**12.1. Oocyte activation**

Oocyte activation is a complex series of events that results in the release of the cortical granules, activation of membrane bound ATPase, resumption of meiosis with the extrusion of the second polar body and finally the formation of male and female pronuclei. The ovulated or retrieved oocyte activates when the sperm enters, by either natural penetration or ICSI. In cases of oocyte activation failure, artificial means of oocyte activation are helpful.

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 129

activation and ICSI restore fertilization, embryo cleavage and development for patients with

Assisted oocyte activation after ICS is very efficient in patients with a suspected oocyterelated activation deficiency and previous total fertilization failure after conventional ICSI. However, when there was a prior history of low fertilization, one should be careful and test the efficiency of assisted oocyte activation on half of the sibling oocytes, because assisted oocyte activation is not always beneficial for patients with previous low fertilization and a suspected oocyte-related activation deficiency. For these patients, a split assisted oocyte activation-ICSI cycle using sibling oocytes can help to distinguish between a molecular oocyte-related activation deficiency and a previous technical or other biological failure [126]. Assisted oocyte activation aims to mimic the action of sperm penetration [127]. Some assisted activation treatments such as strontium chloride [128] and ionomycin [129], promote an increase in intracellular free calcium concentrations by the release of calcium from cytoplasmic stores. Others such as electrical stimulus promote influx of calcium from the extracellular medium and some treatments such as ethanol promote both effects [129].

A birth after oocyte activation by treatment with the calcium ionophore A23187 and ICSI has been obtained in 1994 [130]. Human oocytes injected with round-headed sperm are activated following combination of calcium chloride injection and ionophore treatment. This activation is followed by an apparently normal completion of meiosis, male and female pronuclei formation, embryonic development and successful delivery of a healthy infant [53]. A combination of calcium ionophore A23187 with puromycin stimulates the unfertilized oocytes 20–68 h after ICSI. It results in an activation rate of 91.2% (31/34), a cleavage rate of 64.7% (22/34) and high-quality embryo rate of 44.1% (15/34). Nearly all activated embryos derived from 2PN/2PB had a normal set of sex chromosomes and developed normally [131]. Although calcium ionophore A23187 and puromycin do not appear to be cytotoxic to oocytes and result in pregnancies and the birth of healthy babies when low concentrations are used, the possible teratogenic and mutagenic activity of calcium ionophore A23187 and puromycin needs further investigation in animal models and

Treatment with 10 mM strontium chloride for 60 min, approximately 30 min after ICSl results in activation and fertilization of all injected oocytes [132], development of the embryos to the blastocyst stage and delivery in patients with repeated fertilization failure [133]. Physical and mental development of the children from birth to 12 months is normal [132]. However, further studies are required to substantiate the finding that strontium

An electrical field can generate micropores in the cell membrane of gametes and somatic cells to induce sufficient calcium influx through the pores to activate cytoplasm through calcium dependent mechanisms [134]. Mouse oocytes injected with secondary spermatocytes or spermatids are fertilized when stimulated by electroporation and developed into normal offspring when the resultant embryos are transferred to a recipient

chloride treatment is an effective method of artificial oocyte activation.

globozoospermia [125].

in humans.

The oocytes remain arrested at MII if maturation has been completed. When one sperm contacts the oolemma and penetrates into the ooplasm, intracellular calcium oscillation occurs [114]. This increase in the concentration of calcium underlies oocyte activation and initiation of development. In mammals, growing experimental evidence supports the notion that, following fusion of the gametes, a factor from sperm is responsible for inducing calcium oscillations and stimulating inositol 1,4,5-trisphosphate (IP3) production [115]. Initial evidence stemmed from injection of cytosolic sperm extracts into oocytes that reproduced the calcium responses associated with fertilization regardless of the species of origin [116, 117]. Subsequent biochemical characterization of the extracts revealed that the active component contained a protein moiety [116] that possessed phospholipase C (PLC) like activity capable of inducing production of IP3 [118, 119] and that the PLC activity was highly sensitive to calcium [120]. A screen of expressed sequenced tags from testes identified a sperm-specific phospholipase C, PLCζ. The presence of PLCζ correlates with calcium activity in cytosolic sperm extracts [121]. Moreover, injection of oocytes with the recombinant protein [122] or with the encoding mRNA induces fertilization-like oscillations (Saunders, 2002), whereas depletion of PLCζ from the extracts with specific antisera abrogates PLCζ activity [123] and the calcium oscillatory activity of the extracts [121, 123]. The PLCζ is located in the equatorial region of human sperm. Men whose sperm are unable to initiate calcium oscillations consistently fail to fertilize following ICSI and lack PLCζ [124]. It has to be established that PLCζ is the sole calcium oscillation–inducing factor and how its absence has an impact on male fertility.

The process of natural fertilization encompasses the entry of the sperm, oocyte activation and the first mitotic division resulting in a 2 cell embryo. Two steps are important for successful fertilization following ICSI, namely immobilization of the sperm and rupture of the oolemma in order to facilitate the liberation of the cytosolic sperm factor responsible for the oscillator function [14].

