Introduction

Breastfeeding provides irreplaceable nutritional and nonnutritional factors that influence the postnatal adaptation and development of the infant. In neonatal intensive care practice, three options are available for enteral nutrition: the infant’s own mother’s milk, banked donor milk, and commercial infant formula. Breastfeeding has numerous protective effects compared to formula. It is associated with lower risks of gastroenteritis, respiratory tract infections, sudden infant death syndrome, asthma, atopic dermatitis, diabetes, obesity, and childhood leukemia.1,2 Of particular importance for the preterm infant, breastfeeding reduces the risk of necrotizing enterocolitis and enhances brain development.3,4 Previous investigators have examined the presence of bioactive factors in mature breast milk,5 but little is known about the hormonal content of milk produced by the mothers of preterm infants.

If maternal milk is not available, the recommended feeding for preterm infants is banked donor milk.6 Previous studies have shown that donor milk reduces necrotizing enterocolitis and other morbidities compared to formula.7 Microbiological safety of donor milk is usually ensured by Holder pasteurization (HoP) in human milk banks.6 Several studies have shown that HoP may decrease the levels of bioactive compounds and hormones in term breast milk.8 Maternal hormonal influence is essential for ideal fetal development, first through transplacental circulation, and after birth, breastfeeding is the infant’s only source of maternal hormones. Preterm birth prematurely terminates transplacental hormone delivery; therefore, in the absence of an alternative source, preterm infants may be exposed more briefly to maternal hormones. Thus, it is critically important to determine the extent to which maternally derived hormones can still be transferred to the preterm infant after birth through breast milk. In our study, we focused on pleiotropic hormones that are known to be present in breast milk produced for term infants, including leptin, insulin, cortisol, progesterone and testosterone.

Human leptin promotes cognitive development,9 and leptin supplementation provides neurodevelopmental protection in mice.10 Late preterm and term male infants have lower leptin levels than female infants,11 and limited investigations have shown that leptin levels may be higher in breast-fed versus formula-fed term infants.12 Preterm delivery leads to the early separation from the mother as a source of leptin and predisposes to postnatal leptin deficiency.13

Insulin, an anabolic hormone, promotes the absorption of carbohydrates and increases the blood-to-brain transport of leptin.14 Insulin plays a role in neuronal survival, dendritic arborization, and short- and long-term memory consolidation in the hippocampus.15 Insulin administration can work in concert with endogenous insulin production to modulate blood glucose levels and protect against hyper- and hypoglycemic states.14 In adults with pancreatic insufficiency, oral insulin can be absorbed enough to induce hypoglycemia,16,17 but this has not been demonstrated in preterm infants.18,19 Even with incomplete absorption, enteral insulin can exert local effects, including accelerated intestinal maturation18,19 and improved microbiome diversity.20

Steroid hormones have diverse physiological effects, but their exact roles during early development are not completely understood. Cortisol participates in numerous physiological processes; it contributes to gluconeogenesis, counteracts insulin, and acts as a diuretic.21 Studies suggest that glucocorticoids in milk may influence psychological maturation and growth.22 Progesterone is considered a neurosteroid, as its synthesis in the brain has been documented. Progesterone affects remyelination, plays a role in the development of brain and behavior and suppresses immune responses of the mother to fetal antigens and potentiates T-cell differentiation.23,24 Testosterone is a key hormone in the development of male reproductive tissues. Also, it likely has an important role in the physiology of brain functions.25 An early postnatal androgen elevation, or mini-puberty, contributes to human neurobehavioral sexual differentiation.26 In adults, orally administered progesterone and testosterone are absorbed, especially when given with fats or meals, but their bioavailability is considerably reduced by first pass hepatic metabolism.27,28,29 Given the hormones’ emerging roles in development,30,31 milk-borne progesterone and testosterone could exert biologic effects, especially in the presence of hepatic immaturity, even if they ultimately reach the systemic circulation in relatively low concentrations. The testosterone content within breast milk has not been defined, and progesterone levels in breast milk have only been described for mothers that delivered at term.32

The effects of preterm delivery and specific breast milk processing practices, such as HoP, on the non-nutritive components of breast milk have not been defined. Therefore, our aims were to investigate possible differences in insulin, leptin, cortisol, testosterone, and progesterone concentrations between preterm mother’s own milk and term donor milk and to examine whether HoP affects the levels of these developmentally important hormones in breast milk. We ultimately hypothesized that provision of maternal milk versus pasteurized donor milk significantly alters the hormonal intake of preterm infants.

