Stimulation protocols – what can possibly go wrong?

In this session, Dr Halyna Strelko, Chief physician, Reproductive specialist at IVMED, Kiev, Ukraine, focused on stimulation protocols, she explained what ovarian stimulation is, how it works, what kind of protocols are used, and what happens during this phase of your IVF journey.

The main goal of controlled ovarian stimulation (COS)

Statistics say that only 23% of all embryo transfers lead to delivery. This data is from 38 countries and, 1169 clinics in Europe. The main goal of controlled ovarian stimulation (COS) is to harvest enough eggs with acceptable quality, which can permit to obtain live birth.

The quality of oocytes depends on the female’s age and individual peculiarity. Generally, we need more eggs when the age advances. How many eggs is considered a good number? It depends on the woman’s age. On the graph shown, if a woman is less than 35 years old, she has a 50% of chance to achieve a pregnancy. It’s expected that she would have approximately 7-8 eggs. However, if a woman is 40 years old, she will have the same chance with around 20 eggs. If she is 43 years old, it is around 50 eggs. Normally at this age, we never get to this number. It means that the more advanced reproductive age we have, the more eggs we should receive to have an acceptable pregnancy rate.

What is a ‘normal response’

We need to get around 10-20 eggs until the age of 40 to have a 50% chance to have a live birth. The number of eggs depends on the protocol, Anti-Müllerian hormone (AMH), body mass index (BMI), medication dosage, sensitivity, and a lot of other things.

Dr Strelko then presented a slide with all available protocols used nowadays. It is a short protocol, classical short antagonist protocol, long protocol, short agonist protocol, protocol from luteal phase, natural cycle, mild stimulation, flare-up protocol, stock protocol. What kind of problems we can have during ovarian stimulation?

It may be an excessive response, we call this response ovarian hyperstimulation syndrome (OHSS), and we expect this when the Anti-Müllerian hormone (AMH) is more than 3.5. Sometimes, there is also a possibility for non-expected ovarian hyperstimulation syndrome (OHSS) to occur. We can get the OHSS even without having a high ovarian reserve, it can happen in a natural cycle, it is rare, but it can happen. We can have poor egg quality, it may be expected, as we have mentioned, it mostly depends on a woman’s age. Over 38-40, eggs are not of very good quality more often, but sometimes we can have poor egg quality in younger patients. It may depend on the protocol or some mistakes as well. There is also a chance of having a poor response, it is expected when AMH is quite low, less than 1, although it can also occur in patients with normal AMH and young age.

If the AMH level is less than 1 ng/ml, we can expect a reduced response to stimulation. If, for example, you have 1 ng/ml at 25, it is not good, but if you have the same number at 45, it’s normal. The normal range of AMH depends on age. However, if we’re talking about the ovarian response, there is a different classification.

From the point of COS:

• ≤ 1 ng / ml – reduced response (≤ 0.2 ng / ml – critically reduced prognosis)
• 1 – 2.5 ng / ml – normal response
• ≥ 2.5 ng / ml – very good response
• ≥ 3.5 ng / ml – OHSS
• ≥ 5 ng / ml – PCOS

We can also look at Antral Follicle Count (AFC), and we can see that if we have a low antral follicle count, we can expect a poor response, and if we have a lot, we can expect hyperstimulation syndrome.

Ovarian Hyperstimulation Syndrome (OHSS)

Why is Ovarian Hyperstimulation Syndrome a problem of the protocol? This Ovarian hyperstimulation syndrome is triggered by exogenous administration of hCG (Human chorionic gonadotropin). Two main medications produce the final egg maturation – it is hCG, such as Ovitrel, e.g., this molecule looks like LH (Luteinizing Hormone) it activates the same receptors, but it stays in your body for around 10 days and has, what we call FSH effect. It means that it acts like Follicular Stimulation Hormone (FSH), and during these 10 days, this molecule continues to stimulate the ovary, not only maturate eggs, which we take out during the collection, and that’s why it produces all these symptoms of ovarian hyperstimulation syndrome.

How is OHSS prevented? With hCG, the risk of having mild, moderate or severe ovarian hyperstimulation syndrome is more than 5%. If we use GnRH agonist, it is less than 2%.

Different studies show that the use of a short protocol instead of the classical protocol may decrease the probability of OHSS significantly. The main factor that can prevent OHSS is to use the agonists as a trigger.
In a recent paper published in 2021, 2 groups of patients used agonists and the other hCG. We can see that with agonists, the probability of OHSS is 0.5, so almost 0, and with hCG, it was mostly 1.5 with the symptoms of ovarian hyperstimulation syndrome.
GnRH agonist trigger and freeze-all strategy is the main instrument to prevent OHSS.

To avoid OHSS, it is necessary to identify the patient of high risk. Especially, if there were previous cycles with OHSS, the patient suffers from PCOS, is of a young age, stimulation protocol. Stimulation should be done with antagonists, the trigger should be done with (a-GnRH) antagonist like Diphereline. It is necessary to use a minimal dose of FSH for the stimulation, but even with high response, even if we have 30-40 follicles with agonist triggering, we will never have OHSS. It is also necessary to exclude the chance of pregnancy in this case and choose a freeze-all strategy.

