Sunday, December 14, 2014

Human milk has a microbiome - and the bacteria are protecting mothers and infants!

The human microbiome project was a major undertaking by the National Institutes of Health, with a fairly simple mission: understand the bacterial communities living in and on the human body, and the potential impact these communities may have on health. Hundreds of individuals donated everything from feces to nasal secretions. However, one key system was ignored - human milk. That’s right – the microbiome of human milk was not studied.
Probably some of this had to do with a long standing myth that human milk was sterile. Why study something without bacteria, right? But, as we have quickly learned – human milk is far from sterile. The average baby consuming 800 mL/27 ounces of human milk will received between 100,000 and 10,000,000 million bacteria from human milk per day (Fernandez et al., 2013).

Figure 1: The Human Microbiome Project is not interested in milk. I fixed their image to better reflect this.
Fortunately, research into the human milk microbiome has continued despite this oversight by the Human Microbiome Project. It appears that nine “operational taxonomic units” (generally closely related species based on DNA analysis of the bacteria) are extremely common in most mothers studied to date: Streptococcus, Corynebacteria, Bradyrhizobiaceae, Staphylococcus, Serratia, Ralstonia, Propionibacterium, Pseudomonas, and Sphingomonas. These nine groups typically account for more than 50% of total bacteria. Bififobacterium and Lactobacillus are also common, but less universal (Fernandez et al., 2013).
The microbiota of milk appears to be quite stable (Fernandez et al., 2013), although a few factors appear to shape the composition. First, mothers with higher BMIs (in the obese range) produce colostrum with more Lactobacillus, and mature milk with more Staphylococcus and less Bifidobacterium (Cabera-Rubio et al., 2012). Cabera-Rubio and colleagues (2012) also found that greater pregnancy weight gain predicted more Staphylococcus in the milk in a small study of 18 mothers, half obese and half of normal weight. 

But here is the really neat part – guess what else altered the milk microbiota? Type of delivery. Mothers who had caesarian sections had a different milk microbiota than mothers who had a vaginal delivery. And the variation continued – mothers undergoing emergency caesarians after laboring had milk microbiotas closer to those of women who delivered vaginally than women with elective caesarians.

Where do the bacteria come from? Initially, it was thought that the milk microbiome was really just contamination from the skin microbiome. However, this is WRONG, WRONG, WRONG. While the milk microbiome does contain some of the same families of bacteria as skin, multi-site sampling of mammary skin and milk revealed that these are not the same species and/or genera. Instead, it appears that the microbiome of milk comes from several places, including the maternal gut microflora. Current evidence supports dendritic cells as the likely transfer mechanism. These cells, along with some macrophages, can open the tight junctions between cells forming the gut barrier and take in living bacteria. These cells can then maintain the live bacteria for several days in mesenteric lymph nodes scattered throughout the body (Fernandez et al., 2013). Dendritic cells are also pretty picky about what they take up – dead bacteria or latex beads will not activate immature dendritic cells for bacteria uptake, while commensal species, like Lactobacillus, show high levels of binding (Rescigno et al., 2001).
Figure 2: Dendritic cell (shown in blue). Image from

This allows for oral manipulation of the milk microbiome – mothers given supplemental Lactobacillus from three strands, L. gasseri, L. fermentum, L. salivarius, showed transfer of these strands to the milk (Jimenez et al., 2008). 

This lead to the logical question – could these strands be used to treat mastitis? Arroyo et al., (2010) randomized 352 women with mastitis to three groups – one dosed with L. fermentum, one dosed with L. salivarius, and one given standard antibiotic treatment (4 different drugs were used). Bacterial counts for milk were obtained for all mothers on Day 0 – that is before treatment started. All mothers had bacterial counts of 4.35-4.47 log10 CFU (colony forming units) – a little less than double the recommended bacterial counts for milk of 2.5 log10 CFUs. Mothers received 21 days of treatment, and milk bacterial counts were repeated on day 21. Women who received L. fermentum had mean bacterial counts of 2.61 log10 CFUs; L. salivarius had bacterial counts 2.33 log10 CFUs with clinical relief of mastitis, and all reported reductions in reported breast pain. Mothers who received antibiotics did not fare as well. Mean bacterial count for antibiotic receiving mothers was 3.28 log10 CFUs and pain scores were much higher. Three months later, only 8.8% of mothers receiving either L. fermentum or L. salivarius had experienced recurrent mastitis, while 30.1% of mothers receiving antibiotics had. All differences between antibiotic and probiotic groups were significantly different – the kind of significant difference that makes researchers do their happy dance.
Figure 3: This is how I picture the researchers after making this discovery - just substitute a computer for the piano. Gif by PEANUTS.

