Unravelling the effect of parity on immunoglobulins, cytokines and adipokines in human transitional milk and their association with infant infections during the first 6 months of life

Abstract

Background

Human milk dynamically adapts its composition of immunoglobulins (Igs), cytokines, and other proteins as lactation progresses, influencing the infant’s immune development and protection. Understanding how maternal factors, such as parity, influence the composition of human milk can provide strategies aimed at enhancing infant immune protection and reducing early-life infections. This study aims to investigate whether the immune composition of human milk differs based on parity, and if so, how these changes are related to infections in early life.

Methods

The study included 75 healthy mother-infant pairs from the MAMI cohort (Clinical Trial Registry NCT03552939), with milk samples collected from the same mothers at days 7 and 15 postpartum, during transitional lactation stage. Igs, cytokines, and adipokines were quantified using multiplex immunoassays and ELISA. A comparison was conducted between primiparous and multiparous mothers regarding both the overall and individual composition of immune components in human milk at each time point, as well as their evolution throughout the transitional phase.

Results

Infants from multiparous mothers recorded higher infection rates in early life than those of primiparous mothers. Some human milk immune components also differed by parity, with multiparous mothers exhibiting higher levels of IgA, total IgG, IgG1, IgG2, IgG3, IgE, and IL-23 at the beginning of the transitional phase (day 7), as well as higher IL-18 and IL-21 levels toward its end (day 15), compared to primiparous mothers. Additionally, the evolutionary pattern in levels of Igs, cytokines, and adipokines throughout the transitional milk stage also differed. Moreover, in multiparous mothers, higher levels of IgG, particularly IgG1 and IgG2 (day 7), as well as IL-18 and IL-22 (day 15), were associated with reduced infant infections, highlighting their potential protective role.

Conclusions

Parity is a maternal factor that influences some immune components of human milk during the transitional stage and may be linked to the susceptibility of infants to infections during the first 6 months of life. Future studies aimed at analyzing the impact of the parity factor, among others, on the progression of immune components in human milk may contribute to a better understanding and improved strategies for newborn health.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13006-025-00770-0.

Background

The immunological profile of human milk is dynamic, so the concentrations and proportions of immunoglobulins (Ig), cytokines, adipokines, and other bioactive proteins change as the milk matures and the infant’s immune system develops [1, 2], meeting infant’s needs [3, 4]. Its composition, including macronutrients and microbiota, changes in response to environmental and maternal factors [5, 6]. However, further studies are needed to confirm whether these factors also influence immune components of human milk. Among these immune factors, Igs are the most widely studied, and in particular, IgA, as the predominant Ig in human milk, has been evaluated for a long time. In contrast, less information is available about the presence and functionality of other Igs such as IgM, IgG, and their subtypes (IgG1, IgG2, IgG3, and IgG4), and IgE [7]. It is crucial to investigate not only IgA but also other types of Igs present in human milk, as recent evidence highlights their significant roles in the neonatal gut [7]. In this context, the transitional stage (from day 7 to day 15 of lactation) warrants particular attention, as it remains understudied and is characterized by significant dynamic changes [7, 8].

Ig levels decrease during breastfeeding and are higher in mothers whose infants have a more immature immune system, such as those born preterm. IgA, among other human milk components such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, is increased in the human milk of mothers who deliver preterm to provide protection to the infant and to accelerate the development of the neonatal immune system of the newborn [9–15]. Other maternal factors are key to the composition of human milk [16] and there is an interest in parity as it exerts a relevant influence on other mammalian milk [17, 18], but data on human milk are limited. Recent studies have identified parity-associated variations in the immune cell composition of human milk [19–21].

Newborns who live in environments with older siblings are exposed to a greater variety of viruses and bacteria, thereby increasing their susceptibility to infections [22]. In multiparous mothers, prior exposure of pathogens—often through their older children—may raise the likelihood of maternal infections, also at subclinical level. This, in turn, can stimulate the production of specific antibodies, which are subsequently transferred to the offspring both across the placenta during pregnancy and through human milk during lactation. This transfer could serve to protect the newborn by conferring passive immunity against environmental pathogens. Analysis of plasma samples from a subset of mothers within the MAMI cohort [23] revealed no significant differences in the composition of immune components (such as IgG and its subtypes) in either maternal or umbilical cord plasma between primiparous and multiparous mothers (data not shown). These findings suggest that, according to the parity factor, there is no apparent predominance in maternal transplacental transfer at the level of total IgG and its subclasses. Therefore, this study aims to determine whether the immune composition of human milk differs between primiparous (first-time mothers) and multiparous mothers, and also to assess the relationship between human milk immune components and infant infections during the first 6 months of life.

