The present status of artificial oocyte activation in assisted reproductive technology

Abstract

Intracytoplasmic sperm injection (ICSI) is the most effective treatment for achieving fertilization in assisted reproductive technology (ART). However, fertilization failure occurs. The incidence of fertilization failure after ICSI is 1–5%. Approximately 50% of fertilization failure cases could be attributed to the abnormality of sperm factor. As the fertilization fails after ICSI using mature sperm, round spermatids and globozoospermia, artificial oocyte activation may provide a means of improving fertilization rates in such cases. The oocyte activation treatments used in clinical research include calcium (Ca) ionophore treatment, electrostimulation and strontium treatment. In terms of the efficiency of oocyte activation, electrostimulation and Ca ionophore gave better outcomes than strontium treatment. Strontium treatment causes Ca²⁺ oscillations in mice, so it has been viewed favorably. However, in human oocytes calcium oscillation has not been observed. The fertilization rate after ICSI was low in the case of globozoospermia and wiht round spermatids. Some cases of pregnancy were achieved by ICSI alone and oocyte activation methods were not essential in these cases. Among the various oocyte activation methods currently used, it should be noted that issues of genetic safety have not been addressed for the combined use of these oocyte activation methods. (Reprod Med Biol 2008; 7: 133–142)

INTRODUCTION

OOCYTE ACTIVATION IS the process by which oocytes arrested in metaphase II of meiosis are stimulated to resume meiosis. ¹ This process is marked by pore formation and secretion in the cortical granules, and release of the second polar body. It is necessary for the initiation of fertilization and occurs when the sperm adheres to the cell membrane of the oocyte. However, it can also be triggered by chemical or physical stimulation in a process known as artificial oocyte activation.

Artificial oocyte activation has been used not only to study the mechanism of oocyte activation, ² but also in studies of parthenogenesis ³ and nuclear transplantation. ⁴ Activation of oocytes is precipitated by various types of stimulation, including exposure to ultraviolet light, ⁵ changes in osmotic pressure, ⁶ treatment with calcium (Ca) ionophores, ³ electrostimulation, ⁷ and treatment with strontium, ⁸ puromycin ⁹ or cycloheximide. ⁹

Interestingly, the fertilization rate resulting from intracytoplasmic sperm injection (ICSI) is increased when the oocytes undergo artificial oocyte activation in hamsters ¹⁰ , ¹¹ and humans. ¹² This raises the possibility that artificial oocyte activation might be used to enhance fertilization rates alongside other techniques. As the fertilization fails after ICSI using mature sperm, immature male germ cells and round head sperm, artificial oocyte activation might provide a means of improving the fertilization rates in such cases.

FERTILIZATION BY INTRACYTOPLASMIC SPERM INJECTION

OOCYTE ACTIVATION OCCURS early in the process of fertilization and involves the resumption of meiosis of oocytes arrested at the metaphase stage of meiosis II. The details of the mechanism are not clear, but a transient increase in calcium ions ([Ca²⁺]_i) inside the oocyte is known to play an important role. ¹³ , ¹⁴ In ICSI‐assisted fertilization, the oocyte is activated by a sperm‐derived oocyte activation factor (also known as sperm factor) after the injection. ¹⁴ , ¹⁵ , ¹⁶ A decrease or loss in activity of this factor is a possible cause of fertilization failure with ICSI. When this factor is completely absent, oocyte activation does not occur and the oocyte remains in metaphase II even if ICSI is carried out. We are able to evaluate the ability of sperm to activate oocytes using mouse oocytes. ¹⁷ , ¹⁸ Immobilized sperm injected into the oocyte only undergo decondensation if the sperm nucleoprotein and the oocyte reduction mechanism are normal. ¹⁹ When the activity level of the sperm factor is reduced, the oocyte activation mechanism is stimulated at a low level, resulting in a reduction in the level of maturation‐promoting factor (MPF) in the oocyte and in the release of the second polar body. ²⁰ However, in some cases, MPF is resynthesized and the activation mechanism of the oocyte is halted. In these cases, premature chromatin condensation (PCC) sometimes occurs after the release of the second polar body and decondensation of the sperm injected into the ooplasm (metaphase III).

