In this webinar, Emily Baker, Senior Clinical Embryologist, Deputy Laboratory Manager at GENNET City Fertility, London, UK, has talked about the temperature, security and environment in the embryology/andrology lab.
Emily has explained that in the embryology lab, the conditions are kept as similar to the inside of the uterus as possible. There are strict measures to eliminate external contaminants, and the environment within the lab is always constantly monitored. Should anything go down, any incubators start to malfunction, the embryology team are notified instantly. We get there as quickly as possible to minimize any damage that could happen.
The best environment for an embryo is in a healthy uterus, and we work from what we know about that, so we’ve got things like a new pH, nutrients available, cosy 37C body temperature. There’s no light in the uterus, but most importantly, no UV light, no germs, no contaminants, appropriate water content, and reduced oxygen concentration.
Embryos, eggs, sperm need available nutrients to function, grow. The nutrients that we provide to the embryos, eggs, and sperm comes from the culture media that we prepare them in, so different culture media from different manufacturers will have various energy substrates. Mostly we have glucose, lactate and pyruvate in the culture media that we use. These are used at different stages throughout an embryo’s development. Pyruvate tends to be used throughout. Lactate can be used in the first few days, and glucose uptake is increased as we approach the blastocyst stage, so that’s around day-4 to day-5. Within the culture media, we’ve also got amino acids. These are very important in terms of building blocks for protein development also helps to build DNA. They also have a lot of other roles which are still being explored to this day, we don’t know all of them, we know that they benefit them.
We don’t know the exact concentration and the actual amino acids that are in each culture media. The manufacturers don’t necessarily publish that as freely as we would like. It’s a little of a secret recipe. We know what works, and we know what we need, and we also know what the culture media provides.
When it comes to oxygen, Emily again explains that all of our cells need oxygen to live. The interesting part is that embryos don’t need the same oxygen level as fully grown humans do. Embryos develop better at around about 5 to 6% oxygen levels, it is thought that the uterus will have a lower concentration of oxygen available to it. After the mother is absorbing oxygen into other cells when the blood’s on its way around to the uterus. When it gets there, the partial pressure of the oxygen is slightly reduced.
We manage to reduce the oxygen concentration in our incubators with the help of nitrogen gas to displace the oxygen. Nitrogen is a very inert gas, it doesn’t harm the embryos at all, but by pumping it into the incubators, it will push out the oxygen. Therefore, reduce the concentration down to around about 5 to 6%.
One more also very important thing that Emily added is that to maintain the water content of the culture media. Culture media tends to have quite a high water content. It is estimated that there are even over 99%, but as Emily mentioned, it’s not possible to be sure of the ingredients level that is in there. We need to inhibit any evaporation of this water content while we’re culturing embryos. If we were to do that, then the concentration of any solutes and everything else in the culture media will increase. By increasing those solutes, we will start to harm the embryos, so we inhibit this evaporation in two ways: with the culture media in the dishes, we put a thin layer of mineral oil over the top, and this will reduce any evaporation that can happen.
Other incubators will also have a small tray of sterile water at the bottom, which we keep topped up at all times. This sterile water will evaporate in the heat of the incubator, and it creates a humid environment, which then reduces subsequent evaporation from the culture media in the dishes.
We always try to maintain a neutral pH for the embryos to grow, we use this with two types of buffer systems in the culture media.
The buffer systems prevent hydrogen ions from either being over absorbed or overproduced, and it’s the hydrogen ions that control the pH of the culture media. We can have bicarbonate buffer systems. These tend to be used for the longer-term embryo culture, and they’re used in most incubators.
For anything that has CO2 being pumped into it, we’ll use a bicarbonate buffer system. This buffer system is controlled by CO2 concentration, which is pumped into the incubator. It varies between labs, and between different types of culture media as to which concentration is the best. The general concentration tends to be around 6%, but it can vary. This bicarbonate buffer system is used for the longer-term embryo culture. The living cells will metabolize, they will produce CO2, so anything that then produces CO2, we need to account for because the embryos can essentially increase the pH by accident, by themselves if we don’t account for it. That’s why we use the longer-term bicarbonate buffer system in the longer-term culture.
