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    Issue Date: May 2024

    No Downsides to Donor Milk for Extremely Premature Infants

    • Donor human milk is currently recommended for extremely premature infants
    • Infants fed donor human milk are half as likely to experience necrotizing enterocolitis 
    • Infants fed pre-term formula gained weight more quickly than those who were fed donor milk 

    A typical human pregnancy lasts 40 weeks, and at least 37 weeks of gestation enables a baby’s lungs, heart, and other organs to form and function. Babies defined as ‘extremely premature’—being born before 29 weeks of gestation—are at high risk of disorders because their organs are immature. They are also at higher risk for sepsis, hemorrhage, and the life-threatening intestinal infection necrotizing enterocolitis, which kills up to 50 percent of affected babies.

    Studies have shown that a mother’s milk protects extremely premature infants, who often weigh less than 2 pounds at birth, against necrotizing enterocolitis and improves neurodevelopment until they are of school age [1]. But a mother’s own milk is often unavailable to these infants in part because of their early birth, as pregnancy complications or the infant’s inability to suckle, can reduce the chances of successful lactation. In the absence of a mother’s milk, the American Academy of Pediatrics and the World Health Organization both recommend donor milk over pre-term formula because of studies that suggest that the former offers greater protection against necrotizing enterocolitis. However, no studies had compared the two for their effects on neurodevelopment, weight gain, and other important markers of health in extremely pre-term infants.

    Now, results from a large randomized controlled trial reveal that approximately two years after birth, there is little difference in cognitive scores and other neurodevelopmental markers amongst infants who received donor milk and those who received pre-term formula [2]. The trial was “long awaited” and “well designed and well executed,” according to the authors of an accompanying editorial [3], who were not involved in the study. 

    To compare how infants fared on donor milk and pre-term formula, researchers at 15 hospitals enrolled more than 400 families in a clinical trial between 2012 and 2019. Infants born before 29 weeks of pregnancy and those with a birth weight of less than 1000 grams (2.2 pounds) who had no congenital heart disease, prior infections or other serious illnesses were included. The researchers only included babies who had received little to no of their birthing parents’ own milk in the first three weeks of birth. Approximately 200 received donor milk from human milk banks, and about the same number received pre-term formula, a specialized form of nutrition fortified with additional calories and dietary supplements necessary for extremely pre-term infants. Infants in the study began receiving either donor milk or formula at about 16 days after birth and received the diet for their group for approximately 56 days. The study diet was discontinued at about 37 weeks of age. 

    Babies in both groups were weighed weekly, and their length and head circumference were measured once in two weeks. Infants fed donor milk gained weight more slowly than those who received pre-term formula, but both gained in length and head circumference equally well. 

    At about 22 to 26 months after the date that these infants would have been full-term, clinicians conducted a commonly used test to gauge brain development. This test measures language development, motor skills such as a child’s ability to walk, jump or grasp objects, social and emotional development, cognitive impairment, and more. The researchers found no significant difference between the scores of infants in either group. 

    The greatest benefit of donor milk, it turned out, was reducing the odds of necrotizing enterocolitis: infants who received donor milk contracted the infection half as often as those in the pre-term formula group. 

    The study included a total of 483 children and is likely the largest to compare the benefits of donor milk and pre-term formula. The researchers stopped recruiting infants in part because it became extremely difficult. When they began the work in 2012, less than 25 percent of centers in the Neonatal Research Network, a collaboration of NICUs across the U.S., used donor milk. But just a year later, the AAP, WHO and other organizations officially began to recommend donor milk over pre-term formula. By the time the researchers stopped including infants in 2019, more than 75 percent of centers in the Neonatal Research Network were using donor milk, making it difficult to justify placing infants in the pre-term formula group of the trial. 

    The results are not the last word on the relative benefits of human milk and pre-term formula, according to the authors of the accompanying editorial. Although the study found no benefits of donor milk to cognitive scores and other measures of brain development, it also found no downsides. Considering the protection that donor milk offers against potentially lethal necrotizing enterocolitis, data from this large trial support current recommendations to use donor milk for extremely premature infants. 

