Wisconsin Public Television University Place Forum on Animal Research  David Abbot Polycystic Ovary Syndrome And Animal Resarch  April 4, 2012 UP715  cc >> The biological effectiveness and the ethics of using primates in biomedical research. And it's the latter that brings us here tonight. This is the last in a series of forums, community discussions and presentations, as part of the efforts of the Forum Animal Research Ethics that has created a series of four annual community discussions and presentations in order to facilitate communication between members of the community, including students, general citizens, interested citizens, researchers, etc, in order to create a dialog, a conversation, with the stakeholders and people interested in this particular issue. So, as I mentioned, this is actually the last of the four forums of the year. Over the year we've had, in addition to this one, the four have included two presentations on scientific research that's being done on primates here on campus, and both of those presentations of work that has been somewhat controversial in the community. It involved presentations of the research and then discussions afterward about many of the issues related to that controversy. We had third presentation on empathy, the evolution of empathy and connections between human and nonhuman primates and the ethical implications of those related to primate research. And we had a member of the Humane Society of the United States who gave a presentation advocating for an eventual goal of eliminating any research on primates whatsoever eventually once that was theoretically or actually practical. So tonight we look forward to a presentation by Dr. David Abbott. He's also a member of the committee, by the way, and he's agreed to speak about his own research that also involves primates and has had some controversy because it involves using nonhuman primates studying disease. 

Dr. Abbott, who I'm happy to introduce, is a professor and faculty member studying endocrinology and reproductive physiology at the Wisconsin National Primate Research Center. He got his PhD in zoology from Edinburgh. I asked him earlier what he's doing here. It's the most gorgeous city in the world. He's been doing research related to issues of reproduction, reproductive physiology, for over 22 years. He's published 163 articles on related topics. He uses a variety of primates in his research, focusing mostly on rhesus macaques and marmosets, although he did have a brief foray into the sex lives of the naked mole rat. So if you want to know more about, maybe that would be another talk, maybe we could hear about that. It would be a different one. Perhaps least known, however, is his receipt of the Laurent-Perrier Champagne Award for wild game conservation for the development of a synthetic lion dung product as a deer repellent. 

So if you are in need of lion dung or some kind of artificial synthetic version thereof, now you know who to talk to. But most of his work is not on naked mole rats or lion dung, it's on a metabolic disorder, polycystic ovary syndrome, which is a complex and relatively common metabolic disorder that affects a significant number of women. His work, which he'll describe to you tonight, includes inducing the syndrome in primates in order to study it and has raised some questions, as I mentioned earlier, in the community. We're especially grateful for him for volunteering to talk and to facilitate this kind of community discussion. After his talk, there will be a panel. I'll introduce them and we'll talk to the panel after his talk, but we'll be joined by Dr. Jon Levine, who's the director of the Primate Center here, and Dr. Tracy Bekx, who is a clinician who works with people with this particular disorder. So I will hand it over to Dr. Abbott now, and we'll get back together in the panel afterward. >> Thank you, Tricia. Much appreciate it. [APPLAUSE] Good evening, ladies and gentlemen, and if you ever do want to follow up on the synthetic lion dung, it was actually patented and put into practice in the Austrian Alps. 

The problem is they couldn't synthesize it in a small enough condensed form, so they're hulking around barrels. [LAUGHTER] It worked very well. I didn't think it would work at all, and he had little circles of it around deer food in Whipsnade Park in London. Deer wouldn't go near it. And deer are not easily put off, as if you live in the countryside here you know that only too well. But we are here for another reason tonight. I was actually lured across here to Madison, Wisconsin, from working in London because of the unique populations of monkeys here that had arisen in discussions in London about a highly prevalent and devastating women's health disorder that affects millions of women, polycystic ovary syndrome, or PCOS for short. And I put together a potential research team built around my colleagues here in obstetrics and gynecology and at the Primate Center with the unique population of monkeys because we were sure that at the heart of polycystic ovary syndrome, which is testosterone. And about half or maybe more than half of the audience thinks that's an evil male hormone. Well, I'm sorry but we all have it regardless of sex. 

It just depends when it appears and how high it gets is the key biological question. And in males it's meant to get high, and I know most of you say, yeah, we know what that does. Actually, males don't do too well with low testosterone rather than the other way around. But it's when it gets too high during development that we realized, as you'll see here up in the first slide, we were looking at polycystic ovary syndrome with testosterone at its core as almost starting as an endocrine disruptor where the ovary would make too much testosterone and reprogram and repackage the genes that you see here in diagrammatic form, forming the umbilical cord into an unborn fetus. And together, as the monkey work showed that we weren't expecting, but when you do research and you're asking one question, other information pops up that you weren't expecting but can make perfect sense. And here showing the glucose excess during pregnancy when polycystic ovary syndrome, certainly in monkeys, can be formed and differentiated may act as a metabolic disruptor, maybe in tandem with testosterone, maybe separately, to enable polycystic ovary syndrome. And so at the heart of it we in the team, it was a key blend of scientists and medical doctors, both dedicated to trying to resolve this devastating, heartbreaking disorder that millions of women undergo, could we utilize key monkeys at the Wisconsin National Primate Center and nowhere else at the time to try and resolve perhaps one of the key origins of this syndrome, and therefore balance with about 15 million women in the US in 2010 alone that's 18 and over and under 65 who suffer from PCOS and balance that with the humane and timed and well thought out use of monkeys to target clear experiments to try and drive the origins of this devastating syndrome at its origins during fetal life. At the time, that was revolutionary, and over the 20 years we've been carrying out this work that has involved teams from around the country and I am the front man here, there are many people I am so grateful for, from animal caretakers to veterinarians that are dedicated to keeping the animals healthy and understanding my research subjects. I work with human research subjects and my clinical colleagues keep their health in order. My monkey research subjects are kept in order by our expert team of veterinarians at the Wisconsin Primate Center. 

