In this session, Dr Halyna Strelko, the Co-founder & Leading Reproduction Specialist at IVMED Fertility Center, Kyiv, Ukraine, who has discussed one of her past patient success stories starting from the diagnosis, treatment options and final successful outcome. Dr Strelko has also provided useful advice for advanced maternal age (38+) patients.
Advanced maternal age (38+) – real-life case
- a 39-year-old-woman, with PCOS, 2 previous failed IVF attempts, male partner diagnosed with teratozoospermia
The patient came to the clinic (IVMED) after 2 unsuccessful IVF treatments. The first cycle was cancelled because of OHSS syndrome, and in the second, with mild stimulation, only a few eggs were retrieved, and the embryo arrested at the cleavage stage. In the first cycle done at IVMED, a short agonist (a-GnRH) protocol was used with hCG triggering with a low dose because the patient previously had OSHH. Twenty eggs were retrieved, but those were not of very good quality. Fifteen eggs were mature, we only got 2 blastocysts, it was a fresh embryo transfer because, at this time (2009), cryopreservation programs were not so good. She finally delivered a healthy boy.
After that, the patient continued to do more IVF cycles. In 2013, antagonist protocol (ant-GnRH) with a GnRH trigger was used, a lot of eggs were retrieved, 40 oocytes and 8 embryos in total. They were cryopreserved on day-3. Three embryos were transferred, but the result was negative. In 2015, again, 2 embryos were transferred, but the result was still negative. In 2016, a new stimulation protocol with urinary gonadotrophins was done, and again, we had a very good response. We retrieved 35 oocytes, we got 6 embryos that were cryopreserved after 72 hours because of the quality. The embryologists were afraid that they will not develop into a blastocyst stage. Between 2016-and 2018, 3 cryotransfers were performed, but without success. In 2019, a new protocol with a low dose of gonadotrophin was suggested, but again there was no blastocyst. In 2020, antagonist protocol was done, but we only got a poor quality embryo which was transferred in a fresh transfer, but it ended in a negative result. The patient returned to IVMED in 2021, she had 2 remaining frozen embryos from 2015 that were not of good quality.
Results of examinations
A standard investigation was done, genetic tests were performed, and other tests such as general blood tests, coagulation tests, infections, and hormonal analysis were performed, all tests were more or less normal. The patient’s AMH was quite high, 8.2 ng/ml. The patient’s husband had a problem with the morphology of spermatozoids, only 3% were normal and also he had an increased DNA fragmentation of sperm.
Strategy
We discussed this with the couple and advised against transferring those 2 embryos due to low embryo quality, especially since they had 8 unsuccessful attempts already. It was necessary to create new embryos and try to achieve a better result and receive more eggs of better quality. Try to avoid OHSS, as well as focus on sperm selection methods to see if the embryos are normal or abnormal from a genetic point of view. We wanted to do a frozen transfer in a natural cycle because with
Hormone Replacement Therapy (HRT) the endometrium does not react so well. We also decided to use growth hormone during the stimulation because several scientific studies showed that if we are using growth hormone, we receive better quality eggs, better fertilization, better pregnancy and a live birth rate.
As shown on the slide, there is a significant increase in outcomes when we are using growth hormones, especially in advanced reproductive age patients. The new protocol was used with the short antagonist, Pergoveris, it is the FSH plus LH. We know that in late reproductive age, sensitivity to LH is a bit decreased, and the ovary needs additional LH for normal steroidogenesis. We also used Zomactone growth hormone, and for triggering we used Diphereline to prevent ovarian hyperstimulation syndrome. We finally received a good number of eggs, 35 eggs, 20 were mature, which was not bad however, the quality was very poor. Since the oocytes were so fragile, we decided to do ICSI on 9 eggs and 11 with standard IVF. We also decided to do
Piezoactivation of oocytes after ICIS, where we use a special device where we put eggs just after the fertilization, and there is an increase in piezoelectric activity, and this permits to increase in the concentration of ions of calcium inside the oocytes, and we know that these allow oocytes to fertilize.
All of these processes depend on calcium concentration inside the oocytes. Our embryologist proposed to do a specific method of sperm selection and preparation because the patient’s husband had high DNA fragmentation, so we should select the best sperm cell without fragmentation and improve the probability to have a healthy embryo and a healthy baby.
The embryologists used a specific needle for the ICSI procedure for fragile eggs, and as a result, we received 9 blastocysts in total after the ICSI procedure, but we didn’t receive any blastocysts after IVF. W were surprised because the eggs were fragile, we were afraid that we will not have a good result after ICSI, but it was just the opposite, and it was probably due to Piezoactivation of oocytes. We got 9 embryos, which we biopsied, we got 3 euploid female embryos, and the couple was very happy because they already had a baby boy.
After we prepared the patient with a natural cycle and when her dominant follicle reached 18 millimetres, we have done trigger with hCG, and after the luteal phase was supported with 600 milligrams of micronized progesterone, 2 euploid embryos on the 7th day after triggering were transferred, and we received a positive result, and now the patient is pregnant at 8 weeks.
Causes of age-related fertility decline
After presenting one of her patient cases, Dr Strelko moved on to present some data on the results of IVF treatment depending on the patient’s age. After 38-40 years old, we can see a decrease in pregnancy and live birth rate. Some causes depend on the egg quality, and some of them don’t depend on egg quality, however, they can still decrease the probability of pregnancy and delivery.
