Embryology laboratory environment

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.

The IVF laboratory

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.

Nutrients

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.

Oxygen

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%.

Water content

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.

pH

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.

Temperature

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.

Germs

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.

Class II safety cabinets

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.

Andrology lab

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.

UV light

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.

Incubators

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.

Time-lapse incubator

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.

Security

In the lab, we have two systems:

RI witness

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.

Matcher barcode reader

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.

The cryogenic storage room (Cryo store)

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.