cc
and the ethics of using primates >> The biological effectiveness
And it's the latter that brings in bus here tonight.ch.
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
community discussions and presentations in order to
facilitate communication between
including students, general citizens, interested citizens,
researchers, etc, in order to create a dialog, a conversation,
with the stakeholders and people
issue. interested in this particular
actually the last of the four So, forums of the year.s is
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
presentations of work that has campus, and both of those
the community. been somewhat controversial in
research and then discussions Itafterward about many of thethe
issues related to that
We had a third presentation on controversy.
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
advocating for an eventual goal of eliminating any research on
primates whatsoever eventually
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
Dr. Abbott, who I'm happy to disease.
faculty member studying endocrinology and reproductive
physiology at the Wisconsin National Primate Research
Center. He got his PhD in zoology from
Edinburgh.
doing here. It's the most gorgeous city in
the world.
to issues of reproduction, He's been doing research related
over 22 years. reproductive physiology, for
related topics. He uses a variety of primates in
his research, focusing mostly on
although he did have a brief rhesus macaques and marmosets,
naked mole rat. foSo if you want to know morehe
about, maybe that would be
hear about that. another talk, maybe we could
Perhaps least known, however, is It would be a different one.
Laurent-Perrier Champagne Award for wild game conservation for
the development of a synthetic
repellent. lion dung product as a deer
dung or some kind of artificial synthetic version thereof, now
you know who to talk to.
naked mole rats or lion dung, it's on a metabolic disorder,n
polycystic ovary syndrome, which
common metabolic disorder that is a complex and relatively
women. affects a significant number of
to you tonight, includes His work, which he'll describe
primates in order to study it and has raised some questions,
as I mentioned earlier, in the
We're especially grateful for him for volunteering to talk and
to facilitate this kind of
After his talk, there will be a communitpanel.ussion.
I'll introduce them and we'll talk to the panel after his
talk, but we'll be joined by
director of the Primate Center Dr. Jon Levine, who's the
a clinician who works with here, and Dr. Tracy Bekx, who is
disorder. pSo I will hand it over tor
Dr. Abbott now, and we'll get back together in the panel
afterward.
Much appreciate it. >> Tha[APPLAUSE]icia.
Good evening, ladies and
want to follow up on the gentlemen, and if you ever do
actually patented and put into synthetic lion dung, it was
The problem is they couldn't practice in the Austrian Alps.
condensed form, so they're synthesize it in a small enough
[LAUGHTER] hulking around barrels.
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.
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
polycystic ovary syndrome, or affecPCOS for short.women,
And I put together a potential
colleagues here in obstetrics reand gynecology and at themy
Primate Center with the unique
were sure that at the heart of population of monkeys because we
is testosterone. polycystic ovary syndrome, which
than half of the audience thinks And about half or maybe more
Well, I'm sorry but we all have thait regardless of sex.one.
It just depends when it appears and how high it gets is the key
biological question.
high, and I know most of you And in males it's meant to get
does. Actually, males don't do too
well with low testosterone
around. But it's when it gets too high
during development that we
in the first slide, we were relooking at polycystic ovaryup
syndrome with testosterone at its core as almost starting as
an endocrine disruptor where the
testosterone and reprogram and ovary would make too much
here in diagrammatic form, repackage the genes that you see
an unborn fetus. forming the umbilical cord into
showed that we weren't And together, as the monkey work
research and you're asking one expecting, but when you do
up that you weren't expecting qubut can make perfect sense.ops
And here showing the glucose
polycystic ovary syndrome, excess during pregnancy when
formed and differentiated may act as a metabolic disruptor,
maybe in tandem with
to enable polycystic ovary testosterone, maybe separately,
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
Wisconsin National Primate we utilize key monkeys 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
from PCOS and balance that with athe humane and timed and weller
thought out use of monkeys to target clear experiments to try
and derive the origins of this
origins during fetal life. devastating syndrome at its
revolutionary, and over the 20 At the time, that was
this work that has involved teams from around the country
and I am the front man here,
grateful for, from animal caretakers to veterinarians that
are dedicated to keeping the
understanding my research animasubjects.y and
I work with human research
colleagues keep their health in subjects order. clinical
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
samples or series of blood utilized, commonly for blood
about 200 rhesus monkeys. saAnd that's the balance we're,
looking at in terms of 200
years to try and solve a monkeys humanely used over 20
women in the USA in 2010 alone. devastaThis is worldwide.illion
Those are just those figures
And to try and put this in words there.
