During this event, Dr Harry Karpouzis, Founder and Scientific Director of IVF Pelargos Fertility Group, discussed the potential role of immunotherapy in IVF treatments and recurrent pregnancy loss. Dr Karpouzis shared his expertise on how immune therapies can help achieve a successful pregnancy following previous failures.
Dr Karpouzis started by stating that immunology in reproduction is a complex dance—this is how it is usually characterized. The role of immunology is dual. First, immunology safeguards the fetus from developing threats, but it also allows for the development and implantation of an embryo. There is a very important balance in the endometrium during the implantation window, and conception itself is a process that challenges the immune system.
Why? For example, we know that the sperm carries paternal genetic material, which is foreign to the female body. Even when we are talking about donations, the donor egg is also foreign to the endometrium. The immune system can recognize and tolerate the sperm, and this is very important for natural conception. Then conception happens, and the immune system must exhibit tolerance towards the developing embryo. If it keeps attacking the embryo, it can cause problems like miscarriages, for example.
There is a balance between the regulatory immune cells that suppress immune responses and the natural killer cells. The very famous natural killer cells are a bit different in the blood and the uterus, and their presence is very important for proper implantation. When we hear “natural killer cells,” we may think they are bad, but they are very important for proper implantation. What causes problems sometimes is the overactivity of them.
Many different cells play a role in the immune system. The uterine natural killer cells facilitate tissue remodelling. They help with the trophoblast invasion, which is the placenta invasion, and also with placental growth. On the other hand, if the natural killer cells are overactive, they can inflame and destroy tissues. Many different cells and many different cytokines play a very important role in the implantation window.
15% of the patients who enrol on IVF have a history of recurrent implantation failures, and 1 to 5% of the patients have a history of recurrent miscarriages.
What is recurrent implantation failure? It is the failure to achieve a clinical pregnancy after the transfer of at least four good-quality embryos in a minimum of three fresh or frozen cycles in women under the age of 40 years. This is the definition of recurrent implantation failure. What is the definition of recurrent miscarriages? In the past, it was three or more miscarriages in a row. The definition has changed; even two or more miscarriages are now considered recurrent miscarriages, and it would be good, especially in the private sector, to have them investigated.
Many autoimmune conditions, such as lupus, Hashimoto’s thyroiditis, antiphospholipid syndrome, rheumatoid arthritis, MS, type 1 diabetes, and less common ones like scleroderma and Sjögren’s syndrome, can affect pregnancy. Many times, they are associated with each other or can occur independently. For example, Hashimoto’s and vitiligo or Sjögren’s syndrome are often seen together. All these conditions create an immunological status that can affect pregnancy as well.
Antiphospholipid syndrome is an autoimmune condition that causes hypercoagulation. It is identified by the presence of antiphospholipid antibodies and causes thrombosis or pregnancy loss. Pregnancy loss can even occur as a late miscarriage, which is quite common in antiphospholipid syndrome, in the second or third trimester. It also increases the risk of stillbirths. Sometimes it is associated with primary infertility, and sometimes with pregnancy complications like IUGR (Intrauterine growth restriction), small babies, and preeclampsia.
How does it cause problems? It causes placental thrombosis. It also causes problems by complement activation, which triggers a cascade of thrombosis inside the placenta. Furthermore, it can cause inflammation and can combine with other autoimmune conditions. It is not very common, occurring in about 1 in 2,000 cases, but it is more common in women with pregnancy complications.
What is the treatment? It involves anticoagulant treatment, such as heparin injections and aspirin. In refractory antiphospholipid syndrome or proper antiphospholipid syndrome, hydroxychloroquine can also help. These are the antibodies we usually test for. A person might have one, two, or all three of them. When all three are present, antiphospholipid syndrome is more risky for pregnancy.
It is very important to understand the criteria regarding antiphospholipid syndrome. Many times, you may hear, “You have antiphospholipid syndrome because you have raised antiphospholipid antibodies,” but it’s not that simple.
