Oxidative status in colostrum and mature breast milk related to gestational age and fetal growth

Abstract Introduction The effect of gestational age and fetal growth on the oxidant/antioxidant status of breast milk is poorly understood. Objective To evaluate the oxidative stress biomarkers in colostrum and mature milk according to gestational age and fetal growth. Method A longitudinal study with mothers of premature and term infants, born in a tertiary referral hospital between 2014–2018. Inclusion criteria: postpartum women with a singleton pregnancy, who intended to exclusively breastfeed. Exclusion criteria: maternal diabetes, use of medication, drug addiction, congenital infection or malformation, mastitis, and failure to collect colostrum. Four groups were formed according to gestational age and birth weight (appropriate and small): Preterm small (n = 37), Preterm appropriate (n = 99), Full-term small (n = 65), and Full-term appropriate (control, n = 69). The colostrum samples were collected between 24-72 h and the mature milk was sampled in the 4th week of lactation for malondialdehyde (biomarker for lipid peroxidation) and Glutathione peroxidase, Catalase, and Superoxide dismutase measurements. The data were compared among groups using the Chi-square test or Fisher’s exact test, one-way analysis of variance followed by Wald’s Distribution test and repeated measures analysis of variance. Results We found a lower malondialdehyde level in colostrum in preterm groups and term small for gestational age, and the antioxidant enzymes Superoxide dismutase and Catalase activities were higher for preterm compared to term groups. The malondialdehyde levels differed in mature milk samples (Full-term small > Full-term appropriate > Preterm small > Preterm appropriate). The malondialdehyde levels increased during lactation in all groups except Preterm appropriate, and the levels of Catalase decreased in preterm groups. Conclusion The oxidative status in breast milk is influenced by gestational age and fetal growth, which increased antioxidant defense for preterm infants and decreased oxidative stimuli for small for gestational age infants. These findings contribute to encouraging breastfeeding for newborns.


Introduction
Despite advances in obstetric and perinatal care, the rates of prematurity and fetal growth restriction remain high worldwide, with an incidence !10%, and the highest rates in low-income countries [1,2]. Preterm infants and newborns with fetal growth restriction tend to show oxidative stress since they are frequently exposed to events leading to overproduction of free radicals, such as hypoxia, hyperoxia, ischemia, reperfusion, and infection/inflammation, and have reduced antioxidant defense [2][3][4]. Oxidative stress has been implicated in the pathogenesis of intrauterine growth restriction [5] and has been associated with increased risk of prematurity-related diseases, including bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, intraventricular hemorrhage and periventricular leukomalacia [2][3][4].
The identification of best practices to minimize the risks of damage and improve the prognosis of newborn infants is currently a priority in neonatal care. In this context, breastfeeding and mother's milk are gold standards for infant feeding and nutrition [6]. Evidence shows that breastfeeding is essential to improve neonatal outcome, the infant growth and neurodevelopment in a short and long time, as well as to reduce the risk of future diseases, such as hypertension, diabetes and metabolic syndrome [7][8][9]. Breast milk benefits involve many mechanisms not yet fully understood, and have been related to its complex composition of macro and micronutrients, bioactive factors, such as antioxidants, growth factors, adipokines, cytokines, and antimicrobial compounds [10][11][12].
Breast milk has better antioxidant properties than infant formula and breastfed newborn infants have shown higher levels of antioxidants in plasma [13,14]. Breast milk oxidant/antioxidant status changes according to the stage of lactation, and a higher antioxidant property has been reported in colostrum compared to mature milk [15,16]. The gestational age also influences the oxidant/antioxidant status of breast milk. Although differences between term and preterm breast milk composition remain controversial, some studies showed higher antioxidant capacity in breast milk from mothers of preterm infants [17,18], while others reported lower [15], or no differences in antioxidant properties when compared with the milk from mothers of full-term infants [19,20].
Fetal growth restriction is associated with increased levels of oxidative stress biomarkers in maternal and cord blood [21,22]. Strambi et al. showed that breastfed small for gestational age newborns had higher plasma selenium concentration than formulafed newborns, which suggests that breastfeeding could improve their antioxidant defense [23]. However, the influence of fetal growth on oxidant/antioxidant composition of breast milk is unknown due to scarcity of studies. A small study showed no difference in total oxidant and antioxidant status of breast milk between mothers of preterm and term infants, appropriate and small for gestational age [24].
The data from the literature do not specify how gestational age and fetal growth affect the oxidant/antioxidant status of breast milk. Thus, research is needed to study the possibility that the association between low gestational age and fetal growth restriction may lead to a subgroup of newborns with a higher risk of oxidative stress and its short-and longterm consequences [25].
We hypothesized that milk from mothers of preterm infants, appropriate or small for gestational age, has increased antioxidants and decreased oxidants concentrations. However, fetal growth restriction may reduce the antioxidants of breast milk for full-term small for gestational age infants. For this, we studied the influence of gestational age and fetal growth on the concentration of oxidative stress biomarker and antioxidants enzymes in colostrum and mature breast milk.

