Design matters: structural variation in experimental huts significantly alters entomological endpoints in insecticide treated net (ITN) evaluation -a comparative experimental hut trial using a latin s

The liver, as a central organ in glucose metabolism, is closely linked to the risk of gestational diabetes mellitus (GDM) (–). Early-pregnancy serum hepatic enzymes—including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase (GGT)—have been extensively investigated as potential biomarkers for GDM prediction. However, a persistent scientific puzzle remains: GGT consistently exhibits robust associations with GDM across diverse populations (, ), whereas the associations of ALT and AST with GDM have proven remarkably heterogeneous, with some studies identifying them as significant risk factors and others reporting weak or null associations (–). Of note, a Mendelian randomization study highlights this puzzle: it identified ALT as a causal determinant of GDM yet found no causal effect of GGT (). This discordance suggests that the enzyme-GDM associations may be interrelated rather than independent, and that inconsistent findings across studies may partly reflect the statistical interdependence of these biomarkers. Previous studies, while recognizing the distinct biological functions of these three enzymes, have largely treated them as parallel, independent variables without exploring their potential interdependencies. Although all three enzymes originate from the liver, their biological functions differ substantially: ALT is predominantly cytosolic and serves as a specific marker of hepatocellular integrity (, ); AST exists as both cytosolic and mitochondrial isoforms, with the mitochondrial component (GOT2) directly participating in the malate-aspartate shuttle and cellular energy metabolism (–); GGT, meanwhile, is a critical enzyme in glutathione metabolism and serves as a sensitive indicator of oxidative stress and hepatobiliary dysfunction (–). This functional heterogeneity suggests that these enzymes may occupy distinct roles within an interconnected physiological system, warranting investigation beyond isolated analyses. Of particular relevance, oxidative stress is a key pathophysiological pathway in GDM (, ). As a sensitive marker of oxidative stress and hepatobiliary function, GGT may therefore be biologically positioned to integrate metabolic signals relevant to GDM risk. Accordingly, we hypothesize that inconsistent findings across prior studies may reflect statistical interdependencies among hepatic enzymes—including potential statistical interactions—rather than independent parallel associations. Given GGT’s role in oxidative stress, we explored whether it might statistically account for the associations of ALT and AST with GDM. We examined this hypothesis in a retrospective cohort of 9,077 singleton pregnancies using an exploratory analytical approach.