So the milk microbiome appears to be protecting mothers – but there is also good evidence that it is protecting infants. Little is known about the salivary microbiome of infants, but based on preliminary evidence, it appears to, not surprisingly, have some overlap with the milk microbiome (Nasidze et al., 2009). The milk microbiome also appears to contribute to the microbiome of the infant GI tract, as well as the development of immune function in the infant (Fernandez et al., 2013). Infants supplemented with Lactobacillus fermentum (yes, the same as used for the treatment of mastitis) showed significant reductions in diarrheal and respiratory infections in early infancy compared to control infants (Maldonado et al., 2012). Many of the bacteria in the milk microbiome are protecting both the mother and the infant from infection, and may even be involved in the development of immune tolerance.

Milk remains amazing – even the bacteria in milk!

Arroyo R, Martín V, Maldonado A, Jiménez E, Fernández L, Rodríguez JM. (2010) Treatment of infectious mastitis during lactation: antibiotics versus oral administration of lactobacilli isolated from breast milk. Clinical Infectious Diseases 50:1551–8.

Cabrera-Rubio R1, Collado MC, Laitinen K, Salminen S, Isolauri E, Mira A. (2012) The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery. Am J Clin Nutr. 96(3):544-51.

Fernández L1, Langa S, Martín V, Maldonado A, Jiménez E, Martín R, Rodríguez JM. (2013) The human milk microbiota: origin and potential roles in health and disease. Pharmacol Research 69(1):1-10.

Jiménez E, Fernández L, Maldonado A, Martín R, Olivares M, Xaus J, et al. (2008) Oral administration of lactobacilli strains isolated from breast milk as an alternative for the treatment of infectious mastitis during lactation. Applied and Environment Microbiology 74:4650–5.

Maldonado J, Ca˜nabate F, Sempere L, Vela F, Sánchez AR, Narbona E, et al. (2012) Human milk probiotic Lactobacillus fermentum CECT5716 reduces the incidence of gastrointestinal and upper respiratory tract infections in infants. Journal of Pediatric Gastroenterology and Nutrition 54:55–61.

Martín R, Olivares M, Marín ML, Fernández L, Xaus J, Rodríguez JM. (2005) Probiotic potential of 3 lactobacilli strains isolated from breast milk. Journal of Human Lactation 21:8–17.

Nasidze I, Li J, Quinque D, Tang K, Stoneking M. (2009) Global diversity in the human salivary microbiome. Genome Research 19:636–43.
Rescigno M, Urbano M, Valzasina B, Francolín M, Rotta G, Bonasio R, et al. (2001) Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nature Immunology 2:361–7.

Thursday, October 30, 2014

Biomarkers and milk: Halloween Edition! Old & Scary Milk Myths

Myth #1: Colostrum is poison

The perhaps best known myth about human milk relates to the first milk – colostrum. Many, many cultures have strong beliefs and folklore about colostrum. The most common belief is that colostrum is bad and should not be given to the infant.

As far back as the 1500s, it was regularly suggested that infants should be breastfed by another woman for the first three to fourteen days of life, or sometimes as long as two months, because her milk was “not healthy”. Colostrum especially, because of its sharp visual contrast to mature milk, has had numerous beliefs about it. Some physicians at the time recommended feeding colostrum to a puppy or an adult in order to maintain milk supply. If the mother had to nurse, she should first give the child honey. Gruel was also an acceptable substitute. 

Figure 1: Puppies should not nurse from humans. Dressing them as nurses on Halloween though, is totally okay. Photo: Pinterest.

