DR. ASHLEY VARGAS: …[welcome] you to this…to this workshop. My name’s Ashley Vargas.
I’m a program director at the National Institute for Child Health and Human Development, and
I’m truly honored to serve as one of the co-chairs, along with my colleague, Dr. Krista
Zanetti, for this workshop on Multigenerational Nutritional Influences on Health and Disease.
Nutrition is arguably the central thread in the tapestry of life. Without nutrition, life doesn’t exist.
So, while so much research has focused on how nutrition affects the individual, today
we’re going to really focus on the crucial role nutrition plays in knitting together
families and generations of health. Next slide, please.
I’ll keep going while we’re working on the next slide.
So, this is really a worthy but complex endeavor, and today we have experts from across the globe gathered here in the room with us and virtually to tackle this difficult concept.
As you can see by this diagram here, it’s not very simple, and so you’ll hear different parts of this story today throughout all the presentations on what are…what we’re considering individual-level influences and then again tomorrow on the larger family and societal
influences. So, during these conversations, what we need from you all in the room and online is to ask us the difficult, critical questions.
So, for those of you online, please click on the Zoom Q&A box to ask those critical questions. And for those in the room, we just got an update on how to use the microphones, so please
use them during the discussion. Again, everyone, your role is to ask these hard questions that move us towards a better understanding of research needs and opportunities.
Now, it is my pleasure to introduce leaders in the field. First, we’ll hear from Dr. Diana Bianchi, the Director of NICHD, followed by Dr. Conrad
Choiniere, who is the Director of the Office of Analytics and Outreach for the Center of Food Safety and Nutrition…Applied Nutrition at FDA, and we will also hear from Dr. Christopher
Lynch, who is the Acting Director of the NIH Office of Nutrition Research, and they are going to provide our opening remarks. Dr. Bianchi?
DR. DIANA BIANCHI: Thank you so much, Ashley, and thanks to all of you for attending this workshop.
To echo Ashley’s comments, I’d like to welcome everyone to this very important 2-day workshop focused on the Multigenerational Nutrition Influences on Health and Disease.
The NIH is co-hosting with our sister agency, the FDA. It’s great to be here in person, those of us who are in person this morning to enjoy
each other’s company and to listen and learn from each other. And to those of you who are unable to join us in person, thank you for tuning in via the Webex.
I don’t know about all of you, but during the pandemic, I learned all these amazing
IT skills from home: I can fix my laptop, I can record myself, I now know what a ring
light is, etc.
So for those of you who are online, who didn’t hear my first few welcoming comments, I’m
welcoming you on behalf of our cohost, the FDA.
Multigenerational influences are…are rooted in the developmental origins of health and
disease theory, also known as DOHaD.
And this impacts nearly all the science that NICHD supports.
Moreover, the DOHaD paradigm aligns with NICHD’s mission of “leading research and training
to understand human development, improve reproductive health, enhance the lives of children and
adolescents, and optimize abilities for all.”
And it aligns with our vision of healthy pregnancies, healthy children, healthy and optimal lives.
And it took a long time to come up with that new mission and vision statement.
Importantly, nutrition is a crosscutting theme of the 2020 strategic plan, and we strategically
partner with many institutes, centers, and offices at the NIH, as well as many of our
sister and brother agencies across the government and in the private sector, to advance the
science that supports the nutritional health of families.
We recognize that nutrition is crucial for overall health, growth, and development, as
well as disease prevention. However, nutrition, as you all know, is a complex topic to study,
especially since nutrition can be an exposure, including dietary intake and food access.
It affects health and developmental outcomes, as well as...it’s a health outcome itself.
It’s often assessed with biomarkers of nutritional status.
Historically and currently, the NICHD is very actively engaged in research that addresses
the impact and importance of nutrition, particularly for healthy pregnancies, the postpartum lactation
period for the mother, fetal growth and development, and the well-being of infants throughout childhood
and adolescence.
For example, NICHD has invested in the NICHD Fetal Growth Study to better understand the
dynamics of human fetal growth and to develop standards for fetal anthropometric parameters
for all races, all of which are impacted by maternal nutrition during gestation.
NICHD was also very involved in the overall strategic plan for nutrition research at NIH,
which is a plan for the years 2020–2030.
And I am a co-chair of the NIH Nutrition Research Task Force.
NICHD further serves as a key institute in the ongoing Nutrition for Precision Health,
which is powered by the All of Us Research Program, the goal of which is to develop algorithms
that predict individual responses to food and dietary patterns.
Moreover, we recently engaged the scientific community with the topic-relevant workshops,
including, number one, Breast Milk Ecology, Genesis of Infant Nutrition, with the algorithm—with
the acronym, sorry—BEGIN—it’s the BEGIN Workshop Series—and number two, Biomarkers
of Nutrition for Development Knowledge, Including Dietary Sufficiency, with the acronym BOND-KIDS.
Nothing to do with James Bond.
And the Bridging the Biological and Communication Sciences on Nutrients and Environment…Environmental
Contaminants in Foods to Support Child Development Workshop, which we cohosted again with our
partner at the FDA.
NICHD also coordinates with other funders of nutrition research, such as the Bill & Melinda
Gates Foundation and the Office of Nutrition Research at the NIH, to maximize the support
of maternal and early-life nutrition research.
Over the next two days, you’ll hear from experts across the globe addressing the intersection
of nutrition with the DOHaD paradigm and the impact of nutrition across multiple generations
of families, with an ultimate goal of identifying gaps and research opportunities in this important
area of science.
I’m looking forward to hearing some of the presentations, either live or online, and
the discussions, and seeing the ideas and research opportunities generated from this workshop
put into action.
So, in closing, I’d like to thank the Office of Nutrition Research for sponsoring and co-chairing
this workshop, along with the many staff across the NIH and the FDA who contributed to the
development of this very robust agenda.
So, thanks for your attention, and enjoy the next two days.
And I will hand the podium over to my next presenter.
DR. CONRAD CHOINIERE: Thank you, Dr. Bianchi, and thank you all for…for joining us today
and participating in this workshop.
You know, this is a very important topic for FDA, not just from a nutritional standpoint, but also
from a food safety standpoint.
And I know you might be thinking about food-borne illness, but that’s not the food safety
issue that I’m talking about.
Dr. Bianchi did allude to it in some of the work that we have been doing recently with
respect to environmental contaminants in the food supply and how that can affect childhood
development.
In particular, there are certain subsets of contaminants that are in…they’re naturally
found in the Earth’s crust and/or they may be there from pollution, and so we have…we,
we launched our Closer To Zero plan back in 2021 to focus specifically on reducing the
presence of lead and arsenic and cadmium and mercury in foods that are commonly consumed
by babies and young children.
Now this…what first started out as an action plan has slowly grown into a whole-of-government
effort, not just thinking about FDA’s role in reducing the levels in the foods themselves,
but thinking about: How do we reduce the levels in the commodities that are used to make these
foods?
These are the foods that we want our children to eat, the fruits and vegetables and whole
grains that are important for healthy development.
Thinking about how we can lessen not only the levels in those commodities and foods, but
also how do we lessen exposure to those foods by providing advice to consumers—perhaps
dietary advice.
But this workshop in particular, I think, might get at even one step further, which
is thinking about: How do we reduce the impacts of those exposures?
Because we know that nutrition plays a role in mitigating and even blunting the effects
of exposure to these contaminants.
For example, we know that children with nutrient deficiencies or inadequacies are more likely
to take up lead into their bones.
We know that later…in later stages of life, perhaps during pregnancy and lactation, the
same lead that is in that bone…in those bones can be mobilized and, therefore, potentially
transferred to the next generation.
And if you think about that, those biological processes in the context of issues related
to environmental justice or nutrition security, you can see that we have a role as government
agencies to implement policies to either…to…to break that vicious cycle.
And at least not reinforce that vicious cycle.
So, we’re really looking forward to the scientific discussion today.
Much of what we do—all of what we do at FDA—needs to have strong scientific foundation
to help inform our policies moving forward in this space.
And that’s why we really value our opportunity to…to be here as cohost with NIH...this workshop.
We see this as a great step for us to better understand the biological mechanisms that
are governing the nutrient and contaminant roles in childhood development and…and understanding
how that can affect our lifelong health…health.
So, thank you, again, for having us here today, and I’m really looking forward to hearing
the discussion today.
DR. CHRISTOPHER LYNCH: Hi. Good morning. My name is Christopher Lynch, and I’m the Acting Director of the NIH Office of Nutrition Research.
I want to welcome you to this workshop on Multigenerational Nutritional Influences of
Health.
I thank the scientific leads, Dr. Vargas and Zanetti, for…and the NIH Organizing Committee
for bringing us together.
Around the time I arrived at NIH, the New England Journal of Medicine published a simulation
model that sought to estimate the risk of adult obesity for the United…for the U.S.
population of children.
They projected that 30…57% of those children that were studied in 2016, 57%, would be obese
by age 35, consistent with the rising trends in obesity and other diet-related diseases.
It needs to be a priority that we address these rising trends through…through research.
Nutrition is both a biological variable and as like the air we breathe, a ubiquitous environmental
exposure.
It adds or synergizes with other environmental exposures and social influences of health
to impact our wellness and disease susceptibility.
However, it’s been long unknown, including as outcomes of previous NIH workshops, that
nutrition and other exposures not only affect the adults, but…but act early in development
of their offspring, especially during fetal life, and have profound effects on susceptibility
to…to these later.
And there is strong evidence, as we’ll hear about in this workshop, that these susceptibilities
are being transmitted transgenerationally.
Several studies, both in humans and experimental animals, suggest that altered risk of adult
diseases may be linked to the maternal environment and nutritional status around conception and
implantation.
And other evidence suggests that future risks of disease may be linked to what your father
or grandfather ate, as well.
The specific diseases implicated span the range of interest across many NIH institutes
and call not only for NIH-wide collaboration and coordination, but a trans-federal approach
to address this problem.
Hence, the strategic plan for NIH Nutrition Research prioritizes examining the role of
periconceptional and prenatal nutrition on with other fetal and postnatal exposures in
the development and disease outcomes.
As another important exposure, it also calls upon us to enhance our knowledge of human
milk composition and the translational role of its components.
We are hoping that, through this workshop, we will identify what is already known about
the factors involved in the developmental origins and transgenerational transmittance
of disease risks, but also to identify the gaps and opportunities with key measures and
potential study designs for NIH to reflect upon using preclinical and clinical models.
Thanks again for coming, and welcome.
DR. ASHLEY VARGAS: Well, I want to thank our exceptional leadership for the support of this workshop
and their vision going forward.
And I also want to really thank our keynote speaker, who’s going to speak next.
Dr. Usha Ramakrishnan is going to give us our first introduction into this topic, and
she’s from Emory University.
She’s going to talk about the established evidence of nutrition and diet on multigenerational
effects on health.
And we gave her a very difficult topic, so I want us all to appreciate the coverage that
she’s going to provide over the next 30-40 minutes, and then we’ll have an opportunity
for Q&A and discussion. Dr. Ramakrishnan.
DR. USHA RAMAKRISHNAN: Hello, everyone. And thank you very much and Ashley, it was…I…I felt I was back in school—but I still am,
I guess, when I tell people I’m at a university..."you're still studying?".
But it’s…it’s really a privilege and an honor to have been asked to do this keynote presentation.
And let me open up my presentation.
[Inaudible] Mm-hm. Okay. Yeah. So, we’re all set.
And this is really the work of many generations…many scientists.
And I want to first to start with a caveat.
If I’ve left anyone out, please forgive me for errors of commission and omission.
And I’m really looking forward to the more detailed presentation.
So, it’s going to be a little bit more as an overview.
So, you know, what are, you know, intergenerational effects?
So, the…the first thing, of course, is, you know, what are intergenerational effects?
It’s not a new concept.
If we talk to our grandmothers or grandparents, like, we’ve known this for a long time.
But when we put this into the context of nutrition as way back as almost 100 years ago, this…I
found this useful quote where Mussey said that, “It must be borne that the diet of
a given generation may affect several generations hence.”
So, it’s not a new idea.
It’s been there, but clearly it’s not an easy area to study when we’re sitting
at places like NIH for policymakers or for people right on the ground.
Now, what do you tell a mother?
It’s always the mother’s fault.
I’m talking as a mom, right?
But anyway, the…those are sort of the difficult challenges.
And Emanuel was someone who worked on intergenerational effects.
And I just put a few definitions and these all fit right there.
I’m not going to read the quote with what Dr. Bianchi said.
What’s been the mission of NICHD?
What’s been the career of many people here in this room and people who went before them
that, you know, we’re looking for ways in which we can improve the…develop…human
development for the next generation.
What is it we tell people when you have?
And just broadly, intergenerational effects, it’s…it’s a composite, right?
It reflects the shared…that it’s genetic.
We’ve all heard about nature versus nurture, etc., but it…it…it’s a shared environment,
which now we know gets encoded, and to a certain extent, it’s modifiable.
And I really have had the privilege of being mentored, and I continue to get his advice
of Reynaldo Martorell—many of you know of his work—who just recently retired.
He’s still very active.
He commented on my presentation, as the Woodruff Professor of International Nutrition at Emory
and Chair.
And almost a decade ago, he did this excellent review on intergenerational effects in linear
growth, and this was a quote from that, again, addressing how complex it is when we’re
thinking about, you know, intergenerational effects when looking at the interactions,
looking at genetic predisposition, when looking at the environment, and then obviously, there's some
mechanisms that we’re still trying to unravel when we are looking for potential ways we
can improve the next generation.
And in that construct, I think it’s really important to recognize there are critical
periods, and some of you may have heard about the first 1,000 days.
Folks like me, I’m not a DOHaD person.
I say I’m maternal child nutrition, which is a mouthful, but I think I don’t have
to convince the audience here and the group that, you know, gestation is clearly a very
critical window.
And as you look at this figure on the right, which has really got, you know, all the way
from preconception to the 70s, we can see that, you know, there are different windows
when something which is important may be not so important later on, etc.
But it’s also…it’s not to scale to highlight that gestation and early childhood are very,
very important windows when we…we are concerned about long-term effects.
And I really enjoyed reading the review article.
It’s nice to see Dr. Breton here…you know, definitions, what do we mean?
And this is just, you know, conceptualization.
We talk about intergenerational, transgenerational, keeping our generation straight sometimes
is difficult.
So obviously, with the animal model, which you’re going to hear more of…so, you’re
able to go through several generations.
But if you think of the parents, that’s sort of the F0, and the mother is expecting.
Then that baby is F1, but we know that the baby already is carrying the next generation’s
germ cells, or the F2.
And then, you know, you can go all the way to F3 when we’re studying.
So always, you know, when we think about: Does it work
in maternal and child health? We’re looking at exposures.
When…when do you look at the exposure?
Is it during early childhood of the parent, to the early childhood of the child, and the
next generation?
I thought this was useful just to remind us which…which is intergenerational.
It’s all intergenerational, but we have now seen the literature transgenerational,
and the main point I wanted to make is sometimes these effects skip.
You may not see it in sort of that immediate generation, and then they may manifest later.
And we’re not going to live for 200 years, but I think we have some really exciting developments
that may allow us to examine these effects.
And this is just another conceptual framework to capture the complexity that Rey put together
when they were trying to understand intergenerational effects, you know...I put it as nutrition.
It was really focusing on linear growth, and I’m not going to read through all of it,
but it was just…just to highlight that even when we think about something as simple as,
sort of, child growth and the early birth outcomes, we know maternal nutrition during
pregnancy is very important.
A lot of research has been done.
We know what…you know, weight gain during pregnancy, diet during pregnancy impacts the
child’s immediate birth outcomes and growth and development.
But we also know that there are many factors that are determined prior to conception, whether
it is, you know, the attained size of the mother—that is a function of what her exposures
were much earlier—her preconceptual nutritional status; all of those also influence and may
interact with what she’s consuming during pregnancy, from a nutrition perspective.
And then, of course, there’s the whole environmental factors.
So, there…there are so many layers, and trying to sort this out is complicated.
But we need to be aware that it is a complex issue, and having conceptual frameworks, at
least for me, has been very useful when we try to say, “Okay, I’m trying to address
this piece of it.”
And it truly also emphasizes the importance of team science.
We need people from different areas.
We need different people working.
It’s not, you know, just the pediatricians or the obstetricians, even in that field of
new things.
But it’s…it’s across the lifespan.
You need people who are working in genetics.
You need people who are working in environmental health.
We talk about community.
There’s that bottom line of intergenerational transmission of poverty.
You know, that is not…we don’t know if it’s encoded, but we know environment may
or may not change across generations, and what do we do about it, and what are the implications
of that?
And so, I’m looking forward to the sessions tomorrow on sort of the underlying factors.
So, we have a whole presentation on animal studies.
We’ve learned a lot.
I mean, it’s easier to work with animals.
They’re not inexpensive, so I want to really thank all the funding that’s happened over
the years.
And I’m just going to do a quick summary slide, and you’ll get more details from
my next presenter.
Is…we know that, especially, studies that have been done in rodents, some in mice, that
especially undernutrition food deprivation has long-term effects where, you know, there
have been studies over several generations.
And so they like to see Dr. Galler here, who has done some of the leading work in this.
And so, we know that in the…in the animal model that these effects persist across generations,
and some can be reversed.
But some of the behavioral things don’t get reversed, even when you, you know, come…improve
nutrition, say, several generations later.
In the field of diabetes, there’s been a lot of work showing about, you know, the fetal
programming that happens in animal studies.
There’s quite a bit of interest in showing how, you know, the function of the placenta—not
the placenta…well, the placenta too—but the pancreatic function, the size, insulin,
insufficiency that may start much, much earlier.
And last, but not least, we, you know…I started with, sort of, undernutrition, but
we have… most of the world has experienced the nutrition transition, where we have both
under- and overnutrition.
We have poor diet quality, so the whole thing of what’s happening in overweight and obesity,
where, especially in this country, more than a third of women are entering pregnancy with
many of these metabolic problems, and what are the implications for the next generation?
So, those are some of the areas where we’ve got…we’ve learned a lot, and I’m looking
forward to the next presentation.
So, I’m going to move to human studies—watching my time.
I first want to acknowledge Dr. David Barker.
I mean, he really pioneered in this area, and this quote, which brought back to the
fore that when we talk about what’s happening during pregnancy, it’s not just for that
immediate birth outcome, but it has implications way beyond.
And this is even within that, you know, one F0, F1.
And now we’re wanting to look, and he…he really pioneered and led the way.
Inspired many scientists to start working in this space.
And his early work that show…elegantly showed the increased risk of, you know, chronic disease
later in life as a function of what happens, you know, in gestation has been, you know,
replicated.
The best examples come from the Famine Studies.
My colleague Aryeh Stein at Emory has been part of the scene with the Dutch famine that
happened in…during World War II.
It was short, acute, but has been very carefully followed up.
And we have evidence of, you know, this intergenerational effect that some of these are in the same
cohort, but a lot of these are following the survivors, right?
The babies who were conceived and born.
They’ve been…you know, they were born in the late 40s. And there’s also evidence
that for poor nutrition and low birth weight is associated with increased risk of obesity
and cardiovascular disease.
Now, these are not easy studies to do.
There are challenges.
You have survivor bias.
Epidemiologists can…can rave and rant forever, and it’s an actual experiment, but we’ve
learned a lot.
And I think that’s what’s important to understand what’s the next steps.
This is work by Caroline Fall, who was one of Dr. Barker’s mentees at the University
of Southampton.
Has worked with amazing cohorts in India, which is my country of origin.
And the purpose of sharing this slide is to show how elegantly it’s a combination, right?
I mean, this…if you look at all these graphs, the…the tallest bar is the black one in
the left corner.
And those are children who were born small, so experienced some fetal growth restriction.
But by mid-, you know, by mid-childhood or even as adults, they had…they were in the
upper third of BMI.
So, you see this increased risk where there’s a mismatch, one might say, and that doesn’t
mean we want the small people to remain small.
But how do we adapt?
And it has really important implications for, you know, the advice and the nutritional interventions
that are needed not just during gestation, but during early childhood, during the school-age
years to think in terms of outcomes, like in this case diabetes, which is very, very
rampant, I guess is the word.
But the global burden is very large, and this is showing the developmental origins.
Early work that I did when I started as a postdoc was to look at intergenerational effects
on birth size.
This was a review and, you know, we found about 14 studies.
Most of it was from developed countries and, you know, the effect size was about 10 to
20 grams.
But when we used data from the INCAP study, which I’ll talk a little bit about, which
is an intervention trial, but this was using an observational design.
We found that the intergenerational effect was twofold.
So, there was, you know, for every 100 grams change in maternal birth…birth weight, the
offspring, there was…it…it explained per the gram obviously why it’s complicated,
and this was done at a time when we didn’t have the entire cohort.
So, in the next few slides, I’m going to share some really interesting findings that
Dr. Stein shared.
This is the cohort study.
Some of you may have heard about it.
It really was pulling together cohorts from low/middle-income countries that were…have
been followed up from, you know, early…pretty much from gestation through adulthood.
And it’s from South Africa,
New…it’s the New Delhi Birth Cohort, the Guatemala study, and I’m leaving one more
country out.
Cebu, the Philippines.
So, some of you may have heard.
These studies have individually contributed to our knowledge about the importance of maternal
and child nutrition.
But what I’m focusing on is, you know, what’s the evidence that parental growth during childhood
influences offspring birth weight?
So, this is that generational effect.
And this has been published.
Yaw Addo who worked at Emory and is now at CDC was able to demonstrate that if the mother
was stunted…so these were…these cohorts were done in settings where there was a lot
of undernutrition.
Varying rates of linear growth retardation.
But maternal stunting at age 2 was associated with a 100-gram difference, so they were lighter
at birth.
And there was…this was nonsignificant, but they also looked at paternal because there
was this thing.
So, clearly showing importance of maternal nutritional status.
When they then looked also at conditional weight, so this is not just stopping at birth.
You know, stunting is a composite.
If a child is stunted at age 2, how does that happen?
It could be because the child was born small.
It could be due to results that are experienced during early childhood, first 1,000 days.
And what was great is that these cohorts had enough data that one could partition out,
you know, is it the birth weight?
Does it go up during the first 1,000 days?
And then subsequent, you know, relative weight gain.
So, this is showing you, you know, for parental birth weight and growth…linear growth.
And what you see here is a significant effect of maternal, you know, birth weight on subsequent
generations, but also what happens in linear growth during the first 1,000 days.
Now, interestingly, this analysis did show some effects of fathers.
So, fathers matter.
It’s not only the mothers, and I do want to emphasize it’s smaller, but that also
then begs the question: What are the mechanism?
It’s not just what the mother consumed or breast milk.
It could be other mechanisms involved and that it’s transmitted across generations.
And with all the concerns of, you know, overweight, obesity, we also…they also looked at relative
weight gain.
And what you found here is, you know, mother’s weight gain was positively associated.
Now, one can say, “Is this good or bad?”
It depends on the context.
If you’re working in settings where there’s a lot of people with growth restrictions,
this is good.
So, then the emphasis of, you know, addressing adolescent health, school-age health in settings
where there’s a lot of underweight becomes important.
This next few slides are not intergenerational, but just help you understand: What are the
mechanisms?
Why should maternal or paternal growth influence the next generation?
And these cohort data had very useful information on the…that same generation.
In other words, they have the birth weight of the parents, but they knew what the
attained height of the parents were.
They knew other factors.
And what is shown here is conditional growth and adult height.
So, it’s the parent…I’m still in the same generation here, but it could be mediated
that the mothers were taller.
And we know maternal height’s an important determinant, but the mothers grew better when
they were children.
They resulted in being taller adults and therefore had better birth outcomes.
So, that’s really what’s shown here.
And you…you see that.
And notably, relative weight was not associated.
So, that’s good news in some ways, but the key here is that early part, right?
You see where birth weight is the most important determinant part, which is why it takes generations
to make differences, to, you know, to improve adult height.
Since the interest has been in body mass index, they looked at that, as well, and found that
linear growth had very small effects, but definitely relative weight gain was associated
with higher BMI.
Now, one might say, “Well, is that good or bad?”
It depends on body composition.
The data…there’s not enough information, so this is a gap, but the data—at least
from the cohort—was based on anthropometry, suggests that this was increasing lean body
mass and not fat mass.
But clearly, this is something we need to work more on…on understanding, you know, the limitations
of BMI.
And I’m getting the look.
I don’t want to leave without talking about attained schooling, which is a really important
factor.
You know, mothers are better educated, are able to take better care of themselves, etc.
So, my final sets of slides is really…I was looking for transgenerational effects,
and there are very few studies.
And these are difficult to do.
I mean, just getting two generations is challenging.
Going more than that…but again, there’s some interesting research from the Dutch famine
study.
And finally, I do want to talk about what do we do.
I’m fine?
Okay.
Is what do we know from intervention studies?
Because most of this is observational.
And so the Barbados Study was one…it was, you know, not a randomized controlled trial,
but severely malnourished children received rehabilitation.
They’ve been followed up, and the…the key part here was that there were long-term
detrimental effects.
So, the importance is…okay, it’s fine.
We will just, you know, if we have a severely malnourished child, we need, ethically, to
treat them.
But it’s better to prevent wasting.
It’s better to prevent severe malnutrition because there are these long-lasting effects
on human development.
And the INCAP study is different, in that it is a randomized controlled trial that was
funded in the ʼ70s.
So, this is thanks to NICHD’s investment in maternal child nutrition, and it’s…we’ve
been very fortunate, thanks to Ray’s leadership and several others, and support from NIH,
especially NICHD that this cohort is still being followed up.
We actually are looking at aging outcomes as we speak.
And I say “we”; it’s really…Aryeh is the PI.
But you all may have seen this, but it was, you know, four villages where they got a nutritional
supplement.
This was done in a setting where there was a lot of undernutrition, poor diet quality.
So, this was fortified supplement.
This is something to keep in mind; it had all the micronutrients.
That was…Atole and Fresco was kind of the Kool-Aid of those days, which they didn’t
have saccharin.
They didn’t want to do it for safety reasons, so had some sugar.
And it was actually ad libitum.
You know, pregnant women came in and consumed it lactating, so the exposure was there.
