The impact of breast milk relaxin 2, oxytocin, and insulin‐like peptide 3 on neonatal growth parameters in early life

Abstract

Objectives

This study aims to quantify the levels of relaxin 2 (RLX2), oxytocin (OXT), and insulin‐like peptide 3 (INSL3) in colostrum (postpartum days 1–5, Visit 1) and mature breast milk (postpartum days 21–35, Visit 2), and to evaluate their associations with neonatal growth outcomes.

Methods

During two postpartum visits, anthropometric measurements were obtained from 60 healthy infants, and the concentrations of RLX2, OXT, and INSL3 in both foremilk and hind milk were analyzed.

Results

At both Visit 1 and Visit 2, post‐breastfeeding body weight and body mass index (BMI) were significantly higher than pre‐breastfeeding values. Furthermore, all anthropometric measurements, including head circumference, length, and pre‐ and post‐breastfeeding body weight and BMI, were significantly greater at Visit 2 compared to Visit 1. In foremilk, concentrations of RLX2, OXT, and INSL3 were significantly lower at Visit 2 than at Visit 1 (p = 0.001, p = 0.001, and p = 0.05, respectively). Similarly, in hindmilk, RLX2 and OXT concentrations were significantly lower at Visit 2 than at Visit 1 (p = 0.001 for both). In contrast, INSL3 concentrations in hindmilk did not differ between visits (p > 0.05). Breastmilk OXT and INSL3 were positively correlated with BMI, length, and head circumference, whereas RLX2 concentrations were not significantly associated with these outcomes.

Conclusions

The findings of this study suggest that RLX2, OXT, and INSL3 concentrations in breast milk show significant changes during lactation. Furthermore, it suggests that OXT, and more prominently INSL3, are associated with neonatal growth.

What is Known

• Temporal variations of relaxin 2 (RLX2), oxytocin (OXT), and insulin‐like peptide 3 (INSL3) in foremilk and hindmilk across lactation stages have not been investigated.

• No studies have examined associations between neonatal growth and breast milk RLX2, OXT, and INSL3.

What is New

• Breast milk RLX2, OXT, and INSL3 differ significantly between colostrum (days 1–5) and mature milk (days 21–35).

• RLX2 appears to have no direct impact on neonatal growth.

• OXT, and more prominently INSL3, are associated with early‐life growth parameters, including body mass index, linear growth, and head circumference.

1. INTRODUCTION

Breast milk represents one of the earliest and most effective forms of mother–infant communication. In addition to facilitating mother–infant bonding, it contains biologically active compounds, including various trace elements, proinflammatory cytokines, growth factors, hormones, and peptides, which are likely to have multiple biological effects on infant development. ¹ , ² , ³ In humans, relaxin peptides (RLX1, RLX2, and RLX3) and insulin‐like peptides (INSL3, INSL4, INSL5, and INSL6) are encoded by genes exhibiting homologous structures and generating conserved amino acid sequences. ⁴ Among these peptides, only RLX2 and INSL3 have been identified in human breast milk. ⁵ In both canine and porcine species, RLX derived from breast milk has been demonstrated to enter the neonatal circulation during the lactation period. ⁶ , ⁷ In lactating rats, RLX infusion was found to suppress reflex milk ejection and reduce pup weight gain compared with controls. Following cessation of RLX infusion, maternal plasma oxytocin (OXT) levels increased significantly, intramammary pressure remained persistently elevated, and pups exhibited accelerated weight gain compared with controls, despite no observed difference in the frequency of milk ejection. ⁸ Another study has demonstrated that RLX present in breast milk may play a significant role in the neonatal development of pigs. ⁹ Finally, RLX has been demonstrated to affect drinking behavior in non‐pregnant rats, suggesting that RLX present in breast milk may similarly contribute to neonatal weight gain by modulating this behavior. ¹⁰

