Composition Of Human Breast Milk Research Articles

Profiles of Human Milk Oligosaccharides and Their Relations to the Milk Microbiota of Breastfeeding Mothers in Dubai

The composition of human breast milk is affected by several factors, including genetics, geographic location and maternal nutrition. This study investigated the human milk oligosaccharides (HMOs) of breastfeeding mothers living in Dubai and their relations with the milk microbiota. A total of 30 breast milk samples were collected from healthy Emirati and UAE-expatriates at Latifa Hospital. HMO profiling was performed using UHPLC-MS. Microbiota profiles were determined by sequencing amplicons of the V3-V4 region of the 16S rRNA gene. HMO concentrations were significantly higher in Emirati, and dropped with the lactation period in both groups of mothers. The Le (a−b+)-secretor (Le+Se+) type was the most abundant in Dubai mothers (60%), followed by the Le(a−b−)-secretor (Le−Se+) type (23%). Bifidobacterium and Lactobacillus were considerably lower in Dubai-based mothers, while Pseudomonas and Delftia (Hydrogenophaga) were detected at a higher abundance compared to mothers from other countries. Atopobium was correlated with sialyl-lacto-N-tetraose c, Leptotrichia and Veillonella were correlated with 6’-sialyl-lactose, and Porphyromonas was correlated with lacto-N-hexaose. The study highlights the HMO profiles of breastfeeding mothers in Dubai and reveals few correlations with milk microbial composition. Targeted genomic analyses may help in determining whether these differences are due to genetic variations or to sociocultural and environmental factors.

An examination of breastmilk composition among high altitude Peruvian women.

Relatively little is known about sources of variation in human breastmilk composition. Previous research posits that composition may be affected by the low oxygen environment found at high altitude (HA). Our objective is to compare the milk composition of a sample of HA Andeans to previously published data on HA Tibetans and low altitude (LA) Peruvians. Mid-feed milk samples were collected from 22 women and tested for lipid, carbohydrate and protein content in addition to dietary surveys and anthropometric measures. We examined relationships between maternal and infant characteristics (maternal BMI, adiposity, age, skinfold thickness, infant age) and milk composition using multiple regression analyses. A Welch-Satterthause t-test was used to compare summary measures from Andean sample and Tibetan samples. Milk fat, sugar, and true protein content were correlated with different maternal and infant characteristics, with only waist to hip ratio negatively affecting fat and positively affecting sugar content in full models. Nuñoan and LA Peruvian milk exhibited significantly lower lactose levels (6.01 ± .89 g/100 mL and 6.38 ± 0.03 g/100 mL) than Tibetans (7.25 ± 0.35 g/100 mL). Fat content among Nuñoans was similar to that of Tibetans (4.5 ± 0.78 g/100 mL and 4.6 ± 1.51 g/100 mL, respectively), and higher than those of LA Peruvians. Both HA populations demonstrate relatively high fat content and both Peruvian samples exhibited relatively higher protein and lower lactose than Tibetan samples, suggesting that both population-based differences in feeding behavior and climatic differences may influence composition.

A Review of the Immunomodulating Components of Maternal Breast Milk and Protection Against Necrotizing Enterocolitis.

Breast milk contains immunomodulating components that are beneficial to newborns during maturation of their immune system. Human breast milk composition is influenced by an infant’s gestational and chronological age, lactation stage, and the mother and infant’s health status. Major immunologic components in human milk, such as secretory immunoglobulin A (IgA) and growth factors, have a known role in regulating gut barrier integrity and microbial colonization, which therefore protect against the development of a life-threatening gastrointestinal illness affecting newborn infants called necrotizing enterocolitis (NEC). Breast milk is a known protective factor in the prevention of NEC when compared with feeding with commercial formula. Breast milk supplements infants with human milk oligosaccharides, leukocytes, cytokines, nitric oxide, and growth factors that attenuate inflammatory responses and provide immunological defenses to reduce the incidence of NEC. This article aims to review the variety of immunomodulating components in breast milk that protect the infant from the development of NEC.