colleagues. Andrea Dunaif, and I'm told I
have to use the mouse here and
MD at Northwestern University in don't wander, Andrea Dunaif, an
Chang, in obstetrics and the Department of Medicine; Jeff
California in San Diego; Steve gynecology at the University of
mouse, there we are, Steve Franks, and I've just lost my
London; and Rick Legro at Penn Franks at Imperial College at
forward to a book entitled State in Pennsylvania wrote a
in 2008 bringing together a collection of research works ate
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
factor that somebody else swouldn't be, and environmental
factors.
exposure, I'll call fetal testosterone exposure, to
androgens, the key one being
of the features of the human testosterone, can reproduce most
a paradigm shift, that means a syndrome in primates has led to
understanding, in these clinicians who were at the
forefront of research in human
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
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
A well-informed audience. or polyJust about half.drome?
You're well-informed. Normally about a handful of
hands.
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
girl who right now is born with canPCOS, it's not a lifestyleaby
issue, she is born with it and
whole life, how we might be able has to deal with it for her
being able to change that. to start getting an insight of
monkey work. And that's the power of the
PCOS is a clinically defined What is PCOS?
Even, as you see, medical doctors were calling it a
complex disease.
this was arrived at at the It has a complex definition, and
in 2003. consensus meeting in Rotterdam
this was a key turning point in defining PCOS pretty close as we
have it today.
syndrome, was really clinically formally identified in the
1930s.
things because of its complex And it hunderstanding. so many
And, in fact, before these
of the criticism of the monkey criteria were arrived at, part
monkeys ovulated too well. model was that some of our
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
understanding in these monkeys. The key thing at the heart of
polycystic ovary syndrome in the
are high levels of testosterone, vast majority of women with PCOS
given there. and the clinical definition is
women are just the same as men. HiThey will change the body in
appearance and be more male like.
They'll provide male like body
as on the body. hAnd polycystic ovary syndromell
can be defined from two out of
three. these three criteria or all
types of PCOS, and this is the So you can end up with four
understanding. simplified modernized
types from our treatments, and The monkey represents all four
time as well. wThe second criteria: basicallyn
irregular periods.
less frequently than every 28 The ovdays than it should.le egg
Monkeys, the rhesus monkey is very similar.
It normally ovulates a fertile
we do. egg about every 28 days just as
polycystic ovaries, and this is a very clinical definition, is
defined on ultrasound appearance
In this image here on the left-hand side, you'll see the
normal ovary in the first part
focusing on growing a one single of the menstrual cycle is
the fluid there, the follicle fluid that helps to bathe thee
fertile egg that's inside that
It's concentrating on growing one follicle and egg for
ovulation.
such a large follicle. The other ovary doesn't have
to it, these are images that The polycystic ovary, right next
using a noninvasive imaging cotechnique to show you there
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
Medical science and basic science have evolved that we can
use noninvasive methods a lot these days to actually
understand the pathology we're
There we go. looking at.
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
And so two out of those or all lead to an ovulating egg.
And if you mix and match, you'll three will define you as PCOS.
are there. find the four phenotypes that
also occurs now naturally in PCOS or a PCOS-like disorder
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
clinically defined, is the real health issue, the metabolics
syndrome or the metabolic disorder.
And here you see, and I've just listed some of them, not all of
them, that accompanies PCOS.
you also get with it a four Wtimes increased risk of havinge
type 2 diabetes.
increases your risk of That, not surprisingly,
Gestational diabetes, when mom cardiovascular disease.
levels during pregnancy, there's loses control of her sugar
and that will become important a three times increase in risk,
because that may be part of the wcomponent as well as perhaps a
genetic inheritance.
three times increased in risk. Endometrial cancer is almost
from a land where obesity is Obesity is doubled and I come
Thanks to America we always follow where America goes.
