Original Investigation | Pediatrics

Breast Milk Enema and Meconium Evacuation Among Preterm Infants

A Randomized Clinical Trial
Liqiang Zheng, MD, PhD; Li Gai, MD; Yani Wu, BS; Chaonan Kong, BS; Fangli Sun, BS; Jinyue Gao, MD; Wei Yuan, MD; Min Liu, BS; Hong Jiang, BS;
Nan Tuo, MD, PhD; Fan Yang, MD

Abstract Key Points

Question Does breast milk enema
IMPORTANCE Delayed meconium evacuation and delayed achievement of full enteral feeding
facilitate meconium evacuation
among premature infants are associated with poor short- and long-term outcomes. Identifying a
compared with normal saline for
more effective and safer enema for meconium evacuation is imperative for improving neonatal care.
preterm infants?

OBJECTIVE To examine whether breast milk enemas can shorten the time to complete meconium Findings In this randomized clinical trial
evacuation and achievement of full enteral feeding for preterm infants. of 286 preterm infants with a
gestational age of 23 to 30 weeks, the
DESIGN, SETTING, AND PARTICIPANTS This randomized, open-label, parallel-group, single-center administration of breast milk enema
clinical trial was conducted from September 1, 2019, to September 30, 2022, among 286 preterm resulted in a reduction in the time to
infants with a gestational age of 23 to 30 weeks in the neonatal ward of the Shengjing Hospital of achieve complete meconium evacuation
China Medical University in Shenyang. and the time to achieve full enteral
feeding compared with normal saline.

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INTERVENTIONS Preterm infants were randomly assigned to receive either breast milk enemas or No safety issues emerged.
normal saline enemas 48 hours after birth.
Meaning The results of this trial
demonstrated that breast milk, as a new
MAIN OUTCOME AND MEASURES The primary outcomes were time to complete meconium
enema agent, exhibited both
evacuation and time to achieve full enteral feeding. Secondary outcomes were duration of
effectiveness and safety compared with
hospitalization, weight at discharge, and duration of total parenteral nutrition. Intention-to-treat and
normal saline.
per-protocol analyses were conducted.

RESULTS In total, 286 preterm infants (mean [SD] gestational age, 198.8 [7.9] days; 166 boys + Visual Abstract
[58.0%]) were eligible and included in this study. A total of 145 infants were randomized to the
normal saline group, and 141 were randomized to the breast milk group. The time to achieve
+ Supplemental content
Author affiliations and article information are
complete meconium evacuation was significantly shorter in the breast milk group than in the normal
listed at the end of this article.
saline group (–2.2 days; 95% CI, −3.2 to −1.2 days). The time to achieve full enteral feeding was also
significantly shorter in the breast milk group than in the normal saline group (−4.6 days; 95% CI, −8.0
to −1.2 days). The duration of total parenteral nutrition was significantly shorter in the breast milk
group than in the normal saline group (−4.6 days; 95% CI, −8.6 to −1.0 days). There were no clinically
notable differences in any other secondary or safety outcomes between the 2 groups.

CONCLUSIONS AND RELEVANCE In this randomized clinical trial testing the effects of breast milk
enema on meconium evacuation, breast milk reduced the time to achieve complete meconium
evacuation and achieve full enteral feeding for preterm infants with a gestational age of 23 to 30
weeks. Subgroup analyses highlight the need for tailored interventions based on gestational age
considerations.
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TRIAL REGISTRATION isrctn.org Identifier: ISRCTN17847514

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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

Introduction
For premature infants, establishing enteral feeding is a challenge in the neonatal intensive care
unit,1-3 with delayed full enteral feeding associated with poor short- and long-term prognoses.4-6 This
delay was related to the prolonged meconium excretion time caused by meconium retention7,8 due
to immature intestinal motility mechanisms and neurotransmitter systems. Meconium leads to
intestinal function blockage and gastrointestinal dysfunction.9-14 Furthermore, delayed meconium
excretion prolongs the total parenteral nutrition (TPN) and hospital stay and increases the risk of
infection. Studies have indicated that expediting meconium elimination can enhance feeding
tolerance, facilitating a quicker transition to enteral nutrition and its associated benefits.12
Previous studies have shown many methods for promoting meconium evacuation, including
abdominal massage, prophylactic rectal stimulation, oral contrast, and enema,12,15-18 of which
enemas are the most common in clinical practice. Commonly used enemas include saline and glycerin
solutions or suppositories. However, meta-analyses have shown that prophylactic glycerin
suppositories, small-volume glycerin or normal saline enemas, or oral osmotic contrast agents to
evacuate meconium did not reduce the time to reach full feeding in preterm infants.19,20
Furthermore, a case report described an infantile case of generalized urticaria caused by a glycerin
enema solution,21 and a randomized clinical trial (RCT) found that a contrast agent may increase the
risk of necrotizing enterocolitis (NEC).18 These findings led us to doubt the safety of glycerin and
contrast agents for the enemas of premature infants. Therefore, new enemas must be identified to
promote meconium evacuation among premature infants.
Human milk is an ideal source of nutrition and contains key immune-developing bioactive
ingredients.22 The osmotic pressure of breast milk is appropriate23 because it is a benign stimulus to

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the digestive tract of premature infants. To our knowledge, saline enemas are safer than enemas
with glycerin and contrast agents, and no adverse reactions have been reported. Therefore, this
study used normal saline enemas as the control to explore whether breast milk enemas can shorten
the time to complete meconium evacuation and reach full enteral feeding among premature infants.
To our knowledge, this is the first study to use breast milk as an enema for premature infants. This
RCT tested the hypothesis that, compared with normal saline, breast milk enema would shorten the
time to complete meconium evacuation and reach full enteral feeding among premature infants with
a gestational age of less than 30 weeks.