Study design
This was a prospective observational study with women who had given birth at term or prematurely at the maternity hospital of the São Paulo State University-Botucatu, School of Medicine, from 2014 to 2018. The study was approved by the Institution's Research Ethics Committee and informed consent was obtained from each participant.
Only postpartum women with a singleton pregnancy, who intended to exclusively breastfeed and showed negative serology results for syphilis, HIV, and Hepatitis B were included in the study.
The exclusion criteria were women with diabetes mellitus, use of medication or illicit drugs, presence of mastitis, newborn large for gestational age or with congenital anomalies, and the impossibility to obtain colostrum.
All women who met the inclusion criteria and agreed to participate in the study were distributed into four groups, according to gestational age (GA) and fetal growth: T-AGA-Term infants appropriate for gestational age as a control group; T-SGA-Term infants small for gestational age; PT-AGA-Preterm infants appropriate for gestational age; PT-SGA-Preterm infants small for gestational age.
Gestational age was estimated by the best obstetric estimate (precise date of the last menstruation and/or ultrasound in the first quarter). Birth weight < 10 percentile on Fenton intrauterine growth curve was defined as SGA, and birth weight between 10-90 th percentiles was considered AGA [26]. The independent variables of the study included the mother's data (age, schooling, pre-gestational weight and Body Mass Index (BMI), smoking, parity, preeclampsia, premature rupture of membranes > 18 h, antenatal steroids, and cesarean delivery) and the newborn's data (gestational age, gender, delivery room resuscitation, 1-min Apgar score, birth weight, weight classification for gestational age, neonatal morbidity, and frequency of exclusive breastfeeding during hospitalization). The primary outcomes were the concentrations of oxidant marker (malondialdehyde) and antioxidant substances (glutathione peroxidase, catalase, superoxide dismutase) in colostrum and mature breast milk.

Sample collection and laboratory methods
Colostrum was obtained by manual expression. Samples of 3 ml were collected between 24 and 72 h after delivery and mature milk were sampled at the 4th week of life. Sample collection of colostrum and mature milk was standardized between 9:00-11:00 a.m., during the feeding interval. Mature milk was obtained at the outpatient clinic on the day of the newborn's routine medical appointment. Milk samples were immediately frozen in liquid nitrogen and kept in the freezer at À80 C for 6-9 months, until analysis. The dosages were performed blindly, without identifying the groups.
The lipid peroxidation biomarker, malondialdehyde (MDA), was measured by spectrophotometric absorbance of thiobarbituric acid reactive substances (TBARS), which react with malondialdehyde to produce a pink substance. The amount of TBARS is proportional to the amount of MDA. The absorbance was measured at a wavelength of 535 nm. TBARS concentration was estimated using the standard MDA curve and was expressed in nmol/mg of protein. The measurements were performed using TBARS Assay Kit provided by Cayman Chemical Company V R .
The main antioxidant enzymes were assessed as markers for antioxidant capacity: Glutathione peroxidase (GPX), Catalase (CAT), and Superoxide dismutase (SOD). These measurements were performed by Colorimetric Assay using specific kits: Glutathione Peroxidase Assay Kit, Catalase Assay Kit, and Superoxide Dismutase Assay Kit, provided by Cayman Chemical Company V R .

