Impact of maternal breast development, diet and diabetes on milk production and composition. Video presentation by Professor Donna Geddes 26 November 2024

A research update on the composition of human milk

Professor Donna Geddes is Director, School of Molecular Sciences, Centre for Human Lactation Research and Translation and Senior Principal Research Fellow, School of Molecular Sciences. Donna has dedicated her career to advancing our understanding of human lactation and maternal-infant health. She has had several leadership roles including, Head of the Geddes Hartmann Human Lactation Research Group, Director of the Australian Breastfeeding Research + Lactation Research and Science Translation Network, and Milk Sensor Lead and ARC Industrial Transformation Training Centre for Next-Gen Technologies in Biomedical Analysis.

Topic	Pdf slide number	Time stamp in video
Breast development, lactation cycle, factors which impact	1-9	3:40mins
About milk production	10-13	20:33mins
Gestational Diabetes Mellitus GDM	14-16	24:04mins
Gut & milk microbiomes, diet + allergy	17-41	39:51mins

Transcript

Pam Well, look, I will start right on time. Welcome to everybody, it's so lovely to see you tuning in tonight. And I would like to start by acknowledging the traditional custodians of the lands upon which I live and work, the Jagera and Turrbal peoples, and pay my respect to Elders past, present, and emerging. And it's my Very great pleasure to welcome Professor Donna Geddes, who's presenting to us tonight on a research update on the composition of human milk. As most of you know, actually, Professor Geddes is the director of the Geddes Hartmann Human Lactation Research Group and indeed There are other centers too that Donna's leading, and I'll actually invite Donna to tell you a little bit about what she is doing there. What I have wanted to say before I hand over to Donna is that Donna and her group there at the Geddes Hartmann Human Lactation Research Group really are world leaders in research concerning breastfeeding and lactation. And in fact, it's not possible for any of us to be working with parents or indeed educating others about human lactation and breastfeeding without drawing on what has been a fundamentally paradigm-shifting work that's come out from Donna's team, but from Donna herself. And this is actually something that I think as Australians Australians who really care about the wellbeing of breastfeeding women, their babies and toddlers and their families, something to really celebrate as a national treasure actually on our shores. So with gratitude really, Donna, I'd like to hand over to you and thank you so much for being with the NDC Institute tonight to present to us

Donna That was just such a lovely introduction. Um, all true. I wish if my husband could only hear that, or he might believe me.

And the good thing is I can't see myself, but I can see all of you, um, at the beginning here. Thank you so much for inviting me to talk to you about some of the exciting research that's coming out from my team. I do like— lead a large, good-looking, mainly breastfed when they were young team of researchers that are passionate about providing evidence that can be built upon to understand breastfeeding and then of course being translated into practice as Pam and yourselves do. You can see two logos here. We've formed an Abreast Network, which is really a network that we are formulating to get people to connect across Australia, focusing on breastfeeding, lactation research, and science translation. But more recently I had the opportunity to found the Center of Human Lactation Research and Translation here at UWA, and that I lead with my co-directors. We have the director of the NICU, Dr. Mary Sharp, who's responsible for directing the NICU at King Edward Memorial Hospital as well as Perth Children's Hospital. So that's over 100 beds. We also have Debbie Palmer, Dr. Debbie Palmer, who specializes in RCTs, and we're working with her currently looking at the transfer of cashew and peanut proteins into the milk.

So we'll be doing proper time series studies, which you'll be surprised to hear they don't exist. And we're looking at different stages of lactation and how they might be transferred into the milk as part of a larger trial designed to increase exposure through pregnancy and lactation to prevent nut allergies. And then we also have Richard Voakes, who's an anthropologist, and so he comes with a behavioral lens and is getting more and more involved in breastfeeding research, and that will be— we'll be extending some of our work across to Uganda with some students currently. And then we also have quite an internationally renowned microbiologist, Dr. Mark Nicol, who works with WHO as well, mainly in the upper respiratory tract in infants, but also has a focus on infant feeding and development of that upper respiratory microbiome. So we've got a very interdisciplinary team and we're gaining momentum really to encourage people to study breastfeeding, to study human lactation, to work together to acquire more grant funding so that we can provide evidence to support practice. And a major component of that center is not only the research component, but the education component or pillar, and essentially the consumer, input that we have.

Breast Development [00:05:15.29]

So we've had multiple, consumer workshops, and those women have supported some of our grant applications and informed us about what they really want to see. Us research and how it might impact their breastfeeding journeys. So after that long preamble, um, this is our actual research program here in my group. So we take a holistic view and we study right from pregnancy. We're looking at breast growth, very interested in that, and the secretory differentiation of the breast. In other words, the development and how that development impacts milk production and secretory activation. We're also looking more and more at pregnancy complications because it seems whatever affects the growth of the fetus is obviously also affecting the development of the breast. We study milk composition, of course, from colostrum right through to mature milk and weaning. Some of the work we've done a little while ago now looking at suck, swallow, breathe mechanisms and dynamics, really informative. We still have some work coming out of the preterm nursery that we will hopefully deliver in the next 12 months or so looking at preterm abilities to coordinate that. We obviously look at expression and regimes and we'll look at that in terms of women with GDM later in this presentation, and also how that milk is delivered, for example, to the term and particularly the preterm infant where we use gavage feedings and how we treat the milk prior to delivery.

