Hostname: page-component-78c5997874-dh8gc Total loading time: 0 Render date: 2024-11-10T14:00:09.133Z Has data issue: false hasContentIssue false
  • English
  • Français

Effects of vitrification on blastomere viability and cytoskeletal integrity in mouse embryos

Published online by Cambridge University Press:  07 December 2016

Zenon Oikonomou ,
Katerina Chatzimeletiou ,
Antonia Sioga ,
Louisa Oikonomou ,
Basil C. Tarlatzis  and
Efstratios Kolibianakis
Zenon Oikonomou
Affiliation:
Unit of Human Reproduction, 1st Department of Obstetrics and Gynaecology, Medical School, Aristotle University, Papageorgiou General Hospital, Thessaloniki, Greece.
Katerina Chatzimeletiou*
Affiliation:
Unit for Human Reproduction, 1st Department of Obstetrics & Gynaecology, Medical School, Aristotle University, Papageorgiou General Hospital, Thessaloniki, 56403, Greece.
Antonia Sioga
Affiliation:
Laboratory of Histology and Embryology, Medical School, Aristotle University, Thessaloniki, Greece.
Louisa Oikonomou
Affiliation:
Laboratory of Histology and Embryology, Medical School, Aristotle University, Thessaloniki, Greece.
Basil C. Tarlatzis
Affiliation:
Unit of Human Reproduction, 1st Department of Obstetrics and Gynaecology, Medical School, Aristotle University, Papageorgiou General Hospital, Thessaloniki, Greece.
Efstratios Kolibianakis
Affiliation:
Unit of Human Reproduction, 1st Department of Obstetrics and Gynaecology, Medical School, Aristotle University, Papageorgiou General Hospital, Thessaloniki, Greece.
*
All correspondence to: Katerina Chatzimeletiou. Unit for Human Reproduction, 1st Department of Obstetrics & Gynaecology, Medical School, Aristotle University, Papageorgiou General Hospital, Thessaloniki, 56403, Greece. Tel: +30 231 332 3827; E-mail: katerinachatzime@hotmail.com
Get access Rights & Permissions [Opens in a new window]

Summary

Vitrification is widely used to cryopreserve supernumerary embryos following in vitro fertilization (IVF). The mouse model was used to investigate the effects of vitrification on blastomere viability, using viability markers, and on the cytoskeleton, by analysing spindle/chromosome configurations, using confocal scanning microscopy. Ninety cleavage and morula stage dimethyl sulphoxide (DMSO)/EG vitrified mouse embryos were either processed immediately following warming for viability assessment by labelling with the fluorescent markers carboxyfluorescein-diacetate succinimidylester (CFSE) and propidium iodide (PI) or were cultured to the blastocyst stage and immunostained with α-tubulin antibody to visualize microtubules and DAPI or PI to visualize DNA. Sixty-five fresh embryos were also used as the control. Vitrified embryos showed high survival rates following warming, but they had a higher incidence of damaged blastomeres compared with fresh embryos. Most mitotic spindles examined in all groups were normal, but multivariable analysis revealed that the proportion of abnormal spindles was significantly higher in vitrified/warmed embryos (P < 0.05). This study is the first to examine the immediate effects of vitrification on blastomere viability, using fluorescent markers and shows that although vitrification results in a higher incidence of damaged blastomeres, vitrified embryos may compensate for this limited number of damaged/abnormal cells, as development to the blastocyst stage was not compromised.

