Bacillus is a bacteria genus that is frequently found in DHM, and it represents a special concern for HMBs, as these bacteria are capable of producing heat-resistant toxins and forming spores that are resistant to pasteurization (46, 47). In the HMB of Palma de Mallorca, about 10% of the milk is generally discarded due to the presence of Bacillus sp. In order to test the susceptibility of Bacillus sp to the UV treatment, the researchers used two different batches of donor milk that had been discarded as a result of contamination with Bacillus sp., 3,000 CFU/ml in both cases. The results showed that after 45 min of treatment, the Bacillus sp. were eliminated.

The Spanish group then evaluated the effect on the biological components of HM. One of the main biological components is IgA, which constitutes 90% of all the immunoglobulins present in colostrum and HM. Its importance lies not only in its concentration, but also in its biological activity (48). It has been pointed out that pasteurization affects IgA levels to different extents, depending on the pasteurization temperature (14). In the case of HoP, a clear decrease in IgA concentration was observed, although there were large discrepancies in the range of reduction, from 20 to 60% (9). The results of this group have shown that, after testing seven different batches of DHM, the IgA levels measured by conventional nephelometry techniques, were 96% of the pre-treatment levels, and in five samples, a 100% activity was preserved.

From these results, it can be concluded that the treatment with UV-C light has a number of features that make it a good candidate as an alternative to HoP. In addition to providing a better protection of the biological components than other methods, it is also capable of producing an at least 5-log10 decrease in the number of bacteria (including Bacillus sp) present in DHM. Furthermore, the ability of UV-C radiation to eliminate active forms of Cytomegalovirus in HM has also been demonstrated (45). Unfortunately, until now, there is neither a device nor even a prototype that would enable the use of this technology in an HMB setting.

## EMBA Working Group Recommendations

One important aspect that should be considered when evaluating the processing of human milk is the viral inactivation effect of the new methodologies.

The ability of LTLT pasteurization to inactivate viral pathogens is well-known. The list of human viruses inactivated by HoP includes pathogens for which transmission through breastfeeding has been conclusively demonstrated (i.e., the human immunodeficiency virus, human T-cell lymphotrofic virus, cytomegalovirus), and viruses that can be transmitted via breast milk, such as human papillomavirus, Zikavirus, Ebola and the Marbourg virus (49–55).

On the other hand, virus inactivation still has to be carefully evaluated for each alternative technique and device designed to treat breast milk. This is an important issue for future research.

We can state that fundamental knowledge of new technologies of HM processing is still lacking. Their effects on safety and bioactive components of HM need further evaluation. **Table 2** presents the "state of the art" at the moment, explaining advantages, and disadvantages of the processing techniques described in this paper.

On the basis of evidence taken from the literature and on the personal experience of its members, the Working Group on the Processing of Human Milk makes the following recommendations:

When testing new technologies, the following requirements should be fulfilled:

The final aim of HM processing performed with new technologies should be:

FIGURE 3 | Workflow for assessing the performance of new pasteurization technologies for human milk. \*For the qualification of the Holder pasteurizer, see Buffin et al. (12).

TABLE 2 | Advantages and disadvantages of the processing techniques described in this paper.

TABLE 3 | Parameters to evaluate for validation of new pasteurization technologies.

§ For the design of the microbiological challenge test, see Giribaldi et al. (21).

◦ Or equivalent methods.

A workflow that can be considered suitable to assess the basic performance of new pasteurization technologies for HM is shown in **Figure 3** and **Table 3**. Since Holder pasteurization is not efficient in eradicating spore-forming bacteria, this parameter has not been included in the validation targets. However, any new pasteurization system that could prove to be efficient against spore-forming bacteria (while maintaining all the other aforementioned characteristics) would represent a great advantage for the improvement of HM safety.

# CONCLUSIONS

This paper presents the recommendations of the EMBA Working Group on the "Processing of HM." Although research on the most promising technologies, which will represent a reasonable alternative to HoP in the future (HTST, HPP, UV-C) is progressing, at the moment it is important to recognize that the consistency and quality assurance of the pasteurizers currently available on the market today represent a fundamental approach that was previously lacking in HoP practice.

EMBA recognizes that HoP is at present the safest compromise for the treatment of DHM; however, further studies are needed to improve this technology in order to minimize its effects on the biological components of HM. The new technologies evaluated and studied by the Working Group are being developed rapidly, and EMBA recommends that the final aim of these technologies should be an improved preservation of the nutritional and bioactive components of raw human milk, while assuring microbiological safety of the product, at least at the same level as optimized HoP.

#### AUTHOR CONTRIBUTIONS

GEM wrote the manuscript. CB, BR, JC, LaC, LuC, DE-V, AGa, DL, and AW are components of the EMBA Working Group on Processing of Donor Human Milk and contributed to the content of this manuscript. SA,

#### REFERENCES

EB, AGr, DB, C-YB, CG, GW, and J-CP are components of the Board of Directors of EMBA and made comments and gave suggestions for the final preparation of the manuscript.

#### ACKNOWLEDGMENTS

The authors are grateful to the Italian Association of Human Milk Banks (Associazione Italiana Banche del Latte Umano Donato = AIBLUD) for its continuous efforts to promote research in the field of donor human milk and Human Milk Banks, and for the financial support for the publication of this manuscript.

opportunities for a new treatment option in breast milk banking. PLoS ONE. (2016) 11:1–12. doi: 10.1371/journal.pone.0161116

**Conflict of Interest Statement:** LuC is employed by Carag AG, Switzerland. However, Carag AG did not offer any financial support for this paper.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer MG declared a past co-authorship with the authors JC and AGa to the handling editor.

Copyright © 2019 Moro, Billeaud, Rachel, Calvo, Cavallarin, Christen, Escuder-Vieco, Gaya, Lembo, Wesolowska, Arslanoglu, Barnett, Bertino, Boquien, Gebauer, Grovslien, Weaver and Picaud. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Recommendations for the Establishment and Operation of Human Milk Banks in Europe: A Consensus Statement From the European Milk Bank Association (EMBA)

Gillian Weaver 1 \*, Enrico Bertino2 , Corinna Gebauer 3 , Anne Grovslien4 , Radmila Mileusnic-Milenovic5 , Sertac Arslanoglu6 , Debbie Barnett 7 , Clair-Yves Boquien8 , Rachel Buffin9 , Antoni Gaya10, Guido E. Moro11, Aleksandra Wesolowska12,13 and Jean-Charles Picaud9,14

#### Edited by:

Christoph Bührer, Charité Medical University of Berlin, Germany

#### Reviewed by:

Fernando Cabañas, Hospital Universitario Quirónsalud Madrid, Spain María Gormaz, Agencia Valenciana de Salud, Spain

> \*Correspondence: Gillian Weaver<EMAIL_ADDRESS>
> > Specialty section:

This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics

Received: 31 August 2018 Accepted: 08 February 2019 Published: 04 March 2019

#### Citation:

Weaver G, Bertino E, Gebauer C, Grovslien A, Mileusnic-Milenovic R, Arslanoglu S, Barnett D, Boquien C-Y, Buffin R, Gaya A, Moro GE, Wesolowska A and Picaud J-C (2019) Recommendations for the Establishment and Operation of Human Milk Banks in Europe: A Consensus Statement From the European Milk Bank Association (EMBA). Front. Pediatr. 7:53. doi: 10.3389/fped.2019.00053 1 Human Milk Foundation, Harpenden, United Kingdom, 2 Neonatal Unit of Turin University, City of Health and Science of Turin, Turin, Italy, 3 Abteilung Neonatologie Klinik und Poliklinik für Kinder und Jugendliche, Leipzig, Germany, 4 Neonatal Unit, Milk Bank, Oslo University Hospital, Oslo, Norway, 5 First Serbian Milk Bank, Institute of Neonatology, Belgrade, Serbia, 6 Division of Neonatology, Department of Pediatrics, Istanbul Medeniyet University, Istanbul, Turkey, 7 Greater Glasgow and Clyde Donor Milk Bank, Royal Hospital for Sick Children, Glasgow, United Kingdom, 8 PhAN, Institut National de la Recherche Agronomique (INRA), Université de Nantes, CRNH-Ouest, Nantes, France, 9 Neonatal Intensive Care Unit, Hopital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France, 10 Banc de Teixit, F. Banc de Sang i Teixits de les Illes Balears, Institut d'Investigacio Sanitaria Illes Balears (IdISBa), Barcelona, Spain, 11 Italian Association of Human Milk Banks (AIBLUD), Milan, Italy, 12 Department of Neonatology, Faculty of Health Science, Medical University of Warsaw, Warsaw, Poland, 13 Laboratory of Human Milk and Lactation Research, Holy Family Hospital Regional Human Milk Bank, Warsaw, Poland, 14 CarMeN Unit, INSERM U1060, INRA U1397, Claude Bernard University Lyon 1, Pierre Bénite, France

Objectives: To develop recommendations from the European Milk Bank Association (EMBA) for the establishment and operation of human milk banks (HMB) in Europe.

Method: A working group comprising members of the EMBA was convened in 2015 to develop Europe-wide recommendations for milk banks. Each member had experience of guideline development and/or milk banking operations. An initial survey was agreed using collated published global recommendations. A total of 108 potential recommendations were included in the survey; responders noted which were included in their national guidelines. The responses were collated, compared, and discussed and the group determined where there was consensus and where substantial or minor differences were identified. Where there was consensus or robust published evidence on which to base recommendations these were included. When there was no consensus and no clear evidence base, a statement of explanation based on collective expert opinion was agreed.

Results: Published, internationally available guidelines with recommendations for human milk banks from France, Italy, and the UK, together with guidelines from Austria, Denmark, Germany, Norway, Slovakia, Spain, Sweden, and Switzerland were included as source materials. These covered: General recommendations; Donor recruitment and screening; Expression, handling, and storage of donor human milk (DHM); Pooling of DHM; Milk screening; Milk treatment (pasteurization); Delivery of DHM to recipients.

Conclusions: Evidence based recommendations and consensus statements from the EMBA will now be published on the EMBA website to assist in the safe establishment and operation of HMBs throughout Europe. These have also been used to inform the chapter on human milk to be included in the 2019 edition of the Guide to the quality and safety of tissues and cells for human application, published by the European Directorate for the Quality of Medicines & HealthCare (EDQM).

Keywords: donor human milk (DHM), Human Milk Bank (HMB), breastfeeding, pasteurization, donor screening, bacteriology testing

#### BACKGROUND

Human milk (HM) is the preferred nutrition for preterm infants (1), but not all mothers are able to provide their child with enough milk. There are specific beneficial effects of breastfeeding in these infants as HM feeding reduces the risk of short term and long term complications related to prematurity. Donor HM (DHM) can supplement the supply of maternal breastmilk when it is insufficient or provide the preferred alternative when the mother is not breastfeeding. DHM is beneficial for the health of preterm infants, first because significant properties are preserved following holder pasteurization (2) and secondly because it prevents feeding these infants with a preterm formula. According to the World Health Organization (1) the American Academy of Pediatrics (3) and the European Society of Pediatric Gastroenterology, Hepatology, and Nutrition (4) the feeding of preterm infants with mother's own milk is recommended as the first choice and if this is not available pasteurized DHM from an established milk bank should be the next alternative.

The main benefits for preterm infants that receive DHM instead of formula are faster gastric emptying, faster attainment of full enteral feedings, improved gut growth and maturation, decreased risk of necrotizing enterocolitis and late onset sepsis, improved neurodevelopmental outcomes, less retinopathy of prematurity and improved visual development (5–9).

HMBs have existed since 1909 in Europe and countries that supported the development of a significant number of HMBs developed national guidelines to harmonize practices. The European Milk Bank Association1 (EMBA) was officially launched in October 2010. According to the definitions agreed by the EMBA Board of Directors:

The EMBA constitution is available to view on the EMBA website1 . The Association welcomes membership from HMBs, milk bank associations and individuals who support the aims of the Association. Membership affords the opportunity to access conference presentations from the past 6 years as well as additional materials of interest to those who bank or use DHM.

The EMBA is a non-profit organization that was established to promote milk banking and to encourage international cooperation between the HMBs of the countries of Europe as well as globally. The association's headquarters are in Milan, Italy but association meetings and conferences are held in different locations within Europe. The primary aim of the EMBA is to support and promote exclusive breastfeeding, which is universally acknowledged to be the optimal method of feeding all infants. The use of DHM from HMBs for preterm and sick infants temporarily without access to their own mother's milk has been widely shown to be linked to increased rates of exclusive breastfeeding on discharge from neonatal units (10, 11).