The second buffer system is HEPES. This buffer system is used when we have to take eggs and embryos out of the incubators. They are exposed to atmospheric conditions. For things like egg collection, when we strip the eggs of the cumulus cells and for embryo biopsy, we will use this buffer system to maintain the optimal pH at all times, even though they’re not in their optimal incubation conditions.
We like to maintain egg sperm and embryos at a physiological temperature of 37C. We do this by keeping the incubators at 37 degrees. Any time we have to take the embryos and eggs out of the incubators, they are always placed onto a heated stage.
We must take a good number of steps to reduce any contaminants, any infections that might occur. The ideal embryo culture environment is also the ideal bacterial culture environment, and if there was a bacterial infection, it can be devastating to embryo culture. One of the things that we use is antibiotics in the culture media. It’s supplied by the manufacturer. All the culture media mostly will have antibiotics in it, it’s usually Gentamycin, but this can vary between different brands.
We also make sure that all consumables that we use, so any dishes, pipettes, needles will always come in sterile packaging. It’s always single-use, and we will only ever open that packaging before using it. It won’t be sat on the side, collecting any kind of particles that might land on it. Another potential source of contamination is embryologists. As much as we wouldn’t like to ignite it, we go in and out of the lab, we have things to do outside in the other clinic, in the rest of the clinic, and so we need to make sure that we don’t bring any of that contamination into the lab with us so we will wear scrubs for lab work, we have hats or our hair it doesn’t get in the way. Specific shoes are only worn in the lab, and we’re very strict with hand hygiene, so always washing our hands as we go into the lab, we have a good airflow system in the lab, which helps to keep the air inside sterile, so no particles will go in no airborne toxins etc.
The air is filtered and pumped into the lab at a higher pressure than the air outside the lab, so this has the effect that only filtered air will go into the lab, and it will then push out into the other rooms surrounding the lab. We’ve got one-way vents on the sides of the labs on the walls that will allow the air to flow out, but should the air pressure in the surrounding rooms become higher than that within the lab, they will close to prevent any air to flow back in.
The air is also constantly monitored for volatile organic compounds or VOCs, and these are airborne toxins and chemicals that can have a detrimental effect on embryo development. That’s why during the embryo transfer, they ask not to wear perfume that contains alcohol, and this is an example of a VOC, and it is toxic to embryos, it’s better not to wear perfumes or strongly scented cosmetics.
When we are taking eggs and embryos out of the incubator, it helps to keep purely filtered air on the cells, and any contaminated air won’t make it into contact with the cells.
Seminal fluid is not sterile, so we have to keep it away from the eggs and embryos until the sperm has been processed and ready. Even though it’s not sterile, that’s not a bad thing, it just can contain normal bacteria that are present on somebody’s skin. Sometimes more harmful bacteria could be present, therefore we always need to make sure that we essentially wash the sperm cells. We remove them from the seminal fluid, and then once they’re prepared and clean, they can then be brought into the main lab just before insemination. The eggs and embryos are never taken into the sperm lab, there’s no reason for them to ever go in there, and it’s just too high a risk of contamination.
There’s no light in the uterus, but most importantly, we need to exclude UV light from the embryology lab. The reason for this is that DNA is denatured by UV light. Eggs, embryos and sperm don’t have a protection mechanism against UV light as humans do, so if they’re ever exposed to it, there’s a high chance that their DNA will be denatured, and then they won’t develop. We have UV filtered light coming from the ceilings, the microscopes, and we tend not to have windows in an embryology lab.
Any labs that do have windows in them will have UV filtered glass on them. If they don’t have UV filters in their ceiling lights, they’ll have very dim lighting, so it’s very important, and we always try and control it.
