Aware of the important benefits of human milk, most U.S. women initiate breastfeeding but difficulties with milk supply lead some to quit earlier than intended. Yet, the contribution of maternal physiology to lactation difficulties remains poorly understood. Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA-sequencing to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. The resulting transcriptomes paint an exquisite portrait of human lactation. The resulting transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during similar postpartum time frames could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding β-casein (CSN2) and α-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, protein tyrosine phosphatase, receptor type, F (PTPRF) may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors of sub-optimal lactation in humans.
Serum adiponectin (APN) is associated with lower childhood obesity, and APN concentration in human milk is associated with slower growth during active breastfeeding. Here, we examine infant weight gain in the second year of life after exposure to high or low levels of mother’s milk APN.
Breastfeeding mother-infant pairs were recruited in Mexico City and followed for 2 years; 192 infants with ≥12 months’ follow-up were analyzed. Monthly milk samples were assayed for APN; mothers were classified as producing high or low levels of milk APN. Infant and maternal serum APN were assessed during year 1. Infant anthropometry was measured monthly (year 1) or bi-monthly (year 2), and WHO Z-scores calculated. Longitudinal adjusted models assessed weight-for-age (WEI) and weight-for-length (WFL) Z-score trajectories from 1 to 2 years.
Maternal serum APN modestly correlated with milk APN (r=0.37, p<0.0001) and infant serum APN (r=0.29, p=0.01). Infants exposed to high milk APN experienced increasing WEI and WFL Z-scores between age 1 and 2 years in contrast to low milk APN exposure (p for group*time=0.02 and 0.054, respectively), adjusting for growth in the first 6 months and other covariates. In contrast, infant serum APN in year 1 was not associated with rate of weight gain in year 2.
High human milk APN exposure was associated with accelerated weight trajectory during the second year of life suggesting its role in catch up growth after slower weight gain during the first year of life.
adiponectin; human milk; breastfeeding; weight gain; infancy
To investigate the outcomes following prolonged empirical antibiotic administration to premature infants in the first week of life, concluding subsequent late onset sepsis (LOS), necrotizing enterocolitis (NEC), and death.
Study infants were ≤32 weeks gestational age and ≤ 1500 grams birth weight who survived free of sepsis and NEC for 7 days. Multivariable logistic regression was conducted to determine independent relationships between prolonged initial empirical antibiotic therapy (≥ 5 days) and study outcomes controlling for birth weight, gestational age, race, prolonged premature rupture of membranes, days on high frequency ventilation in 7 days, and the amount of breast milk received in the first 14 days of life.
Of the 365 premature infants surviving 7 days free of sepsis or NEC, 36% received prolonged initial empirical antibiotics, which was independently associated with subsequent outcomes: LOS (odds ratio [OR] 2.45, 95% confidence interval [CI] 1.28–4.67) and the combination of LOS, NEC, or death (OR 2.66, 95% CI 1.12–6.3).
Prolonged administration of empirical antibiotics to premature infants with sterile cultures in the first week of life is associated with subsequent severe outcomes. Judicious restriction of antibiotic use should be investigated as a strategy to reduce severe outcomes for premature infants.
prolonged antibiotic treatment; death; human milk; late-onset sepsis; necrotizing enterocolitis; premature infant
To investigate secretor gene fucosyltransferase2 (FUT2) polymorphism and secretor phenotype in relation to outcomes of prematurity.
Study infants were ≤32 weeks gestational age. Secretor genotype was determined from salivary DNA. Secretor phenotype was measured by H antigen, the carbohydrate produced by secretor gene enzymes, in saliva samples collected on day 9±5. The optimal predictive cut-point in salivary H values was identified by Classification and Regression Tree analysis. Study outcomes were death, necrotizing enterocolitis (NEC, Bell’s stage II/III), and confirmed sepsis.
There were 410 study infants, 26 deaths, 30 cases of NEC, and 96 cases of sepsis. Analyzed by genotype, 13% of 95 non-secretors, 5% of 203 heterozygotes, and 2% of 96 infants who were secretor dominant died (p=0.01). Analyzed by phenotype, 15% of 135 infants with low secretor phenotype died, compared with 2% of 248 infants with high secretor phenotype (predictive value=76%, p<0.001). Low secretor phenotype was associated (P<.05) with NEC, and non-secretor genotype was associated (P=.05) with gram negative sepsis. Secretor status remained significant after controlling for multiple clinical factors.
