Evi Timotheou, BcS, MSc
Senior Clinical Embryologist, Lab Director at Assisting Nature, Assisting Nature – Human Reproduction & Genetics
Category:
Genetics PGS / PGT-A, Success Stories
In this webinar session, Evi Timotheou, BcS, MSc, Senior Clinical Embryologist, Lab Director at Assisting Nature, Thessaloniki, Greece, has talked about the indications for Pre-implantation Genetic Testing (PGT-A). Evi has presented 5 different IVF cases and explained all the steps taken that allowed the couples to achieve a pregnancy.
Evi started her presentation by introducing a brief history of genetic testing. Since 1990, genetic testing worldwide has increased, and indications for its use have expanded. Originally, the goal of PGD was to detect and eliminate the embryos that contained monogenic X linked disorders.
Nowadays, PGS and PGD have expanding roles in reproductive medicine. According to The International Glossary on Infertility and Fertility Care of 2017, Pre-implantation genetic testing is a test performed to analyse the DNA from oocytes or embryos for determining genetic abnormalities. The names have been divided, PGS is now called PGT-A (Preimplantation Genetic Testing for Aneuploidies), while PGD is now called PGT-M (Preimplantation genetic testing for monogenic/single gene defects) and PGT-SR (Preimplantation Genetic Testing for Chromosomal Structural Rearrangements).
The most common indications for the use of PGT-A are:
Around 40% of cases where PGT-A is indicated are because of advanced maternal age. There are different biopsy methods. There is Polar Body Biopsy, Blastomere Biopsy, Trophectoderm Biopsy. Throughout the years, different genetic techniques were used that improved chromosomal analysis. In 1990, FISH PCR was applied, and after 2008, other techniques like aCGH (Array Comparative Genomic Hybridization) were mostly used. Finally, in 2014, with the Next-Generation Sequencing (NGS), a new era began.
On the graph shown where FISH, which was the first method used, we can observe a reduction in implantation and an increase in aneuploidy with advanced material age even though only a few chromosomes were tested. The transition from PGS 0.1 (FISH), which has only 9 chromosomes, to the transition of PGS 2.0 where all 24 chromosomes were analysed using Comprehensive Chromosome Screening (CCS). Numerous studies were presented about the ideal method of biopsy and analysis for each indication.
Nowadays, with the new screening techniques, we can observe and categorize the embryos as normal, mosaic and abnormal. According to The Preimplantation Genetic Diagnosis International Society (PGDIS), less than 20% of aneuploidy in an embryo is considered normal. If it is between 20 to 80%, they are considered mosaic, and more than 80% is categorized as aneuploid and is not suitable for transfer.
Mosaicism is a state in which there is more than one karyotypically distinct cell population arising from a single embryo. It’s a huge area, and it’s not easily covered.
The first woman was 40 years old with a regular cycle. She didn’t have any pregnancies before. Her partner had normal sperm analysis, they had 2 previous IVF cycles in other centres. She started with 2 Intrauterine Inseminations (IUI) and had negative results both times. Then, the couple proceeded with the first IVF, 6 mature oocytes were retrieved, 5 fertilized, fresh 2 day-5 embryos were transferred, she had a positive result that ended in a spontaneous abortion. Then she had a second IVF cycle, another 6 oocytes were retrieved and fertilized, she transferred 2 day-4 embryos that ended in a biochemical pregnancy.
When she came to our clinic (Assisting Nature), we decided to do IVF with PGT-A and freeze all embryos because when we perform NGS we have to freeze them first, and then we wait for the results. We suggested doing a hysteroscopy, and the doctor suggested a long agonist protocol. We got again 6 oocytes, and 5 were fertilized, we had 5 blastocysts for biopsy. We do the file, the blastocyst, the biopsy. The results showed 4 aneuploid embryos and 1 euploid. Therefore, we transferred that 1 euploid embryo and the result was positive. A baby girl was born at 39 weeks.
The patient was 41 with irregular cycles. She had natural pregnancies before, the first one was a 9-week pregnancy, but she lost the baby. And the second, she had an 8-week pregnancy loss and a 3rd biochemical pregnancy. In 2019, she did an endometrial biopsy, and it was discovered that she had chronic endometriosis. Her partner had a normal sperm analysis, they didn’t have any previous IVF cycles.