Keywords

Aseptic vitrificationConfocal laser scanning microscopyCytoskeletal analysisDMSO/EGMouse embryosViability markers
Type
Research Article
Information
Zygote , Volume 25 , Issue 1 , February 2017 , pp. 75 - 84
Copyright
Copyright © Cambridge University Press 2016 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Bettencourt, E.M., Bettencourt, C.M., Silva, J.N., Ferreira, P., de Matos, C.P., Oliveira, E., Romão, R.J., Rocha, A. & Sousa, M. (2009). Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos. Theriogenology 71, 947–58.Google Scholar
Chatzimeletiou, K., Picton, H.M. & Handyside, A.H. (2001). Use of a non-contact, infrared laser for zona drilling of mouse embryos: assessment of immediate effects on blastomere viability. Reprod. Biomed. Online 2, 178–87.Google Scholar
Chatzimeletiou, K., Morrison, E.E., Prapas, N., Prapas, Y. & Handyside, A.H. (2005a). Spindle abnormalities in normally developing and arrested human preimplantation embryos in vitro identified by confocal laser scanning microscopy. Hum. Reprod. 20, 672–82.Google Scholar
Chatzimeletiou, K., Morrison, E.E., Panagiotidis, Y., Prapas, N., Prapas, Y., Rutherford, A.J., Grudzinskas, G. & Handyside, A.H. (2005b). Comparison of effects of zona drilling by non contact infrared laser or acid Tyrode's on the development of human biopsied embryos as revealed by blastomere viability, cytoskeletal analysis and molecular cytogenetics. Reprod. Biomed. Online 11, 697–710.Google Scholar
Chatzimeletiou, K., Morrison, E.E., Prapas, N., Prapas, Y. & Handyside, A.H. (2008). Symposium: Genetic and epigenetic aspects of assisted reproduction. The centrosome and early embryogenesis: clinical insights. Reprod. Biomed. Online 16, 485–91.Google Scholar
Chatzimeletiou, K., Morrison, E.E., Panagiotidis, Y., Vanderzwalmen, P., Prapas, N., Prapas, Y., Tarlatzis, B.K. & Handyside, A.H. (2012). Cytoskeletal analysis of human blastocysts by confocal laser scanning microscopy following vitrification Hum. Reprod. 27, 106–13.Google Scholar
Chen, Y., Zheng, X., Yan, J., Qiao, J. & Liu, P. (2013). Neonatal outcomes after the transfer of vitrified blastocysts: closed versus open vitrification system. Reprod. Biol. Endocrinol. 11, 107.Google Scholar
Chi, F., Luo, C., Yin, P., Hong, L., Ruan, J., Huang, M., Duan, T. & Tong, G. (2015). Vitrification of day 3 cleavage-stage embryos yields better clinical outcome in comparison with vitrification of day 2 cleavage-stage embryos. Zygote 23, 169–76.Google Scholar
Dalcin, L., Silva, R.C., Paulini, F., Silva, B.D., Neves, J.P. & Lucci, C.M. (2013). Cytoskeleton structure, pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos. Cryobiology 67, 137–45.Google Scholar
Dasiman, R., Rahman, N.S., Othman, S., Mustafa, M.F., Yusoff, N.J., Jusoff, W.H., Rajikin, M.H., Froemming, G.R. & Khan, N.A. (2013). Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos. Med. Sci. Monit. Basic Res. 19, 258–66.Google Scholar
Debrock, S., Peeraer, K., Fernandez Gallardo, E., De Neubourg, D., Spiessens, C. & D'Hooghe, T.M. (2015). Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow freezing: a RCT. Hum. Reprod. 30, 1820–30.Google Scholar
Desai, N., Abdelhafez, F., Bedaiwy, M.A., Goldberg, J., Falcone, T. & Goldfarb, J. (2010). Clinical pregnancy and live births after transfer of embryos vitrified on day 3. Reprod. Biomed. Online 20, 808–13.Google Scholar
Devine, K., Connell, M.T., Richter, K.S., Ramirez, C.I., Levens, E.D., DeCherney, A.H., Stillman, R.J. & Widra, E.A. (2015). Single vitrified blastocyst transfer maximizes liveborn children per embryo while minimizing preterm birth. Fertil. Steril. 103, 1454–60.e1.Google Scholar
Fang, Y., Zeng, S., Fu, X., Jia, B., Li, S., An, X., Chen, Y. & Zhu, S. (2014). Developmental competence in vitro and in vivo of bovine IVF blastocyst after 15 years of vitrification. Cryo Lett. 35, 232–8.Google Scholar