The EMBA was also established to support human milk banking in Europe by promoting safe practices and fostering sustainable operations. Hence a further aim of the association, as listed in the EMBA Constitution1 is to "Prepare international and regularly revised guidelines to set standards for the practice of milk banking." The usual methodology for establishing guidelines is to convene appropriate multidisciplinary expertise, consider all of the stages of a process and conduct a review of the evidence before making recommendations linked to the strength of the evidence. In the absence of such a published guideline, aimed at milk banks throughout Europe, it is expected that the development of Europe-wide recommendations by EMBA for HMBs will promote best practice, optimize quality, and safety within the HMBs that are currently operational as well as offer valuable assistance to those who are establishing a new HMB. Evidence and expert opinion based recommendations should be the standard throughout Europe to promote consistent practices both within and between countries.

There are currently 226 HMBs operating in 28 countries in Europe. These include the first milk banks in Estonia, Lithuania, Poland, Portugal, and Russia. A further 16 HMBs are planned. The current status of HMBs in Europe is maintained on the EMBA website1 .

The 226 HMBs in Europe incorporate newly established organizations with a limited history of operational expertise as well as ones that have links to the very early milk banks founded over a century ago. They include national and regionalized services as well as small operations serving a single neonatal unit. The volumes of DHM that are tested and processed and provided mainly to sick and preterm infants vary widely from country to country1 as well as within a single country (12) as do the criteria for receipt of DHM. An EMBA coordinated survey of all the HMBs in Europe will be carried out in 2019. This will gather up to date information about the extent of milk banking

1http://europeanmilkbanking.com

activity including the number of donors recruited, volumes of milk donated and processed, volumes of milk provided to hospitals, and the number of recipients. It will also collate further information about milk banking practices.

There are currently no published Europe-wide guidelines however internationally published guidelines for HMBs are freely available from France2 , Italy (13), and the UK (14). Countries with nationally agreed and recognized guidelines include Austria, Denmark, Germany, Norway, Slovakia, Spain, Sweden, and Switzerland. English translations of recently updated (2017– 2018) national guidelines from Sweden and Spain can be downloaded via links from the EMBA website. Differences exist between national guidelines as a result of variations in practices, regulation, and organization of HMBs in each country. Differences in practices are due to the lack of evidence for some points related to the operation of a HMB. DHM status globally is variable; it is either considered a food, a health product food or a tissue; the latter ones being subject to stricter regulation. Organization of HMBs is different when collecting and delivering DHM only for infants hospitalized in the same building, or over a more or less wide area including several health care institutions.

The HMBs of Europe also have very different historical backgrounds1 and in most cases have operated independently of each other although regular meetings take place between milk banking practitioners in Norway and Sweden and between those of Austria, Germany, and Switzerland.

A major role of the EMBA has, since its inception, been the provision of education, largely via the organization of international congresses. Presentations at these congresses have revealed similarities between the milk banking practices throughout Europe and have also revealed fundamental differences. These have substantiated the results of surveys of HMBs carried out both before and after the establishment of EMBA in 2007 and in 2012. In some cases the official national or local recommendations from various countries lead to significant differences in practice. For example most published guidelines recommend that all DHM should be heat treated [using the standard holder pasteurization method2 (13, 14)]. However, in Norway mainly raw (non-heat treated) DHM is provided by the country's 12 milk banks. This is considered possible and reasonable because (1) the demand can be met due to very high breastfeeding rates, even with extremely strict donor screening, (2) there is complete traceability of milk from donor to consumer, (3) there is a small population with very low HIV and hepatitis rates, (4) extremely rigorous and regularly repeated testing of donors (every 3 months) is practiced (15, 16). However, in almost half of the 20 official HMBs in Germany both raw and pasteurized DHM is available and used in accordance with local clinical judgment based on the gestation and health of the recipient (17).

## OBJECTIVE

One of the clear objectives of the EMBA1 has been to develop Europe-wide recommendations for HMBs. This is especially needed by countries without experience of human milk banking or the use of DHM. Such recommendations should optimize safety and draw upon the available evidence. The processes and practices within human milk banking do not lend themselves to randomized controlled trials and there are few meta-analyses or systematic reviews available to refer to. In the absence of these, expert opinion is required and where this differs, a consensus and in some cases compromise is sought. The EMBA Guideline Working Group was formed in 2015 to undertake this task. EMBA members from 13 countries contributed to the work of the group. Throughout its 3 year existence, members of the Guideline Working Group have included representatives from Austria, France, Germany, Italy, Norway, Poland, Portugal, Serbia, Slovenia, Slovakia, Spain, Switzerland, and the UK.

# METHOD

The three main tasks of the group were:

A useful tool in developing the structure for the survey was the global implementation framework (18) published by the NGO PATH3 . The framework includes a compilation of practices in HMBs from donor recruitment to delivery of DHM to the recipient.

Each of the group members completed the survey in accordance with their local/regional or national guidelines and, if not included in the guidelines, in accordance with local practice. Once completed, the survey responses were collated and highlighted according to whether international consensus was apparent, whether there was near consensus and where practices and recommendations differed significantly. The next step was to note where no published evidence was available. A list of agreed recommendations that were evidence based and that could form the basis of EMBA recommendations was drawn up and presented to the wider EMBA membership at further meetings. Where there was no consensus and no evidence upon which to determine a recommendation, expert opinion within the group was used to decide whether to include a recommendation or to provide an explanation of why no clear recommendation could be made at that time.

# RESULTS

The recommendations that were found to be included in all of the published guidelines or accepted as best practice according to the available evidence or expert opinion and have therefore been agreed by the Working Group are presented below. Many of these are underpinned by principles of good manufacturing

2http://ansm.sante.fr/Activites/Elaboration-de-bonnes-pratiques/Bonnespratiques-des-lactariums

3PATH: http://www.path.org

process (GMP) especially those related to consistency and quality control.

#### EMBA Recommendations for the Establishment and Operation of Human Milk Banks in Europe General Recommendations

the container should be monitored during transport using data loggers or checked on arrival. A record of this should be maintained as part of the HMB records (GMP).

#### Donor Recruitment and Screening

	- Smoke cigarettes or use nicotine containing products including "vaping," gums, and other products
	- Use any recreational or "street" drugs.
	- Are known or found to be infected with HIV, hepatitis B or hepatitis C (13, 14) 2 . Additional screening for HTLV (human T-lymphotropic virus), syphilis, and other viral and bacterial infections may be screened for according to local evaluation.
	- Use medications other than those on the EMBA approved medication list1 .
	- Have received a recent blood transfusion, tattoo or piercing, or needle stick injury. The. determination of "recent" should be locally determined in accordance with blood donation/transfusion services and methods used for serology testing.
	- Follow a vegan diet without supplementation with Vitamin B12.

The extent of the temporary deferral of donors will vary according to the duration of the circumstances and medical advice should be sought to exclude the possibility of acceptance of contaminated, unsuitable, or suboptimal milk.

#### Expression, Handling, and Storage of Human Milk for Donation to the HMB

	- 14.1 The pooling of DHM from the same donor is acceptable prior to any heat processing
	- 14.2 Maintain records of all pooling including the names/ID numbers of the donors, dates the milk was expressed and any medications taken.

#### Milk Screening; Pre- and Post-pasteurization Testing

Within Europe, as well as globally, there is no consensus for recommendations for the microbiological testing of DHM either before or after pasteurization (see **Table 1**). Throughout Europe, local and national guidelines vary both in the timing and frequency of testing and in the acceptance criteria. There is a lack of published evidence to inform decision making and the working group members concluded that to maximize safety of vulnerable immune-compromised recipients, best practice suggests:

	- All pools of milk be tested prior to pasteurization
	- Each batch of milk be tested after pasteurization
	- Acceptance criteria: 105 cfu/ml or less of non-pathogenic organisms and no pathogens for each pool of milk tested prior to pasteurization. Discard all samples of milk from a pool that does not meet this standard.
	- Discard the batch if there is any microbial growth detected in a random sample taken after pasteurization.

It is recognized that most published guidelines include less stringent screening criteria (**Table 1**) as part of the overall recommendations. The EMBA therefore recommend that where milk banks follow nationally or locally agreed guidance, alternative microbiological screening criteria may be adopted if done so as part of the overall adherence to the guideline being followed.

#### Milk Treatment (Pasteurization)

4http://ukamb.org/medication-and-breastfeeding

Frontiers in Pediatrics | www.frontiersin.org

aHuman Milk Bank Association of North America. http://hmbana.org.

#### Delivery of DHM to Recipients

#### DISCUSSION

The Working Group was able to arrive at a consensus for recommendations covering most of the major processes involved in recruiting and screening donors, storing, handling, and transporting DHM and in its testing and processing. In these cases, the evidence, as referenced in the relevant national guidelines was checked and noted. Where published evidence was not available, expert opinion was the basis for the recommendation. The group also highlighted several areas for which consensus could not be achieved and where there was no clear evidence to inform a recommendation. In these instances the group provided a statement of explanation and suggested best practice. The group also acknowledged that not all countries that have milk banks were represented within the guideline group. A further consideration was that HMBs are funded in different ways and have varying resources. It was agreed that to make a single recommendation that may undermine a milk bank's ability to continue to operate would not be in the best interests of the overall population of recipients.

The process by which DHM is tested to provide microbiological safety is an example of where no consensus could be found. Very divergent practices are currently in place throughout Europe and there is no agreement within the published guidelines as to the optimal testing regime either before or after pasteurization or both. There is also no clear evidence base on which to determine the recommendations. However, it is possible to make a recommendation as to which practice will offer optimal safety for all recipients, including the most vulnerable neonates, and through which no untested milk reaches the recipients and the strictest published acceptance criteria are used. Recommended testing regimes exist in Europe that include less frequent sampling [e.g., only testing the first donation and randomly testing subsequent donations as in the Italian guidelines (13)]. There are also recommendations in which the acceptance criteria are less strict (e.g., pre-pasteurization criteria of 105 cfu/ml or less of any organisms and 104 cfu/ml or less of Staphylococcus aureus and Enterobacteriaceae and the presence of <10 cfu/ml of any organism post-pasteurization as per the UK's NICE guideline (14). These are acceptable alternatives but do not provide the same assurance of safety. It should be noted however that less strict testing regimes will lead to more processed milk being available. An additional consideration is that if DHM is fed without any heat treatment (as occurs in some parts of Europe), stricter local microbiological testing protocols should be adopted to increase the safety of the recipients.

The tracking of DHM and the traceability of the milk throughout all the HMB processes is an essential component of safety and quality assurance. This is enhanced by the availability of customized and purpose developed barcode tracking systems and the use of the internationally agreed coding system, ISBT128 (21). Regular strict quality control of pasteurization equipment is necessary to maintain optimum safety and quality of DHM (2, 22, 23).

Whilst safety is at the forefront of all recommendations pertaining to HMBs and the use of DHM it is important to ensure that the ethical considerations of banking and sharing human milk and duties of care to the donors as well as the recipients are not overlooked (24).

A limitation of this consensus statement is that no recommendations were made regarding the analysis of HM. HM analysis is a means of assessing protein and energy contents and to facilitate targeted fortification of HM (i.e., to fortify milk according to its individual composition to reach a target composition as a means of helping to cover the theoretical nutritional needs of each infant). Some HMBs include the assessment of HM composition in their practice. This is done either to perform targeted fortification or to select HM with the highest nutrient content for the smallest babies. However, there is no consensus about the best strategy for fortification. Only adjustable fortification, which does not require HM composition assessment, has been shown to improve postnatal growth.

## CONCLUSIONS

There are no Europe-wide guidelines covering practices within HMBs. Historically national and/or local guidelines have been developed and although the recommendations include many similarities, clear differences also exist. Developing recommendations for HMBs operating throughout Europe was a challenging task because of the diversity within European countries. However, with the input of leading milk

#### REFERENCES

banking experts from a wide range of countries and with additional help from the EMBA Board it was possible to agree a pragmatic approach to where differences could not be resolved through reference to published research. The resultant guidance will now be freely available on the EMBA website to assist in the safe establishment and operation of HMBs throughout Europe. The recommendations have also been used to help inform the chapter on human milk to be included in the 2019 edition of the Guide to the quality and safety of tissues and cells for human application, published by the European Directorate for the Quality of Medicines & Health Care (EDQM).

## AUTHOR CONTRIBUTIONS

GW, EB, CG, AnnG, RM-M, SA, DB, C-YB, RB, AntG, GM, AW, and J-CP contributed to the working group and/or to the discussions of the findings of the working group and all offered amendments to the previous drafts and/or agreed the manuscript.

#### ACKNOWLEDGMENTS

We gratefully acknowledge the valuable contribution made to the Guideline Working Group by the following EMBA members and colleagues: Tanya Cassidy (Canada and Ireland), Elena Darol (Poland), Andreja Domjan (Slovenia), Ivana Letenayova (Slovakia), Israel Macedo (Portugal), Daniel Mumblit (Russia), Ingrid Zittera (Austria). We are also extremely grateful to the Italian Association of Human Milk Banks (Associazine Italiana Banche del Latte Umano Donato—AIBLUD) for its continuous efforts to promote research in the field of human milk banking and for the financial support toward the publication of this manuscript.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped. 2019.00053/full#supplementary-material

bank in an Australian NICU. Early Hum Dev. (2007) 83:667–73. doi: 10.1016/j.earlhumdev.2007.07.012

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer MG declared a past co-authorship with one of the authors AG to the handling Editor.

