Mark D. Cregan

Volume and Frequency of Breastfeedings and Fat Content of Breast Milk Throughout the Day

OBJECTIVE. We aimed to provide information that can be used as a guide to clinicians when advising breastfeeding mothers on normal lactation with regard to the frequency and volume of breastfeedings and the fat content of breast milk. METHODS. Mothers (71) of infants who were 1 to 6 months of age and exclusively breastfeeding on demand test-weighed their infants before and after every breastfeeding from each breast for 24 to 26 hours and collected small milk samples from each breast each time the infant was weighed. RESULTS. Infants breastfed 11 ± 3 times in 24 hours (range: 6–18), and a breastfeeding was 76.0 ± 12.6 g (range: 0–240 g), which was 67.3 ± 7.8% (range: 0–100%) of the volume of milk that was available in the breast at the beginning of the breastfeeding. Left and right breasts rarely produced the same volume of milk. The volume of milk consumed by the infant at each breastfeeding depended on whether the breast that was being suckled was the more or less productive breast, whether the breastfeeding was unpaired, or whether it was the first or second breast of paired breastfeedings; the time of day; and whether the infant breastfed during the night or not. Night breastfeedings were common and made an important contribution to the total milk intake. The fat content of the milk was 41.1 ± 7.8 g/L (range: 22.3–61.6 g/L) and was independent of breastfeeding frequency. There was no relationship between the number of breastfeedings per day and the 24-hour milk production of the mothers. CONCLUSIONS. Breastfed infants should be encouraged to feed on demand, day and night, rather than conform to an average that may not be appropriate for the mother-infant dyad.

Initiation of lactation in women after preterm delivery

Background.  Lactogenesis II describes the onset of copious milk secretion, and the success of lactogenesis II has been determined in women by measuring the changes in the composition of mammary secretion in the immediate postpartum period.

Identification of nestin-positive putative mammary stem cells in human breastmilk.

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.

Importance of Vacuum for Breastmilk Expression

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 mothers maximum comfortable vacuum. CONCLUSIONS Use of the mothers 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.

Computerized Breast Measurement from Conception to Weaning: Clinical Implications

The CBM system has enabled our laboratory to measure breast growth and demonstrate the importance of the short-term local control of milk synthesis in lactating women. Although the specific mechanism by which the short-term control of milk synthesis occurs has yet to be fully understood, it is now apparent that the interaction between storage capacity, degree of fullness, and frequency of milk removal plays a significant role. These factors demonstrate that the breastfeeding mother can take comfort in the individuality of her breast development and feeding pattern, which is uniquely adapted to suit the physiology of her breasts and the developmental requirements of her infant.

Unravelling the Mystery of Stem/Progenitor Cells in Human Breast Milk

Background Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. Methodology/Principal Findings We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6±0.8% (mean±SEM) and 0.7±0.2% of the whole cell population (WCP) were found to be CD133+ and CD34+ respectively, 27.8±9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR), and in 4.17±0.2% and 0.9±0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP) (1.8±0.4% of WCP) as well as CD133+ cells (1.7±0.5% of the WCP). Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM) in the SP cells (50.6±8.6 vs 18.1±6.0, P-value  = 0.02). However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. Conclusions/Significance The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking ones own stem cell for every breastfeeding woman.

Milk prolactin, feed volume and duration between feeds in women breastfeeding their full-term infants over a 24 h period

