Behind the scenes: The embryology lab

Behind the scenes: The embryology lab

by Sarah Lalone, guest author on The Conception Diaries Sarah Lalone 6 May 2018

It’s your big day! The egg retrieval (or embryo transfer) day is the culmination of IVF. All of the injections, hormone treatments and symptoms have led to this, and you are anxious yet excited about what will happen and how you will be helped on your journey to parenthood!

In the fertility operating room or hospital, you’ve probably walked by a large metal door with security-code access that says, Laboratory: restricted access! Here’s what’s happening in that laboratory, where embryologists are working hard to make your embryos. This is what they are seeing and thinking!

People in scrubs, masks, hats, and booties shuffle in and out of the door quietly. It’s easy to feel that what goes on in the embryo lab is a bit of a mystery, but for me, as an embryologist, the lab is my home. Embryologists are scientists who not only are trained in the science of embryo development, but also have the highly developed skill set to perform microscopic tasks with robot-like precision. The lab provides the closest possible approximation to the human body for eggs, sperm and embryos, which enables us to make the healthiest, most competent embryos possible for you to use. It’s full of high-tech incubators, machines, and growth tools, and with help from us, it’s the safest place outside of the body for an embryo to live.

For over a third of the time you are undergoing IVF (from stimulation to transfer), the embryo is the patient we are caring for. Embryologists carefully evaluate patient materials — whether it be egg, sperm, or embryo — and make decisions about how best to use, make and grow them. When the time comes, we select the embryo that’s shown the most competence to become a pregnancy, and we carefully save the rest for future use. Our decisions and actions can have a huge impact on your success with IVF; so, in order to demystify this important aspect of your care, let’s look at some of the work we do in detail.

Sperm and eggs

In IVF, eggs are generated through stimulation of the ovary, under the careful care of a physician team, whereas sperm samples are delivered to the lab fresh on the day of fertilization or from a banked frozen sample. Once these materials arrive in the lab, we assess them for their suitability and determine the course of action for fertilization.


When the physician removes follicles containing eggs from your ovary, they are given to the lab in test tubes. We then search the tubes to find what we call cumulus-oocyte complexes (COCs). A COC is an egg surrounded by several layers of small cells, called cumulus cells. We typically grade each COC as an A (best), B, or C, based on things like how large the mass of cumulus cells is, how well placed they are around the egg itself, and what the texture of the complex is (for example, does it look dark and compact or bright and fluffy)? If we take the cumulus cells off, we can accurately assess the egg itself.

When we are able to see the egg, we are looking for signs of proper maturity. If we see a small circular structure, called a polar body, on the outside of the egg, then we know the egg is ready to be fertilized. If the polar body is not yet present, then we know the egg needs more time to mature, and we can incubate it for a few hours to wait for that to happen. However, if we see signs that it is very immature or overly mature, it will be discarded. Eggs of the appropriate quality are placed in an incubator to wait for the fertilization step.


Sperm samples undergo a set of measurements to determine suitability of the sample for fertilization. Embryologists will calculate how many sperm are in the sample (the count), how well they are moving (motility), and how normal they look (morphology). The sperm cells are then separated from the seminal fluid so that we have a clean population of normal-looking, motile sperm to use for fertilization.


In the lab, we fertilize eggs either by in vitro insemination or intra-cytoplasmic sperm injection (ICSI). If we are performing in vitro insemination, a droplet containing thousands of sperm is placed in the dish of eggs, and the sperm swim in a race to the eggs, where the winning sperm makes its way in. However, during ICSI, the embryologist examines the pool of sperm cells, selects the most appropriate-appearing sperm and physically inserts it into the egg using a microscopic needle. After the in vitro insemination or ICSI process is completed, we place the dishes of eggs with sperm back in the incubator, in a specialized growth fluid called culture medium, and allow them to finish fertilization for 12 to 18 hours.

After this time, they will be checked under a microscope for signs of correct fertilization. We look for what are called pronuclei inside the egg, and if we see two (one from the egg and one from the sperm), that is a sign that the egg was fertilized normally. You might see these marked as “2pn”, which to us means “fertilized”. Now the embryo has formed and will start dividing in time.


An embryo has to complete a set of developmental steps in order to go from being a single cell at the time of fertilization to a special structure called a blastocyst, with 200 to 300 cells. This process, which takes about five days, contains many steps, each of which is required if the embryo is to move to the next step. If an embryo can’t complete a step, we know that something was disorganized within that embryo, and we can infer that it wasn’t competent to become a pregnancy.

In order to go from 1 to over 200 cells, the embryo will rapidly divide over and over again. One cell divides to 2, 2 divide to 4, 4 divide to 8, and so on. We expect divisions to proceed at a regular rate over the five days, and we check the embryos under a microscope once every day to see that they’re on track.

If we look at them on the second day, we should see them at about 2 to 4 cells, and on day three, they should have about 8 to 10 cells. Up to this point, embryos are referred to as cleavage-stage embryos because they are at the stage that we can easily observe cell cleavages (divisions).

On day four, we look for an embryo with more than 16 cells, and now the cells should have started to compact together in a tight structure that we call a morula. Finally, by day five, the embryo should have hundreds of cells, made up of two different types of cells and a fluid-filled compartment inside it. This structure is called a blastocyst. We compare all embryos to this normal growth pattern, and if the embryos are faster or slower than this, we make note of it.

