Human Milk and Lactation Printed Edition of the Special Issue Published in Nutrients www.mdpi.com/journal/nutrients Maria Lorella Gianni Edited by Human Milk and Lactation Human Milk and Lactation Special Issue Editor Maria Lorella Gianni MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Special Issue Editor Maria Lorella Gianni University of Milan Italy Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Nutrients (ISSN 2072-6643) (available at: https://www.mdpi.com/journal/nutrients/special issues/Human Milk Lactation). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. ISBN 978-3-03928-923-3 ( H bk) ISBN 978-3-03928-924-0 (PDF) c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Maria Lorella Gianni, Daniela Morniroli, Maria Enrica Bettinelli and Fabio Mosca Human Milk and Lactation Reprinted from: Nutrients 2020 , 12 , 899, doi:10.3390/nu12040899 . . . . . . . . . . . . . . . . . . . 1 Sara Moukarzel, Alejandra M. Wiedeman, Lynda S. Soberanes, Roger A. Dyer, Sheila M. Innis and Yvonne Lamers Variability of Water-Soluble Forms of Choline Concentrations in Human Milk during Storage, after Pasteurization, and among Women Reprinted from: Nutrients 2019 , 11 , 3024, doi:10.3390/nu11123024 . . . . . . . . . . . . . . . . . . 5 Maria Lorella Gianni, Maria Enrica Bettinelli, Priscilla Manfra, Gabriele Sorrentino, Elena Bezze, Laura Plevani, Giacomo Cavallaro, Genny Raffaeli, Beatrice Letizia Crippa, Lorenzo Colombo, Daniela Morniroli, Nadia Liotto, Paola Roggero, Eduardo Villamor, Paola Marchisio and Fabio Mosca Breastfeeding Difficulties and Risk for Early Breastfeeding Cessation Reprinted from: Nutrients 2019 , 11 , 2266, doi:10.3390/nu11102266 . . . . . . . . . . . . . . . . . . 17 Maria Grunewald, Christian Hellmuth, Franca F. Kirchberg, Maria Luisa Mearin, Renata Auricchio, Gemma Castillejo, Ilma R. Korponay-Szabo, Isabel Polanco, Maria Roca, Sabine L. Vriezinga, Katharina Werkstetter, Berthold Koletzko and Hans Demmelmair Variation and Interdependencies of Human Milk Macronutrients, Fatty Acids, Adiponectin, Insulin, and IGF-II in the European PreventCD Cohort Reprinted from: Nutrients , 11 , 2034, doi:10.3390/nu11092034 . . . . . . . . . . . . . . . . . . . . . 27 Michele R. Machado, Fernanda Kamp, Juliana C. Nunes, Tatiana El-Bacha and Alexandre G. Torres Breast Milk Content of Vitamin A and E from Early- to Mid-Lactation Is Affected by Inadequate Dietary Intake in Brazilian Adult Women Reprinted from: Nutrients 2019 , 11 , 2025, doi:10.3390/nu11092025 . . . . . . . . . . . . . . . . . . 47 Aleksandra Wesolowska, Joanna Brys, Olga Barbarska, Kamila Strom, Jolanta Szymanska-Majchrzak, Katarzyna Karzel, Emilia Pawlikowska, Monika A. Zielinska, Jadwiga Hamulka and Gabriela Oledzka Lipid Profile, Lipase Bioactivity, and Lipophilic Antioxidant Content in High Pressure Processed Donor Human Milk Reprinted from: Nutrients 2019 , 11 , 1972, doi:10.3390/nu11091972 . . . . . . . . . . . . . . . . . . 61 Jiahui Yu, Tinglan Yuan, Xinghe Zhang, Qingzhe Jin, Wei Wei and Xingguo Wang Quantification of Nervonic Acid in Human Milk in the First 30 Days of Lactation: Influence of Lactation Stages and Comparison with Infant Formulae Reprinted from: Nutrients 2019 , 11 , 1892, doi:10.3390/nu11081892 . . . . . . . . . . . . . . . . . . 77 Magalie Sabatier, Clara L. Garcia-Rodenas, Carlos A. De Castro, Peter Kastenmayer, Mario Vigo, St ́ ephane Dubascoux, Daniel Andrey, Marine Nicolas, Janique Richoz Payot, Valentine Bordier, Sagar K. Thakkar, Lydie Beauport, Jean-Fran ̧ cois Tolsa, C ́ eline J. Fischer Fumeaux and Michael Affolter Longitudinal Changes of Mineral Concentrations in Preterm and Term Human Milk from Lactating Swiss Women Reprinted from: Nutrients 2019 , 11 , 1855, doi:10.3390/nu11081855 . . . . . . . . . . . . . . . . . . 91 v Alessandra Mazzocchi, Maria Lorella Giann` ı, Daniela Morniroli, Ludovica Leone, Paola Roggero, Carlo Agostoni, Valentina De Cosmi and Fabio Mosca Hormones in Breast Milk and Effect on Infants’ Growth: A Systematic Review Reprinted from: Nutrients 2019 , 11 , 1845, doi:10.3390/nu11081845 . . . . . . . . . . . . . . . . . . 105 Daniela Hampel, Setareh Shahab-Ferdows, Muttaquina Hossain, M. Munirul Islam, Tahmeed Ahmed and Lindsay H. Allen Validation and Application of Biocrates Absolute IDQ R © p180 Targeted Metabolomics Kit Using Human Milk Reprinted from: Nutrients 2019 , 11 , 1733, doi:10.3390/nu11081733 . . . . . . . . . . . . . . . . . . 117 Adekunle Dawodu, Khalil M. Salameh, Najah S. Al-Janahi, Abdulbari Bener and Naser Elkum The Effect of High-Dose Postpartum Maternal Vitamin D Supplementation Alone Compared with Maternal Plus Infant Vitamin D Supplementation in Breastfeeding Infants in a High-Risk Population. A Randomized Controlled Trial Reprinted from: Nutrients 2019 , 11 , 1632, doi:10.3390/nu11071632 . . . . . . . . . . . . . . . . . . 