The Effect of Maternal Obesity on Breast Milk Fatty Acids and Its Association with Infant Growth and Cognition—The PREOBE Follow-Up

nutrients Article The Effect of Maternal Obesity on Breast Milk Fatty Acids and Its Association with Infant Growth and Cognition—The PREOBE Follow-Up Andrea de la Garza Puentes 1,2,3,* , AdriàMartíAlemany 1, Aida Maribel Chisaguano 4, Rosa Montes Goyanes 5, Ana I. Castellote 1,2,6 , Franscisco J. Torres-Espínola 7,8, Luz García-Valdés7,8, Mireia Escudero-Marín7,8, Maria Teresa Segura 7,8, Cristina Campoy 7,8,9 and M. Carmen López-Sabater 1,2,6,* 1Department of Nutrition, Food Sciences and Gastronomy, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain 2Institut de Recerca en Nutriciói Seguretat Alimentària UB (INSA-UB), 08921 Barcelona, Spain 3Teaching, Research & Innovation Unit, Parc Sanitari Sant Joan de Déu, 08830 Sant Boi, Spain 4Nutrition, Faculty of Health Sciences, University of San Francisco de Quito, 170157 Quito, Ecuador 5Food Research and Analysis Institute, University of Santiago de Compostela, 15705 Santiago de Compostela, Spain 6CIBER Physiopathology of Obesity and Nutrition CIBERobn, Institute of Health Carlos III, 28029 Madrid, Spain 7Centre of Excellence for Paediatric Research EURISTIKOS, University of Granada, 18071 Granada, Spain 8Department of Paediatrics, University of Granada, 18071 Granada, Spain 9CIBER Epidemiology and Public Health CIBEResp, Institute of Health Carlos III, 28029 Madrid, Spain *Correspondence: [email protected] (A.d.l.G.P.); [email protected] (M.C.L.-S.); Tel.: +39-934-024-512 (A.d.l.G.P. & M.C.L.-S.) Received: 6 August 2019; Accepted: 24 August 2019; Published: 9 September 2019   Abstract: This study analyzed how maternal obesity affected fatty acids (FAs) in breast milk and their association with infant growth and cognition to raise awareness about the programming effect of maternal health and to promote a healthy prenatal weight. Mother–child pairs (n=78) were grouped per maternal pre-pregnancy body mass index (BMI): normal-weight (BMI =18.5–24.99), overweight (BMI =25–29.99) and obese (BMI >30). Colostrum and mature milk FAs were determined. Infant anthropometry at 6, 18 and 36 months of age and cognition at 18 were analyzed. Mature milk exhibited lower arachidonic acid (AA) and docosahexaenoic acid (DHA), among others, than colostrum. Breast milk of non-normal weight mothers presented increased saturated FAs and n6:n3 ratio and decreased α -linolenic acid (ALA), DHA and monounsaturated FAs. Infant BMI-for-age at 6 months of age was inversely associated with colostrum n6 (e.g., AA) and n3 (e.g., DHA) FAs and positively associated with n6:n3 ratio. Depending on the maternal weight, infant cognition was positively influenced by breast milk linoleic acid, n6 PUFAs, ALA, DHA and n3 LC-PUFAs, and negatively affected by n6:n3 ratio. In conclusion, this study shows that maternal pre-pregnancy BMI can influence breast milk FAs and infant growth and cognition, endorsing the importance of a healthy weight in future generations. Keywords: maternal obesity; breastfeeding; breast milk; colostrum; mature milk; fatty acids; LC-PUFA; omega-3; omega-6; DHA; AA;children; growth; cognition; earlylife nutrition; programming 1. Introduction In spite of efforts made, as well as existing evidence-based information, for tackling obesity and the burden of the disease, obesity is a societal challenge that is still on the rise, including in women Nutrients 2019,11, 2154; doi:10.3390/nu11092154 www.mdpi.com/journal/nutrients Nutrients 2019,11, 2154 2 of 18 of reproductive age, and this is affecting the health of future generations [ 1 ]. Early-life nutrition plays a key role in infant growth and development and has a programming effect related to the appearance of future non-communicable diseases, such as obesity, diabetes and others [ 2 , 3 ]. Breast milk composition and breastfeeding practice are some of the most influential factors of child outcomes [ 4 – 6 ]. Even though lactation comprises a relatively short period in the average person’s lifespan, the exposure to breast milk in the first months of life occurs during a very critical period of rapid growth and development [ 2 , 7 – 9 ]. Maternal obesity influences the nutritional status of the child through different mechanisms, breastfeeding