Effects of assisted oocyte activation with calcium- ionophore and strontium chloride on in vitro ICSI outcomes

Document Type : Original Article


1 Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

2 Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran

3 Isfahan Fertility and Infertility Center, Isfahan, Iran


Objective(s): Failed fertilization after intra-cytoplasmic sperm injection (ICSI) is mainly attributed to failed oocyte activation and can be overcome by artificial oocyte activation (AOA). The present study aims to compare in vitro outcomes of ICSI following two different assisted oocyte activation chemical procedures (SrCl2 and Ionomycin) in sibling oocytes of ICSI candidates.
Materials and Methods: From March 2015 until February 2016, 105 infertile men with 99–100% abnormal sperm morphology, irrespective of sperm motility, concentration, or origin (semen or testicular) were included in this study. Out of these, 66 couples accepted to be included in the study group (Ionomycin/ SrCl2) and 39 couples requested routine AOA procedure (Ionomycin) as external control group. Primary outcomes of this study (fertilization, embryo quality, and post-implantation development) were compared between these groups.
Results: Significantly higher oocyte activation (67.90±3.6% vs. 51.16±3.6%, P=0.004) and fertilization (65.23±3.63% vs. 49.65±3.63%, P=0.008) rates were observed in sibling oocytes treated with Ionomycin in comparison to the SrCl2 sibling group. Percentage of top quality embryos was insignificantly higher in SrCl2 groups compared to the Ionomycin group (29.90±4.27 vs. 20.65±4.05%, P=0.26).
Conclusion: Ionomycin may be superior to SrCl2 for inducing oocyte activation. However, SrCl2 may be a more efficient means to support the development of better quality embryos following ICSI.


