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Quantification of human milk (HM) fat is important for determining the energy intake of infants. The simplest and most rapid method is the creamatocrit method. However, the reliability of the creamatocrit has not been comprehensively investigated. The aims of this study were to test the inter- and-intra-rater reliability of: 1) HM sampling after hand- or-machine mixing methods and 2) HM fat measurement by the creamatocrit method. Inter-and-intra rater HM sampling after hand- or-machine mixing methods had high intraclass correlation coefficient (0.91). Inter-rater reliability of measurement of HM with low fat (2%) resulted in high variability (median coefficient of variations (CVs) 15%). Intra- and inter-rater reliability of measurement of HM with higher fat (3.5%) had low variability (median CVs 10%). As the greatest variation in the creamatocrit method occurred during the measurement of HM samples with low fat, duplicate readings are necessary to reduce discrepancies in every HM fat determination.

BackgroundInfants breastfed on demand exhibit a variety of feeding patterns and self-regulate their nutrient intake, but factors influencing their gastric emptying (GE) are poorly understood. Despite research into appetite regulation properties of leptin, there is limited information about relationships between human milk leptin and infant GE.MethodsGastric volumes were calculated from ultrasound scans of infants' stomachs (n=20) taken before and after breastfeeding, and then every 12.5 min (median; range: 3-45 min) until the next feed. Skim milk leptin and macronutrient concentrations were measured and doses were calculated.ResultsThe leptin concentration was (mean±SD) 0.51±0.16 ng/ml; the leptin dose was 45.5±20.5 ng per feed. No relationships between both concentration and dose of leptin and time between the feeds (P=0.57; P=1, respectively) or residual stomach volumes before the subsequent feed (P=0.20; P=0.050) were found. Post-feed stomach volumes (GE rate) were not associated with leptin concentration (P=0.77) or dose (P=0.85).ConclusionGE in term breastfed infants was not associated with either skim milk leptin concentration or dose. Further investigation with inclusion of whole-milk leptin and other hormones that affect gastrointestinal activity is warranted.

Objective: We aimed to determine if a novel feeding system where milk only flowed when the preterm infant created a vacuum would influence time to full oral feeds, the length of stay (LOS) in hospital and breastfeeding at discharge. Study Design: This was a randomized controlled trial in the tertiary neonatal intensive care unit at King Edward Memorial Hospital, Perth, Australia. Eligibility criteria were: preterm infants of gestational age 25 to 34 weeks receiving >75% human milk by gastric tube. Infants were randomly assigned to being fed with a novel teat (NT) or conventional teat (CT). Intention to treat analysis was performed. Result: Time to full suck feeds was not different between groups. LOS was shorter (mean: 2.5 days; P=0.026) and less formula was fed at discharge in the NT group (P=0.036). Conclusion: Use of a NT that releases milk when the infant applies vacuum while establishing breastfeeding reduces duration of hospitalization of preterm infants.

Breastfed infants have a reduced risk of becoming overweight and/or obese later in life. This protective effect has been partly attributed to leptin present in breastmilk. This study investigated 24-h variations of skim milk leptin and its relationship with breastmilk macronutrients and infant breastfeeding patterns. Exclusive breastfeeding mothers of term singletons (n = 19, age 10 ± 5 weeks) collected pre- and post-feed breastmilk samples for every breastfeed over a 24-h period and test-weighed their infants to determine milk intake at every breastfeed over a 24-h period. Samples (n = 454) were analysed for leptin, protein, lactose and fat content. Skim milk leptin concentration did not change with feeding (p = 0.184). However, larger feed volumes (&gt, 105 g) were associated with a decrease in post-feed leptin levels (p = 0.009). There was no relationship between the change in leptin levels and change in protein (p = 0.313) or lactose levels (p = 0.587) between pre- and post-feed milk, but there was a trend for a positive association with changes in milk fat content (p = 0.056). Leptin concentration significantly increased at night (p &lt, 0.001) indicating a possible 24-h pattern. Leptin dose (ng) was not associated with the time between feeds (p = 0.232). Further research should include analysis of whole breastmilk and other breastmilk fractions to extend these findings.

MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing, and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises three main fractions: cells, skim milk, and lipids. Typically, the skim milk fraction has been measured in isolation despite evidence that the lipid fraction may contain more microRNA. This study aimed to standardize isolation of microRNA and total RNA from all three fractions of human milk to determine the most appropriate sampling and analysis procedure for future studies. Three different methods from eight commercially available kits were tested for their efficacy in extracting total RNA and microRNA from the lipid, skim, and cell fractions of human milk. Each fraction yielded different concentrations of RNA and microRNA, with the highest quantities found in the cell and lipid fractions, and the lowest in skim milk. The column‐based phenol‐free method was the most efficient extraction method for all three milk fractions. Two microRNAs were expressed and validated in the three milk fractions by qPCR using the three recommended extraction kits for each fraction. High expression levels were identified in the skim and lipid milk factions for these microRNAs. These results suggest that careful consideration of both the human milk sample preparation and extraction protocols should be made prior to embarking upon research in this area. J. Cell. Biochem. 116: 2397–2407, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

The duration of exclusive breastfeeding is affected by maternal confidence and perception of milk supply, but objective measurement of milk supply is rarely used. Mothers of preterm infants have found measuring milk supply by in-home test weighing to be helpful to ascertain their infants' breastfeeding intake. This study aimed to determine if this technique affects breastfeeding confidence of mothers of term infants.Participants filled in a survey of their breastfeeding confidence and perceptions of breastfeeding behavior prior to and after a 24-hour period of recording the times of feeds, amounts of each breastfeed or bottle feed (expressed breastmilk or formula), and amounts of breastmilk expressed.The breastfeeding confidence of at least 66% of mothers who were initially confident was maintained, and the breastfeeding confidence of at least 11% of mothers who were initially not confident was improved after objective measurement of milk supply and breastfeeding behavior.Measurement of actual milk supply and breastfeeding behavior provides objective information that can guide clinicians in the management of lactation. It can also help to maintain or improve breastfeeding confidence.