Copyright © 2019 Weaver, Bertino, Gebauer, Grovslien, Mileusnic-Milenovic, Arslanoglu, Barnett, Boquien, Buffin, Gaya, Moro, Wesolowska and Picaud. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Fortification of Human Milk for Preterm Infants: Update and Recommendations of the European Milk Bank Association (EMBA) Working Group on Human Milk Fortification

Sertac Arslanoglu1 \* † , Clair-Yves Boquien2†, Caroline King3†, Delphine Lamireau4† , Paola Tonetto5†, Debbie Barnett 6 , Enrico Bertino5 , Antoni Gaya7 , Corinna Gebauer 8 , Anne Grovslien9 , Guido E. Moro10, Gillian Weaver 11, Aleksandra Maria Wesolowska12 and Jean-Charles Picaud13,14†

1 Division of Neonatology, Department of Pediatrics, Istanbul Medeniyet University, Istanbul, Turkey, 2 PhAN, Institut National de la Recherche Agronomique (INRA), Université de Nantes, CRNH-Ouest, Nantes, France, 3 Department of Nutrition and Dietetics, Imperial College Healthcare NHS Trust, London, United Kingdom, 4 Lactariums de Bordeaux-Marmande, Pôle Pédiatrique, Centre Hospitalo-Universitaire (CHU) de Bordeaux, Bordeaux, France, 5 Neonatal Unit of Turin University, City of Health and Science of Turin, Turin, Italy, 6 Greater Glasgow and Clyde Donor Milk Bank, Royal Hospital for Sick Children, Glasgow, United Kingdom, 7 Banc de Teixits, Fundaciò Banc Sang i Teixits de les Illes Balears, Palma de Mallorca, Spain, 8 Abteilung Neonatologie Klinik und Poliklinik für Kinder und Jugendliche, Leipzig, Germany, 9 Neonatal Unit, Milk Bank, Oslo University Hospital, Oslo, Norway, 10 Associazione Italiana Banche del Latte Umano Donato (AIBLUD), Milan, Italy, 11 Hearts Milk Bank, Rothamsted Research Institute, Harpenden, United Kingdom, 12 Department of Neonatology, Faculty of Health Science, Medical University of Warsaw, Warsaw, Poland, 13 CarMeN Unit, INSERM U1060, INRA U1397, Claude Bernard University Lyon 1, Pierre Bénite, France, 14 Division of Neonatology, Hôpital de la Croix-Rousse, Lyon, France

Evidence indicates that human milk (HM) is the best form of nutrition uniquely suited not only to term but also to preterm infants conferring health benefits in both the short and long-term. However, HM does not provide sufficient nutrition for the very low birth weight (VLBW) infant when fed at the usual feeding volumes leading to slow growth with the risk of neurocognitive impairment and other poor health outcomes such as retinopathy and bronchopulmonary dysplasia. HM should be supplemented (fortified) with the nutrients in short supply, particularly with protein, calcium, and phosphate to meet the high requirements of this group of babies. In this paper the European Milk Bank Association (EMBA) Working Group on HM Fortification discusses the existing evidence in this field, gives an overview of different fortification approaches and definitions, outlines the gaps in knowledge and gives recommendations for practice and suggestions for future research. EMBA recognizes that "Standard Fortification," which is currently the most utilized regimen in neonatal intensive care units, still falls short in supplying sufficient protein for some VLBW infants. EMBA encourages the use of "Individualized Fortification" to optimize nutrient intake. "Adjustable Fortification" and "Targeted Fortification" are 2 methods of individualized fortification. The quality and source of human milk fortifiers

#### Edited by:

Maximo Vento, Hospital Universitari i Politècnic La Fe, Spain

#### Reviewed by:

Miguel Saenz De Pipaon, University Hospital La Paz, Spain Catherine Mullié, University of Picardie Jules Verne, France Ehsan Khafipour, University of Manitoba, Canada

#### \*Correspondence:

Sertac Arslanoglu<EMAIL_ADDRESS>
†European Milk Bank Association (EMBA) Working Group on Human Milk Fortification

#### Specialty section:

This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics

Received: 31 August 2018 Accepted: 25 February 2019 Published: 22 March 2019

#### Citation:

Arslanoglu S, Boquien C-Y, King C, Lamireau D, Tonetto P, Barnett D, Bertino E, Gaya A, Gebauer C, Grovslien A, Moro GE, Weaver G, Wesolowska AM and Picaud J-C (2019) Fortification of Human Milk for Preterm Infants: Update and Recommendations of the European Milk Bank Association (EMBA) Working Group on Human Milk Fortification. Front. Pediatr. 7:76. doi: 10.3389/fped.2019.00076 constitute another important topic. There is work looking at human milk derived fortifiers, but it is still too early to draw precise conclusions about their use. The pros and cons are discussed in this Commentary in addition to the evidence around use of fortifiers post discharge.

Keywords: nutrition, prematurity, human milk, adjustable fortification, individualized fortification, growth, protein

# INTRODUCTION

Inadequate nutrition during the critical periods of brain development alters the growth trajectory of the brain and can have permanent negative consequences. The most critical period of brain growth and development for humans corresponds to the third trimester of pregnancy and for very low birthweight (VLBW) infants these developmental processes take place in the neonatal intensive care unit (NICU) environment (1, 2). Inadequate nutrition and/or poor postnatal growth during the NICU stay has been associated with neurocognitive impairments (3–11) and poor renal function (12) in preterm infants. Recent studies suggest that not only the growth per se, but also the quality of growth counts. Better linear growth and early gains in fat-free body mass have been found to be associated with improved neurodevelopment in VLBW preterm infants (13, 14). Thus, optimization of the nutritional care for the preterm infants has a key role in improving neurodevelopmental outcomes and has become a priority.

Despite the advancements in nutritional support over 20 years and current focus on "early intense nutrition" in NICU, undernutrition and extrauterine growth restriction (EUGR) are still important problems for VLBW infants (15–18).

Evidence indicates that human milk (HM) is the best source of nutrition for both term and preterm infants conferring health benefits both in the short and long-term (19, 20). Unfortified HM however does not provide sufficient nutrition to VLBW infants when fed at the usual feeding volumes. Human milk should be supplemented (fortified) with the nutrients in short supply, particularly with protein, calcium, and phosphate to meet high requirements of this group of tiny preterm infants as discussed in the next sections. Although HM fortification is widely adopted in the NICUs all over the world, there is still much inconsistency and variability and even some skepticism around this practice. During the last decade optimization of HM fortification- mainly individualization, and the quality of the fortifiers have been the hot topics of discussion.

The European Milk Bank Association (EMBA) Working Group on HM Fortification aims to document the existing evidence on this field, overviews different fortification approaches, clarifies the terminology and definitions, outlines the gaps in knowledge, and gives recommendations for practice and suggestions for future research.

## METHODS

European Milk Bank Association (EMBA) Working Group (WG) on HM Fortification was formed by a group of experts on this field in 2013. In 2016 WG planned to review the related research and to write a position paper with recommendations on HM fortification for preterm infants. The first face-to-face meeting in Milan resulted in organizing the paper into 10 different sections. These sections were then assigned to working subgroups within the WG. The literature review included electronic searches of MEDLINE (1966-30 June 2018), EMBASE (1980-30 June 2018), CINAHL (1981-30 June 2018), the Cochrane Library, and conference proceedings. The electronic search used the following key words: human milk fortification, breast milk fortification, donor milk fortification, banked milk fortification, [human milk OR breast milk] AND [fortification]. All types of articles, including original papers, reviews, and recommendations were considered. Furthermore, the reference lists of the previous reviews and relevant studies were examined. The searches were limited to human studies, and to the published articles written in English. Trials that had been reported only as abstracts were eligible for inclusion if sufficient information was available from the report.

Following the first meeting, a total of 4 face-to-face meetings were held in Milan, Lyon, and Glasgow to formulate and agree on all of the recommendations. All group members interacted during these face-to-face meetings, and by iterative e-mails between them. All conclusions and recommendations were discussed until a full consensus was achieved for each statement.

# THE RATIONALE FOR HUMAN MILK FEEDING AND HUMAN MILK FORTIFICATION

## Human Milk as the Best Feeding Option for Preterm Infants

Evidence-based data show that HM is the best nutritional and normative standard for infant nutrition (19, 20). Its particular composition—"nutrients with optimal bioavailability, hormonal and enzymatic components, anti-infective, trophic and growth factors, stem cells, prebiotics and probiotics and a myriad of bioactive proteins" -makes HM suited not only to term but also to preterm infants (21–26). Feeding preterm infants with HM, indeed, confers protection against the most important NICU challenges such as necrotizing enterocolitis (NEC) and sepsis (27–33), retinopathy of prematurity (ROP) (34–36), bronchopulmonary dysplasia (BPD) (37, 38) and decreases mortality in a dose-dependent manner (31). Human milk feeding improves long-term neurocognitive development (39–41) and cardiovascular health outcomes (29). Studies comparing solely donor human milk vs. formula show that donor human milk confers protection against NEC (27, 29) and improves feeding tolerance (29). That is why HM is the recommended feeding for all neonates including premature infants. The European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) (29), American Academy of Pediatrics (AAP) (19), and Milan EMBA/ESPGHAN/AAP Joint Meeting Consensus (42) in their most recent recommendation papers stated that "mother's own milk (MOM) is the first choice in the feeding of preterm infants. When mother's milk is not available, pasteurized donor human milk (DHM) should be used."

#### The Rationale for Human Milk Fortification

Infants born early in the third trimester miss the placental transfer of nutrients which would create stores for use in the postnatal period. Human milk while acting as "a preventive therapeutic drug," doesn't provide sufficient amounts of many nutrients for premature infants when fed at the usual feeding volumes. The main challenge is to meet the high and variable nutrient requirements of these preterm infants during the whole hospitalization period. Insufficient nutrient intakes place the infant at risk of impaired neurodevelopment. To prevent EUGR, which is associated with poor neurocognitive outcome, and to avoid specific nutrient deficiencies, nutrient fortification of HM is necessary (19, 29, 42–46).

The consequences of intakes falling short of requirements vary from nutrient to nutrient. Evidence suggests that inadequate intake of protein is important for slow growth and it is particularly responsible for decreased fat-free mass (FFM) gains which are directly related to poor neurocognitive outcomes (14, 47). Intake of energy is also clearly important. In a single blinded randomized clinical trial, Bellagamba et al. (48) showed that increasing only protein intake by 1 gram during parenteral and enteral nutrition did not improve growth and neurodevelopment of preterm infants with a birthweight 500–1,249 g. Insufficient intake of some nutrients leads to specific deficiency states, such as osteopenia (due to insufficient intake of calcium and phosphorus) and to various micronutrient deficiencies, such as zinc deficiency. It is important that VLBW infants receive adequate amounts of iron, zinc, copper, selenium, and iodine. The need of fortification is less clear with regard to manganese, chromium, and molybdenum (49). For the great majority of other nutrients, small shortfalls may have less serious effects, especially when they are temporary. With protein however, any shortfall is prone to affect growth and carries the risk of neurocognitive impairment. Thus, protein supply needs special attention in early life and meeting the requirements should be the goal (43, 44).

The objective of fortification is to increase the concentration of nutrients to the levels that at the recommended feeding volumes (135–200 ml/kg/d) preterm infants receive amounts of all nutrients that meet requirements (43, 50). Nutrient requirements of preterm infants are defined as intakes that enable the infant to grow at the same rate as a fetus (44). Requirements for most nutrients have been derived from accretion rates of protein, fat and minerals obtained by the analysis of fetal body composition at various stages of gestation (44, 50, 51). Additionally, empirical methods have been employed to define requirements including those for nutrients such as vitamins (44, 51) (**Tables 1**, **2**). However, these requirements are variable depending on the TABLE 1 | Requirements for protein and energy; best estimates by factorial and empirical methods (44).

clinical condition and characteristics of each infant either present at birth or evolving during NICU stay (such as IUGR or severe BPD). Therefore, HM fortification needs to be adapted to the specific needs of each infant at each time.

The EMBA Working Group on HM Fortification summarizes the latest recommended intakes for protein, carbohydrates, lipids, and energy in **Table 3**. This table comprises the recommendations of the experts and expert panels (50, 52, 53).

## CURRENT HUMAN MILK FORTIFIERS AND SUPPLEMENTS

There are a number of products available for fortifying human milk for preterm babies which differ by the origin of milk used (bovine, human or donkey), and by nutrient composition (multi-nutrient fortifiers or supplements of protein, lipids, carbohydrates).

