Egg quality and imprinting

View the video (requires free Windows Media Player)

Cees Jansen, MD, PhD: I’m with Dr Roger Gosden, who is, at present, Scientific Director of the Howard and Georgeanna Jones Institute for Reproductive Medicine in Norfolk, USA, and who has given very important presentations in the last two days concerning the role of the egg in the follicle, the inter-relationship between the egg and the granulosa cells, and also the consequences that has for the subsequent development in the first days of division. Now, Roger, one of the issues you presented was about the influence of the quality of the egg on the granulosa cell and vice versa. Could you elaborate on this for the audience?

Roger G Gosden, PhD, DSc: Certainly. Well, the egg grows over a long period of time, of course. The follicle has a growth span in humans lasting many months, and throughout that time, it is intimately connected with those granulosa cells; you cannot grow an egg to full size or competence to undergo meiosis or fertilisation without that layer of granulosa cells around it. 

The granulosa cells, of course, are also an endocrine organ, but they are vitally important for the development of that egg; and they determine the timescale of the development of the egg to some extent, although the egg is not a passive passenger in this, so we have to look at the other side of the story -- that they are actually working together, that the egg also instructs the granulosa cells and is required for them to undergo mitosis and also to express certain molecules like the mucilaginous hyaluronic acid.

Cees Jansen, MD, PhD: So, what implications could that have for the subsequent development? For instance, at the moment, the egg is ready to ovulate and has prepared itself for the subsequent development. Could that have an impact in later life if something goes wrong or, for instance, if we interfere with that process?

Roger G Gosden, PhD, DSc: If we interfere with the process at any stage up until the arrival of the LH surge, we are likely to run into big problems. If we interfere with the connection between the granulosa cells after that time, probably the implications are much less because the interactions and instructions that have been sent to and from the egg to the granulosa cells are largely complete; of course, in ICSI we strip off those granulosa cells, although there are still some remaining very fine processes crossing from the granulosa to the egg. We don’t know whether they’re important and probably not by that stage, so the key events are going on much earlier in development. 

And if we disrupt that communication at any stage during the follicle development by breaking those processes or interfering with the signal molecules (e.g. kit ligand from the granulosa cells, or GDF-9 from the egg) -- then follicle development will be aborted. The implications for egg quality are another matter, of course, and that is something that I think we all have to be very focused upon if we really want to improve IVF technology.

Cees Jansen, MD, PhD: The two questions that directly pop up are: first, the implications, for instance, for in vitro maturation, which we do not know enough of in order to be able to use it at present as an acceptable treatment to create human offspring; the second, of course, is the influence of ICSI and maybe the later influence in terms of the development of the babies and the occurrence of rare aspects that have nothing to do with the genetic but more the epigenetic aspects. Could you comment on that?

Roger G Gosden, PhD, DSc: Well, as you know, the safety of IVF and ICSI has been quite extensively studied already by investigating birth defects, by investigating carrier types and by some longer-term follow-ups of the children. The results have been broadly pretty reassuring, just one or two controversies exist, and the major concern I think we all have is with the multiple pregnancies and their implications. 

So that has been the background of research over the last twenty years in follow-up studies. But recently, there’s been much more focus especially in the last year or so on epigenetic factors. We published in the June 7 volume of The Lancet a description of studies, which have reported an association between epigenetic abnormalities and ARTs. Now these abnormalities vary in severity and are rare like Angelman Syndrome and Beckwith-Wiedemann syndrome. It is a matter of concern that the registries for these birth defects show a three- to six-fold increase in frequency over expectation. 

Now, I don’t think that this should be regarded with huge alarm because, in the big picture of IVF with a million babies born around the world, we’re still talking very small numbers. But even if there is a slight increase, we have to be aware and, if it’s connected with any new technologies, we must be very watchful. 