And that's pasteurization, you know, involves pasteurization, storage, UVC. We're very interested in how that's thawed and warmed. And of course how milk components actually are influenced by the mother and the infant. And we'll look at some of that today as well, some new data that we've produced. And essentially it really all comes down to how that impacts our babies, the growth, development, health. Because we measure milk production and milk intake, we can look at dose effects of the milk and how duration of human milk Feeding impacts the infant, but we've taken a big interest in the microbiome recently, as well as allergy and sleep. As you know, you've been studying with Pam, they're so intertwined with feeding, it's really not funny. They're almost synonymous. And then we also look at the health of the mother, and that links back to pregnancy complications, but also later metabolic health and how that impacts lactation and breastfeeding. So if we begin with, uh, the lactation cycle, when we talk about breast development, there are critical windows both at fetal and at puberty. And puberty is something we're focusing on after crunching some of the data that we've had, because we suspect that development here may be impacting the development in pregnancy.

And subsequently having an impact on lactation. So after birth, we move into a phase from secretory differentiation and secretory activation of days 1 to about 5 days postpartum. If we look at the literature, that's literally actually around day 1 to 3 in all reality. And it's quite a complex thing that happens with the tight junctions closing and milk composition dramatically changing and also milk synthesis increasing dramatically as well. And so we have this transitional milk that's produced during the next 5 to 10 days. But if we look at the literature, in all reality, most of the literature shows we should be reaching full milk production by day 8 to 10. So this calls into question some of beliefs around, um, weight loss in the first 2 weeks. After about day 10, milk production surprisingly stays relatively stable, and that's just a reflection of how the baby uses the energy that's consumed, we think. And then we see dramatic changes again as the baby weans in the milk. So after 6 months, but we can still see sustained milk productions as high as 400 to 900 mL in that next 6 to 12 months. So it's not an insignificant amount of milk that the babies are receiving.

When we look at breast development, we of course don't know a lot about that compared to other areas of development such as fetal development. We know some things about the endocrinology and we haven't really advanced that in recent years. And we know a little bit about genetics, but thereby it's clearly obvious that we don't know the full molecular pathways by which milk is produced. We do have evidence in the literature that breast development can be affected by obesity. And increasingly so pregnancy complications, in particular GDM. Polycystic ovarian syndrome is now kind of rising. It's starting to look like it's— it can impact breast development along with hypothyroidism. Hyperplasia is often classified as rare, but by definition is not rare, but it's— and it seems to be less uncommon. So something that might be misleading is the, the image in the top right here is what we expect hypoplastic breasts to look like. But at the moment I've been scanning lots of women with low production, and large breasts with little glandular tissue can have that similar droopy look, but also feel quite different, look quite different on the inside, and be generally composed mainly of adipose tissue.

Zinc transporter mutations have also been discovered in the last few decades, and these can result in accumulation of zinc in the cells, in the epithelial cells, which becomes toxic. Eventually we get turnover of those cells too quickly. I just point out that we know lots more about dairy cattle, and we've known for many, many years that overweight can cause permanent impairment of mammary development. And subsequent milk production. So there's a lot of mechanistic work that suggests this could be happening in women too, particularly with rising incidence of obesity. So, um, one of our PhD students that is still working in this area of milk production and milk composition has been looking at increases in breast volume and see whether that's been associated with the performance of the breast in terms of milk synthesis. And so we know that we have this quite rudimentary gland prior to pregnancy and we get massive changes. So huge proliferation of the ductal system and the lobes of the breast that are lined with epithelial cells that will ultimately produce milk. Now we know that breast volume is composed both of adipose tissue but also glandular tissue. And it's really the glandular tissue that determines lactation capacity.

But we also know that body mass index contributes to adipose tissue. So if we have high BMIs, it's likely to be more tissue and higher volume, breast volume. And this, it can impact lactation capacity as well. Oh, I'll just go back there. You can see here that this is some old data that shows the correlation between increasing BMI and increasing breast volumes. So as our women become more overweight, their breast volume increases. So our student has actually published this now. You can find it online. Most of our work is free online. And this is over 600 women that have measured milk production using the test-weighting method over 24 hours. And you can see that there's about 25%— there's this bimodal distribution. 25% of the moms are producing less than 30 600 mL of milk. Now we rarely see in clinical work, and you'll be aware of Dr. Sharon Perella, our lead in the clinical space, and Dr. Stuart Prosser, they use test weighing a lot. They very rarely see babies thrive with less than 600 mL of milk. So we drew a line in the sand at that, and we're probably quite generous because as you'll see, there are a number of other caveats we need to apply to these definitions and measures to really truly determine whether there's enough milk there for the baby.

You can see 3/4 of the mothers sit above that 600 ml. So after a lot of analysis and looking at different things in the data, What she did was produce the odds ratios here of low milk production. So we have mothers with a low BMI, so less than 25, and high breast volume. So they're designated as the reference group. So you'll see that if you have low BMI and low breast volume, there's not a significant increase in the odds of having low milk production. However, if you have high breast body, high BMI and high breast volume, suddenly that shoots up to 2.8, the odds here that you can see that. And if you have high BMI and low breast volume, you're at the same, almost the same risk So we have quite a large increase in low milk production as we've defined it as just less than 600 mL. As I said, there may be mothers that are feeding formula that are producing more than 600 mL, but it's not adequate for their baby. So that has called us and made us question moms more. Talk about when their BMI increased, what happened at adolescence.

Is there clues in their history that might help you if you don't have ways of measuring milk production to determine if there may be intrinsic causes? Something we do know is that the adipose tissue in the breast does secrete estrogen, so we suspect it will have a a, um, local effect on the mammary epithelial cell and probably breast development, that proliferation. So she was the same student as measuring estrogens and progesterones in the milk to see if our moms with low production and high BMIs actually have more estrogen milk. That way we can be looking for biomarkers that could possibly be measured at point of care to help clinicians determine that the mothers may have low milk production. As I mentioned, there's a rapid increase in milk production. You can see in this data from our group that there's an increase to virtually normal values in the first week to 2 weeks after birth when that remains constant. And then this is literature values here, but these are our values from the breastfeeding center. And you can see ours sit quite high. So the important thing to recognize in the literature, there wasn't a lot of stringency or cleaning of data, if you like, and looking at outcomes of babies and determining these were all, in fact, all normal productions.