#### Multi-Nutrient Fortifiers

Bovine-based multi-nutrient fortifiers contain varying amounts of protein, energy, minerals, trace-elements, vitamins, and electrolytes (**Table 4**). The addition of lipids to multi-nutrient fortifiers with a concomitant reduction in carbohydrate content has allowed a reduction in osmolality of these products (54). In addition, lipids provide a source of essential fatty acids (EFA) which has been shown to improve EFA status in preterm infants (55). As indicated, standard fortification using previously available products was unable to support a satisfactory postnatal growth (See Current Fortification Practices in Neonatal Intensive Care Units: Terminology-Definitions). New fortifiers with higher protein content have been shown to improve short term weight gain (56). Most multi-nutrient fortifiers contain bovine milk protein. Donkey milk was more recently proposed as its composition is very close to human milk (57).

During the past 15 years, some for-profit companies have been set up to collect and buy HM, to manufacture and to sell HM-based products. Prolacta Bioscience is the only one which produces pasteurized HM and HM-based fortifiers. They adhere to the Human Milk Banking Association of North America (HMBANA) guidelines, but test also bacterial content before heat treatment (including pathogens, such as Bacillus cereus, TABLE 2 | Requirements for major minerals and electrolytes determined by factorial method, listed by body weight (51).

TABLE 3 | Recommended enteral protein and energy intakes for clinically stable very low birthweight infants (50, 52, 53).

Escherichia. coli, Pseudomonas aeruginosa, Salmonella spp., yeast and mold), recreational drugs, nicotine, prescription drugs, milk adulteration and breast milk DNA fingerprint for donor identification. To treat huge volumes of HM (1,200 L from 250 donors) they use Vat pasteurization (63◦C, ≥ 30 min). Vat differs from Holder pasteurization which is the commonly used method in non-profit HM banks. Meredith-Dennis et al. (58) showed that Vat pasteurization significantly reduced lactoferrin and total HM oligosaccharide concentrations when compared to Holder pasteurization. Human milk-based fortifier is obtained by concentrating heat-treated donor HM and then adding vitamins and minerals. Various caloric densities of this fortifier allow for individual adjustment based on growth or blood urea nitrogen (BUN). More recently, a novel HM derived cream supplement has been produced by the same company (59, 60).

Although some studies suggested a benefit in terms of morbidity and mortality when babies are fed an exclusively human milk based diet including HM-based fortifier, leading to a reduction of costs (33, 61), much of the work is observational (62–64), and there are still concerns about the efficacy of these products (65). For example, Sullivan et al. (33) showed a significant reduction in NEC rates from 16 to 6%, but this needs to be confirmed in large, independent randomized control trials conducted in units where baseline NEC rates are lower. Sullivan et al. evaluated an exclusive HM-based diet, which consisted of donor HM if no mother's own milk was available and a HM– based fortifier in place of bovine-based formulas and fortifiers. However, the HM-based fortifier was never directly compared with the bovine based fortifier and many of the babies who developed NEC on the bovine fortifier were also on the bovine formula. The OptiMoM study, recently published by O'Connor et al. (66), is the first trial comparing the efficacy of HM-based fortifier to bovine-based fortifier in the absence of formula. There was no difference in feeding tolerance, postnatal growth and morbidity, including NEC ≥ grade 2 (4.7 vs. 4.9%). In 2015, most facilities in US fortified human milk, and approximately one out of five used a HM-based fortifier (67). In summary, HM-based products have been adopted in neonatal care despite being costly and supported by limited efficacy data. Some aspects have not been fully investigated yet, such as metabolic effects and body composition, which are needed before considering these products to be totally safe and effective. It is essential to evaluate the benefit-risk ratio, particularly as these products are very expensive and use large amounts of donated milk to make the fortifier which could be used more directly to feed preterm babies. At the present time these products are available mainly in North America. According to regulations in some European countries, only HM banks in each country are authorized to collect, treat and distribute HM or HM-based products (68, 69). Finally, there could be some ethical concerns. According to available information ethical concerns seem to be well-controlled by present manufacturers but, if the evidence confirms a benefit, the need for these products could increase sharply and ethical questions related to the origin of HM could become a major concern.

In some fortifiers, manufacturers used a hydrolyzed protein source (**Table 4**). There is no evidence supporting the use of such a protein source. It has been shown that preterm infants fed a formula with partially hydrolyzed protein have a shorter transit time, but also a reduced intestinal absorption (70). The rationale cannot be related to the hypothetical prevention of allergy. Indeed, no increased risk of allergy was detected with preterm infants fed on formulas based on cow's milk even those with a high protein content. It has even been suggested that preterm birth reduces the chances of the subsequent development of severe atopic disease (71). Nevertheless, the use of a hydrolyzed protein source is a response to clinicians' preferences, as a lot of professionals are reluctant to add whole bovine protein to HM. This current opinion of professionals comes from a study suggesting that, in a subgroup of preterm infants with a family history of atopy, early exposure to cow's milk increased the risk of

L, lipids; PH, partially hydrolyzed; EH, extensively hydrolyzed; W, whole protein; HMBF, human milk-based fortifier. A-Fortipré®, Nestle; B-Fortema®, Danone; C-FM85®, Nestle; D-Enfamil®, Mead Johnson; E-Similac®, Ross; F-Aptamil PS®, Danone; G-Preemie®, Nestle; H-Beneprotein®, Nestle; I-Pro-Mix®, Corpak; J-Protein instant®, Resource; K- HMBF+4 ®, Prolacta; L- HMBF+6 ®, Prolacta; M- HMBF+8 ®, Prolacta; N- HMBF+10®, Prolacta.

allergic reaction (72). However, more recent studies showed that, compared to exclusively breastfed, preterm infants supplemented with HMF or fed exclusively a preterm formula for 4 months after discharge did not have an increased risk of developing allergic diseases during the first year of life (73). Furthermore, it was previously shown that protein supplementation using wholeprotein is efficient (43, 74, 75). In summary, there is no strong evidence to support the use of hydrolyzed protein source in fortifiers, but it is current practice.

#### Single-Nutrient Supplements

Other products containing only protein, lipids, or carbohydrates are also available. They are useful when individualizing fortification (74–76). Usually, carbohydrate supplements are composed of dextrin maltose, and lipids are composed of medium chain triglycerides**.** More recently, a novel HM-derived cream supplement has been produced to enhance the energy density of feeds. Infants were supplemented with the 2.5 kcal/ml cream supplement whenever their mother's own milk or donor HM was found to be below 67 kcal/dl (20 kcal/oz) (60). When compared to the control group these infants had improved weight and length growth rates and were discharged slightly earlier. This reduction in length of stay was greater in the subgroup of preterm infants with bronchopulmonary dysplasia (59, 60). However, this finding needs to be replicated in other settings to ensure that this can be done without compromising protein to energy ratio.

Protein supplements have been available for years in some countries, but are not specifically designed for neonates (74– 77). One of them contained extensively hydrolyzed protein source (56). Recently a new protein supplement—including partially hydrolyzed protein source–specifically designed for preterm infants, became available in most European countries (54) (Product G, **Table 4**). There is no consensus about how to use these products as studies are scarce. That being said, protein supplements are essential to enable individualized fortification, particularly for Adjustable (ADJ) fortification which has been shown to be associated with clinical benefits (74) (see Individualized Fortification).

# CURRENT FORTIFICATION PRACTICES IN NEONATAL INTENSIVE CARE UNITS: TERMINOLOGY-DEFINITIONS

Following the first introduction of the commercial HM fortifiers in the 1980s, HM fortification has become part of the standard nutritional care for preterm infants in most NICUs. The quality of the fortifiers and the methods of HM fortification have improved over time but nutrient fortification remains suboptimal. An optimal approach to fortification is to provide each individual baby with her/his needs, which might be different from the average of the group (44).

Most of the available commercial fortifiers contain varying amounts of protein, carbohydrate, calcium, phosphate, other minerals, trace elements (zinc, manganese, magnesium, copper), vitamins, and electrolytes and are defined as "multi-nutrient HM fortifiers" (see Current Human Milk Fortifiers and Supplements) (43).

In an attempt to clarify the terminology regarding HM fortification practices, in 2010, World Association of Perinatal Medicine (WAPM) Working Group on Nutrition defined the fortification methods in current practice as follows (43):

	- a. Adjustable (ADJ) HM fortification (74, 77, 78)
	- b. Targeted HM fortification (76, 79–81)

The EMBA Working Group on HM Fortification adopts this terminology and **Table 5** summarizes the characteristics of these methods.

## STANDARD (STD) FORTIFICATION

This is the most widely used fortification method. The standard practice is to add a fixed amount of multinutrient fortifier per 100 ml of HM to achieve the recommended nutrient intakes. This fixed amount has been calculated and determined by the manufacturer assuming a fixed protein content for

#### TABLE 5 | Current human milk fortification methods (43, 74, 76–79).

HM, human milk; VLBW, very low birth weight; BUN, blood urea nitrogen; RCT, randomized controlled trial. \*BUN levels of 10–16 mg/dl correspond to blood urea concentrations of 21.40–34.24 mg/dl (3.57–5.71 mmol/l).

all milk samples without considering intra-, inter-individual and temporal variations. Standard fortification is initiated usually when the fed milk volume is 50–100 ml/kg. Milan EMBA/ESPGHAN/AAP Joint Meeting Consensus recommends fortifying HM for preterm infants with a birthweight <1,800 g (42).

The updated Cochrane review (82) addressed the impact of STD multi-nutrient fortification of HM on growth, development, feeding tolerance and NEC in preterm infants. The systematic review evaluated 1,071 infants in 14 trials. The trials were generally small and weak methodologically. Meta-analyses provided low-quality evidence that STD multi-nutrient fortification of HM, in comparison to the unfortified HM, improved in-hospital weight gain, linear growth, and head circumference growth. Only very little data were available for growth and developmental outcomes beyond infancy and these did not show long-term advantage.

However, when comparisons are made between fortified HM in STD fashion and preterm formula (PF) (83–85) the findings indicate that despite fortification, HM fed preterm infants continue to grow more slowly than PF fed infants. Henriksen et al. (86) reported that 58% of VLBW infants fed predominantly fortified HM had EUGR at discharge. Maas et al. (87) evaluated in-hospital growth of 206 very preterm infants and found that standard deviation score for weight from birth to day 28 decreased more in infants with a cumulative milk intake >75% of all enteral feeds compared to those <25% HM intake. The trend toward poorer weight gain with higher proportions of HM intake persisted also at the time of discharge. Of course these findings cannot be a reason to favor preterm formula vs. HM to promote growth of VLBW infants. Considering all the clinical benefits deriving from the use of HM as already stated in the previous Sections, fortified HM should be the first feeding option for these infants. However, HM fortification should be optimized.

#### Shortfalls With "Standard Fortification"

The reasons for the limited success with STD fortification include:

Undernutrition, particularly protein undernutrition: STD fortification does not take into account the variability of HM macronutrient content and variability of the infants' requirements. Preterm infants fed fortified HM in STD fashion receive less protein than they need due to "customary assumptions" as explained in the following paragraph. Protein is essential for tissue and organ development, and is a rate limiting factor for growth. A rate of postnatal growth similar to the intrauterine growth can be reached only with adequate protein and energy intakes (3.5–4.5 g/kg/d, 110–130 kcal/kg/d, respectively, **Table 3**). Standard fortification usually provides the recommended energy intakes, but cannot provide the adequate protein intakes for many VLBW infants (actual protein intake 2.8–2.9 g/kg/day) (88). Arslanoglu et al. (88) compared the assumed protein content of fortified HM samples and derived protein intakes to actual (measured) protein content/ intakes in a group of preterm infants. Actual protein intakes were consistently and significantly lower than assumed when fortification was performed in STD fashion (range of discrepancy between 0.5 and 0.8 g/kg/day). On the other hand, the differences in energy intake were small and not consistently significant. This observation was important, because it provided a rational basis for simply adding more protein to milk in those infants whose enteral diet came from milk, especially over long periods after birth (89). Similar findings have been reported in the following years by other researchers (75, 90, 91). Picaud et al. (75) showed that one third of extremely low birth weight infants (ELBW) infants needed supplementary protein to reach the expected weight gain. In the recent systematic review and meta-analysis regarding the macronutrient and energy composition of preterm human milk, Mimouni et al. (92) stated that protein content decreased massively (by one-half) and significantly from day 1– 3 at week 10–12. During the same time frame; fat, lactose and energy content showed a significant linear increase. Very recently in PREMATURE MILK study Maly et al. (91) reported that protein content decreased during the first 3 weeks of lactation and the recommended protein intakes couldn't be reached with STD fortification in the majority of the infants.

The main reason for ongoing protein undernutrition despite HM fortification is that the STD regimen is based on assumptions about the protein content of the milk. Usually the assumed protein concentration by the manufacturers is 1.4–1.5 g/dl which only occurs during the first 2–3 weeks of lactation. HM protein concentration decreases with the duration of lactation and drops to around 1 g/dl by week 4–6 (43, 91). Thus, the protein intake would be inadequate most of the time throughout the fortification period (43, 44, 93).

