Microbiological status of donor human milk - A single center study from Poland.

Human milk is considered the most suitable source of nutrition for infants. Donor human milk from human milk banks (HMB) is recommended as the best alternative for infants whose mothers' own milk is unavailable. Microbiological screening of milk donated to HMB is important to ensure the quality and safety of the pasteurised human milk. This article describes the microbiological status of human milk donated to the Regional Human Milk Bank in Torun, Poland. Statistical data regarding the microbiological analysis of milk from 292 donors were collected in the years 2013-2021. Total of 538 milk samples were tested. Only in 6% of human milk samples the bacteria level was above the required standard and/or the milk had potentially pathogenic bacteria. The main core of donors' breastmilk bacteria represents the skin microbiota, and the composition of the microbiota is strictly related to the surrounding environment. The most abundant genera detected in milk samples were the Staphylococcus group. Prolonged hospitalisation of infants' mothers and/or offsprings is associated with potentially pathogenic bacteria colonization in milk. The use of the modern identification method MALDI-TOF resulted in more accurate results compared to the biochemical methods. Our analysis indicates that most of the tested milk samples (94%), both expressing at home and in hospital environments, meet the criteria for admission to the human milk bank. Effective techniques for identifying microorganisms ensure that donor milk from human milk banks meets the guidelines set for these units.

Leptin in Human Milk-One of the Key Regulators of Nutritional Programming.

Breast milk is the optimal food for infants and toddlers, providing basic nutrients. It is also a source of many biologically active substances. Among them are hormones responsible for metabolic balance. One of the hormones taken in with breast milk by a breastfed baby is leptin. This hormone is involved in the regulation of appetite, informing the brain about the body's energy resources. Having the correct mechanisms related to the action of leptin is a factor reducing the risk of obesity. The natural presence of leptin in the composition of breast milk suggests that it has a specific role in shaping the health of a breastfed child. Obesity as a disease of civilization affects more and more people, including children. The development of this disease is multifaceted and determined by many factors, including genetic and environmental factors such as eating habits and low physical activity. Behind obesity, there are complex mechanisms in which many elements of the human body are involved. Understanding the effects of breastfeeding as a natural source of leptin can help prevent childhood obesity and development of this disease in future life.

Nutrients and Bioactive Components of Human Milk After One Year of Lactation: Implication for Human Milk Banks.

OBJECTIVE: Specifying the nutrient content and bioactive compounds in milk from long-term lactation for the purpose of finding additional sources of donors' milk for human milk banks. METHOD: Human milk samples were collected from 43 mothers of term infants (term infant human milk, TIHM) (3-6 weeks of lactation) and 50 mothers who have breastfed for over a year (long-nursing human milk, LNHM). The milk collection time was 24 hours. The analyses of fat, protein, carbohydrate and energy content were performed with human milk analyzer MIRIS (Miris HMA™ ); lactoferrin and vitamin C content using high performance liquid chromatography in reversed phase (RP-HPLC); total antioxidant activity (TAC) and lysozyme by enzyme-linked immunosorbent assay (ELISA); and the activity of glutathione peroxidase (GPx) and catalase (CAT) via spectrophotometric methods. Sociodemographic characteristics of both groups of mothers have been compiled. RESULTS: Higher fat content and energy value was found in LNHM than in TIHM. Protein content in LNHM and TIHM did not differ. Carbohydrate content was lower in LNHM than in TIHM. TAC, CAT and GPx activity were higher in LNHM compared to TIHM. No significant differences in the content of lysozyme, lactoferrin and vitamin C between the studied groups were found. CONCLUSIONS: The findings suggest that after 12 months of lactation, human milk still has significant nutritional value, does not lose bioactive components and can be considered for use in human milk banks.

Effect of convection and microwave heating on the retention of bioactive components in human milk.

