**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 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.



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.


**Table 2.** Human milk fatty acids profile according to maternal pre-pregnancy BMI.



*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.

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 2 also 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 3 shows 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.


**Table 3.** *Cont.*

Associations were evaluated using lineal regression analyses. β and *p* are 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 4 presents 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. On the other hand, the infants of overweight mothers presented a direct association of DHA and n3 LC-PUFA levels in colostrum with cognitive score, while the n6:n3 ratio in colostrum was inversely associated with it. With respect to infants born to mothers with obesity, a positive association was found between ALA levels in mature milk and cognition.



Fatty Acids; LC-PUFA: Long chain Polyunsaturated Fatty Acids.
