Insemination of the Eggs

Insemination refers to the placing of a highly-motile, normal population of sperm into a culture dish with eggs that have been retrieved that same day.

With insemination, 15,000 to 30,000 sperm are placed in a small drop with one or two eggs. The sperm enters the egg by dissolving a path through the outer shell, or zona pellucida, with an enzyme that is located on the head of the sperm. This entry also requires vigorous swimming by the sperm until it gets through the shell.

At that point, the sperm and egg cell membranes chemically bind and the sperm is pulled into the interior, or cytoplasm, of the egg. When the sperm enters the interior of the egg, its tail breaks off. The significance of this is that the cell membrane that surrounds the head and the tail is broken. The breaking of this membrane allows water to enter the sperm head from the cytoplasm of the egg. Then, both the egg and the sperm form a nucleus within the egg. These two nuclei are called pronulei until they merge to form the nucleus of the new embryo. They each contain half of the chromosomes that the embryo will have. The formation of these two pronuclei is what the embryologist looks for when they perform a fertilization check on the morning after the retrieval.

Fertilization takes place by the vigorous movement of the sperm which penetrate the shell of the egg, bind to the inner membrane of the egg and then enter the cell material, or cytoplasm, of the egg. Here, the chromosomes of the sperm and egg combine and produce an embryo.

After the semen sample has been provided and the sperm has been processed, the sperm are placed in a medium that mimics the tissue fluids found in the Fallopian tubes of the woman. This is done because the Fallopian tubes are the site of fertilization when it occurs in the body. This fertilization medium provides the nutrients necessary to keep the sperm alive and to allow them to swim to the eggs at insemination in the lab.

Approximately 6 hours after the egg retrieval, a small volume of the sperm in the fertilization medium is injected into each drop of culture medium containing 1 or 2 eggs. Since the eggs are surrounded by a mass of cells called cumulus cells, the details of the eggs, including quality and maturity, are not visible. These cumulus cells come from the follicle of the ovary and are important in the early maturation of the egg.