Breast Milk: Influence of the Micro-transcriptome Profile on Atopy in Children and Toddlers
Conditions: Atopy, Atopic Dermatitis Eczema, Wheezing, Food Allergy in Infants
Sponsor: Milton S. Hershey Medical Center

TITLE PAGE 
Title: Association between infant consumption of breast milk microRNAs and atopy protection 
 
Authors: Steven D. Hicks, MD, PhD, Desirae Chandran, Kaitlyn Warren, MS, and Alexandra 
Confair, BS 
Author Affiliation: Department of Pediatrics, Penn State College of Medicine, Hershey PA, USA 
 
NCT#: ID: STUDY00008657 
Document Date: 1/26/26 
 
FUNDING 
This study was funded by a grant from the Gerber Foundation to SDH (Grant #204135). The 
funding source had no role in the study design, the collection, analysis or interpretation of data, 
the writing of the manuscript; or the decision to submit the article for publication. 
 
 
 
 
 

ABSTRACT 
Background: Breastfeeding is thought to reduce infant atopy risk. The biologic mechanism for 
this protective effect is not fully understood. 
Objectives: We tested the hypothesis that infant consumption of three breastmilk microRNAs 
(miR-146b-5p, miR-21-5p, miR-375-3p) would be associated with reduced atopy risk. 
Methods: The Breast Milk IMPACT Study involved a cohort of 221 breastfeeding mother-infant 
dyads planning to breastfeed beyond four months. Infant consumption of breastmilk microRNAs 
in the first six months was calculated as the product of breastmilk microRNA concentration and 
the number of breastmilk feeds, across three lactation stages: early milk (0-4 weeks), 
transitional milk (4-16 weeks), and mature milk (16-24 weeks). The primary outcome was infant 
atopy in the first 12 months, defined as atopic dermatitis, food allergies, or wheezing. 
Results: Seventy-three infants developed atopy – 41 atopic dermatitis (25%), 33 food allergy 
(20%), 10 wheezing (6%). Eleven developed more than 1 condition (7%). Infants who did not 
develop atopy consumed higher levels of breastmilk miR-375-3p (d= -0.33, p=0.007) and miR-
146b-5p (d= -0.28, p=0.037). Consumption of miR-375-3p was associated with reduced atopy 
risk (OR = 0.48, 95% CI: 0.23 – 0.98, p = 0.045). Breastmilk levels of miR-375-3p increased 
throughout lactation (F = 132.3, p = 8.4x10-34) and were inversely associated with maternal body 
mass index (t= -2.1, p=0.032). 
Conclusions: This study provides evidence that infant consumption of breastmilk miR-375-3p 
may reduce atopy risk. Such knowledge could be used to develop infant formulas that better 
reflect breastmilk biology, or to develop interventions to enhance breastmilk miR-375-3p levels.   
 
KEY MESSAGES  
• 
Infant consumption of miR-375-3p is associated with a two-fold reduction in atopy risk. 
• 
Increasing levels of miR-375-3p in maternal breastmilk, or adding miR-375-3p to infant 
formula may reduce the risk of allergic conditions. 
 
CAPSULE SUMMARY 
In this cohort study of 221 mother-infant dyads, consumption of breastmilk miR-375-3p was 
associated with a reduction in infant atopy. Breast milk miR-375-3p levels increased throughout 
lactation and were inversely related to maternal body mass.  
 
KEY WORDS: Breastfeeding, microRNA, miR-375, allergies, risk reduction, cohort, body mass 
 
ABBREVIATIONS: AD (Atopic dermatitis), ANOVA (Analysis of variance), BMI (Body mass 
index), DSQ (Dietary screener questionnaire), IFP (Infant feeding practices), IL (Interleukin), 
IMPACT (Influence of the micro-transcriptome profile on atopy in children over time), ISAAC-
WQ (International study of asthma and allergies in childhood – wheezing questionnaire), miRNA 
(microRNA), NSLAH (National survey of lead and allergens in housing). 

