1. Turn off all fluorescent lights and turn on incandescent light.
3. Wipe down the counter top of the hood and microscope stage with 70% ethyl alcohol.
4. Retrieve tubes of media (prepared the day before) from the water bath rack, dry with sterile towel and place in heating block of the embryology laminar flow hood.
5. Place two round bottom 12 X 100 mm tissue culture tubes in block (holder), each containing a sterile short Pasteur pipette with red bulb.
6. Place appropriate number of culture dishes (Falcon) next to the microscope in the laminar flow hood.
7. Place an organ culture dish (Falcon no. 3037) on the stage warmer, in the hood, and using one of the glass Pasteur pipettes add approximately 1.5 mL HH-5% HSA (egg wash) to the inner well and approximately 0.5 mL in the outer well. The organ culture dish containing the egg wash should be kept on the stage warmer to maintain optimum temperature.
8. Each follicle is aspirated. The circulating nurse will label each tube with the follicle number and the side it was aspirated from (for example: R1 or L1). If the aspirate is a rinse the tube is labeled as such: R 1F. The tubes containing the aspirated follicles are then placed in the heating block in the laminar flow hood of the gamete laboratory.
9. Place the lid or the bottom of a culture dish on the dissecting microscope. Remove the aspirate from the heating block, note the volume of the aspirate, uncap the culture tube and decant entire content into the dish.
10. Scan the dish for the cumulus mass while noting other particulate material (i.e. granulosa cells). The size of the expanded cumulus cell-oocyte complex is easily identifiable even in the presence of red blood cells and granulosa cells. It is often easier to see the cumulus mass in slightly bloody fluid than in clear follicular fluid.
11. Once an oocyte is located, remove the cumulus mass containing the oocyte using the small Pasteur pipette. Make sure there is sufficient medium in the pipette prior to aspirating the oocyte-cumulus mass. This will ensure that the mass does not stick to the inner surface of the pipette and that, should it occur, there is sufficient media to be able to dislodge the oocyte.
12. Place the oocyte-cumulus mass in fresh culture medium (HH-5% HSA) for proper identification and scoring.
N ote: If a cumulus mass containing an egg is found, the remainder of the sample should not be discarded. More than one follicle may have been aspirated (biovular follicles have been observed).
13. Score oocyte-cumulus mass: Parameters used to determine maturity include size of the follicle, volume of follicular fluid and direct observation of the cumulus-corona complex.
N ote : Determination of oocyte quality or maturity is important since it dictates the length of time required for in vitro maturation prior to sperm/egg mixing. The more immature the oocyte the longer the maturation period required before the addition of sperm (insemination).
■ Immature oocyte: Characteristically recovered from a follicle in which preovulatory maturation has not been initiated and therefore the oocyte remains arrested in prophase I of meiosis with an intact germinal vesicle (GV). The GV is difficult to visualize directly because of obscuring cell layers but appears as a large, centrally localized nucleus (GV intact oocytes may be
difficult to find in normal follicular aspirates since they are not usually surrounded in a large, highly visible cumulus mass). Immature oocytes will be surrounded by several layers of tightly adhering cumulus-corona cells. ■ Mature oocyte: Usually recovered from follicles of a mean diameter exceeding 15 mm. These follicles contain oocytes (Figure 24.1), which have resumed meiosis from prophase I progressing to metaphase II; the stage normally ovulated in most mammalian species. Mature oocytes will be surrounded by expanded and radiating cumulus-corona cells.
14. Transfer the oocyte to a small tissue culture tube containing 0.5 mL HH-5% HSA. Cap the tube tightly and label with follicle number and side (i.e. R1). Place tube in the heating block and continue scanning other aspirates.
15. Record follicle, volume and oocyte information.
16. Once the retrieval is complete, wash the oocytes and transfer into 30 pL droplets of HH-5% HSA using a short sterile glass Pasteur pipette with a rubber bulb. Aspirate and expel the cumulus-oocytes complex in a sterile organ culture dish containing approximately 1.5 mL HH-10% HSS. It may be necessary to strip the cumulus complex, using two sterile syringes with 30G 1/2" needles to remove excess cells or blood clots.
17. Transfer the oocyte into an organ culture dish, prepared the day before, containing three 30 pL drops of QB XI-5% HSA under mineral oil (one oocyte per drop).
18. Label each drop with the respective egg (i.e. drop no. 1 - R1, etc.), return the organ culture dish to the incubator and allow eggs to equilibrate.
19. Initial the appropriate space on the patient identification flow sheet found in the patient's chart.
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