In this session, Vladimiro Silva, PharmD, Embryologist, Founder & IVF Lab Director at Ferticentro, Coimbra, Portugal, has talked about repeated implantation failures. Dr Silva explained the main causes, treatment solutions and outcomes, all based on real-life patients’ cases.
a complex and broad topic. What is recurrent implantation failure (RIF)? This is a difficult question because there’s no consensus in the scientific community about it. We refer to it as the failure to achieve a pregnancy after at least 3 embryo transfers with good quality embryos, or also after the transfer of 3 or 4 good quality blastocysts. Sometimes we mix things because some patients have tried with their own eggs and had 1 or 2 good embryos and many not-so-good embryos, and then when we try again with 1 or 2 good quality blastocysts, we’re already talking about recurrent implantation failure and starting investigations. It changes a bit from patient to patient. From a more scientific point of view, the most conceptual definition is having at least 3 failed embryo transfers with good-quality embryos.
When we look at statistics, assuming that a certain treatment has a 60% pregnancy rate, after transferring 1 embryo to 100 patients, we will get 60% of them pregnant, and 40% will not be pregnant. If we transfer a second embryo to those remaining 40 patients, we will have 24 patients that will become pregnant and 16 that will not get pregnant. If we transfer the third embryo to those 16, we will end up with 9 patients that are going to be pregnant, and 7 patients who are not going to be pregnant. This means we have a 93% accumulated pregnancy rate, but it was always 60%. Therefore, sometimes when talking about recurrent pregnancy rates, we are just having bad luck within this 60% of pregnancy rate. 60% is brilliant for every IVF clinic for every IVF treatment, but it still represents that 40% of the patients are not getting pregnant for random factors or factors that are difficult to control.
What causes recurrent implantation failure? The reason is usually unknown, there are so many factors involved that it’s sometimes very difficult to pinpoint just a single explanation. It could be a mix of factors, but essentially we can divide the problem into 2 groups of potential causes:
What to do to find a cause for a recurrent implantation failure? Normally, it is best to start with the embryo. It’s important to be as sure as possible about the embryo quality, and the most effective way to evaluate the quality or implantation potential of an embryo is by doing Pre-implantation Genetic Testing for Aneuploidy (PGT-A).
How is it done? The embryos are created in the lab, the eggs are fertilized with sperm, and the embryos created are cultured until day 5 or day 6 to the blastocyst stage. Then an embryologist does a hole in the embryo with the laser system and withdraws a group of about 5 cells. Those cells are analysed in a genetic’s lab and checked to see how many chromosomes are inside those cells, and it is possible to see whether those cells have the right number of chromosomes or an extra chromosome or a missing chromosome.
If an embryo has the correct number of chromosomes, it is called an euploid embryo, and if it has the wrong number of chromosomes, it’s called an aneuploid embryo. A graphic presented on the slide was taken from the Igenomix website, where they compared the implantation rates in the grey bars correspond to patients who have done PGT-A. This means that if you transfer an embryo that has been tested and is considered to be normal, the pregnancy rate will stay more or less the same across all the age groups. However, in the orange bars, the pregnancy results without PGT-A show a steep decline with age.
It’s important to remember that PGT-A doesn’t make the embryos better, it doesn’t increase the chances of embryo implantation, however, it helps us to identify what is the best embryo if the embryo has a good implantation potential or not, and it helps us to select the best embryos for transfer, so it reduces the time for a pregnancy, it keeps us from losing time in low prognosis embryo transfer it saves money for the patients ultimately, but at the same time if the embryos are not good, it will not transform them into good embryos.
This is a very useful tool to find an explanation for recurrent implantation failure cases. If there are negative results and the genetic test is performed, and we don’t find suitable embryos, the problem is likely with the embryo. Therefore, it doesn’t make sense to start looking for other factors because the explanation is probably on the embryo. When we are transferring embryos that have genetic abnormalities, the risk of having a miscarriage increases a lot, but you should take into consideration that even when we transfer embryos that have been tested and proven to be viable, we are not above 60%, so there are still 40% of cases for which we don’t have an explanation. Therefore, the reason for these embryos not to implant is essentially on the uterus side. It could also be other non-genetic issues with the embryo, but this 40% are essential issues with the uterus, the endometrium, or other implantation-related causes.