Causes that are dependent on egg quality are mostly meiotic (abnormal fertilization), abnormal cleavage, a genetic anomaly of eggs, and the problem with eggs’ cytoplasm, as a woman’s age increases, normally the number of mitochondria decreases dramatically. If someone is 30 years old, there are around 250 000 mitochondria in the egg, but if someone is over 38 years old, it is more or less half of this number. Also, ovarian reserve decreases, which means that there are fewer eggs. Normally after 35-37 years old, it is around 1% of the basic number of antral follicles. Other causes not related to egg quality are myomas, endometriosis, immune diseases, and general health issues like diabetes which can decrease the probability of birth rate.
Egg quality is not only a nuclear issue, it is mitochondrial dysfunction, and it may also be shortening the telomere. We know that the cleavage of each cell depends on the structure of this end of the chromosome, and when it becomes short, the division of cells is not possible. Spindle instability is also increasing with age, so it may increase the number of genetically abnormal eggs and embryos.
Main mechanisms of aneuploidy in embryos
It’s important to remember that not only do eggs produce a genetic issue in the embryo. The paternal part is still around 20%, so it’s also necessary to pay attention to the sperm quality, and sperm preparation and perform some additional tests on sperm because it may give, around 20% of the whole genetic issue.
Some scientific studies present that as the male’s age progresses, the sperm concentration, motility, and morphology decrease and DNA fragmentation increases. If we compare the percentage of DNA fragmentation and the blastocyst formation rate and aneuploidy rate, we can see a close relation. The higher DNA fragmentation we have, the fewer blastocysts and more aneuploid embryos we will receive. If the age of the father is more than 50 years old, DNA fragmentation is around 40% and around 75% of genetically abnormal embryos.
When we compare the quality of eggs at IVMED, where we have a big bank of donor oocytes, and we compare donor oocytes and patient oocytes in terms of some morphological problems, we can see that all morphological problems of oocytes increase with the age of women. We can have some morphological issues with donors’ eggs, but with the age, the number of these issues becomes more important.
When we study eggs of patients who are more than 38 years old, we find only 36% of morphologically normal oocytes, and if we don’t have a big number of oocytes, sometimes it’s not possible to get good quality oocytes, and that’s why there is no possibility to receive the good quality embryo.
Another issue that can be related to women’s age is the function of the immune system. The luteal phase should be very active and adequate to support a normal pregnancy, and in advanced reproductive age, the function of the immune system may have incorrect functioning because the immune system is participating in the formation of the corpus luteum. This is the structure in the ovary after ovulation that produces progesterone, therefore, the production of progesterone may be compromised. Progesterone regulates and induces the reduction of pro-inflammatory cytokines and increases the production of interleukin (IL)-4, and it protects the pregnancy from spontaneous abortion. If this mechanism is not working well, it may increase the chance of spontaneous abortion, and we can see that in women of advanced maternal age, spontaneous abortions occur much more often.
Methods that can improve the outcome
Sperm preparation
What can we do to improve the outcome? What we do at IVMED in such cases is use a specific sperm preparation called microfluidic sperm sorting. This helps to avoid centrifugation and additional damage to the DNA of sperm cells. On the slide presented, there is a comparison of the standard sperm selection method with this new method which we call sperm chip. We can see that in this case, with better selection and more gentle preparation, the fertilization rate is around 7% higher, blastulation rate is much higher, and pregnancy rate is mostly 10% higher. In some cases, it might be very helpful, especially in cases discussed during our event.
Piezoelectric oocyte activation
This method permits to improve the concentration of calcium inside of the oocyte, and it gives more possibility to achieve normal fertilization in, the normal cleavage stage. With piezoelectric activation, we receive a much better result in fertilization, in the day-3 cleavage stage and also in blastulation, especially in cases of poor quality or advanced reproductive age.
Morphological oocyte assessment
We are also doing a morphological assessment of the oocytes, we are looking for zona pellucida and detecting a meiotic spindle because if we destroy the spindle during the ICSI procedure, it will decrease the number of viable embryos. In case of a difficult situation or poor egg quality, we detect a meiotic spindle with a specific microscope device, and it permits us to avoid this spindle destruction. Each oocyte is observed before the fertilization process, and the location of the spindle is checked with a specific time-lapse camera which is very helpful for the ICSI procedure.
Conclusions
I would like to say that decrease of pregnancy rate in advanced reproductive age not only depends on aneuploidy rate but also on the problem with spermatozoid, so we need to do a deeper examination of the male partner. We also need a specific strategy for stimulation because the more eggs we receive, the higher possibility to achieve a good quality embryo. We can use artificial intelligence for embryo selection sometimes, it may be very helpful because they analyze thousands of different parameters. Time-lapse technology might be very helpful because that will help us avoid the change in temperature and Co2 concentration. We put the fertilized egg inside of the time-lapse incubator, we follow all stages of development, but we don’t touch the embryo till the moment of transfer. In all cases of advanced reproductive age, we are doing artificial oocyte activation, and it is very helpful, in some cases where we didn’t get any blastocysts before, we were able to receive 5-6 good quality blastocysts. We are also using advanced PGT-A tests, we have also started to use Non-invasive PGT-A, where we don’t take a biopsy, and we don’t need to touch the embryo, it may be also very helpful and improve the probability of this embryo to implant.
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