1 / 35

Artificial Oocyte Activation(AOA) ; Is it beneficial or not?

Artificial Oocyte Activation(AOA) ; Is it beneficial or not?. DoğuFertil Tüp Bebek Merkezi Malatya Embryolog 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).

presley
Download Presentation

Artificial Oocyte Activation(AOA) ; Is it beneficial or not?

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Artificial Oocyte Activation(AOA) ;Is it beneficial or not? DoğuFertil Tüp Bebek Merkezi Malatya Embryolog Koray YILDIZ

  2. 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.

  3. Total Fertilization Failure(TFF) • The average normal fertilization rate in ICSI is approximately 70%, howevercomplete or virtualy complete fertilization failure occurs in 1% to 5% of ICSI cycles. • normal sperm parameters Repeatedly even in good oocyte quality. good ovarian response • The literature reveals fertilization failure after ICSI may be explained by defects in oocyte, sperm, or the ICSI procedure.

  4. Oocyte factors Oocyte cytoplasmic Immaturity(oocyte ageing) (Yildiz,K 2008) Inherited Genetic Defects Expulsion of the injected sperm from the oocytesthrough the ICSI hole accounts for up to 10-20% of unfertilized post-ICSI oocytes(Yanagida,K 2004) Oocyte activation failure (80% of unfertilized oocytes are arrested at the metaphase II stage)(TFF)

  5. Sperm factors Viability Abnormalchromatinstatus Inability of sperm nucleus to decondense(zincsupplydecreased at the semen-prostatitis, largenumber of disulfidbonds in thenucleoproteins, abnormal sperm centrosome,) Inability of sperm to activate oocytes(PLC zetaisoformvirtualorcompletedeficiency)(TFF)

  6. In turn, PLC stimulates the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), a common Ca(2+) releasing compound.

  7. 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)

  8. Artificial OocyteActivation • ICSI+AOA (with repeated fertilization failure) • RescueICSI(RICSI) • Parthenogenesis (stem cell studies) • Nuclear Transplantation • Assisted Oocyte Activation ineffective(Yanagida,K 2004)

  9. 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)

  10. 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.

  11. Mechanical Activation • Membranes of the oocytes are broken using a microneedle to maintain a calcium influx • Direct microinjection of calcium into an oocyte to increase intracellular calcium (effective but it is not common in clinical practice) • Recent findings suggest that the distribution and function ofmitochondria within the oocyte also plays an instrumental role throughthe endoplasmic reticulum and IP3 mediated Ca2+-signalling(Dumollard et al 2006)

  12. Cause an influx of calcium from the surrounding medium Based on the hypothetical accumulation of highly polarized mitochondria, from pericortical regions (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

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

  14. Prospective Routine usage of Modified ICSI 136 sibling oocyte Chi-Square P Value 0,519 Yildiz,K 2011

  15. Chemical Activation • Ionophores • Ethanol (Yi YJ, et al. 2005)(%8 8-15 min in porcine oocytes) • Cycloheximide (CHX)(the protein synthesis inhibitor) • Thimerasol (degenerative to spindles) • 6-dimethylaminopurin(6-DMAP)(The histone kinase inhibitor and blocks the extrusion of second polar body) • Cytochalasin B (CCB)(Blocks the extrusion of second polar body and and may also help prevent fragmentation of embryos (Collas and Robl, 1990; Yang et al., 1992) • SrCl2 (mimics Ca ossilations) • Puromycin(inhibits resythesis of MPF(Cyclin B)

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

  17. There are two broad classifications of ionophores. • Chemical compounds (mobile ion carriers) that bind to a particular ion, shielding its charge from the surrounding environment, and thus facilitating its crossing of the hydrophobic interior of the lipid membrane. • Channel formers that introduce a hydrophilic pore into the membrane, allowing ions to pass through while avoiding contact with the membrane's hydrophobic interior.

  18. A23187 (Ca2+) • Ionomycin (Ca2+) • 2,4-Dinitrophenol (H+) • Beauvericin (Ca2+, Ba2+) • Calixarene (Cs+, Pb2+) • CCCP or Carbonyl cyanide m-chlorophenyl hydrazone (H+) • Crown ether (Na+, K+) • FCCP or Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (H+) • Enniatin (Ammonium) • Gramicidin A (H+, Na+, K+) • Macrocycle (NO3-) • Monensin (Na+, H+) • Nigericin (K+, H+, Pb2+) • Nonactin (Ammonium ionophore I) • Perfluorooctanesulfonamide (H+) • Porphyrin (NO2-) • Proton ionophore II (4-Nonadecylpyridine) • Proton ionophore III (N,N-Dioctadecylmethylamine) • Salinomycin (K+) • Valinomycin (Potassium ionophore I)

  19. A23187 • A23187 is a mobile ion carrier that forms stable complexes with divalent cations and can cause cell activation.(Berridge, M.J. 1993), (Scharff, O. et al. 1993) • It is produced at fermentation of Streptomyces Chartreusensis. • A23187 induces in a concentration and time-dependent manner.(Orrenius, S. et al 2003, Hajnoczky, G. et al. 2003) (2 uM A23187 added to cell culture(Jurkat cells) for 6 hours and apoptosis detected). • It is also known as Calcimycin, Calcium Ionophore, Antibiotic A23187 and Calcium Ionophore A23187.

  20. 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)

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

  22. Strontium chloride(SrCl2) • Strontium chloride action mimiced the effects of sperm on the oocyte, andseemed to be mediated through the inositol trisphosphate receptors Ca release from Endoplasmic reticulum(ER).

  23. 21 srcl2 threated group 55 control group ASRM 2011

  24. Is it Safe or Not? A23187 + Puromycin and strontium chloridedoes not appear to be cytotoxic when used in an optimum concentration and about 80% of unfertilized oocytesrevealed normal chromosomes. (Yamano, S et al., 2000,Lu, Q et al.,2006) Some studies revealed no physical or mentaldevelopmental disorders associated with babies born throughthese procedures, even 12 months after birth(Kyono, K et al.,2008, Lu,Q et al.,2006)

  25. 50 A23187 group+control group 35 SrCl2 group+conrol group ASRM 2011

  26. For mouse oocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation against chemical activation(ionomycin and Srcl2)(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)

  27. Conclusion Artificial oocyte activation may be useful in selected ICSI patientswhen there is no or low fertilization potential and may beconsidered in male factor disorders such as globozoospermia,severeteratozoospermia, and nonobstructiveazoospermia. Calcium ionophore method for AOA has the most reports and the resulting children have no reports of anomalies . Clinical use of these agents in assisted reproduction is limited by insufficient knowledge about their potential cytotoxic, teratogenic, epigenetic and mutagenic effects on oocytes and embryos. 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. However some studies revealed no physical or mental developmental disorders associated with babies born through these procedures, even 12 months after birth.

More Related