**4. Discussion**

The present study analyzed the influence of an infant formula, supplemented with AA + DHA, on LCPUFA levels in infants with different FADS genotype. A number of studies have investigated the association between variants in the FADS gene cluster and FA levels in human tissue [13,14,22–24]; however, little information is available on the neonatal population. Other studies have analyzed the effect of LCPUFA supplementation in early life [9,39–41], but no association with FADS SNPs has been investigated whatsoever. Our study contributes to generating new evidence in this matter.

FADS minor alleles have shown to decrease the desaturase activity [13,16,17,42–46], compromising LCPUFA production. Some authors have demonstrated that FADS SNPs lower proportions of GLA, AA, and EPA, and accumulate the LCPUFA precursors LA and ALA [11,46,47]. Others have demonstrated that FADS minor allele carriers exhibit lower AA:DGLA and EPA:ALA indexes [48,49]. As mentioned before, some studies include indexes of AA to LA, as well as EPA and DHA to ALA in order to use them as markers of the activity of fatty acids desaturation mediated by the enzymes D5D and D6D, respectively; in our study, we use GLA:AA and DGLA:LA to indicate D6D activity, whereas D5D activity is reflected by the ratio of AA to DGLA, and we consider it relevant to record if an infant formula supplemented with LCPUFA can influence the enzymatic activity of infants compared with non-supplemented, to allow a possible comparison for future studies. Moreover, it has been established that the AA is the most severely affected FA [13]. In our study, infants from the BF group carrying FADS minor alleles were not associated with FAs, which suggests that desaturase activity is not affected by FADS genotype when infants are exclusively breastfed.

However, infants carrying FADS minor alleles in the EF and SF groups were associated with decreased D5D and D6D activities, which is in line with the previous studies [46,50–52]. The most affected group was the EF, where infants carrying minor alleles of rs173547 (FADS1) had decreased levels of LA, AA and DHA, and minor allele carriers of rs2072114 (FADS2) were negatively associated with AA and the D5D and D6D desaturases activity (AA:LA index) (*p* < 0.005). Accordingly, the nominal associations (*p* < 0.05) were also inclined to decrease the desaturase activities when infants presented FADS minor alleles. In the EF group this was observed with n6 PUFAs (LA, DGLA, AA, AdA, DPAn6, AA:DGLA), EPA and DHA. Moreover, in the SF group the SNP rs174570 (FADS2) stood out by associating with decreased levels of AA, GLA:LA, DGLA:LA, AA:LA and EPA:ALA. These results sugges<sup>t</sup> that the infants without AA and DHA supplementation were the least affected in terms of FA levels by FADS genetic variants. To make a proper interpretation of the results, it is of interest to take into account the different effect size among the study groups. For instance, the strongest association was found within the EF group, where minor allele carriers of rs2072114 showed the highest negative association with AA (βc −0.522, *p* < 0.001).

In our study, AA and DHA levels, in spite of the genotype, showed a gradient of SF < EF < BF. More specifically, among major homozygotes of FADS SNPs (except rs174570), AA and DHA concentrations were closer between the EF and the BF groups, and higher compared to SF infants, which shows the expected effect of the infant formula supplementation. Nevertheless, when carrying minor alleles, the EF group did not reach similar levels to the BF infants. In summary, Table 4 should be interpreted with caution due to effect size differences among study groups (e.g., the difference between the FA means of EF and BF groups is narrower when infants carry major homozygotes, and these differences increase among minor allele carriers).

This study exposes minor allele carriers as a potential vulnerable group since the same supplementation might not be enough for them, especially in the case of AA that showed the same levels than the SF group, whereas the DHA was at least higher than them.

Increasing the supplemented dose of preformed LCPUFAs may compensate for the effect of FADS minor alleles. According to Miklavcic et al., increasing the formula supplementation to AA 34 mg/100 kcal and DHA 17 mg/100 kcal prevents the reduction in AA by minor alleles [53]. However, in our study, the EF group received a supplementation of 23.2 mg/100 kcal of AA and 15.5 mg/100 kcal of DHA, which could be a reason for their suggested high SNP influence. As for the SF infants, their AA and DHA levels were almost unaffected by minor alleles. Since previous evidence suggested that high supplementation levels of AA and DHA could lessen the influence of FADS SNPs on LCPUFA levels, we somewhat expected a gradient effect of SNPs of SF > EF > BF. However, this expectation was not determined since the SF group was not supplemented at all. The gradient resulted in EF > SF > BF. It is interesting how the LCPUFA levels of the SF group were less influenced by the FADS SNPs than the EF group. We could speculate some kind of protection effect (e.g., a more effective synthesis in the absence of preformed AA and DHA intake [54]) against FADS SNPs when infants have low LCPUFA levels. It is important to remember that, even though the EF group was more influenced by FADS SNPs than the SF group, infants from the EF group still presented higher AA and DHA levels. Corresponding to the theory of the higher the LCPUFA supply the less SNP influence, AA and DHA levels of BF infants were not perceptibly affected by genotype, possibly because they had the highest LCPUFA concentrations related to the high content in breast milk. One should consider that mean fat content of human milk may vary considerably between individuals as well as between study populations from affluent or developing countries [55]. Maternal factors, such as diet and weight gain during pregnancy, and sampling procedures have distinct impact on fat levels [56]. Additionally, DHA levels in breast milk are quite sensitive to maternal diet [4] and maternal FADS genotypes are associated with breast-milk AA concentration and this might therefore influence the supply of breast milk FAs [42].

Several authors have demonstrated that FADS SNPs affect FA levels and are associated with health conditions [13,39,57–62]. The major contribution of our study is providing evidence in infants below 6 months of age, identifying a potential vulnerable group of infants who could benefit from more personalized nutrition, and contributing to defining the ideal AA and DHA supplementation of infant formulas. This line of research merits further attention because early life nutrient exposure can program future health. We acknowledge some limitations, such as the lack of information on maternal dietary intake and supplementation, and the relatively small sample size of our population. We should also take into consideration the estimation of effect size to interpret the influence of SNPs on LCPUFA levels according to the study group. Now that we have evidence for each individual SNP, it is of interest to perform a FADS haplotype analysis to compare the results. The strengths of our study include the double-blinded cohort study design and the participation of three infant groups with different feeding practices. It is important to mention that the infant formulas were created previous to the implementation of the Commission Delegated Regulation (EU) 2016/127 [8] when DHA supplementation was not mandatory.