Materials and methods

Study design and population

We recruited 26 mothers who gave birth to preterm infants who were hospitalized in the neonatal intensive care unit of the University of Iowa Stead Family Children’s Hospital. To avoid sampling colostrum or transitional milk, we collected samples from mothers who were able to continuously breastfeed and whose infants were 3−5 weeks old. Mothers of preterm infants pumped the milk samples in the neonatal intensive care unit to polypropylene tubes, after which the samples were stored at −20 °C until they were thawed for analysis. The donor samples (n = 31) were collected from mothers who delivered at term. The donor mothers expressed their milk manually or by pump at their homes and later donated the samples to the Mother’s Milk Bank of Iowa (Iowa City, IA, USA). The donors from the milk bank were selected from among those who had met the criteria to donate and were screened and tested based on the protocol of the Human Milk Banking Association of North America. The study was approved by the institutional review board of the University of Iowa and the Mother’s Milk Bank of Iowa. The preterm mothers and milk donors consented to the use of their milk samples for this study.

Sample preparation and analysis

The milk samples were sonicated to disrupt milk fat globules and allow proteins to enter the aqueous phase. One aliquot was pasteurized in a preheated water bath at 62.5 °C for 30 min. Then, the pasteurized and nonpasteurized aliquots were centrifuged at 15,000 × g at 4 °C. Consistent with recommendations from prior investigations, the fat layer was discarded, and the skim milk was subjected to analysis.20,33 Milliplex MAP Kit assays were performed to investigate the levels of insulin, leptin, cortisol, progesterone, and testosterone in preterm and donor breast milk. The tests were performed in duplicate. The amounts of leptin and insulin were determined by customized magnetic bead assay (HMHEMAG-34K) (END Millipore Corporation, Billerica, MA, USA). The levels of cortisol, progesterone and testosterone were measured with a separate customized array (MSHMAG-21K) (END Millipore Corporation, Billerica, MA, USA). To measure the leptin and the insulin concentrations, 25 µl of standards and quality controls and 50 µl of milk samples were added to a 96-well plate. After 18 h incubation with mixed beads at 4 °C, we washed the plates then added 50 µl detection antibody followed by 50 µl Streptavidin-Phycoerythrin to each well. In the case of cortisol, progesterone, and testosterone, sample extraction was accomplished using a Savant SpeedVac concentrator (ThermoFisher Scientific, Waltham, MA, USA), and 50 µl of milk was transferred to a 96-well plate and incubated for 18 h with 25 µl HRP conjugate and 25 µl prepared Bead solution. After incubation with secondary antibody and Streptavidin-Phycoerythrin, we added 100 µl of sheath fluid to the wells. We ran the plates on FLEXMAP 3DTM with xPONENT software (Luminex Corp, Austin, TX, USA). Based on prior publications, we anticipated the following minimum concentrations in pg/ml: 320 for insulin,34 270 for leptin,35 290 for cortisol,36 860 for progesterone,32 and no data were available for testosterone. For our chosen assays, the lower limits of quantification (LLOQ) in pg/ml were 44 for insulin, 21 for leptin, 85 for cortisol, 70 for progesterone, and 40 for testosterone. The intraassay coefficients of variation were less than 10%. The interassay coefficients were 8.5% for insulin, 9.3% for leptin, 8.2% for cortisol, 6.4% for progesterone, and 5.2% for testosterone.

Statistical analysis

For statistical analyses, we utilized GraphPad (GraphPad Software, La Jolla, CA, USA) with normal distributions confirmed by Shapiro−Wilks tests. Analyte levels below the LLOQ were replaced by values equal to the LLOQ divided by the square root of 2, as previously performed.13 Data were analyzed by ANOVA or paired Student’s t test, as appropriate, and the sample size was chosen to provide >80% power to detect moderate effect sizes (Cohen’s d = 0.6) with significance set at p < 0.05.