Statistical analysis
In the descriptive analysis of the sample, continuous variables were reported as mean and standard deviation or median and percentiles, as appropriate; and categorical variables were expressed as the number and proportion of events.
The Chi-square test or Fisher's exact test was used to examine the associations between categorical variables, followed by the proportion comparison test. The continuous variables were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test. The oxidative stress biomarker (MDA) and antioxidant enzymes levels were compared among the four groups using ANOVA, followed by the Wald test, adjusting for potential confounders identified in the univariate analysis: preeclampsia, BMI, and mode of delivery. The gamma distribution was used to control for the variability in the values of oxidant and antioxidant substances.
The effect of group and time on the oxidant/antioxidant composition of breast milk was evaluated in paired samples of colostrum and mature milk by repeated measures analysis of variance (ANOVA-RM).
A two-tailed p value considered statistically significant was p < .05. The analyses were performed using SAS for Windows v.9.4 statistical software (Cary, NC, USA).

Maternal characteristics
Among the 430 postpartum women who were approached at random on working days from August 2014 to July 2018, 370 met the inclusion criteria. However, 40 women refused to participate in the study and 61 were excluded, mainly because we could not obtain the necessary volume of colostrum. Thus, we analyzed samples of colostrum and mature milk from 269 women, sub-divided into four groups: 37 preterm infants small for gestational age (PT-SGA); 99 preterm infants appropriate for gestational age (PT-AGA); 64 term infants small for gestational age (T-SGA); and 69 controls (T-AGA).
All women attended antenatal care and had, on average, 25 years of age and 9.5 years of schooling, without difference among groups. Table 1 shows the main maternal and gestational data. Differences among the groups are indicated by different letters. The mean maternal BMI was !25 Kg/m 2 in all groups, which characterizes the sample as overweight. Preeclampsia was the main pregnancy-related complication in the preterm groups (Table 1).

Birth and neonatal characteristics
The groups were similar regarding newborn gender (46% male) and the 1-min Apgar (mean 8 for all groups). Among the 136 preterm infants a total of 73% were late preterm infants (!34 weeks of gestation).
Neonatal morbidity was low, especially in full-term infants. Jaundice requiring phototherapy was the most frequent disease in all groups, followed by respiratory distress syndrome, which occurred in preterm infants only. Table 2 shows that birth and neonatal data were significantly different between preterm and term infants (indicated by different letters). Delivery room resuscitation and use of phototherapy were significantly more frequent in preterm compared to fullterm infants.
Oxidative stress biomarkers Table 3 shows the mean concentration of oxidative stress biomarkers (MDA) and antioxidant enzymes in paired samples of colostrum and mature milk in all four groups, adjusting for confounding factors: preeclampsia, BMI and mode of delivery. Significant differences among the four groups are indicated by different letters.
Overall, mothers of preterm infants had lower levels of the oxidative marker in colostrum and mature milk, and higher concentrations of antioxidants in colostrum, compared to the control group (T-AGA). Mothers of T-SGA had lower levels of the oxidative marker in colostrum, but higher in mature milk, compared to the control group (T-AGA). The concentration of antioxidant enzymes in mature milk did not differ between groups.
Comparing colostrum to mature milk, the concentration of MDA increased during the first month of lactation in all groups except in the PT-AGA in which catalase tended to decrease with significant lower levels in mature milk from mothers of preterm infants (Table 3).