It was in the field for 7 years, but very, very detailed information.
And we’ve had a lot of follow-up studies.
This was where I sort of started when I came to Emory.
I was working on the Thrasher Birth Weight Study, which was looking at birth weight of
these children who had benefited from the INCAP Study.
And some key findings were that across the generations, you can see that certainly there
were improvements.
The linear growth retardation that was seen in the parental generation improved.
But the differences by the intervention group still persisted, if you look at that.
So that’s kind of, you know, comparing the linear growth of generation one to generation
two, and it’s in a longitudinal one…fashion.
And if we look specifically at offspring birth weight and anthropometry.
So, this is…the mother is…the parents received the intervention.
This was their babies.
That’s intergenerational.
There was significant effect.
So, it’s the effect of an intervention is no long…we were able to attribute it to
receiving the atole versus the fresco…to improved birth weight, improved height.
These…they were still children when the follow-ups were done of varying ages, and
also head circumference.
But not many differences in the body composition measures, so that’s something to keep in
mind.
But most importantly, there was also evidence of increased returns to schooling, which is
really important.
So, you know, there are a part…not everything is explained by being heavier and taller,
right?
And there…some of it’s mediated, but there were those investments.
And there have been, of course, the concern about increased chronic disease, and this
is data to suggest that there, you know, there was.
These children were taller.
Adiposity measures were higher, but surprisingly, the prevalence of diabetes was lower.
So again, there’s always this trade-off that we’re concerned…do no harm, but this
is what the data shows in the follow-up of the adults.
And finally, I just want to look like coming back…so, this is an overview on…on mechanisms.
I think work that’s been done by Dr. Yajnik in India, the Barker group.
Their group clearly, especially from animal studies, show that the whole DOHaD paradigm
that undernutrition in the first 1,000 days it’s not just…birth weight’s a crude
indicator.
But really, what’s happening is there are effects across various organs.
And the one which is really exciting as we speak, we’ve been working…is looking at
the brain, you know, the brain…the HPA axis.
The brain for the pituitary to the pancreas, etc.
So, that’s…I think we…we learned a lot from it, and this is not easy to read, but
Caroline—I know her, I’m using her first name—received a very prestigious award in
India and is a part of that publication.
She’s showing how when you’re adapted to something like diabetes, the fetal programming,
and the various mechanisms.
So, it’s very complex, but it again says why looking at intergenerational effects,
even when we’re dealing with chronic disease prevention, is very important.
How does this happen?
As I said, I’m not a genetic expert.
But there’s been a lot of things.
These are the genetic markers, and do they stay?
Do they go away?
But there is quite a bit of emerging evidence in the few cohorts that they have biomarkers
available where they’re able to link that early exposure to poor nutrition to biomarkers
in the next generation.
And some of that’s famine study.
They found some evidence of certain genes that maybe have influenced…explained the
chronic disease risk.
Andrew Prentice from MRC has done work…really elegant work in…in the Gambia, showing how
folate and B-12, so micronutrients…it’s not all about just, you know, how much you
eat, but diet quality is important.
And there’s been…I’m just giving a few examples.
I haven’t shared it, but been very happy to have done a preconception intervention
in Vietnam.
We’re following up the cohort, and we have banked samples.
So, there’s a lot of opportunity to look at some of these mechanisms that may explain
intergenerational effects.
And this is almost my last slide.
I think we always…most of what I shared has come from, you know, cohorts that were
in the ’70s, ’80s.
We’ve dealt a lot with undernutrition.
But we know that we are in the nutrition transition—or sometimes past it—that in pregnancy, you
can have too little of something, too much of something, and diet quality that one really
needs to think of this, you know, very broadly.
But that they coexist.
I think that’s the challenge when you’re talking about obesity, or overweight, which
is affecting so many women in the [inaudible].
You know, they get pregnant.
Diet may be inadequate from other micronutrient perspectives.
It’s not just, “Okay, you’re going to tell the woman to start,” right?
And we’ve had…lot of work in advancing even recommendations for gestational weight
gain.
What is optimal weight gain?
What…what are you going to tell women to eat when they’re pregnant?
It has to be tailored to their individual circumstance.
Yes, it can become burdensome, but people just don’t bother, right?
That’s really what’s important, and that gets me to, you know, thinking, really, you
have to think in terms of a life course.
Pregnancy is very important.
I work on it, but having worked on it for 30 years, I like to say it’s too late, sometimes.
We have to start and work at various points.
And this is what, you know, Dr. Cho had put in as a life-course approach.
And then that really requires everybody to work together.
You don’t have people competing for the same limited resources.
And this is my…just to show that, you know, the burden of micronutrient deficiencies,
of overweight, of underweight persists globally, and they vary by the setting.
This is just using geographic regions, but those of us who work in global health know
that these vary even locally.
Some…right here in the U.S., we know there are disparities.
Similarly, if you go to a country like India or other parts, it’s very diverse.
So, we really need to think about the context that we’re working in as we think about
what to advise to improve the health and development of generations to come.
And I will leave you with some challenges, which I think we will address.
Clearly, you can see what we’ve learned is because of the investment in longitudinal
research, which takes time to follow.
So, thinking creatively how we can harness what comes.
What…you know, we don’t want to wait another 50 years to say, “Oh, this is what you should
do.”
But it still is really important to keep that long-term vision and find ways of…of advancing
our knowledge.
Understanding the mechanisms is clearly something…an area where I think with our…more biomarkers
but interpreting them becomes challenging.
And I think with the dual burden, those are things that I think are going to be the next
frontier as we think about, you know, metabolic abnormalities.
We tend just…to kind of just say “underweight,” “overweight,” “macronutrients,” but
we know it’s much, much more complicated.
But thank you very much, and I look forward to the rest of the presentations in the workshop.
DR. ASHLEY VARGAS: Thank you so much, Dr. Ramakrishnan.
What a wonderful talk and a broad overview of…as we can see, many people’s work,
and many decades of your own work.
So, thank you for that.
I’m joined by my fantastic colleague, Dr. Kellie Casavale from FDA, and we’re going
to move into a moderated discussion for the next 9 or so minutes.
So, we’ll start with a general question, and then for those of you in the room, just
feel free to raise your hand.
Can you…so, your first slide sort of talked about intergenerational, transgenerational…and
we called this workshop very intentionally “multigenerational,” but tried to be inclusive.
But can you talk a little bit about this definition and your interpretation of them?
DR. USHA RAMAKRISHNAN: Right. So, I think definitions are…are important, and multigenerational health…the main point
is when people tend to think of intergenerational, we may stop with just two or a couple because
that transmission is a little more clearer, right?
It’s what…“You are what your mother eats,” kind of language is provided.
But when you get to transgenerational, it is that understanding the mechanisms, you
know, the father’s role, which may not be apparent in that immediate generation.
But it may get…become apparent two generations later, so that’s my understanding.
And again, I…I really would recommend Dr. Breton et al., the ECHOS paper, where you had
a whole box of definitions.
But it’s important to keep that…so, my thing is we just need to keep our generations
straight.
DR. ASHLEY VARGAS: Be consistent.
DR. USHA RAMAKRISHNAN: You know, I like the F0, F1, F2, F3.
I hope that helps.
DR. CHRISTOPHER LYNCH: Yeah, so…yeah. This is Chris Lynch.
So, what I’m wondering about is, you know, given that we have these transgenerational
effects, and since today is Prime Day, how do we get back to prime?
Like, it…it seems to me that if we are even using animal models, the animal models were
affected by the, you know, the previous stresses and things like that that their parents had
in the colony or…or whatever—
DR. CHRISTOPHER LYNCH: —so, how do we get to baseline to identify mechanisms?
Or is that necessary…necessary to do?
DR. USHA RAMAKRISHNAN: I would argue it’s not…it’s a bit like being on a moving train, if…if
that’s an analogy.
It’s…I think go…baseline…you can go back then.
You know, the…you know, the work that I said one of the studies was fascinating from…from
Sweden, where they looked at famine exposure in the 19th century, and then followed the
cohorts in the 20th century and have found…so, it’s good to find data sources, whether
it’s an animal or, you know, human studies that allow you to sort of do those linkages.
But it’s, you know, it…it’s a challenge.
And I tend to be…“What’s the goal?”
What…what do we, you know, what do we want to come up with?
What…and I think that’s going to be important if we understand…if we want to understand
the mechanisms, how is that going to help in…I am a public health person, so…how
is that going to affect what I’m advising a young girl who’s planning to get pregnant
or a woman who’s pregnant or her baby is overweight?
I would argue that you…you can’t control for everything, but characterizing well and
doing good history is important.
I think that would also help.
DR. ASHLEY VARGAS: Excellent.
So, we have questions in the room, but I just want to remind those of you online, please
click the Q&A button on Zoom to ask questions.
We want to make sure we’re including everyone.
So, who’s next in the room?
DR. RICHARD PILSNER: Richard Pilsner, Wayne State University.
Just to clarify on the intergenerational versus the trans.
In the field of environmental health sciences that we…we distinguish the two by the cell
being actually exposed.
So, if F0 mother is exposed to something, then F1 and F2 are considered intergenerational.
F3 would then be the transgenerational.
That animal has not been exposed.
Through the male, the transgenerational will be the F2 offspring.
DR. USHA RAMAKRISHNAN: Thank you.
DR. ASHLEY VARGAS: These are the hard discussions we want to have, so thank you.
AUDIENCE MEMBER: Oh, yeah.
So, thanks very much for that nice overview.
I…I have many questions, but I’m going to just ask you two questions right now, including
lots of methodological questions, as well.
So, my…and I’m going to stick to only the human stuff that you talked about.
One, you mentioned the body mass index and adiposity.
So, you know, if you do simple correlation analyses, these things are highly correlated,
extremely difficult to…to tease them out.
And if you’re going to be doing multigenerational studies using cohorts, it’s probably most
practical to use body mass index.
We have done these things in randomized controlled trials, and it’s adequate when you…when
you try to tease them out, even to predict biological markers.
So that…that’s one thing I’d like to talk about.
The other thing you can comment about: When there is your regional differences in obesity rates,
I wonder whether that is really malnutrition or overnutrition or, really, increasing toxic
and environmental exposures.
And then the final thing is, in terms of randomized controlled trials you talked about, you didn’t
show what the baseline measures were.
People don’t really put on weight over very short period of time…and wonder how you
control for those type of things at baseline or else the…the, you know, but in a short
period of time, you would not expect to see much differences.
I hope I didn’t ask too much questions.
DR. USHA RAMAKRISHNAN: Well, I’ll…I’ll try to answer a couple of the questions.
The methodological one is really important, and I kind of went a bit quickly on it.
One of the ways we got around in the…especially in the cohort analysis the sort of interdependence,
right?—I mean, when a child grows, we know that so much of it is highly correlated—was
to use the statistical approach of conditionals, which allows you—so, I won’t get into
all the details—but it allows you to look at the effect after having accounted for the
correlation.
So that’s as best as we can, and that…that…and that’s been used in various fields as…as
a marker.
So, one way we’ll sort of tease apart the growth.
So, in other words, the weight gain, just say from 2 to 4 years, that is not dependent
on where the child was at 2 years or birth.
It…it…it’s the residue of…[inaudible].
So that’s one of the ways.
Now, your comment about the background is really important.
But these are the…many of the studies that I saw from the humans were done in a background
with…in settings where there was a lot of undernutrition.
Whether it was the Dutch Famine, which is short-term, acute malnutrition, if you want
to think of a very brief period, and then things went back to, you know, food…food
availability…with the INCAP Study in many of these settings where the…the baseline,
there was a lot of undernutrition, poor quality of diet, etc.
But the key here is the trial was done during a period when maternal child nutrition…it
was a maternal and child nutrition study.
They improved…they provided food supplements.
The original study showed in the randomized controlled trial that you could improve birth
weight, something reduced in that short-term.
But these are the follow-ups, so the design remains.
There are challenges with attrition, who remains in the cohorts.
It’s very challenging for those to stay there.
But I hope that helps you, and…which is why that’s the gap that the applicability
of some of those relationships to the baseline today remain.
We still have to keep doing it.
But I think that shouldn’t stop us from understanding mechanisms.
I like to sort of say, pregnancy is still 9 months.
There’s some things about human biology which have remained, but how do we understand
the mechanisms?
I hope that helps address some of the concerns.
MS. KELLIE CASAVALE: All right, this is Kellie Casavale.
We’re going to now take a couple of questions from our virtual audience.
We have two questions in the chat, and they’re…seem to be somewhat related, so I’m going to
ask them both together.
And they’re all…they’re both about really capturing changes over time, you know, in…in
studies.
And so, the audience is interested to hear about how studies are able to tease out what
is nature versus nurture impacts when following participants over their life course, including
things such as the food environment and how that can change across generations pretty drastically.
And also, how to account for those changes in…in things such as changes in socioeconomic
status?
DR. USHA RAMAKRISHNAN: Yeah.
So, the…the good news, especially in studies like the INCAP Cohort or even in the other
cohort study, they just don’t measure, you know, body size.
A lot of information has been collected.
There actually have been a lot of papers to show how change in wealth influences some
of these outcomes.
So, it really comes to having the ability to get that comprehensive picture at various
time points, and then it’s a string of beads, right?
So, absolutely.
And it…it’s expensive to do.
I…I’ve struggled with my own.
I have cohort studies, which have been done more contemporarily and usually the first
thing is, “Well, you didn’t find anything.”
But I’m like, “But I might find something if I continue following these cohorts up.”
And which we are.
I've done a study on omega-3 fatty acids, and we’re finding genetic polymorphisms predict
response to maternal intervention when the children are now adolescents.
So, we have to keep at it and measure all these important developments.
DR. ASHLEY VARGAS: Thank you, Dr. Ramakrishnan.
Everyone join me in thanking her as we move to our next session.
DR. USHA RAMAKRISHNAN: Thank you.
DR. ASHLEY VARGAS: For those of you online, we’re going to take a brief minute break as we transition
people at the podium.
DR. ANDREW BREMER: Are we on?
We’re live?
This is fantastic.
Gosh, it’s great seeing people!
I…I’m…you know, Ashley and Christopher were super nice, and they gave…they gave me a script.
I’m going to still—going to go rogue.
I, you know, it…it is great to see everyone here, and I…and…and for those on campus,
we really appreciate you taking time out of your day to…to be here and join us.
It’s…there’s…there’s…there’s…there’s power and connection, and I…I…I don’t
want to obviate them and minimize the…the beauty of having people online.
But it’s great to have people here in the room.
So…so, we’re…we’re going to continue.
My…my job…Chris is just reminding me I have to stay on time, so I’m not going to
go too rogue.
But, yeah, welcome to Session 1.
It’s…yeah, as you do have in your…that you have in your handout and the…the agenda.
And the session is entitled The Foundational Context for Multigenerational Studies on Nutrition.
As Ashley mentioned, I’m Andrew Bremer.
I had the…the pleasure of working closely with Ashley and Kimberlea and…and many other
of my NIH colleagues.
And so, again, it’s great…it’s great to be here.
And I have the pleasure of moderating the session.
And I…yeah.
I may go rogue.
I call audibles, but I will keep us on time.
And we have three great panelists today.
The…the…the first this morning is Dr. Janne Boone-Heinonen from Oregon Health & Science
University.
And she will begin the session with a presentation on the Historical Data and International Observational
Studies on Multigenerational Effects of and on Nutrition and Diet.
And then Dr. Oliver Rando from UMass Chan Medical School will follow with a presentation
on Historical Animal Studies on Multigenerational Effects of and on Nutrition and Diet.
And then last, my NIH colleague who I have lots of stories about, Sonia Arteaga from
the NIH Environmental influences of Child Health Outcomes Program, otherwise known as
ECHO, will follow and share Relevant Considerations from the ECHO Preconceptional Origins of Child
Health Outcomes Workshop.
I think it was held 2 years ago, Sonia?
And I…I had the privilege of…of being a part of that workshop, as well.
I do…as Ashley mentioned, I would like the virtual participants to…to be engaged, and
certainly feel free to…to…to send your questions to presenters throughout the presentations
to the question and answer box.
And then certainly my…my…my colleague…I want to thank, Lieutenant Commander Kimberlea
Gibbs for keeping me in line and for helping me with the questions.
I think it’s also classic that we’re back in person, and my computer will not connect
even to the NIH Guest Internet.
So, I’m totally relying on Kimberlea because I…even I can’t access anything.
And so, with that, again, welcome, everyone.
It’s…it’s wonderful to see people in person.
And with that, I will turn the floor over to Janne.
Take it away, Janne.
DR. JANNE BOONE-HEINONEN: Well, good morning.
I’m really excited to be here.
This is a incredible workshop, and I’m, I’m really happy to be a part of it.
So, my task today in the next 20 minutes is to provide an overview of the epi research
regarding multigenerational effects of nutrition.
You're ahead of these clips.
Okay.
So, what I’ll do is I will give a summary of kind of how the field has evolved in three
or four main dimensions.
We’ll talk about nutrition-related exposure measures, the types of outcomes that have
been examined, study design, and the types of research questions, and we’ll wrap up
with challenges and opportunities.
My focus here will be on intrauterine effects of maternal nutrition.
Acknowledging that there are other very important topics, including, you know, other exposures,
as well as other multigenerational processes.
All right.
So, we’ll start with nutrition-related exposure measures.
You know, this is, of course, a graph we’ve all seen over and over again.
This is showing higher risk in this case for coronary heart disease with lower birth weight.
This has of course, been replicated for many study populations, countless outcomes.
The key here is that this is interpreted as maternal undernutrition.
So, there is another sort of body of work that has…sort of more directly assesses
maternal undernutrition, examining maternal anthropometry measures, things like short
stature, pelvic dimensions, and these are indicators of undernutrition, especially in
low-resource populations.
On the sort of a lagging, but parallel body of work is the maternal overnutrition.
It’s...sort of, body of work, starting with gestational diabetes, was…was a sort of a
starting point for this.
So…so now, we’ve delved into things like maternal BMI, gestational weight gain, and
body composition.
So…so far, you might have noticed that we still haven’t actually examined maternal
nutrition.
So, I’ll go over a few of those measures of the types of exposures that we have examined.
So, on the maternal undernutrition side, there’s…there’s a really rich body of work, which our previous
presenter talked about.
And this…these are examining, sort of, temporal changes in contextual drivers of maternal
undernutrition.
These are the famine studies.
Also seasonal effects.
This is…shown here is the Dutch Hunger Cohort, showing the very pronounced decrease in food
rations and then the subsequent pronounced, sort of recovery of food rations.
And so, what we have is…is a discrete period of undernutrition.
The colors at the top…those are pregnancy trimesters for multiple pregnancies.
And so, one major contribution of these studies is kind of the recognition that the effects
of undernutrition experienced at different periods of pregnancies have, sort of, distinct
effects.
Many studies also examine actual maternal dietary intake, energy intake, things like
low-dietary protein, micronutrient deficiency, and then on the flip side, supplementation
intervention.
Okay, and we also have biomarker—nutritional biomarkers—the…for circulating blood,
as well as cord blood nutrient levels.
Okay, so this is the type of work that’s been done in human epi populations.
On the overnutrition side, we also sometimes call this high calorie malnutrition, and really
what this is getting at is the…sort of the Western diet that promotes obesity.
The types of dietary-intake measures that kind of comprise the bulk of the field examine
things like macronutrient intake, especially fat intake, different types of food groups;
so, these are…and…and types.
So, things like fruit and vegetable intake, sugar-sweetened beverage intake, dietary patterns.
We see these...sort of Western diet, kind of, overall patterns examined in many of these
cohorts, as well as dietary interventions.
And then, we have the same types of biomarkers that we see in the undernutrition side.
So, this is really important.
The types…the dietary assessment in these studies, you know, these epi studies…and so, typically
they’re measured with food frequency questionnaires.
Sometimes dietary recalls, usually one or two.
Occasionally, we see food diaries.
Those are much more, sort of, resource and time intensive.
And then the exposure period in which diet was—is—assessed.
Typically, we’re seeing one or two assessments in pregnancies, kind of in varying trimesters.
Very few studies examined a preconception diet, and few studies examined postnatal diet.
Oops.
Wrong way.
Okay.
So now, we’ll shift to the types of outcomes that, sort of…that we have seen to date.
So, until reasonably recently, these types of outcomes comprised really the bulk of the
field.
So, clinical is over examining various types of maternal nutrition status, as I just described,
related to clinical risk factors.
These are things like BMI, blood pressure, lipids.
It really…it’s all phases of life starting from childhood all the way through the end
later in adulthood.
In the lower corner on the—just an example—of…of one of these studies, that’s showing higher
child fat mass index with high levels of sugar-sweetened beverage intake for the mother in the second trimester.
So, that’s the red bars compared to the blue bars.
Okay, and then we have a whole host of clinical disease and mortality measures that have been examined
in a sort of cardiometabolic realm, as well as mental health, cognitive function, cancer,
and the list goes on.
Okay.
So, coming in later to the field are…including an examination of these intermediate outcomes.
So, at the top are postnatal factors, lactation and growth.
The…the…it doesn’t quite fit here, I just wanted to note that we could talk about
that for a very long time.
But I won’t.
But those are…those are important, as well.
I’ll focus on the biologic and behavioral mechanisms, and so this is a really interesting,
sort of, crosstalk between the animal and the human studies.
We’re learning more and more about how these processes work.
So, when I talk about biological mechanisms, we’re talking about, you know, epigenetics
metabolomic changes, placental changes, structural changes.
These are things like alterations in kidney volume.
That’s the kind of thing that we can measure in humans.
Functional changes, things like altered stress response.
And behavioral mechanisms, especially in the overnutrition world.
We’re thinking about things like alterations and appetite regulation and reward system.
So, these are the, kind of, the steps, you know, through which we get from maternal nutrition
status to some of these outcomes.
So, just a couple of examples.
So, on the biological mechanisms, this is a example of the study out of the Dutch Hunger
Winter Cohort.
They were examining effects of exposure to that famine during gestation on DNA methylation.
And they’re specifically interested in both methylation patterns as mediators of the effects
of metabolic disease in adulthood.
And then just an example of behavioral mechanisms.
This graph is showing…essentially, we’re seeing worse appetite regulation in girls
born either lower or higher birth weight.
Okay, so, of course, the biological and behavioral mechanisms sort of lead into each other.
But this is kind of one way to separate them out.
Okay.
So, third thing we’ll talk about is study design.
So, there’s a really wide range of, sort of, types of studies that have been examined.
You know, these very long-term studies that really sort of started the field, where we
see the things like birth weight, exposure to famine associated with disease and mortality
many, many decades later.
And so, these all relied on analysis of historical data.
And mostly in countries that have better historical data than the U.S.
I’m coming from the U.S. perspective.
There are a large body of work using linked clinical records for the…the mother and
the child.
So, this…in this way, we can examine...really the you know, any type of measure that’s
recorded in the clinical records.
So, we have, you know, maternal BMI, maternal conditions, birth outcomes, and we can relate
them to growth, BMI, blood pressure, etc., in the offspring.
However, we’re limited in this case in the length of follow-up.
We can really…you know, we’re limited by how long the children stay in the system.
And so, really, it’s…typically, we see these out sort of into early to mid-childhood.
And then, of course, we have prospective studies.
This is a very, very short, sample of the studies that are…are ongoing.
In this case, we can get really detailed measures.
We can measure maternal…actual maternal nutrition.
We can collect cord blood and placenta.
And then follow up on children and collect some of these, sort of, biobehavioral, sort
of, intermediate outcomes that we might be interested in.
Okay?
But this is…you know, we’re even more limited in how far out in the life span we
can follow the offspring.
Okay.
And then there have been a handful of studies that have achieved this three-generation follow-up.
Usually these are kind of combinations of studies, usually sort of anchored in one prospective
cohort.
We can then follow up the…the children of that cohort as they age.
We can link back, for example, to the maternal birth record and learn information about the
grandmaternal characteristic.
But again, we’re limited both in the follow-up time and the types of data that we can obtain
going back in time.
Okay.
And so, in the context of trying to understand intrauterine effects, we also need to recognize
that there are other multigenerational processes at play.
There are ongoing environmental influences.
We have genetic effects.
We have behavioral processes, as well.
So, ways to try to address and kind of isolate these intrauterine effects just kind of…I’m…I’m
putting them into sort of two boxes.
The first is the...sort of the type of research questions that we’re asking.
And so, this is really getting at, you know, it was a couple of slides ago where we’re
kind of probing and testing kind of steps of these theorized processes and…and to
see if they hold up in human populations.
So, examining these…these intermediate outcomes in our studies.
And then, kind of, these analytic causal estimation approaches.
Things like sibling studies, paternal negative exposure controls, Mendelian randomization,
randomized trials that are really trying to address and control for these quote-unquote
“other multigenerational processes.”
Okay.
All right.
So, the last major section here will be just to talk about the types of research questions
we’re asking.
Okay.
So, really, the bulk of the field can be characterized as asking these sort of two main types of
questions: So, does X maternal nutrition status affect Y offspring outcome?
Or, if we’re interested in these mediators, you know, do…does…does an intermediate
mediate the effect of X on Y?
This is really where most of the…the focus is.
So, I’m also coming from a public health perspective, and so the corresponding intervention
for…for these types of studies is, you know, for the exposures where we see higher risk.
The intervention is: We should, of course, be reducing that exposure.
So, if we’re recognizing kind of these ongoing environmental influences, implicitly what
we’re really saying is we need to reduce the exposure in, kind of…in settings with
persistent barriers to healthy nutrition.
You know, we know that the contextual influences on nutrition tend to be constant from one
generation to the next.
Okay.
And so, this next sort of types of research questions, we’re really focusing on the
offspring that have already experienced those exposures.
And really, trying to get them set up to be sort of the next generation of parents.
And so, if we’re kind of stemming from our knowledge of these biological and behavioral
mechanisms, they imply many things, but I’ll talk about two.
About the offspring that experience the…that are…sort of going through these processes.
So, the first is that they may experience differential response to postnatal conditions.
You know, again, things like stress response, exercise response, and there’s a body of
work—early work—that’s…that supports that.
And then the second is…it’s kind of recognizing postnatal contextual effects.
