1. İstanbul Memorial Hospital ART and Genetics Center Section 2

2. İstanbul Memorial Hospital ART and Genetics Center Abnormal oocyte morphology Granular & dark Multiple Vacuol Extracytoplasmic abnormality

3. İstanbul Memorial Hospital ART and Genetics Center Oocyte morphology abnormality

4. İstanbul Memorial Hospital ART and Genetics Center GroupsNormalGranular & dark Extracytopl asmic abnormality Multiple Vacuol & SER Embryo Biop (n) 58312716285 Abnormality (%) 41.352.454.9 71.4 * * p<0.01 Correlation of oocyte morphology and chromosomal abnormalities

5. İstanbul Memorial Hospital ART and Genetics Center Oocyte maturity One of the major causes of TFF after ICSI is a low number of retrieved MII oocytes. About 20% of retrieved oocytes from ovarian stimulation cycles are immature (GV & MI). Despite the use of varying culture techniques and different stimulation protocols, in-vitro matured oocytes have lower fertilization rates, frequent cleavage blocks compared with sibling MII oocytes

6. İstanbul Memorial Hospital ART and Genetics Center Oocyte activation Oocyte activation is a complex series of events that results in: 1. release of cortical granules 2. activation of membrane-bound ATPase 3. resumption of meiosis 4. formation of male and female pronuclei 5. extrusion of 2 nd Pb. The retrieved oocyte is activated when the sperm enters by ICSI.

7. İstanbul Memorial Hospital ART and Genetics Center When one sperm contacts the oolemma & penetrates into ooplasm, intracellular calcium oscillation occurs. This increase in concentration of calcium underlies oocyte activation and initiation of development. In mammals,, following fusion of gametes, a factor from sperm is responsible for inducing calcium oscillations.

8. İstanbul Memorial Hospital ART and Genetics Center Artificial oocyte activation Combination of calcium ionophore A23187 with puromycin stimulates the unfertilized oocytes 20–68 h after ICSI. It resulted in an activation rate of 91.2%, a cleavage rate of 64.7%. Although, ca ionophore and puromycin do not appear to be cytotoxic, and result in pregnancies and birth, the possible teratogenic & mutagenic activity of ca ionophore and puromycin needs further investigation.

9. İstanbul Memorial Hospital ART and Genetics Center Sperm motility and progression Whether sperm motility is slow or rapid has no influence on ICSI results. But, injection of immotile sperm usually results in impaired fertilization. In patients with 100% immotile sperm, the HOS test is a useful to examine sperm viability. It measures the functional integrity of sperm membrane. A mixture of 50% culture medium & 50% deionized water has the least harmful effects on sperm vitality.

10. İstanbul Memorial Hospital ART and Genetics Center Sperm structural defects TEM evaluation of sperm can identify potentially inheritable genetic disorders ( Kartagener’s syndrome), Acrosome agenesis is associated with a spherical shape of head- globozoospermia. The underlying causes of the syndrome remain to be elucidated.

11. İstanbul Memorial Hospital ART and Genetics Center (A) Normal sperm morphology. (B) Round-headed sperm morphology (LM). (C) EM of Round-headed sperm (globozoospermia).

12. İstanbul Memorial Hospital ART and Genetics Center Sperm DNA damage DNA damage in sperm is associated with poor fertilization, defective preimplantation embryonic development and high rates of miscarriage. chromatin rearrangement during spermiogenesis, abortive apoptosis and oxidative stress.DNA damage in sperm includes single- strand nicks and double-strand breaks that can arise because of errors in chromatin rearrangement during spermiogenesis, abortive apoptosis and oxidative stress.

13. İstanbul Memorial Hospital ART and Genetics Center Sperm DNA damage tests TUNEL, comet assay, sperm chromatin dispersion test and sperm chromatin structure assay (SCSA).Most commonly reported tests for indications of sperm nuclear integrity: TUNEL, comet assay, sperm chromatin dispersion test and sperm chromatin structure assay (SCSA). Strategies for treatment of men exhibiting high levels of DNA damage : (i) selective isolation of undamaged sperm (ii) antioxidant treatment

14. İstanbul Memorial Hospital ART and Genetics Center

15. Procedural effects of ICSI technique Not depositing the sperm within the oocyte cytoplasm. In this situation, the oocyte membrane may not be broken during attempts to aspirate the ooplasm into ICSI needle.

16. İstanbul Memorial Hospital ART and Genetics Center During ICSI, the location of first pb is used as an indication of the spindle position, with the assumption that they are located in close proximity. To avoid damage to the spindle, oocytes are injected at 3 o’clock position with the first pb at 6 or 12 o’clock position. However, reports suggest that location of first pb does not necessarily correspond to the spindle position.

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18. ICSI after previous ICSI failure A history of failed fertilization may be related to some gamete abnormality that may be modified or corrected at the next cycle. The differences between fertilization rates are unexplained, although fluctuations in the gamete quality are contributory. 1/3 of patients with TFF achieved pregnancy with their own oocytes in a subsequent ICSI cycle.

19. İstanbul Memorial Hospital ART and Genetics Center Options for patients after repeated ICSI failure Improvements in semen parameters, ovulation induction, and/or oocyte quality may result in fertilization in a subsequent ICSI attempt. Otherwise, the options of donation, adoption and remaining childless should be discussed with the couple.