Abstract Human milk contains diverse live cells that contribute to infant nutrition, immune protection, and maternal-infant health, yet their cellular biology remains difficult to resolve within this lipid-rich and heterogeneous biofluid. Here, we adapted correlative light, electron, and ion microscopy (CLEIM) for nanoscale imaging and metabolic profiling of human milk cells (HMCs) and integrated these spatial measurements with single-cell transcriptomic analysis. This workflow combines confocal microscopy for cell-type identification, electron microscopy (EM) for ultrastructural mapping, nanoscale secondary ion mass spectrometry (NanoSIMS) for spatial elemental and isotope analysis, and single-cell RNA sequencing (scRNA-seq) for transcriptional interpretation. Using this platform, we generated an ultrastructural atlas of HMCs, including lactocytes, neutrophils, macrophages, dendritic cells, and bacteria-like structures. NanoSIMS revealed sulfur- and iron-enriched neutrophil granules, consistent with antimicrobial functions. Stable isotope tracing further uncovered distinct metabolic specializations among HMCs: 13C-glucose-derived carbon was preferentially incorporated into macrophage lipid droplets, supported by lipid-handling transcriptional features, whereas 15N-amino acid tracing identified metabolically active lactocyte subsets associated with translation and secretory programs. These findings reveal cellular and metabolic heterogeneity in human milk that is not captured by conventional profiling methods. More broadly, this work establishes a spatial multimodal framework for linking cell identity, ultrastructure, metabolic activity, and transcriptional state in complex biological fluids, providing a foundation for future studies of human milk biology and maternal-infant health.

Human milk is a complex biofluid containing a diverse array of cells crucial for infant health. Despite their importance, our understanding of these cells remains incomplete due to technical challenges. To fully comprehend human milk cells, high-resolution imaging technologies that can directly measure biological processes are required. We have developed a specialized imaging platform combining light and electron microscopy for human milk cell imaging. To identify different cell types, human milk cells were first stained with several specific cell markers (e.g., EpCAM and MUC1 for lactocytes, CD16 and CD66b for neutrophils, and HLA-DR and CD68 for macrophages) prior to light (confocal) microscopy. Following this, the same cells were processed with osmium staining, resin embedding, and sectioning for electron microscopy, allowing us to observe ultrastructural details. Our imaging workflow has enabled nanoscale visualization of human milk cells, resulting in a first-of-its-kind comprehensive database profiling the organelle-level ultrastructure of different cell types present in human milk. The cells in the human milk are highly heterogenous, featuring a large proportion of lactocytes and lipid droplets, binucleated lactocytes, neutrophil aggregation, neutrophil extracellular traps, dendritic cells/macrophages with bacteria, and immunophagocytosis. This study provides valuable cellular insights contributing to a deeper understanding of human milk biology.

Mastitis is a debilitating condition that can impact around 20% of mothers and is characterized by fever, flu-like symptoms and tender, swollen areas of the breasts. Despite the emerging evidence that breast milk dysbiosis is an underlying cause of mastitis, breast pumps have been implicated as a predisposing risk factor in the pathophysiology of mastitis in breastfeeding mothers. Previous studies have suggested that the use of a breast pump increases a mother's risk for developing mastitis, however, incidence rates of mastitis over the stages of lactation do not match breast pump usage rates. Furthermore, breast pumps, even when used at low vacuum, still promote some breast drainage, thus avoiding milk stasis, which is considered a key factor in the development of mastitis. As a consequence, these data suggest that the literature association of breast pumps with mastitis is more a case of reverse causation and not direct association. Moreover, it is important to note that breast pumps are actually a part of the conservative management of mastitis. In combination, these data show that the breast pump should not be considered a driver in the pathophysiology of mastitis in women.

