Transforming Growth Factor Beta-1 in Human Breast Milk and Its Correlation with Infants' Parameters

Abstract

Background:

Breastfeeding provides optimal nutrition and health protection for the infant; it contains many anti-inflammatory factors, including transforming growth factor beta-1 (TGF-β1). Our study aimed to measure the level of TGF-β1 in human milk and to find its correlation with some infant anthropometric characteristics.

Subjects and Methods:

A milk sample was collected from 84 mothers and the level of TGF-β1 was measured using enzyme-linked immunosorbent assay.

Results:

TGF-β1 was significantly higher in vegetarian mothers compared with nonvegetarian mothers (p = 0.044). Additionally, the mean value of breast milk TGF-β1 was significantly higher in mothers using contraceptive pills compared with those who do not (p = 0.021). Also, the mean value of TGF-β1 was significantly higher in infants 3–6 months than those <3 months (p = 0.010); also there was a significant difference regarding infants' weight and length with average weight and length (p = 0.042) and (p = 0.009), respectively.

Conclusions:

TGF-β1 in human milk may play a role in infants' growth and development; mothers' diet is known to influence TGF-β1 level and its relation to infants' age and weight. Contraceptive method could have an influence on TGF-β1 levels during breastfeeding.

Introduction

Human Breast milk is recommended as an ideal nutrient source for infants, as it has been associated with several short- and long-term benefits for child health, including reduction in gastrointestinal infections, otitis media, and the risks of allergies and autoimmune diseases (e.g., atopic dermatitis, asthma, celiac disease, inflammatory bowel disease, and type 1 diabetes), leukemia, and metabolic syndrome.¹

Human milk is proposed to be ideally adaptive to the needs of the infant, because it contains many bioactive components.^2,3 The World Health Organization recommends that infants should be exclusively breastfed for the first 6 months of their lives. The American Academy of Pediatrics also recommends breastfeeding for at least 12 months.⁴ Moreover, the Academy of Nutrition and Dietetics reaffirms that exclusive breastfeeding provides optimal nutrition and health protection for the first 6 months of infant's life, and that breastfeeding with complementary foods, from the age of 6 months until at least 12 months of age, is an ideal feeding pattern for infants. In addition to its nutritional advantage, breastfeeding is convenient and inexpensive. It is also a bonding experience for the mother and infant. Breast milk has many anti-inflammatory factors such as prostaglandins E2, F2α, lactoferrin, lysozyme, epidermal growth factors and polyamines, cortisol, platelet activating factor acetylhydrolase, α1 antichymotrypsin, uric acid, ascorbate, β-caroten, interleukin (IL)-10, and transforming growth factor beta-1 (TGF-β1).⁵

Cytokines are involved in immune response, health status, and tissue development and can act as tissue hormones in an autocrine and paracrine fashion. They serve as inducers for maturation and development of the immune system.

Multiple cytokines have been identified in colostrum and milk. They are derived from mammary secretions and are involved in maturation of the immune system in the developing gastrointestinal tract. Recently, studies have assessed the interactions between probiotic bacterial strains and cytokines.⁶

One of these is the TGF-β1, which plays a role in the immunological status and development of the infant.⁷

The aim of the study was to measure the level of TGF-β1 in human milk and to find its correlation with infants' anthropometric characteristics.

Subjects and Methods

This study was conducted on 84 lactating mothers from different Egyptian governorates. Mothers were recruited from family health units and central labs in Cairo, from July 2017 till August 2017. All participants provided written informed consent, and the study was approved by the ethical committee of Cairo University.

Inclusion criteria

The study recruited healthy lactating mothers having full-term healthy infants whose ages ranged from 0 to 6 months.

