title: Validation-verification of a highly effective, practical human testicular tissue in vitro culture-cryopreservation procedure aimed to optimize pre-freeze and post-thaw motility.
abstract: OBJECTIVE
  The aim of our paper was to validate a testicular biopsy procedure that simplifies handling, processing, and cryopreservation, while at the same time optimizes sperm motility before freezing and after thawing.
  
  
  METHODS
  Two prospective studies were conducted to verify, optimize, and understand the virtues of pre-freeze testicular tissue IVC at different temperatures (21, 30, or 37 °C). Testicular tissue was obtained from clinical specimens designated for whole tissue cryopreservation (i.e., intact mass of tubules) and/or for fresh use in IVF-ICSI cycles. Whole testicular biopsy pieces (1-3 mm(3)) were diluted in glycerol containing freeze solutions, slow cooled to 4 °C and then rapidly frozen in LN2 vapor. Fresh and post-thaw testicular biopsy tissue were evaluated for changes in the quantity (%) and pattern of motility (I-IV: twitching to rapid progression, respectively) over a 1 week duration. The clinical effectiveness of IVC-cryopreserved whole testicular biopsy tissue was also validated analyzing fresh embryo transfers.
  
  
  RESULTS
  More reliable recovery of motile testicular sperm was achieved using whole tissue freeze preservation combined with IVC (24-96 h) post-acquisition at an incubation temperature of 30 °C compared to ambient temperature (21 °C) or 37 °C. Up to 85 % of the pre-freeze motility was conserved post-thaw (+3 h) for easy ICSI selection. Sperm longevity was optimized to fresh tissue levels by implementing testicular biopsy sucrose dilution post-thaw. Favorable clinical outcomes were proven using frozen-thawed testicular biopsy sperm for ICSI.
  
  
  CONCLUSIONS
  By employing minimal tissue manipulation, integrating pre-freeze IVC processing at 30 °C and the freezing of whole testicular biopsy tissue, we have reduced the labor and improved the efficacy of processing testicular tissue for freeze-preservation and subsequent ICSI use.
authors: Ovagen Fertility/Southern California Institute for Reproductive Sciences (SCIRS), Newport Beach, CA, USA. mcschiewe@verizon.net.|California Cryobank (CCB), Los Angeles, CA, USA. mcschiewe@verizon.net.
authors: M C
authors: http://orcid.org/0000-0002-2979-2932
authors: MC
authors: Schiewe
authors: California Cryobank (CCB), Los Angeles, CA, USA.|Center for Male Reproduction and Vasectomy Reversal, Los Angeles, CA, USA.
authors: C
authors: C
authors: Rothman
authors: Department of Urology, University of California, Irvine (UCI), CA, USA.
authors: A
authors: A
authors: Spitz
authors: Center for Male Reproduction and Vasectomy Reversal, Los Angeles, CA, USA.
authors: P E
authors: PE
authors: Werthman
authors: Department of Urology, University of California, Los Angeles (UCLA), CA, USA.
authors: S I
authors: SI
authors: Zeitlin
authors: Southern California Center for Reproductive Medicine (SCCRM), Newport Beach, CA, USA.
authors: R E
authors: RE
authors: Anderson
mesh_terms: D015925:Cryopreservation / Q000379:methods*; D005307:Fertilization in Vitro / Q000379:methods*; D005615:Freezing; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods; D008297:Male; D009845:Oligospermia / Q000503:physiopathology; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000473:pathology / Q000502:physiology; D013395:Sucrose / Q000494:pharmacology; D013737:Testis / Q000473:pathology / Q000502:physiology*
chemical_list: D013395:Sucrose
keywords: In vitro culture (IVC); Motility; Sperm cryopreservation; TESE; Testicular biopsy