The goal for preterm infants is to achieve full oral feeds quickly and ultimately progress to full breastfeeding. Supplementary oral feeds are often given when the mother is not available to breastfeed. Bottles typically deliver milk in a different fashion compared to breastfeeding, which is thought to hamper transition to full breastfeeding. The aim of this study was to compare the sucking dynamics of preterm infants fed at the breast to feeding with an experimental novel teat (NT) designed to release milk only upon the application of vacuum. Simultaneous ultrasound imaging of the infant oral cavity and measurement of intra-oral vacuum was performed during a breastfeed and a feed with the NT. Test weighs were used to measure milk intake. Linear mixed effects models were performed to investigate differences by feed type, and simultaneous linear regression was performed to investigate individual patterns. Tongue movement was not different between breastfeeding and the NT. Intra-oral vacuums (median (interquartile range: IQR)) were significantly lower with the NT (Baseline vacuum: −5.8 mmHg (−11.0, 0.1); Peak: 40.0 mmHg (−54.6, −27.1)) compared to breastfeeding (Baseline: −31.1 mmHg (−60.0, −12.7); Peak: −106.2 mmHg (−153.0, −65.5)). Milk intake was significantly higher with the NT (33 mL (22.5, 42.5)) compared to the breastfeed (12 mL (3, 15.5)). The novel teat encouraged a similar tongue action to breastfeeding, and infants transferred a greater volume of milk with the novel teat. Intra-oral vacuums were lower in strength with the novel teat compared to the breast. Use of the novel teat for the training of sucking dynamics in preterm infants has the potential to improve breastfeeding success and requires further investigation.

The aim of the present study was to determine whether specific biochemical and energy concentrations influence gastric emptying of unfortified and fortified mother's own milk (MOM) in stable preterm infants, and whether gastric emptying differs between feeds of unfortified MOM and feeds fortified with S-26 or FM 85 human milk fortifier (HMF) when infants are fed the same volume under similar conditions. Influences of infant gestation, age, and weight, and feed characteristics were also explored.Stomach volumes of 25 paired unfortified and fortified MOM feeds were monitored prefeed and postfeed delivery and at 30-minute intervals thereafter. For each feed, MOM samples were analyzed to determine concentrations of total protein, casein, whey, carbohydrate, lactose, fat, and energy. Fortified feed compositions were calculated by adding fortifier biochemical and energy concentrations to unfortified MOM concentrations. Ultrasound images were used to calculate infant stomach volumes. Statistical comparisons were made of paired stomach volume measurements.Higher feed concentrations of casein were associated with faster gastric emptying during feed delivery (P = 0.007). When compared with unfortified MOM, S-26 fortified feeds emptied similarly, whereas FM 85 fortified feeds emptied more slowly both during feed delivery and during the postprandial period (P = 0.002,0.001, respectively). Gastric emptying was slower for 2-hourly feeds compared with that for 3-hourly feeds (P = 0.003) and in supine position compared with that in prone (P = 0.001).Breast milk composition influences gastric emptying in stable preterm infants, with feeds of higher casein concentration emptying faster during feeding than otherwise equivalent feeds, and FM 85 fortified MOM emptying more slowly than unfortified MOM.

BackgroundPreterm infants' meals typically progress to higher volumes over time. Knowledge of gastric emptying (GE) responses to differing meal volumes may inform enteral feeding management. We examined the effect of meal volume and composition on preterm GE.MethodsForty infants were studied at 33.3±1.4 (29.7-35.6) weeks postmenstrual age when fully enteral fed (target 150 ml/kg/day). Intraindividual comparisons of GE were made for paired meals of 100% and 75% prescribed volume and identical composition of mother's own milk (n=21) and pasteurized donor human milk (n=19). Serial stomach ultrasound images were used to calculate gastric residual volumes (GRVs) and remaining meal proportions (% meal).ResultsGE was faster in the early postprandial period and slowed over time (P0.001). Reduced volume meals had slower GE rates and lower GRV (P0.001). Serial postprandial % meal was similar between reduced and full volume meals (P=0.41). Higher milk casein concentration was associated with slower GE (P=0.04). Complete gastric emptying (GRV=0 ml) was more common in infants fed at 3 h intervals compared with those fed every 2 h (P=0.002).ConclusionEarly postprandial GE is more rapid for larger meal volumes. Stable preterm infants may tolerate feeding of a 3 h meal volume at shorter intervals.