127 Agnieszka Bzikowska-Jura, Aneta Czerwonogrodzka-Senczyna, Edyta Jasi ́ nska-Melon, Hanna Mojska, Gabriela Ol ę dzka, Aleksandra Wesołowska and Dorota Szostak-W ę gierek The Concentration of Omega-3 Fatty Acids in Human Milk Is Related to Their Habitual but Not Current Intake Reprinted from: Nutrients 2019 , 11 , 1585, doi:10.3390/nu11071585 . . . . . . . . . . . . . . . . . . 145 Veronique Demers-Mathieu, Robert K. Huston, Andi M. Markell, Elizabeth A. McCulley, Rachel L. Martin and David C. Dallas Antenatal Influenza A-Specific IgA, IgM, and IgG Antibodies in Mother’s Own Breast Milk and Donor Breast Milk, and Gastric Contents and Stools from Preterm Infants Reprinted from: Nutrients 2019 , 11 , 1567, doi:10.3390/nu11071567 . . . . . . . . . . . . . . . . . . 161 C ́ eline J. Fischer Fumeaux, Clara L. Garcia-Rodenas, Carlos A. De Castro, Marie-Claude Courtet-Compondu, Sagar K. Thakkar, Lydie Beauport, Jean-Fran ̧ cois Tolsa and Michael Affolter Longitudinal Analysis of Macronutrient Composition in Preterm and Term Human Milk: A Prospective Cohort Study Reprinted from: Nutrients 2019 , 11 , 1525, doi:10.3390/nu11071525 . . . . . . . . . . . . . . . . . . 173 Sean Austin, Carlos A. De Castro, Norbert Sprenger, Aristea Binia, Michael Affolter, Clara L. Garcia-Rodenas, Lydie Beauport, Jean-Fran ̧ cois Tolsa and C ́ eline J. Fischer Fumeaux Human Milk Oligosaccharides in the Milk of Mothers Delivering Term versus Preterm Infants Reprinted from: Nutrients 2019 , 11 , 1282, doi:10.3390/nu11061282 . . . . . . . . . . . . . . . . . . 185 Malgorzata Witkowska-Zimny, Ewa Kami ́ nska-El-Hassan and Edyta Wr ́ obel Milk Therapy: Unexpected Uses for Human Breast Milk Reprinted from: Nutrients 2019 , 11 , 944, doi:10.3390/nu11050944 . . . . . . . . . . . . . . . . . . . 201 Amanda de Sousa Rebou ̧ cas, Ana Gabriella Costa Lemos da Silva, Amanda Freitas de Oliveira, Lorena Thalia Pereira da Silva, Vanessa de Freitas Felgueiras, Marina Sampaio Cruz, Vivian Nogueira Silbiger, Karla Danielly da Silva Ribeiro and Roberto Dimenstein Factors Associated with Increased Alpha-Tocopherol Content in Milk in Response to Maternal Supplementation with 800 IU of Vitamin E Reprinted from: Nutrients 2019 , 11 , 900, doi:10.3390/nu11040900 . . . . . . . . . . . . . . . . . . . 213 vi Therese A. O’Sullivan, Joy Cooke, Chris McCafferty and Roslyn Giglia Online Video Instruction on Hand Expression of Colostrum in Pregnancy is an Effective Educational Tool Reprinted from: Nutrients 2019 , 11 , 883, doi:10.3390/nu11040883 . . . . . . . . . . . . . . . . . . . 227 Moh` eb Elwakiel, Sjef Boeren, Jos A. Hageman, Ignatius M. Szeto, Henk A. Schols and Kasper A. Hettinga Variability of Serum Proteins in Chinese and Dutch Human Milk during Lactation Reprinted from: Nutrients 2019 , 11 , 499, doi:10.3390/nu11030499 . . . . . . . . . . . . . . . . . . . 239 Beatrice Letizia Crippa, Lorenzo Colombo, Daniela Morniroli, Dario Consonni, Maria Enrica Bettinelli, Irene Spreafico, Giulia Vercesi, Patrizio Sannino, Paola Agnese Mauri, Lidia Zanotta, Annalisa Canziani, Paola Roggero, Laura Plevani, Donatella Bertoli, Stefania Zorzan, Maria Lorella Giann` ı and Fabio Mosca Do a Few Weeks Matter? Late Preterm Infants and Breastfeeding Issues Reprinted from: Nutrients 2019 , 11 , 312, doi:10.3390/nu11020312 . . . . . . . . . . . . . . . . . . . 253 Ida Emilie Ingvordsen Lindahl, Virginia M. Artegoitia, Eimear Downey, James A. O’Mahony, Carol-Anne O’Shea, C. Anthony Ryan, Alan L. Kelly, Hanne C. Bertram and Ulrik K. Sundekilde Quantification of Human Milk Phospholipids: The Effect of Gestational and Lactational Age on Phospholipid Composition Reprinted from: Nutrients 2019 , 11 , 222, doi:10.3390/nu11020222 . . . . . . . . . . . . . . . . . . . 263 Monika A. Zielinska, Jadwiga Hamulka and Aleksandra Wesolowska Carotenoid Content in Breastmilk in the 3rd and 6th Month of Lactation and Its Associations with Maternal Dietary Intake and Anthropometric Characteristics Reprinted from: Nutrients 2019 , 11 , 193, doi:10.3390/nu11010193 . . . . . . . . . . . . . . . . . . . 277 Sagar K. Thakkar, Carlos Antonio De Castro, Lydie Beauport, Jean-Fran ̧ cois Tolsa, C ́ eline J. Fischer Fumeaux, Michael Affolter and Francesca Giuffrida Temporal Progression of Fatty Acids in Preterm and Term Human Milk of Mothers from Switzerland Reprinted from: Nutrients 2019 , 11 , 112, doi:10.3390/nu11010112 . . . . . . . . . . . . . . . . . . . 293 Jing Zhu and Kelly A. Dingess The Functional Power of the Human Milk Proteome Reprinted from: Nutrients 2019 , 11 , 1834, doi:10.3390/nu11081834 . . . . . . . . . . . . . . . . . . 305 Andrea Gila-Diaz, Silvia M. Arribas, Alba Algara, Mar ́ ıa A. Mart ́ ın-Cabrejas, ́ Angel Luis L ́ opez de Pablo, Miguel S ́ aenz de Pipa ́ on and David Ramiro-Cortijo A Review of Bioactive Factors in Human Breastmilk: A Focus on Prematurity Reprinted from: Nutrients 2019 , 11 , 1307, doi:10.3390/nu11061307 . . . . . . . . . . . . . . . . . . 