**4. Discussion**

The present study o ffers the evaluation of human breast milk FA composition during the first month postpartum according to maternal weight, and the impact on child outcomes from 6 months to 3 years of age. This is one of the very few studies analyzing the influence of maternal weight on breast milk FA composition and, to our knowledge, the second one to assess this parameter in both colostrum and mature breast milk. Moreover, we believe this is the first study to address its e ffect on both infant cognitive developmental parameters and growth all in one study.

Upon analysis of the population characteristics, we observed that women with obesity had the lowest GWG, even though no nutritional intervention was carried out. This finding is in line with the results of a systematic review about GWG in women with obesity where they concluded that GWG decreased with each higher BMI classification [39]. In fact, weight loss during pregnancy is more common in women with obesity than non-obese women [40] and GWG decreases as the severity of obesity increases [41]. Nonetheless, we observed that women with obesity had the highest n6:n3 ratio in their dietary intake, which suggests they had the lowest-quality dietary intake, as similarly demonstrated by other studies where a high weight status is related to a high dietary intake of n6 FAs and a low intake of n3 FAs [42].

We did not observe any di fferences in infant characteristics according to maternal BMI. Evidence suggests that infants born to mothers with obesity have an increased risk of having a higher weight and length at birth [1], but this was not the case in our population. Since GWG is directly associated with birth weight [43], a possible explanation for this finding is that women with obesity showed the lowest GWG, and therefore their o ffspring did not present increased weight at birth. This is in the line with the conclusions of the systematic review of Faucher and Barger, in which several studies reported a linear decrease in the prevalence of being large for gestational age (LGA) and less GWG [39], suggesting that women with obesity and low GWG would have some benefits on fetal growth. This could be the reason why in our study children from women with obesity did not present higher weight or length. Nevertheless, our data still showed a tendency in which formula-fed infants and those considered LGA represented a higher percentage in the groups of women with overweight and obesity, according to Lubchenco's curves [44].

Regardless of maternal weight, our data showed that when breast milk transitioned from colostrum (2–4 days postpartum) to mature milk (28–32 days postpartum), the levels of crucial FAs such as AA, DHA, and n3 LC-PUFA were decreased. Other authors have also demonstrated this finding [45,46]. The high content of crucial FAs in colostrum has biological relevance because it is highly associated with child outcomes, possibly because of the nutrient supply during the first few days of life, which are critical for infant health [47]. Nonetheless, we also found higher levels of EPA:AA and DHA:AA in mature milk, which are positively associated with health outcomes as well [48]. Analyzing the breast milk evolution within each weight group, we found that SFA concentrations decreased in the mature milk of women with normal-weight and overweight, but not in the mature milk of women with obesity. As described in other studies, the human milk of these women could present higher levels of SFA [17]. The factors attributed to this increased amount of SFA in breast milk of women with obesity could be the metabolic status and diet. It is well known that obesity is intrinsically a pro-inflammatory state influenced by dietary intake [49], where the ratio of n6:n3 PUFA is a clear factor a ffecting inflammation and obesity development [50]. In our study, the higher dietary intake of n6:n3 PUFA ratio in women with obesity, might be enhancing the pro-inflammatory state, and thereby affecting the levels of breast milk SFAs. Moreover, the fact that women with an increased BMI may have an increased intake of n6:n3 PUFA might also explain the higher n6:n3 PUFA ratio found in the mature milk of overweight women compared to their colostrum, although their increased intake of dietary n6:n3 was not significant.

To the best of our knowledge, eight studies have evaluated the FA composition of breast milk according to maternal BMI, but the results available in the literature are not entirely consistent and the studies di ffer in terms of the weight groups tested and the timing of sample collections. Out of these eight studies, only one shares the same collection timing for colostrum that we used [32]; another one used a similar timing for both colostrum and mature milk [16], but the other 6 studies collected the milk in different times ([10,17,18,29–31]). Regarding the weight groups used, 5 did not share the same groups that we used [10,16,17,29,31], and 2 out of the 3 studies that did [18,30,31] used a different criteria to classify weight according to BMI [30,32].