Main Subjects

1. Neri QV, Lee B, Rosenwaks Z, Machaca K, Palermo GD. Understanding fertilization through intracytoplasmic sperm injection (ICSI). Cell Calcium 2014; 55:24-37.
2. Palermo GD, Neri QV, Takeuchi T, Rosenwaks Z. ICSI: where we have been and where we are going. Semin Reprod Med 2009; 27:191-201.
3. Mahutte NG, Arici A. Failed fertilization: is it predictable? Curr Opin Obstet Gynecol 2003; 15:211-218.
4. Javed M, Esfandiari N, Casper RF. Failed fertilization after clinical intracytoplasmic sperm injection. Reprod Biomed Online 2010; 20:56-67.
5. Swain JE, Pool TB. ART failure: oocyte contributions to unsuccessful fertilization. Hum Reprod Update 2008; 14:431-1446.
6. Amdani SN, Yeste M, Jones C, Coward K. Phospholipase C zeta (PLCζ) and male infertility: Clinical update and topical developments. Adv Biol Regul 2016; 61:58-67.
7. Kashir J, Nomikos M, Swann K, Lai FA. PLCζ or PAWP: revisiting the putative mammalian sperm factor that triggers egg activation and embryogenesis. Mol Hum Reprod 2015; 21: 383-388.
8. Yeste M, Jones C, Amdani SN, Patel S, Coward K. Oocyte activation deficiency: a role for an oocyte contribution? Hum Reprod Update 2016; 22: 23-47.
9. Aghajanpour S, Ghaedi K, Salamian A, Deemeh MR, Tavalaee M, Moshtaghian J, et al. Quantitative expression of phospholipase C zeta, as an index to assess fertilization potential of a semen sample. Hum Reprod 2011; 26: 2950-2956.
10. Chansel-Debordeaux L, Dandieu S, Bechoua S, Jimenez C. Reproductive outcome in globozoospermic men: update and prospects. Andrology 2015; 3:1022-1034.
11. Escoffier J, Yassine S, Lee HC, Martinez G, Delaroche J, Coutton C, et al. Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation. Mol Hum Reprod 2015; 21: 157-168.
12. Heytens E, Parrington J, Coward K, Young C, Lambrecht S, Yoon SY, et al. Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men. Hum Reprod 2009; 24: 2417-2428.
13. Kamali-Dolat Abadi M, Tavalaee M, Shahverdi A, Nasr-Esfahani MH. Evaluation of PLCζ and PAWP expression in globozoospermic individuals. Cell J 2016; 18: 438-445.
14. Amdani SN, Jones C, Coward K. Phospholipase C zeta (PLCζ): oocyte activation and clinical links to male factor infertility. Adv Biol Regul 2013; 53: 292-308.
15. Tavalaee M, Kiani-Esfahani A, Nasr-Esfahani MH. Relationship between potential sperm factors involved in oocyte activation and sperm DNA fragmentation with intra-cytoplasmic sperm injection clinical outcomes. Cell J 2017; 18: 588-596.
16. Vanden Meerschaut F, Nikiforaki D, De Roo C, Lierman S, Qian C, Schmitt-John T, et al. Comparison of pre- and post-implantation development following the application of three artificial activating stimuli in a mouse model with round-headed sperm cells deficient for oocyte activation. Hum Reprod 2013; 28:1190-1198.
17. Darabi MR, Shiravi A, Hojati V. The effects of ethanol and strontium on growth and development of two-cell arrested mouse embryos. Int J Fertil Steril 2012; 5:197-202.
18. Nasr-Esfahani MH, Tavalaee M, Deemeh MR, Arbabian M, Parrington J. Can assessment of total acrosin activity help predict failed or low fertilization rate ICSI for implementation of artificial oocyte activation? The open andrology journal 2010, 2:19-26.
19. Nasr-Esfahani MH, Razavi S, Javdan Z, Tavalaee M. Artificial oocyte activation in severe teratozoospermia undergoing intracytoplasmic sperm injection. Fertil Steril 2008; 90: 2231-2237.
20. Vanden Meerschaut F, Nikiforaki D, Heindryckx B, De Sutter P. Assisted oocyte activation following ICSI fertilization failure. Reprod Biomed Online 2014; 28: 560-571.
21. Chi HJ, Koo JJ, Song SJ, Lee JY, Chang SS. Successful fertilization and pregnancy after intracytoplasmic sperm injection and oocyte activation with calcium ionophore in a normozoospermic patient with extremely low fertilization rates in intracytoplasmic sperm injection cycles. Fertil Steril 2004; 82: 475-477.
22. Deemeh MR, Tavalaee M, Nasr-Esfahani MH. Health of children born through artificial oocyte activation: a pilot study. Reprod Sci 2015; 22: 322-328.