333 vii About the Special Issue Editor Maria Lorella Gianni earned her medical degree in 1993 and completed her residency training in pediatrics in 1998 at the University of Milan, San Paolo Hospital, where she began working on inborn errors of metabolism and infant nutrition and later health. Since 2002, she has been working at Fondazione IRCCS “C` a Granda” Ospedale Maggiore Policlinico, University of Milan, Italy. She is involved in monitoring the health, nutritional status, and neurodevelopmental outcome of high-risk infants. ix nutrients Editorial Human Milk and Lactation Maria Lorella Gianni 1,2, *, Daniela Morniroli 1 , Maria Enrica Bettinelli 2 and Fabio Mosca 1,2 1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, NICU, Via Commenda 12, 20122 Milan, Italy; daniela.morniroli@gmail.com (D.M.); fabio.mosca@unimi.it (F.M.) 2 Department of Clinical Science and Community Health, University of Milan, Via Commenda 19, 20122 Milan, Italy; mebettinelli@gmail.com * Correspondence: maria.gianni@unimi.it; Tel.: + 39-0255-032-483 Received: 9 March 2020; Accepted: 17 March 2020; Published: 26 March 2020 Human milk is uniquely tailored to meet infants’ specific nutritional requirements [ 1 ]. However, it is more than just “milk” since it has emerged as an evolutionary strategy to promote human well-being [ 2 ]. This dynamic and bioactive fluid allows mother–infant signalling over lactation, guiding the infant in the developmental and physiological processes. Human milk exerts protection and life-long biological e ff ects, playing a crucial role in promoting healthy growth and optimal cognitive development [ 3 , 4 ]. For this evidence, the promotion of breastfeeding initiation and duration becomes paramount in all healthcare settings [ 5 ]. The latest scientific advances have provided insight into di ff erent components of human milk and their dynamic and flexible changes over time in response to several biological and environmental triggers. However, the complexity of human milk composition and the synergistic mechanisms responsible for its beneficial health e ff ects have not yet been unravelled [ 4 ]. This special issue has brought together a variety of articles, including original works and literature reviews, further exploring the complexity of the human milk biofluid and the mechanisms underlying the beneficial e ff ects associated with breastfeeding. In this issue, the mounting amount of data regarding human milk proteome and metabolome, gathered using advanced technological achievements such as “omics” techniques, has been reviewed, describing the multitude of bioactive components and their relationship with infants’ cognitive development, growth and immune functions [ 6 , 7 ]. Changes in human milk protein content over the first months of lactation in mothers from di ff erent geographical and ethnic origins have been investigated [ 8 ]. The high abundance of immune active proteins reflects the well-known immunological properties of mothers’ milk [ 8 ]. Authors enhance the importance of passive immunisation through mothers’ antibodies transfer from breast milk, which has a key role for infant immune protection in the first months [ 9 ]. Di ff erences in oligosaccharides content between term and preterm milk have also been examined in view of the potential implications for preterm infants’ clinical outcomes with special regard to their increased vulnerability to infections [ 10 ]. Given the widely known anti-inflammatory and antimicrobial properties of human milk, authors have also explored its implementation as a powerful therapeutic agent for skin issues, suggesting its potential use in settings with limited access to medicine [11]. In this special edition, attention has been focused on the variability of human milk compounds depending on individual di ff erences among mothers and, far more significant, on mothers’ nutritional status and anthropometric characteristics. Authors outline the importance of a healthy lifestyle and a correct micro and macronutrient intake, before and during pregnancy and lactation, in order to promote adequate levels of vitamins and other components in human milk [ 12 – 16 ]. Moreover, author recommendations indicate the need for identifying women at risk for a deficiency, who could, therefore, benefit from an appropriate supplementation aimed at increasing breastmilk micronutrient content [12–16]. The more the exceptional qualities of human milk are brought up, the more the support of breastfeeding initiation and duration becomes fundamental [ 5 ]. However, breastfeeding rates are still Nutrients 2020 , 12 , 899; doi:10.3390 / nu12040899 www.mdpi.com / journal / nutrients 1 Nutrients 