Embryo transfer, implantation window, and uterine lining – how do they depend on each other?

Natalia Szlarb, MD, PhD
Gynaecologist & Fertility Specialist at UR Vistahermosa, UR Vistahermosa

Category:
Advanced Maternal Age, Embryo Implantation, Embryo Transfer, Failed IVF Cycles, Success Rates

From this video you will find out:

Embryo transfer, implantation window, and uterine lining – how do they depend on each other?

Embryo transfer, implantation window, uterine lining guide

When women are born, they have a certain number of quality eggs in their ovaries. Because of the social revolution which has taken place over recent decades and many both personal and professional factors, more women postpone their maternity. The bad news is that the older they get, the lower the quality of embryos they are generated. It is these embryos, as well as the endometrium and the so-called ‘implantation window, which play a crucial role in the successful outcome of any IVF treatment. In this webinar, Dr Natalia Szlarb talks in detail about these factors and explains how they depend on each other.

Dr Szlarb starts by reminding us that young women under 35 years old generate about 50%-60% of genetically healthy embryos. The magic number to work with one’s own eggs is until 40 years old. Between 40 and 43 years old, the majority of embryos are unfortunately genetically abnormal. The most common genetic abnormalities are the ones like trisomy 21 (Down syndrome), trisomy 18 (Edwards’ syndrome) or trisomy 13 (Patau syndrome). If we do not apply pre-implantation genetic screening of blastocysts in women over 35 years old, the pregnancy rates are only 18%. When genetic testing and embryo selection are performed, the pregnancy rates are about 60%.

Embryo selection – the most sensitive methodology

There are different methodologies in the embryo selection process. Generally, after pre-implantation genetic tests (PGD – preimplantation genetic diagnosis and PGS – preimplantation genetic screening), pregnancy rates are cumulatively very high. Embryos generated with donor eggs and selected with Time-Lapse technology can achieve even 70% of pregnancy rates.

Selecting embryos is the most sensitive methodology. However, these perfectly healthy, genetically normal blastocysts have to be put on perfect ground. Dr Szlarb highlights that it is the main reason why every patient is thoroughly examined during the first appointment with a doctor. Among others, they perform a transvaginal ultrasound scan. If on the basis of the transvaginal ultrasound, doctors are not sure what is going on with the uterus lining, they follow up with a hydrosonography. It is an intrauterine instillation of a saline solution aimed at improving the diagnostic accuracy in detecting uterine abnormalities. In other words, it is possible to see if there are polyps or fibroids in the uterus. These are basic anatomical pathologies that have an impact on the uterus lining and they have to be removed before the embryo transfer.

IVF and uterine lining – what’s the standard thickness?

According to Dr Szlarb, another issue to focus on is the thickness of the lining. The standard thickness lining should be 7 millimetres, according to medical literature. It is common to perform the so-called test cycle that mimics a patient’s natural cycle. It includes progesterone and oestrogen supplementing and is conducted in order to see how thick the uterus lining is. It shows what dose of hormones is needed to grow the lining to the desired 7 millimetres and thanks to it, it is easier for the doctors to determine how to proceed with hormone supplementing in the transfer cycle.

However, hormone supplementing is sometimes not enough to achieve the desired thickness of the endometrium. Dr Szlarb says that in such a case, the lining growth has to be supported in an alternative way. We have undertaken an endometrium regeneration study aimed at treating patients with a history of transfer cancellation or failed transfers because of thin endometrium. Doctors are microinjecting patients’ platelet-rich plasma to support the uterus lining growth. It is believed that platelet-rich plasma allows endometrium to regenerate and highly improves its quality – not only in the current cycle but in future cycles as well.

What are endometrial receptivity and implantation window?

Next, Dr Szlarb focuses on the importance of physiology. In fact, the question of how the uterus lining is working seems to be one of the most important issues. It happens that despite a visually perfect 10 mm thick lining and genetically normal embryos transferred, the patient is still not pregnant. Then in case of recurrent implantation failure (3 unsuccessful transfers), it is indicated for a patient to undergo a uterus lining biopsy. There are different ideas on why the endometrium is receptive or not.