But you can see that as we measure it and we define normality, we get less variation than you see in the literature, which is interesting as well. The important thing, and why it's so important, is milk production at 2 weeks is predictive of milk production at 6 weeks. So there seems to be a plastic window when all of their hormones are settling down and women are establishing production, that window is really a critical time period to look at in terms of helping moms increase production if that's necessary. We also know that production accounts for most of the variation in weight gain. So you can see here breast milk intake per kilo body weight and infant weight gain over time here on the yaxis. That's been established in a couple of studies, older studies. This is a newer study that has also shown that. So in one of our case studies, just to illustrate what milk production And when it's low, the impact it can have. This was a small-for-gestational-age baby boy who was born here at a low weight here, less than 2.5 kilos. And you can see that in the first 6 months, the mother was very intent on breastfeeding.

And you can see that the weight gain has tracked along that centile that line here. And you may be reticent to do anything about this, but at 1 year, the baby's jumped to the 50th centile. So we've seen a marked change. If we plot head circumference, you can see that that's been conserved across 6 months. With a low production, baby has conserved head growth at the expense of weight gain. And again, we jump up here at 1 year once complementary foods are started. This mom only had a milk production of 419 mL. If we look at the old data, that would be considered the edge of the range. So what we also did, this was PhD student Alex George, is we measured the fat content of the milk across the first 6 months of lactation. And you can see sometimes it's above the reference range, but in fact the mother was only producing half the amount of milk. So this doesn't compensate for lower volumes. Our proteins in this milk were below the reference. And we've got a publication that's come out more recently that's shown that protein to energy ratio, it seems to drive infant growth in term babies, which is similar to our preterm babies.

And then see the lactose content is in range, but when she calculates the total energy intake, it's half of what it should be for a baby in the first 6 months. Of lactation. So I think it's important to not discount milk volume in favor of suspecting that milk composition is compensating for that. Certainly, it's borne out in the evidence. I mentioned pregnancy complications. So in those 600 and something milk productions, we calculated risk of low supply depending on the actual pregnancy complication that the mom had. And you can see with gestational diabetes, we found a 7 times increase, a 7-fold increase in the risk of low milk supply. But it only got worse with gestational hypertension equating to an 8.8-fold increase and a low birth weight baby are 12 times more likely to have low milk production. And what's interesting, the hypertension and low birth weight babies, whether that's to do with the vascular supply to the baby, which is also compromised to the breast, yet to figure that one out.

Diabetes[00:23:37.12]

So because we saw that gestational diabetes was seemingly increasing risk of low milk production, Sharon had to design a study, or we had to design a study, but Sharon was very passionate about this one, to prospectively track women with GDM and women with normal glycemic levels.

And that was really to be prospective and proactive in management in the GDM groups so that we could see that whether we intervened in the first 2 weeks or that they received really good care, would it make a difference? So we all know that insulin sensitivity steadily declines from mid to late pregnancy and we get that hypoglycemic problem with GDM that's diagnosed by a glucose tolerance test around mid gest— just after mid gestation. And we know the prevalence is increasing dramatically, and that's cut in hand in hand with increasing levels of overweight and obesity. So we know that there are infant risks to long-term health, such as hypoglycemia, development of type 2 diabetes, overweight and obesity, cardiovascular disease, and metabolic disease. But we also know that that mom is also at risk if she has GDM, so she also has an increased risk of type 2 diabetes, cardiovascular disease, metabolic syndrome, as well as eye, renal, and liver disease. The interesting thing is that those risks are diminished by breastfeeding, but In the literature, we see multiple reports of delayed initiation of breastfeeding. So even getting the baby to the breast delays, creamy activation, less exclusive breastfeeding, reports of perceived low milk supply but not measured, and decreased breastfeeding duration.

And we often conclude that these women need more support So that was really the reason or the driver for this study that Sharon's led. So these are new results hot off the press that are looking at early feeding practices in those first 3 weeks to see if that differed between women with GDM and our control group. And you can see we collected a lot of information such as timing of the first breastfeed, first 24 hours, what happened, frequency of breastfeeding, frequency of expressing, timing of secret reactivation, formula in hospital, breastfeeding problems, you name it. And then we got them to do a 24-hour milk production at week 3 to see what those levels were like, because we would expect them to have full milk production by then, but we do have another production at 3 months where you'll see the real impact of GDM in terms of weaning. So these are the participant characteristics. They're all largely similar, mainly primiparous, highly educated women with an intention, a strong intention to breastfeed. We had pre-pregnancy BMI of around 27 in GDM and 25 in our non-GDM moms. And obesity was about a quarter of the moms in GDM group and 17% in the nonGDM group.

Cesarean rates was similar, and we had about 41% of the moms on insulin. And we were slightly lower gestation at birth for our Gini moms. So what did they do? Well, there was no difference in breastfeeding initiation. So two-thirds of our moms were having the baby to the breast within an hour after birth, and another 15 to 20% were within 2 hours. So that didn't differ by group. What did differ was breastfeeding frequency. Now this is likely because the clinic we work in, mothers are likely told that they may have some issues and therefore expression frequency was slightly significantly higher. So if the baby wasn't feeding, they were more likely to be expressing. And so interestingly, our nonGDM group, half of the women expressed as well compared to two-thirds of the GDM group. But the total milk removal frequency was the same. It was around about 9 times in 24 hours. So our GDM group were breastfeeding less, expressing more, but also there was an adequate frequency of milk removal. So what happened in terms of secretory activation? We can see here in the pink, we've got our GDM moms and she's adjusted for, you know, time of day that the baby was born.

