Embryology 101: How Embryos Are Created Through IVF
If you are considering receiving or donating embryos, understanding what happens in the lab to create embryos can make the whole process feel less abstract and a lot less intimidating.
In this walk-through, embryologist Gina McCain of Sanford Women's Health explains how embryos are created through in vitro fertilization (IVF), how they develop over their first several days, and how embryologists assess their quality before freezing or transfer. Whether you are a donor wanting to understand the embryos you are entrusting to another family, or a recipient hoping to learn what those early days looked like, this overview is here to give you a clear picture of the science.
Fertilization and Early Embryo Development
To start with the big picture: fertilization and early embryo development follow the same essential path whether they happen naturally in the body or in an IVF lab.
In the lab, embryologists work to mimic the environment of the fallopian tube. The embryo is cultured in a dish that provides the nutrients and conditions it would normally experience as it travels through the fallopian tube toward the uterus.
The timeline looks like this. On Day 0, eggs are retrieved and fertilized. By Day 3, the embryo has reached the 8-cell stage and can potentially be transferred. If culturing continues to Day 5 or 6, the embryo reaches the blastocyst stage, which is where most transfers happen today.
The Egg at Retrieval (Day 0)
The day of egg retrieval is called Day 0. At this point, each egg is surrounded by a cloud of supportive cumulus cells. Under the microscope, the egg looks soft and clustered, still wrapped in those cells right after it has been retrieved from the follicle.
Once the cumulus cells are removed, the embryologist can assess the egg's maturity. A mature egg, known as an MII oocyte, can be identified by the presence of a polar body, a small structure visible at the edge of the egg.
Immature eggs, which do not yet have a polar body, cannot be fertilized. Only mature eggs move forward to the next step.
How Eggs Are Fertilized: Two Methods of Insemination
Once mature eggs are ready, there are two ways to fertilize them.
The first is regular (conventional) insemination. Here, roughly 100,000 motile sperm are added directly to the culture dish containing the egg, and fertilization is allowed to occur naturally. One sperm penetrates the egg on its own.
The second method is Intracytoplasmic Sperm Injection, or ICSI. With ICSI, the embryologist uses a very fine needle to inject a single sperm directly into each egg. This bypasses any barrier the sperm might face getting through the egg's outer shell, called the zona pellucida.
When ICSI Is Used
There are three main situations where ICSI is the better choice over regular insemination, though it has become the standard for most IVF embryo creation for it’s success rate in creating viable embryos.
Male factor infertility. If the sperm count is low, motility is poor, or the shape of the sperm (its morphology) is abnormal, regular insemination may not give sperm a good chance of fertilizing the egg. ICSI removes that barrier.
Frozen and thawed sperm. Sperm that has been frozen and thawed tends to have lower motility than fresh sperm. ICSI helps ensure fertilization even when sperm function is reduced after thawing.
Frozen and thawed eggs. When an egg has been frozen and thawed, the outer shell, the zona pellucida, can become harder, making it more difficult for sperm to penetrate on its own, so ICSI is used to bypass the hardened shell entirely.
Confirming Fertilization (Day 1)
About 18 hours after insemination, the embryologist checks whether fertilization has occurred. This is Day 1.
A normally fertilized egg is called a 2PN, meaning two pronuclei. It shows two distinct round structures inside, one pronucleus contributed by the egg and one by the sperm, along with two polar bodies at the edge.
Occasionally an egg shows 3PN, meaning three pronuclei. This indicates that two sperm fertilized the egg, leaving an extra set of chromosomes. These embryos are not usable for transfer and are not carried forward.
How Embryos Develop in the Lab (Day 1 to Day 6)
After fertilization is confirmed, the embryos continue to be cultured and observed over the next several days. The progression looks like this:
Day 1: The 2PN fertilized egg. The two pronuclei fuse, and the embryo begins its first cell division.
Day 2: The embryo has divided into 4 cells.
Day 3: The embryo reaches 8 cells. This is the earliest stage at which a transfer can take place.
Day 4: The cells begin to compact together to form what is called a morula.
Day 5 to 6: The embryo develops into a blastocyst. This is the preferred stage for both transfer and freezing.
Understanding the Blastocyst
The blastocyst stage deserves a closer look, because it is the stage most embryos have reached when they are transferred or frozen for donation.
On average, about half of the embryos that are fertilized will make it to the blastocyst stage. Not every embryo survives to this point. Some stop developing along the way, which is completely normal and reflects the natural process of selection.
A blastocyst has two distinct cell types:
Trophectoderm (TE): the outer layer of cells surrounding the blastocyst. These cells go on to become the placenta.
Inner Cell Mass (ICM): the small cluster of cells inside the blastocyst. These cells develop into the fetus and the surrounding embryonic tissues.
How Embryos Are Graded
Before an embryo is transferred or frozen, embryologists grade it to assess its quality. At the blastocyst stage, the grading system has three components.
Stage (1 to 6). This describes how expanded the blastocyst is. A Stage 1 blastocyst is just beginning to form its fluid-filled cavity. A Stage 6 blastocyst has fully expanded and is beginning to hatch out of its shell. Stages 3, 4, and 5 are the most commonly transferred.
Trophectoderm grade (A, B, or C). This grades the quality of the outer cell layer, the cells that become the placenta. A is the best, with many cohesive cells. B is adequate. C is poor, with few or loosely organized cells.
Inner Cell Mass grade (A, B, or C). This grades the quality of the inner cell mass. Again, A is the best.
So a grade of 4AA means a Stage 4 blastocyst (fully expanded) with an A-grade trophectoderm and an A-grade inner cell mass. That is a high-quality embryo.
What Good, Average, and Poor Quality Embryos Look Like
Under the microscope, the differences between quality levels become visible.
Good (“A”) embryos have a cohesive trophectoderm with many organized cells, a clearly visible and tightly packed inner cell mass, and a well-expanded blastocyst.
Fair (“B”) embryos are still very viable. They have fewer trophectoderm cells or a less prominent inner cell mass. These embryos carry good chances of resulting in a successful pregnancy, slightly lower than A embryos.
Poor (“C”) embryos have very few trophectoderm cells, a fragmented or barely visible inner cell mass, or unusual structures within the blastocyst. They have lower implantation rates and would be selected last for transfer. Embryologists note, however, that a euploid PGT-A outcome can override the “C” grading for success probability.
It is very important to note that while there is a standard grading protocol, embryologists and clinics assess embryos within the framework differently! Just like teachers, some are tough graders and never give As, and their C embryos have a good chance for live birth, while others grade more favorably, with more As, and therefore they have less confidence in their C embryos.
What This Means for Embryo Donation
In embryo donation, both donors and recipients benefit from understanding that not every embryo is graded the same. A good or fair graded embryo can carry a strong potential for a healthy pregnancy, and even more likely when paired with evidence that its chromosomes are normal (via age of the egg contributor, testing, and or prior live birth rates).
Knowing how embryos are created, how they develop, and how they are assessed can help you feel more grounded as you make decisions, whether you are donating embryos to another family or hoping to grow your own through donation.
If you have questions about your specific situation, the Embryo Connections RN team and your embryologist at your clinic are great resources. And if you are exploring embryo donation and want to understand your options, the team at Embryo Connections is here to walk alongside you.