INSL3 is a member of the insulin‐like peptide superfamily, characterized by A and B chains connected via three disulfide bonds. Although INSL3 is produced primarily by testicular Leydig cells and ovarian theca cells in mammals, it has also been shown to be secreted from mammary tissue into breast milk. ⁵ , ¹¹ , ¹² Although animal studies have not yet confirmed the passage of INSL3 from breast milk into the neonatal circulation, the 52% identity and 62% similarity of the three‐dimensional structures of RLX2 and INSL3, and the homologous alignment between the human INSL3 A‐chain and RLX2, suggest that INSL3 may similarly cross the neonatal gastrointestinal tract and enter the bloodstream. ⁴ In vivo, INSL3 exerts its physiological actions via its cognate receptor, relaxin/insulin‐like family peptide receptor 2 (RXFP2), which is classified within the G protein‐coupled receptor superfamily. ¹³ RXFP2 expression has been identified in a variety of extragonadal tissues and organs, including seminal vesicles, thyroid gland, kidneys, brain, bone marrow, skeletal muscle, and bone, suggesting that INSL3 may have pleiotropic biological functions that extend beyond its well‐characterized role in reproductive physiology. ¹¹ , ¹⁴ These findings imply the possibility that INSL3 contributes to the regulation of diverse physiological processes such as metabolism, neuroendocrine signaling, and tissue remodeling.

OXT is a neuropeptide produced primarily in the supraoptic and paraventricular hypothalamic nuclei and is released into the bloodstream via the posterior pituitary gland. Human milk is known to contain OXT. ⁵ Animal studies have demonstrated that OXT present in maternal circulation can be stably transferred into breast milk, and that OXT in breast milk can be absorbed in its intact form through the neonatal gastrointestinal tract, subsequently entering the systemic circulation of the newborn. ¹⁵ , ¹⁶ Historically, OXT secretion has been most prominently associated with the roles of facilitating uterine contractions during labor and mediating the milk letdown reflex during breastfeeding. However, recent research suggests that OXT secretion may be related to appetite regulation and satiety. ¹⁷ , ¹⁸ , ¹⁹

In light of the aforementioned findings, it can be assumed that RLX2, OXT, and INSL3 in breast milk may contribute to neonatal growth. To our knowledge, these relationships have never been studied before. The aim of this study was to investigate the variations of RLX2, OXT, and INSL3 levels in foremilk and hindmilk during both the colostrum and mature milk stages, and to evaluate the hypothesis that these breast milk‐derived peptides may contribute to neonatal growth.

2. METHODS

2.1. Ethics statement

This study does not include identifying details of human participants (names, date of birth, identity numbers, biometrical characteristics (such as facial features, fingerprints, deoxyribonucleic acid, or other distinguishing characteristics). The study was conducted in accordance with the ethical principles stated in the “Declaration of Helsinki,” and the study protocol was approved by the ethics committee of the Ataturk University Faculty of Medicine (Approval No. 76, Year: 2023). The written informed consent was obtained from the parents.

2.2. Subjects

The study was conducted at the Department of Pediatrics, Ataturk University, between November 2023 and January 2025. Newborns were stratified as small for gestational age (SGA), appropriate for gestational age (AGA), or large for gestational age (LGA) according to their birth weight plotted against gestational age on the World Health Organization (WHO) Growth Charts. SGA were defined as newborns with a birth weight below the 10th centile, AGA between the 10th and 90th centiles, and LGA above the 90th centile. ²⁰ Inclusion criteria were term deliveries with AGA after uncomplicated pregnancies. Exclusion criteria included multiple gestation, delivery in the last 2 years, pre‐existing maternal disease, fetal malformation, pre‐eclampsia, body mass index (BMI) > 35.0 at enrollment, hypertension, smoking, any hormone or drug therapy, lactation failure, deliveries with LGA or SGA, and any neonates who received formula feeding at any point during the study period.

Milk samples from mothers and anthropometric measurements of infants were collected by the same pediatric endocrinologist during two visits conducted between postpartum days 1–5 (Visit 1) and 21–35 (Visit 2). Milk samples collected at Visit 1 consisted of colostrum, representing the initial phase of lactation, while samples taken at Visit 2 consisted of mature milk, the late phase of lactation characterized by a more balanced composition of nutrients and bioactive factors. ²¹ Milk sample collection was standardized according to milk volume and time: In the morning, mothers were initially allowed to breastfeed their infants, after which they were provided with a breakfast consisting of bread, butter, olives, cheese, and tea. Breastfeeding was then scheduled to occur 3 h after breakfast, and 5.0 mL milk samples were collected from both breasts with a plastic pump, both before (foremilk) and after (hindmilk) breastfeeding. The samples were vortexed and frozen in Eppendorf tubes at −20°C until analysis. During each visit, height and head circumference were measured once. Body weight was recorded twice, both prior to (pre‐breastfeeding) and following (post‐breastfeeding) the feeding session, in order to evaluate whether breast milk hormones are associated with milk intake. Weight change during the feeding session (post–pre‐breastfeeding) was used as an estimate of milk intake. The infants' diapers were examined before feeding to verify dryness. In cases where urination or defecation occurred during feeding, body weight measurements were taken after feeding while the diaper was soiled with urine or feces. Weight, height, and head circumference measurements were performed using an electronic scale (Seca Model 770), infantometer, and tape measure with 5 g, 0.1 mm, and 0.1 mm precision, respectively. Body mass index was calculated as body weight (kg)/square of length (m²). Changes in all parameters between Visit 1 and Visit 2 were defined as delta values. In addition, the pre‐ to post‐feeding changes in body weight and BMI at each visit were defined as visit‐specific delta body weight and delta BMI. Head circumference growth velocity and length growth velocity were calculated by dividing the delta head circumference and delta length values by delta age (days).

