Very Preterm Gestation and Breastmilk Cytokine Content During the First Month of Lactation

Abstract

Objective:

High susceptibility to infection and deficient cytokine production in newborns make breastmilk beneficial, especially for very preterm-born infants. This study was conducted to identify the association between very preterm gestation and levels of pro- and anti-inflammatory cytokines in transitional and mature breastmilk during the first month of lactation after adjustment for mode of delivery and parity.

Study Design:

Four samples of breastmilk (6–8, 13–15, 20–22, and 27–29 days) were collected from each mother after term or very preterm delivery at gestational age of less than 32 weeks. We measured the concentration of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-13, and tumor necrosis factor α (TNF-α) in the milk samples using a multiplex biometric immunoassay.

Results:

Fifteen mothers of very preterm infants and five term-born infants provided 80 samples of breastmilk. We found that delivery at very preterm gestation was linked with lower IL-8 (687 ± 208 pg/dL, p < 0.003) and higher IL-10 (27.8 ± 14.2 pg/dL, p < 0.01) levels in transitional breastmilk. Significantly decreased IL-8 levels were also seen in mature preterm milk samples. Cesarean delivery was associated with increased breastmilk TNF-α and IL-6 (p < 0.01–0.05), but gestational age was not.

Conclusions:

Very preterm gestation does not substantially impact the cytokine content of breastmilk during the first month of lactation, which can be beneficial for the regional and systemic immune response of the very preterm infant with confirmed impairment of innate and adaptive immunity.

Introduction

The physiological role of breastfeeding is correlated with the availability of definitive factors in breastmilk that can be important for the infant's innate immune response during the challenging period of immunological adaptation to extrauterine life.^1,2 Infants born at very preterm gestation are the most vulnerable group among the neonatal population in terms of both high risk for infection-related pathology³ and immune system deficiency.⁴ Among the various components available in human milk are cytokines with essential multifunctional immune activity.^5–7 High susceptibility to infection and deficient cytokine production in newborns^3,4,8,9 make breastmilk beneficial, especially for the very preterm-born infants. Although several studies^10–14 have investigated the relationship between gestational age and cytokine production, knowledge regarding the capability of breastmilk from mothers after very preterm delivery to provide at least the same quantity of cytokines as term milk is incomplete. Different investigators have reported lower,¹² higher,¹⁰ or similar^10,11,14 levels of tested cytokines at different stages of lactation in milk from mothers of preterm-born infants. The present study is the first that compares the concentrations of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-13, and tumor necrosis factor α (TNF-α) in transitional and mature milk from mothers who delivered at term and less than 32 weeks preterm of gestation. In addition, the mode of delivery and parity were included to identify the association between very preterm gestation and breastmilk cytokine content. We believe that this study will improve knowledge regarding the role of gestational age in breastmilk immune composition, which is important for the development of feeding strategies for the most critical population of very preterm-born infants.

Materials and Methods

This study was approved by the Institutional Review Board at Robert Wood Johnson Medical School, New Brunswick, NJ. A signed informed consent form was required for participation in this study. Mothers of any race/ethnicity, age, and parity who delivered preterm infants at 24–31 weeks or term gestation (38–41 weeks) and breastfed their infants were included in this study. We excluded mothers who were smokers or drug users or diagnosed with human immunodeficiency virus.

Serial samples of hind milk were obtained during the first 29 days postpartum from mothers giving birth prematurely and at term. Sample collection was standardized to reduce bias and potential diurnal variability. Hind milk was chosen to produce a uniform sample across participants, as the constituents of milk change from the start to the end of a feeding. The mothers were asked to collect 3 mL of the milk that flowed towards the end of a feeding or a breast pumping session. The samples of transitional (days 6–8 and 13–15) and mature hind milk (days 20–22 and 27–29) were collected in plastic tubes by the mothers in conjunction with the first feeding or by hand/pump expression in the morning (between 8 and 9 a.m.). Skimmed milk (aqueous phase) was prepared by centrifugation (1,500 g, 20 minutes, 4°C), after which the nonfat whey was extracted by pipette, aliquoted into Eppendorf tubes, and frozen at −70°C until further analysis. After the completion of the study enrollment and specimen collection, the milk samples were thawed at room temperature prior to being processed further.