According to Dr Szlarb, around the 19-21 days of a cycle is the moment when the cells of the uterus lining undergo specific changes – the receptors of progesterone are being expressed on the surface of the cells. It would not be possible to discover if not for microarray genetic technology that enabled the development of tests like a receptivity essay or endometrial receptivity map (ER Map). Thanks to these it is easier to determine if the endometrium is ready to accept the embryo or not. The results of the ER Map test are either receptive, pre-receptive or post-receptive. The pre- or post-receptive results are usually corrected by prolonging or shortening the progesterone supplementation. And then the doctors either follow up with a transfer or perform a confirmatory biopsy to know exactly when the receptivity window is.

The importance of endometrium on embryo transfer

The endometrium is extremely important in embryo transfer. However, if the doctors see that the combination of the correct implantation window, good uterus lining and genetically normal embryos does not result in a positive pregnancy test, it is time to move on to egg donation. Egg donation is performed in about 600 patients annually. The first step of the egg donor selection process is the confirmation of donors’ availability. The law in Spain allows for egg donation anonymity. Donors are screened for infectious diseases as well as genetic or heart diseases. The computer programme matches the donors and the recipients on the basis of a phenotype (eyes, hair and skin colour) as well as height and weight. For complicated cases with genetic diseases, we offer to test to match a donor with the recipient genetically. In order to exclude male factor infertility, a sperm analysis is conducted as well. In the case of patients over 35 years old with low ovarian reserve, where AMH is low, and the antral follicle count is less than 6, Dr Szlarb sometimes recommends so-called embryo banking. In such cases, 3 embryos before the blastocyst stage are generated in one cycle, they undergo biopsy and are frozen afterwards. After 3-4 months of the recovery time required for the patient’s body, another cycle is performed.

Summing up, the key to successful implantation and pregnancy is embryo selection. Genetic testing is the most sensitive method and when it is combined with effective endometrium receptivity mapping (ER Map) and reliable determination of the implantation window, the pregnancy rates may be as high as 80%.

According to Dr Szlarb, the biggest advantage of all of these factors is the possibility to “erase” a patient’s age and in this way make pregnancy rates completely age independent.

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- Questions and Answers

Is age-dependent success rate in ART referring to the donor’s age or the patient’s age? For example, donor’s age is under 35 while the patient’s age is over 45 and we’re talking about using donor’s eggs & sperm to create embryo to be transferred to the patient.

In the egg donation cycle, pregnancy rates are due to the donor’s age. As long as I know that in your uterus cavity there are no anatomical abnormalities and your uterus lining is receptive and thick enough, we can bring you to pregnancy rates of ladies who are 30 years old. So in egg donation cycles, pregnancy rates are patient age independent. Let’s say I have a patient who’s 20 years old and she’s in premature ovarian failure, she doesn’t have eggs anymore but she has an amazing uterus. Or I have a patient who is 40 years old, she has an amazing uterus but due to her age, the quality of her eggs is poor and all the embryos are genetically abnormal. Or I have a patient who’s 50 years old and she’s menopausal, she doesn’t have eggs at all, she doesn’t produce them anymore. Now if I put genetically normal donor’s embryos to these patients, the pregnancy rate in each of them is 70% for transfer. So success rates are really patient age independent.

Do you recommend the patient who is over 45 to do PGD testing, even with a high quality blastocyst?

I do recommend PGS in the majority of cycles that are performed. It’s because there is no woman in the world who generates all embryos which are genetically normal. Even young women generate genetically abnormal embryos, maybe just one or two per cycle, but still they are there. I cannot allow myself to have lawsuits because we transferred genetically abnormal embryos. It’s a very good question, especially when you are 45 and you’re defined as recurrent implantation failure. It means that you had cycles in your country with good looking embryos, they were not successful and you’re still trying to be a mom through the assisted reproduction with your own eggs. It’s good to do an IVF cycle with genetic testing because it can turn out that all the embryos you have in embryo banking cycles are genetically abnormal. Then, from the psychological point of view, it’s easier for you to understand that time went by, we did our best to make it happen with your own eggs and now it’s time to move on to egg donation. So I would definitely do PGS, even though we generated one blastocyst per cycle. I would recommend you to do one or two cycles more, in two-three months, to bank the embryos. If you have two-three embryos and all of them are genetically abnormal, we have to move on.

Is there a maximum size for endometrium lining? Can it be too thick for embryo transfer?