Low fertilization rates after ICSI in patients with round-headed sperm, globozoospermia, is a result of reduced ability of round-headed sperm to activate the oocyte. In the literature, the success rates of ICSI in cases of globozoospermia are variable. Assisted oocyte activation combined with ICSI may overcome the infertility associated with globozoospermia. Normal healthy live birth without assisted oocyte activation has also been achieved [54]. Apart from low fertilization rates associated with the use of round-headed sperm, cleavage rates are also compromised and these sperm may lack normal centrosomes [52]. Assisted oocyte activation and ICSI restore fertilization, embryo cleavage and development for patients with globozoospermia [125].

128 Enhancing Success of Assisted Reproduction

**12. Oocyte related factors** 

how its absence has an impact on male fertility.

the oscillator function [14].

Oocyte activation is a complex series of events that results in the release of the cortical granules, activation of membrane bound ATPase, resumption of meiosis with the extrusion of the second polar body and finally the formation of male and female pronuclei. The ovulated or retrieved oocyte activates when the sperm enters, by either natural penetration or ICSI. In cases of oocyte activation failure, artificial means of oocyte activation are helpful.

The oocytes remain arrested at MII if maturation has been completed. When one sperm contacts the oolemma and penetrates into the ooplasm, intracellular calcium oscillation occurs [114]. This increase in the concentration of calcium underlies oocyte activation and initiation of development. In mammals, growing experimental evidence supports the notion that, following fusion of the gametes, a factor from sperm is responsible for inducing calcium oscillations and stimulating inositol 1,4,5-trisphosphate (IP3) production [115]. Initial evidence stemmed from injection of cytosolic sperm extracts into oocytes that reproduced the calcium responses associated with fertilization regardless of the species of origin [116, 117]. Subsequent biochemical characterization of the extracts revealed that the active component contained a protein moiety [116] that possessed phospholipase C (PLC) like activity capable of inducing production of IP3 [118, 119] and that the PLC activity was highly sensitive to calcium [120]. A screen of expressed sequenced tags from testes identified a sperm-specific phospholipase C, PLCζ. The presence of PLCζ correlates with calcium activity in cytosolic sperm extracts [121]. Moreover, injection of oocytes with the recombinant protein [122] or with the encoding mRNA induces fertilization-like oscillations (Saunders, 2002), whereas depletion of PLCζ from the extracts with specific antisera abrogates PLCζ activity [123] and the calcium oscillatory activity of the extracts [121, 123]. The PLCζ is located in the equatorial region of human sperm. Men whose sperm are unable to initiate calcium oscillations consistently fail to fertilize following ICSI and lack PLCζ [124]. It has to be established that PLCζ is the sole calcium oscillation–inducing factor and

The process of natural fertilization encompasses the entry of the sperm, oocyte activation and the first mitotic division resulting in a 2 cell embryo. Two steps are important for successful fertilization following ICSI, namely immobilization of the sperm and rupture of the oolemma in order to facilitate the liberation of the cytosolic sperm factor responsible for

Low fertilization rates after ICSI in patients with round-headed sperm, globozoospermia, is a result of reduced ability of round-headed sperm to activate the oocyte. In the literature, the success rates of ICSI in cases of globozoospermia are variable. Assisted oocyte activation combined with ICSI may overcome the infertility associated with globozoospermia. Normal healthy live birth without assisted oocyte activation has also been achieved [54]. Apart from low fertilization rates associated with the use of round-headed sperm, cleavage rates are also compromised and these sperm may lack normal centrosomes [52]. Assisted oocyte

**12.1. Oocyte activation** 

Assisted oocyte activation after ICS is very efficient in patients with a suspected oocyterelated activation deficiency and previous total fertilization failure after conventional ICSI. However, when there was a prior history of low fertilization, one should be careful and test the efficiency of assisted oocyte activation on half of the sibling oocytes, because assisted oocyte activation is not always beneficial for patients with previous low fertilization and a suspected oocyte-related activation deficiency. For these patients, a split assisted oocyte activation-ICSI cycle using sibling oocytes can help to distinguish between a molecular oocyte-related activation deficiency and a previous technical or other biological failure [126].

Assisted oocyte activation aims to mimic the action of sperm penetration [127]. Some assisted activation treatments such as strontium chloride [128] and ionomycin [129], promote an increase in intracellular free calcium concentrations by the release of calcium from cytoplasmic stores. Others such as electrical stimulus promote influx of calcium from the extracellular medium and some treatments such as ethanol promote both effects [129].

A birth after oocyte activation by treatment with the calcium ionophore A23187 and ICSI has been obtained in 1994 [130]. Human oocytes injected with round-headed sperm are activated following combination of calcium chloride injection and ionophore treatment. This activation is followed by an apparently normal completion of meiosis, male and female pronuclei formation, embryonic development and successful delivery of a healthy infant [53]. A combination of calcium ionophore A23187 with puromycin stimulates the unfertilized oocytes 20–68 h after ICSI. It results in an activation rate of 91.2% (31/34), a cleavage rate of 64.7% (22/34) and high-quality embryo rate of 44.1% (15/34). Nearly all activated embryos derived from 2PN/2PB had a normal set of sex chromosomes and developed normally [131]. Although calcium ionophore A23187 and puromycin do not appear to be cytotoxic to oocytes and result in pregnancies and the birth of healthy babies when low concentrations are used, the possible teratogenic and mutagenic activity of calcium ionophore A23187 and puromycin needs further investigation in animal models and in humans.