2.3. Breast milk INSL3, OXT, and RLX2 concentrations

The milk samples were kept in a refrigerator overnight with the purpose of thawing before analysis. Analyses were performed on skim milk to ensure that the fat content of breast milk did not affect the measurements. For this purpose, milk samples were ultra‐centrifuged at 15,000 rpm for 10 min at +4°C to separate milk fat. The fat layer was removed by using a spatula. Thus, skim milk was obtained. The skim milk samples were used for the assay.

2.4. INSL3 measurement

A commercial enzyme‐linked immunosorbent assay (ELISA) kit (Human Insulin‐Like 3 INSL3 ELISA Kit, E3046Hu; Laboratory of Biological Analysis Technology) designed to measure INSL3 in serum, plasma, and other biological fluids, such as breast milk, was used. The standard curve range is 0.2–60 ng/mL, and the sensitivity limit is 0.11 ng/mL. The inter‐assay and intra‐assay coefficients of variation are <10% and <8%, respectively.

2.5. OXT measurement

A commercial ELISA kit (Human Oxytocin ELISA Kit, E1046Hu, Laboratory of Biological Analysis Technology), produced for measuring the level of human OXT in serum, plasma, cell culture supernatant, acidic fluids, tissue homogenate, and other biological fluids, was used to measure the level of OXT in breast milk. The standard curve range is 2–600 pg/mL, and the sensitivity limit is 1.06 pg/mL. The inter‐assay and intra‐assay coefficients of variation are <10% and <8%, respectively.

2.6. RLX2 measurement

The concentration of RLX2 in breast milk samples was measured using a commercial ELISA kit (Human Relaxin ELISA Kit, E1324Hu; Bioassay Technology Laboratory) produced for the measurement of human RLX‐2 in serum, plasma, cell culture supernatant, acidic fluids, tissue homogenate, and other biological fluids, including breast milk. The standard curve range is 10–3000 ng/mL, and the sensitivity limit is 5.13 ng/mL. The inter‐assay and intra‐assay coefficients of variation are <10% and <8%, respectively.

2.7. Statistical analysis

All calculations were made using SPSS for Windows (Version 25.0 for Windows). The Shapiro–Wilk test was used to analyze whether the data in the study were normally distributed. Normally distributed data at the first and second visits were compared using the paired samples t test, and non‐normally distributed data were compared using the Wilcoxon signed rank test. Correlations between variables were tested with Pearson's or Spearman's correlation coefficient according to whether the data were normally distributed. Data with normal distribution were expressed as mean ± standard deviation (SD), and data without normal distribution were expressed as median (minimum–maximum). p < 0.05 was considered statistically significant.

3. RESULTS

The study was completed with 63 infant–mother pairs (31 [49.2%] boys and 32 [50.8%] girls). All neonates had a healthy appearance, no signs of fetal distress, and Apgar scores of 7 or more at 1 and 5 min. Forty‐nine (77.8%) of the newborns were born by normal spontaneous vaginal delivery, and 14 (22.2%) were born by cesarean section. The mean ages of the infants at the first and second visits were 1.42 ± 0.66 days and 28.73 ± 3.77 days, respectively.

Anthropometric data at Visits 1 and 2 are shown in Table 1. Post‐breastfeeding body weight and BMI increased significantly compared to pre‐breastfeeding at both Visits 1 and 2 (p = 0.024 and p = 0.022 for Visit 1; p = 0.001 and p = 0.001 for Visit 2, respectively). Head circumference and length increased significantly at Visit 2 compared to Visit 1 (p = 0.001 and p = 0.001, respectively) (Table 1). At Visit 2, pre‐breastfeeding and post‐breastfeeding body weight and BMI increased significantly compared to Visit 1 (p = 0.001 and p = 0.001 for pre‐breastfeeding; p = 0.001 and p = 0.001 for post‐breastfeeding, respectively) (Table 1).