So, if we’re thinking about nutrition and diet, you know, there is strong evidence that
maternal nutrition status affects the ability to regulate appetite.
What does this mean in our current food environment, all the food cues, the food marketing that
we’re exposed to every day, all day it seems…it seems sometimes?
Okay.
And so, the kind of the corresponding interventions for this work is things like precision prevention
and public health mitigation.
So, how can we kind of interrupt these processes, mitigate the effects of these prenatal exposures?
Okay.
And I’ll wrap up with challenges and opportunities.
So, there are many, and I picked one, which is our measures.
We have really very blunt measures, both for nutrition, as well as maternal nutrition status.
It’s difficult to measure nutrition at any time point, much less understanding changes
over time.
We have bio...biomarkers.
Many of them can result from multiple exposures.
And so, kind of the improved measures, are…is my…my ask for the field.
And then, in terms of opportunities in the short term, you know, I think that we’re
seeing increased use of methods that can better combine data and…and evidence from, maybe,
different types of study designs.
The things like systems science methods and also the data sharing and collaboration; things
like ECHO, which we’ll hear about next.
And in the long term, I actually have a question mark here, and hopefully the…the…the biology
people in the room can help answer it today and tomorrow.
My impression is that we’re getting closer to identifying biologic signatures that could
indicate either maternal nutrition exposures or some of those intermediate outcomes.
So, things like epigenetics, metabolomics, microbiome, placental measures.
Okay.
And just very quickly, I’d like to acknowledge NIH, my collaborators, staff, and students.
In particular, I would like to thank...or acknowledge the importance of these career development
awards in helping me and others kind of delve into this aspect of the field.
Thank you. Yeah.
DR. ANDREW BREMER: Fantastic. The next one’s going to be Oliver.
DR. OLIVER RANDO: Well, thank you all for having me. I’m excited to be here.
I sort of realize that I pitched my talk a little bit wrong for this audience, but my
goal is to some extent to give you guys a sense for the lack of clarity in the mechanistic
studies of how intergenerational effects work, where we're in rodents, and we have a lot
more access to mechanisms.
So, I sort of come into this field from the field of epigenetics.
Just because that term means many things to many people, I’ll use the 1970s definition
of epigenetics, where something has to be heritable, either mitotically or meiotically,
without a change in DNA sequence.
So, if two organisms have different phenotypes that they pass on to their kids, they differ
in some phenotypes, and they can pass those on even though they’re genetically identical;
that would be an epigenetic…epigenetically heritable trait.
And so, the cocktail party version of this, all your cells…all the cells in your body
come from the fertilized egg.
So, first approximation, they all have the same genome, but when a liver cell divides, it
never makes a skin cell or a kidney cell, even though they share the same genome.
So, state…liverness is an epigenetic state.
Now, decades of genetic analysis, in Drosophila in particular, have identified molecular pathways
that are involved in cell state inheritance, like chromatins, the polycomb trithorax genes
from…from Drosophila screens.
There’s also, of course, DNA modification in small RNAs.
So, these are the three big epigenetic inheritance…pathways.
Now, in terms of thinking about inheritance from one generation to the next, inter- or
transgenerational inheritance, depending on how long it goes on…this is not how we divide,
of course, unlike cell and state inheritance.
So, in order for any information to make it from me to my children, it has to survive
the tremendously disruptive processes of gametogenesis, and then all the information required to sort
of generate a coherent physiological phenotype in the next generation has to be present in
the fertilized zygote, the spark of life.
And all of that information then has to be sufficient through the course of development
to turn into a physiologically meaningful phenotype.
Now, that’s not to say that this doesn’t happen.
There are inter- and transgenerational genetic inheritance paradigms.
The first was discovered…so, this…I’m actually doing sort of history of epigenetics
more than of animal studies, sorry.
So…the first discovery of the transgenerational epigenetic effect was by Bateson and Punnett
in the 1920s in their studies of the rogue garden pea.
The same phenomenon was later called paramutation in the 1950s, in corn, by Brink and Coe.
And so, the idea here is that you have purple corn and green corn in a paramutable maize…system.
They differ in the expression of a transcription factor of B1.
B1 drives the expression of the anthocyanin biosynthesis genes.
Okay?
So, that makes you purple.
So, they differ in whether they have repressed B1 or they have an active copy of B1.
Now, the interesting thing about this genetically is that if you cross purple and green corn,
you’ll get all green babies.
Okay?
Now, if this were a Mendelian Trait, it would mean that green was big B, big B. Purple was
little B, little B. And you have a bunch of heterozygous green kids.
But now, if you cross those green kids, two purple corn, you would expect 50% purple and
50% green in Mendelian genetics.
And what you get is all green offspring.
And this will persist for hundreds of generations.
So, they call this paramutation, as though the inactive copy of the allele was infecting
the active copy of the allele.
Okay?
So, this is a…of course, it’s going through the germ cells, so this is transgenerational,
and it’s more than one generation.
So, this is transgenerational epigenetic inheritance.
And so…decades of genetic analysis of this, starting with Brink and Coe, really pioneered
by Vicki Chandler later in the molecular era have defined a bunch of genetic requirements
for paramutation, and this includes the small RNA pathways, DNA modification and chromatin.
So, all three of the epigenetic pathways, which all talk to each other, are all required
for…for paramutation.
Now, one of the really interesting implications of epigenetic inheritance through the germ
line is that it resurrects an ancient idea, because, unlike DNA sequence, epigenetic marks
are very environmentally responsive.
Right?
So, if you [inaudible]…the entire chromatin landscape changes dramatically.
Small RNAs are induced by various stimuli in the rest of them.
Right?
So, this is in contrast to DNA sequence.
And what this means is that the environment experienced in one generation has the potential
to modify phenotypes in future generations, which is the point of this meeting.
Now, this was tested…historically by August Weismann.
This is the German embryologist who defines the cell line/germ line distinction.
And so, what he did is he cut the tails off of mice and mated them to each other, and…saw
no change in tail length in the offspring.
Okay?
So, this was a very influential refutation of Lamarckian inheritance, or the inheritance
of acquiring characters.
Now, today we would see a number of problems with this experiment.
One is, maybe if your parents tell you that someone’s going around cutting your
tails off, the useful thing to do is not to have a shorter tail, but to run away from
people in lab coats.
The other is that, even at the time, it’s a little bit rigged because…you know, Lamarck
and anyone who thinks about it for 30 seconds recognizes that, you know, soldiers from wars
throughout history who’ve lost their limbs don’t have children with stunted limbs.
So, Lamarck had already invoked this idea of desire or utility in order to gate information
to pass onto kids.
So, mutilation is maybe not the best environmental condition to be looking at.
Okay.
So…so, that’s the end of sort of…epigenetic inheritance history.
We basically and now…thousands of groups have basically revisited this type of paradigm,
where you poke an animal and then you look at its kids to look for inter- or transgenerational
epigenetic effects.
So, we do this in inbred mice, so compared to what all of the human people…all the
human people here…all of the epidemiologists here have to deal with, we at least don’t
have to worry about things like selection on particular alleles, right?
So, if I starve and it kills all my sperm with G in position 1,000 instead of A, this
could be, you know, genetic selection, as opposed to epigenetic inheritance.
We’re using inbred mice.
We studied paternal effects because in mammals, mom is baby’s first environment.
Fetal alcohol syndrome is not magic in epigenetic, you’re just poisoning a baby.
That’s not to say that these things aren’t important for public health and the rest of it.
But as an epigenetic mechanist, it’s much easier to think about paternal effects because
you can set up a cross so that dad just leaves sperm and a memory.
Okay?
And we basically do the Weismann experiment, except we make two big changes.
One is that we…we now study a lot of different paradigms, but the first one we studied was
low protein.
And this fits, and I can get into this, it’s a little bit long-winded to talk about, but
there’s…modeling studies sort of find that under…only under certain environmental
conditions will it be useful to tell your kids about the future, and mutilation is not
one of them.
Whereas…sort of suboptimal diets do make sense.
So, we raise brothers from weaning to sexual maturity on low-protein or controlled diets.
We mate them with females for 1–2 days and take them out.
So, the males are not directly interacting with the kids, and ideally, they’re not
modifying, say the kids’ microbiome through defecation in the cage.
We’re really trying to minimize male involvement to an ejaculate.
We let the females be pregnant, have kids.
At 3 weeks, we grind up the kids, and we…at the time it was micro-raised, we would look
at instead of one quantitative trait, tail length, we’re measuring 30,000.
Okay?
Specifically, we’ll get the list.
And so, what we found…we just signed out of the Zoom.
I don’t know what to do about this.
Yes, it’s going to work here…I don’t know if it’s…is anyone on Zoom and knows
whether it’s…it’s going?
Good?
Okay.
Sorry.
I want those 15 seconds back.
Okay…so, when we look at the kids…so, this is a usual microwave heat map, basically
every column we’re comparing a low-protein offspring…and the offspring, of course,
are…are treated identically.
They’re under controlled conditions, controlled moms, controlled milk.
Every column is a comparison between the liver of one low-protein and one controlled offspring.
Low protein is labeled red.
Controlled is green.
So, all of the green stuff are genes downregulated, depending on bad eating and diet…all of
your red genes are upregulated.
And as you can see, we have not entirely penetrance.
So, if you look across the red, you see still some green on the left.
So, it’s a 90% penetrance…gene expression changes.
Very significant.
The downregulated genes are nothing in particular, the upregulated genes are very highly enriched
for lipid and cholesterol metabolism, specifically cholesterol biosynthesis the most.
And this is not just a gene expression phenotype, when we do metabolomics in these offspring,
we find very significant decreases…significant but small decrease in free cholesterol and
a very significant decrease in cholesteryl ester.
This is the last I’m going to talk about the physiology of the offspring phenotype.
My understanding from metabolism people, which I am not, is that a cholesteryl ester over
free cholesterol effect suggests a bile acid phenotype.
And so, perhaps what’s going on with the kids—and again, we haven’t done any of
this kind of physiology—is that they’re dumping more bile acids to try to extract
more nutrients out of food, and they’re homeostatically upregulating the cholesterol
biosynthesis pathway to keep up with the loss of cholesterol.
Okay?
So, I don’t know…yeah…I don’t need that slide.
This is to show…sorry, this is actually useful.
So, those are just all the genes in the cholesterol biosynthesis pathway, colored by their expression change.
And you can see they’re all coherently upregulated.
Okay.
This is the one slide that sort of hits what you guys kind of wanted me to do a lot of,
which is…so, ours is one of the earlier studies, although not…not the earliest study
in paternal effects in rodents.
There’s been this Anderson study in 2006 before, as well as a lot of work from Michael
Skinner in…endocrine disruptors, but for us, the paper that pushes us out the door
is the top one, there.
So, Margaret Morris found that in rats, males on a high-fat diet sired daughters with glucose
control phenotypes.
The next one down is a low-protein that looks a lot like what ours...you'll see later.
They find that not only do you get lipid metabolism, but also cardiovascular parameters, like vessel
compliance and stuff, change, as well as glucose control.
And glucose control…poor glucose control is probably the most common phenotype people
see in paternal dietary and other kids of paradigms.
The next set down, I highlight because here, what’s going on is that people are either
over- or undernourishing pregnant moms and taking their sons as the paternal generation.
Okay?
And what’s interesting here, these guys were much smarter than us.
Primordial germ cell development occurs during fetal growth.
And so, what they’re trying to do is intervene during cytosine methylation establishment.
So, parental imprint, if you know about imprinting disorders, are established in PGC.
So, these guys are trying to…to interfere with primordial germ cell development.
Whereas the…the top two papers and us are doing sort of postnatal, so during the life
span of things.
And it’ll be very interesting at some point—I don’t have this in my, you know, future
directions, but I’d really like to understand the distinction between effects that are mediated
with PGC perturbations or mature germ cells.
These papers just point out that the dietary effects are not, sort of, permanent.
So, let’s see one from…yeah, Laurie Goodyear.
So, males on high-fat diets, their kids have all sorts of metabolic changes.
But if you exercise males on high-fat diets, you can reverse those paternal effects.
So, you’re not, sort of, doomed by what you did 3 years ago to…to your kids having
a lifetime of…at least in terms of the dietary paradigms.
So, dietary paradigms are one great arm in the paternal effects literature.
A second—and I’m only showing two of…hundreds and hundreds of papers here—are paternal
stressors.
So, things like early maternal…separation, which is what Mansuy studies a lot of.
Or sort of chronic variable stressors or social instability, like [inaudible].
So, all of those things affect anxiety-related traits in the kids.
A third big arm of things, and I…I really ought to have…some of Skinner’s work here,
are environmental, sort of, xenobiotics, whether it be NIEHS-type compounds like, you know,
[inaudible] or BPA, or…or jet fuel, or NIDA-type compounds, like toxicants we take ourselves—so
we said nicotine, cocaine, and ethanol—both have intergenerational effects.
And then the last type, the last big type of thing that affects offspring is the dad’s
paternal age.
And one thing you guys will notice here is that the four kinds of paternal affects out
there are all different NIH institutes.
And this actually makes it very difficult for those of us who want to understand them
broadly, right?
There’s NIDDK for the diets, although they won’t fund anything in this field because
if it touches sperm, they’re not interested.
NIEHS for the toxicants; NIMH for the stress; and NIA for aging.
And so, this is where…Dr. Bianchi isn’t here anymore, but I was sort of heartened
to hear some effort of the NIH to try to get some cross-institute interest in this field.
Okay.
So, these are maybe, like…I showed about 30 of something, like 1,000 papers now, linking
paternal environmental conditions to off…offspring traits.
Okay, we’ve already heard about the Dutch Hunger Winter, so, of course, there is some
evidence for this kind of thing in humans, starting with Hales and Barker.
The point I want to make here, since you guys have already heard about this, is that all
the traits people see in paternal effect studies in rodents also show up in maternal effect
studies in rodents from people like Rebecca Simmons and [inaudible], and also in humans,
despite the fact that the oocyte and sperm could not carry more different information
to the kids.
And so, I think this may give us a mechanistic clue in the sense that I wonder whether or
not paternal effects are acting through altered classifications.
Okay.
So, here’s just a cartoon of how all these things work.
You poke dads, you affect kids.
Since I’m running long per slide, I’ll just very briefly note that while it’s…the
obvious hypothesis is that all these things are mediated by sperm epigenetic marks, it’s
worth pointing out there are many other ways that dads can talk to kids, from seminal fluids—there’s
some great literature on Drosophila from Mariana Wolfner, and a bit less with…in emerging
literature from people like Liz Ra…I’m blanking on her name, in Australia.
Anyway, on seminal fluid effects on offspring development and traits, cryptic maternal effects
where moms judge mate quality and change how they allocate resources to kids, and microbiome
transfer.
And so, in order to deal with these things, now we and a lot of other people…a lot of
the reproduction in the lab goes through IVF, where you take the sperm away from seminal
fluids, the mom never sees the dad, there’s no microbiome transfer.
And we can recapitulate our…our phenotypes through IVF.
So, at least in our system, the information is in sperm or co-purifies with sperm.
The other nice thing about IVF is that it…it allows us to take maternal effects seriously,
which we’re now…as in epigenetically seriously, because you can now separate moms into oocyte
donors and uterus donors.
So, we can separate fetal provisioning from oocyte epigenome.
So, what I wanted to do, and I…I’ll just try to blow through this really quickly, is
point out the state of…everything I’ve told you about so far is correct and true,
and I think as…as much as we really know.
So, there are hundreds of proposed mechanisms for paternal effects on kids, and I just want
to sort of briefly go through the problems with all of them, to point out the state of
where we are in this field.
So, for cytosine methylation, the major problem that people don’t think about is what I
call the “digital sperm” problem.
So, when you see in report an 80% to 90% change in methylation in the liver, that could double
the number of cells that make growth hormone or something, and it could give you a physiological
change.
But that’s a multicellular tissue;every baby is made from one sperm.
And so, when you change from 80% to 90% methylated, what that means is you’ve gone from 8 out
of 10 to 9 out of 10 sperm carrying a methyl group there. And that should only change
the penetrance within the litter.
So, problem one.
Problem two is that dad’s methylation is almost completely erased by mom at fertilization.
The only places that escape this are actually hardwired by having a binding site for protein
called ZFP57.
So, unless you’ve changed methylation near a ZFP57 binding site, the methylation is going
to have to do something very quickly at fertilization before it’s lost to the wind.
So, that’s methylation.
Chromatin I’ll skip in the interests of time.
Basically, nobody has every correctly measured sperm chromatin.
It turns out in mouse, what everyone’s measuring is cell-free DNA that sticks to the sperm.
But again, the issue with even the people who’ve measured histones in various places,
there’s a penetrance problem where the…the enriched locations for nucleosomes seem to
be in only a subset of sperm.
So again, being able to program penetrant changes in offspring through chromatin is
an issue.
We favor small RNAs, but those also favor…face quite a few problems.
One is that sperm, of course, are 1,000 times smaller than the oocyte and carry much less
RNA per femtoliter.
So, do sperm deliver anything meaningful to change the biochemistry of the oocyte?
This is just…we started to get good at…at measuring small RNAs and oocytes from the plantation
embryos, which are very limiting amounts of stuff.
And, you know, if you compare an oocyte and a zygote, which differ by sperm-delivered stuff,
you can barely tell the difference.
So, very little is delivered by sperm.
The other problem is that we now know that we and everyone else have been cloning sperm
RNAs poorly.
So, it turns out sperm have very unusual small RNAs.
In addition to microNAs they carry halves of tRNAs.
We thought only 5 prime halves; we now understand that the 3 primes halves are there, as well.
And so, all of the models that people have been proposing are now complicated by: Are
you just delivering a 5 prime half or a nicked tRNA or two separate halves, and how do they
function molecularly?
So, this is our working model for how…how our low-protein system works.
So as sperm leave the testes, they actually spend 2 weeks in this understudied tube called
the epididymis.
During this time period, the small RNAs they carry change completely.
We think the epididymis ships RNAs to sperm, but that’s…I’m not 100% sure of that.
But certainly, the RNAs change.
So, the epididymal maturation is a really good opportunity for the world to affect your
germ cells.
The levels of specific tRNA fragments change in response to a low-protein diet.
We think what that does is either accelerates or decelerates zygotic gene inactivation.
And in doing so, we speculate that this effects the investment of the embryo in the trophectoderm
versus the inner cell mass and therefore alters investment in embryo both per se versus [inaudible].
So, in other words, a secondary placental effect.
So, with that, I want to raise the questions.
So the first…actually, the first big thing I’d like to emphasize for this audience
is that the paternal effect field is a mess because even people who study the exact same
thing, like high-fat diet, everybody uses a different paradigm, right?
So, some people have high-fat diet for 8 weeks, some for 12 weeks.
Some people are using ZFP57N, and some are using J. I’m…a couple of us in the field
are really trying to get a…a coalition together where we all do the exact same experiment
and sort of control for investigator and mouse facility endemic microbiome, all that kind
of stuff.
It’s been very hard to drum up interest, but I think the field absolutely needs it
because it’s arrogant to think that the diet we feed the mice is the only thing they
listen to.
And so, I’d really like to sort of get just a bunch of people going backwards in time
and doing…but doing the exact same experiments, in order to sort of identify the robust and
less robust aspects of paternal effects.
We don’t know the mechanistic basis, I think I’ve made that clear, so that’s an unanswered
question.
And then, for me, the deepest question about paternal effects is what we call the bandwidth
question.
How much…if you accept that sperm can pass information to kids, then the question is,
how much, right?
So, for cytosine methylation, every CPG can be a bit.
There are 25 million CPGs.
You could tell your kids infinitely complex information about the world.
Alternatively, what we often find, the dietary people see metabolic traits change, the stress
people see anxiety-related traits change.
When you look under other people’s lamp poles, you see their traits.
So, in other words, it could be that you just tell your kids about overall quality of life,
and this is an intergenerational stress response.
And we sort of favor the lower bandwidth side of this.
But I think this is a really deep and important question to understand.
How…if you accept that information is passed on, how much is passed on?
And I’ve already gone a couple minutes over time, so I’ll just skip this.
These are the people in my lab who did all this stuff, and this is sort of a model view
of our lab, and thanks for your time.
DR. ANDREW BREMER: And Oliver, thank you so much.
And actually, we…by…we’re going to bring Sonia up.
But I would…but you…you teed me up beautifully for two things.
You…you know, your…your talks and…and Dan’s, and the…the talks we heard today
really span a breadth of time, and that’s intentional.
And that’s…so, we, you know, it…it underscores the importance of cross-disciplinary science
when we talk about these…these…super important issues.
So…so, you said it’s not what we wanted.
That…that’s exactly what we wanted.
So…so, thank you.
And you also teed me up for the important…I like how you codified institution-specific
science, but I…I want to do a shout out to...to Chris Lynch and…and…and
the Office of Nutrition Research, really, is that body that…that’s trying to coordinate
that…that IC-specific science.
So…so, Chris, thanks to you and your efforts.
And with that, we’ll…we are…we’re running a little bit late, but we will make…make
up for it in…in the Q&A, and Sonia, the floor is yours.
DR. SONIA ARTEAGA: Great.
Thank you so much.
I’m so excited to talk to you today about ECHO and our recent workshop.
You heard two great talks.
One is an overview of observational studies, and also a great talk on epigenetics.
And we’re going to switch gears a little bit now.
I’m going to talk about the ECHO program and our recent workshop.
So, I want to start off by having all of you think about this question as I talk: So, what
are some of the lessons that we learned from the workshop, and how can that inform future
research?
So, I’m going to talk about ECHO, the workshop, how that influenced the next cycle of ECHO.
And if I have time, a little bit on our nutrition research.
So, let me start off by giving you a brief overview of ECHO.
You can go to echochildren.org to find out more in-depth information.
So ECHO started…the first cycle started in 2016, and it ends…the first cycle, in
20…in August 20[23]…so, next month.
And the mission is to enhance the health of children for generations to come.
And the goal is to understand effects of…of a broad range of early environmental exposures
on child health and development.
And really, there are five key ECHO outcomes, and so they are pre-, peri- and postnatal;
upper and lower airways; obesity; neural development; and positive health.
And the idea is to inform solutions to…these common pediatric outcomes.
There are two components to ECHO.
There’s the observational and intervention side.
The intervention side is in IDeA states, and IDeA states are those states that traditionally
receive less NIH funding than other states.
I’m going to focus on the cohort side.
So in ECHO, we really think about the broad range of early environmental exposures from
society to biology and from conception to 5 years.
Those are our exposure periods.
Children in ECHO are enrolled from in utero to age 20, 11 months, 30 days.
The overall scientific goal is to answer solution-oriented questions about the effects of a broad range
of early environmental exposures on child health and development.
And really, in this first cycle, what we tried to do…so we tried to weave together 69 different
maternal–child…dyads that came in…or cohorts that came in across the country.
So, we’re weaving together this cohort.
That was the first cycle, and then I’ll talk about the second cycle.
The first cycle…we have a really diverse cohort, 26% Hispanic, 43% White, 12% Black.
We’re across the country.
What we’ve done so far is we have data on over 100,000…107,000 participants; 65,000
of those are from kids.
About 34,000 are in active follow-up as we speak.
We have extant essay…assay data.
We have HHEAR assay data, and we have biospecimens on…or 83,000 biospecimens.
So, we have a lot we’ve done so far, and this data’s also available to the scientific
community through DASH.
This is a data and specimen hub through NICHD.
So, you can access that data, and currently we have about 1,300 publications, and about
100 of those are related to the ECHO-wide cohort.
Many of them are cohort specific, but every day there are more and more ECHO-wide studies.
And just quickly want to mention, for those predocs and postdocs, we currently have a funding
announcement available to look at the ECHO data through DASH.
So, okay.
I gave you a broad overview of ECHO during the first cycle.
And so, we’re about to start cycle two in September.
But a couple years ago we thought about: Where are we with the science, and where do we want
to go forward?
So we had a workshop on the Preconceptional Origins of Child Health Outcomes, and it was
a trans-NIH workshop.
We had many partners, including Drew and Somdat and many others from different NIH institutes.
And the idea was…the goal really was to assess the state of the science, research
gaps and opportunities, to think about: What are the multiple exposures we should include
in a future study?
How do we…and how do we do that?
So, what is the science, and how do we do it?
So, we had six themes: lifestyles…lifestyle factors, including obesity.
Recruitment and study design—how do we recruit a diverse sample?
What about fathers?
And we’ve talked a little bit about that.
So, we…that was one of our themes.
We were interested in physical and chemical exposures, and psychosocial and societal exposures.
And this was open to the public.
You can go to the website and find our booklet that has all the speaker information.
Okay, so, I’m going to focus on the lifestyle…what we learned from the lifestyle/obesity section.
So, we asked all of our speakers questions.
So, from the evidence of yours and other studies, to what extent do modifiable lifestyle factors
or obesity in women or men before pregnancy cause adverse health outcomes—or lead to
positive health—in their offspring?
How do you recruit moms and dads during the preconception period?
And what are some of the key research gaps?
And here’s a list of our speakers.
So, what are some of the key themes?
So, lifestyle factors.
So, sleep, physical activity, diet, they’re all intertwined with each other…so…and
with obesity.
And so, we…to inform multicomponent interventions…researchers should examine these in combination, not just
in isolation.
We…the need exists to examine the…the benefits and risks of weight loss before pregnancy,
so during the preconception period.
Also, the embryonic period appears to be a critical period for long-term child health,
emphasizing the need to recruit men and women before conception.
So, some of the main themes were about the challenges in recruiting and retaining diverse
populations into a preconception study and following them through pregnancy and into
childhood.
There were a lot of research gaps related to health disparities and equity.
And once again, the role of fathers is really important.
So, these are some of the high-level themes that emerged from this workshop.
So, we had this workshop 2 years ago.
What did we do with the knowledge that we gained from the workshop?
So, with what we learned from the workshop, it informed the next cycle of ECHO.
So, what is the future of ECHO?
So for the next…ECHO will have another, second 7-year cycle.
And so, we’re going to extend and expand the ECHO cohort to further investigate the
roles of a broad range of exposures from society or biology, and we’re including the preconception
period, and we’re going to look at this on ECHO’s five health outcomes among diverse
populations.
