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 a 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 derive 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 ameliorate 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 0.3 to 0.4
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 looked at 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.
What we are now looking into 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 conceptus 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 our 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]