By fertility experts from Spain.
The demand for fertility treatment is increasing and therefore those responsible for the treatment and care of patients need to ensure that the services they provide are the best and most effective available. This article based on a webinar led by Dr Ahmet Ozyigit from North Cyprus IVF outlines options for women over 40 including ovarian PRP (Platelet Rich Plasma) treatment.
Reproductive medicine is always looking for ways to improve pregnancy rates as more people turn to assisted reproduction to have a child. Demand for fertility treatments is rising due to a number of factors including career orientation, the increased age of couples getting married and a greater awareness of the options available for those having difficulty in conceiving or maintaining a pregnancy. However, a significant number of people are also delaying the decision to try for children and this can be both problematic for the patient and the treatment provider.
Age is not the sole factor which determines fertility problems. One factor which can have devastating consequences is so-called Premature Ovarian Failure which can affect women in twenties or thirties. These women have very low AMH (Anti-Mullerian Hormone) levels and very high FSH (follicle-stimulating hormone) levels as well as practically no antral follicles. Ovarian reserve, as well as ovarian function and activity declines naturally with age but premature ovarian failure can mean that women need some assistance in getting pregnant very much earlier in life. Fortunately, there are a number of treatment options available for women who experience premature ovarian failure including cytoplasmic IVF treatment, embryo banking with or without PGS, IVF using donor eggs (or double donation) and ovarian PRP treatment.
As Dr Ahmet Ozyigit explains, the latter has a lot to offer.
Dr. Ozyigit starts the webinar by introducing PRP. Platelets (also known as thrombocytes) are a part of our blood involved in wound healing and tissue regeneration. Platelet-rich plasma (PRP) is the blood product where the blood sample of an individual is subjected to specific laboratory techniques and equipment to separate the platelet-rich part from the part that is platelet poor.
In terms of platelet function, we know they have a lot of growth factors and proteins in them which play a specific role. All of them work in a collective manner in key parts of tissue regeneration such as cell growth, proliferation and differentiation in addition to improved oxygen delivery and circulation. PRP helps with growth and proliferation but since this is a concentrated platelet-rich plasma, it is also is equipped with certain elements (growth factors) that reduce the inflammatory response.
The idea of using platelet rich plasma to rejuvenate is not a far-fetched one. This is something we all witness in our daily life. It happens when we cut or knock ourselves – an initial bruise becomes inflamed with the same oedema. Later on, a yellowish layer is formed that consists of white blood cells, blood clots, platelets and growth factors. All of this helps prevent bleeding and infection and also helps to regenerate connective tissue and repair the damaged area. Dr Ozyigit explains that in assisted reproductive medicine the part of the process which doesn’t cause inflammation is isolated. It focuses on introducing dormant stem cells into ovaries to activate them in order that they go through follicular genesis or oogenesis which means oocyte development.
PRP applications have been shown to have proliferative effects as well as anti-inflammatory effects while working on tissue repair. Currently, PRP is being used in a number of fields and these include anti-ageing, hair growth, dental applications, soft tissue regeneration and endometrial and ovarian rejuvenation.
According to Dr Ozyigit, PRP application has also been associated with increased progesterone receptor activity. Progesterone receptors are the main actors that help maintain a thick and healthy endometrial lining which helps with embryo implantation.
Dr Ozyigit observes that up until recently, it was widely believed that women were born with a limited ovarian reserve which depleted in a non-reversible way. However, recent evidence points to the existence of dormant stem cells in the ovaries. Although it is not sure why they exist, they have been shown to have the potential for activation. This gave the idea of possible ovarian rejuvenation. If these stem cells could be activated to produce new oocytes, menopause could then be potentially stalled allowing a greater opportunity for a woman to achieve a pregnancy.
Based on what is known about platelets and the growth factors involved, Dr Ozyigit explains what to expect from the PRP application. It is increased vascularisation and improved blood supply. Additionally, growth factors in platelets can stimulate the activation of ovarian stem cells which have the potential to mature into oocytes.
The stem cells in the ovary are located in the cortex. For the purpose of PRP treatment, it is very important to know exactly where these stem cells are to target the treatment directly towards them.
According to Dr Ozyigit, the first thing to consider is the method itself. PRP isolation is, for example, one of the main factors that determine success in ovarian PRP treatment. It involves a number of considerations, such as anticoagulation of the blood sample to avoid premature activation of the platelets, appropriate isolation protocols to obtain a highly isolated sample and last but not least – correct timing of activation.
Location in PRP treatment is equally important. As mentioned before, stem cells are found in the ovarian cortex. Therefore, any treatment which involves activation or/and differentiation of these cells, needs to involve the cortex.
