Breast-feeding is the natural and best advisable way of supporting the growth and development of healthy term infants (1). The benefits of breast milk are well recognized as providing health benefits in early infancy and extending into adulthood. In addition, the present research confirms that breast milk with appropriate fortification is the optimal care for the low- and the very-low-birth-weight infants (2). When breast-feeding may not be possible and own mother's milk may not be available, donor human milk becomes the next alternative (3,4).
The benefits of breast milk for preterm infants include faster gastric emptying (5); a faster tolerance to enteral feeding and a reduced need of parental nutrition (6); enhanced nutrient absorption (7); improved visual and cognitive development (8,9); and reduced incidence of necrotizing enterocolitis, sepsis, and other infections (10,11). Such effects are probably because of the combined action of nutrients and a variety of bioactive factors present in colostrum and breast milk, such as inmunoglobulins, inmunocompetent cells, antimicrobial fatty acids, polyamines, oligosaccharides, lysozyme, lactoferrin, and other glycoproteins, antimicrobial peptides and, also, commensal or probiotic bacteria (12,13).
It is long known that the concentration of many, if not all, nutrients and bioactive compounds changes from colostrum to mature milk and, in addition, there is a variability associated to several factors, such as host's genetic background, health status, nutrition, lactation stage (infant's age), “circadian” rhythm, milk fraction (foremilk, hindmilk), geographic location, and the like (12).
Gestational age may also influence the concentration of nutrients and bioactive compounds (14–16). Biochemical and immunological data suggest that mothers’ colostrum or milk feedings may provide the greatest protection from infection for the most immature infants (17) which, in comparison with larger preterm infants, are the most immunocompromised, are exposed routinely to invasive, life-saving procedures and remain in the pathogen-laden neonatal intensive care unit for the longest period. Studies on the composition of colostrum and milk from mothers delivering before the 30th week of gestation are, however, scarce.
In this context, the objective of this work was to study a wide variety of microbiological, biochemical, and immunological parameters in milk of mothers of extremely preterm infants (<27th week of gestation), and to evaluate differences between colostrum and mature milk from these mothers.
Colostrum and Mature Milk Samples
Colostrum samples (n = 17), expressed within 96 hours after delivery, and mature milk samples (n = 34), collected between days 14 and 56 after birth, were obtained from 22 mothers of extremely preterm infants who gave birth at the Hospital Universitario La Paz (Madrid, Spain) (Table 1). Sampling was performed following a specific protocol approved by the local ethical committee, and informed consent was obtained from each donor. To avoid differences between foremilk and hindmilk, an entire feed was manually expressed and, then, a representative aliquot (∼2 mL) was stored at −20°C until analysis.
Bacterial Cultures and Identification of Isolates
Adequate peptone water dilutions of the samples were plated onto Man, Rogosa, and Sharpe (MRS; Oxoid, Basingstoke, UK) supplemented with L-cysteine (0.5 g/L) (MRS-C; a medium for isolation of lactic acid bacteria), TOS-Propionate (TOS; Merck, Whitehouse Station, NJ; a medium for isolation of bifidobacteria), MacConkey (MCK; BioMérieux, Marcy l’Etoile, France; a medium for isolation of enterobacteria), and Columbia Nalidixic Acid (CNA, BioMérieux; a highly nutritious, general-purpose medium for the isolation and cultivation of fastidious microorganisms) agar plates. MCK and CNA plates were aerobically incubated at 37°C for 24 hours, whereas MRS-C and TOS plates were incubated anaerobically (85% nitrogen, 10% hydrogen, and 5% carbon dioxide) in an anaerobic workstation (MINI-MACS; DW Scientific, Shipley, UK) at 37°C for 48 hours.
After incubation and counting, 1 representative of each colony morphology type was selected from each plate. These isolates were grown in brain heart infusion or MRS-C broth and stored at −80°C in the presence of glycerol (30%, v/v). All of the isolates were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry in a Vitek-MS Instrument (BioMérieux) in the facilities of Probisearch (Tres Cantos, Madrid, Spain). Briefly, a portion of a bacterial colony (∼1 μL) was directly spotted onto a MALDI sample plate. Then, it was overlaid with 1 μL of a saturated solution of α-cyano-4-hydroxycinnamic acid in acetonitrile (28%, v/v), and allowed to dry at room temperature. For each isolate, a mean spectrum was constructed with at least 50 m/z spectra profiles and used for the identification by comparison with the spectra contained in the Myla database (BioMérieux). Identification was defined as a 99% to 100% match to the species-specific m/z values in the database.
