In this large observational study of infants between 9 and 24 months old, we found that one out of every five infants has AN. Interestingly, AN in the knuckles was positive in all cases and only a minority of patients (2.2%) had AN in other more classical sites (i.e., neck, axillae). Infants with AN were significantly associated with a higher HOMA-IR, fasting plasma glucose, and serum insulin levels; and to our knowledge, for the first time in the literature, we reliably report a ≥ 90th (≥ 1.04) and ≥ 95th (≥ 1.27) percentile cutoff for HOMA-IR in infants. AN in the knuckles had a high sensitivity (84–90%) and negative predictive value (98–99%) for both ≥ 90th and ≥ 95th HOMA-IR percentiles.
Previous studies have assessed AN in children at age 2 years and older and have reported a range of 4.7–19% with a higher prevalence in overweight and obese children (up to 55%) [20, 26,27,28]. Nevertheless, these studies often limit their assessment of AN to the neck, with a lack of a high inter-observer reliability. Moreover, most of these did not evaluate its association with biochemical markers of IR. The prevalence of AN in our study population was high (21.6%) with the knuckles being the main contributor to this result. When the knuckles and other sites were considered, AN prevalence was only 2.2%. This finding resonates with previous literature in which, in a healthy population of young Latino adults (18–23 years old), the knuckles were the most prevalent site of AN; hence, this suggests that AN in the knuckles might be one of the earliest clinical signs of IR (however, often ignored because of the false notion that the neck or axillae are more common sites) and of paramount importance to direct efforts to the most needed (i.e., individuals at high metabolic risk) in whom early implementation of lifestyle changes could have a profound impact [15,16,17]. This finding is also supported by the fact that AN seems to progress with time as the prevalence found at ages 18–23 years was around 50% (compared to 21% in our study) . Hence, exclusion of the knuckles may delay the clinical diagnosis of IR until its appearance in classical and well-known locations of AN (i.e., the neck or axillae), thereby losing invaluable time to start management of IR.
To date, the association between AN presence and IR in children has been scarcely studied. Kobaissi et al.  aimed to describe the clinical value of AN as a predictor of insulin sensitivity in overweight Hispanic children (8–13 years), concluding that BMI variation was the main predictor of insulin sensitivity, whereas the presence of AN explained only 4% of the estimated variation. Yet children were reported (by a single observer) with a high prevalence of AN (73.3%). The exclusion of patients with normal BMI limits the applicability of AN to a very specific population . In contrast, our study evaluated with high inter-observer reliability the value of AN as a marker of IR at age 2 or younger, independently of their BMI, ensuring its applicability in a boarder range of individuals. Furthermore, we found higher HOMA-IR index values and basal serum insulin levels in participants with AN in the knuckles compared to controls. This suggests that AN is an early and reliable clinical sign to detect patients at high metabolic risk prior to any other usual clinical manifestations (e.g., weight gain, hyperglycemia, hypertension).
Our reported high sensitivity (84–90%) and negative predictive value (> 98%) of AN on the knuckles set this clinical sign as an accurate, non-invasive, and straightforward marker of IR early in life. In perspective, if AN on the knuckles is not detected in a child, he or she would have a greater than 98% probability of not having IR using both HOMA-IR 90th and 95th percentiles cutoff values. On the other hand, according to our AN prevalence (21.6%) and with our reported diagnostic performance, around 80% of the total population at this early stage of life could reliably and easily be recognized as not having IR.
Until now, few studies have described the HOMA-IR index in children and reliable cutoff values have not been widely accepted. Borgoño et al.  compared the HOMA-IR index of 1-year-old children born from mothers with and without GDM and concluded that for infants born from women with GDM, weight gain was positively associated with IR. In contrast to the results of Borgoño et al., the HOMA-IR index values reported in our study were higher (0.21 vs. 0.53, respectively). However, their population consisted mostly of Caucasian patients (75%) and non-specified race/ethnicity (25%) . Besides, the different insulin and glucose laboratory techniques could explain this variation. In fact, compared to Caucasians, higher HOMA-IR index cutoff values have been suggested for Latin-American population . Therefore, as a result of the paucity of evidence, we opted to define the HOMA-IR index cutoff value by percentile and reliably describe the 90th and 95th percentiles.
Our study has several limitations. First, as a result of the study’s design, we cannot accurately predict how higher HOMA-IR or high insulin values will impact infants later in life. However, even though this was not the aim of this study, in an ongoing study, we will follow the participants of this study and assess their complete evolution through time. In fact, the body of evidence is strong regarding the association between metabolic risk and higher HOMA-IR, insulin, and glucose values. Second, we used HOMA-IR to define IR; however, as a result of our large study sample, performing a euglycemic insulin clamp would not have been feasible because of its technical complexity. Moreover, the euglycemic insulin clamp has not been completely validated in this population. At the same time, we followed a strict protocol and, to our knowledge, this is the first study in a large number of infants with a high inter-observer reliability to evaluate AN prevalence, its association with IR, and AN diagnostic accuracy for IR.