Table 1.

Anthropometric data at Visits 1 and 2.

Parameters
Mothers
Age (years)	32.2 ± 1.4
Parity	1.42 ± 0.66
Gestational age (weeks)	38.5 ± 0.3
Infants
	Visit 1 (postnatal 1–5 days)
HC (cm)	34.24 ± 0.95a
Length (cm)	49.59 ± 1.61b
	Pre‐breastfeeding	Post‐breastfeeding	p
Body weight (g)	3073.01 ± 349.49c	3077.46 ± 348.79e	0.024
BMI (kg/m2)	12.46 ± 0.94d	12.48 ± 0.95f	0.022
	Visit 2 (postnatal 21–35 days)
HC (cm)	37.05 ± 0.98a
HC growth velocity (cm/d)	0.10 ± 0.02
Length (cm)	53.85 ± 1.69b
Length growth velocity (cm/d)	0.15 ± 0.03
	Pre‐breastfeeding	Post‐breastfeeding	p
Body weight (g)	4156.58 ± 414.51c	4224.04 ± 422.51e	0.001
BMI (kg/m2)	14.30 ± 1.00d	14.53 ± 1.02f	0.001

Note: Data are presented as mean ± standard deviation.

Abbreviations: BMI, body mass index; HC, head circumference.

^aComparison of HC between visits, p < 0.001.

^bComparison of length between visits, p < 0.001.

^c Comparison of pre‐breastfeeding body weight between visits, p < 0.001.

^d Comparison of pre‐breastfeeding BMI between visits, p < 0.001.

^e Comparison of post‐breastfeeding body weight between visits, p < 0.001.

^f Comparison of post‐breastfeeding BMI between visits, p < 0.001.

For both colostrum and mature milk, there was no difference in RLX2, OXT, and INSL3 levels between foremilk and hindmilk (p > 0.05). While colostrum foremilk INSL3 levels were significantly higher than mature foremilk levels (p = 0.05), colostrum hindmilk INSL3 levels were not different from mature hindmilk levels (p > 0.05) (Table 2). Colostrum foremilk and hindmilk OXT and RLX2 concentrations were significantly higher than mature foremilk and hindmilk concentrations (p = 0.001 and p = 0.001 for foremilk; p = 0.001 and p = 0.001 for hindmilk, respectively) (Table 2).

Table 2.

Foremilk and hindmilk biochemical parameters in colostrum and mature milk.

Parameters	Colostrum	Mature milk		Colostrum	Mature milk
	Foremilk	Foremilk	p	Hindmilk	Hindmilk	p
RLX2 (ng/mL)	541.0 (74.0–1416.0)	90.0 (65.0–896.0)	0.001	459 (81.0–1283.0)	86.0 (61.0–666.0)	0.001
OXT (pg/mL)	152.0 (19.3–331.7)	21.2 (8.6–248.7)	0.001	148.7 (19.0–273.3)	14.1 (8.75–193.3)	0.001
INSL3 (ng/mL)	6.4 (0.68–25.0)	3.12 (0.70–21.6)	0.05	6.87 (0.72–26.4)	6.18 (0.66–18.0)	0.162

Note: Data are presented as median (minimum–maximum) values.

Abbreviations: INSL3, insulin‐like peptide 3; OXT, oxytocin; RLX2, relaxin 2.

No significant correlations were identified between pre‐ and post‐breastfeeding breast milk RLX2 levels at either visit and any neonatal growth parameters, either in visit‐specific analyses or in analyses across visits (all p > 0.05). Likewise, OXT and INSL3 concentrations in colostrum and mature milk (both foremilk and hindmilk) at either visit were not associated with visit‐specific delta body weight or delta BMI (all p > 0.05).