Discussion
The results of this study suggest that the oxidant/antioxidant composition of breast milk changes as a function of gestational age and fetal growth, to match the infant's needs and limitations. These changes in the oxidant/antioxidant status of breastmilk may help counteracting the negative effects of oxidative stress on the most vulnerable newborns: preterm and small for gestational-age infants. Our findings support that breastmilk is the gold standard for infant nutrition, and that breastfeeding should be encouraged for all newborns in daily practice.
Our results showed that gestational age has a greater influence than fetal growth on the oxidant/antioxidant status of breast milk. The colostrum from mothers of preterm infants appropriate or small for gestational age showed a more protective pattern, providing fewer oxidants and more antioxidant enzymes than the colostrum from mothers of full-term infants. We also observed this protective profile in mature milk from mothers of preterm infants with a lower concentration of oxidant biomarkers. Fetal  growth restriction affected the oxidant status of breast milk from mothers of full-term infants, demonstrated by the low MDA concentration in colostrum and high in mature milk from mothers of full-term small for gestational age. However, the concentration of antioxidant enzymes did not change and was similar to breast milk from mothers of full-term appropriate for gestational age (control group).
These findings are essential because prematurity is associated with inflammation/infection and fetal growth restriction is related to hypoxia/ischemia [4]. Both inflammation and hypoxia/ischemia are associated with oxidative stress [4], but the effects of prematurity and fetal growth restriction on breast milk oxidant/antioxidants components have not yet been established.
To our knowledge, this is the first study to assess the influence of gestational age and fetal growth on the oxidant/antioxidant status of colostrum and mature milk.
Since the macronutrient composition of breast milk changes during lactation to offer appropriate nutritional intake to the premature infant [27][28][29], we hypothesized that changes in the oxidant/antioxidant status of breast milk would occur to protect the newborn from oxidative stress, especially the preterm infant, regardless of the fetal growth. This hypothesis was confirmed and showed a high concentration of antioxidant enzymes in colostrum and a low level of oxidative biomarker (MDA) in colostrum and mature milk from mothers of preterm infants, which suggests that the protective effect of their breast milk persisted during the first month of lactation.
The literature on oxidant/antioxidant status of breast milk is still controversial regarding the differences in antioxidant capacity of term and preterm breast milk. Some authors report that the colostrum of mothers of preterm infants has higher total oxidant status and antioxidant capacity than those of mothers of term infants [30]. However, other studies showed no difference between term and preterm colostrum [24] or showed a lower total antioxidant capacity in the colostrum of preterm compared to mothers of full-term infants [15,16]. This heterogeneity may be related to small samples, maternal nutritional status, ethnicity, study selection criteria, differences in the parameters assessed, and measurement methods [12]. Most studies did not evaluate specific oxidant/antioxidant compounds and their balance in breast milk. Thus, more research is needed to clarify differences in antioxidant properties of term and preterm colostrum.
Despite these limitations, evidence shows that breast milk offers antioxidant protection for newborns, especially low birth weight and premature infants, who are more susceptible and exposed to oxidative stress [16,18,20,31].
Regarding the effect of lactation, studies report that the total antioxidant capacity of breast milk is higher in colostrum than in mature milk [12,15,16,20]. However, the antioxidant capacity of breast milk may not decline during lactation for mothers of premature infants [16].
Friel et al. assessed the lipid peroxidation biomarker (MDA) and catalase activity in breast milk from 17 mothers of full-term infants and 28 preterm in the first, second, and 12th weeks of lactation. There was no difference between term and preterm milk at all stages of lactation, and catalase activity increased during lactation [13]. In contrast, Paduraru et al. evaluated the total antioxidant status of breast milk samples from mothers of 60 preterm and 30-term infants, on days 3, 7, and 30, and found that the antioxidant capacity of breast milk increased from day 3 to day 30 in both groups. The study found no difference between Table 3. Oxidative stress marker and antioxidants enzymes in colostrum and mature milk from mothers of the four study groups. term and preterm colostrum, but mature milk from mothers of preterm infants had lower total antioxidant status than term milk [31]. Chrustek et al. showed different results when examining the antioxidant status of mature milk from mothers of term and preterm infants and showed that the antioxidant status of milk from mothers of preterm infants was higher than milk from mothers of full-term infants [18].