As was already mentioned, we should always start with the embryo. When we have good embryos, then we should move our investigation into the uterus. One of the first things we should evaluate is endometrial receptivity. When we are assessing the endometrium receptiveness, we have to think about 3 important factors:
the window of implantation because there is a special moment in time where the endometrium is ready to receive and implant an embryo
the microbiome because all women have bacteria inside their uterus it just has to be the right bacteria because if we don’t have the right bacteria, the uterus is then invaded by pathogenic bacteria or is at risk to be invaded by pathogenic bacteria in chronic endometritis.
The most known test is the Endometrial Receptivity Array (ERA) test, which is a commercial name of the test from Igenomix. There are other tests on the market, the ER-Map, ERPeak, and The Win-Test, there are lots of very similar tests on the market. At Ferticentro, we usually work with the ERA test. How does it work? In that normal cycle, the cycle begins on day 1, in the physiological cycle women ovulate around day 14 and more or less 5 days later, there is this theoretical window of implantation that typically occurs between days 19, then to end 21 of the cycle before that the endometrium is what we call pre-receptive, after that, the endometrium is post-receptive. It is now known that 3 in every 10 implantation failure patients have a displaced window of implantation.
If an embryo is not transferred inside the window of implantation, the odds of pregnancy are significantly lower. What is the window of implantation? It is related to the timing of exposure to progesterone because when preparing for a frozen embryo transfer or an egg donation cycle, or even in a physiological cycle, there are 2 phases in the development of the endometrium. The initial phase is the proliferative phase where the endometrium starts growing, it is very thin at the beginning of the cycle, and it grows thicker, and then there is a moment where progesterone takes section, in a natural cycle, progesterone is produced by the corpus luteum which is originated by the remnants of the ovulating follicle, in an artificial cycle, we add artificial progesterone. In an artificial cycle, after 5 days of exposure to progesterone, the endometrium is ready to receive a day-5 embryo. After 3 days of exposure to the progesterone, the endometrium is ready to receive a day 3 embryo. What happens if there is a deployment in the window of implantation? In some women, for example, we need an extra day for the endometrium to be ready to receive a day 5 embryo transfer, and sometimes we need to do that embryo transfer at 144 hours or 168 hours. The movement in the implantation window is something that we have to control with the ERA test.
How does it work? We do an endometrial preparation like in an artificial cycle or even a natural cycle, and then on the day of the embryo transfer, instead of transferring an embryo, we do a small biopsy. We analyze a piece of tissue in the genetics lab, Igenomix uses an array of 248 genes, to detect the endometrial receptivity. They will tell us whether that corresponds to a viable endometrium or not. That leads to a personalized window of implantation, we should transfer the embryos according to the instructions received from Igenomix.
The live birth rates with or without the personalized embryo transfer are completely different. For example, the study from Igenomix showed that a personalized embryo transfer was 14 points above a frozen embryo transfer done after 120 days, and the cumulative rate was even higher. Therefore, it is a very important way of optimizing uterine conditions. We don’t use it for every patient, we don’t use it as a first-line approach, but it’s a very important resource.
The next thing is the microbiome and chronic endometritis. As Igenomix showed that 30% of infertile women have pathogenic bacteria in their uterus. It is normal to have bacteria inside the uterus, but they have to be the correct bacteria called lactobacillus. When we have the optimal microbiome and the bacteria inside the uterus are the right ones, they keep the bad ones from coming in. When there is an imbalance of pathogenic bacteria in the uterus, the conditions for implantation are not optimal, and so 2 tests can check it. The EMMA and ALICE, the first test check the microbiome, the ALICE detects chronic endometritis, it’s possible to perform both tests at the same time using the same piece of tissue.