INTRODUCTION 
Atopy, the predisposition toward allergic diseases such as atopic dermatitis (AD), food allergies, 
and asthma, occurs in approximately one-third of children. (1) Atopy results from inappropriate 
activation of the immune response to benign environmental exposures. (2) The developmental 
origins of atopy are not fully understood. (3)  
Infants who breastfeed beyond three months may have lower risk for certain atopic conditions. 
(4) (5) Maternal breastmilk contains numerous immunomodulatory components that could 
convey atopy protection. (6) (7) Bioactive factors in breastmilk, called microRNAs (miRNAs), 
may play a role. (8) miRNAs are small, non-coding molecules that regulate gene expression 
across multiple tissues. (9) There are nearly 1,000 different miRNAs in breastmilk. (10) The 
majority are found in the lipid or cellular fractions of milk, (11) and evidence suggests that they 
regulate immune pathways (9) (12). Although breastmilk miRNA composition can vary based on 
maternal weight, diet, or genetics, (13)-(16) breastmilk reliably contains miRNAs, whereas 
formula does not. (14)   
miRNAs are often packaged within protective vesicles, (15) making them stable in breastmilk 
and transferable to the infant gut, where they may be absorbed and functionally incorporated by 
epithelial and immunologic cells in the oropharynx. (16)-(21) Animal studies suggest nutritional 
miRNA influences development of the immune system. (17) (18) Thus, breastmilk miRNA 
exposure may mitigate infant atopy risk.  
Circulating miRNA levels are dysregulated in atopic conditions. (19)-(28) For example, levels of 
miR-21 are elevated in the skin of patients with AD and in the bronchial cells of patients with 
asthma. (19) Up-regulation of miR-375 may prevent inflammatory pathways associated with 
allergic rhinitis. (20) Increases in miR-146 are associated with reduced inflammation in airway 
smooth muscle, and protection from asthma and AD. (21) (22) Notably, all three of these 
miRNAs have a robust presence in breastmilk. (23)  
This study, Breast Milk IMPACT (Influence of the Micro-transcriptome Profile on Atopy in 
Children over Time), followed 221 breastfeeding mother-infant dyads from birth to 12 months to 
test the hypothesis that infant consumption of three breastmilk miRNAs (miR-146b-5p, miR-21-
5p, miR-375-3p) would be associated with reduced atopy risk. To our knowledge, this is the first 
large-scale, prospective cohort study investigating the relationship between longitudinal 
breastmilk miRNA consumption and infant atopy. (24) 
 
 
 