Methods
Study Design and Participants
This study was a randomized, open-label, parallel-group, single-center clinical trial conducted in the
neonatal ward of the Shengjing Hospital of China Medical University in Shenyang from September 1,
2019, to September 30, 2022 (trial protocol and statistical analysis plan in Supplement 1). Neonates
meeting the following criteria were enrolled: gestational age of less than 30 weeks and informed
consent form signed by the guardian. Neonates were excluded if they had congenital malformations,
congenital gastrointestinal anomalies, anorectal deformities, diarrhea, intussusception, NEC, patent
ductus arteriosus, sepsis, neutropenia, and coagulopathy. The trial protocol24 was approved by the
medical ethics committee of Shengjing Hospital, China Medical University. All relevant ethical
regulations were followed. This study followed the Consolidated Standards of Reporting Trials
(CONSORT) reporting guideline and statement.25
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Randomization and Blinding

Randomization was stratified by gestational age (23 weeks to <28 weeks, 28 weeks to <29 weeks,
and 29 weeks to <30 weeks). Computer-generated randomization was conducted by an uninvolved
statistician. The participants were then assigned to the intervention and control groups in a 1:1 ratio
using a randomized block design. Participants were randomly assigned using opaque, sealed

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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

envelopes. Health care professionals administering the enemas were not blinded because of the
nature of the intervention. However, the outcome assessors and statisticians were blinded to the
group assignments.

Procedures
Premature infants in the control and intervention groups received normal saline and breast milk
enemas, respectively. Nurses were trained to ensure the standardization and safety management of
enemas. The specific procedure was as follows:
1. Enema procedure: 48 hours after birth, preterm infants in the intervention and control groups
received enemas twice a day at 9 AM and 9 PM CM;0?>.
2. Material preparation: silicone tube (5 cm, model 3.33 mm [F10]), 5-mL syringe, thermostat
(temperature setting 37 °C), sesame oil, sterile gloves.
3. Implementation: the required volume of the enema (5 mL/kg) was calculated and preheated to 37
°C in a thermostatically controlled water bath. Preheated enemas were extracted using a syringe.
One end of the silicone tube was connected to a syringe, and the other end was lubricated with
sesame oil and then slowly and gently inserted through the anus. The enema was slowly injected
into the rectum for 3 minutes, and then the silicone tube was slowly removed.
Criteria for termination of the intervention included complete meconium evacuation,
discontinuation of the intervention for infants experiencing adverse events (AEs) deemed unsuitable
to proceed, or withdrawal of the guardian’s consent.
The feeding and parenteral nutrition procedures in the control and intervention groups are
detailed in the protocol.24 Other than the different enemas, there were no differences in treatment

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strategies or nursing methods between the control and intervention groups. The Trial Steering
Committee dynamically monitored the rate of loss to follow-up.

Outcomes
Two primary outcomes were assessed. The first was the time to achieve complete meconium
evacuation, defined as the time until the occurrence of yellow stool for 1 day, encompassing both the
complete evacuation of meconium and the transitional stool phase. The second primary outcome
was time to complete enteral feeding, defined as the number of days from birth to full enteral
feeding. More specifically, regarding full enteral feeding, criteria included the feeding volume of 180
mL/kg/d and weight gain exceeding 20 to 25 g/d within a 24-hour time frame. We accurately
recorded the time to the hour and subsequently converted it into days by dividing it by 24 hours. The
secondary outcomes were duration of hospitalization (in days), weight at the time of discharge, and
duration of TPN. Safety outcomes included the incidence of AEs, including retinopathy of
prematurity (any stage), intraventricular hemorrhage (grade 2), NEC, bronchopulmonary dysplasia,
late-onset sepsis, and colorectal and anal injuries, as well as death.

Data Collection, Management, and Quality Control

Trained researchers collected data using standardized questionnaires and measurements to ensure
accuracy. At baseline, the following information regarding the premature infants and their mothers
was collected: gestational age, birth weight, demographic information, history of disease, and
medications used during pregnancy. The following major aspects of outcome information were
collected during the follow-up: meconium evacuation, feeding, laboratory test results, and AEs. The
data were securely managed throughout the trial and reviewed by quality control experts. Reasons
were provided when data was modified. EpiData, version 3.1 (EpiData Association) statistical
software was used to build the database.
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Statistical Analysis
Based on our pilot study, the breast milk enema group exhibited complete meconium evacuation 1.4
days, 1.2 days, and 1.2 days earlier than the saline enema group for the infants aged 23 weeks to less