And over the past 20 years we've utilized, commonly for blood samples or series of blood samples and hormone challenges, about 200 rhesus monkeys. And that's the balance we're looking at in terms of 200 monkeys humanely used over 20 years to try and solve a devastating issue in 50 million women in the USA in 2010 alone. This is worldwide. Those are just those figures there. And to try and put this in words of colleagues, these are MD colleagues. Andrea Dunaif, and I'm told I have to use the mouse here and don't wander, Andrea Dunaif, an MD at Northwestern University in the Department of Medicine; Jeff Chang, in obstetrics and gynecology at the University of California in San Diego; Steve Franks, and I've just lost my mouse, there we are, Steve Franks at Imperial College at London; and Rick Legro at Penn State in Pennsylvania wrote a forward to a book entitled simply Polycystic Ovary Syndrome in 2008 bringing together a collection of research works at the time saying it has become increasingly clear over the past several years that PCOS, that's polycystic ovary syndrome, is a complex genetic disease resulting from the interaction of susceptibility genes, in other words, something you inherit that makes you susceptible to an environmental factor that somebody else wouldn't be, and environmental factors. The insight that prenatal exposure, I'll call fetal testosterone exposure, to androgens, the key one being testosterone, can reproduce most of the features of the human syndrome in primates has led to a paradigm shift, that means a completely revolutionized understanding, in these clinicians who were at the forefront of research in human studies as well as animal studies in concepts, in other words understanding, about the pathogenesis, the origins, of the disorder. That's how they value this work and show the depth of help to understand the changing idea of how PCOS may originate and where we might target future therapies. So tonight, I want to deal with what is polycystic ovary syndrome because until you've read the flier, hands up, how many of you have heard of PCOS or polycystic ovary syndrome? A well-informed audience. Just about half. 

You're well-informed. Normally about a handful of hands. Can we program PCOS in female monkeys and can we learn from those experiments that will tell us about where we need to go to look at the mechanism of programming before birth so we can change the outcome of a baby girl who right now is born with PCOS, it's not a lifestyle issue, she is born with it and has to deal with it for her whole life, how we might be able to start getting an insight of being able to change that. And that's the power of the monkey work. What is PCOS? PCOS is a clinically defined condition. Even, as you see, medical doctors were calling it a complex disease. It has a complex definition, and this was arrived at at the consensus meeting in Rotterdam in 2003. If you know the PCOS literature, this was a key turning point in defining PCOS pretty close as we have it today. PCOS, or polycystic ovary syndrome, was really clinically formally identified in the 1930s. And it has been called so many things because of its complex understanding. 

And, in fact, before these criteria were arrived at, part of the criticism of the monkey model was that some of our monkeys ovulated too well. Okay. It turns out that when they finally met in 2003 and formed a now well accepted definition of polycystic ovary syndrome, ovulating patients are allowed. So the definition of the disease caught up to where the monkey model was to show just how close the monkeys are in this understanding and why clinicians have paid so much attention to what we're learning from the understanding in these monkeys. The key thing at the heart of polycystic ovary syndrome in the vast majority of women with PCOS are high levels of testosterone, and the clinical definition is given there. High levels of testosterone in women are just the same as men. They will change the body appearance and be more male like. They'll provide male like body hair pattern on the face as well as on the body. And polycystic ovary syndrome can be defined from two out of these three criteria or all three. So you can end up with four types of PCOS, and this is the simplified modernized understanding. 

The monkey represents all four types from our treatments, and we can get into that in question time as well. The second criteria: basically irregular periods. The ovary ovulates a fertile egg less frequently than every 28 days than it should. Monkeys, the rhesus monkey is very similar. It normally ovulates a fertile egg about every 28 days just as we do. The final of the three: polycystic ovaries, and this is a very clinical definition, is defined on ultrasound appearance of the ovaries. In this image here on the left-hand side, you'll see the normal ovary in the first part of the menstrual cycle is focusing on growing a one single large follicle, and you'll see the fluid there, the follicle fluid that helps to bathe the fertile egg that's inside that follicle. It's concentrating on growing one follicle and egg for ovulation. The other ovary doesn't have such a large follicle. The polycystic ovary, right next to it, these are images that come from ultrasound that are using a noninvasive imaging technique to show you the ovarian morphology. 

They basically cut the ovary in half but not in the old fashioned way that they did some years ago where they did cut the ovaries in half. Medical science and basic science have evolved that we can use noninvasive methods a lot these days to actually understand the pathology we're looking at. There we go. There's the mouse. This ovary is larger. It is growing many follicles, but none of them are growing to a size that will ovulate. And that's the problem with a polycystic ovary. It overachieves at making testosterone, it overachieves at growing follicles, but it underachieves at growing and focusing on that one that will lead to an ovulating egg. And so two out of those or all three will define you as PCOS. And if you mix and match, you'll find the four phenotypes that are there. 

PCOS or a PCOS-like disorder also occurs now naturally in monkeys. We're studying one of the species that also has this natural occurrence. I'm going to talk to you today about an experimental manipulation and not studying the natural occurrence but it naturally occurs there as well. Now, accompanying this reproductive disorder that's clinically defined, is the real health issue, the metabolic syndrome or the metabolic disorder. And here you see, and I've just listed some of them, not all of them, that accompanies PCOS. With polycystic ovarian syndrome you also get with it a four times increased risk of having type 2 diabetes. That, not surprisingly, increases your risk of cardiovascular disease. Gestational diabetes, when mom loses control of her sugar levels during pregnancy, there's a three times increase in risk, and that will become important when we get to the monkey model because that may be part of the component as well as perhaps a genetic inheritance. Endometrial cancer is almost three times increased in risk. Obesity is doubled and I come from a land where obesity is getting an issue. 