Secretor genotype and phenotype may provide strong predictive biomarkers of adverse outcomes in premature infants.
Recently, we reported the discovery and characterization of Tulane virus (TV), a novel rhesus calicivirus (CV) (T. Farkas, K. Sestak, C. Wei, and X. Jiang, J. Virol. 82:5408-5416, 2008). TV grows well in tissue culture, and it represents a new genus within Caliciviridae, with the proposed name of Recovirus. We also reported a high prevalence of CV antibodies in macaques of the Tulane National Primate Research Center (TNPRC) colony, including anti-norovirus (NoV), anti-sapovirus (SaV), and anti-TV (T. Farkas, J. Dufour, X. Jiang, and K. Sestak, J. Gen. Virol. 91:734-738, 2010). To broaden our knowledge about CV infections in captive nonhuman primates (NHP), 500 rhesus macaque stool samples collected from breeding colony TNPRC macaques were tested for CVs. Fifty-seven (11%) samples contained recovirus isolates. In addition, one NoV was detected. Phylogenetic analysis classified the recovirus isolates into two genogroups and at least four genetic types. The rhesus NoV isolate was closely related to GII human NoVs. TV-neutralizing antibodies were detected in 88% of serum samples obtained from primate caretakers. Binding and plaque reduction assays revealed the involvement of type A and B histo-blood group antigens (HBGA) in TV infection. Taken together, these findings indicate the zoonotic potential of primate CVs. The discovery of a genetically diverse and prevalent group of primate CVs and remarkable similarities between rhesus enteric CVs and human NoVs opens new possibilities for research involving in vitro and in vivo models of human NoV gastroenteritis.
Adiponectin is a protein hormone produced by adipose tissue whose circulating levels are inversely related to adiposity and inflammation. Adiponectin circulates as oligomers, from the low molecular weight trimer to the high molecular weight octodecamer (18mer) Each oligomer has distinct biological activities, which include enhancement of insulin sensitivity and metabolic control, and suppression of inflammation. Adiponectin occurs in human milk at higher concentrations than leptin. The adiponectin in human milk is almost entirely of the high molecular weight form, the form with the highest activity in controlling many types of metabolic processes. Human adiponectin fed to infant mice is transported across the intestinal mucosa into the serum. An inverse relationship between adiponectin levels in milk and adiposity (weight-for-height) of the breastfed infant was observed, and could be due to modulation of infant metabolism by milk adiponectin, and may be related to the observed protection against obesity by breastfeeding. Human milk may be a medium whereby the hormonal milieu (in response to internal factors and the environment) of the mother can be used to communicate with the breastfed infant to modify infant metabolic processes. Transmission of information from mother to infant through milk may allow adaptation to fluctuating environmental conditions.
infant development; body weight; BMI; adiposity
Adiponectin, a circulating adipocyte protein, is associated with lower obesity. We have previously shown that adiponectin is present in human milk. This study determined whether higher milk adiponectin is associated with infant growth and investigated milk adiponectin's oligomeric form.
Design and Methods
This is a study of two parallel longitudinal cohorts of breastfed infants born between 1998 and 2005. Forty-five mother–infant pairs from Cincinnati, OH and 277 mother–infant pairs from Mexico City, Mexico were analyzed. All participants were healthy, term infants breastfed at least 1 month who completed 6 months of follow-up. Monthly milk samples (n = 1,379) up to 6 months were assayed for adiponectin by radioimmunoassay. Infant weight-for-age, length-for-age, and weight-for-length Z-scores up to 6 months of age were calculated using World Health Organization standards. Repeated-measures analysis was conducted. The structural form of human milk adiponectin was assessed by western blot.
In the population studies, initial milk adiponectin was 24.0 ± 8.6 μg/L and did not differ by cohort. Over the first 6 months, higher milk adiponectin was associated with lower infant weight-for-age Z-score (−0.20 ± 0.04, p < 0.0001) and weight-for-length Z-score (−0.29 ± 0.08, p = 0.0002) but not length-for-age Z-score, adjusted for covariates, with no difference by cohort. By western blot, human milk adiponectin was predominantly in the biologically active high-molecular-weight form.
Our data suggest milk adiponectin may play a role in the early growth and development of breastfed infants.
To develop a statistical method for defining clusters of necrotizing enterocolitis (NEC) cases in the neonatal intensive care unit (NICU).