The large incubator is used to set up dishes and tubes of media for treatment for the next day. We want to equilibrate any media that is going to be coming into contact with eggs and embryos and sperm. There are six doors on the internal glass door, so if we have to go into the incubator, and it will hopefully minimize the change in temperature and the atmosphere of the incubator. It’s not possible to eliminate it when we have to take things out, but we minimize it as much as possible. There is an incubator, which has 10 individual compartments. This tends to be used at the beginning or the end of treatment. Any patient’s eggs after egg collection and before insemination will be kept in their compartment. An embryo just before the transfer will be kept in one of these individual compartments. It also allows keeping the eggs and embryos separate from other patients samples. It’s got quite a small volume within, and the benefit of that is that with such a small volume of gas in there, it makes it an awful lot quicker for the appropriate gas concentrations and the appropriate temperature to be resumed.
It is something that enables an embryologist to culture embryos from day 0 up to day 6 without ever having to take them out. The benefit of this is that all of the culture conditions, such as the temperature, the gas concentrations, are all maintained throughout an embryo’s culture. In the uterus, the embryo is not going to be exposed to colder conditions, it’s not going to be exposed to high concentrations of oxygen, and we want to achieve that as well. There are 16 individual wells, and that’s what each embryo will sit in, so when we go to put a dish out, at no point is any other dish exposed to the external environment. It takes pictures every 10 or 20 minutes, and that means we can see everything that an embryo does without ever removing it from its optimal environment, and we can see so much more that we could never see before when we were having to take them out at certain time points throughout the day. We can see it for its entire development and so as well as maintaining the optimal environment to give it its best growth. We can also see everything that it’s doing, and it gives us the ability to select the best embryo from what we’ve seen.
In the lab, we have two systems:
This is where you have a heated stage that reads electronic tags that are put onto each dish. Each electronic tag on each dish is assigned to that exact patient. Any dish that gets assigned while that dish is on the stage has to go to that same patient. If there was ever an accidental mismatch, so two dishes were put on that were assigned to two different patients a big alarm goes off, a red light flashes and the embryologist is notified that something is not right immediately.
These barcode readers are positioned by microscopes. The dishes are labelled with the barcode and the patient details. The barcode readers will detect the barcode, they also take a picture of each dish, and anytime that another barcode is put next to this dish, it must be for the same patient. Otherwise, the embryologist is notified immediately again.
This is the room where all frozen samples are kept. It is separate from the main lab, and it must be separate for the embryologist security. Liquid nitrogen is very dangerous, and it is a bit of a suffocation hazard if something should go wrong, that is why this room is kept entirely separate, and it has an incredibly good ventilation system to make sure nobody will be hurt. These tanks are monitored with remote alarm systems as well, so if anything were to go wrong, if any increase in temperature was detected, the embryologist would be notified immediately.
All the samples are stored at -196 degrees. Once the samples are stored, they can be kept for very long periods and depending on the method of freezing, they can be kept for over 50 years in some instances. It’s an incredibly different environment compared to what we grow the embryos in, but it’s a very good environment for storing them.- Questions and Answers
Our lab doesn’t do in vitro egg maturation, I don’t know if it’s widely done in the UK, and that might just be because of the HFEA and their restrictions. With MI eggs that are frozen, it may survive the thawing, there’s no reason to think it wouldn’t. Hopefully, it can mature, it will depend on when it was frozen and how long it was left to see if it would mature before the freezing.
What I would probably advise is if it matures the day after it’s thawed, for example, so it does mature, it does fertilize, and if it creates a blastocyst, I would probably advise having a genetic test on it, just to make sure that there’s no aneuploidy there.
Some research shows that eggs that mature quite late might have quite a high incidence of aneuploidy, which means the chromosomes aren’t balanced, which could lead to other problems. If it gets to blastocyst, you might want to consider doing some extra tests on it. There’s no reason for me to think that it wouldn’t survive the thawing, everything else is quite dependent on the actual quality of the egg.
