Milk in Life Conditions (MiLC): Characterizing the Bacterial Composition of Human Milk Pumped and Stored in "Real-life" Conditions
Phase: NA
Conditions: Human Milk Microbiome, Bacterial Growth
Sponsor: Cornell University

Study Protocol 
ClinicalTrials.gov Study ID: NCT03123874 
 
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Title: Milk in Life Conditions (MiLC): Bacterial Composition of Human Milk Pumped and 
Stored in "Real-Life" Conditions 
 
ClinicalTrials.gov Study ID: NCT03123874 
 
Study Officials: 
 
Principal Investigators:  
 
Kathleen M. Rasmussen, ScD 
Cornell University 
 
Anthony G. Hay, PhD 
Cornell University 
 
Study Director: 
Sarah M. Reyes 
Cornell University 
 
Internal Review Board at Cornell University  
Unique Protocol ID: 1608006566.  
Approved on: May 2, 2017 
 
 

Study Protocol 
ClinicalTrials.gov Study ID: NCT03123874 
 
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Study Protocol 
Milk in Life Conditions (MiLC) Trial 
Background and Objective: Women use a wide range of practices for pumping their milk (1-4). 
The effects of these practices on the bacterial composition of the milk are unclear. The objective 
of the MiLC trial was to test the effect of women’s own pumping practices on the bacterial 
content of their milk compared to aseptic collection.  
Study Design: The MiLC trial will be a randomized, controlled cross-over trial of 2 breast pump 
set-ups: mother’s own and sterile. Mother’s own pump set-ups will consist of the mother’s own 
electric pump and own collection kits, cleaned at home using her usual practices. Sterile pump 
set-ups will consist of a multi-user electric pump (Medela Symphony) and a sterile collection kit. 
The pump each woman will use first will be randomized using stratified randomization based on 
infant feeding status (human milk only v. human milk and complementary foods).  
Recruitment: We will recruit women in Ithaca, NY and the surrounding area. Participants will 
be recruited on a continuous basis through flyer advertisements at pediatric clinics, day-care 
centers, graduate-student housing at Cornell University, and Ithaca College, in WIC offices, and 
in cafes and local children’s boutiques. Posters will briefly state the purpose of the study and that 
participation is voluntary and anonymous. We will also use snowball, or word-of-mouth, 
sampling, whereby recruited participants may refer other potential participants to the study. We 
will also advertise by word-of-mouth at La Leche League meetings. Written informed consent 
will be obtained according to the study protocol approved by the Institutional Review Board at 
Cornell University (Unique Protocol ID: IRB #: 1608006566). 
 
Inclusion/exclusion criteria: We will include lactating women over the age of 18 years who: i) 
self-report as healthy, ii) pump their milk with an electric pump, iii) are confident of their ability 
to donate 1 oz of milk from one breast during each of two consecutive pumping sessions where 
pumping sessions are 3 hours ± 30 minutes apart and between 0700-1100 hours, iv) are able to 
store their donated milk at home for 30 days, v) describe their infants as being healthy, and vi) 
have infants who do not consume formula or only consume formula episodically as long as the 
most recent formula-feeding occurred > 2 weeks before the day their mothers pump for this 
study. We will exclude women who: i) are not confident of their ability donate 1 oz of milk from 
one breast during each of two consecutive pumping sessions, 3 hours ± 30 minutes apart and 
between the hours of 0700-1100 hours, ii) have an infant who has consumed formula in the past 
2 weeks, iii) have current indication of breast infection (e.g., breast pain, discomfort, lumps, 
mastitis with fever, red streaks, or hard red portions of the breast), iv) have breast pain that the 
woman does not consider “normal” for lactation/breastfeeding, and v) self-reports that she or her 
infant showed signs/symptoms of acute Illness in the past 7 days including: fever (rectal or 
temporal temperature ≥99.5 ºF), dark green nasal discharge, diarrhea (abrupt onset of 3 or more 
excessively “loose” stools in one day), vomiting (where infant vomiting is not associated with 
feeding), or severe cough. 
 
Sample Collection: Lactating women (n=50) will donate 1 oz of pumped milk during each of 
two consecutive pumping sessions, for a total volume of 2 oz donated on one day. Women will 
be asked to fully express one breast during each pumping session. Women will pump once with 

Study Protocol 
ClinicalTrials.gov Study ID: NCT03123874 
 
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their own pumps and bottles and once with the sterile pump and sterile bottles (provided by the 
research team). Women will be randomized to which pump they will use first. Randomization 
will be done using stratified randomization by infant feeding status (human milk only vs. human 
milk and complementary foods). All pumping sessions will occur at participants' homes between 
0700 and 1100 hours. The second pumping session must begin 3 hours ± 30 minutes after the 
beginning of the first pumping session (e.g. the first pumping session at 7:30 am and the second 
at 10:30 am). Women will elect from which breast they donate their milk and that breast will be 
used for both pumping sessions. Women will be asked not to nurse from or pump that breast 
during the 2 hours before to the first pumping session and not until after the second pumping 
session (a total of ~5.5 hours). From the milk produced during each pumping session (which 
could be up to ~6 oz), we will collect 1 oz using a sterile, plastic syringe. Women will keep the 
remaining volume of milk. Each ounce of milk collected will be separated into 5 sterile 
containers that we will provide. Women will store donated milk at their home until it is picked 
up by a researcher 2, 4, and 30 days after pumping. 
 
