi:10.1088/0022-3727/39/11/R01
[37] Lardner, T.J. and Pujara, P. (1980) Compression of spherical cells. Mechanics Today, 5, 161-176.
[38] Liu, K.K., Williams, D.R. and Briscoe, B.J. (1999) A new method to characterize the mechanical properties of a single cell. Medical and Biological Engineering and Computing, 37, 1506-1507.
[39] Ju, B.F., Liu, K.K., Ling, S.F. and Ng, W.H. (2002) A novel technique for characterizing elastic properties of thin biological membrane. Mechanics of Materials, 34, 749-754. doi:10.1016/S0167-6636(02)00182-5
[40] Boulbitch, A. (2000) Deformation of the envelope of a spherical Gram-negative bacterium during the atomic force microscopic measurement. Journal of Electron Microscopy, 49, 459-462. doi:10.1093/oxfordjournals.jmicro.a023829
[41] Arnoldi, M., Fritz, M., Baeuerlein, E., Radmacher, M., Sackmann, E. and Boulbitch, A. (2000) Bacterial turgor pressure can be measured by atomic force microscopy. Physical Review, 62, 1034-1044.
[42] Yao, X., Walter, S., Burke, S., Stewart, M.H., Jericho, M.H., Pink, D., Hunter, R. and Beveridge, T.J. (2002) Atomic force microscopy and theoretical considerations of surface properties and turgor pressures of bacteria. Colloids and Surfaces B: Biointerfaces, 23, 213-230. doi:10.1016/S0927-7765(01)00249-1
[43] Feng, W.W. and Yang, W.H. (1973) On the contact problem of an inflated spherical nonlinear membrane. Transaction of the American Society of Mechanical Engineers: Journal of Applied Mechanics, 40, 209-214. doi:10.1115/1.3422928
[44] Tatara, Y. (1991) On compression of rubber elastic sphere over a large range of displacements-Part I: Theoretical study. ASME Journal of Engineering Material and Technology, 113, 285-291. doi:10.1115/1.2903407
[45] Evans, E. and Yeung, A. (1973) New membrane concept applied to the analysis of fluid shearand micropipettedeformed red blood cells. Biophysics Journal, 13, 941954. doi:10.1016/S0006-3495(73)86036-9
[46] Evans, E. and Yeung, A. (1989) Apparent viscosity and cortical tension of blood granulocytes determined by micropipette aspiration. Biophysics Journal, 56, 151-160. doi:10.1016/S0006-3495(89)82660-8
[47] Hochmuth, R.M. (2000) Micropipette aspiration of living cells. Journal of Biomechanics, 33, 15-22. doi:10.1016/S0021-9290(99)00175-X
[48] Bao, G. and Suresh, S. (2003) Cell and molecular mechanics of biological materials. Nature Materials, 2, 715725.
[49] Richter, K.S., Davis, A., Carter, J., Greenhouse, S.J., Mottla, G.L. and Tucker, M.J. (2006) No advantage of laser-assisted over conventional intracytoplasmic sperm injection: A randomized controlled trial. Journal of Experimental & Clinical Assisted Reproduction, 3, 1-5. doi:10.1186/1743-1050-3-5

Copyright © 2022 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.