Thanks to America we always follow where America goes. You guys are leading the way, we're following. But PCOS women, this is an especial issue, as I'll get to in a minute, because they have a particular issue with not being able to metabolize fat as non-PCOS women can, and they have a lower energy rate. They use energy less for the amount of energy they put out. That wasn't said too well. In other words, they don't burn fat, they store it compared to a non-PCOS woman. So diet and exercise are some ways to -- the issue as we now know, but for PCOS that's not going to be the only solution. And because of all these combinations of issues and others I've left out, depression and anxiety, we have mood disorders that are four times likely to occur in a woman with PCOS because this occurs, this starts to be obvious at adolescence and is a lifelong issue to be dealt with. Not something to be looked like lightly. So PCOS is a perfect storm in women's health, and that's not said in a positive way. 

Here, obesity, PCOS women depending on the study contribute 9% to 28% of women who are obese, and the higher number comes from the study, sorry guys, here in the United States. That's about 10 million women who are obese that PCOS contributes. If we look at type 2 diabetes, up to a third in the USA are because of PCOS. PCOS contributes the largest numbers of young women in their 20s and 30s to becoming type 2 diabetics. And that covers all these three categories. In ovulatory infertility, where women cannot ovulate, PCOS contributes three-quarters to there, and that's across obesity, type 2 diabetes, or non-obese characters. PCOS, its prevalence, is 7% to 15%, which means in the US alone about 15 million women with those metabolic issues because, in this state and many states, reproductive infertility issues are down to you. If we're looking at healthcare costs that are more related to the metabolic issues, they're running over $4 billion a year to deal with PCOS alone. This is a devastating problem at devastating costs. And so PCOS doesn't just change how a woman looks, it changes a woman's whole physiology. 

And there are now PCOS TV dedicated to enabling PCOS women to deal with their many facets of their lifelong condition. And one of them, I'm so thankful to be here at Wisconsin because we have a marvelous biochemistry department with amazing machines with magnetic fields you can't even get close to. So I have to stay outside the yellow lines or dire things will happen to me. Because of the monkey work, I interested my colleagues in biochemistry to study the ability of PCOS women to actually utilize fat and carbohydrate and protein. It turns out that they cannot utilize fat overnight. We normally switch to burning fat as we sleep because our liver runs out of stored sugar. PCOS woman can't do that. They also use less energy every time they expend energy. This is not just a lifestyle issue with these women. It is something they were born with. 

And just to mention, I keep saying diabetes, and just to remind you that it was because of animal research and discovering the role of insulin in diabetes that it's no longer fatal. We can manage it. It still has health issues, cardiovascular disease, strokes, blindness, loss of limbs, and I'm not going to go on because it just gets worse. It used to just be outright fatal until, thanks to animal research, now going back 150 years we now know we can use insulin to actually help treat part of the syndrome and look at insulin's release and action. And I'm going to come back to that in some of the monkey work as well. The problem: the origin of PCOS in women in still unknown. It's only diagnosed after its signs and symptoms appear in adolescence. Physicians can reduce the signs and symptoms, but without understanding the origins we can't really prevent it. So can we program PCOS in female monkeys? And our question, we came here to ask will a female monkey exposed to testosterone as a fetus become PCOS as an adult? In other words, could it be that PCOS is programmed before a girl is born? And our hypothesis that we developed was that the PCOS ovary, as it always is, produces too much testosterone when it's formed during fetal life, during childhood, during adolescence, during reproductive life. And that reprograms the ovary metabolic related tissues, the pancreas, fat cells, the brain. 

As testosterone does, it repackages genes. It's what it's meant to do, but it's not meant to be at such an elevated level in a developing female. We can experimentally mimic that by giving testosterone to mom, overcoming the placenta's amazing ability to inactivate that androgen and get excess levels of testosterone to the fetus in our monkey model, and those levels are in the low fetal male range. The whole thing was to design a developmental program for PCOS to look at genes at an environment that may be causing it in humans but basically resulting in the fetal ovary producing too much testosterone. Will this result in adult PCOS symptoms? And on the way, during our studies of pregnancy, we found that we were elevating maternal and fetal glucose because of this. We were resulting in infants that released too much insulin, and with increased body weight with adolescents with irregular periods now going back 40 years before now, we know this is an adolescent onset. And you'll hear from Dr. Bekx as well about this on the panel, but the monkeys were already showing this many years before it became obvious in people leading to PCOS. Can we come up with a blueprint? And basically we're looking for the seed of a disease that's going to blossom in later life. Can we find where that see is and stop it germinating and giving us some insight? As we started our work, we were harkened by some human studies that were looking at androgen or excess testosterone exposure from two different aspects. One was congenital adrenal hyperplasia where the fetal adrenal causes testosterone exposure. 

Many of those exposed women become PCOS as adults, and in a Chicago study, on fetus with a testosterone secreting tumor that was removed shortly after birth also became PCOS as adult. So we were starting to see as we were beginning our work some indication that there may be outcomes in the human that mirrored the work we were doing in the monkey. How do we create a PCOS monkey? In our model, we inject mom with testosterone. We inject her between, and a monkey pregnancy is about 165 days long, so it's truncated compared to humans, but it follows a similar percentage progression. Not identical, they are monkeys, but very similar to ourselves. And we start about the time the ovaries are forming on day 40 of gestation, and daily inject mom at the time the eggs are normally forming up until the time the ovary can start making androgens. And notice that the ovary, when it can start making hormones, which hormone does it choose to make first? Testosterone and androstenedione before it will mature to making estradiol, and that's when we're giving our injections. And the testosterone levels that we reach are those in the lower male range, about . 3 to . 4 nanograms per mill. 