    Understanding why infants gained weight more slowly on donor milk could help researchers find ways to fortify this crucial food and improve babies’ weight gain in future studies. 

    The authors of the editorial also emphasize that donor milk is not the same as a birthing parent’s milk. A mother’s own milk offers two benefits: it reduces risk of necrotizing enterocolitis and has been linked to better neurodevelopment in extremely pre-term babies. As a result, the authors write that ensuring all extremely preterm infants have access to their mother’s own milk remains crucial “to improve the short-term and long-term outcomes in preterm infants.

    References

    1. Cortez J, Makker K, Kraemer DF, Neu J, Sharma R, Hudak ML. Maternal milk feedings reduce sepsis, necrotizing enterocolitis and improve outcomes of premature infants. Journal of Perinatology. 2018 Jan;38(1):71-4.
    2. Colaizy TT, Poindexter BB, McDonald SA, Bell EF, Carlo WA, Carlson SJ, DeMauro SB, Kennedy KA, Nelin LD, Sánchez PJ, Vohr BR. Neurodevelopmental outcomes of extremely preterm infants fed donor milk or preterm infant formula: a randomized clinical trial. JAMA. 2024 Feb; 331(7):582-591.
    3. Belfort MB, Perrin M. Delivering on the promise of human milk for extremely preterm infants in the NICU. JAMA. 2024 Jan; 331(7):567-569.

    Complement Proteins in Milk Promote Healthy Gut Microbiome

    • Human and some mammalian milks contain complement proteins, but it was not clear what role they played in infant immunity.
    • A new study in a mouse model demonstrates for the first time that milk complement proteins prevent infections by selectively eliminating bacteria from the infant gut.
    • In mice, and presumably humans, milk complement proteins work independently of antibodies to help establish a protective gut microbiome in infants.

     

    Complement proteins were so named because they act as immunological sidekicks to antibodies and other immune cells. Like Robin creating a diversion to help Batman take down the villain, complement proteins from the bloodstream help antibodies identify and attack bacteria. Unlike Robin, however, complement proteins can occasionally destroy bacteria or other pathogens on their own. As a part of the innate, or non-specific, arm of the immune system, complement proteins are one of the body’s first lines of defense in preventing infection—no superhero antibodies needed. 

    In addition to circulating in the blood, complement proteins are also passed from human and other mammalian mothers to their infants in milk. However, the influence of these acquired complement proteins on the infant’s immune system is not well understood. Do maternal complement proteins assist milk antibodies? Are they on the front lines attacking foreign microbes on their own? Or do they serve another immunological role in infants? A new study [1] demonstrates for the first time that milk complement proteins, without the help of antibodies, protect infants from gastro-intestinal infections by modifying the composition of the infant’s gut microbiome. 

    Milk complement protects infants

    To zero in on the immunological role of maternal complement proteins among the multitude of immune factors present in milk, a team of researchers from Johns Hopkins University disabled or “knocked out” the sections of DNA coding for complement proteins in a mouse model [1]. Without genetic instructions for making complement proteins in their liver cells, mouse mothers (aka dams) could not pass complement to their nursing pups in milk.

    The study authors hypothesized that infants receiving complement in their milk would fare better during an immunological challenge than those without milk complement [1]. To test this, their study design had two experimental mouse groups: one with a knocked out C1 protein and another with a knocked out C3 protein. There are over 30 different complement proteins, but these two proteins were selected because of their key roles in activating other complement proteins and immune cells in both innate and adaptive immune responses [1,2]. 

    To tease out the influence of maternal (milk) complement versus infant-produced complement, the researchers designed a cross-fostering system. C1 knockout dams (C1-), C3 knockout dams (C3-), and control (or wild type, WT) dams were bred to produce litters on the same day. Then, pups were moved so that C1- nursed either C1- or WT pups, C3- nursed either C3- or WT pups, and WT nursed WT, C1- or C3- pups. After 21 days of nursing, the pups were removed from their foster mother and challenged with Citrobacter rodentium (CR), a pathogen that causes infection in the guts of mice.