Although a transient increase in Ca ions in the oocyte is necessary for oocyte activation, subsequent Ca²⁺ oscillations are not absolutely required. In many oocyte activation methods, the oocyte is activated by inducing a single transient increase in the concentration of Ca²⁺. Although the functional significance of Ca²⁺ oscillations is not clear, its continuation from the postfertilization to pronuclear fusion stages suggests a role in embryonic development. ²¹ Furthermore, in mice, parthenogenetic oocytes without Ca²⁺ oscillations develop into blastocysts with a smaller inner cell mass. ²² In addition, Ca²⁺ oscillations affect pronucleus formation, ²³ the arrest of early embryo development ²⁴ and postimplantation development. ²⁵

TYPES AND MECHANISMS OF ARTIFICIAL OOCYTE ACTIVATION

OOCYTE ACTIVATION REQUIRES either a transient increase in Ca²⁺ in the oocyte or a decline in MPF, ²⁶ which normally follows such a Ca spike. Methods of artificial oocyte activation are usually based on one of these mechanisms (Table 1).

Table 1.

Types of artificial oocyte activation methods and reported cases in clinical research

Types of artificial oocyte activation methods	Mechanism of oocyte activation	Cases of pregnancy and delivery in clinical research
Calcium ionophore†	Increase in Ca2+ permeability of cell membrane	+ (Hoshi et al. 1995 A23187 27 )
Electrostimulation	Pore formation in cell membrane, entry of extracellular Ca2+	+ (Yanagida et al. 1999 28 )
Strontium	Release of endogenous Ca2+	+ (Yanagida et al. 2006 29 )
Ethanol	Increase in Ca2+ permeability of cell membrane
Puromycin‡	Inhibition of protein synthesis	+ (Murase et al. 2004 30 )
Cycloheximide	Inhibition of protein synthesis

^† Calcium ionophores include A23187 and ionomycin.

^‡ ‡Puromycin is used in combination with A23187.

Calcium ionophores

Calcium ionophores, including A23187 ¹ and ionomycin, ³¹ are commonly used to induce oocyte activation. The first case in which the combined use of A23187 and ICSI resulted in a successful pregnancy and delivery was reported in 1995. ²⁷ This case used the combination treatment to increase the fertilization rate and several subsequent cases have been reported in recent years. ³² , ³³ , ³⁴ , ³⁵ , ³⁶

Mechanism

Calcium ionophores help activate the oocyte by increasing the Ca²⁺ permeability of the cell membrane, thereby letting extracellular Ca²⁺ flow into the cell. ³⁷ The oocyte activating effect of A23187 is weakened by the presence of protein, including human serum albumin. When a strong effect is desired, a Ca^2+/Mg⁺‐free culture medium should be used, and the osmotic pressure should be corrected using polyvinylpyrrolidone. The addition of albumin may be used to stop the ionophore treatment.

For human oocytes, serum‐containing medium is preferred for its gentler effect on the oocyte. Following treatment with A23187, the Ca²⁺ concentration in the oocyte peaks after approximately 1 min and then gradually decreases. ³⁷ The treatment causes a single transient increase in Ca ion concentration, but no Ca²⁺ oscillation.

Method

• 1Dissolve A23187 (C7522; Sigma, St Louis, MO, USA) in dimethyl sulfoxide (DMSO) to make a 1 mmol/L stock solution. The stock solution should be kept frozen at –80°C.
• 2Add 10 µL of the stock solution to 990 µL of culture medium (e.g. human tubal fluid [HTF]) and mix to make 10 µmol/L A23187 treatment solution. Use the treatment solution as soon as possible after preparation and keep it protected from light exposure until use.
• 3Add the A23187 treatment solution to the oocyte and place in a 37°C incubator for 5–15 min.
• 4After treatment is completed, immediately wash the oocyte three times with standard culture medium and resume incubation.