At Visit 1, pre‐ and post‐breastfeeding BMI were positively correlated with colostrum hindmilk OXT (r = 0.302, p = 0.016 and r = 0.304, p = 0.015, respectively). As pre‐ and post‐breastfeeding BMI values showed close agreement, only the association with pre‐breastfeeding BMI is presented (Figure 1A). In addition, delta length was positively correlated with delta foremilk OXT (r = 0.287, p = 0.024) (Figure 1B). However, no significant correlations were identified between OXT concentrations in colostrum foremilk or mature milk (foremilk and hindmilk) and pre‐ and post‐breastfeeding body weight or BMI at either visit (all p > 0.05). In addition, no correlations were found between foremilk and hindmilk OXT levels in colostrum and mature milk and delta body weight, length, delta length, length growth velocity, head circumference, delta head circumference, or head circumference growth velocity (all p > 0.05).

Figure 1.

Correlations between OXT and BMI and delta length. (A) Positive correlation between Visit 1 pre‐breastfeeding BMI and colostrum hindmilk OXT (r = 0.302, p = 0.016). (B) Positive correlation between delta length and delta colostrum foremilk OXT (r = 0.287, p = 0.024). BMI, body mass index; OXT, oxytocin.

At Visit 1, pre‐ and post‐breastfeeding BMI showed positive correlations with colostrum foremilk INSL3 (r = 0.311, p = 0.013 and r = 0.328, p = 0.009, respectively). Given the minimal difference between pre‐ and post‐breastfeeding BMI values, only the association with pre‐breastfeeding BMI is presented (Figure 2A). Delta pre‐ and post‐breastfeeding BMI were negatively correlated with delta foremilk INSL3 (r = −0.260, p = 0.041 and r = −0.277, p = 0.030, respectively). As delta pre‐ and post‐breastfeeding BMI values showed close agreement, only the association with delta pre‐breastfeeding BMI is presented (Figure 2B). Length growth velocity and delta length were positively correlated with colostrum foremilk INSL3 (r = 0.322, p = 0.010 and r = 0.253, p = 0.046, respectively) (Figure 2C,D). Head circumference growth velocity and delta head circumference were negatively correlated with delta hindmilk INSL3 (r = −0.311, p = 0.013 and r = −0.284, p = 0.024, respectively) (Figure 2E,F). However, no significant correlations were identified between INSL3 levels in colostrum hindmilk as well as in mature milk (foremilk and hindmilk) and pre‐ and post‐breastfeeding body weight or BMI at either visit, delta body weight, delta BMI, length, delta length, length growth velocity, head circumference, delta head circumference, or head circumference growth velocity (all p > 0.05). Furthermore, no significant correlations were observed between colostrum foremilk INSL3 concentrations and anthropometric parameters at Visit 2, including pre‐ and post‐breastfeeding body weight and BMI, visit‐specific or overall changes in body weight and BMI, length and length growth velocity, or head circumference and head circumference growth velocity (all p > 0.05).

Figure 2.

Correlations between INSL3 and BMI, length, and head circumference. (A) Positive correlation between Visit 1 pre‐breastfeeding BMI and colostrum foremilk INSL3 (r = 0.311, p = 0.013). (B) Negative correlation between delta pre‐breastfeeding BMI and delta foremilk INSL3 (r = −0.260, p = 0.041). (C) Positive correlation between length growth velocity and colostrum foremilk INSL3 (r = 0.322, p = 0.010). (D) Positive correlation between delta length and colostrum foremilk INSL3 (r = 0.253, p = 0.046). (E) Negative correlation between head circumference growth velocity and delta hindmilk INSL3 (r = −0.311, p = 0.013). (F) Negative correlation between delta head circumference and delta hindmilk INSL3 (r = −0.284, p = 0.024). BMI, body mass index; INSL3, insulin‐like peptide 3.

4. DISCUSSION

The novelty of the four‐sampling approach employed in our study lies in the ability to characterize the profiles of RLX2, OXT, and INSL3 concentrations in foremilk and hindmilk, each with different compositions, in both colostrum and mature milk stages. To the best of our knowledge, no longitudinal study has been conducted to investigate the temporal variations of these peptides in foremilk and hindmilk at different stages of lactation, nor has any study explored the association between their dynamic changes in breast milk composition and neonatal growth. The findings of our study suggest that breast milk RLX2, OXT, and INSL3 levels change significantly with the progression of lactation, and breast milk OXT and, more strikingly, INSL3 are associated with growth parameters of newborns during lactation, but RLX2 levels do not show such a relationship.

