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Noncoding RNAs (ncRNAs) are involved in the pathological processes of various diseases. The aim of the present study was to verify the expression levels and the diagnostic value of two candidate ncRNAs in the blood leukocytes of patients with gestational diabetes mellitus (GDM) compared to healthy controls. The long ncRNA paired box 8 antisense 1 (Pax8-AS1) and the microRNA miR-4646 were selected, which were identified to be associated with GDM by bioinformatics analysis of a dataset from the Gene Expression Omnibus GEO database. By using reverse transcription-quantitative PCR, the expression levels of Pax8-AS1 and miR-4646 were analysed in leukocytes of patients with GDM (n=35) and normal pregnant females (n=35). The results indicated a significant decrease in the expression levels of both Pax8-AS1 and miR-4646 in patients with GDM as compared with those in the healthy controls. In the second trimester, a strong negative correlation between Pax8-AS1/miR-4646 and 2-h glucose levels was detected in patients with GDM. Receiver operating characteristic curve analysis indicated that the levels of Pax8-AS1 and miR-4646 in the second trimester of pregnancy had a significant diagnostic value with high selectivity and specificity for GDM (area under the curve values, 0.902 and 0.891, respectively; P