Microbiome Insights is proud to be the sponsor of season 3 of Ruairi Robertson's Biomes Podcast.
In this third episode, host Ruairi Robertson speaks with Meghan Azad, a professor at the University of Manitoba and a research scientist at the Children's Hospital Research Institute of Manitoba. Meghan delves into her journey into microbiome research, focusing on the impact of breastfeeding on the infant microbiome and the unique components of breast milk that support infant health.
Meghan explains that breast milk is more than just a source of nutrition; it plays a critical role in developing an infant's gut microbiome. She highlights human milk oligosaccharides (HMOs), which are not digested by the baby but feed beneficial gut bacteria. Meghan discusses the genetic and environmental factors influencing HMO composition in breast milk and the potential of adding HMOs to infant formulas.
The conversation also explores antibodies in breast milk, which protect infants from infections and help shape their immune systems. Meghan emphasizes that breast milk composition changes not only over the course of lactation but also within a single day. She emphasizes the importance of promoting breastfeeding while recognizing the need to improve infant formulas for those who cannot breastfeed.
The episode concludes with Meghan discussing her ongoing research projects, including the International Milk Composition Consortium. This project studies milk from diverse populations to understand its impact on infants growth and health. Meghan's work continues to provide valuable insights into the complexities of breast milk and its essential role in early childhood development.
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Ruairi Robertson (Host): Great, so thank you, Meghan, for agreeing to chat today. It's great to have you talking all about breast milk and the infant microbiome, as well as breast milk microbiomes. So maybe we can just start off by you giving us a little insight into who you are and how you got into microbiome research and what it is that you work on today.
Meghan Azad: Sure, well, thanks for having me. This is my favorite thing to talk about, so it's a pleasure. I am a professor at the University of Manitoba in the Department of Pediatrics and Child Health. I'm also a research scientist at the Children's Hospital Research Institute of Manitoba. I co-founded and direct the Manitoba Interdisciplinary Lactation Center, or MILK with a C for short. I lead a bunch of projects related strongly to infant nutrition and microbiome development. I have a Canada research chair in early nutrition and the developmental origins of health and disease.
What brought me here—it's hard to believe how time flies, but I just realized it's been 10 years since I started my lab. We’re planning a little 10-year party in a couple of months. When I started my lab, it was with the intention of understanding how breastfeeding shapes the microbiome. I was interested in that because I did my postdoc with Anita Kaczerski at the University of Alberta. My project there was about the infant microbiome at a time when, thinking back a decade or more, this was a pretty new area of science.
In Canada, there had just been a call from the government to submit applications for team grants in microbiome research. Anita, my advisor, had put in one of those grants and been awarded it. The premise of her grant was to understand how the infant microbiome plays a role in the development or prevention of allergies and asthma in children. It was a bit of a piggyback onto a study that was just getting started in Canada called the Child Cohort, the Canadian Healthy Infant Longitudinal Development Study, or CHILD.
That study was formulated with the idea of understanding why so many more kids in today's generation have allergies and asthma compared to even a few generations ago, with the recognition that genetics are important, but they obviously aren't the whole picture because our genetics haven't changed in a couple of generations. That study was formed to take a really big look at genetics and the environment as the thing that has been changing to understand why there are so many more allergies today.
The microbiome actually wasn't part of the design of that study from the outset because it was designed back in 2005 or 2006 at the planning stage, so it was really before microbiome research came on the scene in a big way. But luckily, someone in designing that study had the foresight to say, "Let's collect stool samples, diapers, amongst all the other many samples we're collecting, blood, urine, house dust." A few years later, the study's getting started. They're recruiting; it's a birth cohort, so recruiting pregnant moms. At that time, the Canadian government put out this call for microbiome research. Anita thought, "Hey, I'm part of this big child cohort study. We have diapers, we could analyze the microbiome." She put in a grant, and it was awarded. Right at the time, I was starting to look for a postdoc, and she was looking for a postdoc to work on this project. So it was really good timing.
I had not done my PhD in anything related to the microbiome or child health, for that matter. I had done it on cell signaling and cancer, but I was sort of not feeling that topic area anymore and looking for a change. This appeared to be a neat way of combining my molecular science background with a new type of research question more applied to population health. So I worked for three years with Anita, learned all about the microbiome. The study was still just getting going. Fun fact: it's still going today. Those babies are now teenagers, and we're still following them. At the time, many were still being born, so we didn't have the ability yet to say how the microbiome was influencing allergies because we still had to wait for them to grow up and get allergies. But we said, "Well, let's just take a first look at the microbiome. What does it look like? What's shaping it?"
My very first paper in this field was with Anita. We looked at 24 babies of this study that now has over 3,500 people. It was pretty small, what we considered a pilot analysis. We just profiled the microbiome using 16S. We saw that the main things shaping the microbiome of these three to four-month-old babies were how they were born—vaginally or by C-section—and how they were fed—breast milk or formula. That was an exciting experience to be part of that research. It was featured on the cover of the journal and in the newspaper. A lot of people were excited about it, which was new to me because the research I'd been doing prior to that was very important but very technical, cell signaling kind of very far from applications. It was fun to me to see that, wow, the average person is interested in babies and their microbes. So I really, I guess, caught the bug, no pun intended, and thought, this is something that I want to pursue. I want to understand at a deeper level why breastfeeding is so important to the microbiome.
Strategically, I guess, I knew that the cohort had also collected breast milk samples. So we had the opportunity to not just look in more detail at how the babies were being fed—because I looked at just breastfeeding, yes or no—but we actually had a lot deeper level questions than that, which I've gone on to explore. But also we have the breast milk itself, so we can start looking at what's in the breast milk and how those different components are related to the gut microbiome.
