**Abstract**

Despite successful treatment of infertility with assisted reproductive technology (ART), total fertilization failure (TFF) after in vitro fertilization (IVF) and even after intracytoplasmic sperm injection (ICSI) still occurs. In the current chapter, the incidence and etiology of TFF after ICSI are described. The literature on physiology of oocyte activation, electrical properties of gametes' membranes, and ion currents is reviewed. Calcium oscillations play an essential role in fertilization, and calcium ions act as secondary messengers in different metabolic pathways and cellular processes during oocyte activation. The contribution of oocyte- and spermrelated causes of fertilization failure is discussed. Many studies on the physiology of fertilization in mammals have shown that oocyte activation is triggered by the sperm factor. Methods for artificial oocyte activation (AOA) try to bypass fertilization failure by influencing physiological processes that are crucial for successful fertilization. Activation can be induced with the use of electrical, mechanical, or chemical stimuli that elevate intracellular concentrations of calcium ions. Different AOA methods and their success and safety are presented.

**Keywords:** oocyte activation, total fertilization failure (TFF), calcium oscillations, artificial oocyte activation (AOA), gamete maturation, ion channels, ion currents, calcium signaling, meiosis, intracytoplasmic sperm injection (ICSI), PLCζ, calcium ionophores

## **1. Introduction**

In vitro fertilization (IVF) techniques enabled conception outside the body and led to the birth of the first child conceived in vitro in 1978 [1]. The first laboratory technique used for conception in vitro was "classic" IVF where a suspension of prepared sperm cells is added to oocytes surrounded by cumulus cells and fertilization occurs naturally. The most discouraging result of such assisted reproduction technology (ART) treatment was fertilization failure, occurring often with the male infertility factor or unexplained infertility. The development of a micromanipulation technique named intracytoplasmic sperm injection (ICSI) in 1990 [2] enabled new treatment possibilities for many couples. Bypassing initial steps in the process of natural fertilization, a single spermatozoon is inserted directly into the cytoplasm. Successful fertilization is thus also achieved with low sperm numbers, surgically retrieved sperm, or frozen sperm samples. ICSI was soon globally accepted as a reliable technique leading to successful fertilization, pregnancy, and healthy offspring. Although ICSI can overcome some fertilization problems, total fertilization failure (TFF) still occurs in some ICSI cycles. In some patients, this failure can

repeat in several ART cycles. Some patients have extremely low fertilization rates, which consequently lowers their chances for successful treatment.

Studies of etiology of fertilization failure after ICSI revealed that the predominant cause is oocyte activation failure [3, 4]. In humans, oocyte activation is the transition of the oocyte into a zygote where a series of intracellular calcium (Ca2+) oscillations following the fusion of the gametes play an essential role. Calcium ions are released from intracellular storage in the endoplasmic reticulum; free in the cytosol, they are intracellular messengers and act as modulators of processes in the early steps of fertilization and embryo development. In humans, oocyte activation thus describes a cascade of events that lead to completion of the meiosis, cortical granule exocytosis for prevention of polyspermy, formation of the male and female pronuclei and progression in the first embryonic cell cycle. Both sperm and oocyte defects can cause failed activation.

Artificial oocyte activation (AOA) methods can be used in clinical practice in reproductive medicine in rare cases of TFF or low fertilization. AOA tries to reproduce elevations of calcium ion concentration in cytosol, which are necessary for triggering downstream processes in oocyte activation.

## **2. Total fertilization failure (TFF) after ICSI**

Total fertilization failure after ICSI is complete lack of fertilization at the standard checking time of 17 ± 1 h post ICSI. This means that the obvious sign, female and male pronuclei, is not visible in any of the injected mature oocytes in the metaphase of meiosis II (MII) of the patient.

According to data from the literature, complete fertilization failure occurs in: 2 [5], 1.3 [6], 3 [7], 3 [8], 5.6 [9], and 4.3% [10] of ICSI cycles.

Complete fertilization failure after ICSI is directly correlated with the number of mature oocytes available [9], so the definition is needed. TFF is not surprising in cases of poor ovarian response, nonmotile spermatozoa, or poor sperm morphology. But even if we have a sufficient number of mature oocytes of normal morphology and sperm of good quality, TFF happens and can reoccur in subsequent cycles.

Another problem that also lowers the chances for successful treatment of infertility is an extremely low fertilization rate.