When oocyte activation with ICSI alone fails, A23187 treatment may be carried out before or after ICSI to improve the fertilization rates. One report describes treating oocytes with A23187 approximately 30 min after performing ICSI. ²⁷

Electrostimulation

Electrostimulation is used in studies of parthenogenesis or for embryo cloning in the field of animal science. Fundamental research ¹¹ , ³⁸ and clinical studies ²⁷ of electrostimulation with ICSI in human oocytes have been reported.

Mechanism

The oocyte is placed between two parallel electrode plates. The electric field generated by a direct current voltage causes charged proteins in the lipid bilayer of the cell membrane to move, thereby forming pores in the membrane. ³⁹ Extracellular Ca²⁺ in the culture medium flow into the oocyte through these pores, transiently elevating the interior Ca²⁺ concentration and activating the oocyte. The lower the electrolyte concentration in the culture medium, the more pore formation is stimulated. The pores are thought to take 10–40 min at 37°C to repair, and longer when the temperature is lower. ⁴⁰ The concentration of Ca²⁺ is elevated immediately after the application of the stimulus, peaks within 1 min, and then gradually decreases and returns to the original level in approximately 5 min, without subsequent oscillation. ¹¹ , ³⁷ When electrostimulation was applied repeatedly to imitate Ca²⁺ oscillations in unfertilized rabbit oocytes, parthenogenesis occurred and fetuses were obtained. ⁴¹

Method

The actual process of electrostimulation is described as follows:

• 1Fill the electrode chamber with Zimmerman solution ³⁹ or Dulbecco's phosphate buffered saline (D‐PBS). ¹¹
• 2Place the oocyte (or multiple oocytes) between the electrodes in the chamber.
• 3Apply a rectangular wave of electrostimulation using an electroporator (e.g. apply one pulse of 100–150 V for 50–100 µsec when the distance between the electrodes is 1 mm).
• 4Incubate the oocyte in standard culture medium.

The conditions for electrostimulation vary according to the electroporator and chamber type (the distance between the electrodes and the material of the electrodes). Conditions must be set in advance using a 1‐day‐old oocyte. Although D‐PBS is used as the pulsing medium in the above method, 0.3 mol/L mannitol solution (containing 100 µmol/L CaCl₂ and 100 µmol/L MgCl₂) is more typically used in basic research. Because D‐PBS contains more electrolytes, the electric current created by electrostimulation would normally be a problem, but the electric resistance between the electrodes is approximately 13 KΩ or higher, and the electric current created is very weak, at approximately 1 mA. Thus, out of concern that changes in the composition of the culture medium might damage the cell, D‐PBS is used.

Strontium treatment method

Because strontium treatment has been shown to cause oocyte activation accompanied with Ca²⁺ oscillations in mouse models, ⁴² this method is preferred when embryonic development is desired. In contrast, only the initial transient increase in cytoplasmic Ca²⁺ concentration is achieved with A23187 treatment and electrostimulation. ³⁷ Another characteristic of strontium treatment is varied efficacy of oocyte activation depending on the species. ²⁹ , ⁴³ , ⁴⁴ , ⁴⁵ , ⁴⁶ It is most effective in mice, but its efficacy has not been sufficiently confirmed in humans. Nonetheless, cases of pregnancy and delivery have been reported in clinical research applications. ²⁹

Mechanism

Sr²⁺ induced Ca²⁺ transients in an activated oocyte. ⁴⁷ However, the mechanism by which Sr²⁺ induces Ca²⁺ oscillations in an oocyte remains unclear. Recently, Ca²⁺ oscillations induced by Sr²⁺ were mediated through inositol trisphosphate receptors. ⁴⁸ Sr²⁺ is thought to move into the oocyte down the concentration gradient, causing Ca to be released from the endoplasmic reticulum.