being one of them. If the mother of the child has obesity, the fatty acid (FA) profile in breast milk can be different, with a prevalence of pro-inflammatory FAs beyond those critical for neurodevelopment [ 10 ]. Thus, the early nutritional status and future health of the child can be affected. Breast milk contains long-chain (LC) polyunsaturated fatty acids (PUFAs), which are crucial nutrients—especially docosahexaenoic (DHA) and arachidonic acid (AA)—involved in growth, the immune system, vision, and cognitive and motor development [ 11 ]. These nutrients are associated with the prevention of obesity [ 12 , 13 ] and other infectious and chronic diseases in the future life [ 14 ]. However, maternal characteristics, such as diet [ 15 ] or obesity [ 10 ], may alter the FA content in human milk. Studies have shown that the breast milk of mothers with overweight and obesity have higher levels of n6 FAs and lower levels of n3 FAs than the breast milk of normal-weight mothers [ 16 – 18 ], and a high ratio of n6:n3 LC-PUFAs in red blood cells membrane phospholipids has been reported as a risk factor for obesity [ 19 ]. In fact, in high-fat rodent models of maternal obesity, lowering the maternal n6:n3 ratio using a novel genetic model or supplemental fish oil has been shown to prevent offspring obesity [20]. Nevertheless, the results appear to be inconsistent [18,21]. The direct impact of maternal weight on the infant cognition has also been studied [ 21 – 24 ]. Mostly, observational, prospective and longitudinal studies correlate a high pre-pregnancy maternal body mass index (BMI) with poorer cognitive performance [ 24 ]. High gestational weight gain (GWG) seems to augment this correlation, as well [ 25 ]. However, three studies have failed to find an association between maternal obesity and cognitive infant deficits [26–28]. Although there are studies that have analyzed the influence of maternal weight on breast milk FA composition [ 10 , 18 , 29 – 35 ], none of these studies have further assessed its effect on infant cognition and growth. Furthermore, there is a lot of variability regarding the timing of breastmilk collection in the existing studies, and most of them focus on the analysis of mature breastmilk, without considering the evolution of the different FAs from colostrum to mature milk. Therefore, the current study aims to analyze the implications of maternal obesity on FA levels in colostrum and mature milk and their association with infant growth and cognition, to raise awareness about the programming effect of maternal nutrition and promote a healthy weight in women. 2. Materials and Methods 2.1. Statement of Ethics This study was carried out in accordance with the ethical standards recognized by the Declaration of Helsinki (2004), the EEC Good Clinical Practice guidelines (document 111/3976/88 of July 1990) and current Spanish legislation governing clinical research in humans (Royal Decree 561/1993 on clinical trials). Additionally, the study was approved by San Cecilio University Hospital Ethics Committee and the Faculty of Medicine at the University of Granada. Written informed consent was obtained from all participants at the beginning of the study. 2.2. Study Population and Design For the present study, a subsample of mother–child pairs (n=78) from the PREOBE cohort was selected and classified according to maternal pre-pregnancy BMI: normal-weight (BMI = 18.5–24.99 Kg/m 2 ,n=34), overweight (BMI =25–29.99 Kg/m 2 ,n=27) and obese (BMI >30 Kg/m 2 , n=17). Nutrients 2019,11, 2154 3 of 18 The PREOBE study (Role of Nutrition and Maternal Genetics on the Programming of Development of Fetal Adipose Tissue) is an observational cohort study of a total of 331 pregnant women that analyzes the impact of maternal obesity and gestational diabetes. The information regarding the PREOBE study has been published elsewhere [ 34 ] and was registered at www.ClinicalTrials.gov (NCT01634464). Figure 1presents the study design and information of the PREOBE study. Nutrients 2019, 11, x FOR PEER REVIEW 3 of 19 PREOBE study (Role of Nutrition and Maternal Genetics on the Programming of Development of Fetal Adipose Tissue) is an observational cohort study of a total of 331 pregnant women that analyzes the impact of maternal obesity and