Researchers have recently called for human lactation research to be conceptualized as a biological framework where maternal and infant factors impacting human milk, in terms of composition, volume and energy content are studied along with relationships to infant growth, development and health. This approach allows for the development of evidence-based interventions that are more likely to support breastfeeding and lactation in pursuit of global breastfeeding goals. Here we summarize the seminal findings of our research programme using a biological systems approach traversing breast anatomy, milk secretion, physiology of milk removal with respect to breastfeeding and expression, milk composition and infant intake, and infant gastric emptying, culminating in the exploration of relationships with infant growth, development of body composition, and health. This approach has allowed the translation of the findings with respect to education, and clinical practice. It also sets a foundation for improved study design for future investigations in human lactation.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), the virus responsible for COVID-19, is a recently discovered coronavirus that as of October 2020 has reached across the globe infecting over 33 million people, of which approximately 1 million have died (1). Coronaviruses are a large family of viruses known to cause respiratory infections in humans. These viruses also include Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The most common symptoms associated with COVID-19 are fever, dry cough, and tiredness. Other less common symptoms include nasal congestion, headache, conjunctivitis, sore throat, diarrhea, loss of taste or smell and general aches and pains. Importantly, some people may experience very mild symptoms whereas others, especially those with existing underlying medical problems, e.g., high blood pressure, heart and lung problems, diabetes, or cancer, are at a higher risk of serious illness and potentially death (2). As the pandemic unfolded, many questions were asked with respect to transmission routes and modes of infection, with particular interest devoted to the mother-infant dyad and the status of breastfeeding and human milk. Whilst the benefits of human milk and breastfeeding to the mother and the infant are well-documented (3) there was uncertainty in the early stages of the pandemic with respect to hospital practices and recommendations. What was clear, however, was the need for evidence-based recommendations addressing the issue of transmission whilst accounting for the value of breastfeeding. Such recommendations would enable new mothers and their babies to continue benefitting from the advantages of breastfeeding and the use of human milk in this COVID-19 era.

Introduction: While many studies have investigated the importance of optimising pumping patterns for milk removal, the influence of breast shield design on milk removal has not been the focus of extensive investigation. This study aimed to determine the effectiveness of breast shields with either a 105 degrees or a 90 degrees flange opening angle on breast drainage and milk volume expressed during pumping. Material and methods: This study was a cross-over, randomised controlled non-inferiority trial (Registration; NCT03091985). Mothers (n = 49) of breastfeeding infants participated in the study over two study sessions. Participants were randomly assigned to pump both breasts simultaneously for 15 minutes with either the 105 degrees or 90 degrees breast shield in the first session, and the other shield in the second session. Effectiveness (breast drainage) and efficiency (volume expressed) of both breast shields were assessed after 15 minutes of pumping. Intention-to-treat and per protocol analyses were performed to determine if the 105 degrees breast shield was non-inferior to the 90 degrees breast shield for breast drainage and volume expressed. Perceived comfort was assessed via questionnaire. Results: The 105 degrees breast shield was both non-inferior and superior compared to the standard 90 degrees shield for breast drainage (intention-to-treat, 3.87% (0.01-7.72), P = .049) and volume expressed (intention-to-treat, 9.14 mL (1.37-16.91), P=.02). In addition, the 105 degrees shield was rated as feeling more comfortable (P < .001) and as having an improved fit to the breast (P < .001) compared to the 90 degrees shield. Conclusions: Expressing with the 105 degrees breast shield was more efficient, effective and comfortable compared to the 90 degrees shield. Breast shield design can significantly impact pumping outcomes, and an opening angle of 105 degrees improves both the dynamics and comfort of milk removal.