Treatment with 10 mM strontium chloride for 60 min, approximately 30 min after ICSl results in activation and fertilization of all injected oocytes [132], development of the embryos to the blastocyst stage and delivery in patients with repeated fertilization failure [133]. Physical and mental development of the children from birth to 12 months is normal [132]. However, further studies are required to substantiate the finding that strontium chloride treatment is an effective method of artificial oocyte activation.

An electrical field can generate micropores in the cell membrane of gametes and somatic cells to induce sufficient calcium influx through the pores to activate cytoplasm through calcium dependent mechanisms [134]. Mouse oocytes injected with secondary spermatocytes or spermatids are fertilized when stimulated by electroporation and developed into normal offspring when the resultant embryos are transferred to a recipient [135]. Clinical pregnancy and delivery after oocyte activation by electrostimulation in combination with ICSI in previously failed to fertilize oocytes has been obtained [80]. Electrical stimulation rescues human oocytes that failed to fertilize after ICSI and stimulates them to complete the second meiotic division, to form pronuclei and to undergo early embryonic development [136]. Although the fertilization rate issimilar regardless of the number of electrical pulses applied, subsequent embryo development is dramatically improved in those oocytes that received three electrical pulses [136]. The embryo formation rate in the electrically activated group is 80% compared to 16% in the control group [137]. Although the fertilization rate is significantly higher in the electroactivated group (68%) as compared with that of the control (60%), a higher miscarriage rate is reported in the electroactivated group (5 of 15 pregnancies) compared to the control (3 of 33) [6]. Like any other new assisted reproductive procedure, the impact of electrical activation on oocyte and embryo health must be evaluated in larger studies before this procedure can be considered for routine clinical purposes. Ideally, karyotyping or fluorescent *in situ* hybridization analysis should be performed to assess the incidences of aneuploidy and mosaicism in the resultant embryos.

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization 131

retrieved compared to cases with ≥5 oocytes [143]. Limited information is available on IVM of immature oocytes retrieved from poor responders in conventional stimulation IVF/ICSI cycles and IVM is not a viable alternative to cancellation of IVF cycles in such patients [10].

Fecundity significantly decreases with increasing maternal age [144]. In a classic study of the Hutterite women, sterility increased from just over 10% at 34 years old to over 85% by the age of 44 years [145]. In women, all germ cells are formed during fetal life. The population of germ cells appears to rise steadily from 600 000 at 2 months post conception, reaching a peak of 6 800 000 at 5 months. By the time of birth, the number declines to 2 000 000 of which 50% are atretic. Of the 1 000 000 normal oocytes in the newborn infant, only 300 000 survive to the age of 7 years [146]. Continuous loss of oocytes occurs through the

The incidence of TFF increases with age [10]. Older women are more likely to undergo multiple cycles, have decreased number of oocytes retrieved and a lower number of

One of the major causes of TFF after ICSI is a low number of retrieved MII oocytes [10]. About 20% of retrieved oocytes from controlled ovarian stimulation cycles are immature, either at metaphase-I (MI) or germinal-vesicle (GV) stage in human IVF [35]. Some of these oocytes may extrude the first polar body during *in vitro* culture and can be injected in ICSI cycles. This may be a useful strategy for patients with low number of retrieved oocytes. However, embryos derived from immature oocytes do not efficiently translate into pregnancies and live births. Therefore, the clinical significance of using immature oocytes in

The injection of MI oocytes immediately after denudation results in a high degeneration rate due to increased fragility of the oolemma. The fertilization rate of retrieved MI oocytes that remained MI at the time of ICSI is lower than the fertilization rate of sibling retrieved MI progressing to MII *in vitro* (25% compared to 62.2%, respectively). It is less than half when compared to the fertilization rate of retrieved sibling MII oocytes (69.5%). A high rate of multinucleated oocytes is also found in fertilized MI oocytes injected immediately after

In cases of poor responders and in patients with an unsynchronized cohort of follicles, where the presence of immature oocytes is frequent after stimulation [149], the use of immature oocytes is important in order to increase the number of embryos obtained in each cycle. Based on the assumption that oocyte maturity is a pre-requisite for obtaining normal fertilization, attempts have been made to mature GV and MI oocytes *in vitro* [147]. Despite the use of varying culture techniques and different stimulation protocols, such IVM oocytes consistently have lower fertilization rates, frequent cleavage blocks and overall retarded cleavage rate compared with sibling MII oocytes [147, 150]. The limited number of transfer cycles makes it difficult to draw solid conclusions about the value of transferring these

physiological process of follicular growth and atresia throughout life [147].

embryos transferred [9].

**14. Oocyte maturity** 

denudation [148].

stimulated cycles needs further investigation [148].