Methods

Participants

The present study included a subgroup of 75 healthy mother-infant pairs from the MAMI birth cohort (Clinical Trial Registry NCT03552939, date of registry: 2018-03-21; Recruitment started in 2015 and the follow-up was completed early 2019) [24]. Informed consent was obtained from all participants involved in the study. The MAMI cohort was conducted in accordance with the Declaration of Helsinki. The MAMI cohort was approved by CSIC and the Hospital Ethics Committees (HECs) (Hospital Clínico Universitario de Valencia).

MAMI cohort subsampling was based on the availability of human milk samples at two time points (days 7 and 15) during the transitional lactational stage. Details on inclusion/exclusion criteria were described elsewhere [24]. In brief, healthy women >18 with no reported chronic health conditions. Exclusion criteria included drug treatment (except for antibiotic use during pregnancy and/or delivery) and the presence of chorioamnionitis, mastitis, or health complications during gestation and breastfeeding.

Maternal-infant clinical, dietary and anthropometric records were available [24]. Data on mode of delivery, parity, maternal body mass index (BMI), antibiotic exposure, neonate sex, and others, were documented. Infections were recorded by clinicians, and data from the first 6 months of life were classified as respiratory-related (bronchiolitis, nasopharyngitis, otitis, upper respiratory tract infection, pneumonia, and colds) and gastrointestinal-related (gastroenteritis and diarrhea). Additionally, the secretor status of the mothers was determined. Total DNA was extracted following a previously described protocol [6] and utilized for FUT2 genotyping through polymerase chain reaction (PCR) combined with random fragment length polymorphisms (RFLPs), as outlined in another study [25]. Mothers were classified as secretors if the FUT2 gene, encoding galactoside 2-alpha-L-fucosyltransferase 2, was positively detected.

Collection of human milk

Milk samples were collected at multiple lactation time points. For this study, only samples corresponding to the transitional stage—collected on days 7 and 15 postpartum—were included. Mothers were instructed by clinicians to adhere to a specific protocol for sample collection, which involved morning collection prior to breastfeeding, cleansing of the breast skin using a 0.5% chlorhexidine solution or soap, and the utilization of a sterile pump as described previously [26, 27]. Moreover, the initial 2–3 drops were discarded to normalize the collection procedure, and approximately 10 mL of milk were collected. These samples were then sent to the biobank and stored at − 80 °C until the whey fraction was obtained through centrifugation at 2,000 × g for 15 min at 4 °C. Following this, the whey fraction was once again frozen at − 80 °C in separate aliquots to perform the different measurements.

Measurement of immunoglobulin, cytokine, and adipokine levels

The quantification of Igs (IgE, IgG1, IgG2, IgG3, IgG4), cytokines (Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interferon (IFN)-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17 A, IL-18, IL-21, IL-22, IL-23, IL-27, TNF-α), and adiponectin was carried out using the ProcartaPlex™ Multiplex immunoassay (Thermo Fisher Scientific, Vienna, Austria), by following established protocols in the manufacturer’s instructions as previously performed [8, 23]. Analysis was conducted on plates using a Luminex instrument and interpreted with ProcartaPlex Analyst Software (MAGPIX^® analyzer, Luminex Corporation) at the Flow Cytometry Unit of the Scientific and Technological Centers at the University of Barcelona (CCiT-UB). The assay sensitivity for each component was as follows: 2.11 ng/mL for IgG1; 16.07 ng/mL for IgG2; 0.08 ng/mL for IgG3; 0.56 ng/mL for IgG4; 0.003 ng/mL for IgE; 1.2 pg/mL for GM-CSF; 0.2 pg/mL for IFN-γ; 0.2 pg/mL for IL-1β; 0.8 pg/mL for IL-2; 1.5 pg/mL for IL-4; 0.3 pg/mL for IL-5; 0.4 pg/mL for IL-6; 0.5 pg/mL for IL-9; 0.1 pg/mL for IL-10; 0.04 pg/mL for IL-12p70; 0.1 pg/mL for IL-13; 0.1 pg/mL for IL-17 A; 0.4 pg/mL for IL-18; 0.6 pg/mL for IL-21; 8.2 pg/mL for IL-22; 0.9 pg/mL for IL-23; 5.1 pg/mL for IL-27; 0.4 pg/mL for TNF-α; and 4.6 pg/mL for adiponectin.

Leptin quantification was carried out using a Quantikine^® Colorimetric Sandwich ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s guidelines, in line with prior research protocols [8]. Data analysis was conducted using Multiskan Ascent v2.6 software (Thermo Fisher Scientific, Vienna, Austria). The assay sensitivity was 7.8 pg/mL.