To date, the variation of RLX levels in human breast milk throughout lactation has not been investigated. In the present study, we observed no significant difference in RLX concentrations between foremilk and hindmilk within a single breastfeeding session, both in colostrum and in mature milk. In contrast, both colostrum foremilk and hindmilk showed significantly higher RLX concentrations than their respective mature milk counterparts. This observation may contribute to a better understanding of RLX dynamics during lactation and serve as a basis for future research in this area. Although RLX2 concentrations in mature foremilk and hindmilk were observed to be significantly lower than those in colostrum, these differences did not exhibit a significant association with any neonatal growth parameters. These results suggest that breast milk RLX2 may not exert a direct influence on neonatal growth.

A study investigating OXT levels in human breast milk found that OXT remained stable until postnatal Day 4 and began to decrease thereafter. ²² In another study, no significant change in OXT levels was observed before and after a breastfeeding session. ²³ With this study, we similarly observed no significant difference in OXT concentrations between foremilk and hindmilk within a single breastfeeding session, both in colostrum and in mature milk. Nevertheless, OXT concentrations in both colostrum foremilk and hindmilk were markedly higher than those observed in the corresponding mature milk samples. These findings highlight a potential physiological difference that warrants further investigation in future studies.

Animal studies have demonstrated that food intake stimulates the release of OXT from hypothalamic OXT neurons. ²⁴ Additionally, OXT secretion can be elicited by sensory stimuli associated with common environmental food cues, including olfactory, visual, and auditory inputs. ¹⁷ Peripheral concentrations of appetite‐regulating hormones show significant interindividual variability. ²⁵ Furthermore, existing studies comparing individuals with elevated BMI to those with healthy weight have reported inconsistent findings regarding OXT levels, observing increases, decreases, or no significant changes. ¹⁷ , ¹⁸ These conflicting findings may be attributable to interactions with comorbidities such as diabetes, metabolic syndrome, and anorexia nervosa or may result from methodological differences between studies. However, studies conducted on men and women, excluding individuals with chronic diseases, found that plasma OXT levels were positively correlated with BMI. ¹⁷ , ²⁶ , ²⁷ Studies investigating the effects of maternal OXT levels on neonatal growth remain limited. One study reported that higher OXT levels in maternal plasma on postpartum Days 4–5 were associated with less weight loss in newborns. ¹⁹ In addition to these findings, the presence of OXT in breast milk, the observed increase in OXT levels in both maternal plasma and breast milk during lactation, and the demonstration of the stable absorption capacity of breast milk‐derived OXT into the neonatal circulation suggest a potential role for breast milk OXT in regulating neonatal growth parameters. ⁵ , ¹⁵ , ¹⁶ , ²⁸ , ²⁹ To the best of our knowledge, this study is the first to explore the association between breast milk OXT levels and growth parameters in exclusively breastfed neonates. In our study, significant positive correlations observed between colostrum hindmilk OXT levels and both pre‐ and post‐breastfeeding BMIs at Visit 1, as well as between delta foremilk OXT levels and delta length, suggest that breast milk OXT may influence neonatal growth.