Well above the normal female range but still within a physiological level that you would see in a fetal male and maybe even not that high for a fetal male. Now, this is well before the follicles that I was showing you in the ovary before our forum around each of those eggs, they mature and now they begin to make estrogen or estradiol and are responsive to the pituitary, and the pituitary is responsive to the hypothalamus in the brain that will regulate how the ovary will stay quiescent until adolescence occurs and then will go through a reproductive lifespan managed by the brain and the pituitary and those two hormones, LH and SFH. Also, at the time we're giving our injections, the part of the brain that regulates reproduction, the hypothalamus, and the neurons that we'll get to, are also migrating to their mature place. And the pancreas is forming well before it will start maturing in later gestation. So our testosterone exposure is when multiple organs are forming in the rhesus monkey, and so we should have a multiple organ change because of our exposure. Key question, though, is will testosterone program PCOS-like signs and symptoms in our monkeys? And here are a series of key items, the height of testosterone, the lack of cycles, the polycystic ovaries, and the metabolic issues that are there in PCOS women, how do the monkeys stack up? And before we just get to the monkeys, when I arrived here the first population of monkeys I worked with had been created for psychology studies, and, taking them over, they were already well into their mid to late reproductive years. So as you'll see here, the monkeys in red are our exposed monkeys. They were age matched to the controls that appear in yellow in the panels. We weight matched them as best we could to controls who didn't have any exposure during pregnancy and gestation. And at the far end there, we tried to match them for BMI. 

And if any of you are aware of BMI, we tried to keep it somewhere between 18 and 24, and then you look at this where there's an average somewhere about 38 or 39. I assure you we're not feeding these animals on bratwurst and cheese and other high fat good Wisconsin foods. No, we actually feed them on low fat, high fiber diet because we are trying to maintain a well-nourished, well-vitaminize, mineralize, I've been in Wisconsin too long, those are nouns that just became verbs. And the issue was our monkeys were not obese because we are giving them a well-balanced diet, in fact the diet you would love individuals with PCOS to be on, but it takes some rigor to stick to. And, as you see, some of the controls were in the obese range because we just couldn't keep them as well matched as we could for the age. Meet some of our adult rhesus monkeys. We socially house them, where we can, in pairs. And, as you see here, they get treats as part of the enrichment. They share. Okay, some of them don't share too well, but they're just like people. 

Some of them get on well, as you see here, and some of them will act singly at times because we try to pair them all the time. But, just like people, they sometimes don't get along, and we have to find compatible pairs as we go through. But we try to house them in pairs. Because I'm studying food intake and how we regulate glucose, we have to separate them when they're on study for about 10 hours a day. Right now the pairs are separated so we can look at their calorie intake per day, and we've now found that our monkeys that we exposed to testosterone eat the same as the monkeys who weren't. So it's not a food intake issue for them, and they get to browse in, this is artificial turf, in various complicated ways. So it's not all that easy to find their food at times. They have breakfast, they have healthy food like fruit and nuts at around lunchtime, and then some chow in the afternoon to provide them with three meals a day. During this period, we calorie count at the end of the day, take what little food there is left so every morning we can get a facet blood sample, and if any of you have been to the doctors and they need to look at your glucose levels and your fat levels, you've got to fast overnight. There's just no way out of it, and we do the same with the monkeys having made sure they've eaten during the day. 

And we also have to make sure we do take all the food out when we put the pair back. If we want to take a blood sample, the animals are accustom to voluntarily entering a transport box. They come to a tabletop restraint. On the top one, you'll see, the animal was offering a leg. It is not being held in restraint. That's there in case the animal will not hold completely still, and we take a blood sample. And then at the end, you will see over in the bottom corner there, the traditional handing over of the grape. And if you saw Franz DeVal's talk, you know how monkeys value grapes. Not cucumbers. They value grapes. 

And we hand the grape over, and you see the monkey is just sitting there after the blood sample quite happy going, yes, I'll take that grape, thank you very much, and when are you going to open that door so I can go home? But there's no stress, there's no panic. We look at stress hormones. We have to. If these animals were stressed, their sugar levels would be high, their fat metabolism would be all wrecked, we couldn't study them for our purposes. They need to be very used to us. They need to realize this is a very straightforward thing to do, as we do when we go get a blood sample. We go, we get a sample taken, and we realize that wasn't too bad. We can train monkeys to offer their legs in cages. In our study we managed to train six of our female monkeys to do this. So in their home cage they offer their leg. 

We can take a blood sample, and then give them a reward. These are old photos, and this is actually an adult male that we were using here because we don't have any female photos to show, but it's the same issue and the same way for all the in-cage sampling. When we're doing any procedures to collect the eggs when we looked at our fertility study that I'll mention to look at their ovaries, to take any biopsies of tissue, we have a top world-class suite of surgeries that you'd find in any top class veterinary hospital here in Wisconsin, and we have expert veterinary surgeons, surgical technicians who can support the individual and anesthesia and surgical support needed for our work. And when we do noninvasive scanning, we do anesthetized the animals because getting monkeys to hold still is just something they won't do. So we lightly anesthetize them to do this, and the animal then goes through about a 20- to 30-minute scan. This is a picture from National Geographic. They came to study and they put a laser on the scanner as it was going backwards and forwards. And the scientist you see beside there was superimposed on the picture just for clarity because you can't be standing there while the animal is scanned. But that is how the animals would go through scans, as you'll see, to understand their fat composition. Okay, now, trying to look at what did we learn from this. 

How well did the animal show PCOS traits because that's the heart of it. If they don't do that, this is not a good model. But if they do do that, this would open the way to understanding at least one cause of PCOS and during fetal life. Do they show high ovarian testosterone? And now, sorry, I'm a biology teacher and if any of my 152 biology students are here, sorry, you're going to get this lesson again but simplified, and there's no exam. [LAUGHTER] Okay, we look at, this is a human head. This is a drawing of a human head. [LAUGHTER] Cut in half so the front is over on your right, the back is over on the left, the top is at the top, and the base is towards the floor. We're interested in the hypothalamus. It's right at the base of the brain. It regulates almost all of your bodily functions, and it communicates by chemical fashion to the anterior pituitary, highlighted in red there, that receives a very private blood supply from they hypothalamus. 