You guys are leading the way, we're following.
But PCOS women, this is an
in a minute, because they have a especial issue, as I'll get to
able to metabolize fat as panon-PCOS women can, and theyg
have a lower energy rate. They use energy less for the
amount of energy they put out.
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
And because of all these solution.
others I've left out, depression combinations of issues and
disorders that are four times likely to occur in a woman with
PCOS because this occurs, this
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
who are obese, and the higher cnumber comes from the study,
sorry guys, here in the United
That's about 10 million women who are obese that PCOS
contributes.
up to a third in the USA are If we look at type 2 diabetes,
PCOS contributes the largest because of PCOS.
20s and 30s to becoming type 2 numbers of young women in their
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
PCOS, its prevalence, is 7% to non-obese characters.
about 15 million women with those metabolic issues because,e
in this state and many states,
are down to you. reproductive infertility issues
costs that are more related to If we're looking at healthcare
running over $4 billion a year the metabolic issues, they're
This is a devastating problem at to deal with PCOS alone.
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
of their lifelong condition. to deal with their many facets
to be here at Wisconsin because we have a marvelous biochemistry
department with amazing machines
even get close to. with magnetic fields you can't
yellow lines or dire things will So I have to stay outside the
Because of the monkey work, I interested my colleagues in
biochemistry to study the ability of PCOS women to
actually utilize fat and
It turns out that they cannot cautilize 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
It is something they were born issue with these women.
And just to mention, I keep saying diabetes, and just to
remind you that it was because
discovering the role of insulin in diabetes that it's no longer
fatal.
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
part of the syndrome and look at insulin to actually help treat
And I'm going to come back to that in some of the monkey work
as well.
in women in still unknown. The problem: the origin of PCOS
signs and symptoms appear in It's only diagnosed after its
Physicians can reduce the signs and symptoms, but without
understanding the origins we
So can we program PCOS in female can't really prevent it.
And our question, we came here monkeys?
exposed to testosterone as a to ask will a female monkey
In other words, could it be that fetus become PCOS as an adult?
is born? PCOS is programmed before a girl
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,
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,
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
it in humans but basically environment that may be causing
producing too much testosterone. resulting in the fetal ovary
symptoms? WiAnd on the way, during ourOS
studies of pregnancy, we found
and fetal glucose because of that we were elevating maternal
We were resulting in infants this.
and with increased body weight that released too much insulin,
periods now going back 40 years wbefore 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
leading to PCOS. it became obvious in people
And basically we're looking for Cathe seed of a disease that'st?
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
from two different aspects. eOne was congenital adrenale
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
birth also became PCOS as adult. that was removed shortly after
were beginning our work some So we were starting to see as we
outcomes in the human that indication that there may be
in the monkey. mirrored the work we were doing
In our model, we inject mom with How do we create a PCOS monkey?
We inject her between, and a testosterone.
days long, so it's truncated monkey pregnancy is about 165
follows a similar percentage compared to humans, but it
Not identical, they are monkeys, progression.
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.
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.
we reach are those in the lower Anmale range, about 0.3 to 0.4at
nanograms per mill. Well above the normal female
range but still within a
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
around each of those eggs, they imature and now they begin to
make estrogen or estradiol and
and the pituitary is responsive to the hypothalamus in the brain
that will regulate how the ovary
adolescence occurs and then will will stay quiescent until
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
later gestation. before it will start maturing in
when multiple organs are forming So our testosterone exposure is
should have a multiple organ in the rhesus monkey, and so we
Key question, though, is will ctestosterone program PCOS-like
signs and symptoms in our monkeys?
And here are a series of key
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
worked with had been created for first population of monkeys I
them over, they were already psychology studies, and, taking
reproductive years. wSo as you'll see here, the
monkeys in red are our exposed
They were age matched to the monkeys.
in the panels. controls that appear in yellow
we could to controls who didn't We have any exposure duringest
pregnancy and gestation.
tried to match them for BMI. And at the far end there, we
BMI, we tried to keep it And if any of you are aware of
then you look at this where sothere's an average somewherend
about 38 or 39. I assure you we're not feeding
these animals on bratwurst and
Wisconsin foods. cheese and other high fat good
fat, high fiber diet because we No, are trying to maintain a low
well-nourished, well-vitaminize,
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
stick to. oAnd, as you see, some of theo
controls were in the obese range
them as well matched as we could because we just couldn't keep
Meet some of our adult rhesus fomonkeys.e.
