TABLE 2.

Troubleshooting table

Step	Problem	Possible reason	Solution
3	Complete absence of or poor spindle image	Incorrect setting of the imaging system Improper position of an oocyte Immature oocytes or premature activation Suboptimal temperature	Make sure that all Oosight parts are properly installed (polarizer, CRi lens, camera port, etc.) and the camera port is open. Run background acquisition and make sure that all Oosight modes are working properly. Refer to the manual The spindle of primate oocytes is much smaller than in rodents and can only be seen at a certain position within an oocyte. Slow rotation of oocytes in different directions is always required to visualize spindles If a meiotic mid-body is present adjacent to the first polar body, the oocyte is still completing meiosis I. Place oocytes back into culture medium for 30–60 min and attempt imaging again. Wash oocytes before culture to remove cytochalasin B residue. If a second PB is observed, meiosis has been completed, possibly due to premature activation The microtubules of the spindle apparatus are temperature sensitive. Always maintain and operate oocytes at 37 °C
7	Difficulties in the laser-assisted zona pellucida drilling	Incorrect setting(s) on the laser system Laser power level set too low	Check all connections of the laser system. Contact Hamilton Thorne representatives for troubleshooting Increase laser output gradually
9	Difficulties in isolation of spindles	Enucleation pipette is out of focus The pipette is too small	Make sure to bring the spindle into an equatorial plane close to the 3 o’clock position and move the pipette tip to the same focal plane. Proper alignment can be confirmed by gently poking the spindles through the zona with the pipette tip. You should be able to push the spindle away The optimal size of the pipette is important. The spindle may not fit easily if the pipette is too small. However, too-large pipettes may remove excess volume of cytoplasm during karyoplast isolation
10	Lysis of cytoplasts during spindle isolation	Incorrect concentration of CB Sharp edges of the enucleation pipette	Check the CB concentrations and make sure to use fresh CB stock The tip of enucleation pipettes must be polished to avoid cutting the membrane. This is normally done by the pipette manufacturer
11	Karyoplast lysis	Incorrect concentration of CB Sticking of the karyoplast membrane to the bottom of the dish, pipette and pipette oil	Check the CB concentrations and make sure to use fresh CB stock Avoid keeping isolated karyoplasts too long within the pipette or in the manipulation drop. Transfer karyoplasts immediately into recipient cytoplasts. Avoid contact between a karyoplast and oil in the pipette
16	‘Leaking’ of the cytoplasm out of the opposite hole in the zona pellucida during karyoplast transfer	Excessive pressure on the zona pellucida during insertion of the pipette Too large a hole in the zona	Avoid squishing the zona during karyoplast transfer. See Figure 4d Minimize the size of the zona slit and always leave a thin intact layer in the zona wall. Review Step 7 and see Figure 4a and b
20	Poor fusion rates	Insufficient contact between karyoplasts and cytoplasts Inactive HVJ-E	Make sure to remove extra fluid surrounding karyoplasts from the perivitelline space to ensure sufficient membrane contact. Review Step 16 and see Figure 4e and f Thaw HVJ-E just before use
34	‘Leaking’ of cytoplasm out of the hole in the zona pellucida during ICSI	Excessive pressure on the zona pellucida during insertion of the pipette Too large a hole in the zona	Poking with injection pipette away from the center of the oocyte may cause cytoplasmic leakage during ICSI. To prevent this, you may insert the pipette through the gap made earlier during chromosome transfer Once again, minimize the size of the zona slit and always leave a thin intact layer in the zona wall as we suggest during Steps 7 and 15. See Figure 4a and b
35	Difficulties of breaking oocyte membrane during ICSI	Excessive softening of oocytes due to CB residue	ICSI should be performed in CB-free media and oocytes must be thoroughly rinsed after chromosome transfer manipulations
37, 38	Poor fertilization and embryo development	Premature activation Inadequate culture condition	Check your reconstructed oocytes carefully before ICSI. If you observe the second PB at the time of ICSI, oocytes are prematurely activated and have completed meiosis Check the quality of all reagents, media and culture conditions