In the present study, we investigated the effects of AOA application with Cai on embryomorphokinetic parameters and embryological development. To the best of our knowledge, our sample population is the largest of any study conducted on this subject to date. Some clinical studies and meta-analyses have reported that AOA may help improve fertilization, embryonic development, and clinical pregnancy outcomes in many clinical conditions [21, 23]. Therefore, we performed AOA mostly in patients with poor prognoses. Also, because changes in intracellular Ca levels influence the entire development process from oocyte to embryo transition, we examined the correlations between Cai application and morphokinetic parameters.
The use of AOA is controversial, as synthetic activating agents do not fully replicate the natural Ca signaling and physiological mechanisms observed in mammalian zygotes [19, 32]. Activation of the embryo by Cai may lead to variation in behavior and division patterns that differ from normal processes. Therefore, this technique is still experimental, and gathering data from a larger number of patient embryos could yield valuable insights.
Few studies have evaluated the relationship between AOA and the morphokinetic behavior of the embryo. Martinez et al. reported that AOA had no effect, with the exception of second polar body extrusion (tPB2) and time to second cell division (t3); however, they concluded that these changes did not seem affect the morphokinetic pattern of the preimplantation embryo and ascribed the findings to a rapid transient nonphysiological increase in free intracytoplasmic Ca2+ [19]. Similarly, Shebl et al. performed a sibling oocyte study in patients with previous fertilization problems. Half of the oocytes were treated with calcimycin. They found that ionophore-treated oocytes underwent significantly earlier pronuclei transformation and had a better synchronized third cell cycle [22]. In both of these studies, the patient population included couples who had experienced fertilization failure and severe male factor infertility similar with our study. However, in contrast to their findings, in our study pronuclei transformation or third cell cycle synchronization were not different and we found a prolonged tb in AOA cycles. Research on humans has highlighted the potential of tb as a determining factor for selecting embryos, surpassing other components of embryo morphological grading [35-37]. Lee et al. and Moustafa et al. noted delayed tb in embryos with chromosome abnormalities [36, 38]. Recently, Quintana-Vehí and colleagues conducted a study comparing groups in donor cycles, including calcimycin, ionomycin, and conventional ICSI cycles. ICSI was not applied in AOA groups, only parthenogenetic activation was examined. According to the results, none of the calcimycin parthenotes formed blastocysts, while an extension in blastulation was observed in the ionomycin group. Although there are some differences on designs of this study and our study we also found an extension in tb [39].
The clinical importance of these changes in embryomorphokinetic parameters is not clear. AOA process results in non-physiological calcium changes in the cells. In normal physiological processes, calcium release during oocyte activation occurs in the form of oscillations from internal stores. However, in artificial oocyte activation, it increases in a peak- shaped manner. This situation may be related to the faster expulsion of the second polar body in the initial fertilization and faster cleavage stages (t3) mentioned in previous reports, while the lack of long-lasting oscillations may be associated with the prolongation of the tb period [19,22]. Previous studies and our study results may suggest the importance of taking caution when applying Cai and the prolonged tb duration may serve as a warning for future clinical practice. It would be more appropriate to consider its utilization in cases where there is a clear indication rather than opting for elective use.
Morphokinetic behavior has also been used to predict euploidy rates in different studies [29,30] because aneuploidy can potentially occur at any stage from the disjunction of chromatids to the mitotic divisions within the embryo [40-43]. We used the morphokinetic embryo grading system that predicts aneuploidy devised by Basile et al. and did not find any differences between the two groups. The literature contains little information on embryo aneuploidy and Cai activation. However, our results are in line with a recent study that found that Cai application did not increase the risk for aneuploidy and there were no developmental differences in live births as a result of transfers made with Cai administration [20]. Thus Ca2 ionophores have the capability to bind Ca2 cations, and due to their hydrophobic properties, they form a complex at the lipid bilayer of the membrane. This structure enables ionophores to transfer Ca2 across the membrane and release them into the cytosol [44]. Consequently, ionophores themselves may not necessarily enter the oocyte, potentially explaining the absence of a detectable effect of ionophores on chromosomal segregation [45]. Reports have indicated that implanted embryos after IVF, using Cai as an artificial oocyte activator, do not exhibit any chromosome abnormalities [7,47]. Moreover, no perinatal morbidity or mortality was reported among the patients involved in these studies. Comparable outcomes have also been observed in children aged 3–10 years, who were born following ICSI cycles using Cai. No differences have been noted compared to their peers [32,48].
The principal limitation of our study lies in its retrospective design. Consequently, definitive conclusions are challenging to establish. However, it is important to acknowledge that this limitation is mitigated by our substantial sample size. Our findings may help understanding the embryomorphokinetic behavior and embryo selection in AOA cycles in future applications.
In conclusion, we did not find significant differences in most of the embryomorphokinetic parameters except prolongation of tb and cavitation time. However, the clinical significance of these changes is controversial because of the lack of correlations between these parameters and clinical outcomes. More studies with larger populations are needed on this subject.