Yes, endometrium lining can be too thick for embryo transfer and this is the moment when you started bleeding. The uterus lining is growing, at the bottom of the uterus lining there are vessels and these vessels are grown with the uterus lining in each cycle. If we see that the lining is too thick, it’s more than 40 millimetres, the vessels are not able to support the upper part of the lining anymore. This is the moment when you start bleeding and this is the moment when I cannot transfer anything. And then I have to cancel the transfer. When you’re flying to Spain from different countries, we cannot allow ourselves to cancel donor cycles because you’re bleeding and your lining is too thick. That’s why we prefer to undergo a test cycle to adjust the dose of hormones specifically to your body.

I am preparing to return frozen embryos from an egg donation. I do not know whether it is worth doing the ER Map test because the embryos were frozen on day 3.

It depends a lot on the indication of a medical director of the place where you perform the receptivity assay. When you are not pregnant with the first good quality blastocyst, I want a receptivity assay immediately. Blastocysts have the potential to implant – more than 70% implantation rate – so if you are not pregnant after the first transfer, I want to know why. I don’t want you to wait three times, for three unsuccessful transfers. For day 3 embryo pregnancy rates are usually lower. So if it was the first unsuccessful transfer with day 3 embryos, maybe you can skip the receptivity assay testing and transfer what you have. If you’re pregnant, then amazing, if not – in the next cycle the endometrium receptivity assay should be performed. Additional explanation: It’s easy to apply the receptivity map to the blastocyst. Of course, we know how to apply the receptivity map to day 3 embryos. Let’s say we have embryos frozen on day 3 and the receptivity assay is being performed with 5.5 days of progesterone. It means that when we transfer the day 3 embryo, the progesterone is calculated as if it was to transfer a blastocyst. So you start three and a half-day before. If the results say: pre-receptive or post-receptive, then we give you progesterone one day more or one day less. With the day 5 receptivity assay, it is very straightforward to calculate, with day 3 it’s also possible to perform.

Which procedure is the best to guarantee the perfect window of implementation?

The best procedure is to perform the uterus lining biopsy on the 21st day of the cycle with 5.5 days of progesterone. If the results say you’re receptive, we can transfer. If the results say you’re pre-receptive, then we will recommend you to get one more day of progesterone and do a confirmatory biopsy. If in the confirmatory biopsy, you’re confirmed 6.5 days receptive, then, after the other transfer, we will give you progesterone longer to open implantation windows. The answer is very straightforward: the best guarantee to the perfect window of implantation is the uterus lining biopsy result which says: receptive. It can say receptive day 4.5, 5.5, 6,7, 8.

Is it suggested to remove a fibroid on the lining? How much fibroid has to be removed? Is it fine if it only has small amount of fibroid? When do we need to do the removal of fibroid? During the embryo transfer cycle or before the transfer cycle?

Uterine fibroids should never be removed in the transfer cycle. In the transfer cycle, we have to focus on the good thickness of the lining and its physiology. We have to include more or less progesterone to open the implantation window. Endometrial polyp removal or uterus fibroids removal should be done before the transfer cycle. It’s a lot easier to do endometrial lining surgeries when somebody’s in their first half of the cycle and the lining is thin. Especially in Europe, you wait a long time for uterus fibroids removal. When your gynaecologist finally has time and they can fit you in, then you’re already in the second half of a cycle. And in the second half of a cycle, you have a lot of thick lining and it’s difficult to operate because you bleed. So what we do while scheduling surgeries, is to put patients on birth control pills. In such way, fibroids or polyps are removed on birth control pills and never ever in the transfer cycle. When the fibroid or polyp is removed, we stop birth control pill, you have a period and then you grow the uterus lining with estrogens. We add five and a half day progesterone in the transfer cycle and then the embryo is transferred.

What can be done if even after trying all the mentioned options, a patient shows no significant endometrial development?

Then we have to do an endometrial regeneration study. We have to do a hysteroscopy, we have to look into the lining to see what is going on. Maybe you have adhesions, and maybe these adhesions are partial. If you have partial adhesions, then we perform the hysteroscopy where you get your platelet-rich plasma microinjected under the uterus lining. This makes the working areas of lining work better and grow more. You get estrogens artificially to let these areas grow, too. You wait 7-10 days for the lining to grow after the subendometrial microinjection of platelet-rich plasma. And then, if we still see that the lining growth is borderline, less than 7 millimeters, we do the lavage. We put the catheter transvaginally into the cervix to put the platelet-rich plasma again in the womb. This way we try to support the lining growth as much as we can.