Method

• 1Dissolve SrCl₂·6H₂O (Sigma) in distilled water to make 1 mmol/L Sr²⁺ stock solution and store in the deep freezer.
• 2Add 20 µL of the stock solution to 1 mL of a Ca²⁺‐free culture medium (such as HTF) to make 20 mmol/L strontium treatment solution.
• 3Place the oocyte into the strontium treatment solution and incubate it for 120 min in an incubator.
• 4Transfer the oocyte into standard culture medium and incubate.

The impact of strontium treatment on the chromosomes of gametes has been studied in mice and no effect has been reported. ⁴⁹

CLINICAL APPLICATION OF OOCYTE ACTIVATION METHODS

Cases of fertilization failure with ICSI

WHEN ICSI DOES not result in oocyte activation, treatments that artificially activate oocytes are used concomitantly with ICSI to promote fertilization. When ICSI is carried out, the incidence of fertilization failure is 1–5%. ⁵⁰ , ⁵¹ , ⁵² , ⁵³ , ⁵⁴ The number of oocytes used in ICSI is closely associated with the fertilization rate. ⁵² The incidence of fertilization failure is 13.3% when ICSI is carried out with one oocyte, but decreases to 3.1% with two oocytes and 1% or lower with three or more oocytes. There was no fertilization failure with six or more oocytes. ⁵² When a small number of oocytes are used in ICSI, the high incidence of failure can be attributed to the poor quality of the collected oocytes or to technical factors (e.g. if a device malfunctioned during the initial ICSI, it would be fixed). Fertilization failure was observed in 5.6% (100/1779) of treatment cycles of ICSI. When fertilization failed in the initial ICSI, the probability of fertilization failure in the second ICSI was 13% and the incidence of such cases is 0.7% in all treatment cycles. ⁵²

Because the steps between capacitation and sperm–egg fusion are bypassed in ICSI, the problem must lie in later steps of the fertilization process. In particular, abnormalities in oocyte activation, decondensation of the sperm head or pronucleus formation may play a role in fertilization failure. The mechanism of oocyte activation in ICSI is thought to involve the action of phospholipase C zeta (PLCzeta), which is present in the sperm. ⁵⁵ When the activity of this factor is low or lacking, fertilization fails. In addition, after fertilization, the sperm needs to undergo decondensation, which depends on the condition of the sperm nucleoprotein as well as the reduction system in the oocyte. ¹⁹ For example, decondensation can be impaired when the zinc supply from the seminal plasma is decreased because of a disorder, such as prostatitis, or when a large number of disulfide bonds are formed in the nucleoproteins. ⁵⁶ The sperm aster also plays an important role in the fusion of male and female pronuclei. Abnormalities in the sperm centrosome that comprise the sperm aster can impair decondensation. ⁵⁷ When oocytes that were not successfully fertilized following ICSI were observed using chromatin staining, oocyte activation failed to occur in 70% of the oocytes, even though the sperm was successfully inserted into the oocyte. ⁵² Moreover, when ICSI was carried out using sperm from volunteers whose sperm had been shown to be capable of fertilizing oocytes, fertilization was successful in 70% of the cases. Therefore, for approximately 50% of the oocytes that were not fertilized following ICSI, the failure of fertilization could be attributed to the sperm. ⁵²

