While the egg retrieval is happening, the sperm is also prepared. A semen sample is provided by the male partner or the donor sperm sample is prepared for use. In the laboratory, a concentrated preparation of the best motile sperm is extracted from the semen sample. This sperm preparation (containing approximately 150,000 sperm) is added to the dishes containing the eggs, and they are incubated together overnight.

In some couples an alternative form of insemination is required called ICSI (Intra-Cytoplasmic Sperm Injection), which involves injecting a single sperm into each egg using a very fine needle, rather than mixing the eggs and sperm in a dish

Regardless of the method of insemination used, on the morning after egg retrieval, the eggs are examined to see which have fertilised.

Fertilised eggs (zygotes) are cultured in the IVF laboratory until day 5 or 6, the blastocyst stage. At that point the embryos are either transferred or frozen, depending on the individual circumstances. Any additional embryos that are not transferred at the blastocyst stage can be frozen.