Optimization of HM fortification is being widely studied. Improvement of the quality and source of the fortifiers, increasing the protein content of the products, early initiation of fortification are all efforts to improve STD fortification. An attempt at earlier initiation of fortification has resulted in better in-hospital head growth and weight gain in a very recent pre-, post- implementation study (94). However, a systematic review and meta-analysis aiming to ascertain whether randomized controlled trials determined the efficacy of early vs. late initiation of fortification on clinical outcomes gave inconclusive results. In this review Mimouni et al. (95) concluded that there is little evidence that early introduction of human milk fortification affects important outcomes.

Individualization of fortification is believed to be a solution to the problem of protein undernutrition with STD fortification and is currently the recommended method by scientific authorities and expert panels (29, 42, 43). The two methods of individualized fortification (**Table 5**); Adjustable and Targeted methods are discussed in the following Sections separately.

# INDIVIDUALIZED FORTIFICATION

## Adjustable (ADJ) Fortification

ADJ method was designed specifically to avoid both protein undernutition and overnutrition. With this method, protein intake is adjusted on the basis of each infant's metabolic response. Human milk fortification is initiated with a multi-nutrient fortifier in a STD fashion and as soon as full strength fortification is tolerated, it is guided by blood urea nitrogen (BUN) levels as a surrogate for assessing protein adequacy. If BUN level is below a pre-defined threshold value (<10 mg/dl according to 2012 protocol) (77), extra protein is added in the form of protein supplement. If BUN level is above a specified value suggesting excessive protein (>16 mg/dl), the level of fortification is reduced (**Tables 6**, **7**) (74, 77).

This model was evaluated in a randomized controlled trial (RCT) by Arslanoglu et al. (74) and was found practical, feasible and effective to provide the preterm infants with adequate protein intakes approximating intrauterine protein intakes and better inhospital growth compared to STD fortification. In this study the mean actual (measured) protein intakes reached 3.5 g/kg/d in ADJ group in the second week of the study, while it remained 2.8–2.9 g/kg/d in the STD group. During the 3 weeks intervention period the infants in ADJ group had better weight and head circumference gains compared to STD group (17 vs. 14 g/kg/d and 1.0 vs. 0.7 cm/wk, respectively).

ADJ fortification; i.e., adding extra protein on the basis of BUN measurements, has been in use since this publication with the protocol being refined in 2012 (77).

#### The Updated Protocol for ADJ Fortification

The threshold range of BUN used to adjust protein supply was selected arbitrarily in the first study (9–14 mg/dl) (74). Adjusting the protein intake according to these values (74), the investigators observed that there was the need to increase the level of fortification during most of the fortification period; and the protein intakes could not reach the recommended intakes at the first week of the fortification. There was need to refine the protocol, and to be cautious only a small increase has been suggested. The threshold values for BUN were modified as 10–16 mg/dl (77, 78). **Tables 6**, **7** show the details of the current ADJ fortification regimen.

ADJ fortification starts as STD fortification when the fed milk volume reaches 50–80 ml/kg/d with multi-nutrient fortifier (Level 0). Protein adequacy is evaluated by twice weekly BUN determinations. Extra-protein is added in the form of protein supplement according to the protocol in 3 levels up to 1.2 g per 100 ml of HM (**Table 7**).

In 2013, Alan et al. (96) utilized a slightly modified form of ADJ fortification in their observational study and compared protein intakes and growth in VLBW infants fed HM fortified according to ADJ regimen to those fed in STD fashion (historical controls). The study replicated similar results in terms of higher protein intake and better in-hospital growth including linear growth with ADJ fortification.

Picaud et al. (75) in their recent retrospective study conducted on the preterm infants weighing <1,250 g at birth reported that 1/3 of extremely low birth weight infants required additional protein to supplement the standard fortification to achieve satisfactory weight gain. According to the practice in their NICU they used weekly measured urea levels and growth together to determine the need for extra protein. They confirmed the findings of Arslanoglu et al. (74) that extra protein supplementation not only improved weight gain but also head circumference gain.

In two observational studies, slightly modified forms of ADJ fortification were associated with both better growth and better neurodevelopmental outcomes. Ergenekon et al. in their retrospective study (97) reported better head growth and weight gain in NICU with ADJ fortification in very preterm infants. TABLE 6 | The products required and the threshold values of the metabolic marker used for the Adjustable (ADJ) fortification method (77).

TABLE 7 | The scheme for adjustable fortification (updated in 2012) (77).

This improvement in growth was associated with significant improvement of Bayley scores at 18 months corrected age. Also, in the observational study of Biasini et al. (98), the improved growth with higher protein intakes in ELBW infants was associated with better neurodevelopment evaluated by Griffiths Mental Development Scores at corrected 12 months of age. At 24 months, small for gestational age (SGA) preterm infants having higher protein intake had higher scores.

Very recently, Mathes et al. (99) showed a highly positive correlation between plasma urea concentrations and actual protein intakes and urinary urea-creatinine ratio. They suggest that urinary urea-creatinine ratio, just like plasma urea concentrations may help to estimate the actual protein supply in preterm infants.

# TARGETED FORTIFICATION

The concept of targeted fortification is to analyze macronutrient composition of HM and to fortify it in such a way that each infant always receives the amount of nutrient that is suggested in population-based recommendations. This method was proposed and studied first by Polberger et al. in 1999 (79) named as "individualized protein fortification of HM." In this study, protein was the only nutrient considered for supplementation in addition to STD fortification. The milk was analyzed periodically and a target nutrient intake (protein) was delivered, which was 3.5 g/kg/day.

Parallel to the introduction of bedside human milk analyzers it has become possible for the researchers and neonatologists to analyze and tailor the macronutrient content based on realtime analysis of HM. In an observational study de Halleux et al. (81) compared standard vs. targeted (mentioned as "individualized" by the authors) fortification approaches; daily breastmilk composition was measured with a mid-infrared milk analyzer.  A fortifier was added to reach a protein intake of 4.3 g/kg/day. As a result, the variability of macronutrients in the individualized approach was significantly decreased, but the average fat intake was 8.6 g/kg/day which exceeded the recommendations (see **Table 3**). Weight gain was superior to the STD fortification group and was similar to the formula fed group. Data regarding head circumference gain and linear growth were not shown.

Using a different approach, Hair et al. (59) in a two-center RCT measured breast milk energy density with a near-infrared analyzer. Infants received HM derived cream in addition to HM derived fortifier if energy density was <20 kcal/oz (67 kcal/100 mL). The HM derived cream was standardized to 25% lipids and contained 2.5 kcal/ml. Infants randomized to the HM derived cream group showed superior weight and length gain vs. the control group without cream. However, the validation studies with infrared analyzers have determined that the measurement of calories is not precise because of the inability to accurately measure lactose with these devices (80).

A pilot study conducted by Rochow et al. (76) has been the first to show the feasibility of targeted fortification of all macronutrients through twice daily breast milk analysis (nearinfrared), using modular products to bring levels of fat to 4.4 g/dl, protein to 3 g/dl and carbohydrates to 8.8 g/dl. Matched pair analysis of 20 infants fed STD fortified milk was performed. Growth rates of the infants with targeted fortification were similar to the group with STD fortification (∼20 g/kg/d). However, the authors showed a high correlation between volume of fed HM and weight gain only in the targeted group. They calculated an additional workload of 5–10 min per milk batch. The similar growth rate could be due to the fact that the STD group had higher milk intake than the targeted group (155 + 5 vs. 147 + 5 ml/kg/d). Another limitation to be improved was the 24 h delay between the milk analysis and the addition of the macronutrients.

Targeted fortification requires a milk analyzer, which is an expensive device, requiring careful calibration. Fusch et al. (100) draw attention to the need of recalibration of these analyzers since they were originally developed for use in the dairy industry and HM has a different matrix and optical characteristics from cow's milk. They conclude infrared analysis seems to be a promising tool for fat and protein with calibration, but lactose and therefore energy cannot be assessed with the current state of technologies.

Buffin et al. (101) compared fat and protein concentrations using two infrared analyzers and reference laboratory methods indicating the same important finding that bedside HM analyzers require recalibration before their use in practice.

In a recent RCT comparing targeted fortification to standard, McLeod et al. (102) did not find any improvement in growth and nutrition in a group of preterm infants born below 30 weeks of gestation. Interestingly, mean measured protein content in the targeted group was higher than the assumed value (1.6 vs. 1.4 g/100 ml), leading to lower amounts of fortifier added to the milk in the intervention group. The authors concluded that targeting fortification on measured composition is labor intensive requiring frequent milk sampling and precision measuring equipment.

### POST DISCHARGE FORTIFICATION

There is no consensus about post discharge nutrition, however there is a position paper from ESPGHAN (103) and recent reviews (104–106), including one focusing on HM supplementation (106). The ESPGHAN position paper evaluated randomized trials published before 2004 and proposed fortifying HM up to at least 40 and possibly up to 52 weeks postconceptional age when infants were small for gestational age at discharge. However, the definition of being small for gestational age was not presented: bodyweight at discharge below 10th (moderate growth restriction) or 3rd percentile (severe growth restriction)? Meta-analysis and reviews suggested that there was evidence to use enriched nutrition [preterm formula (PF) rather than post discharge formula (PDF)] after discharge for formula fed babies. But this evidence was not strong enough to recommend fortification of HM after discharge (105, 106).

The last decade was marked by two trends. Firstly, a decrease in the incidence of extra-uterine growth restriction (16, 17, 107). Secondly, an increase in breastfeeding rates at discharge in these infants, despite significant heterogeneity between different neonatal units suggesting that there is room for improvement (108). Post discharge studies comparing enriched vs. standard nutrition highlighted the ability of some preterm infants -like term counterparts- to regulate their intake volume to compensate for differences in energy density between formulas (109–111). However, this is only true for preterm babies reaching term due date and beyond as it has been reported that many less mature preterm babies are not able to compensate for a low nutrient intake feed due to immature feeding skills (112). Therefore, there is a window of opportunity to optimize nutrition post discharge, which might explain why few studies reported a benefit for growth and mineralization of enriched post discharge nutrition.

Despite the widespread use of human milk fortifiers (HMF) for preterm babies on neonatal units there has been little reporting of its use post discharge. It might seem best practice to mirror the principle behind the use of post discharge formulas for formula fed babies, i.e., the bridge between a nutrient dense milk to one of lower density. In accordance with this there have been recommendations that fortifier is continued in preterm breast fed babies either to term or around 52 weeks post conceptional according to their growth trajectory (103, 113). Although the practicality of putting this into practice has been questioned by some reviewers because of the availability of HMF in different countries and the perceived, but not proven, problems with practicability (106), evidence is accruing that it is possible using many different methods (114).

It is known that babies exclusively breastfed post discharge can have reduced bone mineral density and lower lean body mass than formula fed babies (115), although this may have improved with more recent feeding practices on neonatal units. But it does suggest that some fortification of breast milk post discharge will help nutritional status as well as growth.

There have been 3 reports of randomized controlled trials fortifying breast milk post discharge, to 4 months corrected age (116), for around 5–6 months after discharge (117), and to around 12 weeks after discharge (118). Two used commercial HM fortifiers (116, 118) and one (117) a powdered preterm formula. **Table 8** shows the nutrient intervention and numbers of infants in these trials. No difference in growth was found by Zachariassen et al. (116). However, this group did find better lung function in the fortification group at 6 years old (119). O'Connor et al. (118) found better weight, length and bone mineral density and better head growth in babies <1,250 g birth weight, all of which were maintained to 1 year (120). There is also evidence of better visual function (121). Neurodevelopmental outcome at 12–18

#### TABLE 8 | Nutrient interventions in the randomized controlled trials addressing the effects of fortifying human milk post discharge (116–118).

Arslanoglu et al. Optimization of Human Milk Fortification

months was not found to be different between groups (117, 120). In these studies there may not have been sufficient protein given to achieve an optimal growth and possibly neurodevelopmental outcome. In contrast energy should not be a limiting nutrient for a baby feeding fully responsively at the breast as they will access all the energy rich hind milk they require.

All studies found the HMF to be well-tolerated however each used a different method to administer the fortifier, one adding the entire dose into one bottle per day (116) with no adverse gastrointestinal symptoms reported. Another was by bottle but given spread out over the day (117) and the third by cup twice a day (118). None reported any adverse effect on breast feeding rates in the fortified group. A finger feeding device to administer fortifier has recently been evaluated and was well-accepted (122). Technical improvements are required and a large randomized study will be necessary to evaluate the benefits of such a strategy.

There are 2 reports looking at the effect of fortifier post discharge on breastfeeding. One is a case study where breastfeeding was maintained by the addition of fortifier post discharge rather than formula in a baby failing to thrive on the breast (123). A second suggests an improvement in breastfeeding rates at 6 weeks post conceptional age in a group of babies discharged on fortifier compared to a group on breast milk alone (124).

In theory, a gradual step down from full fortification would seem appropriate for breastfed babies to allow adaption to the lower nutrient intake of unfortified breast milk and to support the rapid growth that occurs around term due date.