The aim of the present study was to determine whether serial gastric volumes and intragastric curding are repeatable within individual preterm infants when given sequential feeds of the same volume and composition. Infant and feeding characteristics that may influence differences in measured gastric volumes, echogenicity, and intragastric curding were also explored.Ultrasound images were used to calculate gastric volumes and to rate echogenicity and intragastric curding for 20 infants. A total of 29 paired feeds of the same volume and composition were monitored prefeed and postfeed and at 30-minute intervals thereafter. Statistical comparisons of paired gastric volume measurements and agreement between echogenicity and curding ratings were made for each time point. Analyses of factors that influence discrepancies between volume measurements and between curding ratings were performed.Paired gastric volume measurements were repeatable (intraclass correlation coefficient [ICC] = 0.971, 0.938 ICC 0.987). Most (75%) discrepancies were2 mL and increased over time, although volume differences were small. Overall moderate levels of consistency were observed for ratings of echogenicity (κ = 0.44), and curd presence (κ ≤ 0.65), density (κ = 0.41), and volume (κ = 0.47). Gastric emptying during feed delivery is influenced with infant positioning, fortification of breast milk, and feeding frequency.For preterm infants serial gastric volumes are repeatable and ratings of intragastric echogenicity and curding are moderately consistent when fed milk of the same volume and composition. Ultrasound has the potential to further explore factors that influence gastric emptying in the preterm infant.

Objectives The aim of this study was to validate 4 sonographic methods of estimating stomach volume for the purpose of monitoring changes in gastric volume over time and to describe the echogenic characteristics of milk feeds. Methods Twenty-four infants were monitored during a single intragastric tube feed, with 2 ultrasound images of the entire stomach and an image of the antral cross-sectional area (ACSA) before, during, and after the feed. Raw measurements, 3 stomach volume calculations, and ACSA were tested for intra- and interrater agreement. Calculated stomach volumes and ACSA were compared with delivered feed volumes, and characteristics of stomach image echogenicity graded at each time point. Results Spheroid calculation of stomach volume was the most reliable and valid measure of stomach volume. Fortified breast milk feeds were more echogenic than unfortified breast milk feeds. Residual stomach volumes (median 2.12 mL, range 0.59-9.27 mL) were identified in 18 of 24 infants. Conclusions Direct ultrasound stomach measurement (spheroid) will provide a useful research tool and a potential clinical tool for assessing gastric emptying and feeding intolerance in preterm infants.

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.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.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.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.

Background:Despite the differences in breastfeeding and bottle-feeding, our understanding of how suck-swallow-breathe (SSwB), oxygenation, and heart rate patterns change as the infant ages is based predominantly on bottle-feeding studies. Therefore, this study aimed to measure how SSwB, oxygenation, and heart rate patterns changed during the first 4 months of lactation in term breastfeeding infants.Methods:Infants less than 1 month postpartum (n = 15) were monitored early in lactation and again later in lactation (2-4 months postpartum). Simultaneous recordings of vacuum, tongue movement, respiration, swallowing, oxygen saturation, and heart rate were made during both nutritive sucking and non-nutritive sucking during breastfeeding.Results:Infants transferred a similar amount of milk ( P = .15) over a shorter duration later in lactation ( P = .04). Compared to early lactation, suck bursts became longer ( P < .001), pauses became shorter ( P < .001), vacuum levels decreased (all P < .05), oxygen saturation increased ( P < .001), and heart rate decreased ( P < .001) later in lactation.Conclusion:This study confirmed that term infants become more efficient at breastfeeding as they age, primarily by extending their suck bursts and pausing less. Although infants demonstrated satisfactory SSwB coordination during early lactation, they showed a level of adaption or conditioning at later lactation by applying weaker vacuum levels and demonstrating improved cardiorespiratory responses.

Background: Breast milk composition can be affected by several factors, and it can exhibit short-term (weekly) variations. Investigating variations in breast milk composition is important to accurately estimate nutrient requirements of the infant. Objective: To investigate short-term changes in breast milk composition between left and right breasts, over a 3-week period within the first 6 months of lactation. Methods: The left and right breasts of the mothers of healthy, term infants (n = 23) were simultaneously expressed with an electric breast pump for 15 minutes, on 3 occasions within 3 weeks. Milk samples (5 mL) were collected from the total expression volume of each breast at each session. The macronutrient contents, total solids, and energy content were determined using a mid-infrared human milk analyzer. Mothers (n = 17) measured their 24-hour milk production, and the average 24-hour fat contents were also determined. Results: Over the 3 weekly sessions, no significant changes were found in macronutrient contents. On average, total solids ( P = .04) and energy ( P = .04) decreased by week 3 of follow-up sessions from 14 to 13 g/100 mL and from 82 to 76 Kcal/100 mL, respectively; however, these changes became insignificant when expression volume was taken into account. The macronutrient concentration was similar for the left and right breasts; however, milk composition varied markedly between mothers. Furthermore, average 24-hour fat content was significantly lower than the mean fat content from a single expression session ( P < .01). Conclusion: Our findings highlight that when determining the nutritional adequacy of a mother’s milk, assuming an average concentration requires caution. The study findings illustrate the importance of using average 24-hour fat content of milk to obtain representative measures of infant energy intake.

