Pronucleate Stage Embryos to Day 3 Culture

All manipulations of oocytes and embryos should be performed using a pulled Pasteur pipette, glass capillary or a displacement pipette. It is important to use a pipette with the appropriate size tip. For example, once the cumulus is removed (day 1 to 3), a bore of around 175-200 mM is required. Using the appropriate size tip minimizes the volumes of culture medium moved with each embryo, which typically should be less than a microliter. Such volume manipulation is a pre-requisite for successful culture.

Around 4 pm on the day of oocyte retrieval, label 60 mm Falcon Primaria dishes with the patient's name. Using a single-wrapped tip, first rinse the tip, then place 6 x 25 ml drops of G1 into the plate. Four drops should be at the 3, 6, 9, and 12 o' clock positions (for embryo culture), the fifth and sixth drops should be in the middle of the dish (wash drops). Immediately cover drops with 9mL of tested oil (such as Ovoil, Vitrolife). Prepare no more than 2 plates at one time. Using a new tip for each drop, first rinse the tip and then add a further 25 ml of medium to each original drop. Place the dish in the incubator at 5% O2 and 6% CO2. Gently remove the lid of the dish and set at an angle on the side of the plate. Dishes must gas in the incubator for a minimum of four hours (this is the minimal measured time for the media to reach correct pH under oil). For each patient, set up a wash dish at the same time as the culture dishes. Place 1 mL of medium G1 into the center of an organ well dish, place 2mL of medium into the outer well, and then place the dish in the incubator. If working outside an isolette, use a MOPS or HEPES buffered medium with amino acids. This should not be placed in a CO2 incubator, but rather warmed on a heated stage.

Following removal of the cumulus cells, embryos are transferred to the organ well dish and washed in the center well drop of medium in the culture dish. Washing involves picking up the embryo 2-3 times and moving it around within the well. Embryos should then be washed in the two center drops in the culture dish and up to 5 embryos placed in each drop of G1. This will result in no more than 20 embryos per dish. Return the dish to the incubator immediately. It is advisable to culture embryos in groups of at least 2. Therefore for example, for a patient with 6 embryos, it is best to culture in 2 groups of 3 and not 4 and 2 or 5 and 1. On day 3, embryos can be transferred to the uterus in a hyaluronan enriched medium (221).

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