We socially house them, where we
And, as you see here, they get can, in pairs.
enrichment. treatThey share.f the
Okay, some of them don't share
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.
sometimes don't get along, and But, just like people, they
as we go through. weBut we try to house them inirs
pairs.
and how we regulate glucose, we Bechave to separate them whenake
they're on study for about
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
monkeys who weren't. testosterone eat the same as the
for them, and they get to browse So it's not a food intake issue
various complicated ways. inSo it's not all that easy ton
find their food at times. They have breakfast, they have
healthy food like fruit and nuts
some chow in the afternoon to provide them with three meals a
day.
count at the end of the day, During this period, we calorie
left so every morning we can get take what little food there is
of you have been to the doctors aand they need to look at yourny
glucose levels and your fat levels, you've got to fast
overnight.
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
If we want to take a blood put the pair back.
to voluntarily entering a sample, ttransport box. accustom
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.
talk, you know how monkeys value And if you saw Franz DeVal's
Not cucumbers. 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
But there's no stress, there's go home?
We look at stress hormones. We have to.
If these animals were stressed, their sugar levels would be
high, their fat metabolism would
study them for our purposes. be all wrecked, we couldn't
They need to realize this is a They need to be very used to us.
do, as we do when we go get a very strblood sample.thing to
We go, we get a sample taken, and we realize that wasn't too
bad.
their legs in cages. We can train monkeys to offer
six of our female monkeys to do In our study this.naged to train
So in their home cage they offer
We can take a blood sample, and then give them a reward.
These are old photos, and this
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
biopsies of tissue, we have a ttop world-class suite ofy
surgeries that you'd find in any
here in Wisconsin, and we have top class veterinary hospital
surgical technicians who can expert veterinary surgeons,
anesthesia and surgical support supneeded for our work.and
And when we do noninvasive
animals because getting monkeys to hold still is just somethinge
they won't do.
to do this, and the animal then Sgoes through about a 20- toem
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
And the scientist you see beside going backwards and forwards.
picture just for clarity because there was superimposed on the
while the animal is scanned. you can't be standing there
would go through scans, as But that is how the animals
fat composition. you'll see, to understand their
what did we learn from this. Okay, now, trying to look at
PCOS traits because that's the How well did the animal show
If they don't do that, this is heart of it.
But if they do do that, this not a good model.
understanding at least one cause would open the way to
Do they show high ovarian of PCOS testosterone?tal life.
And now, sorry, I'm a biology teacher and if any of my 152
biology students are here,
lesson again but simplified, sorry, you're going to get this
[LAUGHTER] and there's no exam.
this is a human head. This is a drawing of a human
head. [LAUGHTER]
Cut in half so the front
is over on the left, the top is over on your right, the back
towards the floor. is at the top, and the base is
hypothalamus. It's right at the base of the
brain.
bodily functions, and it It regulates almost all of your
to the anterior pituitary, communicates by chemical fashion
receives a very private blood supply from they hypothalamus.
And we want to look at a
hypothalamus. parIt's the GnRH neuron. the
It releases a small protein into
anterior pituitary, and cells in that private blood supply to the
receptors for that peptide the anterior pituitary that have
or GnRH, when it binds to them, gothose cells in the pituitary,
release LH. They also release FSH, but we're
really focused on LH today
is the testosterone driver in because thathe ovary.ing hormone
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
It's an electron micrograph. in half.
lost my mouse somewhere. This is a human egg, and I've
And it's a large cell here and There we go.
hormonally active. surrounded by cells that are not
immediate cells surrounding the fluid, the follicular fluid
here. These are the granulosis cells.