2782 infants, between 1996–2004, weighing 401–1500 grams at birth were included. NEC was defined as Bell stage II or III. Two statistical methods used to define “disease clusters” were: (1) modified scan test; (2) comparison of observed and expected incidence density rates (IDR) of NEC at each NICU.
The proportion of infants with NEC was similar between NICUs (7.1% vs. 7.7%; p=0.6) as was the expected IDR of NEC (1.39/1000 patient-days vs. 1.32/1000 patient-days, p=0.72). Twelve temporal clusters of NEC were identified in the NICUs combined, comprising 18% of 203 total NEC cases during the study period. No seasonal/secular trends were noted for NEC rates or identified clusters. Potential NEC clusters of ≥3 cases at either NICU had >75% chance of being a true NEC cluster.
No operational definition of NEC cluster exists. This study introduced methods to be employed for prospective surveillance and guide studies to investigate etiologic relevance. Utilizing the proposed methods, statistically significant clusters (potential outbreaks) of NEC within NICUs can be identified early, providing early opportunity to implement cluster investigation protocols.
disease clusters; outbreaks; neonatal intensive care unit; very low birth weight infants
We evaluated the utility of weight-for-length (defined as gm/cm3, “ponderal index”) as a complementary measure of growth in infants in neonatal intensive care units (NICUs).
Secondary analysis of infants (n=1214) 26-29 weeks at birth, included in a registry database (1991-2003), who had growth data at birth and discharge. Weight-for-age and weight-for-length were categorized as small (<10th percentile), appropriate or large (>90th percentile).
Statistical agreement between the weight-for-age and weight-for-length measures was poor (kappa=0.02 at birth, 0.10 at discharge, Bowker test for symmetry p<0.0001). From birth to discharge, the percent of small-for-age infants increased from 12% to 21%, and the percent of small-for-length infants decreased from 10% to 4%; the percent of large-for-age infants remained similar (<1%), and the percent of large-for-length infants increased from 5% to 17%. At discharge, 92% of small-for-age infants were appropriate or large-for-length, and 19% of appropriate-for-age infants were large-for-length.
Weight-for-age and weight-for-length are complementary measures. Weight-for-length or other measure of body proportionality should be considered for inclusion in routine growth monitoring of infants in the NICU.
Growth status; growth; weight-for-age; weight/length3; ponderal index; weight/length ratio; obesity; overweight; underweight; small-for-gestational age; nutrition
Environmental chemicals are readily measured in human milk. Although it is imperative to conduct studies on frequency of detection and effects of exposures to environmental chemicals in human milk, the potential impact of reporting individual test results to lactating women is poorly understood. The purpose of this study was to determine if mothers want to know if chemicals are in their breastmilk and if knowing the results would alter their breastfeeding practices.
We surveyed 381 mothers who were participating in a longitudinal birth cohort about whether they wanted to receive individual test results for environmental chemicals in their milk and whether they would alter their breastfeeding patterns if they were told that their milk contained “low” or “high” levels of phthalates.
Among the women who breastfed, 68% said that they wanted to know if there were chemicals in their breastmilk. Of breastfeeding women, 78% and 93% of mothers reported that they would either discontinue breastfeeding sooner than intended or pump and discard their milk if they were told they had “low” or “high” levels of phthalates in their milk, respectively. African American women were significantly more likely than Caucasian women to report that they would immediately wean if told of phthalates in their milk.
Concern about environmental chemicals in breastmilk may lead to early termination of breastfeeding. Chemical manufacturers and researchers should recognize the potential implications of isolating and reporting environmental chemicals in breastmilk.
Studies suggest that breastfeeding is protective for later obesity; however, this association has not held among all racial and socioeconomic status groups. Racial and socioeconomic status differences in breastfeeding behavior have also been noted. In this study, we formally test whether breastfeeding mediates the relationship between race and socioeconomic status with adolescent adiposity.
Data were analyzed from 739 black and white 10- to 19-year-old adolescents who participated in a large, school-based study. Parents provided information on parental education, used to measure socioeconomic status, and whether the child was breastfed as an infant. BMI was used to measure adolescent adiposity and was analyzed as a continuous measure (BMI z score) using linear regression and categorically (BMI ≥85th and ≥95th percentile) using logistic regression.