Ruairi Robertson: Right, yeah. And now you're kind of known as the breast milk microbiome person around the world. You've described that so nicely now that we know, partly from your research but other people's research around the world, that breastfeeding is probably the strongest determinant of what an infant's microbiome looks like. Maybe if we can think and discuss a little bit about breastfeeding historically and evolutionarily. Mammals breastfeed, and it's a perfect source of nutrition for us mammals. Can you talk to us a little bit about what you know about breastfeeding in the natural world and breastfeeding historically in humans and how our approaches to breastfeeding have changed or are changing?
Meghan Azad: Yeah, so I think the average person might not realize, although of course it makes a lot of sense once you think about it, mammals all have mammary glands. That's what they're named for, and therefore they all feed milk. They all lactate, make milk, and feed milk to their young. That's like the defining characteristic of mammals. So there are many species, humans included, that rely on lactation. We don't say breastfeeding for other animals that don't have breasts necessarily, but this way of feeding our babies. Every one of us humans alive today really owes our existence to breastfeeding, even if we ourselves weren't breastfed as infants, because in modern history that has changed. But that's obviously then a hugely important physiological system of our species and all mammals. Sometimes I think it's a little wild that we don't know more about it considering how important it is.
I think a good way of putting that in perspective is in medicine, you have a specialty for everything. You have nephrologists that specialize in kidneys. You have people that specialize in brains and kidneys and livers and every part of the body, but there's no specialty for lactation until actually last year. There finally is one now, at least in the US and Canada. It's kind of crazy that it's taken this long. I think there are many kind of historical reasons for that. One of them being that it's a so-called women's health issue. Although I'd argue every baby, regardless of their sex or gender, needs food, so I wouldn't put it in a women's category, but that maybe has been the case historically. I feel like, in a way, I'm lucky to be here in the right place at the right time because talking to my senior colleagues who've been in the field of breast milk and breastfeeding for decades, there's a real sense of frustration. Like, why is nobody understanding that this is important? There's not enough funding for this research. There's not enough people doing it. That is changing, and I'm encouraged to see that. I think we have more progress to make, but there have been, for example, directed calls for research on women's health broadly and then lactation specifically from like the US National Institutes of Health and others in the last five, 10 years, which is great to see.
But yeah, it's hugely important. Our survival as a species depends on it. That's true for all mammals. There are commonalities then in lactation across all mammals. For people who are from like the field of genomics, they'll do things like look across all mammals and see what are the genes involved in lactation? How are they conserved or how are they different? What can that tell us? But at a level of the milk composition itself, I think it's really neat that, you know, we all make milk, all the species, but the milk is very different and kind of optimized for each species, which again makes sense. A baby cow, for example, and a baby human are very different. A cow is going to get up and be wobbling around and it needs really strong muscles really quickly. So cow milk is going to be optimized for that. Whereas a baby human is not really using much of its muscles to walk around for many months. Instead, it's devoting a lot of its energy to brain development. Human milk is optimized for that. It's not just this species specificity, but also the dynamics of milk. A baby cow or a baby human is growing really fast and its needs are changing as it develops over that period that it's consuming milk from its mother. The milk is changing over time as well, which is something that we're studying. We think of day by day, month by month, but even within one day, from the morning to the night, we're learning that milk has differences and that can contribute to infants developing their circadian rhythms. There's a lot of individuality to it too. It's not that every cow is making the same milk or every human is making the same milk. It depends on that mother's genetics and on her environment. A mother living in Canada or the US or the UK, a high-income country, is going to produce different milk from a mother living in a really hot country or a country with other types of pathogens that are sort of endemic in the environment. That's something to keep in mind as well. By learning about the differences and the changes in human milk, I think it is really important. We also have a lot to learn from other species and researchers that have been working in that area. To be honest, you could argue there's more known about cow milk than human milk because cow milk is dairy milk, right? There's money in that, so there's been a lot of research going on.
Ruairi Robertson: I guess one of the main reasons that we have neglected this is because of changing human behavior. We are, in the natural world, probably the only mammal or the only species that can lactate, produce milk, and voluntarily decide not to for whatever reason that may be and shorten that. You mentioned nicely how our genetics take generations to change. However, you could argue that over the last hundred years, our environment has changed drastically. No more so maybe than in the first few months of life. The exposures that children are getting in the first months of life are very different to what they would have been generations or hundreds, if not thousands, of years before. Maybe can you just touch a little bit on changing practices towards breastfeeding in humans with industrialization?
Meghan Azad: Of course. Until maybe 100 or 200 years ago, every baby got breast milk. That's what happened. But then with industrialization and technology, there started to be, well, could we have something a little more convenient? Could we develop a scientific-based formula to feed babies so that mothers aren't having to breastfeed? Maybe we can develop something even better because we have technology and knowledge and science. So infant formulas were developed. They are based on cow milk, which is another thing that I think the average person doesn't necessarily stop to recognize. But formulas are based on cow milk; they're highly processed versions of that. So there's a lot of processing and supplements that go on with making formula from cow's milk with the intention of making it closer and closer to human milk, so better for human babies. That involves adding certain components, making sure that certain essential components, certain vitamins, for example, are there at the recommended level. It's continuously improving. But certainly, the first versions and even the current versions are not human. From what I just said about how human milk changes over time, even from morning to night, and it's different for different moms, that's something we really can't, even with the most advanced technologies and knowledge, replicate.