They make estradiol.
half of this follicle here, We're intthese cells.he outer
These are the theca cells.
bind LH released from the They make tpituitary.e when they
And in this diagram here you can
binds to its receptor. see that what LH does is it
implanted embryo releases a hormone very similar, CG, that
will also bind to this receptor.
convert cholesterol, yes we make And it makes the theca cell
fat, and it makes two androgens. testosterone and estradiol from
another long name here, Yandrostenedione, as well aset
testosterone. Testosterone is almost a
byproduct of the theca cell.
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
are down here, and they need that the egg needs, estradiol,
They can't make estradiol from that androstenedione product.
to make the hormone that will chhelp mature the egg in theat
ovary.
syndrome, those theca cells just The problem in polycystic ovary
testosterone, and if you naturally make too much
CG, they make even more even stimulate them with LH or with
in the first place. tWhat we did here was to see ifh
we give CG, which is a much more
monkeys just as an injection potent hormone than LH, to our
induce their theca cells because into muscle, could we actually
adrenal, not anywhere else. thaIt goes there to stimulatethe
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
that's making the higher testosterone, it's the ovary
Do these monkeys ovulate less tfrequently than they should?
Yes they do.
rhesus monkeys they have Here we have an example where in
humans we need to confirm mhormonally that ovulation doesn
occur each time, before each
We bleed these animals twice a menstrual bleed.
small blood sample each time by wwhich we could measure thea
hormone progesterone and verify that each menstrual cycle was an
ovulatory one.
yellow panel as in us, these And here you'll see, in the
month. monkeys cycle about once a
ovulatory cycles. Our testosterone exposed animals
cycle at about half that rate.
and some of them cycled more And some of them had long gaps,
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
percentage of the regular cycles adolescents and we look at the
testosterone exposed animals are in those adolescents, the
Almost half of their cycles are irregular compared to about 50%
incidence in the normal unexposed adolescent female
monkeys.
as it is in humans. So thiDo they have enlargenset,
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
fluid-filled follicles showing see here some of these
There are multiple follicles in this polycystic or
polyfollicular ovary compared to
taken at the start of a menstrual cycle when they're not
growing a large follicle at that
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
exposed animals that have polycysenlarge ovaries.s the T
The controls, if they have this phenotype or appearance, they
have a normal sized ovary.
they show this multiple growth In our T exposed animals when
their ovaries to accommodate of follicles, they've enlarged
It's just more demonstrative this overgrowth.
Now, like PCOS women, we've not only altered the ovary, which is
the definition of the clinical
we changed the way the brain definition of the disease, have
And the brain works through LH regulates the ovary?
testosterone out of the ovary. to regulate the androgen or
just doesn't get released. In our T exposed monkeys, GnRH
every 50 to 60 minutes. ItThe monkeys do the same, andt
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
And the arrows here indicate and of those pulses.
burst of LH that we are taking episodic release, or basically a
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
frequency we see in the control. exposed animals about double the
the pituitary are driving Wtestosterone from these thecad
cells as well as the theca cells
responsive themselves to our being more testosterone
So we have PCOS-like injection.
monkeys. reproductive traits in our
And that, back in the 1990s when They're there.
results, was a complete novelty. we first published these
other animal models to show that Now it's been replicated in many
similar impact across the what wemammalian kingdom. a very
Now, will these females show the
These are the ones that are more metabolic-like traits?
being diagnostic. Will they show in abdominal fat
increase, particular the
And that's the fat and here's a scan of a rhesus monkey that's
gone through a CT scan just as
bears all. we would, and it reflects, it
not show but can actually grab So all that fat that you like to
that's the non-visceral fat. hold of, and I won't do that,
The visceral fat here surrounds your intestines, your kidneys.
This is a kidney here on the
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. fat, they have more visceral
fat is it impairs insulin And the proaction.th visceral
It's called the bad fat.
meant to be the good fat and the If you put on weight, there's
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.
resistant and does the pancreas fail to release enough insulin?
We do this by giving a glucose
blood glucose and insulin levels injection and following the
through an algorithm that's called insulin sensitivity.t
It's a measure of insulin resistance.
So the higher this number, the
the more effective insulin is in more sensitive an individual is,
so they can use it to make making yourself take up glucose
You'll see our testosterone exposed animals have half the
insulin effectiveness of the
injury, they have maybe about a third or less of an ability to
release insulin, this index of
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
deciliter. humans, above 125 milligrams per
microfractures in blood capillary vessels in ourselves.
