Endometriosis and Embryo Implantation
Thank you Mr. Chairman. Ladies and gentlemen, good morning. It is a great pleasure to be here today and I would like to thank the organisers for giving me this chance to show you in the next 20 or 25 minutes some of our recent data on how this forum has been arguing throughout the morning and probably we’ll keep on discussing with the following speakers, still something that is not answered, whether implantation is or is not affected by endometriosis.
To focus a little bit on the problem, every time we read a review article or any paper from endometriosis we always read two words, it’s a common disease and an enigmatic disease. I think this is just a reflection of very frequent disease and also very little scientific knowledge on this disease and that’s why we keep on having meetings and discussions about this enigmatic and also frequent disease.
So what we do now when we have a problem and we want to answer a question is we go to the internet and we search the web and when we type endometriosis we see that there are over 10,000 papers published from 1966. This database gives us the dimension of this problem, which is a huge problem, and we will review some of these papers, don’t worry we won’t go over of the 10,000, only a few of them.
It seems that from what we have heard this morning there is no question whether severe endometriosis affects infertility or fertility. There is something more intriguing, and this is whether mild or moderate endometriosis affects these couples. So the evidence comes from many different places. We know some of the studies are prevalent from our perspective studies are very valid. We also have data from infertile women treated due to the disease. Some couples are also treated from other causes and also we have many different experimental models, even animal models, that show that this disease decreases fertility.
So going to implantation we know that it’s a delicate balance between these three points, and I think they are equally relevant. First of all we have embryo quality and I think this is a high indicator of how pregnancy rates can be more or less estimated. We also have endometrial receptivity, which is a crucial event and in this process of implantation and both of these are clearly related to a control of hyper stimulation. And third, but not least important, is the transfer efficiency that we are learning recently that it is actually very important step in the implantation rate.
So how does this disease affect fertility? It has been already discussed this morning and throughout all the events from oocyte development to implantation. There is a sequence of different events that may be affected and I’m going to use all the experience from IVF that has shown us how some of these steps may be affected with this oocyte development. We also know that fertilisation may be affected, embryo development, and implantation.
I won't go over this data again because it has been mentioned already this morning that some big groups think that IVF results are exactly the same as was mentioned before by Professor Evers. Similar results do exist. Some other groups, including ourselves, think that patients with endometriosis show a worse result, probably due to a lower number of oocytes or embryos, lower fertilisation rates, and lower implantation rate and as mentioned before, implantation may also be due to a poor oocyte quality which may yield a decrease fertilisation rate, or due to a defective implantation which is again, linked to the lower capacity of these embryos to implant, or maybe an altered endometrial receptivity.
This paper from Carlos Simon has already been mentioned twice this morning so I won’t mention it again but just remember that this was a retrospective study in which we compared IVF patients with and without endometriosis and in this retrospective study, it was published almost 10 years ago, we showed that pregnancy and implantation rates were higher in controlled cases to what is related to any other endometriosis patients. So whether it was early or late stages of the disease, the pregnancy implantation rate were altered by this disease. This is part of the meta-analysis by Christos Coutifaris that has been mentioned by many of the previous speaker. It shows again that there is a lower implantation rate in patients with endometriosis. So we can analyse this IVF results in women with this disease and again look at what is the problem that has been a matter of discussion this morning, whether it’s a lower embryo quality or a poor oocyte quality or the endometrium may be affected.
This reflects on data from this paper that has been mentioned before. This is the data from oocyte donation. These were the results of the general problem. This was whether the recipients were in early menopause or low responder, or a patient with endometriosis and there was no change in the pregnancy implantation rate. But when the donor had endometriosis and she gave eggs to a non-endometriosis patient then we saw a reduced pregnancy implantation rate. This was a retrospective analysis so this may be a little bit biased because it’s a retrospective study but it was clear data that was maintained and we strongly believe that probably this was due to oocyte quality.
