title: Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.
abstract: OBJECTIVE
  Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules.
  
  
  METHODS
  After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy.
  
  
  RESULTS
  Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia.
  
  
  CONCLUSIONS
  Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate.
authors.affiliation: Department of Urology, University of Virginia, P.O. Box 800422, Charlottesville, Virginia 22908-0422, USA. rps2k@virginia.edu
authors.forename: Ryan P
authors.initials: RP
authors.lastname: Smith
authors.forename: Greg J
authors.initials: GJ
authors.lastname: Lowe
authors.forename: Parviz K
authors.initials: PK
authors.lastname: Kavoussi
authors.forename: William D
authors.initials: WD
authors.lastname: Steers
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Costabile
authors.forename: John C
authors.initials: JC
authors.lastname: Herr
authors.forename: Jagathpala
authors.initials: J
authors.lastname: Shetty
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Lysiak
mesh_terms: D000818:Animals; D053713:Azoospermia / Q000139:chemically induced; D002066:Busulfan / Q000009:adverse effects; D004195:Disease Models, Animal; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection / Q000379:methods*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D012671:Seminiferous Tubules / Q000378:metabolism; D053625:Sperm Retrieval; D013737:Testis / Q000166:cytology*; D020858:Tissue and Organ Harvesting / Q000379:methods
chemical_list: D002066:Busulfan