Galliano, D., Garrido, N., Serra-Serra, V. & Pellicer, A. (2015). Difference in birth weight of consecutive sibling singletons is not found in oocyte donation when comparing fresh versus frozen embryo replacements. Fertil. Steril. 30, 1820–30.Google Scholar
Hong, S.W., Sepilian, V., Chung, H.M. & Kim, T.J. (2009). Cryopreserved human blastocysts after vitrification result in excellent implantation and clinical pregnancy rates. Fertil. Steril. 92, 2062–4.Google Scholar
Huang, C.C., Lee, T.H., Chen, S.U., Chen, H.H., Cheng, T.C., Liu, C.H., Yang., Y.S. & Lee, M.S. (2005). Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum. Reprod. 20, 122–8.Google Scholar
Ittner, L.M. & Gotz, J. (2007). Pronuclear injection for the production of transgenic mice. Nat. Protoc. 2, 1206–15.Google Scholar
Kim, H.J., Lee, K.H., Park, S.B., Choi, Y.B. & Yang, J.B. (2015). The effect of artificial shrinkage and assisted hatching on the development of mouse blastocysts and cell number after vitrification. Clin. Exp. Reprod. Med. 42, 94–100.Google Scholar
Kopeika, J., Thornhill, A. & Khalaf, Y. (2015). The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence. Hum. Reprod. Update 21, 209–27.Google Scholar
Larman, M.G. & Gardner, D.K. (2014). Ultrarapid vitrification of mouse oocytes and embryos. Methods Mol. Biol. 1092, 153–65.Google Scholar
Lawitts, J.A. & Biggers, J.D. (1991). Optimization of mouse embryo culture media using simplex methods. J. Reprod. Fertil. 91, 543–56.Google Scholar
Levron, J., Leibovitz, O., Brengauz, M., Gitman, H., Yerushalmi, G.M., Katorza, E., Gat, I. & Elizur, S.E. (2014). Cryopreservation of day 2–3 embryos by vitrification yields better outcome than slow freezing. Gynecol. Endocrinol. 30, 202–4.Google Scholar
Li, Z., Wang, Y.A., Ledger, W., Edgar, D.H. & Sullivan, E.A. (2014). Clinical outcomes following cryopreservation of blastocysts by vitrification or slow freezing: a population-based cohort study. Hum. Reprod. 29, 2794–801.Google Scholar
Liebermann, J. (2015). Vitrification: a simple and successful method for cryostorage of human blastocysts. Methods Mol. Biol. 1257, 305–19.Google Scholar
Liebermann, J. & Tucker, M.J. (2006). Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil. Steril. 86, 20–6.Google Scholar
Linlin, L., Bei, X., Guijin, Z. & Cuilian, Z. (2013). Effect of embryo vitrification on postnatal mouse uterine development. J. Reprod. Med. 58(11–12), 517–28.Google Scholar
Liu, S.Y., Teng, B., Fu, J., Li, X., Zheng, Y. & Sun, X.X. (2013). Obstetric and neonatal outcomes after transfer of vitrified early cleavage embryos. Hum. Reprod. 28, 2093–100.Google Scholar
Lopes, A.S., Frederickx, V., Van Kerkhoven, G., Campo, R., Puttemans, P. & Gordts, S. (2015). Survival, re-expansion and cell survival of human blastocysts following vitrification and warming using two vitrification systems. J. Assist. Reprod. Genet. 32, 83–90.Google Scholar
Martino, N.A., Dell'aquila, M.E., Cardone, R.A., Somoskoi, B., Lacalandra, G.M. & Cseh, S. (2013). Vitrification preserves chromatin integrity, bioenergy potential and oxidative parameters in mouse embryos. Reprod. Biol. Endocrinol. 11, 27.Google Scholar
Mito, T., Yoshioka, K., Noguchi, M., Yamashita, S., Misumi, K., Hoshi, T. & Hoshi, H. (2015). Birth of piglets from in vitro-produced porcine blastocysts vitrified and warmed in a chemically defined medium. Theriogenology 84, 1314–20.Google Scholar
Mukaida, T., Oka, C., Goto, T. & Takahashi, K. (2006). Artificial shrinkage of blastocoeles using either a microneedle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum. Reprod. 21, 3246–52.Google Scholar
Mukaida, T., Takahashi, K., Goto, T. & Oka, C. (2008). Perinatal outcome of vitrified human blastocysts in 7 year experience (2670 attempted cycles). Hum. Reprod. 23, i48.Google Scholar