Study Design

A total of 84 mother–infant pairs were enrolled in this study from different Egyptian governorates, from July 2017 till August 2017. The study was conducted on mothers who were apparently healthy with a mean age of 27.3 ± 5.5 years, and 84 healthy full-term infants (39 males and 45 females). Exclusion criteria included mothers suffering from chronic diseases (hypertension, diabetes mellitus [DM], hepatitis C virus [HCV], and hepatitis B virus [HBV]), endocrinal disorders, mothers taking immunosuppressive agents, premature infants (<37 weeks), and infants with a major birth defect, admitted to neonatal intensive care, and other severe illness.

Medical records and interview

All participants were asked to answer a questionnaire about their medical history. The information collected from the mothers included information about age, residence, parity, smoking status, drug intake, special food habit (vegetarian or nonvegetarian), contact with animals, mode of delivery, use of contraceptive pills, exercise, and whether they chose exclusive breastfeeding or not.

Sample collection, storage, and detection of TGF-β1

Participants were given sterile tubes to collect 2 mL of their own milk. The collected milk samples were to be deposited at the laboratory within 4 hours of collection and soon stored at 2–8°C. Before use, all reagents were prepared at room temperature.

Breast milk samples were used for the estimation of TGF-β1 level by sandwich enzyme-linked immunosorbent assay method (Catalog no.: SG-10060er; SinoGeneClon Biotech Co., Ltd.) and were run according to manufacturer's protocol. The optical density (OD) of each well was determined using a microplate reader. The concentration of TGF-β1 in the samples was then determined by comparing the OD of the samples with the standard curve.

Statistical methods

Data were analyzed using SPSS© Statistics version 17 (SPSS, Inc., Chicago, IL). Normality of numerical data distribution was examined using the Shapiro–Wilk test. Normally distributed numerical variables were presented as mean ± standard deviation, and intergroup differences were compared using Student's t-test and an appropriate one-way analysis of variance. Categorical variables were presented in the forms of numbers and percentages. Correlations among numerical variables were tested using the Pearson product-moment correlation coefficient. A p-value <0.05 was considered statistically significant.

Results

A total of 84 mother–infant pairs were enrolled to this study. Mothers were recruited from family health units and central labs in Cairo from July 2017 till August 2017. The study included mothers who were generally healthy with a mean age of 27.3 ± 5.5 years and 84 healthy full-term infants (39 males and 45 females) with a mean age of 71.5 ± 61.4 days.

We studied the relationship between TGF-β1 levels and their correlation with some environmental and maternal factors.

Among the environmental and maternal factors analyzed, food habits showed a significant influence on TGF-β1 levels, where the mean value of TGF-β1 in vegetarian mothers was much higher compared with nonvegetarian mothers (p = 0.044). Additionally, the mean value of TGF-β1 was much higher for mothers using contraceptive pills than those who do not use them (p = 0.021; Table 1).

Table 1.

Association Between TGF-β1 Levels in Breast Milk and Environmental and Maternal Factors

Variables	TGF-β1 (ng/L)
Smoking
No (41)	356.19 ± 172.18	0.672
Passive (43)	371.90 ± 167.83	0.672
Mode of delivery
CS (68)	367.17 ± 170.56	0.744
Normal (16)	351.75 ± 165.88	0.744
Parity
Primigravida (23)	370.01 ± 158.0	0.612
Multigravida (61)	348.91 ± 197.65	0.612
Using formula
No (73)	369.44 ± 168.41	0.470
Yes (11)	329.68 ± 175.41	0.470
Specific food habit
Nonvegetarian (67)	345.62 ± 159.44	0.044
Vegetarian (17)	437.58 ± 189.14	0.044
Contact with animals
No (58)	378.62 ± 164.58	0.246
Yes (26)	332.15 ± 176.87	0.246
Contraceptive method
No (58)	335.93 ± 153.58	0.021
Yes (26)	427.38 ± 186.58	0.021

Boldface indicates TGF-β1 is significantly increased in infants up to 6 months of age.

TGF-β1, transforming growth factor beta-1.