So, how are we going to do this?
So it begins in September [2023] through May 2030.
We’re going to extend by following the existing kids in ECHO, and we’re going to expand
by recruiting an additional 20,000 women and their partners, recruiting…recruited during
pregnancy and following their kids.
And this will include a preconception pilot.
So, right now, the preconception pilot, we’re thinking potentially about 10,000 individuals
will become part of this pilot.
And so, these are individuals who are at moderate to high probability of a subsequent period…pregnancy.
So, these are individuals that are going to be recruited during the interval pregnancy
period, from the first ECHO pregnancy to the second ECHO pregnancy.
So, this will result in a large 60,000…participant cohort from preconception through adolescence.
In the next cycle, we have a number of expanded scientific opportunities, and I’m not going
to go through all of them.
But just to point out, their preconception exposures is one big area that we’re going
to look at.
We released seven funding announcements a year ago, and this just shows that two of
the funding announcements will directly feed into the preconception pilot.
And as I mentioned, there’s going to be 10,000…at least 10,000 potential pregnant
participants in this pilot and their conceiving partners.
We think it’ll result in about 3,000 live births.
It…it may be higher or lower.
We asked all of the applicants to these funding announcements to provide exploratory aims to
look at these preconception…exposures, and we also in the…in the RFAs and funding announcements,
we asked for them to look at…at the participants during the preconception period for three
visits.
So, there’s a little bit of a timeframe there.
So now, to remind you about the first cycle, we have these 69 different cohorts.
They were coming together, they were…we were weaving together the ECHO-wide cohort.
In the next cycle, we will have…we have the quilt, right?
We’re no longer developing the quilt…quilt, we have the quilt.
There will be one ECHO cohort with many cohort study sites.
All of the ECHO cohort study sites will follow the same protocol.
It is posted, so you can go online right now and look at our current protocols.
It is our core protocol; there will be specialized measures that will be…the preconception
protocol’s not developed yet.
It’s being developed.
And there are specialized measures that will be developed.
And these specialized measures will be either more frequent or more intense measures in
three main areas with…in terms of exposures, so…lifestyle, psychosocial, and physical
and chemical.
So, in the next cycle of ECHO, the investigators will develop the specialized protocol where
they will select measures to go deeper, do a deeper dive in these areas.
Okay.
So, I went pretty fast there, but I gave you a high-level overview of ECHO.
I told you about…we have this preconception workshop.
And then I told you a little bit about how that workshop informs the next cycle of ECHO,
and what we’re doing in…in the next cycle of ECHO, which is about to start in September.
So, now I’m going to give you just some examples of ECHO nutrition research.
As I mentioned, we have over 1,300 publications, so I’m just picking out a couple more…a
couple of examples.
You’ll hear examples of more ECHO research from speakers later in the day and tomorrow,
but just going to mention a couple.
So, this study was about maternal diet.
So, this is from one of our cohorts, the PETALS cohort, about…almost 2,300 participants;
78% ethnic minorities, so very diverse; 57% were overweight and…or obese pre-pregnancy.
And in essence, what they found was that poor maternal diet quality in early pregnancy was
associated with a greater altering birth weight, Z-score, and increased risk of the baby being
large for gestational age at birth.
So, this…this finding, we see this finding over and over in various studies.
Another study, the Healthy Start study, which was mentioned earlier, had about…a little
over 1,200 participants, 13% African American, 26% Hispanic.
And what they did is they developed a maternal diet index, and they looked at allergic diseases.
So, the idea is that maternal diets relate not just to obesity and growth, but also to
other outcomes.
So, in this index that they developed, what they found was that certain foods…so,
vegetables and yogurts were protective, whereas other foods, such as french fries, potatoes,
rice, grains, red meat…were related to negative outcomes.
So, this index during pregnancy was associated with a reduced odds of a combined offspring
diagnosis of any allergy, excluding wheeze.
So, they looked at medical records from…for kids for…from 0 to 4 to assess outcomes.
This next study is from the PRISM Cohort on the Northeast.
And it looked at joint effects—so not just one exposure, but joint effects—of metals
and nutrition on birth weight for gestational age, for Z score.
So, they had about 500 mother–infant dyads: 40% African-American, 36% Hispanic.
And in essence, they found maternal nutrition and metal expose…exposures jointly effected
newborn…newborns’ birth weight.
And interestingly, they found that a better diet can help a male fetus maintain normal
birth weight, and that there were beneficial effects for different dietary vitamins.
And so, what this really means is looking at…this begs the question of looking at
multiple exposures and then the mechanisms.
So, they really talk about, well, what are the mechanisms?
Why are certain vitamins protective?
But we need deeper research.
So, these are examples of cohort-specific studies in ECHO in the first cycle, and these
studies can be replicated in the larger ECHO cohort.
So, an example of an ECHO-wide study is this study by Kate Sauder.
She looked at 15 cohorts, so about 10,000 participants.
She looked at maternal diets in pregnancy.
And she was interested in disparities in risks of inadequate and excessive intake of micronutrients.
And she estimated…she looked at the intake of 19 nutrients from foods alone and foods
and supplements.
And that’s just a map of the different ECHO cohorts where her participants came from.
And so, I’m not going to go through this entire list.
But the headline is: There are many disparities.
So, there’s disparities in…in terms of higher risk of inadequate intake and higher
risk of excessive intake.
And the interesting thing about this particular study is…so, she’s gone on to do more
studies and trying to find the optimal supplement.
And there really are not optimal supplements out there.
And so she’s working with different entities to try and develop optimal supplements.
But this really gets to precision nutrition research.
The next step for this type of research is…so, she saw that there are disparities in excessive
and inadequate intake.
Now, what are the outcomes in kids?
So, that’s the next step to access what…how does this relate to outcomes in…in the children?
So, some of the key takeaways from just…this is just a couple studies that I showed you…is
poor maternal diet in early pregnancy is associated with increased risk for offspring, birth weight;
it’s also associated with…it can be protective in terms of this prenatal diet that…vegetables
and yogurt intake could be associated with a reduce odds of…of allergic outcomes.
Maternal nutrition and metal exposure jointly effect newborns’ birth weight.
So, the importance of looking at more than one exposure and that there are many disparities
that exist in terms…excuse me…in terms of prenatal diet.
So, thinking about considerations.
So, these are really challenges and opportunities.
And the way I think about them is, there’s the science.
What are our research questions?
And then, how do we do it?
So with the science, there are many, many, many research questions we still need to address.
So, to what extent does a mother’s diet and weight trajectory during preconception…how
does it associate with childhood obesity?
How much do epigenetics mediate these associations?
What is the interplay between multiple exposures?
We talked about that and stress during the preconception period.
What is the role of paternal diet?
There’s very little that we know about the role of…of dads in terms of child outcomes.
There hasn’t been much research in this area, and how to do this research is challenging.
And then there’s a lot more that we need to do in terms of addressing health disparities.
In terms of how do we do it, a big question that we face within ECHO is: How and when
do you recruit preconception participants?
So, we’re recruiting them during that interval period, but they’ve already had one baby.
Does that make a difference?
The reason we’re recruiting them during that interval period is because they’re
already a participant, so they’re easier to follow.
But is that ideal?
I…I don’t know.
Right?
That’s…but that’s what was feasible.
What measures do we assess?
So the preconception protocol is currently being developed.
There’s only three visits, and these have to be short visits, so what are the top measures
to assess?
And also, with the dads, how do we assess them?
What do we assess within the dads?
How do we recruit them?
Actually, we don’t…we call them conceiving partners.
How do we recruit the conceiving partners?
There’s a lot of challenges with that.
And then, a big question is: How best do we recruit and retain diverse participants?
We’ve been told that, in many of…with our existing cohorts, that diverse participants
don’t always…or many families don’t always plan pregnancies, so you don’t just
want to enroll the planners, because then you might have a…a biased…sample.
So, how best do we enroll and retain diverse participants is another challenge.
And so, with that, I go back to our discussion question.
Thank you very much.
DR. ANDREW BREMER: Sonia, thank you very much.
And Krista, I totally botched it when it came to time management.
But what I did succeed in is a great…a great discussion.
Thank you for all the speakers.
You know, this in the past…past hour we’ve gone from the single gamete to the population
and back.
But it’s pretty cool.
I do…for those online, please use some…we…we do have a little bit of time.
I…not a lot, but we…we…we do want to capture your questions.
So, for those online, please do submit them in Q&A, and then those in the room…and thank
you, Kimberlea, for keeping me on track.
And those in the room, feel free to raise your…raise your hand.
I have a question, but I didn’t want to do…I’ve already spoken a lot today.
Are there any questions in the room or online?
A few online.
Let’s start with someone online.
DR. KIMBERLEA GIBBS: Okay.
So, this is for Dr. Rando: For the sperm studies, relative to humans, has the data shown that
gastric bypass in male obese fathers improved the outcomes in offspring sperm and child
health?
DR. OLIVER RANDO: So in…are they saying, in humans, gastric bypass—
DR. KIMBERLEA GIBBS: Yes, sir.
DR. OLIVER RANDO: Yeah. So, in…I don’t know that study.
But in rodent models, several groups, Hannon’s group and Laurie Goodyear, have both shown
that high fat effects on offspring can be reversed by exercise in the men.
So again, it is…exposure history is reversible in the parental generation.
It sort of came up, I think in…in the first talk, the question of the extent to which
your program’s predispositions in the offspring can be…can be ameliorated.
That I’m less…I know less about.
But certainly there’s some evidence in rodents that high fat effects can be reversed prior
to conception.
DR. KIMBERLEA GIBBS: Ashley?
DR. ASHLEY VARGAS: Ashley Vargas.
Thank you all for such a fabulous presentation.
I think we started with a high understanding of what’s going on in humans, and we jumped
into what’s going on epigenetically in mice, but…but also some in humans.
And then we ended in lessons learned from ECHO, which sounds disparate.
But the whole point is to think about how we can attack this really difficult question
about nutrition across multigenerations in a new way, because we know it’s very expensive.
It’s multidisciplinary.
It’s really hard.
But I wondered if any of the speakers had any examples of how other studies have intentionally
incorporated both human cohort data and maybe animal model data to help answer these difficult
questions.
And so, the one example I can think of is MoTrPAC.
But I don’t know if the…or if you have suggestions on…on how we might do that intentionally.
We know we need data from both animals and humans.
DR. ANDREW BREMER: And that was my question too.
DR. OLIVER RANDO: Well, I…maybe I’ll start.
It’s certainly much easier in the rodent models to look at multiple generations and
to look at offspring, right?
And it’s also easier to do…and, yeah…to make them eat, basically, a diet that’s
the equivalent of eating butter sticks.
So, you have a lot more power there.
There are a couple people who’ve tried to translate where you can sort of do similar
rodents and human studies at the level of the germ cell molecular contents, right?
So, Larry Feig at Tufts has done a lot of social instability work in mice and he sees
a couple microRNAs change, and then men who have…I think it’s the ACE questionnaire,
Adverse Childhood Experiences, so early life stressors show similar changes to the same
microRNA.
So, I’ve always, for my…my own lab, I’ve always hoped to one day move into humans,
but it would mostly be at the level of looking at molecular stuff and sperm because, you
know, if I hope to look at anything about traits in the kids before I die, I would have
to study those, you know, start those experiments already.
So, these kinds of things, like, our trainees are going to be the ones who start seeing…because,
you know, most of the traits that you see in paternal effects are not, like, things
that show up at 5 years old in humans.
They’re the complex diseases of adulthood.
So, it’s like diabetes, anxiety.
So, a lot of these outcomes in humans are going to show up in the four…you know, people’s
40s and 50s.
So, it’s really starting really well-characterized trials now.
It’s going to be our trainees who end up finding out the answers to these things.
DR. JANNE BOONE-HEINONEN: Oh, sure. No, that sounds good. I’ll just add a couple of things.
I mean, I think that the…just the…the crosstalk across disciplines driving the types
of questions and the measures that we’re examining and…and, you know, collecting
in humans.
I mean, that’s how we’ve been doing it.
But I also think that, you know, as I mentioned, you know, systems and science approaches are…I
mean, they are…they’re really designed to integrate pieces of evidence from multiple
studies and, you know, with the ability to…to conduct simulations.
DR. SONIA ARTEAGA: And I’ll just add, in ECHO, we focus on humans, but I think it would be
great to be able to collaborate with those groups.
DR. ANDREW BREMER: So, I’m going to…I’m going to…I’m going to call an audible
here for the sake of time and our…and our…our participants.
But yeah, I want to keep this kind of conversation going throughout the next 2 days.
Because then, again, it…it’s the power of bringing together lots of disciplines to
this to…to think about these…these…these questions.
Yeah, Usha.
DR. USHA RAMAKRISHNAN: Thank you for a very nice panel.
So, I guess I would love to hear from the group, you know, talking about, you know,
what new things do we have in our toolkit and [inaudible] so we don’t have to wait
another 50 years to figure out something.
Is…I’d love to hear on the role of metabolomics and why there’s been different opinions
on its utility…I’d like to hear…that could be a potential bridge—animal and human
studies.
DR. ANDREW BREMER: Usha, thanks for that question.
We’re going to chew on that because I…I need to get to the next speaker, who has to…who
has to…has to leave.
So with that, I want to thank all of you.
This is the…the start of a discussion, so…so this is…the next 2 days, let’s…let’s
chew on these points because…because that’s super important moving forward.
And with that, I do want to turn…I’m going to introduce Dr. Thad Schug, who—this is
great, I’m seeing him in person and not on screen—from NIEHS, who will moderate
our next session.
Again, sorry to cut everyone off; we need to be respectful of travel time.
Thanks, everyone.
DR. THADDEUS SCHUG: Okay.
So, we’re moving along to session number two now, Individual[-Level] Aspects of Multigenerational
Influences of Nutrition.
I’m Thad Schug of NIEHS.
And we’re excited to be involved with this program and meeting because we’ve been struggling
to better understand the effects of multigenerational exposures for many years now.
So, this session, we'll start off with diet and other exposures to the individual, and
we’ll wrap up the session with parental contributions.
Dr. Kari Nadeau from Harvard University will begin the session with a presentation on allergy.
After Dr. Nadeau’s presentation, we’ll take a break for lunch before moving on to
the rest of the session.
DR. KARI NADEAU Thanks, Dr. Schug.
It’s really a pleasure to be here, and thank you for inviting me.
It sounds like a fantastic symposium, and I’m so glad that people are organizing this.
This is what we really need, and I’m really excited that the ECHO program is listening
and thinking about what to do for the future.
So, I’ve been asked to look at the intersection of allergy, asthma, nutrition, and -omics.
It’s a big topic.
I’ll be focusing mostly on human studies today and looking within, across, and between
families and generations.
I tried to choose sort of the top-level summaries of some really good studies that have been
out there over the next…over the past 2 years to show you today, and then really focusing
on impact on health and disease as it relates to allergy and asthma.
And then finally, I’ll talk about research and opportunities and challenges.
So, the genetics of asthma and allergy have been studied at length.
In humans, there’s about 128 independent SNPs that are associated with asthma and allergies,
and then just 9,700 have been reported in the last 3 years.
Now, gratefully, a lot of these genetic loci actually serve as targets for therapies now.
And it’s a great way to validate whether or not these genes and their influences actually
affect a disease because a lot of these have been targeted in clinical trials and have
been shown to work or not work.
So, I think as we get better at biologics and inhibiting specific pathways with rationally
designed drugs, we’re going to be able to see to what extent these genetic inheritance
patterns meant in terms of disease.
I always like to think about this “nature versus nurture” equation, as was mentioned
by Usha earlier today, when we look at twins, and the concordance interestingly enough with
monozygotic twins is only about 50–60%.
So, if one twin has allergies, the other twin does, as well.
So, there’s a lot of environmental influences than just allergies.
For asthma, it’s even less.
It’s only about 26%.
This was published back by Kathy Barnes, my colleague, back in 1998.
So, again, more influence of environment in asthma and pretty much an equal influence
of allergies with environment.
So, the NIAID actually put a conference together.
A couple of years ago, Wheatley published this—she’s a program officer at NIAID—on
the multigenerational transition of asthma.
And what I want to point out here is that their symposium and their conference concluded
that animal models have shown that there’s up to three generations of progeny that are
involved in asthma inheritance patterns, most of that due to epigenetics and that interplay
of environment and genetics.
And then it’s really important as we think about multigenerational studies to not just
look at parental influences but also grandparental inheritance patterns.
And what I love as an epigenetics researcher to look at is the loci involved in allergy
and asthma pathology are exquisitely sensitive to epigenetic changes.
And why is that?
Well, interestingly enough, the loci sit in key immune cells that direct the pathology
of allergies and asthma—Th2 cells, Th1, Tregs, Th0—and many of these different factors,
these cytokines, are also modulated by epigenetic effects.
So, we think this is why there’s such an interplay between environment and genetics
and allergies and asthma.
These could be modified by vitamins, foods, and other exposures.
And I’ll give you examples of that later on.
And again, I’m going to focus on human studies today.
So, let’s talk at the top.
What are the effects of the grandparents in allergy and asthma disease outcomes?
This is Carrie’s paper actually, in which she…you did a great job summarizing some
of the risks that were published in the literature that children with a parent and grandparent
with asthma over four times the odds of reported asthma compared to those without a parental
or grandparent history.
There’s a lot of environmental explanations for non-genetic risk factors, which I’ll
talk about a little today.
And then importantly, as we think about multigenerational families and those that are underserved in
low socioeconomic neighborhoods and environmental justice issues and red zoning, we really need
to think about those factors as we understand and explain asthma risk factors in multigenerations.
What about the father?
Well, this is a study that was done in our…through our colleagues in Northern Europe, who have
many sites looking at the fathers’ environmental exposures before conception and asthma risk.
And I’m really glad that ECHO is now looking at preconception factors because you can see
here that smoking in the father versus the mother…that smoking only prior to conception,
for example, was more attributed in the father than the mother to early-onset asthma in the
progeny.
And again, that kind of showed itself to be true in the nonallergic asthmatic children,
as well.
So, I’m glad that we’re looking at fathers as well as mothers, because there might be
something attributed here in the germline.
This was also an interesting table that was published in the same article looking at fathers
smoking and the…and the attributions to the smoking exposure of the father but in
relationship to stratification for paternal grandmother smoking.
So, you can see here that there’s an interaction term going on where the father smoked before
the age of 15 and 13.
And so, they put together a model here showing that, in utero, the grandmother smoking alone
increased the risk of asthma and allergies, whereas the father smoking, as well, before
completion of puberty and then post-puberty complications with the father either having
a job of welding or smoking.
And that exposure increased the risk of allergy and asthma in the progeny.
And this is all, again, in humans.
So, what about the role of the mom?
Well, a lot has been done, as was spoken by Sonia, as well, with the maternal dietary
index that Carina Venter published out of ECHO.
But the maternal diet really does play an important role before and during pregnancy
in the risk of allergies and asthma.
This study was done through EDEN cohort by our colleagues in Europe.
They studied at least 1,000…you can see there are 1,140 individuals out of two…2,002
women.
And they did huge amounts of questionnaires—28 pages—to be able to understand the diet
before and around pregnancy and the contributions of the diet to decreased risk.
And this is what’s really interesting.
When you look at how much of a decrease there was for wheezing or for allergic rhinitis—i.e.,
hay fever—as it relates to what the mother ate and the risk of the child at age 3…now,
wheezing at age 3 is a little bit controversial because it doesn’t necessarily mean that
the child’s going to have asthma, because wheezing at age 3 can also mean they have
increase infections, so we need to be a little bit careful as to how to interpret that.
But what is interesting is you can see the raw vegetables and how pertinent they were
to reducing the risk of allergic rhinitis alone compared to cooked vegetables, for example,
and again, compared to meat.
So, this is really something that outside of ECHO has been confirmed in other birth
cohorts, as well.
And what about other maternal dietary risks?
And this is a child cohort study that was done—557 mother–child dyads from the Polish
Mother and Child Cohort looking at the diets of the moms and how that was related to children’s
health at age 1, 2, and 7–9 years.
What I’m going to show you here is the 7–9-year data.
And what they showed was that children of the mothers who were not achieving adequate
intake of vitamin C during pregnancy had a higher risk of wheezing and infections.
And inadequate intake of vitamin C also during pregnancy was related to higher risk of atopic
dermatitis.
Atopic dermatitis is otherwise known as eczema, and that can be a conduit by which episode
barrier defects can increase the atopic allergic phosphorylase, so it’s really important
to try to decrease allergic skin disease.
And then finally, inadequate intake of magnesium was also associated with increased risk of
wheezing.
So, I found this study also very interesting to look at diet-related metabolites because
I was also asked to talk about -omics and how we can examine more detailed, granular
omic-type studies.
And this was one of the first ones that I saw in which this cohort in Denmark looked
at dry blood spots after birth, so it’s the same thing that we do.
We look at the Guthrie card.
And so, this is only after 2 to 3 days of age, and then they followed these children
out, and then they examine the risk of allergies and eczema and asthma at 6 years of age.
So, what did they find?
So, what’s really interesting here is in a large study—and again, in Denmark, so
we have to look at to what degree that it will be validated in different ethnicities
and in different parts of the world.
But they looked at different metabolites that they were able to collect, and you can see
there that if the mother drank a lot of caffeine and…and that was found in the dry blood
spot at 2 to 3 days of age, that it reduced the risk of asthma at 6 years of age.
So, again, these were found to be relatively significant in terms of the nutrition of the
mom as it relates to outcomes later on in life of the child.
Interestingly, they also showed that increasing fruit, green vegetables, and fish decrease
your risk of not only asthma and allergies but also infections.
And, again, this was around pregnancy, 6 years later.
So, what about the child’s side?
So, I want to move forward in the…a field that I worked on very frequently and what
Roduit et al. found—and this was through Europe called the PASTURE study—was that
increased diet diversity, so that before the age of about a year, if the child has eaten
different proteins and been exposed to different types of diverse diets, and this is actually
an index now that Carina Venter has worked on, it actually has been shown to be associated
with a reduced asthma risk, reduced food allergy risk, and reduced atopic dermatitis or eczema
risk.
So, it seems that that imprinting window that Usha spoke about earlier today in which you
have this…one of the main periods, but not the only period of life by which you can imprint
your immune system and create tolerance.
That having that diverse diet, having changes in protein through the gut, that the gut maintains
tolerance and that there might be a reduction in atopic disease in general if we can make
sure that the diet is diverse in children.
And that means well in nutrition, as well.
So, this is a prospective study, instead of the study that I just showed you, which is
the PASTURE cohort.
This is actually a prospective, randomized clinical trial where proactive, preventative
food protein diet was shown to decrease the risk of food allergy in children.
This was published this past year—well, 2022—in Lancet.
And what was really interesting is that just by giving a small dip of peanut butter onto
the pinky of the mother about 2 days out of the week, they were actually able to show
this reduction in food allergy.
And this was peanuts shown here, but they also show that with egg and for milk.
So, you have to give eggs for egg allergy, milk to reduce milk allergy—you have to
be specific of the protein.
But we think this is really interesting in terms of proactive intervention in the diet
to make sure that we can prevent disease.
Finally, many of my patients, as well as my colleagues, have studied pregnancy and prevention
in the maternal diet.
And there was a lot of controversy in the 2000s as to whether or not women should be
eating nuts, should be eating the very same things that a lot of society was worried about
potentially inducing allergies in children to.
But what’s been shown definitively now in larger studies is that there’s a decrease
in the risk of developing nut allergies in children with women who actually take nut
during pregnancy and during breastfeeding.
So, this is really important because, as we think about what proteins need to be shown
to the immune system of not only the pregnant mom, but also the baby, it might be really
important to be able to make sure that the immune system understands and is exposed to
these different proteins in a diverse, healthy way with breastfeeding to be able to help
ensure tolerance later on in life.
And so, I’d like to now sort of move into the -omics part of my talk.
So, I’ve shown some things about multigenerational aspects of how to potentially prevent allergies
and asthma, what studies we’ve had.
I thank our large cohorts, but more needs to be done.
But how are we going to study this at a granular, high-dimensional level?
So, Usha and Sonia talked about some of the tools today to look to epigenetics.
And I think we’re getting better and better.
Obviously, there’s Methyl-Seq.
There’s CpG and pyrosequencing that we’ve done for a while.
But what about getting to epigenetic associations at the single-cell level?
And so, what we’ve done at…at Stanford, and now I’m at Harvard and my laboratory,
thanks to an R01 that we were awarded for the NIEHS that we’d like to understand:
To what extent women’s health is vulnerable during periods of exposure to PM2.5 or air
pollution?
And what we did was we set up four cohorts—pregnancy cohorts—that was exposed to low PM2.5 exposure
versus high PM2.5 exposure.
We’ve been following these women for almost 20 years, so we really know their exposures
in Central Valley in California.
And then we also looked at age-matched women controls that were not pregnant and also low
versus high PM2.5 exposures, same definitions.
And many were Hispanic because we were looking in the Central Valley.
We worked with the communities that are vulnerable there.
And we’ve been following them for almost 20 years.
And what we did was get their blood, and we can do this time-of-flight mass spectrometry
where we can collect and do about 45 different parameters of different cell types and at
the same time look at acetylation patterns in the DNA.
And so, why is that important?
Well, epigenetics is different depending upon different cell types.
We heard that from germ line cells versus somatic cells today.
But we really wanted to get into exquisite detail in terms of these epigenetic changes
and why they’re lasting long for some cells but not for others.
And so, we’re trying to get a glimpse of the immune system in particular, because I
mentioned that the immune system is such an important parameter that relates to allergies
and asthma.
So, we looked at those cells that were most important for allergies and asthma.
And this is, for example, a UMAP study looking at CD8-positive T-cells and how we can look
at acetylation patterns within those cells.
This was, again, part of our R01, and we’re really excited to show you some [of] the unpublished
data here.
This particular method was developed and published in Cell in 2018 by my colleagues.
And here you can see we’ve done the same analysis that they had done in the Cell article,
and this is a heat map using mixed-effect modeling in EpiTOF single-cell level.
