In this session, Björn Heindryckx, PhD, Principle Investigator, Ghent-Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital in Belgium, has been talking about the role of sperm versus oocytes in fertilization. Professor Heindryckx also explained how to diagnose and treat failed fertilization after ICSI.
Professor Heindryckx did engineering studies and started working in the assisted reproductive technology field in 1999. He started as an embryologist but switched over to research quite fast. He defended his PhD in 2006, which was about artificial gametes and assisted oocyte activation. In 2014, Professor Heindryckx became a professor, and nowadays is also a coordinator of the research group called ‘Ghent Fertility and Stem Cell Team (G-FaST)’. The main focus of this group is the patients that are difficult to treat and where we still don’t have available methods to treat them.
There are 2 groups of research: stem cell research for sterile patients. That’s for patients who don’t have any oocytes or sperm, which is a very challenging group, and we still can’t overcome this yet. The second group is more about clinical translational research, and one of the topics involved is failed fertilization after ICSI: diagnosis & treatment via assisted oocyte activation. Other topics include mitochondrial diseases, failed implantation, pre-implantation diagnosis using Next Generation Sequencing, and fertility preservation. For two years, we’re also researching gene editing, we’re trying to correct mutations related to infertility.
We have to realize that when the sperm gets into the egg, some things are happening with the sperm that are normally happening when no assisted reproductive technology is used or when IVF is performed. For example, the capacitation of the sperm happens during normal fertilization in vivo or during IVF. When you do ICSI, you bypass these processes a bit. If you do IVF, the sperm has to go through the cumulus cells around the oocytes, the sperm has to bind to the zona pellucida, so the eggshell around the oocyte, the sperm, and the oocyte membranes will fuse. All these processes are happening during IVF and natural fertilization, but they are not happening during ICSI, and that’s something important to keep in mind.
For the rest of the processes of ICSI, you just inject one sperm directly into the oocyte. These processes of oocyte activation, sperm processing and fertilization, so pronuclei formation, also happen in ICSI. It’s significant to keep in mind that when you do ICSI, you bypass these three first processes.
Fertilization goes together with the start of oocyte activation. Before you have successful fertilization, you need to have oocyte activation. When the sperm enters the oocytes through ICSI or IVF and gets into the oocyte, the sperm’s head content gets released into the oocyte cytoplasma.
One essential factor is called phospholipase C zeta (PLCζ), which is a protein, an enzyme that produces IP3. It will bind to the receptor, the IP3R receptor, which is the oocyte factor and this IP3R receptor contain calcium.
The protein (PLCζ) will provoke calcium oscillations (Ca2 +), and that’s something that has to happen. If these calcium oscillations do not happen, so calcium transients, it’s several calcium oscillations during a certain time, you will not have successful fertilization. That’s the first sign of fertilization, these calcium oscillations need to happen and are triggered by the sperm protein called (PLCζ), which induces the calcium release from the receptor in the oocyte. Calcium oscillations (Ca2 +) are crucial for fertilization, but also, some data shows that a correct calcium pattern also influences embryo development.
In mice and other animal models, they have shown in the literature that this correct calcium pattern is not only necessary for successful fertilization, but it might also affect embryonic development and even a chance of pregnancy. In humans, we still don’t know if this is the case or not.
Along with these calcium oscillations (Ca2 +), lots of other processes are happening in the oocytes. A lot of proteins are present in the oocyte, and they all have to do their own thing. First, you need the calcium oscillations (Ca2 +), but downstream processes need to happen before you have successful fertilization. It can go wrong if you don’t have any calcium rises, but if some proteins are not functional within the oocytes, you will not have successful fertilization as well.
As Professor Heindryckx explained, PLCζ is the sperm factor that triggers the calcium oscillations in the oocytes. It took us a long time before we knew which factor is causing these calcium oscillations. In 2002, in the UK, it was identified that the (PLCζ) is the sperm factor and when we first found a mutation in the (PLCζ) for the first time, we were sure that this (PLCζ) is crucial for fertilization.