Among different age groups of infants, we found that the mean value of TGF-β1 was significantly higher for infants aged 3–6 months than for those aged over 3 months (p = 0.010).

Regarding body weight, infants were divided into two groups, average and below-average groups, depending on the WHO growth charts, where the average weight was after the 50th percentile charts. A comparative study of TGF-β1 among the different body weight groups is shown in Table 2.

Table 2.

Association Between Breast Milk TGF-β1 Levels and Infant Variables

Variables	TGF-β1 (ng/L)	p
Sex
Male (39)	361.42 ± 158.92	0.888
Female (41)	366.67 ± 178.67	0.888
Age
0–3 months (54)	329.12 ± 151.96	0.010
3–6 months (30)	427.45 ± 181.43	0.010
Weight (kg)
Average (42)	379.36 ± 154.41	0.085
Below-average (42)	326.45 ± 122.49	0.085
Length (cm)
Average (47)	380.61 ± 161.2	0.1360
Below-average (37)	327 ± 163.00	0.1360

The mean value of TGF-β1 was significantly higher for infants in the average weight group than in the below-average weight group (p = 0.085). However, no significant correlation was found between infant's length and TGF-β1 (p = 0.13; Table 2).

The correlation between TGF-β1 levels and maternal and infant ages was analyzed. Interestingly, a significant positive correlation was observed between TGF-β1 levels and infant's age, being higher for the 3–6-month age group (p = 0.010). However, no significant correlation was found between TGF-β1 levels and maternal age (p = 0.445).

But when we performed multivariate correlations, we found no significant difference with respect to food habits and contraceptive use (p = 0.14, 0.55, respectively).

In the multivariate linear regression model, we included significant variables from the univariate analysis (mother's age, specific food, contraceptive use, infant's age [days], infant's weight, infant's length) as independent predictors with TGF-β1 as dependent variable to detect which of these factors act as independent predictors of TGF-β1.

We found that the independent predictors of TGF-β1 (ng/L) were infant's age and weight (Table 3).

Table 3.

Multivariate Linear Regression to Detect Independent Predictors of Transforming Growth Factor Beta-1 (ng/L)

Model	Unstandardized coefficients		Standardized coefficients	t	p
Model	B	Standard error	β	t	p
TGF-β1 (ng/L)
Infant's age (days)	1.267	0.294	0.461	4.311	<0.001
Infant's weight (kg)	138.310	35.852	0.412	3.858	<0.001

Discussion

Breast milk provides the newborn with a variety of bioactive factors (such as TGF-β1), which is known to protect the child against infection and inflammation. It is also required for immune maturation, organ development, and healthy microbial colonization.⁸ TGF-β1 appears to be essential for healthy immune maturation. We found much variability in the concentrations of TGF-β1 in breast milk within our participants, with factors such as diet, birth weight, gestational age, infant's age, residence, and administration of probiotics showing significant influence on the concentrations of TGF-β1 in mother's milk.⁹ Environmental and dietary factors may also impact TGF-β1 concentrations in breast milk.

Although few studies have examined the association of diet on TGF-β1 levels, some studies showed that dietary supplementation with TGF-β proved to decrease intestinal damage and allowed regeneration after mucosal injury.¹⁰

We have found a significant influence of maternal dietary habits on the levels of TGF-β1, where the mean value of TGF-β1 was much higher for vegetarian mothers than nonvegetarian mothers.

This is in agreement with a previous study on certain foods or supplements, such as fish oil, black currant seed oil, or probiotic bacteria, which concluded that the type of food might change the immunological composition of human milk.¹¹

Also, in another study, an association between fish consumption and higher levels of TGF-β1 (mean log TGF-β1 for consumption of fish: 6.72 [0.06] for less than once a week, and 6.57 [0.05] for more than once a week) was reported.¹²

Urwin and colleagues also reported a significant increase in TGF-β1 levels in the milk of women who eat salmon fish regularly.¹²

In contrast, other studies demonstrated that a high-fat diet could lead to increased production of IL-1 and IL-6.¹⁴ But when we conducted a multivariate study, we could not find any significance.