Black adolescents and those without a college-educated parent were less likely to have been breastfed for >4 months. Race and parental education were each independent predictors of BMI z score and of having BMI ≥85th percentile or BMI ≥95th percentile. When added to the model, being breastfed for >4 months was also independently associated with lower BMI z score and lower odds of having BMI ≥85th percentile or BMI ≥95th percentile. Inclusion of being breastfed for >4 months resulted in a 25% decrease in racial and parental education differences in adolescent BMI z score, supporting partial mediation.
Having been breastfed for >4 months was associated with lower adolescent BMI z score and lower odds of having a BMI ≥85th percentile or BMI ≥95th percentile, independent of race or parental education. Furthermore, these analyses suggest that being breastfed for >4 months partially explains the relationship between social disadvantage and increased adiposity. Increasing breastfeeding duration could result in lower adolescent adiposity for all racial and socioeconomic status groups and potentially minimize socioeconomic disparities in adiposity.
adolescent obesity; breastfeeding; epidemiology; racial differences; socioeconomic status
Noroviruses, an important cause of acute gastroenteritis, have been found to recognize human histo-blood group antigens (HBGAs) as receptors. Four strain-specific binding patterns to HBGAs have been described in our previous report. In this study, we have extended the binding patterns to seven based on 14 noroviruses examined. The oligosaccharide-based assays revealed additional epitopes that were not detected by the saliva-based assays. The seven patterns have been classified into two groups according to their interactions with three major epitopes (A/B, H, and Lewis) of human HBGAs: the A/B-binding group and the Lewis-binding group. Strains in the A/B binding group recognize the A and/or B and H antigens, but not the Lewis antigens, while strains in the Lewis-binding group react only to the Lewis and/or H antigens. This classification also resulted in a model of the norovirus/HBGA interaction. Phylogenetic analyses showed that strains with identical or closely related binding patterns tend to be clustered, but strains in both binding group can be found in both genogroups I and II. Our results suggest that noroviruses have a wide spectrum of host range and that human HBGAs play an important role in norovirus evolution. The high polymorphism of the human HBGA system, the involvement of multiple epitopes, and the typical protein/carbohydrate interaction between norovirus VLPs and HBGAs provide an explanation for the virus-ligand binding diversities.
Secretory immunoglobulin A (sIgA) is a primary factor responsible for preventing attachment of enteropathogens to gut epithelium in breastfeeding infants. We compared the frequency of sIgA to major surface antigens of enterohemorrhagic Escherichia coli (EHEC) in milk of 123 women from the United States and Mexico to determine whether regional differences existed in the frequency of antibodies to these surface antigens. In both groups of women, milk commonly has sIgA against various EHEC lipopolysaccharides, EspA, EspB, intimin, and less frequently against Shiga toxin. The study suggests that persons living in the U.S. are exposed to attaching/effacing enteropathogens more frequently than is generally assumed. The low frequency of antibodies to Stx1 (in 12% of Mexican and in 22% of U.S. samples) suggests that the rare appearance of hemolytic uremic syndrome in adults is not due to neutralization of toxin at the gut level. Only anti-EspA is found in most milk samples from both populations of women. EspA may represent a useful target for an immunization strategy to prevent EHEC disease in humans.
Shiga toxin; EHEC; Escherichia coli; human milk; colostrum; EspA; research
This paper resulted from a conference entitled “Lactation and Milk: Defining and refining the critical questions” held at the University of Colorado School of Medicine from January 18–20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research. Emerging questions about how milk components affect cognitive development and behavioral phenotype of the offspring are presented in Section II. In Section III we outline the important unanswered questions about regulation of mammary gland development, the heritability of defects, the effects of maternal nutrition, disease, metabolic status, and therapeutic drugs upon the subsequent lactation. Questions surrounding breastfeeding practice are also highlighted. In Section IV we describe the specific nutritional challenges faced by three different populations, namely preterm infants, infants born to obese mothers who may or may not have gestational diabetes, and infants born to undernourished mothers. The recognition that multidisciplinary training is critical to advancing the field led us to formulate specific training recommendations in Section V. Our recommendations for research emphasis are summarized in Section VI. In sum, we present a roadmap for multidisciplinary research into all aspects of human lactation, milk and its role in infant nutrition for the next decade and beyond.
Lactation; Infant nutrition; Human milk; Breastfeeding; Mammary gland development; Human nutrition; Preterm birth; Obesity; Undernutrition; Lactational programming; Milk