gestational diabetes. The information regarding the PREOBE study has been published elsewhere [34] and was registered at www.ClinicalTrials.gov (NCT01634464). Figure 1 presents the study design and information of the PREOBE study. Figure 1. Participants in the PREOBE cohort and classification following BMI and gestational diabetes criteria. Briefly, the study and recruitment of participants were carried out at San Cecilio University Hospital and the Mother-Infant Hospital in the city of Granada, Spain. The inclusion criteria were: singleton pregnancy, gestation between 12 and 20 weeks at enrollment, and an intention to deliver in one of the two obstetrics centers mentioned above. Women were excluded if they were participating in other research studies, receiving drug treatment or supplements of DHA or folate for more than the first three months of pregnancy, suffering from disorders such as hypertension, pre-eclampsia, fetal intrauterine growth retardation, infections, hypoor hyperthyroidism and hepatic renal diseases, or following an unusual or vegan diet. Maternal age, pre-pregnancy BMI, parity, smoking status, diet, alcohol habits, socio-demographic information, education, gestational weight gain, infant anthropometry, gender and feeding practices were recorded. After birth, the women were encouraged to breastfeed their infants. 2.3. Breast Milk Sample Collection Colostrum and mature milk were collected at 2–4 and 28–32 days postpartum, respectively, by an experienced nurse at the hospitals or by the mother at home (after receiving training by the nurse). Samples were collected over the course of an entire day (24 h) from both breasts before and after each feed. Milk samples were gathered in sterile polypropylene tubes by mechanically expressing each breast with a breast pump. Mothers were given 14 tubes with a capacity of 5 mL and the total volume obtained from each mother ranged from 45 to 70 mL. The samples collected at each time were frozen at −20 °C at home, and mothers brought them to the 3-month offspring follow-up visit. Each time, the samples were transported in ice boxes to the laboratory, where they were stored at −80 °C until analysis. All samples from each woman were mixed and aliquoted prior to analysis. 2.4. Fatty Acid Analysis of Breast Milk The FA composition of breast milk was determined according to the method described by Chisaguano et al. [35]. 50 µL human milk samples were used for the analysis. FA methyl esters (FAMEs) were prepared with sodium methylate in methanol (0.5 M) and boron trifluoride methanol solution (14% v/v). They were then separated and quantified by fast gas chromatography (GC)using a HP-6890 Series GC System (Hewlett-Packard, Waldbronn, Germany) equipped with a flame ionization detector (FID), a split/splitless injector, a HP-7683B Series autoinjector, and a fused-silica SP-2560 capillary column (75 m 0.18 mm internal diameter, 0.14 µm thickness) coated with a 100% bis-cyanopropyl polysiloxane stationary phase (Supelco, Saunderton, UK). The chromatographic Figure 1. Participants in the PREOBE cohort and classification following BMI and gestational diabetes criteria. Briefly, the study and recruitment of participants were carried out at San Cecilio University Hospital and the Mother-Infant Hospital in the city of Granada, Spain. The inclusion criteria were: singleton pregnancy, gestation between 12 and 20 weeks at enrollment, and an intention to deliver in one of the two obstetrics centers mentioned above. Women were excluded if they were participating in other research studies, receiving drug treatment or supplements of DHA or folate for more than the first three months of pregnancy, suffering from disorders such as hypertension, pre-eclampsia, fetal intrauterine growth retardation, infections, hypoor hyperthyroidism and hepatic renal diseases, or following an unusual or vegan diet. Maternal age, pre-pregnancy BMI, parity, smoking status, diet, alcohol habits, socio-demographic information, education, gestational weight gain, infant anthropometry, gender and feeding practices were recorded. After birth, the women were encouraged to breastfeed their infants. 