2020 , 12 , 899 lower than recommended, especially in developed countries. Authors highlight the association among breastfeeding di ffi culties in the first months of lactation and early breastfeeding cessation and advocate the provision of continued tailored breastfeeding support also after hospital discharge [ 17 ]. Within this context, the e ff ectiveness of online sources including an expert instructional video in improving maternal knowledge and confidence regarding antenatal colostrum expressing, which in turn may promote long term breastfeeding, has been explored [18]. In this issue, authors have investigated the potential relationship between the presence of unique components of human milk and the positive long-life beneficial e ff ects associated with breastfeeding. In view of the crucial role of neuronic acid in white matter development, its content in human milk through the first month of lactation has been quantified and compared with that of formula milk from three fat sources [ 19 ]. Human milk’s hormonal content, which seems to be involved in infants’ metabolic pathways, including appetite and energy balance, has been also examined in light of the reduced risk of developing overweight and metabolic syndrome in human milk-fed infants [20,21]. Benefits of human milk feeding are indeed even more critical among specific populations at high risk of developing adverse outcomes, as preterm infants [ 22 ]. This value is highlighted not only by the positive e ff ects that human milk has in modulating preterms’ outcomes at every level but also by the results of studies in this issue demonstrating the higher levels of bioactive, micro and macronutrient contents in preterm milk, compared to full-term [ 10 , 23 – 26 ]. Within this context, however, authors have underlined the potential lack of mineral content of preterm milk that should be taken into consideration in the approach to the fortification of milk for the preterm population [27]. Since human milk feeding is associated with several life-long important beneficial health e ff ects, in a dose-dependent relation, its promotion and support should be considered as a public health issue [ 2 ]. Unfortunately, the authors underline that breastfeeding initiation and duration are even more challenging in preterm infants [ 28 ]. Therefore, donor human milk has been studied for its role as a fresh mother’s milk substitute. Even though donor milk has to be processed through pasteurisation for microbiological safety reasons and supplemented with fortifiers, it has been demonstrated to be a better feeding alternative for preterm infants, compared to formula milk, when the own mother’s milk is not available [ 29 ]. The refrigeration, freezing, and pasteurisation of donor milk have a variable impact on vitamin, enzymes and nutrients concentration, resulting in a diminished bioactive function of donor milk [ 30 ]. In this issue, changes in concentrations after pasteurization of water-soluble forms of choline, which is crucial for infants’ development, have been investigated together with the potential for reducing the loss of donor human milk compounds by using innovative techniques including high-pressure processing [31,32]. As the diverse articles in this special issue highlight, commitment towards filling the knowledge gap on the complex and highly dynamic human milk composition and the strictly interrelated mechanisms underpinning its positive long-life biological e ff ects is crucial for a deeper understanding of the biology of the developing infant and the optimisation of infant feeding, particularly that of the most vulnerable infants. Author Contributions: M.L.G., D.M. wrote the editorial, M.E.B., F.M. reviewed and revised the editorial. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. WHO / UNICEF. Global Strategy for Infant and Young Child Feeding ; World Health Organization: Geneva, Switzerland, 2003. 2. Goldman, A.S. Evolution of immune functions of the mammary gland and protection of the infant. Breastfeed. Med. 2012 , 7 , 132–142. [CrossRef] [PubMed] 2 Nutrients 2020 , 12 , 899 3. Mosca, F.; Giann ì , M.L. Human Milk: Composition and Health Benefits. Pediatr. Med. Chir. 2017 , 39 , 155. [CrossRef] [PubMed] 4. Bardanzellu, F.; Peroni, D.G.; Fanos, V. Human Breast Milk: Bioactive Components, from Stem Cells to Health Outcomes. Curr. Nutr. Rep. 2020 , 9 , 1–13. [CrossRef] [PubMed] 5. Rollins, N.C.; Bhandari, N.; Hajeebhoy, N.; Horton, S.; Lutter, C.K.; Martines, J.C.; Piwoz, E.G.; Richter, L.M.; Victoria, C.G. The Lancet Breastfeeding Series Group. Why invest, and what it will take to improve breastfeeding practices? Lancet 2016 , 387 , 491–504. [CrossRef] 6. Zhu, J.; Dingess, K.A. The Functional Power of the Human Milk Proteome. Nutrients 2019 , 11 , 1834. [CrossRef] [PubMed] 