We have about a quarter of our
compared to their age and body adult animal type 2 diabetics
Now, having seen all that wmetabolic 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
It's necessary to cause normal as well.
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.
animals. And here are our T exposed
left-hand side getting an oral In the red panel on the
That's a peanut butter sandwich given before breakfast in the
morning.
with no treatment, and then Aanother six months of peanut
butter sandwiches, but this time
Pioglitazone which is an insulin they contain a drug called
to improve insulin action, and sensitizer that's used in women
regard as well as improving it's usedtheir cycles.n for that
It normalized the menstrual cycles of these monkeys.
In other words, improving insulin's ability to act was
part of the pathophysiological
Not only do they resemble PCOS mechanism in these monkeys.
part of the underlying mechanism women in signs and symptoms,
And, unlike in PCOS, which is the same.
pulled the treatment after six normally doesn't happen, we
reverting to type as insulin months, and you see the animals
lose the ability to undergo actinormal menstrual cycles.hey
What this did for our colony as well as understand how the
mechanism of lack of ovulation
Pioglitazone as a first line worked is we now use
determined as diabetic in our tcolony because this was there
first time this drug had been given for such a long period to
these monkeys.
putting them straight on insulin So we now, rather than just
resistant to that, we can use Pioglitazone and make them use
their own insulin for as long as
humans. So, in our adult monkeys if we
look at all the polycystic
mechanism of lack of ovulation, ovahow the brain is altered,heir
they're just like women with
With the metabolic issues that PCOS.
PCOS, our monkeys are very devastate women's lives who have
We have an amazing comprehensive similar.
The only animal model to date, match.
started, nobody can match this and that's now 20 years after we
monkeys are reproductively comprehension because these
controlled, and age so closely controlled, metabolically
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
restriction along with these sheep, you get fetal growth
You get quite a different metabolic outcome.
So the monkey is very important getting close to humans, where
in PCOS fetal growth restriction
the pathophysiology. is not a major issue in causing
are, what can we learn about the Having shown how PCOS-like they
to people? mechanism that we just cannot do
now with people. We can't go to where we're going
We're going to look at the eggs that these animals are ovulating
when we collect the mature eggs.
colleagues in obstetrics and We do this thanks to my
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,
ovaries are fine for one of them as I mentioned before, both
called the dominate follicle to grow on single large follicle
Just as in humans, we can take that will ovulate that egg.
And this is where FSH comes in, follicle stimulating hormone.
It's now made synthetically, and
humans we can use recombinant bhuman FSH for stimulatingto
monkeys.
mature egg to ovulate, just as And you see instead of one
Somewhere between six and 20 is the common number, very similar
to women as well. Now, we don't let these eggs
naturally ovulate.
single one, just as you would in We go in and we aspirate every
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
We were going to study their embryos as pregnancies.
And their outcomes are quite outcomes.
exposure. The yellow bar, again, are our
controls. These are embryos of the two to
four cell stage all the way to
That's when the embryo was the term called blastocyst.
walks across the endometrial formed enough that it normally
implant here and in it burrows. linWe get up to that stage into
culture before then the embryo
lining. woulAnd we can study them.ine
And in our controls, about 60%
fertilized get to that to 70% of the eggs that were
You look at the testosterone implantable stage.
until they have to turn on the exposed animals doing just fine
here embryonic genome embryonicactivation.we call it
If these were mice and rats,
stage. that would be at the two cell
These are primates just like us. These are not.
five to eight cell stage. They're a primate and they turn
on their genome then, and then
of the success of the fertilized you see the tremendous fall off
so that only about 5% of them eggsend up as blastocysts.oint
There's an impairment.
monkeys is meant to turn on, When that genome from those
So they, at the level of the it has a tremendous impairment.
that we have induced that may be egg, there is something there
generation. Because of the monkey work,
because of this work, we
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
we change. monkey work to see what genes do
monkey, what can we learn directly from the human becausee
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.
eggs from PCOS women We found, compared to normal,
words either have turned up or differentially express, in other
over 300 genes that were turned down genes in the egg,
Most of them were over differently functioning in PCOS.
