Artificial Oocyte Artificial Oocyte Activation(AOA) ;Activation(AOA) ;

Is it beneficial or not?Is it beneficial or not?

DoğuFertil Tüp Bebek Merkezi DoğuFertil Tüp Bebek Merkezi MalatyaMalatya

Embryolog Koray YILDIZEmbryolog Koray YILDIZ

Intracytoplasmic sperm injection (ICSI) has become the method of choice to overcome severe male infertility (Palermo et al.,1992; Van Steirteghem et al., 1993).

However, there are still many couples who have not received the benefits of ICSI.

Total Fertilization Total Fertilization Failure(TFF)Failure(TFF)

The average normal fertilization rate in ICSI The average normal fertilization rate in ICSI is approximately 70%, is approximately 70%, howeverhowever complete complete or or virtualy complete fertilization failure virtualy complete fertilization failure occurs occurs in in 11% to % to 55% of % of ICSI cyclesICSI cycles..

normal sperm parameters Repeatedly even in good oocyte quality. good ovarian response

The literature reveals fertilization failure after The literature reveals fertilization failure after ICSI may be explained by defects in oocyte, ICSI may be explained by defects in oocyte, sperm, or the ICSI procedure.sperm, or the ICSI procedure.

Oocyte factorsOocyte factors

Oocyte cytoplasmic Immaturity(oocyte ageing) Oocyte cytoplasmic Immaturity(oocyte ageing) (Yildiz,K 2008)(Yildiz,K 2008)

Inherited Genetic DefectsInherited Genetic Defects Expulsion of the injected sperm from the oocytes Expulsion of the injected sperm from the oocytes

through the ICSI hole through the ICSI hole accounts for up to accounts for up to 1100-20-20% % of unfertilized post-ICSI oocytesof unfertilized post-ICSI oocytes(Yanagida,K (Yanagida,K 2004)2004)

Oocyte activation failure (Oocyte activation failure (80% of unfertilized 80% of unfertilized oocytes are arrested at the metaphase II stageoocytes are arrested at the metaphase II stage))(TFF) (TFF)

Sperm factorsSperm factors

ViabilityViability Abnormal chromatin statusAbnormal chromatin status IInability of sperm nucleus to nability of sperm nucleus to

decondensedecondense(zinc supply decreased at the (zinc supply decreased at the semen-prostatitis, large number of semen-prostatitis, large number of disulfid bonds in the nucleoproteins, disulfid bonds in the nucleoproteins, abnormal sperm centrosome,)abnormal sperm centrosome,)

IInability of sperm to activate oocytesnability of sperm to activate oocytes(PLC (PLC zeta isoform virtual or complete zeta isoform virtual or complete deficiency)(TFF)deficiency)(TFF)

In turn, PLC stimulates the hydrolysis of phosphatidyl In turn, PLC stimulates the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), a common (DAG) and 1,4,5 inositol trisphosphate (IP3), a common

Ca(2+) releasing compound.Ca(2+) releasing compound.

A single calcium rise is sufficient for oocyte activation, but calcium oscillations in fertilized eggs are believed to regulate not only short-term events such as oocyte activation but also long-term developmental events, early gene expression, and possibly methylation status.

(Ozil JB et al., 1990, Ducibella T et al.,2002)

Artificial Artificial OOocyte ocyte AActivationctivation

ICSI+AOA (with repeated fertilization failure)ICSI+AOA (with repeated fertilization failure) RescueICSI(RICSI)RescueICSI(RICSI) Parthenogenesis (stem cell studies)Parthenogenesis (stem cell studies) Nuclear TransplantationNuclear Transplantation

Assisted Oocyte Activation ineffective(Yanagida,K 2004)

Different procedures for AOA after ICSI have been established ;

1.Electrical 2.Mechanical 3.Chemical

(Yamano,S et al,2000, Alberio, R et al, 2001)

Electrical Activation

Formation of pores in the plasma membrane.

Efficiency depends on : Pore size (voltage potential, application

duration, electrode type) Ionic content(zimmermann solution) Heat Cell type.

Mechanical Activation Membranes of the oocytes are broken using a Membranes of the oocytes are broken using a

microneedle to maintain a calcium influxmicroneedle to maintain a calcium influx Direct microinjection of calcium into an oocyte Direct microinjection of calcium into an oocyte

to increase intracellular calcium (effective but it to increase intracellular calcium (effective but it is not common in clinical practice)is not common in clinical practice)

Recent findings suggest that the distribution Recent findings suggest that the distribution and function ofand function of mitochondria within the oocyte mitochondria within the oocyte also plays an instrumental role throughalso plays an instrumental role through the the endoplasmic reticulum and IP3 mediated Ca2+-endoplasmic reticulum and IP3 mediated Ca2+-signallingsignalling(Dumollard et al 2006)(Dumollard et al 2006)

Cause an influx of calcium from the surrounding mediumBased on the hypothetical accumulation of highly polarized mitochondria, from pericorticalregions (9 o’clock) to the centre of the oocyte, thus, theoretically, supplying more energy (ATP) directly to the place where the spermatozoon was injected.5/15(TFF) pregnant, routine application not efficient