2.3. Breast Milk Sample Collection Colostrum and mature milk were collected at 2–4 and 28–32 days postpartum, respectively, by an experienced nurse at the hospitals or by the mother at home (after receiving training by the nurse). Samples were collected over the course of an entire day (24 h) from both breasts before and after each feed. Milk samples were gathered in sterile polypropylene tubes by mechanically expressing each breast with a breast pump. Mothers were given 14 tubes with a capacity of 5 mL and the total volume obtained from each mother ranged from 45 to 70 mL. The samples collected at each time were frozen at − 20 ◦ C at home, and mothers brought them to the 3-month offspring follow-up visit. Each time, the samples were transported in ice boxes to the laboratory, where they were stored at − 80 ◦ C until analysis. All samples from each woman were mixed and aliquoted prior to analysis. 2.4. Fatty Acid Analysis of Breast Milk The FA composition of breast milk was determined according to the method described by Chisaguano et al. [ 35 ]. 50 µ L human milk samples were used for the analysis. FA methyl esters (FAMEs) were prepared with sodium methylate in methanol (0.5 M) and boron trifluoride methanol solution (14% v/v) . They were then separated and quantified by fast gas chromatography (GC)using a HP-6890 Series GC System (Hewlett-Packard, Waldbronn, Germany) equipped with a flame ionization detector (FID), a split/splitless injector, a HP-7683B Series autoinjector, and a fused-silica SP-2560 capillary column (75 m 0.18 mm internal diameter, 0.14 µ m thickness) coated with a 100% bis-cyanopropyl polysiloxane Nutrients 2019,11, 2154 4 of 18 stationary phase (Supelco, Saunderton, UK). The chromatographic conditions used were: hydrogen as the carrier gas at a constant linear velocity of 22 cm/s (which gave an initial pressure of 39 psi). The detector and injector temperatures were set at 300 ◦ C and 250 ◦ C, respectively; the split ratio was at 1:50 and the injection volume was 1 µ L. Oven temperatures were programmed as follows: the initial temperature was set at 120 ◦ C, which was increased at a rate of 25 ◦ C min −1 to 180 ◦ C. This temperature was held for 6 min and finally increased to 240 ◦C at a rate of 25 ◦C min−1, and held for 9 min. FAs were identified by a comparison of the peak retention times of those of the standard solution Supelco 37-component FAME mix (Sigma-Aldrich, St. Louis, MO, USA). FAs were then quantified by standard normalization (% total fatty acids), and they are therefore expressed as a percentage of the total amount of FAs. FA summatories were derived by adding the corresponding single FAs to saturated FAs (SFAs), monounsaturated FAs (MUFAs), PUFAs, n6 PUFAs, n3 PUFAs, n6 LC-PUFAs and n3 LC-PUFAs. Moreover, n6 to n3 ratios were created for analysis. 2.5. Assessment of Anthropometric Infant Outcomes After birth, the infants received a medical examination during which anthropometric measurements were recorded. Data, such as weight, length and BMI at 6, 18 and 36 months of age were included in the present study. Length and weight (with light clothing and no shoes) were recorded using a Harpenden Infantometer (Model 702) calibrated stadiometer (Holtain, Wales, United Kingdom) and a Multina Comfort calibrated balance scale (SOEHNLE, Backnang, Germany), respectively. Weight, length and BMI measurements were ultimately converted to weight-for-age z-scores (WAZ), length-for-age z-scores (LAZ) and BMI-for-age z-scores (BMIZ) (SD scores), according to World Health Organization (WHO) child growth standards [36,37]. 2.6. Assessment of Infant Cognitive Development Infant cognitive development was assessed at 18 months of age using the Bayley Scales of Infant Development III (BSID III) [ 38 ], by trained psychologists in the presence of the mother of the child. These scales measure the level of motor, language and cognitive or mental development. The present study uses the Cognitive Composite score, which is the global score of the scales and represents the overall cognitive development of the children. 