The impact of cesarean section (CS) birth and pethidine for post-CS epidural analgesia on early breastfeeding behavior is unclear. This study aimed to measure infant sucking and breastfeeding behavior in infants of mothers who delivered by CS (CS group) and used pethidine patient-controlled epidural analgesia (PCEA) after CS with that of infants who were delivered by vaginal birth (V group), during secretory activation and again after the establishment of lactation.Sucking dynamics and milk intake of breastfeeding infants were assessed on approximately 3 and 20 (follow-up) days postpartum (CS group, n=19; V group, n=15). Nipple diameters, tongue movement, and nipple position during sucking were measured from ultrasound scans of the intra-oral cavity during breastfeeding. Time of the first breastfeed and day of breast fullness were recorded, and infant neurobehavior was assessed.CS infants displayed more anterior tongue movement on Day 3 than at follow-up compared with the V group, which showed a similar amount of movement at each assessment (p for interaction0.001). Compared with the V group, the CS group showed faster suck rates, especially on Day 3 (p0.001), later times to first breastfeed (p=0.01) and breast fullness (p=0.03), and lower neurobehavioral scores (p=0.047). Breastfeeding duration and milk intake were similar between groups.Although the observed effect of CS birth followed by pethidine PCEA after CS during the period of secretory activation was small, our results indicate that successful initiation of lactation may require additional breastfeeding support and monitoring at Day 3 postpartum for mothers who undergo CS.

Accurate, noninvasive, and inexpensive methods are required to measure infant body composition. Ultrasound (US) and bioimpedance spectroscopy (BIS) have been validated in adults and introduced in pediatric populations. The aim of this study was to evaluate the performance of both methods in determining percentage fat mass (%FM) in breastfed infants. %FM of 2, 5, 9, and 12 mo-old healthy, breastfed term infants (n = 58) was calculated using BIS-derived total body water equations and skinfold equations then compared with reference models. Skinfolds were measured with US at two and four sites (biceps, suprailiac and/or triceps, and subscapular). %FM differed widely within and between methods, with the degree of variation affected by infant age/sex. Not a single method/equation was consistent with the distributions of appropriate reference values for all age/sex groups. Moderate number of matches with references values (13–24 out of 36) was seen for both types of equations. High number of matches (25–36) was seen for US skinfold-based equations. %FM values calculated from US and BIS were not significantly different (P = 0.35). Both BIS and US are practical for predicting %FM in infants. BIS calculations are highly dependent upon an appropriate set of validated age-matched equations.

Background: Infant sucking problems are frequently implicated in early weaning during breastfeeding, yet our understanding of early sucking dynamics is limited. Objective: This study aimed to describe infant sucking patterns during breastfeeding at secretory activation and determine whether they changed by the time of established lactation. Methods: Sucking patterns and milk intake of 15 breastfeeding infants were assessed on day 3.2 ± 0.8 and later at follow-up, 16.0 (11.3-22.8) days postpartum. Nipple diameters, tongue movement, nipple position, and suck rate during nutritive sucking (NS) and non-nutritive sucking (NNS) were measured from ultrasound scans of the intra-oral cavity during breastfeeding. Milk intake and LATCH scores were also recorded. Results: As the tongue lowered during a suck cycle, the nipple increased in size ( P < .001), milk flowed into the intra-oral space and the nipple moved closer to the hard-soft palate junction ( P < .001). During NS, nipple diameters and the mid-tongue movement were greater than during NNS ( P < .001). As the infant aged, the mid-tongue lowered further ( P = .002), suck rates became faster ( P < .001) and milk intake increased ( P = .004), however, no differences were seen for LATCH scores ( P = .34). Conclusion: Differences in tongue movement between NS and NNS suggest that there is an altered sucking action when milk flow is absent. Similar sucking patterns at day 3 and during established lactation imply that infants have a mature sucking pattern in the early postpartum period.

To investigate the dynamics of milk removal during breast expression.This study used a continuous weighing balance to determine changes in milk flow rate and their relationship to the proportion of milk removed over time and the percentage of the available milk removed during simultaneous expression (15 minutes) of the left and right breasts in 34 mothers of healthy, term, breastfeeding infants.Multiple milk ejections (5.1±2.0; range, two to 14) were detected as increases in milk flow rate. A larger total expression volume was associated with a higher maximum milk flow rate (p0.001). However, 14% less of the available milk was removed for a 100 g increase in available milk (p0.001). After 2.9±1.4 and 6.0±2.5 minutes, 50% and 80%, respectively, of the total expression volume was removed. Milk flow was more active in the first 7 minutes than the last 7 minutes. At the eighth minute (midpoint), 54±25% of the available milk and 86±9% of the total expression volume were removed.The maximum milk flow rate was predictive of the total volume expressed. After the eighth minute of expression the milk flow rate was reduced. Increasing volumes of milk in the breast were associated with less complete breast emptying. These data suggest that long expression sessions are not required for mothers who are not pump dependent and that extending intervals between expressions results in less effective milk removal.

The effect of using a warm breastshield on the efficiency, effectiveness, and comfort of expressing milk with an electric breast pump was assessed. The authors tested a standard breastshield at 25°C and a breastshield warmed to 39°C on 25 mothers using their maximum comfortable vacuum or −125 mm Hg. Using a warm breastshield effectively warmed the nipple and areola and, combined with maximum comfortable vacuum, decreased the time to remove 80% of the total milk yield and increased the percentage of available milk removed after 5 minutes of expression, with no change in the percentage of available milk removed after 15 minutes of expression compared with an ambient-temperature breastshield. The data confirmed that use of the mother’s maximum comfortable vacuum was more efficient than a vacuum of −125 mm Hg. Using a warm breastshield with an electric breast pump was comfortable and improved the efficiency of milk removal.