The criteria require at least one clinical event, either a thrombotic event or a pregnancy complication, along with the detection of one or more antiphospholipid antibodies—such as the lupus anticoagulant, anticardiolipin, and glycoprotein antibodies. The criteria are very important because we cannot label people with antiphospholipid syndrome without them truly meeting the criteria.
Many people do not believe in immunology. At Pelargos, as Dr Karpouzis emphasizes is that we shouldn’t routinely check for immunology in people who do not have a history of recurrent miscarriages, recurrent implantation failures, or unexplained implantation failures. However, in candidates who do have these issues, immunology and a personalized approach can make a difference. To achieve very good results lies in a personalized approach to each case.
The uterine immune reaction occurs in the mid-luteal phase of every cycle—this is the implantation window, about five days after ovulation. During this time, cells such as natural killer cells and dendritic cells invade the endometrium. Additionally, some B lymphocytes and CD8 cells escape into the endometrium.
All of this research is largely based on the work of a French immunologist, Nathalie Lédée, who conducted a very large study on the personalized approach between 2012 and 2018. This study included 1,738 infertile patients and was a randomized controlled study. Based on that, the IMAP test for such candidates is being used. This is a biopsy taken after endometrial preparation at a specific time in the cycle.
IMMAP test examines the lining of the uterus during the implantation window. There are also tests available, such as The MatriceLab which is very similar, and the Chicago tests, which are blood tests. According to Dr Karpouzis, it is better to check the lining of the uterus with a biopsy rather than through blood tests. It should be done after endometrial preparation, as hormones play a role in immunology.
The IMMAP test looks at:
All these factors are involved in the differentiation of the secretory endometrium into a receptive state.
Another important factor is the balance between TH1 and TH2. To calculate this balance, we check interleukin-18. Interleukin-18 plays a role in helping the arteries prepare to receive the trophoblast. However, overexpression of interleukin-18 can be deleterious to the embryo, causing local toxicity and a shift towards TH1 dominance. To understand this better: TH1 causes cytotoxicity, while TH2 promotes the reception of the embryo. A similar balance applies to tumour necrosis factor and its receptor.
What does a personalized approach mean? It means categorizing results after preparing the lining of the uterus and testing at the correct timing—the implantation window. The results can show:
Sometimes, we assume it is an immunological issue and prescribe steroids, intralipids, or IVIG without understanding the problem. However, the issue may not be overactivity but low activity. There are also mixed profiles that require individualized solutions.
What needs to be done when we find out all these things? What we are doing is personalizing the treatment. For example, people ask, “Endometrial scratching, is it good?” Initially, we had very good reviews about endometrial scratching, but the latest reviews say no.
Endometrial scratching is important when we have specific reasons. Endometrial scratching is recommended only in cases of low interleukin-15 ratio and when we have mature uterine natural killer cells, not when we have overactivity. This is because local inflammation when done in the luteal phase of the previous cycle, can trigger a cascade and help balance this ratio.
Even estrogens—many times in endometrial preparation, we give low doses of estrogen or high doses of estrogen. Why are we doing that? The low doses of estrogen in a natural cycle are specific. When we increase estrogens, it is clear that this can downregulate interleukin-18 expression. So, when we already have low interleukin-18 expression, we do not want to decrease it further. In such cases, we prefer to use lower doses of estrogen rather than high doses. All these things, even the doses of estrogen and progesterone, have a reason behind them.
Immunotherapy is used when we have overactivity of the immune system. Steroids, which are the first line of immunotherapy, decrease TH1 levels, reduce natural killer cell toxicity, limit the effects of interleukin-15 overexpression, and modulate the TH1 to TH2 balance. This is when we use steroids. If we give steroids to people with low activity, it will further decrease it and could cause problems rather than benefits.
When we have resistant cases, we can use heparin. Why? Because these clotting injections have an added complement effect, which helps the immune system. They also have some immunosuppressive results apart from the anticoagulant effects. Second-line treatments include intralipids, hydroxychloroquine, and IVIG. These are given depending on the ratios and other specific levels we evaluate to judge what is needed for a personalized approach.