While many studies have investigated the importance of optimizing pumping patterns for milk removal, the influence of breast shield design on milk removal has not been the focus of extensive investigation. This study aimed to determine the effectiveness of breast shields with either a 105° or a 90° flange opening angle on breast drainage and milk volume expressed during pumping. This study was a cross-over, randomized controlled non-inferiority trial (Registration; NCT03091985). Mothers (n = 49) of breastfeeding infants participated in the study over two study sessions. Participants were randomly assigned to pump both breasts simultaneously for 15 minutes with either the 105° or 90° breast shield in the first session, and the other shield in the second session. Effectiveness (breast drainage) and efficiency (volume expressed) of both breast shields were assessed after 15 minutes of pumping. Intention-to-treat and per protocol analyses were performed to determine if the 105° breast shield was non-inferior to the 90° breast shield for breast drainage and volume expressed. Perceived comfort was assessed via questionnaire. The 105° breast shield was both non-inferior and superior compared to the standard 90° shield for breast drainage (intention-to-treat, 3.87% (0.01-7.72), P  = .049) and volume expressed (intention-to-treat, 9.14 mL (1.37-16.91), P =.02). In addition, the 105° shield was rated as feeling more comfortable ( P  < .001) and as having an improved fit to the breast ( P  < .001) compared to the 90° shield. Expressing with the 105° breast shield was more efficient, effective and comfortable compared to the 90° shield. Breast shield design can significantly impact pumping outcomes, and an opening angle of 105° improves both the dynamics and comfort of milk removal.

AimTo determine the impact of the pumping regimes of women with preterm infants on the daily milk production, and on the short-term rate of milk synthesis during early lactation to support evidence-based recommendations for optimising milk production.MethodsMothers of preterm infants (n=25) recorded start time, finish time and expression volumes from every breast expression on days 10, 15–20 postpartum.ResultsExpressing more often than five times per day did not result in a significant increase in daily milk production. Milk volume per expression per breast increased for intervals between expressions of between 2and6 hours then reached a plateau when the interval between expression was 7 hours or longer. The short-term rate of milk synthesis decreased as the interval between expressions increased until about 7.5 hours at which point it begun to increase (p value associated with interval between expressions^2<0.001).ConclusionThe strong inverse association between the short-term rate of milk synthesis and the interval between expressions for intervals up to 7 hours suggest that the maximum interval between expressions should be 7 hours. Data suggest that, on average, the mothers should express at least five times a day to maximise daily milk production. Considering inter-individual variation, determination of an individual mother’s maximum interval between expressions that does not compromise the short-term rate of milk synthesis will help to optimise daily milk production while minimising the demands on the mother’s time.

Milk ejection is a transient episode critical to milk removal and women typically have multiple milk ejections during breastfeeding and pumping. Recently it was found that milk ejection characteristics such as number of milk ejections and periodicity were consistent throughout 12 months of lactation in women who expressed their milk with an electric breast pump. It is not known whether the stimulation of an infant at the breast influences milk ejection patterns or whether this is a programmed event. The aim of this study was to compare milk ejection patterns during breastfeeding and expressing milk with an electric pump within mothers.

Objective: The most common reason given for discontinuation of exclusive breastfeeding is perceived insufficient milk supply. Breastfed infants show more variation in feeding frequency than bottle-fed infants, and this may lead to a mother lacking confidence in her milk supply if the frequency of breastfeeding sessions does not match expectations based on bottle feeding. We aimed to assist clinicians in supporting breastfeeding mothers by providing evidence-based information on expected changes in breastfeeding patterns and milk intake during exclusive breastfeeding for 6 months. Subjects and Methods: Mothers and their healthy infants who were exclusively breastfeeding (total 24-hour milk intake within the normal range) were studied during two to five 24-hour periods between 1 and 6 months of lactation. Results: Between 1 and 3 months of lactation, the frequency of breastfeeding sessions decreased, whereas both the median and maximum breastmilk intakes during each breastfeeding session increased. These parameters remained constant between 3 and 6 months. The duration of each breastfeeding session decreased steadily from 1 to 6 months, but the total 24-hour milk intake remained constant. Conclusions: Breastfeeding becomes more efficient between 1 and 3 months of lactation, although milk intake remains constant. Clinicians can give mothers confidence that these changes in breastfeeding behavior do not indicate insufficient milk supply, but may be a result of the increase in the stomach capacity of the infants and are an expected outcome of a healthy, normal breastfeeding relationship.

