Influence of Newborns’ Sex on Minor and Trace Element Concentrations in Human Milk During Early Lactation

Introduction

The nutritional composition of breast milk is critical for the health and development of infants, providing essential nutrients, immune protection, and growth factors necessary for early life. ¹ While it is well established that milk constituents can vary based on numerous factors, including maternal diet, health status, and environmental exposures, the potential influence of infants’ gender on the value of minor and trace elements of breast milk has not been thoroughly investigated. ² Research indicates that elemental profile of milk in lactating mothers can vary according to the male and female babies. ³ Animal studies demonstrate that offspring sex influences maternal milk constituents, as research in primates and bovines reveals differences in the milk produced for male and female offspring.^4–6 Macaque mothers with male babies produce less milk with greater energy, whereas those with female babies produce more milk that is less energy rich. Despite these variations, the total energy supplied to the offspring remains similar. ⁷ Female offspring also receive milk with higher calcium (Ca). ⁵ In bovines, mothers produce more energy-dense milk for female calves. Similar sex-based variations in nutritional profile of milk, particularly regarding protein, fat, and lactose c, have been observed in other animals, including kangaroos, wallabies, and red deer. Emerging evidence suggests that milk bioactives have sex-specific effects on offspring outcomes, such as elevated cortisol influencing temperament in male macaques. While these studies indicate a link between offspring sex and composition of breast milk, the range of analyzed compounds is limited, and the mechanisms driving these differences remain unclear. ⁴

Human milk is uniquely tailored to meet the nutritional needs of newborns, yet the influence of infant sex on its composition remains an area of limited and mixed research. Previous studies have suggested sex-specific differences in milk constituents, reporting, for example, that energy content may be higher in the milk of mothers with male infants in some populations, whereas others have found the opposite trend for female infants. ⁴ These differences may be influenced by factors such as socioeconomic status and maternal body mass index. ⁴ In addition, certain macronutrients and minerals, such as fat and salt content, have shown sex-specific variations, with higher fat concentrations observed in milk for male infants and elevated salt levels for female infants.^8,9 Amino acid composition, particularly free glutamine levels, has also been linked to infant sex, ¹⁰ further suggesting that human milk may adapt to the distinct nutritional needs of male and female infants.

Sodium (Na) levels in milk are of particular interest, serving as a key indicator of lactation success. ¹¹ A normal decline in Na concentration during early lactation is considered predictive of successful breastfeeding. ¹¹ Studies such as those by Naima Khelouf and colleagues have demonstrated that elements such as Na, potassium (K), and Ca exhibit sex-specific differences, with higher concentrations of these minerals reported in milk produced for female infants. ³ However, such differences are not limited to sex alone; they may also vary within feeds and between breasts, potentially influenced by factors like mastitis. ¹²

Despite these insights, research on the elemental composition of human milk in response to newborn sex remains sparse. Most studies have focused on at-risk populations, such as preterm or small-for-gestational-age infants,^13,14 or have prioritized macronutrients ⁸ and a limited subset of micronutrients. Trace elements, including bromine (Br), iodine (I), and rubidium (Rb), have received minimal attention in this context. Furthermore, no studies to date have investigated sex-specific differences in human milk for elements such as aluminum (Al), Br, chlorine (Cl), I, or Rb.

To address these gaps, our research examines whether newborn sex influences the concentrations of eight essential and nonessential elements—Al, Br, Ca, Cl, I, K, Na, and Rb—in the milk of lactating mothers during early lactation. These elements are vital for infant growth and development, and understanding their variations may provide valuable insights into maternal physiological adaptations and their implications for infant health.

In this study, we utilized neutron activation analysis (NAA), ¹⁵ a highly accurate and nondestructive technique, to analyze milk samples from 75 lactating mothers in Tehran, Iran. NAA is particularly well-suited for complex biological matrices such as human milk, offering exceptional sensitivity for detecting trace elements. By exploring the relationship between newborn sex and the concentrations of these elements, this research aims to enhance our understanding of how human milk composition varies during the critical early stages of breastfeeding. The findings have the potential to inform future studies on infant nutrition and maternal health, shedding light on the biological factors that shape early-life nutrition.