In 2009, in a patient that had failed fertilization after ICSI, we identified a mutation in the PLCζ gene. From then on, we were quite sure that the sperm factor is responsible for these calcium oscillations because there was a mutation and the protein was not there. There can be mutations in the gene that rise to abnormal PLCζ protein so that PLCζ protein is absent or has an abnormal localization in the sperm, and so on. If a mutation is present in the PLCζ, the oocyte activation will fail, which means fertilization will fail. Some research is going on to see if this is the only sperm factor responsible for fertilization, but we still don’t know yet.
Together with the UK group of John Parrington, one of the pioneers in the fertilization process, they have been working on PLCζ knockout mouse models. They knocked out the PLCζ gene, and indeed we see that if the PLCζ is absent, you don’t see any calcium oscillations, and you don’t have any fertilization. The research is still incomplete. However, when we do IVF or natural fertilization with PLCζ, we do see fertilization. That’s why it’s important to keep in mind that when you do ICSI, we don’t see any oscillations, but when we do IVF or in vivo, we do see some fertilization. It’s essential to realize that IVF and ICSI are different things.
When it comes to the role of the oocyte, we know that there are a lot of proteins present that need to work downstream, so it can go wrong in one of these proteins. There is still very little evidence or research done on this. We know for sure this IP3R receptor is responsible for the calcium oscillations (Ca2 +), so if there is something wrong with this receptor, there will be no calcium rises caused by the oocyte. If you have an immature oocyte and you inject sperm into it, the calcium increase is not going to be high. It’s only during the final maturation stage when you inject the sperm into a very mature oocyte, we will see a normal calcium pattern. It’s important to have a very mature oocyte for successful fertilization, for successful oocyte activation.
How do we cope with failed fertilization? We normally check fertilization the next day after we do ICSI, and after 16 hours or 18 hours after ICSI, we should see two nuclei, so one male pronucleus and one female and extrusion of the second polar body. When it’s a failed fertilization, we only see a mature oocyte without any pronuclei or any second polar body, so that’s a failed fertilized oocyte. Luckily, it doesn’t happen often, so ICSI is very efficient, it normally gives an average fertilization rate of 75%, which means of the 10 eggs you inject, you will have 7 to 8 normally fertilized eggs. In about 5% of all ICSI cycles, failed fertilization occurs.
What is the main reason for failed fertilization after ICSI? It can happen if you don’t have a sufficient number of normal or mature oocytes. However, the main reason is the failure of oocyte activation if you have a decent number of oocytes and the partner has motile sperm.
Why do we need to know if a failed fertilization happened because of the sperm or oocytes? If it’s sperm, we will have better success when we do artificial oocyte activation (AOA). On the other hand, if the assisted oocyte activation is not working, we have to send the couple to oocyte donation or sperm donation. For that reason, it’s crucial to know if it’s sperm or oocyte. The third reason is that there could be a mutation present in the oocyte or the sperm. It’s better to counsel the patients and inform them that there is a mutation. Even if we can overcome it if they have a boy or a girl in the future, they might also have this problem of failed fertilization. That’s why it’s important to do a diagnostic test to distinguish sperm from oocytes. In our lab, we do these Mouse Oocyte Activation Test (MOAT) for all the patients that have failed fertilization. We also optimized this test with some other techniques, and it’s called Mouse Oocyte Calcium Analysis (MOCA), but we only do it for some patients. We also perform a lot of genetic screening, so we check if there are mutations present in the PLCζ, that’s one of our research topics.
The first mutation we found in 2009, in a patient that had failed fertilization after ICSI in 2009. When we inject this Mutant sperm, most of the time, we don’t see any oscillations or only one calcium rise, which will not be enough to activate or fertilize the oocytes. Sometimes, there are very small calcium peaks, and these will not be enough to activate human oocytes compared to ‘Control sperm’ with normal fertilization capacity, you see a lot of peaks with a high amplitude.
When we are doing the genetic screening, we find more and more mutations in the PLCζ. We see more and more mutations in the PLCζ in patients that experience failed fertilization.