Bioactive substances are very important components of human milk (HM), especially for premature newborns. The effects of convection (CH) and microwave heating (MWH) at 62.5 and 66 °C, on the level of selected bioactive components of HM: lysozyme (LZ), lactoferrin (LF), secretory immunoglobulin A (sIgA), basal lipase (BL), cytokine TGF-2, vitamin C and total antioxidant capacity (TAC) was compared. Regardless of the used heating methods the TAC of HM, determined by TEAC and ORAC-FL assay, proved to be insensitive to temperature pasteurization, in contrary to BL. MWH in the conditions of 62.5 for 5 min and 66 °C for 3 min are ensuring microbiological safety with a higher retention of most of the tested active HM proteins compared to CH. Only in the case of LZ the MWH had a more degradative effect on its concentration. Controlled conditions of MWH preserve the bioactive components of the HM better than CH.

Tandem Breastfeeding: A Descriptive Analysis of the Nutritional Value of Milk When Feeding a Younger and Older Child.

Breastfeeding is a gold standard of feeding of newborns and infants. Tandem breastfeeding (TBF) is feeding two children of different ages at the same time. The knowledge about the composition of human milk in prolonged lactation is still scarce. Milk from tandem breastfeeding women and after weaning was examined. Milk samples were collected from 13 TBF mothers. A 24-h milk collection was done. Analyses of fat, protein, carbohydrate and energy content were performed using MIRIS. Sociodemographic characteristics of TBF mothers was done. Higher fat content, energy value and total protein concentration was found in TBFM milk during tandem breastfeeding, than in milk after weaning the older child. The carbohydrate content remained stable. The composition of breastmilk, in terms of macronutrients, changes after weaning, taking into account the nutritional requirements of the younger child. The milk of nursing mothers in tandem did not show diurnal variability in individual components. These findings suggest an adaptive role of human milk to nutrient requirements of newborn and older children. The results may support the promotion of long breastfeeding, including tandem breastfeeding.

Donor Human Milk for Preterm Infants: A Cost-Consequence Analysis of Alternative Preservation Methods.

OBJECTIVES: To perform cost-consequence and cost-effectiveness analyses of 2 methods of donor human milk (DHM) preservation-Holder pasteurization (HoP) and high-pressure processing (HPP)-in human milk banks in Poland. METHODS: We used the results of the LACTOTECHnology as an answer to special nutritional requirements of preterm infants (LACTOTECH) preclinical study on the impact of different preservation methods on the content of bioactive milk components. The cost analysis was performed from the hospital perspective. To estimate the Diagnosis-Related Group (DRG) tariff for enteral feeding with DHM preserved by HPP, the pricing process used by the Polish health technology assessment agency (Agencja Oceny Technologii Medycznych i Taryfikacji) was followed. One-way deterministic and probabilistic sensitivity analyses on costs and human milk component parameters were undertaken. RESULTS: HPP maintains an average of 55% more potentially beneficial DHM components than HoP, but is more expensive (€35 750 vs €5066). The DRG tariff relating to milk from human milk banks preserved by the HPP method should be about €54 (130%) higher than with HoP. The cost-effectiveness ratio ranged from €0.84 to €10.27 per 1% gain in the active compound content in a daily portion of DHM. Sensitivity analysis showed that the cost of an HPP device had the most significant impact on pascalization expenses. CONCLUSIONS: HPP is a potentially more beneficial method of DHM preservation than HoP, but it is also about 7 times more expensive. Because of high pascalization costs, the cost-effectiveness analysis based on clinically significant endpoints will play an important role in decision making regarding the implementation of HPP into clinical practice of human milk banking.

Implementation of the Reimbursement Cost of Human-Milk-Based Neonatal Therapy in Polish Health Care Service : Practical and Ethical Background.