In our work, the mean value of TGF-β1 was much higher in mothers using contraceptive pills than those who do not.

This is in agreement with a 2016 study showing a positive correlation between ethinylestradiol pill use and levels of TGF-β1.¹⁵ In 1991, a study tested the effect of using norethisterone and found a marked decrease in TGF-β2 and TGF-β3 mRNA levels, but the level of TGF-β1 mRNA was not affected.¹⁶ This is in contrast to a study conducted in 2014 where a correlation between contraceptive pills and lower levels of serum TGF-β1 and TGF-β2 was reported.¹⁷

Another study suggested that Implanon has no clinical effect on endothelin-1 or TGF-β1.¹⁸ But when we conducted a multivariate study, we could not find any significance.

Infant's age appeared to be a crucial factor on its health outcome. We found a significant positive correlation between TGF-β1 levels and infants' age, being higher for the 3–6-month age group. In an experimental study on healthy newborn rats, the expression of TGF-β1 was not detected before the age of 19 days, and it progressively increased afterward.¹⁹ The same result was reported by another experimental study on rats, which found that the levels of TGF-β1 in rat milk increased toward mid weaning.²⁰

A contrasting result was reported by another study where the concentration of TGF-β2 showed an increase but not for TGF-β1 in breast milk collected when the infants were 3 months old.²¹

However, in a study carried out to measure the level of TGF-β1 in colostrum and the following months, the levels of both TGF-β1 and TGF-β2 declined postnatally in milk samples, wherein the colostrum samples showed higher levels and the 1-month samples showed the lowest.²²

Limited data have been published about examining TGF-β1 levels in breast milk and their association with infant's growth.

To the best of our knowledge, this is the first study to investigate the relationship between TGF-β1 in mature breast milk and the infant's weight and length.

Regarding the infants' body weight and length, they were divided into two groups based on growth charts: average weight and average length after the 50th percentile charts, and below average. A comparative study on different infant body weight and length groups showed that TGF-β1 was much higher in infants with average weight. This means that TGF-β1 could play an important role in the infant's growth. Although some studies found a relationship between infant's growth and level of cytokines (such as IL-8, TNF-α), no study has investigated TGF-β1 particularly in mature milk.²³ Such a result requires further studies to be conducted.

Oppositely, another study showed that TGF-β1 colostrum levels showed an inverse correlation with both birth weight and gestational age.²²

More investigations are needed into the relationship between TGF-β1 and infants' weight.

Conclusions

TGF-β plays an important role in the body's immunity and growth. TGF-β1 levels were higher in infants with average weight than below-average and higher in the first 3 months of age. Mother dietary habits may affect TGF-β1 levels in milk. Contraceptive use could influence TGF-β1 levels during breastfeeding.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Stam

, Sauer

, Boehm

: Can we define an infant's need from the composition of human milk?. Am J Clin Nutr, 2013; 98(suppl):521S–528S.

Kramer

, Chalmers

, Hodnett

, et al. Promotion of Breastfeeding Intervention Trial (PROBIT): A randomized trial in the Republic of Belarus. JAMA, 2001; 285:413–420.

Le Hueërou-Luron

, Blat

, Boudry

. Breast- v. formula-feeding: Impacts on the digestive tract and immediate and long-term health effects. Nutr Res Rev, 2010; 23:23–36.

Johnston

, Landers

, Noble

, et al. Breastfeeding and the use of human milk. Pediatrics, 2012; 129:e827–e841.

Lessen

, Kavanagh

. Position of the academy of nutrition and dietetics: Promoting and supporting breastfeeding. J Acad Nutr Diet, 2015; 115:444–449.