Analysis of Lactose, Glucose, and myo-Inositol
The concentrations of lactose, glucose, and myo-inositol were determined by gas chromatography with flame ionization detector in 17 colostrum and 34 mature milk samples following the method described by Montilla et al (18), with minor modifications. For this purpose, 0.2 mL of sample was made up to 2 mL with methanol in a volumetric flask to remove proteins and fat. Mixtures were gently stirred, followed by standing for at least 1 hour at room temperature until the supernatant became transparent. The clear supernatant was used for carbohydrate analysis and a solution of 0.04% (w/v) phenyl-β-D-glucoside in ethanol/water (70:30, v/v) was added as internal standard.
Before derivatization, equal volumes (0.5 mL) of supernatant and internal standard solution were mixed and dried at 38°C to 40°C in a rotary evaporator. For derivatization 100 μL of N,N-dimethylformamide was added to the dried mixtures and held at 70°C for 1 hour to obtain a constant anomeric composition. Then, 100 μL of N-trimethylsilylimidazole was added to silylate the carbohydrates, and the reaction was completed in 30 minutes at 70°C. Silylated carbohydrates were extracted with 0.1 mL of hexane and 0.2 mL of water. Volumes in the range of 1 to 2 μL of the organic phase containing silyl derivatives were injected into the column.
The trimethylsilyl ethers of carbohydrates were analyzed in an Agilent Technologies (Santa Clara, CA) 7890A gas chromatograph equipped with a commercial 30 m × 0.32 mm inside diameter, 0.5 μm film fused silica capillary column SPB-17, bonded, cross-linked phase (50% diphenyl/50% dimethylsiloxane) (Supelco; Bellefonte, PA). Separation was performed at 235°C for 9 minutes, followed by an increase up to 270°C at rate of 15°C/min and maintenance of this temperature for 15 minutes. Temperatures of the injector and flame ionization detector were 280 and 290°C, respectively. Injections were carried out in split mode 1:30, using 1 mL/min of nitrogen as carrier gas. Data acquisition and integration were performed using Agilent Chem-Station Rev. B.03.01 software (Wilmington, DE). To study the response factor relative to the internal standard, solutions containing lactose, glucose, and myo-inositol were prepared over the expected concentration range in colostrum samples. The identity of carbohydrates present in colostrum samples was confirmed by comparison with relative retention times of standard samples. Quantitative analysis was carried out by the internal standard method. Response factors were calculated after triplicate analysis of standard solutions (glucose, myo-inositol, and lactose) at concentrations ranging from 1 to 6 g/L for lactose and from 1 to 50 mg/L for myo-inositol and glucose.
The concentration of 18 cytokines, chemokines, and growth factors, including interleukin-1β (IL-1β, IL-6, IL-12(p70), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-2, IL-4, IL-10, IL-13, IL-17, IL-8, growth-related oncogene-α (GRO-α), macrophage-monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), IL-5, IL-7, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), was determined in 15 colostrum and 11 milk samples using a Bioplex 200 system instrument (Bio-Rad, Hercules, CA) and the Bio-PlexPro Human Cytokine, Chemokine, and Growth Factor Assays (Bio-Rad). Previously, and to avoid interferences in the immunoassay, the fatty layer and the somatic cells were removed from the samples. Briefly, sample aliquots (1 mL) were centrifuged at 800g for 15 minutes at 4°C and the intermediate aqueous phase was collected and stored at −20°C until analysis. All the determinations were carried out by duplicate following the manufacturer's protocol, and standard curves were performed for each analyte.
The concentration of immunoglobulin (Ig) IgG1, IgG2, IgG3, IgG4, IgM, and IgA was determined in the same samples using the Bio-PlexPro Human Isotyping Assay kit (Bio-Rad) in the Bioplex 200 system instrument. For this purpose, the samples were conditioned as described above for cytokine analysis. Analyses were carried out by duplicate following the manufacturer's protocol, and standard curves were performed for each analyte.
Microbiological data, recorded as colony forming units (CFU) per milliliter, were transformed to logarithmic values before statistical analysis. Biochemical data (sugars and bioactive compounds) were tested for normality of distribution by Shapiro–Wilk tests. Normal data were expressed as the mean and 95% confidence interval (CI) of the mean, whereas not normal data were expressed as the median and interquartile range (IQR).