Sometimes you can, and sometimes you wouldn’t. It will vary between labs, and it will vary based on which brand media you’re using. You can get sequential culture media which is the kind where you will have culture media that’s optimal for embryos to develop from day zero up to day three, and then you would change it to a different type of culture media from day three to day five.
You can also get single-step culture media which is designed to support embryo growth from day zero up to day five, and this is what we currently use in the lab. As I’ve mentioned with the time-lapse incubation that we use, the real benefit of it is that we never have to remove the dishes from the incubator until we get to the final day just before transfer.
If we were to be taking the dishes out and refreshing the media, that could expose the embryos to the conditions we’re trying to avoid, and there’s always a small amount of risk whenever you have to move a dish. It’s not something that we do in our lab, we use the single-step media, and we get good results from it, but every lab will vary based on what they like to use best and what they get the best results from.
I have not advised patients not to wear make-up during transfer. I know you don’t wear it during egg collection, particularly if you have it in sedation because there are signs that the anaesthetist will look out for on your face, which make-up can hide.
I don’t know if make-up during embryo transfers is a particular problem unless there is alcohol in it, high alcohol content should be avoided. If you check your make-up, if you check the ingredients list and if alcohol either isn’t in there or it’s quite far down the ingredients list, it’s probably safe to use. If you’re worried and don’t want to take the risk, then just don’t do it. It is up to you, but I haven’t advised people not to wear makeup during a transfer.
I think skin cream is fine, as long as it’s not heavily scented and doesn’t have a high content of alcohol in it, then it should be fine. I use skin cream on my face daily, not scented obviously, but I think I would dry up if I didn’t, so I use it.
Day-6 blastocysts are fine, I wouldn’t worry about a day-6 blastocyst, we do see good results from transferring day-6 blastocysts. Concerning day-7 blastocyst, we don’t culture to day-7, it’s not something that’s routinely done, I think in the UK. It has been done, but again, I think I would probably recommend performing a genetic test if you have that available to you for any day-7 blastocyst.
It’s not something that we tend to sort of wait to see if it happens, we tend to have day-6 as the cut-off, but again, I wouldn’t write them off if you’ve got good quality day-7 blastocyst and it’s frozen, but I would probably say take extra precautions before having them transferred.
I would say it’s reliable. With everything that we do, it’s not 100% perfect, but I would say that if you have a good reason for wanting your embryos to be genetically tested, for things like recurrent miscarriages or if the female partner is maybe a bit of increased age, then yes, it can be worthwhile. When we’re selecting embryos in the lab, we will watch how they develop, we watch the quality of them on day-5 or day-6. We can never tell what’s going on inside the cells and if you have increased risk factors for aneuploidy or something that could change the chromosomal makeup of the embryos.
I would say it’s worthwhile to at least consider it and have an appointment with a genetic counsellor so that you know what you’re getting into, and then if you think that it’s something that would work for you, I would say it’s a good option to at least give you peace of mind that any embryo that you’re having transferred back is chromosomally normal and it’s just worthwhile finding out if you could be in one of those risk categories.
Egg testing is possible, you can take a small biopsy of what’s called the polar body from the egg and it’s not something that I think is particularly reliable, I’m not sure if we still do it in the UK. The reason is that you can get the quality of the egg, so maybe it could help in terms of ruling out a particular embryo, but you can’t guarantee that an embryo that has developed from a perfectly healthy egg is still perfectly healthy. It’s something that is not as reliable as testing the embryos on day-5 or day-6.
They are developing ways that we can get to know the quality without doing a biopsy of an embryo, so they’re hoping to develop ways of testing the culture media in which the embryo has grown. If you can test that media, you might be able to find out if there are chromosomal abnormalities in that embryo. It’s not perfect yet though, so it might be a way off coming, there is something on the horizon at least, and then it’s just things like if you have access to time-lapse incubation that can help because if you can see the divisions that the embryo has made, it can give you a good idea of everything that is going on.
