**1. Introduction**

In spite of e fforts 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

of reproductive age, and this is a ffecting the health of future generations [1]. Early-life nutrition plays a key role in infant growth and development and has a programming e ffect 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 di fferent mechanisms, breastfeeding being one of them. If the mother of the child has obesity, the fatty acid (FA) profile in breast milk can be di fferent, 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 a ffected.

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 o ffspring 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 augmen<sup>t</sup> 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 e ffect 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 di fferent 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 e ffect 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/m2, *<sup>n</sup>*=34), overweight (BMI=25–29.99 Kg/m2, *<sup>n</sup>*=27) and obese (BMI>30 Kg/m2, *n* = 17). 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 pregnan<sup>t</sup> 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, hypo- or 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 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 μL. Oven temperatures were programmed as follows: the initial temperature was set at 120 ◦C, which was increased at a rate of 25 ◦C min−<sup>1</sup> 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.