Age and poor prognosis

As Dr Strelko mentioned, egg quality is individual, and it depends on a woman’s age. As stated by the American Society for Reproductive Medicine – ASRM, over the age of 35, the quality of eggs and pregnancy rates start to decline. It is mostly related to maternal meiotic abnormality, which means the egg quality. If we calculate the probability of having a genetic abnormality of the embryo, we can see if it is related to eggs and sperm. Mostly it is related to the age of the woman, the more advanced age, the probability of genetic abnormality is higher. Paternal age is not so directly related, and the percentage of meiotic abnormality is mostly the same in all ages. Therefore, the main problem is woman’s age because, with age, the number, and quality of mitochondria decrease dramatically due to some damage, and as we know, mitochondria are the main battery of eggs. When the number and quality of mitochondria decreases, we have less potential for fertilization, cleavage stage and so on.

Other factors may also be involved in this process, such as shortening of the telomere, coenzyme dysfunction, spindle degradation and abnormality. This increases the number of genetically abnormal eggs, and in this case, we will have low egg quality.

Low response & AMH

The prognosis of potential response of COS is mostly based on AMH and AFC levels. Both of the parameters are not very stable. Sometimes, we have a good prognosis patient, we have a good number of antral follicles, we have a young patient, we have a good level of AMH, and yet we retrieve only 1 egg. Why does it happen? We have a lot of mechanisms of discordance between ovarian response and AMH levels which we have measured. We can see that the follicular AMH and AMH levels we receive from the lab are two different numbers. We have different sources of variability of AMH, for example, there might I be some systemic problem like physiological, e.g. day of the cycle, it may also be a pathological problem like some tumours, some abnormalities. It could be the administration of some hormonal medication like estrogens, progesterone, contraceptive pills during ovarian stimulation. Another thing would be pre-analytic variations like temperature, or it may be some preparation protocols that are not correct, be freezing and thawing of blood or plasma before they analyze it at the laboratory. Finally, there may be some analytical variability due to some kind of machines and so on. In this case, we will have some level of AMH in blood circulation, but we will receive another number in your analysis, and it may be quite a big difference.

In her next slide, Dr Strelko focused on physiological variability. The graph represented a different colour for each patient. In the study, AMH levels were checked every 7 days during the menstrual cycle. It shows that some patients have quite stable levels of AMH, but some others have a very big difference in different phases of the cycle. It may be due to waves of follicle growing, it means that follicles grow by some cohorts, and in some cycles, you have more or fewer antral follicles. This may be due to the issue we have mentioned before. It happens quite often, and the difference may reach 20-30% in the same cycle. We should also be aware that when women take oral contraceptive pills, it may decrease AMH levels up to 30%. The pregnancy rate can also decrease, on the other hand, ovarian tumours may increase AMH levels. Surgical interventions and chemo or radiotherapy can also influence AMH levels. When we get an AMH level, we get the prognosis of a good or bad ovarian response. In 30% of cases, our prognosis may not be correct. It is not because the protocol was wrong, it is because the prognosis was only based on AMH, due to its variability, it would not be very correct.

The next slide showed that different conditions may change AMH levels dramatically. For example, the effect of long-term storage at -20, AMH level may increase by 230%. It means that if at the beginning you have an AMH level at 1, you would get 3, and after with low dose stimulation protocol, you will receive 2 follicles, and it will be a big problem. So as you can see quite a lot of things may influence the AMH level, and it may change the final result. Therefore, it is necessary to know that, and for example, if your doctor sees that you have a good follicle number and you receive a bad AMH level or just the opposite, it is probably necessary to change laboratory and repeat the analysis once more.

Unexpected low ovarian response

If you expect to have 10 eggs and receive 3 it is a problem. Therefore, a group of doctors divided patients with the low response into 4 groups. This classification is called the Poseidon group. Two first groups are patients under 35 years old and over 35 with good AMH levels, low response to normal standard stimulation. What is the reason behind it? It may be due to mutation of FSH receptors, and in these cases, women may have delayed puberty, decreased breast development, sometimes primary amenorrhea. Other factors include FSH gene variant, LH receptor mutation, polymorphism of FSH and LH, also polymorphism of their reception. There are a lot of mutations and polymorphisms that were described. It is quite difficult to know about that before, and even if we suspect it, we would need to do 10 or 20 different genetic tests, which would be quite difficult from a practical point of view. On the other hand, we should be aware that sometimes it is not related to a bad protocol or bad product, sometimes patients say that they had a bad response, for example, with Puregon, and they want to change it to Gonal-F. We can change it, but this may be due to this kind of thing and not medications.