Quantification of IgA and IgM was also performed utilizing ELISA kits from Bethyl Laboratories (Montgomery, TX, USA) and Cloud-Clone Corp. (Houston, TX, USA), respectively. Procedures followed the manufacturers’ instructions, in line with previous studies [8]. These kits enabled the measurement of both the secretory and non-secretory forms. The IgM assay sensitivity was 14.1 ng/mL, and that of the IgA assay was 1.03 ng/mL. Data analysis was performed using Multiskan Ascent v2.6 software (Thermo Fisher Scientific, Vienna, Austria).

Analysis and processing of data

The results are presented as mean ± SEM (standard error of the mean), unless otherwise stated. Normality and homogeneity of variance were assessed using the Shapiro-Wilk and Levene’s tests, respectively. The Student’s T-test and Mann-Whitney U test were employed to determine significant group differences, while the chi-square test (χ²) was utilized to compare frequencies. Spearman’s correlation coefficient was used to explore correlations between variables. To assess whether immune components levels changed over time within each parity group, Wilcoxon signed-rank tests were performed. Paired comparisons of levels between time points (d7 vs. d15) were conducted using the Wilcoxon signed-rank test with 0 as the hypothetical median of differences. Additionally, one-sample Wilcoxon signed-rank tests were used to compare the calculated d15/d7 ratios of each component against a hypothetical median of 1, representing no change over time.

The statistical analyses were conducted using IBM SPSS Statistics 27 (IBM, USA). Canonical correspondence analysis (CCA) was performed using the vegan package [28] in R version 4.4.0 (R foundation, Austria). Additionally, permutational analysis of variance (PERMANOVA) was conducted with the Adonis2 function from the same package.

Results

Description of maternal and infant characteristics

A total of 75 mother-infant pairs were classified into two groups according to parity: primiparous (n = 41) for first-time mothers and multiparous (n = 34) for mothers with previous childbirth experience. A total of 150 human milk samples were included in the study, n = 75 from day 7 and n = 75 from day 15.

The multiparous mothers had a higher BMI before birth (but within the normal weight range, BMI < 25) than the primiparous mothers (Table 1). In addition, the infants from multiparous mothers also weighed more and had a higher BMI z-score during the studied period (although in all cases within the normal range) (Table 1). With regard to the prevalence of infant infection, the offspring belonging to first-time mothers had significantly fewer infections and received fewer treatments with antibiotics in the first six months of life than those belonging to multiparous mothers (Fig. 1). Other maternal and infant characteristics — including maternal antibiotic use during pregnancy and on the day of delivery, gestational age, mode of delivery, exclusive breastfeeding, maternal secretor status, and infant sex — were comparable between parity groups.

Table 1.

Maternal and infant characteristics

Maternal characteristics	Primipara	n	Multipara	n	p value
Pre-gestational BMI (Kg/m2), 1 mean ± S.E.M.	21.75 ± 0.37	41	23.68 ± 0.69 *	34	0.017
Pregnancy weight gain (Kg), 1 mean ± S.E.M.	12.09 ± 0.61	41	11.51 ± 0.81	34	0.566
Antibiotic during pregnancy, 2 yes (%)	13 (31.71)	41	11 (32.35)	34	0.952
Intrapartum antibiotic, 2 yes (%)	13 (31.71)	41	16 (47.06)	34	0.174
Antibiotic during breastfeeding, 2 yes (%)	9 (21.95)	41	9 (26.47)	34	0.645
Gestational age (weeks), 1 mean ± S.E.M.	39.36 ± 0.22	41	39.75 ± 0.17	34	0.177
Mode of delivery: vaginal birth, 2 yes (%)	29 (70.73)	41	20 (58.82)	34	0.281
Exclusive maternal breastfeeding before 15th day, 2 yes (%)	33 (80.49)	41	29 (85.29)	34	0.584
Exclusive maternal breastfeeding until 6-month,2 yes (%)	29 (70.73)	41	28 (82.35)	34	0.241
Secretor status, 2 yes (%)	29 (70.73)	41	25 (73.53)	34	0.632
Infant characteristics
Sex: Female, 2 yes (%)	22 (53.66)	41	20 (58.82)	34	0.654
Birth weight (kg), 1 mean ± S.E.M.	3.13 ± 0.07	41	3.33 ± 0.08 #	34	0.061
7th day weight (kg), 1 mean ± S.E.M.	3.09 ± 0.05	41	3.34 ± 0.08 *	34	0.010
15th day weight (kg), 1 mean ± S.E.M.	3.35 ± 0.06	41	3.58 ± 0.09 *	34	0.034
1st month weight (kg), 1 mean ± S.E.M.	3.99 ± 0.08	41	4.17 ± 0.10	34	0.161
6th month weight (kg), 1 mean ± S.E.M.	7.31 ± 0.13	41	7.49 ± 0.14	34	0.353
Birth BMI z-score, 1 mean ± S.E.M.	-0.06 ± 0.14	41	-0.05 ± 0.16 *	34	0.005
7th day BMI z-score, 1 mean ± S.E.M.	-0.86 ± 0.12	41	-0.28 ± 0.16 *	34	0.005
15th day BMI z-score, 1 mean ± S.E.M.	-0.86 ± 0.13	41	-0.23 ± 0.17 *	34	0.004
1st month BMI z-score, 1 mean ± S.E.M.	-0.93 ± 0.13	41	-0.32 ± 0.17 *	34	0.006
6th month BMI z-score, 1 mean ± S.E.M.	-0.55 ± 0.13	41	-0.18 ± 0.1 4#	34	0.057