These formulas came on the market. If you look historically at trends in breastfeeding, you see everyone's breastfed, then a dip because now we have these alternatives, and then an uptick. There was this, okay, we have an alternative, no need to breastfeed. Then it took a couple of decades for research to catch up and people to start thinking, well, we're assuming this is better, but is it really? Collecting the data. One of my epidemiology heroes is Dr. Cesar Vectorra. He's an epidemiologist from Brazil. He's been recognized with some really large international awards for his work on breastfeeding. He's done lots of other things too, but he was one of the first people around the early 80s to start collecting data and showing that actually formula-fed babies in his home country were at higher risk of things like diarrheal disease and infections and even mortality. First of all, notice that and say, why is that? Is there something in human milk that we missed that we need to be adding? He even took it a step beyond that to take it to his government and policymakers in Brazil and say, look, I have this evidence that breastfeeding is ideal for babies. What are we going to do to promote this and turn this shift around because people are assuming that formula is better or at least sufficient? He's done a lot of great work and was inspirational to me, I would say, when he won that award. The first one was a Gairdner Award in 2017, so I had just started my lab. We can talk a little bit about, you know, there's a lot of emotion and controversy in the field of breastfeeding. As a new PI, I was thinking, is this where I want to focus all my efforts? It's difficult. It was also before these new grants in the area. I thought, is this going to be too challenging to pit myself in as a field? Seeing that recognition was like, okay, people are starting to catch on. This is important. There was definitely a downtrend in breastfeeding. It's now, in many countries, Canada and the UK included, on an uptrend, but we have a lot of ground to make up because of this evidence coming out and showing, yeah, there are a lot of important things in a mother's own milk that we can't replicate in a formula, and that are important, at least for a certain amount of time to give to your baby. The public health messaging and things that go with that are in progress. But yeah, there have definitely been some historical changes and trends over the last 50 years, 100 years.
Ruairi Robertson: Still, there are large geographical differences around the world. As you mentioned, in non-industrialized countries, it's a free source of food and nutrition for babies. So the babies tend to be breastfed for much longer there and tend to be breastfed exclusively rather than maybe mixed feeding. The WHO recommendations are for exclusive breastfeeding, and babies don't technically need anything else other than breast milk for the first six months of life. Then they're weaned onto solid foods, but the recommendation is to continue breastfeeding up until at least a year, maybe even two years and beyond if wanted.
Meghan Azad: Yeah, and that's different in different countries too, in terms of the practices. In high-income countries today, the trend tends to be mothers with more education, maybe more opportunities, are more likely to breastfeed longer, possibly because they've received the education and knowledge that this is really beneficial, possibly because they have more opportunity to take time off work when they have a baby and therefore be at home and be breastfeeding. It tends to be the lower socioeconomic status segment of the population that has lower breastfeeding rates. But if you look in the past or in other countries today, that can be reversed. In some countries, it's sort of like, well, being able to afford formula is a status symbol. So people associate feeding babies formula with better wealth. You might see the opposite trends. Similarly, in countries like ours, several generations ago, it was again, well, working moms are busy and they can afford it and they'll feed formula. So it was the opposite of what we see now.
Ruairi Robertson: Before we stray too much into the social science behind breastfeeding, maybe we should come back to the science of it all. You touched on that kind of historical research where people were looking at what components were in breast milk and realizing that maybe these replacements, we had in formula didn't have all the ingredients that were there. We know that there are sugars and fats and basic things like that in breast milk, in human milk, that are similar to what you could get in cow's milk. But there's also plenty of other things there, which we are only maybe beginning to replicate, or in some cases, haven't been able to replicate. Maybe we can start off talking about human milk oligosaccharides, because anyone in this field will be aware of them. Maybe you can tell us what these are, what HMOs are, and why they are such an integral component of breast milk when we're thinking about a baby's microbiome?
Meghan Azad: Sure. As you mentioned, there are components in milk, I'd say the nutrients, that we're going to find in formula. These are fats, lipids, sugars, mainly lactose, as the one that the baby will digest, and then micronutrients, vitamins, and minerals. Then I think of everything else on top of that as the non-nutritive bioactive. So things that the baby is not digesting for food or energy, but that are having an important influence on the baby's immune system, for example, or brain development, or other things besides just metabolism and calories. HMOs are one of those. HMOs are the third most abundant solid component of milk. If we sucked out the water and then measured what's left, you would first have fats and lactose. But even before proteins, which you think, well, proteins are an important nutrient, you actually have more HMOs than protein in human milk. That's one thing that's different in other species of milk, perhaps. In human milk, we have more HMOs than protein. These are human milk oligosaccharides. They are complex carbohydrates. They are not digested by the baby. For a while, it was maybe, well, these things must be important. The mother is devoting a lot of energy to produce them and put them in the milk. But what are they doing there if the baby is not digesting them? With the surgence of interest in the microbiome, it's become very clear that, oh, they're there to feed the baby's microbes. Because the microbes in the gut can digest them, at least certain microbes, which is another interesting point. It's not every microbe that can digest these HMOs, but certain ones that turn out to be beneficial for infant health. I don't want to discredit the early researchers of HMOs. I think since HMOs have been known, it's been known that certain bacteria can eat them. But it's