And we want to look at a particular nerve cell in the hypothalamus. It's the GnRH neuron. It releases a small protein into that private blood supply to the anterior pituitary, and cells in the anterior pituitary that have receptors for that peptide gonadotropin releasing hormone, or GnRH, when it binds to them, those cells in the pituitary release LH. They also release FSH, but we're really focused on LH today because that luteinizing hormone is the testosterone driver in the ovary. It's the testosterone driver in the testes, but in the ovary it is targeting on part of the follicle. Now the follicle is the functional unit of the ovary. As I said before, this is a follicle close to ovulation, and you'll see the egg here nestled in the catcher's mitt. This is a follicle, again, cut in half. It's an electron micrograph. This is a human egg, and I've lost my mouse somewhere. 

There we go. And it's a large cell here and surrounded by cells that are not hormonally active. We're interested in not the immediate cells surrounding the fluid, the follicular fluid here. These are the granulosis cells. They make estradiol. We're interested in the outer half of this follicle here, these cells. These are the theca cells. They make testosterone when they bind LH released from the pituitary. And in this diagram here you can see that what LH does is it binds to its receptor. CG, peptide released by an implanted embryo releases a hormone very similar, CG, that will also bind to this receptor. 

And it makes the theca cell convert cholesterol, yes we make testosterone and estradiol from fat, and it makes two androgens. Yes, I'm going to give you yet another long name here, androstenedione, as well as testosterone. Testosterone is almost a byproduct of the theca cell. The theca cell doesn't really want to make testosterone because that's not good use in a female. It wants to make androstenedione, a bio inactive androgen. And that is key because the cells that are close to the egg and need to make the hormone that the egg needs, estradiol, are down here, and they need that androstenedione product. They can't make estradiol from cholesterol, and they use that to make the hormone that will help mature the egg in the ovary. The problem in polycystic ovary syndrome, those theca cells just naturally make too much testosterone, and if you stimulate them with LH or with CG, they make even more even though they were making too much in the first place. What we did here was to see if we give CG, which is a much more potent hormone than LH, to our monkeys just as an injection into muscle, could we actually induce their theca cells because that's where LH goes, not to the adrenal, not anywhere else. It goes there to stimulate testosterone release. 

And here, these are hours after that injection that's driving that LH receptor. And you see the red panels from the testosterone exposed monkeys, those testosterone levels are much higher than the yellow bars made by the controls. These animals not only have high testosterone, it's the ovary that's making the higher testosterone just as in PCOS. Do these monkeys ovulate less frequently than they should? Yes they do. Here we have an example where in rhesus monkeys they have menstrual cycles, but just as in humans we need to confirm hormonally that ovulation does occur each time, before each menstrual bleed. We bleed these animals twice a week for six months taking a small blood sample each time by which we could measure the hormone progesterone and verify that each menstrual cycle was an ovulatory one. And here you'll see, in the yellow panel as in us, these monkeys cycle about once a month. So in six months, they have six ovulatory cycles. Our testosterone exposed animals cycle at about half that rate. And some of them had long gaps, and some of them cycled more regularly, but there was a statistical significance, and 67% of them type as PCOS in terms of their irregularity and lack of cycles. 

Just to point out that in these animals, those were the adults. If we look at those animals as adolescents and we look at the percentage of the regular cycles in those adolescents, the testosterone exposed animals are on the right-hand side. Almost half of their cycles are irregular compared to about 50% incidence in the normal unexposed adolescent female monkeys. So this is an adolescent onset, as it is in humans. Do they have enlarge polyfollicular ovaries? And we can show it here on ovaries that are illuminated. In other words, when there's a follicle present you'll see a red circle here, and in the enlarged polycystic ovary you'll see here some of these fluid-filled follicles showing up. There are multiple follicles in this polycystic or polyfollicular ovary compared to a normal ovary, and these were taken at the start of a menstrual cycle when they're not growing a large follicle at that time. It hasn't developed to that stage. And you'll see that here 40%, compared to 14% of the controls, had polycystic ovaries, but interestingly, of our animals that did develop those polycystic ovaries, it's the T exposed animals that have enlarge ovaries. The controls, if they have this phenotype or appearance, they have a normal sized ovary. 

In our T exposed animals when they show this multiple growth of follicles, they've enlarged their ovaries to accommodate this overgrowth. It's just more demonstrative than in the controls. Now, like PCOS women, we've not only altered the ovary, which is the definition of the clinical definition of the disease, have we changed the way the brain regulates the ovary? And the brain works through LH to regulate the androgen or testosterone out of the ovary. In our T exposed monkeys, GnRH just doesn't get released. It's released in episodes about every 50 to 60 minutes. The monkeys do the same, and here you'll see LH is a faithful, reliable reflector of GnRH pulsatile release. And those theca cells read the pulses so they respond to each of those pulses. And the arrows here indicate and episodic release, or basically a burst of LH that we are taking now, we're just taking a blood sample from a vein here and we're just taking a peripheral vein. This is actually a leg vein here, and we're showing in the T exposed animals about double the frequency we see in the control. We've changed how the brain and the pituitary are driving testosterone from these theca cells as well as the theca cells being more testosterone responsive themselves to our injection. 

So we have PCOS-like reproductive traits in our monkeys. They're there. And that, back in the 1990s when we first published these results, was a complete novelty. Now it's been replicated in many other animal models to show that what we're seeing here is a very similar impact across the mammalian kingdom. Now, will these females show the metabolic-like traits? These are the ones that are more health concerning rather than being diagnostic. Will they show in abdominal fat increase, particular the visceral fat? And that's the fat and here's a scan of a rhesus monkey that's gone through a CT scan just as we would, and it reflects, it bears all. So all that fat that you like to not show but can actually grab hold of, and I won't do that, that's the non-visceral fat. That's in gray. The visceral fat here surrounds your intestines, your kidneys. This is a kidney here on the rhesus monkey. 

And we colorize it in red so we can quantify it. Just as using human technology, using human ability to quantify it. And we find in our monkeys, they not only have more abdominal fat, they have more visceral fat. And the problem with visceral fat is it impairs insulin action. It's called the bad fat. If you put on weight, there's meant to be the good fat and the bad fat. And the good fat is the non-visceral fat when it's here. The bad fat is the visceral fat because as it increases your insulin's ability to enable yourself to take up glucose so they can actually use that to make energy goes down and down and down with this. And we'll come back to that point. Now, are these animals insulin resistant and does the pancreas fail to release enough insulin? We do this by giving a glucose injection and following the blood glucose and insulin levels they're after and running it through an algorithm that's called insulin sensitivity. 