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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

than 28 weeks, 28 weeks to less than 29 weeks, and 29 weeks to less than 30 weeks, respectively.
With the use of a 2-sided significance level of .05, power of 0.8, and SD of 2, with a sampling ratio of
1 and factoring in a 20% dropout rate, the planned total sample sizes for infants aged 23 weeks to
less than 28 weeks, 28 weeks to less than 29 weeks, and 29 weeks to less than 30 weeks were 78,
108, and 108 preterm infants, respectively (PASS, version 15.0; NCSS Co). Intention-to-treat (ITT) and
per-protocol (PP) analyses were conducted. We present the per-protocol results in eTable 4,
eTable 5, and eTable 6 in Supplement 2. Descriptive statistics for all baseline characteristics are
reported for each group. Categorical variables are presented as counts and percentages, while
continuous variables are presented as mean (SD) or median (IQR) values. The Mann-Whitney test was
used to compare the 2 groups according to the intention-to-treat and per-protocol principles for the
primary and secondary outcomes. Outcomes are presented as median (IQR) values. For outcome
differences between the treatment and control arms, the median difference and 95% CI were
assessed using the Mann-Whitney test and the Hodges-Lehmann method. The primary outcomes
were analyzed using Kaplan-Meier curves and log-rank tests. Safety was assessed by the number of
AEs. Given that some infants discontinued due to AEs, sensitivity analyses were performed using
worst outcome imputation and last observation time in the Mann-Whitney test.
Statistical significance was set at a 2-tailed P < .05. Due to the 2 primary end points, a Bonferroni
correction was applied to our analyses of the primary outcomes. All statistical analyses were
performed using IBM SPSS Statistics for Windows, version 22.0 (IBM Corp) and R Studio, version 4.1.2
(R Project for Statistical Computing).

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Results
The recruitment and retention of infants are shown in Figure 1. Among the 433 preterm infants
initially screened, 12 parents declined to participate, and 135 infants met at least 1 exclusion criterion.
Ultimately, 286 preterm infants (mean [SD] gestational age, 198.8 [7.9] days; 166 boys [58.0%] and
120 girls [42.0%]) with a mean (SD) birth weight of 1115.5 (237.8) g were eligible and included in this
study (Table 1). A total of 145 infants (81 boys [55.9%] and 64 girls [44.1%]) were randomized to the
normal saline group and 141 to the breast milk group (85 boys [60.3%] and 56 girls [39.7%]). The
actual sample sizes at each gestational age were 78 for infants aged 23 to less than 28 weeks, 100 for
infants aged 28 to less than 29 weeks, and 108 for infants aged 29 to less than 30 weeks. In the
breast milk and normal saline groups, 22 and 12 preterm infants were lost to follow-up, respectively.
The sociodemographic characteristics, the clinical features of the mothers, birth
anthropometric measures, infant sex, birth type, Apgar score, and gestational age at birth in the
normal saline and breast milk groups are shown in Table 1. As shown in Table 2, the time to achieve
complete meconium evacuation was significantly shorter in the breast milk group than in the normal
saline group (11.4 days [IQR, 9.6-14.1 days] vs 13.7 [IQR, 10.6-16.9 days] days; difference, –2.2 days
[95% CI, −3.2 to −1.2 days]; P < .001). Subgroup analysis showed sustained significance in the age
groups of 28 to less than 29 weeks and 29 to less than 30 weeks (28 to <29 weeks: breast milk group,
12.5 days [IQR, 10.0-15.3 days]; normal saline group, 14.8 days [IQR, 11.5-17.9 days]; P = .008; 29 to
<30 weeks: breast milk group, 11.0 days [IQR, 8.8-13.7 days]; normal saline group, 13.7 days [IQR,
10.3-17.8 days]; P = .01). However, the advantage in the breast milk group was not statistically
significant for infants aged 23 to less than 28 weeks (breast milk, 10.7 days [IQR, 9.4-12.7 days] vs 12.8
days [IQR, 9.5-15.8 days]; P = .14). Figure 2A illustrates the distribution of the time to achieve
complete meconium evacuation according to the gestational age subgroups. The median time to
achieve complete meconium evacuation was calculated as the point at which 50% of the participants
accomplished this outcome. The breast milk group exhibited a significantly shorter median time to T.ME/NEONATOLOGY
complete meconium evacuation than the normal saline group (11.4 days [IQR, 9.6-14.1 days] vs 14.1
days [IQR, 10.7-17.6 days]; P < .001) (Figure 2B). This trend persisted among the infants aged 28 to
less than 29 weeks (median, 12.4 days [IQR, 9.7-15.1 days] vs 15.0 days [IQR, 11.8-18.0 days]; P = .01)
and among the infants aged 29 to less than 30 weeks (median, 11.0 days [IQR, 8.8-13.7 days] vs 13.7

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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

[IQR, 10.1-16.7 days] days; P = .01), although not among the infants aged 23 to less than 28 weeks
(Figure 2C).
The time to achieve full enteral feeding was also significantly shorter in the breast milk group
than in the normal saline group for all participants (29.5 days [IQR, 21.5-42.2 days] vs 35.4 days [IQR,
25.6-46.8 days]; P = .02) and for infants aged 23 to less than 28 weeks (31.6 days [IQR, 24.7-48.7
days] vs 46.5 days [IQR, 31.9-65.0 days]; P = .03) (Table 2). Breast milk enemas facilitated total
enteral feeding 4.6 days (95% CI, −8.0 to −1.2 days) earlier for all participants and 10.1 days (95% CI,
–18.6 to –1.7 days) earlier for infants aged 23 to less than 28 weeks. Among infants aged 28 to less
than 29 weeks, the intervention and control groups achieved full enteral feeding in 31.6 days (IQR,
24.9-42.8 days) and 35.5 days (IQR, 27.5-44.8 days), respectively (P = .69). Among infants aged 29
to less than 30 weeks, the intervention and control groups achieved full enteral feeding in 22.7 days
(IQR, 18.7-32.8 days) and 26.8 days (IQR, 21.8-36.7 days), respectively (P = .10). Although both
intervention groups experienced a significant reduction, the difference was not statistically
significant. However, the observed differences may still be clinically significant.
Violin plots in Figure 3A indicate the distribution of time to achieve full enteral feeding
according to the gestational age subgroups. Similar to previous analyses, for all participants, the
breast milk group exhibited a significantly shorter median time to achieve full enteral feeding
compared with the normal saline group (30.2 days [IQR, 21.6-42.8 days] vs 35.6 days [IQR, 25.7-47.5
days]; P = .02) (Figure 3B). This finding suggests that the use of breast milk enemas accelerates the
attainment of both complete meconium evacuation and full enteral feeding.