And you can see the contributions of air pollution—for example, in pregnancy versus contributions
of air pollution alone—and then importantly, the impact of pregnancy alone on these epigenetic
profiles in the cell types.
So, we’re really getting a lot more granular in terms of how these epigenetic changes might
affect these cell types.
And we’re going to be following them now longitudinally over time, which is what we’re
doing as part of the R01, which we’re really grateful for.
So, again, this is just unpublished data, a spattering of what the possibilities are,
and I think this will help us understand more as to the contributions of epigenetic changes,
environmental exposures, nutrition, and disease outcomes later to follow these cells over
time.
So, again, moving forward from epigenetic to more omic-type research to metabolomics,
we’ve been doing a lot of work in allergies and asthma in metabolomics.
This is thanks to a P01 that we have with NHLBI.
We’ve looked at sample collections across two countries: the U.S. and France.
We looked at preschool-age as well as school-age children with allergies, allergic asthma,
or asthma, or healthy controls.
And then we basically used agnostic clustering to examine their plasma with full blown metabolomics.
And we had 470 children involved in this cohort.
And what we did CCA analysis, you can see the structures which differentiated themselves
in terms of metabolomics.
And I just want to make sure that we clarify that metabolomics is a great tool if it’s
used wisely and carefully and thoughtfully and making sure that all of the samples were
collected the same, making sure that there weren’t any technical issues, and making
sure that there are appropriate controls to compare to and validation sets to be able
to test with.
So, what we found, interesting enough—and this is somewhat based on nutrition and also
xenobiotics—is that there were differences in peptide metabolism, lipid metabolism, and
xenobiotics metabolism between those that had allergy, asthma, allergic asthma, and
healthy.
And this is, again, between 0 and school age.
So, we’re trying to understand now more…now looking retrospectively at their nutrition
and how this could perhaps discern why or why not they have that disease.
We can’t really say in a cause-and-effect relationship because some of these children
were already on medication, but we can at least try to use metabolomics to understand
the differences in disease in these children.
What about metabolomics in fecal samples?
Well, we actually took identical twins raised in the same household where you have a discordance
for allergies.
So, one was completely healthy, and the other had allergies.
Those are hard to find.
And…and they had to have grown up in the same household.
And so, what we found using metabolomics with…again, understanding nutritional differences that
were very, very minor, we actually found diacylglycerol to be important for associations with healthy
twins versus other food components which are actually found to be more in the allergic
twin.
So, again, these are associations, but we need to understand more about the contributions
of nutrition to the diet.
Finally, I’ll end with research challenges and opportunities.
I’m really grateful for this group for giving us, as scientists, the ability to talk about
what we think might be part of the future as you move forward at the NIH and with the [ONR].
I believe that multiexposure research can be a challenge, but much can be tracked now
and measured in urine, blood, hair, nails, teeth, cells, skin samples, test samples over time.
And this can be an opportunity for multigenerational exposomics.
Long-term cohorts are needed across generations to perform exposomics and epigenetics studies.
There’s a lot of tools now.
There’s also a lot of personal exposure monitoring, and I’m glad ECHO is involved
in that, as well.
We need to really separate out nutrition on the father and the mother of peri- and preconception.
I’m so glad that ECHO is looking at that now, Sonia.
Metabolomic studies are appropriate, but we have to make sure they’re controlled correctly
and controlled for the time of day when the blood samples were collected.
And good nutrition improves quality of life, and studying the effects of environmental
exposure in the diet at any point in time over the life course and through multigenerations
is very important.
So, thank you so much for your time.
Appreciate it.
DR. THADDEUS SCHUG: All right. Thanks for the excellent presentation, Kari.
So, we’re going to take a break for lunch, and we’ll reconvene for our next presentation
at 1:40.
Thank you.
DR. THADDEUS SCHUG: During the session version…during this session, virtual parti participants are
invited to ask questions through the Q&A box.
And I want to thank Dr. Crystal Barksdale, who’s here up front with me, from NIMHD,
who assists in the moderation.
And so let’s start things off here with Dr. Friedman, who’s coming in virtually.
DR. JACOB FRIEDMAN Okay. Thanks everybody.
And I apologize for not being able to be in Bethesda.
It’s an important conference, well organized. I thank Kimberlea for the opportunity to present virtually.
I’m…I’m actually coming to you from Big Sky, Montana, so I didn’t mean to interrupt
my vacation, but I wanted to participate, so thanks for the invite.
So we’re going to move on, I think, in this program now to a more reductionist view.
I’m not an epidemiologist, but I work with epidemiologists.
I’ve been working for around 25, 30 years on this problem of transgenerational nutrition
from the standpoint of gestational diabetes and maternal obesity.
So, this is my disclosures, really, nothing too dangerous here.
As you can see, most of my funding is out of DK, right, with a little bit of the ECHO
projects.
And then we just got this U54, which is really exciting; it's prevention of maternal mortality
and morbidity.
So where I’m going to go with this talk is the studies that we’ve done, intense
phenotyping studies in humans, mothers with nutrition, but I’ve also worked for the
last 20 years or so on nonhuman primates— nobody’s talked about them—as a model for
developmental programming, particularly understanding fetal development.
And you’ll see one of the key outcomes of these studies will be the fatty liver.
And if you haven’t heard about fatty liver disease in…in kids, it’s just as rampant
as obesity, and it’s kind of where we’re…we’re really focused with a lot…a lot of interest
in prevention.
We also work in stem cells—umbilical cord stem cells—and epigenetics.
I’ll show you one final paper that just got accepted from the ECHO Project that you
might be interested near the end.
My disclosures are, I’m not an obstetrician, and I’ve never been pregnant, so you can
ask me lots of questions about…about pregnancy, because I work with obstetricians all the
time, but not one.
So the way I present this to the Harold Hamm Diabetes Center, where I operate now, is prevention
in that first 1,000 days is so critical.
We know that there’s, you know, a genetic risk, probably 40, 50%. There’s probably
700 alleles for diabetes, and they all contribute a little bit.
So, there is a genetic history of how you get diabetes and obesity.
We know that body weight runs in families, but mostly what we’ve been concerned with
is that genes haven’t changed.
So, the gestational risk, in our opinion, in our estimation, is what’s really driving
this epidemic.
And if you think about it, probably 30% of women are obese.
Probably 50% are obese or overweight.
And so, there’s one in six women who are exposing approximately 2 million kids a year
to this gestational risk, which is just about body weight; it’s not about nutrition.
We’ll talk about that.
The lifestyle risk that kids encounter, you know, we’ve all talked about, once you’re
born, you know, whether it’s environmental, for the food or for the chemical exposures,
the problem that we have is that all of the…the…the…the chicken has come home to roost.
So, all those adult diseases, including youth type 2 diabetes, is up by 300%, as well as
type 1 diabetes.
So both of those things are going up dramatically.
Here’s an example of the youngest child to be diagnosed with type 2 diabetes is a
3-year-old.
This was not genetic.
This was as soon as the parents changed the diet of the kid, diabetes went away.
But it’s this kind of a thing that it’s really perpetuating our obesity epidemic,
which drives all the other things that we’ve been talking about.
And it’s estimated today that one in three children born in 2020 will develop diabetes
as an adult.
So, it’s not hard for me as a director of a diabetes center to talk about prevention
for the cure.
We know that if we don’t do anything, these babies are not built to face today’s challenges.
They’re not going to be as resilient.
They won’t do as well intellectually, physically, and socially, as you’ve all heard about.
And as adults, they’ll die too soon.
So, how do I think about diabetes?
I came into this field actually working on type 2 diabetes, and now it’s really focused
on gestational diabetes and the risk for kids.
So, it’s really gene function and protein.
And as we’ve been talking about epigenetics, there’s a small genetic component, which
we get into later, but it’s the environments and the obesity, and probably something to
do with the gut microbiome, that’s driving the insulin resistance that is part and parcel
for diabetes.
And that’s caused by too much lipid, too much inflammation, which drives insulin resistance,
along with changes in insulin…or glucose sensing and a reduction in insulin secretion.
So, it all comes down to here, but it really plays out through the pancreas and a lot of
other tissues.
Now, we talk about maternal obesity as a strong risk factor, and it’s actually more prevalent,
of course, than gestational diabetes.
And when you look longitudinally, we know that maternal obesity predicts childhood obesity,
but what about GDM versus just garden-variety obesity?
So, this is a study by Pat Catalano, where actually measured body composition in 9-year-olds
who were offsprings of 89 women who are either GDM or normal glucose tolerance.
And really, the strongest predictor for the percent fat at 9 years old was the maternal
BMI, which odds ratio was over 5.
But we know that mothers who have type 2 diabetes, it’s not the same as GDM.
These are women who have diabetes coming into pregnancy…actually have the highest risk
for diabetes.
I…I wanted to make the point about maternal triglycerides.
So, if you don’t have gestational diabetes, but you have obesity, you generally have metabolic
syndrome, which involves both maternal triglycerides and maybe a little bit of glucose, which independently
predict adiposity at birth.
So, we always talk about targeting gestational diabetes, but I think we’re going to have
to start talking about a way to get at the fuel source, which is the triglycerides.
We actually have a pilot study, which we’re doing randomized control trial intervention
on women who come in the first trimester with high postprandial TGs because we know fat
makes fat.
And we’re using a point-of-care meter, just like you use a glucose meter to stick your
finger, measure your triglycerides, and then we’re using something called Vascepa, which
is high-dose omega-3 fatty acids that will reduce triglycerides outside of pregnancy,
to pilot this study, both here and in…both in Oklahoma and in Colorado with my…my collaborators.
So, we’re trying to take a stab at…just see if we can lower those triglycerides, what
will happen.
Now, to…to the point at hand, we…we know that there’s overnutrition hypothesis.
So, this Western diet, high in sugar additives, low in fiber, high in fat, and it’s…it’s
part of our…our part and parcel of what we eat.
And so, mothers who enter pregnancy with obesity or diagnosed with GDM, they frequently have
a metabolic syndrome, which is higher TGs, higher glucose, more inflammation.
And even if we fix their diet for a few days, give them a meal, this is what they have.
And this only advances with gestation, so it only gets worse.
Now, if you talk to neonatologists, which I worked in neonatology for quite a while,
the fetus is extremely vulnerable to this overnutrition.
So, it has fewer mitochondria, has low antioxidants, and low pO2; it’s almost like living at
Mount Everest.
And so, when you expose the fetus to these fuels, you’re going to alter the TCA cycle
activity, de novo lipogenesis, the epigenotypes, and mitochondrial health.
We just don’t know quite where that happens and exactly what the actors are, particularly
in humans.
There’s also this inflammatory component, which may come from the microbiome, which
I’ll have something to say about further on.
So, first of all, the biggest scientific challenge is what has been said already, and that is:
Diet is really difficult to measure accurately.
And this window of when the exposures are may not be known or may vary by the outcomes
that you’re studying.
In other words, some nutrition exposures affect one organ; others affect another.
So, even for these well-described prospective cohort studies, these nutritional measures
probably begin after some of the windows of exposure.
And if you’re just doing it at a single point in time, it’s really confounded, particularly
by the subjective measures of dietary recall.
So, this is an area that’s our scientific challenge, and I think everybody recognizes
this at this point.
So, how I come at this is the following: So, we know diets, obesity, the microbiome, placenta,
inflammation, and lipids, they all drive fetal growth.
And we know that this changes fetal gene expression, alters growth, and increase adiposity, and
result in metabolic syndrome, and plenty of animal and epidemiology evidence as we’ve
heard about.
But what we’re missing is this human evidence—like, where is it and how does it actually occur?
So I’ve taken this…this approach the last 10 or 15 years, which is to use a lot of invasive
tools, both in mother and baby.
And this is a project that we published on and continue to publish on where we’re actually
fixing the…the diets at the time of gestational diabetes and giving all the meals that we…we
know would be important for fetal health.
And we’re measuring the microbiome. We’re measuring insulin secretion. We’re doing
fat biopsies and then harvesting the placenta.
But on the fetal side, we’re developing these tools where we can take the stem cells
out of the umbilical cord, we can measure infant fat, and we can put babies in an MRI,
which I’ll show you here in a second.
This is a…a newborn who is going to get wrapped up in a papoose and stuck in an infant
MRI in order to measure its liver fat.
This is a Pea Pod, which you’re probably familiar with, measures total body fat.
And this is Kristen Boyle and Kathleen Shapiro, who worked in the lab isolating the stem cells
from these babies after these diets.
What we published back in 2016, probably the first studies to show that the Wharton’s
jelly in that umbilical cord gives you an MSC that epigenetically and metabolically
is more prone to be a fat cell than a muscle cell.
And if it’s a muscle cell, it has low mitochondria and low respiration if the mother was obese.
So there is this biological he, or I call it a canary in the coal mine, that actually
can help us understand, if we tried to prevent something, what’s the outcome that has this
predictive power?
Here was the data that we published in 2013 on the…on the liver fat of these babies.
This is a papoose with intralipid as a standard.
And what we showed in this study was that the higher the maternal pre-pregnancy BMI,
the higher the liver fat in these babies.
It wasn’t how fat the babies were, it was how fat the mothers were, which kind of gives
you a clue why they’re developing liver fat.
There’s nowhere else for it to go for the most part except to the sub-Q compartment.
But why is this important
is because this might be part of the genesis of this NAFLD disease.
And if you look at kids…teenagers with biopsy-confirmed NAFLD, even if you adjust for their BMI, if
they’re born with a low birth weight or a high birth weight, they begin to develop
NASH, or nonalcoholic steatohepatitis, and advanced fibrosis based only on birth weight.
So, there’s a key indicator of where we start for a disease that’s rampant.
So, this occurs in 10% of all children, probably 30% of all obese youth or even more, and they’re
more likely to have this advanced disease and its symptoms.
So, I talk a lot of times to gastroenterologists, pediatric gastroenterologists, that this poor
child needs a liver biopsy because he has NASH and there is no…there’s no drugs
for this—no pharmaceutical therapy—and it accelerates type 2 diabetes.
So, when I think about programming, yeah, I think about obesity, but I think about this
disease a lot more because it’s more severe and has more higher consequences.
And there’s a lot to learn.
I won’t bore you with all of it, but we don’t know where the epigenetics are, the
inflammation, the cell death, fatty acids that are released that activate the liver
and cause this injury.
And we don’t know anything about the…the repair of this liver.
So, anytime the liver gets overloaded with fat, if it proceeds, all of these things,
whether it’s hypoxia, gut microbiome, lipotoxic liver, lipids, contribute to this inflammation
and advancing towards fibrosis.
So, we’ve got to figure out how to stop this because this is what we’re faced with.
We wrote this editorial—or a review article—about epigenetics in the micro…in the mitochondria.
So, when we did the mitochondrial metabolomics and the epigenetics, we found that these neonates
had…not only were obesity prone if mother was exposed to the high-fat diet epigenetically,
but they had all these signs and symptoms of NAFLD going forward.
So, it leads me to the next part of this talk, which is primates.
So, we can’t really, with good control, study diet in humans.
And so, we’ve been down this road the last 15 years to study this primate model, which
we overfeed the…the mothers in their juvenile years before they become pregnant,
and then we ask the question: What happens in utero?
What happens to these 3-year-olds as they grow up?
So, this is the model.
Japanese macaques at the Oregon National Primate Research Center are either fed a control diet
or a high-fat diet.
We call it Western.
It’s not that high, actually, but it does have components of fructose and cholesterol
and saturated fat.
Some of the animals get very obese, but actually some of the animals are like humans, resistant
to this diet, but they’re still on the diet, which allows us to study if it’s diet or
obesity.
And then we could also take some of the animals off the high-fat diet the next pregnancy to
find out: Is it the obesity or the diet?
So, I’ll just summarize about 10 or 15 years of work here.
So, we’ve identified that high fatty acids delivers more triglycerides to the placenta,
which causes inflammation and mitochondrial changes, and fatty acid transporters that
then lead to fat accumulation primarily in the liver, but in many tissues, as well:
oxidative stress, inflammation, and premature gluconeogenesis.
And essentially, these fetal livers, these animals are born with this lifelong risk for
the pro-inflammatory response to overnutrition.
And so, as you know, obese kids, they’re more likely to get this NAFLD NASH, and it
may actually be coming from the get-go, from the actual mother’s diet.
We’ve also looked—and I’ll show you in a minute—at the bone marrow.
So, in the fetus, the stem cells that come out of your bone marrow make macrophages, they
migrate into the liver and causes inflammation.
And a paper we just got done publishing will show you the epigenetics, actually, in the
bone marrow.
And this is [inaudbile] reports that just came out in Cell.
And so, this Western diets is skewing the bone marrow and the liver towards pro-inflammatory
gene expression.
And then, when we took the offspring off the high-fat diets at weaning and at 3 years old,
2 1/2 years after being on a chow diet, you still go into the bone marrow and you find
epigenetic programming and open chromatin or macrophages that were derived from the
stem cell that are now inflamed.
So, it kind of gives us a hint at any inflammatory disease might actually be hiding in the bone
marrow because of the mother’s diet in utero.
People always ask then, “Well, where are the abnormalities?”
And just like in humans from our epi studies, we’ve looked in detail in the fetus in the
brain, and we can see changes in the brainstem circuits that program behavior.
So, the females are more depressed, the males are more aggressive.
We’ve measured the placental function or dysfunction.
We’ve measured the steatosis and inflammation.
We’ve actually shown an early collagen deposition, which proceeds towards NASH.
The muscle is less sensitive. There’s reduced mitochondria.
We haven’t done much on the fat cell at this point, but the beta cell is also less
apt to be proliferative, and it’s lost the alpha cell component of this mass.
We’ve published all of these papers, you know, in the last decade or so about this
model.
And the microbiome is persistent.
So, we switched the offspring off the diets that the mother is eating, and their microbiome
is persistent, and it actually hypertrophs the organoids in the gut.
And I just got done telling you about bone marrow.
I just want to…I don’t want to take away the next speaker’s thunder about the microbiome.
We talk about this all the time, about how the maternal diet is modifying maternal microbiome
and then trying to get these babies what is a healthy start.
And so, I took a look at the baby’s stool from moms who are obese, and I transplanted
it into mice.
And this was the paper that we published in Nature Communications.
So, I know that these stool samples from a 2-week-old infant will increase inflammation
susceptibility to NAFLD if you put it in a germ-free mouse.
So, we are now doing studies with bacteriologists to try and identify which species—the early
Pioneers—are really the drivers of this response.
I’ll…I’ll…I don’t have enough time to talk about this.
I’ll leave you with a couple other studies that we’ve published recently.
This is the one that talks about that diet that we fixed for gestational diabetes.
So, we compared mothers where we fed every meal, at the time of diagnosis, a high-complex
carb diet, which allowed them to pass their glucose tolerance test, but actually, it was
an isocaloric diet, and it increased the health Bifidobacteria and the early life acquisition
of the infant microbiome in these babies born to gestational diabetes.
So, we can do something about this.
This is a really hard study to do, by the way, but that’s what it showed.
And this paper is about to be released probably in the next month or so.
And the last I’ll show you, it’s about cord blood DNA methylation in the Healthy
Start cohort from the ECHO study.
So, over 400 infants in which we measure the cord blood DNA methylation and found changes
in the pattern of the immune cells and lipid metabolism.
And further on, it was associated in mediation studies with maternal triglycerides.
And then adiposity at these babies measured mostly at 4 to 5 months and at 4 to 5 years.
So to me, this is still…it’s impugning maternal triglycerides, gene methylation,
and childhood adiposity.
So I…I wrote this editorial some years back about why this is happening.
I think it’s subcutaneous fat.
I think it’s hepatic fat, as I’ve shown you.
This microbiota gut–liver axis is probably contributing to inflammatory programming of
our innate immune system, along with mitochondrial dysfunction.
When you add the postnatal Western diets, you run through all these metabolic disorders
that wind up as liver disease and other things, as well, any inflammatory disease that you
can think of.
So, what is the most promising scientific or technological opportunity?
We were asked to talk about that.
And I…I think at this moment, we’ve seen some data on metabolomics, some on epigenetics,
but I think it’s this diets impacting the composition and function of these fuels and
food additives, by the way, to the mother and fetus.
I think we’re at the point now where we can use machine learning; if we have careful
infant phenotyping, we can probably reveal specific mechanisms for nutrients and fetal
health.
At least, that’s what I hope to see in the future.
In the long term, I think our problem is this nutrient delivery across the placenta.
There’s been a lot of talk about the release of these exosomes and the cargo that they
carry affecting both maternal health and fetal health.
So, we’re now working with bioengineering to target these exosomes to try and deliver
changes in tissues and cells during pregnancy, hopefully in the future to deliver either
healthy mitochondria or reduce the fetal overload and maybe placental inflammation–involved
fatty acid transport and all the things that we know are probably part and parcel of that
fetus exposure.
And just…here’s a couple of examples, you know, in the literature.
Exosomes carrying microRNAs.
Exosomes in healthy pregnancies are protecting against hypoxia and so on and so forth.
And that’s where I think the future might be.
So I’ll just stop there and acknowledge my collaborators, both in Colorado and in…now
in University of Oklahoma, and then at Oregon National Primate Research Center.
And thank you for the invitation, and I’ll…I’ll take any questions if there’s time later.
DR. THADDEUS SCHUG: Okay. We’ll do questions at the end of the session. Next up is Dr. Orjuela-Grimm.
DR. MANUELA ORJUELA-GRIMM: Thank you very much, everyone, and particularly the organizers,
Drs. Zanetti and Vargas, and I’m thrilled to be able to be here and be back at NIH.
I am going to give you a…a particular view of cancer—so, not cancer in general, but
just using one example from the tumors that I’ve worked on for a while—and…and talk
about diet- and…and nutrient-related exposures.
Also, just from…particularly from one pathway.
So, I’m going to talk a bit about from the perspective of using your framework—or the
conference framework—thinking of multigenerational influences, maternal characteristics, maternal
diet, and…and individual influences, from the perspective of thinking about methyl donors
and then how that also…we need to account in…within those at global food policies
and thinking about diet and feeding practices as environmental exposures and geographic
variation.
So, the image on the left-hand side is a child with retinoblastoma.
This is…for those not familiar, that’s a…something called leukocoria—
basically, a cat’s eye reflex.
You’ll see a few images of that.
It’s…the image on the bottom is where I take it to measurements of food.
So, retinoblastoma is a primitive neuro-ectodermal tumor.
So, my perspective on carcinogenesis is thinking from cancer like a neurodevelopmental instance,
but it…it certainly still is carcinogenesis.
It arises…just a quick summary of retinoblastoma.
It arises in the retina in cone cells.
During eye development, one or both eyes can be affected, and it exists in three forms.
A familial form in which it’s inherited, as you can see there on the left-hand side.
The first generation has it, and then subsequent generations, as well.
That’s a very rare form, and not the form that I focus on.
The ones I focus on are the two remaining ones, where the first generation isn’t affected,
and then the second generation will either have both eyes affected or one and basically
a germline variant, or both hits happening in somatic cells.
And I mention that because retinoblastoma is actually the paradigm for the two-hit model
of carcinogenesis.
And the really, the two-hit model of carcinogenesis came from Al Knudson, who was really looking
at this mathematically from looking at the incidents in families affected by retinoblastoma.
And that has really been the paradigm for our understanding of cancer.
However, it is a rare disease.
It’s one in 15,000 live births.
The median age at diagnosis is 22 months, which means when we think about when could
relevant exposures be happening, we have a pretty narrow window.
It has a higher incidence in the Global South, and incidents within countries where it’s
been examined is higher in four regions.
The biology is fairly well understood.
There’s a retinoblastoma gene.
It was the first tumor suppressor gene to be described.
It sits on chromosome 13.
The disease manifestations are determined by changes in the RB1—the retinoblastoma gene.
It encodes protein pRb, which is a nuclear phosphoprotein, and really, the product function
of the retinoblastoma gene and carcinogenesis is well understood and mutations in it tend
to be nonsense mutations,
so, leading the stop codons, truncated protein.
And because of the function of the retinoblastoma gene, it kind of tells us very clearly where
cancer is coming from.
The mutations happen preferentially on methylated cytosine, and sometimes the gene can be silenced
through a methylated promoter.
So, it makes one think about the possible role of methylation and methyl donors.
It is a key regulator in the cell cycle.
So, really thinking about retinoblastoma protein is…is really relevant for carcinogenesis
in general.
But…and…and thinking, as well, in…from the perspective of methylated cytosines.
So, is there a role for methylation?
The image on the top is a RetCam image of what the tumor actually looks like when it’s
causing the retina to be elevated.
So, the issue is we understand the biology of retinoblastoma really well; we don’t
understand the origin of the disease.
And so, what are the risk factors for development of…of mutations in RB1 that then we know
lead to carcinogenesis?
There’s some evidence from epidemiologic studies and some evidence on the mutation
distribution that suggests maybe methylation, methyl donors could play a role.
So, from an epidemiologic perspective, what are the relevant questions?
Well, when does the tumor actually develop?
So, as we’re thinking of this from an intergenerational…this is key from…from my perspective, and is
the key period of interest tumor initiation, or is it progression that we’re interested
in?
And then, when does the retina actually start to form?
It’s just thinking about how we formulate what we’re interested in, and when does
the cell of origin replicate?
And for a long time, cell of origin wasn’t actually known.
So, from my perspective, this…this is really thinking about pregnancy, but really, period…time
period within pregnancy, and then the first 2 years of life, because the median age of
diagnosis is 22 months.
So…so from a nutritional perspective, really thinking about those periods.
And our first work was looking at a case-control study.
All of my work on retinoblastoma—or almost all of it—has been based in Mexico and Central
Mexico.
This was our first study where we really looked very crudely at diet and saw that there seemed
to be an increased risk of having a child with retinoblastoma if you had low vegetable
intake during…during your…during pregnancy, and particularly in the second trimester of
pregnancy.
And…and really, when we looked at what were the vegetables, then it actually seemed that
it was low folate intake and maybe also carotenoids that…specifically lutein, zeaxanthin.
But it was also important to account for other factors.
So, we…we had found that low maternal education, and because we were looking at…at a setting
in central Mexico being in an urban area versus being outside of the sort of food delivery
patterns of…of being in a major urban area were contributors.
And so, we went on to follow this.
