Background: The association between human milk (HM) feeding in the NICU and neurodevelopmental (ND) outcome in very low birth weight (VLBW) infants is unclear. Limitations of previous studies include a lack of exact estimates of HM dose and of generalizability to minority populations. Objective: To determine the impact on ND outcome of an exact dose of HM received in the NICU in a diverse, contemporary cohort of VLBW infants. Methods: We included 430 VLBW infants born in the period 2008-2012 for whom the mean daily dose (DD) of HM received during the stay in the NICU (NICU HM-DD) was calculated prospectively from the daily nutritional intake from admission to discharge. Outcomes included Bayley-III index scores at 20 months' corrected age (CA) as assessed upon ND follow-up, which were collected retrospectively. Multivariable linear regression analyses controlled for neonatal and social risk factors. Results: Each 10 mL/kg/day increase in NICU HM-DD was associated with a 0.35 increase in cognitive index score (95% CI [0.03-0.66], p = 0.03), but no significant associations were detected for the language or motor indices. Conclusions: There is a significant dose-dependent association between NICU HM intake and cognitive scores at 20 months' CA. Further follow-up will determine whether these findings persist at school age, and could help alleviate the special-education and health-care burden in this population.

Very low birth weight (VLBW; <1,500 g BW) infants are at significant risk for neurodevelopmental (ND) problems and their associated costs long after discharge from the NICU [1,2,3]. Thus, a priority in NICU care is the identification and implementation of best practice that reduces the risk of ND problems which are burdensome for families, health-care and educational systems, and society at large. Although human milk (HM: from the infant's own mother, excluding donor milk) feeding during the NICU stay has been associated with improvements in ND outcome in premature infants, earlier studies are limited by older cohorts and imprecise definitions of HM intake [4,5,6,7,8,9]. Studies that have found no association between HM and ND outcome have been limited by the calculation of HM dose being only for portions of the NICU stay [10,11,12]. A number of these studies comprise homogeneous cohorts and/or lower rates of HM use in minority populations, limiting the generalizability to patients who might most benefit from a higher HM intake [4,5,6,7,8,9,10,11,12]. We therefore sought to assess the relationship between the daily measured HM intake during the NICU stay and the ND outcome data at 20 months in a diverse, contemporary cohort of VLBW infants in the LOVE MOM (Longitudinal Outcomes in Very Low Birthweight Infants Exposed to Mothers' Own Milk) cohort.

Subjects

The LOVE MOM cohort consisted of 430 VLBW infants born in the period 2008-2012 who were admitted to the Rush University Medical Center (RUMC) NICU and enrolled in a prospective study examining NICU health outcomes and cost of HM feedings for VLBW infants. Inclusion criteria for the original study included admission to the RUMC within 24 h of birth and the initiation of feedings prior to 14 days of life. Additional criteria for the original study and the associations between HM dose and neonatal morbidities have already been published [13,14,15]. Although ND follow-up was not incorporated into the original study design, all VLBW infants were scheduled for ND assessments in the RUMC follow-up clinic as per standard care. Infants who attended the 20-month ND assessment (n = 251) were included in this study. Both the original and the retrospective ND follow-up studies were approved by the institutional review board and written informed consent was obtained.

Design

This study combined the prospectively collected NICU data for the LOVE MOM cohort with retrospective data from each infant's visit to the follow-up clinic. Clinicians performing ND assessments were blinded to NICU HM intake.

HM Dose Data

The daily volume of HM intake was prospectively collected for the NICU hospitalization and used to calculate the daily dose (DD) of NICU HM, i.e., the NICU HM-DD, as a weight-adjusted mean dose (mL/kg/day). HM feedings were fortified with powdered, multinutrient, protein-intact HM fortifier (Similac Human Milk Fortifier, Abbott Nutrition) as per standard NICU practice. Protein supplementation beyond standard fortification was not provided during the study period. Donor milk was not used in the NICU and infants received preterm formula when HM was unavailable. After discharge from the NICU, HM intake was measured as “any” or “no” HM at each follow-up visit.

