Sleep disorders are prevalent occupational health problems among shift workers, especially healthcare workers with long shifts. Serotonin is a neurotransmitter related to circadian variations accompanied by shift work. A cross-sectional study was performed on 73 nurses at a tertiary hospital in Cairo, Egypt, to assess sleep quality among shift work nurses (SWNs), to determine blood serotonin level, and its relation to shift work and sleep quality. A demographic and occupational history questionnaire, Pittsburgh Sleep Quality Index (PSQI) questionnaire, and measurement of blood serotonin were carried out to the studied group. The data were analyzed using SPSS 25, and descriptive statistics, unpaired t-test, ANOVA, Kruskal–Wallis Test, Chi-square, Spearman correlation, and multivariate regression analysis were utilized. The results showed that the mean PSQI global score was significantly higher among SWNs than non-shift work nurses (NSWNs) and was the highest (10.32 ± 3.56 and 10.22 ± 2.4, respectively) among rotatory and fixed night shift nurses. Blood serotonin showed highly significant differences between SWNs over NSWNs (p = 0.001), and mostly reduced among rotatory and fixed night shift nurses (66.7% and 65%, respectively). Moreover, there were highly significant differences in serotonin levels between poor and good sleep quality nurses (p < 0.001), and most of the poor sleep quality nurses (62.7%) had low serotonin levels. Abnormal serotonin level (odds = 246.5) and working years (odds = 1.2) were statistically significant predictors of poor sleep quality. In conclusion, SWNs, especially rotating and night shift nurses, suffer from poor sleep quality associated with abnormal levels of blood serotonin.

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