Individual differences in haemoglobin concentration influence bold fMRI functional connectivity and its correlation with cognition

Resting-state connectivity measures the temporal coherence of the spontaneous neural activity of spatially distinct regions, and is commonly measured using BOLD-fMRI. The BOLD response follows neuronal activity, when changes in the relative concentration of oxygenated and deoxygenated haemoglobin cause fluctuations in the MRI T2* signal. Since the BOLD signal detects changes in relative concentrations of oxy/deoxy-haemoglobin, individual differences in haemoglobin levels may influence the BOLD signal-to-noise ratio in a manner independent of the degree of neural activity. In this study, we examined whether group differences in haemoglobin may confound measures of functional connectivity. We investigated whether relationships between measures of functional connectivity and cognitive performance could be influenced by individual variability in haemoglobin. Finally, we mapped the neuroanatomical distribution of the influence of haemoglobin on functional connectivity to determine where group differences in functional connectivity are manifest.In a cohort of 518 healthy elderly subjects (259 men), each sex group was median-split into two groups with high and low haemoglobin concentration. Significant differences were obtained in functional connectivity between the high and low haemoglobin groups for both men and women (Cohen's d 0.17 and 0.03 for men and women respectively). The haemoglobin connectome in males showed a widespread systematic increase in functional connectivity correlation values, whilst the female connectome showed predominantly parietal and subcortical increases and temporo-parietal decreases. Despite the haemoglobin groups having no differences in cognitive measures, significant differences in the linear relationships between cognitive performance and functional connectivity were obtained for all 5 cognitive tests in males, and 4 out of 5 tests in females.Our findings confirm that individual variability in haemoglobin levels that give rise to group differences are an important confounding variable in BOLD-fMRI-based studies of functional connectivity. Controlling for haemoglobin variability as a potentially confounding variable is crucial to ensure the reproducibility of human brain connectome studies, especially in studies that compare groups of individuals, compare sexes, or examine connectivity-cognition relationships.