23. D’haeseleer E, Vanden Meerschaut F, Bettens K, Luyten A, Gysels H, Thienpont Y, et al. Language development of children born following intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA). Int J Lang Commun Disord 2014; 49: 702-709.
24. Eldar-Geva T, Brooks B, Margalioth EJ, Zylber-Haran E, Gal M, Silber SJ. Successful pregnancy and delivery after calcium ionophore oocyte activation in a normozoospermic patient with previous repeated failed fertilization after intracytoplasmic sperm injection. Fertil Steril 2003; 79 Suppl 3:1656-8
25. Vanden Meerschaut F, D’Haeseleer E, Gysels H, Thienpont Y, Dewitte G, Heindryckx B, et al. Neonatal and neurodevelopmental outcome of children aged 3-10 years born following assisted oocyte activation. Reprod Biomed Online 2014; 28: 54-63.
26. Ebner T, Köster M, Shebl O, Moser M, Van der Ven H, Tews G, et al. Application of a ready-to-use calcium ionophore increases rates of fertilization and pregnancy in severe male factor infertility. Fertil Steril 2012; 98:1432-1437.
27. Mortimer D. Comparison of the fertilizing ability of human spermatozoa preincubated in calcium- and strontium-containing media. J Exp Zool 1986; 237: 21-4.
28. Mortimer D, Curtis EF, Dravland JE. The use of strontium-substituted media for capacitating human spermatozoa: an improved sperm preparation method for the zona-free hamster egg penetration test. Fertil Steril 1986; 46: 97-103.
29. Kline D, Kline JT. Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev Biol 1992; 149: 80-89.
30. Suganuma R, Walden CM, Butters TD, Platt FM, Dwek RA, Yanagimachi R, et al. Alkylated imino sugars, reversible male infertility-inducing agents, do not affect the genetic integrity of male mouse germ cells during short-term treatment despite induction of sperm deformities. Biol Reprod 2005; 72: 805-813.
31. Tesarik J, Mendoza C, Greco E. The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei. Fertil Steril 2000; 74:1245-1247.
32. Yanagida K, Morozumi K, Katayose H, Hayashi S, Sato A. Successful pregnancy after ICSI with strontium oocyte activation in low rates of fertilization. Reprod Biomed Online 2006; 13:801-806.
33 Borges E Jr, de Almeida Ferreira Braga DP, de Sousa Bonetti TC, Iaconelli A Jr, Franco JG Jr. Artificial oocyte activation with calcium ionophore A23187 in intracytoplasmic sperm injection cycles using surgically retrieved spermatozoa. Fertil Steril 2009; 92:131-136.
34. Kang HJ, Lee SH, Park YS, Lim CK, Ko DS, Yang KM, et al. Artificial oocyte activation in intracytoplasmic sperm injection cycles using testicular sperm in human in vitro fertilization. Clin Exp Reprod Med 2015; 42:45-50.
35. Mansour R, Fahmy I, Tawab NA, Kamal A, El-Demery Y, Aboulghar M, et al. Electrical activation of oocytes after intracytoplasmic sperm injection: a controlled randomized study. Fertil Steril 2009; 91:133-139.
36. Moaz MN, Khattab S, Foutouh IA, Mohsen EA. Chemical activation of oocytes in different types of sperm abnormalities in cases of low or failed fertilization after ICSI: a prospective pilot study. Reprod Biomed Online 2006; 13:791-794.
37. World Health Organization. Examination and processing human semen. (5th Ed.), Cambridge University press, WHO, 2010.
38. Levron J, Munné S, Willadsen S, Rosenwaks Z, Cohen J. Male and female genomes associated in a single pronucleus in human zygotes. Biol Reprod 1995; 52:653-657.
39. Sultan KM, Munné S, Palermo GD, Alikani M, Cohen J. Chromosomal status of uni-pronuclear human zygotes following in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod 1995; 10:132-136.
40. Giorgetti C, Terriou P, Auquier P, Hans E, Spach JL, Salzmann J, et al. Implantation:Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers. Hum Reprod 1995; 10: 2427-2431.
41. Terriou P, Giorgetti C, Hans E, Salzmann J, Charles O, Cignetti L, at al. Relationship between even early cleavage and day 2 embryo score and assessment of their predictive value for pregnancy. Reprod Biomed Online 2007; 14: 294-299.
42. Yang XY, Wang J, Liu JY, Gao Y, Zhou ZM, Sha JH, et al. Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient. Asian J Androl 2012; 14: 341-343.
43. Heindryckx B, De Gheselle S, Gerris J, Dhont M, De Sutter P. Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection. Reprod Biomed Online 2008; 17:662-668.
