The IVF journey consists of a number of steps. One of the most mysterious and fascinating of these occurs in the Embryology Lab. . In fact, the whole magic happens there and it’s the people who make the IVF laboratory what it is. It’s their knowledge which makes it all possible. During this webinar, Laura Van Os, an embryologist from IVF Spain, presents the Complete guide to the IVF Lab which includes techniques and procedures.
Sperm selection techniques
At the beginning of the presentation Laura reminds us of a crucial fact: eggs are always limited in number so basically it is only the valuable mature eggs which will be fertilised whereas every sperm sample has millions of sperm cells and in the end, only a few will be needed. For this reason, several
sperm selection techniques have been developed in the latest years for different reasons: to find the best way to select the best sperm cells in the sample and to use this best sperm cells to fertilise the eggs. Usual and mandatory techniques used in the laboratory while processing a sperm sample are density gradients and the swim-up technique.
Laura pays attention to the fact that there are some patients who have some more specific problems, like a higher rate of apoptotic cells among the sample. That’s why they need specific techniques. One technique that can be used to overcome this problem is MACS (magnetic-activated cell sorting). Apoptotic cells have started the process of dying and no-one wants to fertilise the eggs with these sperm cells. With MACS , embryologists can remove these sperm cells from a sample. Another technique that is useful to overcome some molecular problems is piCSI or Spermslow. These two techniques they are based on the fact that mature sperm cells will be able to bind to hyaluronic acid whereas the sperm cells which have not undergone this maturation process will be moving around freely. It will give embryologists a hint to distinguish these two kind of cells and it help them to choose the best sperm cell for fertilisation in the laboratory.
Another extra technique to select sperm cells which have morphologic problems is IMSI. Basically you look at the sperm cells with a really big magnification – the sperm cell is more than 6000 times amplified. The theory says that thanks IMSI it is possible to select sperm cells which have a best morphology.
On the other hand, there are alternative sperm sources to main source which is obviously ejaculation. These alternative sources are the testicles and the epididymis. Sperm cells are produced in the testicles in a process that lasts around two and a half months. They then later mature in the epididymus. Sperm cells can be extracted from there by aspiration with a needle or by a little surgery. And this is indicated in case of patients for example who have undergone vasectomies, which might have difficulties collecting the sperm sample, there are some genetic mutations (like the absence of vas deferent in case of cystic fibrosis). There are also some cases in which the quality of the sperm in the testicles seems to be better than the quality of the sperm cells in the ejaculation.
Fertilisation process and ICSI
Once the eggs have matured and the sperm cells have been selected, the next step is fertilisation. It can happen by one of two main techniques. It can happen through ICSI. ICSI stands for intracytoplasmic sperm injection and through this technique, the sperm cell is directly microinjected into the egg. The other technique is conventional insemination (IVF). In this technique there are some droplets of media with sperm cells. The egg is placed in these droplets and the sperm cells will compete to naturally fertilise the eggs. IVF, despite seeming more natural and less invasive, has more risks because no one is really sure if cell reaches the place that it should reach. Usually there’s also a higher rate of abnormal fertilisation so viable eggs may be missed out. These are always the embryologists who decide which of the two techniques should be used depending on the egg and sperm quality as well as the previous history of the patient.
Artificial oocyte activation
Artificial oocyte activation is starting to be used more frequently with patients who have had recurrent fertilisation failure meaning that they has less than 1/3 of their mature eggs fertilised. This is sometimes due to a failure in oocyte activation, both due to egg and sperm factors. To provoke this artificial activation embryologists are using calcium ionofore and have very good results. In this protocol you basically have to culture embryos in droplets of calcium ionofore rich media for 15 or 10 minutes after ICSI. We apply this media in all 100% of eggs in case of case of previous complete fertilisation failure and if it’s just a low fertilisation rate we consider applying it only in half of the eggs to see if this is really helping.
If the egg is correctly fertilised, the embryo will start dividing: on day 2 it will have four cells, on day 3 – around eight cells, then 16 cells. At that point the embryo will turn into morula stage. A morula will start to expand expand until we find the blastocyst. The blastocyst is a very defined structure of an embryo. It’s composed of two parts there is some mass in the middle of the embryo this is called the inner cell mass and it will become the future baby. It is surrounded by a layer of cells this is called the trophectoderm and it will become the future placenta. At this point the embryo usually has around 1 hundred – 2 hundred cells. The shell surrounding the embryo becomes thinner at the blastocyst stage.
Embryo culture till day 5
Laura admits that it is more and more common to leave embryos in culture until the day five of development. The point is that even if the embryo looks nice on day 3, it does not mean that it will become a blastocyst. Not all eggs are programmed to do so and it’s like a natural selection process . Embryologists tend to leave embryos to day 5 because if you transfer embryos from day 3 and you’re not becoming pregnant you might be wasting your time because maybe they are not even able to reach the blastocyst stage. Laura highlights that quantity is not the same as quality. Some patients have many eggs but very few of them turn into blastocysts of good quality, the others may have less eggs but most of their eggs will turn to good quality blastocysts.
Embryo monitoring – time-lapse
Laura says there is a very powerful tool to evaluate embryo development called time-lapse. In this process, the camera inside the incubators takes pictures of the embryos every five minutes. This allows embryologists to have a continuous recording of the embryos without disturbing their stable culture conditions. In addition, morphokinetic analysis of the embryo can be obtained and this information can be used to predict implantation and to select the best embryo for transfer.
Embryo selection via PGT / PGS
Another way to select which embryo to transfer is PGT (pre-implantation genetic testing). It is based on the fact that some embryos have genetic errors. In the laboratory, when the embryo which is the blastocyst stage, the embryologists remove a few cells, extract a genetic material from these cells, amplify it and we sequence it. In this way they are able to know if the embryo has the correct number of chromosomes. PGT is used in order to transfer only the genetically normal embryos and thus, increase the implantation rate per transfer. As the percentage of abnormal embryos is linked to maternal age, PGT is especially recommended for older patients.
Embryo transfer – increasing the implantation rate
When it is decided which embryo to transfer, there are also some techniques used to increase the implantation rate of these particular embryos. The embryos can only implant when they have broken the shell around them. This happens at the blastocyst stage around day 5 or day 6 of the embryo development. In the laboratory this process can be assisted. According to Laura, embryologists find little benefit in performing assisted hatching in fresh embryo transfers. However, the implantation potential increases when assisted hatching is performed before transferring frozen embryo. Assisted hatching is performed by the use of chemical or mechanical ways, like with a little laser that is very easy to control and very safe for the embryos.
After a blastocyst or an embryo is transferred it will come out of the shell, contact the endometrium (the wall of the uterus), adhere to it and finally invade the endometrium. This process implies complex molecular interactions between the embryo and the endometrium. The stage of endometrium when it is capable of performing these interactions is called the implantation window. In other words, it is the point at which the endometrium is receptive and ready to accept the embryo. That’s why some women perform biopsies to check when exactly their implantation window is.
Last but not least, Laura mentions one more technique which is related to embryo transfer: embryoglue. It is basically a media containing hyaluronic acid that makes the media little bit more viscous and thus, helps the embryo stick to the endometrium. So it does make some scientific sense to think that this media could slightly improve the adherence of the embryo to the endometrium.