2.7. Statistical Analysis Statistical analyses were performed using the SPSS statistical software package for Windows (version 23.0; SPSS Inc., Chicago, IL, USA). The Kolmogorov-Smirnov test was used to study the normal distribution of the data and non-normally distributed data were natural log-transformed. Means and standard deviations (SD) were used to describe continuous variables. The characteristics of the population were analyzed using the ANOVA and Bonferroni post-hoc test. To analyze the FA evolution from colostrum to mature milk, a paired Student’s t-test was used. The independent Student’s t-test was used to compare the breast milk FA composition between maternal weight groups. The associations between breast milk FAs and child anthropometric measurements and cognitive scores were determined using linear regression analyses and corrected for potential confounders such as maternal BMI, smoking, education, GWG and parity, and infant characteristics, such as gender and feeding practices. The Bonferroni correction (0.05/(48 FAs ×3 study groups =144 analyses)) was applied to take multiple testing into account and p-value thresholds were set at 0.002. In the tables, p-values ≤0.05 are highlighted in bold, while those ≤0.002 are additionally marked by stars. 3. Results 3.1. Characteristics of the Population The characteristics of the population are shown in Table 1. Normal-weight women presented the highest GWG, followed by overweight and finally mothers with obesity. The latter group had Nutrients 2019,11, 2154 5 of 18 the highest n6:n3 ratio in dietary intake, while normal-weight mothers had the lowest intake of AA. No significant differences were found in infant characteristics according to maternal BMI. Table 1. Characteristics of the population. Characteristic Normal-Weight Overweight Obesity p Mean (SD) Mean (SD) Mean (SD) Maternal characteristics nnn Age (years) 34 31 (4) 27 32 (4) 17 32 (4) 0.492 Pre-pregnancy BMI (kg/m2)34 22.14 (1.54) a27 27.59 (1.35) b17 33.40 (2.65) c<0.001 * Weight Gain (kg) 25 13.17 (3.55) 23 10.32 (5.20) 15 9.14 (7.06) 0.042 Education (%) 0.660 <High school 26 14.71 19 11.11 11 23.53 High school 3 8.82 5 18.52 2 11.76 >High school 5 76.47 3 70.37 4 64.71 Smoking during pregnancy (%) 0.415 No, never 17 77.27 15 71.43 10 73.68 Yes 3 13.64 5 23.81 1 7.14 Quit 2 9.09 1 4.76 3 21.43 Maternal dietary intake Energy (Kcal/day) 27 2066.37 (261.93) 23 2089.59 (542.07) 12 2058.08 (469.97) 0.961 Lipids (g) 27 86.89 (17.29) 23 85.25 (25.86) 12 93.23 (20.95) 0.468 Lipids (%) 27 37.76 (5.26) 23 39.04 (7.73) 12 41.17 (5.38) 0.307 SFA(g/d) 27 30.81 (6.36) 23 30.05 (8.67) 12 34.19 (5.87) 0.203 MUFA (g/d) 27 36.53 (10.92) 23 39.33 (17.63) 12 36.32 (12.08) 0.926 PUFA(g/d) 27 12.10 (3.17) 23 13.34 (6.59) 12 14.63 (3.95) 0.285 n6 PUFA (g/d) 27 2.48 (1.95) 23 2.89 (2.53) 12 3.44 (1.50) 0.122 n3 PUFA (g/d) 27 0.18 (0.11) 23 0.21 (0.13) 12 0.20 (0.09) 0.427 n-3 from fish (g/d) 27 0.36 (0.31) 23 0.28 (0.34) 12 0.45 (0.34) 0.690 AA (g/d) 27 0.11 (0.06) a23 0.17 (0.08) b12 0.16 (0.08) ab 0.005 EPA(g/d) 27 0.12 (0.11) 23 0.09 (0.11) 12 0.16 (0.12) 0.213 DHA (g/d) 27 0.24 (0.18) 23 0.22 (0.21) 12 0.31 (0.21) 0.269 n6:n3 27 12.99 (2.98) a23 13.53 (3.40) a12 19.12 (8.94) b0.004 Infant characteristics Sex, male (%) 14 41.18 11 40.74 7 41.18 0.999 Birth weight (g) 32 3359.06 (352.35) 27 3340.37 (511.85) 16 3532.35 (389.61) 0.277 Birth length (cm) 31 50.52 (1.57) 27 50.30 (1.88) 16 51.22 (1.80) 0.245 Birth head Circumference (cm) 26 34.31 (1.36) 22 34.36 (1.39) 16 34.69 (1.40) 0.674 Placenta (g) 30 496.67 (144.11) 25 509.20 (130.25) 16 568.13 (143.17) 0.332 Newborn according Lubchenco curves #(%) 0.627 SGA 0 0.00 1 4.55 0 0.00 AGA 26 81.25 16 72.73 11 73.33 LGA 6 18.75 5 22.73 4 26.67 Breastfeeding †(%) 0.290 Exclusive 16 53.33 16 66.67 8 50.00 Mixt 10 33.33 3 12.50 3 18.75 Artificial 4 13.33 5 20.83 5 31.25 Different superscript letters indicate differences among BMI groups, according to ANOVA and the Bonferroni post-hoc test. Chi-square test was applied to qualitative variables. p-values ≤ 0.05 are highlighted in bold and those ≤ 0.002 are additionally marked by stars. # The newborns were divided into three groups according to the Lubchenco curves: SGA: Small for Gestational Age; AGA: Appropriate for Gestational Age; LGA: Large for Gestational Age (LGA). † Breastfeeding practice information was collected at 3 months of age of the child. SFA: Saturated Fatty Acids; MUFA: Monounsaturated Fatty Acids; PUFA: Polyunsaturated Fatty Acids; AA: Arachidonic Acid; EPA: Eicosapentaenoic Acid; DHA: Docosahexaenoic Acid. 3.2. Breast Milk Fatty Acid Evolution The FA evolution from colostrum to mature milk