We take into account not only how fast or slow the embryo grows, but also how it looks. During the cleavage stage, we assess the embryos for how healthy the cells appear. For example, are the cells smooth, round, and symmetrical? Are parts of cells broken off (called fragments)? Are all the cells dividing, or are some not? In later embryonic stages (blastocysts), we look to see that all of the necessary structures are present (including the inner cell mass (ICM), which will grow into the fetus, and the outer layer of cells (TE), which will grow into the placenta), as well as how expanded the fluid-filled compartment is. These factors together are how we assign a morphological grade.

Grading methods and terms differ between labs, but in all clinics, embryo grades are meant to communicate a complicated assessment to a colleague or for records in a simple and standardized way. For cleavage-stage embryos, grades show the cell number and morphological score. You might see grades like 8G1, which means it’s an 8-cell embryo with a good appearance, whereas a 4G3 is a 4-cell embryo with a poor appearance. For blastocysts, grading takes into account expansion, ICM quality and TE quality. For example, 4AA is a fully-expanded blastocyst with a good-appearing ICM and a good-appearing TE, but a 1CC is a relatively unexpanded blastocyst with a poor-appearing ICM and TE.

Embryo transfer

On day three, embryologists will look at the group of embryos you have and decide whether it would be prudent to continue growing them to day five for a blastocyst transfer or to transfer them as cleavage-stage embryos on day three. Many factors are taken into account, but generally the embryologists are looking at how many are competent on day three, how well the embryos are tolerating living in the laboratory environment, and how likely some will reach the blastocyst stage.

Whether on day three or five, when it’s time for transfer, we will take the embryo’s entire developmental history into account before choosing which embryo is the best candidate for transfer. We pour over this information and test it against rigorous selection decision trees and often ask our colleagues to help us decide. In the end, we make the best decision we can.

Beyond-morphological grade

Embryo selection is often referred to as a beauty contest because all we have to go on is how the embryo looks and a little bit about how it acts. This analogy is somewhat true because while morphological selection helps us find a competent embryo, looks can be deceiving. To get a fuller picture of embryo competency, we need to know things such as the genetic, cytoplasmic, and metabolic health of the embryo. Embryo competency is a puzzle made of many pieces, and one piece alone won’t tell us everything. Scientists are working hard to develop ways to see inside the embryo without disturbing it, but not all of the tools are available to us in the clinic yet. What’s interesting is that as we integrate these techniques into the lab, we’ve learned that sometimes we can have a very good-looking embryo with very abnormal underlying processes, and vice versa.

Your clinic might be able to determine whether the embryo has the right number of chromosomes through a test called preimplantation genetic screening (PGS). PGS works by removing 5 to 10 cells from the embryo and sending them to a lab, which looks at whether those cells have the right number of chromosomes. This technique is still developing, and we don’t have the ability to test the complete genetic makeup of the whole embryo yet; however, transferring an embryo with the correct chromosomal complement is more likely to result in a pregnancy than result in one that is not correct.

Tests to determine cytoplasmic health are based on developmental timing algorithms that watch the embryo grow and that predict how competent it is. This emerging method of analysis relies on imaging incubation, which means that an incubator takes a picture every five minutes of the embryo growing. When these images are stitched together, the embryologist can watch a movie of the embryo developing without missing any milestones of this dynamic process, and they can make measurements and predictions with that information. Different algorithms have been proposed, but a single preferred algorithm has yet to be determined.

Metabolic health can be assessed by looking at the culture media droplets that embryos grow in to see what nutrients embryos have taken from it versus what they’ve left in it. Currently, no easily accessed tests are available for clinical use, but this technique is being developed for future use.

The more information embryologists have about an embryo, the better equipped they will be to determine its capabilities.

Embryo preservation

If you are undergoing a freeze-all cycle or have more embryos than were transferred to you, then the remaining embryos need to be preserved by freezing. Embryos can be kept in an inanimate state in cold storage for years until you need them, through a method called vitrification. Embryologists must determine which embryos are competent enough to withstand the freezing and subsequent thawing process, which can be stressful. It wouldn’t be useful to preserve embryos that would be unable to survive, so a general standard is applied to embryos, taking into account their development and any beyond-morphology testing results to determine which to freeze and which to discard. When it’s time to use your frozen embryos, the embryologist will select from the bank of embryos those that are the most competent to achieve pregnancy, and the rest remain in storage.

The embryology team works over many steps to optimize the performance of your embryo cohort. The laboratory is an important facility for this work, and if you look inside, you’ll see it is full of scientific, high-quality, highly engineered equipment; but to an embryologist, the lab feels warm, quiet, and calm. Not many people are allowed in because conditions are tightly monitored, making it a privilege to enter and especially to work in. Rest assured that the embryologists behind those doors are highly trained and, more importantly, love their job and work tirelessly to help you achieve your goals.

Sarah Lalone, guest author on The Conception Diaries Guest author: Sarah Lalone is both a Research and Clinical Embryologist who has been active in the field for over 10 years. She is currently the Clinical Embryology Liaison for EMD Serono Canada where she acts as an educator, trainer and scientific consultant.