really in the last 10, 15 years that people are paying so much attention to the microbiome that it's like, oh, that's what these are there for. Let's understand the HMOs. I got interested in them for that same reason because I was interested in the baby's gut microbiome. I was at a microbiome conference where my now collaborator Lars Bodi was giving a talk about HMOs, which he was studying in rat models and also in some human samples. That was my first real introduction to the fact that, first of all, these things exist in human milk and they're highly abundant. Essentially, we don't know a lot about them, what they do, like how variable they are, what drives that variation, how are they important to infant health? So I immediately went up to Lars after his talk and said, you have this technology to measure these important things. I have a cohort of babies where we've already measured their gut microbiome and I have lots of milk samples, we should work together. So we did, and we now work on many projects together, profiling HMOs. One of the fascinating things about them is in human milk, we still don't have the official number, but there are arguably a hundred, if not 200 different HMOs produced by women. These are complex sugars in slightly different physical formations. They're all HMOs, but they're structurally different. That structure can impact their function, of course. They can have different functions based on their structure. Some of them are present at high amounts, some of them at low amounts, and we're still learning about that. But in other species, for example, it's estimated that cows make about 40 different, we wouldn't call them HMOs, BMOs, bovine milk oligosaccharides. Mice, for example, which of course are also mammals, only make two oligosaccharides in their milk. This seems to be one of the things that, as arguably more complex or higher evolved species developed, they also increased the complexity of their human milk oligosaccharide profile. It's one of these components of the milk that is very species specific. Maybe it's one of the things that helps milk be optimized for different species. For me, it's a fascinating reason to study it. We think a lot about what they do for the gut microbiome because we do know that certain microbes in the baby can consume them. But I also have to acknowledge that's not their only function. We know they're consumed by the gut microbes, but they also, we are learning, have other functions. They can interact, for example, directly with the infant's gut epithelium. They can also be absorbed in small amounts into the baby's circulation, which allows them access to other organs of the body. We even know now that they are found in a mother's amniotic fluid and in her blood when she's pregnant towards the end of pregnancy. Maybe the word human milk oligosaccharide is a misnomer in a way because it's not only in milk, although that's certainly where we found it first and we study it most.
Ruairi Robertson: Babies are almost getting breast milk before they're born, they're getting some of these components.
Meghan Azad: Exactly. They're ingesting them through the amniotic fluid, which makes me think maybe that is kind of priming their gut before they even reach the world and have microbes present to receive these HMOs.
Ruairi Robertson: They’re produced in the mammary gland and being passed through the placenta, wow.
Meghan Azad: Yeah, which makes sense why they're found in the mother's blood as well because they have to get from the mammary gland through the placenta. They're in the blood to a certain degree. There's someone, I think, from Lars's lab years back who's been studying them in circulation and amniotic fluid and showing they're different maybe in mothers with different metabolic conditions. So looking at what do they look like before the baby's even born and then what do they look like in the milk. With Lars, one of the things we chatted about after that first encounter was, in the child cohort where we have a pretty large sample size, we know a lot about the moms, like what is their diet, what is their lifestyle, what is their ethnicity, we have their genetics. Could we use that information to understand more about what drives the huge inter-individual variability in HMO profiles? Because it's highly individualized in different people. So we've been working, for example, to link the genetic data we have with the HMO data to understand which genes are driving the variation in HMO profiles.
Ruairi Robertson: What is the headline of that? We know what the main environmental influences are on the gut microbiome. We know there's a small genetic effect. There isn't a very strong effect, genetic effect on the gut microbiome. What are the main influences on HMO composition? Is it genetic or is it?
Meghan Azad: Genetic is a big one. Almost maybe a little bit the reverse of what you just described for the microbiome, where there's a small genetic effect, but it's highly environmental. For HMOs, we see the opposite, where it's highly genetic and there's a bit of environmental influence as well. One gene that we know a lot about is called FUT2, or Flocosal Transferase 2. This is an enzyme that contributes to glycosylation pathways, not only for HMOs but for all the glycans in your body. It's a gene that has a common SNP, so a single nucleotide polymorphism, one letter difference in your DNA. Based on that SNP, you either have this enzyme that's functional or you don't. It's truncated, it doesn't work. It's kind of like one of those rare traits that's either on or off. You are, it's called a secretor if you have it, a non-secretor if you don't. You can see that black and white in a milk profile or other types of tests where either you make certain HMOs or you do not make them at all. I mention this because one of the HMOs affected by this gene is called 2-Fecosolactose or 2-FL. If you're a secretor, it's the most abundant HMO that you make. It's there in huge quantities. If you don't, then you don't make it at all. You make other HMOs in different quantities to almost make up for it. It's a very big signal. This is not a rare mutation. About 25% of people are non-secretors. We talked a little bit earlier about evolution. Evolutionarily speaking, you don't find 25% of people with a mutation or DNA difference if it's not good for something.
Ruairi Robertson: OK, right.
Meghan Azad: We don't know from historical evidence that people who are non-secretors are less fit, there's no fitness.
Ruairi Robertson: So non-secretors are completely resistant to norovirus infection because the virus uses one of these glycans, sort of controlled by that gene, and enzyme to infect host cells. This is unrelated to milk, but that's an advantage that non-secretors have. They cannot get infected at all.
Ruairi Robertson: This is winter vomiting both norovirus.
