ICSI - Intracytoplasmic Sperm Injection

Intra-Cytoplasmic Sperm Injection (ICSI)

• 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.

Intra-Cytoplasmic 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