How does the Mouse Oocyte Activation Test (MOAT) test work? We collect sperm from the patient, then we inject it into mouse oocytes. Ideally, we should do it in human oocytes, but we don’t have thousands of human oocytes to research on. That’s why we use mouse oocytes, and human sperm can activate mouse oocytes, and then we check how many of the mouse oocytes were activated. We check two cell formations, so we check the next day, and we see the two cell formations, that’s the activation rate. Then we classify our patients into three groups, so in MOAT group 1, it means that less than 20% of the mouse oocytes were activated, so that’s a sperm problem because most of the sperm cannot activate even a mouse oocyte.
MOAT, group 2 until recently, we were not sure if this was sperm or oocyte related because some sperm can activate mouse oocytes, but some sperm cannot. After doing calcium analysis (MOCA), we now know that this MOAT group 2 is also related to a sperm problem. MOAT group 3 means all the sperm is capable of activating a mouse oocyte. In this group, it’s an oocyte problem because the sperm can activate the mouse oocytes.
We also have this specific group of patients that have globozoospermia. Normally, a sperm head has an oval head with the nucleus and an acrosome, and in globozoospermia, they are missing the acrosome. PLCζ is located in the acrosome, so they are missing the PLCζ. Patients with globozoospermia can never fertilize an egg naturally, or through ICSI, so we have to overcome this.
On one of the slides, Professor Heindryckx showed an overview of the MOAT tests that they have done between 2001- 2016. 40% of the patients had the sperm problem. 10% in the MOAT group 1 and around 30% in MOAT group 2. In 60% of the cases, we identified an oocyte related problem.
We can artificially activate an oocyte by inducing a single calcium Ca2+ raise or several calcium raises in the oocyte. Different products do this, people have used a lot of products to activate mouse or human oocytes. We use ionomycin because it’s a very powerful product to induce a big calcium peak in the oocytes. In the UK and a lot of European countries, they use another product called calcimycin. However, for us, this is not working efficiently. There are different products with different efficiency, for example, some people use Strontium chloride (SrCl2) to activate oocytes to overcome fertilization failure but again, it doesn’t work for us. We have published some data on this, and if we expose, for example, 10 oocytes to this Strontium, we don’t see any fertilization happening. We don’t see any calcium oscillations when we expose the oocytes to Strontium.
In our hospital, we inject an amount of calcium together with sperm, and we expose the oocytes to ionomycin two times, and we see 3 calcium increases. The first one is due to the calcium injection together with the sperm, and then a second and third peak is caused by exposure to ionomycin.
How is the technique of assisted oocyte activation performed? It’s ICSI but with something extra. We first pick up sperm with a needle, and normally, we inject it into an oocyte. For assisted oocyte activation, we first take an amount of calcium inside the needle, so now there is calcium in the needle, and then we do normal ICSI, we go to the oocytes, and then we inject the calcium solution together with the sperm inside the oocyte to get this first calcium increase to enable successful fertilization.
We did the first study of assisted oocyte activation (AOA) in 2008. We treated 30 patients, and with standard ICSI, the fertilization rate was 14%, that’s much lower than the average 70%, no pregnancy in 50 cycles occurred. When we applied AOA, we restored the fertilization rate up to 75%, and we also got a very satisfactory pregnancy rate of 33%.
Nowadays, we have updated our results, so instead of 30 patients, we now have described 122 patients. 19 belonged to the MOAT group 1, so a sperm factor. 56 belonged to the MOAT group 2, also a sperm factor. 47 belonged to the MOAT group 3, oocyte related factor. If we look at the efficiency, we see that the fertilization is still very high. In MOAT group 1, we can increase the fertilization rate from 10% with normal ICSI to 70%, which is very normal after assisted oocyte activation. When we did a standard ICSI, it was 0, and when we did assisted oocyte activation, it went up very high, close to 50%, which is a very high success rate, at least in MOAT group 1. In MOAT group 2, we increased the fertilization rate up to 63% and the pregnancy rate from 7% to 35%, which are normal success rates. In MOAT group 3 where we suspect an oocyte factor, we do see that the fertilization rate is not so high after AOA, so it’s 57% instead of 18% with normal ICSI. The pregnancy rate is significantly higher, up to 29%, so even if there is an oocyte factor, it’s efficient but a little bit less efficient than when there is a sperm problem.