Results

Maternal age and infant sex did not significantly differ between the two cohorts. As expected, the milk bank donors delivered at later gestational ages and they were further removed from the time of delivery when the samples were collected (Table 1). While additional demographics were not available for the milk bank donors, 24 of the preterm mothers were Caucasian (including two Hispanic), one was African-American, and one multiracial. Mean body mass index (BMI) for the preterm cohort was 29.4 ± 8.3 kg/m2, and 10 (38%) had a BMI > 30. None of the women in the preterm cohort had diabetes mellitus, two (8%) had chronic hypertension, and four (15%) had preeclampsia or HELLP syndrome.

Table 1 Maternal characteristics were obtained for women who donated their own breast milk to the Mothers’ Milk Bank of Iowa and women who were providing their own breast milk to their premature infants within the University of Iowa neonatal intensive care unit.
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We detected all five hormones of interest, insulin, leptin, cortisol, progesterone, and testosterone in term donor and preterm milk. All samples had insulin levels in excess of the LLOQ. Leptin was quantifiable in all 26 maternal and 28/31 donor samples; cortisol was quantifiable in 23/26 maternal and all 31 donor samples; progesterone was quantifiable in 5/26 maternal and 11/31 donor samples; and testosterone was quantifiable in 13/26 maternal and 24/31 donor samples. While nonpasteurized preterm milk contained threefold more leptin than nonpasteurized term donor milk (p < 0.01), there were no significant differences in insulin, cortisol, progesterone, or testosterone concentrations between nonpasteurized term donor and preterm milk samples (Table 2).

Table 2 The concentration of hormones in nonpasteurized term donor milk and preterm mother’s own milk, expressed as mean ± SEM, was analyzed by ANOVA.
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HoP significantly decreased insulin and leptin levels, by 13 and 81% respectively (Table 3). Cortisol, progesterone, and testosterone levels were not significantly influenced by HoP (Table 3). During hospitalization preterm infants receiving human milk are fed their own mother’s milk or pasteurized donor milk. While the insulin, progesterone, and testosterone concentrations do not statistically differ between those two options, the preterm infant’s own mother’s milk contains 16 times as much leptin as donor milk and about one-third as much cortisol as donor milk (Table 4).

Table 3 Hormones concentrations in breast milk samples obtained from both term donors and preterm mothers were determined before and after pasteurization with the results expressed as mean ± SEM.
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Table 4 Hormone concentrations are contrasted for the two main types of preterm infant enteral nutrition, pasteurized term donor milk and raw preterm mother’s own milk.
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Exploring potential contributory or confounding variables, we found no effect of infant gender on milk hormone levels (data not shown). We next examined whether maternal BMI is associated with the hormonal content of preterm breast milk. Cortisol, progesterone, and testosterone were not influenced by maternal BMI. However, the milk samples of obese mothers (BMI 30 or above) had significantly increased insulin and leptin levels compared to samples from mothers without obesity, and those associations persisted after HoP (Fig. 1).

Fig. 1: Women providing breast milk for the own premature infant were classified as obese if their body mass index (BMI) was 30 or higher.
figure 1

Using two-way ANOVA, breast milk insulin levels (a) and leptin levels (b), determined before (gray bars) and after (black bars) Holder pasteurization, were compared for mothers with or without obesity. *p < 0.05 or **p < 0.001 for raw milk from mothers with BMI < 30 versus BMI 30 or higher; †p < 0.05 or ††p < 0.001 for pasteurized milk from mothers with BMI < 30 versus BMI 30 or higher.