When we look at anatomical anomalies, we need to keep in mind that some women can be born with such anomalies, and others can acquire that through life. We can use hysteroscopy to check for:
Some of these can be corrected surgically, some don’t, and some can, but we shouldn’t do it because we always have to balance between trying to optimize the uterine conditions as much as we can or taking the risk of being too invasive. Some studies suggest that generally speaking, congenital uterine abnormalities can lower the reproductive outcome. However, for example, in another study from the Cochrane library, they have seen, for example, in the case of hysteroscopic septum resections, there is no evidence that this kind of surgical procedure can improve the reproductive outcome. Therefore, it is so important to evaluate every patient individually.
On the third level of importance and likelihood, we have the immune and haematological tests. We have the NK cells, in France, for example, there is the MatriceLAB test which tests for the immunity environment at the uterine level, we have the KIR/HLA tests that many patients do, and we have packages of tests designed to screen for autoimmune diseases or autoimmune factors or parameters we also have screening packages for thrombophilia either acquired, genetic or hereditary. There are a lot of tests that can be done, in our opinion, these are not first-line tests. When we have 3 negative results in the past, it could make sense to do these tests, we request them a lot, we’re not against it, we ask for these tests quite often. Sometimes when patients get here, even before they start treatment, when they tell us stories like I’ve tried 5 times elsewhere, and so on, before doing any kind of treatment, we can start by asking them to do these tests, this can be very important, but we have to use them to target these tests. It is not necessary to do all these tests on all patients.
The first case presented a patient who was 42 years old when she came to the clinic, she already had 5 years of infertility, she was married, she had 2 previous miscarriages, and she had multiple fibroids and low ovarian reserve.
The first thing we had to do was to remove fibroids because her fibroids were interfering with the uterine cavity, and so we had to remove them surgically. It was not only a laparoscopy but also a laparotomy because she had so many fibroids. We did a couple of cycles to accumulate eggs and then do the PGT-A. We ended up having 1 blastocyst, and unfortunately, that blastocyst was not viable, it had 4 different genetic abnormalities. Therefore, we did an egg donation treatment, since she already had 2 miscarriages and a case of an abnormal blastocyst in a previous PGT-A case, we filed for an authorization to do PGT-A in the egg donation case as well, and we got 3 blastocysts, and unfortunately, all were aneuploid. We discussed the situation with the patient, and we considered the possibility of doing a double donation, the couple didn’t want it, we also discussed the possibility of this being just bad luck, and we agreed to proceed once again because this was just 1 attempt and it could be just bad luck. We did the egg donation cycle, and PGT-A and everything went well. We had 6 blastocysts, and 5 of them were euploid, this was a wonderful result, a lot better than we expected, however, we did the first frozen embryo transfer of a euploid blastocyst, and the result was negative.
We did another embryo transfer without further testing because, finally, we had viable embryos and were disappointed because the result was negative again. Then we moved on with the ERA test, and then we thought that the endometrium was only receptive at 144 hours, so this was good news because there was something in which we could intervene. At the same time, we prescribed lots of tests, but in the thrombophilia screening panel, there were multiple positive results, so we referred the patient to the haematologist and the haematologist during her consultation developed an individualized prophylactic protocol. This patient had a family history of strokes and haematological disorders, so the haematologist developed her protocol, and then we moved on with the next frozen embryo transfer respecting the indications from the ERA test and using the prophylactic protocol from the haematologist. We immediately got a healthy pregnancy with the birth of a baby boy, and the patient with her husband still has 3 great embryos that allow them to have another baby.
The second case presented a young younger patient of 30 years old, she came to the clinic after having 2 previous miscarriages and also because she had PCOS, so she could spend 2 or 3 months without having her menstruation, on top of that, there was also a male factor. The couple didn’t want to do PGT-A because they were against it for ethical reasons.