A lot of UK clinics and also other European IVF labs use a commercial activating medium, which is called CultActive, and we have done a study in 2015 on this, but we see that CultActive in human oocytes doesn’t increase calcium. When we compare it to ionomycin, which we are using, we see a calcium increase. Therefore, we are not using this commercial CultActive medium.
We performed ICSI to avoid dispermic fertilization with two sperm, with ICSI, you only inject one sperm. However, no fertilization happened after ICSI. We did some testing, the MOAT result was normal, but we saw in calcium pattern analysis (MOCA) that the calcium patterns were abnormal, and we found a mutation in PLCζ. Then after assisted oocyte activation (AOA), we could restore the fertilization, and she got 4 children after AOA.
There were several other couples with this problem, it seems that two sperm contain enough of PLCζ to activate the egg, but of course, resulting in an abnormal chromosomal embryo and an abnormal pregnancy which will lead to miscarriage. One sperm does not contain enough PLCζ to induce a pregnancy.
We performed all the testing, and everything proved to be normal. We still suspected a low oocyte factor, so we did AOA, but it resulted in failed fertilization. We discussed this, and as they had some fertilization after IVF, we decided to do half IVF because she had some fertilization in the past and half ICSI with AOA in the next cycle.
In the end, IVF worked. From 4 oocytes, 4 were fertilized, and she had good embryo development, but unfortunately, it didn’t result in pregnancy. When we did ICSI on the other half of the oocytes, it again resulted in failed fertilization. That’s why we have to realize there is a difference between IVF and ICSI, and sometimes in some couples, we don’t have to use any fancy technique such as AOA, we should rather think of going one step back, and for example, do IVF. It will not be helpful for all the couples, but it’s an interesting case that might help others.
We have high success rates with assisted oocyte activation (AOA), but not in all couples. 90% of the couples we treat will be successful, they will have fertilization and a chance of pregnancy. In less than 10% of patients, AOA does not lead to fertilization. It means that something downstream goes wrong, we know that a lot of proteins in the oocytes have to be functional, so it can go wrong in one of these proteins that we still don’t know. There are sometimes mutations present in these genes that should make these proteins, and if something goes wrong there, we cannot help the couple, and we have to go to oocyte donation at the moment.
We already did some experiments with the nuclear transfer. We are quite convinced that if there is a problem with the proteins in the cytoplasm, the nuclear transfer could overcome this problem. You take out the nucleus of a diseased oocyte or a low-quality oocyte, and you bring it into an egg from a donor where you first remove the nucleus, the DNA from the egg donor, so you have reconstructed a new oocyte containing the nucleus, so the genetic information from the patient with the good cytoplasma, with all good proteins. We are quite sure that in these cases where there is an oocyte problem, this technique might be useful in the future. We are doing a lot of research on this topic, and we already tried it on a couple who had failed fertilization after ICSI with AOA.
We did the spindle transfer, we removed the nucleus and put it in another patient’s oocyte and finally, we achieved fertilization for the first time in this couple. It’s still in a research context, we can’t transfer these embryos because we are not allowed to transfer them back. However, we will start up a new study to get more evidence that this nuclear transfer technique might help some oocyte problem cases. By applying the technique of assisted oocyte activation, we know that some patients are having, for example, a mutation in PLCζ, we can overcome it efficiently with assisted oocyte activation. However, we know that, for example, if boys will be born, they will inherit this mutation, and they will also have problems reproducing in the future.
That’s why we investigate if we can use CRISPR, which is a tool to modify the DNA. We are researching to see if we can correct the mutation and avoid it for those boys to deal with infertility, this is of course, still in a research context.
We think it’s very important to distinguish between sperm and oocytes for different reasons. Genetic screening should be done, and we do it to warn patients that if they have children in the future with AOA, they might inherit the infertility condition. In general, we can say that AOA, so using calcium rises is very efficient and it can overcome failed fertilization after ICSI in most patients, more than 90 % of the patients if the right protocol is used as several protocols are being used in different clinics.
I think we have a powerful product – ionomycin to induce efficient fertilization, and of course, future research is necessary and ongoing in our department. There are other members of our research team involved, two clinical doctors, two gynaecologists Professor Dominic Stoop and Doctor Vanden Meerschaut, excellent experts on the topic of failed fertilization after ICSI.