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Discussion

We studied the levels of relevant hormones in preterm and term donor breast milk and assessed the effect of HoP. Utilizing the latest technology, we were able to detect the biologically active forms of the hormones and demonstrate the capacity to quantify key hormones with emerging roles in neurologic development and metabolic adaptation. Leptin, cortisol, progesterone, and testosterone may be absorbed from the gastrointestinal tract and, through the circulation, affect organ development and postnatal adaptation. The investigated hormones can also act directly in the gastrointestinal tract, influencing the composition of the microbiome, which affects development through the microbiome−gut−brain axis. Presumably, the protective factors of breast milk both influence the composition of gut microbiota and contribute to maturation of gut-associated lymphatic tissue.20,37 Furthermore, these hormones can act like growth factors, influencing the differentiation of gut epithelia.38

Leptin is a pleiotropic hormone with multifunctional effects during human development. Its early neurotrophic effect on the hypothalamus has been previously described.39 Milk leptin correlates positively with infant serum leptin, which correlates with infant BMI and weight.12 Leptin is not present in formula, but it is present in breast milk, and breast-fed infants have elevated plasma leptin levels.12 Our findings confirmed previous investigations, which have shown that leptin is highly affected by HoP.8 Likewise, the leptin levels we measured in preterm milk are very similar to those reported by both Bielicki and Eilers.35,40 In Bielicki’s study, term colostrum had higher leptin levels than preterm colostrum, but leptin levels quickly decreased in term but not preterm milk, such that term and preterm levels were similar when the final samples were collected at 6 weeks.35 In Eiler’s investigations, preterm and term milk leptin content did not differ, but they again noted a longitudinal decrease specifically among the term cohort.40 Compared to the prior investigations, the lower leptin levels we detected in mature term milk may reflect the much longer postpartum interval that preceded milk collection in our study, designed to reflect the nutritional options typically available for preterm infants.

The intact form of leptin is transported from blood into the parenchymal compartment of the brain. Several factors, such as decreased breast milk intake, male sex, preterm birth and intrauterine growth restriction increase the risk of perinatal leptin deficiency.41 Because preterm infants are often born prior to the third-trimester leptin surge, they are at the greatest risk of neonatal leptin deficiency.42 Previous studies suggest that leptin replacement influences feeding behavior and endocrine functions,43 and the prolonged hypoleptinemia observed in preterm infants may lead to increased susceptibility to diet-induced obesity.44 Even if leptin is incompletely absorbed following enteral administration, gut epithelial cell proliferation can be enhanced by exogenous leptin.45 Our findings are consistent with previous studies reporting that obese mothers have elevated insulin and leptin levels in their breast milk,40,46 and leptin levels are decreased in mature milk donated for use in preterm infants.46

Given that insulin is actively transported into human milk and is protected from degradation, it presumably plays a functional or developmental role in the infant.47 In preclinical models, Koldovský and coworkers showed that intact insulin could retain biological activity when ingested, cross from breast milk into the bloodstream, and decrease blood glucose levels.47,48 Epithelial cell insulin receptors exist in the intestine of both piglets and calves, and insulin has been suggested to play a role in influencing growth and development of the small intestine.38 Shehadeh et al.34 recruited nondiabetic mothers of preterm or term infants, and consistent with our data, they noted no effect of gestational age on breast milk insulin levels. Our results revealing increased insulin levels in the breast milk of women with increased BMI identifies a potential etiology for the development of hypoglycemia in infants of obese mothers,49 but limited bioavailability may minimize the impact oral insulin has on the blood glucose levels of most infants.18,19 Like leptin, insulin is a peptide hormone, and it was not surprising that HoP significantly decreased insulin levels. Utilizing a similar donor milk bank population and the same HoP process, Ley et al.50 also noted a significant postpasteurization reduction in insulin (46% decrease). The smaller effect size we detected (13% decrease) could reflect differences in sample processing (e.g., sonication) or method of detection (electrochemiluminescence versus microbead array).

