- Ensure that the stage is heated
- Remove all air bubbles from the tube of the manipulator by emptying the syringe in a oil bottle.
- Fill the syringe back with the oil
- Wipe the needle holder with tissue paper and mount it back on the holder
- Make sure the holder is in the centre f the field
- The holding pipette is aligned first in low power and then in high power
- The alignment is done parallel to the microscope stage
- Adjust the injection control the oil should reach the proximal end of pipette
- Take the icsi dish from the incubator and add sperm in one of the droplet of pvp.
- To immobilize sperm localize the sperm near the edge f the pvp drop and bring both the tail of the sperm and injecting pipette in sharp focus
- Select normal looking sperm and try to hit the tail of the sperm to immobilize it.
- Aspirate the tail first by slowly turning the injector anticlockwise
- Focus the egg
- Now lower the holding pipette in to droplet containing egg
- Move the pipette alng side the egg the polar body in to focus
- Bring it close to the egg and make sure the polar body is at 6 or 12 o clock position
- Attach the egg to the holding pipette
- Slowly bring down the injection pipette to the same droplet
- Keep the sperm close to the end of the pipette
- Align the pipette close to the egg at 3o clock position
- Push the pipette in to the egg
- Once it has sucked cytoplasm in and mix with the sperm and sperm is slowly released in to the egg
- Slowly withdraw the pipette out of the egg as gently as possible ,making sure that the sperm remains inside
- When all the injections are done transfer the egg to fresh culture medium.
Intracytoplasmic Sperm Injection (ICSI) is often recommended if :
- The male partner has a very low sperm count
- Other problems with the sperm have been identified, such as poor morphology (abnormally shaped) and/or poor motility (poor swimmers)
- At previous attempts at In Vitro Fertilization (IVF) there was either failure of fertilisation or an unexpectedly low fertilisation rate
- Te male partner has had a vasectomy and sperm have been collected from the testicles or epididymis (sperm reservoir)
- Other situations where the sperm count is zero and donor insemination is not wanted
- The male partner does not ejaculate any sperm but sperm have been collected from the testicles
- The male partner has had problems obtaining an erection and ejaculating. This includes men with spinal cord injuries, diabetes and other disorders.
Prepration For Intra-Cytoplasmic Sperm Injection (ICSI)
- Prepare a dish with small droplet of flushing medium in a circle with two droplets in the middle with PVP.
- Layer the droplet with oil and put in the incubator for 30mins
- Prepare a dish with 0.8ml hyase and put in the incubator.
Denudation Of Oocyte For Intra-Cytoplasmic Sperm Injection (ICSI)
- Remove the oocyte and hyase dishes from the incubator together and immers oocyte in hyase dish.
- Gently aspirate oocyte with the pipette 300micrns so as to loosen the cumulus cells.the oocyte are further denuded with 170 n 140 micrometer needle till all cumulus cells are removed and transferred t another dsh containing washing media.
- The oocyte should not remain in hyase for more than 30 seconds.
- The oocytes are then washed in washing medium and placed in the icsi plate and keep it in the incubator
Oocyte Cumulus Complex
Denuded Oocyte