But you can see that this is the actual numbers here that 32% of GDMs had not initiated by— initiated after day 4 as opposed to 8%. We've got a delay in secretory activation in the pink here of our GDM mothers. So they're definitely confirming a delay there. The formula supplementation in hospital is hugely different. We've got 57% of the mothers' babies getting formula as opposed to 25% in the control group. At 1 week, 7 days after there, we can see that the breastfeeding frequency is still lower in our GDM group, but it's 7 versus 9 breastfeeds. So it's not largely different. And you can see our moms are still expressing 5 times, 5.5 times on average. So they're really still lower breastfeeding, higher expressing frequency, but total milk removal is essentially the same. So we can't say these moms aren't feeding enough or frequently enough or removing milk enough. What they are is a reflection of that. They are giving more formula. So we've got 39% here giving breast milk and formulas as opposed to 6%. And in our non-GDM group, we've got 94% just giving breast milk. So about two-thirds managing breast milk only, but only 28% breastfeeding only.

So because breastfeeding and EBM, so we've got more mixed feeding and less breastfeeding only. The difficulties at week 1 was similar between the groups except the nipple damage for GDMs was higher, 20% versus 6%, and may will have been coming with the extra, you know, attempts to breastfeed. We also see reported by the mothers already low milk supply. So 35% of them were reporting that versus 6% in the control group at 3 weeks. We now look at the milk production here and you can see again our GDM mothers are breastfeeding on average 7 breastfeeds a day versus 9. And the expression frequency is higher still in our GDM group compared to our non-GDM. But total milk removal frequency is up around 10 and 11. For both groups. Again, we see this time 88% of the GDMs that were breastfeeding were— 88% were giving breast milk only versus the non-GDMs, but only 35% were breastfeeding only. So it was more EBM and breastfeeding compared to our control group. Here we have the frequencies plotted. You can see the GDM paralleling our non-GDM mothers. And you can see the expression frequencies holding about the same for the GDM group.

But interestingly, the control group for whatever reason are still expressing milk as well. Then we come to the kicker here, which is the production at 3 weeks. So in the pink we see the control group. And if we draw the line at 600, you can see that a lot of our GDMs are making virtually nothing here. These are the ones that eventually wean. Anywhere from 100 mils to just on 600. And we've got a few more that we've just collected the control group on, but you can see, yeah, our GDMs, 46% have low milk production at 3 weeks. If we look at who We had 4 women who weaned or did not measure due to low milk production. So they were more stressed about their productions. Then begs the question, so we see this low milk production, there's a lot of talk about milk composition being different if mothers have GDM or if they don't. So we have looked at all of those milk samples across the 6 months and looked at milk hormones. So adiponectin, that's responsible for energy balance, protein here, lactose, leptin, another appetite control hormone, glucose, and total lipids. And if you compare the pink to the blue, there is no difference across time in any of these components.

So the macronutrients don't change, and that's how the babies grow. And we don't see any changes in the appetite hormones, which has also been surmised in some publications. If we look at actual intakes, which is probably more important than the concentration because we've measured how much the baby's getting, you can see in the control in the orangey color and blue for GDM, that insulin intakes, glucose intakes, adiponectin, lactose, leptin, and protein don't differ. So we can be reassured, at least the mothers, that their milk is not inadequate or of different quality or whatever term we might like to use when describing composition. So that is reassuring. So what have we found so far in this study? We found that women with GDM have an increased likelihood of a delayed first breastfeed, delayed screen reactivation, and low milk production. But we're not seeing any reflections yet in changes in milk composition as a result. So what can we do? Well, we can set expectations of lactation and pregnancy. We basically in the clinic have this conversation in midgestation. The midwives have the conversation with the moms. They educate on the fact that there might be some problems breastfeeding.

So if they do it, then they can talk about it again later. Basically, what to look for if things aren't going— this is what it looks like when things are going well. This is what it looks like when problems occur. What to do, simple instructions, where to get help, and to really impress the fact in getting help in that 2 to 3 week window where intense milk removal may have an impact. And to act quickly. What is encouraging and what I didn't have here was that despite the high incidence of low milk production, 80% or more at every time point of the women in the GDM group was still breastfeeding at 6 months across that time. So if anything, the actual intervention of having the conversation and preparing women, we think is actually helping these women, um, breastfeed for longer. And we think that's a good thing since we haven't figured out why exactly there is low milk production and who it might affect. And we do know insulin is critical to breast development. So finally, I just want to talk a little bit about some of our new data looking at the impact of diet and the impact of other things on milk composition, because there's a lot of talk of isolating or supplementing the baby with components in the event of milk not being adequate.

And you can see with GDM, that's not the case. It is the case in terms of probiotics and prebiotics, though. And so we have done a dietary intervention study and collected a lot of samples. There are a lot of people involved in this study, and so I acknowledge them here, particularly the 2 students that have— 3 students that have worked on this, but the 2 that I'll show today in particular.