AIM:To document post-discharge feeding practices of preterm infants with chronic lung disease (CLD) and determine if sufficient protein and energy is consumed for optimal growth.METHOD:Protein and energy intakes of preterm infants with CLD were quantified through detailed analysis of measured food and fluid intakes at four corrected age (CA) assessments, post-discharge. Most of the infants were in hospital for the term assessment. Milk intake from breastfeeding was determined by test weighing. Protein and energy intakes were compared with the Australian and New Zealand Nutrient Reference Values (NRV) for healthy term-born infants, and CA z-scores for weight, length and head circumference were calculated using Australian national gestational growth data and Centre for Disease Control 2000 growth data.RESULTS:Ten of the 28 CLD infants who were exclusively receiving expressed breast milk in hospital were transitioned to infant formula within 1 month of discharge. Complementary foods were introduced at a median CA of 3.6 months. Protein intakes almost always exceeded the NRV for healthy term-born infants, and at each assessment, at least 63% of infants met the energy NRV. Longitudinal growth data are available for 20 infants, four of whom had been small for gestational age. At the 12-month assessment, 10 of these infants weighed less than the 10th percentile.CONCLUSION:Preterm infants who develop CLD do not always achieve reference growth in their first year following discharge, despite protein and energy intakes being mostly comparable to those recommended for healthy term-born infants.

Adequate calcium intake is vital for infant health, and some cases of rickets have been associated with a low concentration of calcium in breastmilk. The concentration of calcium in breastmilk has been shown to vary widely both between mothers, and over the course of lactation. To address potential concerns about the adequacy of calcium intake for infants who are exclusively breastfed, we discuss the factors likely to be affecting the concentration of calcium in breastmilk. We review and provide new evidence for a physicochemical model of the interactions of calcium with other components of breastmilk, particularly phosphate, citrate and casein. A proposed mechanism for the control of the concentration of calcium in milk is described that highlights the influence of the concentrations of citrate and casein. Understanding these interactions clarifies why the concentration of calcium in breastmilk is not affected by manipulations of maternal dietary calcium and vitamin D.

OBJECTIVE:To determine the effect of the strength of applied vacuum on the flow rate and yield of breastmilk using an electric breast pump.STUDY DESIGN:Twenty-one breastfeeding mothers and two expressing mothers expressed their breastmilk for 15 minutes using an electric breast pump set at their own maximum comfortable vacuum, and at one to three softer vacuums. Milk yield and flow rate were measured.RESULTS:At the maximum comfortable vacuum (-190.7 +/- 8.8 mm Hg) 4.3 +/- 0.4 milk ejections occurred during 15 minutes of expression and yielded 118.5 +/- 11.4 mL of milk (65.5 +/- 4.1% of the available milk). Softer vacuums yielded less milk volume (p < 0.05) and less of the available milk (p < 0.01). Milk flow rate was greater during the first milk ejection than the third or subsequent milk ejections (p < 0.001). Cream content of the milk was highest after expressing for 15 minutes using the mother's maximum comfortable vacuum.CONCLUSIONS:Use of the mother's maximum comfortable vacuum enhances milk flow rate and milk yield. The cream content of the milk at the end of the expression period was an indicator of how effectively the breast had been drained.

OBJECTIVE:The mechanism by which the breastfeeding infant removes milk from the breast is still controversial. It is unclear whether the infant uses predominantly intra-oral vacuum or a peristaltic action of the tongue to remove milk from the breast. The aim of this study was to use ultrasound to observe movements of the tongue during breastfeeding and relate these movements to both milk flow and simultaneous measurements of intra-oral vacuum. METHODS:Submental ultrasound scans of the oral cavity of 20 breastfed infants (3-24 weeks old) were performed during a breastfeed. Intra-oral vacuums were measured simultaneously via a milk-filled supply line (SNS) connected to a pressure transducer. RESULTS:Vacuum increased during the downward motion of the posterior tongue and at the same time milk flow and milk ducts in the nipple was observed. Peak vacuum (-145+/-58 mmHg) occurred when the tongue was in the lowest position. CONCLUSIONS:Ultrasound imaging demonstrated that milk flow from the nipple into the infant's oral cavity coincided with both the lowering of the infants tongue and peak vacuum. Therefore vacuum is likely to play a major role in milk removal from the breast.