INSL3 is produced by Leydig cells in male mammals and by ovarian theca cells derived from antral follicles and luteal cells, which are considered the functional counterparts of Leydig cells, in female mammals. ¹⁴ , ³⁰ INSL3 is constitutively secreted by Leydig cells, independently of acute regulation by the hypothalamic–pituitary–gonadal axis, and is therefore considered a reliable biomarker of Leydig cell function and differentiation status, as well as a reflection of Leydig cell activity and population size. Accordingly, INSL3 levels exhibit a transient surge during fetal development, followed by a second peak approximately 3 months postnatally—referred to as “mini‐puberty”—a third elevation during puberty, and a gradual decline with advancing age. ¹⁴ Although mutations in the INSL3 or RXFP2 genes have been associated with cryptorchidism, experimental studies suggest that reduced INSL3 expression is a consequence rather than a cause of this association and serves as a surrogate marker for the underlying factor responsible for the endocrine disorder. ¹⁴ Similar to males, circulating INSL3 levels in females have been shown to rise during late adolescence, fluctuate throughout the menstrual cycle in response to follicle‐stimulating hormone, decline with advancing age, and become undetectable after menopause. Additionally, elevated INSL3 concentrations have been identified as a marker of theca cell activity in pathological conditions such as polycystic ovary syndrome. ³⁰ Although INSL3 is primarily associated with reproductive function, the detection of its receptor RXFP2 in non‐gonadal tissues such as seminal vesicles, thyroid, kidneys, brain, bone marrow, muscle and bone, and the expression of INSL3 in tubuloalveolar epithelial cells of human breast tissue at both messenger ribonucleic acid and protein levels, suggest that INSL3 may exert additional endocrine functions beyond the reproductive system. ⁵ , ¹¹ , ¹⁴ Indeed, stimulation of primary osteoblast cell lines derived from mice or humans with INSL3 has been shown to induce intracellular cyclic adenosine monophosphate accumulation and activate mitogen‐activated protein kinase and extracellular signal‐regulated kinases 1 and 2 signaling pathways, which are involved in promoting cell proliferation and upregulating genes associated with bone mineralization, including Collagen‐6A1, osteonectin, osteocalcin, and alkaline phosphatase. ³¹ , ³² Recent findings have shown that INSL3 exerts direct, concentration‐dependent effects on osteocytes by reducing the expression and secretion of sclerostin, a glycoprotein produced by osteocytes that acts as a negative regulator of bone formation. ³³ , ³⁴ Conversely, decreased INSL3 expression or genetic ablation of its receptor RXFP2 has been associated with reduced bone mass in both experimental models and human studies. ³¹ , ³⁴ , ³⁵ , ³⁶ Similar to bone tissue, in vitro studies have demonstrated that INSL3/RXFP2 signaling plays a pivotal role in dose‐dependently enhancing cell size and protein synthesis in mature myotubes through activation of the Akt, protein kinase B/mechanistic target of rapamycin/ribosomal protein S6 signaling pathway, whereas ablation of the RXFP2 gene exacerbates muscle atrophy. ³⁷ , ³⁸ To date, no studies have directly demonstrated the clinical impact of INSL3/RXFP2 signaling on skeletal muscle mass in humans. However, sarcopenia and muscle loss are commonly observed comorbidities associated with overweight and obesity. ³⁹ In this context, recent independent studies in human males have demonstrated a significant negative correlation between serum INSL3 levels and both BMI and waist‐to‐hip ratio, two widely recognized markers of body adiposity. ³⁶ , ⁴⁰ Given that INSL3/RXFP2 signaling results in increased bone and muscle mass, it seems likely that breast milk INSL3 contributes to the growth of neonates. However, to date, there are no direct studies in the literature investigating how INSL3 levels in human breast milk change throughout lactation. To our knowledge, this is the first study to investigate this aspect. In our study, several statistically significant correlations were observed: first, positive correlations between colostrum foremilk INSL3 levels and both pre‐ and post‐breastfeeding BMI measurements at Visit 1; second, negative correlations between delta pre‐ and post‐breastfeeding BMI and delta foremilk INSL3 levels; third, negative correlations between delta hindmilk INSL3 levels and both head circumference growth velocity and delta head circumference; and finally, positive correlations between colostrum foremilk INSL3 levels and length growth velocity as well as delta length. These associations were statistically significant but generally modest in strength. These findings suggest that higher INSL3 levels in colostrum and the maintenance of relatively elevated levels in mature milk—which implies minimal fluctuations in INSL3 concentrations—may exert a positive influence on neonatal growth parameters.

This study has some limitations. First, mothers with a BMI > 35.0 at the time of enrollment were excluded. Beyond this criterion, maternal height, body weight, and BMI were not recorded, as the study population was considered sufficiently standardized. Nevertheless, had these anthropometric data been available, they could have been incorporated as covariates to adjust for potential confounding in the statistical analyses. Second, although RLX2, OXT, and INSL3 levels are higher in colostrum and lower in mature milk, the volume of milk consumed by infants per feeding is smaller in early lactation and greater in late lactation. Therefore, breast milk hormone concentrations do not necessarily reflect the total amount of hormones ingested by the infant, and these findings should be interpreted with caution.

5. CONCLUSIONS

The findings of this study suggest that RLX2, OXT, and INSL3 concentrations in breast milk undergo significant changes throughout lactation. Furthermore, OXT—and more prominently INSL3, but not RLX2—may positively impact neonatal growth parameters, including BMI, linear growth, and head circumference during early life. Future studies involving larger cohorts are warranted to elucidate the long‐term effects of various nutrients on growth parameters in infants.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Doneray H, Ozden A, Tavlas G, Ozkan HI. The impact of breast milk relaxin 2, oxytocin, and insulin‐like peptide 3 on neonatal growth parameters in early life. J Pediatr Gastroenterol Nutr. 2026;82:1479‐1487. 10.1002/jpn3.70438