The contingency tables of the detection frequencies of bacterial species, cytokines, and immunoglobulins were obtained, and then differences were evaluated with the χ2 test; when required the Yates correction was applied. Relations between epidemiological information were evaluated with the χ2 test; when required the Yates correction was applied. Differences in mayor bacterial species counts, Shannon-Weaver diversity indexes, sugar concentrations, and immunocompounds in colostrum and mature milk were evaluated using 1-way analysis of variance when the variable was normally distributed, and the Kruskal-Wallis test when not. Differences were considered significant at P < 0.05. Statgraphics Centurion XVI version 16.1.15 (Statpoint Technologies Inc, Warrenton, VA) and R 2.15.3 (R Project, http://www.r-project.org) software were used to perform these analyses.
A total of 22 women participated in this study and provided 17 colostrum and 34 mature milk samples. The characteristics (age, week of pregnancy, way of delivery, single/twins pregnancy, antibiotic and corticosteroid treatment, and chorioamnionitis) of study participants are presented in Table 1. All of them gave birth between weeks 24 and 27 (14% at week 24, 18% at week 25, 36% at week 26, and 32% at week 27). Some relations were observed when frequencies of these characteristics’ categorical factors were cross-tabulated and an independency test was carried out (Table 2). Week of birth was related to mother's age (P = 0.045), those women being younger who delivered at the shortest gestational ages; similarly, a diagnosis of chorioamnionitis was more frequent as the gestational age was shorter (P = 0.012), and as the mother's age was lower (P = 0.038). Finally, antibiotic treatment was related with C-section deliveries (P = 0.000).
Bacterial Counts and Identification of the Isolates
Viability of bacteria using both general and selective growth media was evaluated in the 17 colostrum and 34 mature milk samples. Low detection frequencies were observed in both types of samples because bacteria could only be isolated from a relatively low percentage of them (Table 3). When bacterial growth was detected, the bacterial counts oscillated between 2.00 and 3.28 log10 CFU/mL and between 2.00 and 4.19 log10 CFU/mL in colostrum and mature milk samples, respectively (Table 3). The main genera isolated from colostrum were Staphylococcus, Streptococcus, and Lactobacillus, whereas, in addition to these genera, Enterococcus and enterobacteria were also among the main microorganisms isolated from mature milk (Table 3). The detection frequencies of enterococci (P = 0.000), lactobacilli (P = 0.041), and enterobacteria (P = 0.038) in mature milk samples were statistically higher than in the colostrum ones (Table 3). The mean for staphylococci counts was higher in colostrum, whereas those for streptococci and lactobacilli were higher in mature milk, although such differences did not reach a statistically significant level. In relation to the Shannon-Weaver diversity index, no significant differences were observed between the colostrum and the mature milk samples analyzed in this study.
Analysis of Lactose, Glucose, and myo-Inositol
The mean values and ranges of lactose, myo-inositol, and glucose concentrations found in the colostrum and mature milk samples are shown in Fig. 1. Mean (95% CI) concentration values of lactose were 56.90 (52.08–61.71) and 62.54 (60.37–64.71) g/L in the colostrum and the milk samples, respectively. myo-inositol and glucose were also detected and quantified; myo-inositol concentrations were 300.59 (249.70–351.48) and 194.27 (172.51–216.02) mg/L in the colostrum and the mature milk samples, respectively, whereas those of glucose, expressed as median (IQR), were 109.29 (44.24–154.41) and 588.79 (152.80–845.11) mg/L, respectively.
Although the levels of these 3 compounds showed a certain degree of variability depending on the women, the mean concentrations of colostrum's lactose, myo-inositol, and glucose were statistically different from those found in the mature milk samples (Fig. 1); glucose (P = 0.000) and lactose (P = 0.013) concentrations were significantly higher in mature milk whereas that of myo-inositol (P = 0.000) was significantly higher in the colostrum ones.
The concentrations of a variety of cytokines, chemokines, growth factors, and immunoglobulins in 15 colostrum and 11 mature milk (milk obtained ≥21 days after birth) samples were measured in this study (Table 4). Globally, the values obtained for all these immune factors showed a high degree of variability depending on the donor, a fact that is reflected in the CI or IQR values obtained for some of the analyzed parameters, such as IgG1, IgG2, IgM, IgA, IL-1β, IL-6, IFN-γ, IL-17, IL-8, IL-7, and G-CSF.