Brenmoehl

, Ohde

, Wirthgen

, et al. Cytokines in milk and the role of TGF-beta. Best Pract Res Clin Endocrinol Metab, 2018; 32:47–56.

Garofalo

. Cytokines in human milk. J Pediatr, 2010; 156(2 Suppl):S36–S40.

Hawkes

, Bryan

D-L

, Gibson

. Variations in transforming growth factor beta in human milk are not related to levels in plasma. Cytokine, 2002; 17:182–186.

Ben-Lulu

, Pollak

, Mogilner

, et al. Dietary transforming growth factor-beta 2 (TGF-β2) supplementation reduces methotrexate-induced intestinal mucosal injury in a rat. PLoS One, 2012; 7:e45221.

10.

Ruiz

, Espinosa-Martos

, García-Carral

, et al. What's normal? Immune profiling of human milk from healthy women living in different geographical and socioeconomic settings. Front Immunol, 2017; 8:696.

11.

Munblit

, Treneva

, Peroni

, et al. Colostrum and mature human milk of women from London, Moscow, and Verona: Determinants of immune composition. Nutrients, 2016; 8:pii:E695.

12.

Urwin

, Miles

, Noakes

, et al. Salmon consumption during pregnancy alters fatty acid composition and secretory IgA concentration in human breast milk. J Nutr, 2012 Aug;142(8):1603–1610.

13.

Cortez

, Carmo

, Rogero

, et al. A high-fat diet increases IL-1, IL-6, and TNF-α production by increasing NF-κB and attenuating PPAR-γ expression in bone marrow mesenchymal stem cells. Inflammation, 2013; 36:379–386.

14.

Chen

, Gokhale

, Schofield

, et al. Inhibition of TGF-beta-induced apoptosis by ethinyl estradiol in cultured, precision cut rat liver slices and hepatocytes. Carcinogenesis, 2000; 21:1205–1211.

15.

Jeng

, Jordan

. Growth stimulation and differential regulation of transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 messenger RNA levels by norethindrone in MCF-7 human breast cancer cells. Mol Endocrinol, 1991; 5:1120–1128.

16.

Memon

AA1

, Sundquist

, Wang

, et al. Transforming growth factor (TGF)-β levels and unprovoked recurrent venous thromboembolism. J Thrombosis Thrombolysis, 2014; 38:348–354.

17.

Merki-Feld

GS1

, Imthurn

, Seifert

. Effects of the progestagen-only contraceptive implant Implanon on transforming growth factor beta1 and endothelin-1. Horm Metab Res, 2008; 40:692–696.

18.

Penttila

, van Spriel

, Zhang

, et al. Transforming growth factor-beta levels in maternal milk and expression in postnatal rat duodenum and ileum. Pediatr Res, 1998; 44:524–531.

19.

Zhang

, Zola

, Read

, et al. Localization of transforming growth factor-beta receptor types I, II, and III in the postnatal rat small intestine. Pediatr Res, 1999; 46:657–665.

20.

Prescott

, Wickens

, Westcott

, et al. Supplementation with Lactobacillus rhamnosus or Bifidobacterium lactis probiotics in pregnancy increases cord blood interferon-gamma and breast milk transforming growth factor-beta and immunoglobin A detection. Clin Exp Allergy, 2008; 38:1606–1614.

21.

Young

, Morrow

, Davidson

, et al.: Inflammatory cytokines in human milk are inter-correlated and may be related to infant growth characteristics and maternal weight status. FASEB J, 2013; 27: https://www.fasebj.org/doi/abs/10.1096/fasebj.27.1_supplement.629.10 (accessed April 8, 2019).

22.

Frost

, Jilling

, Lapin B

MPH

, et al. Maternal breast milk transforming growth factor beta and feeding intolerance in preterm infants. Pediatr Res, 2014; 76:386–393.

23.

Ballard

, Morrow

. Healthy composition: Nutrients and bioactive factors. Pediatr Clin North Am, 2013; 60:49–74.