7. Hampel, D.; Shahab-Ferdows, S.; Hossain, M.; Islam, M.M.; Ahmed, T.; Allen, L.H. Validation and Application of Biocrates AbsoluteIDQ ® p180 Targeted Metabolomics Kit Using Human Milk. Nutrients 2019 , 11 , 1733. [CrossRef] [PubMed] 8. Elwakiel, M.; Boeren, S.; Hageman, J.A.; Szeto, I.M.; Schols, H.A.; Hettinga, K.A. Variability of Serum Proteins in Chinese and Dutch Human Milk during Lactation. Nutrients 2019 , 11 , 499. [CrossRef] 9. Demers-Mathieu, V.; Huston, R.K.; Markell, A.M.; McCulley, E.A.; Martin, R.L.; Dallas, D.C. Antenatal Influenza A-Specific IgA, IgM, and IgG Antibodies in Mother’s Own Breast Milk and Donor Breast Milk, and Gastric Contents and Stools from Preterm Infants. Nutrients 2019 , 11 , 1567. [CrossRef] 10. Austin, S.; De Castro, C.A.; Sprenger, N.; Binia, A.; A ff olter, M.; Garcia-Rodenas, C.L.; Beauport, L.; Tolsa, J.F.; Fischer Fumeaux, C.J. Human Milk Oligosaccharides in the Milk of Mothers Delivering Term versus Preterm Infants. Nutrients 2019 , 11 , 1282. [CrossRef] 11. Witkowska-Zimny, M.; Kami ́ nska-El-Hassan, E.; Wr ó bel, E. Milk Therapy: Unexpected Uses for Human Breast Milk. Nutrients 2019 , 11 , 944. [CrossRef] 12. Machado, M.R.; Kamp, F.; Nunes, J.C.; El-Bacha, T.; Torres, A.G. Breast Milk Content of Vitamin A and E from Early- to Mid-Lactation Is A ff ected by Inadequate Dietary Intake in Brazilian Adult Women. Nutrients 2019 , 11 , 2025. [CrossRef] [PubMed] 13. Zielinska, M.A.; Hamulka, J.; Wesolowska, A. Carotenoid Content in Breastmilk in the 3rd and 6th Month of Lactation and Its Associations with Maternal Dietary Intake and Anthropometric Characteristics. Nutrients 2019 , 11 , 193. [CrossRef] [PubMed] 14. Bzikowska-Jura, A.; Czerwonogrodzka-Senczyna, A.; Jasi ́ nska-Melon, E.; Mojska, H.; Ol ̨ edzka, G.; Wesołowska, A.; Szostak-W ̨ egierek, D. The Concentration of Omega-3 Fatty Acids in Human Milk Is Related to Their Habitual but Not Current Intake. Nutrients 2019 , 11 , 1585. [CrossRef] [PubMed] 15. Dawodu, A.; Salameh, K.M.; Al-Janahi, N.S.; Bener, A.; Elkum, N. The E ff ect of High-Dose Postpartum Maternal Vitamin D Supplementation Alone Compared with Maternal Plus Infant Vitamin D Supplementation in Breastfeeding Infants in a High-Risk Population. A Randomized Controlled Trial. Nutrients 2019 , 11 , 1632. [CrossRef] 16. de Sousa Rebouças, A.; Costa Lemos da Silva, A.G.; Freitas de Oliveira, A.; Thalia Pereira da Silva, L.; de Freitas Felgueiras, V.; Cruz, M.S.; Silbiger, V.N.; da Silva Ribeiro, K.D.; Dimenstein, R. Factors Associated with Increased Alpha-Tocopherol Content in Milk in Response to Maternal Supplementation with 800 IU of Vitamin E. Nutrients 2019 , 11 , 900. [CrossRef] 17. Gianni, M.L.; Bettinelli, M.E.; Manfra, P.; Sorrentino, G.; Bezze, E.; Plevani, L.; Cavallaro, G.; Ra ff aeli, G.; Crippa, B.L.; Colombo, L.; et al. Breastfeeding Di ffi culties and Risk for Early Breastfeeding Cessation. Nutrients 2019 , 11 , 2266. [CrossRef] 18. O’Sullivan, T.A.; Cooke, J.; McCa ff erty, C.; Giglia, R. Online Video Instruction on Hand Expression of Colostrum in Pregnancy is an E ff ective Educational Tool. Nutrients 2019 , 11 , 883. [CrossRef] 19. Yu, J.; Yuan, T.; Zhang, X.; Jin, Q.; Wei, W.; Wang, X. Quantification of Nervonic Acid in Human Milk in the First 30 Days of Lactation: Influence of Lactation Stages and Comparison with Infant Formulae. Nutrients 2019 , 11 , 1892. [CrossRef] 20. Mazzocchi, A.; Giann ì , M.L.; Morniroli, D.; Leone, L.; Roggero, P.; Agostoni, C.; De Cosmi, V.; Mosca, F. Hormones in Breast Milk and E ff ect on Infants’ Growth: A Systematic Review. Nutrients 2019 , 11 , 1845. [CrossRef] 21. Grunewald, M.; Hellmuth, C.; Kirchberg, F.F.; Mearin, M.L.; Auricchio, R.; Castillejo, G.; Korponay-Szabo, I.R.; Polanco, I.; Roca, M.; Vriezinga, S.L.; et al. Variation and Interdependencies of Human Milk Macronutrients, 3 Nutrients 2020 , 12 , 899 Fatty Acids, Adiponectin, Insulin, and IGF-II in the European PreventCD Cohort. Nutrients 2019 , 11 , 2034. [CrossRef] 22. Verduci, E.; Giann ì , M.L.; Di Benedetto, A. Human Milk Feeding in Preterm Infants: What Has Been Done and What Is to Be Done. Nutrients 2019 , 12 , 44. [CrossRef] [PubMed] 23. Fischer Fumeaux, C.J.; Garcia-Rodenas, C.L.; De Castro, C.A.; Courtet-Compondu, M.C.; Thakkar, S.K.; Beauport, L.; Tolsa, J.F.; A ff olter, M. Longitudinal Analysis of Macronutrient Composition in Preterm and Term Human Milk: A Prospective Cohort Study. Nutrients 2019 , 11 , 1525. [CrossRef] [PubMed] 24. Thakkar, S.K.; De Castro, C.A.; Beauport, L.; Tolsa, J.F.; Fischer Fumeaux, C.J.; A ff olter, M.; Giu ff rida, F. Temporal Progression of Fatty Acids in Preterm and Term Human Milk of Mothers from Switzerland. Nutrients 2019 , 11 , 112. [CrossRef] [PubMed] 25. Ingvordsen Lindahl, I.; Artegoitia, V.M.; Downey, E.; O’Mahony, J.A.; O’Shea, C.A.; Ryan, C.A.; Kelly, A.L.; Bertram, H.C.; Sundekilde, U.K. Quantification