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

PURPOSE: Male infertility is a complex health condition. To our knowledge there are no molecular biomarkers of male infertility. Sperm RNA is a potential biomarker for detecting sperm abnormalities and viability at infertility clinics. However, RNA use is hindered by its inconsistent quantity, quality, multiple cell types in semen and condensed sperm structure. MATERIALS AND METHODS: We tested the usefulness of high quality RNA isolated from mature sperm and whole semen by our protocol, which reduces RNA degradation by maintaining semen and protocol components at 37 C and decreasing processing time. We isolated RNA from 83 whole semen samples, 18 samples of motile sperm prepared by the swim-up protocol and 18 of sperm prepared by the standard Percoll gradient method. RESULTS: Electrophoretic and spectral analysis of RNA revealed high quality 18S and 28S rRNAs in 71 of 83 whole semen samples (86%) and 15 of 18 mature sperm swim-up samples (83%). However, high quality RNA was isolated from only 7 of 18 Percoll gradient sperm samples (39%). Interestingly quantitative reverse transcriptase-polymerase chain reaction analysis of 4 somatic and 10 germ cell markers showed that whole semen and swim-up samples had similar RNA profiles. RNA sequencing revealed that most encoded proteins were involved in mature sperm function, regulation of DNA replication, transcription, translation, cell cycle and embryo development. CONCLUSIONS: 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 of the in vitro fertilization prognosis.

Testicular touch preparation cytology in the evaluation of male infertility.

BACKGROUND: Male infertility is traditionally evaluated by tissue core biopsies of the testes. Touch preparations (TP) of these biopsies have been infrequently used. The aim of this study is to report our experience with using testicular biopsy TP for the evaluation of male infertility. MATERIALS AND METHODS: A retrospective search was performed for cases of testes biopsies with concurrent TP. These cases were evaluated for clinical information, specimen adequacy, and cytological-histological correlation. RESULTS: A total of 39 cases were identified from men with a mean age of 34 years (range 23 to 50 years). TP slides were satisfactory for evaluation in 31 (89%) cases, and less than optimal in four due to low cellularity, obscuring blood or air drying artifact. Cytopathology showed concordance with the biopsy in almost all cases. In one discordant case where the biopsies showed no active spermatogenesis, a rare sperm were identified on the TP. CONCLUSIONS: TP of the testis is a helpful adjunct to biopsy because of its ability to clearly evaluate all stages of spermatogenesis. These data demonstrate that TP cytopathology of the testes in our experience has an excellent correlation with both normal testicular biopsies and those showing pathological spermatogenesis, and in rare cases may provide added benefit in evaluating the presence of spermatogenesis for male infertility. Albeit uncommon, cytopathologists may be required to identify and evaluate spermatogenic elements in cytology specimens being submitted from men with infertility.