*5.2.2 Strontium chloride (SrCl2)*

It is reported as very efficient in mice and induces not only single calcium concentration elevation, but oscillations [80]. The mechanism by which it induces oscillations is not fully understood.

A study that investigated efficacy of SrCl2 in human oocytes showed significantly increased fertilization rates, when compared with conventional ICSI or calcium ionophore treatment [81].

#### *5.2.3 PLCζ*

*Embryology - Theory and Practice*

not in another.

mechanical.

**5.1 Electrical methods**

**5.2 Chemical methods**

*5.2.1 Ionophores*

through mitochondrial level is discussed.

**5. Artificial oocyte activation (AOA)**

is only one large calcium concentration increase.

series of calcium spikes after spermatid injection [75].

transient, while others cause multiple oscillations.

oocyte quality. In a review of [72], the environmental impact on oocyte function

Artificial oocyte activation methods try to induce oocyte activation by using physiological properties of the gametes and in this way interfere in different levels of the cascade of events during fertilization. In general, they try to alleviate intracellular calcium concentration and mimic oscillations. As we are well aware by now that there is big species-specific variability in the mechanisms of oocyte activation, it is not surprising that different AOA methods can be successful in one species, but

By influencing gamete physiological properties such as electrical excitability and

By applying direct electrical current within a Petri dish with oocytes, the electrical field stimulates charged proteins to move toward the plasma membrane, and by this, the number of pores in the plasma membrane increases [30]. Calcium conductivity increases, and more calcium enters the oocyte from the surroundings. There

Chemical activation is the most commonly used method. Oocytes are exposed to chemical agents that lead to an increase in intracellular calcium concentration in the oocyte. Some agents, such as calcium ionophores cause a single, prolonged calcium

Calcium ionophores, such as ionomycin, A23187 (calcimycin), and gm508 are molecules soluble in lipids, synthesized by microorganisms; today several synthetic compounds are known. They can transport ions across lipid bilayers. They increase membrane permeability for Ca2+ ions, thus allowing calcium influx in the oocyte from the surrounding medium and intracytoplasmic rise of calcium concentration. It has been recently established that intracytoplasmic rise of calcium concentration in human and mouse oocytes is not only the consequence of the influx from the surroundings but also from intracellular stores, since this rise appears also in calcium-free medium. However, they are not able to induce calcium oscillations

In the prospective randomized study of [73], an electrical pulse in a special chamber with electrodes 30 minutes after ICSI in 0.3 M glucose drops was used to activate oocytes, and a small increase in the fertilization rate after ICSI was achieved. Successful pregnancy and birth were achieved and reported in the case report [74]. In a study with round spermatid injection coupled with electrostimulation, the electrical pulse triggered not only a single calcium concentration increase, but a

plasma membrane conductivity, the aim is to increase intracytoplasmic calcium concentrations and mimic the frequency and amplitude of the oscillations. Basically, there are three types of AOA methods: electrical, chemical, and

**48**

Soon after the discovery of the role of PLCζ as a trigger of oocyte activation, the ideas of using the protein as an artificial activator emerged. The synthesis of the first recombinant human PLCζ protein was published [82]; when injected into mouse oocytes, calcium oscillations were evoked that closely resembled those initiated by the sperm after fertilization. Later, a study where human recombinant PLCζ was used on human and mouse oocytes was published [83] describing dose-dependent manner of calcium oscillations. These authors also showed that by injecting recombinant human PLCζ the next day in oocytes that failed to fertilize after ICSI, five of eight oocytes were rescued.

Earlier, it was established that PLCζ complementary RNA (cRNA) injection in MII oocytes also triggered oscillations [52] with a time lag that enables protein to synthesize.

The commercial use of recombinant human PLCζ still has to be validated in terms of safety.

#### *5.2.4 Ethanol*

In veterinary medicine, ethanol is often used for parthenogenetic oocyte activation. Parthenogenesis is development of an embryo from an unfertilized oocyte, naturally occurring in invertebrates or even some vertebrates. By inhibiting the second polar body extrusion, diploid parthenotes with two maternal genomes can be created and embryos can develop normally for several days, but later die. In several species, artificial parthenogenetic activation was described to be caused by ethanol [84].

#### **5.3 Mechanical methods**

Some data from the literature suggest that the modified ICSI technique can give better fertilization in patients with a history of TFF or low fertilization [85, 86]. Vigorous aspiration of cytoplasm and a different position of the pipette tip when ejecting sperm in the oocyte is supposed to increase calcium levels during injection and enable better contact of sperm with intracellular storage of calcium.

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

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 option for some couples [87].

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 sperm defects are excluded indicating oocyte defects.

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 suspected oocyte-related activation deficiency.

#### **7. Success rates of AOA**

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

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 developmental potential .

#### **8. Safety of AOA methods**

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 important to verify all possible negative effects of such procedures.

**51**

*Oocyte Activation Failure: Physiological and Clinical Aspects*

The number of children born after AOA is relatively small for statistical analysis,

but there are accumulating data on the safety of these methods. The study from Ghent analyzed neonatal and neurodevelopmental outcomes of 21 children born after cycles with AOA [90]. For all tests and questionnaires, the mean outcomes lay within the expected ranges, but since the number of studied cases is small, the authors state that AOA should still be performed only in selected couples. In another study, 79 children born following AOA-ICSI and 89 born by ICSI were compared in terms of intrauterine fetal death, preterm delivery, birth weight, growth rate, hospitalization in neonatal intensive care units, abnormal behavior according to age, and the physical and mental health of children born and no significant differences were found [91]. In a study, genetic content of donated oocytes in metaphase II artificially activated with calcium-ionophore was analyzed. By using array comparative genomic hybridization, single-nucleotide polymorphism genotyping and maternal haplotyping chromosome segregation errors in meiosis II were not increased

There are concerns about the effects of AOA on gene expression and later embryonic development coming from animal studies [33, 93]. In the case of the use of SrCl2 for AOA, data in mice show that birth weight of male pups is reduced [80].

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.

The authors confirm that there are no known "conflicts of interest" associated

The publication is a part of research program P3-0327 and research project

*DOI: http://dx.doi.org/10.5772/intechopen.83488*

compared to the control group [92].

**9. Conclusions**

**Conflict of interest**

with this publication.

**Notes/thanks/other declarations**

J3-7177 funded by the Slovenian Research Agency.

The number of children born after AOA is relatively small for statistical analysis, but there are accumulating data on the safety of these methods. The study from Ghent analyzed neonatal and neurodevelopmental outcomes of 21 children born after cycles with AOA [90]. For all tests and questionnaires, the mean outcomes lay within the expected ranges, but since the number of studied cases is small, the authors state that AOA should still be performed only in selected couples. In another study, 79 children born following AOA-ICSI and 89 born by ICSI were compared in terms of intrauterine fetal death, preterm delivery, birth weight, growth rate, hospitalization in neonatal intensive care units, abnormal behavior according to age, and the physical and mental health of children born and no significant differences were found [91].

In a study, genetic content of donated oocytes in metaphase II artificially activated with calcium-ionophore was analyzed. By using array comparative genomic hybridization, single-nucleotide polymorphism genotyping and maternal haplotyping chromosome segregation errors in meiosis II were not increased compared to the control group [92].

There are concerns about the effects of AOA on gene expression and later embryonic development coming from animal studies [33, 93]. In the case of the use of SrCl2 for AOA, data in mice show that birth weight of male pups is reduced [80].
