title: Density gradient centrifugation prior to cryopreservation and hypotaurine supplementation improve post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia.
abstract: OBJECTIVE
  Can selection of spermatozoa by density gradient centrifugation prior to cryopreservation and/or hypotaurine supplementation improve the post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia?
  
  
  CONCLUSIONS
  Sperm selection by density gradient centrifugation before freezing and supplementation of the media by hypotaurine is beneficial for the cryopreservation of semen samples of patients with oligoasthenoteratozoospermia.
  
  
  BACKGROUND
  Sperm from men with oligoasthenoteratozoospermia are more susceptible than normal to cryoinjury. Density gradient centrifugation before sperm freezing may allow the selection of a subpopulation of spermatozoa more resistant to cryopreservation. Hypotaurine is an antioxidant with a protective effect on sperm functions.
  
  
  METHODS
  The experiment was carried out according to a factorial design involving two binary factors resulting in four treatment combinations which were randomly allocated in oligoasthenoteratozoospermia sperm samples from 64 patients recruited between January 2009 and June 2010.
  
  
  METHODS
  Semen was provided by 64 men undergoing evaluation for infertility at the Centre for Reproductive Medicine of the University Hospital in Clermont-Ferrand, France, between January 2009 and June 2010. Four treatment combinations were tested: sperm freezing before selection without (F-S/H-; n = 16) and with hypotaurine supplementation (F-S/H+; n = 16); sperm selection before freezing without (S-F/H-; n = 16) and with hypotaurine supplementation (S-F/H+; n = 16). Measurements of sperm recovery rates and markers of apoptosis (externalization of phosphatidylserine (PS), mitochondrial membrane potential and DNA fragmentation) were compared in recovered spermatozoa after each procedure.
  
  
  RESULTS
  Higher recovery rates of progressive and total motile spermatozoa were observed when sperm selection was performed before freezing (P < 0.05). The protective effect of hypotaurine was only observed on the percentage of live spermatozoa with PS externalization among total live spermatozoa (AN+ PI-/((AN+ PI-) + (AN- PI-)) when the sperm selection by density gradient centrifugation was performed before freezing (S-F/H+ versus S-F/H-: 6.8 ± 1.09 versus 11.8 ± 2.03%, P = 0.04). The percentage of mitochondrial membrane potential (DiOC6(3) (high)) spermatozoa was higher (P = 0.001) when sperm selection was done before freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 58.1 ± 3.50 versus 46.7 ± 5.48%) or without (S-F/H- versus F-S/H-: 57.0 ± 5.18 versus 35.4 ± 4.99%) hypotaurine supplementation. The percentages of TUNEL+ spermatozoa were significantly lower (P = 0.001) when sperm selection was done before sperm freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 38.6 ± 9.59 versus 55.7 ± 5.88%) or without hypotaurine supplementation (S-F/H- versus F-S/H-: 37.2 ± 7.91 versus 71.0 ± 5.66%).
  
  
  CONCLUSIONS
  The ICSI outcomes were not assessed and the fertility of the spermatozoa remains unknown.
  
  
  CONCLUSIONS
  Sperm selection by density gradient centrifugation before freezing and hypotaurine supplementation could improve the cryopreservation of sperm from oligoasthenoteratozoospermic men and make a larger number of functional spermatozoa available for ICSI.
  This work was supported by a hospital grant (Projet Hospitalier Recherche Clinique, CHU Clermont Ferrand, France). None of the authors has any conflict of interest to declare.
authors.affiliation: Assistance Médicale á la Procréation, CECOS, CHU Clermont Ferrand, CHU Estaing, place Aubrac, 63000 Clermont Ferrand, France. fbrugnon@chu-clermontferrand.fr
authors.forename: F
authors.initials: F
authors.lastname: Brugnon
authors.forename: L
authors.initials: L
authors.lastname: Ouchchane
authors.forename: H
authors.initials: H
authors.lastname: Pons-Rejraji
authors.forename: C
authors.initials: C
authors.lastname: Artonne
authors.forename: M
authors.initials: M
authors.lastname: Farigoule
authors.forename: L
authors.initials: L
authors.lastname: Janny
mesh_terms: D000328:Adult; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000494:pharmacology*; D006801:Humans; D007248:Infertility, Male; D008297:Male; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods*; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology; D013654:Taurine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D015415:Biomarkers; D003451:Cryoprotective Agents; D013654:Taurine; C003949:hypotaurine
keywords: Cryopreservation; apoptosis; hypotaurine; oligoasthenoteratozoospermia; spermatozoa