Birth, Neonatal, and Sociodemographic Data

Collected birth and neonatal data included: antenatal steroids, delivery mode, BW, gestational age (GA), small for gestational age (SGA) at birth, multiple gestation, 5-min Apgar score, severe brain injury (grades 3-4 intraventricular hemorrhage, periventricular leukomalacia, or hydrocephalus), sepsis (culture-proven), necrotizing enterocolitis (NEC, Bell's stage 2/3), bronchopulmonary dysplasia (BPD; oxygen or positive pressure ventilation at 36 weeks' corrected age [CA]), and SGA at discharge [16,17,18]. Collected sociodemographic data included: maternal race/ethnicity, maternal education level, and eligibility for WIC (Special Supplemental Nutrition Program for Women, Infants, and Children; a federal assistance program for low-income women and children) [19].

ND Outcome Data

Infants were evaluated at 20 months' CA by a neonatologist and psychologist. Assessments included a medical history, neurologic exam according to Amiel-Tison and Stewart [20 ]and the Bayley Scales of Infant and Toddler Development III (BSITD-III) [21]. The BSITD-III provides age-adjusted standard scores for cognitive, language, and motor skills (mean 100 ± 15) and subtest scores for cognitive, receptive language, expressive language, fine motor, and gross motor subscales (10 ± 3) based on nationally representative normative data [21]. Primary outcomes included cognitive, language, and motor index scores.

Data Analysis

Subjects were divided into quintiles based on NICU HM-DD for descriptive statistics. Multivariable linear regression analyses were performed to examine the association between NICU HM-DD (mL/kg/day) as a continuous variable and BSITD-III scores (SPSS v23.0, IBM Corp. Armonk, NY, USA). The following confounders that might potentially impact the relationship between HM and ND were adjusted for in the analyses: gender, GA, birth SGA, multiple gestation, 5-min Apgar score, severe brain injury, sepsis, NEC, BPD, SGA at discharge, maternal race/ethnicity, maternal education level, and WIC eligibility.

Sociodemographic, Neonatal, and HM Data

Two-hundred and fifty-one subjects (58%) attended the ND follow-up at 20 months' CA. Infants who did not attend the follow-up had a greater BW (1,076 vs. 1,024 g) and a shorter NICU stay (66 vs. 78 days) and had received a lower NICU HM-DD (52 vs. 65 mL/kg/day). There were no differences in the rates of neonatal morbidities or social risk factors between infants who did and those who did not attend the 20-month visit. The follow-up cohort was racially and ethnically diverse, and predominantly low income, reflecting the demographics of our urban NICU (Table 1). Of note, 98% of the subjects received some HM during the NICU hospitalization. The mean NICU HM-DD for the cohort was 65 ± 49 mL/kg/day and 35% of the cohort was receiving HM at discharge. At 4, 8, and 20 months' CA, however, only 10, 6, and 0.4%, respectively, of the follow-up cohort were receiving HM.

Table 1

Neonatal and sociodemographic characteristics by HM quintile

Neonatal and sociodemographic characteristics by HM quintile
Neonatal and sociodemographic characteristics by HM quintile

ND Outcome Data

Table 2 reports the unadjusted BSITD-III index scores for the cohort, divided into NICU HM-DD quintiles. There was a trend for increasing scores from the first/lowest to the fifth/highest HM quintiles. A significant positive association between NICU HM-DD and cognitive scores remained after adjusting for confounding variables, such that every 10 mL/kg/day increase in NICU HM-DD was associated with an increase of 0.35 in cognitive index score (95% CI [0.03-0.66]; p = 0.03; Table 3). There was no significant effect of NICU HM-DD for either motor or language index scores.