of Human Milk Phospholipids: The E ff ect of Gestational and Lactational Age on Phospholipid Composition. Nutrients 2019 , 11 , 222. [CrossRef] 26. Gila-Diaz, A.; Arribas, S.M.; Algara, A.; Mart í n-Cabrejas, M.A.; L ó pez de Pablo, Á .L.; S á enz de Pipa ó n, M.; Ramiro-Cortijo, D. A Review of Bioactive Factors in Human Breastmilk: A Focus on Prematurity. Nutrients 2019 , 11 , 1307. [CrossRef] 27. Sabatier, M.; Garcia-Rodenas, C.L.; Castro, C.A.; Kastenmayer, P.; Vigo, M.; Dubascoux, S.; Andrey, D.; Nicolas, M.; Payot, J.R.; Bordier, V.; et al. Longitudinal Changes of Mineral Concentrations in Preterm and Term Human Milk from Lactating Swiss Women. Nutrients 2019 , 11 , 1855. [CrossRef] 28. Crippa, B.L.; Colombo, L.; Morniroli, D.; Consonni, D.; Bettinelli, M.E.; Spreafico, I.; Vercesi, G.; Sannino, P.; Mauri, P.A.; Zanotta, L.; et al. Do a Few Weeks Matter? Late Preterm Infants and Breastfeeding Issues. Nutrients 2019 , 11 , 312. [CrossRef] 29. American Academy of Pediatrics. Section on Breastfeeding. Breastfeeding and the use of human milk. Pediatrics 2012 , 129 , e827–e841. [CrossRef] 30. Peila, C.; Emmerik, N.E.; Giribaldi, M.; Stahl, B.; Ruitenberg, J.E.; van Elburg, R.M.; Moro, G.E.; Bertino, E.; Coscia, A.; Cavallarin, L. Human Milk Processing: A Systematic Review of Innovative Techniques to Ensure the Safety and Quality of Donor Milk. J. Pediatr. Gastroenterol. Nutr. 2017 , 64 , 353–361. [CrossRef] 31. Moukarzel, S.; Wiedeman, A.M.; Soberanes, L.S.; Dyer, R.A.; Innis, S.M.; Lamers, Y. Variability of Water-Soluble Forms of Choline Concentrations in Human Milk during Storage, after Pasteurization, and among Women. Nutrients 2019 , 11 , 3024. [CrossRef] 32. Wesolowska, A.; Brys, J.; Barbarska, O.; Strom, K.; Szymanska-Majchrzak, J.; Karzel, K.; Pawlikowska, E.; Zielinska, M.A.; Hamulka, J.; Oledzka, G. Lipid Profile, Lipase Bioactivity, and Lipophilic Antioxidant Content in High Pressure Processed Donor Human Milk. Nutrients 2019 , 11 , 1972. [CrossRef] [PubMed] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ). 4 nutrients Article Variability of Water-Soluble Forms of Choline Concentrations in Human Milk during Storage, after Pasteurization, and among Women Sara Moukarzel 1, ‡ , Alejandra M. Wiedeman 2,3, ‡ , Lynda S. Soberanes 2,4 , Roger A. Dyer 2,3 , Sheila M. Innis 2,3, † and Yvonne Lamers 2,4, * 1 Larsson-Rosenquist Foundation Mother-Milk-Infant Center of Research Excellence, University of California, San Diego, CA 92093, USA; smoukarzel@ucsd.edu 2 British Columbia Children’s Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada; awiedeman@bcchr.ca (A.M.W.); lynda.soberanes@gmail.com (L.S.S.); radyer@mail.ubc.ca (R.A.D.); sheila.innis@ubc.ca (S.M.I.) 3 Department of Pediatrics, Faculty of Medicine, The University of British Columbia, Vancouver, BC V5Z 4H4, Canada 4 Food, Nutrition, and Health Program, Faculty of Land and Food Systems, The University of British Columbia, Vancouver, BC V6T 1Z4, Canada * Correspondence: yvonne.lamers@ubc.ca; Tel.: + 1-604-827-1776 † Deceased. ‡ Shared first authorship. Received: 30 October 2019; Accepted: 6 December 2019; Published: 11 December 2019 Abstract: Choline is critical for infant development and mother’s milk is the sole source of choline for fully breastfed infants until six months of age. Human milk choline consists to 85% of water-soluble forms of choline including free choline (FC), phosphocholine (PhosC), and glycerophosphocholine (GPC). Donor milk requires safe handling procedures such as cold storage and pasteurization. However, the stability of water-soluble forms of choline during these processes is not known. The objectives of this research were to determine the e ff ect of storage and pasteurization on milk choline concentration, and the diurnal intra- and inter-individual variability of water-soluble choline forms. Milk samples were collected from healthy women who were fully breastfeeding a full-term, singleton infant < 6 months. Milk total water-soluble forms of choline, PhosC, and GPC concentrations did not change during storage at room temperature for up to 4 h. Individual and total water-soluble forms of choline concentrations did not change after storage for 24 h in the refrigerator or for up to one week in the household freezer. Holder pasteurization decreased PhosC and GPC, and thereby total water-soluble choline form concentrations by < 5%. We did not observe diurnal variations in PhosC and total water-soluble forms of choline concentrations, but significant di ff erences in FC and GPC concentrations across five sampling time points throughout one day. In conclusion, these outcomes contribute new knowledge for the derivation of evidence-informed guidelines for the handling and storage of expressed human milk as well as the development of