Methods

Breast milk samples were collected from 75 breastfeeding mothers, aged 18 to 41, receiving care at three hospitals in Tehran, Iran. Before collection, informed consent was obtained from all participants. Milk was gathered during the early lactation period, specifically within the first five breastfeeding sessions. Among the newborns, 28 were girls and 47 were boys. A detailed checklist was completed for each participant, capturing information on sample number, contact details, maternal age, education, place of residence, socioeconomic status (monthly income), number of pregnancies, number of breastfed children, dietary habits (e.g., vegetarian), supplement and medication use during pregnancy, illnesses, and infant details such as gender, gestational age, birth weight, and preterm or term status.

A minimum of 10 mL of milk was collected from each mother in sterile, standard storage containers, labeled with unique identifiers to ensure traceability. The samples were immediately frozen to preserve their integrity and were transported under strict cold chain protocols to the Cellular and Molecular Research Center at Iran University of Medical Sciences. Upon arrival, the samples were stored at −80°C for at least 5 hours before processing. To prepare the samples for analysis, they were freeze-dried at −40°C under vacuum for 21 hours using an EDWARD freeze dryer, converting them into a powdered form. These powdered samples were securely stored for subsequent analysis of minor and trace elements.

NAA based on the relative method was used to measure element constituents in the dry milk powder samples. ¹⁴ Gamma-ray emissions of neutron-irradiated samples were analyzed to identify and quantify the elements. The accuracy and precision of the measurements were assessed by analyzing A-11 milk powder reference material, which was used as the standard for quality control. The relative deviation (RD) in the average value of elements between the milk of mothers with female infants and those with male infants was determined.

Ethical approval for the present work was obtained from the Iran University of Medical Sciences (IRB Approval ID: IR.IUMS.REC.1395.9221216310), and all participant data were kept confidential.

Results

This study analyzed essential minor elements—Ca, K, Na, and Cl—and the essential trace element I, as well as nonessential trace elements such as Al, Br, and Rb, in breast milk samples from 75 lactating mothers in Tehran, Iran, during early breastfeeding. Among these, 28 mothers had female newborns, and 47 had male newborns. Elemental analysis was performed on powdered milk samples using NAA, with statistical comparisons (t tests) performed to assess relationships between elements and the sex of newborn babies.

Sex-based differences were observed in the levels of Br, Cl, and Na in milk samples (p < 0.05). Br concentrations were notably greater in the milk of mothers with girls (14.8 ± 1.3 mg/kg) in contrast to those with boys (11.3 ± 0.58 mg/kg; p = 0.011). The amounts of Cl in breast milk followed a similar pattern, being higher in mothers nursing daughters compared with those nursing sons. Specifically, the milk from mothers with daughters contained 10.16 ± 1.18 mg/g of Cl, whereas milk from mothers with sons had lower values at 7.10 ± 0.51 mg/g (p = 0.009). This suggests a noteworthy variation in Cl based on the infant’s gender. Na in human milk varied by the newborns’ gender, with milk for female babies containing 6.18 ± 0.60 mg/g and milk for male babies having a lower concentration of 4.45 ± 0.41 mg/g (p = 0.017).

For the remaining elements, no statistically notable differences were observed between the two groups. The average Al content in all samples was 7.7 ± 0.5 mg/kg, with higher values observed in the milk of mothers with daughters (8.6 ± 1.0 mg/kg) compared with those with sons (7.2 ± 0.5 mg/kg; p = 0.199). The mean value of I element in milk was greater for mothers of female babies (1.34 ± 0.24 mg/kg) than for those of male babies (0.98 ± 0.11 mg/kg; p = 0.121). The value of Ca element averaged 2.90 ± 0.14 mg/g in all samples, with no meaningful differences detected between female (3.06 ± 0.24 mg/g) and male (2.77 ± 0.18 mg/g) infants (p = 0.329). K and Rb values remained nearly steady across the groups, with K showing an average of 5.84 ± 0.16 mg/g (p = 0.81) and Rb averaging 3.6 ± 0.2 mg/kg (p = 0.762).

Table 1 provides a summary of the mean values, standard errors (SEs), results of statistical comparisons based on newborn infants’ sex, and RDs for each element analyzed.

Discussion

This research highlights significant gender-specific differences in the elemental composition of human milk from lactating mothers in early lactation. Mothers of newborn girls produced milk with higher concentrations of Br, Cl, and Na compared with mothers of newborn boys. These findings suggest that the elemental profile of milk may adapt in a sex-specific manner to meet the distinct physiological and developmental requirements of male and female infants during early growth stages.

