Evaluation of housekeeping gene stability in placental tissues: comparative expression studies in first-trimester, preeclamptic and control placenta

Molecular studies of the human placenta may reveal gene expression alterations responsible for impaired placentation and the subsequent development of pregnancy complications ( e.g. , preeclampsia, fetal-growth restriction, pre-term birth) characterised by a significantly increased risk of maternal and newborn mortality. Accurate qPCR normalization is crucial in placental research; however, reference gene stability across different gestational stages, pathological conditions, and experimental models remains poorly validated. This study systematically evaluated six candidate housekeeping genes ( ACTB , GAPDH , HPRT1 , RPS18 , TBP , YWHAZ ) in human placental tissues (first/third-trimester, preeclampsia) and trophoblast cell lines (BeWo, HTR-8/SVneo, JAR, JEG-3). Expression stability was assessed using four algorithms: Delta-Ct, BestKeeper, NormFinder, and RefFinder. Validation was performed by analyzing target genes ( CD46 , PGF , HLA-G , CD93 ). Strong transcriptional divergence was identified between tissue samples and cell lines; restricting the analysis to placental tissue samples significantly improved stability values. Among the six candidate genes tested, the RefFinder comprehensive ranking identified GAPDH and HPRT1 as the most stable reference pair for placental tissues. Normalization with this pair successfully detected significant differences in CD93 expression across groups, whereas the least stable combination ( TBP/YWHAZ ) failed to do so. The geometric mean of GAPDH and HPRT1 provides a robust normalization strategy among the candidates evaluated here for qPCR studies in human placental tissues across physiological and pathological conditions.