    As predicted, milk complement proteins offered a protective effect to nursing mice pups [1]. Pups fostered by C1- and C3- dams demonstrated signs of infection from CR, including diarrhea, reductions in body growth, and even death, whereas pups fostered by WT dams had limited or no signs of infection, regardless of their genetic make-up [1]. With no noted differences across knockout and WT dams in the concentration of milk nutrients, total milk energy, or important milk antibodies (e.g., secretory immunoglobulin A), susceptibility to CR was presumed to be mediated by the presence of milk C1 and C3 complement proteins [1]. 

    Milk complement influences the infant’s gut microbiome

    Now came the tricky part: determining how milk complement proteins prevent CR infection. Studies on milk complement from human cell cultures suggested these proteins selectively killed or inhibited the growth of certain types of commensal gut bacteria, including Escherichia coli and Helicobacter pylori—could milk complement behave the same way in a living mouse pup? 

    To investigate this, the research team first profiled the gut microbiomes of WT, C1-, and C3- pups. They applied a sequencing technique called 16s ribosomal RNA that identifies specific types of gut microbes and found that each pup type had a distinct microbiome composition. Of particular interest were the elevated levels of Staphylococcus bacteria in the guts of pups from C1- and C3- fosters compared with pups from WT fosters [1].

    Intrigued by this difference, the researchers gave C1- and C3- dams a broad-spectrum antibiotic that would kill gut Staphylococcus bacteria in both mothers and nursing pups. The antibiotic treatment changed the composition of the gut microbiome by reducing the abundance of Staphylococcus in C1- and C3- pups while also reducing symptoms associated with CR infection [1]. The exact mechanism by which Staphylococcus promotes CR infection is still unknown, but the study team did observe that the reduction of Staphylococcus bacteria was associated with a decrease in infant gut inflammation. This finding suggests that a non-inflammatory gut environment may prevent the growth of pathogenic microbes such as CR [1].

    Milk complement selectively kills bacteria

    The study team had demonstrated an association between milk complement proteins and CR susceptibility and between Staphylococcus abundance and CR susceptibility, but still needed to link them functionally. To find the smoking gun—evidence that milk complement proteins were directly involved in reducing Staphylococcus populations in the infant gut—they turned to in vitro experiments. 

    The whey fraction from milk from WT, C1-, and C3- dams was applied to cell cultures on agar plates containing a particular species of Staphylococcus, S. lentus. The plates with C1- and C3- milk had continued growth whereas the plate with WT milk stopped the growth of S. lentus. They then repeated this experiment using the whey fraction from human milk and found that it also stopped the growth of S. lentus. The time it took for both human and mouse milk complement proteins to stop the growth of S. lentus was indictive of bactericidal activity—complement was killing the bacteria by poking holes in their cell membrane [1]. And the complement continued to kill the S. lentus bacteria even after the researchers removed antibodies such as immunoglobulin G (IgG) and IgA from the whey fraction—milk complement is nobody’s sidekick. 

    Altering the Gut Microbe Environment

    This study demonstrated that milk complement proteins protect infants against infection, but also went to the trouble to demonstrate how they do this, down to the molecular level. And in doing so, the researchers identified a novel molecular role for maternal complement proteins of selectively killing gut bacteria, independent of antibodies. The benefits of passive immunity almost exclusively focus on the role maternally derived antibodies, but this study is a good reminder that innate immune factors in milk, like complement, can be equally important in protecting infants from infection. 

    References

    1. Xu D, Zhou S, Liu Y, Scott AL, Yang J, Wan F. Complement in breast milk modifies offspring gut microbiota to promote infant health. Cell. 2024 Feb;187(3): 750-63.
    2. Ricklin D, Reis ES, Mastellos DC, Gros P, Lambris JD. Complement component C3–The “Swiss Army Knife” of innate immunity and host defense. Immunological reviews. 2016 Nov;274(1): 33-58.