Add-ons have had such bad press recently, some of them probably rightfully so, and some of them they’re just getting a bad time because the gold standard of scientific trials was randomized, controlled tests just can’t be performed on it. Things like assisted hatching can be very useful. If you’re in the risk category if you’ve had repeated failed implantation, for example, if you had a few transfers of frozen embryos and again if you’re in an advanced stage category, sometimes it can be useful to have something like assisted hatching.
Assisted hatching, it’s such a simple process, and when it’s done properly, it doesn’t damage the embryos, and yes, at least we know then that the embryos will be able to hatch out of their shell. It’s difficult for me to say if they work because all the scientists, all the embryologists in the country can’t come up with scientific proof that it does. It’s something that we do in the lab, we do believe that there is good reasoning behind it. If somebody has had the repeated cycles, why not try something different that won’t harm the embryos, and it might just increase your chances, so it’s something that I think is worthwhile.
We use PICSI more routinely than we use IMSI. PICSI is basically where you do ICSI, and you use a dish that allows the sperm or only mature sperm to bind to hyaluronan, which is on the dish, so this is something found in the shell of the egg. The theory behind this is that any mature sperm which would have the physical capability of fertilizing an egg will be bound to the bottom of the dish, and we then go along and select the best-looking one. Anything that’s not bound to the dish, we know it’s not mature, so it gives us an extra level of knowing which sperm to pick while we’re doing an ICSI.
If you have had a sperm test, which has suggested that you have a higher concentration of immature sperms, the sperm that doesn’t bind to hyaluronan, then yes, they might suggest PICSI. If you’ve got the higher concentration, there is a higher chance of an embryologist selecting an immature sperm by accident because otherwise, without it, we can’t necessarily tell.
IMSI is doing ICSI but with a higher magnification. If there are things like vacuoles in the sperm head or if the sperm tends to misshapen, then we might use IMSI. To make sure that we are looking at each sperm cell that we’re selecting. It depends on the semen analysis that you might have had if it’s recommended for use.
I suppose you’d want to know things mostly about success rates and you can go into all the details, you could ask for all of their service reports, all of their qualifications, but at the end of the day, that doesn’t mean that they’re going to be doing the best work for you. I think you can look at the individual success rates of a clinic, that’s going to give you a much better picture. At the end of the day, it’s not just the embryologists that will contribute to your treatment. It’s so important to take all into account, the nurses, the doctors, the stimulation that they give you, the procedures that they do,
I probably wouldn’t advise you to only look at the embryologists when you’re selecting a clinic. Look at the success rates as a whole, and if you can look at the treatments that they offer in terms of IMSI or PICSI and genetic testing, anything that you think you might want to look into. Then at least you have the option if you need to without having to change clinics.
It will vary between clinics. Generally speaking, ICSI and IVF have comparable success rates. It will vary between clinics, and I can’t comment on everyone’s success rates, but certainly, the clinics I’ve worked at, ICSI and IVF, do tend to be very comparable.
I have no idea, and as incompetent as that probably makes me sound, the reality is we know that our culture media works, we validate it for the processes that we do, but in terms of the ingredients, that’s not something that the manufacturers published to us. We know that they’ve got antibiotics and glucose, pyruvate, and much more than that, but they don’t tend to share with us, so it’s very difficult for me to answer that one, I’m afraid.
In your lab, we have good rates, so embryo thawing, I think we’re at about 90 to 95% survival rate, which is good. The egg survival rate is always slightly lower just because of the nature of egg cells and how they survive, I’m not sure of the exact survival rates of eggs after thawing, it’s usually around 60 to 70%.
It’s a nice, comfy temperature, about 23 degrees generally. We don’t keep the lab at 37 degrees because we’d all pass out, but we keep a nice, warm temperature to minimize any effects on taking any dishes out.
Our pH tends to be around about 7.2, it will vary between labs, but it’s what we calibrate our media.