Yeah, you read that right. Women’s colostrum, the thick, wonderful substance commonly called “liquid gold” because of its richness in immune factors and benefits to the infant was fed to puppies. It is almost certain this contributed to both infant and maternal morbidity and mortality, as newborn infants received consider immunological support from the immunoglobulins in milk and mothers nursing puppies may have been at increased risk of mastitis. “Milk fever” was at one point considered a stage of lactation (Obladen, 2012). 

Fortunately, by around the 1750s, a few physicians figured out that this was probably not the best advice, and started recommending infant breastfeeding within a few hours of birth. This had a beneficial effect on infant mortality rates, as it turns out puppies aren’t very good at removing milk, and too often, by the time the 3-14 days had passed, the woman had no milk and would have to rely on animal milks or gruels and paps. 

The scary part? In many parts of the world, myths persist about colostrum. In my work in Tibetan communities in the highlands of Nepal, more than half the mothers did not breastfeed their infant for the first 3 days and about 10% told us that the first milk was poisonous. When we asked why, several reported that a nurse had provided that piece of information. 

Myth #2: Bad behavior makes bad milk

If you are what you eat, then you are what you make for someone else to eat, or so the story goes. It was long believed that maternal characteristics were in the milk, and while an upstanding lady with a good husband might make good, upstanding milk, she probably wasn’t nursing the baby – a wet nurse was. And finding a good, morally upright wet nurse (that is, someone to nurse your child instead of their own) was a bit complicated. If her baby had died, was her milk any good? If her baby was born out of wedlock, would she pass “moral looseness” onto the child? The ideal candidate was a morally upstanding, lower middle class woman with her own child . . .who could be farmed out to a lower class woman for cheaper wet nursing . . .who could feed her child gruel or other substitutes.  And of course, the wet nurse had to be not only morally upright but free from hysteria, angry thoughts, and “violent afflictions of the mind” that might give the baby rickets, or epilepsy.
Figure 2: Darth Vader as a baby. Likely candidate for moral failings, but blaming milk seems a bit over the top. Unless the Emperor was nursing.

Rickets of course, comes from a lack of Vitamin D, not afflictions of the mind, and epilepsy is not linked with milk in any way shape or form. What is described here is control over women, and specifically control over women’s bodies and reproductive potential. Delicate upper class women could certainly nurse – but because of high childhood mortality, were often instead relegated to repeated, closely spaced pregnancies while infants were fed by wet nurses or artificial means. Their capacity to bear numerous legitimate heirs for a husband was more important than their role in keeping the child alive. Additionally, the idea that bad behavior in milk could affect the infant was a means of social control, limiting women’s capacity to act, circumscribing their behavior and using infant morbidity and mortality as a means of social control over women. 

Sadly, as with the colostrum myths, ideas about maternal behavior and milk quality continue to this day in many parts of the world. An example of this comes from rural Bangladesh, where young married women’s low status within their husband’s families are often associated with limited food and harsh treatment, especially from mother in laws. Many women will substitute paps or gruels for milk, told that their milk is bad because they are disobedient or abused by their mother in law.

Myth #3: Breastfeeding makes your breasts sag

This one is one of my favorites, and is one you will often hear whispered among contemporary American women. Breastfeeding makes your boobs sag. You may have heard it. You may have said it. You may have thought it. It may have been said to you.

But is it true?

Figure 3: This boob hat might sag during breastfeeding . . .from a milk coma! Hats by:

NOPE!!!!!! Everyone take a deep breath and dance around with that joyful news, because we once again have science on our side. Rinker et al., (2007) looked at changes in the position of the mammary glands in women who breastfed and women who did not. The results? There was no difference in the amount of structural loss integrity (a nice way of saying sag) between the women that breastfed and the women that used formula. It was pregnancy, not breastfeeding that lead to the changes in collagen and support for the breasts!
On that happy note, have a nice Halloween, and remember: colostrum = good, behavior doesn’t influence milk, and breastfeeding does not cause breasts to sag.