optimized milk collection and storage protocols for research studies. Keywords: human milk; donor milk; choline; phosphocholine; storage; pasteurization; milk banking; pumping; breastfeeding; lactation 1. Introduction Choline is an essential nutrient with crucial roles in brain function, neurodevelopment, and growth [ 1 , 2 ]. Biological roles of choline include neurogenesis and synapse formation in the form of acetylcholine, membrane biogenesis, cell division, lipid transports, and myelination in Nutrients 2019 , 11 , 3024; doi:10.3390 / nu11123024 www.mdpi.com / journal / nutrients 5 Nutrients 2019 , 11 , 3024 the form of choline phospholipids, and as a methyl donor in the form of its oxidation product betaine [ 3 – 6 ]. Betaine contributes to the generation of S -adenosylmethionine, which is the main methyl donor involved in creatine and phosphatidylcholine synthesis, and DNA methylation, among other biochemical reactions [ 7 ]. Choline adequacy in early infancy and rapid stages of growth is critical to support membrane formation, cell proliferation, and parenchymal growth [6]. Exclusive breastfeeding ad libitum is the recommended feeding practice for the first six months of life, with continued breastfeeding up to two years of age [ 8 ]. Human milk contains various forms of the essential nutrient choline. The three water-soluble forms of choline, i.e., free choline (FC), phosphocholine (PhosC), and glycerophosphocholine (GPC), contribute to approximately 85% of total choline in human milk; the lipid-soluble phosphatidylcholine and sphingomyelin account for the remaining 15% [9–13]. The practice of human milk pumping and storage for later use is on the rise both at home and in clinical settings to accommodate various situations when feeding human milk at the breast is not possible, e.g., mothers returning to work; insu ffi cient volume of mother’s own milk, and need for donor milk [ 14 , 15 ]. Current guidelines for the safe handling of expressed human milk focus largely on microbiological safety, whereby Holder pasteurization (at 62.5 ◦ C for 30 min) of donor milk is mandatory in all North American hospitals [ 16 ]. Additionally, it is recommended that milk be stored in the refrigerator at ≤ 4 ◦ C for no more than eight days, and in the freezer at − 17 ◦ C for up to 12 months [ 17 ]. Whilst evidence suggests that refrigeration, freezing and pasteurization impact human milk concentration of folate, vitamin B6, vitamin C, and other nutrients to various degrees [ 18 – 21 ], the e ff ect of pasteurization and short- and long-term storage at di ff erent temperatures on water-soluble forms of choline in expressed human milk is not known. Water-soluble forms of choline concentrations in human milk may vary within and between women. Higher maternal dietary choline intake increases milk concentrations of water-soluble, yet not fat-soluble, forms of choline, as shown in a 12-week dose-response feeding study and a supplementation trial from 18 gestational weeks to 90 days postpartum [ 11 , 13 ]. No consistent changes in total concentration of water-soluble forms of choline in human milk were observed in six women over a time period of 72 h [ 22 ]. The diurnal changes and variability of individual water-soluble choline forms among women have not been studied to date. Understanding these variabilities has important implications for designing study protocols (e.g., sampling techniques) that focus on identifying the determinants of choline in human milk and infant outcomes due to milk choline consumption. The objectives of this research were to determine the e ff ect of cold storage and pasteurization on the concentrations of water-soluble forms of choline in human milk, and to determine the intra- and inter-individual variability of water-soluble forms of choline concentrations in human milk within a day. Water-soluble forms of choline seem stable during short-term cold storage and most forms remained unchanged after four hours of storage at room temperature and six months of storage at ultra-low freezing temperatures, as employed in research settings. Holder pasteurization significantly impacted milk concentrations of water-soluble forms of choline, but to a small extent that is outweighed by the microbiological safety benefits of pasteurization. We observed intra-individual variability of individual but not total water-soluble forms of choline throughout one day, and recommend standardized sampling protocols for research studies. 