¹ p values were calculated with Student’s t-test. ² p values were calculated using a χ² test. *p < 0.05 comparing two groups. BMI, body mass index; S.E.M., standard error of the mean. #p < 0.1 comparing two groups

Fig. 1.

Number of infant accumulated infections (A) and number of antibiotic intake (B) from birth to 6 months of life. The total number of infections includes both gastrointestinal and respiratory infections. Results are expressed as mean ± S.E.M. n = 41 (Primipara group) and n = 34 (Multipara group). p values were obtained by Mann-Whitney U test

Variation of human milk immune composition according to parity-dependent factor

Multivariate canonical correspondence analysis (CCA) revealed that, when considering all immune components, the parity groups did not show a statistically significant separation at either day 7 or day 15 (Fig. 2A and B). However, Igs on day 7 tended to be affected by the parity factor but without statistically significance (F = 3.5208, p = 0.08) (Fig. 2C). With regard to cytokines and adipokines, these appeared to be more grouped on day 15, but without reaching statistical significance (Fig. 2F).

Fig. 2.

The impact of the parity factor on the global human milk immune composition. Canonical correspondence analysis (CCA) of the human milk samples according to parity factor based on all human milk immune components on day 7 (A) and on day 15 (B). CCA of the human milk samples according to parity factor based on human milk Igs on day 7 (C) and on day 15 (D). CCA of the human milk samples according to parity factor based on human milk cytokines and adipokines on day 7 (E) and on day 15 (F). PERMANOVA test is indicated in each figure

To study the changes in depth, each bioactive component was analyzed individually (Figs. 3, 4 and 5 and Table 2) and several associations were found between the immune components of human milk and the parity factor. First, having given birth once previously had an influence on human milk IgA levels (Fig. 3A). First-time mothers had significantly lower IgA levels than multiparous ones on day 7. Moreover, the primiparous mothers also showed significantly lower levels of IgG1, IgG2, IgG3, total IgG, and IgE on day 7 than the multiparous ones. Except for a lower IgG2 concentration in multiparous mothers, no changes were observed on day 15 compared to primiparous mothers (Fig. 3A).

Fig. 3.

Ig profiles in transitional milk stratified by parity. Concentration of Ig (mg/L) in human milk at day 7 and at day 15 from the same mothers (n = 75) regarding parity factor (primipara (n = 41) and multipara (n = 34) groups) (A). Changes of the human milk Ig over time in each parity group (from day 7 to day 15) (B). Relative proportions (%) of Ig in human milk at day 7 and at day 15 in each parity group (C). Data is expressed as mean ± S.E.M. *p < 0.05 and #p < 0.1 by Mann-Whitney U test comparing primipara vs. multipara groups. ^αp < 0.05 and ^βp < 0.1 by Wilcoxon test comparing time points within each parity group

Fig. 4.

Adipokine composition in transitional milk by parity. Concentration of adipokine-type cytokine (pg/mL) in human milk at day 7 and at day 15 from the same mothers (n = 75) regarding parity factor (primipara and multipara groups) (A). Changes of the human milk adipokines over time (from day 7 to day 15) in each parity group (B). Data is expressed as mean ± S.E.M. *p < 0.05 and #p < 0.1 by Mann-Whitney U test. ^αp < 0.05 and ^βp < 0.1 by Wilcoxon test comparing time points within each parity group. L/A, Leptin/Adiponectin

Fig. 5.

Changes of the human milk cytokines over time (from day 7 to day 15) in each parity group. Data is expressed as mean ± S.E.M. *p < 0.05 and #p < 0.1 by Mann-Whitney U test. ^αp < 0.05 and ^βp < 0.1 by Wilcoxon test comparing time points within each parity group

Table 2.