#### **2.1 Results of retrospective analysis of data from our center**

We analyzed all consecutive ICSI cycles in the period between years 2011 and 2016. Results are presented in **Table 1**. In this period, we performed 7474 ART cycles (IVF and ICSI) in our center. The majority of these cycles were stimulated with gonadotropins (recombinant FSH or highly purified human menopausal gonadotropin) with pituitary suppression using agonists or antagonists, followed by hCG administration for 36–37 h before ultrasound-guided follicle aspiration. Some of these cycles were natural cycles, as previously described in [11].

In this period, we performed 4533 ICSI cycles with at least one mature oocyte in the metaphase stage of meiosis II (MII) available. Complete fertilization failure (TFF) occurred in 247 (5.5%) of ICSI cycles.

We compared standard characteristics of these cycles regarding the number of oocytes. There were 3550 cycles with 3 or more MII oocytes, TFF occurred in 76 among the (2.14%) cycles. There were 983 cycles with 1 or 2 MII oocytes available, TFF occurred in 171 among them (17.4%). A total of 35 of these TFF cycles were natural cycles.

**37**

obtain more oocytes.

*Oocyte Activation Failure: Physiological and Clinical Aspects*

**Cycles with ≥3MII**

N of all cycles 3550 983 175 1980 N of TFF cycles (%) 76 (2.14%) 171 (17.4%) / / Woman age (years) 35.62 ± 4.42 37.26 ± 4.69 34.70 ± 4.96 34.14 ± 4.65

• Natural cycles / 35 (20.5%) / /

• Other 5 12 12 88 Duration of stimulation 10.40 ± 1.95 8.77 ± 4.85 9.96 ± 1.76 9.98 ± 1.8

N oocytes 8.04 ± 6.10 2.02 ± 2.46 10.16 ± 5.4 10.16 ± 5.95 MII 5.80 ± 4.13 1.25 ± 0.44 7.91 ± 4.41 8.51 ± 5.07 2PN 0 0 1.35 ± 1.21 7.37 ± 4.33 1PN 0.07 ± 0.25 0.09 ±0.30 0.16 ± 0.40 0.12 ± 0.37 3PN 0.16 ± 0.49 0.26 ± 1.11 0.22 ± 0.64 0.10 ± 0.35 Damaged 0.29 ± 0.82 0.26 ± 1.11 0.57 ± 1.06 0.22 ± 0.55

**Cycles with <3MII**

26 (34.2%) 23 (13.5%) 58 (33.1%) 595 (30.6%)

45 (59.2%) 101 (59.1%) 105 (60.0%) 1297 (65.1%)

32.80 ± 11.84 31.53 ± 20.72 28.86 ± 10.75 27.34 ± 9.92

15 (19.74%) 61 (35.67%) 31 (17.7%) 371 (18.7%)

18 (23.68%) 17 (9.94%) 45 (25.7%) 214 (10.8%)

17.7%

702/1822 38.5%

**Cycles with >0% and <30% fertilization (≥3MII)**

**Cycles with >70% fertilization (≥3MII)**

We also analyzed the characteristics of cycles with 3 or more MII oocytes and low fertilization; in doing so, we took into account cycles with more than 0% and

ET 0 0 1.04 ± 0.76 1.57 ± 0.69

Birth rate / / 13.2% 30.5%

*Analysis of all consecutive ICSI cycles performed in our clinic in the period between years 2011 and 2016 (N = 4533); cycles with at least one mature oocyte in the metaphase stage of meiosis II (MII) are included. Data* 

Woman's age is greater in cycles with fewer oocytes, which can be explained with lower ovarian reserve in greater age. Regarding the stimulation protocol of the ART cycles, there were 35 natural cycles in the group with <3 MII oocytes. In our center, natural cycles are mainly performed in patients with extremely low ovarian response, where increasing gonadotropin dosage does not increase the chance to

In TFF cases where 3 or more MII oocytes are available, there is a higher proportion of severe male infertility cases (22.68%) compared to the cycles with less than

less than 30% MII oocytes fertilized. There were 175 such cycles (3.9%).