METHODS 
This study was approved by the Independent Review Board at the Penn State College of 
Medicine (STUDY00008657). Written informed consent was obtained from all participant at 
enrollment. The study was registered on clinicaltrials.gov (NCT04017520).  
Participants 
This longitudinal cohort study involved a convenience sample of 221 mother-infant dyads 
followed from birth to 12 months of age. This sample size was determined by a-priori power 
analysis estimating that approximately 33% of infants (n = 73) would develop atopy and provide 
>80% power to detect a 1.5-fold difference in miRNA consumption across atopy and non-atopy 
groups on Mann Whitney U-testing (α = 0.05). Inclusion criteria were mothers who delivered at 
term (>35 weeks), and intended to breastfeed beyond four months. Exclusion criteria were: 1) 
maternal morbidities that could impact breastfeeding success or influence breastmilk miRNA 
composition (e.g. cancer, drug addiction, human immunodeficiency virus infection); 2) plan for 
infant adoption; 3) presence of neonatal condition that could impact ability to breastfeed (e.g. 
cleft lip, metabolic disease, NICU admission >7 days); 4) plan for pediatric care outside the 
medical center, or 5) non-English speaking. Between April, 2018 and October, 2020 research 
staff screened 2,487 potential participants through the electronic medical record, approached 
359 eligible participants (14%), and enrolled 221 participants (61% of those eligible). 
Recruitment occurred at four pediatric outpatient clinics affiliated with the academic medical 
center. All participants had access to on-site lactation support for the duration of the study. 163 
dyads completed the study (Figure 1). The primary medical outcome was presence or absence 
of atopy in the first 12 months, defined by parent report of AD, food allergies, or wheezing on 
standardized questionnaires, (25)-29) in accordance with guidelines from the American 
Academy of Allergy, Asthma, and Immunology. (26) Atopy cases were confirmed through review 
of the medical record. 
Survey collection 
Medical and demographic characteristics were collected for all dyads through nurse-
administered survey at enrollment. Maternal age, tobacco use, body mass index (BMI), and 
atopy history were recorded. Infant sex, gestational age, delivery route, birth weight, and race 
were recorded. Duration of breastfeeding and the proportion of feeds consisting of breastmilk 
were assessed at 4, 16, and 24 weeks using the Infant Feeding Practices (IFP)-II survey. (27) 
Infant AD was assessed by trained research nurses at 4, 16, 24 and 48 weeks using the 
Scoring Atopic Dermatitis (SCORAD) tool. (25) The SCORAD was developed by the European 
Task Force on Atopic Dermatitis for rapid utility in outpatient clinics and has been validated in 
infants and children. Infant food allergies were assessed through the IFP-II survey at 4, 16, 24, 
and 48 weeks. (27) Infant wheezing during the first 12 months was assessed through 
administration of a standardized wheezing questionnaire from the International Study of Asthma 
and Allergies in Childhood (ISAAC-WQ) at 48 weeks. (28) The ISAAQ-WQ was developed to 
measure the prevalence of recurrent wheezing and related risk factors in infants during the first 
12 months. Allergens in the infant environment were reported at 4 weeks using a modified form 
of the National Survey of Lead and Allergens in Housing (NSLAH). (29) This survey was 
developed by the National Institute of Environmental Health Sciences to assess environmental 
allergens within the home. In order to assess the relationship between breastmilk miRNAs and 
maternal diet, the Dietary Screener Questionnaire (DSQ) was administered at each breastmilk 

collection (0, 4, and 16 weeks). (30) Missing survey data (42/2608, 1.6%) was imputed using the 
mean cohort value. 
Sample collection 
Breastmilk (5 ml) was manually expressed into RNAse-free tubes from a sterilized nipple 
surface, as we have previously described. (13) (31) Foremilk (pre-feed) samples were collected 
in weeks 0, 4, and 16. These time points were chosen to reflect important changes in breastmilk 
miRNA levels that occur during the course of lactation, (23) and to capture the period when 
atopic benefits attributed to breastfeeding occur. (4) To control for differences between breasts, 
mothers utilized the same breast for each collection. Time of collection was recorded for all 
samples. The 163 mothers who completed the study contributed 442 breastmilk samples: 153 
“early milk” samples at 0 weeks (4 ± 2 days post-partum), 155 “transitional milk” samples at 4 
weeks (39 ± 11 days post-partum), and 134 “mature milk” samples at 16 weeks (128 ± 8 days 
post-partum). There were 127 mothers that contributed 3 samples, 25 mothers that contributed 
2 samples, and 11 mothers that contributed 1 sample. Samples were immediately transferred to 
-20° C for storage and placed at -80° C within four weeks, undergoing exactly one freeze-thaw 
cycle prior to RNA extraction. 
Sample processing 
Breastmilk RNA was purified from the lipid fraction using a Norgen Circulating and Exosomal 
RNA Purification Kit (Norgen Biotech; Ontario, Canada), as previously described. (13) (23) Lipid 
fractions were selected for their robust concentration of immunologic miRNAs with high potential 
for maternal-infant transfer (due to the protective effects of lipid encapsulation and micro-
vesicles). (10) (11) (13) Samples were processed in batches containing matched ratios of atopic 
and non-atopic dyads. RNA was sequenced at the SUNY Molecular Analysis Core using the 
Illumina TruSeq Small RNA Prep protocol and a NextSeq500 instrument (Illumina; San Diego, 
CA, United States) at a targeted depth of ten million, 50 base, paired-end reads per sample. 
RNAseq was selected to permit estimation of miRNA concentrations as parts per million, rather 
than a relative value yielded by polymerase chain reactions. FASTQ files were deposited into 
the Gene Expression Omnibus repository (GSE192543). Reads were aligned to the hg38 build 
of the human genome using Partek Flow (Partek; St. Louis, MO, United States) and the Bowtie2 
aligner. Samples with raw miRNA counts <10,000 were excluded (n = 10, 2.2%). Individual 
miRNA features with sparse counts (<10 in >10% of samples) were filtered. Within each 
sample, the concentration of the three miRNAs of interest (miR-146b-5p, miR-21-5p, miR-375-
3p) was determined as reads per million, and mean-center scaled.  
Statistical analysis 
The primary exposure for the study was infant miRNA consumption in the first six months. 
Consumption was calculated as the product of milk microRNA concentration and the number of 
breastmilk feeds, across three lactation stages. For example, consumption of miR-375-3p was 
calculated as follows: [miR-375T0] x DT0 x PT0 + [miR-375T4] x DT4 x PT4 + [miR-375T16] x DT16 x 
PT16; where T represents the time period (T0: 0-4 weeks, T4: 4-16 weeks, T16: 16-24 weeks), D 
represents the number of breastfeeding days during the time period, and P represents the 
proportion of feeds that included breast milk (eFigure 1). There were 24 missing samples (4% 
of all samples), due to collection failure (i.e., missed appointment, or COVID-related 
interruptions). For these samples, miRNA concentration was imputed using the mean miRNA 
value for the specific lactation stage.  