Saenz-de-Juano, M.D., Marco-Jimenez, F., Schmaltz-Panneau, B., Jimenez-Trigos, E., Viudes-de-Castro, M.P., Peñaranda, D.S., Jouneau, L., Lecardonnel, J., Lavara, R., Naturil-Alfonso, C., Duranthon, V. & Vicente, J.S. (2014). Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level. Reproduction 147, 789–801.Google Scholar
Shi, W., Xue, X., Zhang, S., Zhao, W., Liu, S., Zhou, H., Wang, M. & Shi, J. (2012). Perinatal and neonatal outcomes of 494 babies delivered from 972 vitrified embryo transfers. Fertil. Steril. 97, 1338–42.Google Scholar
Vajta, G., Holm, P., Kuwayama, M., Booth, P.J., Jacobsen, H., Greve, T. & Callesen, H. (1998). Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev. 51, 53–8.Google Scholar
Van Landuyt, L., Van de Velde, H., De Vos, A., Haentjens, P., Blockeel, C., Tournaye, H. & Verheyen, G. (2013). Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos. Hum. Reprod. 28, 2943–9.Google Scholar
Van Landuyt, L., Polyzos, N.P., De Munck, N., Blockeel, C., Van de Velde, H. & Verheyen, G. (2015). A prospective randomized controlled trial investigating the effect of artificial shrinkage (collapse) on the implantation potential of vitrified blastocysts. Hum. Reprod. 30, 2509–18.Google Scholar
Vanderzwalmen, P., Bertin, G., Debauche, C., Standaert, V., van Roosendaal, E., Vandervorst, M., Bollen, N., Zech, H., Mukaida, T., Takahashi, K. & Schoysman, R. (2002). Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum. Reprod. 17, 744–51.Google Scholar
Vanderzwalmen, P., Bertin, G., Debauche, Ch., Standaert, V., Bollen, N., van Roosendaal, E., Vandervorst, M., Schoysman, R. & Zech, N. (2003). Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum. Reprod. 18, 1504–11.Google Scholar
Vanderzwalmen, P., Ectors, F., Grobet, L., Prapas, Y., Panagiotidis, Y., Vanderzwalmen, S., Stecher, A., Frias, P., Liebermann, J. & Zech, N.H. (2009). Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM. Reprod. Biomed. Online 19, 700–7.Google Scholar
Vanderzwalmen, P., Zech, N., Lejeune, B., Wirtleitner, B., Zech, M. & Ectors, F. (2010). Vitrification and the use of high concentrations of cryoprotectants: is it a justified argument to prefer slow freezing? Gynecol. Obstet. Fertil. 38, 536–40.Google Scholar
Vanderzwalmen, P., Connan, D., Grobet, L., Wirleitner, B., Remy, B., Vanderzwalmen, S., Zech, N. & Ectors, F.J. (2013). Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions. Hum. Reprod. 28, 2101–10.Google Scholar
Varago, F.C., Moutacas, V.S., Carvalho, B.C., Serapião, R.V., Vieira, F., Chiarini-Garcia, H., Brandão, F.Z., Camargo, L.S., Henry, M., Lagares, M.A. (2014). Comparison of conventional freezing and vitrification with dimethylformamide and ethylene glycol for cryopreservation of ovine embryos. Reprod. Domest. Anim. 49, 839–44.Google Scholar
Wikland, M., Hardarson, T., Hillensjö, T., Westin, C., Westlander, G., Wood, M. & Wennerholm, U.B. (2010). Obstetric outcomes after transfer of vitrified blastocysts. Hum. Reprod. 25, 1699–707.Google Scholar
Xue, Y., Tong, X., Jiang, L., Zhu, H., Yang, L. & Zhang, S. (2014). Effect of vitrification versus slow freezing of human day 3 embryos on β-hCG levels. J. Assist. Reprod. Genet. 31, 1037–43.Google Scholar
Zhao, X.M., Du, W.H., Hao, H.S., Wang, D., Qin, T., Liu, Y. & Zhu, H.B. (2012). Effect of vitrification on promoter methylation and the expression of pluripotency and differentiation genes in mouse blastocysts. Mol. Reprod. Dev. 79, 445–50.Google Scholar
Zhu, D., Zhang, J., Cao, S., Zhang, J., Heng, B.C., Huang, M., Ling, X., Duan, T. & Tong, G.Q. (2011). Vitrified-warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles—time for a new embryo transfer strategy? Fertil. Steril. 95, 1691–5.Google Scholar