*are presented in means ± SD or number of cases (percentage of all cases in a group).*

Clinical pregnancy / / 39/220

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

*Stimulation protocol*

• Long protocol with agonists GnRHa

• Short protocol with antagonists antGnRH

Gonadotropin dose (ampoules)

*Male diagnosis*

TFF cycles)

**Table 1.**

• Normozoospermia (% of

• Krypto- and azoospermia (% of TFF cycles)


#### *Oocyte Activation Failure: Physiological and Clinical Aspects DOI: http://dx.doi.org/10.5772/intechopen.83488*

#### **Table 1.**

*Embryology - Theory and Practice*

defects can cause failed activation.

triggering downstream processes in oocyte activation.

**2. Total fertilization failure (TFF) after ICSI**

[5], 1.3 [6], 3 [7], 3 [8], 5.6 [9], and 4.3% [10] of ICSI cycles.

**2.1 Results of retrospective analysis of data from our center**

these cycles were natural cycles, as previously described in [11].

metaphase of meiosis II (MII) of the patient.

tility is an extremely low fertilization rate.

(TFF) occurred in 247 (5.5%) of ICSI cycles.

subsequent cycles.

repeat in several ART cycles. Some patients have extremely low fertilization rates,

Studies of etiology of fertilization failure after ICSI revealed that the predominant cause is oocyte activation failure [3, 4]. In humans, oocyte activation is the transition of the oocyte into a zygote where a series of intracellular calcium (Ca2+) oscillations following the fusion of the gametes play an essential role. Calcium ions are released from intracellular storage in the endoplasmic reticulum; free in the cytosol, they are intracellular messengers and act as modulators of processes in the early steps of fertilization and embryo development. In humans, oocyte activation thus describes a cascade of events that lead to completion of the meiosis, cortical granule exocytosis for prevention of polyspermy, formation of the male and female pronuclei and progression in the first embryonic cell cycle. Both sperm and oocyte

Artificial oocyte activation (AOA) methods can be used in clinical practice in reproductive medicine in rare cases of TFF or low fertilization. AOA tries to reproduce elevations of calcium ion concentration in cytosol, which are necessary for

Total fertilization failure after ICSI is complete lack of fertilization at the standard checking time of 17 ± 1 h post ICSI. This means that the obvious sign, female and male pronuclei, is not visible in any of the injected mature oocytes in the

Complete fertilization failure after ICSI is directly correlated with the number of mature oocytes available [9], so the definition is needed. TFF is not surprising in cases of poor ovarian response, nonmotile spermatozoa, or poor sperm morphology. But even if we have a sufficient number of mature oocytes of normal morphology and sperm of good quality, TFF happens and can reoccur in

According to data from the literature, complete fertilization failure occurs in: 2

Another problem that also lowers the chances for successful treatment of infer-

We analyzed all consecutive ICSI cycles in the period between years 2011 and 2016. Results are presented in **Table 1**. In this period, we performed 7474 ART cycles (IVF and ICSI) in our center. The majority of these cycles were stimulated with gonadotropins (recombinant FSH or highly purified human menopausal gonadotropin) with pituitary suppression using agonists or antagonists, followed by hCG administration for 36–37 h before ultrasound-guided follicle aspiration. Some of

In this period, we performed 4533 ICSI cycles with at least one mature oocyte in the metaphase stage of meiosis II (MII) available. Complete fertilization failure

We compared standard characteristics of these cycles regarding the number of oocytes. There were 3550 cycles with 3 or more MII oocytes, TFF occurred in 76 among the (2.14%) cycles. There were 983 cycles with 1 or 2 MII oocytes available, TFF occurred in 171 among them (17.4%). A total of 35 of these TFF cycles were

which consequently lowers their chances for successful treatment.

**36**

natural cycles.

*Analysis of all consecutive ICSI cycles performed in our clinic in the period between years 2011 and 2016 (N = 4533); cycles with at least one mature oocyte in the metaphase stage of meiosis II (MII) are included. Data are presented in means ± SD or number of cases (percentage of all cases in a group).*

We also analyzed the characteristics of cycles with 3 or more MII oocytes and low fertilization; in doing so, we took into account cycles with more than 0% and less than 30% MII oocytes fertilized. There were 175 such cycles (3.9%).

Woman's age is greater in cycles with fewer oocytes, which can be explained with lower ovarian reserve in greater age. Regarding the stimulation protocol of the ART cycles, there were 35 natural cycles in the group with <3 MII oocytes. In our center, natural cycles are mainly performed in patients with extremely low ovarian response, where increasing gonadotropin dosage does not increase the chance to obtain more oocytes.