BACKGROUND: A human-milk-based diet is the best option for nutritional therapy for preterm and/or sick newborns. RESEARCH AIM: The study aims were to restructure the reimbursement rates to hospitals in Poland for infants' tube feedings to favor the use of donor human milk over formula for newborns who required supplementation of expressed mother's milk and evaluate the results of the financing change during the first year of implementation (2018). METHODS: Financial data from hospitals were collected (2015-2016) by the Human Milk Bank Foundation using a data sheet designed by the Agency for Health Technology Assessment and Tariff System. We used data to restructure the reimbursement rates to hospitals for infants' tube feedings and implemented the changes in late 2017. The National Health Fund was requested to share reported data in 2018 concerning tube feeding services. RESULTS: More than half (61%) of NICUs introduced human milk tube feeding for newborns. It was provided to participants (N = 5,530), most frequently to seriously ill preterm infants (66.6%). Of these infants, 2,323 were fed donor human milk. Only 1,925 newborns received formula tube feeding. However, there were large differences in frequency of services reported among various parts of the country. CONCLUSIONS: Based on our knowledge, Poland is the only European country where the reimbursement cost for human-milk-based nutritional therapy has been implemented in a manner intended to increase the quality of health care services for preterm newborns. Equal reimbursement for expressed mother's milk and donor milk did not appear to cause overuse of donor milk based on our analysis of the 2018 data.

QuEChERS extraction coupled to GC-MS for a fast determination of polychlorinated biphenyls in breast milk from Polish women.

Polychlorinated biphenyls (PCBs) in breast milk has been determined. Therefore, it was necessary to develop and adapt an analytical method to analyze PCB compounds. The whole procedure was applied to 31 breast milk samples, which were collected from Polish mothers. The QuEChERS method was optimized as a fast and cheap sample preparation method. The procedure allowed us to obtain recovery values between 96.46% and 119.98% with acceptable relative standard deviations (3.36-12.71%). Gas chromatography with mass spectrometry (GC-MS) was used for final determination. The method was validated using parameters such as linearity, limit of detection and quantification, intra-day precision, and reproducibility. The mean concentration of ∑iPCBs in this study was 30.94 ng/g of lipid. Assigned daily intake of PCBs was lower than the tolerable daily intake, which shows that the analyzed milk is safe to the infants. However, the monitoring of PCBs in milk is still important, and the QuEChERS method with GC-MS can be an effective tool for tracking organic impurities in breast milk.

Innovative Techniques of Processing Human Milk to Preserve Key Components.

Human milk not only contains all nutritional elements that an infant requires, but is also the source of components whose regulatory role was confirmed by demonstrating health-related deficiencies in formula-fed children. A human milk diet is especially important for premature babies in the neonatal intensive care unit (NICU). In cases where breastfeeding is not possible and the mother's own milk is insufficient in volume, the most preferred food is pasteurized donor milk. The number of human milk banks has increased recently but their technical infrastructure is continuously developing. Heat treatment at a low temperature and long time, also known as holder pasteurization (62.5 °C, 30 min), is the most widespread method of human milk processing, whose effects on the quality of donor milk is well documented. Holder pasteurization destroys vegetative forms of bacteria and most viruses including human immunodeficiency virus (HIV) herpes and cytomegalovirus (CMV). The macronutrients remain relatively intact but various beneficial components are destroyed completely or compromised. Enzymes and immune cells are the most heat sensitive elements. The bactericidal capacity of heat-pasteurized milk is lower than that of untreated milk. The aim of the study was for a comprehensive comparison of currently tested methods of improving the preservation stage. Innovative techniques of milk processing should minimize the risk of milk-borne infections and preserve the bioactivity of this complex biological fluid better than the holder method. In the present paper, the most promising thermal pasteurization condition (72 °C-75 °C,) and a few non-thermal processes were discussed (high pressure processing, microwave irradiation). This narrative review presents an overview of methods of human milk preservation that have been explored to improve the safety and quality of donor milk.

The impact of environmental pollution on the quality of mother's milk.

Breastfeeding is a gold standard of neonate nutrition because human milk contains a lot of essential compounds crucial for proper development of a child. However, milk is also a biofluid which can contain environmental pollution, which can have effects on immune system and consequently on the various body organs. Polychlorinated biphenyls are organic pollutants which have been detected in human milk. They have lipophilic properties, so they can penetrate to fatty milk and ultimately to neonate digestive track. Another problem of interest is the presence in milk of heavy metals-arsenic, lead, cadmium, and mercury-as these compounds can lead to disorders in production of cytokines, which are important immunomodulators. The toxicants cause stimulation or suppression of this compounds. This can lead to health problems in children as allergy, disorders in the endocrine system, end even neurodevelopment delay and disorder. Consequently, correlations between pollutants and bioactive components in milk should be investigated. This article provides an overview of environmental pollutants found in human milk as well as of the consequences of cytokine disorder correlated with presence of heavy metals. Graphical abstract.