The primary medical outcome was presence or absence of atopy in the 12 months after 
delivery. Medical, demographic, and environmental characteristics were compared between 
atopic and non-atopic groups with chi-square or student’s t tests. Mann-Whitney U-tests were 
used to assess differences in breastmilk miRNA consumption between groups. Binomial 
regression with an analysis of variance (ANOVA) omnibus test was used to assess the 
contribution of breastmilk miRNA consumption to atopy risk, while controlling for relevant 
medical, demographic, and environmental characteristics. Co-linearity was assessed. Odds 
ratios with 95% confidence intervals were reported. 
The following secondary analyses were used to assess expression patterns for each miRNA 
candidate: 1) A Kruskal-Wallis test was used to investigate differences in breastmilk miRNA 
exposure between atopy sub-groups (e.g., AD, food allergy, wheezing); 2) A Friedman repeated 
measures ANOVA with post-hoc Durbin Conover pairwise comparisons was used to determine 
whether miRNA candidates displayed significant changes across the three stages of lactation; 
3) A mixed effects model was fit by restricted maximum likelihood to examine the impact of 
modifiable maternal characteristics (i.e., dietary scores, body mass index, and tobacco use) on 
breastmilk miRNA levels over time. Breastmilk miRNA concentration served as the dependent 
variable, participant ID was the clustering variable, and maternal characteristics served as 
covariates. Effects of maternal characteristics were assessed with fixed effects omnibus tests.      
 
 