As mentioned earlier, progesterone also has immunological effects. For example, it inhibits natural killer cell activity and leads to TH2-dominant cytokine production. When we have local overactivity, high levels of progesterone before embryo transfer can modulate this and cause immunosuppressive effects. So, even the dose of progesterone we give can affect the results.
KIR genotyping is performed in cases of recurrent implantation failures or recurrent miscarriages. Natural killer cells recognize the embryo through the KIR receptors, which attach to the HLA-C of the embryo. There are many combinations of HLA-C (C1, C2) and KIR receptors (like KIR AA). We have noticed that specific combinations, such as HLA-C2 and KIR AA, can cause miscarriages.
When we find these issues, the main recommendation is a single embryo transfer rather than transferring two embryos. In some cases, immunomodulatory treatments, especially G-CSF (Neupogen), can help.
Endometriosis is another factor. There are many theories about its cause, and one of them involves immunology. We know that macrophages, lymphocytes, and natural killer cells are present in the peritoneum and that this can also affect the endometrium. Women with endometriosis often have deranged immunological activity. When a patient with endometriosis has recurrent implantation failures or recurrent miscarriages, we are quicker to consider immunology. Endometriosis is also associated with chronic infection, and in such cases, antibiotic or immunosuppressive treatments can help.
Lastly, the implantation window mechanisms are not fully clear. Many local factors affect the balance, and each person has their own immunological profile. This profile can change, even in six months. Blood immunology does not always correspond to local immunology in the endometrium, which is why we prefer to check inside the endometrium. Hormones, such as progesterone and estrogen, also play a role and can affect immunology.
Because of all this, a personalized approach is very important.
Do not blindly proceed with treatments, especially in cases of recurrent implantation failures. Find out the issue first. And, in all cases, it is crucial to ensure a healthy embryo. If we transfer embryos without knowing that they are chromosomally normal (e.g., by PGT-A), we will not know the reason for failure.
Because you started doing IVF at the age of 37, to be honest, when you have had six transfers, and they are not working, yes, checking other things, including immunology, is something that could be important. However, if you haven’t done PGT-A on the embryos, and you are now 42 years old, the first thing before spending money and time on all these tests is to do PGT-A. You need to get a chromosome analysis of the embryos to find out if the embryo is chromosomally normal or not. If you don’t know that, even if the embryo is of good quality, when you transfer it back, you will not know what chromosomes are inside.
PGT-A is not allowed in Germany, but it is allowed in other countries. It’s important because, at 42, the benefits far outweigh the risks. With a history of recurrent implantation failure like yours, it is the most important thing to know if you have a healthy embryo, especially if it is just one precious embryo.
Then, yes, you can check for other things as well, like hysteroscopy, immunology, and chronic infections—not only immunology but other factors too. I hope that helps. If you have any follow-up questions, feel free to ask.
Many factors can cause a miscarriage. First of all, it’s not only immunology that causes miscarriages. As I explained before, what tests exactly have you done to find out about your immunology problems, and what protocol are you following? Is it a personalized approach, or are you just taking steroids because you have raised natural killer cells in the blood? That might not be the perfect way to deal with it. But, even if you are taking all the medication and even if you have overactivity inside the uterus, other things can cause a miscarriage. For example, the chromosomes of the embryo, chronic infections, sperm DNA fragmentation that goes into the embryo and causes chromosomal problems, scar tissue, diaphragms, septums identified by hysteroscopy, and fibroids. Many things can cause a miscarriage, and the most common one is the embryo itself.
Regarding antibiotics, if someone has a history of recurrent miscarriages and recurrent implantation failure, we prefer to do a hysteroscopy and identify elements of chronic infection. The cultures we do on the vagina do not always correspond to what is happening inside the lining of the womb. We take PCR samples from inside the lining of the womb regarding chronic infection. When we find a reason, we give antibiotics, usually a triple scheme or something else, and many times it is combined with probiotics.