All the immunoglobulins (with the exception of IgG2 detected in 47% of the samples), together with IL-1β, IL-6, IL-8, GRO-α, MCP-1, MIP-1β, IL-7, and G-CSF, could be detected in all the colostrum samples. In contrast, GM-CSF (1 sample), IL-2 (3 samples), and IL-10 (5 samples) showed the lowest detection frequencies in these samples (Table 4).
In relation to mature milk, all the immunoglobulins were also detected in all the samples with the exception, again, of IgG2, which could not be detected in any sample. IL-8, MCP-1, and MIP-1β could also be detected in all the samples, whereas in the case of IL-1β, IL-6, GRO-α, IL-7, and G-CSF, there was only a single sample in which these could not be detected (Table 4). In addition, IgG2, IFN-γ, IL-2, IL-4, and IL-17 were not detected in any mature milk sample (Table 4).
The mean concentration of all the immune compounds in the colostrum samples was higher than that in the mature milk ones, with the exceptions of IL-13 and GRO-α (Table 4). Despite the high degree of variability depending on the donor, statistically significant differences between colostrum and mature milk samples were found for the concentrations of IgG3 (P = 0.017), IgG4 (P = 0.031), IL-6 (P = 0.036), IFN-γ (P = 0.000), IL-4 (P = 0.000), IL-13 (P = 0.000), IL-17 (P = 0.000), MCP-1 (P = 0.006), and MIP-1β (P = 0.000) (Table 4). Statistically significant differences were also observed in relation to the detection frequencies of IgG2 (P = 0.045), IL-12(p70) (P = 0.000), IFN-γ (P = 0.005), TNF-α (P = 0.012), IL-4 (P = 0.000), IL-13 (P = 0.001), IL-17 (P = 0.000), IL-5 (P = 0.003), and GM-CSF (P = 0.001), all of them being more frequently detected in colostrum than in mature milk samples, except for IL-13 whose detection frequency was higher in mature milk (Table 4).
Staphylococci, streptococci, and lactobacilli were the main bacterial groups isolated from colostrum and they could be also isolated, together with enterococci and enterobacteria, from some mature milk samples. In the last decade, breast milk has been recognized as a source of commensal and potential probiotic bacteria, including the bacterial groups cited above (13). The bacterial concentration (2–4 log CFU/mL) was similar to the values previously reported from colostrum and milk of healthy women (19–21). In contrast, bacteria could be detected only in a relatively low percentage (<67%) of the colostrum and mature milk samples analyzed in this study, whereas previous studies have shown that milk of most (if not all) women contain detectable levels of viable bacteria (22–26). The low bacterial detection frequency observed among colostrum and mature milk of mothers of extremely preterm babies may be for 2 reasons. First, it has been pointed that a specific mammary microbiota is formed during late pregnancy through an enteromammary mechanism involving gut monocytes (27); this process, involving a physiological bacterial translocation from the gut to mesenteric lymph nodes and mammary gland, seems to be exacerbated in the last weeks before the term delivery (19). Therefore, bacterial colonization of the mammary glands of extreme preterms’ mothers may be, at least, minimum, and it may account for the lower detection frequency found in this study. Some culture-dependent and -independent studies have confirmed a vertical mother-to-infant bacterial transfer of maternal gut bacteria via breast milk (28–31). In addition, 2 studies that focused on the oral administration of 3 lactobacilli strains isolated from human milk provided new evidences that show the existence of a bacterial enteromammary pathway during lactation (32,33).
The second reason for a low bacterial detection frequency is the high percentage of women who received antibiotherapy among those who participated in this study. It has been reported that the number of lactobacilli- or bifidobacteria-positive milk samples was significantly lower in women who had received antibiotherapy during pregnancy or lactation (26). It is well known that antibiotics are responsible for dysbiosis processes in the human microbiota, leading to antibiotic-associated diarrhea and gastroenteritis, genitourinary, and oral infections. In the last years, it is becoming evident that antibiotherapy during pregnancy, intrapartum, or lactation alters the maternal microbiota, a fact that may have negative consequences to infant health (34). The use of antibiotics during pregnancy is associated to an increased risk of infant asthma exacerbation and hospitalization, thereby supporting a role for bacterial ecology in pre- or perinatal life for the development of asthma (35).
A cultivation-independent assessment of the bacterial diversity of the samples would have been an excellent complement to the culture-based analysis, particularly for the detection of strict anaerobe bacteria (25); however, the bacterial concentration of hygienically collected human milk samples is typically low (<103 CFU/mL) and, therefore, a relatively high quantity (5–10 mL) of sample is required to obtain a concentration of bacterial DNA suitable for pyrosequencing or other DNA-related analysis. In this case, only 2 mL of each sample was available because the rest of the colostrum/milk volume was used to feed the baby.