Br levels in the milk of mothers with girls showed a notable 31% increase (Table 1) compared with the milk of mothers with boys. Br is a trace element involved in tissue development, particularly as a cofactor in type IV collagen scaffolds, which are critical during periods of rapid growth. ¹⁶ This is the first study to report Br differences based on infants’ sex, indicating a need for further investigation into its biological role and implications for infant development.

Cl and Na, both key electrolytes,^17–19 also exhibited substantial differences, with levels being 43% and 39% greater (Table 1), respectively, in the milk of mothers of females versus mothers of males. Na plays a vital role in cellular proliferation, protein synthesis, and overall growth. ²⁰ Increased Na may reflect the more significant hydration and electrolyte needs of newborn girls during this stage. Notably, deficiencies in sodium chloride (NaCl) can impair growth and cognitive development, particularly in preterm infants. ²⁰ Our findings on Na align with recent studies reporting similar trends in Na based on the infant’s sex. ³ The observed relationship between Na and Cl suggests a potential connection between these electrolytes. ²¹ However, further research is needed, as the variability of Cl in human milk is still not well studied or fully understood.

For other elements—Ca, I, Al, K, and Rb—no meaningful gender-specific differences were observed. However, trends suggest potential subtle variations. The milk of lactating mothers with girls contained approximately 10% more Ca and 36% more I compared with the milk of lactating mothers with boys. Although these differences were not statistically significant, they are consistent with previous studies reporting slightly higher Ca amounts in milk for girls.^3,22 The Al content was approximately 20% higher in milk from mothers of female infants, possibly influenced by maternal diet or environmental factors.^2,23,24 K and Rb showed little variation between the breast milk of mothers with male and female infants, suggesting that the regulation of these elements in breast milk is independent of the infant’s sex. Rb, which is associated with neurocognitive development, ²⁵ and K, essential for cellular functions and fluid balance, ²⁶ require further investigation to clarify their roles in newborn development during early feeding stages.

The observed differences in elemental content based on infant sex may stem from physiological and hormonal variations in the mother influenced by the baby’s gender. For example, hormonal factors like estrogen, which can vary depending on fetal sex, might affect maternal nutrient metabolism and the nutritional constituents of milk. ²⁷ In addition, variations in hormones such as testosterone and glucocorticoids, depending on the infant’s sex, could impact the transfer of certain elements from mother to child. ²⁸ Differences in placental transport mechanisms may explain the variations in elemental content, as male and female placentas have distinct gene expression profiles and transporter activity patterns. For instance, male fetuses are associated with increased Ca transporter activity in the placenta, a factor that may continue to impact nutrients in breast milk during lactation.^13,29 The interaction between maternal and fetal physiology is likely a key factor influencing sex-based differences in breast milk composition, particularly for elements such as Br, Cl, and Na.

This work is among the first to report sex-specific differences in Al, Br, Cl, I, and Rb concentrations in human milk during early lactation. Although previous research has examined Ca, Na, and K in relation to newborns’ sex, ³ information on other elements is still limited. Larger studies incorporating sample size expansion, maternal dietary assessments, environmental exposure data, and genetic analysis are needed to validate these findings and better understand their significance for newborn nutrition and development. By expanding our understanding of the factors influencing milk composition, these findings provide a foundation for improving breastfeeding recommendations and strategies tailored to optimize infant health and maternal well-being.

Conclusion

This research provides new insights into how infant sex influences the concentration of minor and trace elements in breast milk during the early feeding stage. Using NAA, we measured the levels of key elements—both minor (Ca, K, Na, and Cl) and trace (I, Al, Br, and Rb)—in milk samples from 75 lactating mothers in Tehran, Iran. Our findings reveal significantly higher concentrations of Cl, Na, and Br in the milk of mothers of female infants compared with those with male infants, with relative increases of 43%, 39%, and 31%, respectively (p-values of 0.009, 0.017, and 0.011). These results suggest a biological mechanism linking infant sex to the regulation of specific elements in breast milk. While levels of Al, Ca, and I were also higher in mothers of female infants, these differences were not statistically significant, and no variation in K or Rb was observed based on infant sex. These findings highlight the need to consider infant sex when studying human milk, pointing to potential sex-based differences in elemental concentrations during early lactation. Further research in larger sample size and more diverse populations is needed to confirm these observations.