Infant feeding problems are extremely common during breastfeeding establishment. To objectively assess infant sucking, consistent methods to analyze ultrasound images of the infant's oral cavity are required. We developed and assessed the reliability of an extensive ultrasound measurement protocol by measuring nipple diameter and placement. Midline submental ultrasound scans of 30 term breastfed infants were analyzed by two raters. Nipple diameter, nipple hard-soft palate junction distance and tongue hard-soft palate junction distance were measured on two frames: tongue-up and tongue-down. No evidence of measurement bias was found between raters and inter-rater agreement and consistency scores were high. The changes in nipple diameter and placement were consistent with previous descriptions; however, the diameter of the nipple was not consistent in either position. This method provides objective measurements representative of tongue movement, and further investigation is required to ensure usefulness when examining sucking difficulties.

A mid-infrared human milk analyzer (HMA) is designed to measure the macronutrients in human milk over a wide range of concentrations. Human milk samples (N = 30, 4 different dilutions each) were used to compare the macronutrient levels determined by the HMA to those derived from traditional laboratory methods. There was a small but statistically significant difference in the levels of fat, protein, lactose, total solids, and energy for all samples. These differences were consistent with subtle differences in the chemical principles governing the assays. For higher macronutrient levels, a trend to greater differences between the HMA and the laboratory method was seen, particularly in samples with high fat concentration. The intra-assay variation for the HMA for all macronutrients was less than 4%. It is concluded that that with appropriate sample preparation, the mid-infrared HMA can provide a practical measurement of macronutrients in human milk. J Hum Lact. 26(4):376-383.

Background: Infants of breastfeeding mothers with persistent nipple pain have been shown to apply stronger vacuums to the breast and transfer less milk during one monitored feed. This may be associated with differences in the movement of the tongue. The aim was to analyse the intra-oral nipple shape and movement of the tongue of infants of mothers with and without nipple pain. Methods: Breastfeeding infants of mothers with or without nipple pain were monitored using ultrasound and intra-oral vacuum during one breastfeed. From cine clips of the ultrasound scans measurements were made of the depth of the intra-oral space between the hard-soft palate junction (HSPJ) and the mid-tongue, the distance of the tip of the nipple to the HSPJ, and nipple diameters from the tip to the base. Results: During nutritive sucking, tongue movements of infants of mothers with nipple pain resulted in a smaller intra-oral space (p = 0.040) and restricted nipple expansion compared to controls (p &lt, 0.012). Stronger baseline and peak vacuums compared to controls were confirmed (p = 0.002). Conclusion: In these mothers, nipple pain was associated with restricted infant tongue movement. Ultrasound may complement measurement of intra-oral vacuum in monitoring treatment strategies in breastfeeding women experiencing nipple pain.

Breastmilk is a rich source of cells with a heterogeneous composition comprising early-stage stem cells, progenitors and more differentiated cells. The gene expression profiles of these cells and their associations with characteristics of the breastfeeding mother and infant are poorly understood. This study investigated factors associated with the cellular dynamics of breastmilk and explored variations amongst women. Genes representing different breastmilk cell populations including mammary epithelial and myoepithelial cells, progenitors and multi-lineage stem cells showed great variation in expression. Stem cell markers ESRRB and CK5, myoepithelial marker CK14 and lactocyte marker α-lactalbumin were amongst the genes most highly expressed across all samples tested. Genes exerting similar functions, such as either stem cell regulation or milk production, were found to be closely associated. Infant gestational age at delivery and changes in maternal bra cup size between pre-pregnancy and postpartum lactation were associated with expression of genes controlling stemness as well as milk synthesis. Additional correlations were found between genes and dyad characteristics, which may explain abnormalities related to low breastmilk supply or preterm birth. Our findings highlight the heterogeneity of breastmilk cell content and its changes associated with characteristics of the breastfeeding dyad that may reflect changing infant needs.

Bioimpedance spectroscopy is an accurate non-invasive method for measuring body composition in adults, but in infants it requires further testing and validation. Of the few studies of bioimpedance conducted in infants, none have comprehensively investigated the effect of milk intake volume. This study assessed the effect of the milk intake, feed duration and the volume of the infant's stomach and bladder on the resistance values pre-/post-feed to establish the feasibility of using these values interchangeably during data collection.Forty-eight breastfeeding infants were measured at 2, 5, 9 and/or 12 months (n=62 sessions) within 1-2 min before the start and after the end of breastfeed. Median (IQR) time between measurements was 24 (20.0-30.0) min. Resistance measurements at 0 and 50 kHz, and infinite frequency (R0, R50 and Rinf) and resistance of intracellular water (Ricw) were analysed with customised infant settings. Milk intake was measured by test weights. Free-water volumes and free-water change were determined from stomach and bladder volumes calculated from ultrasound images.Small pre-to-post-feed changes (median (IQR): R0 -3.7 (-14.8, 14.3); R50 0.3 (-10.4, 15.0); Rinf 2.8 (-13.3, 35.5); Ricw 20.8 (-98.1, 290.9)) were not significantly different from zero (R0: P=0.92; R50: P=0.48; Rinf: P=0.32; Ricw: P=0.097). No significant effect of milk intake or free-water change was detected.The lack of consistent change in resistance across a breastfeed provides flexibility in the timing of measurements of infants in the research setting, such that typically pre- and post-feed measures of resistance can be used interchangeably.