The transition from colostrum to mature breast milk during early puerperium is associated with significant concentration changes of numerous compounds, including concentrations of free sugars and polyols. It has been repeatedly observed that, after the first days of a term postpartum, the concentrations of lactose and glucose increase significantly (36–38). The results of this work showed that the glucose and lactose concentrations in the examined population had also a significant upward trend from colostrum to milk. Although previous studies reported that preterm milk generally has lower lactose levels than term milk (36,39), the colostrum and mature milk concentrations of lactose observed in this study were similar to those obtained with the same methodology in mothers of term neonates (21,40).
In contrast to lactose or glucose, the concentrations of myo-inositol decrease significantly during the first 4 days of lactation (38,40,41), a fact that has been observed in this study, too. myo-Inositol promotes maturation of several components of surfactant and may play a critical role in fetal and early neonatal life (42). In a pioneer study, Bromberger and Hallman (43) found striking differences in the myo-inositol concentration of infant feedings (preterm colostrum, term colostrums, mature milk, infant formulas, parenteral nutrition). More specifically, colostrum from mothers who delivered prematurely had the highest myo-inositol concentration, which was significantly higher than that in colostrum from mothers who delivered at term. myo-Inositol concentrations in colostrum were significantly higher than those in mature milk, whereas preterm and mature milk myo-inositol levels did not differ significantly from each other. The results obtained in our work confirm such findings since the myo-inositol concentration in colostrum samples (mean 300.59 mg/L) was significantly higher than that in the mature milk ones (mean 194.27 mg/L). In addition, the mean colostrum concentration found in this study (from samples provided by mothers of extreme preterms) was notably higher than that reported in a previous work (40) when colostrum samples from mothers of term babies were analyzed using the same procedure and technology (mean 243.28 mg/L). The administration of inositol to premature infants with respiratory distress syndrome who are receiving parenteral nutrition during the first week of life is associated with increased survival without bronchopulmonary dysplasia and with a decreased incidence of retinopathy of prematurity (44,45). A Cochrane revision concluded that inositol supplementation results in statistically significant and clinically important reductions in important short-term adverse neonatal outcomes, and that a multicenter randomized controlled trial of appropriate size is justified to confirm such findings (42). Therefore, the results of this study highlight the importance of own's mother colostrum for preterm neonates.
In agreement with our findings, previous studies have reported that the amounts of several cytokines, chemokines, growth factors, and immunoglobulins, with anti-infectious, anti-inflammatory, and immunomodulatory roles, are notably higher in colostrum than in milk (46). The colostrum of mothers of extremely preterm infants analyzed in this study showed higher concentrations of relevant immunocompounds, such as IgA, IL-6, TNF-α, IL-4, IL-17, MCP-1, MIP-1β, IL-5, IL-7, or G-CSF, than those observed in term colostrum samples using the same procedure (40). It has been previously reported that, globally, immunoprotection provided by colostrum and milk increases as the gestational age (and, as a consequence, the neonate maturity) decreases (14,15,47). Colostrum collected at such a low gestational age (24–27 weeks) may be considered as a “prepartum milk” from the first stage of lactogenesis. Therefore, its peculiar composition may be because the junctions between alveolar cells are leaky at such pregnancy stage, allowing fluids and solutes (including large proteins) to flow between the milk space and the interstitial fluid of the mammary gland. So, it is more likely that the specific composition of colostrum in this extremely low period of gestation is related more to the immaturity of the mammary gland than to a physiological adaptation to the different metabolic/immunological needs of the extremely premature infants. These immune components that are unique to preterm colostrum may be especially protective during the first week of life and, particularly, to extremely preterm infants, a population at the highest risk for feeding intolerance and nosocomial infection (48,49). Therefore, it should be strongly recommended to start, as soon as possible, trophic feeds using own mother colostrum in extremely preterm infants because it is an easy and inexpensive procedure and well tolerated by even the smallest and sickest extremely preterm infants (50).
Analyses of correlations between all the parameters tested in this study were carried out but no statistically significant relations could be identified. Having in account the high number of factors that may affect the analyzed parameters, it is possible that a higher number of samples are required to observe significant relations among some of them.
In conclusion, a better knowledge on the composition of preterm colostrum and milk will help neonatologists and human milk banks to improve and optimize existing feeding strategies and to design novel alternatives.
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