Wednesday, September 10, 2014

The rise of milk volume measurement products and the implied lack of confidence in maternal bodies

Today, I was stunned to see a press release for a new breastfeeding measurement tool, the MilkSense. From a research standpoint, I will admit that any handheld device that allows me to accurately measure milk transfer and is small enough to fit in my backpack makes me excited. Those highly accurate baby scales are heavy when they have to be moved by hand at altitudes greater than >10,000 feet. But, when I see devices like this, I, the researcher, want to be the product’s intended audience. Sadly, I, and other researchers, am never the target audience.

It is always mothers.

And that is a huge problem. 

Why? Because what devices like the MilkSense and the recently discontinued Milk Screen test strips actually serve to do is not to increase maternal confidence in the capacity to produce milk, but to call into question the ability of breastfeeding to meet an infant’s needs. 
Figure 1: MilkScreen. These were test strips used to calculate milk volume. Now discontinued.

Figure 2: MilkSense. This device measures electrical changes in the breast and calculates volume based on the changes. It is now packaged with a scale for weighing the baby.
Human milk and human babies evolved together, and hands down, human milk is the best option for human babies. Certainly, there may be individual instances that differ, but for the global whole, human milk is the best.  Commercial infant formula is a fairly recent development, and before that, while the use of milk substitutes and supplements was common, breastfeeding was the de facto method of feeding infants.  It is incredibly unlikely we as a species would have survived and produced some 7 billion humans if human milk wasn’t so good at meeting infant needs. So why then, are we so convinced that women can’t breastfeed?

Well, as discussed in the multi-part series “who manages themammaries”, “I didn’t make enough milk” is the single most common reason women in the United States give for cessation of breastfeeding.  However, making enough milk is, in itself, a problematic concept. How do you define not making enough milk? Too often, common behaviors such as closely spaced feedings, are used as a yardstick for “not enough milk”.  
Baby wants to nurse every two hours. Mother uses MilkSense. Sees she is only transferring 60cc of milk to the infant (2.02 ounces) per feeding.  Googles this, and sees that formula fed and babies receiving expressed human milk usually take 3-4 ounces per feeding. Decides she does not have enough milk, and starts supplementing the infant with formula. 

Except, if we do the math, she had plenty of milk! Two ounces every two hours is 24 ounces a day -  and this is within the range of normal milk intake for a breastfed baby (19-30 oz).  Why then, is she only making 2 ounces per feeding?
Because that day, or that feeding, that was likely all the infant wanted. Figure 3 shows the average stomach size of an infant at three different ages. Two ounces may perfectly fit that tiny stomach. For another baby, four ounces may be the perfect amount, and chances are, this baby may be taking 120 ccs (4 ounces) at each feeding.  Breastfeeding is the ultimate supply and demand system: the infant demand typically sets the supply. 

There are of course, some exceptions. Mothers with biological insufficient milk syndrome, often the result of insufficient glandular tissue or similar, may never meet the infant’s demand. This is, as I have said elsewhere, part of normal human biological variation. 2-5% of people’s pancreases fail to make insulin. Two to five percent of people cannot make milk. But when the population levels are at >50%, then we have a problem. If 50% of people suddenly developed Type 1 diabetes over the next decade, we’d assume that something was terribly, terribly wrong in the environment and not that it was a biological reality that 50% of people cannot make insulin. Why then, do we buy this argument for milk synthesis?

If you answered “it’s the economy, stupid,” you’d be about half right. Breastfeeding has become a major business. A market that used to start and end with breast pumps and boppies now has a tremendous number of additional products and “tools” available to mothers. And while numerous options of pumps have allowed many women to meet their breastfeeding goals while engaging in other activities, much of the industry thrives on the construction of maternal anxieties about making enough milk to feed a baby. MilkSense, MilkScreen, and other products like this attempt to quantify milk transfer without a complete picture of the breastfeeding relationship between the mother and baby.  Feeding frequency is as important as volume transferred per feeding – and volume transferred may have more to do with stomach capacity and hunger than production capacity of the breast. Maybe yesterday was 90F, and in attempt to stay hydrated, the infant that usually nurses every 3-4 hours wanted to nurse every hour to eliminate thirst. Today, the infant is less interested in nursing, because it is cooler, a tooth aches, older sibling is distracting. But the volume tests don’t take into account normal infant behavior. Instead, breastfeeding is re-framed as milk production. The objective is uniform production across multiple days, similar to factory production of goods. But breastfeeding is not factory production, rather it is a biological practice, informed by maternal behavior, infant demand, and social factors. 
Figure 3: Normal variation in milk production across a single day. Photo by Megan Hart.

