In this week, what we want to start thinking about, is how broadly the environment can influence epigenetic state. So we're going to consider this broad consideration of the environment in two ways. The first being altered environment due to changed scientific or medical practices, and so this includes assistive reproductive technologies, like IVF, cloning and somatic cell reprogramming. And then what you might more likely consider is the environment, so changes to diet, chemical exposures, so chemicals you might come across in the environment and changes to mothering style. In this instance, we're thinking about rats. So, for most of these topics for this week, we're going to consider human examples. Next week, when we'll move on and think more about mouse models. What's really important to think about and can keep in the back of your mind for all of this discussion that we're going to go through this week, is that there are really, sensitive periods of exposure. So, periods when changes in the environment are going to have the biggest effect. While epigenetic reprogramming is occurring is of course two of these periods. Because it's during germ cell development and early embryonic development. And that's what's relevant to our first topic which are these assistive reproductive technologies, cloning and somatic cell reprogramming. But also for any of these changes which broadly the environment can bring about, if they're to have an influence on the organism, then really we require for them to have some mitotic heritability. Otherwise, that will only really effect the cells that are exposed to this environmental influence at the time and be lost when those cells divide. Now, the ideas that we're talking about. These ideas of how the environment can influence epigenetic state, also relate to something that we're going to talk about next week predominantly, and that is trans-generational epigenetic inheritance through the gametes. I guess because what we're thinking about in humans is, if we're exposed to a particular environment and that it might alter our epigenotype, we'd like to know, is this going to affect future generations. And so that's when we start to think about transgenerational epigenetic inheritance through the gametes. So, I’ll touch on what I mean by transgenerational epigenetic inheritance this week, and then we'll come to more details about it next week. What I think is really important to remember at this stage right at the beginning of dealing with these topics is that all of these topics are highly controversial. This is actually why I want to talk about them in this course, because they are very controversial and the studies that have been done in humans, by definition can't be controlled in the same way as we’d control, mouse studies or rat studies, or any other model organism studies. And yet, they do still receive a lot of public press. And so, what I'd like you to be able to do by the end of the week or the end of next week, is really start to think about these controls that you would need to be able to best interpret the information that's there. And be able to perhaps temper your interpretation of the data that's present. So while many studies have been done in mouse model models we will cover next week, as I said very few exist for human and we don't yet really know how to interpret the data that's there. What is important to keep in mind the whole time is that there are three questions that we really don't have the answers to yet. So, if we believe all the studies that have been performed and perhaps believe that what we show in a mouse is true in a human. Even then, we don't really know the answer to the first of these questions here. What proportion of the genome is actually sensitive to the environment? So, is every site in the genome going to be able to be manipulated by the environment? Or is it in fact only some selective sites which are likely to be changed? The second question that we don't really know the answer to yet is what proportion of people are genetically sensitive to environmental disruption? So in other words, while there might be particular sites in the genome that are very sensitive, or that are going to display the largest possible effect sizes, do we all have the right genetic makeup to be able to show this effect? Or in fact, is it only a smaller proportion of the population? And finally, of these changes which might occur, what proportion can actually be inherited mitotically, as I mentioned earlier? And perhaps even more importantly, meiotically, in other words, through meiosis and passed on to the next generation. So keep these questions in mind, and the controversial nature of what we've been talking about for this coming week as we think about how the environment influences epigenetic state. So for the next two lectures we're going to think about how epigenetic reprogramming can be disrupted. And the consequences for imprinting disorders, and also for other epigenetic reprogramming spread throughout the genome. We're going to talk about three particular cases. We're going to start out by thinking about the assisted reproductive technologies or ART. And these are IVF, for example, or intracytoplasmic sperm injection, ICSI. And then in lecture we'll think about two other instances. The first is somatic cell nuclear transfer, or more commonly known as cloning. And the last is reprogramming of somatic cells, back to pluripotent cells, which are called IPS cells. And we'll come to these next time. So let's first think about what happens during assisted reproductive technologies. So I think it's very important to start out by saying that, although there seems to be some evidence that imprinting disorders arise more frequently in children that are born from assisted reproductive technologies. The jury is certainly still out. And so it's not yet entirely clear what the effect of ISCI or IVF is on epigenetic reprogramming. And so, keep in mind that when I talk about these studies. We don't have a definitive answer as yet. But, there are a considerable number of studies that are now showing, that there seems to be an increase in the occurrence of Beckwith Wiedemann Syndrome, and Angelman syndrome. These two imprinted disorders, I've mentioned that are maternally transmitted, following ICSI, in particular, so, this intracytoplasmic sperm injection. So, what I'm showing you in these pictures here is, first of all, on the left-hand side a human oocyte, which is being stripped of the cumulus cells, which would normally surround it And so it's ready for IVF. So, IVF would just be, that the oocyte would be harvested from the female. The female would be made to release more eggs than would normally be released in a normal ovulation cycle. They'd be placed in a dish, after stripping away these cumulous cells and the sperm added so that the fertilisation can happen in-vitro. Then following on from that, the embryo is cultured for a period, until it gets to the blastocyst stage, and then it's implanted back into the mother again. By contrast, ICSI, or intra-cytoplasmic sperm injection is shown in this right-hand picture here. So the oocyte is being held by a holding needle on the left-side, which doesn't damage the oocyte but just keeps it in one particular location. And then the sperm, because in this case the sperm is unable to swim. Is injected directly into the egg. So, even if a