P-211 Significant differences in efficiency between two commonly used ionophore solutions for assisted oocyte activation (AOA): a prospective comparison of ionomycin and A23187

Abstract

Abstract Study question Are there differences in oocyte activation rate and developmental morphokinetic after assisted oocyte activation (AOA) with either ionomycin or A23187? Summary answer Ionomycin produces greater oocyte activation rate than A23187 (43.1% vs 15.9%), while the morphokinetic pattern of parthenotes’ development is similar between both Ca2+ ionophores. What is known already Fertilization failure (FF) after ICSI is often due to a male factor, such as the alteration of sperm-borne oocyte activating factors or globozoospermia. AOA can restore fertilization rate by stimulating transient spikes of Ca2+ in the cytoplasm of the oocyte, but its efficiency and effect on embryo development are not fully characterized. Here, the efficiency of two AOA preparation and protocols commonly used in clinical practice is compared using parthenogenetically activated human oocytes. The objective is to identify the most appropriate protocol for AOA and to compare the morphokinetic pattern of the generated parthenotes up to expanded blastocyst (tB). Study design, size, duration Prospective study involving 120 human oocytes from 66 women from March 2019 to November 2021. Oocytes were activated with two AOA protocols after mock ICSI: i) A23187 (ready-to-use solution, GM508 CultActive (Gynemed) n = 69), and ii) Ionomycin (homemade solution, 10 µmol/L, n = 51). Oocyte activation and development were analyzed in both groups; further, the morphokinetic patterns were compared; videos of embryos obtained with donor oocytes and sperm (n = 39) were used as comparator of normal developmental kinetics. Participants/materials, setting, methods Oocytes were injected with latex microspheres to simulate ICSI, followed by AOA. A23187 was used according to manufacturer specification. For ionomycin, three incubations of seven minutes each were performed. Resulting parthenotes (1PN) were incubated in a time-lapse system for 160h. Activation and developmental rates, tPNf (pronucleus fading), t2, t3, t4, t5, t8 (from 1st division to 8-cell), tsB (blastulation onset) and tB (blastocyst expansion) were compared using Student’s T-test and ANOVA. Statistical significance: p-value <0.05. Main results and the role of chance Ionomycin resulted in a significantly higher oocyte activation rate (22/51, 43.1%) than A23187 (11/69, 15.9%), p = 0.0009. In the ionomycin group, 81.8% (18/22) of parthenotes reached the 2-cell stage, 45.5% (10/22) reached the 5-cell stage, and 18.2% (4/22) reached the pseudo-blastocyst stage, as expected for these pseudo-embryos lacking the contribution of the sperm. In the A23187 group, 54.5% (6/11) reached the 2-cell stage, 27.3% (3/11) the 5-cell stage, and none formed pseudo-blastocysts. While the number of parthenotes progressing through development is much lower for A23187, the expected poor development of human parthenotes past activation and corresponding low numbers did not allow to reach statistical significance (p > 0.05). tPNf was significantly different among the 3 groups compared: 47.4±37.5h (n = 11, A23187), 27.7±23.8h (n = 21, ionomycin) and 23.3±4.6h (n = 39, control), p = 0.0019; with ionomycin presenting an average tPNf similar to the one obtained by ICSI. Among activated oocytes, the morphokinetic pattern in the ionomycin and A23187 groups was very similar to the one obtained in the control group (p > 0.05 at all timings). As an example, the t5 in the 3 groups was 58.5±12.6h (n = 3, A23187), 45.6±21.8h (n = 10, ionomycin), and 49.5±12.4h (n = 34, control), p = 0.29. Limitations, reasons for caution The low number of parthenotes progressing past 2-cells limit the possibility to extract solid conclusions regarding the morphokinetic patterns after AOA. The efficiency of activation using the two tested protocol is however confirmed. Wider implications of the findings The use of homemade ionomycin solutions is an effective option for the treatment of fertilization failures where assisted oocyte activation is indicated. Caution should be exerted when using GM508 Cultactive to investigate fertilization failures of oocyte origin. Trial registration number not applicable