Concentrations of cytokines in human milk at day 7 and at day 15

	Primipara				Multipara
	d7		d15		d7		d15
	pg/mL	IQR	pg/mL	IQR	pg/mL	IQR	pg/mL	IQR
GM-CSF	1.85 ± 1.24	0.00–0.00	0.00 ± 0.00 α	0.00–0.00	0.00 ± 0.00 *	0.00–0.00	0.10 ± 0.10	0.00–0.00
IFN-γ	0.86 ± 0.39	0.00–0.00	0.65 ± 0.35	0.00–0.00	0.17 ± 0.11	0.00–0.00	0.81 ± 0.32 α	0.00–0.613
IL-1β	7.60 ± 4.25	0.00–3.76	1.71 ± 0.90 α	0.00–0.37	2.65 ± 1.11	0.00–1.37	16.10 ± 13.98	0.00–0.05
IL-2	2.46 ± 1.00	0.00–0.00	0.21 ± 0.15 α	0.00–0.00	0.39 ± 0.28 *	0.00–0.00	0.06 ± 0.06	0.00–0.00
IL-4	0.07 ± 0.05	0.00–0.00	0.01 ± 0.01	0.00–0.00	0.44 ± 0.30	0.00–0.00	0.00 ± 0.00	0.00–0.00
IL-5	0.91 ± 0.43	0.00–0.00	1.22 ± 0.81	0.00–0.00	0.35 ± 0.23	0.00–0.00	0.14 ± 0.14	0.00–0.00
IL-6	58.20 ± 26.54	0.00–57.02	31.24 ± 19.73 α	0.00–15.19	35. 16 ± 11.07	0.00–39.13	28.87 ± 11.09	0.00–21.57
IL-9	0.77 ± 0.43	0.00–0.00	0.20 ± 0.11	0.00–0.00	0.48 ± 0.31	0.00–0.00	0.01 ± 0.01	0.00–0.00
IL-10	1.96 ± 1.09	0.00–0.83	0.15 ± 0.06 α	0.00–0.15	0.24 ± 0.08	0.00–0.23	1.19 ± 0.45	0.00–1.17
IL-12	0.19 ± 0.08	0.00–0.14	0.11 ± 0.03	0.00–0.14	0.74 ± 0.68	0.00–0.02	0.11 ± 0.03	0.00–0.21
IL-13	0.08 ± 0.08	0.00–0.00	0.06 ± 0.04	0.00–0.00	0.00 ± 0.00	0.00–0.00	0.00 ± 0.00	0.00–0.00
IL-17	1.00 ± 0.43	0.00–0.00	0.11 ± 0.08 α	0.00–0.00	0.10 ± 0.06	0.00–0.00	0.22 ± 0.14	0.00–0.00
IL-18	21.11 ± 5.23	3.32–24.19	18.22 ± 4.68	2.42–22.26	10.97 ± 2.15	2.42–13.07	25.08 ± 4.56 # α	6.18–36.44
IL-21	5.63 ± 1.63	0.42–8.30	2.79 ± 0.49	0.097–3.86	18.40 ± 7.46	0.80–9.96	9.28 ± 2.71*	1.52–9.62
IL-22	8.61 ± 1.38	0.26–15.88	5.93 ± 1.13	0.00–10.56	7.23 ± 1.78	0.00–10.75	8.77 ± 2.31	1.79–9.24
IL-23	0.00 ± 0.00	0.00–0.00	0.25 ± 0.14	0.00–0.00	0.50 ± 0.25 *	0.00–0.00	0.00 ± 0.00 β	0.00–0.00
IL-27	0.52 ± 0.39	0.00–0.00	0.00 ± 0.00	0.00–0.00	0.00 ± 0.00	0.00–0.00	4.01 ± 4.01	0.00–0.00
TNF-α	3.03 ± 0.68	0.05–3.75	1.90 ± 0.31	0.51–2.05	2.04 ± 0.52	0.51–2.14	1.41 ± 0.25	0.01–2.31

Data shown are expressed as mean ± S.E.M., and interquartile ranges (IQRs). Mann-Whitney U test was used to determine significant differences and trends (*p < 0.05, #p < 0.1) between groups in the same sampling day (indicated in bold). Wilcoxon test was used to determine significant differences and trends between sampling days in the same parity group (^αp < 0.05 and ^βp < 0.1). GM-CSF, Granulocyte Macrophage Colony-Stimulating Factor; IFN, Interferon; IL, Interleukin; TNF, Tumor necrosis factor

