Multi‐stage screening cell‐free seminal mRNAs to diagnose completion of meiosis and predict testicular sperm retrieval in men with non‐obstructive azoospermia

Differential diagnosis of men with subtypes of non-obstructive azoospermia (NOA) is important for their treatment. Many genes are transcripted during meiosis. We hypothesized that some of these genes can be detected in cell-free seminal message RNA (mRNAs) (cfs-mRNA) and be developed as non-invasive biomarkers for diagnosing NOA subtypes.To screen cfs-mRNA to diagnose the completion of meiosis and predict successful sperm retrieval (SR) in men with NOA.NOA patients who visited our institutes from September 2018 to December 2020 for testicular histopathological diagnosis (n = 109) or testicular SR (n = 92) were screened for participation in the study. Microarray and real-time quantitative polymerase chain reaction were used in five stages to obtain candidate cfs-mRNAs for comparisons between patients with early maturation arrest (eMA, meiosis not completed) and late MA or hypospermatogenesis (meiosis completed) and between NOA patients with successful SR and SR failure.Twelve cfs-mRNAs were selected based on this comparison between men with eMA and hypospermatogenesis and their gene expression and function information. Of these, AKAP1, BOLL, TCP11 and SETX predominantly derived from testes and germ cells were proposed as candidate cfs-mRNAs. Further quantification in men with NOA demonstrated significantly higher levels of BOLL cfs-mRNA (p < 0.0001) in men with late MA or hypospermatogenesis (n = 23), compared with men with eMA (n = 51); and significantly higher levels (p < 0.0001) in patients with successful SR (n = 44) when compared with patients with SR failure (n = 37). Interestingly, with a similar cutoff value, BOLL cfs-mRNA showed high sensitivity and specificity in diagnosing late MA and hypospermatogenesis (>404 copies/ml) and predicting successful SR (>415 copies/ml). Correlation of BOLL mRNA levels was observed in paired semen and testicular tissues.We propose that BOLL cfs-mRNA is a promising non-invasive marker for diagnosing the completion of meiosis and predicting successful testicular SR in men with NOA.