So, what is needed now, I think, are two things: one is that we need to do prospective clinical studies to verify if there is an association, and whether it is a causal relationship; so we need to follow-up large numbers of IVF children probably on a multi-national basis for statistical validity and to test for cluster effects. So that’s one part of the equation. 

The other part is that we need to investigate the developmental biology of the egg. So I come back to the egg and egg quality, because during that developmental span of many months when the egg is growing, it is accumulating mRNAs, which are either stored for later translation or translated immediately. Among those transcripts, and of course there are thousands of them, there are some which are involved in this imprinting phenomenon where one of the alleles of imprinted genes is repressed by methylation or another process in the chromatin, such as histone modification. 

For Concerns have been expressed whether the imprinting process is influenced by ART procedures, perhaps raising the risk of syndromes like Beckwith-Wiedemann Syndrome . One is, does the condition or specific conditions of infertility in themselves predispose to imprinting abnormalities in which the DNA is not appropriately methylated. In that case, you get expression of imprinted genes from both the paternal and the maternal sides (alleles) in the embryo of the child, leading to the abnormal phenotype? 

So we have to focus very much, I think, upon the expression of those molecules, which were involved in the imprinting process. There are certain enzymes which are expressed during egg development, called DNA methyltransferases, which seem to be key for this process and they have to be expressed at the appropriate time. 

And then the question of whether there are influences of other factors -- in our culture conditions, in our hormone stimulation? There is a seeming excess of BWS cases associated with ART, but we simply don’t know if they are related to the technology or the clinical history of the patient and their infertility condition? 

Therefore, we need the prospective clinical studies and to understand whether culture conditions might perturb the imprinting process in human eggs and embryos, and the expression of the enzymes responsible for it.

Cees Jansen, MD, PhD: Well, that’s quite a lot, I must say, and I totally agree with you. I would like to summarize this by saying that there are a lot of these subtle changes in the oocyte and also in the fertilised egg and the embryo in terms of the selective methylation and demethylation and other processes that we still don’t understand. Would you agree with me that it is imperative that we gain more knowledge in this aspect in order to especially look at the subtle and not very often occurring changes that may have large consequences for the offspring?

Roger G Gosden, PhD, DSc: I absolutely agree. There has, of course, been a huge emphasis upon chromosomal anomalies in PGD with aneuploidy screening, because we know that a lot of chromosomal anomalies arise during oogenesis and so that has been the focus for some years, very appropriately. But I think in the next few years we will be looking for these more subtle modifications, as you say. It’s going to be quite difficult to investigate them in human embryology because the material is always very precious, but I think we do have the tools for that now and I hope we may even have other ways of screening embryos in the future, whereby we can avoid any risk of some of these epigenetic phenomena, just as we screen at the moment for single gene defects.

Cees Jansen, MD, PhD: Well, I’m really looking forward to research in that area. Are you going to participate in that yourself actively in your centre?

Roger G Gosden, PhD, DSc: Yes, we are investigating this at the cell biology level, looking at these enzymes and when the imprinting process takes place in human eggs, because there’s very little data on that. We also are interested in embryo stem cells, and I raise that because I think human embryo stem cells, can provide a potential model for looking at factors that influence the blastocyst. We can never get enough blastocysts for all the studies we might want to do; for instance, to look at how culture conditions might alter imprinting processes, and we know that does happen in animal studies. 

This is where the connections between reproductive medicine and stem cell technology are very strong. Besides, there is evidence from animals that this imprinting process is quite unstable in the mouse embryo stem cell. 

So we have to watch this in the human embryo stem cell too, because there would be problems potentially with embryo stem cell technology if the imprinting marks were lost. Biallelic expression could lead to tumour formation in the transplant. But, in the same way as we provide information relevant to transplantation medicine through these studies of imprinting in embryo stem cells, we may gain information about what could potentially destabilise imprints in the blastocyst before it is transferred.

Cees Jansen, MD, PhD: Well, really, we are all are looking forward to this research and we have high expectations of your input on this. Thank you very much.