And really, here we were looking at intake from food, not from supplements.
And so, we became interested in really understanding the folate pathway, folate metabolism, and
thinking initially about, really, neural tube defects, and was this a role of…a really
folate deficiency in methylenetetrahydrofolate reductase?
And so, we obtained our first NCI funding and had a case-control study, and it was cases
and friend controls in central and southern Mexico, recruiting from two hospitals that
are major referral centers from very different systems: one the public system of, essentially,
the uninsured, and one…a very large national system that includes…or that…that requires
having at least one parent employed within the formal sector.
And so, we had trio blood sample collections, and this was thinking about nutrient levels
and genotyping, and also for the cases being able to collect their tumors.
And we had information on the perinatal environment and then diet and nutrient intake in pregnancy
in the first 2 years of life.
And here’s some images from data collection, really throughout central and southern Mexico.
And what we found was that, actually, there had been a natural change—or not a natural
change—but there was a change within Mexico with the onset of fortification.
And so, the…we actually needed to change our focus to not just think about methylenetetrahydrofolate
reductase, but actually think about what was the important enzyme within the rate-limiting
step for the metabolism of folate
because now we were actually thinking of a different source.
It was no longer just coming from natural sources, but coming from multivitamins and
from fortified foods.
So, that dihydrofolate reductase reduce…reduces folic acid.
And…and it needs to go through that step of being…before being metabolized in the
body.
And this meant we needed to think about different polymorphisms that were the rate-limiting
steps and that were prevalent within the population.
And so, essentially, this changed our…it…it turned our entire model upside-down.
But we found that, actually, we were looking at a…a really much greater risk coming from
women who were being exposed, who…who had less functional metabolism of folic acid,
and specifically, then, these women who were taking supplements.
So, we were no longer looking at deficiency.
We were actually looking at…at having an excess intake of a different form that is
not the natural substrate of…of the metabolizing enzyme, and we needed to account, as well,
for the child’s genotype.
So, when we accounted for both the maternal and the child’s, then, is where we started
to see the most relevant findings.
So, just a little bit about the polymorphism we were looking at, which had also been looked
at initially through neurodevelopment and neural tube defects.
This is a deletion and a non-coding part of the gene within an intron, but it is the critical
rate-limiting step for folic acid to enter the methyl donor pool.
And so, then we really wanted to understand: Where was the folic acid coming from in our
population?
And supplements was a part of it, but actually, supplements were not so commonly used.
And so, we needed to think about food…food sources.
And this really brought us into thinking about the fact that we rely on…when we think about
diet, we assume that when we take it to nutrients, that we actually know what the food contains.
And…and sometimes that’s questionable.
And so, we found, really, that we needed to think about fortification.
And so, Mexico fortifies wheat and corn flour with about 50% higher rate than the U.S. does.
They started a few years after we did.
It was legislated in 1999, implemented in 2000, was made mandatory, but not enforced.
And then you can see there, in the U.S., it’s 1.4 milligrams per kilogram fortification
of flour; in Canada, it’s 1.5.
And basically, as you go south, it increases within the Western hemisphere.
So, the southern cones says, “Let’s do it even more.
More is better.”
But in the EU, there’s actually no fortification.
And then, as I mentioned, supplement intake in Mexico is not universal among women of
childbearing age.
So, this is just a…a map of fortification globally—countries with industrially milled
flour and a rice fortification legislation.
And so, you can see, there…countries choose which product…which staple they fortify,
and not everybody fortifies at the same rate.
And so, we actually found that we needed to learn how…the whole industry of bread.
So, thank you—my program officer is sitting here—for…for the…for these studies with…NCI
was generous enough to allow us to really understand where the…in order to understand
the measures of the exposure, we needed to understand where the folate was coming from.
And we realized that measuring intake…measuring biomarkers was only part of the story.
We also needed to measure the food.
So, we became experts at how food was being fortified in Mexico.
And we used our retinoblastoma study really to get a lens at understanding staples—dietary
staples—in Mexico, quantified concentrations of…of folic acid precursors, and link these
geographically.
And then, we’re able to work with our wonderful colleagues at the Public Health Institute
in Mexico with the NHANES equivalent in Mexico to really see what happens to folate measures
in the population when you account for actual food measurements.
And so, this graph is basically showing that in the red line is when you don’t account
for bread and tortilla contents, and then when you bring in how much bread contains,
when you bring in how much tortillas and…and corn flour, corn masa-based foods contain,
then…then you start to see that you’re really underestimating your food content.
And we also found that place of residence made a difference.
And so, we were able to see that, not surprisingly, in more urban areas, we were more likely to
rely on fortified flour to make your dietary staples rather than grinding your own corn.
So…so, this really introduced us.
One of the things that we found was that your folic acid intake…this measure for the population
was…it turned out to be not reaching the target population, which are women of childbearing
age, where they are much more likely to be under-consuming.
And instead, we were potentially over-fortifying children of small surface areas.
So, potentially unintended consequences.
So, from starting from trying to understand carcinogenesis in a very rare tumor—that’s
a model we found ourselves really looking at—maybe trying to look more deeply at some
global food policies.
This is just mapping out sources of folic acid within our population—gray is tortillas—and
the bottom too, you’ll see actually our…our breads.
So, we see that these are making up…these dietary staples are making up a large amount
of folic acid, particularly in the younger ages, but really throughout the lifespan.
And this was…the Public Health Institute in Mexico made an infogram to really educate
people about thinking about their sources of folate and folic acid.
So, what we found was that the amount you ingest depends not just on what you eat, but
also on governance.
But, you know, folate and folic acid for carcinogenesis is really, potentially, a double-edged sword.
So, one size may not fit all.
It’s important to think of really what’s the right amount.
There’s literature—which I’m not going to discuss because of time—but, really,
ample evidence on colon, breast, and pancreatic cancer suggesting an increased risk with both
insufficient and excess levels of intake.
So, sort of both extremes, but it’s very much about thinking who’s too little or
too much, and just the polymorphism that we looked at had really been defined within the
Framingham cohort in terms of how it…it is actually functional.
So, really looking at it, not necessarily from a carcinogenesis perspective, but the
polymorphism matters only when your intake is moderately high or very low and not in
between.
This polymorphism has been relevant also for neurocognitive work in…for memory loss.
And so, really coming back to our thinking of carcinogenesis from a neurodevelopmental
perspective because we…because of the tissue of origin, but we also think that our platform
may be relevant for also thinking about cognition.
And in fact, we’re using the same approach with the collaborators in Mexico City with
a birth cohort who are looking at effects of lead on…on cognition and just thinking
about accounting for ideal amounts of…of folate.
And just to show that this isn’t relevant just for Mexico.
In fact, in the U.S. in 2016, fortification went beyond wheat to also specifically including
corn masa, really targeting the Latino populations that use corn flour as part of their staples.
So, this is relevant in the U.S.
And then just a little bit…a tiny bit of our other work that is nutrient related that
comes from retinoblastoma was actually thinking about sunlight exposure.
And then that model, we actually had to think that when this eventually brought us to thinking
about vitamin D, but we needed to think about practices of covering children.
How much of a child’s skin is exposed to sun, turned out to…actually, sunlight was
protective for retinoblastoma.
We had anticipated actually UV exposure would increase carcinogenesis, but we actually found
ourselves in…in a different model.
And where we also needed to think about geography because we needed to think about altitude
in terms of thinking about vitamin D exposure.
And then this eventually then led to thinking about…to…to finding that, in fact, vitamin
D was protective,
and this has been replicated in an unrelated study in the U.S.
So, multigenerational impact of individual-level diet and feeding practices.
And from our perspective, retinoblastoma has served as a model for us to understand the
effects of early prenatal exposure and one-carbon donors.
That’s led to us thinking about the examination of an impact of a food additive on diet and
early childhood exposure and neural development.
And then it’s actually led in our work that I haven’t talked about here, examination
of these exposures in the context of migration, resettlement, living conditions, and food
and nutrition security, which turned out to be quite related to these exposures and the
children’s diet.
So, I’d like to just acknowledge my many collaborators in Mexico and…and in some
institutions in the U.S., especially at Tufts, and some images from our studies and the scientific
challenges.
For me, the biggest scientific challenge is…is measurements, but measurements that can approximate
the relevant time of exposure and the relevant tissue.
And then, most promising scientific or technological opportunities for me, and the short term,
it’s the widespread availability of mobile technology and thinking about how to be creative
with its use.
And then long term, the ability to examine indirect and functional markers of exposure
through -omics, including examination of impact on the microbiota.
Thank you very much.
DR. ANDREW BREMER: Okay, next up is Dr. Cowardin.
DR. CARRIE COWARDIN: Good afternoon, everyone, and thank you to the organizers for inviting me
to speak today.
I’ve learned so much already, and I’m really looking forward to the rest of the
program, as well.
So, today I’ll be talking about the gut microbiome, particularly the maternal and
early-life microbiome and how that influences child growth and immunity.
So, I’d like to begin at the beginning and start talking about the maternal microbiome
and…and birth outcomes and what’s known.
And then a little bit later in the talk, I’ll get into what we’ve done to…to study this.
And so I think the maternal microbiome can influence pregnancy and…and fetal development
in a variety of ways.
I think one of the primary is…is through influencing metabolism and nutrition.
And so, the microbiome has sort of been increasingly recognized as an endocrine organ.
And it influences adipose tissue composition, nutrient processing and absorption, and lipid
and glucose metabolism and, through those functions, can influence obesity and weight
gain, as well as undernutrition,
and…and, of course, gestational diabetes.
I think the other really important function of the microbiota is influencing immunity.
And so, we know that the gut microbiome can regulate maternal immunity and inflammation.
And this is driven by sort of microbial structural components.
So, those can include, like, pathogen-associated molecular patterns, as well as metabolites,
and then, actual microbial function, such as adherence to the intestinal epithelium.
And through this role can influence preterm birth, as well as cognitive development.
And then finally, I think there’s some really exciting emerging evidence about placental
development and how microbial metabolites that originate from the gut actually can regulate
placental development in using murine models.
So, particularly, vascularization and nutrient transport seem to be impacted by the presence
of the microbiome.
And, of course, this can have major impacts on fetal growth.
So, I think one of the challenges—and I’ll talk more about other challenges later—but
one of the challenges is defining what normal is because there’s so much variation in
individuals with…with microbiota composition.
And so, there’s been some studies looking at the composition of the microbiome throughout
pregnancy.
And a few studies have noted increased diversity in the third trimester, and interestingly,
if you, if you take microbiota from the third trimester and actually transfer it to gnotobiotic
mice, this third trimester microbiota can confer…increase inflammatory cytokines,
adiposity, and insulin insensitivity in mice when you compare it to microbiota from someone
in the first trimester.
And so, this may be a…a process of normal microbiota changing during pregnancy, but,
of course, this can also be associated with disease states, as well.
And I think there’s a couple of different likely mechanisms underlying this.
And so, one is hormonal changes.
So, you know, it’s been shown that progesterone and estrogen can alter microbiota composition,
as well as microbiota activity.
And then, maternal diet is, of course, a huge factor.
So, high-fat diet, fiber deprivation, and undernutrition can all have adverse effects
on the maternal microbiome.
And there’s certainly been demonstrated links between the maternal microbiome and
birth outcomes.
And so, this has been shown in high-income countries.
So, links between microbiome alpha diversity and newborn head circumference and preterm
birth.
But it’s also been shown in low- and middle-income countries.
And I think we’re really starting to develop an appreciation for the potential role of
the…the maternal microbiome in undernutrition.
So, there was a recent study out of Zimbabwe that actually implicated specific taxa in
regulating birth weight or…or showed associations between these taxa and birth weight.
And then one of the nice things they did was they really got into microbial function.
And so, they were looking at starch metabolism and vitamin B metabolism being associated
with weight for age G scores and length for G scores at 1 month.
And then they found, interestingly, that markers of biofilm formation in the microbiota were
associated with reduced birth weight.
So, I think this is one of the opportunities to getting past microbial taxonomy and really
looking at functions.
And, of course, the maternal microbiome is also critically linked to immune outcomes,
as many of the speakers have already discussed today.
But there’s links between maternal antibiotic exposure, dietary exposures to things like
asthma, atopic dermatitis, food allergy, inflammatory bowel disease, and…and childhood obesity.
And then, we’ve seen from…from preclinical models that maternal…maternal antibiotic
exposure can also alter offspring microbiota even in animals that haven’t been themselves
exposed to those…those antibiotics.
And so, just a few of the…the changes that have been shown are increased splenic T and
B cells linked with asthma susceptibility after maternal treatment with vancomycin;
cefoperazone led to decreased regulatory T cells and increased colitis susceptibility
in offspring.
And then, of course, maternal antibiotic exposure is known to predispose to diet-induced obesity.
So, beyond direct maternal microbiome effects in utero, I think it’s also important to
talk about intergenerational microbial transmission.
And one of the reasons is because infants really inherit a lot of their microbiome from
their mothers.
So, we know that vaginal- and skin-derived strains transiently colonized neonates.
And then, over time, actually, maternal gut–derived strains really tend to dominate the infant
gut.
And so, these strains actually are thought to be more persistent in the infant gut when
they’re maternally derived than if they’re picked up from environmental sources.
So, you can see a large percentage of that infant microbiome is actually derived from
mom.
And I think there’s also some really interesting data about microbes within breast milk.
And so, I think this is an active area of investigation that we don’t know a ton about
right now: how maternal microbes get into breast milk, and then how much those actually
shape the infant microbiome, but there’s a lot of excitement in this area.
We also know that beyond sort of direct colonization with breast milk microbes, human milk oligosaccharides
can act as sort of prebiotics for the microbiota.
And so, they’re actually—I’ll talk more about them in a minute—but they’re really
not absorbed by the infant directly, so they actually act on the microbiota itself.
There’s some interesting and…and consequential studies on cesarean section.
And so, infants delivered by C-section have altered microbial communities relative to
vaginally delivered infants.
And so, one of the effects of…of the C-section delivery is sort of reduced similarity to
the maternal microbiome.
And those differences gradually decrease over the first few years of life, but they…they…they
do have sort of long-term consequences for some immune-linked pathologies.
So, there have been multiple attempts to seed infants with maternal microbiota using vaginal
microbes.
And so, there’s mixed results in this, but I think there’s some recent studies showing
that—or a…a very recent study showing—that they actually had some success with restoring
sort of a…a… a vaginally-derived infant microbiota in a c-section infant using this
vaginal microbiota transplantation.
And it actually was associated with improved cognitive outcomes.
So, I think there’s some pretty interesting work going on in this area.
We know that at least one study has shown that you can actually transplant fecal microbiota
in infants, which is…it’s a little bit scary, but it’s quite interesting showing
that the fecal microbiota transplant, there’s extensive screening that they did on the samples,
but they were actually able to restore a more normal microbiota.
Although it’s not clear whether…how this is linked with…with outcomes.
So, I think there’s definitely some active investigation going on in this area.
And then, just to talk a little bit about the sort of microbiota development in the
first few years of life, we know that neonates are born with minimal or no microbiota.
This is also an area of debate, but regardless of which…which side you fall on, I think
we can appreciate that the biomass at birth is very low in terms of microbiota.
So, most microbes are acquired from mom, from family, from the environment during the first
days of life, and then this maturation proceeds over the first 3 years.
So, if we look at just the number of OTUs or…or number of different taxonomic units,
we can see within the microbiota over the first 3 years, there’s a sort of progressive
increase over time.
And beyond this, or I guess more specifically than this sort of first 3-year window, we
also know that there’s a major shift during the weaning period.
So, the transition from breastfeeding to introduction of solid foods results in reduction in taxa
that are primary consumers of milk sugars.
Those are things like Bifidobacteria, and increases in those that are consuming plant
polysaccharides.
So things like Bacteroides are sort of the classic example.
And so, you can see that over time in this…in this heat map that the first 6–12 months
of life, there’s a lot of Bifidobacteria that are present.
And then in this transition when breastfeeding stops and complimentary foods are introduced,
you get an expansion of…of a more diverse microbiota that more closely resembles an
adult community.
So, what are some of the postnatal factors that influence microbiota assembly?
I think breastfeeding is a huge one, obviously, as…as you can see, some…from that transition
from milk consumers to…to polysaccharide consumers.
There are tons of benefits to breastfeeding.
We know there’s lots of bioactive components of breast milk.
So, in addition to those HMOs, they’re antimicrobial peptides, hormones, immunoglobulin cytokine
cells themselves, microbes themselves, cells with the mediators.
And so, all of those things can potentially have an effect on the intestinal microbiota.
And as I said, these…these human milk oligosaccharides, they’re really interesting example of nature’s
prebiotic.
So, they’re not digested in the infant gut, and they’re minimally absorbed by the host.
They actually make it to a large intestine, where they’re fermented by the…the microbiota.
And there’s all kinds of…of benefits to growth and immunity that have been demonstrated
for HMOs.
I think one area that is less well understood is what really dictates the composition…the
HMO composition in breast milk.
So, we know there’s certain things that are…play a…a really big role, like the
time of…the stage of lactation, there’s very…a lot of variations between individuals,
but I think there’s some, like, interesting data coming out showing that there could be
links with nutrition, other environmental exposures.
And I think this is an area that we don’t understand very well yet, but there’s a
lot of opportunity here.
And so, one of the things that got me into…into…interested in this is, this is a study from mothers in
Malawi actually showing that mothers of children with healthy growth had higher levels of sialylated
oligosaccharides in their breast milk than mothers of children with…with growth stunting.
So, we don’t know why that is.
It could be many different things, but the fact that there was this difference identified,
I think suggests that there’s more going on here.
So, in addition to breast milk, additional factors that influenced microbiota assembly
in early life include diet after weaning.
So, that’s an obvious one.
So, we know that children who have under…experience undernutrition in early life actually have
microbiota that look immature for their…for their age.
So, if you take a healthy child look at their microbiota, let’s say 6 months of age, and
you take a child with undernutrition, a lot of times their microbiota will look more similar
to that of a younger child.
And then we can see from a couple of different studies that treatment with therapeutic foods
does improve this microbiota composition, but it’s not always very durable.
And so, if you look in the figure here, you can see the microbiota shifting along with…with
therapeutic food treatment, and it’s becoming closer to that healthy configuration, but
it doesn’t quite make it there.
So, of course, early-life diet has an effect…it influences obesity, as well.
We know there’s differences in microbiota composition in…during obesity.
And studies have shown that if you take these microbiota and you transplant it to gnotobiotic
mice, you can actually see them gain more weight.
We do think there’s a sort of causal role for the microbiome in this…in this area,
as well.
And then, as I mentioned, antibiotics either a maternal antibiotic treatment or antibiotics
in early life can also predispose to diet-induced obesity.
So, I think I…I just want to emphasize what are the consequences of these differences
in microbial colonization and why does it matter that you get a…appropriate microbiota
maturation and early life.
And I think, you know, for me, it always comes back to the immune system and development
of the immune system.
And so, from preclinical models, we can see that exposure to the microbiota during this
weaning phase is really critical for immune development.
So, if you don’t have a microbiota during this time period, you really miss out on a
lot of immune regulatory cell development.
And so, you’re actually much more susceptible to pathological inflammation later in life.
So, what are things that are linked to pathological inflammation?
Everything, most things that we care about.
So, infectious disease, metabolic disease, cognitive development, even obesity, cardiovascular
disease, all kinds of different…different diseases have this sort of immune component.
So, I think this is a really important aspect of…of the functions of the microbiota in
early life.
And there’s a lot of interest in understanding how this sort of weaning reaction that occurs
in mice is…is translated to…to humans.
So, I want to talk very briefly about some of the work that we’re doing investigating
microbial mechanisms in childhood undernutrition.
So, to…to do that, I’ll give you a brief overview of what we know about undernutrition.
And I think the biggest thing is just, it’s not about insufficient diet alone; that’s
a component of it, but it’s also…reflects these diverse environmental challenges.
And so, that’s pathogen exposure, that’s intestinal inflammation, reduced absorption,
and intestinal inflammation that ties all of these things together.
So, we know that environmental enteric dysfunction, or EED, is really strongly linked to undernutrition,
and particularly linear growth stunting.
So, EED is a…is a subclinical syndrome of…of its enteropathy.
And that’s…basically, we think that EED is causing this failure of nutrient absorption,
and it’s driven by intestinal inflammation.
So, it’s been sort of challenging to diagnose because the gold standard is endoscopy and…and
biopsy.
So, it’s a little bit hard to study, but we know it’s really prevalent in areas where
undernutrition is common.
And it may be one reason why therapeutic foods are not as effective as they should be, is
because these kids just can’t absorb the nutrients that we’re giving them.
And I think there’s also a really important component of maternal undernutrition, particularly
in linear growth stunting in children, which is really strongly linked to later life outcomes
that we care about.
So, things like overall mortality, but also cognitive development, susceptibility to infectious
diseases, all kinds of negative outcomes of being stunted in early life can be tied back
to maternal undernutrition.
So, we know that mothers who are…experienced growth stunting in early life are more likely
to give birth to stunted children themselves.
And so, we are really focused on understanding how intergenerational transmission of the
gut microbiota can tie into this and contribute to this intergenerational transmission of
growth stunting.
And I think this has partly been inspired by observations in other enteropathies in
high-income countries.
So, things like celiac disease and inflammatory bowel disease.
We know that in…gut inflammation in those disorders is linked with negative outcomes,
as well.
So, things like preterm birth and pregnancy loss, as well as, size and fetal development.
So, how do we go about modeling intergenerational growth stunting?
We know that all of these different factors are contributing and that they’re all sort
of interrelated, so it’s not just a unidirectional interaction; all of these things are in this
complicated network.
So, the way that we’re approaching this is using gnotobiotic mice.
And we’re actually taking microbiota samples from children with linear growth stunting
and undernutrition, and we’re using them to colonize gnotobiotic mice.
We then breed these mice, and we look at their offspring.
And so, we’re actually studying both, sort of, the paternal components, as well as the
offspring components of undernutrition.
And just to give you an example for some of the data I’m going to show, this is a…a
length-for-age Z scores from the four donors that we’ve been using in our gnotobiotic
mouse models.
So, we’re using samples that are collected from these children when they were 6 months
of age.
These are children living in Malawi, and we have two donors that have healthy growth trajectories
and two donors that have moderate to severe stunting.
So, the way that we do this is we actually…we take germ-free mice, and we colonize them
when they’re 4 weeks old, and we maintain them on a malnourished diet for an additional
4 weeks.
So, they’re getting this sort of period of malnutrition in early life.
At…at…when they’re 8 weeks old, we transfer, transfer them back to a nutrient-sufficient
diet, and then we actually breed them, look at their offspring, and their offspring are
weaned onto a malnourished diet, as well.
So, we’re capturing both windows.
We compare these to mice that are just malnourished and colonized in early life alone, so they
don’t have that parental exposure component.
So, generally, what we find when we do this is that, in terms of…of weight, we don’t
see huge differences between…so, here we’re comparing between the healthy microbiota donors
and the stunted microbiota donors in both conditions.
We don’t see massive differences in weight between the sort of donor health status, but
we do see big differences in linear growth.
So, when we look at tail length, which is a surrogate for linear growth in mice, we
see a significant reduction, but it only occurs when the mice are born to colonized parents.
It doesn’t occur when you colonize the mice directly.
So, what are some of the other things that we care about with undernutrition?
We’re really interested in intestinal physiology.
And so, in addition to linear growth reductions that are seen, we use this intergenerational
model.
We also see reductions in intestinal…small intestinal villus lengths, which is one of
those EED-associated phenotypes.
And then we see a reduction in the thickness of the…of the muscularis in the intestine,
as well.
And I think one of the most interesting changes to me is that we see a lot of immune signatures
in the intestine that look a lot like what we see in humans with EED.
So, there’ve been some recent single-cell studies showing what immune changes are happening
in the gut during EED.
And we see similar things here.
So, we see an…a big increase in intraepithelial lymphocytes, as well as IgA+ plasma cells.
So, those have both been shown in humans with EED.
And I’m not going to go into this a lot, other than to say all four of our microbiota
donors are distinct communities.
So, we don’t think this is just a question of having the same microbes.
We think there’s conserved microbial functions that are being performed in these different
communities, in addition to probably conserved host responses to those functions that are
then having these effects.
So, I will move on to challenges and opportunities.
So, I think one of the biggest scientific challenges for this area of research is just
the scale and complexity of the microbiome and…and how many different taxa are there
and how…and all of their different functions.
So, I…I think this makes moving beyond description of the microbiota…so, like, this group is
different from this group, to actually understanding what those microbes are doing really important
but also really challenging.
And to get at that, I think microbial function is becoming much more of a focus than just
describing which microbes are there.
We really want to understand what they’re doing.
There are a lot of different options for therapeutic interventions.
So, there’s prebiotics that can nurture a different microbiome community; probiotics
that give the microbes themselves; symbiotics, which are combinations of the two; and then
postbiotics, which are things like microbial metabolites, giving those directly.
There’s lots of different options here, but we don’t really know what the best approach
is.
And then promising scientific and technological opportunities.
In the short term, I think there’s been a lot of progress in developing these models
that actually harness the complexity of the microbiota—so, using gnotobiotic mice.
It’s really important that we can manipulate those systems so we can actually find functional
differences.
So, having a model where we see these immune changes gives us the opportunity to understand
what those immune cells are doing.
And then there’s, of course, the opportunity to shape the microbiota through diet and beneficial
microbes.
Long term, I think this concept of precision medicine is really appealing in this…in
this area.
Of course, when we’re talking about undernutrition in low- and middle-income countries, we’re
really concerned about cost.
So, I think there’s a…there’s a sort of trade off there that we have to be aware
of.
And so, whether it’s something like a…a geographically specific therapy, even if it’s
not an individual level, if we can tailor it to a…a broad dietary context, that could
be helpful.
And then, I want to just end by talking about what I think is a really cool story.
So, this is actually the lab where I did my postdoc.
They designed a microbiota-directed food intervention.
And I think the…the most interesting thing about this story, to me, is that it’s based
on really extensive preclinical investigation about the microbiota and undernutrition.
And so, they didn’t just jump in and give different foods; they identified taxa that
were associated with growth.