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Figure 1. Study Flowchart for Screening, Randomization, and Follow-Up

433 Participants assessed for eligibility

147 Excluded
12 Declined to participate
135 Met at least 1 exclusion criterion
14 Congenital cardiovascular anomalies
7 Congenital gastrointestinal anomalies
5 Anorectal deformities
16 Necrotizing enterocolitis
21 Shock
43 Sepsis
11 Neutropenia
18 Coagulopathy

286 Randomized according to gestational age

78 Aged 23 to <28 wk and randomized 100 Aged 28 to <29 wk and randomized 108 Aged 29 to <30 wk and randomized
(ITT population) (ITT population) (ITT population)

39 Received breast 39 Received normal 48 Received breast 52 Received normal 54 Received breast 54 Received normal
milk enema saline enema milk enema saline enema milk enema saline enema
(intervention group) (control group) (intervention group) (control group) (intervention group) (control group)

9 Intervention and
4 Intervention and 4 Intervention and 9 Intervention and
assessment not 4 Intervention and
completed assessment not assessment not 4 Intervention and assessment not
assessment not
completed completed assessment not completed
4 Withdrew completed
2 Withdrew 3 Withdrew completed 3 Withdrew
4 Discontinuation 3 Withdrew
2 Discontinuation 1 Discontinuation 4 Withdrew 6 Discontinuation
due to AEs 1 Death
due to AEs due to AEs due to AEs
1 Death

30 Included in 35 Included in 44 Included in 48 Included in 45 Included in 50 Included in

the per-protocol the per-protocol the per-protocol the per-protocol the per-protocol the per-protocol
analysis analysis analysis analysis analysis analysis

AE indicates adverse event; ITT, intention-to-treat.

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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

As for secondary outcomes, the duration of TPN was found to be significantly shorter in the
breast milk group than in the normal saline group for all participants (30.5 days [IQR, 21.8-42.8 days]
vs 35.8 days [IQR, 25.7-50.1 days]; difference, −4.6 days [95% CI, −8.6 to −1.0 days]; P = .01) and
among infants aged 23 to less than 28 weeks (31.5 days [IQR, 24.1-49.7 days] vs 47.5 days [IQR, 34.0-
65.0 days]; P = .01); however, no significant differences were observed among infants aged 28 to
less than 29 weeks and those aged 29 to less than 30 weeks (Table 2). Moreover, neither the total
number of participants nor subgroup analyses revealed statistically significant differences in duration
of hospitalization for infants or weight at discharge. The median duration of hospitalization for the
intervention and control groups were 62.0 days (IQR, 50.0-77.0 days) and 63.5 days (IQR, 50.0-77.8
days), respectively (P = .13). The median infant weight at discharge in the intervention and control
groups were 2270 g (IQR, 2020.0-2500.0 g) and 2240 g (IQR, 1990.0-2557.5 g), respectively
(P = .94).
In the safety analysis, no significant differences were observed between the intervention and
control groups across all safety outcomes, including bronchopulmonary dysplasia, late-onset sepsis,
retinopathy of prematurity, intraventricular hemorrhage, NEC, bloody stools, and mortality. These
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findings remained consistent in subgroup analyses, as presented in eTable 1 in Supplement 2.
However, all 4 cases of colorectal and anal injuries occurred in the breast milk group and were
confined to infants aged 29 to less than 30 weeks’ gestational age.
Sensitivity analyses were generally consistent with the main analysis (eTable 2 and eTable 3 in
Supplement 2). In addition to the intention-to-treat analysis, a reanalysis using the per-protocol
dataset yielded results consistent with the main findings, with no statistically significant disparities
(eTable 4, eTable 5, and eTable 6 in Supplement 2).