Trace mineral composition of human breast milk from Brazilian mothers

Background: Human milk is a dynamic food and some important differences in composition can be found between the milk from preterm and terms infants. Additionally, in some situations, the mother’s own milk is unavailable and the use of milk from human milk banks is considered as the most appropriate substitute. In this way, concentrations of trace elements (Ba, Cu, Fe, Mn, Mo, Se, Sr, and Zn) were determined in human milk, considering the differences about preterm and term human milk and its processing in a human milk bank.Methods: A total of 156 samples were analyzed, which were divided in three groups: samples collected at the hospital at bedside (BS, 60 samples) from mothers of preterm infants and samples from mothers of term infants collected in a human milk bank without pasteurization (WP, 49 samples) and pasteurized by the Holder procedure (P, 47 samples). The analyzes were conducted by inductively coupled plasma mass spectrometry (ICP-MS) after the treatment of the samples with acid mineralization assisted by microwave radiation.Results: Concentrations varied in a range of 0.6–88.2 μg/L for Ba, 78.6–954.5 μg/L for Cu, 24.2–5229.2 μg/L for Fe, 0.4–42.6 μg/L for Mn, 0.1–39.1 μg/L for Mo, 2.5–70.6 μg/L for Se, 8.9–187.5 μg/L for Sr and 76.3–17727.2 μg/L for Zn. Significant differences (p < 0.05) were found between preterm (BS) and term human milk (WP and P) for Ba, Cu, Mo, Se, and Zn, whereas the processing of the donated milk by Holder pasteurization did not influence the concentration of the studied trace elements. The milk of term infants does not attend the recommended daily intake (RDI) of Zn and for preterm infants the RDI of Fe and Mn is not achieved.Conclusions: The higher concentrations of Cu, Mo, Se and Zn observed in milk from mothers of preterm infants indicate that the milk to be offered for these high-risk neonates in neonatal intensive care units should contain higher levels of these trace elements. Besides, considering the RDI, the milk of term infants should be fortified with Zn, whereas the milk of preterm infants should be fortified with Fe.

Human Breast Milk NMR Metabolomic Profile across Specific Geographical Locations and Its Association with the Milk Microbiota.

The composition of human breast milk is highly variable, and it can be influenced by genetics, diet, lifestyle, and other environmental factors. This study aimed to investigate the impact of geographical location and mode of delivery on the nuclear magnetic resonance spectroscopy (NMR) metabolic profile of breast milk and its relationship with the milk microbiome. Human milk metabolic and microbiota profiles were determined using NMR and 16S rRNA gene sequencing, respectively, in 79 healthy women from Finland, Spain, South Africa, and China. Up to 68 metabolites, including amino acids, oligosaccharides, and fatty acid-associated metabolites, were identified in the milk NMR spectra. The metabolite profiles showed significant differences between geographical locations, with significant differences (p < 0.05) in the levels of galactose, lacto-N-fucopentaose III, lacto-N-fucopentaose I and 2-fucosyllactose, 3-fucosyllactose, lacto-N-difucohexaose II, lacto-N-fucopentaose III, 2-hydroxybutyrate, 3-hydroxybutyrate, proline, N-acetyl lysine, methyl-histidine, dimethylamine, kynurenine, urea, creatine and creatine phosphate, formate, lactate, acetate, phosphocholine, acetylcholine, LDL, VLDL, ethanolamine, riboflavin, hippurate, spermidine, spermine and uridine. Additionally, the effect of caesarean section on milk metabolome was dependent on the geographical region. Specific interrelations between human milk metabolites and microbiota were also identified. Proteobacteria, Actinobacteria, and Bacilli were most significantly associated with the milk metabolites, being either positively or negatively correlated depending on the metabolite. Our results reveal specific milk metabolomic profiles across geographical locations and also highlight the potential interactions between human milk’s metabolites and microbes.

Preclinical safety evaluation of the human-identical milk oligosaccharide lacto-N-tetraose

Lacto-N-tetraose (LNT) is one of the most abundant oligosaccharides that are endogenously present in human breast milk. To simulate the composition of human breast milk more closely, commercial infant formula can be supplemented with human-identical milk oligosaccharides, which are manufactured structurally identical versions of their naturally occurring counterparts. As part of the safety evaluation of LNT, in vitro genotoxicity tests and a subchronic oral gavage toxicity study (in neonatal Sprague-Dawley rats) were conducted. In the subchronic study, LNT was administered at dose levels of 0, 1,000, 2500 or 4000 mg/kg body weight (bw)/day, once daily for at least 90 days, followed by a 4-week treatment-free period. An identically comprised reference control group received fructooligosaccharides powder (a non-digestible oligosaccharide used in infant formula) at 4000 mg/kg bw/day, to allow for direct comparison against the high-dose LNT group. LNT was non-genotoxic in the in vitro tests. There were no compound-related adverse effects in the 90-day study; therefore, 4000 mg/kg bw/day (the highest feasible dose) was established as the no-observed-adverse-effect-level. These results support the safe use of LNT in infant formula and as a food ingredient, at levels not exceeding those found naturally in human breast milk.