It's a measure of insulin resistance. So the higher this number, the more sensitive an individual is, the more effective insulin is in making yourself take up glucose so they can use it to make energy. You'll see our testosterone exposed animals have half the insulin effectiveness of the controls, and to add insult to injury, they have maybe about a third or less of an ability to release insulin, this index of pancreatic insulin release. They mimic, amazingly, PCOS women in terms of the degree of decreased insulin sensitivity and the degree of reduced release of insulin from the pancreas. What about type 2 diabetes? If they're showing these pre-diabetic predispositions, what about type 2 diabetes? It's defined, just as it is in humans, above 125 milligrams per deciliter. When it starts to cause microfractures in blood capillary vessels in ourselves. We have about a quarter of our adult animal type 2 diabetics compared to their age and body weight and body match controls. Now, having seen all that metabolic issue together with the reproductive components, in PCOS we know that the two actually interact, and that reduced ability for insulin to act is a problem for the ovary as well. It's necessary to cause normal ovulatory menstrual cycles. And our monkeys, we wanted to test do they suffer from that same mechanistic issue that humans do having shown how similar they are to PCOS to get at the mechanistic understanding. 

And here are our T exposed animals. In the red panel on the left-hand side getting an oral placebo. That's a peanut butter sandwich given before breakfast in the morning. And then there's three months with no treatment, and then another six months of peanut butter sandwiches, but this time they contain a drug called Pioglitazone which is an insulin sensitizer that's used in women to improve insulin action, and it's used in PCOS women for that regard as well as improving their cycles. It normalized the menstrual cycles of these monkeys. In other words, improving insulin's ability to act was part of the pathophysiological mechanism in these monkeys. Not only do they resemble PCOS women in signs and symptoms, part of the underlying mechanism is the same. And, unlike in PCOS, which normally doesn't happen, we pulled the treatment after six months, and you see the animals reverting to type as insulin action become impaired and they lose the ability to undergo normal menstrual cycles. What this did for our colony as well as understand how the mechanism of lack of ovulation worked is we now use Pioglitazone as a first line treatment for monkeys that are determined as diabetic in our colony because this was the first time this drug had been given for such a long period to these monkeys. So we now, rather than just putting them straight on insulin and eventually they can become resistant to that, we can use Pioglitazone and make them use their own insulin for as long as they can, just as it is in humans. 

So, in our adult monkeys if we look at all the polycystic ovarian syndrome criteria, their mechanism of lack of ovulation, how the brain is altered, they're just like women with PCOS. With the metabolic issues that devastate women's lives who have PCOS, our monkeys are very similar. We have an amazing comprehensive match. The only animal model to date, and that's now 20 years after we started, nobody can match this comprehension because these monkeys are reproductively controlled, metabolically controlled, and age so closely to us. Their genes are so similar. That's why we're getting such a close fit and such a good understanding. If we did this in rats and sheep, you get fetal growth restriction along with these outcomes. You get quite a different metabolic outcome. So the monkey is very important getting close to humans, where in PCOS fetal growth restriction is not a major issue in causing the pathophysiology. Having shown how PCOS-like they are, what can we learn about the mechanism that we just cannot do to people? We can't go to where we're going now with people. 

We just can't do it. We're going to look at the eggs that these animals are ovulating when we collect the mature eggs. We do this thanks to my colleagues in obstetrics and gynecology who are whizzes at infertility and can education a basic scientist no problem in what's needed in terms of looking at how to undergo infertility treatment for monkeys compared to women. Normally in the menstrual cycle, as I mentioned before, both ovaries are fine for one of them to grow on single large follicle called the dominate follicle that will ovulate that egg. Just as in humans, we can take the process over. And this is where FSH comes in, follicle stimulating hormone. It's now made synthetically, and because we are so similar to humans we can use recombinant human FSH for stimulating monkeys. And you see instead of one mature egg to ovulate, just as in women, you can get many. Somewhere between six and 20 is the common number, very similar to women as well. Now, we don't let these eggs naturally ovulate. 

We go in and we aspirate every single one, just as you would in a human condition to then mix them with sperm in a Petri dish for in vitro fertilization to then, in humans, try to replace that as the pregnancy. We weren't replacing these embryos as pregnancies. We were going to study their outcomes. And their outcomes are quite different depending on their exposure. The yellow bar, again, are our controls. These are embryos of the two to four cell stage all the way to the term called blastocyst. That's when the embryo was formed enough that it normally walks across the endometrial lining and decides I'm going to implant here and in it burrows. We get up to that stage in culture before then the embryo would burrow into the uterine lining. And we can study them. And in our controls, about 60% to 70% of the eggs that were fertilized get to that implantable stage. 

You look at the testosterone exposed animals doing just fine until they have to turn on the embryonic genes, or we call it here embryonic genome activation. If these were mice and rats, that would be at the two cell stage. These are not. These are primates just like us. That doesn't happen until the five to eight cell stage. They're a primate and they turn on their genome then, and then you see the tremendous fall off of the success of the fertilized eggs and embryos at that point so that only about 5% of them end up as blastocysts. There's an impairment. When that genome from those monkeys is meant to turn on , it has a tremendous impairment. So they, at the level of the egg, there is something there that we have induced that may be being passed on to the next generation. Because of the monkey work, because of this work, we collaborated with clinicians at U Penn in Philadelphia to study PCOS women and control women who very kindly volunteered their eggs for gene typing. 