The plan was again to do IVF with PGT-A and freeze the embryos. Hysteroscopy was performed before embryo transfer. classical antagonist protocol was done. We retrieved only 3 oocytes, but all 3 were fertilized. We got 2 blastocysts, and with the result, we had 1 euploid and 1 aneuploid. We transferred the euploid embryo and the couple got a positive result with a live birth at 38 weeks.
The couple where a woman was 42 and her partner was 47. The partner had preserved sperm before he underwent chemotherapy for non-Hodgkin’s lymphoma. They had previous IUIs cycles in other centres that were negative, and they had an IVF cycle with 7 oocytes, from which 7 were fertilized, and they had 6 blastocysts. They proceeded with 2 blastocysts and had the first embryo transfer. The result was positive, but ended in an 11-week pregnancy loss. Then they proceeded with the frozen embryos, again she had a positive pregnancy test, but a 6-week pregnancy loss and then the last 2 frozen embryos were transferred, but she had a biochemical pregnancy.
When they came to our clinic, we suggested PGT-A because of her age, it was normal to proceed with a PGT-A. We planned IVF with classical antagonist protocol. In our clinic, we had 6 oocytes, 6 were fertilized and we had 4 blastocysts. We only got 1 euploid embryo, the rest were aneuploid. We proceeded with the embryo transfer, and the result was positive, and we had an ongoing pregnancy. Now, she is at 31 weeks, and she is well.
The couple had a pregnancy through natural conception and a 2-week pregnancy loss. Then, they proceeded with IVF cycles because of her age, she had 3 consecutive IVF cycles in other centres. Firstly, she had 5 mature oocytes, 4 were fertilized with 2 blastocysts, she had an embryo transfer, but with a negative result. Then she had another IVF cycle with 4 oocytes, 2 fertilised, and 1 blastocyst was transferred, which resulted in a negative. In the last embryo transfer, she had 4 oocytes, 3 fertilized, and they got 2 blastocysts, resulting in a biochemical pregnancy.
We planned the protocol and PGT-A. We proceeded with the antagonist protocol, she had the first IVF cycle where we retrieved 4 mature oocytes, 4 fertilized, 2 blastocysts were biopsied, but unfortunately, we didn’t have any normal embryos. All blastocysts were abnormal. They proceeded with a second IVF cycle where 5 mature oocytes were retrieved, 4 fertilized, and we got 3 blastocysts. This time, they had 1 euploid embryo and 2 aneuploid embryos, and she had an embryo transfer. She had a positive result and an ongoing pregnancy, currently at 13 weeks of pregnancy.
A 44-years-old woman with irregular cycles and normal sperm analysis. She had natural conceptions with 2 biochemical pregnancies, and then she started the IVF cycles. Firstly, she had 5 mature oocytes, 4 were fertilized, 2 blastocysts with a fresh embryo transfer, and she had a negative result. Secondly, she had another cycle with 4 mature oocytes, 2 were fertilized, 1 blastocyst was transferred, and she had a biochemical pregnancy.
Once again, we planned PGT-A, hysteroscopy, appropriate protocol for her. The doctor also discussed the egg donation option if there were no euploid embryos. In her first IVF cycles at our clinic, we got 4 mature oocytes, 3 were fertilized, 2 blastocysts were biopsied, but the result showed no euploid embryos. They had a second IVF cycle with 4 mature oocytes, all 4 were fertilized with 3 blastocysts. Unfortunately, all embryos were abnormal.
The couple decided to proceed with the last 3rd attempt, 3 oocytes, 2 fertilized, and we got 1 blastocyst. The result was also an abnormal embryo. Finally, the couple decided not to proceed with other IVF cycles and proceeded with an oocyte donation program.
On the graph shown, in the last 3 years in our clinic (Assisting Nature), between 2018-2020, the pregnancy rate is 90%. The clinical pregnancy rate is 85%, and the live birth rate is 70%. In most cases, a single embryo transfer was performed. In 65%, it was a single embryo transfer, and we can see the live birth rates are very high if somebody proceeds with the PGT-A method.
We believe in the non-invasive biopsy technique, but it’s still early to use it. Until now, we have 80% accuracy. We hope in the future that we will be able to use only this technique.