It depends on the sperm quality, so we have some minimum parameters, the motility should be at least so much percentage, the concentration should be at least so many millions. We have criteria that almost every IVF clinic is Rusing, and if a sperm sample does not meet the minimum criteria, we will do ICSI in the first cycle. Nowadays, ICSI is a little bit overused, sperm quality has decreased during the last decades, that’s for sure. The reason for this is that most people say it’s environment-related or the lifestyle of the patients. We know it’s a fact that sperm quality has decreased immensely during the last decades, so we are using a lot of ICSI. For example, in Belgium, 70 to 80% of all the cycles use ICSI, and only 20 to a maximum of 30% use IVF. It’s because the sperm quality is not good enough to be sure that IVF will work, that’s why we prefer to do ICSI in the first cycle to prevent fertilization failure after IVF because of bad sperm quality. We have our criteria that everyone is using in all the IVF labs worldwide, and then we decide if it’s IVF or ICSI depends on the sperm quality. I’m quite sure that in a lot of ICSI cases, where the sperm is like on the edge of not being good enough for IVF and if we would do IVF with this couple that it would also work, but we try to avoid failed fertilization after IVF, that’s why we go ahead with ICSI a bit quicker.
If no fertilization happens after ICSI, it’s mostly due to the oocyte activation problem. We do artificially induced calcium rises, and in the majority of the patients, it works. It’s only in less than 10% of the oocytes that we still don’t have fertilization yet. There is no other technique at the moment next to ICSI because that’s the last resort technique that is now existing, but if it’s an oocyte problem, we are quite sure that we could maybe rescue it with this nuclear transfer technology, but it’s too premature to use it in the clinic. We still have to do a lot more experiments and research to know if this nuclear transfer technique would be the next step after ICSI to overcome some fertilization problems. It will only help if it’s an oocyte, probably because we are transferring the nucleus from bad quality oocytes to donor oocytes, and then we have to fertilize it with sperm, so it would only be beneficial for patients with oocytes problem. In most cases, when we identify a sperm problem, this assisted activation always works, that’s a fact, and it’s working very efficiently.
In our clinic, we shifted to day-5 transfer, and our success rates have improved. We are doing day-5 transfer for 5-6 years already. I would go to day-5 because when the embryos don’t make it to day-5, it’s very likely that they won’t make it. It’s a good natural selection when you culture your embryos to day-5. We know in human embryology that the first 3 days of development are very much directed from the oocyte. It has all the proteins present in the oocyte that ensure embryo development until day-3. It’s only from day-3 onwards that the embryo awakes those new proteins, which are produced by the embryo itself. It’s also good if you culture until day-5, that way you know that the last two days, so from day-3 to day-5 that they are working. If they are not working from day-3 to day-5, if you have failed blastocyst development, failed embryo development, we suspect that there is something wrong with the paternal DNA, so with the father.
PICSI, it’s a kind of selection technology to select sperm with normal DNA, without DNA fragmentation, but I’m not a believer in the technique. The studies that have been published are not convincing to me and the experts in the field. If it would be promising, everyone would do PICSI. I don’t see a lot of publications anymore on PICSI outcomes, so I think it was hype for a certain time. People thought that if you select sperm, which contains less DNA fragmentation that you have a higher success rate resulting in pregnancy, but all the studies that have been published are not reassuring, not convincing. We also did some studies, on PICSI and we didn’t see any benefit of using this technology, so I would not recommend it. The literature is now quite sure that it doesn’t give any benefits for any indication of any sperm abnormality.
We don’t use it in our clinic, it’s a technique to increase the maturation status of the oocytes. When we have this failed fertilization after ICSI, and we see that the mouse tests point to an oocyte factor, we wait longer to do the ICSI with assisted oocytes activation (AOA). We know that the maturation stage of the oocytes is very crucial. If you suspect an oocyte factor, the cytoplasm can be a little bit too immature, so we wait for a very long time to remove the cumulus cells around the oocytes. The oocyte is getting matured after collection even longer than what we normally, do in other normal patients. When we then denuded the oocytes, we also wait an extra time before we do this assisted activation, so it might not be a bad idea to start the upper study to increase the maturation stage of the oocytes, but we don’t do it in our clinic, and we have done a study, but it has not been done in the context of failed fertilization, it might be a good suggestion to dig into further.
