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OBJECTIVE To investigate microRNA (miRNA) expression profiles in the seminal plasma of nonobstructive azoospermia (NOA) patients with different histopathologic patterns and evaluate potential noninvasive diagnostic biomarkers of NOA. DESIGN Sequencing and validation using quantitative reverse transcription polymerase chain reaction (qRT-PCR). SETTING Reproductive center and research institute. PATIENT(S) Thirteen patients with NOA (7 Sertoli cell-only syndrome [SCOS] and 6 hypospermatogenesis to spermatogenesis arrest [SA]) and 7 normal fertile controls for sequencing, six samples per group for validation; 54 patients with NOA (27 SCOS and 27 SA) and 19 normal fertile controls for large-sample qRT-PCR analysis. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) MicroRNA expression profiles in the seminal plasma of patients with NOA with different histopathologic patterns were assessed using high-throughput sequencing and validated using qRT-PCR. RESULT(S) There were 78 overexpressed and 132 underexpressed miRNAs in patients with SCOS and 32 up-regulated and 90 down-regulated miRNAs in patients with SA compared with fertile men with normozoospermia. Two down-regulated and one up-regulated miRNA were validated using qRT-PCR, which indicated that the qRT-PCR and sequencing results were basically consistent. Hsa-miR-34c-5p expression was significantly lower in the seminal plasma of patients with NOA than normal fertile controls. The area under the receiver operating characteristic curve(AUC) for hsa-miR-34c-5p was 0.979 and 0.987 in the seminal plasma of patients with SA and patients with SCOS, respectively, compared with normal fertile controls. The AUC was 0.799 for hsa-miR-34c-5p in the seminal plasma between patients with SA and patients with SCOS. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed miRNA target genes revealed that the Notch signaling pathway was one of the most abundant signaling pathways. The expression of Hes5, an effector of the Notch signaling pathway, was significantly higher in the seminal plasma of patients with NOA than normal fertile controls. CONCLUSION(S) MicroRNA expression profiles in seminal plasma were altered in patients with NOA compared with normal fertile controls. The profiles differed in patients with NOA with different pathologic patterns. We speculate that miR-34c-5p in seminal plasma could be a potential noninvasive biomarker to diagnose patients with NOA and distinguish different pathologic types of NOA. The Notch signaling pathway may be involved in the pathogenesis of NOA.