Although our results and the ones available in the literature sugges<sup>t</sup> that a high maternal weight status alters human milk nutrient content, there is an inconsistency regarding which FAs are the most influenced according to BMI groups. This could be attributed to numerous factors, such as sample size, population, methods, FAs included in the analysis, weight group classification and the timing of breast milk collection. However, it is important to highlight that, even without a clear consistency among studies, an increased BMI is found to alter FA concentrations in breast milk, generally increasing SFA and n6 PUFAs and decreasing FAs from the n3 series. An important factor that could explain the differences found among weight groups could be related to dietary intake during late pregnancy, since several studies have demonstrated that this affects breast milk composition [51]. This suggests that women with overweight and obesity could have an increased dietary intake of n6 FAs and SFAs and a poor intake of n3 FAs. As previously mentioned, this happened in our population, where we found that the n6:n3 ratio of dietary intake was higher in women with an increased BMI, especially those with obesity. Since a maternal pro-inflammatory diet is positively associated with increased concentrations of SFA and MUFA in breast milk [10], specific maternal metabolic markers could be an interesting approach to predicting the predominance of certain FAs in breast milk.

This study also analyzed the possible association between breast milk FA composition and infant growth and cognition. It is well known that many nutrients are critical for proper infant growth and neurodevelopment. Animal models and epidemiological studies sugges<sup>t</sup> that PUFAs such as AA and DHA are particularly important [52,53]. Thus, we evaluated the association between the PUFA levels in breast milk and infant anthropometric measurements at 6, 18 and 36 months of age. For this analysis, we used the z-score values WAZ, LAZ and BMIZ to evaluate with greater accuracy which children were within or outside the normal range [1,36,54]. Our findings showed that LC-PUFAs—especially AA, EPA, DHA, n3 and n6 LC-PUFAs—in colostrum had a negative association with infant BMIZ at 6 months. In accordance with these results, a recent review that analyzed the association between n3 PUFAs and growth suggested that DHA during pregnancy, lactation and early life may be associated with significant benefits in infant growth and development [55]. Similarly, Pedersen et al. observed a negative association between DHA levels in breast milk and BMI in children from 2 to 7 years of age. They also found an overall inverse association between breast milk DHA and body fat percentage [56]. Although it is important to mention that BMI is not the best method to quantify body composition, and especially to assess body fat in children [57,58], DHA content in breast milk could have some benefits in postponing the age of adiposity rebound [56], which is the second rise in adiposity that usually occurs between 3 and 7 years of age [59]. It is known that the age that rebound occurs predicts later fatness, meaning that an earlier rebound would be a risk factor for later obesity [59]. On the other hand, our data also indicated that n6 PUFA levels may contribute to a fat mass increase in children [59], since LA in colostrum and the n6:n3 ratio in both mature milk and colostrum could influence WAZ and BMIZ at 6 months of age. Since the n6 PUFAs in mature milk were generally increased in overweight and obese mothers, their children could be more susceptible to developing obesity [17,20,50]. Indeed, children from overweight and obese mothers presented a tendency to be LGA. In contrast, Much et al. found that AA and n6 PUFAs in mature breast milk were negatively associated with infant weight and BMI (up to 4 months of age) [33], suggesting that the role of these n6 FAs (including AA) might be age-dependent and serve as important regulating factors for growth in early postnatal life. Due to the low variability of AA contents in breast milk across populations (0.24–1% of FAs) [7], a possible explanation of this discrepancy could be the quantitative amount of milk intake by the breastfed infant, meaning that depending on the daily ingested volume of milk, AA would have its growth-regulatory effects or not [33]. Further studies are needed to look into such quantitative aspects. In our study, we only found significant associations between FAs and infant growth at 6 months of life, but not at 18 nor 36. This finding may be due to the child's own diet, lifestyle and metabolism. However, the associations found at 6 months are relevant, because it is a crucial age that represents a critical period in the child's development and programming [3]. A curious result that we found is that length was not correlated to any PUFA, which again is in disagreement with Much et al. They inversely correlated DHA, EPA and n3 PUFA with length at 1 year of age. Their milk collection was at 6 weeks and 4 months postpartum [33], whereas in our study it was at 2–4 days and 28–32 days postpartum. Therefore, the possible evolution of FA species over time would be a possible explanation for the di fferent results. From our study and the evidence gathered, we can see that PUFAs in breastmilk influence infant growth; however, there is a high variability in existing results. Further studies are needed to obtain more conclusive outcomes [2].