We're not gong to fertilize any eggs here. We form no embryos. We just loop to the eggs and their genes because of the monkey work to see what genes do we change. And rather that just look at the monkey, what can we learn directly from the human because the monkey told us where to look, and it told us where to look in the eggs. And this is an amazing story condensed down. It's already published now five years ago. We found, compared to normal, eggs from PCOS women differentially express, in other words either have turned up or turned down genes in the egg, over 300 genes that were differently functioning in PCOS. Most of them were over expressed. They were producing too much product, and in trying to target what those products were, our colleagues at U Penn found that they altered genes were the ones that regulate how the cells divide, when they know when to divide and how they divide and how they regulate the chromosomes during that division. That's what was different about the eggs from the PCOS women to normal to realize this is a cell cycle change, and that's going to become important when we look at some of the tissue that comes from the monkeys. 

What did we learn from the fetal monkeys in doing this? We learned that we had engaged the mother more than we thought when we gave the testosterone injections. And this is something, again, we can't do with humans because here we're looking at manipulating the pregnancy outcome. And giving testosterone now to normal mothers that were, the red group here is a normal group that will get testosterone. The yellow group here is, again, a normal group that will get an oil injection control or placebo. And you'll find that the testosterone injected moms here increase their body weight dramatically to that that's normally there when they give birth by mid-gestation. The controls normally get there, but our testosterone injected moms got there by mid-gestation. When obstetricians see this greater weight gain in humans, that means they are going to have problems with usually increased birth size of the baby, other birthing complications, and those offspring are commonly obese and diabetic because of this outcome. So we have now our mothers showing signs, because we're giving them an anabolic steroid, of losing their ability to control glucose in addition to that fetus having a higher testosterone exposure. What happened to mom during this time? And we capture that not only with her body weight but by giving her a glucose injection at mid-gestation here at day 80 and watching her ability to clear glucose. And we measure that by a measure called KG, to cut a long story short. 

And KG basically measures the rate of glucose lose, and the higher that bar is, the faster the glucose is being cleared. And we'll see here that the red bar is about half the size of the yellow bar. Our testosterone injected moms are slow at clearing glucose. In fact, they've just become as slow at clearing glucose as obese rhesus monkey mothers are that are two to three kilos heavier. So these monkeys, because the testosterone injections, the mothers are losing their ability to control glucose, and the worse the mothers were at controlling glucose because of the testosterone injections, the higher the level of glucose in their fetuses at this time in mid-gestation. And here you'll see those fetal glucose levels here on this Y-axis. And you'll see all the red, there are four red dots here. It's 44% that are completely exceeding the control range, the yellow dots here, because their moms are so poor at clearing glucose. It has an impact of the fetus just as it does in human pregnancies, and that means the baby is likely going to be bigger and there are going to be birth complications. We can measure that in our monkeys just as you can in humans by looking at head size. 

The head size in our testosterone injected pregnancies was about 5% bigger than the controls, and in diabetic pregnancies, this is a similar complication in terms of the baby getting larger than it otherwise would be. Another common complication from such a pregnancy outcome is increased body weight as the infants grow. And here even by eight weeks you'll see our red bars again are heavier, about 10% heavier than the controls by eight weeks. And we know this sort of increased growth in weight is certainly, again, linked to obesity and diabetic outcomes in humans. And we know these animals are going to grow up to be the adults I just told you about. So we know we're looking at the early onset, and it may be that the glucose may be a key part of what was going on. Here we're looking at the insulin measures I showed you in the adults. They look nothing like the adults. We're not seeing a mini version of the adults when these testosterone exposed monkeys are born. We learned something completely new here that we didn't even appreciate was there and medical doctors didn't even appreciate was there but they did in diabetic pregnancies but not in PCOS pregnancies. 

The monkeys showed that the two may be very, very similar because of the monkey work. And what we're seeing here is that insulin sensitivity, on the left side again, the yellow bar is the controls, look at where the red bar is. It's towering above the controls. These are very, very insulin responsive animals. If we look at the right panel, again the red bar is towering over the controls. It was exactly the opposite in the adults. These animals are over releasing insulin when we give them an injection of glucose. And they're clearing their sugar far too fast. When we look at their blood glucose values down here, you'll see that they're clearing about 20% faster than the controls. In other words, they're over releasing insulin. 

Their problem is keeping normal levels of glucose, rather than low levels of glucose. In fact, half of these animals were born hypoglycemic, just as can happen after diabetic pregnancies. And that was a complete new revelation to PCOS understanding. It doesn't start out with insulin resistance. It doesn't start out with a failed pancreas. It starts out with a pancreas that had to cope with mom not regulating her sugar. So it is much more robust in releasing insulin to control its own sugar. So when these infants are born, just as happens in human pregnancies, they are far too robust at releasing insulin. What does that mean? Well, insulin is one of our growth hormones. It drives growth. 

And what we are now looking in to is if part of the problem is PCOS and in our monkey mimic of PCOS is that increased glucose during pregnancy leading to increased insulin release in the infants, promoting weight gain, that's certainly going to be fat as well as muscle, so by the time they get to adolescence, they've got, at least in the monkey, increase abdominal fat, or in adulthood, abdominal fat insulin resistance and all the accompanying diminished insulin components of the adult because now this high fat component impairs the ability of insulin to act and is driven all the way from infancy. And that may have been driven because mom couldn't regulate her sugar levels too well during pregnancy. All come from understanding the monkey. And I could keep going on this because I think the monkeys have given us such an insight into what occurs in humans. I'm going to take one more advantage of your time here today to tell you a more recent insight that we've had that makes an even bigger leap to actually looking at maybe the different genes that are packaged in a monkey, and they come very close to some studies genetically inherited in the human. And here I am shamelessly thankful to colleagues who are whizzes at microarrays that actually can detect because rhesus monkey genes are so similar to humans, we can use human arrays, okay don't look at all this biology. You want to see the compare to current controls over on the far right. This is where we want to look. We can use human technology because we can look at genes that are over 94% identical to ourselves. And how they're differently packaged in the infant monkeys at two months of age and in the adults and what tissue did we choose because we had one shot to do this to begin with? The visceral fat. 