Lipids for infant formulas

Recommendations for infant formulas (IF) had been established on the basis of human breast milk composition, still considered as “the gold standard”. Historically, till the 20th century, cow milk-based formulas have been used to feed infants when breast feeding was not possible. Later, infant formulas based on cow’s milk proteins but only vegetable oils blends as lipid source became the usual rule in most countries. However, considering“the gold standard”, a lot of changes occurred since the sixties that considerably modified lipid composition of human breast milk which is correlated to the diet of the mothers, who increased their consumption of n-6PUFA to replace saturated fat considered as proatherogenic. This introduced an imbalance in the ratio of linoleic/alpha-linolenic acids (18:2n-6/18:3n-3), limiting the bioconversion to long-chain-n-3 docosahexaenoic acid (DHA, 22:6n-3). Compared to pure vegetable blends and following the usual recommendations for IF, linoleic (LA 15% of total fatty acids), alpha-linolenic acids (ALA 1.5%) and LA/ALA ratio (10), the use of dairy fat blend was beneficial in terms of brain DHA accretion in young rats and a further increase of brain DHA was obtained by using pure dairy fat (LA 2%, ALA 0.8%, LA/ALA 2.3). Cow’s milk presents naturally some similarities (lipid quality, cholesterol, globule structure…) with human breast milk and cannot be compared to pure vegetable blends. Utilization of dairy fat in infant formula should be reconsidered, as well as the absolute amount of polyunsaturated LA and ALA: at least a reduction of LA for IF as well as for lactating women to improve breast milk quality.

Macro- and Micronutrients of Human Milk Composition: Are They Related to Maternal Diet? A Comprehensive Systematic Review.

This study aims at systematically reviewing the observational and interventional studies on the association of maternal macro- and micronutrient intake with breast milk content. We systematically searched the Medline via PubMed, Scopus, and ISI Web of Knowledge till October 2016 with the following search strategy: ("human milk" OR "breast milk" OR "breast milk composition" OR "human breast milk composition" OR "composition breast milk" OR "mother milk" OR "human breast milk") AND ("maternal diet" OR "maternal nutrition"). We also searched Google scholar for increasing the sensitivity of our search. The search was not limited to title and abstract due to the possibility that the desired outcome might have been considered a secondary aim. We excluded conference papers, editorials, letters, commentary, short survey, and notes. The search was refined to English language, and we did not consider any time limitation. To increase the sensitivity and to select more studies, the reference list of the published studies was checked as well. This review included 59 observational and 43 interventional studies on maternal diet related to breast milk composition. Different studies determined the associations and effects of some maternal dietary intake of micro and macronutrients and its reflection in human milk. Maternal dietary intake, particularly fatty acids, and some micronutrients, including fat soluble vitamins, vitamin B1, and vitamin C, was related to their content in breast milk composition.

Human Milk and Allergic Diseases: An Unsolved Puzzle.

There is conflicting evidence on the protective role of breastfeeding in relation to the development of allergic sensitisation and allergic disease. Studies vary in methodology and definition of outcomes, which lead to considerable heterogeneity. Human milk composition varies both within and between individuals, which may partially explain conflicting data. It is known that human milk composition is very complex and contains variable levels of immune active molecules, oligosaccharides, metabolites, vitamins and other nutrients and microbial content. Existing evidence suggests that modulation of human breast milk composition has potential for preventing allergic diseases in early life. In this review, we discuss associations between breastfeeding/human milk composition and allergy development.

Rapid Characterization of the Human Breast Milk Lipidome Using a Solid-Phase Microextraction and Liquid Chromatography-Mass Spectrometry-Based Approach.