Diet [00:40:12.25]

So we, we have evidence, right, that diet affects our gut microbiome, and we have evidence, however, usually from cross-sectional studies where we know that there's a difference in the gut microbiome with increased fiber diets versus what we call a Western diet of decreased fiber and high fat and sugar diet. So when we come to our intervention, this is what we've tackled. We see differences between rural and urban environments and exposure to farm and pet animals and family size. We often forget actually that a big impact of our gut microbial profile is determined by our genes, and that might be the reason we see variation across populations rather than diet. So there has been some diets, of course, that have looked at extreme diets, so really heavily based animal-based diet versus a plant-based diet here.

And just check if I've got the animations. So this is, um, if we go back, these are just groups of bacteria. So it's not one bacteria looking at— and it would have been statistically or biologically determined, these groups. And then if we zoom up, the red dots on here are the changes in the gut microbiome that significantly changed, I should say, according to the animal diet. You see green and red indicating that these bacteria have changed in response to both diets. But interestingly, we didn't see anything change solely based on the plant-based diet. So that makes us think a little bit more about that, because we think that the gut microbiome is quite dynamic. I'm not sure that holds out in the literature. However, there has been a vegan and keto diet study where it's crossed over, so the participants were on both for a while. And the interesting thing that we saw was, um, in this study, an increase in bacteria associated with the vegan diet and a decrease in a particular genre in the animalbased diet. They were different. And this actually affected the microbial amino acid metabolism, and it increased in the vegan diet and decreased in the keto diet.

But we saw changes in immunity, and this was verified in animal models. So you see an increase in innate immunity and antiviral pathways with the vegan diet. and an increase in adaptive immune pathways with keto diet. I think there's some good evidence that bacteria may change, but sometimes it's the metabolites that enact a change in our immune function. What affects our baby's gut microbiome? This is a study of thousands of babies in Europe, and at the top is microbial composition across time. And below is the gut functional potential here. So the actual function, and it's pretty clear in the pink that breast milk and introduction of solid food had the most dramatic impact on our baby's gut microbiome up to about the age of, I think that's 1.5. And functional potential, exactly the same thing. Thing. So this outstrips maternal BMI, it outstrips things like mode of delivery. So that's, um, quite interesting. We also think that the gut microbiome is, um, dynamic, but actually there are stages. The most dynamic phase is from birth to 1 month, and then it stays constant for the full period of breastfeeding. You introduce solids and we get another change to 12 months, and then we have more solids and less breast milk, and we see progression to a more adultlike microbiome.

So whilst it's highly dynamic, supposedly there are periods of stability, particularly around the breastfeeding period, that are probably critical to gut maturation and immunity. So our hypothesis, starting backwards, working backwards with the diet, was we wanted to see if changing diet would affect the infant's gut microbiome. So we thought that maybe the milk would be mediating that impact on the microbiome, and that the maternal gut would be mediating the impact on the human milk. And that by changing diet, we would change maternal gut, thereby change the milk and the infant outcomes. So what we did was we delivered light and easy for 2 weeks. Mothers loved that. After they had recorded their habitual diet a week before. And so we took samples pre-diet, post-diet, 4 weeks, and 8 weeks after the diet. And we put milk, infant stool, and maternal stool. So these are the ones that were analyzed first. And what we of course looked at was whether our dietary intervention was successful because it was a low-fat. So indeed, the fat was reduced, sugar was reduced, fiber was increased, saturated fat was also decreased. So a deviation from the typical Western diet.

We also saw as a result in 2 weeks, a reduction by about 1.5 kilos of body weight reflected in a decrease in BMI. And we also saw a reduction in fat mass post-diet, which was nice to see. So what did we see with the maternal diet and the gut microbiome? We looked that first. We did some fancy meta shotgun metagenomics. But what we can see here in 10 very clean, what I call clean samples, because it was extremely expensive, healthy babies, same mode of delivery, you know, as much as we could exclude out of this. You can see the composition, these colors represent proportions of bacteria, doesn't change much from pre to post-diet. So no significant changes in microbial composition, but if we looked at the functional potential— so we're just looking at this row down here, this is all stuff that can be done in these boxes— and then this row, which is post-diet, you can see a big change in the colors, which means things are changing. It's a function So synthesis of B vitamins, for example, could be changing, or carbohydrate biosynthesis. So we're seeing functional potential change, but not really microbes at this stage.

So then we next saw, well, did the diet affect the milk? What we found was a significant increase in Cutibacterium acnes and a decrease in Haemophilus parainfluenza. So two bacteria that are relatively low abundance that changed significantly, as you can see here. It's not a great huge change, but a change nonetheless. Then we looked at the— finally, the diet and how that impacted the maternal gut microbiome. And again, we saw decrease in Bacteroides casei and an increase in called Bacillus intestinalis. So again, two relatively small, low abundance bacteria decreasing significantly across the diet. So, it begs the question whether maternal gut, the maternal gut microbiome is able to be impacted dramatically during the phase of lactation. Which is interesting. So our hypothesis, we did see small significant changes in the maternal gut microbiome and the human milk, but not much really at the baby there in terms of microbial composition. So then we looked at human milk immune proteins because they're antibacterial and antiviral and highly bioactive in the milk. And did they affect the infant's gut potential. So what we wanted to know was, did the diet actually impact these levels? That's the first thing.

And we know lactoferrin sequesters iron and, and stops bacteria growing, that lysozyme affects the bacterial cell wall and allows macrophages and immune cells in to kill the bacteria or viruses. Bacteria. And we know that a low-fat, low-sugar diet that we'd given the mothers actually results in lower inflammation. So this time, just looking at daily sampling. So these models were very good, took samples every day of their milk. So we looked at the habitual diet for a week and then 2 weeks. This is a beautiful colorful graph. But all you need to look at is the fact that this is the first week, lactoferrin and lysozyme. You've got lots of these lines here indicating lots of significant differences, daily differences. They reduce in the first intervention week, they disappear, lysozyme is very constant here, and they remain about the same in week 2 for lactoferrin. This might indicate that some level of lowering of inflammation might be impacting the immune proteins in the milk. But really, we are missing some puzzle pieces. The next thing is to look at whether diet— there is some evidence, not super strong, that might impact the HMOs in the milk, which would obviously affect the bacteria in the milk.