While the use of these products may serve to increase maternal anxieties – another legitimate concern may be that they may become substitutes for far more successful breastfeeding interventions - namely social support. Peer-based breastfeeding support, WIC breastfeeding support, professional breastfeeding support, and even web-based breastfeeding support groups focus on the mother and the baby, not quantifying production. The motto “watch the baby, not the clock” may be “watch the baby, not the device, the scale, the pump, the app.” Further, the substitution of such devices for expert care may identify low milk supply in some instances, but cannot offer solutions. Instead, you have a mother in isolation with a device or test telling her she does not make enough milk. Breastfeeding support however, would be able to investigate why – and refocus the “problem” as not one of production. 

MilkScreen has been pulled from the US market, and it is unclear what the fate of MilkSense will be.  MilkSense is not currently available in the United States and only recently became available in Israel.  It will be interesting to see the public response to MilkSense. 

Monday, August 11, 2014

Swirled or shaken? Does shaking actually damage milk - the scientific evidence


Perhaps one of the most widespread pieces of advice women expressing milk will hear is about the best way to remix milk after expression. Human milk separates after expression (Figure 1) and needs to be remixed before feeding. 

Figure 1: Milk samples (1.5 mL) from 3 different mothers allowed to separate to show the variation in milk fat. Photo: EA Quinn/Biomarkers & Milk.

Many, many websites and books have strict recommendations for the remixing: swirl, never shake. 

As an anthropologist and a bench scientist, I am always interested in the natural history of advice, Where did this advice to swirl, never shake, come from? Upon investigation, I found 3 primary reasons given for why expressed milk should be swirled, never shaken:
1)      Shaking denatures proteins
2)      Swirling helps to remove fat globules stuck to the side of the container
3)      Shaking damages cells.

But, like many before me, I can’t find any actual scientific evidence. I started with PubMed, the national, searchable database of scientific literature ( Figure 2).   

Figure 2: Screenshot of my PubMed search for shaking breast milk. Stirring breast milk looked similar, but with less hits. None were relevant. Image: me.
Here is what I found – and how I went about trying to solve this issue.
Let’s start with #1: shaking denatures proteins. There are many, many different types of proteins in human milk and these are highly variable in size. In addition to size variations, there are also going to be major differences in the way in which proteins are folded – with denaturing being the unfolding of these proteins. 

There are no published papers on this topic. Since the literature was not an option, I turned instead, to math and physics. The idea that shaking denatures proteins is based on the shear force the proteins would be exposed to during shaking.  We need two pieces of information here: what level of force is generated by shaking and what level of force denatures proteins. 

Several reference values for the shear force necessary to denature proteins were available in the literature. Most data however, were based on experimental models of the protein in isolation, when micro-tweezers could be used to literally rip the protein apart. This model is not valid here – what we need is a measure of the shear force necessary to denature a protein in a liquid medium.  Again, we don’t have any studies in human milk, so we will have to substitute water as a medium – and given the composition of human milk, this is a reasonable substitute. In a highly viscous medium, similar to milk, α-amylase (a protein involved in starch digestion found in breast milk), requires a force of 3 x 10^4 Pa to denature the protein.
Figure 3: Alpha-amylase, of pancreatic origin. Image from:
Proteins with beta folds, it is estimated, would be much more resistant to shear force. The predicted force (in a highly viscous medium) necessary to shear a beta protein would be 2 x 10^5 to 10^7 Pa. 