The ratio of each component (d15/d7) was tested against the hypothetical value of 1 using a one-sample Wilcoxon signed-rank test (Fig. 3B). The IgA d15/d7 ratio was significantly lower than 1, suggesting a decline in IgA levels over time in both parity groups, associated with lactation progression [7]. Furthermore, the IgA d15/d7 ratio was significantly higher in primiparous mothers than in the multiparous group (Fig. 3B). Similarly, the d15/d7 ratio of total IgG, IgG1, IgG2 and IgE levels were also significantly higher in primiparous mothers than in the multiparous group (Fig. 3B). Moreover, in the primiparous group, the ratio of these components was significantly different from 1, indicating a significant increase in these components during the transition stage. In contrast, no significant change was observed in the multiparous group, suggesting stable levels over time (Fig. 3B). However, absolute levels of IgG, IgG2, IgG3, and IgE were significantly lower at day 15 compared to day 7 in multiparous mothers (Fig. 3A). The observed discrepancy may be due to the different analytical approaches: while the ratio d15/d7 reflects individual longitudinal changes (Fig. 3B), the direct comparison between time points (day 7 vs. day 15) captures overall group-level differences (Fig. 3A).

As regards the relative proportions of Igs normalized to total Igs levels in human milk, the primiparous group exhibited a higher percentage of IgG and IgE than the multipara group on day 15 (Fig. 3C). Additionally, relative IgM levels tended to be higher in the primiparous group on day 7, whereas the relative proportions of IgE and IgG1 tended to be lower in the primiparous group than in the multipara one on day 7 (Fig. 3C). These latter trends align with observations based on total concentrations (Fig. 3A).

With respect to the adipokines analyzed in human milk, similar levels were observed in both groups on the two sampling days (Fig. 4A). However, when evaluating changes from day 7 to day 15, significant differences emerged within and between the groups. Specifically, leptin levels increased from day 7 to day 15 in multiparous mothers (Fig. 4A and B), whereas the concentration remained stable over time in primiparous mothers (Fig. 4A and B). The evolution of adiponectin levels displayed opposite profiles, although this was not statistically significant. Consequently, the leptin/adiponectin (L/A) ratio also increased over time in the multiparous group and the d7/d15 ratio was significantly higher compared to the primipara group. (Fig. 4B).

In regard to cytokine levels, as expected there was generally low detectability, with IL-18, IL-21, IL-22, TNF-α, and IL-6 being the most frequently detected cytokines in both groups (Additional file 1). Furthermore, only specific differences were observed between groups (Table 2, Additional file 1, and Fig. 5). Notably, higher detectability of GM-CSF and IL-2 was observed on day 7 in the primiparous group than in the multipara one, with the former being statistically significant and the latter showing a trend. Additionally, the detectability of IL-23 and IL-21 was higher in the multipara group than in the primipara one on day 7 and on day 15, respectively (Additional file 1). In line with these results, GM-CSF and IL-2 levels were higher in the primiparous group than in the multiparous one on day 7, whereas IL-23 levels were higher in the multiparous group on day 7 and IL-21 levels were higher in the multiparous group than in the primiparous one on day 15 (Table 2). With regard to IL-18, although the detectability percentages were similar when comparing groups at both time points (Additional file 1), a trend toward increased levels was observed in the multiparous group compared to the primiparous group on day 15 (Table 2).

Furthermore, when analysing the evolution from day 7 to day 15 with the d7/d15 ratios, additional differences were observed within and between parity groups. For instance, IL-1β, IL-6, IL-10 and IL-12 levels decreased over time in the primiparous group, whereas they remained stable in the multiparous group, although with high inter-individual variability (Fig. 5). Importantly, IL-18 levels increased significantly over time in the multiparous group (Table 2) and the corresponding d7/d15 ratio was significantly higher than in the primiparous group (Fig. 5).

The relationship between immune component levels at both breastfeeding time points and the number of infant infections was assessed using Spearman correlation analysis (Fig. 6). When the participants were stratified by parity, significant correlations were only observed in the multiparous group. Notably, negative correlations were identified: Higher concentrations of IgG, IgG1, and IgG2 on day 7, as well as IL-18 and IL-22 on day 15, were linked to a reduced number of infections in infants. These findings highlight the potential importance of these immune components in protecting against infections during early infancy.

Fig. 6.

Correlations of human milk Ig and cytokines with infant infections from 0–6 months. The Spearman correlation coefficient is represented in the heat map following the color in the legend. Bold frames represent correlations with statistical significance (p < 0.05). M, month; d, day

Discussion

This study describes the effect of parity, a low studied factor in human milk research, on the immune composition of human milk, which in turn has a relevant impact on infant infections during the first 6 months of life.

