Eggs (oocytes) are retrieved from patients by transvaginal aspiration directly from the ovarian follicles, approximately 36 hours after an injection of HCG. At that time, the follicular fluid is taken into the laboratory so that the eggs can be identified by the embryologists As oocytes mature within the developing follicle, they are surrounded by cumulus cells which participate in the nourishment of the egg. The egg and the cumulus are referred to as the oocyte-cumulus complex.
Immediately after retrieval, each complex is identified under a microscope and then transferred to a special solution ("insemination media") that is specially designed to provide all of the nutrients and other substances necessary to maximize the likelihood of successful fertilization of eggs by the sperm.
The dishes containing the eggs are then placed into an incubator, so that the environmental conditions surrounding the eggs can be tightly controlled with regard to light, oxygen and carbon dioxide concentrations, as well as the pH and temperature. The cumulus cells are left attached to the oocyte for at least 4 hours post-retrieval to allow the oocytes to complete their maturation. As different procedures regarding oocyte and embryonic development occur on specific days, we refer to the day of the oocyte retrieval as "Day zero".
The assessment of fertilization takes place by evaluating each egg the day after the oocyte retrieval and looking for the presence of two pronuclei. One pronucleus contains the genetic material contributed by the egg; the other contains the genetic material contributed by the sperm. The presence of two pronuclei denotes normal fertilization. The pre-embryo at this one cell stage is referred to as a zygote, which is the first stage in embryo development. The presence of only one pronucleus generally means that the egg has been activated, but there is no sperm contribution.
The presence of more than two pronuclei means either that more than one sperm has entered the oocyte or that the oocyte did not complete its final maturation process (which occurs after the sperm enters the egg). Consequently, there is too much maternal genetic material present for a normal embryo to develop. These zygotes that contain more or fewer than two pronuclei are chromosomally abnormal and, therefore, can never develop into a normal baby.
Fertilized egg with polar body
Embryo Culture with Sequential Media
In nature, fertilization typically occurs in the fallopian tube. The resulting embryo then travels slowly down the tube and enters the uterus, where it floats for several days before attaching to the uterine wall. Along this journey, the embryo is exposed to different biochemical environments – each of which contains the particular nutrients that the developing embryo requires at each stage of its growth prior to implantation. We attempt to recreate this same environment in our IVF laboratory through the use of different media (culture solutions) at different stages of embryonic development.
Fertilization takes place in culture media that is specifically designed to mimic the same environment that an egg would be exposed to in the fallopian tube. Embryos at each subsequent stage of development are grown in media similar to what they would encounter as they make their way down the tube into the uterus. As embryos develop, their metabolic requirements change. Therefore, the nutrients and other substrates required for their growth are sequentially changed to meet these needs.
After the 8-cell stage of development, cellular division becomes more rapid and the embryos require additional resources from the environment. Since the requirements of the embryos have changed, a different media formulation is required to better meet the needs of these later-stage, highly active embryos. By having specific media for the different stages of embryo development (Fertilization, Early Embryo, and Blastocyst Development) the laboratory is better able to mimic what the embryos would be experiencing if they were still in the woman's body, ie. "in vivo". Since implementing the use of sequential media in the laboratory, Austin IVF has seen significantly improved embryo quality resulting in a greater number of embryos developing to the blastocyst stage.
Day 3 Embryo Day 5 Embryo
Typically because you have a large number of nicely developing embryos - the embryos are allowed to continue to grow in the lab and the selection of the best embryos is made on day 5. If the transfer is on day 3, the embryos chosen for transfer are usually at the 6-8 cell stage. By day 5, the embryos should be at the blastocyst stage at which time each embryo contains approximately 100-120 cells. At this stage the embryo is beginning to differentiate. The cells destined to become the baby are identified as the "inner cell mass". The cells around the periphery of the embryo are called the trophectoderm and become the placenta. There is a clear space within the blastocyst called the blastocele.
The development of the trophectoderm and the inner cell mass is equally important for a healthy pregnancy to develop.
Although you and your physician will decide on the number of embryos to transfer, Texas Fertility Center and Austin IVF strictly comply with the embryo transfer guidelines established by the Society of Assisted Reproductive Technology (SART). These guidelines were developed based on an extensive review of the published literature in the hope of maximizing a couple's chance for pregnancy while minimizing their risk of a multiple pregnancy. The number of embryos to transfer is determined by multiple factors, including the age of the egg and the development of the embryos. Once the decision regarding the number of embryos to transfer has been made, the physician informs the embryologist. The embryologists are the ones who actually choose the specific embryos to be transferred.