**9. Conclusions**

*Embryology - Theory and Practice*

option for some couples [87].

sperm defects are excluded indicating oocyte defects.

suspected oocyte-related activation deficiency.

**7. Success rates of AOA**

mental potential .

**8. Safety of AOA methods**

**defects**

**6. Diagnostic tools for assessing sperm- or oocyte-dependent activation** 

A proper diagnostic procedure is very important prior to the decision to use artificial oocyte activation, and oocyte or sperm donation is a reasonable treatment

There are several diagnostic methods available, but not always accessible to all clinics since legislation can prohibit the use of heterologous human-animal models. The mouse oocyte activation test (MOAT) is a heterologous ICSI model where mouse oocytes are fertilized with the patient's sperm [78]. As a negative control, mouse oocytes are injected with the medium and as a positive control, they are injected with donor sperm with proven fertilizing ability. It allows discrimination between sperm- and oocyte-borne causes for fertilization failure. According to the ratio of fertilized mouse oocytes, three groups are described: MOAT1 indicating sperm-borne defects, MOAT2 fertilization failure of unknown origin, MOAT3

In some patients from groups MOAT2 or MOAT3, capacity to activate mouse oocytes is demonstrated but later ICSI-AOA results in TFF. In these cases, assessment of calcium oscillations can give better answers as to whether the underlying reason is the presence of human sperm activation deficiencies or oocyte-related activation deficiency. Mouse oocyte Ca2+ analysis (M-OCA) or even more sensitive human oocyte Ca2+ analysis (H-OCA) can be performed before using AOA [88]. In the M-OCA test, the patient's sperm is used and frequency patterns of calcium oscillations are analyzed. H-OCA yields higher sensitivity than M-OCA to detect the presence of human sperm activation deficiencies. It helps detect cases with

The systematic review and meta-analysis of RCTs that compared ICSI-AOA and conventional ICSI first established that there is insufficient evidence available from RCTs to judge the efficacy and safety of ICSI-AOA for couples with previous fertilization failure [89]. A total of 14 articles were assessed and 9 included in meta-analysis. It cannot be concluded that the outcomes are improved using ICSI followed by artificial oocyte activation compared with conventional ICSI. The fertilization rate, cleavage rate, and likelihood of blastocyst formation seem to improve

Recently, important evidence appeared that the conditions in which activation takes place are very important for the success rate and can vary a lot. Varying concentrations of both ionomycin and calcium ions in culture media used during AOA can have significant effects on calcium release and further embryonic develop-

Although AOA methods have been proven efficient to overcome some cases of TFF, the concern around using them in clinical practice is quite big. By artificially increasing intracellular calcium levels we interfere with cellular mechanisms that normally would not occur. Nature has regulatory mechanisms to eliminate errors and when we force events that would not happen spontaneously it is always impor-

tant to verify all possible negative effects of such procedures.

according to some studies, but it is difficult to make a general conclusion.

**50**

The problem of failed fertilization is a big burden for patients and clinicians and the pressure to help these patients is enormous. Today, ART methods are generally easily accessible and patients' expectations are very high. In Europe alone, there have been 1,308,289 children born from IVF treatments between the years 1997 and 2013 according to data collected in European IVF monitoring [94]. Global data collection on IVF is a difficult task, but there are reports that in a three-year period, more than a million babies are born worldwide [95]. Despite the great success of ART, there are always some patients facing fertilization failure and the emotional burden of inability to achieve pregnancy is great for these couples. For successful fertilization, sperm must activate a quiescent oocyte to complete meiosis and progress toward embryonic development characterized with repeated mitotic divisions. Oocyte activation is a complex cascade of intracellular processes. Sperm or oocyte abnormalities can contribute to activation failure. In clinical practice, there is a need for safe methods of artificial oocyte activation based on the physiological properties of the gametes that closely imitate calcium oscillations triggered naturally by sperm.