They were producing too much expressed.
what those products were, our colleagues at U Penn found thatt
they altered genes were the ones
divide, when they know when to that regulate how the cells
how they regulate the divchromosomes during that and
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
at some of the tissue that comes to become important when we look
What did we learn from the fetal from the monkeys.
We learned that we had engaged monkeys in doing this?
when we gave the testosterone the motherinjections.we thought
And this is something, again, we
here we're looking at cmanipulating the pregnancye
outcome.
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.
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
increased birth size of the havbaby, other birthinglly
complications, and those
diabetic because of this offspring are commonly obese and
So we have now our mothers outcome.
giving them an anabolic steroid, showing signs, because we're
control glucose in addition to of losing their ability to
testosterone exposure. that fetus having a higher
time? WhAnd we capture that not onlyis
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.
called KG, to cut a long story And we measure that by a measure
And KG basically measures the rate of glucose lose, and the
higher that bar is, the faster
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
worse the mothers were at controlling glucose because of
the testosterone injections, the
their fetuses at this time in higher tmid-gestation.ucose in
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
moms are so poor at clearing yellow dots here, because their
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
We can measure that in our birth complications.
humans by looking at head size. monThe head size in our in
testosterone injected pregnancies was about 5% bigger
than the controls, and in diabetic pregnancies, this is a
similar complication in terms of
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
And we know this sort of weeks.
certainly, again, linked to obesity and diabetic outcomes in
humans. And we know these animals are
going to grow up to be the
So we know we're looking at the early onset, and it may be that
the glucose may be a key part of
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
born. We learned something completelye
new here that we didn't even
doctors didn't even appreciate appreciate was there and medical
diabetic pregnancies but not in was PCOS pregnancies.d in
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
is the controls, look at where left side again, the yellow bar
It's towering above the thecontrols.is.
These are very, very insulin
If we look at the right panel, responsive animals.
over the controls. again the red bar is towering
the adults. These animals are over releasing
insulin when we give them an
And they're clearing their sugar injection of glucose.
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
Their problem is keeping normal levels of glucose, rather than
low levels of glucose.
were born hypoglycemic, just as Incan happen after diabeticals
pregnancies.
revelation to PCOS And that was a complete new
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
So when these infants are born, just as happens in human
pregnancies, they are far too robust at releasing insulin.
What does that mean?
growth hormones. Well, insulin is one of our
What we are now looking into It drives growth.
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
they've got, at least in the time they get to adolescence,
or in adulthood, abdominal fat monkey, increase abdominal fat,
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
And that may have been driven because mom couldn't regulate
her sugar levels too well during
All come from understanding the pmonkey.y.
And I could keep going on this
given us such an insight into becauwhat occurs in humans. have
I'm going to take one more
today to tell you a more recent insight that we've had that
makes an even bigger leap to
different genes that are actually looking at maybe the
come very close to some studies pgenetically inherited in they
human.
thankful to colleagues who are And here I am shamelessly
actually can detect because wrhesus 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
This is where we want to look. right.
because we can look at genes We can use human technology
ourselves. that are over 94% identical to
packaged in the infant monkeys And how they're differently
adults and what tissue did we achoose because we had one shot
to do this to begin with? The visceral fat.
The fat that is most involved in
amasses in size. impairing insulin action as it
160 genes were differently And we found in the infants over
That means they were over expressing or under expressing
compared to controls. And in the adults, over 300
genes.
genes that were differentially And the thing about it is the
different between the two. changed were completely
All the rest were different. Only two in common.
development in humans. And this is not unusual through
similarly packaged genes until Even identical twins will have
and then their gene methylation signatures, as they're called,
differ. Even though the genes are
identical, the way the genes are
differ even in identical twins. exprAnd that comes with age.ss,
And that's what we're looking at
The most significantly altered here.
adults and infants. paI'm going to be shameless inh
telling you about transforming
To cut it short, it controls how growth factor beta, or TGF-beta.
mature. Remember those eggs before?