As studies that evaluated post discharge HM fortification showed no deleterious effect on breastfeeding rates, it is proposed that HMF post discharge is considered in breastfed babies where a post discharge formula would be used were they formula fed, particularly if they have not grown well while on the neonatal unit. One group who might benefit being babies with bronchopulmonary dysplasia BPD (125). More work is needed to assess an optimal amount, length of time and method of administering fortifier in a breast feeding baby post discharge.

# CONCLUSIONS-COMMENTS, RECOMMENDATIONS

#### Conclusions-Comments

#### Recommendations

#### FUTURE RESEARCH DIRECTIONS

# AUTHOR CONTRIBUTIONS

SA is leading the EMBA WG on Human Milk Fortification. J-CP, C-YB, CK, DL, and PT are the components of the WG. All contributed to the construction and writing of the manuscript. GM, EB, GW, AW, AGa, AGr, CG, and DB commented on the manuscript as the components of the EMBA Board.

#### REFERENCES

## ACKNOWLEDGMENTS

The authors thank the Italian Association of Human Milk Banks (Associazione Italiana Banche del Latte Umano Donato = AIBLUD) for its continuous efforts to promote research in the field of donor human milk and human milk banks, and for the financial support given to the publication of this manuscript.

preterm infants from the NICHD Neonatal Research Network. Pediatrics. (2010) 126:443–56. doi: 10.1542/peds.2009-2959

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Arslanoglu, Boquien, King, Lamireau, Tonetto, Barnett, Bertino, Gaya, Gebauer, Grovslien, Moro, Weaver, Wesolowska and Picaud. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Mother's Own Milk and Bronchopulmonary Dysplasia: A Systematic Review and Meta-Analysis

#### Eduardo Villamor-Martínez 1 , Maria Pierro2 , Giacomo Cavallaro3 , Fabio Mosca3 and Eduardo Villamor 1 \*

1 Department of Pediatrics, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Center (MUMC+), Maastricht, Netherlands, 2 UOC TIN e Neonatologia, Dipartimento Salute Mamma e Bambino, Fondazione Poliambulanza, Brescia, Italy, 3 Neonatal Intensive Care Unit, Department of Clinical Sciences and Community Health, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy

#### Edited by:

Guido Eugenio Moro, Italian Association of Donated Milk Banks (AIBLUD), Italy

#### Reviewed by:

Hercília Guimarães, Universidade do Porto, Portugal Jegen Kandasamy, University of Alabama at Birmingham, United States

> \*Correspondence: Eduardo Villamor<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics

Received: 06 August 2018 Accepted: 20 May 2019 Published: 06 June 2019

#### Citation:

Villamor-Martínez E, Pierro M, Cavallaro G, Mosca F and Villamor E (2019) Mother's Own Milk and Bronchopulmonary Dysplasia: A Systematic Review and Meta-Analysis. Front. Pediatr. 7:224. doi: 10.3389/fped.2019.00224 Background: Bronchopulmonary dysplasia (BPD) is the most common complication of very preterm birth and can lead to lifelong health consequences. Optimal nutrition is a cornerstone in the prevention and treatment of BPD. In very preterm infants, mother's own milk (MOM) feeding is associated with lower risks of necrotizing enterocolitis, retinopathy of prematurity, and sepsis. Although several studies have shown that MOM may protect against BPD, a systematic analysis of the evidence has not been performed to date.

Methods: A comprehensive literature search was conducted using PubMed/MEDLINE and EMBASE, from their inception to 1 December 2017. Longitudinal studies comparing the incidence of BPD in preterm infants fed with exclusive MOM, MOM supplemented with preterm formula (PF), and/or exclusively fed with PF were selected. A random-effects model was used to calculate the Mantel Haenszel risk ratio (RR) and 95% confidence interval (CI).

Results: Fifteen studies met the inclusion criteria (4,984 infants, 1,416 BPD cases). Use of exclusive MOM feedings was associated with a significant reduction in the risk of BPD (RR 0.74, 95% CI 0.57–0.96, 5 studies). In contrast, meta-analysis could not demonstrate a significant effect on BPD risk when infants fed with more than 50% MOM were compared with infants fed with <50% MOM (RR 0.98, 95% CI 0.77–1.23, 10 studies) or when infants fed with MOM supplemented with PF were compared with infants fed with exclusive PF (RR 1.00, 95% CI 0.78–1.27, 6 studies). Meta-regression showed that differences in gestational age were a significant confounder of the effect of MOM.

Conclusion: To our knowledge, this is the first systematic review and meta-analysis that specifically evaluates the role of MOM on BPD. Our data indicate that MOM may reduce the incidence of BPD when used as an exclusive diet, but this result needs to be interpreted with caution. We did not find the same difference in analyses with other dosages of MOM. Further studies adequately powered to detect changes in BPD rates and that adjust for confounders are needed to confirm the beneficial effects of MOM on BPD.

Keywords: mother's own milk, human milk, bronchopulmonary dysplasia, preterm formula, meta-analysis, systematic review, meta-regression

## INTRODUCTION

Mother's own milk (MOM), fresh or frozen, is the normative standard for preterm infant feeding and nutrition (1–4). If MOM is unavailable despite significant lactation support, pasteurized donor human milk (DHM) is the recommended alternative over the use of bovine milk-based preterm formula (PF) (1– 4). However, it is increasingly recognized that numerous MOM components which could contribute to its protective effects against adverse outcomes of prematurity are reduced or absent in DHM (5).

Bronchopulmonary dysplasia (BPD) is one of the most common complications of prematurity, and it predicts multiple adverse outcomes including chronic respiratory impairment and neurodevelopmental delay (6, 7). Optimal nutritional support is considered a cornerstone in the treatment/prevention of BPD (8). Recently, we performed a systematic review and metaanalysis on the effects of DHM on BPD (9). Meta-analysis of randomized controlled trials (RCTs) could not demonstrate that supplementation of MOM with DHM had a significant effect on BPD risk when compared to supplementation with PF. However, meta-analysis of observational studies showed a protective effect of DHM supplementation on BPD (9). Two very recent systematic reviews confirmed that the protective effects of human milk (i.e., MOM and/or DHM) on BPD are only observed in meta-analysis of observational studies (10, 11). Using the GRADE-system (12), the authors of these meta-analyses consider the evidence to be inconclusive.

Despite the important differences between DHM and MOM, the umbrella term "human milk" is frequently used to encompass both MOM and DHM, implying that the beneficial effects of MOM can be directly extrapolated to DHM (5, 13). Moreover, many of the studies and meta-analyses have compared PF feedings with various combinations of PF, MOM, and DHM. A recent meta-analysis evaluated the effects of MOM on retinopathy of prematurity (ROP) (14). This analysis excluded data on DHM and showed that the overall incidence of ROP was reduced among infants fed MOM compared with those fed PF. To the best of our knowledge, no systematic review has focused on the role of MOM in the development of BPD. The analysis of exclusive MOM vs. PF was beyond the scope of our previous study (9), and Miller et al. and Huang et al. did not study the effect of MOM separately from that of DHM (10, 11). Therefore, we aimed to conduct a systematic review and metaanalysis on the association between MOM/PF feeding and BPD development. The present meta-analysis does not include data on DHM.

### METHODS

This study is a continuation of our previous review on DHM and BPD (9), and shares much of the same methodology. We expanded on the protocol of our earlier study, and specified the objectives, criteria for study inclusion, method for evaluating study quality, outcomes and statistical methodology. We report this study according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) (15). The PRISMA checklist for this report can be found in the **Supplementary Material**.

#### Data Sources and Search Strategy

We modified and expanded on the search strategy of our earlier review (9). We carried out a comprehensive literature search using PubMed/Medline and EMBASE, from their inception to March 1, 2018. The search strategy for PubMed used the following terms, including Mesh terms: (breast milk OR infant feeding OR mother's own milk) AND (preterm infant OR very low birth weight infant) AND (outcome OR bronchopulmonary dysplasia OR BPD) AND (observational study OR cohort study OR case-control). We used a similar strategy in EMBASE. We applied no language restrictions. We translated articles when needed. We included cohort and case-control studies in this review, as well as RCTs with an observational arm. Other types of studies were excluded, but when considered relevant, they were read to identify additional studies to include. We also used the "cited by" tool in Google Scholar and Web of Science to identify studies for inclusion. Moreover, we included articles which we came across in the elaboration of our earlier review (9).

# Eligibility Criteria and Study Selection

We included studies if they were original cohort or case-control studies, which examined very preterm (gestational age, GA <32 weeks) or very low birth weight (VLBW, BW <1,500 g) infants receiving either MOM or PF, and which included at least two groups divided according to feeding policy. Only full-length published studies were considered for inclusion. Studies were included if they reported results on the incidence of BPD. We defined BPD as oxygen dependence at 28 days of life (BPD28) or as oxygen dependence at 36 weeks adjusted gestational age (BPD36). Studies were excluded if the group receiving MOM or PF also received DHM. Two reviewers (EV-M, EV) independently screened the results of the searches, and included studies according to the inclusion criteria using EndNote (RRID:SCR\_014001), using the methodology of Bramer et al. (16). Studies on which reviewers disagreed for inclusion were identified, and discrepancies were resolved through discussion or by consulting the other authors.

#### Data Extraction

We collected the following information per study: citation information, study design, number of patients, number of centers, location of study, inclusion and exclusion criteria, patient characteristics (GA, BW), type of feeding received (MOM, PF, combination of MOM and PF, and type of fortifier), and incidence of BPD per group. Two researchers (EV-M, EV) extracted the data using an Excel sheet designed for this review. We resolved discrepancies in data extraction through discussion, or by consulting the other authors. Another researcher (MP) independently validated the accuracy of the data extracted.

## Assessment Risk of Bias

Two researchers (EV-M, MP) assessed the risk of bias in included studies. We used the Newcastle-Ottawa Scale (NOS) for quality assessment of cohort and case-control studies. The NOS is used to assign a score to studies on selection (0–4 points), comparability (0–2 points), and outcome/exposure (0–3 points), for a total score of up to 9 points. Discrepancies were resolved through discussion.

#### Statistical Analysis

We used Comprehensive Meta-Analysis V3.0 software (RRID:SCR\_012779) to combine and analyze studies. The Mantel Haenszel (MH) risk ratio (RR) for BPD with 95% confidence interval (CI) was calculated in each study. Due to anticipated heterogeneity, we used a random-effects model to combine studies. This model accounts for heterogeneity between and within studies and it does not assume that "true" effect sizes are identical across studies. Subgroup analyses were conducted according to the mixed-effects model (17). In this model a random-effects analysis is used to combine studies within each subgroup, and a fixed-effect model is used to combine subgroups and yield the overall effect. The model does not assume studyto-study variance (tau-squared) to be the same for all subgroups. We assessed statistical heterogeneity using the Cochran's Q statistic, and the I 2 statistic which is derived from it. We planned to evaluate the risk of publication bias through visual inspection of the funnel plot and with Egger's regression test (18). We decided a priori to analyze the effect of GA as a confounding factor, by analyzing the mean difference in this covariate between groups, and through subgroup analysis, by removing studies with large differences in GA from analysis. We decided to use the group with the higher MOM intake as the reference group in all our analyses. We carried out sensitivity analyses by removing one study from analyses at a time. We used an α = 0.05 for statistical significance (α = 0.10 for statistical heterogeneity).

## RESULTS

After removing duplicates, our comprehensive search found 965 articles, of which we identified 84 as potentially relevant, and 15 met our inclusion criteria (19–33) after full-text review. The PRISMA search diagram is shown in **Figure 1**. The characteristics of the included studies are shown in **Supplementary Table 1**. Fourteen included studies were observational cohorts, of which seven were prospective (20, 21, 23, 25, 30, 31, 33) and seven were retrospective (19, 22, 24, 26, 28, 29, 32), and one study was a retrospective case-control (27). One study was excluded from meta-analysis because it did not group by type of feeding (22). We divided studies according to the proportion of feeding that was MOM or PF in each group, and we made three comparisons for analysis: (1) Exclusive MOM vs. Any PF; (2) Mainly MOM vs. Mainly PF; (3) Any MOM vs. Exclusive PF.

#### Quality Assessment

Three studies scored six points on the NOS, 10 studies scored seven points, and two studies scored the maximum of 9 points. We downgraded studies in quality for not adjusting for confounders (k = 13), for excluding infants who were lost to follow-up (k = 2) and for not defining BPD clearly (k = 1).

#### Exclusive MOM vs. Any PF

Five studies (19–21, 27, 30) compared infants who received a diet of exclusive MOM to infants who received MOM and any supplementation with PF. Meta-analysis of these studies found that the exclusive MOM group had a reduced risk of BPD (RR 0.74, 95% CI 0.57–0.96, p = 0.021, **Figure 2**). When we excluded the study of Fewtrell et al. which used a different definition of BPD (BPD28), the effect of MOM on BPD remained significant (RR 0.71, 95% CI 0.54–0.93 p = 0.014). Sensitivity analysis showed that removing the study of Madore et al. (27) or the study of Schanler et al. (30) made the overall association no longer significant (**Supplementary Figure 1**).