I have had 2 failed cycles with my own eggs, we are now trying donor eggs. I asked about testing my implantation window but I was told it was not worth the money as the chances of donor eggs working were so high. Is that right?

We have to differentiate three reasons why embryos are getting implanted or not. The first reason is very bad quality of the embryos that we are transferring from the patient’s own eggs. There’s no blastocyst or all the blastocysts are genetically abnormal. The second reason is transferring outside of the implantation window. The third reason is immunology. There are women that see HLA of their husband on the surface embryos as a foreign body and no matter what we’re going to do, they will always reject the embryos of a husband. So we cannot mix these three reasons. When you have had two failed cycles with your own eggs, we have to see if it was the problem of an egg, the endometrium or immunology. When I see that I cannot develop a genetically normal healthy blastocyst with your own eggs, no matter what I’m going to do and no matter how receptive the lining is, it will never work. So first we have to review protocols, we have to see what happened in your previous cycles. Have you had blastocysts? If yes, then don’t give up. Try with your own eggs. If the blastocysts that you were supposed to have are very bad quality, then we have to move on to egg donation. Of course, if you had three-four cycles and never a positive pregnancy test, you can have the implantation window moved. This is why we would indicate to do an endometrium lining biopsy and see the implantation window.

How tiring it is to collect banking embryos? The full process of taking progesterone up until the retrieval of the eggs was very tiring for me in France. I don’t know if the doses of medicine were too much. But having an IVF made my belly bloat a lot and my body so tired.

I can understand you. I hate progesterone, too. I can only sleep and eat and grow which is not fun. In reproductive medicicne, when we talk about euploid rate and genetically normal embryos to select from, the magic number is 6-8 blastocysts. When I have a lady and her AMH is more than 2 and we put a middle dose stimulation 2.25, recombinant gonadotropin, then I’m about to generate 20-30 eggs. If somebody has AMH between 1 and 2, I’m able to generate 10 eggs on the same protocol. When AMH is under 1, then it’s difficult, it’s a low ovarian reserve. Then only 50% of eggs which I’m achieving (if I have great sperm quality) is going to develop into blastocysts. If the blastocyst rate is 50%, it means that I expect 8 blastocysts out of 16 eggs. And then it’s age dependent. If somebody is 20 years old, 6 out of 8 embryos are genetically normal. If somebody is 35 years old, 5 out of 8 embryos are genetically normal. If a girl is 40 years old, 2-3 out of 8 blastocysts are genetically normal. At the age of 43, 1 blastocyst is genetically normal. If somebody has a good ovarian reserve, if somebody has high AMH, you can have these numbers in one cycle. In one cycle, I can do the entire family planning. If you have AMH under 2 and in the first cycle you generate 2-3 blastocysts, in two months you have to go back and we do one cycle more to generate 2-3 more blastocysts. So the timeframe where your body has to recover between cycles is 2-3 months. If after the first cycle somebody generates zero blastocysts, or instead of beautiful blastocysts I have creatures with a couple of cells, then we’ll have to talk about egg donation probably.

Does PGD or PGS testing have to be done to embryo prior to freezing?

Yes, it has to be done prior to freezing. We wait for the blastocyst culture for 7 days. Days 5 and 6 embryos are the best, they are biopsied and frozen. We have higher success rates with genetically normal frozen embryos than with the fresh ones. It’s because the selection of them is so sensitive.

I’m 32. I have undergone 2 embryo bankings. Firstly, egg collection was 13, 10 mature eggs and 2 blastocysts. The second embryo banking was in August this year, I had15 eggs: 13 mature but only 1 made it to blastocyst. Now we have decided to do third embryo-banking in the end of October. Is this okay? What should I do to improve my eggs as I felt i have done as much as I could. I can produce loads of eggs but very little to none blastocysts.

