• 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