Human breast milk (HBM) is a biofluid consisting of various biomolecules such as proteins, lipids, carbohydrates, minerals and bioactive substances. Due to its unique and complex composition, HBM provides not only nutritional components required for the growth of the infant, but also additional protection against infections. Global insight into the composition of HBM is crucial to understanding the health benefits infants receive from breastfeeding and could be used to improve the composition of milk formula for babies that cannot be breastfed. To improve global profiling of the HBM lipidome, a new analytical approach based on solid-phase microextraction (SPME) and liquid chromatography-mass spectrometry (LC-MS) was developed. The new extraction method allows for the rapid and simple extraction of a broad range of lipids directly from HBM samples. Moreover, the optimized two-step lipid extraction protocol ensures high lipidome coverage without using toxic solvents such as chloroform. The use of liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) and an automated search of a lipid database allows comprehensive identification of the lipids contained in HBM. The demonstrated analytical approach based on SPME sample preparation and LC-Q-TOF-MS is rapid, free of toxic solvents, and suited for the qualitative analysis of the HBM lipid composition.

Composition of human breast milk in acute kidney injury.

Breastfeeding is a widely encouraged practice due to its benefits for mother and the infant. Little is known about the impact of disease states, such as kidney dysfunction and childbirth complications, on the composition of breast milk. We describe a case of a 35-year-old woman who suffered a postpartum hemorrhage, was administered a contrast dye prior to computer tomography, and developed an acute kidney injury. Using nuclear magnetic resonance spectrometry, we measured composition of milk in acute kidney injury. The amount of dye secreted into milk was determined using a spectroscopic assay. Here we show that acute kidney injury results in changes in milk composition, but it does not significantly affect major macronutrients. We also determine that iodinated computer tomography contrast dye does not accumulate in milk in appreciable amounts. Acute kidney injury has impact on breast milk. Intravenous administration of computer tomography contrast dye does not result in significantly elevated levels in milk.

On the Human Species Re-Engineering

An art of artificial feeding human infants has been evolved since 1860, many different ways of feeding infants have been introduced till 1960. In 1960, a precursor form of modern infant formula was introduced and since then the formula makers have been fortifying formulae with additional substances to mimic the composition of human breast milk. This implies that human infants who were fed formula were brought up without all the benefit of the nutrients that comprised human breast milk. Brains of human infant were established to develop within initial few years after birth. The effects of artificial feeding formula on the brains of infant as compared with those fed human breast milk remained a topic of great interest. So as experiments of human species re-engineering has begun and the consequences of this human experiment are less than desirable.

Comparing the fatty acid levels of preterm and term breast milk in Turkish women

Lipids are the main source of calories and considered very important in infant growth. We aimed to compare fatty acid composition of term and preterm breast milk. This is the first study that compares the fatty acid levels of preterm and term breast milk in Turkish women. Breast milk samples were obtained from mothers of term (n = 15) and preterm (n = 15) infants on postnatal days 3, 7, and 28. Fatty acid composition of human breast milk was determined longitudinally by gas-chromatography/mass spectrometry. There Were 31 fatty acids measured in the milk samples. In the first month, 17 fatty acid levels had significant differences. In group comparison, some fatty acids (C14:0, C16:0, C18:1 and C20:5) had significantly increased in the preterm group (P = 0.041, P = 0.046, P = 0.027, P = 0.033, respectively), whereas myristoleic acid (C14:1) and eicosanoic acid (C20:0) had significantly increased in the term group (P = 0.015, P = 0.048, respectively). Term and preterm milk have different compositions of fatty acids. Breast milk composition changes over time. As a general conclusion, breast milk provides the lipid requirements of infants.

Pre-clinical safety evaluation of the synthetic human milk, nature-identical, oligosaccharide 2′-O-Fucosyllactose (2′FL)

In order to match the composition of human breast milk more closely, it is now possible to supplement commercial infant formula (IF) with synthesised oligosaccharides that are chemically identical to human milk oligosaccharides. The safety data generated on a new human-identical milk oligosaccharide (HiMO), 2′-O-Fucosyllactose (2′FL), are presented. Standard in vitro genotoxicity tests were performed. To investigate the toxicological profile in a model representative of the intended target population, 2′FL was administered via gavage in a juvenile adapted sub-chronic rat study at dose levels of 0, 2000, 5000 and 6000mg/kgbw/day. Fructooligosaccharide (FOS), currently acknowledged as safe and approved for use in IF, was used as a reference high-dose control at 6000mg/kgbw/day. 2′FL was non-mutagenic in the in vitro assays. Oral administration up to 5000mg/kgbw/day to rats over 90days was not associated with any adverse effects based on clinical observations, body weight gain, food consumption, ophthalmoscopy, clinical pathology, organ weights and histopathology findings. Based on this 90-day study, a No Observed Adverse Effect Level (NOAEL) of 5000mg/kgbw/day for both male and female rats was established for 2′FL. These findings support the safety of synthetic 2′FL for possible use in infant food.