44. Heindryckx B, Van der Elst J, De Sutter P, Dhont M. Treatment option for sperm- or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI. Hum Reprod 2005; 20: 2237-2241.
46. Nasr-Esfahani MH, Deemeh MR, Tavalaee M. Artificial oocyte activation and intracytoplasmic sperm injection. Fertil Steril 2010; 94: 520-526.
46. Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P, Dhont M. Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity. Fertil Steril 1997; 68: 1144-1147.
47. Kim JW, Kim SD, Yang SH, Yoon SH, Jung JH, Lim JH. Successful pregnancy after SrCl2 oocyte activation in couples with repeated low fertilization rates following calcium ionophore treatment. Syst Biol Reprod Med 2014; 60: 177-182.
48. Zhang D, Pan L, Yang LH, He XK, Huang XY, Sun FZ. Strontium promotes calcium oscillations in mouse meiotic oocytes and early embryos through InsP3 receptors, and requires activation of phospholipase and the synergistic action of InsP3. Hum Reprod 2005; 20: 3053-3061.
49. Grasa P1, Coward K, Young C, Parrington J. The pattern of localization of the putative oocyte activation factor, phospholipase Czeta, in uncapacitated, capacitated, and ionophore-treated human spermatozoa. Hum Reprod 2008; 23:2513-25122.
50. Kashir J, Jones C, Mounce G, Ramadan WM, Lemmon B, Heindryckx B, et al. Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability. Fertil Steril 2013; 99:107-117.
51. Young C, Grasa P, Coward K, Davis LC, Parrington J. Phospholipase C zeta undergoes dynamic changes in its pattern of localization in sperm during capacitation and the acrosome reaction. Fertil Steril 2009; 9:2230-2242.
52. Tavalaee M, Nasr-Esfahani MH. Expression profile of PLCζ, PAWP, and TR-KIT in association with fertilization potential, embryo development, and pregnancy outcomes in globozoospermic candidates for intra-cytoplasmic sperm injection and artificial oocyte activation. Andrology 2016; 4: 850-856.
53. Heytens E, Parrington J, Coward K, Young C, Lambrecht S, Yoon SY, et al. Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men. Hum Reprod 2009; 24: 2417-2428.
54. Swann K, Ozil JP. Dynamics of the calcium signal that triggers mammalian egg activation. Int Rev Cytol 1994; 152:183-222.
55. Tateno H, Kamiguchi Y. How long do parthenogenetically activated mouse oocytes maintain the ability to accept sperm nuclei as a genetic partner? J Assist Reprod Genet. 2005; 22: 89-93.
56. Nikiforaki D, Vanden Meerschaut F, de Roo C, Lu Y, Ferrer-Buitrago M, de Sutter P, et al. Effect of two assisted oocyte activation protocols used to overcome fertilization failure on the activation potential and calcium releasing pattern. Fertil Steril 2016; 105:798-806.
57. Bilodeau-Goeseels S. Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes. Mol Reprod Dev 2011; 78: 734-743.
58. Hosseini SM, Hajian M, Moulavi F, Shahverdi AH, Nasr-Esfahani MH. Optimized combined electrical-chemical parthenogenetic activation for in vitro matured bovine oocytes. Anim Reprod Sci 2008; 108:122-33.
59. Schatten H, Sun QY. New insights into the role of centrosomes in mammalian fertilization and implications for ART. Reproduction 2011; 142:793-801.
60. Méo SC, Yamazaki W, Ferreira CR, Perecin F, Saraiva NZ, Leal CL, et al. Parthenogenetic activation of bovine oocytes using single and combined strontium, ionomycin and 6-dimethylaminopurine treatments. Zygote 2007; 15:295-306.
61. Rogers NT, Hobson E, Pickering S, Lai FA, Braude P, Swann K. Phospholipase Czeta causes Ca2+ oscillations and parthenogenetic activation of human oocytes. Reproduction 2004; 128: 697-702.
62. Chen J, Qian Y, Tan Y, Mima H. Successful pregnancy following oocyte activation by strontium in normozoospermic patients of unexplained infertility with fertilisation failures during previous intracytoplasmic sperm injection treatment. Reprod Fertil Dev 2010; 22:852-855.
63. Kyono K, Kumagai S, Nishinaka C, Nakajo Y, Uto H, Toya M, et al. Birth and follow-up of babies born following ICSI using SrCl2 oocyte activation. Reprod Biomed Online 2008; 17:53-58.
64. Tripathi A, Chaube SK. High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro. Apoptosis 2012; 17:439-448.