In TFF cases where 3 or more MII oocytes are available, there is a higher proportion of severe male infertility cases (22.68%) compared to the cycles with less than

#### **Figure 1.**

*Number of available oocytes and incidence of TFF. The proportion of ICSI cycles with total fertilization failure regarding the number of mature oocytes available; x = number of injected MII oocytes; y = % of cycles with TFF. 4533 ICSI cycles analysed.*

3 MII oocytes (9.94%). When there are more oocytes available, there is statistically less probability for TFF, and more cases of TFF are due to gamete defects. A similar proportion of low fertilization cycles are those with severe male infertility (25.7%). Severe male infertility is described as diagnosis of cryptozoospermia and azoospermia, where individual sperms have to be extracted from semen sediments or testis aspirates/biopsies.

In cases where only one mature oocyte was available, total fertilization failure occurred more often (30.8%) than in those with more oocytes.

In **Figure 1**, the correlation between the number of available mature oocytes and occurrence of TFF is presented. With more oocytes available, there is less probability for TFF.

#### **3. Etiology of failed fertilization after ICSI**

Soon after implementation of the ICSI technique, some investigations of possible reasons for unsuccessful fertilization began. It was first speculated that perhaps the proportion of unfertilized oocytes arises from technical limitations of the method itself that cannot deliver the sperm in the cytoplasm, or ejection of the sperm from the cytoplasm occurs after injection. It was established that in only 7 [12], 16.7 [7], 10.6 [13], and 12.6% [14] of unfertilized oocytes after ICSI the sperm DNA was outside the oocytes.

With different staining techniques, visualization of the chromatin, spindle, and other structures was possible, and this enabled a better understanding of at what stage unfertilized oocytes are (**Table 2**). It soon became evident that the majority of unfertilized oocytes are arrested in the metaphase of the meiosis II with different levels of sperm chromatin decondensation, which suggested that oocyte activation and sperm decondensation run independently [15]. In the majority of these oocytes, sperm chromatin is in a decondensed state, which indicates that protamines are usually successfully replaced by histones [14], so unsuccessful decondensation of sperm chromatin can be the underlying cause for only a relatively small proportion of unfertilized oocytes. Premature chromosome condensation (PCC) is a condition when sperm chromosomes are getting condensed in the cytoplasm of oocyte too early and the right synchronization between sperm and oocyte genetic material is compromised. Up to 33% of studied unfertilized oocytes had PCC [15], but it is difficult to conclude whether this indicates sperm- or oocyte-borne defect.

**39**

*Oocyte Activation Failure: Physiological and Clinical Aspects*

**Study Method Number of** 

Hoechst 33342: fluorescent stain for adenine-thymine rich regions in DNA

Giemsa: stain for adeninethymine rich regions in

chromatin staining

Chromomycin A3: binds to G-C rich DNA regions, does not bind to DNA coupled with protamines Propidium iodide: fluorescent DNA stain

Immunofluorescence analysis with immunoglobulins and monoclonal antibodies

DNA

Yanagida K [13] Aceto-orcein stain:

**analyzed specimens**

n = 1005 NF MII

n = 82 NF MII

n = 76 NF MII

n = 93 NF MII

n = 150 NF MII

MII

**Findings**

cytoplasm

the cytoplasm

27.3% intact SC

place.

division

82% of oocytes are arrested in metaphase MII; of these, 74% decondensed SC, 11% intact SC, 15% without sperm in the

only 17% of oocytes activated; of these 56% decondensed SC, 20% intact SC, 15% without sperm in

93% oocytes having sperm in cytoplasm and MII chromosomes of the oocyte present; of these 51% SC, 41% intact SC, 8% premature chromosome condensation (PCC)

86.8% oocytes having sperm in cytoplasm; of these 68.2% decondensed SC, 4.5% PCC,

74.8% metaphase MII oocytes; of these 63.6% decondensed SC, 23.4% condensed SC, and 13% no sperm in the cytoplasm. In majority of spermatozoa, successful replacement of protamines with histones took

13.3% oocytes with no sperm, 39.9% activation failure, 22.6% defects of pronuclear formation/ migration, 13.3% arrest in metaphase of the 1st mitotic

69% oocytes arrested in metaphase MII, 11% completed meiosis, but no PN development