The clinical (gestational age, birth weight, parity, mode of delivery, and intrapartum complications and medications) and demographic (maternal age, race/ethnicity) data of the enrolled mothers were collected.

Analytical methods

A multiplex biometric immunoassay, containing fluorescent-dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to the manufacturer's instructions (Bio-Plex™ Human Cytokine Assay, Bio-Rad Laboratories, Hercules, CA). The limit of detection of this assay is ≤1 pg/mL, intra-assay coefficient of variation is <10%, inter-assay coefficient of variation is <15%, accuracy (percentage recovery) is 80–120%, and cross-reactivity is <1%. Cytokines (IL-1β, IL-6, IL-8, IL-10, IL-13, and TNF-α) were measured in 20 μL of skimmed human milk samples that after appropriate dilution were incubated with antibody-coupled beads. Complexes were washed and then incubated with biotinylated detection antibody and, finally, with streptavidin–phycoerythrin prior to assessment of cytokine concentration titers. Concentrated human recombinant cytokine was provided by the vendor (Bio-Rad Laboratories). A range of 1.95–32,000 pg/mL recombinant cytokines was used to establish standard curves and to maximize the sensitivity and the assay dynamic range. Cytokine levels were determined using a Luminex™ (Austin, TX) Instrumentation System multiplex array reader (Bio-Plex Workstation from Bio-Rad Laboratories). Analyte concentration was calculated using software provided by the manufacturer (Bio-Plex Manager Software).

Statistical analysis

Data were analyzed with respect to the gestational age and stage of lactation (transitional and mature). We used analysis of variance statistics to identify the difference in the tested cytokines levels in transitional and mature breastmilk samples from mothers after very preterm delivery (preterm breastmilk [PBM]) and from mothers who delivered at term (term breastmilk [TBM]). Friedman's analysis of variance was used to compare the tested parameters in association with the different stages of lactation (transitional versus mature milk) in each gestational age group. We used χ² analysis to compare the categorical data. In addition, multiple regression analysis was performed to control the parity and mode of delivery as confounding variables in order to identify the true association between gestational age and quantity of cytokines in breastmilk samples obtained during the first month of lactation. The data are presented as mean ± SD and range and β ± SE. All the p values presented are two-tailed with significance <0.05.

Results

Overall, 60 breastmilk samples were collected from 15 mothers who delivered at very preterm gestation and 20 samples from five mothers who delivered at term. We found no difference in maternal age and race/ethnicity between the mothers who delivered at very preterm and term gestation. Among the mothers who delivered at very preterm gestation, 33% were diagnosed with pre-eclampsia and/or premature rupture of membranes. The majority (80%) of the mothers who delivered at very preterm gestations had completed prophylaxis with corticosteroids prior to delivery, and 60% were treated with antibiotics. Nulliparity was recorded in all the mothers who delivered at term and 40% of the mothers who delivered at very preterm gestation (p < 0.02). Among the mothers in the preterm gestational group, 66.7% were delivered by cesarean section compared with 40% of the mothers who delivered at term (p > 0.05).

We found no statistically significant difference in the levels of tested cytokines between the transitional and mature milk samples obtained from mothers after term delivery. Mature breastmilk samples from mothers after very preterm delivery had lower levels of IL-6, IL-8, IL-10, and IL-13 (p < 0.05–0.01) compared with transitional milk and similar levels of TNF-α and IL-1β.

A comparison of the cytokine quantitative data between preterm and term milk in transitional and mature samples is presented in Table 1. We found lower levels of IL-8 in transitional and mature PBM. The observed difference in IL-8 was confirmed in regression analysis after adjustment for parity and mode of delivery (0.693 ± 0.169, p < 0.0002). Although the TNF-α and IL-6 levels in PBM were higher than in the TBM samples (Table 1), data from multiple regression analysis showed that it was not the gestational age at birth but cesarean delivery that is associated with increased breastmilk TNF-α (0.404 ± 0.148, p < 0.01) and IL-6 (0.352 ± 0.157, p < 0.05). The increased level of IL-10 in association with very preterm gestation (Table 1) was confirmed after adjusting for mode of delivery and parity (0.524 ± 0.177, p < 0.005). In the present study, gestational age, parity, and cesarean delivery did not seem to affect the concentration of IL-13 and IL-1β in transitional and mature breastmilk.