Glucocorticoids are known to be present in breast milk and absorbed from the gastrointestinal tract.51 Previous studies have similarly shown reduced mature breast milk cortisol levels for mothers that delivered preterm versus term infants,52 although no difference has been detected within transitional milk produced within a week of term or preterm delivery.53 Glucocorticoids play a crucial role in maintaining the delicate hormonal equilibrium that controls metabolism in mammals.54 Cortisol exposure via human milk may provide protection against childhood obesity, and early glucocorticoid exposure may be a regulator of infant metabolism.55 The reduced cortisol levels we detected in fresh milk from mothers of preterm infants versus HoP milk from mothers of term infants are consistent with the results of van der Voorn et al.,52 and we show that the choice of nutrition has implications for infant cortisol intake. As a steroid, cortisol is a relatively thermostable, and previous investigations have confirmed a lack of denaturation following HoP.36

After delivery, maternal progesterone levels rapidly decrease, helping to trigger milk production.32 Preterm infants are born at a time they would typically be exposed to relatively high progesterone concentrations; therefore, postnatal progesterone intake may be particularly important to the preterm infant, and progesterone absorption from the gastrointestinal tract is known to occur.56 Unfortunately, the levels of progesterone that we measured in milk from preterm mothers were similar to the low levels measured in mature milk,32 suggesting targeting collection at an even shorter interval from delivery may be needed to increase progesterone delivery via breast milk.32

Testosterone is a neuromodulator and immunomodulator capable of reversing environmentally induced memory deficits.57 Mini-puberty, the postnatal elevation of testosterone in male infants,58 has been associated with sex-typed behavior at 14 months.59 Little is known about the presence of testosterone in breast milk, and our data are the first to demonstrate similar content in term and preterm breast milk.

Neonatal intensive care units are moving away from the use of formula for preterm infants, and our investigations comparing hormone levels in mother’s own milk to HoP donor milk are novel. Our study does have some limitations. For each mother of a preterm infant, we had a milk sample at only one point in time, which precluded analysis of postpartum changes in milk hormone levels. Given the expected postpartum decline in maternal serum progesterone, longitudinal changes in milk progesterone content could warrant further investigation. While not close to statistical significance, in part due to low levels of progesterone seen in both cohorts and variability in our preterm cohort, we did note higher progesterone levels in the milk of preterm mothers that had a shorter postpartum interval than the milk bank donors. We did not have access to data about the mother’s socioeconomic status, which might have an impact on breast milk hormone concentrations. We also were unable to obtain detailed information about milk bank donors, including BMI. Finally, although each of the hormones were detectable, both progesterone and testosterone were often present in levels below the lower limit of quantification. Removal of the fat layer prior to analysis may have reduced recovery of fat-soluble compounds, including testosterone and progesterone, but the assay we utilized has not been validated for nonaqueous samples. Future studies, potentially with spiked samples and alternative methodologies, could assess differential recovery of hormones in the fat and aqueous layers of milk.

The bioassays applied in this study are the preferred method to measure biologically active hormone levels in breast milk and assess the effects of milk preparation methods, like HoP. We have shown significant differences in the hormonal composition of preterm mother’s own milk and term donor milk and confirmed that the leptin and insulin content of a preterm mother’s own milk is increased with obesity. HoP dramatically decreases the levels of leptin, further magnifying the reduction in leptin content of donor breast milk feedings. Given the decrease in leptin concentration following HoP and the absence of leptin in commercial formulas, targeted leptin administration should be investigated for preterm infants that are not receiving their mother’s own milk.

For preterm infants, breast milk is the exclusive source of maternal hormones, and these infants are the main recipients of pasteurized donor human milk. We detected remarkable differences in the concentration of leptin and cortisol between milk produced by the mothers of preterm infants and pasteurized term donor milk. It is not established that preterm milk levels are ideal or that relatively high levels of leptin and low levels of cortisol are preferred. We have previously demonstrated profound leptin deficiency in preterm infants, with plasma levels often falling below the lowest limits of detection (41 pg/ml) at a time when leptin is believed to have an important role in evolving neuromaturation.13 Cortisol levels of preterm infants are variable, but generally exceed 10,000 pg/ml.60 Those plasma ratios are approximated by the content of donor but not preterm milk. Studies in monkeys and humans have correlated lower milk cortisol levels with increased impulsiveness and social withdrawal,61 an outcome reminiscent of the behavioral phenotype described for premature infants.62 Our findings highlight important differences in the delivery of leptin and cortisol to premature infants based on the source of milk with potential implications for breast milk processing and feeding guidelines.