On the basis of the studies listed in **Table 2**, we can conclude that failed oocyte activation seems to be the predominant reason for fertilization failure. However, it is unclear whether the cause is sperm or oocyte defect, since proteins, organelles,

*SC = sperm chromatin, MII = mature oocyte in metaphase of meiosis II, NF = non-fertilized oocyte, and PCC =* 

Perhaps, in the future, genetic data will give us more information on the etiology of fertilization failure. An interesting case report where researchers investigated possible reasons for fertilization failure on genetic levels analyzed gene expression

Oocyte activation failure being the main problem was also confirmed by electron microscopy, where unreleased cortical granule at periphery, maternal chromosomes in the metaphase plate, and paternal intact or partially decondensed chromatin were found [3]. These are all signs of failed activation, but it is difficult

Hoechst 33258, FITC n = 180 NF

profiles in unfertilized oocytes of a patient with previous TFF history [17].

and metabolic paths of both gametes are involved in the activation.

to conclude on which level in the cascade there is a failure.

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

Flaherty SP, Payne D, and Matthews CD [7]

Dozortsev D, Sutter PD, and Dhont M [12]

Pitsos MA, Nicolopoulou-Stamati P [14]

Rawe VY, Olmedo SB, Nodar FN, Doncel GD, Acosta AA, and Vitullo AD [16]

Kovacic B and Vlaisavljevic V [15]

**Table 2.**

*premature chromosome condensation.*

*Studies of the etiology of fertilization failure.*

The studies are summarized in **Table 2**.


*Oocyte Activation Failure: Physiological and Clinical Aspects DOI: http://dx.doi.org/10.5772/intechopen.83488*

*SC = sperm chromatin, MII = mature oocyte in metaphase of meiosis II, NF = non-fertilized oocyte, and PCC = premature chromosome condensation.*

#### **Table 2.**

*Embryology - Theory and Practice*

aspirates/biopsies.

*TFF. 4533 ICSI cycles analysed.*

ity for TFF.

**Figure 1.**

3 MII oocytes (9.94%). When there are more oocytes available, there is statistically less probability for TFF, and more cases of TFF are due to gamete defects. A similar proportion of low fertilization cycles are those with severe male infertility (25.7%). Severe male infertility is described as diagnosis of cryptozoospermia and azoospermia, where individual sperms have to be extracted from semen sediments or testis

*Number of available oocytes and incidence of TFF. The proportion of ICSI cycles with total fertilization failure regarding the number of mature oocytes available; x = number of injected MII oocytes; y = % of cycles with* 

In cases where only one mature oocyte was available, total fertilization failure

Soon after implementation of the ICSI technique, some investigations of possible reasons for unsuccessful fertilization began. It was first speculated that perhaps the proportion of unfertilized oocytes arises from technical limitations of the method itself that cannot deliver the sperm in the cytoplasm, or ejection of the sperm from the cytoplasm occurs after injection. It was established that in only 7 [12], 16.7 [7], 10.6 [13], and 12.6% [14] of unfertilized oocytes after ICSI the sperm

With different staining techniques, visualization of the chromatin, spindle, and other structures was possible, and this enabled a better understanding of at what stage unfertilized oocytes are (**Table 2**). It soon became evident that the majority of unfertilized oocytes are arrested in the metaphase of the meiosis II with different levels of sperm chromatin decondensation, which suggested that oocyte activation and sperm decondensation run independently [15]. In the majority of these oocytes, sperm chromatin is in a decondensed state, which indicates that protamines are usually successfully replaced by histones [14], so unsuccessful decondensation of sperm chromatin can be the underlying cause for only a relatively small proportion of unfertilized oocytes. Premature chromosome condensation (PCC) is a condition when sperm chromosomes are getting condensed in the cytoplasm of oocyte too early and the right synchronization between sperm and oocyte genetic material is compromised. Up to 33% of studied unfertilized oocytes had PCC [15], but it is difficult to conclude whether this indicates sperm- or oocyte-borne defect.

In **Figure 1**, the correlation between the number of available mature oocytes and occurrence of TFF is presented. With more oocytes available, there is less probabil-

occurred more often (30.8%) than in those with more oocytes.

**3. Etiology of failed fertilization after ICSI**

The studies are summarized in **Table 2**.

DNA was outside the oocytes.