Mechanical disruption of endoplasmic reticulum during the Mechanical disruption of endoplasmic reticulum during the movements of the microinjection needle in the oocyte cytoplasm movements of the microinjection needle in the oocyte cytoplasm and repeated aspiration releases calcium stored in this organelle.and repeated aspiration releases calcium stored in this organelle.Case report: 5/6 pregnancy in TFFCase report: 5/6 pregnancy in TFF

Prospective Routine Prospective Routine usage of Modified ICSI usage of Modified ICSI

136 sibling oocyte136 sibling oocyte

Chi-Square P Value 0,519 Chi-Square P Value 0,519

Yildiz,K 2011 Yildiz,K 2011

Modified ICSI Classical ICSI

Oocyte (n) 73 63

Fertilized (2PN)Oocyte(n)

60 49

Fertilization ratio (%)

83 77

Chemical Activation IonophoresIonophores Ethanol (Yi YJ, et al. 2005)(%8 8-15 min in porcine Ethanol (Yi YJ, et al. 2005)(%8 8-15 min in porcine

oocytes)oocytes) Cycloheximide (CHX)(the protein synthesis inhibitor)Cycloheximide (CHX)(the protein synthesis inhibitor) Thimerasol (degenerative to spindles)Thimerasol (degenerative to spindles) 6-dimethylaminopurin(6-DMAP)(The histone kinase 6-dimethylaminopurin(6-DMAP)(The histone kinase

inhibitor and blocks the extrusion of second polar body)inhibitor and blocks the extrusion of second polar body) Cytochalasin B (CCB)(Blocks the extrusion of second Cytochalasin B (CCB)(Blocks the extrusion of second

polar body and and may also help prevent polar body and and may also help prevent fragmentation of embryosfragmentation of embryos

(Collas and Robl, 1990; Yang et al., 1992)(Collas and Robl, 1990; Yang et al., 1992) SrCl2 (mimics Ca ossilations)SrCl2 (mimics Ca ossilations) PuromycinPuromycin (inhibits resythesis of MPF(Cyclin B)(inhibits resythesis of MPF(Cyclin B)

IonophoreIonophore

An ionophoer is a lipid-soluable An ionophoer is a lipid-soluable molecule usually synthesized by molecule usually synthesized by microorganisms to transport ions microorganisms to transport ions across the lipid bilayer of the cell across the lipid bilayer of the cell membrane. membrane.

There are two broad classifications of

ionophores. Chemical compounds (mobile ion Chemical compounds (mobile ion

carriers) that bind to a particular ion, carriers) that bind to a particular ion, shielding its charge from the surrounding shielding its charge from the surrounding environment, and thus facilitating its environment, and thus facilitating its crossing of the hydrophobic interior of crossing of the hydrophobic interior of the lipid membrane. the lipid membrane.

Channel formers that introduce a Channel formers that introduce a hydrophilic pore into the membrane, hydrophilic pore into the membrane, allowing ions to pass through while allowing ions to pass through while avoiding contact with the membrane's avoiding contact with the membrane's hydrophobic interior. hydrophobic interior.

A23187 (Ca2+) A23187 (Ca2+) Ionomycin (Ca2+) Ionomycin (Ca2+) 2,4-Dinitrophenol (H+) 2,4-Dinitrophenol (H+) Beauvericin (Ca2+, Ba2+) Beauvericin (Ca2+, Ba2+) Calixarene (Cs+, Pb2+) Calixarene (Cs+, Pb2+) CCCP or Carbonyl cyanide CCCP or Carbonyl cyanide mm-chlorophenyl hydrazone (H+) -chlorophenyl hydrazone (H+) Crown ether (Na+, K+) Crown ether (Na+, K+) FCCP or Carbonyl cyanide-FCCP or Carbonyl cyanide-pp--

trifluoromethoxyphenylhydrazone (H+) trifluoromethoxyphenylhydrazone (H+) Enniatin (Ammonium) Enniatin (Ammonium) Gramicidin A (H+, Na+, K+) Gramicidin A (H+, Na+, K+) Macrocycle (NO3-) Macrocycle (NO3-) Monensin (Na+, H+) Monensin (Na+, H+) Nigericin (K+, H+, Pb2+) Nigericin (K+, H+, Pb2+) Nonactin (Ammonium ionophore I) Nonactin (Ammonium ionophore I) Perfluorooctanesulfonamide (H+) Perfluorooctanesulfonamide (H+) Porphyrin (NO2-) Porphyrin (NO2-) Proton ionophore II (4-Nonadecylpyridine) Proton ionophore II (4-Nonadecylpyridine) Proton ionophore III (Proton ionophore III (NN,,NN-Dioctadecylmethylamine) -Dioctadecylmethylamine) Salinomycin (K+) Salinomycin (K+) Valinomycin (Potassium ionophore I) Valinomycin (Potassium ionophore I)

A23187

A23187 is a mobile ion carrier that forms stable complexes A23187 is a mobile ion carrier that forms stable complexes with divalent cations and can cause cell activation.(Berridge, with divalent cations and can cause cell activation.(Berridge, M.J. 1993), (Scharff, O. et al. 1993)M.J. 1993), (Scharff, O. et al. 1993)

It is produced at fermentation of Streptomyces It is produced at fermentation of Streptomyces Chartreusensis.Chartreusensis.