And the short-chain fatty acids from the mother's change in her gut may travel via the circulation and also transfer into the milk, which would all impact the gut microbiome and the functional potential, but not necessarily in microbial composition, as we saw. Finally, just to finish off, this has been a pet thing for me. There's a lot of talk about infant sex impacting human milk composition. I guess it's been irking me because it doesn't make sense for twins. I couldn't imagine how that might be working. And some mothers do get quite concerned about having girl milk for girl babies and boy milk for boy babies. This is some unpublished data. You can see that this bar looks very similar to this bar. This is the female babies in the group of 283 in the male babies. So this looks like there's no microbial composition difference so far. We look at macronutrients which grow our babies. These are concentrations in colostrum, transitional, and mature milk. The blue and the male, you can see they don't really differ at all for lactose, protein, which decreases across time, and fat. So we also have looked at intakes. We've looked at all of those hormones I've told you about, as well as minerals and immune proteins and casein and whey, and we can't find a difference.

So that should be quite reassuring for the moms. Finally, we looked at the impact of allergy, um, maternal allergy on infant allergy by breastfeeding with samples from an RCT of fish oil intervention. And the actual fish oil didn't impact the outcomes of infant allergy, which allowed us to use this cohort where 50% of the infants were sensitized or allergic at 1 year and 44% of them were allergic or sensitized 2 to 3 years. What we found was what we expected potentially, that the diversity of the human milk microbiome was decreased in the milk fed to the babies that developed allergies. 7 of these bars here, 7 of the highest, most prevalent bacteria in the milk were associated with allergy. And you can see lower amounts or proportions of these bacteria in the blue were associated with multiple allergies or sensitization at 1 or 2 to 3 years. And Acinobacter, for example, was positively related with this, and Pseudomonas. So, we can see quite a complex shifting landscape. In terms of what's in the milk and how it relates to allergy. So to finish up, just in case you've forgotten anything, milk production, no breast growth seems to be associated with increased risk of low production.

In GDM and type 2 diabetics, which I haven't shown you today, secretory activation is often delayed. Around 2 in 5 women don't seem to make enough milk for their babies no matter what they do if they've had GDM. And that frequent feeding and milk removal may not resolve this deficit in production in our GDM mothers. So we really need to elucidate the mechanisms of what's going on in terms of production and development of the breast. With milk composition, it doesn't differ between women that have had GDM It didn't— doesn't seem to differ by infant sex. It can be potentially affected by diet depending on what we're looking at, and it may be related to infant allergy. So with that, I think I've taken enough time.

Questions [00:56:51:07]

I'm happy to answer any questions you might have about the studies or the results. Pip said, do you think the earlier gestation for GDM babies has a role to play? I think a week is probably not enough to maybe explain the severe deficits we're seeing in some of the mothers, and no matter what they do, it seems to not be resolved. When we've been trying to tease out the insulin versus non-insulin, we've hit the wall.

We don't really see, you know, we've got to do some more modeling. We don't really see anything about the treatment with that. So we typically see some mothers, as you say, and, uh, slim with GDM and will potentially be the ones that don't produce enough milk versus somebody who's overweight and GDM. So that's why we're looking at that critical window of adolescence as well to see if it's a chronic issue. Who wants to go next, Beth?

Beth I'm actually at PCH. I'm an OT and LC there, so I've been keen to catch up and ask a few questions actually. So I guess, like, I think we're probably, or I'm probably struggling a little bit at the moment. I think in particularly even just using that GDM study that Sharon did with our prems and our earlier bunnies, I guess I think it was, I think about a 2018 study where you looked at prems and babies with laryngomalacia and T21, and that looked at the vacuum. Do you think, I mean, I guess I just wanted to kind of ask a little bit about that and whether you think kind of our preemie babies, we do really need to think realistically about how to support those mummies to be breastfeeding. I'm not sure if exclusively without any mixed feeding as possible. But in the context then of the GDM mums on top of that, how do you— yeah, how do you think we can best support them? Yeah, so how— like, because they're perhaps more likely to be a bit earlier, then they're going to be a bit— a little bit weaker in their ability to transfer milk. How do you think we kind of support them in the best way?

Donna I think it's all about realistic expectations and positive reinforcement. But I think that for you guys, yeah, we saw that the preterm babies, two-thirds the strength of the vacuum. Again, you couldn't tell that from the outside. I think, you know, test weighing, Sharon's also shown that guessing how much they get's not been very good for top-ups. And then you end up with not enough topping up for these specifically medical babies that need that. So I think that's really important. And I think that some of that would allay women's concerns and fragility around the NICU. You know, I really think you measure everything else and perhaps it's actually potentially more reassuring to know that their babies have got enough milk or that they can feed the top up. I think to preterm on top of— so you're looking at pregnancy complications plus prematurity of the baby. It's a perfect storm for getting direct breastfeeding going. I think taking the pressure off what we've found with the term moms, taking the pressure off the direct feeding at the beginning and making it okay to pump and progressing towards more, you see an increase in breastfeeding in that group, in the GDM group, um, mapping out that journey.