We evaluated the effects of fortification and composition on gastric emptying and curding in un/fortified pairs of mother's own milk (MOM, n = 17) and pasteurized donor human milk (PDHM, n = 15) in preterm infants. Retained meal proportions (%) and curding were determined from sonography. Immediate and subsequent postprandial % were higher for PDHM (23%, P = 0.026; 15%, P = 0.006) and fortified meals (31.5%; 8.8%, both P < 0.001), whereas higher casein, whey, and lactose concentrations were associated with lower immediate postprandial % (all P < 0.006). Curding did not affect emptying. Influences of fortification, pasteurization, and differing breast milk compositions are small and unlikely implicated in preterm feeding intolerance.

Breastfeeding has been implicated in the establishment of infant appetite regulation, feeding patterns and body composition (BC). A holistic approach is required to elucidate relationships between infant and maternal BC and contributing factors, such as breastfeeding parameters. Associations between maternal and breastfed term infant BC (n = 20) and feeding parameters during first 12 months of lactation were investigated. BC was measured at 2, 5, 9 and/or 12 months postpartum with ultrasound skinfolds (US; infants only) and bioimpedance spectroscopy (infants and mothers). 24-h milk intake (MI) and feeding frequency (FFQ) were measured. Higher FFQ was associated with larger 24-h MI (p ≤ 0.003). Higher 24-h MI was associated with larger infant fat mass (FM) (US: p ≤ 0.002), greater percentage FM (US: p ≤ 0.008), greater FM index (FMI) (US: p ≤ 0.001) and lower fat-free mass index (FFMI) (US: p = 0.015). Lower FFQ was associated with both larger FFM (US: p ≤ 0.001) and FFMI (US: p < 0.001). Greater maternal adiposity was associated with smaller infant FFM measured with US (BMI: p < 0.010; %FM: p = 0.004; FMI: p < 0.011). Maternal BC was not associated with FFQ or 24-h MI. These results reinforce that early life is a critical window for infant programming and that breastfeeding may influence risk of later disease via modulation of BC.

Infants rely on their innate immune systems to protect them from infection. Human milk (HM) contains fatty acids (FAs) and monoacylglycerols that are known to exhibit antiviral and antibacterial properties in vitro. The specific fat content of HM may potentially affect the efficacy of this antimicrobial activity. This preliminary study investigates whether the proportions of FA in HM change in response to infections, leading to cold-like symptoms in the mother or infant. Milk samples were obtained from mothers (n = 26) when they and their infants were healthy, and when mother, infant, or both suffered cold-like symptoms. The milk was hydrolysed and FA proportions were measured using gas chromatography. Fifteen FAs were recorded, of which eight were detected in sufficient quantities for statistical analysis. The proportions of capric (C10:0) and lauric acids (C12:0) in HM were significantly lower, and palmitic acid (C16:0) was higher when mothers and infants were ill compared to healthy samples. Palmitoleic (C16:1, n-7) and stearic acid (C18:0) proportions were higher in HM when the infant was unwell, but were not related to maternal health. Whilst the differences detected were small (less than 0.5%), the effects may be additive and potentially have a protective function. The value of further studies is certainly indicated.

Human milk contains both free and encapsulated RNA and microRNA in its cell, lipid and skim milk fractions. Recently, the microRNA content of human milk has attracted much attention due to its high...

Large inter- and intra-individual variations exist in breastmilk composition, yet factors associated with these variations in the short-term are not well understood. In this study, the effects of breastfeeding on breastmilk cellular and biochemical content were examined. Serial breastmilk samples (∼5 mL) were collected from both breasts of breastfeeding women before and immediately after the first morning breastfeed, and then at 30-minute intervals for up to 3 hours post-feed on 2-4 mornings per participant. The infant fed from one breast only at each feed. Effects of pump versus hand expression for samples were evaluated. A consistent response pattern of breastmilk cell and fat contents to breastmilk removal was observed. Maximum fat and cell levels were obtained 30 minutes post-feed (P0.05), although large intra-individual variability was noted for protein. Expression mode for samples did not influence breastmilk composition (P>0.05). It is concluded that breastmilk fat content, and thus breast fullness, is closely associated with breastmilk cell content. This will now form the basis for standardization of sampling protocols in lactation studies and investigation of the mechanisms of milk synthesis and cell movement into breastmilk. Moreover, these findings generate new avenues for clinical interventions exploring growth and survival benefits conferred to preterm infants by providing the highest in fat and cells milk obtained at 30 min post-expression.