Previous studies have suggested that the uptake of prolactin from the blood into the milk may be restricted when the alveolus is distended with milk. Therefore the aim of this study was to determine the relationship between prolactin in milk and milk production by measuring the concentration of prolactin in samples of fore‐ and hind‐milk as well as the volume of milk removed for each breast, at each breastfeed over a 24 h period. The mean (± S.D.) concentration of prolactin in milk for all women (n = 15) over the 24 h period was 18.5 ± 11.6 μg l−1 (fore‐milk) and 16.8 ± 12.8 μg l−1 (hind‐milk). The variation between women masked small changes within women in the concentration of prolactin in milk over the 24 h period, therefore a prolactin ratio (individual fore‐ or hind‐milk value divided by the mean for all fore‐ or hind‐milk samples collected over a 24 h period) was determined. The concentration of prolactin was highest in milk between 02.01 and 06.00 h (prolactin ratio for fore‐ to hind‐milk, 1.5), and lowest between 10.01 and 18.00 h (prolactin ratio for fore‐ to hind‐milk, 0.8). Furthermore, we observed that the difference in prolactin concentration between the fore‐ and hind‐milk (fore‐hind gradient) was greatest between 06.01 and 10.00 h (4 μg l−1). To ensure that this effect was not due to permeability in the paracellular pathway, the concentrations of serum albumin and sodium in milk were measured. No significant (P > 0.05) changes over the 24 h period, or with increasing time since last feed were observed. We therefore concluded that when the breast is most drained of milk (in the late evening), and the rate of milk synthesis is greatest, the fore‐hind prolactin gradient in the milk of the following feed (in the early morning) is highest. This is consistent with the observation that prolactin uptake from the blood by the lactocyte is dependent on the fullness of the breast, such that prolactin uptake may be inhibited in full alveoli.

14-3-3σ (sigma) regulates proliferation and differentiation of multipotent p63-positive cells isolated from human breastmilk

The mammary gland is a dynamic organ that only undergoes complete differentiation during pregnancy. Differentiation is fuelled by asymmetric division of stem cells that reside in normally quiescent niches in the resting gland in response to pregnancy-associated hormones. Loss of regulation of stem cells is believed to underlie some breast cancers. This process is poorly understood in humans since it is difficult to extract stem cells from the lactating gland. We have identified a p63-positive population in breastmilk that proliferates and differentiates into at least two separate mammary lineages in culture. Nuclear translocation of p63 coincides with expression of the cell-cycle arrest protein 14-3-3σ (Sigma) and precedes differentiation. Transient down-regulation of Sigma promotes maintenance of the p63-positive population without affecting normal differentiation. We propose that p63-postive cells from breastmilk represent a novel source of cells to model regulation of mammary gland development and tumorigenesis.

Pre-term delivery and breast expression: consequences for initiating lactation.

This paper determined the success of lactogenesis II in women expressing milk for their pre-term (31-35 weeks gestation) infants. Milk citrate lactose sodium and total protein on day 5 postpartum were measured and compared with women breast-feeding full-term infants. Findings noted no difference between the means of the lactogenesis markers for expressing and breast-feeding women. However there was a greater variation of the mean in expressing women compared to the mean in breast-feeding women. Statistical analysis also revealed that 80% of expressing women had at least 1 lactogenesis II marker at pre-initiation concentrations but none had all 4 markers. Furthermore it was indicated that the delay in lactogenesis II partly resulted to compromised initiation among pre-term expressing women. No significance difference was noted in the prolactin concentration of milk and mean variation between expressing and breast-feeding women suggesting that prolactin deficiency was not responsible for the compromised initiation in pre-term expressing women.

Maternal Modulation of Specific and Non-Specific Immune Components of Colostrum and Mature Milk

The neonates of most mammalian species cannot survive on an adult diet or exposure to an array of adult pathogens. Lactation provides a transitional period for the immature newborn to adapt to the adult environment. However, there is immense variation in the metabolic maturity of mammals at birth, as well as in the rate of postnatal growth and development during the lactation period. As a result, large differences in milk composition have evolved among mammals (Oftedal and Iverson, 1995). Furthermore, large progressive changes in milk composition occur during lactation to meet the changing metabolic needs of the young (Nicholaset al.1997). For example, the Tammar Wallaby has a relatively short gestation period, during which Phase 1 of mammary development occurs. The animal weighs only 400 mg at birth and remains permanently attached to the teat for the next 130 days, while growing to 100 g (Phase 2A). The pouch young (PY) then detaches from the teat and sucks intermittently to reach a weight of 400500 g by 200 days of age (Phase 2B). The PY then leaves the pouch intermittently until 260 days when it exits the pouch permanently, finally weaning at 300 days, at a weight of 1.6-2.5 kg (Phase 3). The increase in body weight of the young from about 0.5 g to 2000 g during lactation is associated with major changes in milk composition that define phases 2A, 2B and 3 of lactation. Both mammary gland size and milk production increase commensurate with the rate of growth of the young.