Human milk (HM) contains a wide array of peptide hormones including leptin and adiponectin, which are involved in the regulation of infant growth and development. These essential hormones might play an important role in the regulation of metabolic reprogramming of the new-born infant. However, HM hormone studies are sparse and heterogeneous in regard to the study design, sample collection, preparation and analysis methods. This review discussed the limitations of HM hormone analysis highlighting the gaps in pre-analytical and analytical stages. The methods used to quantify HM metabolic hormones (leptin, adiponectin, ghrelin, insulin, obestatin, resistin and apelin) can be classified as immunoassay, immunosensor and chromatography. Immunoassay methods (ELISA and RIA) have been predominantly used in the measurement of these HM hormones. The relative validity parameters of HM hormones analysis are often overlooked in publications, despite the complexity and differences of HM matrix when compared to that of plasma and urine. Therefore, appropriate reports of validation parameters of methodology and instrumentation are crucial for accurate measurements and therefore better understanding of the HM metabolic hormones and their influences on infant outcomes.

There is an inadequate understanding of the daily variations in hormones and macronutrients in human milk (HM), and sample collection protocols vary considerably from study to study. To investigate changes in these milk components across 24 h, 22 lactating women collected small milk samples before and after each breastfeed or expression from each breast. Test weighing was used to determine the volume of HM consumed in each feed. The concentrations of leptin, adiponectin, insulin, fat, and glucose were measured, and the intakes were calculated. A linear mixed model was fitted to assess within-feed and circadian variation in HM feed volume and concentration, and intakes of several components. The average infant intake of HM was 879 g/24 h. Significantly higher pre-feed concentrations were found for adiponectin and glucose and lower post-feed concentrations were found for insulin and fat. Significant circadian rhythms were displayed for leptin, adiponectin, insulin, glucose (both concentration and intake), fat concentration, and milk volume. These findings demonstrate the necessity for setting up standardised and rigorous sampling procedures that consider both within-feed and circadian variations in HM components to gain a more precise understanding of the impacts of these components on infant health, growth and development.

Gestational diabetes mellitus (GDM) is a common pregnancy complication with short- and long-term health consequences for the infant and mother. Breastfeeding is the recommended mode of feeding as it offers an opportunity to reduce the risk of GDM consequences, likely partially mediated through changes in human milk (HM) composition. This review systematically reviewed 12 identified studies that investigated the impact of GDM on concentrations of HM metabolic hormones. Meta-analysis was not possible due to significant heterogeneity in study designs and hormone measurement techniques. The risk of bias was assessed using the National Institute for Clinical Excellence (NICE) tool. The methodological qualities were medium in half of the studies, while 25% (3/12) of studies carried a high risk of bias. Significant relationships were reported between GDM and concentrations of HM ghrelin (3/3 studies), insulin (2/4), and adiponectin (2/6), which may play an integral role in infant growth and development. In conclusion, preliminary evidence suggests that GDM may alter HM metabolic hormone concentrations; however, these relationships may be limited to the early lactation stage.

Human milk (HM) composition, including metabolic hormones and lipids, is influenced by various factors, including lactation stage and, potentially, infant sex, which may affect infant body composition (BC) development. We aimed to: (a) characterize the longitudinal concentration and intake profiles of HM leptin, adiponectin, insulin, and total lipids; (b) determine if their concentrations and intakes differ by infant sex; and (c) explore the intakes relationships with the development of infant BC. Milk samples (n = 501) were collected from 82 mother–infant dyads during the first 6 months postpartum. Infant 24 h HM intake was measured, and the average cumulative HM component intakes were calculated. The statistical analysis used linear mixed modeling. Intakes of HM leptin, adiponectin, insulin, and total lipids increased to 1 month postpartum and then remained stable. HM intake and total lipids intake but not hormone intakes were positively associated with infant BC (fat-free mass, fat-free mass index, fat mass, fat mass index, percentage fat mass, and fat mass to fat-free mass ratio). HM component concentrations and intakes did not differ by sex. These findings advance our understanding of the temporal nature of HM components, emphasizing the role of infant 24 h HM and total lipids intake in development of infant lean and adipose tissue.

Limited attention is given to the efficacy of protocols for the estimation of infant intake of milk components when investigating their impact on infant outcomes. We compared the actual measured intake of human milk components with estimations derived from 15 protocols to determine the most reliable approach for estimating intake of HM leptin, adiponectin, insulin, glucose, and total lipid. Twenty mothers who were 3–5 months postpartum completed a 24 h milk profile study with pre-/post-feed milk samples collection. The true infant intake (control group) based on 24 h milk intake (MI) was compared to estimated infant intakes using concentrations from five sampling protocols that were multiplied by one of true infant MI, considered mean MI (800 mL), or global mean MI (766 mL). The mean measured concentrations of six samples (three sets of pre- and post-feed samples, from morning (06:00–09:00), afternoon (13:00–16:00), and evening (19:00–22:00)) multiplied by the true infant MI, mean considered MI, and global mean MI produced the most accurate estimates of infant intake of these components. Therefore, in the absence of 24 h measurements and sampling, a sampling protocol comprising three sets of pre-/post-feed samples provides the most reliable infant intake estimates of HM leptin, adiponectin, insulin, glucose, and total lipid.

Human milk (HM) components are highly variable, and infants consume different volumes of milk. There has been little focus on evaluating the effectiveness of protocols in estimating infant intake of milk components before studying their impact on infant outcomes. Our goal was to compare 24 h measured intake with estimated intakes from different sampling protocols in order to determine the most accurate method for estimating infant intakes of milk leptin, adiponectin, insulin, glucose and total lipids. Mothers of term infants (n = 20) collected pre- and post-feed samples and measured their infant milk intake during each feed over a 24 h period using the test weighing method at 3–6 months postpartum. Infant true intakes of HM leptin, adiponectin, insulin, glucose and total lipids were calculated by averaging the measured pre- and post-feed concentrations and multiplying by the milk intake for the corresponding feed. Intakes were then summed to provide total intake of each component over 24 h. The estimated intakes were calculated with concentrations determined using five different sampling protocols, designed to be representative of sampling protocols used in previous HM component studies: (a) morning pre-feed sampling, (b) morning post-feed sampling, (c) average of morning pre- and post-feed sampling, (d) average of three pre-feed samples from the morning (06:00–09:00), afternoon (13:00–16:00) and evening (19:00–22:00) and (e) average of six pre- and post-feed samples from the morning (06:00–09:00), afternoon (13:00–16:00) and evening (19:00–22:00). The concentration from each protocol was further multiplied by true measured intake, a constant average intake of 800 mL/24 h and a global average milk intake of 766 mL/24 h to obtain the estimated intakes (15 protocols). The average intake of HM was 791 ± 212 mL. Comparison revealed that using the average measured concentration from three sets of pre- and post-feed samples, taken in the morning (06:00–09:00), afternoon (13:00–16:00) and evening (19:00–22:00), multiplied by either true infant 24 h measured intake, a constant estimate of milk intake (800 mL) or global average of milk intake (766 mL) provided the most accurate estimation of the infant’s intake for all components (p > 0.05). To obtain accurate estimates of HM leptin, adiponectin, insulin, glucose and total lipid intake in the absence of 24 h sampling, it is recommended to use a sampling protocol that involves taking samples before and after at least three breastfeeding sessions in a 24 h period.