We became surer of that with the next study. We’ve probably seen 2,000 women with the following design. These are also patients from the oocyte donation program. These were healthy donors that had a high response and placed their eggs with two recipients. Half of the eggs went to a healthy recipient and the other half of the eggs went to a recipient with severe endometriosis. So we are talking about good quality eggs and patients with or without severe endometriosis and these are the outcomes; here you have exactly similar implantation and pregnancy rates. Which means that the problem was not in the recipient having endometriosis, but probably the successful cycle due to a good oocyte quality.
So, severe endometriosis undergoing ART is as likely to conceive as women without endometriosis undergoing oocyte donation. The uterus receptivity it’s not impaired in these cases and this might be an effect as mentioned by Dr. Blumenfeld of the GnRH antagonist treatment; whether this is true or not should be tested in a natural cycle.
So looking at the embryo quality we also analysed some of the data from IVF cycles in patients with endometriosis. We published this a few years ago also and we looked at the embryo cleavage stayed 72 hours and analysed the fertilisation rate, the number blastocysts, the number of arrested embryos and this is what we found. When we compared this data to two well patients we saw that the number of blastocysts in patients with endometriosis was lower, which means that it’s lower cleavage rate and also the number of embryos that were arrested was higher in patients with endometriosis and this shows statistical significance.
So this was in our line of thought that probably this was a problem with the oocyte and not with endometriosis activity. We hypothesised then that a lower embryo quality originates poor oocyte quality due to the endometriosis and I’m going to show you some data that may convince you of this.
We know that for a good oocyte donor quality we need adequate aromatisation and adequate oestrogen production. Also low androgen concentrations are good in the oocyte as you all know and also we a good progesterone production. So we tested this particular environment in patients with endometriosis and this has also been mentioned this morning with a case controlled study in 50 women ; we tested this follicular fluid in different esteroids such as progesterone and testosterone and we found that progesterone was higher in follicular fluid. This was opposed to the paper to previous publication by Cahill showing just the opposite result. When we look at these esteroids as I mentioned, progesterone was significantly higher all the time in patients with endometriosis compared to control.
So we thought that maybe progesterone was accumulated in vivo in these patients. This was the data not from follicular fluid as before, but from granulosa cell cultures. Again high progesterone was seen in patients with endometriosis and if we gave hCG, again this increased the rise and progesterone production was higher in patients with endometriosis.
So the idea was that probably that these patients they have a different steroidogenesis, which may indicate an altered intra-follicular health but it wasn’t true at the final stage. We thought that the progesterone was increased due to in vivo follicular fluid secretion. We also tested this in in vitro in high concentrations in the granulosa cell culture and we further hypothesised that this might be due to difference in schematic activity in these granulosa cells or probably this progesterone was being sequestered. It was probably with a corticosteroid binding globulin or maybe interacting with the immune system. We also went forward and tested these patients for some of this interleukin-6 and growth factors and in a similar setting, what we found is that IL-6 again was higher in these patients compared to the other diseases and VEGF was increased. This was true also in this spontaneous cycle but we tested for unstimulated cycles. So something was going on inside of the follicle and still we don’t know what it is. If we test for the cytokines we know that this follicular fluid is different in concentrations with many different variables than when we looked. If we used granulosa cell cultures again, we have this concentration of hCG and that changed the complete production of these cytokines.
We were only looking at this follicular fluid and serum at the protein level or the steroid level but we know that we have to go farther and we have to check whether this protein secretions is ok but again we have to look at the protein production and of course at the mRNA expression and just make sure that these findings are consistent.
And here is where we became very frustrated because we tested for progesterone and also for cortico steroid binding globulin and for 3-beta hydroxy steroid dehydrogenase, the enzyme that metabolises the progesterone and we couldn’t see using competitive RT-PCR, no difference at the mRNA level of these hormones and we also tested this for RT-PCR and flow cytometry for IL-6 production. And again we didn’t find any significant difference. Same thing also for VEGF, as it was shown in this study, this was a little bit disappointing because it was the opposite of what we found in previous data. We thought that probably there were alterations in the follicular micro-environment but they are not related to either cortico steroid binding globulin or this enzyme that metabolises progesterone, which were the three beta hydroxies steroid hydrogenate and we also know that the granulosis cell functions in terms of IL-6 or VEGF production is not altered in women with endometriosis.