RESULTS 
Participants  
Participating infants were predominantly female (92/163, 56%), Caucasian (132/163, 131/163, 
80%), and born via vaginal delivery (132/163, 81%) (Table 1). Nearly half had a history of 
maternal atopy (67/163, 41%), or atopy in a first-degree relative (74/163, 45%). Approximately 
one-third attended daycare (57/163, 35%). Most families reported pets in the home (99/163, 
60%), one-third reported local atmospheric pollution (56/163, 34%), and few reported employing 
allergen mitigation techniques at home (26/163, 16%). Most families introduced solids prior to 
six months of age (142/163, 87%) and breastfed for at least six months (132/163, 81%).   
Atopy characteristics   
Seventy-three infants developed atopy. AD was the most common atopic condition (41/163, 
25%), followed by food allergy (33/163, 20%), and wheezing (10/163, 6%). Eleven infants (6%) 
developed more than one atopic condition. The majority of atopic infants (42/73, 57%) 
experienced symptom onset after six months of age. There were no differences (p < 0.05) in 
maternal traits, infant traits, or environmental exposures between atopic (n = 73) and non-atopic 
(n = 90) infants.  
Breastmilk miRNA consumption 
Breastmilk samples collected from atopic and non-atopic dyads did not differ (p < 0.05) in 
collection time (12:39 ± 3:33 vs. 12:38 ± 3:07), total RNA counts (6.4 x 106 ± 5.0 x 105 vs. 7.1 x 
106 ± 6.0 X 105), or RNA quality (34.6 ± 0.1 vs. 34.6 ± 0.2). Infants who did not develop atopy 
consumed higher levels of breastmilk miR-375-3p (d = -0.33, p = 0.007) and miR-146b-5p (d = -
0.28, p = 0.037), but not miR-21-5p (d = 0.01, p = 0.21) (Figure 2A). Atopy subgroups displayed 
no difference in consumption of miR-375-3p (X2 = 8.07, p = 0.089), miR-146b-5p (X2 = 9.51, p = 
0.050), or miR-21-5p (X2 = 4.25, p = 0.37) (Figure 2B). Consumption of breastmilk miR-375-3p 
was associated with reduced atopy risk (Z = -2.0, p = 0.045, OR = 0.48, 95% CI: 0.23 – 0.98) 
(Figure 2C). Timing of solid food introduction (Z = 1.9, p = 0.048, OR = 3.16, 95% CI: 1.01 – 
9.94) was the only other factor associated with atopy risk (eTable 1).  
Factors impacting breastmilk miRNA levels 
All three candidate miRNAs displayed significant changes (p < 0.05) in breastmilk levels over 
the course of lactation (Figure 3A-C). Levels of miR-375-3p increased from early milk (0 weeks) 
to transitional milk (4 weeks), and remained elevated in mature milk (16 weeks; F = 132.3, p = 
8.4 x 10-34). In contrast, levels of miR-21-5p were highest in early milk (F = 23.7, p = 1.4 x 10-9), 
whereas miR-146b-5p levels peaked in transitional milk (F = 55.4, p = 4.5 x 10-14). Only miR-
375-3p displayed an interaction between lactation stage and atopy status (F = 6.5, p = 0.002). 
(Figure 3D). 
The relationship between breastmilk miRNA levels and modifiable maternal characteristics (i.e., 
tobacco use, diet, BMI) were assessed with linear mixed effects models (eTable 2). Levels of 
miR-375-3p were inversely associated with maternal BMI (F = 4.7, t = -2.1, p = 0.032) (Figure 
4A). Levels of miR-146b-5p were directly associated with maternal calcium intake (F = 5.3, t = 
2.3, p = 0.021) and inversely associated with maternal tobacco use (F = 6.8, t = -2.6, p =0.009) 
(Figure 4B-C). There were no associations between maternal traits and miR-21-5p levels. 