If abnormalities in oocyte activation factor activity are the cause of fertilization failure, the combined use of oocyte activation treatments and ICSI can be considered. However, if the source of the problem lies elsewhere, there is currently no treatment method. The recurrence rate of fertilization failure following ICSI is 13%, which means that in many cases, fertilized oocytes can be obtained from a second round of ICSI. ⁵² When the risk of fertilization failure is high, however, an oocyte activation treatment combined with ICSI has been used. The oocyte activation treatments used in clinical research include Ca ionophore treatment, electrostimulation and strontium treatment. Table 2 shows the results of clinical research on the combined use of oocyte activation and ICSI in patients with fertilization failure and low fertilization cases following an ICSI attempt, excluding cases using spermatid and those with globozoospermia. We reported the first case of pregnancy and delivery after ICSI with electrostimulation. Since then there have been 10 delivery cases using the same treatment as the fertilization failure case. In terms of the efficiency of oocyte activation, electrostimulation and A23187 gave better outcomes than strontium treatment. Strontium treatment showed variable efficacy. This treatment was developed in studies using mice and has been viewed favorably because it causes Ca²⁺ oscillations and is similar to the physiological stimulus that activates oocytes. ⁴¹ However, in human oocytes, Ca²⁺ oscillations have not been observed following treatment and its role in human oocyte activation is not clear. ²⁹ For clinical research, methods with known mechanisms should be selected. Chromosomal analysis of oocytes fertilized using both ICSI and oocyte activation did not show an increased risk from electrostimulation ³⁶ and A23187 + puromycin. ⁵⁹

Table 2.

Reports on the combined use of oocyte activation methods and intracytoplasmic sperm injection^†

Reference	Indication	Types of oocyte activation methods	Cases of pregnancy and delivery
Tesarik et al. (1994) 12	Fresh oocytes	A23187	Research‡
Hoshi et al. (1995) 27	Low fertilization	A23187	Two cases of pregnancy
Yanagida et al. (1999) 28	Fertilization failure	Electrical stimulation	One case of pregnancy and delivery
Yamano et al. (2000) 57	1‐day‐old unfertilized oocytes	A23187 + puromycin	Research‡
Nakagawa et al. (2001) 58	1‐day‐old unfertilized oocytes	A23187 + puromycin	Research‡
Tesarik et al. (2002) 59	ICSI patients	Mechanical	One case of pregnancy and delivery
Eldar‐Geva et al. (2003) 32	Fertilization failure	A23187	One case of pregnancy and delivery
Murase et al. (2004) 30	Fertilization failure	A23187 + puromycin	One case of pregnancy and delivery
Ebner et al. (2004) 60	Fertilization failure	Mechanical	One case of pregnancy and delivery
Chi et al. (2004) 33	Low fertilization	A23187	One case of pregnancy and delivery
Heindryckx et al. (2005) 34	Fertilization failure	A23187	Three cases of pregnancy
Lu et al. (2006) 61	1‐day‐old unfertilized oocytes	A23187 + puromycin	Research‡
Manipalviratn et al. (2006) 62	1‐day‐old unfertilized oocytes	Electrostimulation	Research‡
Ahmady et al. (2007) 35	Immotile testicular sperm	A23187	One case of pregnancy and delivery
Moaz et al. (2006) 63	Low fertilization and fertilization failure	Ionomycin	Research‡
Yanagida et al. (2006) 29	Low fertilization	Strontium	One case of pregnancy and delivery
Nasr‐Esfahani et al. (2008) 36	Low fertilization (teratozoospermia)	Ionomycin	Two cases of pregnancy

^† Excluding cases using spermatids and globozoospermia.

^‡ ‘Research’ means that this report was carried out for the purpose of research not a therapeutic purpose. ICSI, intracytoplasmic sperm injection.

The timing of electrostimulation relative to ICSI is another important factor to consider. When electrostimulation is carried out prior to ICSI, the oocyte tends to degenerate. It is possible that the cell membrane is not sufficiently repaired after electrostimulation ⁴⁰ and is further damaged by ICSI. Because the cell membrane can also be damaged by Ca ionophore treatment, we treat oocytes with Ca ionophore 30 min after ICSI. In addition, in mice, when the sperm is present during electrostimulation, the incidence of chromosomal aberration (structural abnormality) in the sperm is approximately 50% higher. In such cases, the chromosomes of the oocyte were unaffected. In contrast, when electrostimulation was carried out 30–60 min before ICSI, there were no chromosomal abnormalities, but the oocyte still tends to be damaged.