is shown in Table 2. In spite of maternal pre-pregnancy BMI, mature breast milk presented lower levels of C16:1n9, C20:1n9, AA, C22:1n9, C22:4n6, C22:5n6, C22:5n3, DHA, C24:0, C24:1n6 and n3 LC-PUFAs, and higher levels of C8:0, C10:0, medium-chain FAs (MCFAs), eicosapentaenoic acid (EPA):AA and DHA:AA ratios than those found in colostrum. Nutrients 2019,11, 2154 6 of 18 Table 2. Human milk fatty acids profile according to maternal pre-pregnancy BMI. Normal-Weight Overweight Obesity Colostrum (n=26) Mature Milk (n=20) p Colostrum (n=21) Mature Milk (n=23) p Colostrum (n=16) Mature Milk (n=14) p Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) C6:0 0.06 (0.04) 0.08 (0.03) 0.19 0.05 (0.03) 0.10 (0.04) <0.001 * 0.05 (0.03) 0.08 (0.07) 0.002 * C8:0 0.13 (0.09) 0.23 (0.07) 0.002 * 0.13 (0.08) 0.24 (0.09) <0.001 * 0.08 (0.03) #0.24 (0.10) <0.001 * C10:0 0.86 (0.57) 1.29 (0.37) 0.010 0.86 (0.47) 1.36 (0.33) 0.002 * 0.70 (0.57) 1.47 (0.32) <0.001 * C12:0 4.10 (2.07) 4.86 (1.80) 0.039 4.18 (1.97) 4.86 (1.92) 0.14 3.67 (2.49) 5.32 (1.65) 0.029 C14:0 5.15 (1.41) 4.79 (1.71) 0.88 5.38 (1.92) 4.67 (1.66) 0.38 5.04 (2.31) 5.23 (1.22) 0.32 C14:1 0.08 (0.02) 0.10 (0.03) 0.20 0.11 (0.03) †0.11 (0.04) 0.51 0.10 (0.05) 0.12 (0.06) 0.10 C15:0 0.21 (0.03) 0.21 (0.06) 0.80 0.24 (0.04) †0.20 (0.05) 0.016 0.19 (0.04) #0.22 (0.08) 0.32 C16:0 21.13 (2.32) 19.56 (2.29) 0.006 21.24 (1.46) 19.53 (2.02) 0.001 * 21.96 (2.11) 21.20 (2.55) #0.42 C16:1t0.12 (0.03) 0.13 (0.04) 0.59 0.14 (0.04) 0.11 (0.05) 0.06 0.13 (0.04) 0.13 (0.06) 0.98 C16:1n9 0.50 (0.05) 0.43 (0.06) <0.001 * 0.54 (0.13) 0.45 (0.07) 0.010 0.54 (0.10) 0.46 (0.10) 0.024 C16:1n7 1.48 (0.45) 1.83 (0.62) 0.11 1.63 (0.36) 1.80 (0.49) 0.014 1.78 (0.60) 1.88 (0.58) 0.61 C17:0 0.30 (0.04) 0.29 (0.08) 0.74 0.34 (0.03) †0.29 (0.06) 0.026 0.29 (0.05) #0.32 (0.07) 0.28 C17:1 0.15 (0.03) 0.17 (0.03) 0.28 0.18 (0.04) †0.18 (0.04) 0.88 0.17 (0.07) 0.17 (0.05) 0.83 C18:0 5.74 (0.67) 5.74 (0.41) 0.20 5.58 (0.85) 5.88 (0.67) 0.12 5.44 (0.81) †6.03 (0.49) <0.001 * C18:1n9 38.47 (3.51) 39.69 (4.23) 0.41 38.16 (4.57) 38.73 (5.53) 0.87 37.36 (3.11) 36.63 (0.96) †0.30 C18:1n9t0.33 (0.14) 0.28 (0.09) 0.12 0.31 (0.09) 0.26 (0.08) 0.24 0.24 (0.05) †#0.22 (0.04) †0.15 C18:1n7 1.69 (0.22) 1.58 (0.24) 0.02 1.69 (0.28) 1.63 (0.23) 0.81 1.89 (0.42) 1.59 (0.32) 0.022 C18:1n-7t11 0.28 (0.12) 0.30 (0.12) 0.95 0.28 (0.09) 0.26 (0.11) 0.15 0.27 (0.14) 0.24 (0.08) 0.72 C18:2n6 (LA) 13.03 (2.42) 13.60 (3.21) 0.31 13.31 (3.20) 15.20 (3.86) 0.050 12.98 (2.74) 13.90 (3.14) 0.49 C18:2n7c9t11 0.12 (0.03) 0.13 (0.05) 0.69 0.14 (0.04) 0.12 (0.04) 0.32 0.13 (0.05) 0.14 (0.06) 0.67 C18:3n6 0.09 (0.04) 0.17 (0.06) 0.010 0.11 (0.07) 0.18 (0.05) <0.001 * 0.13 (0.09) 0.17 (0.05) 0.15 C18:3n3 (ALA) 0.54 (0.18) 0.59 (0.21) 0.61 0.53 (0.13) 0.58 (0.16) 0.14 0.41 (0.04) †#0.46 (0.08) †#0.16 C20:0 0.20 (0.03) 0.18 (0.01) 0.012 0.18 (0.02) 0.17 (0.03) 0.005 0.19 (0.04) 0.18 (0.04) 0.75 C20:1n9 0.77 (0.20) 0.50 (0.07) <0.001 * 0.74 (0.26) 0.46 (0.05) <0.001 * 0.77 (0.24) 0.48 (0.06) <0.001 * C20:3n6 0.60 (0.15) 0.47 (0.11) <0.001 * 0.65 (0.21) 0.48 (0.06) 0.004 0.68 (0.24) 0.52 (0.14) 0.05 C20:4n6 (AA) 0.67 (0.19) 0.49 (0.05) 0.005 0.66 (0.13) 0.49 (0.12) <0.001 * 0.67 (0.23) 0.47 (0.10) 0.004 C20:5n3 (EPA) 0.05 (0.02) 0.07 (0.03) 0.07 0.04 (0.02) 0.06 (0.02) <0.001 * 0.05 (0.03) 0.07 (0.02) 0.10 Nutrients 2019,11, 2154 7 of 18 Table 2. Cont. Normal-Weight Overweight Obesity Colostrum (n=26) Mature Milk (n=20) p Colostrum (n=21) Mature Milk (n=23) p Colostrum (n=16) Mature Milk (n=14) p Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) C22:0 0.08 (0.03) 0.06 (0.02) 0.007 0.09 (0.03) 0.08 (0.03) 0.20 0.08 (0.02) 0.07 (0.02) 0.039 C22:1n9 0.18 (0.06) 0.09 (0.02) <0.001 * 0.19 (0.08) 0.09 (0.02) <0.001 * 0.18 (0.06) 0.09 (0.01) <0.001 * C22:2n6 0.07 (0.03) 0.04 (0.01) 0.003 0.08 (0.03) 0.06 (0.03) 0.22 0.06 (0.02) 0.06 (0.02) †0.22 C22:4n6 0.25 (0.13) 0.10 (0.02) <0.001 * 0.28 (0.15) 0.12 (0.04) <0.001 * 0.37 (0.27) 0.12 (0.03) <0.001 * C22:5n6 0.12 (0.04) 0.05 (0.01) <0.001 * 0.12 (0.05) 0.07 (0.03) †0.048 0.12 (0.05) 0.09 (0.05) †0.040 C22:5n3 0.16 (0.08) 0.11 (0.03) 0.012 0.14 (0.05) 0.11 (0.03) 0.002 * 0.19 (0.09) 0.12 (0.04) 0.007 C22:6n3 (DHA) 0.41 (0.14) 0.28 (0.11) <0.001 * 0.35 (0.08) 0.22 (0.06 †<0.001 * 0.39 (0.13) 0.25 (0.07) 0.002 * C23:0 0.12 (0.05) 0.05 (0.02) <0.001 * 0.12 (0.05) 0.06 (0.03) <0.001 * 0.13 (0.05) 0.09 (0.07) †0.06 C24:0 0.10 (0.03) 0.05 (0.02) 0.002 * 0.10 (0.04) 0.06 (0.03) 0.007 0.09 (0.03) 0.06 (0.03) 0.031 C24:1 0.20 (0.11) 0.06 (0.02) <0.001 * 0.19 (0.13) 0.06 (0.04) <0.001 * 0.19 (0.09) 0.07 (0.03) <0.001 * EPA:AA 0.07 (0.03) 0.12 (0.03) 0.005 0.06 (0.03) 0.09 (0.04) †<0.001 * 0.07 (0.03) 0.12 (0.07) 0.004 DHA:AA 0.63 (0.22) 0.77 (0.19) 0.005 0.55 (0.13) 0.76 (0.24) <0.001 * 0.61 (0.21) 0.90 (0.34) 0.004 SFA 27.45 (2.66) 25.83 (2.48) 0.008 27.43 (1.93) 25.95 (2.49) 0.004 27.96 (2.34) 27.80 (2.62) †,# 0.96 MCFA 5.14 (2.69) 6.46 (2.19) 0.021 5.23 (2.46) 6.56 (2.27) 0.037 4.50 (3.06) 7.11 (1.99) 0.010 MUFA 42.80 (3.51) 43.73 (4.17) 0.87 42.61 (4.91) 42.76 (5.70) 0.74 42.17 (3.56) 40.74 (1.37) †0.12 n6 PUFA 15.41 (2.53) 15.24 (3.31) 0.77 15.79 (3.23) 16.89 (3.93) 0.29 15.67 (3.01) 15.64 (3.19) 0.93 n3 PUFA 1.16 (0.30) 1.05 (0.24) 0.018 1.07 (0.15) 0.96 (0.21) 0.12 1.04 (0.23) 0.90 (0.13) 0.06 n6 LC-PUFA 2.30 (0.59) 1.48 (0.22) <0.001 * 2.36 (0.63) 1.52 (0.24) <0.001 * 2.56 (0.93) 1.58 (0.34) <0.001 * n3 LC-PUFA 0.62 (0.22) 0.45 (0.16) <0.001 * 0.54 (0.12) 0.38 (0.11) <0.001 * 0.63 (0.23) 0.44 (0.09) 0.008 n6:n3 PUFA 14.28 (4.76) 15.08 (3.91) 0.06 15.13 (4.37) 18.28 (5.33) †0.033 15.67 (4.22) 17.80 (4.97) 0.15 n6:n3 LC-PUFA 4.00 (1.16) 3.62 (1.18) 0.08 4.49 (0.95) 4.28 (1.24) 0.41 4.23 (1.05) 3.73 (0.92) 0.14 Means are expressed as percentages of total FAs. The p-value shown refers to the breast milk evolution within each group of weight, according to Student’s paired t-test. p-values ≤ 0.05 are highlighted in bold and those ≤ 0.002 are additionally marked by stars. † Indicates differences (p ≤ 0.05) compared with normal-weight group and its corresponding breast milk, according to Student’s independent t-test. # Indicates differences (p ≤ 0.05) between overweight and obese groups and corresponding breast milk, according to Student’s independent t-test; LA: Linoleic Acid; AA: Arachidonic Acid; ALA: α -linolenic Acid; EPA: Eicosapentaenoic Acid; DHA: Docosahexaenoic Acid; SFA: Saturated Fatty Acids; MCFA: Medium-chain Fatty Acids; MUFA: Monounsaturated Fatty Acids; PUFA: Polyunsaturated Fatty Acids; LC-PUFA: Long-Chain Polyunsaturated Fatty Acids. Nutrients 2019,11, 2154 8 of 18 Regarding other biologically important FAs, and always compared to colostrum levels, the mature milk of normal-weight mothers showed higher levels of C12:0 and C18:3n6, and lower levels of C16:0, C20:0, C20:3n6, C22:0, C22:2 n6, C23:0, saturated fatty acids (SFAs) and n3 PUFAs; the mature milk of overweight mothers showed increased levels of C6:0, C16:1n7, linoleic acid (LA), C18:3n6, EPA and n6:n3 ratio, and decreased concentrations of C15:0, C16:0, C17:0, C20:0, C23:0 and SFAs; and finally, the mature milk of mothers with obesity had higher levels of C6:0, C12:0 and C18:0 and lower levels of C18:1n7 and C22:0. 3.3. Breast Milk FAs According to Maternal Weight Group Table 2also shows the differences in breast milk FAs between weight groups. Compared to normal-weight women, the overweight group had higher levels of C14:1, C15:0, C17:0 and C17:1 in colostrum; and higher levels of C22:5n6 and n6:n3 ratio and lower levels of DHA and EPA:AA in mature milk. On the other hand, compared to normal-weight mothers, mothers with obesity had lower levels of C18:0, C18:1n9t and ALA in colostrum; and lower levels of C18:1n9, C18:1n9t, ALA and MUFAs and higher levels of C22:2n6, C22:5n6, C23:0 and SFAs in mature milk. We also compared overweight with mothers with obesity and found that the group with obesity had lower concentrations of C8:0, C15:0, C17:0, C18:1n9t, ALA in colostrum; and higher levels of C16:0 and SFAs and lower levels of ALA in mature milk. 3.4. Association of Breast Milk FAs with Infant Growth Table 3shows the associations between breast milk FAs and infant growth. All associations were observed after adjusting for potential confounders, which included maternal pre-pregnancy BMI, maternal smoking, weight gain during pregnancy, maternal education, gender of the child and type of infant feeding practice. Table 3. Associations between breast milk PUFA levels and anthropometric measurements in infants. Fatty Acid BMIZ WAZ LAZ Colostrum Mature Milk Colostrum Mature Milk Colostrum Mature