Meghan Azad: Exactly. You can go on that cruise and not worry. You will not get norovirus. Yeah. Eat all of the dodgy food that you want, even if it's gone off for a few days. On the flip side of that, because there's always trade-offs, non-secretors have a somewhat higher risk for certain autoimmune conditions like type 1 diabetes and IBD. It's an interesting question to say, I wonder if that's related to the microbiome because the microbiome is starting to be related to everything. When we come back to milk, 25% of moms do not make 2-FL, have a different microbiome or a different HMO profile in their milk. What does that mean to their babies? There's an inclination to say, oh, it must not be good. They must be missing this HMO for them. But I would say, well, they've been gestating for nine months with this mom who's a non-secretor, eventually consuming these non-secretor HMOs through amniotic fluid. Maybe they're just kind of primed to receive that non-secretor milk. Two questions that emerged from that knowledge for me. One is around HMOs and their addition to formulas. As we talked about earlier, there's lots of additions being made to formula to make them closer to human milk. No surprise, one of these additions is HMOs in the last decade or so. Companies are catching on that these HMOs are really important. Let's start putting them in formula. They are not putting 200 different HMOs. That would be really complex and expensive. So they're sort of like, well, which are the most important ones? Oh, it must be 2FL because that's the most abundant, which is true on average, but for 25% of people, they make none of this. Should babies of non-secretor mothers get formula with this 2-FL that they would never otherwise receive? Maybe, maybe not. The newer formulas are adding more than just 2-FL. They're adding three or four HMOs, which is a step in the right direction. But that's something interesting to consider.
Ruairi Robertson: Isn't the complexity of that in that we can actually synthesize, these are such complex chemical structures. They're very, very hard to isolate or to synthesize chemically.
Meghan Azad: Yeah, exactly. 2FL, in addition to being the most abundant, if you're a secretor, is also the simplest structure to make. Yes, the more complex they get structurally, the more difficult it is to either chemically synthesize them, or there are approaches now, like could we engineer bacteria to create these things if we give them the right genes. Now that there's a motivation to create them for commercial purposes, there's a lot of work going on. How could we better make these more efficiently and higher yields and also more different versions of them? That's certainly in progress. The other question it raises for me is the scenario of donor milk. For babies whose mothers are not able to breastfeed or babies who cannot breastfeed or be breastfed, what do we feed them? For most part, the answer is formula. But for babies who are really fragile and vulnerable and born early and in the hospital, there's a recognition that they're at very high risk for gut diseases like necrotizing enterocolitis and we know that giving them breast milk is the best way to prevent that. Ideally, their mother's own milk, but if that's not available, then rather than move straight to formula based on cow's milk, they will provide donor milk. So that's milk from another human mother who's got extra for whatever reason and decided to donate it, and it will get processed and pasteurized and then fed to those new babies. There's lots of fascinating research to be done in that area. On the topic of HMOs, I wonder, should we be matching the donor mothers to their recipient babies in the same way that we do for blood? If you go donate blood, they don't mix that blood with other people's blood and then just give it to everyone. They say, what's your blood type? Let me give it to someone with a matching blood type. Right now, we don't do that for milk, but I wonder if we should. The question is, how do you match it? There's all sorts of things you could match on.
Ruairi Robertson: It's not just secretive stages.
Meghan Azad: But secretive status would be a place to start. We actually have a trial starting where we're going to do that and say, is it better for babies if we actually match their milk type on secretive status?
Ruairi Robertson: Cool. Can one who is breastfeeding or lactating change the composition of their HMOs through diet, through probiotics or something environmentally? Is there any evidence to show that you can intentionally change it other than just these natural genetic effects?
Meghan Azad: It's a tricky question. We didn't find, for example, in our child study much evidence at all of diet, for example, being related to the HMOs. But in our study, we didn't have mothers assigned to different diets. We just said, what do you normally eat? At that level, there wasn't a signal. But there have been a few studies where they actually did say, okay, you mothers eat a high-fat diet and you eat a high-carb diet, and they do see some shifts in the HMOs. So it's certainly not as dramatic as something like, for example, the fatty acids in breast milk. Omega-3 fatty acids are something that you can take as a supplement, a fish oil, or you can eat lots of fish. If you do that and you are lactating, you will almost immediately see that reflected in your milk. You'll have higher levels of these fatty acids in your milk and that will get passed on to your baby. So those lipids are much more diet-based or derived than something like HMOs.
Ruairi Robertson: We don't completely talk about HMOs, because there's probably an ingredient list about 200 or 300 long, but yeah, exactly, who we could go on to. I think the other one that's quite interesting, mainly because you'll be able to tell us more about this, because we can't yet replicate it in milk, is antibodies. We know that babies are protected from certain diseases because they're getting these antibodies through milk. Maybe you can tell us about antibodies in breast milk a little bit.
Meghan Azad: There's a reason I'm cross-appointed to so many departments. I mentioned I'm in pediatrics, but I have affiliation to immunology. I now work with immunologists, have immunology students doing projects on this type of thing, and then microbiology and nutrition. I'm more fascinated the more I learn about antibodies and milk. When a baby is born, certainly if they're preterm, but even a normal, healthy, full-term baby, they have a very immature immune system. They're not yet producing large levels of their own antibodies. That's something they have to develop over the first sort of year of life. That is also the period when they're drinking breast milk. They are getting antibodies from their mother. They're relying on breast milk to get those antibodies to help them fend off different infections. The antibodies in their mother's milk are going to be a reflection of that mother's own sort of disease history and also her environment. If she's exposed to a particular pathogen, we could take the COVID pandemic as an example. If moms had COVID or someone in the household has had it, her immune system is going to respond, create antibodies against that virus, and then the baby's going to receive them for milk. In many cases, those antibodies are active in the baby and able to offer protection. Like you said, that's something that we can't replicate in milk. Even if we could, which ones would you put? It would depend on what environment this baby lives in.
Ruairi Robertson: Antibodies are so much more diverse than we've talked about the diversity of HMOs, but antibodies are specific to individual bacteria, individual strains of bacteria. It's almost impossible to replicate unless you put very generic IgA or whatever's in there. Sorry.