Special cases of ICSI include cases of round‐headed spermatozoa (globozoospermia) and the use of spermatids. Round‐headed spermatozoa possess round heads lacking an acrosome. ⁶⁵ In spermatozoa, the level of sperm factor appears to be low and there are reported cases of fertilization failure associated with it, for which clinical application of ICSI has been reported (Table 3). The reported fertilization rates were 0–42% with ICSI alone and approximately 70% in conjunction with oocyte activation methods. With round‐headed spermatozoa, many cases of pregnancy were achieved by ICSI alone ⁶⁶ , ⁶⁸ , ⁷¹ , ⁷² , ⁷³ , ⁷⁴ , ⁷⁷ and oocyte activation methods were not essential. From clinical research using round spermatids, 14 cases of pregnancy have been reported to date (Table 4). ⁷⁸ , ⁷⁹ , ⁸¹ , ⁸² , ⁸⁴ , ⁸⁵ , ⁸⁶ , ⁹³ , ⁹⁴ Because the activity level of the sperm factor is low in human round spermatids, oocyte activation in conjunction with ICSI has been considered. However, the reported fertilization rates were between 16 and 69% with ICSI alone. ⁷⁸ , ⁷⁹ , ⁸¹ , ⁸² , ⁸³ , ⁸⁴ , ⁸⁵ , ⁸⁶ , ⁸⁷ , ⁸⁸ , ⁸⁹ , ⁹² , ⁹⁵ , ⁹⁶ Oocyte activation methods were used along with ICSI in a small number of reported cases and the fertilization rate was approximately 40%. ⁸⁰ , ⁸¹ In our experience, the fertilization rate with round spermatids was 17%. ⁹⁷ Elongated spermatids seem to have a similar oocyte activation capacity as mature sperm.

Table 3.

Reported cases of intracytoplasmic sperm injection for globozoospermia

Reference	Oocyte activation treatment	Fertilization rate (%)	Cases of pregnancy and delivery
Liu et al. (1995) 66	None	–	Two cases of pregnancy
Trokoudes et al. (1995) 67	None	50	One case of pregnancy
Bourne et al. (1995) 68	None	–	None
Battaglia et al. (1997) 69	None	10	None
	A23187	75	None
Rybouchkin et al. (1997) 70	A23187	–	One case of pregnancy
Stone et al. (2000) 71	None	10–42	One case of pregnancy and delivery
Kim et al. (2001) 72	A23187	60	One case of pregnancy and delivery
Zeyneloglu et al. (2002) 73	None	31	One case of pregnancy and delivery
Nardo et al. (2002) 74	None	–	One case of pregnancy and delivery
Kilani et al. (2004) 75	None	38	One case of pregnancy and delivery
Heindryckx et al. (2005) 34	A23187	71–77	Five cases of pregnancy
Khalili et al. (2007) 76	None	0 (four cases)	None
Dirican et al. (2007) 77	None	9, 33 (two cases)	Two cases of pregnancy and delivery

Table 4.

