Sequential Growth Media
All of the media in our laboratory can be classified as one of two basic types. One type of medium is chemically buffered to keep a constant pH of 7.3 to 7.4 while it is exposed to room air. It cannot be used in the incubator environment. The other type of medium is designed to keep this same pH of 7.3 to 7.4 while it is exposed to the gas concentration within the incubators, which is very different from room air. It cannot be used in room air. The reason for the two types of media is that it is essential to keep sperm, eggs and embryos at a pH of 7.3 to 7.4 in order for them to be healthy and to carry out their normal metabolism. While the sperm, eggs and embryos are usually kept within the controlled environment of the incubator, they must be taken out occasionally in order to perform necessary procedures and to move them from one culture dish to another. The two types of media keep the embryos exposed to a stable pH while they are in each environment.
In our laboratory, the Fertilization Medium used on the day of egg retrieval and insemination (or ICSI) provides a basic environment with a minimal amount of energy sources. The main energy source provided by the media is pyruvate and lactate in small amounts. This is because at this stage of development the egg uses mostly internal reserves of nutrients as energy sources. During this time the egg is fertilized by the sperm and organizes its chromosomes. It is still too early for cell division.
The following morning (24 hours after the egg retrieval), the eggs are checked for fertilization. All of the fertilized eggs and the unfertilized, mature eggs are transferred into new Petri dishes containing small drops of Cleavage Medium. The eggs and the embryos that they become are kept in individual drops from this point until they are transferred, frozen or discarded. This Cleavage Medium must support the metabolism of the embryos during their initial cell division. In the body, the embryos would still be in the fallopian tubes while they divide and grow to around eight cells. The major sources of energy, both in the fallopian tubes and in our medium, are larger amounts of pyruvate and lactate with small amounts of sucrose. The embryos will stay in this medium for two days as they grow from one cell to eight cells.
Early on the third day of culture in the lab, the embryos are removed from the incubator and observed for growth. They should have divided and become six to eight cells by this time. This is the first day on which embryos might be transferred into the uterus of the intended parent. They are assessed, the physician is notified as to their growth status and then the physician decides if the transfer will take place on day3 or on day 5. If embryos are to be transferred on day 3, the embryos for transfer are separated from the others and the remaining embryos are moved into new dishes containing Blastocyst Medium. Since the embryos would be entering the uterus on this day if they were growing in the woman’s reproductive tract, this medium mimics the tissue fluids that are present in the uterus. The embryos are supported by a higher concentration of sucrose while the levels of pyruvate and lactate are much lower. This medium must provide the energy necessary for the embryos to grow from eight cells to more than forty cells within 24 hours. At that point in their development (day four), the embryo growth stage is known as morula. This same dish of Blastocyst Medium must continue to support the growth of the embryos for one more day of culture. By this day, day 5, the healthy embryos will have grown to more than 100 cells and are known as blastocysts.
The fifth day of the embryos’ development in the lab is a very important one. If the embryo transfer did not take place on day 3, the embryologist will remove the embryos from culture and assess their growth. The physician will be notified and decide how many embryos will be transferred. The embryologist will select and separate the highest quality embryos for transfer and move the remaining embryos to a new dish containing more