DISCUSSION 
This study is the first to demonstrate that infant consumption of breastmilk miRNA may provide 
protection against atopy development. Specifically, consumption of miR-375-3p over the first six 
months was associated with a two-fold risk reduction for AD, food allergies, and wheezing in the 
first year of life. Consumption of miR-375-3p was more strongly associated with atopy outcomes 
than oft-cited risk factors, such as family history and maternal tobacco use. (3) This knowledge 
has important applications for infant health, including the optimization of formula to better reflect 
the biologic characteristics of breastmilk.  
Currently, infant formula contains no human miRNA, (32) and what little bovine miRNA survives 
the pasteurization process is unlikely to have a bioactive impact on the developing infant. (33) 
Addition of synthetic miR-375-3p to formula might ameliorate the disparities in atopy outcomes 
reported between formula- and breast-fed infants, (4)-(7) In contrast to formula, miR-375-3p is 
present in over 99% of all breastmilk samples and constitutes just under 1% of all miRNA in 
breastmilk. (23)  
This study shows that breastmilk levels of miR-375-3p increase over the course of lactation, 
which may explain why sustained breastfeeding is important for atopy protection. (5) Intriguingly, 
miR-375-3p levels were lower in mothers with elevated BMI. This could explain previous 
findings linking maternal weight with infant AD and wheezing outcomes. (34) It could also 
provide an opportunity to enhance the atopic benefits associated with breastfeeding through 
targeted interventions aimed at increasing miR-375-3p levels via maternal weight control. (35)  
A growing body of literature suggests that orally administered exosomal miRNAs survive 
digestion, impact immunologic responses in local mucosa, and are readily absorbed into 
circulation. (8) (16) (21) (32) (36) These foundational investigations have predominantly relied on 
cell culture, animal models, or bovine milk consumption. The current study adds to this body of 
evidence by demonstrating that consumption of miR-375-3p in human milk is associated with 
reduced atopy risk.  
Several prior studies have established the importance of miR-375 in atopy pathophysiology. (21) 
(37)-(43) A study of cultured esophageal tissue from children with eosinophilic esophagitis found 
that miR-375 levels were repressed by interleukin (IL)-13, and that miR-375 was inversely 
related to the level of eosinophils and the expression of mast-cell-specific genes. (37) Another 
study employing a mouse model of allergic rhinitis showed that miR-375 expression was 
decreased in nasal mucosa, but administration of miR-375 could prevent epithelial inflammation 
by inhibiting IL-6. (21) Although the precise mechanisms connecting miR-375 with atopy are still 
being defined, it appears that frequent miR-375 exposure (through breastmilk ingestion) is a 
plausible explanation for reduced inflammation in the oropharynx, and reduced atopy risk.     
To our knowledge, only one prior study has investigated the relationship between breastmilk 
miRNAs and child atopy. (38) A retrospective study by Simpson and colleagues (2015) used 
RNA sequencing to measure breastmilk miRNA levels in mature milk from 54 women, and 
found no association with infant AD at 2 years of age. However, this important study established 
that atopy related miRNAs (i.e., miR-21-5p, miR-146b-5p, miR-375-3p) were highly present in 
breastmilk and formed the basis for our hypothesis-driven investigation. Our ability to detect a 
relationship between breastmilk miRNAs and infant atopy outcomes may be attributed to a 
larger sample size, longitudinal collection of milk samples at various lactation stages, inclusion 

of multiple atopic conditions, or a novel approach to control for total milk consumption in the first 
six months.   
There are, however, several limitations of this study. We did not include allergic rhinitis in the 
definition of atopy due to the limited prevalence of this condition among infants. Impacts of miR-
375-3p on allergic rhinitis or atopic outcomes beyond 12 months cannot be inferred. The study’s 
drop-out rate (24%) was exacerbated by the COVID-19 pandemic, and may have contributed 
attrition bias. Missing data (4%) was imputed using mean values from the entire cohort. Such an 
approach may enhance the likelihood of false-negative results. Despite the importance of 
exosomes in miRNA transport, (39) (32) we did not specifically isolate exosomal RNA. However, 
we note that the majority of miRNAs in breastmilk are contained within exosomes, (33) and that 
lipid fractions are likely to contain additional sources of encapsulated miRNAs that may survive 
the digestive tract. Finally, we recognize that our approach for estimating miRNA consumption 
assumes that total RNA content is relatively stable across milk samples. In the absence of 
technologies capable of measuring absolute miRNA levels, this assumption is unavoidable, but 
likely provides a more accurate estimate than approaches that ignore duration and frequency of 
breastfeeding completely.    
In conclusion, this study demonstrates that infant consumption of breastmilk miR-375-3p is 
associated with reduced atopy risk in the first year of life. Increases in miR-375-3p over the 
course of lactation support the idea that sustained breast feeding enhances atopy protection. 
Additional studies are necessary to confirm this important relationship and determine if oral 
administration of synthetic miR-375 conveys atopy protection in translational models. 
 
