Selection and Evaluation of Optimal Reference Genes for Quantitative Reverse Transcription-Polymerase Chain Reaction Analyses of Gene Expression in Human Spermatozoa

Objective: Optimal reference genes are critical for accurate normalization and reliable interpretation of gene expression quantification data. Recently, several strategies have been utilized for validating reference genes in different human tissues. However, no universal reference genes have been described that accurately summarize transcriptional activity in human spermatozoa. Methods: Using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), we evaluated ten commonly used candidate reference genes between two groups of human cryopreserved donor sperm with different pregnancy rates. We assessed the stability of reference genes using three different algorithms, namely geNorm, NormFinder, and BestKeeper. We then identified the most stable reference genes. Results: Male-enhanced antigen 1 (MEA1) was identified as the most stably expressed reference gene, followed by testis-enhanced gene transcript (TEGT). Conclusions: We comprehensively identified MEA1 and TEGT as the most stably expressed reference genes for the normalization of gene expression data in human spermatozoa.