title: Optimizing human semen cryopreservation by reducing test vial volume and repetitive test vial sampling.
abstract: OBJECTIVE
  To investigate optimal test vial (TV) volume, utility and reliability of TVs, intermediate temperature exposure (-88°C to -93°C) before cryostorage, cryostorage in nitrogen vapor (VN2) and liquid nitrogen (LN2), and long-term stability of VN2 cryostorage of human semen.
  
  
  METHODS
  Prospective clinical laboratory study.
  
  
  METHODS
  University assisted reproductive technology (ART) laboratory.
  
  
  METHODS
  A total of 594 patients undergoing semen analysis and cryopreservation.
  
  
  METHODS
  Semen analysis, cryopreservation with different intermediate steps and in different volumes (50-1,000 μL), and long-term storage in LN2 or VN2.
  
  
  METHODS
  Optimal TV volume, prediction of cryosurvival (CS) in ART procedure vials (ARTVs) with pre-freeze semen parameters and TV CS, post-thaw motility after two- or three-step semen cryopreservation and cryostorage in VN2 and LN2.
  
  
  RESULTS
  Test vial volume of 50 μL yielded lower CS than other volumes tested. Cryosurvival of 100 μL was similar to that of larger volumes tested. An intermediate temperature exposure (-88°C to -93°C for 20 minutes) during cryopreservation did not affect post-thaw motility. Cryosurvival of TVs and ARTVs from the same ejaculate were similar. Cryosurvival of the first TV in a series of cryopreserved ejaculates was similar to and correlated with that of TVs from different ejaculates within the same patient. Cryosurvival of the first TV was correlated with subsequent ARTVs. Long-term cryostorage in VN2 did not affect CS.
  
  
  CONCLUSIONS
  This study provides experimental evidence for use of a single 100 μL TV per patient to predict CS when freezing multiple ejaculates over a short period of time (<10 days). Additionally, semen cryostorage in VN2 provides a stable and safe environment over time.
authors.affiliation: Department of Urology, University of Michigan, Ann Arbor, Michigan; Department of Urology, Herlev University Hospital, Herlev, Denmark; Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Christian F S
authors.initials: CF
authors.lastname: Jensen
authors.affiliation: Department of Urology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Dana A
authors.initials: DA
authors.lastname: Ohl
authors.affiliation: Department of Urology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Walter R
authors.initials: WR
authors.lastname: Parker
authors.affiliation: Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Andre M
authors.initials: AM
authors.lastname: da Rocha
authors.affiliation: Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Keller
authors.affiliation: Department of Urology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Schuster
authors.affiliation: Department of Urology, Herlev University Hospital, Herlev, Denmark; Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Sonksen
authors.affiliation: Department of Urology, University of Michigan, Ann Arbor, Michigan; Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan; Department of Physiology, University of Michigan, Ann Arbor, Michigan. Electronic address: smithgd@umich.edu.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Smith
mesh_terms: D002138:Calibration; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods* / Q000592:standards; D006801:Humans; D008297:Male; D012661:Semen / Q000166:cytology / Q000502:physiology; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods* / Q000592:standards; D013048:Specimen Handling / Q000379:methods*; D053625:Sperm Retrieval / Q000592:standards; D013696:Temperature; D013997:Time Factors
keywords: Semen cryopreservation; fertility preservation; liquid nitrogen vapor; test vial