We started the ovarian stimulation, but we couldn’t do the embryo transfer immediately because of PCOS, we ended up freezing 6 very nice blastocysts, and we froze them all and then prepared the patient for the embryo transfer. We transferred the first blastocyst, we got an ongoing pregnancy and then she miscarried around 8 or 9 weeks or so. This was her third miscarriage, when we get to the third miscarriage, there is an indication for further tests, we discussed this with the patient and her husband they didn’t want to do further tests, and so we moved on with frozen embryo transfer number 2. We’ve transferred them to another blastocyst, and the result was negative. We talked with the patients again, there were some lifestyle corrections in terms of nutrition, they were not living a very healthy lifestyle, and the patients rejected further tests, so we decided to give them a prophylactic protocol with probiotics and antibiotics because there were already 3 miscarriages, so we couldn’t exclude the risk of any underlying infection completely. We used the high dosage progesterone protocol because we were not sure whether the progesterone levels after the embryo transfer were borderline, so we decided to increase the dosage of progesterone, and then we immediately got an ongoing pregnancy, and the healthy baby is already born. We still have some embryos for them, so it’s also a very interesting success story.
The last case was about a 44-year-old with a very low ovarian reserve, so we advised her to go through egg donation. We obtained 4 blastocysts which are a normal and very good result, we did the elective single embryo transfer, and the result was negative.
We thought this was probably just bad luck, and we’ve transferred her to another fantastic blastocyst, and the result was negative again. She didn’t yet fill the criteria for recurring implantation failure, but we were not comfortable with the situation because we only had 2 more embryos, and actually, the first 2 were probably the best 2 embryos. We did the EndomeTrio test, which is a mix of the ERA test for the window of implantation, the EMMA test for the microbiome, and the ALICE test for chronic endometritis. We found that her endometrium was only receptive after 146 hours of progesterone, and she only had 26% of lactobacilli, on top of that, we screened her for immune factors, and we accidentally found out that she’s a carrier of an autoimmune disease we had to refer her to a specialized consultation for that, she had no symptoms nor any knowledge that she was a carrier for that condition and we also found a mutation in factor V in the thrombophilia screening.
We ended up finding a lot of abnormal factors in this case, and so we corrected everything, we did a 3rd elective single embryo transfer respecting the indications from the ERA test, using the protocol of probiotics and antibiotics recommended by the EMMA test. We used the treatment protocol adapted for thrombophilia and autoimmune patients, and a healthy baby girl was immediately born, it was a very successful story. The patient still has another embryo, she is now 48 years old, and she has to move on if she wants to do the embryo transfer before the age of 50, which is the legal limit in Portugal.
I don’t know if you’re doing IVF with your own eggs or with donor eggs, but the first thing we have to do is assess your ovarian reserve to see if your ovaries are still working. If they are still working, we should use an aggressive ovarian stimulation cycle as soon as possible. If you are willing to do that, we can even do more than one stimulation to accumulate eggs. With donor eggs, the odds immediately climb up to 60% or maybe 70%, depending on the context of the patients. You have two positive factors in your favour. First, you already have two daughters, so you have proven that you’re capable of receiving an embryo and implanting an embryo and taking a pregnancy to term, and you have done that twice. I don’t know if the father is the same or not, but if so, your partner has already proven that he’s a fertile person. In principle, I wouldn’t advise you to do any further tests because you have everything to become a good prognosis patient. My advice would be to just move on with egg donation, create the embryos, do a standard protocol because, in principle, this seems to be a good prognosis case.
You had 2 day-3 embryos, we don’t know whether they would make it to the blastocysts stage or not. Assuming they wouldn’t because the pregnancy result was negative, I think the only way is to try. On the other hand, with 0.83 of AMH and 4.7 of FSH despite being 40, I think it still makes sense to try conventional IVF with a more aggressive ovarian stimulation. We can also perform pre-implantation genetic testing for aneuploidies (PGT-A), but if you can’t do this or don’t want to do it or if it’s too expensive, we can also try the mini IVF. Mini IVF was a technique that everybody was talking about 10 years ago or so. Theoretically speaking, we could obtain just 1 or 2 eggs of better quality instead of 7, for example, as you mention here with the lower quality. Nowadays, the tendency worldwide is the more eggs we can get, the better. I think it’s a respectable strategy, at our clinic (Ferticentro), we usually don’t do it, we prefer to try a more aggressive stimulation in these circumstances because, in our experience, it works better.
