Preimplantation genetic diagnosis for treatment of RIF in IVF

Evangelos Papanikolaou, MD, PhD
Founder & Reproductive Medicine Specialist, Assisting Nature – Human Reproduction & Genetics

Genetics PGS / PGT-A, Miscarriages and RPL

PGD for patient with recurrent implantation failure
From this video you will find out:
  • How often implantation failure happens?
  • What medical intervention can be taken to diagnose the cause?
  • When and why aneuploidy embryos happen?
  • What are the reasons for frequent aneuploidy?
  • How to assess the quality of the embryos?
  • What are the results of the genetic analysis of the embryo?
  • What is PGT-A? Is it invasive?
  • What are the real patient cases at Assisting Nature?

Is PGD a solution if I have had recurrent implantation failures?

In this session, Dr. Evangelos Papanikolaou, MD, Ph.D., Founder of Assisting Nature, Thessaloniki, Greece has been discussing RIF patients’ cases and talked about preimplantation genetic diagnosis and when it is indicated.

Firstly, according to Dr Papanikolaou, the aim of higher success in IVF is the standard goal for reproductive specialists. It is mentioned that although the establishment of pregnancy is perceived, there is a prevalent 50% percent of implantation failure or miscarriages. Therefore, the goal is not only limited to pregnancies but to live births.

Implantation failure – definition

Two scenarios of implantation failure are prevalent. On the one hand, real implantation failure implies no implantation with a negative HCG test. On the other hand, there is no visible formation of a gestational sac despite detectable HCG, which is called chemical pregnancy. Thus, it is common knowledge that recurrent implantation failure encompasses the transfer of three high-quality embryos at least or more than six blastocysts.

Implantation failure – reasons

The first reason is said to be mother-related as age plays an essential factor in implantation since the higher the age of the patient, the higher the chances of abnormalities occurring because of genetic factors. Anatomical reasons, such as the uterine septum, are also said to be a second reason.

Some other factors include immunological reasons, infections, subclinical endometritis, and others that can also alter the endometrium receptivity. For instance, the ability of the uterus to allow implantation of the blastocyst can be improved either by performing endometrial scratching or infusion of growth factors into the cavity.

Factors arising from the male part involve DNA sperm fragmentation and a low number of sperm.


The correct term for this situation is termed as aneuploidy, which implies the abnormal number of chromosomes, which is normally 46. Aneuploidy is the most significant single factor affecting early pregnancy failure.

Aneuploidy can result in:

  • Implantation failure
  • Miscarriage (65% of abnormal embryos end in spontaneous miscarriages)
  • Congenital disabilities
Aneuploidy: when and why?

The reason for abnormal embryos in 80% of the cases is due to advanced maternal age. As regards sperm, only an evident 2% is present, while errors in embryo cell divisions after fertilization encompass 20%.

Advanced Maternal Age

Looking at the graph shown, we can see that the higher the maternal age, the higher the percentage of aneuploidy. The higher the aneuploidy rate, the lower the pregnancy rate. Even if a good-quality blastocyst is transferred, the pregnancy rate decreases with age.
What are the most frequent aneuploidies?

  • Trisomy 21 (54%)
  • Trisomy 18 (13%)
  • Trisomy 13 (5%)
  • Klinefelter syndrome
  • Triple X syndrome

Until 2000, only morphological criteria were available for the selection of embryo transfer, as a shortage of knowledge about the genetic background of the embryos was evident. In addition, 44% of good morphology embryos are said to be aneuploid after genetic testing, although this has changed due to preimplantation genetic diagnosis (PGT-A) arrival.

PGT- A (Preimplantation Genetic Testing for aneuploidies)

Only a very small part of the embryo is used, and it is called the trophectoderm, which later on will become the placenta. After the testing, the results will show whether the embryo is euploid, aneuploid, or a mosaic embryo (euploid and aneuploid cell lines). After many studies performed, nowadays, mosaic embryos can be transferred as well, as long as up to 40% of mosaic embryos are transferred which can result in a live birth. Trophectoderm (day 5) biopsy followed by vitrification of the embryos for later selection of embryos after PGT-A testing with NGS is a gold standard nowadays.