2. Materials and Methods 2.1. Participants and Study Design The research consisted of two cross-sectional studies, i.e., the stability and variability study, as well as the secondary analysis of bio-banked milk samples for the pasteurization study. For all studies, healthy women who were exclusively breastfeeding a healthy, full-term and singleton infant < 6 months of age were eligible to participate. Exclusion criteria included: su ff ering from diabetes mellitus Type 1 6 Nutrients 2019 , 11 , 3024 or 2, or any chronic disease involving fat metabolism, taking routine medications known to influence fat metabolism, or consumption of more than 1 alcoholic drink per day. 2.1.1. Stability Study A convenience sample of 6 mothers of 2- to 6- month old infants was included in this study. Women were recruited through active and passive recruitment methods in the Greater Vancouver area in 2014. Signed informed consent was obtained prior to enrolment. Information on maternal and infant age, sociodemographic status, supplement use, and general infant health, was collected using a self-administered questionnaire. Ethical approval was granted by the University of British Columbia and the British Columbia Children’s and Women’s (C&W) Hospital Research Ethics Board (H12-03191). Women provided a fresh complete milk expression collected in the morning (between 9:00–10:00 a.m.) using a commercial pump (Medela); samples were immediately transferred on ice to the lab to be aliquoted and analyzed. One milk aliquot was analyzed immediately, with the rest of the samples being stored at di ff erent temperatures for di ff erent durations (see Supplemental Table S1), prior to analysis. 2.1.2. Pasteurization Study Mid-feed human milk samples (milk collected after breastfeeding or pumping milk for approximately 3 min) from mothers of 1-month old infants were used in the pasteurization study; these bio-banked samples were derived from a completed, randomized controlled trial studying the e ff ect of DHA supplementation during pregnancy on the cognitive and visual outcomes on infants [ 23 ]. The study was approved by C&W REB (H03-70242). A subset of milk samples was randomly selected from the bio-banked samples that had been stored for 5–9 years at − 80 ◦ C. In the original study, milk samples were immediately frozen at home for a maximum time of 3 days, and transferred on ice to the lab where they were frozen at − 80 ◦ C until analysis. Original milk samples obtained from the participants were thawed on ice, homogenized by gentle mixing, and divided in two aliquots. The first aliquot was stored at − 20 ◦ C for 1–2 h, and the second aliquot underwent Holder pasteurization in a water bath. Water-soluble forms of choline were quantified before and after pasteurization on the same day. 2.1.3. Variability Study Twenty women were enrolled in this cross-sectional study. Recruitment and enrolment procedures were similar to those described for the stability study. Sociodemographic, health and supplement use data were collected using the same self-administered questionnaire as in the stability study. Ethical approval was granted by the University of British Columbia and the British Columbia Children’s and Women’s Hospital Research Ethics Board (H12-03191). Each participant provided 5 mid-feed milk samples at di ff erent times throughout one day. For feasibility purposes, the five di ff erent time points were flexible as follows: before breakfast, before lunch, 45–60 min after lunch, 45–60 min after dinner, and before bedtime. Participants were instructed to place the vials in their home freezers immediately after pumping. Frozen samples were transferred to the lab on ice the day after collection and stored at − 80 ◦ C for 2 weeks, when choline analysis was completed. 2.2. Human Milk Choline Quantitation Concentrations of water-soluble forms of choline in milk were determined using isotope dilution liquid chromatography tandem mass spectrometry as previously described [ 24 ]. In brief, aliquots of 20 μ L of human milk were transferred to Eppendorf tubes containing 10 μ L of deuterium-labeled internal standards (choline-d9, PhosC-d9, GPC-d9) and vortexed. Protein was precipitated with 30 μ L of methanol with 0.1% formic acid. The supernatant was recovered after centrifugation at 18,000 × g at 4 ◦ C for 10 min, transferred to an autosampler vial and mixed with acetonitrile with 0.1% formic acid in 7 Nutrients 2019 , 11 , 3024 dilutions of 1:5. The inter-assay and intra-assay coe ffi cient of variation (CV) based on 5 replicates were as follows: For FC, 5.5% and 4.1%; for PhosC, 6.4% and 5.2%; and for GPC 9.5% and 2.3%; respectively. 2.3. Statistical Analysis Participant characteristics are presented using descriptive statistics. Normality of data distribution was assessed using Shapiro–Wilk test. Di ff erences in milk concentrations of water-soluble forms of choline under di ff erent storage conditions were determined using the related-samples Friedman test, followed by Wilcoxon signed-ranks test as post-hoc analysis and adjusted using the Bonferroni correction. Di ff erences in water-soluble forms of choline concentrations after pasteu