When we analyzed the difference in GA between groups, meta-analysis did not find a significant difference (MD GA −0.06 weeks, 95% CI −0.38–0.25, p = 0.689, **Supplementary Figure 2**). None of the included studies had a mean difference in GA between groups which was larger than 0.3 weeks.

Out of the five studies which had an exclusive MOM group, three studies (19–21) also provided data on a group of infants receiving exclusive PF. We carried out a subgroup analysis of these studies. When pooled, meta-analysis could not find a significant difference in BPD risk between groups (RR 1.08, 95% CI 0.63–1.87, p = 0.770 **Figure 3**). When we excluded the study of Fewtrell et al., which defined BPD as BPD28 instead of BPD36, the effect of MOM did not change in significance (RR 1.10, 95% CI 0.58–2.09, p = 0.777). When we removed the study of Assad et al. for having a MD in GA (of 1.5 weeks) between groups ≥0.5 weeks, the results did not change in significance (RR 0.88, 95% CI 0.57–1.37, p = 0.583). An analysis on the difference in GA between groups did not find a significant difference overall (MD −0.41 weeks, 95% CI −1.19–0.36, p = 0.295, **Supplementary Figure 3**).

# Mainly MOM vs. Mainly PF

Ten studies compared infants receiving mainly MOM vs. infants receiving mainly PF. We included studies which had stricter criteria for comparison (i.e., exclusive MOM vs. exclusive PF) in this analysis as well. Meta-analysis could not find a significant difference in risk of BPD between the mainly MOM and the

mainly PF group (RR 0.98, 95% CI 0.77–1.23, p = 0.833, **Figure 4**). When we excluded the study of O'Connor et al. for using a definition of BPD at 28 days of life instead of at 36 weeks PMA, the effect of MOM on BPD development remained nonsignificant (RR 0.99, 95% CI 0.75–1.31, p = 0.938). Excluding any study one at a time did not change the significance of the effect of MOM on BPD (**Supplementary Figure 4**).

We used meta-analysis to study the differences in GA between the MOM and PF groups in each study. Metaanalysis found no significant MD in GA when pooling all studies together (MD −0.31 weeks, 95% CI −0.78–0.17, p = 0.204, **Supplementary Figure 5**). However, individual studies showed significant differences in GA between groups, and the heterogeneity was very high (p < 0.001, I 2 = 92.1%), which indicated that GA could be a significant confounder. When we used subgroup analysis to exclude studies where the difference in GA between groups was larger than 0.5 weeks, we were left with 6 studies, but the effect of MOM on BPD did not change significantly (RR 0.99, 95% CI 0.82–1.18, p = 0.890).

We used meta-regression to explore the role of GA in potentially modifying the effect of MOM on BPD development. Meta-regression found a significant association between MD in GA and the risk of BPD in the MOM group (Coefficient: −0.59, 95% CI −0.95 to −0.23, p = 0.001, R 2 = 1.00, **Figure 5**). This indicates that in studies where the MOM group had a higher risk of BPD, this group was also more premature than the PF group, and in studies where the MOM group had a lower risk of BPD, this group was also more mature than the PF group.

#### Any MOM vs. Exclusive PF

Six studies (19, 20, 23–25, 33) compared infants who received any MOM to infants who received exclusive PF. We also included

FIGURE 2 | Meta-analysis of exclusive MOM vs. any PF and risk of BPD. MOM, mother's own milk; PF, preterm formula; BPD, bronchopulmonary dysplasia; CI, confidence interval; MH, Mantel-Haenszel.

FIGURE 3 | Meta-analysis of exclusive MOM vs. exclusive PF and risk of BPD. MOM, mother's own milk; PF, preterm formula; BPD, bronchopulmonary dysplasia; CI, confidence interval; MH, Mantel-Haenszel.

FIGURE 4 | Meta-analysis of mainly MOM vs. mainly PF and risk of BPD. MOM, mother's own milk; PF, preterm formula; BPD, bronchopulmonary dysplasia; CI, confidence interval; MH, Mantel-Haenszel.

studies in this comparison where the infants of the MOM group received larger proportions of MOM (e.g., infants receiving mainly MOM or exclusive MOM). Meta-analysis could not find a significant effect of any MOM on the risk of developing BPD (RR 1.00, 95% CI 0.78–1.27, p = 0.975, **Figure 6**). When we removed the study of Hylander et al. from the analysis, which did not clarify their definition of BPD, the effect of any MOM remained non-significant (RR 1.07, 95% CI 0.80–1.42, p = 0.665). Removal of any one study did not affect the significance of the results (**Supplementary Figure 6**).

Meta-analysis found that there was a significant difference in mean GA between the any MOM group and the exclusive PF group, with the infants receiving any MOM being born earlier (MD −0.50 weeks, 95% CI −0.99 to −0.01, p = 0.045, **Figure 7**). Removing studies where the groups differed by more than 0.5 weeks in GA left us with three studies (20, 24, 33), but the effect of MOM on BPD remained non-significant (RR 0.93, 95% CI 0.82–1.05, p = 0.236, **Supplementary Figure 7**).

# Publication Bias

We tested the three comparisons for publication bias (**Supplementary Figure 8**), but neither visual inspection of the funnel plot nor Egger's regression test could find significant evidence of publication bias. A small number of studies made the analyses on "Exclusive MOM vs. Any PF" and "Any MOM vs. Exclusive PF" inconclusive (**Supplementary Figure 8**).

# Adjusted Data

Two studies (23, 26) reported data on BPD incidence that was adjusted for confounders. Furman et al. (23) reported incidence of BPD by amount of maternal milk received, and they adjusted for several confounders (BW, sex, and ethnicity). They found no significant difference in BPD risk for varying levels of MOM intake, compared to receiving exclusive PF. Maayan-Metzger et al. (26) used logistic regression to adjust for confounders including GA, BW and sex. They found that receiving only or mainly MOM, compared to receiving only or mainly PF, did not significantly affect the risk of developing BPD. They found the same result in the subgroup of infants with GA 24–28 weeks.

## Other Studies

One study (22) did not group according to MOM or PF intake. Instead they compared infants with BPD to infants without BPD and studied median intake of MOM in the first 6 weeks of life. In their study infants were given MOM, supplemented by PF when necessary. They found infants with BPD had a significantly lower median daily MOM intake compared to infants without BPD (2.3 mL/kg/d vs. 10.8). The protective effect on BPD of a higher MOM intake at 42 days remained after adjustment for confounding factors (RR 0.98, 95% CI 0.96–0.99, p = 0.030).

## DISCUSSION

RCTs are widely regarded to provide the highest degree of evidence (34). However, the random allocation of infants to a group receiving PF instead of MOM is not ethical and, therefore, evidence must be based on observational studies (14, 35). To our knowledge, this is the first systematic review and meta-analysis that specifically evaluated the role of MOM on BPD. We found that MOM reduced the risk of developing BPD but only when used as an exclusive diet. In contrast, meta-analysis could not find significant changes in BPD risk when comparing infants fed mainly with MOM with those fed mainly with PF, or when comparing any MOM vs. exclusive PF.

The reduction of BPD rates when MOM is used as an exclusive diet may have various explanations. The major pathogenetic clue of BPD is the arrest in the alveologenesis and vasculogenesis of the lung due to very preterm birth (36).  Prenatally, in the setting of chorioamnionitis, the overwhelming inflammatory cascade may interfere with lung development (37). Postnatally, the intensive care support needed by very preterm infants, including resuscitation, mechanical ventilation, and oxygen administration, carries a high grade of inflammation to the immature lung, leading to the establishment of BPD (38). When postnatal infections occur, the incidence of BPD sharply increases (39–41). Finally, inadequate nutrition can further worsen BPD (42). MOM may reduce the incidence of BPD thanks to nutritional and bioactive components, counteracting oxidative stress (43), inflammation (44, 45), and nutritional flaws involved in the BPD pathogenesis (46, 47). In addition, MOM may also impact the risk of BPD indirectly by reducing the incidence of necrotizing enterocolitis (NEC) and late-onset sepsis (LOS).

Due to the observational character of the studies included in the meta-analysis, the MOM and PF groups may differ in a number of maternal and infant characteristics which may affect the development of BPD. Previous studies have shown associations between characteristics such as ethnicity, socioeconomic status, maternal education, pregnancy hypertensive disorders, smoking during pregnancy, GA, BW, infant sex, Apgar score, or respiratory distress syndrome, and rate of MOM feeding in preterm infants (32, 48–54). We evaluated one possible major confounder: difference in GA between groups. GA played a role in modifying the association

FIGURE 6 | Meta-analysis of Any MOM vs. Exclusive PF and risk of BPD. MOM, mother's own milk; PF, preterm formula; BPD, bronchopulmonary dysplasia; CI, confidence interval; MH, Mantel-Haenszel.

between MOM and BPD, as we have shown through metaregression and sub-group analyses. This is relevant since the incidence of all the complications of prematurity, including NEC, LOS, and BPD, is inversely related to GA. In studies which compared mainly MOM vs. mainly PF, the comparison that included the highest number of studies, meta-regression showed a significant correlation between difference in GA and the protective effect of MOM on BPD (**Figure 5**). In other words, in the studies where the mainly MOM group had a higher BPD risk, this group was also more premature than the mainly PF group. Interestingly, in the comparison where we found a significant positive result (exclusive MOM vs. any PF), the differences in GA between groups were small. This suggests that the protective effects of exclusive MOM are not affected by GA as confounder in this analysis.

Several studies have reported that the effects of human milk in reducing the incidence of adverse outcomes of prematurity are dose-dependent (14, 23, 31, 55–58). It has been suggested that at least 50% of the infant's total enteral intake should be MOM in order to achieve a decreased incidence of NEC (13). With regards to BPD, Patel et al. have shown a 9.5% reduction in the risk of BPD for each 10% increase in MOM received from birth to 36 weeks PMA. This may generate a reduction in BPD risk up to 63% when an exclusive MOM diet is compared with an exclusive PF diet (55). Surprisingly, the present meta-analysis could not demonstrate a different rate of BPD in infants fed exclusive MOM when compared with infants fed exclusive PF. However, this analysis was based on only three studies (**Figure 3**). Moreover, in one of the studies the infants in the PF group had a markedly higher GA (1.5 weeks) than the infants of the MOM group. To date, there are no exact limits set in the amount of MOM that would produce benefits in terms of BPD reduction (59). The studies that we analyzed documented a high variability of MOM amount in their study groups. Since the relation between MOM and BPD, may not be as direct as for NEC and LOS, it is possible that higher minimum amounts of MOM may be needed to detect significant differences. In addition, the conditions of storage and the use of fresh, refrigerated, frozen, or deep-frozen MOM may affect the antioxidant as well as other biological properties of MOM (60).

# CONCLUSION

Our data indicate that MOM may reduce the incidence of BPD when used as an exclusive diet, but this result needs to be interpreted with caution. We did not find the same difference in analyses with other dosages of MOM, which may be related to the high variability in the available studies and the dose-dependent beneficial effects of MOM. It may also be due to differences in GA between infants who receive MOM and infants who receive PF, which we found had modified the protective effects of MOM against BPD. Moreover, there may be other differences in infant and maternal characteristics that play a role and which we could not account for. Further studies, adequately powered to detect changes in BPD rates, and that adjust for the different characteristics of infants who receive MOM and PF are needed to confirm the beneficial effects of MOM on BPD.

# AUTHOR CONTRIBUTIONS

EV-M designed the study, performed the search, selected studies for inclusion, collected data, performed the statistical analyses, contributed to the interpretation of the results, and drafted the

## REFERENCES

initial manuscript. MP revised collected data, contributed to statistical analysis and interpretation of the results, and reviewed and revised the manuscript. GC contributed to interpretation of results and reviewed and revised the manuscript. FM contributed to interpretation of results and reviewed and revised the manuscript. EV conceptualized and designed the study, performed the search, selected the studies for inclusion, supervised data collection, contributed to the statistical analyses and interpretation of the results, and reviewed and revised the manuscript. All authors approved the final manuscript as submitted.

