**1. Introduction**

Long chain polyunsaturated fatty acids (LCPUFA) have an important role in the immune system regulation, blood clots, neurotransmitters, cholesterol metabolism, and in the structure of membrane phospholipids in the brain and the retina [1]. Attention has been devoted especially to arachidonic acid (AA) and docosahexaenoic acid (DHA), due to their key role for optimal health, cognition and development during fetal and early postnatal life [2]. Breastmilk is usually the only external source of AA and DHA for infants during the first months of life [3,4]. When breastfeeding is not possible, infants require breast milk substitutes [5], which are usually supplemented with nutrients to match the breast milk content [6]. However, there is still debate and different opinions in regards of DHA and AA supplementation in infant formulas. The European Food Safety Authority (EFSA) [7] and the Commission Delegated Regulation (EU) 2016/127 [8] have proposed DHA supplementation as mandatory for infant formulas, while no minimum amount of AA was determined to be necessary, setting AA supplementation as an optional ingredient. However, it has been observed that when infants receive both AA and DHA supplementation they have better outcomes in cognitive performance than receiving DHA alone [9]. Other authors have even found that DHA alone did not influenced cognitive development at all [10]. Therefore, whether DHA should be supplemented alone or with AA remains controversial. Additionally, there is no agreed specific dose for supplementation, and there is little evidence of the long-term effect.

LCPUFAs can also be endogenously synthesized from the essential fatty acids (FAs): linoleic acid (LA) and alpha-linolenic acid (ALA). LCPUFA synthesis requires desaturation and elongation reactions. D6 and D5 desaturases (D6D and D5D) are two key enzymes that catalyze the synthesis by introducing *cis* double bonds at specific positions. Fatty acid desaturase genes FADS1 and FADS2 encode D5D and D6D, respectively, making them a rate limiting factor in LCPUFA conversion [11]. However, the endogenous synthesis of AA and DHA from their precursors is limited in humans as Demmelmair et al. observed in women that only 1.2% of the AA is directly derived from LA intake [12]. Likewise, both blood and tissue levels of PUFAs are influenced to a large extent by genetic heritability [13]. There are many studies showing the major impact of gene variants of the FADS gene cluster on the FA composition of blood, tissues and human milk [14–16]. For instance, single nucleotide polymorphisms (SNPs) in the FADS gene modulate the capacity for endogenous synthesis of LCPUFAs by compromising the desaturase activity of the involved enzymes [17–21]. It has been observed that up to 28% of variation of AA blood levels is due to FADS genetic variants, while LA is affected in 9% [13]. Furthermore, Baylin et al. observed that an impaired desaturase activity may induce an unbalanced proportion of n3 (e.g., the FADS2 deletion could prevent the conversion of the precursor ALA into LCPUFAs) [22]. Likewise, Schaeffer et al. observed that variants in the FADS1 and FADS2 genes showed strong associations with levels of n6 (e.g., LA, gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), AA, and adrenic acid (AdA)), and n3 FAs (ALA, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) [13], and DHA [23]). It has also been observed that variants in the FADS cluster can also influence total LDL, and DHL cholesterol, triglycerides, phospholipids, C-reactive protein, proinflamatory eicosanoids and cardiovascular disease endpoints [24,25].

Even though there is wide evidence of the effect of FADS genetic variants in FA concentrations of different biological tissues, there is little evidence for infants during their first year of life [19,26–28], which is a critical period of early life programming in which LCPUFAs play an important role [29]. Studying this could identify vulnerable groups in the pediatric population and contribute to the refinement of current recommendations and legislations in regards to infant AA and DHA supplementation. Given the low rates of exclusive breastfeeding in Europe, and while efforts are still being made to promote it, it is important to secure an appropriate source of LCPUFAs for infants who are not breastfed to promote their development. Therefore, the aim of this study was to determine the effect of an infant formula supplemented with AA and DHA on fatty acid levels of three-month-old infants with different FADS genotypes.

#### **2. Materials and Methods**

## *2.1. Ethics Statement*

This study was carried out in accordance with the ethical standards established by the Declaration of Helsinki (2004), the Good Clinical Practice recommendations of the EEC (document 111/3976/88 July 1990) and the current Spanish legislation governing clinical research in humans (Royal Decree 561/1993 on clinical trials). In addition, the study was approved by the San Cecilio University Hospital Ethics Committee and the Faculty of Medicine at the University of Granada.

#### *2.2. Study Population and Design*

We analyzed 176 infants from the total of 220 participants in the COGNIS study (A Neurocognitive and Immunological Study of a New Formula for Healthy Infants), which is an interventional, randomized, and double-blinded study registered at www.ClinicalTrials.gov (NCT02094547). Full-term infants were recruited at the University Hospitals (Clinical San Cecilio and Mother-Infant Hospital) in the city of Granada (Spain), where samples and data were also collected. Each parent or legal guardian signed a written informed consent before the recruitment. To be eligible for enrolment of COGNIS study, infants had to meet the following inclusion criteria: 0–2 month old full-term infants, adequate birth weight for gestational age, normal Apgar score, umbilical pH ≥ 7.10 (normal range), availability to continue throughout the entire study period, signed informed consent, and for infants in the SF and EF groups, a maximum of 30 days of exclusive breastfeeding was considered and a minimum of 70% of infant formula consumption was required afterwards. Participants were excluded if they were participating in other studies, if they had nervous system or gastrointestinal disorders, and if the mother had a disease history or had received harmful drug treatment during pregnancy. Questionnaires and medical records were used to obtain maternal characteristics, including maternal age, gestational age, pre-pregnancy BMI, pre-pregnancy weight, smoking status during pregnancy, educational level, and Edinburgh scale. Likewise, infant characteristics such as gender, birth weight, birth length, and birth head circumference were obtained. The study design and information of the COGNIS participants are given in Figure 1.

After inclusion, infants were randomly allocated to the Standard Formula (SF) or the Experimental Formula (EF) group. Later, a third group was added with infants who were Exclusively Breastfed (BF) for at least 2 months to function as the control group.

After infant randomization, the research team decided to withdraw from the trial those infants who met the following criteria: Infants fed with infant formulas unrelated to COGNIS study, breastfed infants with formula intake >25% before 6 months, formula fed infants with human milk intakes higher than 25% beyond the 3rd month of life, any adverse event that could interfere with study follow-up, cow's milk protein allergy/intolerance or lactose intolerance, infant formula intake rejection or neurological disorder.

All infants from COGNIS study were followed up at 2, 3, 6, 12, 18 months of life and 2.5 years of age. During the follow-up visits, and depending on the subject age, different assessment procedures and data collection were carried out; however, those will be explored elsewhere. Nevertheless, for the purpose to obtain a better control of records avoiding possible twists on data due to factors that involved other time lines, such as mixed feeding in the 1st month of life or initiation of complementary feeding at 6 months, this study only takes into account infants from the second visit (3 months of age).

**Figure 1.** Participants in the COGNIS study and classification following type of feeding.
