EFFECTIVENESS OF CALCIUM IONOPHORE ARTIFICIAL OOCYTE ACTIVATION IN ART PROGRAMS FOR PATIENTS WITH ADVERSE REPRODUCTIVE HISTORY: EMBRYOLOGICAL AND CLINICAL OUTCOMES

Background. Oocyte competence is a determinant of successful fertilization and embryo development. Poor oocyte quality may lead to fertilization failure or in vitro embryo developmental arrest. Artificial oocyte activation (AOA) using calcium ionophores has been proposed to overcome oocyte activation deficiency in patients with fertilization failure and unsatisfactory embryo quality. The effectiveness of this technology is of interest to fundamental medicine specialists and ART clinics.
Aim. To evaluate the effectiveness of calcium ionophore-mediated oocyte activation at the embryological stage of assisted reproductive technology (ART) programs in patients with previous unsuccessful infertility treatments.
Materials and Methods. A retrospective cohort comparative study was conducted at the Clinical Hospital "Mother and Child" (Samara) from January to December 2024. Study group (n=35): cycles with AOA using calcium ionophore (A23187, Sigma) after ICSI by medical indications (low fertilization rate in history). Control group (n=61): cycles with standard ICSI procedure without activation. Inclusion criteria: age 18–43 years, partner sperm use, complete data on culture up to day 5. Vitrolife media (Sweden) were used for cell preparation and ICSI. Cells were obtained by transvaginal follicular aspiration 36–37 hours after ovulation trigger. After ICSI, cells were in-cubated in medium with Ca²⁺ ionophore (10 mM concentration) for 8–10 minutes at 6% CO₂, 5% O₂, and +37°C. Embryo quality was assessed using a scoring system based on Gardner et al. (1999) criteria, modified with additional morphological parameters.
Results. Main parameters–fertilization rate, cleavage rate, and blastocyst formation rate–showed no significant differences between groups (p>0.05). A significant difference was observed only in mean embryo score at trans-fer (2.91±2.09 vs 3.98±1.41, p=0.008). Clinical pregnancy rate confirmed by ultrasound (36.0% vs 30.4%) and implantation rate (27.3% vs 23.8%) remained comparable (p>0.05). A clinically significant trend toward re-duced difference between positive hCG and clinical pregnancy rate (4% vs 10.7% in control) was revealed, sug-gesting higher embryo competence in the AOA group and reduced early reproductive loss.
Conclusion. Calcium ionophore artificial oocyte activation does not lead to statistically significant improvement in main embryological parameters but demonstrates a trend toward reducing the gap between biochemical and clinical pregnancy, which may indicate improved embryo quality in patients with adverse reproductive history. Data accumulation and multicenter randomized studies with increased sample size are required for more reliable conclusions.

1. Smeenk J, Wyns C, Kupka M, et al. ART in Europe, 2020: results generated from European registries by ESHRE European IVF Monitoring Consortium (EIM) for the European Society of Human Reproduction and Embryology (ESHRE). Hum Reprod. 2024;39(6):1268–1285. https://doi.org/10.1093/humrep/deae080

2. Glatthorn HN, Decherney A. The efficacy of add-ons: selected IVF "add-on" procedures and future directions. J Assist Reprod Genet. 2022;39(4):827–838. https://doi.org/10.1007/s10815-022-02420-7

3. Vanden Meerschaut F, Nikiforaki D, De Roo C, et al. Assisted oocyte activation following ICSI fertilization failure. Reprod Biomed Online. 2012;24(6):621–631. https://doi.org/10.1016/j.rbmo.2012.02.010

4. Adamson GD, de Mouzon J, Chambers GM, et al. International Committee for Monitoring Assisted Reproductive Technology: world report on assisted reproductive technology, 2018. Hum Reprod. 2023;38(2):164–179. https://doi.org/10.1093/humrep/deac243

5. Kashir J, Ganesh D, Jones C, et al. Oocyte activation deficiency and assisted oocyte activation: mechanisms, obstacles and prospects for clinical application. Hum Reprod Open. 2022;2022(2):hoac003. https://doi.org/10.1093/hropen/hoac003

6. Ruan JL, Liang SS, Pan JP, et al. Artificial oocyte activation with Ca²⁺ ionophore improves reproductive outcomes in patients with fertilization failure and poor embryo development in previous ICSI cycles. Front Endocrinol (Lausanne). 2023;14:1244507. https://doi.org/10.3389/fendo.2023.1244507