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Table 1. Baseline Characteristics of Infants and Their Mothersa
Infants aged 23 to <28 wk Infants aged 28 to <29 wk Infants aged 29 to <30 wk
All infants (N = 286) (n = 78) (n = 100) (n = 108)
Normal saline Breast milk Normal saline Breast milk Normal saline Breast milk Normal saline Breast milk
Characteristic (n = 145) (n = 141) (n = 39) (n = 39) (n = 52) (n = 48) (n = 54) (n = 54)
Maternal characteristics
Mother’s age, mean (SD), y 32.2 (4.5) 32.0 (5.0) 32.7 (4.5) 30.9 (4.8) 31.8 (4.2) 31.3 (4.1) 32.2 (4.8) 33.4 (5.6)
Educational level, No. (%)
Less than a high school 39 (26.9) 30 (21.3) 9 (23.1) 8 (20.5) 17 (32.7) 8 (16.7) 13 (24.1) 14 (25.9)
diploma
High school degree 30 (20.7) 36 (25.5) 10 (25.6) 8 (20.5) 10 (19.2) 15 (31.3) 10 (18.5) 13 (24.1)
Associate’s degree 30 (20.7) 26 (18.4) 7 (17.9) 6 (15.4) 11 (21.2) 9 (18.8) 12 (22.2) 11 (20.4)
Bachelor’s degree and above 46 (31.7) 49 (34.8) 13 (33.3) 17 (43.6) 14 (26.9) 16 (33.3) 19 (35.2) 16 (29.6)
Cesarean delivery, No. (%) 91 (62.8) 86 (61.0) 22 (56.4) 17 (43.6) 32 (61.5) 32 (66.7) 37 (68.5) 37 (68.5)
Pregnancies, median (IQR), No. 2.0 (1.0-3.0) 2.0 (1.0-3.0) 2.0 (1.0-3.0) 2.0 (1.0-3.0) 2.0 (1.0-2.0) 2.0 (1.0-3.0) 2.0 (1.0-3.0) 2.0 (1.0-3.0)
Parity, median (IQR), No. 1.0 (1.0-2.0) 1.0 (1.0-2.0) 1.0 (1.0-2.0) 1.0 (1.0-2.0) 1.0 (1.0-2.0) 1.0 (1.0-2.0) 1.0 (1.0-2.0) 1.5 (1.0-2.0)
Pregnancy-induced 37 (25.5) 37 (26.2) 8 (20.5) 8 (20.5) 12 (23.1) 18 (37.5) 17 (31.5) 11 (20.4)
hypertension, No. (%)
Diabetes mellitus, No. (%) 25 (17.2) 22 (15.6) 6 (15.4) 3 (7.7) 10 (19.2) 8 (18.8) 9 (16.7) 10 (18.5)
Medication use during 62 (42.8) 78 (55.3) 17 (43.6) 22 (56.4) 21 (40.4) 31 (64.6) 24 (44.4) 25 (46.3)
pregnancy, No. (%)
Infant characteristics
Male infants, No. (%) 81 (55.9) 85 (60.3) 13 (33.3) 14 (35.9) 24 (46.2) 31 (64.6) 31 (57.4) 29 (53.7)
Gestational age, mean (SD), d 198.6 (7.7) 198.9 (8.1) 189.5 (5.8) 188.1 (5.7) 198.8 (1.8) 199.3 (2.1) 205.8 (2.2) 206.3 (2.1)
Birth weight, mean (SD), g 1109.6 (219.9) 1121.5 (255.6) 953.9 (151.5) 928.2 (191.6) 1097.3 (207.2) 1119.8 (245.8) 1233.9 (199.6) 1262.6 (211.9)
Apgar score, median (IQR)
1 min 7.0 (6.0-8.0) 7.0 (6.0-8.0) 6.0 (4.0-7.0) 6.0 (5.0-7.0) 7.0 (6.0-8.0) 7.0 (6.0-8.0) 8.0 (6.0-9.0) 7.0 (6.0-8.0)
5 min 9.0 (8.0-9.0) 9.0 (8.0-9.0) 8.0 (7.0-9.0) 8.0 (8.0-9.0) 9.0 (8.0-9.0) 8.0 (8.0-9.0) 9.0 (8.0-9.0) 9.0 (8.0-9.0)
a
Data include all patients in the full analysis set, unless indicated otherwise.

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 Table 2. Effect of Breast Milk Enema on Primary and Secondary Outcomes Among Overall Participants and in Subgroup Analysesa

All infants Infants aged 23 to <28 wk Infants aged 28 to <29 wk Infants aged 29 to <30 wk
Normal Normal Breast Normal Normal
saline, Breast milk, Estimated saline, milk, Estimated saline, Breast milk, Estimated saline, Breast milk, Estimated
median median difference median median difference median median difference median median difference
Outcome (IQR) (IQR) (95% CI) P value (IQR) (IQR) (95% CI) P value (IQR) (IQR) (95% CI) P value (IQR) (IQR) (95% CI) P value
Time to achieve 13.7 (10.6 11.4 (9.6 −2.2 (−3.2 <.001 12.8 (9.5 10.7 (9.4 −1.8 (−3.5 .14 14.8 (11.5 12.5 (10.0 −2.4 (−4.0 .008 13.7 (10.3 11.0 (8.8 −2.4 (−4.0 .01
complete to 16.9) to 14.1) to −1.2) to 15.8) to 12.7) to 0.2) to 17.9) to 15.3) to −0.8) to 17.8) to 13.7) to −0.6)
meconium
evacuation, d
Time to achieve 35.4 (25.6 29.5 (21.5 −4.6 (−8.0 .02 46.5 (31.9 31.6 (24.7 −10.1 (−18.6 .03 35.5 (27.5 31.6 (24.9 −2.6 (−8.3 .69 26.8 (21.8 22.7 (18.7 −4.1 (−8.3 .10
full enteral to 46.8) to 42.2) to −1.2) to 65.0) to 48.7) to −1.7) to 44.8) to 42.8) to 2.9) to 36.7) to 32.8) to 0.1)
feeding, d
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Duration of TPN, d 35.8 (25.7 30.5 (21.8 −4.6 (−8.2 .01 47.5 (34.0 31.5 (24.1 −10.4 (−19.1 .01 35.5 (27.4 32.9 (24.9 −1.5 (−7.5 .60 27.6 (21.7 22.9 (18.8 −4.3 (−9.0 .06
to 50.1) to 42.8) to −1.0) to 65.0) to 49.7) to −2.2) to 45.0) to 42.8) to 4.1) to 39.6) to 36.0) to 0.2)
Hospitalization 63.5 (50.0 62.0 (50.0 −4.0 (−10.0 .13 81.0 (68.0 84.0 (73.0 −5.0 (−17.0 .41 65.0 (50.0 64.0 (53.0 −3.0 (−10.0 .51 51.0 (44.8 50.0 (42.5 −3.0 (−9.0 .27
for infant, d to 77.8) to 77.0) to 1.0) to 93.0) to 94.0) to 6.0) to 76.0) to 75.0) to 4.0) to 60.8) to 61.0) to 3.0)
Weight at 2240.0 2270.0 0.0 (−90.0 .94 2550.0 2400.0 −160.0 .20 2140.0 2240.0 55.0 .48 2165.0 2230.0 60.0 .28