ACKNOWLEDGEMENTS 
SDH had full access to all the data in the study and takes responsibility for the integrity of the 
data and the accuracy of the data analysis. Data from this study has been made publicly 
available through the Gene Expression Omnibus repository (GSE192543).  
All authors made substantial contributions to design of the work, or the acquisition, analysis, or 
interpretation of data. All authors played a role drafting the work or revising it critically for 
intellectual content. All authors approve of the manuscript and agree to be accountable for all 
aspects of the work. 
The authors thank Faoud Ishmael, MD, PhD (Penn State College of Medicine, Hershey, PA) 
and Nicole Hackman, MD (Penn State College of Medicine, Hershey, PA) for assistance with 
study design. We wish to acknowledge Jessica Beiler, MS (Penn State College of Medicine, 
Hershey, PA) for assistance with data and sample acquisition. We thank Frank Middleton, PhD 
(SUNY Upstate Medical University, Syracuse, NY), Karen Gentile, MS (SUNY Upstate Medical 
University, Syracuse, NY), and Susan DiAngelo, BS (Penn State College of Medicine, Hershey, 
PA) for assistance with sample processing. 

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Table 1. Participant Characteristics 
 
All  
(n = 163) 
Atopy  
(n = 73) 
No Atopy 
(n = 90) 
P-val  
(d or X2) 
 
Maternal Traits 
 
 
 
 
Maternal age in years, mean (SD) 
30 (4) 
30 (4) 
30 (3) 
0.61 (0.5) 
Tobacco use, n (%) 
19 (11) 
7 (9) 
12 (13) 
0.45 (0.5) 
BMI in kg/m2, mean (SD) 
27.6 (6) 
28.1 (6) 
27.2 (6) 
0.50 (0.6) 
Maternal atopy, n (%) 
67 (41) 
35 (48) 
32 (36) 
0.11 (2.5) 
 
Infant Traits 
 
 
 
 
Female sex, n (%) 
92 (56) 
42 (57) 
50 (56) 
0.80 (0.06) 
Gest. age in weeks, mean (SD) 
39.0 (1) 
39.0 (1) 
38.9 (1) 
0.50 (0.1) 
Vaginal delivery, n (%) 
132 (81) 
56 (77) 
76 (84) 
0.21 (1.5) 
Birth weight in grams, mean (SD) 
3358 (442) 
3362 (440) 
3355 (447) 
0.35 (4.4) 
Caucasian, n (%) 
131 (80.4) 
55 (75) 
76 (84) 
0.88 (0.1) 
African American, n (%) 
7 (4.3) 
3 (5) 
4 (5) 
0.96 (0.04) 
Asian, n (%) 
8 (4.9) 
6 (8) 
2 (2) 
0.089 (1.7) 
Bi-racial, n (%) 
8 (4.9) 
5 (7) 
3 (3) 
0.26 (1.1) 
Other, n (%) 
9 (5.5) 
4 (5) 
5 (6) 
0.88 (0.1) 
Atopy in 1st degree relative, n (%) 
74 (45) 
37 (50) 
37 (41) 
0.22 (1.4) 
 
Environment 
 
 
 
 
Daycare attendance, n (%) 
57 (35) 
24 (33) 
33 (37) 
0.61 (0.2) 
Allergen mitigation, n (%) 
26 (16) 
13 (18) 
13 (14) 
0.56 (0.3) 
Pet(s) in home, n (%) 
99 (60) 
44 (60) 
55 (61) 
0.91 (0.01) 
Atmospheric pollution, n (%) 
56 (34) 
24 (33) 
32 (36) 
0.72 (0.1) 
Solid food intro. <6 months, n (%) 
142 (87) 
60 (82) 
82 (91) 
0.091 (2.8) 
Breastfeeding ≥6 months, n (%) 
132 (81) 
57 (78) 
75 (83) 
0.39 (0.7) 
 
Maternal atopy includes self-reported atopic dermatitis, food allergies, asthma, or allergic 
rhinitis. Daycare attendance includes any daycare in the first 12 months. Presence or absence 
of allergen mitigation techniques and household pet(s) was self-reported on the National Survey 
of Lead and Allergens in Housing. Atmospheric pollution was self-reported on the International 
Survey of Allergies and Asthma in Children – Wheezing Questionnaire. Abbreviations: Body 
mass index (BMI), Gestational (Gest.) 