Reports on round spermatid injection

Reference	Oocyte activation treatment	Fertilization rate (%)	Cases of pregnancy and delivery
Tesarik et al. (1995,96) 78 , 79	None	36	Two cases of pregnancy and delivery
Tanaka et al. (1996) 80	Electrostimulation	42	One case of pregnancy
Vanderzwalmen et al. (1997) 81	None	16	None
	A23187	36	One case of pregnancy
Antinori et al. (1997) 82	None	56	Two cases of pregnancy
Yamanaka et al. (1997) 83	None	69	None
Amer et al. (1997) 84	None	25	Four cases of pregnancy
Kahraman et al. (1998) 85	None	26	One case of pregnancy
Barak et al. (1998) 86	None	27	One case of pregnancy and delivery
Al‐Hasani et al. (1999) 87	None	18	None
Ghazzawi et al. (1999) 88	None	22	None
Levran et al. (2000) 89	None	44.9	None
Vicdan et al. (2001) 90	None	–	None (0/6)
Khalili et al. (2002) 91	None	–	None (0/7)
Sousa et al. (2002) 92	None	16	None (0/33)
Saremi et al. (2002) 93	None	–	One case of pregnancy and delivery
Amarin et al. (2002) 94	None	–	One case of pregnancy
Ulug et al. (2003) 95	None	42	None
Benkhalifa et al. (2004) 96	None	36	None

ASSISTED ACTIVATION

ASSISTED ACTIVATION IS based on the idea of using oocyte activation methods in conjunction with ICSI in patients without fertilization failure. These patients do not exhibit a low fertilization rate, but this approach is used to further raise the fertilization rate and increase the number of embryos obtained. As this method works on the oocytes that are still unfertilized after ICSI, the number of fertilized oocytes should increase. However, fertilized oocytes would also undergo treatment. Because the techniques of oocyte activation are still at the clinical research stage, they should not be used for such a purpose. When two or fewer oocytes are used in ICSI, the risk of fertilization failure is higher. When oocyte activation methods were concomitantly used with ICSI in such cases, the fertilization rate, embryonic development rate and pregnancy rate did not show any significant difference and, therefore, these methods were evaluated to be ineffective. ⁵²

RESCUE ACTIVATION

IN RESCUE ACTIVATION, oocyte activation is used to promote fertilization in oocytes that remain unfertilized following ICSI when the fertilization status is evaluated. The time interval between the collection of the oocyte and the oocyte activation treatment is important. Ontogenesis cannot be expected after aging of the oocyte. With 1‐day‐old oocytes, the pregnancy rate is extremely low. When rescue activation was carried out for such oocytes in 52 cases, the fertilization rate was 78% and two cases of pregnancy were obtained, but both resulted in miscarriage. ⁵² Therefore, this method should not be used clinically for 1‐day‐old oocytes. ⁹⁸ , ⁹⁹ Rescue ICSI 6 h after insemination has already been shown to be effective in cases of fertilization failure with in vitro fertilization. ¹⁰⁰ In the same way, rescue activation should be carried out within 6 h after ICSI. When oocyte activation does not occur following ICSI, the head of the injected sperm may undergo decondensation and PCC may occur over time. Premature chromatin condensation is the abnormal condensation of chromatin and can induce damage in the chromosome or DNA. Therefore, an evaluation of fertilization should be done before PCC takes place. ¹⁰¹ Because the incidence of PCC is elevated starting 4 h later when oocyte activation does not occur following ICSI, it is recommended that the fertilization status be evaluated and oocyte activation treatment carried out on unfertilized oocytes within 4 h. ¹⁰² If at least one fertilized oocyte is found at the evaluation, oocyte activation treatment is not necessary.

CONCLUSION

ARTIFICIAL OOCYTE ACTIVATION is frequently used in basic research in the field of reproductive technology. Oocyte activation occurs at the initial stage of fertilization and is essential for fertilization. In some cases of fertilization failure with ART, oocyte activation did not occur because of abnormalities in the sperm‐derived oocyte activation factor. In 50% of the unfertilized oocytes following ICSI, the failure can be attributed to problems with the sperm. ⁵² When fertilization failure is predicted with ICSI, oocyte activation methods can be used along with ICSI in a clinical research setting. Oocyte activation methods can also be used as a rescue procedure when fertilization failure is detected within 4 h of ICSI. ¹⁰¹ Among the various oocyte activation methods currently used, Ca ionophore treatment is the easiest to use. However, it should be noted that issues of genetic safety and abnormal imprinting have not been addressed for the combined use of these oocyte activation methods. ¹⁰²