They screened ingredients in gnotobiotic mice to find ingredients that boosted those taxa.
They assembled those ingredients in a couple different formulations and tested them out
extensively in gnotobiotic mice and pigs before they took them into humans.
And they actually recently published this paper showing that this might provide a directed
food that actually increases weight gain significantly in undernourished kids.
I think there’s a lot of questions that come out from this.
So, could these therapies be more effective if they’re given earlier?
Rather than in early life, what if we started with mothers?
Could they be given for longer and could they be tailored to individual populations?
So, I will stop with that and just acknowledge my lab members who’ve worked really hard
on this project and our collaborators and funding from NICHD, of course.
DR. THADDEUS SCHUG: Okay, our next speaker is Dr. Shelly Buffington.
DR. SHELLY BUFFINGTON: Hello. And thank you so much for the invitation. It’s really wonderful to be here.
I’ve very much enjoyed the content so far, and I’m…I’m excited about all of the
talks tomorrow, as well.
Okay. So as we’ve already covered today, in the U.S., roughly three in 10 children are born
to women with pregestational obesity.
And as you can see here, it’s not only a problem in the U.S., but also in…throughout
the world and in certain areas of the world, there’s even a greater increase over the
last, you know, 30 years percentage wise of the population.
So this is really a, a global issue.
And you might ask yourself, why is a neuroscientist in my interest in this problem?
Well, of course, the growing prevalence of maternal diet associated obesity does present
a mental health challenge.
And that’s because maternal pregestational obesity predisposes children to neurodevelopmental
disorders, including autism spectrum disorder.
And there’s a growing epidemiological literature that reflects this.
One study here, published in Pediatrics in 2018, shows a significant increase in the
odds ratio for autism spectrum disorder when you’ve got pregestational diabetes mellitus.
And there’s also this link, of course, to obesity.
And what one…one study I really liked in 2019 was published in…in Autism Research,
and this is on the Boston Birth Cohort.
And what this study shows is, it gets even more into…into the weeds looking at how
offspring sex.
So, what they found is that if it was a male child that was born to a woman that was obese
prior to pregnancy, and mom had elevated branched chain amino acids—serum branched chain amino
acids—postpartum, this led to a tenfold increase in odds ratio.
So that means, like, if we’re talking about a one in 59 children in the U.S. having autism,
that’s a risk of one in six kiddos born in that particular parameter.
I think, importantly, what we can see is that this increased risk for adverse neurodevelopmental
outcomes due to maternal overnutrition is recapitulated in preclinical animal models.
And so, Elinor Sullivan up at the OHSU has done a lot of great work in nonhuman primates,
which is shown in one of her recent studies using rhesus macaque.
And what she showed is that when mom is fed a Western-style diet—that’s going to be
about 40% kcals from fat—that does decrease the amount of social interaction that two
stranger interacting juveniles will have.
So, they…they spend a lot less time in proximity to each other—let me see if I can get this…there
we go—less time in proximity to each other.
They’re initiating less contact.
And these animals that were born to moms on a Western-style diet, have more idiosyncratic—so,
abnormal—behaviors in this social interaction paradigm.
So…and this is true not only in nonhuman primates, but also in rodents.
So, this is some of my postdoctoral work, where we looked at the effects of maternal
high-fat diet on offspring outcomes, and we were specifically interested in these neurodevelopmental
autism-like phenotypes in mice.
And so, what we showed was that in…especially in…in male offspring born to a high-fat
diet–fed mother, we’ve got a decreasing amount of time spent in interaction, with
a novel interacting partner in the reciprocal social paradigm, as well as the Crowley three-chamber
test.
And so, what you can see here is when we do a…a familiar interaction, you know, the
animals are much more interested in exploring the box than they are in interacting with
each other.
However, when there’s a stranger, a new interaction partner, we’ve got about 2 out
of 10 minutes spent interacting in this paradigm via a normal…a mouse born to a…a mom on
a regular diet.
But we lose that social novelty preference and interest and interaction, and it really
reduces the overall quality of these interactions, as you can see by a significant reduction
in contact duration.
And so we wanted to go in…and again, we’re interested, really, in the brain and what’s
underlying these phenotypes.
What is the physiological correlate of what we’re seeing here?
And so, there’s…there’s a…basically a social reward circuit in the brain.
This is a mesocorticolimbic dopaminergic reward circuit that, of course, is targeted by addictive
drugs, but really, it’s normally responding to natural stimulating, including social behavior,
even affiliative interactions.
And so we’ve got—coming out of the hypothalamus shown there, light blue with this rodent brain—we’ve
got oxytocinergic output on to VTA dopaminergic neurons that then released dopamine, a salient
signal on the nucleus accumbens that drives action.
And what we found in our maternal high-fat diet offering shown here in the red is that
whereas normally after a stranger action, you’ve got this potentiation of the dopaminergic
response, which you can see in the blue curve, has the larger area under the curve.
You lose that in our maternal high-fat diet mice.
So, these animals cannot interpret social interaction as rewarding.
And so…and so, not only have we found this, others have been looking into the underlying
mechanisms by which maternal high-fat diet can change brain function and behavior.
There’s a lot of great data showing that gut increased circulating cytokines and chemokines
leading to immune activation that drives activation of microglia and all of the synaptic changes
and pruning that can happen there, as well as reduced dopamine signaling similar to what
we found with the, the physiological correlative to reduced social interaction.
And all of these, none of them are mutually exclusive, right?
But so many of them tie into this idea of the dysbiosis, the gut microbiome could be
really driving these deficits in brain function and behavior.
And so, we see this not only in animal models, but really parallel data coming out in the
human literature.
So, when we’ve got a maternal high-fat diet drives the altered firmicutes, the Bacteroidetes,
these are the two most common phyla of gut microbes to each other.
And you see that both in mice and in humans.
We see decrease in short-chain fatty acids, which are metabolites that are exclusively
derived from microbes, microbial... creation of, or short chain fatty acids—excuse
me.
And so, then we’ve also got altered gut microbiome, not only in mom, but also in…in
offspring.
So, in the lab, we primarily focus on neurodevelopment, neurodevelopmental disorders, and one of these
is autism.
We know that it’s highly heritable.
However, even as the genes that we know, including these synaptic proteins, chromatin remodelers
that do predispose individuals to autism.
Those that we know have account for less than 7% of autism cases.
There’s this huge unaccounted for liability.
And this is where our two high…hit hypothesis is coming in, that these environmental factors
could be a tipping point towards phenotype manifestation in genetically predisposed individuals
and autism.
And so, what we see in the lab is maternal nutrition, as well as maternal infection with
live infectious agents.
And we’re also getting into maternal toxin exposure.
But what I’ll tell you about today, and obviously the purpose of this meeting is for
maternal nutrition effects.
And so, previously in the study that we published in 2016, we found…and I…I briefly went
into it.
We’ve got maternal high-fat diet.
It leads the dysbiosis of the gut microbiome of the offspring.
As Carrie was covering, we’ve got this vertical transmission from mom to baby of a dysbiotic
gut microbiome driven by high-fat diet maternal intake.
And now we’ve got a…oh, so…and we have brain…brain and behavioral dysfunction.
However, what we found was that there’s this ninefold loss of a, a probiotic species
called L. reuteri in the gut of these maternal high-fat diet males that, once restored, we
could restore normal brain function and behavior.
So, we had…you know, whereas we had a…a loss of normal social preference, now we can
restore that with lack of Limosilactobacillus reuteri, but it’s not just any Lactobacillus
because L. johnsonii didn’t do the trick, right?
And so, we figured out mechanistically that L. reuteri is working by driving the oxytocinergic
system, and actually, we can not only rescue the behavior, but the underlying physiological
correlates.
You see this elevated VTA dopaminergic plasticity following social interaction with a stranger.
And this is, you know, 24 hours later.
So, it’s…this is a really strong hit.
It’s on the level of what you see with addictive dose of cocaine in these VTA dopaminergic
neurons.
In subsequent studies, we showed that L. reuteri restoration, it’s not…it’s not a one
trick pony; it doesn’t only work in the…in the context of the maternal high-fat diet.
It can actually rescue social deficits in the Shank3B genetic mouse model for autism,
as well as the idiopathic model called the BTBR mice.
And subsequently, we also found that this is true in the Cntnap2 mouse model for, for
autism, where we found that there’s a combination of microbial and host genetics that drives
the overall phenotypic profile of the Cntnap2 knockout.
So, in a recent study out of my lab, we were really…came back to the question of whether
it was the…the state of the juvenile gut microbiome that was driving these behavioral
deficits, or really because these neurodevelopmental disorders, they’re really founded in utero.
Was it moms?
Was it dysbiosis of mom’s gut microbiome that was creating an adverse entry uterine
environment that basically set these F1 offspring off on the adverse neurodevelopmental trajectory?
So, this work that was performed by Claudia Di Gesù, a graduate student in lab who…who
defended last summer, and a current postdoc, Lisa Matz, in the lab.
So again, we wanted to really focus on: How is this diet changing the maternal gut microbiome
and how is that altering fetal neuro…fetal neurodevelopment?
And the idea for the study came from some really strong work out of the Elaine Hsaio’s
lab, showing that there’s differential gene expression in the embryonic brain of animals
that are…whose moms are exposed to antibiotics or in gnotobiotic mice.
And what she pointed out here is this loss of expression of netrin-G1.
This is an…an important gene for axon pathfinding in the brain.
And what…the way you see this manifested here is this is an embryonic brain immunostain
for the internal capsule, which is a bundle of axons that’s important for…it’s
[inaudible] cortical projection important for normal sensory processing.
It’s really thinned out in these animals that were born to an antibiotic-treated dam.
Now, this has repercussions in juvenile behaviors, gut-altered sensory behaviors.
Now, when Elaine and her group reintroduced Clostridium [inaudible] spore forming gut
microbiota that could rescue the thickness of this internal capsule, and it also led
to a rescue of the behavior.
And not only was it these microbiota themselves, but just four microbially derived metabolites
was sufficient to do the same thing.
So, these…these maternal microbes are really critical to early-life neurodevelopment, fetal
neurodevelopment of offspring.
We also were familiar with work from Justin Sonnenburg’s lab at…at Stanford, showing
that normally in…in your typical mouse vivaria, from one generation to the next on a regular,
you know, high-fiber diet, there’s really no change in gut microbiome composition across
generations.
However, when these mice are on a low-fiber diet, you’ve got this progressive loss of
taxa, leading even to extinction of certain species across generations.
And so, we asked, so if it’s really the maternal high-fat diet that’s altering neuro…early-life
neurodevelopment leading this brain and behavioral dysfunction offspring then—and creating
this adverse in utero environment—the best way we could test this was to use the sisters
of these animals, these F1 males that we found.
These were found social deficits in, because we hypothesized that they would have a similar
dysbiotic gut microbiome.
So, follow me here.
This is a, you know, these are 16S ribosomal RNA sequencing data from animals that were
6 months old.
So that’s about 5 months after weaning.
And they’ve got this persistent long-term dysbiosis of the gut microbiomes, similar
to what you’ve been hearing earlier, that these dysbiotic gut microbiomes, they are…that
they endure.
And so, now we took the sisters of these animals, and so they’ve got the F1 females, and at
weaning, we…we weaned everybody onto a regular diet.
So, the only exposure to a high-fat diet was during gestation and lactation.
However, they had this remnant dysbiotic gut microbiota from their mom having had the high-fat
diet.
So, we had the dysbiosis in the maternal gut microbiome, even though mom was on a regular
diet in this case.
We had an altered serum metabolome and immune dysregulation, which basically recapitulated
this adverse in utero environment.
And we hypothesized that that would lead to brain and behavioral dysfunction in F2.
And that’s exactly what we thought.
So, with just grandma on a high-fat diet, we’ve got profound social dysfunction.
These animals…they don’t prefer a mouse interaction with a mouse over an empty cup.
They don’t appreciate social novelty.
There’s no preference for interacting with the new interaction partner versus a…a familiar
one.
We saw that also mildly in the females, but a stronger phenotype in the males, as you
can see shown here, reciprocal social interaction.
We got significant deficit specifically in the males.
And this does reflect the stronger phenotypes in the male human population in the…in the
autism spectrum disorder community.
When we looked at the gut microbiome composition using 16S ribosomal RNA gene sequencing, we
indeed saw the…the same change in dysbiosis of the maternal gut microbiome in these F1
females, just like we saw in the males.
Interestingly, in F2, we began to see a convergence of the gut microbiota.
When we broke this down to the parameters that were driving the…the disparate clusters
on that principal coordinate analysis, what we saw, just like in the males, we saw a significant
decrease in alpha diversity.
So, a loss of species in the maternal gut microbiome and the F1 gut microbiome that
was reflected here in beta diversity.
So, we’ve got changes not only in the total number of species, but what is present.
And many of these are known short-chain fatty acid producers that were lost in our F1 maternal
high-fat diet females.
Interestingly, when we looked at the F2 gut microbiome, there was no change in alpha diversity.
However, we still had altered beta diversity levels.
But again, those short-chain fatty acid producers were…were normalized.
So, we hypothesized.
If now…if we go transgenerational and we breed our F2 animals, right?
These have regular diet, but they’ve got this partially restored maternal gut microbiome,
would that create a more normal and uterine environment leading to neurotypical fetal
brain development that we’d see neurotypical social behavior in F3?
And that’s…that’s precisely what we saw.
We saw normal reciprocal social interaction, normal performance in the three chamber tests
for sociability and preference for social novelty and…and even increased sociability
or preference for social novelty in our females.
Now, this didn’t preclude us from trying to do a rescue of this F2 phenotype with our
L. reuteri, which worked in F1.
And so, what we did was we treated these animals with L. reuteri, right at that weaning period,
where this…there’s this transition of the gut microbiota that Carrie was telling
us about in the last talk.
L. reuteri was likewise efficient to rescue the social deficits that we’ve observed
in our F2 animals.
And really interesting, now we’ve got this increased sociability and preference for social
novelty in our females that was really specific and…and is still puzzling us mechanistically,
but we’re super excited about this response of the female gut microbiome driving female
social behavior.
When we did perform 16S ribosomal RNA gene sequencing, we identified the microbes that
were present in these L. reuteri–treated animals.
We saw now…now we see this significant change in alpha diversity in our downregulation or
L. reuteri–treated maternal high-fat diet animals.
So, what we think this is doing is getting rid of some of these opportunistic pathogens.
So, L. reuteri, I’d like to think of it as kind of like the sheriff in town that comes
and clears out the bad guys.
And what’s interesting to me, you remember initially we had no change in alpha diversity,
and now we’ve got this negative collection; that’s not too surprising, given that L.
reuteri produces reuterin, which is an antimicrobial peptide.
Now, when we break this down by sex, we saw some moderate changes in the…in the male
gut microbiome, but when we looked at literally their sisters—so, these are, you know, male
versus females, they’re coming from the same litters, multiple litters, clearly based
on the numbers that we were showing here—there was very significant response of the female
gut microbiome to L. reuteri.
So, very strong difference in alpha diversity, and about 39 differentially expressed species.
And so, we’re really excited about this idea that you could think of it as a female
vulnerability to modification with a probiotic or an…a therapeutic opportunity.
And so, it…it does make me think that, you know, we’ve talked about methyl donors,
folate metabolism, etc.
So, what prenatal vitamins did in the 1960s to reduce fetal malformations, maybe we’re
on a precipice of realizing prenatal probiotics modifying the maternal gut microbiome to reduce
risk for neurodevelopmental disorders or other inflammation associated disorders that originate
in the field of neurodevelopment.
And I think this can be extended beyond diet, although that’s what we’re clearly focused
on today to maybe reduce risk for other…other causes of these disorders, as well.
So, opportunities.
Thanks in no small part to the advances made possible by the NIH Human Microbiome Project,
I think the cost of metagenomic sequencing is substantially reduced, making this technique
very accessible to many labs that…throughout the globe that previously it wasn’t.
So, now we can—and must, just like Carrie was saying—we must move from a compositional
to a truly functional understanding of how microbial communities contribute to host health.
And we are beginning to harvest the power of microbial genetics as we engineer microbes
for the benefit of host health.
Challenges.
Integrated multi-omics will be key to providing clinical advances toward a risk diagnosis
and therapeutic dietary guidelines and interventions.
So, like…like Carrie was saying earlier, we’ve got to have microbiomes, we’ve got
to have microbiome sequencing, metabolomics, we’ve got to have epigenomics.
And all of this will really inform mechanism, as well as therapeutic pathways.
A good goal would be to harness these integrated multi-omics to yield early life biomarkers
for neurodevelopmental disorders, for minimally invasive specimens, and also to establish
multi-omics as a clinical standard for care of mothers and infants.
So, thank you very much, especially the NICHD for supporting our work and…as well as our
other funders.
DR. THADDEUS T. SCHUG: Okay, thanks to the speakers for keeping mostly on time.
We’re a little bit behind time, mainly because my fumbling through the intro.
So, we’re going to give you a little extra time and reconvened at 3:30.
And I was reminded to inform folks that there’s a store to the left of the cafeteria for coffee
and snacks if you want to refill before the final presentation in the conclusion of the day.
So 3:30, we’ll begin the last presentation of today.
[Room noise]
DR. THADDEUS SCHUG: Okay, we’re going to continue with the final presentation of the day, and for that, we
have Dr. Rick Pilsner from Wayne State University.
DR. RICHARD PILSNER: All right, I’d first like to thank all the organizers for inviting me
here.
Just a…a note that I’m not a nutritionist.
I’m a toxicologist, so hopefully the framework that I’m going to present today can certainly
be applied to nutritional studies and add some value in…in this workshop here.
So, the outline of my talk: We have background, and we’re going to go over windows of susceptibility
in a few slides, epigenetic reprogramming of the male germ cells, and phthalates.
And then, for results, I’m going to present preconception phthalate exposure in mice and
men and something new we’ve been working on…on sperm epigenetic aging or creating
an epigenetic clock specific to sperm.
And then, of course, conclusions and relevance to nutrition.
So, modern toxicology, and when we talk about determinants of the health, we know that it’s…it’s
a combination of genetics (inherited susceptibility) and the environment (the modifiable susceptibility),
and nutrition certainly falls in this category.
The third thing people think less about is time.
So, what is really time?
As a toxicologist, we can think of time as duration of exposure.
This again could sit in a realm of nutrition, of acute versus chronic, but time is also
age and the windows of susceptibility.
What brings all these three factors, the three-way interaction together, at least in my view,
is epigenetics.
All right, so we’ve had an introduction of DOHaD in the morning, so won’t really
go into that, but it really, in the context of DOHaD, how do we define development, right?
And most of the people have been looking in utero effects, but there’s also different
windows of susceptibility across the life course, obviously, during pregnancy, early
childhood, puberty, and menopause.
But one thing people have really not considered is the preconception period, and that’s
when germ cells mature, and in males, that is spermatogenesis.
So, just want to go through some of the reprogramming steps in male germ cells.
The first on the very left there is in utero reprogramming.
This is the primordial germ cell specification, where you see the really large wave of epigenetic
reprogramming.
And this is where a lot of the nutritional and especially a lot of the tox studies kind
of focus on this window.
What’s less appreciated is the preconception period, or during spermatogenesis, sperm takes
about, in humans, about 74 days to fully mature; in mice, about 35 days.
And they have, really, three distinct stages of windows of susceptibility.
The first one is the DNA methylation patterns are…are finally set.
They’re…the final methylation patterns are acquired.
The next is a histone protamine exchange.
So, the sperm head has to be condensed, so 80–90% of all the histones are replaced
by very basic protamines that cause a condensation of the nuclear head.
So, it’s been thought that the remaining nucleosomes are associated with genes that
are important for early life development, as well.
Once sperm leaves the testes, they’re not really fully capable of fertilization.
They go through a 2-week migration through the epididymis.
And here, there’s a lot of exchange, or interaction with extracellular vesicles, or
EVs.
And there’s an exchange of cargo…small non-coding RNA…protein and everything.
So, there’s a way that, during this last 2 weeks, the sperm still can respond to the
current environment.
So, when I started this preconception research line, you know, writing grants, I got killed
in the beginning, right?
Because they’re like, “Why do you care about methylation in sperm?"
"Because we know right after fertilization, everything’s reprogrammed, as well.”
Right?
So we know that imprinted genes…escape the reprogramming event, as well as some repetitive
sequences, but we kind of have to revisit this dogma, and what we’re looking for is
looking at these escapees, these DNA methylation or epigenetic, program, escapees whereby the
environmental cues that are in sperm can be directly transferred into the offspring.
So, some examples of what we’ve been doing.
Our hypothesis was that sperm DNA methylation mediates the effects of male preconception
phthalate exposure on early-life development.
So, we have the exposure on early-life development, and that’s potentially mediated by sperm
methylation.
So, phthalates is my toxic of choice, largely due to Thad here, as well, telling me that
it would be a good exposure to look at Phthalates are everywhere.
I’m sure most people are familiar with phthalates who love the new car smell.
Most people do.
They don’t realize that’s actually off-gassing of a lot of the phthalates.
So, open up your windows when you have a new car for a couple of weeks is always a good
thing.
But a lot of people get exposed to high molecular weight phthalates through more processed foods,
so nutrition kind of ties in with phthalates, as well.
What you see on the right is a list of the phthalates that we actually measure, and what’s
in red are the anti-androgenic phthalates.
So, most of these are anti-androgenic, and that could have consequences in…when we
talk about male reproductive health.
So, the next slide is a really nice review.
Dr. Joe Braun was one of the co-authors on this, looking at the evidence of phthalate
exposure on male reproductive effects.
And if we look at preconception or the adult, again, these anti-androgenic phthalates are
really associated with decline in semen parameters, affects the time to pregnancy, as well as
effects in testosterone, as well.
So, for our clinical study, this is out of Baystate Medical study—excuse me, Medical
Center—in Springfield, Massachusetts.
We called it the Sperm Environmental Epigenes and Development Study, or SEEDS.
Every good cohort has to have a good acronym, and it took me a while to come up with this
one.
But what we ended up doing is measuring phthalates in both males and females and looking at embryo
development, and what we found is with blastocyst quality, we found that DEHP, DBP, BBzP, and
DINCH metabolites, which is a replacement for DEHP. They were all associated with diminished
blastocyst quality.
We next looked at these specific metabolites on sperm DNA methylation, and we found 128
sperm DMRs are differentially methylated regions; 100 of these came from DEHP metabolites itself.
And finally, we then looked at those sperm methylation sites associated with diminished
blastocyst quality, and we found 17 DMRs out of those 128 that were also associated with
blastocyst quality.
And when we ran oncology on these, we actually came up with hits for in utero…embryonic
development.
So, given that we were…that most of our hits were from DEHP, we were motivated to
replicate these results in more animal models.
Epidemiologists have a hard time sometimes with a lot of cofounding, and a lot of the
basic scientists want to really have a more of a defined exposure and controlled experiment,
so we did that, with male mice.
We exposed male mice to 2.5 or 25 milligrams per kilogram per day of DEHP.
Just a note that the NOAEL for DEHP is…is 4.8, so our low dose is actually below the…the
allowable, exposure regimen.
We collected sperm from the F0, as well as the embryonic and extra-embryonic tissue from
F1 mice.
Looking at the effect of male preconception DEHP exposure on the sperm methylome, this
is via RRBS, or reduced restriction bisulfite sequencing.
We saw a whole host of changes, about 400 to 500 DMRs.
There was only 20% overlap with the high and low exposure, and we had a whole host of different
pathways.
Nothing terribly specific that we were hoping to see, but what was really fascinating is
what we…what we observed in the F1 tissue.
And so, in the embryonic tissue, we actually saw a doubling of the amount of DMRs that
we found.
So instead of 400, we saw 800, 900, and what’s even more interesting in the extra-embryonic
tissue, which will eventually make up the placenta, we saw three- to fourfold increase
in the amount of methylation changes.
And when we look at oncology in these, what we see is most everything pointing to developmental
terms.
So, this is pretty interesting.
We also ran RNAseq on the same tissues, and we found a lot of differential expressions.
Again, the extra-embryonic seemed like it was pushing a little bit higher, but there
was a lot of changes in key developmental genes.
Hox genes, GATA genes, other things.
So, not surprisingly, embryonic development and pattern specification, all these things
popped up.
So, I know that was a lot of different slides.
This is kind of a summary of our animal model, and what we found is around 400 DMRs, depending
on the dose of the F0 sperm, and then this amplified effect of DMRs.
So, we had a two- to fourfold increase in the conceptus epigenome changes.
These were also associated with changes in gene expression and gene…developmental genes.
Again, the Hox family, Sox family, GATA family, CDX1, and this all resulted in pattern specification,
embryo development, key terms.
So again, the animal model kind of corroborated our findings in the human studies, which is
nice for an epidemiologist to kind of say, “Hey, this is…this is…these two are
kind of working in sync.”
What I want to do next is introduce something that we’ve published on—and we’re re-inventing
this, where we have a version 2 coming out—is looking at sperm epigenetic aging.
A few people have commented on advanced paternal age, but the question is: How do…how should
we define aging or age, right?
Most people use chronological age.
You know, is that really a good measure of age or the aging process?
Not really, because it does not capture the intrinsic or extrinsic factors that impact
the rate of aging.
So, a great quote from my childhood hero, Indiana Jones—I don’t know if anyone’s
seen the recent movie, but I just saw it with my kids, and it was…it was a good time.
“It’s not the years, it’s the mileage,” right?
So, all…all the people you went in high school with, you’re the same age, right?
But everyone’s aging at a different rate, depending on what they’re exposed to, their
nutrition, exercise, all of these things.
So, if we could capture, an…an estimate of the biological age of…of sperm, that
would be really cool.
And that’s what we did.
We know that there’s…there’s two different kind of ideas of…of sperm methylation changes.
We went over epigenetic reprogramming, but there’s also something called epigenetic
drift, that as you age, you get this accumulation of epigenetic errors that you could kind of
leverage to build biological clocks.
So, biological clocks are not new.
Horvath and Hannum have developed really great clocks for somatic tissue.
They…they predict cancer, chronic diseases, mortality, but they are…they have no predictive
value of sperm, so we went ahead and built our own sperm epigenetic clock.