**38**

*Studies of the etiology of fertilization failure.*

On the basis of the studies listed in **Table 2**, we can conclude that failed oocyte activation seems to be the predominant reason for fertilization failure. However, it is unclear whether the cause is sperm or oocyte defect, since proteins, organelles, and metabolic paths of both gametes are involved in the activation.

Oocyte activation failure being the main problem was also confirmed by electron microscopy, where unreleased cortical granule at periphery, maternal chromosomes in the metaphase plate, and paternal intact or partially decondensed chromatin were found [3]. These are all signs of failed activation, but it is difficult to conclude on which level in the cascade there is a failure.

Perhaps, in the future, genetic data will give us more information on the etiology of fertilization failure. An interesting case report where researchers investigated possible reasons for fertilization failure on genetic levels analyzed gene expression profiles in unfertilized oocytes of a patient with previous TFF history [17].

## **4. Oocyte activation**

Oocyte activation is a downstream cascade triggered by sperm that causes progression of the oocyte from meiosis arrested in metaphase II toward its completion and beginning of embryonic development. It is a serial of biochemical reactions, organelle redistribution, changes in metabolism, transmembrane potentials, mRNA translation, gene transcription, and cytoskeletal rearrangements.

The role of calcium in fertilization was established very early with a series of experiments on sea urchin eggs where the amount of bound and free calcium was measured in fertilized and nonfertilized eggs [18]. Later, calcium-specific lightemitting protein aequorin injected in fish oocytes enabled visualization of light flash after fusion of oocyte and sperm [19]. It soon became evident that calcium ions play an essential role in activation of the animal oocyte and that the frequencies and amplitudes of these elevations of calcium ions in cytoplasm are species-specific [20].

The term "oocyte activation" probably evolved on the basis of these evident sudden changes that happen during the transition from oocyte to embryo. It describes not only calcium waves that occur but also other processes and morphological changes that happen during fertilization. Intracytoplasmic calcium elevation is essential for fertilization, but it is not always the sperm that triggers it. In some animal species such as fruit flies (*Drosophila*) the calcium wave occurs prior to oocyte-sperm fusion, during ovulation [21]. The focus of our text will be human oocyte activation, but since nonhuman biological material is usually more available or even easier to study, many data on fertilization come from studies on sea organisms such as starfish and sea urchins or different mammalian species. Early studies of the role of calcium in the process of fertilization and even use of ionophores are well documented in the review of Epel [22]. The source of an intracellular rise of calcium ion concentration can be external—calcium enters the cell by influx through calcium channels in the plasma membrane or can be released in cytoplasm from intracellular stores in the endoplasmic reticulum [23].

But it is important to understand that the details vary a lot through the animal kingdom and that these differences can be the reason why the ICSI method can be successful in humans but not in other species. However, the animal studies are the foundation for the development of assisted reproduction techniques in human medicine.

Early studies of fertilization in mammals are well reviewed by Miyazaki [24]; in sum, there is the first hyperpolarization of membrane potential as a result of a change in potassium conductivity across the plasma membrane. This coincides with an increase of free calcium in cytosol; there is no electrical block of polyspermy and a serial of intracytoplasmic rises of calcium concentration follow continuously (oscillations) at intervals of different frequencies and amplitudes, which depends on the species studied. Intracellular calcium first rises near the site of the sperm attachment and spreads like a wave over the entire egg [24]. The model of generating calcium spikes from intracellular stores in the endoplasmic reticulum was described by Igusa and Miyazaki [25]. The techniques used for revealing these processes were measurements with calcium-sensitive microelectrodes, the voltageclamp technique, aequorin injections, injection of calcium ion chelators, and injection of different compounds that interact with the calcium-releasing system.

The first study of calcium measurements at fertilization in human oocytes showed that the first rise in intracytoplasmic calcium concentration appears 20–35 min after adding sperm suspension in a chamber with oocytes; spikes appear every 10–35 min, with a single spike of amplitude up to 2.25 μM calcium concentration and duration of 100–120 s [26].