New Achievements in High-Pressure Processing to Preserve Human Milk Bioactivity.

High-pressure processing (HPP) is a non-thermal technology that is being increasingly applied in food industries worldwide. It was proposed that this method could be used as an alternative to holder pasteurization (HoP; 62.5°C, 30 min) in milk banks but its impact on the immunologic, enzymatic and hormonal components of human milk has not yet been evaluated in detail. The aim of our study was to compare the effects of HPP in variants: (1) 600 MPa, 10 min (2) 100 MPa, 10 min, interval 10 min, 600 MPa, 10 min (3) 200 MPa, 10 min, interval 10 min, 400 MPa, 10 min (4) 200 MPa, 10 min, interval 10 min, 600 MPa, 10 min in temperature range 19-21°C and HoP on the leptin, adiponectin, insulin, hepatocyte growth factor (HGF), lactoferrin and IgG contents in human milk. HoP was done at the Regional Human Milk Bank in Warsaw at the Holy Family Hospital on S90 Eco pasteurizer (Sterifeed, Medicare Colgate Ltd). Apparatus U4000/65 (Unipress Equipment, Poland) was used for pascalization. Milk samples were obtained from women during 2-6 weeks of lactation. Post-treatment culture showed no endogenous bacterial contamination in any tested option. Concentrations of selected components were determined using ELISA tests. The level of all analyzed components were significantly decreased by HoP: leptin 77.86%, adiponectin 32.79%, insulin 32.40%, HGF 88.72%, lactoferrin 60.31@.%, IgG 49.04%. All HPP variants caused an increase in leptin concentration, respectively (1) 81.79% (2) 90.01% (3) 86.12% (4) 47.96%. Retention of insulin after HPP was (1) 88.20% (2) 81.98% (3) 94.76% (4) 90.31% HGF (1) 36.15% (2) 38.81% 97.15% (3) 97.15% (4) 43.02%, lactoferrin (1) 55.78% (2) 57.63% (3) 78.77% (4) 64.75%. Moreover, HPP variant as 200 + 400 MPa preserved IgG (82.24%) better than HoP and resulted not statistically significant change of adiponectin level (38.55%) compare to raw milk. Our results showed that HPP leads to preservation of adipokines, growth factor, and lactoferrin, IgG much better or comparable with HoP.

The LITAF/SIMPLE I92V sequence variant results in an earlier age of onset of CMT1A/HNPP diseases.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) represent the most common heritable neuromuscular disorders. Molecular diagnostics of CMT1A/HNPP diseases confirm clinical diagnosis, but their value is limited to the clinical course and prognosis. However, no biomarkers of CMT1A/HNPP have been identified. We decided to explore if the LITAF/SIMPLE gene shared a functional link to the PMP22 gene, whose duplication or deletion results in CMT1A and HNPP, respectively. By studying a large cohort of CMT1A/HNPP-affected patients, we found that the LITAF I92V sequence variant predisposes patients to an earlier age of onset of both the CMT1A and HNPP diseases. Using cell transfection experiments, we showed that the LITAF I92V sequence variant partially mislocalizes to the mitochondria in contrast to wild-type LITAF which localizes to the late endosome/lysosomes and is associated with a tendency for PMP22 to accumulate in the cells. Overall, this study shows that the I92V LITAF sequence variant would be a good candidate for a biomarker in the case of the CMT1A/HNPP disorders.

Clinical, electrophysiological, and molecular findings in early onset hereditary neuropathy with liability to pressure palsy.