And most of them were still breastfeeding by the ones that weaned, and they're the ones that could only pump 50ml of milk in 24 hours. You know, this was seriously concerning. So I think, you know, telling mums the baby's tone might be low. I've seen twins at something like 2 to 3 months preterm, one sucking flat out and the other's just this weak vacuum, but you can't tell. So I guess growth is, you know, I'm not sure if I'm helping with that, but I think mothers are open to the expectation and saying, you know, so there's also the ones that are really low. Look, let's go hard, let's do this for 3 weeks. If you're not there, let's map out what's sustainable, and you get that longevity and enjoying the breastfeeding. You know, along with that comes all the help around breastfeeding and positioning and attachment as well, but I guess that's what we're finding. Yeah, I think, and I think this in pregnancy, you know, I don't know if that's possible with preterm, obviously not always, but you know, like you could have some problems and we'll just, you know, this is what they'll look like and then they're not kind of caught by surprise, I think. — I think women are more and more anxious, but I think our experience has been the more information they have, the more likely they are to continue breastfeeding. And to— for them to know that this might just be something metabolic, something to do with their pregnancy complications, something that is potentially beyond our control, then they— it kind of encourages them to keep going without— not being concerned if things don't, you know, take off rapidly. Is there anyone else?

Pam At the beginning of your presentation, which I've just— it's just been so good for us to hear, and I would say thank you so much for sharing some of that— so much unpublished data there. I've just hung off every study that you've been talking about. It's so interesting. Thank you. But I was going to ask, You said early on, 8 to 10 days is when we're seeing perhaps full milk production kicking in for that woman, and you flagged this may in fact mean that we need to be rethinking how we're managing those early days. Big topic. I just wondered if you had further reflections to offer there. On that, because it's such a challenging time, isn't it?

Donna Yes, it is. I— it is. In terms of reflections, it's been a big concern, um, just for us, that we don't want to miss a window that's potentially, um— so, so good help early I guess, and our mothers aren't getting that right, so we're not getting as many child health visits. They're not seeing the child health nurse till later. The education of the people seeing them might not be great. And whilst we have this, I guess it's recognizing, as you well know, is when the baby's not doing a great job as the great pretender and things aren't going well, that expression for a short period of time doesn't have to be forever. And if that helps establish the milk production, um, they also, you know, when I first saw our antenatal screening data, it's like, these moms are still feeding, you know. It— we need to tackle these things in pregnancy before they're exhausted and and, you know, beyond not sure what's happening, secondguessing themselves, it's their first baby, and, and really encouraging them to come back if you were multidisciplinary, or who to go to, and do that early. Um, and so I think the intervention itself was actually telling the woman, and then also Giving very just the basic information again in late pregnancy.

So Stuart's integrating this late pregnancy complications. So if we've got hypertension going on, that's concerning. We talk to Stephen Tong about that, a very prominent obstetrician that deals with preeclampsia, and he said, well, you know, if the vascular system is bad in the placenta, you've probably got a knock-on effect to the breast. Um, so I think, I think it's interesting that that might work for us until we figure out what we can possibly— who, who would be the woman that are going, because the other half are making plenty of milk for their babies, right? So our next step is, can we, can we help you identify which women might need to be called, you know, 2 days after birth and checked closely for that weight gain, um, issues in the first 2 weeks? Um, so, so yeah, I, I know it's quite contentious. But if you know the mother's not fluid loaded, she's had, you know, she's not suffering any of those things, the baby's not a sleepy baby. Yeah, I just reflecting on that, I think we just have better outcomes for the 6 months to 12 months. We tackle things early and make sure that Yeah, I met a mom the other day that's been triple feeding for almost 5 months. It's like, I came in to scan her and I think, how are you doing this? This is amazing. Well, she didn't have a toddler, but a toddler, and she had very, um, you know, hyperplastic breasts really. They had that different kind of shape. And when we look at the milk flow, the milk flow The waveforms, Doppler waveforms, are often a high resistance. So, you know, I think, uh, it's, it's amazing if a woman can do that, but often our women can't. And so therefore, that, you know, you've really done all this, you're doing this 10 times a day, now let's figure out how you can sustain the lactation without completely weaning. And yeah.

Pam Thank you. Thank you. You know, when we're looking in those first days, are you reflecting that even though we need the formula for safety on occasions, that we're coming in too quickly with formula but not quickly enough with the intensive lactation support is what I hear you saying.

Donna Yes, that's a nice synopsis, right? There's a jump in formula which we haven't seen for years, and in the last 5 years we've seen a sudden increase in formula supplementation, um, and women leaving hospital early without any education on how to pump or express if their babies aren't gaining weight, right? So then they're kind of left looking for that. Um, so I think you, you've, you know, summarized that quite nicely, that, um, that— and I, and I think there's this tug of war between direct breastfeeding, right? That's what we all really want. But if we've got, you know, this rising case of complications, you know, we're just going to have to find the right— the right balance of, yeah, getting human milk into that baby and producing as much as— does the frequency of feeding impact the total milk intake if someone has lower glandular tissue? You know, that's the mom who's triple feeding 10 times a day but still has to feed formula. So it depends on the capacity, I think. Um, so that's what we, we hold on to, that local feedback control. But in all reality, if you're not increasing the number of mammary epithelial cells there, that mother's stuck on that having to feed every 2 hours, 24 hours around the clock.