Different calcium ionophores are being used as a technique to induce this calcium raises. In the UK, as far as I know, they are only allowed to use this commercial one, it’s called Cult-Active, well the product is called calcimycin. Because of the regulations in the UK, they can only use this commercial one, which is EU approved, but in our hands, it’s not working. When we exposed oocytes to this calcium ionophore that is used in the UK, we didn’t see any activation happening. We only saw as I showed you in the picture a very small calcium raise. This is not enough to activate an oocyte. I’m not in favor of this commercial calcium, and I know the company is shifting. They will start producing this commercial calcium ionophore that we are using shortly, the ionomycin. It is much more powerful and gives a very big calcium raise, and is very efficient in our hands. I heard it’s already 2 or 3 years that the company is doing this since we published our study and showed that this Cult-Active is not very efficient and not activating human oocytes. The company has developed a commercial version of the ionophore that we are using, however, it’s still not on the market, unfortunately. In Belgium, all the labs use ionomycin, and they all use the protocol to induce efficient calcium rises to overcome failed fertilization.
It gives some calcium raise, as we have seen in our study, but it doesn’t activate the oocyte. I think it has to do with the stability of the product, so maybe because some publications mention that they can rescue failed fertilization with this Cult-Active, but the efficiency rate is always much lower than the efficiency rate. If you use it quite freshly, you have to keep it in the refrigerator, this commercial one and it’s ready to use, so I think if you buy it, and you use it immediately, it still has some power to work efficiently. We used it after several weeks of being in the refrigerator. It doesn’t expire so fast, so you can use it whenever you want, and we didn’t see any activation, any fertilization with this product. It might work if you use it when it is very fresh, and that’s why some clinics get some activation and success but with less efficiency than the protocol that we are using. I heard a lot of complaints about this commercial Cult-Active and a lot of labs contacted us to ask why this Cult-Active is not working, but in our hands, it also was not working. We didn’t do a big study on that yet, for example, study when it expires or when the activity is losing its effects. I do think it has to do with the stability of the ready-to-use-product. The ionomycin that we are using, we make it fresh for each patient. We make it just before the treatments, we make the solution, and we apply it on the day for one patient, and then we throw it away, so we make it fresh for every patient that has to undergo AOA because the ionophores we know are very unstable so if you keep it for a long time, it will lose its activity, and it will lose its efficiency. I would not recommend using this commercial Cult-Active that is used in the UK.
We always use motile sperm to inject because it’s a sign that the sperm is alive, you should never select immotile sperm because if it’s immotile, it can already be dead for a long time, and then you have DNA degradation, which will affect your embryonic developmental potential. We have to base on the morphology, the head if there are no gross abnormalities that we see but we cannot see, the DNA inside, so the DNA might be more damaged in one sperm than the other. There are no techniques to examine this or no selection procedures like this magnetic sorting that can help to increase the efficiency of selecting sperm. Not all the embryos that we produce make it to day-5, so it could happen that some sperm is not capable of supporting embryo development, and then it’s the wrong sperm, but we cannot avoid it, there are no techniques or microscopes at the moment to choose the perfect sperm, that’s not possible.
This is a little bit out of my expertise field, but I know a lot is going on with nutrition and antioxidants for sperm quality now, as well as, other stuff. For me, there is no concrete evidence that there is something magic to improve your sperm or egg quality because if there would be, then we would recommend it for every patient, so it’s also a commercial angle. There are some companies specialized in all these antioxidants and healthy foods for IVF people, so like I said, if there would be something magical again, we would be obligated to mention it to patients and give the advice of eating some kinds of food or some supplements in their food to increase their pregnancy chances. Some things are very detrimental for the egg or sperm quality like smoking, smoking will reduce 50% of your pregnancy chances, especially, in female but also male, too much alcohol will also decrease your sperm quality and your egg quality, so that’s the obvious things that we know that are reducing your success rates immensely but apart from smoking and drinking, I would not say there is concrete evidence that there is something magic that can improve your fertility apart from your lifestyle, a healthy BMI is also important.