As for doxycycline, without really having checked before, it’s relevant because if you don’t know if you have, for example, ureaplasma, mycoplasma, chlamydia, or anything like that, it’s better to prevent the spread of infection and cover with doxycycline. But when we have a patient with recurrent implantation failures, it is better to know what infection we have inside the lining of the womb and then give the relevant antibiotics.
Regarding the first question, yes, we are big fans of checking inside the uterus, not just in the blood. But not everyone needs this. I need to say that it’s more important for people with unexplained failures, recurrent implantation failures, or recurrent miscarriages.
It’s again, you know, what I was saying before. I don’t know where you’ve checked your natural killer cells, most probably in the blood. But your natural killer cells are normal, so why take Intralipid? Intralipid is actually used to reduce the overactivity of the immune system. If the immune system is working properly, and you’ve done the tests, why use Intralipid? The benefit is not clear to me, as I don’t know your case. But again, our approach is to do things knowing why we are doing them.
It’s a very complicated case. After six failed embryo transfers with good-quality embryos, I would again recommend PGT-A as the first step. It’s important to know that you have a chromosomally normal embryo. Stage 3 endometriosis, unfortunately, affects the ovarian reserve and the quality of the eggs sometimes. Many times, we do not have chromosomally normal embryos, so the issue might be the embryo itself.
The first thing I would do after all these failed transfers is to ensure that I have a chromosomally normal embryo. Then, I would break down all the things that can cause miscarriages: check for thrombophilia, check if you need blood thinning injections and aspirin, do tests about immunology to create a personalized approach if needed and check your progesterone levels because of your endometriosis. In severe endometriosis patients, there is often progesterone resistance before embryo transfer. After breaking everything down, we will proceed with the transfer after creating a personalized protocol for you.
Usually, the combination of all these things is not used. At least in our practice, we don’t use all of them. We have first-line treatments and second-line treatments. If you take hydroxychloroquine, which is Plaquenil, it’s a very potent medication and a very good medication for the really increased activity of natural killer cells. We don’t combine it; we usually use either one or the other. But of course, you should follow the advice of your doctor as well because clinics might have different protocols.
The immunology testing we usually use is the IMAP testing. During a hysteroscopy or an outpatient procedure, we can take a biopsy for the endometrial flora as well, to see if we have a predominant lactobacillus or not. We can also test for chronic infections. We used to test for the implantation window, but the latest studies regarding the implantation window are in favour of not doing it anymore. However, in our practice, when we cannot find anything else, we’ve had some cases with success with implantation window displacement as well, and we are still using it.
You can do them in your country, possibly, if you find a unit that will collaborate with that.
You can do that with the pipelle. You can do it at the time of the hysteroscopy by taking small biopsies with scissors, so it’s not a problem. We don’t have any issues. We usually take all three of the tests together, and it doesn’t affect the endometrium. Instead of scraping the lining of the womb massively, you can use scissors to take the biopsy, and you don’t affect anything.
Adenomyosis is something you cannot completely stop. Sometimes it is associated with endometriosis, sometimes it is not. Adenomyosis is a very difficult condition, and there aren’t many things you can do. Usually, when we have adenomyosis in a woman who still has her cycles and hormones, meaning she’s not in menopause, we prefer to downregulate before the embryo transfer to make things a bit better.
As for the inflammation, I don’t know what kind of inflammation you mean. How did you find out about this inflammation? Is it a specific inflammation? Usually, you would need to take antibiotics for inflammation inside the lining of the womb.
Well, it depends on why you are doing them. If you have suspicions that there is a polyp, a saline test might tell you if you have a polyp, but it cannot treat it at the same time. The most accurate thing you can do is put the camera inside because at the same time, you can treat as well if there is a polyp, and you can also see the lining of the womb. You can get information that you might not be able to find out with HSG or ultrasound scans, for example, signs of a chronic infection or a small degree of scar tissue.
Now, regarding checking the tubes with HSG, if we have a suspicion of hydrosalpinx, we need to check the tubes. If not, we don’t need to check the tubes with IVF, as we are not interested in whether the tubes are blocked or not, as long as they don’t have flow inside.