Who should have IVF-PGT-A?

  • Women of Advanced maternal age (AMA)
  • Couples with severe male factor (sperm FISH, high DNA fragmentation (SMF)
  • Couples with recurrent miscarriages (RM)
  • Couples with recurrent implantation failure (RIF)
  • Couples with a known chromosomal abnormality

It is of the utmost importance to know exactly in which lab PGT-A, which is a complex technique, is performed, as it is an invasive procedure. In case performance is well executed, precise genetic results and an improvement in IVF outcomes are expected.

Real-life cases

Case 1 – a 36-year-old woman, with a partner who had normal sperm parameters, had gone through 5 frozen embryo transfers, they had 9 blastocysts, 2 were the negative result, 1 chemical pregnancy, and 1 spontaneous abortion

They came to the clinic (Assisting Nature), for their next cycle, and this time PGT-A testing has been suggested. They got 7 blastocysts, however, only 3 blastocysts were euploid. After the first single embryo transfer, the patient got positive results that ended in a live birth.

Case 2 – a 29-year-old woman, with a partner (33) had azoospermia, ICIS with TESE procedure was performed, and 5 embryos have become blastocysts, they had gone through 3 embryo transfers, which resulted in 2 negatives and 1 spontaneous abortion.

Once again, PGT-A testing has been suggested, only 3 blastocysts were obtained that were tested, 1 embryo was euploid, and 1 was a transferrable mosaic embryo. The first euploid embryo was transferred, and the patient got pregnant.

Case 3 – a 37-year-old woman with autoimmune syndrome and relatively normal AMH level, her partner had normal sperm parameters, they had gone through 2 previous IUIs, that were negative and 3 frozen embryo transfers that did not work as well

After coming to the clinic, they were advised to go for PGT-A, they got 5 blastocysts, from which 4 were aneuploid and 1 transferable mosaic embryo. After the transfer of this 1 transferable mosaic, she got pregnant (ongoing pregnancy at 7 weeks).


Recurrent implantation failure (RIF) is a complex problem with various etiologies and mechanisms. It’s crucial to reevaluate the couple, only after identifying the problem, it’s possible to proceed with the right protocol.


- Questions and Answers

What are the causes of more than 46 chromosomes in embryos? Or, causes of less than 46? Could high stimulation drugs be a cause?

The causes, as we’ve discussed earlier, are mainly the incapability of the egg to divide those 46 chromosomes into 2 pairs of 23. There is eternal incapability of the egg to divide strictly into those 2 parts to become a polar body. In the past, there were a lot of discussions about whether high stimulation might be the cause. And there were some papers back in 200-2004 regarding this issue. The truth is that the higher the stimulation the doctor retrieves more not mature eggs that at the end may divide into a blastocyst, but this blastocyst will be abnormal. The problem is not per se the drugs, the high stimulation, but that we retrieve all the eggs, all the follicles that we have in the ovaries and in the end we up with higher aneuploidy rate in that particular patient, but you should always to remember that if you’re a good responder If I’m a good responder and I produce a lot of eggs at the end, I  increase my chances of having normal eggs and in the end having normal embryos. High stimulation is not the real causative agent, but only it can change this result statistically. That’s why we need rational stimulation, so produce as many eggs as we can without any harm to your health, and we know that the perfect number of eggs is around fifteen. If we have more than this, there’s no benefit, less than this will cause lower pregnancy chances. So the maximum of eggs is around 15, and we should aim for this number if it’s possible.

In your experience what was the oldest woman, you have treated with euploid embryos and healthy live birth?

The oldest woman where we had 1 euploid embryo and live birth was 45. The chances of having a euploid embryo at the age of 45, as you know, it’s less than 1%. If you have 100 ladies at 45, in the end, one of them will become pregnant.

What are the costs for PGD testing?