Usually, we don’t recommend it because it’s risky to thaw it. And if we thaw it, if the couple insists on thawing and testing it, we prefer to do the Array CGH. It’s the technique that we use, we have the result the day after. So we transfer it directly. And we don’t refreeze it.
No, it’s also for the repeated implantation failure, the severe male factor. We have couples mostly of advanced maternal age. 50% of the cases are of advanced maternal age, so over 40. There are also couples with some chromosomal rearrangements, but mostly of advanced maternal age.
Yes, we put all the embryos that we culture for the PGT-A in the time-lapse incubators, and we try to check if the genetic results are by the embryologists. It’s not 100%, so we cannot rely only on morphokinetics. We use it and the time-lapse, which are both helpful. However, we prefer to wait for the PGT-A results to be sure.
I think the analysis is about 250 on the embryo, I think. But I am not sure, it’s better to contact our customer team to make sure.
In our clinic (Assisting Nature), we do only day-5 embryo transfers, especially at the age of 40 plus. It’s normal to put 2 embryos on day-3 because we don’t know if they will arrive at day-5. I would prefer to test them first and then accumulate them to be sure that they are healthy. We could have 10 embryos, and only 2 could be healthy.
I think it’s not recommended because usually, the donors are very young. They are less than 32 years old. We don’t recommend it. We typically recommended it when there is a severe male factor, repeated miscarriages or advanced maternal age, but not in embryo donation.
The geneticist suggests if the mosaic embryo can have a positive pregnancy test or not. We discuss it first with a geneticist and the couple, and then the geneticist decides with the couple if this mosaic embryo is proper to transfer. We had a lot of couples that transferred mosaic embryos. We have live births from these embryos, and we know that they are healthy. We don’t transfer aneuploid embryos.
The rates, in general, are about 60% to 65%.
A lot of factors affect the results. Starting from the stimulation, the sperm, and then all the culture media that we use, the lab, the embryologists that proceed with PGT-A and do a biopsy of the embryos. That’s why all embryologists want to work in the best labs and try to minimalize any potential harm to the embryos.
I don’t know if you’ve done PGT-A on the embryos. It would be good to know, because if it is not about the embryos, then the doctors should perform more tests on the womb, e.g., ERA test, etc. If you haven’t done PGT-A before, possibly this is the reason for miscarriage, not necessary the womb. At your age, I would advise performing PGT-A on the embryos.
Nowadays, we have lots of programs, but I don’t know if it is better to only rely on Artificial Intelligence programs or not. New technologies always help, for sure.
Morphologically, the embryos could be AA, this doesn’t mean however that they are genetically normal. We sometimes see an embryo with Down’s syndrome in the lab, and it’s a very good blastocyst, but it is not a normal embryo. If we don’t do PGT-A we can say that morphologically the embryo is perfect, but genetically it is not healthy.
We prefer day-5 biopsy because then we have better results. Sometimes the embryos can autocorrect on day-5. It’s not worth doing a biopsy on day-3 as it can also harm the embryo.
This decision needs to be made by the couple. We suggest performing 1-3 cycles, but the number of cycles depends on the couple and how they want to proceed. There are a lot of factors, including psychological, financial, involved when it comes to deciding. If you have 1-2 blastocysts, it would be worth trying possibly, this 1 blastocyst is normal and could give a pregnancy.
Depending on the age, we perform a day-5 biopsy, freeze the embryos, and we wait for the results. We perform embryo transfer in the next cycle when we have a normal embryo.
It does not increase the pregnancy rates, as it does not change the result. If we have 10 embryos and 1 is normal after PGT-A analysis, possibly you’ll get pregnant in the first attempt. We don’t need to do 5 embryos transfers to find 1 normal embryo that will implant. It gives us the option to achieve pregnancy sooner without wasting more time.
We suggest transferring 1 embryo. If the couple insists on transferring more because they had gone through a couple of transfers already and they don’t want to wait, we sometimes agree to transfer 2 embryos, but not more.
I do the biopsy with a laser, and it didn’t affect the PGT-A outcome, so I don’t think it affects it. I think this depends on the embryologist and the experience, if it is going to be done badly, then yes, it can affect the result.
We use it at our clinic (Assisting Nature), and we believe it helps the implantation. We suggest doing that 1 month before the embryo transfer, the patient discusses this with a doctor, and he/she decides when is the best time to do it.
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