When injecting the PRP, oogenesis is the most important consideration of all. If one doesn’t know how eggs grow, how they develop and go into ovulation, then the whole procedure is not going to be successful. In terms of oocyte development, timing is everything. Dr Ozyigit reminds us that the timeline to grow oocytes from scratch is four months.
One more thing which is very important is supportive treatment. When initiating the growth of cells, we need to support them at the cellular level. Dr Ozyigit says they use a human growth hormone which helps to increase the number of the preantral follicles from the primordial ones. They also use aromatase inhibitors to suppress the estrogen and lock the negative feedback mechanism between the ovaries and the brain. At a later stage, they also include gonadotropins to produce more eggs than in a natural cycle.
Dr Ozyigit admits that in terms of success rates, it is too soon to tell if the method has the potential to be ground breaking. It usually works for women with premature ovarian failure who still have primordial follicles. However, it has low success rates in women who are over 50 years of age and after menopause. According to Dr Ozyigit, there is a considerable difference between the first IVF cycle before the PRP and the second IVF cycle after the PRP. The difference is not seen just in terms of the number of collected eggs but also in the AMH measurements as well as the number of antral follicles seen in the ultrasound scan. The antral follicle count before the PRP and after the PRP varies greatly. However, this still does not guarantee a successful pregnancy. Dr Ozyigit concludes, PRP treatment is still a work in progress and there are a lot of modifications and improvements which will need to be introduced and made in the future.
Yes, usually we do have to have an age limit for cytoplasmic IVF treatments. Cytoplasmic IVF treatment requires you to have your own eggs. The principle behind cytoplasmic IVF is that the women go through an IVF cycle and we collect the eggs. The eggs themselves could have problems with the mitochondria, they could have DNA related issues or cytoplasm related issues. And after a certain age, after the ovaries are highly depleted of eggs, we’re not able to obtain enough eggs from the IVF cycle. However, we still need to have eggs from women. And that’s a problem for cytoplasmic IVF. So we have to make sure that we are still able to get at least three or four eggs after an IVF cycle. In that way, we could use the cytoplasm from the donor’s eggs and inject that cytoplasm into the recipient’s eggs to provide a better host for these eggs. So the intended parent still needs to be able to produce a few eggs during the IVF cycles We usually play by ear, because not every woman is the same. Someone will have a depleted ovarian function at the age of 35 and the other will still have some ovarian activity at the age of 49. As long as there is an ovarian function, as long as the AMH is not totally zero, we can definitely consider cytoplasmic IVF treatments.
We have been performing PRP for around four years now and it does not work for every woman on the first go. Sometimes a second attempt is done. However, we have had women where we did not receive results even after the second attempt. So it’s not all rainbows and unicorns. But if you do your protocol correctly, you do the PRP infusion, you follow up with the HGH, you do a proper protocol for the IVF cycle, then you will definitely see a difference. So in terms of the success of the PRP injection, I would say 80% of the time it is successful in increasing the antral follicle count and improving the AMH. We usually don’t measure FSH anymore because it’s not very indicative. We measure AMH and we measure the antral follicle count before proceeding with the IVF cycle. We do see improvements. In most women who have stopped having menstrual cycles, we do see the cycles coming back. But sometimes we do not get a positive pregnancy after the IVF cycle that follows the PRP. Sometimes we may need to do a second cycle of PRP and get success with that. However, sometimes even with that, we may not be able to get success. So it’s very case-based, I’d say.
Yes, we can. Antral follicle count is the number of antral follicles that you can see in the ultrasound. If you don’t see something, that either means that you have nothing or you do have some primordial follicles. You cannot see the primordial follicles in an ultrasound scan. So even if you don’t see anything and the AMH is very close to zero, you can still proceed with PRP. You can still inject the PRP and get the primordial follicles to grow, the stem cells to activate themselves and grow into primordial follicles. If we’re talking about zero AFC and older women, someone who’s entered menopause or who’s almost menopausal, we will definitely wait for four months after the PRP infusion. We’ll do the PRP in both ovaries and then use human growth hormone for a slightly longer period of time. The protocols are adjusted based on age, AMH levels, AFC and generally on how we expect the patients to respond to the treatment. We do modify our protocols slightly. In this case with zero AFC, I would expect a higher length of time for oocyte development to take place. We’re not looking here at any preantral follicles, we’re looking at primordial only and we’re looking at stem cells only. They will take a longer time to grow and develop into the preantral and antral follicles. So for this specific question, I will say yes. We can do the PRP and the PRP should be followed by a higher length of human growth hormone usage. We would retest after four months and I would expect to actually see something then.