    Gut Microbes Boost Dairy Tolerance in Adults Lacking Lactase

    • Without the lactase enzyme, adults with lactase non-persistence (LNP) cannot digest milk sugars. The lactose sugars are fermented by bacteria in the large intestine, leading to gas production and gastrointestinal (GI) symptoms such as bloating, flatulence, and cramping.
    • Bifidobacteria are beneficial microbes found in the gut that can digest lactose without producing gas.
    • In this clinical trial, repeated consumption of lactose by individuals with LNP led to an increase in bifidobacteria in the gut and a decrease in intestinal gas, as measured by a hydrogen breath test.

    Approximately 70% of adults across the world are lactase nonpersistent (LNP), meaning they cannot digest the milk sugar lactose [1]. They lack the lactase enzyme, which breaks down lactose into glucose and galactose in the small intestine. Without lactase, the milk sugar travels through the gut to the large intestine, where bacteria then ferment the sugar, producing gasses that can cause abdominal cramps, bloating, and flatulence.

    The threshold for experiencing these lactose intolerance symptoms varies among individuals with LNP. Many people with the LNP genotype can digest 12 g of lactose, equivalent to a full glass of milk, without symptoms [2]; however, many still choose to avoid dairy, reaching instead for alternatives, such as oat, almond, or soy milk or lactose-free products. Others choose to consume lactase capsules with their dairy to aid digestion. New studies suggest individuals with LNP can build a tolerance for lactose [3, 4]. 

    In a new clinical trial, published in The American Journal of Clinical Nutrition, researchers show that repeated daily consumption of lactose in LNP individuals alters the gut microbiome and improves their lactose tolerance. These individuals had an increase in beneficial gut microbes called bifidobacteria and produced less intestinal gas, as measured by a hydrogen breath test [5].

    “Bifidobacteria are able to break down lactose into glucose and galactose in such a way that no gas is formed,” says Lonneke Janssen Duijghuijsen, a lead researcher on the study and a Clinical Research Scientist at Wageningen Food & Biobased Research. The bacteria use an enzyme called β-galactosidase to digest lactose. “If you have lots of these beneficial bacteria, you may still be able to consume lactose without impactful intolerance symptoms.”

    In this single-blind study, the researchers gave LNP individuals, who carry two copies of the LNP gene, a daily dose of lactose. The daily dose gradually increased from 6 to 24 g over the course of 12 weeks. To determine the effect of increasing lactose on the gut microbiome, the researchers collected fecal samples from participants before and after the intervention. They performed shotgun metagenomic sequencing to determine the types of bacteria present as well as their abundance. The researchers also measured β-galactosidase activity in the fecal samples and intestinal gas using a hydrogen breath test. Finally, they tracked classic lactose intolerance symptoms, such as bloating, diarrhea, and flatulence, over time [5].

    The researchers found that repeated daily consumption of lactose led to an increase in beneficial bifidobacteria. β-galactosidase activity also increased, suggesting that the lactose was properly digested. This finding was confirmed with a hydrogen breath test, which showed that the LNP individuals produced significantly less intestinal gas while taking lactose. Interestingly, the lactose intolerance symptoms were mild, both at the start and end of the intervention, and variable. Therefore, the participants did not experience a significant improvement [5]. 

    “Symptoms are always subjective and will, therefore, always show high variability,” says Janssen Duijghuijsen. “Moving forward, it would be valuable to explore the factors contributing to this variability … and determine individual thresholds for lactose consumption.”

    She says that in hindsight, the researchers could have used a higher dose of lactose to induce symptoms at the start of the intervention. They could have also screened patients at the beginning of the study and only included individuals with clear lactose intolerance symptoms. 

    “It’s important to note that these results were backed by the relevant and significant finding that the production of gas after the intake of the high dose of lactose was substantially decreased,” says Janssen Duijghuijsen. “This is an objective indication for lactose intolerance.”