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discharge, g (1990.0 (2020.0 to 90.0) (2220.0 (2100.0 (−410.0 (1940.0 (2010.0 (−110.0 (1957.5 (2015.0 (−60.0 to
to 2557.5) to 2500.0) to 2850.0) to 2840.0) to 80.0) to 2440.0) to 2525.0) to 210.0) to 2365.0) to 2400.0) 170.0)

Abbreviation: TPN, total parenteral nutrition.

a
The median difference and 95% CIs were assessed using the Mann-Whitney test and the Hodges-
Lehmann method.

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T.ME/NEONATOLOGY

April 22, 2024

Breast Milk Enema and Meconium Evacuation Among Preterm Infants

7/13
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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

Discussion
This RCT compared the administration of breast milk enemas with that of normal saline enemas
among preterm infants and revealed significant benefits. The breast milk group exhibited a shorter
time to achieve complete meconium evacuation, faster attainment of full enteral feeding, and a
reduced duration of TPN. Various methods have been used in routine neonatal care to promote
meconium evacuation; however, a literature review indicates that there is no consensus on the
agents used and the frequency of applications. To our knowledge, this is the first study to use breast
milk as an enema agent, representing a novel approach.

Figure 2. Time to Achieve Complete Meconium Evacuation

Group
Breast milk Normal saline

A Time to achieve complete meconium evacuation B Event rates for achieving complete meconium evacuation
50 1.0

40 0.8
Time to achieve complete

Cumulative event rate, %

meconium evacuation, d

30 0.6

20 0.4

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Breast milk,
day 11.4
10 0.2
Normal saline, day 14.1
P <.001
0 0

23 to <28 28 to <29 29 to <30 0 10 20 30 40

Gestational age, wk Time, d
No. at risk
Breast milk 141 90 5 2 1
Normal saline 145 116 18 2 0

C Complete meconium evacuation by age group

23 to <28 wk 28 to <29 wk 29 to <30 wk
1.0 1.0 1.0

0.8 0.8 0.8

Cumulative event rate, %

Cumulative event rate, %

Cumulative event rate, %

0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0 0 0
0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
Time, d Time, d Time, d

A, Violin plots represent the entire range of the time to achieve complete meconium evacuation across 3 age subgroups for both the intervention and control groups. The dots
represent median values, and lines represent the first and third quartiles. The results were as follows for the breast milk vs normal saline groups: median, 10.7 days (IQR, 9.4-12.7 days)
vs 12.8 days (IQR, 9.5-15.8 days) for infants aged 23 to less than 28 weeks; P = .14; median, 12.5 days (IQR, 10.0-15.3 days) vs 14.8 days (IQR, 11.5-17.9) for infants aged 28 to less than
29 weeks; P = .008; and median, 11.0 days (IQR, 8.8-13.7 days) vs 13.7 (IQR, 10.3-17.8 days) for infants aged 29 to less than 30 weeks; P = .01. B, Kaplan-Meier estimates for event rates
for achieving complete meconium evacuation in the intervention and control groups for all participants. The dotted horizontal line represents an event rate of 50%. The dotted
vertical lines represent specific time points. The shaded areas represent 95% CIs. The breast milk group exhibited a significantly shorter median time to achieve complete meconium
evacuation compared with the normal saline group (11.4 vs 14.1 days; P < .001). C, Subgroup analysis revealed that the breast milk group achieved complete meconium evacuation
significantly faster than the normal saline group among infants aged 28 to less than 29 weeks (median, 12.4 vs 15.0 days; P = .01) and those aged 29 to less than 30 weeks (median,
11.0 vs 13.7 days; P = .01). However, there was no significant difference among infants aged 23 to less than 28 weeks (median, 10.8 vs 12.8 days; P = .22).

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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

Our findings differed from a prior investigation by de Pipaón Marcos et al,1 which used rectal
stimulation and normal saline enemas. Varied definitions of complete meconium evacuation
contributed to a longer observed duration in our study than in the study by de Pipaón Marcos et al1
(10.7 vs 9 days). Specifically, we defined this period as the time until the occurrence of 1 day of yellow
stool, covering both complete evacuation of the meconium and transition stools. Our study also T.ME/NEONATOLOGY
deviates from the findings by de Pipaón Marcos et al1 regarding the time to achieve full enteral
feeding (31.6 vs 16 days). Discrepancies may arise from variations in the definition of full enteral
feeding; we used 180 mL/kg/d and weight gain more than 20 to 25 g/d within 24 hours, while the
criteria in the study by de Pipaón Marcos et al1 were 120 mL/kg/d for full enteral feeding.