Meghan Azad: The most abundant antibody of all the ones found in human milk is IgA, immunoglobulin A. It's very high levels in breast milk and it's different than the IgA you would find in blood, even from the same mother, in the way that it's configured. We call it secretory IgA. It's sort of designed to be ingested early by a baby and survive that trip through the digestive tract and then be active in the gut. It's doing lots of things beyond protecting from infections necessarily. It's actually helping shape the microbiome. This IgA can bind to bacteria, even like our good bacteria in our gut, but then help them do different functions. This is something I'm working with Brett Finley and his team on because a lot of the technology we have to study the microbiome is about sequencing the DNA of the microbes. Get the microbes out of your sample, whether it's stool or milk, and then sequence their DNA and say, oh, these are the microbes that are there. It matters a lot whether those microbes are bound to IgA because that's going to allow them to do or not do certain functions like bind to the epithelium or interact with other bacteria. It's sort of a whole layer of information that's really important, that has not been paid much attention to. The project I have with Brett is to look at, let's not only sequence the bacteria, but also sort them into whether or not they have IgA bound and then see what difference that makes to the gut microbiome community and the different health outcomes.
Ruairi Robertson: You spoke a little bit about antibodies against infections and pathogens. This is a way to protect the baby. If the mother becomes infected, she'll create the antibody, pass it through her breast milk so that the baby is protected. It's a beautiful evolutionary mechanism. But as you've mentioned, antibodies also target the microbiome as well, the healthy microbes. Does that mean that there are antibodies in breast milk that are specific towards the mother's microbiome, which then are passed to the baby and determine what types of microbes, normal microbes, colonize the baby? Is there an indirect way of changing the microbiome through these antibodies?
Meghan Azad: This is an area that still has a lot of work to be done, but that's the general idea. The mother's own complement of these microbiome-targeted antibodies are going to be reflecting her own microbiome. These are then passed on in the milk to her baby. It's going to be one way of her signaling microbiome to the baby. The other way that we think of more directly is that when a baby is born, it's exposed to its mother's microbes, both vaginal and fecal microbes, so it's a direct transfer. We can also talk about the microbes that are present in breast milk and moms kissing and cuddling their babies and having skin-to-skin time. There are ways of microbes directly transferring from a mom to a baby, but then there's also this way of the indirect communication through the IgA complement.
Ruairi Robertson: I think that you've segued perfectly into what I was going to ask next. We've talked about the sugars and these lovely antibodies in breast milk, but a lot of your research is looking at the live microbes, the microbiome of breast milk itself. As you've mentioned, babies are exposed to microbes for the first time. Some people argue that this isn't the case, but we know that microbiome starts developing from birth. A lot of people try to track where most of these microbes come from. It seems like most of them come from the mother's gut, some from vaginal microbes. You have shown that a lot of these actually come from breast milk itself. Maybe you can tell us a little bit about what you found in the breast milk microbiome.
Meghan Azad: Studying microbes in breast milk has an added layer of complexity compared to studying them in something like stool, just because of the sheer number of bacteria. Stool is full of plenty of bacteria. Breast milk, as you would expect, isn't full of lots of bacteria. It has some, but they are relatively low amounts compared to the human cells you find in that milk from the mother's mammary gland sloughing off. If we want to use a method like DNA sequencing to understand them, we have the challenge of there's going to be a lot more human DNA in a breast milk sample than bacterial DNA. That's not the case with something like stool. That means we have to be very careful about things like contamination. Also, just because it's such low levels, different reagents in the lab can be contaminated with bacterial DNA. Those are definitely issues to be wary of when you're studying this type of low biomass sample. We're very aware of that and doing the appropriate controls. When I started doing this research, Shelly McGuire's lab in the US had published, I think, the first paper on the breast milk microbiome. Up until that point, it had been presumed that breast milk is sterile because it's coming directly from the mom, unless she has some sort of infection like mastitis, it's sterile. Shelly's lab was the first to really analyze some samples and show, no, actually there's microbes here. There's live microbes. Her lab studies not only humans but also cow milk, actually her husband's lab. They're a good partner, one doing human milk, one doing cow milk. They had done some work to show, well, there are microbes in milk. They're there at a very low level, but they are there. Also, that it's different between different moms.
Following on the methods that she had applied, I did this in the child study in a larger cohort and also found we detect bacterial DNA. It does look quite different between moms in a similar way that HMOs are different between moms. So are microbes. We started asking similar questions about what is driving the difference in these microbiomes in the milk and also what does that mean for the baby. First, we were just describing and we thought, what's driving the differences? We started with a list of things that we knew were important for the gut microbiome. C-sections, did it matter if a mom delivered by C-section or vaginally? That wasn't really important to her milk microbiome. Maybe that's not a surprise, but we checked. We checked other things like the mother's age and her BMI. None of these were lighting up the charts, but the one thing we found that was the most important of anything was how they were feeding their babies. They were all giving breast milk to their babies, but some of them were doing that directly at the breast all of the time, and some of them were giving pumped milk some of the time. That was an interesting clue to us about where these microbes actually come from. There are two theories. One is they come from the mother's own gut microbiome and somehow migrate their way into the mammary gland and into the milk. The other idea is, well, actually, they come from the outside, the environment on the mom's skin. Our finding lends credence to that hypothesis because if the baby is always sucking at the breast, then the baby's mouth could actually be a source of bacteria. Whereas if the baby's never there and it's always a pump, then you would get a different kind of exposure. That's also been confirmed by other groups now. I think it gives, and I don't think it has to be one or the other of these pathways. I think both play a role. I think there are some internal microbes migrating, but also there are external microbes getting into the breast milk. That was our first study looking at what's there and what's affecting it. Then we wanted to know how this is related to the baby's microbiome. We worked with other colleagues in the child study who had already done the gut microbiome sequencing of the babies. We compared the milk microbiome from the mom's milk to the gut microbiome from the baby's stool samples. These were collected on the same day, which is nice. Then we said, to what degree do these overlap? It's not surprising that they didn't overlap a whole lot because the niche of breast milk versus stool or a gut is very different. A lot of different microbes in these two niches. We did find a short list of about a dozen microbes that we found in both places. They were in the milk and they were in the gut. What's more is they seem to be correlated. In a mother and her own baby, if the mom had more of microbe A, then the baby tended to have more of that same microbe. We called that microbe sharing. We were careful to say that because it's not clear whether the microbes came from the mom to the baby or the baby to mom. Regardless, there seems to be some sharing of those microbes going on. It's still an open question, what does that mean for the baby's health? But we do think those microbes are one way of moms sharing microbes with their babies.