FIGURE LEGENDS 
Figure 1. CONSORT Diagram 
There were 2,487 mother-infant dyads screened for eligibility, and 359 eligible dyads were 
approached. 221 dyads consented to participate, and 163 completed the 12 month longitudinal 
study. There were 73 infants who developed atopy – 41 had atopic dermatitis (AD), 33 had food 
allergy, and 10 had wheezing. Eleven infants had more than one atopic condition. In total, 442 
breastmilk samples were collected from the 163 participants and underwent RNA sequencing. 
Ten samples were excluded for insufficient microRNA read counts. This left 190 samples from 
mothers of atopic infants and 242 samples from mothers of non-atopic infants. Each mother 
provided up to three samples: at 0 weeks (“early milk”), 4 weeks (“transitional milk”) and 16 
weeks (“mature milk”).    
 
Figure 2. Increased consumption of breastmilk miR-375 is associated with reduced atopy risk 
The boxplots display average consumption of breastmilk miR-375-3p, miR-146b-5p, and miR-
21-5p for atopic (n = 73) and non-atopic (n = 90) infants (A). Mann-Whitney U testing revealed 
that infants without atopy consumed higher levels of miR-375-3p (d = -0.33, p = 0.007) and miR-
146b-5p (d = -0.28, p = 0.037). Across atopy sub-groups, there was no difference (p > 0.05) in 
consumption of miR-375-3p, miR-146b-5p, or miR-21-5p (B). Sub-groups were defined as 
atopic dermatitis (AD), food allergy (FA), wheezing (W), or infants with more than one atopic 
condition (>1). A binomial regression controlling for medical, demographic, and environmental 
risk factors related to infant atopy revealed that consumption of miR-375-3p was associated with 
reduced atopy risk (Z = -2.0, p = 0.045, OR = 0.48, 95% CI: 0.23 – 0.98) (C). The marginal 
means plot is shown with 95% CI. 
 
Figure 3. Breastmilk miRNA levels change throughout lactation  
The spaghetti plots display maternal breastmilk levels of miR-375-3p (A), miR-146b-5p (B), and 
miR-21-5p (C) across three lactation stages: early milk (0 weeks), transitional milk (4 weeks), 
and mature milk (16 weeks). Repeated measures ANOVA revealed a significant effect of 
lactation stage on miR-375-3p (F = 132.3, p = 8.4 x 10-34), miR-146b-5p (F = 55.4, p = 4.5 x 
10-14), and miR-21-5p (F = 23.7, p = 1.4 x 10-9). Only miR-375-3p displayed an interaction (F = 
6.5, p = 0.002) between lactation stage and atopy status (D). 
 
Figure 4. Breastmilk miRNA levels are impacted by maternal body mass, diet, and smoking 
Linear mixed effects models were used to assess the relationship between breastmilk miRNA 
levels and modifiable maternal traits over time. The scatter plot and trend line display an inverse 
relationship between maternal body mass index (BMI, kg/m2) and breastmilk miR-375-3p levels 
(F = 4.7, t = -2.1, p = 0.032) (A). Levels of miR-146b-5p were directly associated with maternal 
calcium intake (mg/day), as reported on the Dietary Screener Questionnaire (F = 5.3, t = 2.3, p 
= 0.021) (B). Whisker box plots display breastmilk miR-146b-5p levels for mothers who reported 
prior tobacco use and those who had never used tobacco (C). Milk miR-146b-5p level were 
higher among mothers who had never used tobacco (F = 6.8, t = -2.6, p =0.009).