Table 2

Unadjusted Bayley Index scores at 20 months' CA by NICU HM-DD quintile

Unadjusted Bayley Index scores at 20 months' CA by NICU HM-DD quintile
Unadjusted Bayley Index scores at 20 months' CA by NICU HM-DD quintile
Table 3

Multiple linear regression model for Bayley Index scores at 20 months' corrected age

Multiple linear regression model for Bayley Index scores at 20 months' corrected age
Multiple linear regression model for Bayley Index scores at 20 months' corrected age

Our findings reveal a dose-dependent relationship between mean HM-DD received in the NICU and improved ND outcome at 20 months' CA in a diverse, contemporary cohort of VLBW infants, with prospectively collected HM feeding data for the entire NICU hospitalization. Specifically, every 10 mL/kg/day increase in NICU HM-DD was associated with an increase of 0.35 in cognitive index score after controlling for confounding variables. This translates into a 5-point increase in cognitive index score between infants who received no HM and those who received the highest volumes of HM in the NICU.

The relationship between NICU HM intake and subsequent ND outcome was first studied systematically in a randomized cohort of preterm infants from 9 months of age to adolescence [4,5]. Although these serial reports showed a significant association between early HM intake and ND outcome, the data are limited in that they included heavier preterm infants and the composition of the milk formula used was different from contemporary formulas. Nonetheless, a subset of their cohort was evaluated in adolescence, and dose-dependent differences were detected in the brain white matter and ND outcome based on the proportion of HM feedings received in the NICU, especially in males [5]. In contrast, we did not find gender-based differences in the relationship between HM and ND outcome at 20 months.

Subsequent studies have not consistently shown an association between NICU HM intake and ND outcome, but have been limited by variations in measurements of NICU HM intake. For example, Furman et al. [10 ]found no effect of HM on ND outcome at 20 months' CA in a cohort of VLBW infants born in the 1990s. However, they measured HM intake only in the first 4 weeks after birth, so they may have underestimated the range of HM doses and, consequently, the impact on ND outcome. Consistent with their study, we did not find an association between the HM-DD during the first 28 days of life and the ND outcome, despite there being a dose-dependent relationship between early HM intake and a reduction in NEC and sepsis [13,14]. Similarly, Pinelli et al. [11 ]studied a prospective cohort of VLBW infants for whom NICU HM data was recorded once weekly by the mother, and they found no difference in ND scores at 6 and 12 months' CA. We speculate that these negative findings may have been due not only to differences in HM measurements, but also due to the ND assessment at a younger age when cognitive and language deficits may not have been evident. These findings are in contrast to Gibertoni et al. [9], who recently found a positive association between HM intake and ND outcome at 24 months in VLBW infants. However, their study measured HM only at NICU discharge and for 3 categories only (exclusive, mixed, or none), which is significantly different from measuring the actual intake throughout NICU hospitalization.

Our findings also differ from a recent study of preterm infants who were part of the DHA for Improvement of Neurodevelopmental Outcome (DINO) trial, in which HM dose was estimated from HM volume data collected once weekly throughout the NICU stay; no association was found between HM intake and ND outcome [12]. However, these infants had an older GA, a greater BW, and lower rates of most NICU morbidities, and were born to predominantly Caucasian, educated mothers who had a much higher rate of continuing HM feeding of their infants after discharge from the NICU than our cohort (73 vs. 35%). It is possible that parenting practices and home environment after discharge had a greater influence on these infants than the NICU HM dose (which may play a more significant role for higher-risk populations such as ours). Although 98% of our cohort received HM feedings in the NICU, there was a significant decline in feedings in the first year of life; this limited our ability to analyze the HM intake after discharge and the ND. However, we would argue that this may allow for a more reliable assessment of the role of NICU HM-DD on later ND outcome.