Aim: The objective of this study was to determine whether infants of mothers experiencing persistent nipple pain exerted very strong intraoral vacuums during a breastfeed.Methods: Thirty mothers experiencing persistent pain during breastfeeding (Pain group; infants aged 49.4 +/- 35.5 days) were compared to 30 successfully breastfeeding mothers (Control group; infants aged 55.0 +/- 22.7 days). Infant intraoral vacuums were measured via a small milk-filled tube taped alongside the nipple and connected to a pressure transducer. Milk intake was measured using the test weigh method.Results: Infants in the Pain group applied significantly stronger baseline (-90.8 +/- 54.5 vs. -56.4 +/- 31.4 mmHg, p = 0.004), peak (-214.3 +/- 60.5 vs. 163.2 +/- 62.4 mmHg, p = 0.002) and pause vacuums (-104.8 +/- 67.9 vs. -45.8 +/- 30.3 mmHg, p < 0.001). Despite similar active sucking times (377.5 +/- 175.2 vs. 349.4 +/- 184.0 sec, p = 0.554) the mean milk intake was significantly lower for infants of mothers with nipple pain (41.6 +/- 31.3 vs. 70.7 +/- 30.7 g, p = 0.001).Conclusion: Infants of breastfeeding mothers experiencing persistent nipple pain applied significantly higher vacuum to the breast during breastfeeding despite assistance with positioning and attachment from a lactation consultant. Further investigation into the cause of the abnormally high vacuums is essential to develop successful interventions for these mother-infant dyads.

Stem cells in mammary tissue have been well characterised by using the mammary stem cell marker, cytokeratin (CK) 5 and the mature epithelial markers CK14, CK18 and CK19. As these markers have never been reported in cells from breastmilk, the aim of this study has been to determine whether mammary stem cells are present in expressed human breastmilk. Cultured cells from human breastmilk were studied by using immunofluorescent labelling and reverse transcription/polymerase chain reaction (RT-PCR). We found a heterogeneous population of cells with differential expression of CK5, CK14, CK18 and CK19. Further, by using the multipotent stem cell marker, nestin, we identified cells in culture that were positive only for nestin or double-positive for CK5/nestin, whereas no co-staining was observed for CK14, CK18 and CK19 with nestin. When cells isolated from breastmilk were analysed by using RT-PCR prior to culture, only nestin and CK18 were detected, thereby indicating that breastmilk contained differentiated epithelial and putative stem cells. Furthermore, fluorescence-activated cell-sorting analysis demonstrated, in breastmilk, a small side-population of cells that excluded Hoechst 33342 (a key property of multipotent stem cells). When stained for nestin, the cells in the side-population were positive, whereas those not in the side-population were negative. The presence of nestin-positive putative mammary stem cells suggests that human breastmilk is a readily available and non-invasive source of putative mammary stem cells that may be useful for research into both mammary gland biology and more general stem cell biology.

This study evaluated the longitudinal effect of fish oil in pregnancy on breast milk fatty acid composition and infant outcomes. In a randomized, controlled trial, 98 women received 2.2 g docosahexaenoic acid (DHA) and 1.1 g eicosapentaenoic acid (EPA) or olive oil from 20 wk of gestation until delivery. Fatty acid composition in breast milk (at 3 d, 6 wk, and 6 mo) and infant erythrocyte membranes (at 1 y) were determined by gas liquid chromatography. Breast milk fatty acids were examined in relationship to growth and development. Compared with control group, breast milk from women who received fish oil had proportionally higher DHA and EPA levels at 3 d and 6 wk after delivery, but this difference was no longer apparent by 6 mo. Infant DHA status at 1 y of age was directly related to DHA levels at 3 d, 6 wk, and 6 mo postpartum (but not to antenatal supplementation). Both EPA and DHA in breast milk were positively correlated with Griffith's developmental scores including hand and eye coordination. Thus, supplementation in pregnancy was associated with increased n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in breast milk, particularly in early lactation, and this was positively associated with infant DHA status at 1 y.