We know that in this follicle something is happening and we are still not sure what it is. We know there are some non-genomic effects that may affect folliculogenesis modulation and there also some genomic effects that have not been tested yet and probably others will show us what’s going on inside. We are moving towards this new technology and we’re trying to test with difference of display whether there is any difference in women with or without endometriosis. This, as you know, will compare two DNA populations arrays or DNA chips, looking for specific genes that may be altered in these cell population.
This was about the oocyte development and what’s happening in the endometrium. Well there are many receptivity markers and some of them have been mentioned already this morning and I won’t go over all of them but when you read about this subject pinopodes are always mentioned as a good marker for endometriosis activity, integrins especially by the group of Bruce Lessey have been repeatedly shown as a very good marker although not all of us have been able to replicate their findings. Hugh Taylor by the group at Yale University also has shown good results and good correlation between HOXA genes and implantation. New genes like the endometrial bleeding associated factor, e-baf, may also be implicated here. Again the free radicals activity may be also relevant.
So I’ll just mention some of these markers and first of all I’ll talk a little bit about pinopodes. We know that they are expressed in the luminal epithelium of endometrium at the very specific time point where the embryos should be implanting and what we did is to check the expression of these pinopodes in women undergoing oocyte donation. Again, a good model to separate endometrium and oocyte quality. We did a mock cycle endometrial biopsy to make sure that this endometrium may have a different expression in pinopodes or not. So, with this perspective and observational study in 12 women with severe endometriosis, we also biopsied 12 women with premature ovarian failure looking at the pinopode expression and also at the pregnancy rates.
This work was done in collaboration with Gorgos Nikas who is an expert in pinopodes. What we look at the different type of expression of the pinopodes and also then we tried to time the embryo transfer according to the pinopodes expression whether it was one day before or one day later and we didn’t find any difference in the pregnancy rate between these two groups and the implantation rate. So we concluded that pinopodes were not expressed differentially in women with endometriosis.
Again, more data is on intergreens. This is work done by Maria Illera in collaboration with Bruce Lessey and this experimental model shows conflicting data that is not as happening in humans like sometimes it happens. What they did was to inject human peritoneal fluid from women with or without the disease into mice and checked the implantation sites and they found reduction in implantation sites in these animals receiving peritoneal fluid from an endometriosis patient. As you can see here they tested not only the implantation sites that were reduced when peritoneal fluid was injected into the animals but also the expression of the alpha v-beta 3 integrin and observed a reduction in the expression.
The new technology as I mentioned before is moving forward and this research is going fast so we can check at two different time points, LH+2, LH+7, the different expression. This is a differential display showing that genes are differentially expressed at the implantation time and previous to implantation time. Now we are trying to check this data in the endometriosis and non-endometriosis model using biopsies from women with or without endometriosis to ascertain whether the endometrium is important or not to this question that is still unanswered.
To conclude, clinical evidence using the oocyte donation model strongly suggests that the endometrium is not altered in these women. Functional studies from the endometrium of an endometriosis patient do not support the hypothesis of an altered endometrium environment, which is controversial as I showed, in animal models. Rather decreased oocyte and embryo quality seems to be the cause of endometriosis related infertility. Probably what is happening with this disease, why we all not agree in this question, is because we are looking at different parts of the puzzle, different puzzle pieces, and we are still not able to see the full picture together.
I’d like to acknowledge all of my collaborators, especially Carmela and Nicolás and Jankusho for their competitive RTPCR . Enrique OConnor for the flow cytometry and specilly Carlos Simon, Pepe Remohi and Toni Pellicer - my good friends.
Thank you very much.
Question:
May I ask you if you have put your fingers on this very nice puzzle in the contradictory results – do you have an explanation why you find this progesterone accumulation?
Answer:
Well at the moment we don’t. We thought that it was a problem with the corticosteroid-binding globulin. We thought that also again the main catalyser of progesterone, 3beta hydroxisteroid dehydrogenase was reducing the expression. But we couldn’t find any correlation; we did some experiments with flow cytometry and RT-PCR and didn’t see any difference in expression so we are still looking for that.
Thank you.
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