High-quality RNA in semen and sperm: Isolation, analysis, and potential application in clinical testing.

Journal of Urology (White)

PubMedID: 25088949

Georgiadis AP, Kishore A, Zorrilla M, Jaffe TM, Sanfilippo JS, Volk E, Rajkovic A, Yatsenko AN. High-quality RNA in semen and sperm: Isolation, analysis, and potential application in clinical testing. J Urol. 2015;193(1):352-9.
PURPOSE
Male infertility is a complex health condition. There are no molecular biomarkers of male infertility. Sperm RNA is a potential biomarker for detecting abnormalities and viability of sperm in infertility clinics. However, the use of RNA is hindered by its inconsistent quantity, quality, multiple cell types in semen, and condensed sperm structure.

METHODS
We tested the utility of high-quality RNA isolated from mature sperm and whole semen (WS) by our protocol, which reduces RNA degradation by maintaining semen and protocol components at 37°C and decreasing the processing time. We isolated RNA from WS (n=83), motile sperm prepared by the swim-up (SU) protocol (n=18), and sperm prepared by standard Percoll gradient (PG) method (n=18).

RESULTS
Electrophoretic and spectral analysis of RNA revealed high-quality 18S and 28S rRNAs in the majority of WS (71/83, 86%) and mature sperm SU samples (15/18, 83%). However, high-quality RNA was isolated from a minority of PG sperm (7/18, 39%). Interestingly, our quantitative RT-PCR analyses of 4 somatic and 10 germ cell markers show that WS and SU samples have similar RNA profiles. RNA sequencing revealed that a majority of encoded proteins are involved in mature sperm function, regulation of DNA replication, transcription, translation, cell cycle, and embryo development.

CONCLUSION
We believe that semen- and sperm-specific RNAs are highly informative biomarkers for germ cell stages and somatic cell contribution. Therefore, these RNAs could be valuable diagnostic indicators of sperm survival, fertilization, and early embryogenesis, and could serve as a predictor for in vitro fertilization prognoses.