The window of implantation is more about the progesterone, it’s not so much about the estrogen. The principle behind the ERA test and other tests are that under the same type of protocol, the women’s endometrium will behave the same way. Before they started doing this test, they were tested in thousands of patients, they have confirmed that the cycles were reproductive. The women’s uterus will respond the same way to the same type of strategies. Our advice is to try to do things the same, so if you start with transdermal patches, you should stay with them. If you start with tablets, you should stay with tablets and so on. It is never good to change the way of administration of the estrogen from one cycle to the other, but still, the estrogen is not the critical factor. What I would change is the way of implantation of the progesterone. If you’re using injectable progesterone, you should always keep injectable progesterone. If you’re using it in gel, you should keep it in the gel. If you are using it on tablets, as we do in Portugal, you should stay with tablets. If you can, keep the same strategy. If you cannot because medicines are not available, don’t worry too much, as long as you can keep the progesterone way of administration in steady dosage and the way of administration.
There is no problem with that. We can do artificial cycles, and so we do what we call hormonal replacement cycles. Everything is artificial, you don’t need to ovulate since you’re doing treatment with donor eggs. Since we are using donor eggs, the eggs are already there. We will create the embryos in the lab, so we just need to prepare your endometrium to receive and implant an embryo. IVF with egg donation can even be used in menopausal women. By doing ultrasound scans and programming the cycle with the right hormones, we can control timings. Your doctors will be able to identify the best moment we need the endometrium to be at least 8 millimetres thick, sometimes some patients can never get to that, but there are different strategies to improve the endometrial conditions.
We will never know. I mean, if we’re transferring a day-3 embryo, we would recommend for the embryo transfer to be done after 3 days of progesterone unless a window of implantation test said otherwise. Assuming that your window of implantation is not displaced, I would say that you shouldn’t think about this, you should think about hours as day-3 embryos should be transferred after 72 hours of progesterone that’s the most important. If it was transferred after 96 hours, for instance, then maybe it was too late, I would have to know the results of your window of implantation, but if that was the case and the embryo transfer was not done at the right moment, this could be a reason.
In principle, prednisolone is a steroid and a powerful anti-inflammatory drug. We sometimes use it in cases where there is a risk of immune rejection of the embryo. Sometimes we use it just as a prophylactic approach, and yes, we use it a lot in frozen embryo transfer cycles, and as far as I know, there is no relationship with lactose intolerance. However, I’m not a specialist in this field, I can only say that I’ve never heard about it.
Scratching was something that we were doing a lot until more or less 4 or 5 years ago, and we did it a lot for maybe 10 years or 8 years. Initially, scratching had very promising results. These days and especially in recent years, there was some research published that in most patients, it didn’t improve results, for certain patients, it could be worse. The group of patients for whom this could be beneficial was very short and very specific, so at our clinic, we’re not doing it anymore, we stopped doing scratching like 5 years ago or so.
PRP procedure is a little the opposite, it’s a new treatment, and it stands for platelet enriched plasma, it can be used either inside the uterus for women with very thin endometrium, they are injected directly in the ovaries. When it comes to low ovarian reserve, I don’t have any experience with this personally or in our clinic. We’ve had patients who have done this in other clinics in collaboration with us, and the results were not so great. There are a lot of recent publications mentioning very good results using it, so I would say that PRP could be the future. Published studies are all very recent and include low numbers of patients, but the results seem promising. I don’t think scratching will be done for much longer, it tends to be abandoned.