**41**

*Oocyte Activation Failure: Physiological and Clinical Aspects*

Other researchers studied changes of membrane potential across the plasma membrane in human oocytes at fertilization and showed that the increase of potassium ion conductivity of the plasma membrane and outward current of ions, which causes hyperpolarization, is calcium dependent [27]. A study on bovine oocytes gave more information about the relationship between hyperpolarization of the plasma membrane potential and calcium release from intracellular stores and targeted calcium-activated potassium channels as membrane proteins involved in

Soon after introducing ICSI, it was of great interest to compare these responses with the classic IVF method, where events such as sperm capacitation and activation, acrosome reaction, and sperm-oocyte membrane fusion happen first. From the work of Tesarik et al. [29], we can see that when performing ICSI in human oocytes, the first intracytoplasmic rise of calcium ion concentration happens immediately; the peak is 10–15 s after penetration with the needle. Sperm then evokes intracellular calcium oscillations. They described that oscillations follow the lag period that lasts 4–12 h. Oscillations are in the form of spikes that last 20 s; the intervals between spikes are 1–5 min. The duration of the oscillatory phase is 30 min–1 h; at the end of the period, the amplitude of calcium spikes gets smaller. The proposed mechanism through which calcium oscillations are maintained is through the phosphoinositide signaling pathway, where inositol 1,4,5-trisphosphate (InsP3) is generated from phosphatidylinositol 4,5-bisphosphate (PIP2) [23]. The positive feedback cycle involving calcium-dependent InsP3 generation and InsP3 induced calcium release seem to be responsible for the oscillations [23]. The main protein is InsP3 receptor (InsP3R), a ligand-gated channel found in the membrane of

the endoplasmic reticulum that allows calcium release from the ER [30].

**4.1 The role of free calcium ions in cytoplasm**

That calcium oscillations have a role in long-term embryonic events and provide more than merely a stimulus for meiotic resumption was shown in experiments with different activating agents and subsequent measurements of cell mass of the

Calcium is the secondary messenger that regulates different events during fertilization, such as progression of the cell cycle from metaphase II arrest toward chromatid segregation, extrusion of the second polar body and completion of the second meiotic division, and cortical granule exocytosis [32]. The role of calcium in reproduction is preserved through evolution; it is important in plants and animals. Species-specific calcium signatures, like oscillations in mammals, have evolved, which are optimal for activation and development of a specific type of organism [33]. The variations in amplitude, duration, and frequency of oscillations over time are coordinated with the cell cycle, and experimentally changing them also affects development in the later stages when blastocyst forms [31]. Experiments with injecting calcium (Ca2+) chelators in the cytosol of frog eggs demonstrated the

Calcium rises in cytosol are converted in different cellular responses.

Cortical granule exocytosis enables polyspermy block by altering the zona pellucida with the content of the granule (proteases, peroxidases, and glycosaminoglycans) and prevent more sperms from fertilizing oocytes. The proposed model is that calcium stimulates Ca2+/calmodulin (CaM)-dependent protein kinase

*4.1.1 Ca2+-dependent process of cortical granule exocytosis*

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

the process [28].

blastocyst [31].

blockage of activation [34].

Oocyte activation is a downstream cascade triggered by sperm that causes progression of the oocyte from meiosis arrested in metaphase II toward its completion and beginning of embryonic development. It is a serial of biochemical reactions, organelle redistribution, changes in metabolism, transmembrane potentials, mRNA

The role of calcium in fertilization was established very early with a series of experiments on sea urchin eggs where the amount of bound and free calcium was measured in fertilized and nonfertilized eggs [18]. Later, calcium-specific lightemitting protein aequorin injected in fish oocytes enabled visualization of light flash after fusion of oocyte and sperm [19]. It soon became evident that calcium ions play an essential role in activation of the animal oocyte and that the frequencies and amplitudes of these elevations of calcium ions in cytoplasm are species-specific [20]. The term "oocyte activation" probably evolved on the basis of these evident sudden changes that happen during the transition from oocyte to embryo. It describes not only calcium waves that occur but also other processes and morphological changes that happen during fertilization. Intracytoplasmic calcium elevation is essential for fertilization, but it is not always the sperm that triggers it. In some animal species such as fruit flies (*Drosophila*) the calcium wave occurs prior to oocyte-sperm fusion, during ovulation [21]. The focus of our text will be human oocyte activation, but since nonhuman biological material is usually more available or even easier to study, many data on fertilization come from studies on sea organisms such as starfish and sea urchins or different mammalian species. Early studies of the role of calcium in the process of fertilization and even use of ionophores are well documented in the review of Epel [22]. The source of an intracellular rise of calcium ion concentration can be external—calcium enters the cell by influx through calcium channels in the plasma membrane or can be released in cytoplasm