A23187 induces in a concentration and time-dependent A23187 induces in a concentration and time-dependent manner.(Orrenius, S. et al 2003, Hajnoczky, G. et al. 2003)manner.(Orrenius, S. et al 2003, Hajnoczky, G. et al. 2003)

(2 uM A23187 added to cell culture(Jurkat cells) for 6 hours (2 uM A23187 added to cell culture(Jurkat cells) for 6 hours and apoptosis detected).and apoptosis detected).

It is also known as Calcimycin, Calcium Ionophore, It is also known as Calcimycin, Calcium Ionophore, Antibiotic A23187 and Calcium Ionophore A23187.Antibiotic A23187 and Calcium Ionophore A23187.

Ionomycin Ionomycin is an ionophore produced by the

bacterium Streptomyces conglobatus. Ionomycin, is a narrow spectrum antibiotic for

Gram-positive bacteria. In mammals cells ionomycin acts as a potent

and selective Ca2+ionophore, more effective than A23187.

Ionomycin induces hydrolysis of phosphoinositides, activation of PKC in human T cells and apoptosis in various cells lines.

(Takei, N et al 1994, Aagaard Tillery KM et al 1995)

Ionomycin induced cytosolic  Ca2+ increase.  Ca2+ traces from Fluo-3 AM loaded C6 glioma cells treated with 1mM Ionomycin.

Strontium chloride(SrCl2)

Strontium chloride Strontium chloride action mimiced the effects of action mimiced the effects of sperm on the oocyte, andsperm on the oocyte, and seemed to be mediated seemed to be mediated through the inositol trisphosphatethrough the inositol trisphosphate receptors Ca receptors Ca release from Endoplasmic reticulum(ER).release from Endoplasmic reticulum(ER).

21 srcl2 threated group21 srcl2 threated group

55 control group55 control group

ASRM 2011ASRM 2011

Is it Safe or Not?Is it Safe or Not?

A23187 A23187 + P+ Puromycin and strontium chlorideuromycin and strontium chloride does does not appear to be cytotoxic when used in an not appear to be cytotoxic when used in an optimumoptimum concentration and concentration and about 80% of about 80% of unfertilized oocytesunfertilized oocytes revealrevealeded normal normal chromosomes.chromosomes. (Yamano, S et al., 2000,Lu, Q et (Yamano, S et al., 2000,Lu, Q et al.,2006)al.,2006)

SomeSome studies revealed no physical or mental studies revealed no physical or mental developmental disorders associated with babies developmental disorders associated with babies born throughborn through ththeseese procedure proceduress, even 12 months , even 12 months after birthafter birth(Kyono, K et al.,2008, Lu,Q et al.,2006)(Kyono, K et al.,2008, Lu,Q et al.,2006)

50 A23187 group+control group35 SrCl2 group+conrol group

ASRM 2011

For mouse oocytes and human failed-For mouse oocytes and human failed-fertilized oocytes, blastocyst development fertilized oocytes, blastocyst development was significantly higher after electrical was significantly higher after electrical activationactivation against chemical against chemical activation(ionomycin and Srcl2)activation(ionomycin and Srcl2) (Versieren, K et al 2010)(Versieren, K et al 2010)

There is a tendency toward an increase in fertilization and embryo grade rates as a result of ICSI with combined electrical activation. (Baltaci,V 2010)

ConclusionConclusion Artificial oocyte activation may be useful in selected Artificial oocyte activation may be useful in selected ICSI ICSI patientspatients when there is no or low fertilization potential and may bewhen there is no or low fertilization potential and may be considered considered in male factor disorders such as globozoospermiain male factor disorders such as globozoospermia,,severesevere teratozoospermia, and nonobstructive azoospermiateratozoospermia, and nonobstructive azoospermia..

Calcium ionophore method for AOA has the most reports and the Calcium ionophore method for AOA has the most reports and the resulting children have no reports of anomalies .resulting children have no reports of anomalies .

Clinical use of these agents in assisted reproduction is limited by Clinical use of these agents in assisted reproduction is limited by insufficient knowledge about their potential cytotoxic, teratogenic, insufficient knowledge about their potential cytotoxic, teratogenic, epigenetic and mutagenic effects on oocytes and embryos. It should epigenetic and mutagenic effects on oocytes and embryos. It should be noted that issues of genetic safety and abnormal imprinting have be noted that issues of genetic safety and abnormal imprinting have not been addressed for the combined use of these oocyte activation not been addressed for the combined use of these oocyte activation methods.methods.

However someHowever some studies revealed no physical or mental developmental studies revealed no physical or mental developmental disorders associated with babies born through thdisorders associated with babies born through theseese procedure proceduress, , even 12 months after birth.even 12 months after birth.