*3.3. Certainty of Evidence (GRADE)*

The certainty of the evidence was overall low to very low, as there were problems with inaccurate effect estimates in small studies (very serious risk of imprecision) as well as serious risk of bias. The risk of bias was primarily due to problems with blinding of the observers that assessed the effect (parent and teachers). However, the studies generally suffered from methodological flaws and were largely poorly described, especially regarding how the randomization sequence was generated, how the allocation was concealed, and how incomplete data were handled.

#### **4. Discussion**

The objective of this systematic literature review and meta-analysis was to provide clinicians, caregivers and guideline panels with an updated overview and critical assessment of the evidence, investigating the effect of PUFA among children aged 6 to 18 years diagnosed with ADHD. Based on a substantial body of evidence, there is no indication that

supplementation with PUFA has a positive effect on core symptoms of ADHD, behavioral difficulties, or quality of life. The present review suggests that there is no substantial increase in side effects following the use of PUFA, including the occurrence of diarrhea, gastrointestinal discomfort, or nausea.

Based on our findings, there is insufficient evidence to support patients, parents, clinicians, and caregivers in their decision on whether to use PUFA as a supplementation in the treatment of ADHD. Consequently, the patient preferences are expected to be unambiguous, in the sense that some parents will prefer dietary changes rather than pharmacological treatment, and other parents will find it difficult and relatively intrusive to implement dietary changes in children and adolescents. Effective treatment with PUFA would supposedly depend on the presence of an initial PUFA deficiency observed in the patient at baseline. A significant decrease in PUFA levels has indeed been observed in patients with ADHD as compared to healthy controls [57]. However, it still needs to be investigated what role PUFA plays in the pathology of ADHD. It remains unknown whether PUFA deficiency represents a neuropathological finding directly potentiating symptom outbreak, or rather a compensatory mechanism due to long-standing disease manifestation. In addition, an evaluation of the long-term effects of providing PUFA as a supplement is needed. For now, the effect has only been tested in patients over a time period ranging from 8 weeks to 12 months.

The amount of RCTs investigating the use of PUFA as a treatment for ADHD has increased since the latest Cochrane review by Gilles et al. was published in 2012 [58]. Despite an increase in the number of available studies, the conclusion and quality of the evidence remains unchanged. Thus, the evidence still indicates an inability of PUFA to effectively alter the symptomatology of ADHD. The evidence remains of a low to very low quality, thus reflecting a high degree of uncertainty of the effect estimates.

The risk of bias in the identified studies includes an inadequate random sequence generation and allocation concealment, which in RCTs is mandatory to ensure that intervention and control groups are kept as identical as possible. Thus, the inability to perform sufficient random allocation may induce systematic errors, which may have a considerable impact on the final results. Other major sources of bias were due to incomplete outcome reporting, thus reflecting an increased risk of reporting bias and attrition. Regarding incomplete data, a review has previously described in which problems with drop-out seem to be common in n-3 long chain PUFA supplementation trials in children and adolescents in general [59]. This indicates that dropouts may be a common, inherent issue when seeking to investigate the effect of PUFA in a research setting.

Blinding of the observers evaluating the effect is essential. The included studies generally displayed an unclear level of blinding, as blinding in the majority of studies was inadequately described. Blinding of participants may once again constitute an inherent problem when investigating the effect of PUFA due to the distinct smell and taste [60]. This may unmask the allocation to the respective groups, as patients and parents over time may become aware if they indeed are receiving the active PUFA component. Nevertheless, adequate blinding of observers not directly related to the child should be possible, including the researcher and teachers. Collectively, these problems lead to a high risk of bias, which may essentially have an impact on the results and thereby lead to a distortion in the final conclusion.

Our findings displaying an inadequate effect of PUFA in the treatment of ADHD is in line with many previous reviews on the subject [57,61–65], but not all [66–69].

When comparing our review to others reporting a beneficial effect, it becomes evident that there is a discrepancy in the methodology used to evaluate treatment effects, which may explain the discrepancy in results. In three of the reviews [66,67,69], no specific analysis to obtain pooled estimate of effects was performed, as results from the individual trials were only narratively described. This prevents a direct comparison between trials, including an assessment of the overall magnitude of effect. Common for all reviews reporting a positive effect was a lack of quality assessment of the included studies. This hinders an evaluation of the extent of trust in the data and may essentially mask some issues that otherwise would lead to a downscaling of the certainty in the evidence, and thereby the confidence in the results. It should be mentioned, apart from ADHD core symptoms, the reviews reporting a positive effect also include other outcomes not evaluated in the present review. The inclusion and exclusion criteria furthermore varied across the reviews. This may in part also explain some of the discrepancy in the reported results.

As mentioned above, several inherent methodological issues due indeed exist when it comes to investigating the effect of PUFA in research settings. Nevertheless, this does not unconditionally prevent the possibility of performing high-quality research on the matter. As such, future research should focus on conducting clinical trials of high-quality, following the CONSORT (Consolidated Standards of Reporting Trials) Statement [70]. This is essential if we wish to move forward and be able to conclusively evaluate the role of PUFA in the treatment of ADHD.