PUFAs are also critical for an adequate brain growth and function in aspects such as neurogenesis, nerve impulse transmission, neuronal integrity, and vitality and gene expression in the brain [52,53,60]. Thus, we explored the association between breast milk PUFA levels and cognitive score at 18 months of life. On the one hand, when we analyzed the total population, we found no association between any FA in breast milk and child development. Similarly, there have been observational studies that found no strong evidence for a beneficial role of LC-PUFAs in order to explain the positive relationship between breastfeeding and cognition [61]. This raises the question as to whether LC-PUFA levels may only be beneficial in children's mental development when breastfeeding levels are high [62]. Although we corrected the analysis by the type of breastfeeding, this information was collected at 3 months of age, so we do not know which e ffect could have had a longer period of exclusive breastfeeding.

We also explored the association between breast milk FA levels and infant cognition according to maternal BMI. In general, we found a direct association between n3 and n6 PUFA levels in colostrum and infant cognition at 18 months of age. The colostrum from overweight mothers was the one that presented more relevant associations, specifically, a high n6:n3 ratio was negatively associated with cognition, whereas higher DHA concentrations were directly associated with better cognitive scores, which is in line with Bernard et al. [63]. This suggests that the cognition of infants born to overweight women could be enhanced by promoting n3 FAs, more specifically DHA, in the maternal diet. These results are in line with meta-analyses, animal and epidemiologic studies [60,64,65], and support WHO recommendations on breastfeeding for the two first years of life or beyond [66]. We must consider that, in our study, the cognitive score was assessed at 1.5 years of life, and at this age, there are many factors related to the child that could influence their cognition. The potential cofounders that we have used to adjust this analysis were mainly related to the mother, and only the gender and type of feeding practice were related to the child. Important factors such as infant diet or physical activity are lacking and could have a huge influence in the results because intake of micronutrients, such as n3 FAs, vitamin B12, folic acid, zinc, iron and iodine, together with malnutrition and general dietary patterns and other lifestyle habits, influence child cognitive development as well [67–69].

Overall, our study highlights the importance of the maternal health before, during and after pregnancy, since it could have a grea<sup>t</sup> impact in the breast milk FA composition and, in consequence, in the offspring's growth and cognition which a ffects their future health. Many women start developing healthy habits when they are pregnan<sup>t</sup> or planning a pregnancy. However, as presented in our study, the pre-pregnancy health status has an important e ffect in the quality of the human milk, consequently affecting the health of the child. Therefore, bigger e fforts must be put in place to promote and guarantee a healthier lifestyle and nutritional status in the general population to pursuit healthier future generations.

We acknowledge some limitations in our study, such as the small sample size. However, it is important to understand that, even though PREOBE is a larger cohort, we were not able to include all the participants in the present study due to lack of data or samples, possibly related to indisposition to participate given the complexity and sensitivity of the periods involved: childbirth and breastfeeding. Although risk factors, such as socio-demographic information and maternal diet, allowed us to adjust our statistical models for potential confounders, we cannot rule out residual confounding, especially coming from data related to the infants at 1.5 and 3 years of age because data on their dietary intake, lifestyle and other characteristics, could be greatly influencing the results. Another limitation is that women receiving supplements of DHA for over 3 months were excluded, but we do not know the possible effect of that initial supplementation in the breast milk FA profile. Moreover, recording the timing between sample collection or the last meal, collecting information on what was consumed before and after each sample was taken, and analyzing the different breast milk samples of one day without mixing them, would provide valuable data to assess the human milk nutrient content and impact. In general, further research is required to provide a better understanding of the role that FAs play in obesity development and management, paying special attention to the methods used for analysis and promoting the comparison of results between cohorts.