Figure 3. Time to Achieve Full Enteral Feeding

Group
Breast milk Normal saline

A Time to achieve full enteral feeding B Event rates for achieving full enteral feeding
150 1.0
Time to achieve full enteral feeding, d

0.8
Cumulative event rate, %
100
0.6

Breast milk,
0.4

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day 30.2
50

0.2
Normal saline, day 35.6
P =.02
0 0

23 to <28 28 to <29 29 to <30 0 25 50 75 100 125

Gestational age, wk Time, d
No. at risk
Breast milk 141 78 13 3 2 1
Normal saline 145 106 33 8 3 0

C Full enteral feeding by age group

23 to <28 wk 28 to <29 wk 29 to <30 wk
1.0 1.0 1.0

0.8 0.8 0.8

Cumulative event rate, %

Cumulative event rate, %

Cumulative event rate, %

0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0 0 0
0 25 50 75 100 125 0 25 50 75 100 125 0 25 50 75 100 125
Time, d Time, d Time, d

A, Violin plots represent the entire range of the time to achieve full enteral feeding across 3 age subgroups for both the intervention and control groups. The dots represent median
values, and lines represent the first and third quartiles. The results were as follows for the breast milk vs normal saline groups: median, 31.6 days (IQR, 24.7-48.7 days) vs 46.5 days
(IQR, 31.9-65.0 days) for infants aged 23 to less than 28 weeks; P = .03; median 31.6 days (IQR, 24.9-42.8 days) vs 35.5 days (IQR, 27.5-44.8 days) for infants aged 28 to less than 29
weeks; P = .69; and median, 22.7 days (IQR, 18.7-32.8 days) vs 26.8 days (IQR, 21.8-36.7 days) for infants aged 29 to less than 30 weeks; P = .10. B, Kaplan-Meier estimates for event
rates for achieving full enteral feeding in the intervention and control groups for all participants. The dotted horizontal line represents an event rate of 50%. The dotted vertical lines
represent specific time points. The shaded areas represent 95% CIs. The breast milk group exhibited a significantly shorter median time to achieve full enteral feeding compared with
the normal saline group (30.2 vs 35.6 days; P = .02). C, Subgroup analysis revealed no significant difference among the 3 subgroups. The results were as follows for the breast milk vs
normal saline groups: median, 31.6 vs 46.5 days for infants aged 23 to less than 28 weeks; P = .12; median, 31.8 vs 36.6 days for infants aged 28 to less than 29 weeks; P = .46; and
median, 23.6 vs 26.9 days for infants aged 29 to less than 30 weeks; P = .24.

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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

Furthermore, our feeding approach was cautious, whereby feeding was halted when the residual
gastric volume surpassed half of the feeding volume. This strategy was guided by a comprehensive
evaluation26 that highlighted gastric residual volume as the predominant factor impeding the
progression of feeding. Another study27 further supported this perspective and indicated that
routine evaluation of residual gastric volume prolonged the attainment of full enteral feeding and the
duration of TPN.
Subgroup analyses discerned nuanced differences in the intervention’s efficacy among different
gestational age subgroups, providing a more detailed insight into its effect. Primarily, the time to
achieve complete meconium evacuation showed statistical significance only among infants aged 28
to less than 29 weeks and those aged 29 to less than 30 weeks. This discrepancy may stem from the
heterogeneous developmental trajectories in the gastrointestinal milieu of preterm neonates.28 For
the relatively more mature subgroup of infants aged 28 to less than 30 weeks, breast milk enemas
are postulated to augment gastrointestinal motility, expediting meconium evacuation. Conversely,
neonates aged 23 to less than 28 weeks might require a more nuanced and tailored stimulus given
their relatively nascent gastrointestinal milieu. This finding emphasizes the need to consider the
effectiveness of the intervention, considering the distinctive physiological variations present among
preterm infants across different gestational age categories. Conversely, the time to achieve full T.ME/NEONATOLOGY
enteral feeding followed a different trajectory. A significant intervention effect was found only
among infants aged 23 to less than 28 weeks. This differential response could reflect the disparate
effect of the intervention on feeding tolerance and gastrointestinal maturation across diverse
gestational age cohorts.29 Among infants aged 23 to less than 28 weeks, breast milk enemas likely
enhanced feeding tolerance by promoting gastrointestinal motility and augmenting maturation for a

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quicker transition to comprehensive enteral nutrition. However, among infants aged 28 to less than
30 weeks, for whom gastrointestinal functionality approached maturation, the effects of the
intervention may have dwindled.
Human milk is a complex biological substance containing not only macronutrients and
micronutrients but also living cells, growth factors, and immunoprotective substances.30,31 Many of
these factors are resistant to digestive enzymes in the infant gastrointestinal tract and are biologically
active on the mucosal surfaces, stimulating gastrointestinal growth and motility. Breast milk is a
natural oil-in-water emulsion,32 which can soften meconium to a certain extent and lubricate and
stimulate the intestinal wall, making it easier to excrete the meconium. Simultaneously,
oligosaccharides in human breast milk can promote the colonization of probiotics in the intestinal
tract of newborns,33 contribute to the integrity of the gastrointestinal epithelial barrier in preterm
infants, promote intestinal maturation,34 enhance feeding tolerance, and achieve total enteral
feeding as soon as possible. Although evidence suggests that breast milk can improve the immunity
of preterm infants,35 there was no significant difference in most safety outcomes, except colorectal
and anal injuries between the normal saline and breast milk groups in all age subgroups.
Consequently, the confirmed safety of breast milk enemas remains noteworthy. Future studies are
warranted to further explore and highlight the necessity for continued safety monitoring.