    The study adds to the growing body of evidence that links daily lactose consumption with beneficial gut microbes and improved tolerance [4, 5]. Together, these results suggest that individuals with LNP do not necessarily need to avoid dairy products. This is important regarding food security and maintaining good health, particularly in developing countries. Milk is affordable and nutrient-dense, consisting of high-quality proteins, vitamins, minerals, and antioxidants

    However, it is still unclear how long the lactose tolerance persists. “The intestinal microbiota is highly dynamic,” Janssen Duijghuijsen says. “The beneficial effects will not be long-lasting, and continuous exposure is a prerequisite in preventing lactose intolerance symptoms in the long run. It would be valuable to find out how long the observed effects would last.” 

    “We’ve learned a lot about the community of microorganism living in our intestines and their impact on our health,” Janssen Duijghuijsen says. “There is much to discover in this field, which may ultimately lead to more personalized approaches in nutrition and health research.” 

    Janssen Duijghuijsen adds, “One thing is certain — a ‘one-size fits all’ approach does not apply.” 

    References

    1. Storhaug CL, Fosse SK, Fadnes LT. Country, regional, and global estimates for lactose malabsorption in adults: A systematic review and meta-analysis. Lancet Gastroenterol Hepatol. 2017;2(10):738–46.
    2. Savaiano DA, Levitt MD. Milk intolerance and microbe-containing dairy foods. J Dairy Sci. 1987;70(2):397–406.
    3. Szilagyi A. Adaptation to lactose in lactase non persistent people: Effects on intolerance and the relationship between dairy food consumption and evalution of diseases. Nutrients. 2015;7(8):6751–79.
    4. Kable ME, Chin EL, Huang L, Stephensen CB, Lemay DG. Association of estimated daily lactose consumption, lactase persistence genotype (rs4988235), and gut microbiota in healthy adults in the United States. J Nutr. 2023;153(8):2163–73.
    5. JanssenDuijghuijsen L, Looijesteijn E, van den Belt M, Gerhard B, Ziegler M, Ariens R, et al. Changes in gut microbiota and lactose intolerance symptoms before and after daily lactose supplementation in individuals with the lactase nonpersistent genotype. Am J Clin Nutr. 2024;119(3):702–10.

    Amphibian Species Responds to Offspring’s Demands for Milk

    • Siphonops annulatus, a type of egg-laying amphibian called a caecilian, produces milk for its young on demand, a parental care and feeding system that’s not shared by its closest relatives.
    • Many animals besides mammals produce milk for their young, but this caecilian is the first amphibian found to do so in response to signals from its young. 
    • This discovery enables researchers to study the traits and evolutionary significance of different parental feeding and care strategies.

    A worm-like, egg-laying amphibian is the latest addition to the list of animals that produce milk to feed their young, according to a new study in Science [1]. The researchers observed that in the two months after hatching, the young of this species, Siphonops annulatus, crowded around the mother, consuming a secretion produced from her body. The hatchlings also emitted sounds that appeared to act as signals to elicit its production.

    “What seems quite spectacular about this paper is that the young are signaling for the parents to release the milk,” says Katie Hinde, an evolutionary biologist who studies lactation at Arizona State University and who was not involved in the work.

    S. annulatus is the first egg-laying amphibian observed to produce milk for its young on demand—that is, in response to cues from infants—begging the fascinating question of this why this arrangement for feeding and parental care evolved in this species and not other, closely related ones, Hinde says.

    Mammals may be the animals most famous for producing milk for their young, but many other animals do so, including some spiders [2], cockroaches [3], fish [4] and birds [5].  Members of the worm-like group of amphibians called caecilians, to which S. annulatus belongs, are among the least-studied vertebrates. They are known to exhibit another type of parental feeding behaviors, called skin-feeding [6]. In these caecilian species, maternal skin changes consistency and becomes enriched with lipids, and offspring feed on this substance during their first two months of life. Unlike S. annulatus, most caecilian species are viviparous – that is, females give birth to live young. Viviparous caecilians – and other amphibians – are thought to produce milk that is consumed by embryos in the oviducts – the tubes in which embryos develop [7].