Milk 6mo n=37 6mo n=39 6mo n=37 6mo n=39 6mo n=38 6mo n=39 18mo n=38 18mo n=37 18mo n=38 18mo n=38 18mo n=38 18mo n=38 36mo n=16 36mo n=13 36mo n=16 36mo n=13 36mo n=18 36mo n=14 βpβpβpβpβpβp C18:3n3 (ALA) 6mo −0.11 0.63 −0.15 0.40 −0.06 0.77 −0.22 0.24 0.05 0.80 −0.12 0.49 18mo 0.32 0.13 −0.11 0.57 0.11 0.63 −0.13 0.51 −0.20 0.26 −0.06 0.69 36mo −0.44 0.21 0.01 0.99 −0.27 0.52 0.06 0.95 −0.17 0.63 −0.86 0.22 C18:2n6 (LA) 6mo 0.33 0.12 −0.13 0.43 0.42 0.027 0.16 0.36 0.15 0.40 0.09 0.60 18mo −0.19 0.34 0.04 0.85 0.18 0.37 0.05 0.79 0.06 0.73 0.17 0.26 36mo 0.12 0.75 −0.22 0.50 0.02 0.96 − 0.136 0.75 0.01 0.98 −0.33 0.41 C20:4n6 (AA) 6mo −0.44 0.016 0.02 0.91 −0.20 0.26 0.04 0.84 0.25 0.13 0.03 0.86 18mo −0.03 0.89 −0.12 0.57 0.06 0.77 −0.13 0.52 0.10 0.50 0.15 0.39 36mo 0.30 0.30 0.09 0.81 0.42 0.20 0.07 0.86 0.20 0.48 0.12 0.75 C20:5n3 (EPA) 6mo −0.51 0.012 0.00 0.99 −0.36 0.07 −0.13 0.49 0.18 0.31 −0.18 0.29 18mo −0.30 0.12 0.00 0.98 −0.13 0.51 0.08 0.66 0.15 0.35 0.09 0.62 36mo −0.74 0.155 −1.08 0.30 −0.58 0.34 −1.13 0.28 0.14 0.69 −0.59 0.61 Nutrients 2019,11, 2154 9 of 18 Table 3. Cont. Fatty Acid BMIZ WAZ LAZ Colostrum Mature Milk Colostrum Mature Milk Colostrum Mature Milk 6mo n=37 6mo n=39 6mo n=37 6mo n=39 6mo n=38 6mo n=39 18mo n=38 18mo n=37 18mo n=38 18mo n=38 18mo n=38 18mo n=38 36mo n=16 36mo n=13 36mo n=16 36mo n=13 36mo n=18 36mo n=14 βpβpβpβpβpβp C22:6n3 (DHA) 6mo −0.37 0.043 −0.16 0.38 −0.31 0.07 −0.29 0.10 0.00 0.99 −0.23 0.17 18mo 0.14 0.42 0.03 0.88 0.08 0.66 0.00 0.99 −0.05 0.74 −0.03 0.84 36mo 0.42 0.29 0.33 0.46 0.65 0.13 0.38 0.39 0.46 0.22 0.58 0.14 n6 PUFA 6mo 0.21 0.32 0.13 0.45 0.34 0.07 0.16 0.35 0.20 0.27 0.10 0.55 18mo 0.16 0.41 −0.06 0.776 0.18 0.40 0.04 0.83 0.08 0.65 0.18 0.23 36mo 0.20 0.587 −0.19 0.64 0.15 0.72 −0.11 0.79 0.07 0.84 −0.31 0.44 n3 PUFA 6mo −0.38 0.047 −0.19 0.27 −0.33 0.07 −0.32 0.07 −0.00 0.991 −0.22 0.18 18mo 0.16 0.38 −0.11 0.56 0.04 0.84 −0.11 0.53 −0.12 0.427 −0.05 0.75 36mo −0.20 0.60 0.17 0.78 0.05 0.90 0.21 0.74 0.05 0.897 −0.18 0.77 n6 LC−PUFA 6mo −0.38 0.047 −0.06 0.77 −0.17 0.36 0.00 0.98 0.19 0.253 0.09 0.65 18mo −0.05 0.77 −0.27 0.19 0.03 0.88 −0.17 0.41 0.10 0.508 0.19 0.25 36mo 0.40 0.22 0.11 0.78 0.60 0.09 0.12 0.76 0.25 0.390 −0.03 0.95 n3 LC−PUFA 6mo −0.43 0.020 −0.19 0.28 −0.34 0.05 −0.33 0.06 0.03 0.866 −0.24 0.16 18mo 0.07 0.70 −0.04 0.82 0.05 0.78 −0.02 0.90 −0.01 0.955 0.02 0.89 36mo 0.28 0.44 0.19 0.63 0.53 0.18 0.21 0.59 0.42 0.211 0.40 0.31 n6:n3 6mo 0.42 0.031 0.30 0.10 0.45 0.011 0.45 0.013 0.11 0.519 0.30 0.08 18mo −0.04 0.82 0.05 0.78 0.06 0.74 0.14 0.47 0.14 0.369 0.21 0.19 36mo 0.30 0.34 −0.23 0.55 0.05 0.88 −0.18 0.65 0.01 0.978 −0.26 0.58 LC n6:n3 6mo 0.12 0.56 0.14 0.41 0.22 0.24 0.29 0.09 0.15 0.373 0.24 0.13 18mo −0.13 0.48 −0.08 0.66 −0.04 0.86 −0.05 0.77 0.11 0.490 0.07 0.65 36mo 0.35 0.54 −0.07 0.86 0.16 0.81 −0.08 0.85 −0.31 0.516 −0.30 0.46 Associations were evaluated using lineal regression analyses. β and pare corrected values after adjustment for potential confounders: maternal pre-pregnancy BMI, maternal smoking, weight gain during pregnancy, maternal education, sex of the child and type of infant feeding practice. p-values ≤ 0.05 are highlighted in bold and those ≤ 0.002 are additionally marked by stars. mo: month; LA: Linoleic Acid; AA: Arachidonic Acid; ALA: α -linolenic Acid; EPA: Eicosapentaenoic Acid; DHA: Docosahexaenoic Acid; PUFA: Polyunsaturated Fatty Acids; LC-PUFA: Long chain Polyunsaturated Fatty Acids. At 6 months of age, we found that colostrum levels of AA, EPA, DHA, n3 PUFAs, n6 LC-PUFAs and n3 LC-PUFAs were inversely associated with infant BMIZ, while the n6:n3 ratio was positively associated with it. Also, at 6 months of age, LA and the n6:n3 ratio in both colostrum and mature milk were positively associated with WAZ. No associations were found between mature milk and any variable at 1.5 or 3 years of age. 3.5. Associations of Breast Milk FAs with Infant Cognition Table 4presents the associations between breast milk PUFAs and infant cognition at 18 months of age. When the whole population was analyzed, no associations were found. However, infants born to normal-weight mothers presented a positive association between cognition scores and LA and n6 PUFA levels in colostrum. 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