And this is, kind of, just a schematic of kind of a life course approach, that you could
have this normal epigenetic drift, but there’s this epigenetic drift acceleration, depending
on your lifetime exposures.
And we consider that sperm epigenetic age.
So, constructing the clock, we used the LIFE study.
This was an NICHD, study done 10–20 years ago.
It was a prospective cohort of couples, 501, from the general population.
The group stopped contraception and were trying to actively become pregnant.
We had 379 semen samples from that.
We used the EPIC array, which is 850,000.
Male age was, between 19 and 50 years old, and we used machine learning…we won’t
get into that…to, kind of, gather the methylation sites that best predict age.
So again, after pre-processing, we were left with 800,000 CpG sites.
When we ran associations with chronological age, we had 85,000 that were, FDR significant.
If we use a more stringent cutoff, we had 22,000 CpGs that were associated with chronological
age.
Using machine learning, it came down to 120 CpGs out of the 800,000 that predicted male
age, and what we did is we used the regressions, the residuals from linear regression between
chronological age and predicted age to kind of get an understanding of the rate of aging
of sperm.
So, if your dot was above this line, that means you have accelerated aging of sperm.
There’s a mismatch of that.
All right, so, looking at kind of the metrics on this, we had very low error.
We had a correlation of 0.91 with an MAE of 1.6 years.
This is within the LIFE cohort.
We validated this in a clinical IVF cohort, SEEDS.
It still performs really well.
So, our first thing is we looked at sperm epigenetic age and time of pregnancy using
Cox proportional hazard model, we found a highly significant effect of sperm epigenetic
age on couples’ time-to-pregnancy, so lower FOR, or fecundability odds ratios, equals
longer time to pregnancy.
This is seen in this graph where we have tertiles with different sperm epigenetic age of being
young, equivalent, or older, and you can see a clear dose response at 3 months, 6 months,
and 12 months of trying to achieve pregnancy.
So, looking at pregnancy outcomes, we didn’t see anything with birth weight, length, head
circumference, but we did find a significant association with gestational age, such that
there was a 2-day shorter gestational age for every year increase in sperm epigenetic
age, as well.
So, this is kind of interesting, that can sperm really impact, kind of, the timing of
delivery.
We’re still trying to sort that one out.
So, the final thing I’m…I’m leaving you with is, so now that we know that sperm
epigenetic age is associated with time to pregnancy, what could be some of the environmental
factors that are associated with that?
We found that current smoking—those men who have…are current smokers—have a higher
sperm epigenetic age.
Not surprising.
We also found a host of phthalates that showed higher…those men exposed to higher levels
of phthalates had an increased sperm epigenetic age, as well.
Some more of the modern modeling.
We looked at mixture, so instead of analyzing one phthalate at a time, we did a mixture
analysis, which kind of combines all the phthalates, and we do see this clear dose response curve,
and this is validated with another method, quantile G-comp analysis.
So overall conclusions.
Sperm…male preconception exposures to anti-androgenic phthalates influence sperm epigenetics, early-life
development, and sperm epigenetic age.
Timing is really important.
The preconception period is a susceptible window of germ cell development.
Men have an environmental responsibility, too, and that starts at least 3 months prior
to conception, okay?
So, we know sperm epigenesis is 74 days, so at least 3 days—excuse me, 3 months—before
conception.
And irrespective of your chronological age, the biological age of your sperm can impact
pregnancy success.
So just two…two slides left.
These are some of the papers that I kind of captured that I thought were interesting,
in terms of diet.
There some studies looking at sugar intake here, showing methylation changes, as well
as small, non-coding RNA changes.
I think we had a question from the audience or online about bariatric surgery, and that
actually changes sperm epigenetic profiles, both in methylome and small, non-coding RNA,
so that’s really interesting.
There was another NICHD study, the FAZST trial.
Here, they looked at methylation, but as a group.
Over 6 months, there was no significant group changes in sperm methylation from folic acid
or zinc.
And then there’s…Jacquetta Trasler’s group found here that…they actually did
find some changes in in sperm DNA methylation with folate exposure.
All right, so future needs.
What is the biggest challenge?
Well, we know it takes two to tango.
So, most birth cohort studies ignore the paternal contribution; we need to change that.
I’m very glad that ECHO 2 is doing the preconception cohort.
I’m now going to be part of the ECHO 2 through Michigan State University, looking at…focusing
on the preconception period, but also, not even just the data, but we need to collect
the biologicals, really, to get this going.
Promising scientific opportunities.
The short term is, I think we could leverage these biological clocks—not just mine, but
there’s a lot, the Hannum and Horvath clocks to look at biological aging.
And then the long term, I think, is…is—we’ve heard this a few times now in the past hour
or two—is the integration of the -omic data through machine learning.
We kind of have to put all this together, and hopefully…and at least what we’re
trying to do is kind of derive risk scores, so we’re working on something of a fecundity
risk score, using all of the -omics we have with sperm at this point.
So with that, I just want to thank our lab folks, and I won’t take questions, but we’ll
do group questions, right?
All right, thank you.
DR.
THADDEUS SCHUG: Hey, can we have the speakers from the session back up front here?
And Dr. Nadeau had to go to a prior commitment, so we’ll be missing her for the question-and-answer
session.
So, please, for folks who are attending the work…the workshop online, ask your questions
in the Q&A box, and we’ll take questions from participants in the room, as well.
UNIDENTIFIED SPEAKER: So, while we wait, it looks like we do have a question already in
the queue from the Q&A, and this is actually, I believe, for Dr. Friedman.
Dr. Friedman, have you done analyses of fatty livers in childhood NASH, specifically linoleic acid? Thanks.
DR. JACOB FRIEDMAN: So, the lipidomic changes that have been done in most of the nutritional
studies have proven that cholesterol pathway and the ceramide pathway are the major drivers
of the fatty liver disease, at least correlatively.
Knocking out some of these genes have actually been helpful in established causes of…established
models of disease.
And so, the toxic lipids that I discussed really revolve around ceramides, [inaudible]
ceramides, and the cholesterol pathway that seems to be driving the inflammation.
That is one of the key steps to moving from simple steatosis to steatohepatitis, if that’s helpful.
UNIDENTIFIED SPEAKER: Okay. Great, thank you.
Any questions in the audience?
Yes.
DR. JOE BRAUN: This…okay. You guys can hear me. Joe Braun, Brown University.
So, these were great talks, and I heard a lot about, you know, using innovative technologies
and tools.
A lot of calls for, you know, more -omics, more -omes, right?
And I think, you know, that’s warranted, but one thing I think that we do is we get
really excited about these things and tend to forget about some of the basic, describing
characteristics of them—the validity, reliability, reproducibility.
I think about this with microbiome, right?
Like, you know, the question of whether the fetus is born with a microbiome, you know,
and doing really well-controlled studies to see, is it contamination?
Or, if we’re talking about metabolomes, does it vary with within a day…day to day, etc.?
And just sort of what your thoughts are and all of you having used these technologies,
you know, and particularly, you know, from the scale of, you know, rodents to humans,
and even single cells, like sperm, you know, what…what do you think we need to do for
that, particularly when we’re talking about, you know, going to a multi-omics-type platform?
DR. SHELLY BUFFINGTON: Hi, this is Shelly.
Yeah, so, I was one of the ones that was saying, yeah, multi-omics for sure.
And so, I think we’re really driving home the idea that, you know, composition can be
variable, even between you and I, but, you know, say we’re both healthy.
[inaudible] the functional output of these microbes and how they affect host physiology
is what’s important.
So even if there is, like, diurnal flux or etc., what is the actual function?
So, that’s where I think, you know, with the microbial genetics combined with metabolomics
is actually really informative as far as, like, therapeutics.
And so…so, that’s why I think it’s really important to keep doing it, you know, at least
at the level of, like, genetic investigation, you know, whole genome, you know, shotgun
sequencing, as opposed to just, you know, 16S, which will give you true, you know, metabolomic
information, rather than just predictive metabolomics.
So, I think that really would be quite valuable.
And, you know, day-to-day variation, yes, it’s there.
I think that, you know, longitudinal studies are going to be really important, you know,
especially in humans, but certainly in mouse models.
And so, often, we only pick one or two, you know, time points, and that isn’t really
the full picture.
So, I think that you’re making an excellent point.
DR. JACOB FRIEDMAN: I’ll just jump in and say from my point of view—this is Dr. Friedman. The
multi-omics pathways are going to lead, I think, ultimately to algorithms and machine
learning.
And just like when you do a diagnosis and you take a medical record, you know, if you
can plug in enough data points, you can…begin to identify what we…really are biomarkers.
So, we’re not talking about causative effects here; we’re mostly talking about correlative
effects.
But it’s going to require large sample sizes.
And I think the last speaker who gave the caveats of when a sample is taken, what the
diet was of the person who gave the sample, and what their genetic background is all kind
of figure into whether these things can actually be made better as predictors.
And I think that’s going to happen eventually because that’s the way that the field is going.
UNIDENTIFIED SPEAKER: I wanted to comment, as well.
I really liked your question, and I really liked your answer, Shelly.
And I think it’s…the way that I think about it, personally, is that these are tools,
and they’re really powerful tools, and they can point us in a direction, but we still
have to do the experiments to test them.
So, we need both approaches, right?
And we can’t just do one or the other.
And that takes all kinds of scientists and all kinds of models, and so…but yeah, I
think you can get sort of caught up in the excitement of -omics and maybe not do sort
of all the due diligence that you need to really show…those effects experimentally,
and I think that part is really important.
And that’s…when we talk about, like, getting to mechanisms, like, that’s kind of we’re…what
we’re talking about.
AUDIENCE MEMBER: And since the microphone is on, I’ll just…just to add, just…despite
all the wonders of -omics, I think in addition to really being aware, as Rick was pointing
out, about really thinking the periods that we’re…to be certain that we’re capturing
the right timing in terms of what’s the vulnerable period, but the other thing is
to make certain…all of these -omics require the samples, and certainly for the humans,
and the relative invasiveness of being able to collect the samples at the right times.
And so, we also have to be mindful that we’re really capturing the populations that are
at greatest risk.
And so, I just want to put out that, in addition, there’s the complexities of…through the
different development stages, the challenges of capturing the samples and…and of being
inclusive in…in terms of the populations that we sample.
UNIDENTIFIED SPEAKER: Okay. We have one question coming in virtually, and then we’ll go back to the…sorry, and
then we’ll go back to the live.
What is the strength of evidence that these methylation changes are positive?
So many sites and the genes themselves can be silenced or turned on based on methylation.
Is it possible that RNA gene expression is more meaningful?
DR. RICHARD PILSNER: Hi, there. Richard Pilsner here.
Yes, there’s…you know, in the study I presented in mouse, there was only about 10%
of the sperm DMRs that were kind of consistent with…from F0 sperm to F1.
We did see this amplified effect, but in only 10% of those DMRs and sperm overlaps with
the embryonic tissue.
So, certainly the methylation, I think, is one piece of the puzzle, but it’s not explaining
everything.
We have a series of publications we just…just published looking at small non-coding RNA
in seminal plasma—extracellular vesicles, too—and how that’s related to live birth,
phthalate exposure, as well as semen parameters.
So, you know, methylation is just one aspect of kind of this epigenetic transfer of information.
DR. JACOB FRIEDMAN: I asked the question.
I guess I want to follow up.
And that is, if people ask you, “Prove that methylation matters,” what do you tell them?
How do you…how do you actually…you got a mouse.
You got these sites, and they’re all over the genome, and far away from the genes, often.
And I…that’s my problem.
I’m trying to figure out, like, what would you do to actually prove that 10 or 12 of
these sites are causing disease?
That’s what I want to know.
DR. RICHARD PILSNER: A hard question from one of the speakers, I tell you.
You’re supposed to be kind to people like me here.
DR. JACOB FRIEDMAN: No softballs here.
I’m…I don’t even…I’m not there, so I can ask a tough one. Sorry.
DR. RICHARD PILSNER: Yeah. Right, right, yeah. No, that…it’s a…it’s a very good point.
I mean, what we have is…you know, we could validate, you know, the findings from the
-omic approaches using targeted, you know, pyrosequencing, techniques like that, but,
you know, if you’re thinking actual causative, you know, I guess a CRISPR approach, removing
those methylation marks may be a good way to go.
But, you know, I would ask that same question back to people that do small, non-coding RNA
and, you know…you know, there’s been a big push to get away from sperm methylation
and saying that sperm RNA is more important or have a big effect.
I think Dr. Rando commented that there’s so little sperm in—or RNA in sperm—that
does it really have an effect on the oocyte?
So, that’s what I have.
AUDIENCE MEMBER: If I can just sort of stand up for small RNAs, because…I mean, I don’t
think anyone knows how any paternal effect works, so I don’t mean to say this like
small RNAs aren’t the answer, but what…part of the reason they’re quite popular in the
paternal effects field is that they’re much easier to test sufficiency than for methylation
or chromatin changes because you can microinject RNAs into a zygote, whereas in order to do
methylation stuff, you’d have to do a dCas9, you know, like, basically make a transgenic
to install methylation or a chromatin change in sperm, so testing…and many people have
injected small RNAs and seen outcomes in the offspring, so that’s…whether or not people
are injecting a physiologically relevant amount—these are the kinds of questions that one could
raise against small RNAs, but at least they sort of structurally have built in an advantage
that it is very easy to test sufficiency.
And in many cases, they have been shown to induce phenotypes.
Now, reconciling that with the amount of RNA sperm carry and so on and so forth is a whole
other question, but anyway, those…but with your kind of stuff, it’s like…it can be
very hard to induce just one methylation change in sperm, much less, like, 50 if you think
it’s not, like, a mono-epigenetic effect.
DR. RICHARD PILSNER: Right.
And just to add on that…it’s “one takes all” for sperm.
Dr. Rando mentioned it’s a digital thing, so for methylation, it’s just…it’s either
on or off, but one sperm, too—they have very little.
The concentrations of the small, non-coding RNA is super small. So, yes, future research, for sure.
DR. JACOB FRIEDMAN: Love the concept of epigenetic aging. I just want to know, is that my true age?
You know.
How do you use those information, you know?
It’s…those clocks are really fascinating, but not sure how to use it yet.
DR. THADDEUS SCHUG: Dr. Barker?
DR. MARY BARKER: Thank you. I have absolutely nothing to contribute to that conversation at all—I’m a psychologist.
I don’t know about this stuff—except to say that the science that you guys have spoken
about just completely blows my mind.
It is amazing what you’re doing and fantastic even just to sit and listen and understand
the edges of it.
But all the time I’m listening, I’m thinking from, obviously, from my perspective, and
I guess from the wider perspective of maybe [inaudible] perspective, too, which is about:
So, where’s the benefit?
Where’s the human benefit from this?
What are we going to do?
Everything you’re doing is predicated on the basis that nutrition needs sorting out,
but undernutrition, overnutrition are having terrible consequences for poor populations.
Where do…how much time do you guys spend thinking about where this goes, this work,
or what we can do with it?
That’s a genuine question. It’s not a challenge. It’s a question.
DR. JACOB FRIEDMAN: I will jump in, just because I also work on the social determinants of
health in Native Nations in Oklahoma.
And as I tell people, you know, I can do the biology, but I don’t solve the sociology
of what prevents prenatal care; what prevents proper nutrition in a food swamp, as they
call it; and what affects families, the social dynamics of eating.
There are many things on the psychological side that I think if we…we spend all our
time doing biology, we’re going to…we’re still going to be, at the end of the day,
trying to figure out, like you say, the low-resource persons.
The people who really need our help, they have other problems that go along with their
biology, so in answer to your question, that’s probably another symposium, but there are
both sides to this phenomenon that we’re doing. Just my opinion.
DR. THADDEUS SCHUG: [inaudible]
AUDIENCE MEMBER: Yes. Great set of speakers. I have some…well, one question, really.
You know, in addition to the issue of reproducibility, validity, timing of exposure, and so forth,
as I listen to all the speakers, you know, I feel that there is a kind of a crosstalk
among the different topics.
So, my question relates to, you know, considerations regarding obesity; adiposity; folic acid;
folates, which are different; and the microbiome—because if you think about it…and I’m asking this
because I think different individuals and different researchers have a different perspective.
So, if you think about obesity—and I didn’t hear this mentioned at all—and the folate
metabolic pathway, for example, obesity has an independent effect on folates, and we have
done this in controlled feeding studies.
And this has been established, it is reproducible, it is found in the literature.
The microbiome has a connection with folate because the bacteria needed to create nucleotides
for DNA synthesis and to produce short-chain fatty acids.
Similarly, you can think about the, you know…you want to go to the genetic aspect and things
like that, so I’m just thinking in terms of the studies, like, obesity is very important.
We are seeing that.
Should we be thinking about…when we are looking about the generational effects of
nutrition, should we be saying, “Okay, we need ideas or account for differences in body
size?”
That’s one.
I’ll leave it at that, but I have more.
DR. SHELLY BUFFINGTON: So, in our mouse work, you know, the first study that we published
in 2016, we did, like, an 8-week exposure for a high-fat diet.
We saw a significant fat mass added to the dams, but we actually cut that down for the…to
4 weeks’ exposure, and we saw the same phenotypes, the F1 after 4 weeks of diet, but that did
not make the dams obese, and so we’re really trying to start to separate this dietary exposure
driving the dysbiosis of the gut microbiome from the obesity effects in our animals.
And I haven’t done the study yet, but I think probably just a week of a high-fat diet
exposure would be sufficient to drive the phenotypes that we see, and that’s, you
know, because we start to…that’s when the microbiome starts to really shift.
We don’t see phylum-level changes until about 4 weeks on diet, but I’m really interested
in that question: Can we separate the two?
And kind of like what you’re getting into, and so that also has therapeutic implications,
right?
Because basically, that means, like, even if a woman’s not obese, there could be dietary
changes that we’re all interested in here that could be still targeted, and so we can’t
exclusively select patients to be within a, you know, a therapeutic group just based on BMI, right?
AUDIENCE MEMBER: Just to follow up on that, I’ll say we see something sort of similar
in our undernutrition studies where, you know, 4 weeks of an undernourished diet is not enough
to really change, like, parental body size, and so we really don’t actually see the
effects until the F1 generation.
And even in conventional mice, when we give the malnourished diet, we actually don’t
see a significant difference in body size over about a 3-week treatment.
But interestingly, if we disrupt the microbiota with antibiotics, then we do.
So, I think, you know, it’s complicated teasing all of those things apart.
So, there does seem to be something that is really dependent on having that maternal environment
that is, I think, separable from just sort of body size, at least in these sort of controlled experiments that we’re doing.
DR. THADDEUS SCHUG: Thank you.
DR. JACOB FRIEDMAN: Yeah, you can…you can also say that the AMA has just released a statement
about BMI, just so y’all know, that when you’re considering a history of physical…of
a patient, the BMI is now no longer considered to be the accurate description of their obesity
status.
You know, there’s the waist circumference, there’s the comorbidities, but BMI really,
in my estimation, is just gravity.
It’s just…that’s all it is because we’re…we all respond different to diet, and your BMI
could be a lot healthier than mine at the same number.
So, I think the BMI problem is…is one of just convenience, but I think it, you know,
it’s being recognized, as I said, by, you know, medical societies as not the driver
of the problem.
These big obesity drugs, that’s a good example of which…you know, you’re not going to
give it to somebody who has a healthy metabolic state, who maybe has a BMI of 30.
You’re going to look to see: Do they have hypertension?
Are they hyperlipidemic, and what is their diabetes status?
And then you’re going to challenge the weight.
And we may need to do the same thing in pregnancy at the end of the day.
A person comes to the…the clinic the first time, they didn’t plan to be pregnant, but
they are pregnant, and you look at them, and you say, “You know, what are your risk factors
for an unhealthy pregnancy?”
And your BMI is just one, but then when you have to think about what comes along with
that BMI, that’s the more important thing I think we need to start focusing on is, you
know, it’s not the BMI, per se.
It’s…you know, doing clinical research, that’s what we bump into all the time.
You do a fat biopsy of these women at the same BMI, the gene expression pattern could be totally different, for example.
AUDIENCE MEMBER: So, thank you. I learned a lot, and I have one very specific question probably for Richard.
Interesting, you know, in a reproductive biology perspective, you think it’s just one sperm
that gets the job done, but we know that’s not true.
So, I was just curious: How do you factor in that, you know, the old-fashioned thing
for people who can’t conceive is looking at sperm count, but does that matter?
In other words, the milieu in which fertilization occurs, would that be another factor?
Am I making sense?
So you know, it’s…the genetic package is in that one sperm that makes it through,
but then there’s all the other stuff around.
Has that been looked at?
DR. RICHARD PILSNER: Right, and that’s where we’re doing a lot of…we just finished
a metabolomic study in seminal plasma, too, so we’re looking at a lot of different factors
outside of the sperm.
So, it may not, as you said, just be the genetic or epigenetic material that’s in the sperm,
but it could be kind of its nourishment after ejaculation or during the migration through
the epididymis, so there could be a lot of factors that are kind of pushing the sperm
in one direction or in the other in terms of sperm fitness.
AUDIENCE MEMBER: And the other…I think Dr. Friedman commented, and I did want to add to that concern.
Like, well…how…what can we do better?
And then we know enough.
BMI has been useful.
It’s a good public health tool, but we know it’s not the best measure of body composition
or metabolic health, and so we have terms of metabolically healthy, obese, unhealthy,
so I think…I didn’t hear that, but from the thinking of biomarkers of metabolic health
or nutrition, etc., that’s…I didn’t see that, and how do we integrate that?
And a question for those of you from the sort of environmental health or even the micro…you
know, the microbiome, etc.: Would love to get your thoughts, but we talk about nutrition—and
as a nutritionist, myself included—as this micronutrient and as this micronutrient, etc.,
but people eat food; they don’t eat nutrients.
And food is a vehicle for so many other things, and how does…how could we do that integral,
so to speak, that it’s also delivering?
I mean, the additives of the high-fructose corn syrup and the list goes on, right?
So, just wanted to hear your thoughts and your evaluating these other factors.
How does one capture that?
UNIDIENTIFIED SPEAKER: I’m not going to answer your question, but just to add to it
to say…just to say that…that I think it’s…just one additional element that I think we need
to grapple with is not just the inadequacy of BMI, but also thinking of all the different
developmental stages that we’re thinking about as we’re thinking about children or
life course, in general.
Just, you know, Z scores doesn’t actually either really get around the issue of just
what should we be considering as we’re…if we’re thinking about anthropometry or an
alternative to it for children beyond head circumference and waist circumference.
But, you know…but we need to somehow consider variability in shape and…and we need better
tools for that, so I can’t answer it, but just wanted to ask.
DR. THADDEUS SCHUG: Okay, we’ll do one final question here and then wrap things up so you
can catch the bus back to the hotel.
AUDIENCE MEMBER: Hi, so, thank you all for the wonderful presentations and very…I enjoyed it.
My question is for Dr. Pilsner.
I’m thinking about the…your findings where you said sperm can impact the timing of delivery,
and from a public health implications perspective, I was also wondering if your clock…if you
see your work moving towards loose guidelines for conception because right now, as we know,
a lot of the recommendations are very…have a very heavy emphasis on maternal age.
And so, you know, I see your clock kind of playing a role in guiding fathers, as well.
DR. RICHARD PILSNER: Yeah. Thank you for that question.
Yeah, we’re really hoping that our clock will inform clinical decisions in terms of
if we could potentially get people…couples to submit a semen sample, and we could develop
a fecundity risk score to kind of estimate the probability of them getting pregnant,
say, within 12 months, which is, you know, the cutoff for clinical infertility.
You know, so that could kind of move people into infertility treatment, IVF, sooner than
waiting, you know, a year or year and a half of trying to naturally become pregnant.
So, that’s kind of our hope.
DR. THADDEUS SCHUG: Okay, so thank you to the…let’s give a round of thanks to the speakers here,
and hand it back over to Krista and Ashley to close things out.
DR. ASHLEY VARGAS: Great, so I will be brief.
I think today we have seen the complexity of an area of research where we haven’t
really seen so many disciplines and people brought together in one room to talk about,
so I think today was maybe a little jarring, seeing the different topics that we’ve covered
today.
And that was intentional because we wanted to bring you guys in this room to get all
you great minds together—and virtually, I know some of you aren’t in the physical
room—to really think about how we can move this field forward as a group, and maybe in
different directions, but as a group to think together about the complexity of this problem.
And so, I don’t know if I can get up our figure slide.
Oh, it’s coming.
Okay.
Hopefully it’ll come next…that I showed you guys at the beginning.
I just want to reorient ourselves to that figure slide.
Thank you.
And so today, we learned just about individual-level factors, if you can believe it or not.
Tomorrow, we are going to learn about all the things down here that are different colors
besides red, and we’re going to learn about not only the individual-level factors, but
also the family- and societal-level factors that contribute to nutrition’s effects across
generations.
And so, just to go back to that idea of nutrition being a thread, and we’re building…I think
someone showed a quilt today, which I thought was beautiful.
Sonia, I think, showed a quilt today.
So, we’re building the quilt of families and, really, generations of families.
And so, we know it’s complex.
We thank you for hanging in there, and we thank you for asking the tough questions.
And tomorrow, I really want you guys to think about how we can move this field forward,
now knowing at least a big piece of the complexity.
Tomorrow we’re going to add more, but we’ve really dabbled across so many different disciplines.
We’ve used so many different model systems today: humans, primates, computerized models,
mice.
We’ve talked about so many different disciplines, and that’s what it’s going to take to
move this field forward.
So, I want to thank all of our speakers today.
I’m going to end with some…just Cliffs Notes on what’s going to happen next.
So, for those of you that are going back to the hotel for the small get-together, the
shuttle will be at the Gateway Center at 5 p.m.
You have a little bit of time to walk there, but not a huge amount.
And we will start tomorrow morning at 10 a.m. as planned.
So, I believe the shuttle will be picking you guys up in the hotel at 9 or 9:30.
We will get clarity on that…9; 9:00 tomorrow morning to make sure you guys are here in
enough time to get some coffee and join us for tomorrow’s session.
All right, thank you so much. We’ll close for today.