During In Vitro Fertilization (IVF), eggs and sperm are taken from the couple and are incubated together in a dish in a laboratory to produce an embryo. A health care provider places the embryo into the woman's uterus, where it may implant and result in a successful pregnancy.
The steps of In Vitro Fertilization (IVF) are :
- Stimulation of Egg Maturation
- Egg Retrieval
- Fertilization
- Embryo Transfer
Stimulation of Egg Maturation
In this process, also known as ovarian stimulation or ovulation induction, a woman takes medication to stimulate the ovaries to make many mature eggs at one time.
These medications are given by injection for 8 to 14 days. A health care provider closely monitors the development of the eggs using transvaginal ultrasound. Blood is drawn frequently to assess estrogen production by the ovaries. When the eggs are mature—as determined by the size of the ovarian follicles and the level of estrogen—an hCG injection initiates the ovulation process. A health care provider takes out (egg retrieval) the eggs 34 to 36 hours after the injection
Egg Retrieval(Day-0)
This is the process used to remove the eggs from the ovaries so they can be fertilized. The procedure is performed in In Vitro Fertilization (IVF) OT as an outpatient procedure. A mild sedative and painkiller are often used during the procedure, and it normally takes about 30 minutes. The steps for egg retrieval are:
- An ultrasound probe is inserted into the vagina to visualize the ovaries and the follicles, which contain the eggs.
- A needle is inserted through the wall of the vagina to the ovaries.
- Suction is used to pull the eggs from the ovaries into the needle
Oocyte Handling
- The follicular aspirates are collected and passed through the hatch to the embryologist
- The foliicular aspirates are scanned on a dissecting microscope with heated stage
- Alliquot each aspirate from the test tube in to two or three petridishes
- once the oocyte is identified ,picked it up with sterile pipette and transfer in to flushing medium
- Give one wash to all the OCCs(oocyte cumulus complex) in flushing medium
- This is then transferred in to the fertilization medium and keep it inside the incubator for 2-3 hr
Insemination For In Vitro Fertilization (IVF)
- 0.8ml of pre equilibrated fertilization medium is put in a center well dish
- 4-5 OCC placed per dish to be inseminated and cultured together
- Meanwhile sperm is washed and prepared for insemination either by swim up or density gradient method.
- 1,00,0000 sperms are introduced in this dish
- keep it in the incubator over night
Fertilization Check(Day-1)
- The dish is removed from the incubator 16-18 hrs after insemination
- Use 170 um denudation pipette to pick the egg and transferred to fresh culture medium
- This is then picked with a 140um denuding pipette,gently aspirate and expel the egg until enough of the cumulus and corona is removed to get a clear visualization of the pronuclei
- This is then checked for fertilization
- Two pronuclei and the two polar bodies are visible when the egg has fertilized
- For PN grading-placement of PN and alignment of nucleoli are considered.
- collect all the egg showing 2PN and transfer in to preincubated cleavage medium and put back in to the incubator.