Breast cancers with lactating features, some of which are associated with pregnancy and lactation, are often poorly differentiated, lack estrogen receptor, progesterone receptor, and HER2 expression and have high mortality. Very little is known about the molecular mechanisms that drive uncontrolled cell proliferation in these tumors and confer lactating features. We have recently reported expression of OCT4 and associated embryonic stem cell self-renewal genes in the normal lactating breast and breastmilk stem cells (hBSCs). This prompted us to examine OCT4 expression in breast cancers with lactating features and compare it with that observed during normal lactation, using rare specimens of human lactating breast. In accordance with previous literature, the normal resting breast (from non-pregnant, non-lactating women) showed minimal OCT4 nuclear expression (0.9%). However, this increased in the normal lactating breast (11.4%), with further increase in lactating adenomas, lactating carcinomas, and pregnancy-associated breast cancer (30.7–48.3%). OCT4 was expressed in the epithelium and at lower levels in the stroma, and was co-localized with NANOG. Comparison of normal non-tumorigenic hBSCs with OCT4-overexpressing tumorigenic breast cell lines (OTBCs) demonstrated upregulation of OCT4, SOX2, and NANOG in both systems, but OTBCs expressed OCT4 at significantly higher levels than SOX2 and NANOG. Similar to hBSCs, OTBCs displayed multi-lineage differentiation potential, including the ability to differentiate into functional lactocytes synthesizing milk proteins both in vitro and in vivo. Based on these findings, we propose a hypothesis of normal and malignant transformation in the breast, which centers on OCT4 and its associated gene network. Although minimal expression of these embryonic genes can be seen in the breast in its resting state throughout life, a controlled program of upregulation of this gene network may be a potential regulator of the normal remodeling of the breast toward a milk-secretory organ during pregnancy and lactation. Deregulation of this gene network either within or outside pregnancy and lactation may lead to aberrant breast cell proliferation and malignant transformation, suggesting a role of these genes in both normal lactation and breast oncogenesis.

Human milk (HM) components influence infant feeding patterns and nutrient intake, yet it is unclear how they influence gastric emptying (GE), a key component of appetite regulation. This study analyzed GE of a single breastfeed, HM appetite hormones/macronutrients and demographics/anthropometrics/body composition of term fully breastfed infants (n = 41, 2 and/or 5 mo). Stomach volumes (SV) were calculated from pre-/post-feed ultrasound scans, then repeatedly until the next feed. Feed volume (FV) was measured by the test-weigh method. HM samples were analyzed for adiponectin, leptin, fat, lactose, total carbohydrate, lysozyme, and total/whey/casein protein. Linear regression/mixed effect models were used to determine associations between GE/feed variables and HM components/infant anthropometrics/adiposity. Higher FVs were associated with faster (−0.07 [−0.10, −0.03], p < 0.001) GE rate, higher post-feed SVs (0.82 [0.53, 1.12], p < 0.001), and longer GE times (0.24 [0.03, 0.46], p = 0.033). Higher whey protein concentration was associated with higher post-feed SVs (4.99 [0.84, 9.13], p = 0.023). Longer GE time was associated with higher adiponectin concentration (2.29 [0.92, 3.66], p = 0.002) and dose (0.02 [0.01, 0.03], p = 0.005), and lower casein:whey ratio (−65.89 [−107.13, −2.66], p = 0.003). FV and HM composition influence GE and breastfeeding patterns in term breastfed infants.

The effect of pethidine as patient-controlled epidural analgesia (PCEA) on specific biochemical components in breast milk in relation to the timing of secretory activation is not well investigated. The aim of this study was to compare biochemical timing of secretory activation between women who had a vaginal (V) or Caesarean birth with pethidine-PCEA (CBP). Several milk samples were collected daily from 36 mothers (17 V, 19 CBP) for the first 265 h post-partum. Protein and lactose concentrations and Na+ and K+ ion levels were measured. Samples were assigned to three time periods: 0-72, >72-165 and >165-265 h post-partum for statistical analyses. Data were analyzed using linear mixed effect models. In the first 72 h post-partum, the mean difference in lactose concentration was 5 gL-1 higher in group V (P 72-165 h post-partum, protein and Na+ concentrations were lower in group V (P = 0.05, P = 0.02), and K+ levels were higher in group V (P 165-265 h post-partum, there were no significant differences between the groups. Biochemically, secretory activation had occurred by 72 h post-partum in both groups. There were greater variations in measured biochemical components observed within group CBP initially. However, by 165 h post-partum, there were no differences in the biochemical components between the groups. This suggests that effects of pethidine-PCEA are diminished by 72 h post-partum and undetected by 165 h.

Background: Data regarding the association between breast milk composition and infant feeding patterns (frequency and amount of breast milk taken) would help in understanding the regulation of food intake in breastfed infants. Objective: This study examined the relationship between breast milk macronutrient concentration and patterns of milk intake in breastfeeding infants over a 24-hour breastfeeding period. Methods: Mothers of healthy term infants (n = 15) collected pre- and postfeed breast milk samples from each feed at each breast over a 24-hour period. Breast milk samples were analyzed for fat, lactose, total protein, casein, and whey protein content. The energy content for each feed was calculated. Results: Breastfeeding patterns and milk composition varied greatly between individuals. The fat content of milk significantly differed over 24 hours ( P = .01), whereas the concentration of lactose and protein content remained the same. The mean 24-hour total protein, whey, and casein intake was inversely ( P < .01), whereas lactose concentration was positively ( P = .03) related to the number of breast feeds per day. No relationship was seen either between fat or energy content and feeding patterns. The mean (SD) concentration of fat, lactose, and total protein over the 24-hour period was 43 (12) g/L, 68 (7) g/L, and 13 (2) g/L, respectively. Conclusion: The association between milk protein intake and the breastfeeding frequency suggests that the protein intake may play a role in infant appetite control.

