I just had the best shift at my internship thus far. This is the experience that has made the hours upon hours of filing, scanning, and typing worth it. The second I got home, I got on my computer and just word vomited everything that happened so that I wouldn’t forget anything. Feel free to skip reading this post since it doesn’t involve food in the least! However, if you are interested in the process of in vitro fertilization and whatnot, read on!
Egg Retrieval, Insemination & Cryopreservation
Once a woman has come to a certain point in her cycle of fertility drugs, it is time to retrieve the eggs that have been collecting in the follicles in her ovaries. The procedure, called an egg retrieval, is done in a room adjacent to the embryology lab. I have watched an egg retrieval, but I have been wanting to be on the other side of the wall for ages! I wasn’t allowed into the embryology lab for the first 4 months of my internship because you have to take a class offered once a month by the head embryologist before you can enter. In the class, you learn all about how the lab works and what the embryologists do (grossly summed up, they receive the eggs and sperm, prepare them for procedures, incubate them and cryopreserve them). The lab is really cool! Because eggs and sperm have to be kept at certain temperature, humidity, % oxygen, and pH ranges, the lab is kept at body temp and has special incubating storage units for the eggs and sperm. To ensure that no toxins or anything gets into the lab, it is pressurized so that when you open the lab door, air is only pushed out and nothing comes inside.
Anywho, during egg retrieval, the woman has her follicles emptied by the doctor, in hopes of collecting as many eggs for later insemination and implantation back into her uterus. Basically, the doctor takes a long needle and punctures each follicle in both of her ovaries and sucks the liquid from them. The liquid is tubed into a beaker and handed through a passageway to the embryology lab. The embryologist pours the bright red, or sometimes clear-ish, liquid from the test tube into a petri dish and looks at it under the microscope. She is looking for eggs, which look like a tiny black dots surrounded by greyish clouds floating in the liquid amongst some debris. The grey clouds are the cumulus cells. When she finds an egg, she sucks it up with a pipet and empties it into a new petri dish that is labeled and contains a medium that simulates the environment in the uterus. The eggs are to be cleaned up a bit before being inseminated later in the day.
After a woman has had her eggs removed, inseminated, and incubated (which is a whole other story) some are to be implanted and the extras (if there are any) are usually cryopreserved. That way, in case she doesn’t achieve pregnancy during this cycle, she has backup eggs to try again. To cryopreserve the eggs, the embryologist first makes the freezer by pouring liquid nitrogen into a styrofoam box and inserting the mini freezer tube. Liquid nitrogen looks SO COOL!
To prepare the eggs to go into the freezer, which is set at (I think) -196 degrees Celsius, the eggs must go through a series of dilutions. As you know, water expands as it freezes, so the water must be removed from the egg so that it doesnt burst during freezing. But you dont want a shriveled up egg, so the water must be replaced with cryopreservent. To get the cryopreservent in and the water out of the eggs, the eggs are placed in droplets of increasing molarity (concentration) of cryopreservant, which pulls out the water and lets the preservent seep in. Under the microscope you can actually see them shrink a bit as you move them into different solutions!
The embryos that are not cryopreserved are to be incubated until they are ready to be implanted back into the patient’s uterus. Today, I got to look at some fertilized eggs under the microscope, and see the differences between a normal looking embryo and an abnormal embryo. The normal ones contain two little spheres inside the larger sphere, one is a set of chromosomes from the egg (the mother) and the other is a set of chromosomes from the sperm (the father). You can’t actually see the individual chromosomes, though. Today, we saw that one of a patient’s embryos was abnormal. Instead of having two spheres (and thus 46 chromosomes), she had three, which means that embryo has 69 chromosomes. Definitely not good.
Because this particular egg was inseminated with ICSI (where the embryologist chooses one good looking sperm and manually injects it into the egg), we knew that the extra set of chromosomes was not from an extra sperm making its way into the egg, but from the egg itself. Eggs have two sets of chromosomes, and the egg is supposed to eliminate one set so that the set of chromosomes from the sperm can combine and make a complete set of 46 chromosomes. The eliminated chromosome set is discarded in what is called a polar body.
In the case of this abnormal embryo, the polar body was made but without the chromosomes inside of it. Thus, the egg still had 46 chromosomes and the sperm addition brought the count to 69. That embryo clearly will not be implanted.
Whether doing in vitro fertilization (fertilizing the egg in the lab) or intra-uterine insemination, the woman’s partner’s sperm (or donor) must be checked to make sure it is viable and able to create a pregnancy. Once the man has given his semen sample to the lab it needs to be analyzed and possibly prepared for use. 10 microliters of his sample are looked at under a microscope for the percent motility (how many of the little guys can swim). When you look in the microscope, you see THOUSANDS of tiny little sperm swimming around. The microscope has a 10×10 grid of squares on it. To determine the percent motile, you choose 10 squares and count how many are swimming and how many are immotile.
They use a little handheld clicker to keep track, but it is still hard! I tried, but my numbers were pretty off. The swimmers are constantly moving in and out of the boxes, so really, it is a fairly raw estimate. Then, you look at the numbers and determine the percent motile versus immotile, and then it is time to “wash” the semen to try to increase the percent motile. The raw specimen is placed in a test tube on top of two layers of liquid of increasing density. The sample is centrifuged for 20 minutes at 21,000 Rotations Per Minute so that the live semen are concentrated into a pellet at the bottom of the beaker and the debris and dead semen are at the top. The top is removed and the process is repeated. Then, you do the motility count again and if the numbers are good, you further prepare the sample for insemination of the egg. This time, you centrifuge the semen in medium and they are again concentrated into a pellet on the bottom of the tube. The strongest semen are those that are able to dislodge from the pellet and swim up, and those are what are used to inseminate the egg.
One of the embryologists also showed me how to perform a morphology analysis. Basically, you have the image of normal semen imprinted in your mind (like a cookie cutter) and you place that cookie cutter over each semen that you see in the sample under the microscope. If it fits the cookie cutter, it is normal, if it doesn’t, it isn’t.
You pay attention to the shape of the head, the size of the acrosome compared to the rest of the head, the shading, and probably a few other things I don’t know about. You can ignore if the tail looks a bit off (unless its a double tail or something) because you can alter the tail when doing your smear (placing the sample between two glass pieces for the microscope).
Normal parameters for a semen analysis, from my clinic’s website:
|Total Sperm Count||> 40 million||Morphology (WHO)||> 30% normal shape|
|Volume||> 2 milliliters||Morphology (Kruger)||> 14% normal shape|
|Motility||> 50% motile||Liquefaction||complete by 60 min|
All images taken from google!
Hopefully I didn’t weird y’all out too much! Probably did. Whateva – I’ll be making jokes all day about what I did this morning…
I will post a delicious recipe tomorrow