There are some patients that are very hardworking and you are one of them. I respect you a lot for this. Your blastocyst rate is very low. As I said, the blastocyst rate should be 50 %. This is something that we see in egg donation cycles when the eggs are amazing. If patients have lower blastocyst rate, we have to see why. Embryology says: if embryos are fragmented, then between day 0 and day 3 it is the problem of an egg. If embryos are bad between day 3 and day 5, then it’s a problem of a sperm. So this is something that we have to work on to improve the blastocyst rate. Sometimes patients work on things very hard, take vitamins, DHA, some of them are even on a growth hormone protocol, but we still do not generate quality eggs that are good enough to develop more blastocysts. So you just have to work hard and after 3-4 embryo banking cycles, it’ll be the time to put the cards on the table and say: I want to know if they are genetically normal or not. You’re young and you’re fighting so much, so if we generate 1-2 genetically normal embryos, you still have 70% chance for transfer to be pregnant with this kind of embryos. Even though your blastocyst rate is low, you still generate 1 genetically normal and healthy embryo and you still have the same pregnancy rate as everybody else.

I would like to ask about the quality of pre-implanted embryos. I have embryos graded 5BB. Is it good enough for transfer?

In order to answer this question I would need to know how old you are. I’m famous for saying that it is the same story with good-looking embryos as with good-looking men. I still believe that when you are older than 35 years old, there is no time anymore for experiments. Especially when you’re older than 35 years old and only every other embryo is good. Sometimes this BB quality embryo is healthy and sometimes it’s not healthy. That is something nobody knows. So in order to answer this question you will need to add your age and then I’ll tell your chances. I’m not a fortune-teller, it’s just evidence-based medicine.

What are your thoughts on Neupogen? My neutrophil levels are quite low and I wonder if this could be affecting implantation. Would Neupogen prior to transfer help?

We’d have to see the neutrophils in the blood or in the lining. We know that results in the blood are not as sensitive as in the lining. So maybe it’s worth doing the uterus lining biopsy for immunology to see what is going on there. I had to do a PhD in immunology a couple of years ago, and I introduced all this immunological knowledge into the work. We have quite a good study that we use. We do not use Neupogen, we use Granulocyte and it’s for repeated implantation failure. We have a protocol when patients are putting colony-stimulating factors with subcutaneous injections every day in the first week after the transfer. And then twice a week, from the second week until week 8. The results with recurrent implantation failure are quite promising. So the majority of our patients undergo immune modulation with this medication after a couple of unsuccessful transfers. But usually, the results are in the biopsy and it’s not about neutrophils in the uterus lining biopsy but it’s about Th1/Th2 ratios. If you say neutrophils, I think you’re talking about blood. So take it one step at a time, verify the result through the uterus lining biopsy and let’s take it from there.

Is there an average rate at which endometrium grows per day on estrogen?

No, there is not. What we usually do is we monitor the first scan after a patient starts with estrogens (day 7 – day 10) of a cycle to see how thick the lining is. There is a formula to follicles that they grow 2 millilitres every 2 days. There is a formula in gynaecology about hCG, the pregnancy test. If hCG doubles every two days, it means that the pregnancy develops correctly. I haven’t heard about any formula to the uterus lining growth. But if we expect endometrium to be seven millimetres on day 7 or day 10 of a cycle, the lining has to grow 1-1,5 millimetre every day. But of course, there’s no formula, it’s just fun now.

Does adding progesterone make the lining plateau?

Yes, it does. What we do is we grow the lining with estrogens. We want the result to be more than 7 milimeters. I know that progesterone will freeze the lining, it will stop the growth. There is only one medication that doesn’t stop the growth of the lining with progesterone. It’s Viagra vaginally, tried by patients. So yes, progesterone will stop the growth of the lining. It’s definitely true.

With egg donation, do you choose genetically close eggs?

You’re asking me if we match HLA? No, not at all. We match HLA to KIR (killer cell immunoglobulin-like) receptors. When we have patients coming and we know that they’re carriers of sickle-cell or thalassemia or some other diseases, we either match them with the donors through the HLAC test or if there are specific genes with a very high polymorphism, we do the gene sequencing of the donor to make sure that donor is not a carrier of the same mutation. We do not choose genetically close eggs. The donor has to be matched specifically to certain cases and certain patients.

Is there anything one can do to help the lining of the womb for embryos to implant properly?