Exploring human breast milk composition by NMR-based metabolomics

Breast milk is a complex fluid evolutionarily adapted to satisfy the nutritional requirements of growing infants. In addition, milk biochemical and immunological components protect newborns against infective agents in the new environment. Human milk oligosaccharides, the third most abundant component of breast milk, are believed to modulate the microbiota composition, thus influencing a wide range of physiological processes of the infant. Human milk also contains a number of other bioactive compounds, the functional role of which has not yet been clearly elucidated. In this scenario, NMR-based metabolic profiling can provide a rapid characterisation of breast milk composition, thus allowing a better understanding of its nutritional properties.

A Simple and Efficient HPLC Method for Benznidazole Dosage in Human Breast Milk

Due to migration, Chagas disease is a significant public health problem in Latin America, and in other nonendemic regions. The 2 drugs currently available for the treatment, nifurtimox and benznidazole (BNZ), are associated with a high risk of toxicity in therapeutic doses. Excretion of drug into human breast milk is a potential source of unwanted exposure and pharmacologic effects in the nursing infant. However, this phenomenon was not evaluated until now, and measurement techniques for both drugs in milk were not developed. In this work, we described the development of a simple and fast method to quantify BNZ in human milk using a pretreatment that involves acid protein precipitation followed by tandem microfiltration, and reverse phase high-performance liquid chromatography/ultraviolet analysis. It is simple because it takes only 3 steps to obtain a clean extracted solution that is ready to inject into the high-performance liquid chromatography equipment. It is fast because a complete analysis of a sample takes only 36 minutes. Although the human breast milk composition is very variable, and lipids are one of the most difficult compounds to clean up on a milk sample, the procedure has proven to be robust and sensitive with a limit of detection of 0.3 μg/mL and quantization of 0.9 μg/mL. Despite a 70% recovery value, which could be considered a relatively low result, this recovery is reproducible (coefficient of variation <10%) and the analytical response under the linear range is very good (r = 0.9969 adjusted). Real samples of human breast milk from patients in treatment with BNZ were dosed to support the validation process of the method. The method described is fast, specific, accurate, precise, and sufficiently sensitive in the clinical context for the quantification of BNZ in human milk. For all these reasons, it is suitable for clinical risk evaluation studies.

The cellular hierarchy and composition of human breastmilk

During pregnancy and lactation, mammary stem cells remodel the breast into a milk‐secretory organ by proliferating and subsequently differentiating into both myoepithelial cells and lactocytes. Thus, we reasoned that the lactating gland, which is the fully mature organ, can offer a more comprehensive view of the mammary cellular hierarchy and dynamics than the resting gland. To non‐invasively access cells from the human lactating breast, we used freshly expressed breastmilk from which cells were isolated and characterized ex vivo for expression of markers of various lineages and differentiation stages. These included markers of pluripotent and multipotent stem cells, epithelial and mammary‐specific markers, hematopoietic and mesenchymal markers. Rare specimens of human lactating breast tissues were used to examine the origin of the different cell types identified in breastmilk. The ex vivo and in situ analyses were coupled by in vitro studies, which showed that along with the more differentiated cells, breastmilk contains progenitor cells and stem cells, some of which display pluripotency. These findings reveal a novel cellular hierarchy in breastmilk, characteristic of the lactating breast. Importantly, the presence of stem cells in breastmilk raises the question of the role of these cells for the breastfed infant.Grant Funding Source: Women and Infants Research Foundation; Medela AG