So how much force can a human arm generate? Again, there is no direct measurement for a human shaking a highly viscous medium (but there is plenty of data on ketchup).  If you’ve goggled this (or seen Mythbusters) you know an elite boxer can punch with 5000 pounds of force, or more than 22,000 Newtons. 
Figure 3: The action of boxing, as demonstrated by Manny Pacquiao, is very, very different than the action of shaking breast milk in a container. Image:

But boxing, pitching, and shaking are very different actions – and this causes some interesting differences in the way in which force must be calculated.
When you pitch or punch, the entire body is involved in the action. Punching involves rotation at the waist, shoulder, and elbow. Pitching involves the same rotation, plus the fingers. But shaking is typically done with a stationary shoulder and body and the primary point of movement at the elbow. This is going to limit the force the arm is generating – and the forces extended to the container.  The best analogy in the literature for shaking a container is, remarkably, swinging a hammer, as the hammer swing comes mostly from the elbow. Even a hammer swing is probably an over-estimation, as the shoulder may be involved. 

The average speed for swinging a hammer is 4 meters per second, with maximum times closer to 10 meters per second. The average hammer weights about 3 pounds – the average container of breast milk will weigh a little bit more than 4 ounces.  Now, one thing about a liquid medium is that the forces within the fluid may vary considerably – but it is still unlikely that the human arm will generate enough force through shaking to damage the proteins.  Earlier studies (Thomas and Dunnill 1979) reported that proteins were often stable under shear forces exceeding 9000 s-1 for more than 15 hours. 

One additional factor serves to protect the proteins in human milk, particularly those proteins that are hormones or immune factors rather than more nutritional proteins. We know for example, that many of the hormone proteins are bioactive infant circulation, and thus survive digestion in the infant stomach. Many of these protein hormones are found in a glycosylated form – that is, with the protein has added sugars attached to it that protect the protein structure and serve to reduce the risk of denaturing.  Other proteins may be packaged within the membrane bound fat globules, which will further act to protect the proteins from damage. 

Skipping ahead to #3 – shaking damages cells – the math from above remains important. Again, it is unlikely that the human arm is capable of generating enough force to damage the cells in the milk. Most of the research looking at shear forces and cell damage uses a platelet cell model (Christi 2001). Platelets are not found in human milk, and are also more prone to cell damage and death than many of the other cells commonly found in human milk. Again, human milk specific data are not available – except for spinning in a centrifuge – and we are substituting a leukocyte model for the reference cell. Moazzam et al., (1997), in a study of leukocytes exposed to shear forces in a rat model, found that leukocytes incurred very little damage from shear forces.  Breast milk cells are likely exposed to high shear force at multiple points in their normal life course – from milk ejection to swallowing to digestion, and may be more resistant to cell damage (Papoutsakis 1991). 

Concern #2: Swirling helps remove the fat stuck to the side of the contained.
Again, there are no available data. However, in a study of ultrasonic mixing versus stirring, Garcia-Lara et al., (2013) found that samples mixed by ultrasonic waves had higher fat, suggesting that the ultrasonic mixing was better at removing fat adhering to the sides of the container compared to manual mixing. Current research protocols for measuring milk fat in samples have used multiple inversion techniques to mix milk to ensure adequate mixing – and inversion is a lot closer to shaking than swirling.

So what is the final verdict? There is no published evidence to support that shaking actually damages breast milk when compared to swirling. Many of the issues identified with shaking are better described as myths, and simply do not hold up when the actual shear forces are calculated. Certainly, it would be awesome if we could do an in depth study of this – have women swirl and shake milk with sensors on the hand and in the milk cup and actually measure the acceleration of the hand and then analyze the milk. I suspect however, that we wouldn’t find much damage. 

Sarah and I were discussing the origins of this myth while I was working on this post over the last several days.  She made a really excellent point about this myth – “Really I think it's just one more way to make breastfeeding seem super hard and easy to mess up.” And it seems to be one piece of advice that while well meaning, may contribute to the persistent idea that human milk is fragile, easily damaged, and requires a high degree of care. It serves as one more perceived “threat” mothers (and fathers and caregivers) pose to human milk – the “if you aren’t careful, you’ll damage it and you can’t damage formula*” underlying subtext that serves to undermine breastfeeding mothers.

*see all the recalls and allowable insect parts

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Papoutsakis ET. Fluid-mechanical damage of animal cells in bioreactors. Trends Biotechnol. 1991 Dec;9(12):427-37.

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