Table 1.

Levels of Cytokines in Transitional and Mature Breastmilk in Association with Very Preterm and Term Gestational Age

	Level (pg/mL)
	Transitional breastmilk			Mature breastmilk
Cytokine	PBM (n = 30)	TBM (n = 10)	p value	PBM (n = 30)	TBM (n = 10)	p value
IL-1β	30.7 ± 22.4 (10–88)	18.2 ± 7.1 (10–32)	0.09	24.5 ± 17.8 (10–63)	17.3 ± 7.3 (10–32)	0.25
IL-6	54.7 ± 22.9 (21–98)	39.0 ± 11.9 (22–55)	<0.05	40.5 ± 17.9 (10–72)	32.6 ± 8.1 (23–45)	0.19
IL-8	687 ± 208 (306–997)	910 ± 697 (786–996)	<0.003	565 ± 206 (282–937)	843 ± 121 (584–996)	<0.001
IL-10	27.8 ± 14.2 (11–65)	15.1 ± 3.1 (11–21)	<0.01	21.1 ± 10.1 (11–54)	15.2 ± 4.9 (11–25)	0.106
IL-13	63.4 ± 17.5 (32–92)	48.4 ± 7.0 (34–62)	<0.02	50.6 ± 17.4 (22–85)	42.0 ± 8.4 (32–52)	0.165
TNF-α	32.3 ± 17.3 (11–69)	17.2 ± 4.2 (13–25)	<0.04	25.2 ± 13.8 (10–58)	15.3 ± 5.2 (10–26)	<0.02

Data are mean ± SD (range) values.

IL, interleukin; PBM, preterm breastmilk; TBM, term breastmilk; TNF-α, tumor necrosis factor α.

Discussion

Several studies that were designed to investigate the association between preterm delivery and breastmilk cytokine content at different stages of lactation, mostly without specification of gestational age at preterm delivery,^10–15 reported no difference in IL-8^11–14 and IL-10¹⁵ and either no difference or lower levels of IL-1β^11–13 and IL-6.^11–13 None of the studies compared the level of IL-13 with respect to gestational age at delivery.

The present study was designed to clarify whether milk from mothers delivering preterm at less than 32 weeks of gestation is able to provide major pro- (IL-1β, IL-6, IL-8, and TNF-α) and anti- (IL-10 and IL-13) inflammatory cytokines at levels comparable to term milk. We found that very preterm delivery independently from parity and mode of delivery was associated with lower IL-8 and higher IL-10 in breastmilk but not IL-1β, IL-6, IL-13, and TNF-α. Relative deficiency of IL-8 may impact the maturation of intestinal tissue because IL-8 is not only an important pro-inflammatory chemokine¹⁶ but also a cytokine that has a dose-dependent trophic and cytoprotective function in the developing human intestine.¹⁷ However, the higher concentration of IL-10 that was observed in transitional preterm milk may be beneficial for controlling the production of an excessive inflammatory response.¹⁸

Our finding of increased breastmilk IL-6 and TNF-α in association with cesarean delivery independently from parity and gestational age is important because of the paucity of research regarding the role of the mode of delivery in the composition of breastmilk. There is only one study that reported an increased level of TNF-α¹⁹ despite the expected interrelationship between human milk IL-6 and TNF-α.^20–22

Notwithstanding the discrepancy with other reports (perhaps due to differences in the stage of lactation, degree of prematurity, parity, and mode of delivery), we have to conclude that very preterm gestation does not substantially impact the cytokine content of transitional and mature breastmilk during the first month of lactation. Although the newborn's requirements for these cytokines need further clarification, the ability of very PBM to provide comparable quantities of cytokines can be beneficial for the regional and systemic immune response of the very preterm infant with confirmed quantitative and qualitative impairment of innate and adaptive immunity.

Footnotes

Acknowledgments

The authors thank all the benevolent mothers who took the time to set aside some of their own milk for research purposes and Uday Nadgir, Nitin Goyal, and Zindadil Gandhi who assisted with the sample processing and cytokine assay.

Disclosure Statement

No competing financial interests exist.

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