What to do in this case? The recommendation for the Poseidon’s group I and II for patients with normal AMH levels and bad responses is to increase the dose of FSH and LH and add Growth Hormone to use what we call double or dual triggering. Sometimes we also suggest doing the egg banking program, so we want to collect the eggs in several cycles, 2-3 times and then use them for fertilization. We can also use advanced embryological techniques for egg maturation to improve the probability of fertilization and cleavage.

In situations where we have an unexpected low egg quality and maturity, the cause might be the product, the dosage, protocols, and the triggering. Premature ovulation may also be due to late triggering. Normally it is 3-4%, but in some groups of patients, if we don’t have a sufficient maturity of oocytes of more than 50% of immature eggs, normally it should be 75% or more MII eggs, but if we have less than 50%, it means that something was wrong or the trigger was premature, or it was an insufficient dose, or it was not a good type of triggering. We also can have postmature oocytes, which means late triggering, and we would have what we call empty follicle syndrome, so no eggs at all. The empty follicle syndrome is a very rare condition, and in most cases, it is due to not appropriate triggering. We see Empty Follicle Syndrome in clinical practice much more often, and it is due to some errors and mostly triggering mistakes.

Suboptimal response to an a-GnRH trigger

In this group, we can include:

• low maturity rate
• low fertilization rate
• cleavage arrest
• low blastulation rate, normally, it should be around 40%
• low pregnancy rate and live birth rate

When it comes to a low maturity rate, it means that the nucleus and cytoplasm should be matured at the same time, but those are two different processes. When we have a lot of non-mature eggs, some parts of eggs we consider as mature have immature cytoplasm. In this case, we will have a problem with fertilization cleavage and so on. Statistics and scientific papers show that when we have low maturity rates, even eggs considered mature end up with lower fertilization and worse quality. One of the risk factors for suboptimal response to a-GnRH is the contraceptive pill. When can we expect this not adequate response to a-GnRH triggering? It is when we see a very low LH, and quite often, it occurs when women take oral contraceptive pills for a long time.

Patients with low LH levels on the day of the trigger had a 25% chance of suboptimal LH surge. In the past, it was quite often that doctors prescribed contraceptive pills a few months before IVF treatment. Nowadays, it is changing, and in most cases, we can start stimulation any day of the cycle, and there is no need to take these pills during 2-3 months and decrease LH dramatically. Other possible causes of suboptimal response to triggering maybe some inner conditions such as early oocyte atresia due to folliculogenesis dysfunction, a mutation of LH or hCG receptors, some abnormal activity of some medication. Also, increased hepatic hCG clearance where the body destroys hCG more quickly. There also may be a very low LH level at the beginning of the stimulation, as it was mentioned, it may be due to taking contraceptive pills, as well as possible mistakes during the administration of medication, especially self-administration.

How can we check if trigger injection was not done or done incorrectly? The next day after trigger injection, so after 8-12 hours, we can do the ovulation test, if it is positive, it means that your LH level is high and all is fine, but if it is low, it means that the medication is not doing its job or you have done it incorrectly. We can repeat a new triggering, it is a good method to control. Other risk factors of low egg quality and maturity can be due to high body mass index (BMI) and the dosage of stimulation. In patients with a very high dosage of stimulation, this problem of maturity occurs 4 or 5 times more often than in patients with a normal response. Patients with very low BMI and patients with low LH levels more often can have not a good response to the agonist triggering. Sometimes, oocyte maturation failure can be caused by some inner inherited genetic problem. Lots of mutations and mistakes are hereditary, and we cannot change them with the stimulation protocol, triggering or medication. When we don’t have a good maturity of oocytes, there are some ways to deal with that. If the patient is a poor responder, we can do what we call double triggering, it means that we are using hCG and agonists at the same time. If we have a normal response, we can do the same, and it may increase the number of mature eggs. The difference between poor and normal responders is that in poor responders, there is a higher risk of premature ovulation. It would be better to do the triggering a bit later, a few hours earlier before the egg retrieval, around 34 hours earlier.

The third category is abnormal oocytes maturation, and in this case, we can get agonists 40 hours before the egg retrieval and hCG 34 hours before. It lets us stimulate the LH receptors for some more hours, and sometimes it can improve the number of mature eggs.

There are a few treatment options that we can use for oocyte maturation failure and these are:

• dual or double triggering
• LH control in 12 hours after agonists of GnRH triggering
• discover mistakes
• repeat the triggering if the LH level after triggering is not good
• advanced embryological techniques and approaches for oocyte maturation

Conclusions

Theoretically, we can divide poor results into 2 categories: expected and unexpected poor prognosis. The unexpected poor prognosis may be due to some objective patterns like receptor mutations, genetic problems, age and whatever we would do, we wouldn’t be able to change a lot. Some problems with Controlled Ovarian Stimulation (COS) we can improve and resolve by some supplements addition, change of the final trigger injection, using advanced embryological techniques. It is also crucial to avoid mistakes because, unfortunately, quite a lot of problems may be due to some simple technical mistakes and miscommunication between medical staff and patients.