With NGS, which is the technique that we perform our biopsy, the cost is 1,000 EUR for the biopsy itself, in Greece, plus 250 EUR per embryo. If you have one blastocyst, then you will pay 1,250 Euros. If you have 4 blastocysts, you will pay 2000 EUR.

In the beginning, we had 18 oocytes that fertilized, however, on day 6, only 3 are suitable for PGS. Why would only 3 be suitable, for PGS? Have they got to be very good embryos for PGS testing?

We do not biopsy everything, of course. The blastocyst should be above B or A to be tested. The problem here is that those blastocysts were already on day-6, which means that they were already very late in development. If you have a day-6 blastocyst, and it is perfect, the chances of this blastocyst to be euploid is around 10%, so it does not mean that if you have an excellent blastocyst on day-6, that this blastocyst is normal.  In this particular couple, the problem might arise either from the high number of eggs, maybe you have PCOS, or from the poor quality of the sperm or the poor quality of the stimulation. If they retrieved the eggs earlier, then you might end up with this late development as the blastocyst stage.

What are the criteria you use to select a mosaic embryo for transfer?

Mosaic means that if the blastocyst has 100 cells, 20 of them might be abnormal and 18 normal, or the opposite 18 might be abnormal and 20 normal. Mosaicism, in some way, is a normal procedure in the development of the blastocyst. It looks strange, but actually, it is a normal procedure. So the embryo tries to avoid the abnormal cells because mistakes do happen in the best families. They try to abort those abnormal cells to the trophectoderm, unfortunately, we do the biopsy, so actually, the trophectoderm sometimes might be the protection shield for the embryo itself where abnormal cells are deposited. This is the explanation of mosaicism.

On the other hand, of course, mosaicism does indicate the embryo incompatibility to divide itself correctly. This might be the problem that in the end, half are abnormal, and half are normal, so these embryos cannot give life to a human.

The criteria that we use to select a mosaic embryo is that if we have less than 10% of mosaicism and specific chromosomes, our geneticist advises for them to be transferred. You can find this information in our leaflets on the Internet.

Do women over 42 often have enough embryos to perform PGS? Did you talk about 5 embryos?

I talked about 5 or 6 embryos, in total, from different IVF to define that someone has recurrent implantation failure. So actually, we only need 1, as you know, the more we have if we are over 42, of course, the better chances for finding euploid embryos. On the other hand, if you’re 42 and your AMH is low, and if you take medications, but you won’t produce more than 2 eggs, we can do several natural cycles. We can deposit 2 or 3 blastocysts after 9 natural cycles, for example, and to do genetic testing on the 3 blastocysts after 1 year of trying that should be the maximum, so a doctor needs to put the threshold for how long you should try and out of this 2-3 blastocysts if you’re lucky, you can have a normal blastocyst. At the age of 42, this probability is around 15%.

I’m choosing between doing NGS and another method of PGS where the transfer is the same month. How much higher is the risk of Down Syndrome if I do regular PGS instead of the new NGS? And the miscarriage and implantation failure rates?

There is no reason for fresh transfer anymore. As we know, the endometrium is always better outside the simulation. On the other hand and NGS is an excellent technique at the moment for identifying abnormalities. The only reason to perform it the old way with micro ways of PGS is when I have frozen embryos, for example, from another trial or another center, and I do transfer them to another center, and I want to know if I have 4 blastocysts, for example. Let’s say I have 7 blastocysts in another center, and I transfer 4 of them and I have no pregnancy and I don’t want to continue, and I still have those 3 blastocysts. If I want to know if these 3 blastocysts are genetically normal, only then we can do this old way of PGS on day-5 and transfer them the next day on day-6.

Would clinics be willing to do regular PGS on some embryos (for immediate transfer) and NGS on the rest and freeze? (I have PCOS, so I might get many)

It’s up to you, and you should have a thorough discussion with your fertility specialist and your senior embryologist. Let’s say you have 10 blastocysts, then we would freeze, let’s say 4 of the blastocysts without doing PGS, and we do NGS on the rest of them, waiting for the result.  I don’t agree with doing a fresh embryo transfer, and in women, especially in a woman like you with PCOS, it is malpractice.