Breastfeeding and risk of overweight and obesity at nine-years of age

Whether breastfeeding is protective against the development of childhood overweight and obesity remains the subject of considerable debate. Although a number of meta-analyses and syntheses of the literature have concluded that the greater preponderance of evidence indicates that breastfeeding reduces the risk of obesity, these findings are by no means conclusive. The present study used data from the Growing Up in Ireland study to examine the relationship between retrospectively recalled breastfeeding data and contemporaneously measured weight status for 7798 children at nine-years of age controlling for a wide range of variables including; socio-demographic factors, the child's own lifestyle-related behaviours, and parental BMI. The results of the multivariable analysis indicated that being breastfed for between 13 and 25 weeks was associated with a 38 percent (p < 0.05) reduction in the risk of obesity at nine-years of age, while being breastfed for 26 weeks or more was associated with a 51 percent (p < 0.01) reduction in the risk of obesity at nine-years of age. Moreover, results pointed towards a dose–response patterning in the data for those breastfed in excess of 4 weeks. Possible mechanisms conveying this health benefit include slower patterns of growth among breastfed children, which it is believed, are largely attributable to differences in the composition of human breast milk compared with synthesised formula. The suggestion that the choice of infant feeding method has important implications for health and development is tantalising as it identifies a modifiable health behaviour that is amenable to intervention in primary health care settings and has the potential to improve the health of the population.

Genetic influence on fatty acid composition of human breastmilk

ObjectiveThe cognitive advantage conferred by breastfeeding may be dependent on maternal FADS2 genotype. In a Canadian sample (Xie &amp; Innis, 2008), fatty acid content of human milk related to genotype at rs174575 on the FADS2 gene. This study was undertaken to explore this in a sample from the USA.MethodsBreastfeeding dyads were enrolled when infant was 3–4 months of age (n=200). Mothers collected a morning sample by emptying one entire breast, agitating, aliquoting, and transporting to the lab on ice. Samples were stored at −20 F until lipids were extracted and saponified. Fatty acids were trans‐methylated to methyl esters that were analyzed using capillary gas chromatography. DNA was extracted from saliva using a QIAcube robotic station (Qiagen), as per manufacturer's protocol. 20 ng DNA was used with validated primers from Applied Biosystems. The real time PCR assay was run on an Eppendorf thermal cycler.ResultsMaternal genotype is related to fatty acids: homozygous recessive (GG) had lower fatty acid levels than wildtype (CC). Levels of arachidonic acid, docosapentaenoic acid (n‐3), and docosahexaenoic acid reached statistical significance (p&lt;0.05).ConclusionThe desaturases used in biosynthesis of fatty acids from their precursors may be in limited supply in certain genotypes. These mothers should insure exogenous fatty acids are being consumed.Support: Nutrition Obesity Research Center, UNC

Smoking in Pregnancy

Zanardo et al. (2005) raise an interesting issue in their article, namely, the influence of smoking on immunologic parameters and especially on the composition of human breast milk. The findings deserve notice and further investigation. Because of the importance of the findings, the errors and inconsistencies in the article (Zanardo et al. 2005) should be corrected. The first inconsistency is related to the number of participants: In the “Materials and Methods,” Zanardo et al. (2005) gave two different numbers: Of the 1,217 eligible participants, 25 of 26 self-identified as smokers (≥ 5 cigarettes per day through pregnancy until last trimester) were recruited for study participation. One was excluded from the final analysis because of maternal fever. Control participants included consecutive women without history of smoking and matched a smoking participant on the basis of overall inclusion criteria. We grouped human milk samples into third postpartum day smoker and nonsmoker mother groups, with 42 and 40 samples per group, respectively, and into 10th postpartum day smoker and nonsmoker mother groups, with 42 and 40 samples per group, respectively. The latter numbers (42 and 40) are also repeated in the first sentence of “Results” and in Table 1. Also in Table 1 the average number of cigarettes smoked per day by the 42 smokers is given as 3.2 ± 0.7 (mean ± SD). This is inconsistent with their selection criterion of “≥ 5 cigarettes per day.” Table 1 also provides information on birth weight, gestational age, and APGAR score. As expected from numerous previous studies, the children of the smokers scored lower in all these respects. Under “statistical analysis” the authors write that they “used the Student t-test for the analysis of [these] data.” To apply the Student t-test for the birth weight data seems a sensible choice. When doing this using the figures provided in Table 1, the difference of the birth weight between the children of smokers and nonsmokers is clearly highly significant (t = 9.45). In the “Results,” Zanardo et al. (2005) stated erroneously that “the birth weight of the newborn infants of smoker mothers was not significantly lower.”