MRI is good that you have done it. It can show if you have any spots of endometriosis or fibroids. AMH hormone tests and sperm analysis should be checked nicely. These are tests that you need to do. The next step is to start stimulating your ovaries and creating some good embryos. Given your history, I don’t think it’s 100% important to check for immunology as well. You don’t fall into the category of recurrent implantation failure or recurrent miscarriages based on your history.
Yes, it is possible. We have seen it many times. We have many cases where there was a previous normal pregnancy, and then miscarriages occurred in the next pregnancy. So yes, I would check for immunology. Immunology is associated with many autoimmune conditions, and you could have developed an autoimmune condition that you didn’t have before.
Wow, that’s a very complicated medical history. Well, things are very difficult. I don’t know about your FSH or AMH, but at 41, the chances of getting pregnant naturally are very low. Even if you get pregnant, you will need to manage your thyroid problems. You might need some immunology treatments as well. Regarding the HLA KIR issues, to be honest, the chances are extremely low, and egg donation would be the best option for you right now.
It depends on how much scar tissue there is. When there is a lot of scar tissue, we usually put an IUD in place to keep the walls of the uterus dilated, which helps prevent adhesions from coming back. Sometimes we also give estrogen treatment to help proliferate the endometrium, preventing the adhesions from coming back. However, many times, adhesions do return. The timeline isn’t immediate, but it depends on the degree of adhesions and whether it was a septum or actual adhesions. Each case is different, but with scar tissue inside, it often comes back.
If you’re interested in IVF with your own eggs at 43 with low AMH and borderline FSH, the first step is to create some embryos and biopsy them to see if you can manage to create a chromosomally normal embryo. The chances are low. If you don’t manage to create a chromosomally normal embryo, then we’re talking about egg donation. Regarding your HLA situation, you will most likely undergo a single embryo transfer. There are some theories suggesting G-CSF treatments as well. Regarding adenomyosis and endometriosis, if you have a regular cycle, after creating embryos, we would downregulate before transferring the embryo. But the first thing is to manage to create a chromosomally normal embryo, which is difficult.
You need an endocrinological input. You may need to increase your L-thyroxin dosage. The ideal TSH levels before embryo transfer should be less than 2.5, so we generally aim for these levels. It also depends on your anti-TPO antibodies. If it’s related to thyroiditis or a similar condition, since you don’t have a thyroid and are on replacement treatment, talk to your endocrinologist. You may need to increase the dose or monitor the levels before IVF stimulation, and if it’s higher, adjust it to drop it below 2.5.
The more embryos, the better, because you never know if it will work. Even if you have one, it’s a very precious embryo that gives you very good chances. Thrombophilia is managed nowadays with injections and aspirin, so it shouldn’t be a problem. The more embryos you have, the more chances you get if the first one fails to have a backup. However, even one embryo is important, and it should be transferred under the best circumstances.
It depends on how severe the endometriosis is and its stage. Usually, we give a single downregulation injection in the luteal phase of the previous cycle. Sometimes we do a double injection, one per month for two months, and then start the preparation. After waiting for a period, we begin the preparation. That’s usually what we do, but of course, every clinic might have a different approach.
I don’t think it’s really associated. You might have some fluid retention with all this medication, but regarding inflammation, I wouldn’t say it would cause inflammation of the uterus.
If we’re talking about medical tourism and people coming from abroad, medicated cycles are much easier to coordinate. In a natural cycle, there’s a risk of ovulation, and you need to measure blood tests, etc. We usually prefer medicated cycles for better control. However, a natural cycle can also work, especially in women with completely regular periods. When periods are irregular, it’s harder to control.
If we manage to do a natural cycle and understand the risks, like missed ovulation or travel timing issues, it’s a good option. Sometimes, when medicated cycles fail or result in biochemical pregnancies, a natural cycle might increase the chances of a normal pregnancy. It’s all about balancing the advantages and disadvantages.