The fat that is most involved in impairing insulin action as it amasses in size. And we found in the infants over 160 genes were differently packaged. That means they were over expressing or under expressing compared to controls. And in the adults, over 300 genes. And the thing about it is the genes that were differentially changed were completely different between the two. Only two in common. All the rest were different. And this is not unusual through development in humans. Even identical twins will have similarly packaged genes until they get through adolescence, and then their gene methylation signatures, as they're called, differ. Even though the genes are identical, the way the genes are expressed, either more or less, differ even in identical twins. 

And that comes with age. And that's what we're looking at here. The most significantly altered pathway that was linked in both adults and infants. I'm going to be shameless in telling you about transforming growth factor beta, or TGF-beta. To cut it short, it controls how and when cells divide and mature. Remember those eggs before? Some of the issues they had is controlling how they divide. What pathway is most significantly altered in how the genes are packaged in our monkeys? It's a pathway that regulates how cells divide and mature. And this is in multiple organ systems. Why is that so important? Because in humans it's the only gene candidate to date that's been reliably found associated with PCOS in three independent studies, and replication in science as well as medicine is huge. Three studies, only one candidate has managed that to date, and it's a TGF-beta related gene. 

The same pathway that's genetically linked to PCOS is the same pathway that we have epigenetically linked in our monkeys. From two separate studies, we're arriving in the same place. And these pathways operate in almost every cell system and organ system in our body, and in studies inspired by the monkey work, because when investigators first went to look at the human gene in mature adult tissue, nonfunctional, nonactive. And they went wait a minute, there's a monkey model and there are fetal origins for PCOS. Let's look at fetal studies from -- that they obtained, and these are studies in Australia, and in there they found a gene in the fetal ovary. This gene was functional only from early to mid-gestation, right about the time we're giving our testosterone treatments to out monkeys and at the time the ovary starts to make androgens. The genetic link in human is related to a fetal active gene only right at the time we give our testosterone in the monkeys. We didn't know this. We just had a hypothesis of hitting organ systems as they matured, and that may be one of the genetic pieces of the puzzle in humans as well. Now that we know that, we can start, and it's not as if okay, there's the answer. 

This is a multi-molecule pathway. So we may have a target but it's a gargantuan one that is going to take, in the human studies for those with related genes have involved 4,000 research subjects just in one of those studies alone to give you an idea of the scale that you have to get to in the human studies. How in the human have we come to relate, just finishing off here, fetal testosterone exposure with PCOS outcome? And here is a picture, as you'll see in a minute, of the hair clipping that's occurring in a newborn baby. This is a mom with PCOS with a newborn baby in the birthing center at Meriter Hospital. We are looking at hair because hair is a historical record of your hormones. This is thanks to the drug and doping testing in professional athletics. [LAUGHTER] You can take these hormones because they do draw tissue, they change your genes. And you can get a metabolic advantage. So you can abuse them for personal gain. Yes, they also make you insulin resistant and diabetic, but that's another matter. 

But we can use the hair as a historical record of what went on during fetal life. But some studies in human are also just using blood at birth well past the time that it should be elevated. But one of the studies in the UK had found higher testosterone in the newborn samples in those baby girls born to PCOS moms than non-PCOS women, daughters. A study in the US from Chicago showed an abnormality but the other way around. The androstenedione was too low. Both of them were abnormal, just one's high and one's low, but it's at birth. It's not the time we're aiming for, and we're awaiting our hair outcome to see if we can get a better understanding of the historical elevation of testosterone because hair starts to grow about mid-gestation, and hormones get laid down as the hair grows. And you can literally score where the hormone was produced by where it is in the hair, length from the follicle. Okay, in a study in Chile on two-month old infant girls born to women with PCOS they have abnormal ovarian hormone levels. In prepubertal adolescents of women with PCOS, they have high insulin levels before they start to convert to high testosterone during adolescence. 

Does that ring a bell? The high insulin levels, again mimicking what we're seeing in our immature PCOS-like monkeys, are starting to be picked up now in human adolescent studies, and before you think I've gone off the deep end here, our finger lengths are controlled by testosterone. And the story is that male fetal testosterone makes your second one shorter than you fourth. Don't worry, there's tons of overlap so if yours isn't, no problem. [LAUGHTER] Or the other way around. And two studies out of Cornell associated high testosterone in PCOS women with longer second finger length. So it can't possibly be due to testosterone exposure during fetal life. Well, thanks to a resourceful student in my lab looking at finger lengths in our monkeys, or actually two resourceful students, in our monkeys their second fingers are longer than normal. They match the PCOS studies and they mismatch with the rat and the mouse studies and the assumed data from humans because you can't do this in humans. You can't go, we'll give you testosterone and then we'll see you in 20 years and we'll measure your fingers. We can do that in monkeys, and what we're finding is very similar the higher the testosterone level gets in PCOS women, the longer that finger is. 

This is fixed in fetal life. Sorry, oops. That is fixed in fetal life. So it's a postnatal marker of testosterone exposure. Our monkeys are showing what really happens when you're exposed to testosterone and you're a primate and you're not a rat and you're not a mouse. It's not the same outcome. So, lessons learned from our monkeys? Testosterone and maybe high glucose that nobody ever was bringing into the picture may program PCOS before birth. It certainly does in monkeys; it certainly does in other mammals. PCOS alters gene expression, and we've shown in the monkey we can mimic that or we can mimic what is inherited in women by these manipulations during fetal life. So we may now have molecular targets or we can perhaps target lifestyle interventions with this knowledge that will manipulate these systems. 

So the whole thing behind this is a personal drive, a personal empathy with women who have a devastating health disorder that we have an animal model that provides an insight you can't get from other animals, let alone cells and let alone from human studies because you can only go so far into looking at the origins, and it comes back to this. We are trying to get insight from the humane use of monkeys to help over about 15 million women in the USA alone, certainly over that worldwide, to get an understanding that one day will result in effective treatment and prevention in the clinic, and we hope we played our part in that and humanely derived the work to do it. Thank you very much for listening. [APPLAUSE]