The largest study to date on HM intake and ND focused on extremely low BW (ELBW) infants enrolled in the NICHD Glutamine trial, for whom enteral feeding data were collected daily until the infants were on full feedings, and then 3 times weekly [7,8]. The investigators reported that for every 10 mL/kg/day increase in HM-DD received in the NICU, there was a 0.53- and 0.59-point increase in the BSITD-II Mental Development Index and a 0.63- and 0.56-point increase in the Psychomotor Developmental Index at 18 and 30 months' CA, respectively [7,8]. However, a disproportionate number of black infants in the study received no HM, confounding the subsequent generalizability of results. With 98% of our diverse, urban cohort receiving HM during the NICU stay with no racial/ethnic differences across HM quintiles, we were able to examine the association between HM intake and ND outcome across a spectrum of VLBW infants considered to be at a higher risk of both a low provision of HM and a worse ND outcome. Interestingly, we did not find any association between HM intake and motor index scores. However, our population had a greater BW and older GA and, overall, had much lower rates of the neonatal morbidities known to adversely impact motor development. We speculate that our population was likely at a lower risk for motor impairments commonly seen in ELBW infants, particularly those born in previous decades. Our study also differs in that we utilized the BSITD-III, which has been shown to have higher scores than earlier versions, which may also account for differences in results [21,22].

There are multiple mechanisms by which HM potentially impacts ND outcome, especially in VLBW infants whose immature brains are undergoing rapid growth and development, representing a critical window in which white-matter structures are susceptible to inflammation, oxidative stress, and suboptimal nutrition [2,23]. For these infants, HM likely exerts both a direct and indirect influence on the ND outcome. Directly, HM feedings provide a unique nutritional substrate and bioactive components that stimulate and/or program growth and maturation, while preventing and/or moderating biologic insults during this critical developmental window [2,23]. These include anti-inflammatory components, antioxidants, growth factors, HM-borne commensal bacteria, HM oligosaccharides, and HM stem cells [24,25]. Indirectly, high-dose HM intake during the critical period after birth reduces the risk of NEC, sepsis, and BPD, morbidities that increase the risk of subsequent ND problems [13,14,15].

Our study has several limitations. Although 95% of the eligible infants were enrolled in the original prospective study, the ND outcomes were obtained retrospectively from routine care visits and we had significant attrition. Infants who did not complete ND follow-up had a greater BW and a shorter NICU stay and received a lower NICU HM-DD than infants who did the follow-up; it is possible that including these infants would have changed our results. However, there were no differences in morbidity rates or sociodemographic differences between infants who did and those who did not complete the follow-up. Another limitation is the association between NICU HM-DD and maternal and infant characteristics which possibly impact ND, such as maternal education, WIC eligibility, and GA. Although almost all mothers initiated HM feeding of their infants, mothers with an education level lower than high school and WIC eligibility provided significantly less HM than mothers with a higher level of education and a greater income, who were also more likely to have infants with a younger GA. While we adjusted for these variables in our analysis, our results may still have been subjected to residual confounding, thus limiting the generalizability to all populations.

Another limitation of our study is that we did not specifically measure maternal long-chain polyunsaturated fatty acid (LCPUFA) supplementation, which was shown to have a beneficial impact on the 18-month ND in preterm girls in the DINO trial [26]. Our subjects' mothers were, however, instructed to continue prenatal supplements containing LCPUFA after delivery. There is also the potential for unmeasured confounding in this cohort particularly with respect to post-NICU discharge factors. We did not have accurate measurements of HM intake after discharge for our subjects, but given that only 6% of the cohort was receiving any HM by 8 months' CA, it is unlikely that this affected our results. In addition, we did not collect information on the home environment, which may impact ND outcome in childhood. Nevertheless, this study is the first to have prospectively collected HM doses for the entire NICU stay in a recent cohort of racially and economically diverse US-born VLBW infants. As such, we address a frequent limitation of many HM cohort studies consisting of homogeneous populations or reporting disparate HM intakes based on maternal race/ethnicity.

We have shown in this contemporary, diverse, high-risk cohort of VLBW infants that there is a dose-dependent relationship between increasing HM intake during the NICU hospitalization and improved cognitive outcome at 20 months' CA. These findings persisted even after adjusting for relevant neonatal and sociodemographic factors. Thus, high-dose HM intake should be prioritized as part of a NICU care bundle that reduces the risks and costs of ND problems in this population. Longer-term ND evaluation will determine whether these findings persist at school age and the extent to which they are influenced by parenting and environmental factors.

This work was funded by an NIH grant (NR010009) and a Rush University Pilot Program Grant 2011.

The authors declare no conflicts of interest.

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