Some of the issues they had is
What pathway is most controlling how they divide.
genes are packaged in our significantlmonkeys?d in how the
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
science as well as medicine is studies, and replication in
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.
arriving in the same place. FAnd these pathways operate inre
almost every cell system and
studies inspired by the monkey organ system in our body, and in
first went to look at the human wogene in mature adult tissue,rs
nonfunctional, nonactive. And they went wait a minute,
there's a monkey model and there
Let's look at fetal studies from are fetal origins for PCOS.
and these are studies in conceptus that they obtained,
found a gene in the fetal ovary. Australia, and in there they
from early to mid-gestation, This gene was functional only
giving our testosterone treatments to our monkeys and at
the time the ovary starts to
The genetic link in human is make androgens.
only right at the time we give related to a fetal active gene
We didn't know this. our testosterone in the monkeys.
hitting organ systems as they matured, and that may be one of
the genetic pieces of the puzzle
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.
a gargantuan one that is going So we may have a target but it's
for those with related genes to take, in the human studies
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
fetal testosterone exposure with relate, just finishing off here,
And here is a picture, as you'll PCOS outcome?
clipping that's occurring in a see in a minute, of the hair
This is a mom with PCOS with a newborn baby.
center at Meriter Hospital. We are looking at hair because
hair is a historical record of
This is thanks to the drug and your hormones.
athletics. doping testing in professional
You can take these hormones because they do draw tissue,
they change your genes. And you can get a metabolic
advantage.
personal gain. So you can abuse them for
resistant and diabetic, but Yes, that's another matter.ulin
But we can use the hair as a
on during fetal life. But some studies in human aret
also just using blood at birth
should be elevated. But one of the studies in the UK
had found higher testosterone in
baby girls born to PCOS moms the newborn samples in those
A study in the US from Chicago than non-PCOS women, daughters.
other way around. The androstenedione was too low.
Both of them were abnormal, just
it's at birth. one's high and one's low, but
for, and we're awaiting our hair It's not the time we're aiming
better understanding of the outhistorical elevation ofet a
testosterone because hair starts
hormones get laid down as the to grow abhair grows.tation, and
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
to women with PCOS they have two-month old infant girls born
In prepubertal adolescents of women with PCOS, they have high.
insulin levels before they start
during adolescence. to coDoes that ring a bell?rone
The high insulin levels, again
our immature PCOS-like monkeys, are starting to be picked up now
in human adolescent studies, and
the deep end here, our finger belengths are controlled byoff
testosterone. And the story is that male fetal
testosterone makes your second
Don't worry, there's tons of one shorter than you fourth.
no problem. overlap [LAUGHTER]s isn't,
Or the other way around.
associated high testosterone in PCOS women with longer secondl
finger length.
testosterone exposure during So it can't possibly be due to
Well, thanks to a resourceful student in my lab looking at
finger lengths in our monkeys,
students, in our monkeys their or actually two resourceful
normal. They match the PCOS studies and
they mismatch with the rat and
assumed data from humans because the mouse studies and the
You can't go, we'll give you you can't do this in humans.
you in 20 years and we'll testomeasure your fingers.l see
We can do that in monkeys, and
similar the higher the what we're finding is very
women, the longer that finger testosterone lis.l gets in PCOS
This is fixed in fetal life.
That is fixed in fetal life. Sorry, oops.
testosterone exposure. So it's a postnatal marker of
really happens when you're Our monkeys are showing what
you're a primate and you're not a rat and you're not a mouse.
It's not the same outcome.
monkeys? So, lessons learned from our
glucose that nobody ever was Testosterone and maybe high
program PCOS before birth. bringing into the picture may
certainly does in other mammals. It certainly does in monkeys; it
we've shown in the monkey we can PCOS alters gene expression, and
is inherited in women by these mimic that or we can mimic what
So we may now have molecular manipulations during fetal life.
lifestyle interventions with targets or we can perhaps target
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
provides an insight you can't get from other animals, lett
alone cells and let alone from
only go so far into looking at human studies because you can
to this. the origins, and it comes back
from the humane use of monkeys We are trying to get insight
women in the USA alone, certainly over that worldwide,
to get an understanding that one
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]