Normally, when we work with Igenomix, they give a list of probiotics available in many places around the world. Probiotics don’t have the same name everywhere, it depends on the country, there are the ones used either for 7 days or 14 days. They are used interventionally at night, and we don’t make a distinction between different types of probiotics, so all of them can be suitable. We cannot say that a certain probiotic is better than the other. Regarding antibiotics, it will depend on the bacteria that are causing endometritis. Normally, when we get the results from the biopsies that I’ve identified the presence of pathogenic bacteria, we also get the list of antibiotics that work against them. It’s not always the same, sometimes we use Amoxicillin, sometimes Metronidazole or Azithromycin, so there are plenty of antibiotics that can be used.
Regarding Thrombophilia protocol is a little the same because we work with haematologists, we don’t have a standard protocol for all patients, but typically these protocols use heparin aspirin, and they use steroids. The dosages and the regimes are variable according to many factors, including the type of Thrombophilia that is identified. Not all thrombophilia is the same. The dosage of this medication is not always the same from the beginning to the end of the cycle. There are plenty of things that can be used.
We don’t recommend PGT-A for donor eggs. The example I gave was a very particular situation because it was a patient who had 2 miscarriages, and she was the same age as the egg donor. She miscarried twice, and then we did a treatment with her own eggs, and none of the embryos were viable. We did PGT-A, and they were all abnormal, and so it was because of this very particular situation and context that we did PGT-A in the egg donation case. In the first cycle that she has done, out of 3 blastocysts, none was viable. In the second cycle, out of six, five embryos were viable. We have seen that PGT-A below the age of 35 doesn’t make much of a difference, and all egg donors are less than 35 years old, so I don’t recommend PGT-A for egg donation.
I don’t think it makes a difference, maybe it is useful when we have a lot of embryos, and we need to prioritize them, then there are regulatory issues. For example, in Portugal, to do PGT-A with egg donation, we need a special permit. In the case of the first patient, we had to file for that permit, and the Portuguese IVF authority authorized this, but we had to explain why we were requesting that. Without the proper explanation they wouldn’t have allowed us, so it was a very specific situation. It’s not something that we can generalize for all patients. Globally speaking, I would say PGT-A is not recommended for donor eggs.
It will depend on the bacteria and the recommendations that we have. Typically, it’s about a week, and honestly, I don’t remember, but I believe it was 500 milligrams every 8 hours, I would have to take a look because this is these protocols are tailor-made, they are different according to the patient. There are situations where we have to do longer therapies, it’s variable.
here are other tests, there is ERmap, the WinTest, I’m talking about the ERA test because that’s the one we work with. We’ve been working with this for five years now, but they are all very similar. They test a lot of genes, they include 248 genes. We know how to interpret their results, so this is why we’ve been working with this test for quite a while now. It is important because this kind of problem affects 3 in every 10 cases of recurrent implantation failure. We find a lot of women with displacements in the window of implantation. There is a certain consensus in the scientific community about the importance of these tests, whether it’s the ERA test, ERmap or the WinTest.
I will certainly recommend them, and I think among all of these tests for recurrent implantation failures. This is probably the number one test to be made because it’s the one we are more likely to find. Sometimes, we don’t know why treatments are not working. We have to start with the issues that we are more likely to find and then go to the ones that we are less likely to find. The ERA test is the one that’s more likely to be there.
There is a logistics problem because when we are doing an ERA test, it takes more or less 21 days to prepare the endometrium, we do the biopsy, we get the result in about 2 to 3 weeks. When we stop the medication, the patient has her menstruation more or less a week to 10 days later. When she bleeds, we still don’t have the result, so we lose this cycle, we have to wait for the next cycle. If the patient is on menopause, we don’t lose that cycle, we can manipulate things with the pill, we can even start the cycle and wait for the result to arrive in the middle of it and start immediately. There are ways to solve this problem, but typically we would lose a cycle between the cycle where we did the ERA test and the cycle where we do the embryo transfer, but we can lose more time. These results are typically valid for 9 months. After 9 months, we should repeat it because these profiles can sometimes change for multiple different reasons.