man has a fertility problem such that his sperm is unable to swim. In this case, you can allow fertilisation to happen. And then again, just like with IVF, these resultant embryos are cultured to the blastocyst stage and are implanted back into the mother again. So, when there are these reports that suggest that perhaps you get a higher rate of, imprinting disorders, at least with Beckwith Wiedemann syndrome and Angelman syndrome, following ICSI or IVF, in all of these cases, these haven't been due to genetic abnormalities of the clusters that are involved, but rather due to epigenetic abnormalities. And you'll remember that I said that the epigenetic disruption, the cases that come from epigenetic disruption are extremely rare in comparison to those that are caused by genetic disruption. And so, this was a surprising finding, that you should have exclusively these epigenetic cases following ART or assisted reproductive technologies. Again, think that Angelman syndrome and Beckwith Wiedemann syndrome are both maternally transmitted. I'd also like to point out at this stage that even if you believe that there is some increase in the incidence of the imprinting disorders, Beckwith Wiedemann syndrome and Angelman syndrome following ART, assistive productive technologies, then the absolute risk for the IVF children is still extremely low. So we notice that imprinted disorders related to epigenetic disruption occur extremely rarely, one in 300,000 births, for example if it was Angelman syndrome or Beckwith Wiedemann syndrome alone. So if we increase the risk of these particular imprinting disorders by maybe three or five fold, they are still extremely rare. And so in the vast majority of cases, absolutely the majority of cases, then the children born from assisted reproductive technologies would still be normal. It's a very rare occurrence for these to occur, even if we believe all of the current evidence. I also want to mention at this point that although, we're talking about imprinted disorders in this case, there's also evidence now that perhaps there are broader epigenetic abnormalities that can result. So at other locations spread throughout the genome, not just at these particular imprinted clusters. So, when considering the data from human studies that people have been through ICSI or IVF. The question arises, why is it that these things would have raised? Why would these abnormalities have arised? Is it possibly because the original patients have an underlying fertility defect? So we know that people, patients that undergo IVF or ICSI tend to be of increasing maternal age. We know that in itself can really result in an increased number of particular disorders. Or could it be that underlying reason why they have a fertility defect could result in the increased incidence of the epigenetic and abnormalities and, and therefore the imprinting disorders. So, while there certainly seems to be some contribution from each of these factors, we know that if they're looking at the non-humans, so we're looking at cattle or mice, if you take cattle or mice through assistive reproductive technologies, you can still result in some epigenetic abnormalities. So assistive reproductive technologies or IVF in particular are used for cattle for husbandry reasons. You can have a prize bull, and you want him to pass on his genes to as many offspring as possible, then you could send his sperm around the world, but the bull himself doesn't have to travel and he could Have a bull that lives in England, but is used to sire calves that are in Australia for example. In mice we do these for experimental purposes. Partly to study IVF, but again for a similar reason, for husbandry reasons like is performed in cattle. Because we have some abnormalities resulting in these cases as well, this suggests that it's not just to do with the underlying defects that may occur in the people that need to undergo IVF in humans. But rather there might be some procedural issue, something to do with the way that ARTs are performed. So, let's think back to epigenetic reprogramming. We know there are, these two waves of reprogramming that occur, first of all during germ cell development, and second of all during early development. So these are two very sensitive periods, for epigenetic reprogramming, but they are also the times when IVF or ICSI are coming into play and are being effectively rather disruptive. So, the reason that primordial germ cell development or germ cell development is disrupted, is because of the way that gem cells are harvested. So, rather than naturally ovulating just one egg to try in IVF. Instead the females are what's called superovulated, so they're induced to release many eggs at one time. This means that eggs that may not be completely mature yet, they may be only partway through their maturity, are rapidly matured, and ovulated. And this, of course, disrupts this final stage of epigenetic reprogramming. We also know, although we don't know as much about it, that sometimes in the case of patients, male patients, that have a blockage in their reproductive system. Sometimes the sperm are actually physically removed from the testes, rather than being ejaculated. And presumably this may have, similar sorts of consequences for epigenetic reprogramming. So then if we think about the epigenetic reprogramming that happens in early development, we know that there's the procedure of IVF, so the fact that the fertilisation is actually happening in vitro or ICSI where it's more invasive and you have the injection of the sperm. But then there's this long culture period, between fertilisation and blastocyst stage. And what it appears through many studies is that, exactly the components of the media in which this culture occurs, can influence epigentic reprogramming and the efficiency of IVF. And finally, of course is embryo handling. Normally in vivo we don't handle an embryo at all, the embryo is stuck inside. But if we move it around, and the way that you move it around, this may have some consequences as well. But essentially we're disrupting both of these time points both of these times of epigenetic reprogramming in this IVF, or assistive reproductive technologies. So, what we think is happening is that the imprinted genes, which need to be reprogrammed properly here and then need to maintain their level of DNA methylation. The imprinted clusters that are set up in the parental origin specific way, may be disrupted during this process. So the germ cell harvest may disrupt their resetting and then importantly during early development it seems that there might be some erosion of those imprinted, those DNA menthylation imprints found in the imprint control regions during this culture period. So, at the moment it's thought that perhaps this is because the maternal affected proteins, those ones that protect those imprint control regions from this rapid clearing of DNA methylation during this period, they seem to be mis-expressed. So if you don't have the correct level of expression of the maternal effect proteins, then they're unable to properly protect those imprint control regions. And so this is what's thought to happen at the moment. However, as I said the jury is still out as to what exactly happens in the human population. So in the next lecture, I'd like to think about how epigenetic reprogram is disrupted in cloning, and somatic cell reprogramming.