So that's why we think that first 2 to 3 weeks is your biggest bang for your buck to put pressure on the breast if it is going to proliferate. What worries me is we don't really know if that's what happens, if we're not just kicking the synthesis and So yeah, so when moms are doing that and they're not getting anywhere, it's not that they're not trying, it's something to do. So we're asking also, so something we had to go and relook at, if mothers, if you ask a mother if she had breast growth in pregnancy and I've been down in our research room and a mother goes, oh, A little bit, like, that's a no, you know. Oh, my husband said they changed a bit. I'm like, uh, that's no. Like, a whole cup size is like, you know. And so we're asking more about density and tension. Did you— so when you scan the moms, you know, you can obviously see the, the adipose tissue. So I think there's quite a— it's quite amazing that women, um, report breast growth, but maybe because they've never experienced substantial breast growth, they're not sure, right? So it's a, it's a matter of asking the right question and then looking for that response, um, and whether they had growth, how much growth they had in purity. It's quite different for some mums.

Pam Yeah. And Donna, when you talk about impressions of, um, glandular tissue volume, this is not something that's been formally— we can't really quantify this, can we? So you're talking about your clinical experience with ultrasound, your impressions, but in fact we don't really know what— this is going to be very sort of variable, isn't it? We don't really know what amount of glandular tissue that you're visualizing is, is going to correlate with good supply, do we?

Donna So it's, so it's really hard to measure volume because the heterogeneity of the breast and the amount of adipose tissue and glandular tissue. So that's why you don't see CT studies or MRI studies that are measuring volume, because that would be the sensible way to go. It's still not that simple when you're taking lots of slices through a breast and then you try to figure out what's glandular tissue and what's not to get a good volume. And even measuring breast volume, we've tried all sorts of ways and it's not easy. It's not an easy organ to do that with. So yeah, my— it's just come from, I guess, that's where I grew up. An ultrasound of breast, so it's very semi-quantitative and objective— subjective, I should say. Nevertheless, the appearances are quite dramatic sometimes between breasts. And I think— I know there's been a hands-off approach, but I come from a background of examining breasts for breast cancer, you know, and we'd be looking for lumps, and I did mammograms. And, you know, it might be a case of— I don't know what you think, Pam— of you would definitely feel it's like when you put a probe on, it's soft, you know, there's no real tension.That kind of examination and encouraging the mothers to, to not just handle their breasts but feel them and see if they feel different.

Pam Renee has a question

Renee I'm just wondering about the 5x5 paradigm , it was in the Journal of Human Lactation. One of my colleagues pointed it out to me and I was just a bit like, well, yeah, I was reading a little bit about it. I guess it would probably help to, if you all knew about it, but it was just shifting the paradigm for establishing and maintaining milk production for the setting of mother-infant separation. And it was really just talking about— it sounded like reducing the amount of milk removal. It talks about expressing milk at least 5 times a day, um, early, at least one of those expressings between 1 and 5 AM, and then don't go longer than 5 hours between pumping sessions. And I was a bit like, it sounded just not enough for me, especially with separation. And yeah, but I guess if nobody's heard of it yet, um, it's hard to depend on that. — I just wondered if anyone had heard about it because it was so concerning.

Donna I haven't heard of any of my colleagues that work in the preterm whatever say it's okay to do 5 times a day. Yeah, that's not enough, is it? Often you will see averages of 6 which is a bit concerning. 8 or more in 24. So it will be different for every mom. There will be a mom that can pump 4 times a day, which is ridiculous, and she's the tail of the distribution, and everybody thinks everyone should be like that. And it's the same as women that, you know, I've scanned women that can hand express 30ml before they let down, and they huge milk ducts. Like, that is also the end of the spectrum. So, um, there will be women that can sustain a, like, quite large lactation, but they've got a large storage capacity. So they've got large glandular tissue. Yeah. So they can store enough milk before they downregulate. So they might store 200 mils. Like, most of us could only pump— I would only pump 70, you know, like babies were taking 90. Yeah, so that's where most people sit in that curve. In terms of the overwhelm, again, it's expectations. So, so babies don't feed regularly, so there's some leeway around the 2 to 3 hours, right? There's 2 and a half if it's going to suit before you go to bed versus 4 at night, but then pumping again at 3. Um, so power pump before a longer, a little bit longer stretch. Yeah. Um, yeah, we struggle a bit with what, what's, what's exactly power pumping as well, but you know, most of our studies would show that— well, it depends. I suppose it depends on the pump again. So if your mother is pumping most of their milk out, you don't want to keep pumping, um, even if for sure a short period if she's feeling empty because she doesn't probably need to. So again, it's, it's quite individualized in terms of that.

Renee I think it's all just coming down as counseling our moms, you know, whatever, and doing the best that they can and then counseling them around what will be and what may not be.

Donna Yeah, and I think, um, yeah, exactly. And you say, you know, this would be ideally every 3 hours, but, um, it's going to depend on what's going on at home and stuff like that. As well. So I think it's encouraging. Yeah, and encouraging, you know, to be able to reduce frequency, you really need quite a big production. So then you want to go hard in those first 3 weeks and set that, set that expectation for 3 weeks, and then say, well, look, and then after that, we might be more flexibility around So maybe that takes the pressure off knowing there's an end in sight if things are— if she's not pumping much for some reason, or she can't produce as much. We're very evidence data driven, obviously being in a research world, so you need to track everything you change and, you know, follow up and see that it, it's positive and how many mothers it's positive for versus perhaps negative.

Pam Well, Donna, you've been so generous with your time, generous to present to us tonight. I have really enjoyed what you've had to offer, and I'm confident that that's the case with our participants and with those who will be listening to the recordings. So thank you so much. And, um, take so much care, and thank you for all your amazing work.

Donna There's more to come, Pam. Oh, good, good. We've got to get it out there. Um, yeah, but, uh, it's a pleasure. It's a great space to work in, a bit, um, challenging sometimes, but I think, um, we're all trying to do the right thing. By the mums. And, you know, as long as we keep doing that, we can't go wrong.