But it is important to understand that the details vary a lot through the animal kingdom and that these differences can be the reason why the ICSI method can be successful in humans but not in other species. However, the animal studies are the foundation for the development of assisted reproduction techniques in

Early studies of fertilization in mammals are well reviewed by Miyazaki [24]; in sum, there is the first hyperpolarization of membrane potential as a result of a change in potassium conductivity across the plasma membrane. This coincides with an increase of free calcium in cytosol; there is no electrical block of polyspermy and a serial of intracytoplasmic rises of calcium concentration follow continuously (oscillations) at intervals of different frequencies and amplitudes, which depends on the species studied. Intracellular calcium first rises near the site of the sperm attachment and spreads like a wave over the entire egg [24]. The model of generating calcium spikes from intracellular stores in the endoplasmic reticulum was described by Igusa and Miyazaki [25]. The techniques used for revealing these processes were measurements with calcium-sensitive microelectrodes, the voltageclamp technique, aequorin injections, injection of calcium ion chelators, and injection of different compounds that interact with the calcium-releasing system. The first study of calcium measurements at fertilization in human oocytes showed that the first rise in intracytoplasmic calcium concentration appears 20–35 min after adding sperm suspension in a chamber with oocytes; spikes appear every 10–35 min, with a single spike of amplitude up to 2.25 μM calcium

translation, gene transcription, and cytoskeletal rearrangements.

from intracellular stores in the endoplasmic reticulum [23].

concentration and duration of 100–120 s [26].

**4. Oocyte activation**

**40**

human medicine.

Other researchers studied changes of membrane potential across the plasma membrane in human oocytes at fertilization and showed that the increase of potassium ion conductivity of the plasma membrane and outward current of ions, which causes hyperpolarization, is calcium dependent [27]. A study on bovine oocytes gave more information about the relationship between hyperpolarization of the plasma membrane potential and calcium release from intracellular stores and targeted calcium-activated potassium channels as membrane proteins involved in the process [28].

Soon after introducing ICSI, it was of great interest to compare these responses with the classic IVF method, where events such as sperm capacitation and activation, acrosome reaction, and sperm-oocyte membrane fusion happen first. From the work of Tesarik et al. [29], we can see that when performing ICSI in human oocytes, the first intracytoplasmic rise of calcium ion concentration happens immediately; the peak is 10–15 s after penetration with the needle. Sperm then evokes intracellular calcium oscillations. They described that oscillations follow the lag period that lasts 4–12 h. Oscillations are in the form of spikes that last 20 s; the intervals between spikes are 1–5 min. The duration of the oscillatory phase is 30 min–1 h; at the end of the period, the amplitude of calcium spikes gets smaller.

The proposed mechanism through which calcium oscillations are maintained is through the phosphoinositide signaling pathway, where inositol 1,4,5-trisphosphate (InsP3) is generated from phosphatidylinositol 4,5-bisphosphate (PIP2) [23]. The positive feedback cycle involving calcium-dependent InsP3 generation and InsP3 induced calcium release seem to be responsible for the oscillations [23]. The main protein is InsP3 receptor (InsP3R), a ligand-gated channel found in the membrane of the endoplasmic reticulum that allows calcium release from the ER [30].

That calcium oscillations have a role in long-term embryonic events and provide more than merely a stimulus for meiotic resumption was shown in experiments with different activating agents and subsequent measurements of cell mass of the blastocyst [31].

#### **4.1 The role of free calcium ions in cytoplasm**

Calcium is the secondary messenger that regulates different events during fertilization, such as progression of the cell cycle from metaphase II arrest toward chromatid segregation, extrusion of the second polar body and completion of the second meiotic division, and cortical granule exocytosis [32]. The role of calcium in reproduction is preserved through evolution; it is important in plants and animals. Species-specific calcium signatures, like oscillations in mammals, have evolved, which are optimal for activation and development of a specific type of organism [33]. The variations in amplitude, duration, and frequency of oscillations over time are coordinated with the cell cycle, and experimentally changing them also affects development in the later stages when blastocyst forms [31]. Experiments with injecting calcium (Ca2+) chelators in the cytosol of frog eggs demonstrated the blockage of activation [34].

Calcium rises in cytosol are converted in different cellular responses.