Background: Nipple pain is a major cause of early weaning. The causes of nipple pain are diverse, and most treatments involve experience-based assessment. There is little knowledge of the intensity or variation in pain experienced by breastfeeding women. Given the high breastfeeding initiation rates, it is important to evaluate pain experienced by lactating women in detail. Objective: To investigate and compare the pain experienced by breastfeeding women using objective measures. Methods: The type, effect, and severity of pain were measured using the McGill Pain Questionnaire, Brief Pain Inventory, and Visual Analogue Scale, respectively, for 2 groups of breastfeeding women. One group were experiencing persistent nipple pain despite treatment, and the other had obvious signs of nipple trauma. Results: Pain intensity and interference scores were highly variable for both groups. Mothers with nipple trauma reported significantly higher mean pain intensity and breastfeeding interference. Higher pain intensity scores were related to higher interference scores. After accounting for pain intensity, higher interference with general activity, mood, and sleep interference was related to longer duration of pain. There was no difference in MPQ class scores. Conclusions: The ramifications of nipple pain extend far beyond the act of breastfeeding, particularly for women whose pain lasts several months. Given the lack of evidence-based treatments, it is not surprising that pain is a major contributor to premature weaning. Further research into the causes of nipple pain is necessary to enable the implementation of effective interventions, thus reducing further complications such as infection and postnatal depression. Detailed pain analysis may assist in assessing the success of these interventions.

Nipple pain and insufficient milk supply are major causes of early weaning. We have found that persistent nipple pain was associated with strong infant sucking vacuums during breastfeeding. Several studies indicate that nipple pain and abnormal infant sucking have the potential to reduce milk transfer. We aimed to determine whether women with persistent nipple pain had low milk supply.The 24-hour milk production and feeding characteristics of mothers with persistent nipple pain (n=21) were compared with those mothers without nipple pain (n=21). Milk productions were measured by test-weighing the infant before and after every feed from each breast over a 24-26-hour period. Comparisons were made using Student's t tests and linear mixed models as appropriate.Lower milk productions were associated with longer meal durations for mothers with pain. There were no significant differences in the average 24-hour milk production or any feeding characteristics between the groups. However, four women with persistent nipple pain had milk production levels below 500 mL/day.The majority of breastfeeding women experiencing persistent nipple pain were able to achieve normal milk production levels. Feeding duration and frequency were similar to those of women not experiencing pain. However, longer meal durations in the pain group were associated with lower levels of milk production. Further investigation is necessary to identify mothers most affected by maternal nipple pain.

Objectives We investigated changes in breast milk whey and casein proteins, between fore and hind milk during breast expression, between breasts and within 24-hour period during breast-feeding. This has implications for developing an appropriate sampling protocol for investigating the influence of milk composition on gastric emptying and infants' feeding behaviour. Methods Breast milk samples were collected from mothers (n = 25) of healthy term infants ages 1 to 8 months. A total of 17 mothers provided fore and hind milk samples, which were collected during simultaneous expression of both breasts. Fifteen mothers provided samples from each breast-feed during 24-hour period, of which samples were selected from 4 time points (morning, day, evening, night). Whey and casein were isolated from skim milk, and protein concentration of the skim, whey, and casein fractions were determined. Results Mean protein concentrations were found to be 13.5 ± 2.1 (skim milk), 7.6 ± 1.5 (whey), and 3.4 ± 0.97 g/L (casein). Protein concentrations were not significantly different between fore and hind milk. During a 24-hour period, no significant differences were found in protein concentration of any fraction at the 4 time points or between left and right breasts. Large variations were seen between mothers with coefficient of variances of 15.5%, 19.8%, and 28.4% for skim milk, whey, and casein, respectively. Conclusions Although there was wide variation between mothers, the small variations within mothers indicate that for sampling purposes, a single breast milk sample (fore or hind from each breast at any time point of the day) will be representative of that mother's protein concentration of skim, whey, and casein fractions for that day.

Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).

This study aimed to investigate the consistency of milk ejections and milk expression characteristics within mothers at repeated expression sessions.Twenty-five breastfeeding mothers expressed their breasts simultaneously on three occasions within 3 weeks, and follow-up visits were performed at 6, 9, and 12 months of lactation. During the 15-minute expression, milk was collected onto a continuous weigh balance to measure milk flow rate.The number of milk ejections was similar at the three sessions (5.1±2.0), decreasing at the 12-month follow-up (3.3±1.2). Mothers had a similar pattern of milk ejection at each session. The time that each milk ejection occurred was consistent for the first 9 months of lactation. Of the four milk ejection patterns identified, each removed a similar percentage of available milk but varied in the time to reach 80% of the total expression volume. The first two milk ejections produced the greatest percentage (62%) of total milk volume during breast expression.For each individual mother, the timing, pattern, and number of milk ejections were consistent, suggesting a predetermined release of oxytocin. In light of the innate oxytocin release and milk removal characteristics in women, there is potential for individual tailoring of the duration of expression.

Breastmilk protects infants against infections; however, specific responses of breastmilk immune factors to different infections of either the mother or the infant are not well understood. Here, we examined the baseline range of breastmilk leukocytes and immunomodulatory biomolecules in healthy mother/infant dyads and how they are influenced by infections of the dyad. Consistent with a greater immunological need in the early postpartum period, colostrum contained considerable numbers of leukocytes (13–70% out of total cells) and high levels of immunoglobulins and lactoferrin. Within the first 1–2 weeks postpartum, leukocyte numbers decreased significantly to a low baseline level in mature breastmilk (0–2%) (P