Ruairi Robertson: What are those ones that are most commonly shared? You've mentioned about these totally different niches. You'd expect Lactobacilli to grow in milk, whereas you get more enterococci or these firmicutes, things that are more anaerobic, which ones live in stool. What is PAS most commonly?
Meghan Azad: You're going to test my memory now. Maybe I'll open the paper. What I can say is lactobacillus you'd think, because it has that name lacto, like milk. But it wasn't one of the big ones we found in, so maybe again, a little bit of a misnomer. We found, for example, and we were excited that on this short list of a dozen microbes, two of them were familiar to us. That was because we'd seen them come up before in the child study in research we did on the gut microbiome and asthma in children. We saw that there were four microbes that seemed to be missing from the babies that went on to develop asthma, which made us think, maybe they're important in setting up the immune system correctly in early life. This was Stuart Turvey and Brett Finley's group. They nicknamed those four bacteria Flavr, FLVR for the first initials. The V and the R are Veinilla and Rothia. Those were two of our bacteria that we found shared between milk and gut. We knew from other research still in the child cohort and others that breastfeeding seems protective against asthma. The question is how does that work? Is it through the microbiome or could be other things? We've always thought that microbiome was a likely pathway because we see breastfeeding related to the microbiome, microbiome related to asthma, is this a causal pathway? This provides some evidence for that. Bifidobacteria is another, and that's one that we hear a lot about in infant microbiome research. We know that breastfed babies have microbiomes with high levels of bifidobacteria, depending on where you look, even highly predominant bifidobacteria. That was one that we did find in the milk as well. Staph and strep are commonly found in the milk and maybe they come from skin, but we find those also in the baby's guts. Kind of an interesting list. For me, I thought too, it hasn't been my field of research to develop products, but if I was looking for what bacteria should we use as probiotics, like ingestible probiotics, I would think milk would be a good inspiration for that. Just in the same way we talked about the IgA antibodies in milk are sort of configured in a way that's designed to withstand digestion. So might these particular microbes, just because you eat a microbe doesn't mean it's going to survive the trip and set up shop in your gut. But maybe these ones do. Whether that would work for adults is another question because baby guts are very different than adults.
Ruairi Robertson: I can picture, it's probably like the gut microbiome when people ask, what is a healthy microbiome or is my gut microbiome right? It's probably similar in milk, I'm imagining, that we don't know for sure what is healthy or not yet. We know there are different microbes there. If we take your pumping example, you found different microbes in the milk from women who pumped versus those who breastfed. Could you say that was a worse microbiome? Could you say, what were the species there that maybe would allow you to say how bad it was or whether it was bad?
Meghan Azad: You said, could you say that it's worse? I wouldn't say that it's worse, but the media would say that. I learned that lesson the hard way. We put out this paper and the headlines were things like moms who pump have worse bacteria or are causing asthma in their babies, which is not what we said, but things can get out of hand in the media. I'm always careful when I'm giving a talk on this topic to take a pause and a PSA slide and say I'm not saying pumping is bad. I know secondhand from friends who pumped, it's a lot of work. Our own data shows that babies who receive pumped milk are still receiving benefits from that milk as compared to being fully formula-fed, for example. We see differences and I think that's important to study to understand, is it that pumped milk? You gotta put it in the fridge. Are some of the microbes or other bioactive elements losing their activity? Once we understand that, could we give better instructions for storage? Or is it about the lack of that skin-to-skin contact you get through breastfeeding? If that's the case, maybe even if you have to pump, you could just make sure you have lots of skin-to-skin time with your baby. There's lots to learn there and I think it's important. We know that for some moms, whether it's because they need to be at work or the babies don't latch, pumped milk is what their babies are going to get. With that out of the way, I will say we did find some higher levels of potential pathogens in the milk from moms who pumped. We speculate that could be an issue. Again, it's an actionable advice of not cleaning the pump and apparatus appropriately and having those microbes feed back into the milk. The other thing is we call them potential pathogens. They are bugs that have the potential to be pathogenic, but that doesn't mean they always are. Some babies can carry this and not get sick. But that's something we're looking at. I had a former postdoc, Sarah Reyes, in the lab, who had done her PhD specifically focused on pumps and pump hygiene and how pumps were cleaned and what that meant for babies and moms. I think it's a really important area, especially in certain countries with little or no paid maternity leave where that's the only option.