With respect the clinical data of this study, it was observed that infants of multiparous mothers had higher BMI z-scores than those of primiparous mothers. This aligns with previous literature, in which a retrospective human cohort study reported that increasing parity is associated with a reduced risk of low birth weight [29]. It was also observed that multiparous mothers had a higher BMI than primiparous ones. While this observation is not fully established in the literature, previous studies have shown that maternal weight gain can influence the birth weight of their offspring [30]. It is worth noting that, although differences in BMI were observed in both mothers and offspring in this study, values remained within the normal range in all cases. The absence of data on milk macronutrients, represents a limitation of the present study, as these factors could potentially contribute to the observed differences in offspring BMI. Nonetheless, previous research has reported associations between parity and variations in lipid and human milk oligosaccharide concentrations [31–33]. Regarding infant infections, infants born from multiparous mothers showed a significantly higher incidence of infections and antibiotic use during the first six months of life compared to those born from primiparous mothers. This difference could be associated with a higher exposure to pathogens and the transmission of infections from older siblings, a risk that is not present in firstborn children [34].

As human milk adapts to meet the infant’s needs, it is reasonable to hypothesize that its immune components may vary depending on the parity factor. Some studies have reported higher levels of IgA, IgG isotypes, IgM, TGF-α, and other factors in colostrum samples from primiparous mothers than in those from multiparous ones [35, 36]. An association has also been found between the parity factor and the concentration of human milk oligosaccharides in colostrum [33]. However, in transitional and mature milk, a positive association between parity and lipid concentration has been observed, with no significant differences in total protein or IgA levels [31, 32].

In line with these findings, which suggest that the impact of the parity factor on human milk immune components may be more pronounced in early milk, our study demonstrated that parity had a stronger influence on the immune components, particularly Igs, on day 7, closer to the colostrum phase, than on day 15. Indeed, Ig levels in human milk are the highest in colostrum, and it is during this stage that their influence on the infant is expected to be most significant [7]. Therefore, observing a greater impact of the parity factor on Ig concentrations at the beginning of the transitional stage is consistent with this pattern. Specifically, higher levels of IgA, IgE, IgG, and its subtypes IgG1, IgG2 and IgG3 were observed on day 7 in multiparous than in primiparous mothers. This contrasts with findings from previous studies, which reported higher levels of IgA and IgG in primiparous mothers [35, 36]. However, those studies focused on colostrum, whereas the present study examined milk from the transitional phase. This distinction is relevant, as the timing of human milk analysis is critical due to its highly dynamic composition. Currently, few studies address this topic, and additional research is needed to draw robust conclusions. This area of study is important as these Igs, particularly IgA, play a critical role in neonatal defense and the maturation of the immune system [7]. This explains why IgA levels naturally decline throughout lactation, with the highest concentrations being found in colostrum, when the newborn’s immune system is developing [7, 8].

In the present study, we observed that in infants with siblings, who require greater early passive immunity, the physiological decline in human milk IgA levels during the transitional phase was more pronounced than in infants from the primiparous group, due to initially higher IgA concentrations Regarding total IgG and some of its subclasses, it is also noteworthy that their evolutionary profiles vary according to the parity factor. In this study, total IgG levels increased during the transitional stage in primiparous mothers, whereas they decreased in multiparous mothers. This pattern may be partly explained by the higher initial IgG concentrations observed in the latter group at the onset of the transitional phase. Maternal infections and even vaccinations can alter the levels of pathogen-specific IgA and IgG in human milk [37–41]. Thus, while this study focused on total Ig levels, it would be valuable to investigate their specificity to particular pathogens, especially those responsible for infections in the mother’s environment and among older siblings.

IgE is the primary Ig involved in allergic processes and is extensively studied in infant plasma to better understand the mechanisms of tolerance acquisition and its association with allergic manifestations in early life. However, limited information is available regarding IgE concentrations in human milk and its potential role in these contexts [7]. Additionally, IgE is present at lower concentrations in human milk than in other Igs [7]. In this study only two cases of infants with dermatitis/allergy were recorded and were from the primipara group. Even though, the observation that IgE levels were also elevated in the milk of multiparous mothers is intriguing and warrants further investigation.

The human milk adipokines leptin and adiponectin are known to play critical roles in development and growth [42, 43], as well as in immunomodulation by regulating both innate and adaptive immune responses [44–46]. In this study, human milk leptin levels increased over time in the multiparous group, whereas they remained stable in primiparous mothers. These findings are particularly interesting due to their potential association with body weight and growth data observed in both mothers and infants from the multiparous group. Similarly, a recent study involving 67 mother-infant pairs found that human milk provided to newborns classified as small for their gestational age (SGA) contained lower levels of leptin than that provided to non-SGA infants [47].