We assessed the three main antioxidant enzymes (SOD, CAT, GPx) and a biomarker of oxidative stress. Overall, our results agree with previous studies [15,32], showing a greater protective effect of colostrum when compared with mature milk for preterm and term infants. SOD and GPx concentrations were stable in all groups during the first month of lactation and, although the catalase levels decreased in breast milk for preterm infants, MDA concentration did not increase, which suggests that oxidant-antioxidant balance is preserved in mature milk for preterm infants. These findings emphasize the importance of breastfeeding, especially for preterm infants.
Few studies have assessed the influence of fetal growth on the oxidant/antioxidant status of breast milk. Sandal et al. assessed the total oxidant status, total antioxidant capacity, and index of oxidative stress in colostrum of 84 mothers divided into four groups: ¼ 19), and did not find significant differences among the groups, which suggest that mother's milk could not improve antioxidant mechanisms for SGA neonates [24]. Our study, with a larger sample, showed that oxidant content of milk from mothers of T-SGA was lower in colostrum and higher in mature milk compared to the control group. We hypothesize a possible adaptive mechanism in the mother's mammary gland to protect neonates with fetal growth restriction during the first days of life, when the infant would be at high oxidative risk, since fetal growth restriction relates to placental insufficiency and intrauterine hypoxia, which may induce free radical generation and fetal oxidative stress, expressed by increased levels of biomarkers, including MDA in maternal and cord blood [4,5]. However, we did not find a protective antioxidant profile in mature breast milk from mothers of term-SGA. This was an unexpected finding that will require further investigation to better understand the influence of fetal growth restriction on the oxidant/antioxidant status of breast milk and its changes during lactation. Our data showed that besides the greater influence of gestational age on the oxidant/antioxidant status of breast milk, colostrum was beneficial for SGA at term due to its lower oxidant content. These results suggest that the antioxidant protection of colostrum may occur by different mechanisms: mainly decreasing reactive oxygen species and oxidative damage in SGA at term, and increasing the antioxidant defense systems of preterm infants.
Our study contributes to the literature despite some limitations. We cannot comment on the total oxidant and antioxidant status of breast milk since we only evaluated one oxidative stress biomarker (MDA) and the three main antioxidant enzymes. We chose to study these biomarkers because few previous studies have evaluated specific compounds and their balance in breast milk, thus, we offered new information on the oxidant/antioxidant status of breast milk. Gestational weight gain was not evaluated. Another limitation was the mothers' food consumption and its influence on breast milk. However, when we collected colostrum, the diet was similar because the women were hospitalized.
The strength of the study was the comparison of the oxidant/antioxidant status of breast milk between mothers of term and preterm infants, appropriate and small for gestational age, thus, we could study the influence and possible interaction of gestational age and fetal growth on oxidant/antioxidant status of breast milk. Few studies assessed the effect of fetal growth restriction on oxidant/antioxidant properties of breast milk, and only one previous small study could not detect differences in the colostrum [24]. Our study, with higher sample size and comparison between colostrum and mature milk, allowed us to add new knowledge on this issue by showing the influence of fetal growth on oxidant/antioxidant status of breast milk, particularly the lower oxidant content of colostrum which may improve the antioxidant protection of breast milk for the small for gestational age newborn infant. To our knowledge, this is the first study to show differences in breast milk from mothers of small for gestational age infants, and further studies would benefit to confirm and better explain these results.

Conclusion
Oxidative status in breast milk is influenced by gestational age and fetal growth, providing increased antioxidant defense for preterm infants and decreased oxidative stimulus for SGA infants. Colostrum is a good strategy to protect preterm infant and full-term small for gestational age infant from oxidative stress, and breastfeeding must be stimulated for all newborns.