Bacterial Composition of Human Milk: The bacterial content of milk will be assessed via 
aerobic culturing and high-throughput sequencing of the bacterial 16S rRNA gene from DNA 
extracted from the milk. Total aerobic bacterial counts will be reported as colony-forming unit 
(CFU)/mL estimated using standard methods. Sequence data from 16S rRNA analysis will be 
reported as bacterial community richness and diversity using observed taxa (amplicon sequence 
variants) and the Shannon diversity index, respectively.  
 
Sample size calculations: Inasmuch as the variability of our outcomes differs widely, we 
decided to base our sample size calculations on 16S rRNA data from our bacterial community 
analysis because these data are more variable than those from culture-related assays. However, 
there is not sufficient evidence on which to base sample size calculations. We are aware of one 
on-going multi-site study in which each site has 40 participants (5). We have sufficient resources 
to analyze the milk of 50 women. This sample size is also within the range of most culture-based 
investigations; thus, this will be our target sample size. 
 
Outcomes: 
• Bacterial Community Richness: Richness is the total number of different bacterial taxa 
(amplicon sequence variants) detected in the sample. This metric will be assessed on data 
derived from high-throughput sequencing of the bacterial 16S rRNA gene present in 
milk. 
• Bacterial Community Diversity: Bacterial community diversity will be assessed using 
the Shannon diversity index. The Shannon diversity index is a type of entropy measure 
and is a function of the distribution of the total number of organisms across all of the 
species. If S is the total number of species in the sample and pi is the number of 
organisms in the i-th species divided by the total number of organisms, then Diversity = 
−Σ pi log(pi). This metric will be assessed on data derived from high-throughput 
sequencing of the bacterial 16S rRNA gene present in milk. 
• Total Live Aerobic Bacterial Counts: Number of live total aerobic bacteria in milk 
assessed by aerobic culturing of milk on plate count agar. Reported as colony-forming 
units (CFU)/mL. 
 

Study Protocol 
ClinicalTrials.gov Study ID: NCT03123874 
 
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Statistical Analysis: Statistical analyses will be based on our primary hypothesis that the 
bacterial content of expressed human milk pumped with mothers’ own pump set-up differs from 
the bacterial content of expressed human milk pumped with a sterile pump set-up. To test this 
hypothesis, we will use linear mixed effects regression. Our 3 primary outcomes (e.g., bacterial 
counts, observed taxa, and Shannon diversity index) will serve as response variables in separate 
models. For all models, the fixed effect of interest will be pump set-up (own v. sterile pump set-
up). In addition, models will be adjusted for covariates relevant to our study design, namely 
infant feeding status (human milk feeding only v. human milk and complementary foods) and 
randomization schedule (which pump each woman used first). For all models, the random effect 
will be participant ID to account for multiple observations per participant. 
 
 

Study Protocol 
ClinicalTrials.gov Study ID: NCT03123874 
 
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References 
 
1. 
Felice JP, Geraghty SR, Quaglieri CW, Yamada R, Wong AJ, Rasmussen KM. 
"Breastfeeding" without baby: A longitudinal, qualitative investigation of how mothers 
perceive, feel about, and practice human milk expression. Matern Child Nutr 
2017;13(3):e12426. 
2. 
Felice JP, Geraghty SR, Quaglieri CW, Yamada R, Wong AJ, Rasmussen KM. 
"Breastfeeding" but not at the breast: Mothers' descriptions of providing pumped human 
milk to their infants via other containers and caregivers. Matern Child Nutr 
2017;13(3):e12425. 
3. 
Labiner-Wolfe J, Fein SB, Shealy KR, Wang C. Prevalence of breast milk expression and 
associated factors. Pediatrics 2008;122(suppl 2):S63-8. 
4. 
Labiner-Wolfe J, Fein SB. How US mothers store and handle their expressed breast milk. 
J Hum Lact 2013;29(1):54-8. 
5. 
McGuire MK, Meehan CL, McGuire MA, Williams JE, Foster J, Sellen DW, Kamau-
Mbuthia EW, Kamundia EW, Mbugua S, Moore SE, et al. What's normal? 
Oligosaccharide concentrations and profiles in milk produced by healthy women vary 
geographically. Am J Clin Nutr 2017;105(5):1086-100.