title: High quality RNA in semen and sperm: isolation, analysis and potential application in clinical testing.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Georgiadis
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Archana
authors.initials: A
authors.lastname: Kishore
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Michelle
authors.initials: M
authors.lastname: Zorrilla
authors.affiliation: Department of Urology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Jaffe
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Sanfilippo
authors.affiliation: Magee-Womens Hospital, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.
authors.forename: Etta
authors.initials: E
authors.lastname: Volk
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Molecular and Cellular Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Human Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Rajkovic
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. Electronic address: yatsenkoan@mwri.magee.edu.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Yatsenko
mesh_terms: D005307:Fertilization in Vitro; D006801:Humans; D007248:Infertility, Male / Q000175:diagnosis*; D008297:Male; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification*; D012661:Semen / Q000737:chemistry*; D013094:Spermatozoa / Q000737:chemistry*
chemical_list: D012313:RNA
keywords: RNA; biological markers; fertilization; infertility; male; spermatozoa
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