Can ubiquinol help to fortify the eggs?

I don’t think that all these additives, in the end, can improve the quality of the eggs. I think that in the coming years, we will have a real pharmaceutical agent that makes these mitotic errors not to happen anymore in the egg, and a lot of women within the next 50 years will not need any IVF because they would produce euploid eggs and euploid embryos.

For which reasons sometimes embryos do not reach the blastocyst stage? Is it because of the quality of the egg? I did 4 IVFs, in the first one, I had 4 blastocysts (I was 30), and on the 3 other IVFs, I only got 4 embryos in total, and they always transferred them on day-2 or 3. All of them were negative. I never got them tested, but for the next IVF, I would ask to test them.

Sometimes it’s the sperm, and sometimes it’s the egg, so they don’t know if the eggs don’t look really well if they’re dark with a lot of micro vacuoles. They can say that this lady does not produce good eggs from the beginning and say they would not expect to have good blastocyst, whatever is the fertilization rate. On the other hand, if the sperm is bad, especially morphologically, they are looking at it for 1 or 2 hours to identify morphologically good sperm, then they inject it into the egg, which indicates a male factor. Until the day-3, the embryos develop with the machinery of the egg, and only after day-3 it comes from both the female and male genetics, so the combine and work together to divide into a blastocyst. We say many times that if after day-3, we don’t have a blastocyst, it indicates bad sperm. But we don’t say this if the embryologist sees that the eggs are bad morphologically from the beginning. You were young when you had those 4 IVFs and getting only 4 blastocysts is a very bad number. I cannot give more explanations having only this information, but definitely, you should do the genetic testing of your embryos in your next cycle or one after that, and only then you can decide if going for egg or sperm donation is an option.  Do not decide that after 1 abnormal genetic testing right away because it can only be a bad coincidence of a bad cycle.

Is there anything that can be done to improve sperm morphology? Would you have any recommendations?

Yes, with sperm it is much easier.  Firstly because if the embryologist is experienced, she or he can choose a good morphological sperm.  So the first one is the selection per se on the day of ICSI. Another thing that we can do is PICSI, we do sometimes select morphologically better sperm.  We can, of course,  give some antioxidants to help the sperm morphology, but I sometimes laugh because if someone is still smoking, and he’s taking all those antioxidants, it is useless to take all this medication, there is no improvement of the sperm.

Do you tend to get higher quality embryos with the short protocol than the long one?

I don’t believe that we have better quality embryos with any of these protocols and the best protocol is when we prepare it individually for the patient. From the studies that we’ve done on patients from the same category, let’s say women who are less than 39 with both protocols, the percentage of the euploid embryos is the same. I don’t think that there is any interference in the protocol with the genetic quality in the end.

What would be the maximum size of the follicle at trigger?

The optimal size for the follicle to be triggered is 17-20. But keep in mind that one doctor will measure it 17, and the other one 21. There is a lot of deviation from doctor to doctor. However, answering your question, I think that the follicle that is more than 22 or 24 does not have a chance to produce a good quality embryo.

What could be the reason for empty follicles or follicles that grow fast, but the eggs are not mature?

90% of empty follicles, it’s a mistake of the patient or the doctor or the ovulation triggering injection. Empty follicle syndrome (EFS) is what we call when we have empty follicles repeatedly, it happens very rarely.  
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Evangelos Papanikolaou, MD, PhD

Evangelos Papanikolaou, MD, PhD

Evangelos Papanikolaou, MD, PhD, is a Reproductive Medicine Specialist as well as a world-class recognized specialist in fertility treatment. Dr. Papanikolaou is the author of dozens of medical publications and has been at the forefront of several innovations and improvements in IVF treatment and is the main visionary and founder of Assisting Nature IVF Unit. He is ESHRE and EBCOG accredited as a Reproductive Specialist.
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