Ruairi Robertson: Is there any evidence, you spoke about this fascinating potential for microbes from a mom's gut to go through what they call this entero-mammary axis and travel through the body potentially up into the mammary gland. As far as I know, there's no evidence for this in humans. Maybe there's a little bit of evidence in mice. Does that suggest potentially that if mothers take oral probiotics, some sort of strain, which probably doesn't exist right now, that could change the microbiome of milk if they wanted to do that for whatever reason?
Meghan Azad: Like you said, there's not much evidence for this yet, but certainly the possibility is there. There's been a bit of evidence in mice or maybe cows tracking orally administered bacteria, finding it in the milk. There's been some work in mice with fluorescent tagging and tracking of immune cells from the gut and seeing them physically go to the mammary gland, which I think is fascinating. To find out if this is the case in humans is an important question and really interesting. There is the possibility of that. It would be a matter of finding the right microbes that are able to survive this different translocation and doing more research for us to understand how certain microbes get selected out of the mother's complex microbiome to then get translocated to the mammary gland.
Ruairi Robertson: Or is it a case, with all the caveats you're saying that we don't know what is a good breast milk microbiome or not at the moment. You're still at a stage in your research of just identifying these differences first. But looking a few years down the line, maybe from this research, you'll find something that suggests that maybe something is missing in certain women and maybe there will be something that needs to be replaced. That could be orally, equally it could be some sort of topical skin probiotic. You said that some microbes come from the environment, you don't exactly know where, maybe from the skin. Is there any evidence for that, showing that skin microbes, topically applied if they're probiotics, can have beneficial effects or make their way into breast milk?
Meghan Azad: That is a great question. I'm not aware of any work going on in that area, but for example, there are antibiotic nipple creams for if you have a mastitis infection. I could see a world where there's probiotic nipple creams that are gonna help, maybe help the breast milk microbiome. It's possible. We could think about if the goal is to get this ideal healthy microbiome formulated in the mom and then in her milk and then to the baby, maybe you skip part of that step and say, let's try and give some microbes right to the baby. Certainly, there are people working on that, probiotics for babies. One of the things to keep in mind then is the way a baby naturally ingests microbes in breast milk is that many of those microbes are going to be IgA bound, for example, like we talked about earlier. If you just give them the naked microbes, maybe that won't have the same effect. We have to think about, if this baby is not being breastfed, then they're not getting HMOs. Do we need IgA-bound microbes with HMO, like what would naturally be in milk, which is so complex and we're still learning about? That's only three components we're talking about. There are also certain lipids and other metabolites and short-term fatty acids. These are all found in breast milk. We're still sort of getting the full ingredient list.
Ruairi Robertson: What is the other exciting thing? What have we not touched on? You've mentioned a few there. What's next going to be added to the list of formulas to be replaced? It's not going to be antibodies. It might be another HMO. Is it a lipid? Is it a metabolite? What's there and is very important?
Meghan Azad: I'll answer that in two ways. One is that I always like to say, rather than what's the other thing we need to add into formula, let's dedicate some energy to promoting breastfeeding and doing what we can to support moms who want to do that but maybe don't have the support they need. I have a quote from a colleague that I always love who was asked, if we put this in breast milk, will it be better, closer to humans? He goes, yes, in the same way that if I stand on my roof, I will be closer to the moon. I will be closer, but I'm really never going to get there with that strategy. We need to keep that in mind. At the same time, we do need to study and learn more about human milk for the reason of making better alternatives for babies who need it. Also for many other reasons, there's a lot we can learn from milk. A good example, since we talked about antibodies and COVID. There are ideas of, well, could we from donor milk, extract the antibodies and then use them for treating COVID patients or preventing COVID in elderly people who are at high risk? COVID's a little bit behind us now, but for the next pandemic or for other diseases. It's been found that milk is supporting immune system development for babies who have a very naive immune system. Who else has a very naive immune system? People who get bone marrow transplants, they first have to wipe out their immune system so that they won't reject the new donor marrow. That brings with it a lot of problems when you wipe out your immune system. There are studies going on, could we feed breast milk to those people to support their immune system in a time of need? For all these reasons, I think there are lots of reasons to study what's in breast milk. It has a lot to teach us. One of the projects I'm really excited about is I have a project right now called the International Milk Composition Consortium, or IMIC. We're studying milk from women in Canada, but also Tanzania, Burkina Faso, and Pakistan. Very, very different environments to try and look at what's similar and what's different in these different contexts. We're measuring growth outcomes in the babies. Three of those places are quite different from Canada in their environments where stunting and wasting, poor growth is a problem for babies. Understanding if we can identify profiles of milk related to optimal growth, that can teach us how to support other babies. I have projects on food allergy prevention and donor milk I mentioned and other ones, antenatal milk. The idea that moms can actually start producing milk towards the end of pregnancy. Right now that's recommended in certain scenarios, but it's also gaining a lot of traction, which in a way is exciting to me. People are figuring out that breast milk is important. If we can get some of it stockpiled before the baby, maybe that's good. I also think, well, no one's really analyzed that milk. I don't know what the composition of it is. I want to look into that. There's never-ending projects around milk.
Ruairi Robertson: It sounds like you have a long career yet to go. Surely, none of it will all get answered. It sounds like you're doing some very exciting stuff and you've given us an amazing insight into breast milk. I think we'll leave it there. Thank you very, very much, Meghan, for giving us that insight. We'll surely hear some more in the future from your ongoing research. Thank you.
Meghan Azad: Awesome. Thank you for the work you're doing to spread the word about microbiome science. I think that's a great service to the community. Great.