7. Caglar Aytac P, Bulgan Kilicdag E, Haydardedeoglu B, et al. Can calcium ionophore "use" in patients with diminished ovarian reserve increase fertilization and pregnancy rates? A randomized, controlled study. Fertil Steril. 2015;104(5):1168–1174. https://doi.org/10.1016/j.fertnstert.2015.07.1157

8. Swann K, Yu Y. The dynamics of calcium oscillations that activate mammalian eggs. Int J Dev Biol. 2008;52(5-6):585–594. https://doi.org/10.1387/ijdb.072530ks

9. Backs J, Backs T, Neef S, et al. The delta isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload. Proc Natl Acad Sci USA. 2009;106(7):2342–2347. https://doi.org/10.1073/pnas.0813013106

10. Madgwick S, Jones KT. How eggs arrest at metaphase II: MPF stabilisation plus APC/C inhibition equals cytostatic factor. Cell Div. 2007;2:4. https://doi.org/10.1186/1747-1028-2-4

11. Ducibella T, Fissore R. The roles of Ca²⁺, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Dev Biol. 2008;315(2):257–279. https://doi.org/10.1016/j.ydbio.2007.12.012

12. Kashir J, Deguchi R, Jones C, Coward K. Comparative biology of oocyte activation in mammals: new insights from the sperm factor paradigm. Reproduction. 2016;152(1):R1–R14. https://doi.org/10.1530/REP-16-0057

13. Nomikos M, Kashir J, Lai FA. The role and mechanism of action of sperm PLC-zeta in mammalian fertilisation. Biochem J. 2017;474(21):3659–3673. https://doi.org/10.1042/BCJ20160521

14. Yoon SY, Jellerette T, Salicioni AM, et al. Human sperm devoid of PLC, zeta 1 fail to induce Ca²⁺ release and are unable to initiate the first step of embryo development. J Clin Invest. 2008;118(11):3671–3681. https://doi.org/10.1172/JCI36942

15. Heytens E, Parrington J, Coward K, et al. Reduced amounts and abnormal forms of phospholipase C zeta (PLCζ) in spermatozoa from infertile men. Hum Reprod. 2009;24(10):2417–2428. https://doi.org/10.1093/humrep/dep207

16. Darwish E, Magdi Y. A preliminary report of successful cleavage after calcium ionophore activation at ICSI in cases with previous arrest at the pronuclear stage. Reprod Biomed Online. 2015;31(6):799–804. https://doi.org/10.1016/j.rbmo.2015.08.012

17. Ebner T, Oppelt P, Wöber M, et al. Treatment with Ca²⁺ ionophore improves embryo development and outcome in cases with previous developmental problems: a prospective multicenter study. Hum Reprod. 2015;30(1):97–102. https://doi.org/10.1093/humrep/deu285

18. Yakh′yarova M.P., Zastavskiy I.A., Nastupov A.V. Nastupleniye beremennosti i rody u patsiyentok posle korrektsii raspo-lozheniya pronukleusov v ootsitakh s ispol′zovaniyem ionofora kal′tsiya. Reproduktivnaya meditsina. 2022;3:87–92.

19. https://doi.org/10.37800/RM.3.2022.87-92 In Russian

20. Sfontouris IA, Kolibianakis EM, Lainas TG, et al. Clinical applications of artificial oocyte activation: a systematic review and meta-analysis of randomized controlled trials. Hum Reprod Update. 2015;21(6):695–706. https://doi.org/10.1093/humupd/dmv034

21. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril. 2000;73(6):1155–1158. https://doi.org/10.1016/s0015-0282(00)00518-5

22. Shurygina OV, Yuldasheva SZ, Ivanova OV, Demidova NN. Otsenka vyzhivayemosti vitrifitsirovannykh embrionov v zavisimosti ot stadii razvitiya. Journal of Biomimetics, Biomaterials and Biomedical Engineering. 2020;45:22–27. In Russian

23. ESHRE Special Interest Group of Embryology and Alpha Scientists in Reproductive Medicine. The Vienna consensus: report of an expert meeting on the development of ART laboratory performance indicators. Reprod Biomed Online. 2017;35(5):494–510. https://doi.org/10.1016/j.rbmo.2017.06.015

24. 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):1270–1283. https://doi.org/10.1093/humrep/der037

25. Ferraretti AP, Goossens V, Kupka M, et al. Assisted reproductive technology in Europe, 2009: results generated from European registers by ESHRE. Hum Reprod. 2013;28(9):2318–2331. https://doi.org/10.1093/humrep/det278

26. Montag M, Toth B, Strowitzki T. New approaches to embryo selection. Reprod Biomed Online. 2013;27(5):539–546. https://doi.org/10.1016/j.rbmo.2013.05.013