INTRODUCTION: The first episode of hereditary neuropathy with liability to pressure palsy (HNPP) in childhood is rare. METHODS: We analyzed retrospectively the data of 7 patients with a deletion in PMP22 and onset of symptoms before age 18 years. Direct sequencing of the LITAF (lipopolysaccharide-induced tumor necrosis factor) gene was performed in patients and family members. RESULTS: Clinical presentations varied from mononeuropathies to brachial plexopathy, with recurrent episodes in 4 patients. Electrophysiological abnormalities characteristic for HNNP were found in most subjects. Analysis of the LITAF gene revealed an Ile92Val polymorphism in 6 of 7 (86%) probands and 5 of 7 (83%) family members, over 4 times greater frequency than in the general population. CONCLUSIONS: Clinical suspicion of HNPP even when nerve conduction study results do not fulfill HNPP criteria should indicate genetic testing. In our patients, early-onset HNPP was associated frequently with isoleucine92valine LITAF polymorphism.

Charcot-Marie-Tooth type 1C disease coexisting with progressive multiple sclerosis: a study of an overlapping syndrome.

Charcot-Marie-Tooth type 1C disease (CMT1C) is a rare form of hereditary demyelinating neuropathy caused by mutations in the LITAF (lipopolysaccharide-induced tumor necrosis factor-) gene. CMT1C disease was mapped to chromosome 16p12-p 13.3. To date only a few mutations in the LITAF gene have been reported. Due to a small group of CMT1C reported patients, the phenotype of CMT1C is poorly characterized. CMT1C disease is a pure demyelinating neuropathy limited to the peripheral nervous system with a mild clinical course, manifesting without any additional symptoms. To the best of our knowledge, in this study, for the first time we present a three generational CMT1C family in which in the proband, CMT1C disease coexists with central demyelination fulfilling criteria of primary progressive multiple sclerosis (PPMS). The coexistence of PPMS and CMT1C in one family may not result from a common pathogenetic trait, however only in the proband with central demyelination and CMT1C we have detected a -308G>A sequence variant in the promoter of the TNF-α gene.

The 5' regulatory sequence of the PMP22 in the patients with Charcot-Marie-Tooth disease.

Little is known about the molecular background of clinical variability of Charcot-Marie-Tooth type 1A (CMT1A) disease and hereditary neuropathy with liability to pressure palsies (HNPP). The CMT1A and HNPP disorders result from duplication and deletion of the PMP22 gene respectively. In a series of studies performed on affected animal transgenic models of CMT1A disease, expression of the PMP22 gene (gene dosage) was shown to correlete with severity of CMT course (gene dosage effect). In this study we hypothesized that single nucleotide polymorphisms (SNPs) located within the 5' regulatory sequence of PMP22 gene may be responsible for the CMT1A/HNPP clinical variability. We have sequenced the PMP22 5' upstream regulatory sequence in a group of 45 CMT1A/HNPP patients harboring the PMP22 duplication (37) /deletion (8). We have identified five SNPs in the regulatory sequence of the PMP22 gene. Three of them i.e. -819C>T, -4785G>T, -4800C>T were detected both in the patients and in the control group. Thus, their pathogenic role in the regulation of the expression of the PMP22 gene seems not to be significant. Two SNPs i.e. -4210T>C and -4759T>A were found only in the CMT patients. Their role in the regulation of the PMP22 gene expression can not be excluded. Additionally we have detected the Thr118Met variant in exon 4 of the PMP22 gene, which was previously reported by other authors, in one patient. We conclude that the 5' regulatory sequence of the PMP22 gene is conserved at the nucleotiode level, however rarely occurring SNPs variant in the PMP22 regulatory sequence may be associated with the gene dosage effect.

A newly identified Thr99fsX110 mutation in the PMP22 gene associated with an atypical phenotype of the hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) is manifested by a spectrum of phenotypes, from the classical HNPP course associated with intermittent nerve palsies to a neuropathy resembling Charcot-Marie-Tooth type 1 (CMT1) disease. The majority of HNPP cases are associated with submicroscopical deletions in the 17p11.2-p12 region containing the PMP22 gene, while PMP22 point mutations are rare, representing about 15% of HNPP cases. In this study, we present a patient manifesting with atypical HNPP phenotype associated with a new Thr99fsX110 mutation in the PMP22 gene. We conclude that all patients who fulfill the electrophysiological criteria of HNPP, even if they lack the typical HNPP phenotype, should be tested for point mutations in the PMP22 gene.