    S. annulatus is known to engage in skin feeding. But researchers led by Carlos Jared at the Butantan Institute in Sao Paulo, Brazil, were studying the species’ reproductive biology, they noticed that offspring also consumed a viscous substance secreted from a slit near the mother’s tail. They also saw that the hatchlings were getting nourishment from this substance, which they then referred to as milk.

    The researchers then observed the feeding dynamics of 16 adult females and their offspring in detail. Several times a day, the hatchlings nibbled at the mother’s tail area while moving their bodies and producing high-pitched sounds. These actions seemed to stimulate the release of milk from the mothers’ vents. The researchers aligned video and recordings of hatchlings feeding and determined that their vocalizations were strongest in the minute before the mother’s milk was released. This suggested that the vocalizations act as a biological signal that acts on the mother’s body, much like the sounds of mammalian infants stimulate maternal milk let-down

    Additionally, when S. annulatus hatchlings stopped consuming milk, the mothers seemed to stop producing it, Jared and his colleagues found.  “That lets us know that it is costly, to some extent, for her to be producing this fluid for them,” says Hinde. The energetic cost suggests that the system confers an evolutionary advantage, she says. 

    An analysis of the composition of S. annulatus milk revealed it to be rich in lipids and carbohydrates. The lipid portion contained long-chain fatty acids – primarily palmitic acid and stearic acid. “Lipids are such a rich fuel that it makes sense as a key nutrient for energy for the young,” says Hinde. 

    “We demonstrate that the secretion from the oviducts released through the vent is an important food source provided in response to hatchlings’ signaling,” the researchers write in the paper. “This context can explain why the mother remains entirely dedicated to the hatchlings during the 2 months of parental care, not leaving them even to feed herself.” 

    The study raises a host of fascinating questions, says Hinde. Millions of years ago, there were lots of mammal-like animals that did not produce milk, but we milk-producers outcompeted them during the course of evolution, she says. 

    References

    1. Mailho-Fontana PL, Antoniazzi MM, Coelho GR, Pimenta DC, Fernandes LP, Kupfer
      A, Brodie ED Jr, Jared C. Milk provisioning in oviparous caecilian amphibians. Science. 2024 Mar ;383(6687):1092-1095.
    2. Chen Z, Corlett RT, Jiao X, Liu SJ, Charles-Dominique T, Zhang S, Li H, Lai R, Long C, Quan RC. Prolonged milk provisioning in a jumping spider. Science. 2018 Nov ;362(6418):1052-1055.
    3. Stay B., Coop A.C. “Milk” secretion for embryogenesis in a viviparous cockroach. Tissue and Cell.1974; 6: 669–693.
    4. Sato K, Nakamura M, Tomita T, Toda M, Miyamoto K, Nozu R. How great white
      sharks nourish their embryos to a large size: evidence of lipid histotrophy in
      lamnoid shark reproduction. Biol Open. 2016 Sep ;5(9):1211-1215.
    5. Wang L, Zhu J, Xie P, Gong D. Pigeon during the Breeding Cycle: Behaviors,
      Composition and Formation of Crop Milk, and Physiological Adaptation. Life
      (Basel). 2023 Sep ;13(9):1866.
    6. Kupfer A, Müller H, Antoniazzi MM, Jared C, Greven H, Nussbaum RA, Wilkinson
      M. Parental investment by skin feeding in a caecilian amphibian. Nature. 2006
      Apr ;440(7086):926-929.
    7. Jared C, Mailho-Fontana PL, Jared SGS, Kupfer A, Delabie JHC, Wilkinson M, Antoniazzi MM. Life history and reproduction of the neotropical caecilian Siphonops annulatus (Amphibia, Gymnophiona, Siphonopidae), with special emphasis on parental care. Acta Zoologica. 2019 July;100(3): 292-302.

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