Strengths and Limitations

Our study has several strengths. It stands as the first RCT to investigate the efficacy and safety of
breast milk as an enema agent, providing robust support for this novel approach. The use of an RCT
design enhances objectivity and credibility in drawing conclusions. In addition, this study
innovatively categorizes preterm infants according to gestational age. Furthermore, to our
knowledge, our sample size on facilitating meconium excretion is the largest to date, allowing for
stratified analyses by gestational age and thereby augmenting statistical power.
However, our study also has some limitations. First, stratified analysis based on birth weight was
not conducted. Nonetheless, the results from the stratified analysis based on gestational age
remained consistent with the overall analysis. Second, this study was limited to a single center,
thereby reducing the generalizability of its findings compared with multicenter studies.

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 JAMA Network Open | Pediatrics Breast Milk Enema and Meconium Evacuation Among Preterm Infants

Conclusion
This RCT found that, in comparison with normal saline enemas, breast milk enemas have been
demonstrated to be both effective and safe, resulting in a reduction in the time to achieve complete
meconium evacuation, time to achieve full enteral feeding, and duration of TPN among preterm
infants. Subgroup analyses highlight the complex nature of neonatal development, emphasizing the
need for tailored interventions based on gestational age considerations. To corroborate our findings
further, a multicenter RCT is crucial, providing broader insights into the potential benefits of breast
milk enemas for preterm infant care.

ARTICLE INFORMATION
Accepted for Publication: February 20, 2024.
Published: April 22, 2024. doi:10.1001/jamanetworkopen.2024.7145
Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2024 Zheng L
et al. JAMA Network Open.
Corresponding Authors: Liqiang Zheng, MD, PhD, Clinical Research Centre, The International Peace Maternity and
Child Health Hospital, Shanghai Jiao Tong University School of Medicine, No.910 Hengshan Road, Xuhui District,
Shanghai 200030, China (liqiangzheng@126.com); Fan Yang, MD, Department of Nursing, Shengjing Hospital of
China Medical University, Shenyang, Liaoning Province, China (yangfan@sj-hospital.org).
Author Affiliations: Clinical Research Centre, The International Peace Maternity and Child Health Hospital,
Shanghai Jiao Tong University School of Medicine, Shanghai, China (Zheng, Tuo); MOE-Shanghai Key Laboratory
of Children’s Environmental Health, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of

t.me/neonatology
Medicine, Shanghai, China (Zheng); School of Public Health, Shanghai Jiao Tong University School of Medicine,
Shanghai, China (Zheng, Wu); Department of Pediatrics, Shengjing Hospital of China Medical University, Shenyang,
Liaoning Province, China (Gai, Kong, Sun, Jiang, Yang); Department of Nursing, Shengjing Hospital of China
Medical University, Shenyang, Liaoning Province, China (Gai, Kong, Sun, Jiang, Yang); School of Public Health,
China Medical University, Shenyang, China (Gao, Yuan, Liu).
T.ME/NEONATOLOGY
Author Contributions: Dr Zheng had full access to all of the data in the study and takes responsibility for the
integrity of the data and the accuracy of the data analysis. Dr Zheng, Ms Gai, and Ms Wu made equal contributions
to this manuscript and should be considered joint first authors.
Concept and design: Zheng, Gai, Jiang, Tuo, Yang.
Acquisition, analysis, or interpretation of data: Zheng, Gai, Wu, Kong, Sun, Gao, Yuan, Liu.
Drafting of the manuscript: Zheng, Gai, Wu, Yuan, Liu.
Critical review of the manuscript for important intellectual content: Zheng, Gai, Kong, Sun, Gao, Jiang, Tuo, Yang.
Statistical analysis: Wu.
Administrative, technical, or material support: Gai, Kong, Sun, Jiang.
Supervision: Gai, Yuan, Liu, Jiang, Tuo, Yang.
Conflict of Interest Disclosures: None reported.
Data Sharing Statement: See Supplement 3.

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2022.849012

SUPPLEMENT 1.
Trial Protocol and Statistical Analysis Plan

SUPPLEMENT 2.
eTable 1. Safety Outcomes Among Overall Participants and in Subgroup Analyses for the ITT Set
eTable 2. Sensitivity Analyses on Primary Outcomes
eTable 3. Adverse Events Leading to Discontinuation Among All Participants
eTable 4. Baseline Characteristics of Infants and Their Mothers
eTable 5. Effect of Breast Milk Enema on Primary and Secondary Outcomes Among Overall Participants and in

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Subgroup Analyses
eTable 6. Safety Outcomes Among Overall Participants and in Subgroup Analyses for the PP Set

SUPPLEMENT 3.
Data Sharing Statement

T.ME/NEONATOLOGY

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