Cytokines are minor components of human milk that are present in low concentrations, often with limited detection rates [8, 10, 48]. Current knowledge regarding the influence of external factors on cytokine levels is still limited. In this study, we also aimed to assess the impact of the parity factor on cytokine levels. Our findings indicate that it significantly influences the cytokine profile in human milk during the transitional phase. Multiparous mothers exhibited higher IL-23 levels at day 7 compared to primiparous mothers, as well as elevated IL-18 and IL-21 levels at day 15. Detection of IL-23 was limited in some cases, in contrast to IL-18 and IL-21, which showed good detectability in the human milk samples of this study. Additionally, a pronounced effect is seen in the progression between day 7 and day 15 for certain cytokines. Specifically, the most notable change in progression was observed in IL-18, which exhibited a significantly higher increase over the transitional period in multiparous mothers compared to primiparous mothers, and was also the most consistently detected cytokine across all samples. This cytokine was suggested to play an important role in infant defense against pathogens [49]. In line with this, higher levels of IL-18 have been observed in colostrum than in mature milk [49], reflecting the greater need for immune protection in newborns during the early stages of life. The elevated levels observed in multiparous mothers on day 15, along with the increase during the transitional phase, may suggest that these mothers provide higher amounts of this immune component to their infants, potentially due to their exposure to an environment with a greater prevalence of infections.

Furthermore, in multiparous mothers, higher levels of IgG—particularly the IgG1 and IgG2 subtypes—in human milk on day 7, as well as elevated levels of IL-18 and IL-22 on day 15, were associated with a reduced incidence of infections in their infants during the first six months of life. This finding highlights the potential key role of these immune components in human milk, thereby warranting further investigation.

Although the present study offers novel insights into the factors that may influence the immunological composition of human milk, it had some limitations. A limitation of this study is the absence of longitudinal data on the progression of these immune components and their potential association with maternal parity, as well as the lack of extended follow-up regarding infant infections. Further research including additional sampling time points and longer clinical monitoring would be valuable to address these aspects. Furthermore, the cellular fraction of breast milk—an essential contributor to the infant’s immune development—was not characterized. Moreover, the antigen specificity of the Igs was not determined, which limits our understanding of how these antibodies may contribute to protecting infants at mucosal surfaces.

Conclusion

These findings indicate that, beyond IgA, other Ig types, cytokines, and adipokines in human milk can be influenced by maternal characteristics. Specifically, multiparous mothers exhibited a distinct immune profile in their human milk during the transitional lactation period, with higher levels of IgA, IgE, total IgG, its subtypes IgG1, IgG2, and IgG3, and IL-23 early in this stage and with higher levels of IL-18 and IL-21 at the end of the transitional phase. Moreover, the progression of these immune components during the transition stage differs between multiparous and primiparous mothers. Furthermore, among multiparous mothers, higher IgG levels in human milk on day 7 and higher levels of IL-18 and IL-22 on day 15 were associated with fewer infections in their infants during the first six months of life, suggesting a key role for these immune components. However, the contribution of human milk immune factors to the relationship between parity, infant growth, and infections is still not fully understood, thus warranting further research in this area.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

BMI

Body mass index

CCA

Canonical correspondence analysis

GM-CSF

Granulocyte-macrophage colony-stimulating factor

IFN

Interferon

Ig

Immunoglobulin

IL

Interleukin

PERMANOVA

Permutational analysis of variance

SEM

Standard error of the mean

TNF

Tumor necrosis factor

Author contributions

K.R.-A. conducted the experiments, analyzed the data, and wrote the main manuscript text. C.M.-C., M.C., and M.J.R.-L. contributed to the investigation and reviewed the manuscript. M.C.C. and F.J.P.-C. conceptualized the study, acquired funding, and supervised the project. All authors reviewed and approved the final manuscript.

Funding

This research was supported by La Marató-TV3 (DIM-2-ELI, ref. 2018-27/30–31), the European Research Council under the European Union’s Horizon 2020 research and innovation program (ERC Starting Grant, no. 639226, MAMI), and Spanish Ministry of Science and Innovation (MCIN) research grant (PID2022-139475OB-I00, (MAMI+). M.C.C. acknowledges the support from Horizon Europe Program (ref. 101094099, INITIALISE). K.R.-A. was the recipient of a fellowship from the Spanish Ministry of Economy, Industry, and Competitiveness (FPU 19/05150).

Data availability

All data are available from the corresponding author upon request.

Declarations

Competing interests

The authors declare no competing interests.