#### ACKNOWLEDGMENTS

We thank Paula M. Sisk for kindly providing additional clarification on their study.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped. 2019.00224/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Villamor-Martínez, Pierro, Cavallaro, Mosca and Villamor. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Human Milk–A Valuable Tool in the Early Days of Life of Premature Infants

#### Ekhard E. Ziegler 1,2 \*

*1 The University of Iowa, Iowa City, IA, United States, 2 Department of Pediatrics, University of Iowa, Iowa City, IA, United States*

The objective of early premature infant nutrition is to maintain, during the turbulent early days of life, a flow of nutrients that differs only minimally from that which would have prevailed had the infant remained *in utero*. Out of necessity, nutrients have at first to be provided mainly via the parenteral route. While that is going on, the feeding of small amounts of human milk (gut priming) is initiated as soon as practical. As mother's own milk is not available in sufficient quantity at this time, donor milk needs to be used temporarily. If not available, formula should be used. Gastric residuals are physiologic at this stage and are monitored to guide the increase of the size of feedings. As the volume of milk is gradually increased, nutrient fortification is initiated when the milk volume reaches around 20 ml/kg/day. There is no need to start with less than full-strength fortification. Fortification should employ one of the liquid fortifiers. Adjustable fortification may be employed but is labor-intensive and is not a necessity as long as full feeding volumes of around 170 ml/kg/day are maintained. As the infant grows beyond 1,500 g the level of fortification can be reduced gradually by omitting fortification first from one, and then from more feedings. After discharge there is still a need for fortification, which requires the mother to express some of her milk so it can be fortified. Nutrient supplementation directly to the infant would obviate the need for milk expression.

#### Edited by:

*Sertac Arslanoglu, Istanbul Medeniyet University, Turkey*

#### Reviewed by:

*Ulrich Herbert Thome, Leipzig University, Germany Guido Eugenio Moro, Italian Association of Donated Milk Banks (AIBLUD), Italy Carol L. Wagner, Medical University of South Carolina, United States*

# \*Correspondence:

*Ekhard E. Ziegler<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics*

Received: *30 June 2018* Accepted: *12 June 2019* Published: *09 July 2019*

#### Citation:

*Ziegler EE (2019) Human Milk–A Valuable Tool in the Early Days of Life of Premature Infants. Front. Pediatr. 7:266. doi: 10.3389/fped.2019.00266* Keywords: human milk, early, premature infant, parenteral nutrition, avoidance of nutrient deficit

# INTRODUCTION

There is no time in the premature infant's life that is more important than the first few days. It is a time when the provision of nutrients meets with technical difficulties and nutrient intakes frequently fall short of needs. It is a time when the rich flow of nutrients that has been supporting rapid growth and development in utero has just been cut off abruptly. The trauma of this disruption is intense and has severe consequences unless the flow of nutrients is restored promptly. Restoring the flow of nutrients is challenging, technically, and otherwise, and that is the reason why prompt restoration is so often not occurring. But this is a time of exquisite vulnerability to the lack of adequate nutrients and prompt restoration is mandatory. Failure to provide adequate amounts of protein and energy during this early period has serious consequences (1). Whether and to what extent nutrient deficits can be made up later is not known exactly, but the widespread occurrence of impaired cognitive development among former preterm infants suggests that the ability to make up is limited (2). Fortunately, we have the techniques for promptly restoring the flow of key nutrients. If nevertheless the restoration of nutrient intakes does not always occur in a timely fashion, it is because of misperceptions and misinterpretation of physiologic signs, such as gastric residuals. While restoration of the nutrient flow has absolute primacy, it is closely followed in urgency by the need to establish enteral feedings. This is where human milk with its unique properties plays a crucial role, a role that is not always fully utilized.

The benefits of human milk for the premature infant are well-established. They are of particular importance during the early days and weeks of life. The property of human milk that makes it so valuable at this time is its strong trophic effect on the immature gut. This maturational effect not only enables earlier establishment of full feedings, it also provides protection against necrotizing enterocolitis (NEC) and against late-onset sepsis. But for these effects to come to bear, human milk must be fed, and not be withheld, as is too often the case, for various reasons. That is why sound feeding practices are of such crucial importance during the early days of life. One essential condition is the acceptance of gastric residuals as what they are, namely mere manifestations of gut immaturity rather than as signs of "feeding intolerance" or, worse, "impending NEC." Gastric residuals eventually subside, the faster so if the necessary stimulation by feeding of human milk is provided. The only disadvantage of human milk is its low nutrient content relative to the high needs of the premature infant that must be overcome by nutrient fortification.

The objective of the present essay is to foster feeding practices that maximize the utilization of the beneficial effects of human milk. Good feeding practices are part of the overall effort to minimize the hazards that premature birth presents to the baby. The present discussion makes the assumption that parenteral nutrition is being provided starting within hours of birth, that it is providing adequate amounts of amino acids and energy, and that it is not terminated until enteral nutrition has reached near-full levels.

The opinions offered are derived largely from personal experience and anecdotal reports, but are overall consistent with the current literature.

To begin with, it is useful to review some basic facts about the preterm infant and to agree on some of the principles that govern nutrition of the premature infant:

# PARENTERAL NUTRITION

As the gut is at the outset unable to absorb nutrients, the provision of nutrients must of necessity be via the parenteral route, which has been shown to be effective and safe (3). Parenteral nutrition administered through an umbilical vessel is initiated within a few (preferably <2) hours of birth. It should provide protein/amino acids of at least 2.0 g/kg/day but preferably 3.0 g/kg/day. Blood glucose concentration must be monitored at regular intervals, and hyperglycemia and hypoglycemia, if present, must be treated appropriately. Monitoring of blood urea is not necessary, but monitoring of acid-base status is. After a few days, the administration of parenteral nutrition is shifted to a peripherally placed central venous catheter. As the volume of enteral feedings increases, parenteral nutrition is tapered while maintaining at all times an adequate total nutrient intake. Parenteral nutrition should not be discontinued prematurely as that practice leads to a temporary slowdown or cessation of growth. Parenteral nutrition should be continued until enteral feeds have reached at least 90% of full feedings.

# THE INITIATION OF FEEDINGS (GUT PRIMING)

The main reason for the common hesitation to provide enteral feedings during the early days of life is the frequent occurrence of gastric residuals. It is therefore, important to recognize that residuals are merely a manifestation of immaturity of the GI tract and have nothing to do with NEC. Immature motility needs to be overcome by GI priming, which is done better by human milk than by anything else. Until immaturity is overcome, full feedings are not possible. The manifestations of gut immaturity are physiologic and subside in time with proper management. The risk of NEC, while ever present, is not eliminated by withholding of feedings.

The sole purpose of providing enteral feedings early on, also known as trophic feedings or gut priming, is to facilitate maturation of the immature gastrointestinal tract. The progress of gut maturation is, for lack of a better parameter, judged by the decrease of the size of gastric residuals. The goal of gut priming is to reach a rate of gastric emptying that permits the administration of full or near-full feedings. Until that goal

is reached, parenteral nutrition must be continued to ensure an adequate supply of nutrients at all times. In practice, the initiation of feedings is often delayed by one or more days after birth due to the belief that the risk of NEC is thereby reduced. Practice for many years has shown that such a delay does not reduce the risk of NEC and only causes a delay in reaching full feeds. Thus, gut priming should always be initiated within a few hours of birth, at the latest within the first 24 h of life, and should not be interrupted due to the occurrence of gastric residuals.

There is no evidence that the frequency of early feedings or the strength of feedings are of importance. What is important is that gastric residuals are monitored because the rate of advancement of feeds needs to be based on the occurrence and size of gastric residuals. Historically, before the phenomenon of gut immaturity was fully appreciated, feedings were often advanced too rapidly, and the ensuing gastric residuals were interpreted as constituting NEC grade I (incipient NEC) and prompted the withholding of feedings for 24 h or longer. Today we know that gastric residuals are physiologic, as long as they are of modest size and of normal color, and may require a reduction in the size of feedings but not a cessation of feedings. There is no evidence that the volume of feedings or the rate of advancement of feedings impacts the risk of NEC.

The choice of feeding for GI priming is important because human milk offers several strong advantages, including that it enhances the maturation of the GI tract. Every effort must therefore be made to secure the mother's milk for gut priming. Freezing of milk entails loss of all cellular components and diminution of some of the valuable components of human milk. But the logistics of providing fresh milk are such that much milk is provided after a shorter or longer period of freezing.

Since the availability of mother's own milk is typically limited in the first few days after birth, the temporary (until mother's milk comes in) use of donor milk is necessary. Donor milk is pooled milk from multiple donors who are typically in advanced stages of lactation with its decreases of some components of breast milk. It is always frozen and heat treated, which causes a decrease of the content of nutrients and other components, and the complete elimination of components such as lipase. Despite these decreases, donor milk is the preferred feeding during the first few days after birth when mother's own milk is not available in sufficient quantity. It is also the preferred feeding whenever a mother experiences diminishing milk supply in spite of efforts to maintain her milk supply, which, unfortunately, is a rather common occurrence. If donor milk is not available, formula is a second choice for gut priming. NEC is extremely uncommon in the first few days of life and the feeding of formula temporarily is preferred over not giving any feedings at all.

## FORTIFICATION OF HUMAN MILK

Growth and nutrient needs of premature infants are shown in **Table 1**. Infants weighing <1,200 g require an intake of protein of around 4.0 g/kg/day in order to grow like the fetus. If human milk is fed at 150 ml/kg/day, it provides only about 2 g/kg/day of protein (except during the first 2 weeks of lactation when the protein content of human milk tends to be higher). The need for extra protein is thus evident. Although their requirements are not shown in **Table 1**, the intakes of electrolytes, minerals, and vitamins from human milk are similarly not adequate to cover the needs of the small premature infant. Most of the nutrients in short supply are provided by fortifiers. Powder fortifiers have been available for some time but were woefully inadequate mainly in the amount of protein they provide. If used, the addition of extra protein is mandatory (4). The newer liquid fortifiers provide more protein so that the total intake from fortified human milk approaches the required 4.0 g/kg/day.

# Initiation of Fortification

Fortification with a liquid multicomponent fortifier should be initiated when the feeding volume reaches around 25 ml/day, which has been the practice at the author's institution for many years. There is nothing gained by waiting any longer, nor is there any advantage from starting at less than full strength fortification. Concerns about an increase in osmolality of feedings caused by the fortifier are unfounded. It has been the practice in many NICU's to initiate fortification when feedings reach a volume of around 100 ml/kg/day. The origin of this practice is murky as is its rationale. The disadvantage from delaying fortification is a deficit in nutrient intakes and a delay in the achievement of a full nutrient intake. Whether the early addition of fortifier delays the attainment of full feedings is a matter of debate. Individualized fortification, if used, is usually initiated only after the first week of life. Fortification with one of the liquid fortifiers in standard fashion is recommended as individualized methods have not been shown to yield superior nutrient intakes.

# MONITORING OF GROWTH

As growth provides the ultimate proof of nutritional adequacy (or inadequacy), the monitoring of growth assumes central importance. It is complicated by the fact that all infants undergo a physiologic weight loss, which is not caused by nutritional inadequacy but which is due to constriction of the infant's body water after birth. The decrease of body water is somewhat variable but usually is equivalent to about −0.6 weight z-scores. A loss of 0.6 weight z-scores is therefore physiologic, but any greater weight loss is abnormal and is associated with a risk of neurocognitive impairment.

Daily weight is routinely monitored in every unit, and length and head circumference are measured periodically in most units. The problem lies in the interpretation of measurements. The calculation of average weight gain over some period of time is not an appropriate method because normative values decrease with increasing body weight and are generally not known. An acceptable way of interpreting growth measurements is to display them on charts of fetal growth, where growth in parallel to percentile lines is the expected norm (**Figure 1**). A fall-off from percentile lines indicates growth failure. The preferred way to interpret growth measurements is to transform them into zscores derived from current references of fetal growth (5) and to express them as deviations from the birth z-score (**Figure 2**). The weight z-score should not drop by more than 0.6, and if it does, every effort should be made to make up the deficit as soon as possible. The z-scores for length and head circumference should not drop at all.

The widespread notion that some degree of postnatal weight loss is unavoidable is slowly giving way to the notion that loss of more than water is avoidable and loss of water only should be the norm. Examples of avoidance of weight loss are provided by Senterre and Rigo (6).

TABLE 1 | Estimated protein intakes needed for weight gain like the fetus.

#### TAPERING OF FORTIFICATION

Human milk fortified with a liquid fortifier meets approximately the high nutrient needs of the infant weighing <1,500 g. As the infant grows beyond 1,500 g, this regimen provides a surfeit of nutrients that becomes larger as the infant grows larger. While no adverse consequences of the nutrient surfeit in the short run are known, it is counterintuitive to burden the infant with an excess of nutrients. An excess of a comparable size is not routinely provided to any other group of infants. And, if data in full term infants may be used as guidance, the provision of unduly high intakes of protein in early life has been shown to lead to an increase in adiposity and to a greater risk of obesity later in life (7). It is, of course, not known whether the same mechanism may be at work in premature infants, but prudence would suggest that an excess of protein ought to be avoided.

It is suggested that nutrient intakes be reduced as the infant grows. There are no established methods or schedules for the tapering of fortification. A decrease of nutrient intakes could be brought about by diminishing the amount of fortifier added to each feeding. Alternatively, omitting fortification from some feedings and not from others seems to be a simpler way of accomplishing tapering. A suggested schedule would be to omit fortification initially from one feeding per day, then from two feedings per day, and so forth, until at the time of discharge only about 30% of feedings are fortified.