Definitely yes. Firstly, it’s anatomy: no fibroids, no polyps. Secondly: the implantation window biopsy. Thirdly: proper thickness, meaning a proper adjustment of estrogens to your body to achieve lining of more than 7 millimetres. There are no more secrets.

How long is the ER Map valid? Forever, like genetic tests, or are there any factors that can change the implantation window and the ER Map result?

Usually, the receptivity assay is valid for a year. If it’s more than a year, we would like to confirm it. What is changing the receptivity assay results? Surely delivery and D&C procedures on the uterus.

I had two cycles of egg donation (fresh and frozen). I got pregnant with one frozen embryo. It developed to heartbeat. But I started bleeding at that point. My doctor considers the fibroids in my uterus not to be in the endometrium cavity. But there are many. I finally lost the baby. What can I do to improve my situation. Could there be another reason, why it did not work? You mentioned clotting, for instance.

We’ll have to do a second egg donation cycle and do the workup from the beginning. We have to see if the fibroids have an impact or they do not have an impact. How do I confirm or exclude something in medicine results? I have to work to prove that it is the truth. So, in this case, we have to do hydrosonography. If we see that fibroids do not have an impact, we do the uterus lining receptivity to see your implantation window and then we transfer. If we see that fibroid has an impact, they have to be surgically removed. This is anatomy. Then there are the embryos, they have to develop to the blastocyst stage. With the second egg donation, I would test them genetically. Then we have to see if your immunology is average. It means that you either accept HLA of majority donors or we have to match them to you specifically. When blood is too thin, it creates problems. But it’s super easy to exclude or confirm by blood clotting tests. If there are too low platelets, because you have ITP and haematological issues, then we send you to a specialist with blood diseases. And then we know what to do with you. You’ll probably have to take Prednisone. But this doesn’t happen often, maybe once or twice annually. On the other hand, too thick blood means there’s a factor V mutation or there are prothrombin factor II mutations. Then you have to take Heparin until the end of pregnancy. So blood clotting it super easy to confirm or exclude through blood tests.

If my lining is healthy (10 millimetres) and ERA results moved my window an extra day and I have no other complications. If my 5BA donor egg did not implant, what would be other possible reasons?

Sometimes BA embryos look amazing but they are genetically abnormal. Sometimes even genetically normal embryos do not implant because they are too weak to make it through. So I wouldn’t cry that I had one transfer and it failed. I’d just work more. I would go for one more transfer. With healthy, good quality embryos you have a chance to be pregnant in the next cycle.

What is the D&C procedure? The one that can change the implantation window.

D&C stands for dilation and curettage. If somebody has had a miscarriage or if somebody has abnormal uterine bleeding or if embryos are genetically abnormal and we see that we have to terminate the pregnancy, this changes the implantation window.

Will you recommend to have 1 or 2 embryos transferred in the first implantation? What are the risks of transferring 2 embryos when you’re over 40?

Blastocyst culture, genetically normal embryos, single embryo transfer – it’s a key answer. The embryo selection is so sensitive that the implantation rate of this kind of embryos is over 70%. As I say, through genetic pre-testing of embryos I erase your age. I bring you to pregnancy rates of somebody who’s twenty-thirty years old. If I transferred two genetically normal embryos at the same time, I’d do a selective double embryo transfer, your twin rate is more than 50%. I would like to avoid that because twin pregnancies are always high-risk pregnancies for you. Your cervix can dilate. You can stay in bed till the end of pregnancy and I have to do the C-section. So these are the reasons why we recommend single embryo transfer. If you’re over 40 and your embryos are not tested genetically, you have to be aware that the euploid rate is 10%. So we can discuss double your transfer but with blastocyst culture, I’d still prefer a single one.

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Authors

Natalia Szlarb, MD, PhD

Dr Natalia Szlarb a Gynaecologist & Fertility Specialist at UR Vistahermosa, Alicante. She graduated from a medical university in Poland in 2002 and then worked in gynaecology and obstetrics wards at several German hospitals. She also participated in international internships in Egypt, Brazil and Poland during her medical studies. In 2011 Dr Szlarb obtained her PhD in Immunology in the United States of America. She has extensive experience in IVF with donor eggs and is known by patients as a friendly and warm doctor. Dr Szlarb speaks fluent English, Polish, German and Russian.

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