We have not had any patients with side effects so far. But technically yes, you could have side effects. These could be a little bit of inflammation, a little bit of bleeding that’s not really menstrual bleeding, but just some bleeding that doesn’t really belong. Possibly you could experience some cramps and a little bit of pain. But we have not really had a single patient complaining about any side effects. Essentially, what we’re doing is injecting a very tiny part of your own blood, just one ml, into your ovaries. If this was technically a significant amount of fluid injected into the ovaries, then we would expect some sort of a side effect, some sort of inflammation. But this is not large enough to induce any side effects really. I would not expect anything else.
Yes, the cost of a PRP cycle is EUR1,500.
We were kind of concerned about that initially because you don’t know how those stem cells are going to turn out. You don’t know how you’re going to wake up those stem cells and convince them into being oocytes. These stem cells are also living in the ovaries for as long the patient has been living so they’re getting older as well. So we weren’t sure if this would succeed. We weren’t sure if you would actually get good quality oocytes or embryos. But again it’s very case based . In some cases, we do see quite a large number of oocytes compared to what it would be without the PRP. We do observe that the quality of the oocytes is good enough to give us embryos, to go into the cleavage stage or into the blastocyst stage. That’s how we grade them. We don’t really run any tests on the oocytes themselves. It’s based on the appearance, the physiology and the morphology of the oocytes. So they’re either graded as a germinal vesicle or an M1 grade or an M2 grade oocyte. M2 oocyte is what we would like to see. Most of the time, we are able to get M2 grade oocytes. So to answer this question: yes, we do see good quality oocytes that are able to give us embryos.
I’m not sure whether FDA needs to approve this or not because essentially you’re not doing anything that comes unnaturally. This is part of your blood. For example, PRP can be applied into your skin, into the cheekbones, into the lips. There are a lot of applications of PRP in cosmetics, there are a lot of applications of PRP in orthopaedics. So I am not sure if they do need the approval of the FDA. I would need to look into that. But if it requires FDA approval, it is probably approved, because this is something that’s being performed in the US as well. There is one specific clinic in New York that is performing PRP injections for as long as have – for about four years. So it is probably approved as the centres in the US are able to offer this.
I would say probably about three hundred and fifty, four hundred? This therapy is used for over four years. On average, we perform five or six treatments each month and then we follow up with them for up to four months. Some women get pregnant naturally so they don’t need the IVF. Other women don’t get pregnant naturally, even though we tell them to try. If you can get pregnant naturally, why go for the IVF? If they do get pregnant, it’s perfect, they don’t need IVF. If they don’t get pregnant, then we do the tests and we prepare them for the IVF cycle. Sometimes we do get pregnancies, sometimes we don’t get pregnancies. Sometimes we do get a pregnancy but it ends up being miscarried. Even if you do get eggs and perfect embryos, not all the IVF cycles will give you a 100% pregnancy. We are able to get eggs and embryos but unfortunately not all the cycles result in a positive pregnancy.
Stem cells come from several lineages and they do migrate to specific places. We don’t guide them because they’re already committed to being there. If they’re existing in the cortex of the ovary, they’re already committed to be ovarian, reproductive type of cells. So we just giving them a nudge to proliferate and multiply and then differentiate into the type of cells that already exist in their vicinity. To be what they’re supposed to be. However, if you were talking about embryonic stem cells that are not differentiated and not committed, then you would be talking about a whole set of issues. If you inject embryonic stem cells into any organ, you’re potentially looking at something that can have catastrophic side effects or end results. But in our case, it is much safer because we’re just using the stem cells that are existing in the ovaries and are already committed to being there.
I’m usually honest with the patients. We see the best results in patients who still have some ovarian activity. It ensures that they also have some primordial follicles that we can recruit. That usually gives us the best kind of outcome. The patients with the lowest rates of success are those who are already in menopause and who haven’t had a cycle for a few years. For example, someone who’s entered menopause at the age of 45 and they’re trying this treatment at the age of 55. In such a case, it’s been ten years since any kind of activity in the ovary. However, the patients who had menstrual cycles up until recently or patients who still have their menstrual cycles but have a significantly reduced ovarian function can still have better results. Age does have a significant impact here. Someone who is 35 years old with premature ovarian failure is going to have a better outcome than someone who is 50 and has a natural course of ovarian failure. PRP is usually considered to be very safe for everybody. Technically, it should not do something that it’s not supposed to do. However, if there is a history of ovarian cancer or reproductive cancers, we do not advise PRP. It’s uncharted territory for us as well and we do not want to take any risk.
Endometriosis is something different. In that case, you’re looking at a totally different pathology in the endometrium. I wouldn’t expect PRP to help with endometriosis, to be honest. If we’re talking about women having problems getting a thicker lining for their embryo transfer, then yes. It definitely helps. But we’ve actually never considered using PRP for endometriosis and I don’t see how it could help.