Study Shows Hormones and Bacteria in Breast Milk Change Daily

Breastfeeding carries countless health benefits for both the baby and the mother. The nourishment a baby receives through breastfeeding is tailored to its needs, and at the same time, it supports the mother’s own health. What makes breast milk even more remarkable is that it contains numerous bioactive components that change over the course of the day and influence the infant’s circadian rhythm, for example, signaling when it is time to sleep. Yet, it is not always possible for mothers to breastfeed at the same time every day, so researchers studied how breast milk changes over 24 hours. In a new study, “Day/Night Fluctuations of Breast Milk Bioactive Factors and Microbiome,” scientists analyzed 24-hour changes in hormones and bacterial composition.

Scientists have noticed differences in the levels of these bioactive compounds depending on the time of day. This discovery highlights the importance of not only breastfeeding when possible, but also of labeling expressed breast milk according to the time it was pumped. By doing so, even in situations when direct breastfeeding is not feasible, the baby can still receive milk that matches the natural rhythms of day and night.

As explained, the scientists collected 10 mL of breast milk from 21 participants at 6 a.m., 12 p.m., 6 p.m., and midnight on two separate days, approximately one month apart. An additional 17 participants provided samples collected at the same time, resulting in 236 samples.

The results showed that some components of breast milk, especially melatonin and cortisol, changed throughout the day. Melatonin peaked at midnight, while cortisol was at its highest in the early morning. The bacteria in breast milk remain largely the same throughout the day and night, meaning that milk always has a similar diversity of microbes. However, some types of bacteria change in number at different times of the day. At night, the number of bacteria that normally live on the skin, such as Prevotella and Finegoldia, increases, while during the day, the number of bacteria from the environment, such as Chryseobacterium and Clostridiales, increases. The composition of bacteria is also affected by the age of the child and the mother’s body weight, with these changes being more pronounced in mothers with higher body weight.

The results suggest that feeding expressed milk can be timed to help preserve natural biological rhythms and support healthy infant development.

The first author, Dr. Melissa Woortman, a recent PhD graduate from the Department of Nutritional Sciences at Rutgers University, answered a few questions for Unknown Focus.

“We decided to collect 10 mL of breast milk at each time point to ensure we had enough breast milk for our analyses.”

Why was 10 mL of breast milk collected? Why was there about a one-month gap between the two sampling days, and why did 17 participants provide samples only once?

Dr. Melissa Woortman: We decided to collect 10 mL of breast milk at each time point to ensure we had enough breast milk for our analyses. The timing of the sampling instances was due to this study being part of a larger study that ended up stopping due to the COVID-19 pandemic. The one-month separation aligned with the original study visit timing, and we were able to continue this smaller part of the study after some of the restrictions were lifted. Some of the mothers only provided one sampling instance due to the timing of the pandemic; the second sampling could not be obtained due to the shutdown.  For the other mothers who provided only one sample, they were a part of a different study that focused on the impact of COVID-19 infection and the time-of-day variation of breast milk components in mothers who just gave birth. Since that study only focused on the immediate postpartum period (first week after giving birth), only one sampling instance was collected for each subject.

The scientists collected the samples within 48 hours and transported them on ice or dry ice to the laboratory, where they were stored at −80°C until analysis.

Does this mean that storing breast milk at such low temperatures preserves it without affecting its hormones or bacterial content?

Dr. Melissa Woortman: Storing samples at -80°C is standard for laboratory microbiome analyses to essentially get a “snapshot” of the bacterial communities that are present in the samples. At this temperature, bacterial growth is slowed down greatly, and many milk components remain stable in terms of concentration.

“This research could potentially inform women who do not breastfeed about the complexity of breast milk and that it is a dynamic food for infants.”

How can these data help women who, for example, do not or cannot breastfeed, and could this research contribute to developing baby formulas that align with the mother’s circadian rhythm?

Dr. Melissa Woortman: This research could potentially inform women who do not breastfeed about the complexity of breast milk and that it is a dynamic food for infants. It is possible that this research could inform infant formula development since formula usually does not account for changes in breast milk composition that occur over 24 hours.

The researchers note that, due to the limited number of participants, the study did not cover all important factors, such as delivery method and maternal diet. Larger and more diverse cohorts will be needed for more reliable results, and it is also important to study how infants respond to the observed changes in milk.

Image: Breastfeeding Class